NIH GUIDELINES FOR RESEARCH

INVOLVING

RECOMBINANT OR SYNTHETIC

Nucleic Acid MOLECULES

(NIH GUIDELINES)

 

April 2016

DEPARTMENT OF HEALTH AND HUMAN SERVICES

National Institutes of Health

 

 

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Visit the NIH OSP Web site at:

http://www.osp.od.nih.gov

For current information on Guidelines, Protocols, Principal Investigators, Meetings,
and information about upcoming Gene Therapy Policy Conferences

 

NIH Office of Science Policy Contact Information:

 

Office of Science Policy, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda, MD  20892-7985 (20817 for non-USPS mail), (301) 496-9838; (301) 496-9839 (fax).

 

For inquiries, information requests, and report submissions:                    NIHGuidelines@od.nih.gov

Human gene transfer protocol submissions:                                           HGTprotocols@mail.nih.gov

 

 

These NIH Guidelines shall supersede all earlier versions until further notice.

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FEDERAL REGISTER NOTICES

 

Effective June 24, 1994, Published in Federal Register, July 5, 1994 (59 FR 34472)

Amendment Effective July 28, 1994, Federal Register, August 5, 1994 (59 FR 40170)

Amendment Effective April 17, 1995, Federal Register, April 27, 1995 (60 FR 20726)

Amendment Effective December 14, 1995, Federal Register, January 19, 1996 (61 FR 1482)

Amendment Effective March 1, 1996, Federal Register, March 12, 1996 (61 FR 10004)

Amendment Effective January 23, 1997, Federal Register, January 31, 1997 (62 FR 4782)

Amendment Effective September 30, 1997, Federal Register, October 14, 1997 (62 FR 53335)

Amendment Effective October 20, 1997, Federal Register, October 29, 1997 (62 FR 56196)

Amendment Effective October 22, 1997, Federal Register, October 31, 1997 (62 FR 59032)

Amendment Effective February 4, 1998, Federal Register, February 17, 1998 (63 FR 8052)

Amendment Effective April 30, 1998, Federal Register, May 11, 1998 (63 FR 26018)

Amendment Effective April 29, 1999, Federal Register, May 11, 1999 (64 FR 25361)

Amendment Effective October 2, 2000, Federal Register, October 10, 2000 (65 FR 60328)

Amendment Effective December 28, 2000, Federal Register, January 5, 2001 (66 FR 1146)

Amendment Effective December 11, 2001, Federal Register, December 11, 2001 (66 FR 64051)

Amendment Effective December 19, 2001, Federal Register, November 19, 2001 (66 FR 57970)

Amendment Effective January 10, 2002, Federal Register, December 11, 2001 (66 FR 64052)

Amendment Effective January 24, 2002, Federal Register, November 19, 2001 (66 FR 57970)

Amendment Effective September 22, 2009, Federal Register, September 22, 2009 (74 FR 48275)

Amendment Effective January 19, 2011, Federal Register, January 19, 2011 (76 FR 3150)

Amendment Effective May 12, 2011, Federal Register, May 12, 2011 (76 FR 27653)

Amendment Effective October 11, 2011, Federal Register, October 11, 2011 (76 FR 62816)

Amendment Effective February 21, 2013, Federal Register, February 21, 2013 (78 FR 12074)

Amendment Effective March 5, 2013, Federal Register, September 5, 2012 (77 FR 54584)

Amendment Effective November 6, 2013, Federal Register, November 6, 2013 (78 FR 66751)

Amendment Effective April 27, 2016, Federal Register, March 22, 2016 (81 FR 15315)

Amendment Effective April 15, 2016, Federal Register, April 15, 2016 (81 FR 22286)

 


 

TABLE OF CONTENTS

 

SECTION I........................ SCOPE OF THE NIH GUIDELINES. 9

Section I-A................... Purpose. 9

Section I-B................... Definition of Recombinant and Synthetic Nucleic Acid Molecules. 9

Section I-C................... General Applicability. 10

Section I-D................... Compliance with the NIH Guidelines. 10

Section I-E................... General Definitions. 11 

SECTION II....................... SAFETY CONSIDERATIONS. 12

Section II-A.................. Risk Assessment 12

Section II-A-1................ Risk Groups. 12

Section II-A-2................ Criteria for Risk Groups. 12

Section II-A-3................ Comprehensive Risk Assessment 12

Section II-B.................. Containment 13

SECTION III...................... EXPERIMENTS COVERED BY THE NIH GUIDELINES. 15

Section III-A.................. Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation (See Section IV-C-1-b-(1), Major Actions). 15

Section III-A-1............... Major Actions under the NIH Guidelines. 15

Section III-B.................. Experiments That Require NIH OSP and Institutional Biosafety Committee Approval Before Initiation. 15

Section III-B-1............... Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms per Kilogram Body Weight 16

Section III-B-2............... Experiments that have been Approved (under Section III-A-1-a) as Major Actions under the NIH Guidelines. 16

Section III-C.................. Experiments that Require Institutional Biosafety Committee and Institutional Review Board Approvals and RAC Review Before Research Participant Enrollment 16

Section III-C-1............... Experiments Involving the Deliberate Transfer of Recombinant or Synthetic Nucleic Acid Molecules, or DNA or RNA Derived from Recombinant or Synthetic Nucleic Acid Molecules, into One or More Human Research Participants. 16

Section III-D................. Experiments that Require Institutional Biosafety Committee Approval Before Initiation. 17

Section III-D-1............... Experiments Using Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems (See Section II-A, Risk Assessment) 17

Section III-D-2............... Experiments in Which DNA From Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems. 18

Section III-D-3............... Experiments Involving the Use of Infectious DNA or RNA Viruses or Defective DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems. 18

Section III-D-4............... Experiments Involving Whole Animals. 19

Section III-D-5............... Experiments Involving Whole Plants. 19

Section III-D-6............... Experiments Involving More than 10 Liters of Culture. 20

Section III-D-7............... Experiments Involving Influenza Viruses. 20

Section III-E.................. Experiments that Require Institutional Biosafety Committee Notice Simultaneous with Initiation. 21

Section III-E-1. ............. Experiments Involving the Formation of Recombinant or Synthetic Nucleic Acid Molecules Containing No More than Two-Thirds of the Genome of any Eukaryotic Virus. 22

Section III-E-2............... Experiments Involving Whole Plants. 22

Section III-E-3............... Experiments Involving Transgenic Rodents. 23

Section III-F.................. Exempt Experiments. 23

SECTION IV..................... ROLES AND RESPONSIBILITIES. 24

Section IV-A................. Policy. 24

Section IV-B................. Responsibilities of the Institution. 24

Section IV-B-1.............. General Information. 24

Section IV-B-2.............. Institutional Biosafety Committee (IBC) 25

Section IV-B-2-a........... Membership and Procedures. 25

Section IV-B-2-b........... Functions. 27

Section IV-B-3.............. Biological Safety Officer (BSO) 28

Section IV-B-4.............. Plant, Plant Pathogen, or Plant Pest Containment Expert 28

Section IV-B-5.............. Animal Containment Expert 28

Section IV-B-6.............. Human Gene Therapy Expertise. 28

Section IV-B-7.............. Principal Investigator (PI) 28

Section IV-B-7-a........... General Responsibilities. 29

Section IV-B-7-b........... Information to Be Submitted by the Principal Investigator to NIH OSP. 29

Section IV-B-7-c........... Submissions by the Principal Investigator to the Institutional Biosafety Committee. 30

Section IV-B-7-d........... Responsibilities of the Principal Investigator Prior to Initiating Research. 30

Section IV-B-7-e........... Responsibilities of the Principal Investigator During the Conduct of the Research. 30

Section IV-C................. Responsibilities of the National Institutes of Health (NIH) 31

Section IV-C-1.............. NIH Director 31

Section IV-C-1-a........... General Responsibilities. 31

Section IV-C-1-b........... Specific Responsibilities. 31

Section IV-C-1-b-(1)...... Major Actions. 31

Section IV-C-1-b-(2)...... Minor Actions. 32

Section IV-C-2.............. Recombinant DNA Advisory Committee (RAC) 32

Section IV-C-3.............. Office of Science Policy (OSP) 33

Section IV-C-4.............. Other NIH Components. 34

Section IV-D................. Voluntary Compliance. 34

Section IV-D-1.............. Basic Policy - Voluntary Compliance. 34

Section IV-D-2.............. Institutional Biosafety Committee Approval - Voluntary Compliance. 35

Section IV-D-3.............. Certification of Host-Vector Systems - Voluntary Compliance. 35

Section IV-D-4.............. Requests for Exemptions and Approvals - Voluntary Compliance. 35

Section IV-D-5.............. Protection of Proprietary Data - Voluntary Compliance. 35

Section IV-D-5-a........... General 35

Section IV-D-5-b........... Pre-submission Review. 35

SECTION V...................... FOOTNOTES AND REFERENCES OF SECTIONS I THROUGH IV. 36

APPENDIX A.................... EXEMPTIONS UNDER SECTION III-F-6--SUBLISTS OF NATURAL EXCHANGERS. 37

Appendix A-I................ Sublist A. 37

Appendix A-II............... Sublist B. 38

Appendix A-III............... Sublist C. 38

Appendix A-IV.............. Sublist D.. 38

Appendix A-V............... Sublist E. 38

Appendix A-VI.............. Sublist F. 38

APPENDIX B.................... CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF HAZARD. 38

Appendix B-I................ Risk Group 1 (RG1) Agents. 39

Appendix B-II............... Risk Group 2 (RG2) Agents. 39

Appendix B-II-A............ Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia. 39

Appendix B-II-B............ Risk Group 2 (RG2) - Fungal Agents. 40

Appendix B-II-C............ Risk Group 2 (RG2) - Parasitic Agents. 40

Appendix B-II-D............ Risk Group 2 (RG2) - Viruses. 41

Appendix B-III............... Risk Group 3 (RG3) Agents. 42

Appendix B-III-A........... Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia. 42

Appendix B-III-B........... Risk Group 3 (RG3) - Fungal Agents. 43

Appendix B-III-C........... Risk Group 3 (RG3) - Parasitic Agents. 43

Appendix B-III-D........... Risk Group 3 (RG3) - Viruses and Prions. 43

Appendix B-IV.............. Risk Group 4 (RG4) Agents. 44

Appendix B-IV-A........... Risk Group 4 (RG4) - Bacterial Agents. 44

Appendix B-IV-B........... Risk Group 4 (RG4) - Fungal Agents. 44

Appendix B-IV-C........... Risk Group 4 (RG4) - Parasitic Agents. 44

Appendix B-IV-D.......... Risk Group 4 (RG4) - Viral Agents. 44

Appendix B-V............... Animal Viral Etiologic Agents in Common Use. 44

Appendix B-V-1............ Murine Retroviral Vectors. 45

APPENDIX C.................... EXEMPTIONS UNDER SECTION III-F-8. 45

Appendix C-I................ Recombinant or Synthetic Nucleic Acid Molecules in Tissue Culture. 45

Appendix C-I-A............. Exceptions. 46

Appendix C-II............... Escherichia coli K-12 Host-Vector Systems. 46

Appendix C-II-A............ Exceptions. 46

Appendix C-III............... Saccharomyces Host-Vector Systems. 46

Appendix C-III-A........... Exceptions. 46

Appendix C-IV.............. Kluyveromyces Host-Vector Systems. 47

Appendix C-IV-A........... Exceptions. 47

Appendix C-V............... Bacillus subtilis or Bacillus licheniformis Host-Vector Systems. 47

Appendix C-V-A........... Exceptions. 47

Appendix C-VI.............. Extrachromosomal Elements of Gram Positive Organisms. 47

Appendix C-VI-A........... Exceptions. 48

Appendix C-VII............. The Purchase or Transfer of Transgenic Rodents. 48

Appendix C-VIII............ Generation of BL1 Transgenic Rodents via Breeding. 48

Appendix C-IX.............. Footnotes and References of Appendix C. 49

APPENDIX D.................... MAJOR ACTIONS TAKEN UNDER THE NIH GUIDELINES. 49

APPENDIX E.................... CERTIFIED HOST-VECTOR SYSTEMS (See Appendix I, Biological Containment) 68

Appendix E-I................ Bacillus subtilis. 68

Appendix E-I-A............. Bacillus subtilis Host-Vector 1 Systems. 68

Appendix E-I-B............. Bacillus subtilis Host-Vector 2 Systems. 68

Appendix E-II............... Saccharomyces cerevisiae. 68

Appendix E-II-A............ Saccharomyces cerevisiae Host-Vector 2 Systems. 68

Appendix E-III............... Escherichia coli 69

Appendix E-III-A........... Escherichia coli (EK2) Plasmid Systems. 69

Appendix E-III-B........... Escherichia coli (EK2) Bacteriophage Systems. 69

Appendix E-IV.............. Neurospora crassa. 69

Appendix E-IV-A........... Neurospora crassa Host-Vector 1 Systems. 69

Appendix E-V............... Streptomyces. 69

Appendix E-V-A........... Streptomyces Host-Vector 1 Systems. 69

Appendix E-VI.............. Pseudomonas putida. 69

Appendix E-VI-A........... Pseudomonas putida Host-Vector 1 Systems. 69

APPENDIX F.................... CONTAINMENT CONDITIONS FOR CLONING OF GENES CODING FOR THE BIOSYNTHESIS OF MOLECULES TOXIC FOR VERTEBRATES. 70

Appendix F-I................ General Information. 70

Appendix F-II................ Cloning of Toxin Molecule Genes in Escherichia coli K-12. 70

Appendix F-III............... Cloning of Toxic Molecule Genes in Organisms Other Than Escherichia coli K-12. 70

Appendix F-IV.............. Specific Approvals. 70

APPENDIX G.................... PHYSICAL CONTAINMENT. 71

Appendix G-I................ Standard Practices and Training. 71

Appendix G-II............... Physical Containment Levels. 71

Appendix G-II-A............ Biosafety Level 1 (BL1) (See Appendix G-III-M, Footnotes and References of Appendix G) 72

Appendix G-II-A-1......... Standard Microbiological Practices (BL1) 72

Appendix G-II-A-2......... Special Practices (BL1) 72

Appendix G-II-A-3......... Containment Equipment (BL1) 72

Appendix G-II-A-4......... Laboratory Facilities (BL1) 72

Appendix G-II-B............ Biosafety Level 2 (BL2) (See Appendix G-III-N, Footnotes and References of Appendix G) 72

Appendix G-II-B-1......... Standard Microbiological Practices (BL2) 72

Appendix G-II-B-2......... Special Practices (BL2) 73

Appendix G-II-B-3......... Containment Equipment (BL2) 74

Appendix G-II-B-4......... Laboratory Facilities (BL2) 74

Appendix G-II-C............ Biosafety Level 3 (BL3) (See Appendix G-III-P, Footnotes and References of Appendix G) 74

Appendix G-II-C-1......... Standard Microbiological Practices (BL3) 74

Appendix G-II-C-2......... Special Practices (BL3) 75

Appendix G-II-C-2-t....... Alternative Selection of Containment Equipment (BL3) 76

Appendix G-II-C-3......... Containment Equipment (BL3) 76

Appendix G-II-C-4......... Laboratory Facilities (BL3) 77

Appendix G-II-C-5......... Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses.  (See Appendices G-II-C-2-n, G-II-C-2-r, and G-II-C-4-i for additional guidance for facilities, waste handling, and serum collection for research involving mammaliantransmissible HPAI H5N1 virus. 77

Appendix G-II-C-5-a...... Containment, Practices, and Training for Research with Risk Group 3 Influenza Viruses (BL3 Enhanced). 78

Appendix G-II-C-5-b...... Containment for Animal Research. 79

Appendix G-II-C-5-c...... Occupational Health. 79

Appendix G-II-D............ Biosafety Level 4 (BL4) 80

Appendix G-II-D-1......... Standard Microbiological Practices (BL4) 80

Appendix G-II-D-2......... Special Practices (BL4) 80

Appendix G-II-D-2-m..... Alternative Selection of Containment Equipment (BL4) 82

Appendix G-II-D-3......... Containment Equipment (BL4) 82

Appendix G-II-D-4......... Laboratory Facilities (BL4) 83

Appendix G-III.............. Footnotes and References of Appendix G.. 85

APPENDIX H.................... SHIPMENT. 86

Appendix H-III............... Footnotes and References of Appendix H. 87

APPENDIX I..................... BIOLOGICAL CONTAINMENT (See Appendix E, Certified Host-Vector Systems) 87

Appendix I-I.................. Levels of Biological Containment 87

Appendix I-I-A.............. Host-Vector 1 Systems. 87

Appendix I-I-A-1............ Escherichia coli K-12 Host-Vector 1 Systems (EK1) 87

Appendix I-I-A-2............ Other Host-Vector 1 Systems. 87

Appendix I-I-B.............. Host-Vector 2 Systems (EK2) 87

Appendix I-II................. Certification of Host-Vector Systems. 88

Appendix I-II-A.............. Responsibility. 88

Appendix I-II-B.............. Data to be Submitted for Certification. 88

Appendix I-II-B-1........... Host-Vector 1 Systems Other than Escherichia coli K-12. 88

Appendix I-II-B-2........... Host-Vector 2 Systems. 88

Appendix I-III................ Footnotes and References of Appendix I 89

APPENDIX J..................... BIOTECHNOLOGY RESEARCH SUBCOMMITTEE. 90

APPENDIX K.................... PHYSICAL CONTAINMENT FOR LARGE SCALE USES OF ORGANISMS CONTAINING RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULES. 90

Appendix K-I................ Selection of Physical Containment Levels. 91

Appendix K-II............... Good Large Scale Practice (GLSP) 91

Appendix K-III............... Biosafety Level 1 (BL1) - Large Scale. 92

Appendix K-IV.............. Biosafety Level 2 (BL2) - Large Scale. 92

Appendix K-V............... Biosafety Level 3 (BL3) - Large Scale. 94

Appendix K-VI.............. Footnotes of Appendix K. 98

Appendix K-VII............. Definitions to Accompany Containment Grid and Appendix K. 98

APPENDIX L.................... GENE THERAPY POLICY CONFERENCES (GTPCS) 99

APPENDIX M.................... POINTS TO CONSIDER IN THE DESIGN AND SUBMISSION OF PROTOCOLS FOR THE TRANSFER OF recombinant or synthetic NUCLEIC ACID MOLECULES INTO ONE OR MORE HUMAN RESEARCH PARTICIPANTS (POINTS TO CONSIDER) 99

Appendix M-I................ Requirements for Protocol Submission, Review, and Reporting – Human Gene Transfer Experiments. 100

Appendix M-I-A............ Requirements for Protocol Submission. 100

Appendix M-I-B............ Selection of Individual Protocols for Public RAC Review and Discussion. 101

Appendix M-I-C............ Reporting Requirements. 102

Appendix M-I-C-1.......... Initiation of the Clinical Investigation. 102

Appendix M-I-C-2.......... Additional Clinical Trial Sites. 102

Appendix M-I-C-3.......... Annual Reports. 103

Appendix M-I-C-4.......... Safety Reporting. 103

Appendix M-I-C-4-a....... Safety Reporting: Content and Format 104

Appendix M-I-C-4-b....... Safety Reporting: Time frames for Expedited Reports. 104

Appendix M-I-C-5.......... Confidentiality. 104

Appendix M-I-D............ Safety Assessment in Human Gene Transfer Research. 105

Appendix M-II............... Long-Term Follow-Up. 105

Appendix M-III.............. Footnotes of Appendix M.. 105

APPENDIX P.................... PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULE RESEARCH INVOLVING PLANTS. 105

Appendix P-I. .............. General Plant Biosafety Levels. 106

Appendix P-II............... Physical Containment Levels. 106

Appendix P-II-A............ Biosafety Level 1 - Plants (BL1-P) 106

Appendix P-II-A-1......... Standard Practices (BL1-P) 106

Appendix P-II-A-1-a....... Greenhouse Access (BL1-P) 106

Appendix P-II-A-1-b...... Records (BL1-P) 107

Appendix P-II-A-1-c....... Decontamination and Inactivation (BL1-P) 107

Appendix P-II-A-1-d...... Control of Undesired Species and Motile Macroorganisms (BL1-P) 107

Appendix P-II-A-1-e....... Concurrent Experiments Conducted in the Greenhouse (BL1-P) 107

Appendix P-II-A-2......... Facilities (BL1-P) 107

Appendix P-II-A-2-a....... Definitions (BL1-P) 107

Appendix P-II-A-2-b...... Greenhouse Design (BL1-P) 107

Appendix P-II-B............ Biosafety Level 2 - Plants (BL2-P) 107

Appendix P-II-B-1......... Standard Practices (BL2-P) 107

Appendix P-II-B-1-a....... Greenhouse Access (BL2-P) 107

Appendix P-II-B-1-b...... Records (BL2-P) 108

Appendix P-II-B-1-c....... Decontamination and Inactivation (BL2-P) 108

Appendix P-II-B-1-d...... Control of Undesired Species and Motile Macroorganisms (BL2-P) 108

Appendix P-II-B-1-e....... Concurrent Experiments Conducted in the Greenhouse (BL2-P) 108

Appendix P-II-B-1-f....... Signs (BL2-P) 108

Appendix P-II-B-1-g...... Transfer of Materials (BL2-P) 108

Appendix P-II-B-1-h....... Greenhouse Practices Manual (BL2-P) 109

Appendix P-II-B-2......... Facilities (BL2-P) 109

Appendix P-II-B-2-a....... Definitions (BL2-P) 109

Appendix P-II-B-2-b...... Greenhouse Design (BL2-P) 109

Appendix P-II-B-2-c....... Autoclaves (BL2-P) 109

Appendix P-II-B-2-d...... Supply and Exhaust Air Ventilation Systems (BL2-P) 109

Appendix P-II-B-2-e....... Other (BL2-P) 109

Appendix P-II-C............ Biosafety Level 3 - Plants (BL3-P) 109

Appendix P-II-C-1......... Standard Practices (BL3-P) 109

Appendix P-II-C-1-a....... Greenhouse Access (BL3-P) 109

Appendix P-II-C-1-b...... Records (BL3-P) 110

Appendix P-II-C-1-c....... Decontamination and Inactivation (BL3-P) 110

Appendix P-II-C-1-d...... Control of Undesired Species and Motile Macroorganisms (BL3-P) 110

Appendix P-II-C-1-e....... Concurrent Experiments Conducted in the Greenhouse (BL3-P) 110

Appendix P-II-C-1-f....... Signs (BL3-P) 110

Appendix P-II-C-1-g...... Transfer of Materials (BL3-P) 110

Appendix P-II-C-1-h....... Greenhouse Practices Manual (BL3-P) 111

Appendix P-II-C-1-i........ Protective Clothing (BL3-P) 111

Appendix P-II-C-1-j........ Other (BL3-P) 111

Appendix P-II-C-2......... Facilities (BL3-P) 111

Appendix P-II-C-2-a....... Definitions (BL3-P) 111

Appendix P-II-C-2-b...... Greenhouse Design (BL3-P) 111

Appendix P-II-C-2-c....... Autoclaves (BL3-P) 112

Appendix P-II-C-2-d...... Supply and Exhaust Air Ventilation Systems (BL3-P) 112

Appendix P-II-C-2-e....... Other (BL3-P) 112

Appendix P-II-D............ Biosafety Level 4 - Plants (BL4-P) 112

Appendix P-II-D-1......... Standard Practices (BL4-P) 112

Appendix P-II-D-1-a...... Greenhouse Access (BL4-P) 112

Appendix P-II-D-1-b...... Records (BL4-P) 112

Appendix P-II-D-1-c...... Decontamination and Inactivation (BL4-P) 113

Appendix P-II-D-1-d...... Control of Undesired Species and Motile Macroorganisms (BL4-P) 113

Appendix P-II-D-1-e...... Concurrent Experiments Conducted in the Greenhouse (BL4-P) 113

Appendix P-II-D-1-f....... Signs (BL4-P) 113

Appendix P-II-D-1-g...... Transfer of Materials (BL4-P) 114

Appendix P-II-D-1-h...... Greenhouse Practices Manual (BL4-P) 114

Appendix P-II-D-1-i....... Protective Clothing (BL4-P) 114

Appendix P-II-D-2......... Facilities (BL4-P) 114

Appendix P-II-D-2-a...... Greenhouse Design (BL4-P) 114

Appendix P-II-D-2-b...... Autoclaves (BL4-P) 115

Appendix P-II-D-2-c...... Supply and Exhaust Air Ventilation Systems (BL4-P) 115

Appendix P-II-D-2-d...... Other (BL4-P) 115

Appendix P-III............... Biological Containment Practices. 115

Appendix P-III-A........... Biological Containment Practices (Plants) 115

Appendix P-III-B........... Biological Containment Practices (Microorganisms) 116

Appendix P-III-C........... Biological Containment Practices (Macroorganisms) 116

APPENDIX Q.................... PHYSICAL AND BIOLOGICAL CONTAINMENT FOR RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULE RESEARCH INVOLVING ANIMALS. 116

Appendix Q-I................ General Considerations. 116

Appendix Q-I-A............. Containment Levels. 117

Appendix Q-I-B............. Disposal of Animals (BL1-N through BL4-N) 117

Appendix Q-II............... Physical and Biological Containment Levels. 117

Appendix Q-II-A............ Biosafety Level 1 - Animals (BL1-N) 117

Appendix Q-II-A-1......... Standard Practices (BL1-N) 117

Appendix Q-II-A-1-a...... Animal Facility Access (BL1-N) 117

Appendix Q-II-A-1-b...... Other (BL1-N) 117

Appendix Q-II-A-2......... Animal Facilities (BL1-N) 117

Appendix Q-II-B............ Biosafety Level 2 - Animals (BL2-N) (See Appendix Q-III-A, Footnotes and References for Appendix Q) 117

Appendix Q-II-B-1......... Standard Practices (BL2-N) 118

Appendix Q-II-B-1-a...... Animal Facility Access (BL2-N) 118

Appendix Q-II-B-1-b...... Decontamination and Inactivation (BL2-N) 118

Appendix Q-II-B-1-c...... Signs (BL2-N) 118

Appendix Q-II-B-1-d...... Protective Clothing (BL2-N) 118

Appendix Q-II-B-1-e...... Records (BL2-N) 118

Appendix Q-II-B-1-f....... Transfer of Materials (BL2-N) 119

Appendix Q-II-B-1-g...... Other (BL2-N) 119

Appendix Q-II-B-2......... Animal Facilities (BL2-N) 119

Appendix Q-II-C............ Biosafety Level 3 - Animals (BL3-N) (See Appendix Q-III-B, Footnotes and References for Appendix Q) 120

Appendix Q-II-C-1......... Standard Practices (BL3-N) 120

Appendix Q-II-C-1-a...... Animal Facility Access (BL3-N) 120

Appendix Q-II-C-1-b...... Decontamination and Inactivation (BL3-N) 120

Appendix Q-II-C-1-c...... Signs (BL3-N) 120

Appendix Q-II-C-1-d...... Protective Clothing (BL3-N) 121

Appendix Q-II-C-1-e...... Records (BL3-N) 121

Appendix Q-II-C-1-f....... Transfer of Materials (BL3-N) 121

Appendix Q-II-C-1-g...... Other (BL3-N) 121

Appendix Q-II-C-2......... Animal Facilities (BL3-N) 122

Appendix Q-II-D............ Biosafety Level 4 - Animals (BL4-N) (See Appendix Q-III-C, Footnotes and References for Appendix Q) 123

Appendix Q-II-D-1......... Standard Practices (BL4-N) 123

Appendix Q-II-D-1-a...... Animal Facility Access (BL4-N) 123

Appendix Q-II-D-1-b...... Decontamination and Inactivation (BL4-N) 124

Appendix Q-II-D-1-c...... Signs (BL4-N) 124

Appendix Q-II-D-1-d...... Protective Clothing (BL4-N) 125

Appendix Q-II-D-1-e...... Records (BL4-N) 125

Appendix Q-II-D-1-f....... Transfer of Materials (BL4-N) 125

Appendix Q-II-D-1-g...... Other (BL4-N) 126

Appendix Q-II-D-2......... Animal Facilities (BL4-N) 127

Appendix Q-III.............. Footnotes and References for Appendix Q.. 128

 

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LIST OF TABLES

 

Appendix B - Table 1.         Basis for the Classification of Biohazardous Agents by Risk Group. 43

 

Appendix G - Table 1.        Possible Alternate Combinations Of Physical And Biological Containment Safeguards  90

 

Appendix K - Table 1.        Comparison of Good Large Scale Practice (GLSP) and Biosafety Level (BL) - Large Scale (LS) Practice. 103

 

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SECTION I.       SCOPE OF THE NIH GUIDELINES

 

Section I-A.      Purpose

 

The purpose of the NIH Guidelines is to specify the practices for constructing and handling:  (i) recombinant nucleic acid molecules, (ii) synthetic nucleic acid molecules, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, and (iii) cells, organisms, and viruses containing such molecules.

 

Section I-A-1.  Any nucleic acid molecule experiment, which according to the NIH Guidelines requires approval by NIH, must be submitted to NIH or to another Federal agency that has jurisdiction for review and approval.  Once approvals, or other applicable clearances, have been obtained from a Federal agency other than NIH (whether the experiment is referred to that agency by NIH or sent directly there by the submitter), the experiment may proceed without the necessity for NIH review or approval.  (See exception in Section I-A-1-a regarding requirement for human gene transfer protocol registration.)

 

Section I-A-1-a.  For experiments involving the deliberate transfer of recombinant or synthetic nucleic acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules, into human research participants (human gene transfer), no research participant shall be enrolled (see definition of enrollment in Section I-E-7) until the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion); Institutional Biosafety Committee (IBC) approval (from the clinical trial site) has been obtained; Institutional Review Board (IRB) approval has been obtained; and all applicable regulatory authorization(s) have been obtained.

 

For a clinical trial site that is added after the completion of the NIH protocol registration process, no research participant shall be enrolled (see definition of enrollment in Section I-E-7) at the clinical trial site until IBC approval and IRB approval from that site have been obtained.  Within 30 days of enrollment (see definition of enrollment in Section I-E-7) at a clinical trial site, the following documentation shall be submitted to NIH OSP:  (1) Institutional Biosafety Committee approval (from the clinical trial site); (2) Institutional Review Board approval; (3) Institutional Review Board-approved informed consent document(s); and (4) NIH grant number(s) if applicable.

 

Section I-B.      Definition of Recombinant and Synthetic Nucleic Acid Molecules

 

In the context of the NIH Guidelines, recombinant and synthetic nucleic acids are defined as:

 

(i)             molecules that a) are constructed by joining nucleic acid molecules and b) that can replicate in a living cell, i.e., recombinant nucleic acids;

(ii)            nucleic acid molecules that are chemically or by other means synthesized or amplified, including those that are chemically or otherwise modified but can base pair with naturally occurring nucleic acid molecules, i.e., synthetic nucleic acids, or

(iii)           molecules that result from the replication of those described in (i) or (ii) above.


 

 

Section I-C.      General Applicability

 

Section I-C-1.  The NIH Guidelines are applicable to:

 

Section I-C-1-a.  All recombinant or synthetic nucleic acid research within the United States (U.S.) or its territories that is within the category of research described in either Section I-C-1-a-(1) or Section I-C-1-a-(2).

 

Section I-C-1-a-(1).  Research that is conducted at or sponsored by an institution that receives any support for recombinant or synthetic nucleic acid research from NIH, including research performed directly by NIH.  An individual who receives support for research involving recombinant or synthetic nucleic acids must be associated with or sponsored by an institution that assumes the responsibilities assigned in the NIH Guidelines.

 

Section I-C-1-a-(2).   Research that involves testing in humans of materials containing recombinant or synthetic nucleic acids developed with NIH funds, if the institution that developed those materials sponsors or participates in those projects.  Participation includes research collaboration or contractual agreements, not mere provision of research materials.

 

Section I-C-1-b.  All recombinant or synthetic nucleic acid research performed abroad that is within the category of research described in either Section I-C-1-b-(1) or Section I-C-1-b-(2).

 

Section I-C-1-b-(1).  Research supported by NIH funds.

 

Section I-C-1-b-(2).  Research that involves testing in humans of materials containing recombinant or synthetic nucleic acids developed with NIH funds, if the institution that developed those materials sponsors or participates in those projects.  Participation includes research collaboration or contractual agreements, not mere provision of research materials.

 

Section I-C-1-b-(3).  If the host country has established rules for the conduct of recombinant or synthetic nucleic acid molecule research, then the research must be in compliance with those rules.  If the host country does not have such rules, the proposed research must be reviewed and approved by an NIH-approved Institutional Biosafety Committee or equivalent review body and accepted in writing by an appropriate national governmental authority of the host country.  The safety practices that are employed abroad must be reasonably consistent with the NIH Guidelines.

 

Section I-D.      Compliance with the NIH Guidelines

 

As a condition for NIH funding of recombinant or synthetic nucleic acid molecule research, institutions shall ensure that such research conducted at or sponsored by the institution, irrespective of the source of funding, shall comply with the NIH Guidelines.

 

Information concerning noncompliance with the NIH Guidelines may be brought forward by any person.  It should be delivered to both NIH OSP and the relevant institution.  The institution, generally through the Institutional Biosafety Committee, shall take appropriate action.  The institution shall forward a complete report of the incident recommending any further action to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

In cases where NIH proposes to suspend, limit, or terminate financial assistance because of noncompliance with the NIH Guidelines, applicable DHHS and Public Health Service procedures shall govern.

 

The policies on compliance are as follows:

 

Section I-D-1.  All NIH-funded projects involving recombinant or synthetic nucleic acid molecules must comply with the NIH Guidelines.  Non-compliance may result in:  (i) suspension, limitation, or termination of financial assistance for the noncompliant NIH-funded research project and of NIH funds for other recombinant or synthetic nucleic acid molecule research at the institution, or (ii) a requirement for prior NIH approval of any or all recombinant or synthetic nucleic acid molecule projects at the institution.


 

 

Section I-D-2.  All non-NIH funded projects involving recombinant or synthetic nucleic acid molecule conducted at or sponsored by an institution that receives NIH funds for projects involving such techniques must comply with the NIH Guidelines.  Noncompliance may result in:  (i) suspension, limitation, or termination of NIH funds for recombinant or synthetic nucleic acid molecule research at the institution, or (ii) a requirement for prior NIH approval of any or all recombinant or synthetic nucleic acid molecule projects at the institution.

 

Section I-E.      General Definitions

The following terms, which are used throughout the NIH Guidelines, are defined as follows:

 

Section I-E-1.  An "institution" is any public or private entity (including Federal, state, and local government agencies).

 

Section I-E-2. An "Institutional Biosafety Committee" is a committee that:  (i) meets the requirements for membership specified in Section IV-B-2, Institutional Biosafety Committee (IBC), and (ii) reviews, approves, and oversees projects in accordance with the responsibilities defined in Section IV-B-2, Institutional Biosafety Committee (IBC).

 

Section I-E-3.  The "Office of Science Policy (OSP)" is the office within the NIH that is responsible for:  (i) reviewing and coordinating all activities relating to the NIH Guidelines, and (ii) performing other duties as defined in Section IV-C-3, Office of Science Policy (OSP).

 

Section I-E-4.  The "Recombinant DNA Advisory Committee" is the public advisory committee that advises the Department of Health and Human Services (DHHS) Secretary, the DHHS Assistant Secretary for Health, and the NIH Director concerning recombinant or synthetic nucleic acid molecule research.  The RAC shall be constituted as specified in Section IV-C-2, Recombinant DNA Advisory Committee (RAC).

 

Section I-E-5. The "NIH Director" is the Director of the National Institutes of Health, or any other officer or employee of NIH to whom authority has been delegated.

 

Section I-E-6.  "Deliberate release" is defined as a planned introduction of recombinant or synthetic nucleic acid molecule-containing microorganisms, plants, or animals into the environment.

 

Section I-E-7.  “Enrollment” is the process of obtaining informed consent from a potential research participant, or a designated legal guardian of the participant, to undergo a test or procedure associated with the gene transfer experiment.

 

Section I-E-8.  A “serious adverse event” is any event occurring at any dose that results in any of the following outcomes:  death, a life-threatening event, in-patient hospitalization or prolongation of existing hospitalization, a persistent or significant disability/incapacity, or a congenital anomaly/birth defect.  Important medical events that may not result in death, be life-threatening, or require hospitalization also may be considered a serious adverse event when, upon the basis of appropriate medical judgment, they may jeopardize the human gene transfer research subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition.

 

Section I-E-9.  An adverse event is “associated with the use of a gene transfer product” when there is a reasonable possibility that the event may have been caused by the use of that product.

 

Section I-E-10.  An “unexpected serious adverse event” is any serious adverse event for which the specificity or severity is not consistent with the risk information available in the current investigator’s brochure.

 

Section I-E-11.  An “oversight body” is an institutional entity (an Institutional Biosafety Committee or an Institutional Review Board) that must review and approve a human gene transfer trial.

 

Section I-E-12.  A “regulatory authority” in the context of human gene transfer research is a federal entity that by statute has oversight over research involving human subjects.

 

 

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SECTION II.       SAFETY CONSIDERATIONS

 

Section II-A.       Risk Assessment

 

Section II-A-1.    Risk Groups

 

Risk assessment is ultimately a subjective process.  The investigator must make an initial risk assessment based on the Risk Group (RG) of an agent (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard).  Agents are classified into four Risk Groups (RGs) according to their relative pathogenicity for healthy adult humans by the following criteria:  (1) Risk Group 1 (RG1) agents are not associated with disease in healthy adult humans.  (2) Risk Group 2 (RG2) agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.  (3) Risk Group 3 (RG3) agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.  (4) Risk Group 4 (RG4) agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

 

Section II-A-2.    Criteria for Risk Groups

 

Classification of agents in Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, is based on the potential effect of a biological agent on a healthy human adult and does not account for instances in which an individual may have increased susceptibility to such agents, e.g., preexisting diseases, medications, compromised immunity, pregnancy or breast feeding (which may increase exposure of infants to some agents).

 

Personnel may need periodic medical surveillance to ascertain fitness to perform certain activities; they may also need to be offered prophylactic vaccines and boosters (see Section IV-B-1-f, Responsibilities of the Institution, General Information).

 

Section II-A-3.    Comprehensive Risk Assessment

 

In deciding on the appropriate containment for an experiment, the first step is to assess the risk of the agent itself.  Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, classifies agents into Risk Groups based on an assessment of their ability to cause disease in humans and the available treatments for such disease.  Once the Risk Group of the agent is identified, this should be followed by a thorough consideration of how the agent is to be manipulated.  Factors to be considered in determining the level of containment include agent factors such as:  virulence, pathogenicity, infectious dose, environmental stability, route of spread, communicability, operations, quantity, availability of vaccine or treatment, and gene product effects such as toxicity, physiological activity, and allergenicity.  Any strain that is known to be more hazardous than the parent (wild-type) strain should be considered for handling at a higher containment level.  Certain attenuated strains or strains that have been demonstrated to have irreversibly lost known virulence factors may qualify for a reduction of the containment level compared to the Risk Group assigned to the parent strain (see Section V-B, Footnotes and References of Sections I-IV).

 

While the starting point for the risk assessment is based on the identification of the Risk Group of the parent agent, as technology moves forward, it may be possible to develop an organism containing genetic sequences from multiple sources such that the parent agent may not be obvious.  In such cases, the risk assessment should include at least two levels of analysis.  The first involves a consideration of the Risk Groups of the source(s) of the sequences and the second involves an assessment of the functions that may be encoded by these sequences (e.g., virulence or transmissibility).  It may be prudent to first consider the highest Risk Group classification of all agents that are the source of sequences included in the construct.  Other factors to be considered include the percentage of the genome contributed by each parent agent and the predicted function or intended purpose of each contributing sequence.  The initial assumption should be that all sequences will function as they did in the original host context.

 

The Principal Investigator and Institutional Biosafety Committee must also be cognizant that the combination of certain sequences in a new biological context may result in an organism whose risk profile could be higher than that of the contributing organisms or sequences.  The synergistic function of these sequences may be one of the key attributes to consider in deciding whether a higher containment level is warranted, at least until further assessments can be carried out.  A new biosafety risk may occur with an organism formed through combination of sequences from a number of organisms or due to the synergistic effect of combining transgenes that results in a new phenotype.

 

A final assessment of risk based on these considerations is then used to set the appropriate containment conditions for the experiment (see Section II-B, Containment).  The appropriate containment level may be equivalent to the Risk Group classification of the agent or it may be raised or lowered as a result of the above considerations.  The Institutional Biosafety Committee must approve the risk assessment and the biosafety containment level for recombinant or synthetic nucleic acid experiments described in Sections III-A, Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation; III-B, Experiments that Require NIH OSP and Institutional Biosafety Committee Approval Before Initiation; III-C, Experiments that Require Institutional Biosafety Committee and Institutional Review Board Approvals and NIH OSP Registration Before Initiation; III-D, Experiments that Require Institutional Biosafety Committee Approval Before Initiation.

 

Careful consideration should be given to the types of manipulation planned for some higher Risk Group agents.  For example, the RG2 dengue viruses may be cultured under the Biosafety Level (BL) 2 containment (see Section II-B); however, when such agents are used for animal inoculation or transmission studies, a higher containment level is recommended.  Similarly, RG3 agents such as Venezuelan equine encephalomyelitis and yellow fever viruses should be handled at a higher containment level for animal inoculation and transmission experiments.

 

Individuals working with human immunodeficiency virus (HIV), hepatitis B virus (HBV) or other bloodborne pathogens should consult the applicable Occupational Safety and Health Administration (OSHA) regulation, 29 CFR 1910.1030, and OSHA publication 3127 (1996 revised).  BL2 containment is recommended for activities involving all blood-contaminated clinical specimens, body fluids, and tissues from all humans, or from HIV- or HBV-infected or inoculated laboratory animals.  Activities such as the production of research-laboratory scale quantities of HIV or other bloodborne pathogens, manipulating concentrated virus preparations, or conducting procedures that may produce droplets or aerosols, are performed in a BL2 facility using the additional practices and containment equipment recommended for BL3.  Activities involving industrial scale volumes or preparations of concentrated HIV are conducted in a BL3 facility, or BL3 Large Scale if appropriate, using BL3 practices and containment equipment.

 

Exotic plant pathogens and animal pathogens of domestic livestock and poultry are restricted and may require special laboratory design, operation and containment features not addressed in Biosafety in Microbiological and Biomedical Laboratories (see Section V-C, Footnotes and References of Sections I through IV).  For information regarding the importation, possession, or use of these agents see Sections V-G and V-H, Footnotes and References of Sections I through IV.

 

Section II-B.    Containment

 

Effective biological safety programs have been operative in a variety of laboratories for many years.  Considerable information already exists about the design of physical containment facilities and selection of laboratory procedures applicable to organisms carrying additional recombinant or synthetic nucleic acid molecules (see Section V-B, Footnotes and References of Sections I-IV).  The existing programs rely upon mechanisms that can be divided into two categories:  (i) a set of standard practices that are generally used in microbiological laboratories; and (ii) special procedures, equipment, and laboratory installations that provide physical barriers that are applied in varying degrees according to the estimated biohazard.  Four biosafety levels are described in Appendix G, Physical Containment.  These biosafety levels consist of combinations of laboratory practices and techniques, safety equipment, and laboratory facilities appropriate for the operations performed and are based on the potential hazards imposed by the agents used and for the laboratory function and activity.  Biosafety Level 4 provides the most stringent containment conditions, Biosafety Level 1 the least stringent.

 

Experiments involving recombinant or synthetic nucleic acid molecules lend themselves to a third containment mechanism, namely, the application of highly specific biological barriers.  Natural barriers exist that limit either:  (i) the infectivity of a vector or vehicle (plasmid or virus) for specific hosts, or (ii) its dissemination and survival in the environment.  Vectors, which provide the means for recombinant or synthetic nucleic acid molecule and/or host cell replication, can be genetically designed to decrease, by many orders of magnitude, the probability of dissemination of recombinant or synthetic nucleic acid molecule outside the laboratory (see Appendix I, Biological Containment).

 

Since these three means of containment are complementary, different levels of containment can be established that apply various combinations of the physical and biological barriers along with a constant use of standard practices.  Categories of containment are considered separately in order that such combinations can be conveniently expressed in the NIH Guidelines.

 

Physical containment conditions within laboratories, described in Appendix G, Physical Containment, may not always be appropriate for all organisms because of their physical size, the number of organisms needed for an experiment, or the particular growth requirements of the organism.  Likewise, biological containment for microorganisms described in Appendix I, Biological Containment, may not be appropriate for all organisms, particularly higher eukaryotic organisms.  However, significant information exists about the design of research facilities and experimental procedures that are applicable to organisms containing additional recombinant or synthetic nucleic acid molecules that are either integrated into the genome or into microorganisms associated with the higher organism as a symbiont, pathogen, or other relationship.  This information describes facilities for physical containment of organisms used in non-traditional laboratory settings and special practices for limiting or excluding the unwanted establishment, transfer of genetic information, and dissemination of organisms beyond the intended location, based on both physical and biological containment principles.  Research conducted in accordance with these conditions effectively confines the organism.

 

For research involving plants, four biosafety levels (BL1-P through BL4-P) are described in Appendix P, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants.  BL1-P is designed to provide a moderate level of containment for experiments for which there is convincing biological evidence that precludes the possibility of survival, transfer, or dissemination of recombinant or synthetic nucleic acid molecules into the environment, or in which there is no recognizable and predictable risk to the environment in the event of accidental release.  BL2-P is designed to provide a greater level of containment for experiments involving plants and certain associated organisms in which there is a recognized possibility of survival, transmission, or dissemination of recombinant or synthetic nucleic acid molecule containing organisms, but the consequence of such an inadvertent release has a predictably minimal biological impact.  BL3-P and BL4-P describe additional containment conditions for research with plants and certain pathogens and other organisms that require special containment because of their recognized potential for significant detrimental impact on managed or natural ecosystems.  BL1-P relies upon accepted scientific practices for conducting research in most ordinary greenhouse or growth chamber facilities and incorporates accepted procedures for good pest control and cultural practices.  BL1-P facilities and procedures provide a modified and protected environment for the propagation of plants and microorganisms associated with the plants and a degree of containment that adequately controls the potential for release of biologically viable plants, plant parts, and microorganisms associated with them.  BL2-P and BL3-P rely upon accepted scientific practices for conducting research in greenhouses with organisms infecting or infesting plants in a manner that minimizes or prevents inadvertent contamination of plants within or surrounding the greenhouse.  BL4-P describes facilities and practices known to provide containment of certain exotic plant pathogens.

 

For research involving animals, which are of a size or have growth requirements that preclude the use of conventional primary containment systems used for small laboratory animals, four biosafety levels (BL1-N through BL4-N) are described in Appendix Q, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals.  BL1-N describes containment for animals that have been modified by stable introduction of recombinant or synthetic nucleic acid molecules, or DNA derived therefrom, into the germ-line (transgenic animals) and experiments involving viable recombinant or synthetic nucleic acid molecule-modified microorganisms and is designed to eliminate the possibility of sexual transmission of the modified genome or transmission of recombinant or synthetic nucleic acid molecule-derived viruses known to be transmitted from animal parent to offspring only by sexual reproduction.  Procedures, practices, and facilities follow classical methods of avoiding genetic exchange between animals.  BL2-N describes containment which is used for transgenic animals associated with recombinant or synthetic nucleic acid molecule-derived organisms and is designed to eliminate the possibility of vertical or horizontal transmission.  Procedures, practices, and facilities follow classical methods of avoiding genetic exchange between animals or controlling arthropod transmission.  BL3-N and BL4-N describe higher levels of containment for research with certain transgenic animals involving agents which pose recognized hazard.

 

In constructing the NIH Guidelines, it was necessary to define boundary conditions for the different levels of physical and biological containment and for the classes of experiments to which they apply.  These definitions do not take into account all existing and anticipated information on special procedures that will allow particular experiments to be conducted under different conditions than indicated here without affecting risk.  Individual investigators and Institutional Biosafety Committees are urged to devise simple and more effective containment procedures and to submit recommended changes in the NIH Guidelines to permit the use of these procedures.

 

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SECTION III.     EXPERIMENTS COVERED BY THE NIH GUIDELINES

 

This section describes six categories of experiments involving recombinant or synthetic nucleic acid molecules:  (i) those that require Institutional Biosafety Committee (IBC) approval, RAC review, and NIH Director approval before initiation (see Section III-A), (ii) those that require NIH OSP and Institutional Biosafety Committee approval before initiation (see Section III-B), (iii) those that require Institutional Biosafety Committee and Institutional Review Board approvals and RAC review before research participant enrollment (see Section III-C), (iv) those that require Institutional Biosafety Committee approval before initiation (see Section III-D), (v) those that require Institutional Biosafety Committee notification simultaneous with initiation (see Section III-E), and (vi) those that are exempt from the NIH Guidelines (see Section III-F).

 

Note:  If an experiment falls into Sections III-A, III-B, or III-C and one of the other sections, the rules pertaining to Sections III-A, III-B, or III-C shall be followed.  If an experiment falls into Section III-F and into either Sections III-D or III-E as well, the experiment is considered exempt from the NIH Guidelines.

 

Any change in containment level, which is different from those specified in the NIH Guidelines, may not be initiated without the express approval of NIH OSP (see Section IV-C-1-b-(2) and its subsections, Minor Actions).

 

 

Section III-A.    Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation (See Section IV-C-1-b-(1), Major Actions).

 

Section III-A-1.    Major Actions under the NIH Guidelines

 

Experiments considered as Major Actions under the NIH Guidelines cannot be initiated without submission of relevant information on the proposed experiment to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov, the publication of the proposal in the Federal Register for 15 days of comment, review by RAC, and specific approval by NIH.  The containment conditions or stipulation requirements for such experiments will be recommended by RAC and set by NIH at the time of approval.  Such experiments require Institutional Biosafety Committee approval before initiation.  Specific experiments already approved are included in Appendix D, Major Actions Taken under the NIH Guidelines, which may be obtained from the Office of Science Policy, National Institutes of Health, preferably by submitting a request for this information to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section III-A-1-a.  The deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally (see Section V-B, Footnotes and References of Sections I-IV), if such acquisition could compromise the ability to control disease agents in humans, veterinary medicine, or agriculture, will be reviewed by the RAC.

 

Consideration should be given as to whether the drug resistance trait to be used in the experiment would render that microorganism resistant to the primary drug available to and/or indicated for certain populations, for example children or pregnant women.

 

At the request of an Institutional Biosafety Committee, NIH OSP will make a determination regarding whether a specific experiment involving the deliberate transfer of a drug resistance trait falls under Section III-A-1-a and therefore requires RAC review and NIH Director approval.  An Institutional Biosafety Committee may also consult with NIH OSP regarding experiments that do not meet the requirements of Section III-A-1-a but nonetheless raise important public health issues.  NIH OSP will consult, as needed, with one or more experts, which may include the RAC.

 

Section III-B.    Experiments That Require NIH OSP and Institutional Biosafety Committee Approval Before Initiation

 

Experiments in this category cannot be initiated without submission of relevant information on the proposed experiment to NIH OSP.  The containment conditions for such experiments will be determined by NIH OSP in consultation with ad hoc experts.  Such experiments require Institutional Biosafety Committee approval before initiation (see Section IV-B-2-b-(1), Institutional Biosafety Committee).

 

Section III-B-1.   Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms per Kilogram Body Weight

 

Deliberate formation of recombinant or synthetic nucleic acid molecules containing genes for the biosynthesis of toxin molecules lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight (e.g., microbial toxins such as the botulinum toxins, tetanus toxin, diphtheria toxin, and Shigella dysenteriae neurotoxin).  Specific approval has been given for the cloning in Escherichia coli K-12 of DNA containing genes coding for the biosynthesis of toxic molecules which are lethal to vertebrates at 100 nanograms to 100 micrograms per kilogram body weight.  Specific experiments already approved under this section may be obtained from the Office of Science Policy, National Institutes of Health, preferably by submitting a request for this information to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section III-B-2.   Experiments that have been Approved (under Section III-A-1-a) as Major Actions under the NIH Guidelines

 

Upon receipt and review of an application from the investigator, NIH OSP may determine that a proposed experiment is equivalent to an experiment that has previously been approved by the NIH Director as a Major Action, including experiments approved prior to implementation of these changes.  An experiment will only be considered equivalent if, as determined by NIH OSP, there are no substantive differences and pertinent information has not emerged since submission of the initial III-A-1-a experiment that would change the biosafety and public health considerations for the proposed experiments.  If such a determination is made by NIH OSP, these experiments will not require review and approval under Section III-A.

 

 Section III-C.   Experiments that Require Institutional Biosafety Committee and Institutional Review Board Approvals and RAC Review Before Research Participant Enrollment

 

Section III-C-1.   Experiments Involving the Deliberate Transfer of Recombinant or Synthetic Nucleic Acid Molecules, or DNA or RNA Derived from Recombinant or Synthetic Nucleic Acid Molecules, into One or More Human Research Participants

 

Human gene transfer is the deliberate transfer into human research participants of either:

1.     Recombinant nucleic acid molecules, or DNA or RNA derived from recombinant nucleic acid molecules, or

2.     Synthetic nucleic acid molecules, or DNA or RNA derived from synthetic nucleic acid molecules, that meet any one of the following criteria:

a.     Contain more than 100 nucleotides; or

b.    Possess biological properties that enable integration into the genome (e.g., cis elements involved in integration); or

c.     Have the potential to replicate in a cell; or

d.    Can be translated or transcribed.

 

No research participant shall be enrolled (see definition of enrollment in Section I-E-7) until the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion).

 

In its evaluation of human gene transfer protocols, the NIH will make a determination, following a request from one or more oversight bodies involved in the review at an initial site(s), whether a proposed human gene transfer experiment has one or more of the characteristics that warrant public RAC review and discussion (See Appendix M-I-B).  The process of public RAC review and discussion is intended to foster the safe and ethical conduct of human gene transfer experiments.  Public review and discussion of a human gene transfer experiment (and access to relevant information) also serves to inform the public about the technical aspects of the proposal, the meaning and significance of the research, and any significant safety, social, and ethical implications of the research.

 

Public RAC review and discussion of a human gene transfer experiment will be initiated in two exceptional circumstances:  (1) Following a request for public RAC review from one or more oversight bodies involved in the review at an initial site(s), the NIH concurs that (a) the individual protocol would significantly benefit from RAC review and (b) that the submission meets one or more of the following NIH RAC review criteria: i) the protocol uses a new vector, genetic material, or delivery methodology that represents a first-in-human experience, thus presenting an unknown risk; ii) the protocol relies on preclinical safety data that were obtained using a new preclinical model system of unknown and unconfirmed value; or iii) the proposed vector, gene construct, or method of delivery is associated with possible toxicities that are not widely known and that may render it difficult for oversight and federal regulatory bodies to evaluate the protocol rigorously.  However, if one or more oversight bodies involved in the review at an initial site(s) requests public RAC review, but the NIH does not concur that (a) the individual protocol would significantly benefit from RAC review and (b) that the submission meets one or more of the RAC review criteria (listed in i, ii, or iii), then the NIH OSP will inform, within 10 working days, the requesting and other oversight bodies involved in the review at an initial site(s) that public RAC review is not warranted.  (2) The NIH Director, in consultation (if needed) with appropriate regulatory authorities, determines that the submission:  (a) meets one or more of the NIH RAC review criteria (listed in i, ii, or iii) and that public RAC review and discussion would provide a clear and obvious benefit to the scientific community or the public; or (b) raises significant scientific, societal, or ethical concerns.

 

For a clinical trial site that is added after the completion of the NIH protocol registration process, no research participant shall be enrolled (see definition of enrollment in Section I-E-7) at the clinical trial site until IBC approval and IRB approval from that site have been obtained.  Within 30 days of enrollment (see definition of enrollment in Section I-E-7) at a clinical trial site, the following documentation shall be submitted to NIH OSP:  (1) Institutional Biosafety Committee approval (from the clinical trial site); (2) Institutional Review Board approval; (3) Institutional Review Board-approved informed consent document(s); and (4) NIH grant number(s) if applicable.

 

In order to maintain public access to information regarding human gene transfer (including protocols that are not publicly reviewed by the RAC), the NIH OSP will maintain the documentation described in Appendices M-I through M-II.  The information provided in response to Appendix M should not contain any confidential commercial or financial information or trade secrets, enabling all aspects of RAC review to be open to the public.

 

Note:  For specific directives concerning the use of retroviral vectors for gene delivery, consult Appendix B-V-1, Murine Retroviral Vectors.

 

 

Section III-D.   Experiments that Require Institutional Biosafety Committee Approval Before Initiation

 

Prior to the initiation of an experiment that falls into this category, the Principal Investigator must submit a registration document to the Institutional Biosafety Committee which contains the following information:  (i) the source(s) of DNA; (ii) the nature of the inserted DNA sequences; (iii) the host(s) and vector(s) to be used; (iv) if an attempt will be made to obtain expression of a foreign gene, and if so, indicate the protein that will be produced; and (v) the containment conditions that will be implemented as specified in the NIH Guidelines.  For experiments in this category, the registration document shall be dated, signed by the Principal Investigator, and filed with the Institutional Biosafety Committee.  The Institutional Biosafety Committee shall review and approve all experiments in this category prior to their initiation.  Requests to decrease the level of containment specified for experiments in this category will be considered by NIH (see Section IV-C-1-b-(2)-(c), Minor Actions).

 

Section III-D-1.   Experiments Using Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems (See Section II-A, Risk Assessment)

 

Section III-D-1-a.  Experiments involving the introduction of recombinant or synthetic nucleic acid molecules into Risk Group 2 agents will usually be conducted at Biosafety Level (BL) 2 containment.  Experiments with such agents will usually be conducted with whole animals at BL2 or BL2-N (Animals) containment.

 

Section III-D-1-b.  Experiments involving the introduction of recombinant or synthetic nucleic acid molecules into Risk Group 3 agents will usually be conducted at BL3 containment.  Experiments with such agents will usually be conducted with whole animals at BL3 or BL3-N containment.

 

Section III-D-1-c.  Experiments involving the introduction of recombinant or synthetic nucleic acid molecules into Risk Group 4 agents shall be conducted at BL4 containment.  Experiments with such agents shall be conducted with whole animals at BL4 or BL4-N containment.

Section III-D-1-d.  Containment conditions for experiments involving the introduction of recombinant or synthetic nucleic acid molecules into restricted agents shall be set on a case-by-case basis following NIH OSP review.  A U.S. Department of Agriculture - Animal and Plant Health Inspection Service (USDA/APHIS) permit is required for work with plant or animal pathogens (see Section V-G and V-M, Footnotes and References of Sections I-IV).  Experiments with such agents shall be conducted with whole animals at BL4 or BL4-N containment.

 

Section III-D-2.    Experiments in Which DNA From Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems

 

Section III-D-2-a.  Experiments in which DNA from Risk Group 2 or Risk Group 3 agents (see Section II-A, Risk Assessment) is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BL2 containment.  Experiments in which DNA from Risk Group 4 agents is transferred into nonpathogenic prokaryotes or lower eukaryotes may be performed under BL2 containment after demonstration that only a totally and irreversibly defective fraction of the agent's genome is present in a given recombinant.  In the absence of such a demonstration, BL4 containment shall be used.  The Institutional Biosafety Committee may approve the specific lowering of containment for particular experiments to BL1.  Many experiments in this category are exempt from the NIH Guidelines (see Section III-F, Exempt Experiments).  Experiments involving the formation of recombinant or synthetic nucleic acid molecules for certain genes coding for molecules toxic for vertebrates require NIH OSP approval (see Section III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram Body Weight) or shall be conducted under NIH specified conditions as described in Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates.

 

Section III-D-2-b.  Containment conditions for experiments in which DNA from restricted agents is transferred into nonpathogenic prokaryotes or lower eukaryotes shall be determined by NIH OSP following a case-by-case review (see Section V-L, Footnotes and References of Sections I-IV).  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV).

 

Section III-D-3.    Experiments Involving the Use of Infectious DNA or RNA Viruses or Defective DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems

 

Caution:  Special care should be used in the evaluation of containment levels for experiments which are likely to either enhance the pathogenicity (e.g., insertion of a host oncogene) or to extend the host range (e.g., introduction of novel control elements) of viral vectors under conditions that permit a productive infection.  In such cases, serious consideration should be given to increasing physical containment by at least one level.

 

Note:  Recombinant or synthetic nucleic acid molecules or nucleic acid molecules derived therefrom, which contain less than two-thirds of the genome of any eukaryotic virus (all viruses from a single Family (see Section V-J, Footnotes and References of Sections I-IV) being considered identical (see Section V-K, Footnotes and References of Sections I-IV), are considered defective and may be used in the absence of helper under the conditions specified in Section III-E-1, Experiments Involving the Formation of Recombinant or Synthetic Molecules Containing No More than Two-Thirds of the Genome of any Eukaryotic Virus.

 

Section III-D-3-a.  Experiments involving the use of infectious or defective Risk Group 2 viruses (see Appendix B-II, Risk Group 2 Agents) in the presence of helper virus may be conducted at BL2.

 

Section III-D-3-b.  Experiments involving the use of infectious or defective Risk Group 3 viruses (see Appendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions) in the presence of helper virus may be conducted at BL3.

 

Section III-D-3-c.  Experiments involving the use of infectious or defective Risk Group 4 viruses (see Appendix B-IV-D, Risk Group 4 (RG4) - Viral Agents) in the presence of helper virus may be conducted at BL4.

 

Section III-D-3-d.  Experiments involving the use of infectious or defective restricted poxviruses (see Sections V-A and V-L, Footnotes and References of Sections I-IV) in the presence of helper virus shall be determined on a case-by-case basis following NIH OSP review.  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV).

 

Section III-D-3-e.  Experiments involving the use of infectious or defective viruses in the presence of helper virus which are not covered in Sections III-D-3-a through III-D-3-d may be conducted at BL1.

 

Section III-D-4.    Experiments Involving Whole Animals

 

This section covers experiments involving whole animals in which the animal's genome has been altered by stable introduction of recombinant or synthetic nucleic acid molecules, or nucleic acids derived therefrom, into the germ-line (transgenic animals) and experiments involving viable recombinant or synthetic nucleic acid molecule-modified microorganisms tested on whole animals.  For the latter, other than viruses which are only vertically transmitted, the experiments may not be conducted at BL1-N containment.  A minimum containment of BL2 or BL2-N is required.

 

Caution - Special care should be used in the evaluation of containment conditions for some experiments with transgenic animals.  For example, such experiments might lead to the creation of novel mechanisms or increased transmission of a recombinant pathogen or production of undesirable traits in the host animal.  In such cases, serious consideration should be given to increasing the containment conditions.

 

Section III-D-4-a.  Recombinant or synthetic nucleic acid molecules, or DNA or RNA molecules derived therefrom, from any source except for greater than two-thirds of eukaryotic viral genome may be transferred to any non-human vertebrate or any invertebrate organism and propagated under conditions of physical containment comparable to BL1 or BL1-N and appropriate to the organism under study (see Section V-B, Footnotes and References of Sections I-IV).  Animals that contain sequences from viral vectors, which do not lead to transmissible infection either directly or indirectly as a result of complementation or recombination in animals, may be propagated under conditions of physical containment comparable to BL1 or BL1-N and appropriate to the organism under study.  Experiments involving the introduction of other sequences from eukaryotic viral genomes into animals are covered under Section III-D-4-b, Experiments Involving Whole Animals.  For experiments involving recombinant or synthetic nucleic acid molecule-modified Risk Groups 2, 3, 4, or restricted organisms, see Sections V-A, V-G, and V-L, Footnotes and References of Sections I-IV.  It is important that the investigator demonstrate that the fraction of the viral genome being utilized does not lead to productive infection.  A U.S. Department of Agriculture permit is required for work with plant or animal pathogens (see Section V-G, Footnotes and References of Sections I-IV).

 

Section III-D-4-b.  For experiments involving recombinant or synthetic nucleic acid molecules, or DNA or RNA derived therefrom, involving whole animals, including transgenic animals, and not covered by Section III-D-1, Experiments Using Human or Animal Pathogens (Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems), or Section III-D-4-a, the appropriate containment shall be determined by the Institutional Biosafety Committee.

 

Section III-D-4-c.  Exceptions under Section III-D-4, Experiments Involving Whole Animals

 

Section III-D-4-c-(1).  Experiments involving the generation of transgenic rodents that require BL1 containment are described under Section III-E-3, Experiments Involving Transgenic Rodents.

 

Section III-D-4-c-(2).  The purchase or transfer of transgenic rodents is exempt from the NIH Guidelines under Section III-F, Exempt Experiments (see Appendix C-VII, The Purchase or Transfer of Transgenic Rodents).

 

Section III-D-5.    Experiments Involving Whole Plants

 

Experiments to genetically engineer plants by recombinant or synthetic nucleic acid molecule methods, to use such plants for other experimental purposes (e.g., response to stress), to propagate such plants, or to use plants together with microorganisms or insects containing recombinant or synthetic nucleic acid molecules, may be conducted under the containment conditions described in Sections III-D-5-a through III-D-5-e.  If experiments involving whole plants are not described in Section III-D-5 and do not fall under Sections III-A, III-B, III-D or III-F, they are included in Section III-E.

 

NOTE - For recombinant or synthetic nucleic acid molecule experiments falling under Sections III-D-5-a through III-D-5-d, physical containment requirements may be reduced to the next lower level by appropriate biological containment practices, such as conducting experiments on a virus with an obligate insect vector in the absence of that vector or using a genetically attenuated strain.


 

 

Section III-D-5-a.  BL3-P (Plants) or BL2-P + biological containment is recommended for experiments involving most exotic (see Section V-M, Footnotes and References of Sections I-IV) infectious agents with recognized potential for serious detrimental impact on managed or natural ecosystems when recombinant or synthetic nucleic acid molecule techniques are associated with whole plants.

 

Section III-D-5-b.  BL3-P or BL2-P + biological containment is recommended for experiments involving plants containing cloned genomes of readily transmissible exotic (see Section V-M, Footnotes and References of Sections I-IV) infectious agents with recognized potential for serious detrimental effects on managed or natural ecosystems in which there exists the possibility of reconstituting the complete and functional genome of the infectious agent by genomic complementation in planta.

 

Section III-D-5-c.  BL4-P containment is recommended for experiments with a small number of readily transmissible exotic (see Section V-M, Footnotes and References of Sections I-IV) infectious agents, such as the soybean rust fungus (Phakospora pachyrhizi) and maize streak or other viruses in the presence of their specific arthropod vectors, that have the potential of being serious pathogens of major U.S. crops.

 

Section III-D-5-d.  BL3-P containment is recommended for experiments involving sequences encoding potent vertebrate toxins introduced into plants or associated organisms.  Recombinant or synthetic nucleic acid molecules containing genes for the biosynthesis of toxin molecules lethal for vertebrates at an LD50 of <100 nanograms per kilogram body weight fall under Section III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram Body Weight, and require NIH OSP and Institutional Biosafety Committee approval before initiation.

 

Section III-D-5-e.  BL3-P or BL2-P + biological containment is recommended for experiments with microbial pathogens of insects or small animals associated with plants if the recombinant or synthetic nucleic acid molecule-modified organism has a recognized potential for serious detrimental impact on managed or natural ecosystems.

 

Section III-D-6.    Experiments Involving More than 10 Liters of Culture

 

The appropriate containment will be decided by the Institutional Biosafety Committee.  Where appropriate, Appendix K, Physical Containment for Large Scale Uses of Organisms Containing Recombinant or Synthetic Recombinant or synthetic nucleic acid Molecules, shall be used.  Appendix K describes containment conditions Good Large Scale Practice through BL3-Large Scale.

 

Section III-D-7.    Experiments Involving Influenza Viruses

 

Experiments with influenza viruses generated by recombinant or synthetic methods (e.g., generation by reverse genetics of chimeric viruses with reassorted segments, introduction of specific mutations) shall be conducted at the biosafety level containment corresponding to the Risk Group of the virus that was the source of the majority of segments in the recombinant or synthetic virus (e.g., experiments with viruses containing a majority of segments from a RG3 virus shall be conducted at BL3).  Experiments with influenza viruses containing genes or segments from 1918-1919 H1N1 (1918 H1N1), human H2N2 (1957-1968) and highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI H5N1), including, but not limited to, strains of HPAI H5N1 virus that are transmissible among mammals by respiratory droplets, as demonstrated in an appropriate animal model or clinically in humans (hereinafter referred to as mammalian-transmissible HPAI H5N1 virus), shall be conducted at BL3 enhanced containment (see  Appendix G-II-C-5, Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses) unless indicated below.

 

Section III-D-7-a.  Human H2N2 (1957-1968).  Experiments with influenza viruses containing the H2 hemagglutinin (HA) segment shall be conducted at BL3 enhanced (see Appendix G-II-C-5, Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses).  Experiments with the H2 HA gene in cold-adapted, live attenuated vaccine strains (e.g., A/Ann Arbor/6/60 H2N2) may be conducted at BL2 containment provided segments with mutations conferring temperature sensitivity and attenuation are not altered in the recombinant or synthetic virus.  Experiments with Risk Group 2 influenza viruses containing genes from human H2N2 other than the HA gene can be worked on at BL2.

 

Section III-D-7-b.  Highly Pathogenic Avian Influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI H5N1).  Experiments involving influenza viruses containing a majority of genes and/or segments from a HPAI H5N1 influenza virus shall be conducted at BL3 enhanced containment, (see Appendix G-II-C-5, Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses).  Experiments involving influenza viruses containing a minority of genes and/or segments from a HPAI H5N1 influenza virus shall be conducted at BL3 enhanced unless a risk assessment performed by the IBC determines that they can be conducted safely at biosafety level 2 and after they have been excluded pursuant to 9 CFR 121.3(e).  NIH OSP is available to IBCs to provide consultation with the RAC and influenza virus experts when risk assessments are being made to determine the appropriate biocontainment for experiments with influenza viruses containing a minority of gene/segments from HPAI H5N1.  Such experiments may be performed at BL3 enhanced containment or containment may be lowered to biosafety level 2, the level of containment for most research with other influenza viruses.  (USDA/APHIS regulations and decisions on lowering containment also apply.)  In deciding to lower containment, the IBC should consider whether, in at least two animal models (e.g., ferret, mouse, Syrian golden hamster, cotton rat, non-human primates), there is evidence that the resulting influenza virus shows reduced replication and virulence compared to the parental RG3 virus at relevant doses.  This should be determined by measuring biological indices appropriate for the specific animal model (e.g., severe weight loss, elevated temperature, mortality or neurological symptoms).

 

Section III-D-7-c.  1918 H1N1.  Experiments involving influenza viruses containing any gene or segment from 1918 H1N1 shall be conducted at BL3 enhanced containment (see Appendix G-II-C-5, Biosafety Level 3 Enhanced for Research Involving Risk Group 3 Influenza Viruses).

 

Section III-D-7-d. Antiviral Susceptibility and Containment.  The availability of antiviral drugs as preventive and therapeutic measures is an important safeguard for experiments with 1918 H1N1, HPAI H5N1, and human H2N2 (1957-1968).  If an influenza virus containing genes from one of these viruses is resistant to both classes of current antiviral agents, adamantanes and neuraminidase inhibitors, higher containment may be required based on the risk assessment considering transmissibility to humans, virulence, pandemic potential, alternative antiviral agents if available, etc.

 

Experiments with 1918 H1N1, human H2N2 (1957-1968) or HPAI H5N1 that are designed to create resistance to neuraminidase inhibitors or other effective antiviral agents (including investigational antiviral agents being developed for influenza) would be subject to Section III-A-1 (Major Actions) and require RAC review and NIH Director approval.  As per Section I-A-1 of the NIH Guidelines, if the agent is a Select Agent, the NIH will defer to the appropriate Federal agency (HHS or USDA Select Agent Divisions) on such experiments.

 

Section III-E.    Experiments that Require Institutional Biosafety Committee Notice Simultaneous with Initiation

 

Experiments not included in Sections III-A, III-B, III-C, III-D, III-F, and their subsections are considered in Section III-E.  All such experiments may be conducted at BL1 containment.  For experiments in this category, a registration document (see Section III-D, Experiments that Require Institutional Biosafety Committee Approval Before Initiation) shall be dated and signed by the investigator and filed with the local Institutional Biosafety Committee at the time the experiment is initiated.  The Institutional Biosafety Committee reviews and approves all such proposals, but Institutional Biosafety Committee review and approval prior to initiation of the experiment is not required (see Section IV-A, Policy).  For example, experiments in which all components derived from non-pathogenic prokaryotes and non-pathogenic lower eukaryotes fall under Section III-E and may be conducted at BL1 containment.


 

 

Section III-E-1.    Experiments Involving the Formation of Recombinant or Synthetic Nucleic Acid Molecules Containing No More than Two-Thirds of the Genome of any Eukaryotic Virus

 

Recombinant or synthetic nucleic acid molecules containing no more than two-thirds of the genome of any eukaryotic virus (all viruses from a single Family being considered identical [see Section V-J, Footnotes and References of Sections I-IV]) may be propagated and maintained in cells in tissue culture using BL1 containment.  For such experiments, it must be demonstrated that the cells lack helper virus for the specific Families of defective viruses being used.  If helper virus is present, procedures specified under Section III-D-3, Experiments Involving the Use of Infectious Animal or Plant DNA or RNA Viruses or Defective Animal or Plant DNA or RNA Viruses in the Presence of Helper Virus in Tissue Culture Systems, should be used.  The DNA may contain fragments of the genome of viruses from more than one Family but each fragment shall be less than two-thirds of a genome.

 

Section III-E-2.    Experiments Involving Whole Plants

 

This section covers experiments involving nucleic acid molecule-modified whole plants, and/or experiments involving recombinant or synthetic nucleic acid molecule-modified organisms associated with whole plants, except those that fall under Section III-A, III-B, III-D, or III-F.  It should be emphasized that knowledge of the organisms and judgment based on accepted scientific practices should be used in all cases in selecting the appropriate level of containment.  For example, if the genetic modification has the objective of increasing pathogenicity or converting a non-pathogenic organism into a pathogen, then a higher level of containment may be appropriate depending on the organism, its mode of dissemination, and its target organisms.  By contrast, a lower level of containment may be appropriate for small animals associated with many types of recombinant or synthetic nucleic acid molecule-modified plants.

 

Section III-E-2-a.  BL1-P is recommended for all experiments with recombinant or synthetic recombinant or synthetic nucleic acid molecule-containing plants and plant-associated microorganisms not covered in Section III-E-2-b or other sections of the NIH Guidelines.  Examples of such experiments are those involving recombinant or synthetic nucleic acid molecule-modified plants that are not noxious weeds or that cannot interbreed with noxious weeds in the immediate geographic area, and experiments involving whole plants and recombinant or synthetic nucleic acid molecule-modified non-exotic (see Section V-M, Footnotes and References of Sections I-IV) microorganisms that have no recognized potential for rapid and widespread dissemination or for serious detrimental impact on managed or natural ecosystems (e.g., Rhizobium spp. and Agrobacterium spp.).

 

Section III-E-2-b.  BL2-P or BL1-P + biological containment is recommended for the following experiments:

 

Section III-E-2-b-(1).  Plants modified by recombinant or synthetic nucleic acid molecules that are noxious weeds or can interbreed with noxious weeds in the immediate geographic area.

 

 

Section III-E-2-b-(2).  Plants in which the introduced DNA represents the complete genome of a non-exotic infectious agent (see Section V-M, Footnotes and References of Sections I-IV).

 

Section III-E-2-b-(3).  Plants associated with recombinant or synthetic nucleic acid molecule-modified non-exotic microorganisms that have a recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).

 

Section III-E-2-b-(4).  Plants associated with recombinant or synthetic nucleic acid molecule-modified exotic microorganisms that have no recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).

 

Section III-E-2-b-(5).  Experiments with recombinant or synthetic nucleic acid molecule-modified arthropods or small animals associated with plants, or with arthropods or small animals with recombinant or synthetic nucleic acid molecule-modified microorganisms associated with them if the recombinant or synthetic nucleic acid molecule-modified microorganisms have no recognized potential for serious detrimental impact on managed or natural ecosystems (see Section V-M, Footnotes and References of Sections I-IV).

 

Section III-E-3.    Experiments Involving Transgenic Rodents

 

This section covers experiments involving the generation of rodents in which the animal's genome has been altered by stable introduction of recombinant or synthetic nucleic acid molecules, or nucleic acids derived therefrom, into the germ-line (transgenic rodents).  Only experiments that require BL1 containment are covered under this section; experiments that require BL2, BL3, or BL4 containment are covered under Section III-D-4, Experiments Involving Whole Animals.

 

Section III-E-3-a.  Experiments involving the breeding of certain BL1 transgenic rodents are exempt under Section III-F, Exempt Experiments (See Appendix C-VIII, Generation of BL1 Transgenic Rodents via Breeding).

 

Section III-F.    Exempt Experiments

 

The following recombinant or synthetic nucleic acid molecules are exempt from the NIH Guidelines and registration with the Institutional Biosafety Committee is not required; however, other federal and state standards of biosafety may still apply to such research (for example, the Centers for Disease Control and Prevention (CDC)/NIH publication Biosafety in Microbiological and Biomedical Laboratories).

 

Section III-F-1.  Those synthetic nucleic acids that:  (1) can neither replicate nor generate nucleic acids that can replicate in any living cell (e.g., oligonucleotides or other synthetic nucleic acids that do not contain an origin of replication or contain elements known to interact with either DNA or RNA polymerase), and (2) are not designed to integrate into DNA, and (3) do not produce a toxin that is lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight.  If a synthetic nucleic acid is deliberately transferred into one or more human research participants and meets the criteria of Section III-C, it is not exempt under this Section.

 

Section III-F-2.  Those that are not in organisms, cells, or viruses and that have not been modified or manipulated (e.g., encapsulated into synthetic or natural vehicles) to render them capable of penetrating cellular membranes.

 

Section III-F-3.  Those that consist solely of the exact recombinant or synthetic nucleic acid sequence from a single source that exists contemporaneously in nature.

 

Section III-F-4.  Those that consist entirely of nucleic acids from a prokaryotic host, including its indigenous plasmids or viruses when propagated only in that host (or a closely related strain of the same species), or when transferred to another host by well-established physiological means.

 

Section III-F-5.  Those that consist entirely of nucleic acids from a eukaryotic host including its chloroplasts, mitochondria, or plasmids (but excluding viruses) when propagated only in that host (or a closely related strain of the same species).

 

Section III-F-6.  Those that consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent.  A list of such exchangers will be prepared and periodically revised by the NIH Director with advice of the RAC after appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), Major Actions).  See Appendices A-I through A-VI, Exemptions under Section III-F-6--Sublists of Natural Exchangers, for a list of natural exchangers that are exempt from the NIH Guidelines.

 

Section III-F-7.  Those genomic DNA molecules that have acquired a transposable element, provided the transposable element does not contain any recombinant and/or synthetic DNA.

 

Section III-F-8.  Those that do not present a significant risk to health or the environment (see Section IV-C-1-b-(1)-(c), Major Actions), as determined by the NIH Director, with the advice of the RAC, and following appropriate notice and opportunity for public comment.  See Appendix C, Exemptions under Section III-F-8 for other classes of experiments which are exempt from the NIH Guidelines.

 

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SECTION IV.    ROLES AND RESPONSIBILITIES

 

Section IV-A.    Policy

 

The safe conduct of experiments involving recombinant or synthetic nucleic acid molecules depends on the individual conducting such activities.  The NIH Guidelines cannot anticipate every possible situation.  Motivation and good judgment are the key essentials to protection of health and the environment.  The NIH Guidelines are intended to assist the institution, Institutional Biosafety Committee, Biological Safety Officer, and the Principal Investigator in determining safeguards that should be implemented.  The NIH Guidelines will never be complete or final since all conceivable experiments involving recombinant or synthetic nucleic acid molecules cannot be foreseen.  The utilization of new genetic manipulation techniques may enable work previously conducted using recombinant means to be accomplished faster, more efficiently, or at larger scale.  These techniques have not yet yielded organisms that present safety concerns that fall outside the current risk assessment framework used for recombinant nucleic acid research.  Nonetheless, an appropriate risk assessment of experiments involving these techniques must be conducted taking into account the way these approaches may alter the risk assessment.  As new techniques develop, the NIH Guidelines should be periodically reviewed to determine whether and how such research should be explicitly addressed.

 

It is the responsibility of the institution and those associated with it to adhere to the intent of the NIH Guidelines as well as to their specifics.  Therefore, each institution (and the Institutional Biosafety Committee acting on its behalf) is responsible for ensuring that all research with recombinant or synthetic nucleic acid molecules conducted at or sponsored by that institution is conducted in compliance with the NIH Guidelines.  The following roles and responsibilities constitute an administrative framework in which safety is an essential and integral part of research involving recombinant or synthetic nucleic acid molecules.  Further clarifications and interpretations of roles and responsibilities will be issued by NIH as necessary.

 

Section IV-B.    Responsibilities of the Institution

 

Section IV-B-1.    General Information

 

Each institution conducting or sponsoring recombinant or synthetic nucleic acid molecule research which is covered by the NIH Guidelines is responsible for ensuring that the research is conducted in full conformity with the provisions of the NIH Guidelines.  In order to fulfill this responsibility, the institution shall:

 

Section IV-B-1-a.  Establish and implement policies that provide for the safe conduct of recombinant or synthetic nucleic acid molecule research and that ensure compliance with the NIH Guidelines.  As part of its general responsibilities for implementing the NIH Guidelines, the institution may establish additional procedures, as deemed necessary, to govern the institution and its components in the discharge of its responsibilities under the NIH Guidelines.  Such procedures may include:  (i) statements formulated by the institution for the general implementation of the NIH Guidelines, and (ii) any additional precautionary steps the institution deems appropriate.

 

Section IV-B-1-b.  Establish an Institutional Biosafety Committee that meets the requirements set forth in Section IV-B-2-a and carries out the functions detailed in Section IV-B-2-b.

 

Section IV-B-1-c.  Appoint a Biological Safety Officer (who is also a member of the Institutional Biosafety Committee) if the institution:  (i) conducts recombinant or synthetic nucleic acid molecule research at Biosafety Level (BL) 3 or BL4, or (ii) engages in large-scale (greater than 10 liters) research.  The Biological Safety Officer carries out the duties specified in Section IV-B-3.

 

Section IV-B-1-d.  Appoint at least one individual with expertise in plant, plant pathogen, or plant pest containment principles (who is a member of the Institutional Biosafety Committee) if the institution conducts recombinant or synthetic nucleic acid molecule research that requires Institutional Biosafety Committee approval in accordance with Appendix P, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants.

 

Section IV-B-1-e.  Appoint at least one individual with expertise in animal containment principles (who is a member of the Institutional Biosafety Committee) if the institution conducts recombinant or synthetic nucleic acid molecule research that requires Institutional Biosafety Committee approval in accordance with Appendix Q, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals.

 

Section IV-B-1-f.  Ensure that when the institution participates in or sponsors recombinant or synthetic nucleic acid molecule research involving human subjects:  (i) the Institutional Biosafety Committee has adequate expertise and training (using ad hoc consultants as deemed necessary), (ii) all aspects of Appendix M have been appropriately addressed by the Principal Investigator; and (iii) no research participant shall be enrolled (see definition of enrollment in Section I-E-7) in a human gene transfer experiment until the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion), Institutional Biosafety Committee approval has been obtained, Institutional Review Board approval has been obtained, and all applicable regulatory authorizations have been obtained.  Institutional Biosafety Committee approval must be obtained from the clinical trial site.

 

Section IV-B-1-g.  Assist and ensure compliance with the NIH Guidelines by Principal Investigators conducting research at the institution as specified in Section IV-B-7.

 

Section IV-B-1-h.  Ensure appropriate training for the Institutional Biosafety Committee Chair and members, Biological Safety Officer and other containment experts (when applicable), Principal Investigators, and laboratory staff regarding laboratory safety and implementation of the NIH Guidelines.  The Institutional Biosafety Committee Chair is responsible for ensuring that Institutional Biosafety Committee members are appropriately trained.  The Principal Investigator is responsible for ensuring that laboratory staff are appropriately trained.  The institution is responsible for ensuring that the Principal Investigator has sufficient training; however, this responsibility may be delegated to the Institutional Biosafety Committee.

 

Section IV-B-1-i.  Determine the necessity for health surveillance of personnel involved in connection with individual recombinant or synthetic nucleic acid molecule projects; and if appropriate, conduct a health surveillance program for such projects.  The institution shall establish and maintain a health surveillance program for personnel engaged in large-scale research or production activities involving viable organisms containing recombinant or synthetic nucleic acid molecules which require BL3 containment at the laboratory scale.  The institution shall establish and maintain a health surveillance program for personnel engaged in animal research involving viable recombinant or synthetic nucleic acid molecule-containing microorganisms that require BL3 or greater containment in the laboratory.  The Laboratory Safety Monograph discusses various components of such a program (e.g., records of agents handled, active investigation of relevant illnesses, and the maintenance of serial serum samples for monitoring serologic changes that may result from the employees' work experience).  Certain medical conditions may place a laboratory worker at increased risk in any endeavor where infectious agents are handled.  Examples cited in the Laboratory Safety Monograph include gastrointestinal disorders and treatment with steroids, immunosuppressive drugs, or antibiotics.  Workers with such disorders or treatment should be evaluated to determine whether they should be engaged in research with potentially hazardous organisms during their treatment or illness.  Copies of the Laboratory Safety Monograph are available from the Office of Science Policy, National Institutes of Health,  preferably by submitting a request for this information to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-1-j.  Report any significant problems, violations of the NIH Guidelines, or any significant research-related accidents and illnesses to NIH OSP within thirty days, unless the institution determines that a report has already been filed by the Principal Investigator or Institutional Biosafety Committee.  Reports shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-2.    Institutional Biosafety Committee (IBC)

 

The institution shall establish an Institutional Biosafety Committee whose responsibilities need not be restricted to recombinant or synthetic nucleic acid molecule research.  The Institutional Biosafety Committee shall meet the following requirements:

 

Section IV-B-2-a.    Membership and Procedures

 

Section IV-B-2-a-(1).  The Institutional Biosafety Committee must be comprised of no fewer than five members so selected that they collectively have experience and expertise in recombinant or synthetic nucleic acid molecule technology and the capability to assess the safety of recombinant or synthetic nucleic acid molecule research and to identify any potential risk to public health or the environment.  At least two members shall not be affiliated with the institution (apart from their membership on the Institutional Biosafety Committee) and who represent the interest of the surrounding community with respect to health and protection of the environment (e.g., officials of state or local public health or environmental protection agencies, members of other local governmental bodies, or persons active in medical, occupational health, or environmental concerns in the community).  The Institutional Biosafety Committee shall include at least one individual with expertise in plant, plant pathogen, or plant pest containment principles when experiments utilizing Appendix P, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants, require prior approval by the Institutional Biosafety Committee.  The Institutional Biosafety Committee shall include at least one scientist with expertise in animal containment principles when experiments utilizing Appendix Q, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals, require Institutional Biosafety Committee prior approval.  When the institution conducts recombinant or synthetic nucleic acid molecule research at BL3, BL4, or Large Scale (greater than 10 liters), a Biological Safety Officer is mandatory and shall be a member of the Institutional Biosafety Committee (see Section IV-B-3, Biological Safety Officer).  When the institution participates in or sponsors recombinant or synthetic nucleic acid molecule research involving human research participants, the institution must ensure that:  (i) the Institutional Biosafety Committee has adequate expertise and training (using ad hoc consultants as deemed necessary); (ii) all aspects of Appendix M have been appropriately addressed by the Principal Investigator; (iii) no research participant shall be enrolled (see definition of enrollment in Section I-E-7) in a human gene transfer experiment until the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion); and (iv) final IBC approval is granted only after the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion).  Institutional Biosafety Committee approval must be obtained from the clinical trial site.

 

Note:  Individuals, corporations, and institutions not otherwise covered by the NIH Guidelines, are encouraged to adhere to the standards and procedures set forth in Sections I through IV (see Section IV-D, Voluntary Compliance.  The policy and procedures for establishing an Institutional Biosafety Committee under Voluntary Compliance, are specified in Section IV-D-2, Institutional Biosafety Committee Approval).

 

Section IV-B-2-a-(2).  In order to ensure the competence necessary to review and approve recombinant or synthetic nucleic acid molecule activities, it is recommended that the Institutional Biosafety Committee:  (i) include persons with expertise in recombinant or synthetic nucleic acid molecule technology, biological safety, and physical containment; (ii) include or have available as consultants persons knowledgeable in institutional commitments and policies, applicable law, standards of professional conduct and practice, community attitudes, and the environment, and (iii) include at least one member representing the laboratory technical staff.

 

Section IV-B-2-a-(3).  The institution shall file an annual report with NIH OSP which includes:  (i) a roster of all Institutional Biosafety Committee members clearly indicating the Chair, contact person, Biological Safety Officer (if applicable), plant expert (if applicable), animal expert (if applicable), human gene therapy expertise or ad hoc consultant (if applicable); and (ii) biographical sketches of all Institutional Biosafety Committee members (including community members).

 

Section IV-B-2-a-(4).  No member of an Institutional Biosafety Committee may be involved (except to provide information requested by the Institutional Biosafety Committee) in the review or approval of a project in which he/she has been or expects to be engaged or has a direct financial interest.

 

Section IV-B-2-a-(5).  The institution, that is ultimately responsible for the effectiveness of the Institutional Biosafety Committee, may establish procedures that the Institutional Biosafety Committee shall follow in its initial and continuing review and approval of applications, proposals, and activities.

 

Section IV-B-2-a-(6).  When possible and consistent with protection of privacy and proprietary interests, the institution is encouraged to open its Institutional Biosafety Committee meetings to the public.

 

Section IV-B-2-a-(7).  Upon request, the institution shall make available to the public all Institutional Biosafety Committee meeting minutes and any documents submitted to or received from funding agencies which the latter are required to make available to the public.  If public comments are made on Institutional Biosafety Committee actions, the institution shall forward both the public comments and the Institutional Biosafety Committee's response to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-2-b.    Functions

 

On behalf of the institution, the Institutional Biosafety Committee is responsible for:

 

Section IV-B-2-b-(1).  Reviewing recombinant or synthetic nucleic acid molecule research conducted at or sponsored by the institution for compliance with the NIH Guidelines as specified in Section III, Experiments Covered by the NIH Guidelines, and approving those research projects that are found to conform with the NIH Guidelines.  This review shall include:  (i) independent assessment of the containment levels required by the NIH Guidelines for the proposed research; (ii) assessment of the facilities, procedures, practices, and training and expertise of personnel involved in recombinant or synthetic nucleic acid molecule research; (iii) ensuring that all aspects of Appendix M have been appropriately addressed by the Principal Investigator; (iv) ensuring that no research participant is enrolled (see definition of enrollment in Section I-E-7) in a human gene transfer experiment until the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion), Institutional Biosafety Committee approval (from the clinical trial site) has been obtained, Institutional Review Board approval has been obtained, and all applicable regulatory authorizations have been obtained; (v) for human gene transfer protocols selected for public RAC review and discussion, consideration of the issues raised and recommendations made as a result of this review and consideration of the Principal Investigator’s response to the recommendations; (vi) ensuring that final IBC approval is granted only after the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion); and (vii) ensuring compliance with all surveillance, data reporting, and adverse event reporting requirements set forth in the NIH Guidelines.

 

Section IV-B-2-b-(2).  Notifying the Principal Investigator of the results of the Institutional Biosafety Committee's review and approval.

 

Section IV-B-2-b-(3).  Lowering containment levels for certain experiments as specified in Section III-D-2-a, Experiments in which DNA from Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems.

 

Section IV-B-2-b-(4).  Setting containment levels as specified in Sections III-D-4-b, Experiments Involving Whole Animals, and III-D-5, Experiments Involving Whole Plants.

 

Section IV-B-2-b-(5).  Periodically reviewing recombinant or synthetic nucleic acid molecule research conducted at the institution to ensure compliance with the NIH Guidelines.

 

Section IV-B-2-b-(6).  Adopting emergency plans covering accidental spills and personnel contamination resulting from recombinant or synthetic nucleic acid molecule research.

 

Note:  The Laboratory Safety Monograph describes basic elements for developing specific procedures dealing with major spills of potentially hazardous materials in the laboratory, including information and references about decontamination and emergency plans.  The NIH and the CDC are available to provide consultation and direct assistance, if necessary, as posted in the Laboratory Safety Monograph.  The institution shall cooperate with the state and local public health departments by reporting any significant research-related illness or accident that may be hazardous to the public health.

 

Section IV-B-2-b-(7).  Reporting any significant problems with or violations of the NIH Guidelines and any significant research-related accidents or illnesses to the appropriate institutional official and NIH OSP within 30 days, unless the Institutional Biosafety Committee determines that a report has already been filed by the Principal Investigator.  Reports to NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-2-b-(8).  The Institutional Biosafety Committee may not authorize initiation of experiments which are not explicitly covered by the NIH Guidelines until NIH (with the advice of the RAC when required) establishes the containment requirement.

 

Section IV-B-2-b-(9).  Performing such other functions as may be delegated to the Institutional Biosafety Committee under Section IV-B-2, Institutional Biosafety Committee.

 

Section IV-B-3.      Biological Safety Officer (BSO)

 

Section IV-B-3-a.  The institution shall appoint a Biological Safety Officer if it engages in large-scale research or production activities involving viable organisms containing recombinant or synthetic nucleic acid molecules.

 

Section IV-B-3-b.  The institution shall appoint a Biological Safety Officer if it engages in recombinant or synthetic nucleic acid molecule research at BL3 or BL4.  The Biological Safety Officer shall be a member of the Institutional Biosafety Committee.

 

Section IV-B-3-c.  The Biological Safety Officer's duties include, but are not be limited to:

 

Section IV-B-3-c-(1).  Periodic inspections to ensure that laboratory standards are rigorously followed;

 

Section IV-B-3-c-(2).  Reporting to the Institutional Biosafety Committee and the institution any significant problems, violations of the NIH Guidelines, and any significant research-related accidents or illnesses of which the Biological Safety Officer becomes aware unless the Biological Safety Officer determines that a report has already been filed by the Principal Investigator;

 

Section IV-B-3-c-(3).  Developing emergency plans for handling accidental spills and personnel contamination and investigating laboratory accidents involving recombinant or synthetic nucleic acid molecule research;

 

Section IV-B-3-c-(4).  Providing advice on laboratory security;

 

Section IV-B-3-c-(5).  Providing technical advice to Principal Investigators and the Institutional Biosafety Committee on research safety procedures.

 

Note:  See the Laboratory Safety Monograph for additional information on the duties of the Biological Safety Officer.

 

Section IV-B-4.    Plant, Plant Pathogen, or Plant Pest Containment Expert

 

When the institution conducts recombinant or synthetic nucleic acid molecule research that requires Institutional Biosafety Committee approval in accordance with Appendix P, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Plants, the institution shall appoint at least one individual with expertise in plant, plant pathogen, or plant pest containment principles (who is a member of the Institutional Biosafety Committee).

 

Section IV-B-5.    Animal Containment Expert

 

When the institution conducts recombinant or synthetic nucleic acid molecule research that requires Institutional Biosafety Committee approval in accordance with Appendix Q, Physical and Biological Containment for Recombinant or Synthetic Nucleic Acid Molecule Research Involving Animals, the institution shall appoint at least one individual with expertise in animal containment principles (who is a member of the Institutional Biosafety Committee).

 

Section IV-B-6.    Human Gene Therapy Expertise

 

When the institution participates in or sponsors recombinant or synthetic nucleic acid molecule research involving human subjects, the institution must ensure that:  (i) the Institutional Biosafety Committee has adequate expertise and training (using ad hoc consultants as deemed necessary) and (ii) all aspects of Appendix M, Points to Consider in the Design and Submission of Protocols for the Transfer of Recombinant or Synthetic Nucleic Acid Molecules into One or More Human Subjects (Points to Consider) , have been appropriately addressed by the Principal Investigator prior to its approval.

 

Section IV-B-7.    Principal Investigator (PI)

 

On behalf of the institution, the Principal Investigator is responsible for full compliance with the NIH Guidelines in the conduct of recombinant or synthetic nucleic acid molecule research.  A Principal Investigator engaged in human gene transfer research may delegate to another party, such as a corporate sponsor, the reporting functions set forth in Appendix M, with written notification to the NIH OSP of the delegation and of the name(s), address, telephone, and fax numbers of the contact.  The Principal Investigator is responsible for ensuring that the reporting requirements are fulfilled and will be held accountable for any reporting lapses.

 

Section IV-B-7-a.    General Responsibilities

 

As part of this general responsibility, the Principal Investigator shall:

 

Section IV-B-7-a-(1).  Initiate or modify no recombinant or synthetic nucleic acid molecule research which requires Institutional Biosafety Committee approval prior to initiation (see Sections III-A, III-B, III-C, III-D, and III-E, Experiments Covered by the NIH Guidelines) until that research or the proposed modification thereof has been approved by the Institutional Biosafety Committee and has met all other requirements of the NIH Guidelines;

 

Section IV-B-7-a-(2).  Determine whether experiments are covered by Section III-E, Experiments that Require Institutional Biosafety Committee Notice Simultaneous with Initiation, and ensure that the appropriate procedures are followed;

 

Section IV-B-7-a-(3).  Report any significant problems, violations of the NIH Guidelines, or any significant research-related accidents and illnesses to the Biological Safety Officer (where applicable), Greenhouse/Animal Facility Director (where applicable), Institutional Biosafety Committee, NIH OSP, and other appropriate authorities (if applicable) within 30 days.  Reports to NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-7-a-(4).  Report any new information bearing on the NIH Guidelines to the Institutional Biosafety Committee and to NIH OSP (reports to NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-7-a-(5).  Be adequately trained in good microbiological techniques;

 

Section IV-B-7-a-(6).  Adhere to Institutional Biosafety Committee approved emergency plans for handling accidental spills and personnel contamination; and

 

Section IV-B-7-a-(7).  Comply with shipping requirements for recombinant or synthetic nucleic acid molecules (see Appendix H, Shipment, for shipping requirements and the Laboratory Safety Monograph for technical recommendations).

 

  Section IV-B-7-b.  Information to Be Submitted by the Principal Investigator to NIH OSP

 

The Principal Investigator shall:

 

Section IV-B-7-b-(1).  Submit information to NIH OSP for certification of new host-vector systems;

 

Section IV-B-7-b-(2).  Petition NIH OSP, with notice to the Institutional Biosafety Committee, for proposed exemptions to the NIH Guidelines;

 

Section IV-B-7-b-(3).  Petition NIH OSP, with concurrence of the Institutional Biosafety Committee, for approval to conduct experiments specified in Sections III-A-1, Major Actions Under the NIH Guidelines, and III-B, Experiments that Require NIH OSP and Institutional Biosafety Committee Approval Before Initiation;

 

Section IV-B-7-b-(4).  Petition NIH OSP for determination of containment for experiments requiring case-by-case review; and

 

Section IV-B-7-b-(5).  Petition NIH OSP for determination of containment for experiments not covered by the NIH Guidelines.

 

Section IV-B-7-b-(6).  Ensure that all aspects of Appendix M have been appropriately addressed prior to submission.  No research participant shall be enrolled (see definition of enrollment in Section I-E-7) in a human gene transfer experiment until the NIH protocol registration process has been completed (see Appendix M-I-B, Selection of Individual Protocols for Public RAC Review and Discussion); IBC approval (from the clinical trial site) has been obtained; Institutional Review Board (IRB) approval has been obtained; and all applicable regulatory authorization(s) have been obtained.

 

For a clinical trial site that is added after the completion of the NIH protocol registration process, no research participant shall be enrolled (see definition of enrollment in Section I-E-7) at the clinical trial site until IBC approval and IRB approval from that site have been obtained.  Within 30 days of enrollment (see definition of enrollment in Section I-E-7) at a clinical trial site, the following documentation shall be submitted to NIH OSP:  (1) Institutional Biosafety Committee approval (from the clinical trial site); (2) Institutional Review Board approval; (3) Institutional Review Board-approved informed consent document(s); and (4) NIH grant number(s) if applicable.

 

Section IV-B-7-c.    Submissions by the Principal Investigator to the Institutional Biosafety Committee

 

The Principal Investigator shall:

 

Section IV-B-7-c-(1).  Make an initial determination of the required levels of physical and biological containment in accordance with the NIH Guidelines;

 

Section IV-B-7-c-(2).  Select appropriate microbiological practices and laboratory techniques to be used for the research;

 

Section IV-B-7-c-(3).  Submit the initial research protocol and any subsequent changes (e.g., changes in the source of DNA or host-vector system), if covered under Sections III-A, III-B, III-C, III-D, or III-E (Experiments Covered by the NIH Guidelines), to the Institutional Biosafety Committee for review and approval or disapproval; and

 

Section IV-B-7-c-(4).  Remain in communication with the Institutional Biosafety Committee throughout the conduct of the project.

 

Section IV-B-7-d.    Responsibilities of the Principal Investigator Prior to Initiating Research

 

The Principal Investigator shall:

 

Section IV-B-7-d-(1).  Make available to all laboratory staff the protocols that describe the potential biohazards and the precautions to be taken;

 

Section IV-B-7-d-(2).  Instruct and train laboratory staff in:  (i) the practices and techniques required to ensure safety, and (ii) the procedures for dealing with accidents; and

 

Section IV-B-7-d-(3).  Inform the laboratory staff of the reasons and provisions for any precautionary medical practices advised or requested (e.g., vaccinations or serum collection).

 

Section IV-B-7-e.    Responsibilities of the Principal Investigator During the Conduct of the Research

 

The Principal Investigator shall:

 

Section IV-B-7-e-(1).  Supervise the safety performance of the laboratory staff to ensure that the required safety practices and techniques are employed;

 

Section IV-B-7-e-(2).  Investigate and report any significant problems pertaining to the operation and implementation of containment practices and procedures in writing to the Biological Safety Officer (where applicable), Greenhouse/Animal Facility Director (where applicable), Institutional Biosafety Committee, NIH OSP, and other appropriate authorities (if applicable) (reports to NIH OSP shall be sent to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

Section IV-B-7-e-(3).  Correct work errors and conditions that may result in the release of recombinant or synthetic nucleic acid molecule materials; and

 

Section IV-B-7-e-(4).  Ensure the integrity of the physical containment (e.g., biological safety cabinets) and the biological containment (e.g., purity and genotypic and phenotypic characteristics).

 

Section IV-B-7-e-(5).  Comply with reporting requirements for human gene transfer experiments conducted in compliance with the NIH Guidelines (see Appendix M-I-C, Reporting Requirements).

 

Section IV-C.     Responsibilities of the National Institutes of Health (NIH)

 

Section IV-C-1.    NIH Director

 

The NIH Director is responsible for:  (i) establishing the NIH Guidelines, (ii) overseeing their implementation, and (iii) their final interpretation.  The NIH Director has responsibilities under the NIH Guidelines that involve OSP and RAC.  OSP's responsibilities under the NIH Guidelines are administrative.  Advice from RAC is primarily scientific, technical, and ethical.  In certain circumstances, there is specific opportunity for public comment with published response prior to final action.

 

Section IV-C-1-a.    General Responsibilities

 

The NIH Director is responsible for:

 

Section IV-C-1-a-(1).  Promulgating requirements as necessary to implement the NIH Guidelines;

 

Section IV-C-1-a-(2).  Establishing and maintaining RAC to carry out the responsibilities set forth in Section IV-C-2, Recombinant DNA Advisory Committee (RAC membership is specified in its charter and in Section IV-C-2);

 

Section IV-C-1-a-(3).  Establishing and maintaining NIH OSP to carry out the responsibilities defined in Section IV-C-3, Office of Science Policy;

 

Section IV-C-1-a-(4).  Conducting and supporting training programs in laboratory safety for Institutional Biosafety Committee members, Biological Safety Officers and other institutional experts (if applicable), Principal Investigators, and laboratory staff.

 

Section IV-C-1-a-(5).  Establishing and convening Gene Therapy Policy Conferences as described in Appendix L, Gene Therapy Policy Conferences.

 

Section IV-C-1-b.    Specific Responsibilities

 

In carrying out the responsibilities set forth in this section, the NIH Director, or a designee shall weigh each proposed action through appropriate analysis and consultation to determine whether it complies with the NIH Guidelines and presents no significant risk to health or the environment.

 

Section IV-C-1-b-(1).     Major Actions

 

To execute Major Actions, the NIH Director shall seek the advice of RAC and provide an opportunity for public and Federal agency comment.  Specifically, the Notice of Meeting and Proposed Actions shall be published in the Federal Register at least 15 days before the RAC meeting.  The NIH Director's decision/recommendation (at his/her discretion) may be published in the Federal Register for 15 days of comment before final action is taken.  The NIH Director's final decision/recommendation, along with responses to public comments, shall be published in the Federal Register.  The RAC and Institutional Biosafety Committee Chairs shall be notified of the following decisions:

 

Section IV-C-1-b-(1)-(a).  Changing containment levels for types of experiments that are specified in the NIH Guidelines when a Major Action is involved;

 

Section IV-C-1-b-(1)-(b).  Assigning containment levels for types of experiments that are not explicitly considered in the NIH Guidelines when a Major Action is involved;

 

Section IV-C-1-b-(1)-(c).  Promulgating and amending a list of classes of recombinant or synthetic nucleic acid molecules to be exempt from the NIH Guidelines because they consist entirely of DNA segments from species that exchange DNA by known physiological processes or otherwise do not present a significant risk to health or the environment;

 

Section IV-C-1-b-(1)-(d).  Permitting experiments specified by Section III-A, Experiments that Require Institutional Biosafety Committee Approval, RAC Review, and NIH Director Approval Before Initiation;

 

Section IV-C-1-b-(1)-(e).  Certifying new host-vector systems with the exception of minor modifications of already certified systems (the standards and procedures for certification are described in Appendix I-II, Certification of Host-Vector Systems).  Minor modifications constitute (e.g., those of minimal or no consequence to the properties relevant to containment); and

 

Section IV-C-1-b-(1)-(f).  Adopting other changes in the NIH Guidelines.

 

Section IV-C-1-b-(2).     Minor Actions

 

NIH OSP shall carry out certain functions as delegated to it by the NIH Director (see Section IV-C-3, Office of Science Policy).  Minor Actions (as determined by NIH OSP in consultation with the RAC Chair and one or more RAC members, as necessary) will be transmitted to RAC and Institutional Biosafety Committee Chairs:

 

Section IV-C-1-b-(2)-(a).  Changing containment levels for experiments that are specified in Section III, Experiments Covered by the NIH Guidelines (except when a Major Action is involved);

 

Section IV-C-1-b-(2)-(b).  Assigning containment levels for experiments not explicitly considered in the NIH Guidelines;

 

Section IV-C-1-b-(2)-(c).  Revising the Classification of Etiologic Agents for the purpose of these NIH Guidelines (see Section V-A, Footnotes and References of Sections I-IV).

 

Section IV-C-1-b-(2)-(d).  Interpreting the NIH Guidelines for experiments to which the NIH Guidelines do not specifically assign containment levels;

 

Section IV-C-1-b-(2)-(e).  Setting containment under Sections III-D-1-d, Experiments Using Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents as Host-Vector Systems, and III-D-2-b, Experiments in which DNA from Risk Group 2, Risk Group 3, Risk Group 4, or Restricted Agents is Cloned into Nonpathogenic Prokaryotic or Lower Eukaryotic Host-Vector Systems;

 

Section IV-C-1-b-(2)-(f).  Approving minor modifications of already certified host-vector systems (the standards and procedures for such modifications are described in Appendix I-II, Certification of Host-Vector Systems);

 

Section IV-C-1-b-(2)-(g).  Decertifying already certified host-vector systems;

 

Section IV-C-1-b-(2)-(h).  Adding new entries to the list of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates); and

 

Section IV-C-1-b-(2)-(i).  Determining appropriate containment conditions for experiments according to case precedents developed under Section IV-C-1-b-(2)-(c).

 

Section IV-C-2.    Recombinant DNA Advisory Committee (RAC)

 

The RAC is responsible for carrying out the functions specified in the NIH Guidelines, as well as others specified in its charter or assigned by the Secretary of Health and Human Services or the NIH Director.  The RAC membership and procedures, in addition to those set forth in the NIH Guidelines, are specified in the charter for the RAC which is filed as provided in the General Services Administration Federal Advisory Committee Management regulations, 41 CFR part 101-6, and is available on the OSP web site, http://www.osp.od.nih.gov/sites/default/files/resources/RAC_2015-2017_Charter_Updated.pdf.  In the event of a conflict between the NIH Guidelines and the charter, the charter shall control.

 

The RAC will consist of not less than 15 voting members, including the Chair, appointed under the procedures of the NIH and the Department of Health and Human Services.  The maximum number of voting members will be established in the charter of the RAC.  At least a majority of the voting members must be knowledgeable in relevant scientific fields, e.g., molecular genetics, molecular biology, recombinant or synthetic nucleic acid molecule research, including clinical gene transfer research.  At least 4 members of the RAC must be knowledgeable in fields such as public health, laboratory safety, occupational health, protection of human subjects of research, the environment, ethics, law, public attitudes or related fields.  Representatives of the Federal agencies listed in the charter shall serve as non-voting members.  Nominations for RAC members may be submitted to the Office of Science Policy, National Institutes of Health, preferably by e-mail to:  NIHGuidelines@od.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).

 

All meetings of the RAC shall be announced in the Federal Register, including tentative agenda items, 15 days before the meeting.  Final agendas, if modified, shall be available at least 72 hours before the meeting.  No item defined as a Major Action under Section IV-C-1-b-(1) may be added to an agenda following Federal Register publication.

 

RAC shall be responsible for:

 

Section IV-C-2-a.  Advising the NIH Director on the following actions:  (1) Adopting changes in the NIH Guidelines.  (2) Assigning containment levels, changing containment levels, and approving experiments considered as Major Actions under the NIH Guidelines, i.e., the deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally, if such acquisition could compromise the use of the drug to control disease agents in humans, veterinary medicine, or agriculture.  (3) Promulgating and amending lists of classes of recombinant or synthetic nucleic acid molecules to be exempt from the NIH Guidelines because they consist entirely of DNA segments from species that exchange DNA by known physiological processes or otherwise do not present a significant risk to health or the environment.  (4) Certifying new host-vector systems.

 

Section IV-C-2-b.  Transmitting to the NIH Director specific comments/ recommendations about:  (i) a specific human gene transfer experiment, or (ii) a category of human gene transfer experiments;

 

Section IV-C-2-c.  Publicly reviewing human gene transfer clinical trial data and relevant information evaluated and summarized by NIH OSP in accordance with the annual data reporting requirements;

 

Section IV-C-2-d.  Identifying broad scientific, safety, social, and ethical issues relevant to gene therapy research as potential Gene Therapy Policy Conference topics;

 

Section IV-C-2-e. Identifying novel social and ethical issues relevant to human applications of gene transfer and recommending appropriate guidance in the preparation of Informed Consent documents; and

 

Section IV-C-2-f.  Identifying novel scientific and safety issues relevant to human applications of gene transfer and recommending appropriate guidance in the design of human gene transfer clinical trials.

 

Section IV-C-3.    Office of Science Policy (OSP)

 

OSP shall serve as a focal point for information on recombinant or synthetic nucleic acid molecule activities and provide advice to all within and outside NIH including institutions, Biological Safety Officers, Principal Investigators, Federal agencies, state and local governments, and institutions in the private sector.  OSP shall carry out such other functions as may be delegated to it by the NIH Director.  OSP's responsibilities include (but are not limited to) the following:

 

Section IV-C-3-a.  Serving as the focal point for public access to summary information pertaining to human gene transfer experiments;

 

Section IV-C-3-b.  Serving as the focal point for data management of human gene transfer experiments;

 

Section IV-C-3-c.  Administering the annual data reporting requirements (and subsequent review) for human gene transfer experiments (see Appendix M-I-C, Reporting Requirements);

 

Section IV-C-3-d.  Transmitting comments/recommendations arising from public RAC discussion of a novel human gene transfer experiment to the NIH Director.  RAC recommendations shall be forwarded to the Principal Investigator, the sponsoring institution, and other DHHS components, as appropriate.

 

Section IV-C-3-e.  Collaborating with Principal Investigators, Institutional Biosafety Committees, Institutional Review Boards, and other DHHS components (including FDA and the Office for Human Research Protections), to ensure human gene transfer experiment registration compliance in accordance with Appendix M-I, Requirements for Protocol Submission, Review, and Reporting-Human Gene Transfer Experiments of the NIH Guidelines.

 

Section IV-C-3-f.  Administering Gene Therapy Policy Conferences as deemed appropriate by the NIH Director (see Appendix L, Gene Therapy Policy Conferences).

 

Section IV-C-3-g.  Reviewing and approving experiments in conjunction with ad hoc experts involving the cloning of genes encoding for toxin molecules that are lethal for vertebrates at an LD50 of less than or equal to 100 nanograms per kilogram body weight in organisms other than Escherichia coli K-12 (see Section III-B-1, Experiments Involving the Cloning of Toxin Molecules with LD50 of Less than 100 Nanograms Per Kilogram Body Weight, Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates);

 

Section IV-C-3-h.  Serving as the executive secretary of RAC;

 

Section IV-C-3-i.  Publishing in the Federal Register:

 

Section IV-C-3-i-(1).  Announcements of RAC meetings and tentative agendas at least 15 days in advance (Note:  If the agenda for a RAC meeting is modified, OSP shall make the revised agenda available to anyone upon request in advance of the meeting);

 

Section IV-C-3-i-(2).  Announcements of Gene Therapy Policy Conferences and tentative agendas at least 15 days in advance;

 

Section IV-C-3-i-(3).  Proposed Major Actions (see Section IV-C-1-b-(1), Major Actions) at least 15 days prior to the RAC meeting; and

 

Section IV-C-3-j.  Reviewing and approving the membership of an institution's Institutional Biosafety Committee, and where it finds the Institutional Biosafety Committee meets the requirements set forth in Section IV-B-2, Institutional Biosafety Committee (IBC), giving its approval to the Institutional Biosafety Committee membership.

 

Section IV-C-4.    Other NIH Components

 

Other NIH components shall be responsible for certifying maximum containment (BL4) facilities, inspecting them periodically, and inspecting other recombinant or synthetic nucleic acid molecule facilities as deemed necessary.

 

Section IV-D.    Voluntary Compliance

 

Section IV-D-1.    Basic Policy - Voluntary Compliance

 

Individuals, corporations, and institutions not otherwise covered by the NIH Guidelines are encouraged to follow the standards and procedures set forth in Sections I through IV.  In order to simplify discussion, references hereafter to “institutions” are intended to encompass corporations and individuals who have no organizational affiliation.  For purposes of complying with the NIH Guidelines, an individual intending to carry out research involving recombinant or synthetic nucleic acid molecules is encouraged to affiliate with an institution that has an Institutional Biosafety Committee approved under the NIH Guidelines.

 

Since commercial organizations have special concerns, such as protection of proprietary data, some modifications and explanations of the procedures are provided in Sections IV-D-2 through IV-D-5-b, Voluntary Compliance, in order to address these concerns.


 

 

Section IV-D-2.    Institutional Biosafety Committee Approval - Voluntary Compliance

 

It should be emphasized that employment of an Institutional Biosafety Committee member solely for purposes of membership on the Institutional Biosafety Committee does not itself make the member an institutionally affiliated member.  Except for the unaffiliated members, a member of an Institutional Biosafety Committee for an institution not otherwise covered by the NIH Guidelines may participate in the review and approval of a project in which the member has a direct financial interest so long as the member has not been, and does not expect to be, engaged in the project.  Section IV-B-2-a-(4), Institutional Biosafety Committee, is modified to that extent for purposes of these institutions.

 

Section IV-D-3.    Certification of Host-Vector Systems - Voluntary Compliance

 

A host-vector system may be proposed for certification by the NIH Director in accordance with the procedures set forth in Appendix I-II, Certification of Host-Vector Systems.  In order to ensure protection for proprietary data, any public notice regarding a host-vector system which is designated by the institution as proprietary under Section IV-D, Voluntary Compliance, will be issued only after consultation with the institution as to the content of the notice.

 

Section IV-D-4.    Requests for Exemptions and Approvals - Voluntary Compliance

 

Requests for exemptions or other approvals as required by the NIH Guidelines should be submitted based on the procedures set forth in Sections I through IV.  In order to ensure protection for proprietary data, any public notice regarding a request for an exemption or other approval which is designated by the institution as proprietary under Section IV-D-5-a, Voluntary Compliance, will be issued only after consultation with the institution as to the content of the notice.

 

Section IV-D-5.    Protection of Proprietary Data - Voluntary Compliance

 

Section IV-D-5-a.   General

 

In general, the Freedom of Information Act requires Federal agencies to make their records available to the public upon request.  However, this requirement does not apply to, among other things, “trade secrets and commercial or financial information that is obtained from a person and that is privileged or confidential.”  Under 18 U.S.C. 1905, it is a criminal offense for an officer or employee of the U.S. or any Federal department or agency to publish, divulge, disclose, or make known “in any manner or to any extent not authorized by law any information coming to him in the course of his employment or official duties or by reason of any examination or investigation made by, or return, report or record made to or filed with, such department or agency or officer or employee thereof, which information concerns or relates to the trade secrets, (or) processes...of any person, firm, partnership, corporation, or association.”  This provision applies to all employees of the Federal Government, including special Government employees.  Members of RAC are “special Government employees.”

 

In submitting to NIH for purposes of voluntary compliance with the NIH Guidelines, an institution may designate those items of information which the institution believes constitute trade secrets, privileged, confidential, commercial, or financial information.  If NIH receives a request under the Freedom of Information Act for information so designated, NIH will promptly contact the institution to secure its views as to whether the information (or some portion) should be released.  If NIH decides to release this information (or some portion) in response to a Freedom of Information request or otherwise, the institution will be advised and the actual release will be delayed in accordance with 45 Code of Federal Regulations, Section 5.65(d) and (e).

 

Section IV-D-5-b.    Pre-submission Review

 

Any institution not otherwise covered by the NIH Guidelines, which is considering submission of data or information voluntarily to NIH, may request pre-submission review of the records involved to determine if NIH will make all or part of the records available upon request under the Freedom of Information Act.

 

A request for pre-submission review should be submitted to NIH OSP along with the records involved to the Office of Science Policy, National Institutes of Health, preferably by e-mail to: HGTprotocols@mail.nih.gov; additional contact information is also available here and on the OSP website (www.od.osp.gov).  These records shall be clearly marked as being the property of the institution on loan to NIH solely for the purpose of making a determination under the Freedom of Information Act.  NIH OSP will seek a determination from the responsible official under DHHS regulations (45 CFR Part 5) as to whether the records involved, (or some portion) will be made available to members of the public under the Freedom of Information Act.  Pending such a determination, the records will be kept separate from NIH OSP files, will be considered records of the institution and not NIH OSP, and will not be received as part of NIH OSP files.  No copies will be made of such records.

 

NIH OSP will inform the institution of the NIH Freedom of Information Officer's determination and follow the institution's instructions as to whether some or all of the records involved are to be returned to the institution or to become a part of NIH OSP files.  If the institution instructs NIH OSP to return the records, no copies or summaries of the records will be made or retained by DHHS, NIH, or OSP.  The NIH Freedom of Information Officer's determination will represent that official's judgment at the time of the determination as to whether the records involved (or some portion) would be exempt from disclosure under the Freedom of Information Act if at the time of the determination the records were in NIH OSP files and a request was received for such files under the Freedom of Information Act.

 

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SECTION V.    FOOTNOTES AND REFERENCES OF SECTIONS I THROUGH IV

 

Section V-A.  The NIH Director, with advice of the RAC, may revise the classification for the purposes of the NIH Guidelines (see Section IV-C-1-b-(2)-(e), Minor Actions).  The revised list of organisms in each Risk Group is reprinted in Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard.

 

Section V-B.  Section III, Experiments Covered by the NIH Guidelines, describes a number of places where judgments are to be made.  In all these cases, the Principal Investigator shall make the judgment on these matters as part of his/her responsibility to "make the initial determination of the required levels of physical and biological containment in accordance with the NIH Guidelines" (see Section IV-B-7-c-(1)).  For cases falling under Sections III-A through III-E, Experiments Covered by the NIH Guidelines, this judgment is to be reviewed and approved by the Institutional Biosafety Committee as part of its responsibility to make an "independent assessment of the containment levels required by the NIH Guidelines for the proposed research" (see Section IV-B-2-b-(1), Institutional Biosafety Committee).  The Institutional Biosafety Committee may refer specific cases to NIH OSP as part of NIH OSP's functions to "provide advice to all within and outside NIH" (see Section IV-C-3).  NIH OSP may request advice from the RAC as part of the RAC's responsibility for "interpreting the NIH Guidelines for experiments to which the NIH Guidelines do not specifically assign containment levels" (see Section IV-C-1-b-(2)-(f), Minor Actions).

 

Section V-C.  U.S. Department of Health and Human Services, Public Health Service, Centers for Disease Control and Prevention and the National Institutes of HealthBiosafety in Microbiological and Biomedical Laboratories, 5th Edition, 2007.  Copies are available from:  Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20401-0001, Phone (202) 512-1800 [http://www.gpo.gov/].

 

Section V-D.  Classification of Etiologic Agents on the Basis of Hazard, 4th Edition, July 1974, U.S. Department of Health, Education, and Welfare, Public Health Service, Centers for Disease Control, Office of Biosafety, Atlanta, Georgia 30333.

 

Section V-E.  Chin, James ed., Control of Communicable Diseases Manual, 17th Edition, 2000.  ISBN:  087553-242-X, American Public Health Association, 800 I Street, N.W., Washington, D.C. Phone:  (202) 777-2742.

 

Section V-F.  World Health Organization Laboratory Biosafety Manual, 2nd edition.  1993.  WHO Albany, NY.  Copies are available from:  WHO Publication Centre, USA, (Q Corp) 49 Sheridan Avenue, Albany, New York 12210; Phone:  (518) 436-9686 (Order # 1152213).

 

Section V-G.  A U.S. Department of Agriculture permit, required for import and interstate transport of plant and animal pathogens, may be obtained from the U.S. Department of Agriculture, ATTN:  Animal and Plant Health Inspection Service (APHIS), Veterinary Services, National Center for Import-Export, Products Program, 4700 River Road, Unit 40, Riverdale, Maryland 20737.  Phone:  (301) 734-8499; Fax:  (301) 734-8226.

 

Section V-H.  American Type Culture Collection Catalogues of plant viruses, animal viruses, cells, bacteria, fungi, etc. are available from American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209.  Phone:  (703) 365-2700.

 

Section V-I.  U.S. Department of Labor, Occupational Safety and Health Administration, 29 CFR 1910.1030, Bloodborne Pathogens.  See also, Exposure to Bloodborne Pathogens, OSHA 3127, 1996 (Revised).

 

Section V-J.  As classified in the Virus Taxonomy:  The Classification and Nomenclature of Viruses. The Seventh Report of the International Committee on Taxonomy of Viruses, Academic Press, 2000 (0123702003) San Diego, CA.

 

Section V-K.  i.e., the total of all genomes within a family shall not exceed two-thirds of the genome.

 

Section V-L.  Organisms including alastrim, smallpox (variola) and whitepox may not be studied in the United States except at specified facilities.  All activities, including storage of variola and whitepox, are restricted to the single national facility (World Health Organization Collaborating Center for Smallpox Research, Centers for Disease Control and Prevention, Atlanta, Georgia).

 

Section V-M.  In accordance with accepted scientific and regulatory practices of the discipline of plant pathology, an exotic plant pathogen (e.g., virus, bacteria, or fungus) is one that is unknown to occur within the U.S. (see Section V-G, Footnotes and References of Sections I-IV).  Determination of whether a pathogen has a potential for serious detrimental impact on managed (agricultural, forest, grassland) or natural ecosystems should be made by the Principal Investigator and the Institutional Biosafety Committee, in consultation with scientists knowledgeable of plant diseases, crops, and ecosystems in the geographic area of the research.

 

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APPENDIX A.   EXEMPTIONS UNDER SECTION III-F-6--SUBLISTS OF NATURAL EXCHANGERS

 

Certain specified recombinant or synthetic nucleic acid molecules that consist entirely of DNA segments from different species that exchange DNA by known physiological processes, though one or more of the segments may be a synthetic equivalent are exempt from these NIH Guidelines (see Section III-F-6, Exempt Experiments).  Institutional Biosafety Committee registration is not required for these exempt experiments.  A list of such exchangers will be prepared and periodically revised by the NIH Director with advice from the RAC after appropriate notice and opportunity for public comment (see Section IV-C-1-b-(1)-(c), NIH Director--Specific Responsibilities).  For a list of natural exchangers that are exempt from the NIH Guidelines, see Appendices A-I through A-VI, Exemptions under Section III-F-6 Sublists of Natural ExchangersSection III-F-6, Exempt Experiments, describes recombinant or synthetic nucleic acid molecules that are:  (1) composed entirely of DNA segments from one or more of the organisms within a sublist, and (2) to be propagated in any of the organisms within a sublist (see Classification of Bergey's Manual of Determinative Bacteriology; 8th edition, R. E. Buchanan and N. E. Gibbons, editors, Williams and Wilkins Company; Baltimore, Maryland 1984).  Although these experiments are exempt, it is recommended that they be performed at the appropriate biosafety level for the host or recombinant/synthetic organism (see Biosafety in Microbiological and Biomedical Laboratories, 5th edition, 2007, U.S. DHHS, Public Health Service, Centers for Disease Control and Prevention, Atlanta, Georgia, and NIH Office of Biosafety, Bethesda, Maryland).

 

Appendix A-I.   Sublist A

 

Genus Escherichia

Genus Shigella 

Genus Salmonella - including Arizona

Genus Enterobacter

Genus Citrobacter - including Levinea

Genus Klebsiella - including oxytoca

Genus Erwinia

Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas mendocina

Serratia marcescens

Yersinia enterocolitica

 

Appendix A-II.    Sublist B

 

Bacillus subtilis

Bacillus licheniformis

Bacillus pumilus

Bacillus globigii

Bacillus niger

Bacillus nato

Bacillus amyloliquefaciens

Bacillus aterrimus

 

Appendix A-III.    Sublist C

 

Streptomyces aureofaciens

Streptomyces rimosus

Streptomyces coelicolor

 

Appendix A-IV.    Sublist D

 

Streptomyces griseus

Streptomyces cyaneus

Streptomyces venezuelae

 

Appendix A-V.     Sublist E

 

One way transfer of Streptococcus mutans or Streptococcus lactis DNA into Streptococcus sanguis

 

Appendix A-VI.     Sublist F

 

Streptococcus sanguis

Streptococcus pneumoniae

Streptococcus faecalis

Streptococcus pyogenes

Streptococcus mutans

 

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APPENDIX B.   CLASSIFICATION OF HUMAN ETIOLOGIC AGENTS ON THE BASIS OF HAZARD

 

This appendix includes those biological agents known to infect humans as well as selected animal agents that may pose theoretical risks if inoculated into humans.  Included are lists of representative genera and species known to be pathogenic; mutated, recombined, and non-pathogenic species and strains are not considered.  Non-infectious life cycle stages of parasites are excluded.

 

This appendix reflects the current state of knowledge and should be considered a resource document.  Included are the more commonly encountered agents and is not meant to be all-inclusive.  Information on agent risk assessment may be found in the Agent Summary Statements of the CDC/NIH publication, Biosafety in Microbiological and Biomedical Laboratories (see Sections V-C, V-D, V-E, and V-F, Footnotes and References of Sections I through IV.  Further guidance on agents not listed in Appendix B may be obtained through:  Centers for Disease Control and Prevention, Biosafety Branch, Atlanta, Georgia 30333, Phone:  (404) 639-3883, Fax:  (404) 639-2294; National Institutes of Health, Division of Safety, Bethesda, Maryland 20892, Phone:  (301) 496-1357; Biosafety Manager, National Animal Disease Center, U.S. Department of Agriculture - ARS, Ames, Iowa 50010, Phone:  (515) 337-7772.

 

A special committee of the American Society for Microbiology will conduct an annual review of this appendix and its recommendation for changes will be presented to the Recombinant DNA Advisory Committee as proposed amendments to the NIH Guidelines.


 

 

Appendix B - Table 1.      Basis for the Classification of Biohazardous Agents by Risk Group (RG)

 

Risk Group 1 (RG1)

Agents that are not associated with disease in healthy adult humans

Risk Group 2 (RG2)

Agents that are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available

Risk Group 3 (RG3)

Agents that are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available (high individual risk but low community risk)

Risk Group 4 (RG4)

Agents that are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available (high individual risk and high community risk)

 

Appendix B-I.   Risk Group 1 (RG1) Agents

 

RG1 agents are not associated with disease in healthy adult humans.  Examples of RG1 agents include asporogenic Bacillus subtilis or Bacillus licheniformis (see Appendix C-IV-A, Bacillus subtilis or Bacillus licheniformis Host-Vector Systems, Exceptions); adeno- associated virus (AAV – all serotypes); and recombinant or synthetic AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus.  A strain of Escherichia coli (see Appendix C-II-A, Escherichia coli K-12 Host Vector Systems, Exceptions) is an RG1 agent if it (1) does not possess a complete lipopolysaccharide (i.e., lacks the O antigen); and (2) does not carry any active virulence factor (e.g., toxins) or colonization factors and does not carry any genes encoding these factors.

 

Those agents not listed in Risk Groups (RGs) 2, 3 and 4 are not automatically or implicitly classified in RG1; a risk assessment must be conducted based on the known and potential properties of the agents and their relationship to agents that are listed.

 

Appendix B-II.    Risk Group 2 (RG2) Agents

 

RG2 agents are associated with human disease which is rarely serious and for which preventive or therapeutic interventions are often available.

 

Appendix B-II-A.   Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia

 

--Acinetobacter baumannii (formerly Acinetobacter calcoaceticus)

--Actinobacillus

--Actinomyces pyogenes (formerly Corynebacterium pyogenes)

--Aeromonas hydrophila

--Amycolata autotrophica

--Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum)

--Arizona hinshawii - all serotypes

--Bacillus anthracis

--Bartonella henselae, B. quintana, B. vinsonii

--Bordetella including B. pertussis 

--Borrelia recurrentis, B. burgdorferi

--Burkholderia (formerly Pseudomonas species) except those listed in Appendix B-III-A (RG3))

--Campylobacter coli, C. fetus, C. jejuni

--Chlamydia psittaci, C. trachomatis, C. pneumoniae

--Clostridium botulinum, C. chauvoei, C. haemolyticum, C. histolyticum, C. novyi, C. septicum, C. tetani

--Coxiella burnetii – specifically the Phase II, Nine Mile strain, plaque purified, clone 4

--Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale

--Dermatophilus congolensis

--Edwardsiella tarda

--Erysipelothrix rhusiopathiae

--Escherichia coli - all enteropathogenic, enterotoxigenic, enteroinvasive and strains bearing K1 antigen, including E. coli O157:H7

--*Francisella tularensis specifically *F. tularensis subspecies novicida [aka F. novicida], strain Utah 112; *F. tularensis subspecies holarctica LVS; *F. tularensis biovar tularensis strain ATCC 6223 (aka strain B38)

   *For research involving high concentrations, BL3 practices should be considered (see Appendix G-II-C-2. Special Practices (BL3)).

--Haemophilus ducreyi, H. influenzae

--Helicobacter pylori

--Klebsiella - all species except K. oxytoca (RG1)

--Legionella including L. pneumophila

--Leptospira interrogans - all serotypes

--Listeria

--Moraxella

--Mycobacterium (except those listed in Appendix B-III-A (RG3)) including M. avium complex, M. asiaticum, M. bovis BCG vaccine strain, M. chelonae, M. fortuitum, M. kansasii, M. leprae, M. malmoense, M. marinum, M. paratuberculosis, M. scrofulaceum, M. simiae, M. szulgai, M. ulcerans, M. xenopi

--Mycoplasma, except M. mycoides and M. agalactiae which are restricted animal pathogens

--Neisseria gonorrhoeae, N. meningitidis

--Nocardia asteroides, N. brasiliensis, N. otitidiscaviarum, N. transvalensis

--Pseudomonas aeruginosa

--Rhodococcus equi

--Salmonella including S. arizonae, S. choleraesuis, S. enteritidis, S. gallinarum-pullorum, S. meleagridis, S. paratyphi, A, B, C, S. typhi, S. typhimurium

--Shigella including S. boydii, S. dysenteriae, type 1, S. flexneri, S. sonnei

--Sphaerophorus necrophorus

--Staphylococcus aureus

--Streptobacillus moniliformis

 

--Streptococcus including S. pneumoniae, S. pyogenes

--Treponema pallidum, T. carateum

--Vibrio cholerae, V. parahaemolyticus, V. vulnificus

--Yersinia enterocolitica

--Yersinia pestis specifically pgm() strains (lacking the 102 kb pigmentation locus) and lcr() strains (lacking the LCR plasmid)

 

Appendix B-II-B.   Risk Group 2 (RG2) - Fungal Agents

 

--Blastomyces dermatitidis

--Cladosporium bantianum, C. (Xylohypha) trichoides

--Cryptococcus neoformans

--Dactylaria galopava (Ochroconis gallopavum)

--Epidermophyton

--Exophiala (Wangiella) dermatitidis

--Fonsecaea pedrosoi

--Microsporum

--Paracoccidioides braziliensis

--Penicillium marneffei

--Sporothrix schenckii

--Trichophyton

 

Appendix B-II-C.   Risk Group 2 (RG2) - Parasitic Agents

 

--Ancylostoma human hookworms including A. duodenale, A. ceylanicum

--Ascaris including Ascaris lumbricoides suum

--Babesia including B. divergens, B. microti

--Brugia filaria worms including B. malayi, B. timori

--Coccidia

--Cryptosporidium including C. parvum

--Cysticercus cellulosae (hydatid cyst, larva of T. solium)

--Echinococcus including E. granulosis, E. multilocularis, E. vogeli

--Entamoeba histolytica

--Enterobius

--Fasciola including F. gigantica, F. hepatica

--Giardia including G. lamblia

--Heterophyes

--Hymenolepis including H. diminuta, H. nana

--Isospora

--Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruviana, L. tropica

--Loa loa filaria worms

--Microsporidium

--Naegleria fowleri

--Necator human hookworms including N. americanus

--Onchocerca filaria worms including, O. volvulus

--Plasmodium including simian species, P. cynomolgi, P. falciparum, P. malariae, P. ovale, P. vivax

--Sarcocystis including S. sui hominis

--Schistosoma including S. haematobium, S. intercalatum, S. japonicum, S. mansoni, S. mekongi

--Strongyloides including S. stercoralis

--Taenia solium

--Toxocara including T. canis

--Toxoplasma including T. gondii

--Trichinella spiralis

--Trypanosoma including T. brucei brucei, T. brucei gambiense, T. brucei rhodesiense, T. cruzi

--Wuchereria bancrofti filaria worms

 

Appendix B-II-D.   Risk Group 2 (RG2) - Viruses

 

Adenoviruses, human - all types

 

Alphaviruses (Togaviruses) - Group A Arboviruses

--Chikungunya vaccine strain 181/25

--Eastern equine encephalomyelitis virus

--Venezuelan equine encephalomyelitis vaccine strains TC-83 and V3526

--Western equine encephalomyelitis virus

 

Arenaviruses

--Junin virus candid #1 vaccine strain

--Lymphocytic choriomeningitis virus (non-neurotropic strains)

--Tacaribe virus complex

--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV)

 

Bunyaviruses

--Bunyamwera virus

--Rift Valley fever virus vaccine strain MP-12

--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV)

 

Caliciviruses

 

Coronaviruses

 

Flaviviruses - Group B Arboviruses

--Dengue virus serotypes 1, 2, 3, and 4

--Japanese encephalitis virus strain SA 14-14-2

--Yellow fever virus vaccine strain 17D

--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV)

 

Hepatitis A, B, C, D, and E viruses

 

Herpesviruses - except Herpesvirus simiae (Monkey B virus) (see Appendix B-IV-D, Risk Group 4 (RG4) - Viral Agents)

--Cytomegalovirus

--Epstein Barr virus

--Herpes simplex types 1 and 2

--Herpes zoster

--Human herpesvirus types 6 and 7

 

Orthomyxoviruses

--Influenza viruses types A, B, and C (except those listed in Appendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions)

--Tick-borne orthomyxoviruses

 

Papilloma viruses

--All human papilloma viruses

 

Paramyxoviruses

--Newcastle disease virus

--Measles virus

--Mumps virus

--Parainfluenza viruses types 1, 2, 3, and 4

--Respiratory syncytial virus

 

Parvoviruses

--Human parvovirus (B19)

 

 

Picornaviruses

--Coxsackie viruses types A and B

--Echoviruses - all types

--Polioviruses - all types, wild and attenuated

--Rhinoviruses - all types

 

Poxviruses - all types except Monkeypox virus (see Appendix B-III-D, Risk Group 3 (RG3) - Viruses and Prions) and restricted poxviruses including Alastrim, Smallpox, and Whitepox (see Section V-L, Footnotes and References of Sections I through IV)

 

Reoviruses - all types including Coltivirus, human Rotavirus, and Orbivirus (Colorado tick fever virus)

 

Rhabdoviruses

--Rabies virus - all strains

--Vesicular stomatitis virus non exotic strains: VSV-Indiana 1 serotype strains (e.g. Glasgow, Mudd-Summers, Orsay, San Juan) and VSV-New Jersey serotype strains (e.g. Ogden, Hazelhurst)

 

Rubivirus (Togaviruses)

--Rubella virus

 

Appendix B-III.    Risk Group 3 (RG3) Agents

 

RG3 agents are associated with serious or lethal human disease for which preventive or therapeutic interventions may be available.

 

Appendix B-III-A.    Risk Group 3 (RG3) - Bacterial Agents Including Rickettsia

 

--Bartonella

--Brucella including B. abortus, B. canis, B. suis

--Burkholderia (Pseudomonas) mallei, B. pseudomallei

--Coxiella burnetii (except the Phase II, Nine Mile strain listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia)

--Francisella tularensis (except those strains listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia)

--Mycobacterium bovis (except BCG strain, see Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia), M. tuberculosis

--Orientia tsutsugamushi (was R. tsutsugamushi)

--Pasteurella multocida type B -"buffalo" and other virulent strains

--Rickettsia akari, R. australis, R. canada, R. conorii, R. prowazekii, R. rickettsii, R, siberica, R. typhi (R. mooseri)

--Yersinia pestis (except those strains listed in Appendix B-II-A, Risk Group 2 (RG2) - Bacterial Agents Including Chlamydia)

 

Appendix B-III-B.    Risk Group 3 (RG3) - Fungal Agents

 

--Coccidioides immitis (sporulating cultures; contaminated soil)

--Histoplasma capsulatum, H. capsulatum var. duboisii

 

Appendix B-III-C.    Risk Group 3 (RG3) - Parasitic Agents

 

None

 

Appendix B-III-D.    Risk Group 3 (RG3) - Viruses and Prions

 

Alphaviruses (Togaviruses) - Group A Arboviruses

--Chikungunya virus (except the vaccine strain 181/25 listed in Appendix B-II-D Risk Group2 (RG2) – Viruses)

--Semliki Forest virus

--St. Louis encephalitis virus

--Venezuelan equine encephalomyelitis virus (except the vaccine strains TC-83 and V3526, see Appendix B-II-D (RG2) – Viruses)

--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV)

 

Arenaviruses

--Flexal

--Lymphocytic choriomeningitis virus (LCM) (neurotropic strains)

 

Bunyaviruses

--Hantaviruses including Hantaan virus   

--Rift Valley fever virus

 

Coronaviruses

--SARS-associated coronavirus (SARS-CoV)

--Middle East respiratory syndrome coronavirus (MERS-CoV)

 

Flaviviruses - Group B Arboviruses

--Japanese encephalitis virus (except those strains listed in Appendix B-II-D Risk Group2 (RG2) - Viruses)

--West Nile virus (WNV)

--Yellow fever virus

--Other viruses as listed in the reference source (see Section V-C, Footnotes and References of Sections I through IV)

 

Orthomyxoviruses

-- Influenza viruses 1918-1919 H1N1 (1918 H1N1), human H2N2 (1957-1968), and highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/96-like H5 lineage (HPAI H5N1).

 

Poxviruses

--Monkeypox virus

 

Prions

--Transmissible spongiform encephalopathies (TSE) agents (Creutzfeldt-Jacob disease and kuru agents)(see Section V-C, Footnotes and References of Sections I through IV, for containment instruction)

 

Retroviruses

--Human immunodeficiency virus (HIV) types 1 and 2

--Human T cell lymphotropic virus (HTLV) types 1 and 2

--Simian immunodeficiency virus (SIV)

 

Rhabdoviruses

--Vesicular stomatitis virus (except those strains listed in Appendix B-II-D Risk Group2 (RG2) - Viruses)

 

Appendix B-IV.    Risk Group 4 (RG4) Agents

 

RG4 agents are likely to cause serious or lethal human disease for which preventive or therapeutic interventions are not usually available.

 

Appendix B-IV-A.    Risk Group 4 (RG4) - Bacterial Agents

 

None

 

Appendix B-IV-B.    Risk Group 4 (RG4) - Fungal Agents

 

None

 

Appendix B-IV-C.    Risk Group 4 (RG4) - Parasitic Agents

 

None

 

Appendix B-IV-D.    Risk Group 4 (RG4) - Viral Agents

 

Arenaviruses

--Guanarito virus

--Lassa virus

 

--Junin virus (except the candid #1 vaccine strain listed in Appendix B-II-D Risk Group2 (RG2) – Viruses)

--Machupo virus

--Sabia

 

Bunyaviruses (Nairovirus)

--Crimean-Congo hemorrhagic fever virus

 

Filoviruses

--Ebola virus

--Marburg virus

 

Flaviruses - Group B Arboviruses

--Tick-borne encephalitis virus complex including Absetterov, Central European encephalitis, Hanzalova, Hypr, Kumlinge, Kyasanur Forest disease, Omsk hemorrhagic fever, and Russian spring-summer encephalitis viruses

 

Herpesviruses (alpha)

--Herpesvirus simiae (Herpes B or Monkey B virus)

 

Paramyxoviruses

--Equine Morbillivirus (Hendra virus)

 

Hemorrhagic fever agents and viruses as yet undefined

 

Appendix B-V.     Animal Viral Etiologic Agents in Common Use

 

The following list of animal etiologic agents is appended to the list of human etiologic agents.  None of these agents is associated with disease in healthy adult humans; they are commonly used in laboratory experimental work.

 

A containment level appropriate for RG1 human agents is recommended for their use.  For agents that are infectious to human cells, e.g., amphotropic and xenotropic strains of murine leukemia virus, a containment level appropriate for RG2 human agents is recommended.

 

Baculoviruses

 

Herpesviruses

--Herpesvirus ateles

--Herpesvirus saimiri

--Marek's disease virus

--Murine cytomegalovirus

 

Papilloma viruses

--Bovine papilloma virus

--Shope papilloma virus

 

Polyoma viruses

--Polyoma virus

--Simian virus 40 (SV40)

 

Retroviruses

--Avian leukosis virus

--Avian sarcoma virus

--Bovine leukemia virus

--Feline leukemia virus

--Feline sarcoma virus

--Gibbon leukemia virus

--Mason-Pfizer monkey virus

--Mouse mammary tumor virus

--Murine leukemia virus

 

--Murine sarcoma virus

--Rat leukemia virus

 

Appendix B-V-1.    Murine Retroviral Vectors

 

Murine retroviral vectors to be used for human transfer experiments (less than 10 liters) that contain less than 50% of their respective parental viral genome and that have been demonstrated to be free of detectable replication competent retrovirus can be maintained, handled, and administered, under BL1 containment.

 

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APPENDIX C.     EXEMPTIONS UNDER SECTION III-F-8

 

Section III-F-8 states that exempt from these NIH Guidelines are "those that do not present a significant risk to health or the environment (see Section IV-C-1-b-(1)-(c), NIH Director--Specific Responsibilities), as determined by the NIH Director, with the advice of the RAC, and following appropriate notice and opportunity for public comment.  See Appendix C, Exemptions under Sections III-F-8, for other classes of experiments which are exempt from the NIH Guidelines."  The following classes of experiments are exempt under Section III-F-8:

 

Appendix C-I.     Recombinant or Synthetic Nucleic Acid Molecules in Tissue Culture

 

Recombinant or synthetic nucleic acid molecules containing less than one-half of any eukaryotic viral genome (all viruses from a single family being considered identical -- see Appendix C-IX-E, Footnotes and References of Appendix C), that are propagated and maintained in cells in tissue culture are exempt from these NIH Guidelines with the exceptions listed in Appendix C-I-A.


 

Appendix C-I-A.   Exceptions

 

The following categories are not exempt from the NIH Guidelines:  (i) experiments described in Section III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents, (iii) experiments involving the deliberate introduction of genes coding for the biosynthesis of molecules that are toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates), and (iv) whole plants regenerated from plant cells and tissue cultures are covered by the exemption provided they remain axenic cultures even though they differentiate into embryonic tissue and regenerate into plantlets.

 

Appendix C-II.    Escherichia coli K-12 Host-Vector Systems

 

Experiments which use Escherichia coli K-12 host-vector systems, with the exception of those experiments listed in Appendix C-II-A, are exempt from the NIH Guidelines provided that:  (i) the Escherichia coli host does not contain conjugation proficient plasmids or generalized transducing phages; or (ii) lambda or lambdoid or Ff bacteriophages or non-conjugative plasmids (see Appendix C-IX-B, Footnotes and References of Appendix C) shall be used as vectors.  However, experiments involving the insertion into Escherichia coli K-12 of DNA from prokaryotes that exchange genetic information (see Appendix C-IX-C, Footnotes and References of Appendix C) with Escherichia coli may be performed with any Escherichia coli K-12 vector (e.g., conjugative plasmid).  When a non-conjugative vector is used, the Escherichia coli K-12 host may contain conjugation-proficient plasmids either autonomous or integrated, or generalized transducing phages.  For these exempt laboratory experiments, Biosafety Level (BL) 1 physical containment conditions are recommended.  For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the host organism unmodified by recombinant or synthetic nucleic acid molecule techniques; the Institutional Biosafety Committee can specify higher containment if deemed necessary.

 

Appendix C-II-A.   Exceptions

 

The following categories are not exempt from the NIH Guidelines:  (i) experiments described in Section III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human

 

Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I

through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval, (iii) large-scale experiments (e.g., more than 10 liters of culture), and (iv) experiments involving the cloning of toxin molecule genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).

 

Appendix C-III.    Saccharomyces Host-Vector Systems

 

Experiments involving Saccharomyces cerevisiae and Saccharomyces uvarum host-vector systems, with the exception of experiments listed in Appendix C-III-A, are exempt from the NIH Guidelines.  For these exempt experiments, BL1 physical containment is recommended.  For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the unmodified host organism; the Institutional Biosafety Committee can specify higher containment if deemed necessary.

 

Appendix C-III-A.    Exceptions

 

The following categories are not exempt from the NIH Guidelines:  (i) experiments described in Section III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval, (iii) large-scale experiments (e.g., more than 10 liters of culture), and (iv) experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).

 

Appendix C-IV.    Kluyveromyces Host-Vector Systems

 

Experiments involving Kluyveromyces lactis host-vector systems, with the exception of experiments listed in Appendix C-IV-A, are exempt from the NIH Guidelines provided laboratory-adapted strains are used (i.e. strains that have been adapted to growth under optimal or defined laboratory conditions).  For these exempt experiments, BL1 physical containment is recommended.  For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the unmodified host organism; the Institutional Biosafety Committee may specify higher containment if deemed necessary.

 

Appendix C-IV-A     Exceptions

 

The following categories are not exempt from the NIH Guidelines:  (i) experiments described in Section III-B, which require NIH OSP and Institutional Biosafety Committee approval before initiation; (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval; (iii) large-scale experiments (e.g., more than 10 liters of culture), and (v) experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).

 

 

Appendix C-V.     Bacillus subtilis or Bacillus licheniformis Host-Vector Systems

 

Any asporogenic Bacillus subtilis or asporogenic Bacillus licheniformis strain which does not revert to a spore-former with a frequency greater than 10-7 may be used for cloning DNA with the exception of those experiments listed in Appendix C-V-A, Exceptions.  For these exempt laboratory experiments, BL1 physical containment conditions are recommended.  For large-scale fermentation experiments, the appropriate physical containment conditions need be no greater than those for the unmodified host organism; the Institutional Biosafety Committee can specify higher containment if it deems necessary.

 

Appendix C-V-A.     Exceptions

 

The following categories are not exempt from the NIH Guidelines:  (i) experiments described in Section III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval, (iii) large-scale experiments (e.g., more than 10 liters of culture), and (iv) experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).

 

Appendix C-VI.    Extrachromosomal Elements of Gram Positive Organisms

 

Recombinant or synthetic nucleic acid molecules derived entirely from extrachromosomal elements of the organisms listed below (including shuttle vectors constructed from vectors described in Appendix C), propagated and maintained in organisms listed below are exempt from these NIH Guidelines.

 

Bacillus amyloliquefaciens

Bacillus amylosacchariticus

Bacillus anthracis

Bacillus aterrimus

Bacillus brevis

Bacillus cereus

Bacillus globigii

Bacillus licheniformis 

Bacillus megaterium

Bacillus natto

Bacillus niger

Bacillus pumilus

Bacillus sphaericus

Bacillus stearothermophilus

Bacillus subtilis

Bacillus thuringiensis

Clostridium acetobutylicum

Lactobacillus casei

Listeria grayi

Listeria monocytogenes

Listeria murrayi

Pediococcus acidilactici

Pediococcus damnosus

Pediococcus pentosaceus

Staphylococcus aureus

Staphylococcus carnosus

Staphylococcus epidermidis

Streptococcus agalactiae 

Streptococcus anginosus

Streptococcus avium

Streptococcus cremoris

Streptococcus dorans

Streptococcus equisimilis

Streptococcus faecalis

Streptococcus ferus

Streptococcus lactis

Streptococcus ferns

Streptococcus mitior

Streptococcus mutans

Streptococcus pneumoniae

Streptococcus pyogenes

 

Streptococcus salivarius

Streptococcus sanguis

Streptococcus sobrinus

Streptococcus thermophilus

 

Appendix C-VI-A.  Exceptions

 

The following categories are not exempt from the NIH Guidelines:  (i) experiments described in Section III-B which require NIH OSP and Institutional Biosafety Committee approval before initiation, (ii) experiments involving DNA from Risk Groups 3, 4, or restricted organisms (see Appendix B, Classification of Human Etiologic Agents on the Basis of Hazard, and Sections V-G and V-L, Footnotes and References of Sections I through IV) or cells known to be infected with these agents may be conducted under containment conditions specified in Section III-D-2 with prior Institutional Biosafety Committee review and approval, (iii) large-scale experiments (e.g., more than 10 liters of culture), and (iv) experiments involving the deliberate cloning of genes coding for the biosynthesis of molecules toxic for vertebrates (see Appendix F, Containment Conditions for Cloning of Genes Coding for the Biosynthesis of Molecules Toxic for Vertebrates).

 

Appendix C-VII.   The Purchase or Transfer of Transgenic Rodents

 

The purchase or transfer of transgenic rodents for experiments that require BL1 containment (See Appendix G-III-M, Footnotes and References of Appendix G) are exempt from the NIH Guidelines.

 

Appendix C-VIII.           Generation of BL1 Transgenic Rodents via Breeding

 

The breeding of two different transgenic rodents or the breeding of a transgenic rodent and a non-transgenic rodent with the intent of creating a new strain of transgenic rodent that can be housed at BL1 containment will be exempt from the NIH Guidelines if:
(1) Both parental rodents can be housed under BL1 containment; and

(2) neither parental transgenic rodent contains the following genetic modifications:  (i) incorporation of more than one-half of the genome of an exogenous eukaryotic virus from a single family of viruses; or (ii) incorporation of a transgene that is under the control of a gammaretroviral long terminal repeat (LTR); and

(3) the transgenic rodent that results from this breeding is not expected to contain more than one-half of an exogenous viral genome from a single family of viruses.

 

Appendix C-IX.     Footnotes and References of Appendix C

 

Appendix C-IX-A.  The NIH Director, with advice of the RAC, may revise the classification for the purposes of these NIH Guidelines (see Section IV-C-1-b-(2)-(b), Minor Actions).  The revised list of organisms in each Risk Group is located in Appendix B.

 

Appendix C-IX-B.  A subset of non-conjugative plasmid vectors are poorly mobilizable (e.g., pBR322, pBR313).  Where practical, these vectors should be employed.

 

Appendix C-IX-C.  Defined as observable under optimal laboratory conditions by transformation, transduction, phage infection, and/or conjugation with transfer of phage, plasmid, and/or chromosomal genetic information.  Note that this definition of exchange may be less stringent than that applied to exempt organisms under Section III-F-6, Exempt Experiments.

 

Appendix C-IX-D.  As classified in the Third Report of the International Committee on Taxonomy of Viruses:  Classification and Nomenclature of Viruses, R. E. F. Matthews (ed.), Intervirology 12 (129-296), 1979.

 

Appendix C-IX-E.  i.e., the total of all genomes within a Family shall not exceed one-half of the genome.

 

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APPENDIX D.   MAJOR ACTIONS TAKEN UNDER THE NIH GUIDELINES

 

As noted in the subsections of Section IV-C-1-b-(1), the Director, NIH, may take certain actions with regard to the NIH Guidelines after the issues have been considered by the RAC.  Some of the actions taken to date include the following:

 

Appendix D-1.  Permission is granted to clone foot and mouth disease virus in the EK1 host-vector system consisting of E. coli K-12 and the vector pBR322, all work to be done at the Plum Island Animal Disease Center.

 

Appendix D-2.  Certain specified clones derived from segments of the foot and mouth disease virus may be transferred from Plum Island Animal Disease Center to the facilities of Genentech, Inc., of South San Francisco, California.  Further development of the clones at Genentech, Inc., has been approved under BL1 + EK1 conditions.

 

Appendix D-3.  The Rd strain of Hemophilus influenzae can be used as a host for the propagation of the cloned Tn 10 tet R gene derived from E. coli K-12 employing the non-conjugative Hemophilus plasmid, pRSF0885, under BL1 conditions.

 

Appendix D-4.  Permission is granted to clone certain subgenomic segments of foot and mouth disease virus in HV1 Bacillus subtilis and Saccharomyces cerevisiae host-vector systems under BL1 conditions at Genentech, Inc., South San Francisco, California.

 

Appendix D-5.  Permission is granted to Dr. Ronald Davis of Stanford University to field test corn plants modified by recombinant DNA techniques under specified containment conditions.

 

Appendix D-6.  Permission is granted to clone in E. coli K-12 under BL1 physical containment conditions subgenomic segments of rift valley fever virus subject to conditions which have been set forth by the RAC.

 

Appendix D-7.  Attenuated laboratory strains of Salmonella typhimurium may be used under BL1 physical containment conditions to screen for the Saccharomyces cerevisiae pseudouridine synthetase gene.  The plasmid YEp13 will be employed as the vector.

 

Appendix D-8.  Permission is granted to transfer certain clones of subgenomic segments of foot and mouth disease virus from Plum Island Animal Disease Center to the laboratories of Molecular Genetics, Inc., Minnetonka, Minnesota, and to work with these clones under BL1 containment conditions.  Approval is contingent upon review of data on infectivity testing of the clones by a working group of the RAC.

 

Appendix D-9.  Permission is granted to Dr. John Sanford of Cornell University to field test tomato and tobacco plants transformed with bacterial (E.coli K-12) and yeast DNA using pollen as a vector.

 

Appendix D-10.  Permission is granted to Drs. Steven Lindow and Nickolas Panopoulos of the University of California, Berkeley, to release under specified conditions Pseudomonas syringae, pathovars (pv.) syringae, and Erwinia herbicola carrying in vitro generated deletions of all or part of the genes involved in ice nucleation.

 

Appendix D-11.  Agracetus of Middleton, Wisconsin, may field test under specified conditions disease resistant tobacco plants prepared by recombinant DNA techniques.

 

Appendix D-12.  Eli Lilly and Company of Indianapolis, Indiana, may conduct large-scale experiments and production involving Cephalosporium acremonium strain LU4-79-6 under less than Biosafety Level 1 - Large Scale (BL1-LS) conditions.

 

Appendix D-13.  Drs. W. French Anderson, R. Michael Blaese, and Steven Rosenberg of the NIH, Bethesda, Maryland, can conduct experiments in which a bacterial gene coding for neomycin phosphotransferase will be inserted into a portion of the tumor infiltrating lymphocytes (TIL) of cancer patients using a retroviral vector, N2.  The marked TIL then will be combined with unmarked TIL, and reinfused into the patients.  This experiment is an addition to an ongoing adoptive immunotherapy protocol in which TIL are isolated from a patient's tumor, grown in culture in the presence of interleukin-2, and reinfused into the patient.  The marker gene will be used to detect TIL at various time intervals following reinfusion. 

Approval is based on the following four stipulations:  (I) there will be no limitation of the number of patients in the continuing trial; (ii) the patients selected will have a life expectancy of about 90 days; (iii) the patients give fully informed consent to participate in the trial; and (iv) the investigators will provide additional data before inserting a gene for therapeutic purposes. (Protocol #8810-001)

 

Appendix D-14.  U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) may conduct certain experiments involving products of a yellow fever virus originating from the 17-D yellow fever clone at the Biosafety Level 3 containment level using HEPA filters and vaccination of laboratory personnel.

 

In addition, USAMRIID may conduct certain experiments involving vaccine studies of Venezuelan equine encephalitis virus at the Biosafety Level 3 containment level using HEPA filters and vaccination of laboratory personnel.

 

Appendix D-15.  Drs. R. Michael Blaese and W. French Anderson of the NIH, Bethesda, Maryland, can conduct experiments in which a gene coding for adenosine deaminase (ADA) will be inserted into T lymphocytes of patients with severe combined immunodeficiency disease, using a retroviral vector, LASN.  Following insertion of the gene, these T lymphocytes will be reinfused into the patients.  The patients will then be followed for evidence of clinical improvement in the disease state, and measurement of multiple parameters of immune function by laboratory testing.

 

Approval is based on the following two stipulations:  (I) that intraperitoneal administration of transduced T lymphocytes not be used before clearance by the Chair of the Recombinant DNA Advisory Committee; and (ii) that the number of research patients be limited to 10 at this time.

 

In addition to the conditions outlined in the initial approval, patients may be given a supplement of a CD-34+-enriched peripheral blood lymphocytes (PBL) which have been placed in culture conditions that favor progenitor cell growth.  This enriched population of cells will be transduced with the retroviral vector, G1NaSvAd.  G1NaSvAd is similar to LASN, yet distinguishable by PCR.  LASN has been used to transduce peripheral blood T lymphocytes with the ADA gene.  Lymphocytes and myeloid cells will be isolated from patients over time and assayed for the presence of the LASN or G1NaSvAd vectors.  The primary objectives of this protocol are to transduce CD 34+ peripheral blood cells with the adenosine deaminase gene, administer these cells to patients, and determine if such cells can differentiate into lymphoid and myeloid cells in vivo.  There is a potential for benefit to the patients in that these hematopoietic progenitor cells may survive longer, and divide to yield a broader range of gene-corrected cells. (Protocol #9007-002)

 

 

Appendix D-16.  Dr. Steven A. Rosenberg of the National Institutes of Health, Bethesda, Maryland, can conduct experiments on patients with advanced melanoma who have failed all effective therapy.  These patients will be treated with escalating doses of autologous tumor infiltrating lymphocytes (TIL) transduced with a gene coding for tumor necrosis factor (TNF).  Escalating numbers of transduced TIL will be administered at three weekly intervals along with the administration of interleukin-2 (IL-2).  The objective is to evaluate the toxicity and possible therapeutic efficacy of the administration of tumor infiltrating lymphocytes (TIL) transduced with the gene coding for TNF. (Protocol #9007-003)

 

Appendix D-17.  Dr. Malcolm K. Brenner of St. Jude Children's Research Hospital of Memphis, Tennessee, can conduct experiments on patients with acute myelogenous leukemia (AML).  Using the LNL6 retroviral vector, the autologous bone marrow cells will be transduced with the gene coding for neomycin resistance.  The purpose of this gene marking experiment is to determine whether the source of relapse after autologous bone marrow transplantation for acute myelogenous leukemia is residual malignant cells in the harvested marrow or reoccurrence of tumor in the patient.  Determining the source of relapse should indicate whether or not purging of the bone marrow is a necessary procedure. (Protocol #9102-004)

 

Appendix D-18.  Dr. Malcolm K. Brenner of St. Jude Children's Research Hospital of Memphis, Tennessee, can conduct experiments on pediatric patients with Stage D (disseminated) neuroblastoma who are being treated with high-dose carboplatin and etoposide in either phase I/II or phase II trials.  All the patients in these studies will be subjected to bone marrow transplantation since it will allow them to be exposed to chemoradiation that would be lethal were it not for the availability of stored autologous marrow for rescue.  The bone marrow cells of these patients will be transduced with the gene coding for neomycin resistance using the LNL6 vector.  The purpose of this gene marking study is to determine whether the source of relapse after autologous bone marrow transplantation is residual malignant cells in the harvested marrow or residual disease in the patient.  Secondly,

it is hoped to determine the contribution of marrow autographs to autologous reconstitution. (Protocol #9105-005/9105-006)

 

Appendix D-19.  Dr. Albert B. Deisseroth of the MD Anderson Cancer Center of Houston, Texas, can conduct experiments on patients with chronic myelogenous leukemia who have been reinduced into a second chronic phase or blast cells.  The patients in these studies will receive autologous bone marrow transplantation.  Using the LNL6 vector, the bone marrow cells will be transduced with the gene coding for neomycin resistance.  The purpose of these marking studies is to determine if the origin of relapse arises from residual leukemic cells in the patients or from viable leukemic cells remaining in the bone marrow used for autologous transplantation. (Protocol #9105-007)

 

Appendix D-20.  Drs. Fred D. Ledley and Savio L. C. Woo of Baylor College of Medicine of Houston, Texas, can conduct experiments on pediatric patients with acute hepatic failure who are identified as candidates for hepatocellular transplantation.  Using the LNL6 vector, the hepatocytes will be transduced with the gene coding for neomycin resistance.  The purpose of using a genetic marker is to demonstrate the pattern of engraftment of transplanted hepatocytes and to help determine the success or failure of engraftment. (Protocol #9105-008)

 

Appendix D-21.  Dr. Steven A. Rosenberg of the National Institutes of Health, Bethesda, Maryland, can conduct experiments on patients with advanced melanoma, renal cell cancer, and colon carcinoma who have failed all effective therapy.  In an attempt to increase these patients' immune responses to the tumor, the tumor necrosis factor gene or the interleukin-2 gene will be introduced into a tumor cell line established from the patient.  These gene-modified autologous tumor cells will then be injected into the thigh of the patient.  To further utilize the immune system of the patient to fight the tumor, stimulated lymphocytes will be cultured from either the draining regional lymph nodes or the injected tumor itself.  The patients will be evaluated for antitumor effects engendered by the injection of the gene modified tumor cells themselves as well as after the infusion of the cultured lymphocytes. (Protocol #9110-010/9110-011)

 

Appendix D-22.  Dr. James M. Wilson of the University of Michigan Medical Center of Ann Arbor, Michigan, can conduct experiments on three patients with the homozygous form of familial hypercholesterolemia.  Both children and adults will be eligible for this therapy.  In an attempt to correct the basic genetic defect in this disease, the gene coding for the low-density lipoprotein (LDL) receptor will be introduced into liver cells taken from the patient.  The gene-corrected hepatocytes will then be infused into the portal circulation of the patient through an indwelling catheter.  The patients will be evaluated for engraftment of the these treated hepatocytes through a series of metabolic studies; three months after gene therapy, a liver biopsy will be taken and analyzed for the presence of recombinant derived RNA and DNA to document the presence of the gene coding for the normal LDL receptor. (Protocol #9110-012)

 

Appendix D-23.  Dr. Michael T. Lotze of the University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, can conduct experiments on 20 patients with metastatic melanoma who have failed conventional therapy.  A gene transfer experiment will be performed, transducing the patients' tumor infiltrating lymphocytes (TILs) with the gene for neomycin resistance.  Through the use of this gene marking technique, it is proposed to determine how long TIL cells can be detected in vivo in the peripheral blood of the patients, and how the administration of interleukin-2 and interleukin-4 affects localization and survival of TIL cells in tumor sites. (Protocol #9105-009)

 

Appendix D-24.  Dr. Gary J. Nabel of the University of Michigan Medical School, Ann Arbor, Michigan, can conduct gene therapy experiments on twelve patients with melanoma or adenocarcinoma.  Patient population will be limited to adults over the age of 18 and female patients must be postmenopausal or have undergone tubal ligation or orchiectomy.  The patient's immune response will be stimulated by the introduction of a gene encoding for a Class I MHC protein, HLA-B7, in order to enhance tumor regression.  DNA/liposome-mediated transfection techniques will be used to directly transfer this foreign gene into tumor cells.  HLA-B7 expression will be confirmed in vivo, and the immune response stimulated by the expression of this antigen will be characterized.  These experiments will be analyzed for their efficacy in treating cancer. (Protocol #9202-013)

 

Appendix D-25.  Kenneth Cornetta of Indiana University, Indianapolis, Indiana, can conduct gene transfer experiments on up to 10 patients with acute myelogenous leukemia (AML) and up to 10 patients with acute lymphocytic leukemia (ALL).  The patient population will be limited to persons between 18 and 65 years of age.  Using the LNL-6 vector, autologous bone marrow cells will be marked with the neomycin resistance gene.  Gene marked and untreated bone marrow cells will be reinfused at the time of bone marrow transplantation.  Patients will then be monitored for evidence of the neomycin resistance gene in peripheral blood and bone marrow cells in order to determine whether relapse of their disease is a result of residual malignant cells remaining in the harvested marrow or inadequate ablation of the tumor cells by chemotherapeutic agents.  Determining the source of relapse may indicate whether or not purging of the bone marrow is a necessary procedure for these leukemia patients.  Further studies will be performed in order to determine the percentage of leukemic cells that contain the LNL-6 vector and the clonality of the marked cells. (Protocol #9202-014)

 

Appendix D-26.  Dr. James S. Economou of the University of California, Los Angeles, can conduct gene transfer experiments on 20 patients with metastatic melanoma and 20 patients with renal cell carcinoma.  These patients will be treated with various combinations of tumor-infiltrating lymphocytes and peripheral blood leukocytes, including CD8 and CD4 subsets of both types of cells.  These effector cell populations will be given in combination with interleukin-2 (IL-2) in the melanoma patients and IL-2 plus alpha interferon in the renal cell carcinoma patients.  The effector cells will be transduced with the neomycin resistance gene using either the LNL6 or G1N retroviral vectors.  This "genetic marking" of the tumor-infiltrating lymphocytes and peripheral blood lymphocytes is designed to answer questions about the trafficking of these cells, their localization to tumors, and their in vivo life span. (Protocol #9202-015)

 

Appendix D-27.  Drs. Philip Greenberg and Stanley R. Riddell of the Fred Hutchinson Cancer Research Center, Seattle, Washington, may conduct gene transfer experiments on 15 human immunodeficiency virus (HIV) seropositive patients (18-45 years old) undergoing allogeneic bone marrow transplantation for non-Hodgkin's lymphoma and 15 HIV-seropositive patients (18-50 years old) who do not have acquired immunodeficiency syndrome (AIDS)-related lymphoma and who are not undergoing bone marrow transplantation to evaluate the safety and efficacy of HIV-specific cytotoxic T lymphocyte (CTL) therapy.  CTL will be transduced with a retroviral vector (HyTK) encoding a gene that is a fusion product of the hygromycin phosphotransferase gene (HPH) and the herpes simplex virus thymidine kinase (HSV-TK) gene.  This vector will deliver both a marker gene and an ablatable gene in these T cell clones in the event that patients develop side effects as a consequence of CTL therapy.  Data will be correlated over time, looking at multiple parameters of HIV disease activity.  The objectives of these studies include evaluating the safety and toxicity of CTL therapy, determining the duration of in vivo survival of HIV-specific CTL clones, and determining if ganciclovir therapy can eradicate genetically modified, adoptively transferred CTL cells. (Protocol #9202-017)

 

Appendix D-28.  Dr. Malcolm Brenner of St. Jude Children's Research Hospital, Memphis, Tennessee, can conduct gene therapy experiments on twelve patients with relapsed/refractory neuroblastoma who have relapsed after receiving autologous bone marrow transplant.  In an attempt to stimulate the patient's immune response, the gene coding for Interleukin-2 (IL-2) will be used to transduce tumor cells, and these gene-modified cells will be injected subcutaneously in a Phase 1 dose escalation trial.  Patients will be evaluated for an anti-tumor response. (Protocol #9206-018)

 

Appendix D-29.  Drs. Edward Oldfield, Kenneth Culver, Zvi Ram, and R. Michael Blaese of the National Institutes of Health, Bethesda, Maryland, can conduct gene therapy experiments on ten patients with primary malignant brain tumors and ten patients with lung cancer, breast cancer, malignant melanoma, or renal cell carcinoma who have brain metastases.  The patient population will be limited to adults over the age of 18.

Patients will be divided into two groups based on the surgical accessibility of their lesions.  Both surgically accessible and surgically inaccessible lesions will receive intra-tumoral injections of the retroviral Herpes simplex thymidine kinase (HS-tk) vector-producer cell line, G1TkSvNa, using a guided stereotaxic approach.  Surgically accessible lesions will be excised seven days after stereotaxic injection, and the tumor bed will be infiltrated with the HS-tk producer cells.  The removed tumor will be evaluated for the efficiency of transduction.  Ganciclovir (GCV) will be administered beginning on the fifth postoperative day.  In the case of surgically inaccessible lesions, the patients will receive intravenous therapy with GCV seven days after receiving the intra-tumoral injections of the retroviral HS-tk vector-producer cells. (Protocol #9206-019)

 

Appendix D-30.  Dr. Albert D. Deisseroth of MD Anderson Cancer Center, Houston, Texas, can conduct gene transfer experiments on ten patients who have developed blast crisis or accelerated phase chronic myelogenous leukemia (CML).  The retroviral vectors G1N and LNL6 which code for neomycin resistance will be used to transduce autologous peripheral blood and bone marrow cells that have been removed and stored at the time of cytogenetic remission or re-induction of chronic phase in Philadelphia chromosome positive CML patients.  Following reinduction of the chronic phase of CML and preparative chemotherapy, patients will be infused with the transduced autologous cells.

 

This protocol is designed to determine the cause of relapse of CML.  If polyclonal CML neomycin marked blastic cells appear at the time of relapse, their presence will indicate that relapse arises from the leukemic CML blast cells present in the autologous cells infused following chemotherapy.  If residual systemic disease contributes to relapse, the neomycin resistance gene will not be detected in the CML leukemic blasts at the time of relapse.

 

This study will compare the relative contributions of the peripheral blood and bone marrow to generate hematopoietic recovery after bone marrow transplantation and evaluate purging and selection of peripheral blood or bone marrow as a source of stem cells for transplant.  The percentage of neomycin resistant CML cells which are leukemic will be determined by PCR analysis and detection of bcr-abl mRNA. (Protocol #9206-020)

 

Appendix D-31.  Dr. Cynthia Dunbar of the National Institutes of Health, Bethesda, Maryland, can conduct gene transfer experiments on up to 48 patients with multiple myeloma, breast cancer, or chronic myelogenous leukemia.  The retroviral vectors G1N and LNL6 will be used to transfer the neomycin resistance marker gene into autologous bone marrow and peripheral blood stem cells in the presence of growth factors to examine hematopoietic reconstitution after bone marrow transplantation.  The efficiency of transduction of both short and long term autologous bone marrow reconstituting cells will be examined.

 

Autologous bone marrow and CD34+ peripheral blood stem cells will be enriched prior to transduction.  Myeloma and CML patients will receive both autologous bone marrow and peripheral blood stem cell transplantation.  These separate populations will be marked with both the G1N and LNL6 retroviral vectors.  If short and long term marking experiments are successful, important information may be obtained regarding the biology of autologous reconstitution, the feasibility of retroviral gene transfer into hematopoietic cells, and the contribution of viable tumor cells within the autograft to disease relapse. (Protocol #9206-023/9206-024/9206-025)

 

Appendix D-32.  Dr. Bernd Gansbacher of the Memorial Sloan-Kettering Cancer Center, New York, New York, can conduct gene therapy experiments on twelve patients over 18 years of age with metastatic melanoma who are HLA-A2 positive and who have failed conventional therapy.  This is a phase I study to examine whether allogeneic HLA-A2 matched melanoma cells expressing recombinant human Interleukin-2 (IL-2) can be injected subcutaneously and used to create a potent tumor specific immune response without producing toxicity.  By allowing the tumor cells to present the MHC Class I molecule as well as the secreted IL-2, a clonal expansion of tumor specific effector cells is expected.  These effector populations may access residual tumor at distant sites via the systemic circulation. (Protocol #9206-021)


 

 

Appendix D-33.  Dr. Bernd Gansbacher of the Memorial Sloan-Kettering Cancer Center, New York, New York, can conduct gene therapy experiments on twelve patients over 18 years of age with renal cell carcinoma who are HLA-A2 positive and who have failed conventional therapy.  This Phase I study will examine whether allogeneic HLA-A2 matched renal cell carcinoma cells expressing recombinant human Interleukin-2 (IL-2) can be injected subcutaneously and used to create a potent tumor specific immune response without producing toxicity.  By allowing the tumor cells to present the MHC Class I molecule as well as the secreted IL-2, a clonal expansion of tumor specific effector cells is expected.  These effector populations may access residual tumor at distant sites via the systemic circulation. (Protocol #9206-022)

 

Appendix D-34.  Dr. Michael T. Lotze, University of Pittsburgh, Pittsburgh, Pennsylvania, can conduct experiments on twenty patients with metastatic, and/or unresectable, locally advanced melanoma, renal cell carcinoma, breast cancer, or colon cancer who have failed standard therapy.  Patients will receive multiple subcutaneous injections of autologous tumor cells combined with an autologous fibroblast cell line that has been transduced in vitro with the gene coding for Interleukin-4 (IL-4) to augment the in vivo antitumor effect.  Patients will be monitored for antitumor effect by PCR analysis and multiple biopsy of the injection site. (Protocol #9209-033)

 

Appendix D-35.  Dr. Friedrich G. Schuening, Fred Hutchinson Cancer Research Center, Seattle, Washington, can conduct human gene transfer experiments on patients ≥ 18 years of age with breast cancer, Hodgkin's disease, or non-Hodgkin's lymphoma.  A total of 10 patients per year will be enrolled in the studies over a period of four years.  Patients will undergo autologous bone marrow transplantation with a selected population of Interleukin-3 (IL-3) or granulocyte colony-stimulating factor (G-CSF) stimulated CD34(+) peripheral blood repopulating cells (PBRC) that have been transduced with the gene coding for neomycin resistance (neoR) using the retroviral vector, LN.  Patients will be continuously monitored for neoR to determine the relative contribution

 

of autologous PBRCs to long-term hematopoietic reconstitution.  Demonstration of long-term contribution of autologous PBRC to hematopoiesis will enable the use of PBRC alone for autologous transplants and suggest the use of PBRC as long-term carriers of therapeutically relevant genes. (Protocol #9209-027/9209-028)

 

Appendix D-36.  Dr. Friedrich G. Schuening, Fred Hutchinson Cancer Research Center, Seattle, Washington, can conduct human gene transfer experiments on patients ≥ 18 years of age with breast cancer, Hodgkin's disease, or non-Hodgkin's lymphoma.  A total of 5 patients per year will be enrolled in the study over a period of four years.  Patients will undergo allogeneic bone marrow transplant with granulocyte colony-stimulating factor (G-CSF) stimulated CD34(+) PBRC harvested from an identical twin that have been transduced with neoR using the retroviral vector, LN.  Patients will be continuously monitored for neoR to determine the relative contribution of G-CSF stimulated allogeneic PBRCs to long-term bone marrow engraftment.   Demonstration of long-term contribution of allogeneic PBRC to hematopoiesis will enable the use of PBRC alone for allogeneic transplants and suggest the use of PBRC as long-term carriers of therapeutically relevant genes. (Protocol #9209-029)

 

Appendix D-37.  Dr. Malcolm K. Brenner of St. Jude Children's Hospital, Memphis, Tennessee, and Dr. Bonnie J. Mills of Baxter Healthcare Corporation, Santa Ana, California, can conduct a multicenter uncontrolled human gene transfer experiment on 12 patients ≤ 21 years of age with Stage D Neuroblastoma in first or second marrow remission.  Autologous bone marrow cells will be separated into two fractions, purged and unpurged.  Each fraction will be transduced with the neoR gene by either LNL6 or G1Na.  Patients will be monitored by the polymerase chain reaction (PCR) for the presence of neoR.  The protocol is designed to evaluate the safety and efficacy of the Neuroblastoma Bone Marrow Purging System following high dose chemotherapy. (Protocol #9209-032)

 

Appendix D-38.  Drs. Carolyn Keierleber and Ann Progulske-Fox of the University of Florida, Gainesville, Florida, can conduct experiments involving the introduction of a gene coding for tetracycline resistance into Porphyromonas gingivalis at a physical containment level of Biosafety Level-2 (BL-2).

 

Appendix D-39.  Dr. Scott M. Freeman of Tulane University Medical Center, New Orleans, Louisiana, can conduct experiments on patients with epithelial ovarian carcinoma who have clinical evidence of recurrent, progressive, or residual disease who have no other therapy available to prolong survival.  Patients will be injected intraperitoneally with the irradiated PA-1 ovarian carcinoma cell line which has been transduced with the herpes simplex thymidine kinase (HSV-TK) gene.  The patients will then receive ganciclovir therapy.  Previous, data indicates that HSV-TK+ tumor cells exhibit a killing effect on HSV-TK- cells when exposed to ganciclovir therapy.  Patients will be evaluated for safety and side effects of this treatment. (Protocol #9206-016)

 

Appendix D-40.  Dr. Michael J. Welsh, Howard Hughes Medical Institute Research Laboratories, University of Iowa College of Medicine, Iowa City, Iowa, may conduct experiments on 3 cystic fibrosis (CF) patients ≥ 18 years of age with mild to moderate disease.  This Phase I study will determine the:  (1) in vivo safety and efficacy of the administration of the replication-deficient type 2 adenovirus vector, Ad2/CFTR-1, to the nasal epithelium; (2) efficacy in correcting the CF chloride transport defect in vivo; and (3) effect of adenovirus vector dosage on safety and efficacy. (Protocol #9212-036)

 

Appendix D-41.  Dr. Ronald G. Crystal, National Institutes of Health, Bethesda, Maryland, may conduct experiments on 10 cystic fibrosis (CF) patients ≥ 21 years of age.  Patients will receive an initial administration of the replication-deficient type 5 adenovirus vector, AdCFTR, to their left nares.  If no toxicity is observed from intranasal administration, patients will receive a single administration of AdCFTR to the respiratory epithelium of their left large bronchi.  Five groups of patients (2 patients per group) will be studied based on increased dosage administration of AdCFTR.  This study will determine the:  (1) in vivo safety and efficacy of the administration of AdCFTR into the respiratory epithelium; (2) efficacy of the correction of the biologic abnormalities of CF in the respiratory epithelium;  (3) duration of the biologic correction; (4) efficacy of the correction of the abnormal electrical potential difference of the airway epithelial sheet; (5) clinical parameters relevant to the disease process; and (6) if humoral immunity develops against AdCFTR sufficient to prevent repeat administration. (Protocol #9212-034)

 

Appendix D-42.  Dr. Kenneth Culver, Iowa Methodist Medical Center, Des Moines, Iowa, and Dr. John Van Gilder, University of Iowa, Iowa City, Iowa, may conduct experiments on 15 patients ≥ 18 years of age with recurrent malignant primary brain tumors or lung, melanoma, renal cell carcinoma, or breast carcinoma brain metastases who have failed standard therapy for their disease.  Patient eligibility will be limited to those patients who have measurable residual tumor immediately following the post-operative procedure as demonstrated by imaging studies.  The number of patients treated will be equally divided between the Iowa Methodist Medical Center and the University of Iowa.  If a positive response is observed in any of the first 15 patients, the investigators may submit a request to treat an additional 15 patients.

Following surgical debulking, patients will receive a maximum of 3 intralesional injections of the G1TkSvNa vector- producing cell line (VPC) to induce regression of residual tumor cells by ganciclovir (GCV) therapy.  Patients who demonstrate stable disease for a minimum of 6 months following this treatment will be eligible for additional VPC injections and subsequent GCV therapy. (Protocol #9303-037)

 

Appendix D-43.  Drs. Malcolm Brenner, Robert Krance, Helen E. Heslop, Victor Santana, and James Ihle, St. Jude Children's Research Hospital, Memphis, Tennessee, may conduct experiments on 35 patients ≥ 1 year and ≤ 21 years of age at the time of initial diagnosis of acute myelogenous leukemia (AML).  The investigators will use the two retroviral vectors, LNL6 and G1Na, to determine the efficacy of the bone marrow purging techniques:  4-hydroxyperoxicyclophosphamide and interleukin-2 (IL-2) activation of endogenous cytotoxic effector cells, in preventing relapse from the reinfusion of autologous bone marrow cells. (Protocol #9303-039)

 

Appendix D-44.  Drs. Helen E. Heslop, Malcolm Brenner, and Cliona Rooney, St Jude Children's Research Hospital, Memphis, Tennessee, may conduct experiments of 35 patients ≤ 21 years of age who will be recipients of mismatched-related or phenotypically similar unrelated donor marrow grafts for leukemia.  In this Phase I dose escalation study, spontaneous lymphoblastoid cell lines will be established that express the same range of Epstein-Barr Virus (EBV) encoded proteins as the recipient.  These EBV-specific cell lines will be transduced with LNL6 or G1Na and readministered at the time of bone marrow transplant.  This study will determine:  (1) survival and expansion of these EBV-specific cell lines in vivo, (2) the ability of these adoptively transferred cells to confer protection against EBV infection, and (3) appropriate dosage and administration schedules. (Protocol #9303-038)

 

Appendix D-45.  Drs. Robert W. Wilmott and Jeffrey Whitsett, Children's Hospital Medical Center, Cincinnati, Ohio, and Dr. Bruce Trapnell, Genetic Therapy, Inc., Gaithersburg, Maryland, may conduct experiments on 15 cystic fibrosis (CF) patients who have mild to moderate disease ≥ 21 years of age.  The replication-deficient type 5 adenovirus vector, Av1CF2, will be administered to the nasal and lobar bronchial respiratory tract of patients.  This study will demonstrate the:  (1) expression of normal cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in vivo, (2) synthesis of CFTR protein, and (3) correction of epithelial cell cAMP dependent Cl- permeability.  The pharmacokinetics of CFTR expression and ability to re-infect the respiratory tract with AvCF2 will be determined.  Systemic and local immunologic consequences of Av1CF2 infection, the time of viral survival, and potential for recombination or complementation of the virus will be monitored. (Protocol #9303-041)

 

Appendix D-46.  Dr. James M. Wilson of the University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, may conduct experiments on 20 adult patients with advanced cystic fibrosis lung disease.  An isolated segment of the patients' lung will be transduced with the E1 deleted, replication-incompetent adenovirus vector, Ad.CB-CFTR using a bronchoscope for gene delivery.  Ad.CB-CFTR contains the human gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein.  Pulmonary biopsies will be obtained by bronchoscopy at 4 days, 6 weeks, and 3 months following treatment.  Patients will be monitored for evidence of CFTR gene transfer and expression, immunological responses to CFTR or adenovirus proteins, and toxicity. (Protocol #9212-035)

 

Appendix D-47.  Dr. Hilliard F. Seigler of Duke University Medical Center, Durham, North Carolina, may conduct experiments on 20 patients with disseminated malignant melanoma.  Autologous tumor cells will be transduced with a retroviral vector, pHuγ-IFN, that contains the gene encoding human γ-IFN.  Following lethal irradiation, the transduced cells will be readministered to patients for the purpose of generating cytotoxic T cells that are tumor specific along with the up-regulation of Class I major histocompatibility antigens.  Patients will be monitored for clinical regression of tumors and generation of tumor-specific cytotoxic T lymphocytes. (Protocol #9306-043)

 

Appendix D-48.  Drs. Stefan Karlsson and Cynthia Dunbar of the National Institutes of Health, Bethesda, Maryland, and Dr. Donald B. Kohn of the Children’s Hospital of Los Angeles, Los Angeles, California, may conduct experiments on 10 patients with Gaucher disease.  CD34(+) hematopoietic stem cells will be isolated from bone marrow or from peripheral blood treated with granulocyte-colony stimulating factor.  CD34(+) cells will be transduced with a retrovirus vector, G1Gc, containing cDNA encoding human glucocerebrosidase and administered intravenously.  Patients will be monitored for toxicity and glucocerebrosidase expression. (Protocol #9306-047)

 

Appendix D-49.  Dr. Gary J. Nabel of the University of Michigan Medical Center, Ann Arbor, Michigan, may conduct experiments on 12 patients with AIDS to be divided into 4 experimental groups.  CD4(+) lymphocytes will be isolated from peripheral blood and transduced with Rev M10, a transdominant inhibitory mutant of the rev gene of the human immunodeficiency virus (HIV).  Transduction of the rev mutant will be mediated either by the retrovirus vector, PLJ-cREV M10, or by particle-mediated gene transfer of plasmid DNA.  Patients will be monitored for survival of the transduced CD4(+) cells by polymerase chain reaction and whether Rev M10 can confer protection against HIV infection to CD4(+) cells. (Protocol #9306-049)

 

Appendix D-50.  Dr. Gary J. Nabel of the University of Michigan Medical Center, Ann Arbor, Michigan, may conduct experiments on 24 patients with advanced cancer.  Patients will undergo in vivo transduction with DNA/liposome complexes containing genes encoding the HLA-B7 histocompatibility antigen and beta-2 microglobulin in a non-viral plasmid.  These DNA/liposome complexes will be administered either by intratumoral injection or catheter delivery.  Patients will be monitored for enhanced immune responses against tumor cells, and safe and effective doses will be determined. (Protocol #9306-045)

 

Appendix D-51.  Dr. John A. Barranger of the University of Pittsburgh, Pittsburgh, Pennsylvania, may conduct experiments on 5 patients with Gaucher disease.  The CD34(+) hematopoietic stem cells will be isolated from peripheral blood and transduced in vitro with the retrovirus vector, N2-Sv-GC, encoding the glucocerebrosidase (GC) enzyme.  Following reinfusion of the transduced cells, patients will be monitored by PCR analysis for GC expression in peripheral blood leukocytes.  Patients currently receiving GC replacement therapy and who demonstrate clinical responsiveness will be withdrawn from exogenous GC therapy.  Patients not previously treated with exogenous GC, will be monitored for clinical reversal of lipid storage symptoms. (Protocol #9306-046)

 

Appendix D-52.  Dr. Robert Walker of the National Institutes of Health, Bethesda, Maryland, may conduct experiments on 12 HIV-infected patients who have a seronegative identical twin.  CD4(+) and CD8(+) cells will be isolated from the seronegative twin and induced to polyclonal proliferation with anti-CD3 and interleukin-2.  The enriched population of cells will be transduced with either LNL6 or G1Na, which contain the neoR gene.  The transduced cells will be expanded in tissue culture and administered to the HIV-infected twin.  Patients will be monitored for immune function and the presence of marked cells. (Protocol #9209-026)

 

Appendix D-53.  Dr. Corey Raffel of the Children’s Hospital Los Angeles, Los Angeles, California, and Dr. Kenneth Culver of Iowa Methodist Medical Center, Des Moines, Iowa, may conduct experiments on 30 patients between 2 and 18 years of age with recurrent malignant astrocytoma.  Fifteen patients will be accrued into this study initially.  If at least one patient responds to therapy, an additional 14 patients will be treated.  Patients with either surgically accessible or non-accessible tumors will be treated with the vector producing cell line (PA317) carrying the retrovirus vector, G1TkSvNa.  This vector will transduce tumor cells in vivo with the Herpes simplex thymidine kinase (HS-tk) gene that renders the cells sensitive to killing by ganciclovir.  Surgically accessible patients will undergo surgical debulking of their tumor followed by repeated administration of the HS-tk vector producer cells into the tumor bed.  Children with unresectable tumors will undergo stereotaxic injection of vector producer cells into tumors. (Protocol #9306-050)

 

Appendix D-54.  Dr. Jeffrey E. Galpin of the University of Southern California, Los Angeles, California, and Dr. Dennis A. Casciato of the University of California, Los Angeles, California, may conduct experiments on 15 HIV(+) asymptomatic patients.  Patients will receive 3 monthly intramuscular injections of the retrovirus vector (N2IIIBenv) encoding the HIV-1 IIIB envelope protein.  Patients will be monitored for acute toxicity, CD4 levels, HIV-specific CTL responses, and viral burdens. (Protocol #9306-048)

 

Appendix D-55.  Drs. Charles Hesdorffer and Karen Antman of Columbia University College of Physicians and Surgeons, New York, New York, may conduct experiments on 20 patients with advanced breast, ovary, and brain cancer.  CD34(+) hematopoietic stem cells will be isolated from bone marrow, transduced with the retrovirus vector, PHaMDR1/A, and readministered to patients.  Patients will be monitored for the presence and expression of the MDR-1 gene.  The investigators will determine whether MDR-1 expression increases following chemotherapy. (Protocol #9306-051)

 

Appendix D-56.  Dr. Enzo Paoletti of Virogenetics Corporation, Troy, New York, may conduct experiments with poxvirus vectors NYVAC, ALVAC, and TROVAC at Biosafety Level 1.

 

Appendix D-57.  Drs. Richard C. Boucher and Michael R. Knowles of the University of North Carolina, Chapel Hill, North Carolina, may conduct experiments on 9 patients (18 years old or greater) with cystic fibrosis to test for the safety and efficacy of an E1-deleted recombinant adenovirus containing the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, Ad.CB-CFTR.  A single dose of 108, 3 x 109 or 1011 pfu/ml will be administered to the nasal cavity of 3 patients in each dose group.  Patients will be monitored by nasal lavage and biopsy to assess safety and restoration of normal epithelial function. (Protocol #9303-042)

 

Appendix D-58.  Dr. Joyce A. O'Shaughnessy of the National Institutes of Health, Bethesda, Maryland, may conduct experiments on 18 patients (18-60 years old) with Stage IV breast cancer who have achieved a partial or complete response to induction chemotherapy.  This study will determine the feasibility of obtaining engraftment of CD34(+) hematopoietic stem cells transduced by a retroviral vector, G1MD, and expressing a cDNA for the human multi-drug resistance-1 (MDR-1) gene following high dose chemotherapy, and whether the transduced MDR-1 gene confers drug resistance to hematopoietic cells and functions as an in vivo dominant selectable marker.  Patients will be monitored for evidence of myeloprotection and presence of the transduced MDR-1 gene." (Protocol #9309-054)

 

Appendix D-59.  Drs. Larry E. Kun, R. A. Sanford, Malcolm Brenner, and Richard L. Heideman of St. Jude Children's Research Hospital, Memphis, Tennessee, and Dr. Edward H. Oldfield of the National Institutes of Health, Bethesda, Maryland, may conduct experiments on 6 patients (3-21 years old) with progressive or recurrent malignant supratentorial tumors resistant to standard therapies.  Mouse cells producing the retroviral vector containing the herpes simplex thymidine kinase  gene (G1TKSVNa) will be instilled into the tumor areas via multiple stereotactically placed cannulas.  Patients will be treated with ganciclovir to eliminate cells expressing the transduced gene.  Patients will be monitored for central nervous system, hematologic, renal or other toxicities, and for anti-tumor responses by magnetic resonance imaging studies. (Protocol #9309-055)

 

Appendix D-60.  The physical containment level may be reduced from Biosafety Level 3 to Biosafety Level 2 for a Semliki Forest Virus (SFV) vector expression system of Life Technologies, Inc., Gaithersburg, Maryland.

 

Appendix D-61.  Dr. Albert B. Deisseroth of the University of Texas MD Anderson Cancer Center, Houston, Texas, may conduct experiments on 10 patients (≥ 16 to ≤ 60 years of age) with chronic lymphocytic leukemia.  Autologous peripheral blood and bone marrow cells will be removed from patients following chemotherapy and marked by transduction with two distinguishable retroviral vectors, G1Na and LNL6, containing the neomycin resistance gene.  The gene marked cells will be reinfused into patients to determine the efficiency of bone marrow purging and the origin of relapse following autologous bone marrow transplantation. (Protocol #9209-030)

 

Appendix D-62.  Dr. Jonathan Simons of the Johns Hopkins Oncology Center, Baltimore, Maryland, may conduct experiments on 26 patients (≥ 18 years of age) with metastatic renal cell carcinoma to evaluate the safety and tolerability of intradermally injected autologous irradiated tumor cells transduced with the retrovirus vector, MFG, containing the human granulocyte-macrophage colony stimulating factor gene.  Acute and long-term clinical toxicities and in vitro and in vivo induction of specific anti-tumor immune responses will be monitored. (Protocol #9303-040)

 

Appendix D-63.  Dr. Albert B. Deisseroth of the University of Texas MD Anderson Cancer Center, Houston, Texas, may conduct experiments on 20 patients (≥ 18 and ≤ 60 years old) with ovarian cancer.  A murine viral vector was constructed from the third generation of L series retroviruses with the insert of the human multi-drug resistance-1 (MDR-1) transduced gene.  The investigators will assess the safety and feasibility of administering CD34 (+) autologous peripheral blood and bone marrow cells.  Patients will be monitored for the presence of the MDR-1 gene and for the effect of gene transfer on hematopoietic function following the transplantation. (Protocol #9306-044)

 

Appendix D-64.  Dr. Joseph Ilan of the Case Western Reserve University School of Medicine and University Hospital of Cleveland, Cleveland, Ohio, may conduct experiments on 12 patients (≥ 18 years of age) with advanced brain cancer.  Human malignant glioma tumor cells will be cultured, transfected with Epstein-Barr virus (EBV)-based vector, anti-Insulin growth factor-I, lethally irradiated, and injected subcutaneously into patients in an attempt to express antisense Insulin growth factor-1.  Patients will be monitored for toxicity and immunologic responses to the vector. (Protocol #9306-052)

 

Appendix D-65.  Drs. James S. Economou and John Glaspy of the University of California, Los Angeles, California, may conduct experiments on 30 patients (≥ 18 to ≤ 70 years of age) with metastatic melanoma.  A human melanoma cell line (M-24) will be transduced with the retroviral vector, G1NaCvi2, expressing the human interleukin-2 (IL-2) gene.  The IL-2 producing cells will be mixed with the patient's autologous tumor cells, irradiated, and injected subcutaneously in an attempt to enhance the tumor-specific immunologic response.  Patients will be monitored for toxicity, in vitro and in vivo immunologic responses, and clinical anti-tumor effects. (Protocol #9309-058)

 

Appendix D-66.  Drs. Peter Cassileth, Eckhard R. Podack, and Kasi Sridhar of the University of Miami, and Niramol Savaraj of the Miami Veterans Administration Hospital, Miami, Florida, may conduct experiments on 12 patients (≥ 18 years of age) with limited stage small cell lung cancer.  Autologous tumor cells will be removed, expanded in culture, and transduced by lipofection with the BMG-Neo-hIL2 vector (derived from bovine papilloma virus).  The objective of this protocol is to demonstrate the safety and efficacy of administering IL-2 transduced autologous tumor cells in an attempt to stimulate a tumor-specific cytotoxic T lymphocyte (CTL) response, and to determine the quantity and characteristics of the CTL that have been generated. (Protocol #9309-053)

 

Appendix D-67.  Drs. Edward H. Oldfield and Zvi Ram of the National Institutes of Health, Bethesda, Maryland, may conduct experiments on 20 patients (≥ 18 years of age) with leptomeningeal carcinomatosis.  The patients will receive intraventricular or subarachnoid injection of murine vector producing cells containing the retroviral vector, G1Tk1SvNa.  Tumor cells expressing the herpes simplex thymidine kinase gene will be rendered sensitive to killing by subsequent administration of ganciclovir.  Patients will be monitored for safety and anti-tumor response by magnetic resonance imaging (MRI) and cerebral spinal fluid cytological analysis. (Protocol #9312-059)

 

Appendix D-68.  Drs. Tapas K. Das Gupta and Edward P. Cohen of the University of Illinois College of Medicine, Chicago, Illinois, may conduct experiments on 12 subjects who differ in at least 3 out of 6 alleles at the Class I histocompatibility locus (≥ 18 years of age) with Stage IV malignant melanoma.  The subjects will be immunized with a lethally irradiated allogeneic human melanoma cell line transduced with the human interleukin-2 expressing retroviral vector, pZipNeoSv-IL-2.  Subjects will be monitored for toxicity, induction of B and T cell antitumor responses in vitro and in vivo, and any clinical evidence of antitumor effect. (Protocol #9309-056)

 

Appendix D-69A.  Dr. Michael J. Welsh of the Howard Hughes Medical Institute, Iowa City, Iowa, may conduct experiments on 20 patients (≥ 18 years of age) with cystic fibrosis.  The investigator will administer increasing doses of either of the two adenovirus vectors, AD2/CFTR-1 or AD2-ORF6/PGK-CFTR, to the nasal epithelium of 10 patients (1 nostril) or maxillary sinus epithelium of 10 patients (1 maxillary sinus).  Patients will be isolated for a period of 24 hours following vector administration; however, if 1 patient demonstrates secreted virus at 24 hours, the investigator will notify the Recombinant DNA Advisory Committee for reconsideration of the isolated period.  Patients will be assessed for the safety and efficacy of multiply administration of adenovirus vectors encoding the cystic fibrosis transmembrane conductance regulator (CFTR) gene.  (Protocol #9312-067)

 

Appendix D-69B.  Dr. Richard Haubrich of the University of California at San Diego Treatment Center, San Diego, California, may conduct experiments on 25 human immunodeficiency virus (HIV)-infected, seropositive, asymptomatic subjects (≥ 18 to ≤ 65 years of age).  Subjects will receive 3 monthly intramuscular injections of the retroviral vector, N2/IIIB env/rev, which encodes for HIV-1 IIIB env/rev proteins.  The objective of the study is to induce HIV-1- specific CD8(+) cytotoxic T lymphocyte and antibody responses in order to eliminate HIV-infected cells and residual virus.  This Phase I/II study will evaluate acute toxicity, identify long-term treatment effects, and evaluate the disease progression. (Protocol #9312-062)

 

Appendix D-70.  Dr. Mario Sznol of the National Institutes of Health, Frederick, Maryland, may conduct experiments on 50 subjects (≥ 18 years of age) with advanced stage melanoma.  Subjects will receive subcutaneous injections of lethally irradiated allogeneic melanoma cells that have been transduced by lipofection with the plasmid DNA vector, CMV-B7, derived from bovine papilloma virus to express the human B7 antigen.  The B7 antigen, which binds to the CD28 receptor of T cells, will serve as a co-stimulatory signal to elicit an antitumor immune response.  Subjects will be monitored for induction of cytotoxic T lymphocyte, antitumor responses in vitro and in vivo and any clinical evidence of antitumor effect. (Protocol #9312-063)

 

Appendix D-71.  Dr. Joseph Rubin of the Mayo Clinic, Rochester, Minnesota, may conduct experiments on 15 subjects with hepatic metastases from advanced colorectal cancer (≥ 18 years of age).  Subjects will receive intratumoral hepatic injections of the plasmid DNA/lipid complex, pHLA-B7/β-2 microglobulin, expressing a heterodimeric cell surface protein consisting of the HLA-B7 histocompatibility antigen and β-2 microglobulin.  Subjects must be HLA-B7 negative.  The objective of this study is to determine a safe and effective dose of the DNA/lipid complex.  Subjects will be monitored for antigen-specific immune responses and in vivo HLA-B7 expression. (Protocol #9312-064)

 

Appendix D-72.  Dr. Nicholas J. Vogelzang of the University of Chicago Medical Center, Chicago, Illinois, may conduct experiments on 15 subjects with metastatic renal cell carcinoma ≥ 18 years of age.  Subjects will receive intratumoral injections of the plasmid DNA/liposome vector pHLA-B7/β-2 microglobulin, expressing a heterodimeric cell surface protein consisting of the HLA-B7 histocompatibility antigen and β-2 microglobulin.  Subjects must be HLA-B7 negative.  Subjects will be monitored for antigen-specific immune responses and in vivo HLA-B7 expression. (Protocol #9403-071)

 

Appendix D-73.  Dr. Evan M. Hersh of the Arizona Cancer Center and Drs. Emmanuel Akporiaye, David Harris, Alison T. Stopeck, Evan C. Unger, and James A. Warneke of the University of Arizona, Tucson, Arizona, may conduct experiments on 15 subjects with metastatic malignant melanoma ≥ 18 years of age.  Subjects will receive intratumoral injections of the plasmid DNA/liposome vector, pHLA-B7/β-2 microglobulin, expressing a heterodimeric cell surface protein consisting of the HLA-B7 histocompatibility antigen and β-2 microglobulin.  Subjects must be HLA-B7 negative.  Subjects will be monitored for antigen-specific immune responses and in vivo HLA-B7 expression. (Protocol #9403-072)

 

Appendix D-74.  Dr. Ralph Freedman of MD Anderson Cancer Center, Houston, Texas, may conduct gene marking experiments on 9 subjects with ovarian carcinoma or peritoneal carcinomatosis (≥ 16 years of age).  Autologous CD3(+)/CD8(+) tumor infiltrating lymphocyte derived T cells will be transduced with the retroviral vector G1Na that encodes for neoR.  Subjects will receive intraperitoneal administration of bulk expanded transduced and nontransduced T cells and recombinant interleukin-2.  Previously documented tumor sites and normal tissues will be monitored for neoR and the proportion of CD3(+)/CD8(+) T cells will be determined.  (Protocol #9406-075)

 

Appendix D-75.  Drs. Helen Heslop, Malcolm Brenner, and Robert Krance of St. Jude Children’s Research Hospital, Memphis, Tennessee, may conduct gene marking experiments on 20 subjects undergoing autologous bone marrow transplantation for therapy of leukemia or solid tumor (< 21 years of age).  The distinguishable retroviral vectors, LNL6 and G1Na (both encoding neoR), will be used to determine the rate of reconstitution of untreated versus cytokine expanded CD34(+) selected autologous bone marrow cells.  (Protocol #9406-076)

 

Appendix D-76.  Drs. Albert Deisseroth, Gabriel Hortobagyi, Richard Champlin, and Frankie Holmes of MD Anderson Cancer Center, Houston, Texas, may conduct experiments on 10 fully evaluable subjects (maximum of 20 entered) with Stage III or IV breast cancer (≥ 18 and ≤ 60 years of age).  Subjects will receive autologous CD34(+) peripheral blood cells that have been transduced with the retroviral vector, pVMDR-1, which encodes the multi-drug resistance gene.  The objective of this study is to evaluate the safety and feasibility of transducing early hematopoietic progenitor cells with pVMDR-1 and to determine in vivo selection of chemotherapy resistant hematopoietic cells.  (Protocol #9406-077)

 

Appendix D-77.  Drs. Johnson M. Liu and Neal S. Young of the National Institutes of Health, Bethesda, Maryland, may conduct experiments on 6 patients with Fanconi anemia (≥ 5 years of age).  Subjects will receive autologous CD34(+) cells that have been transduced with the retroviral vector, FACC, which encodes the normal Fanconi anemia complementation group C gene.  The objective of this study is to determine whether autologous FACC transduced hematopoietic progenitor cells can be safely administered to subjects, the extent of engraftment, and correction of cell phenotype.  (Protocol #9406-078)

 

Appendix D-78.  Drs. Robert E. Sobol and Ivor Royston of the San Diego Regional Cancer Center, San Diego, California, may conduct experiments on 15 subjects with recurrent residual glioblastoma multi-forme (≥ 18 years of age). Subjects will receive subcutaneous injections of autologous tumor cells that have been lethally irradiated and transduced with the retroviral vector, G1NaCvi2.23, which encodes for interleukin-2.  Subjects will be monitored in vitro for cellular and humoral antitumor responses and in vivo for antitumor activity.  (Protocol #9406-080)

 

Appendix D-79.  Dr. Alfred E. Chang of the University of Michigan Medical Center, Ann Arbor Michigan, may conduct gene marking experiments on 15 subjects with metastatic melanoma (≥ 18 years of age).  Subjects will undergo adoptive immunotherapy of anti-CD3/interleukin-2 activated lymph node cells that have been primed in vivo with tumor cells that have been transduced with the retrovirus vector, GBAH4, encoding the gene for interleukin-4.  The investigator will evaluate the antitumor efficacy and in vivo immunological reactivity in subjects receiving adoptively transferred T cells, and the in vitro immunological reactivities of the activated T cells that might correlate with their in vivo antitumor function.  (Protocol #9312-065)

 

Appendix D-80.  Dr. Robert Walker of the National Institutes of Health, Bethesda, Maryland, may conduct gene marking experiments on 40 HIV(+) subjects (≥ 18 years of age).  The investigator may also enter an additional number of subjects (to be determined by the investigator) who will receive a single administration of 1 x 107 HIV-specific CD8(+) cells.  The investigator will:  (1) Assess the safety and tolerance of the adoptive transfer of anti-HIV cytotoxic, syngeneic, CD8(+) peripheral blood lymphocytes that have been transduced with the retrovirus

vector, rkat4svgF3e-, that encodes for a universal chimeric T cell receptor.  (2) Determine the longevity of the genetically marked CD8(+) lymphocytes in the subject's peripheral blood.  (Protocol #9403-069)

 

Appendix D-81.  Dr. Joseph Rosenblatt of the University of California, Los Angeles, California, and Dr. Robert Seeger of Children’s Hospital, Los Angeles, California, may conduct gene transfer experiments on 18 subjects with neuroblastoma (≤ 21 years of age).  Patients at high risk of relapse with minimal or no detectable disease following myeloablative therapy and autologous bone marrow transplantation, or patients with progressive or persistent disease despite conventional therapy will be serially immunized with autologous or allogeneic neuroblastoma cells transduced to express γ interferon.  Cells will be transduced with the retroviral vector, pHuγ-IFN, encoding the human gene for γ interferon and lethally irradiated prior to use as an immunogen.  The objectives of the study are: (1) to determine the maximum tolerable dose of transduced cells; (2) to determine the local, regional, and systemic toxicities of injected cells; and (3) to determine the antitumor response in vivo as measured by standard clinical tests and immunocytologic evaluation of marrow metastases.  (Protocol #9403-068)

 

Appendix D-82.  Dr. Kenneth L. Brigham of Vanderbilt University, Nashville, Tennessee, may conduct gene transfer experiments on 10 subjects (≤ 21 years of age) in two different patient protocols (5 for each protocol).  Both protocols will use the same DNA/liposome preparations to deliver a plasmid DNA construct, pCMV4-AAT, encoding human alpha-1 antitrypsin gene driven by a cytomegalovirus promoter.  In patients scheduled for elective pulmonary resection, the DNA/liposome complexes will be instilled through a fiber optic bronchoscope into a subsegment of the lung.  Tissues of the lung will be obtained at the time of surgery.  Transgene expression will be assessed by immunohistochemistry, in situ hybridization, and Western and Northern blot analyses.  The effect of DNA/liposome complex administration on the histological appearance of the lung will also be evaluated.  In patients with alpha-1 antitrypsin deficiency, the DNA/liposome complexes will be instilled into the nostril.  Transgene expression will be determined in cells obtained by nasal lavage and nasal scraping, and the time course of transgene expression will be measured.  The secretion of the alpha-1 antitrypsin protein in nasal fluid will be determined.  Histological appearance of nasal mucosa will also be examined.  The study will assess safety and feasibility of gene delivery to the human respiratory tract.  (Protocol #9403-070)

 

 

Appendix D-83.  Dr. H. Kim Lyerly of Duke University Medical Center, Durham, North Carolina, may conduct gene transfer experiments on 20 subjects with refractory or recurrent metastatic breast cancer (≥ 18 years of age).  Autologous breast cancer cells will be transduced with the DNA/liposome complex, pMP6-IL2, containing a plasmid DNA vector derived from adeno-associated virus (AAV) that expresses the gene for human interleukin-2.  Subjects will receive 4 subcutaneous injections of lethally irradiated tumor cells transduced with the DNA/liposome complex prior to injection.  The objective of this study is to:  (1) evaluate the safety and toxicity of the treatment, (2) determine the immunological effects, (3) determine the duration of clinical responses, and (4) measure patient survival.  (Protocol #9409-086)

 

Appendix D-84.  Drs. Flossie Wong-Staal, Eric Poeschla, and David Looney of the University of California at San Diego, La Jolla, California, may conduct gene transfer experiments on 6 subjects (≥ 18 and ≤ 65 years of age) infected with human immunodeficiency virus-1 (HIV-1).  Autologous CD4(+) T lymphocytes will be transduced ex vivo with the retroviral vector, pMJT, expressing a hairpin ribozyme that cleaves the HIV-1 RNA in the 5' leader sequence.  The transduced cells will be expanded and reinfused into the patients.  The objectives of the study are: (1) to evaluate safety of reinfusing the transduced lymphocytes, (2) to compare (in vivo) the kinetics and survival of ribozyme-transduced cells with that of cells transduced with a control vector, (3) to determine in vivo expression of the ribozyme sequences in transduced lymphocytes, (4) to determine whether host immune responses directed against the transduced cells will occur in vivo, and (5) to obtain preliminary data on the effects of ribozyme gene therapy on in vivo HIV mRNA expression, viral burden and CD4(+) lymphocyte levels.  (Protocol #9309-057)

 

Appendix D-85.  Dr. Friedrich Schuening of the Fred Hutchinson Cancer Research Center, Seattle, Washington, may conduct gene transfer experiments on 10 subjects (≥ 18 years of age) with Type I Gaucher's disease.  The peripheral blood repopulating cells (mobilized by patient pretreatment with recombinant granulocyte colony-stimulating factor) will be harvested and CD34(+) cells selected.  CD34(+) cells will be transduced ex vivo with the retroviral vector, LgGC, that encodes human glucocerebrosidase cDNA.  Following transduction, the transduced cells will be infused into the patient without myeloablative treatment.  The primary endpoint of this study is to examine the safety of infusing CD34(+) cells transduced with the human glucocerebrosidase cDNA.  Patients will be monitored for persistence and expression of the glucocerebrosidase gene in hematopoietic cells.  (Protocol #9312-061)

 

Appendix D-86.  Dr. Terence R. Flotte of the Johns Hopkins Children's Center, Baltimore, Maryland, may conduct gene transfer experiments on 16 subjects (≥ 18 years of age) with mild cystic fibrosis (CF).  An adeno-associated virus (AAV) derived vector, encoding cystic fibrosis transmembrane conductance regulator (CFTR) gene, (tgAAVCF), will be administered to nasal (direct) and airway (bronchoscope) epithelial cells.  This is a dose escalation study involving 8 cohorts.  Each subject will receive both intranasal and bronchial administration of the adenoviral vector at 4 escalating doses.  Nasal doses will range between 1 x 106 and 1 x 109 pfu.  Lung administration will range between 1 x 107 and 1 x 1010 pfu.  The primary goal of the study is to assess the safety of vector administration.  Respiratory and nasal epithelial cells will be evaluated for gene transfer, expression, and physiologic correction.  (Protocol #9409-083)

 

Appendix D-87.  Drs. Jeffrey M. Isner and Kenneth Walsh of St. Elizabeth's Medical Center, Tufts University, Boston, Massachusetts, may conduct gene transfer experiments on 12 subjects (≥ 40 years of age) with peripheral artery disease (PAD).  A plasmid DNA vector, phVEGF165, encoding the human gene for vascular endothelial growth factor (VEGF) will be used to express VEGF to induce collateral neovascularization.  Percutaneous arterial gene transfer will be achieved using an angioplasty catheter with a hydrogel coated balloon to deliver the plasmid DNA vector to the artery.  The objectives of the study are:  (1) to determine the efficacy of arterial gene therapy to relieve rest pain and/or heal ischemic ulcers of the lower extremities in patients with PAD; and (2) to document the safety of the phVEGF arterial gene therapy for therapeutic angiogenesis.  Subjects will undergo anatomic and physiologic examination to determine the extent of collateral artery development following phVEGF arterial gene therapy.  (Protocol #9409-088)

 

Appendix D-88A.  Dr. Ronald G. Crystal of New York Hospital-Cornell Medical Center, New York, New York, may conduct gene transfer experiments on 26 patients (≥ 15 years of age) with cystic fibrosis (CF).  A replication deficient recombinant adenovirus vector will be used to transduce epithelial cells of the large bronchi with the E1/E3 deleted type 5 adenovirus vector, AdGVCFTR.10, which encodes the human cystic fibrosis transmembrane conductance regulator (CFTR) gene.  The objective of this study is to define the safety and pharmacodynamics of CFTR gene expression in airway epithelial cells following single administration of

 

escalating doses to the vector.  If single administration is determined to be safe, subjects will undergo repeat administration to localized areas of the bronchi.  (Protocol #9409-085)

 

Appendix D-88B.  Drs. Eric J. Sorscher and James L. Logan of the University of Alabama, Birmingham, Alabama, may conduct gene transfer experiments on 9 subjects (≥18 years of age) with cystic fibrosis (CF).  The normal human cystic fibrosis transmembrane conductance regulator (CFTR) gene will be expressed by a plasmid DNA vector, pKCTR, driven by the simian virus-40 (SV40) early gene promoter.  The CFTR DNA construct will be delivered by cationic liposome-based gene transfer to nasal epithelial cells.  The objectives of the study are to:  (1) evaluate the safety of lipid-mediated gene transfer to nasal epithelial cells (including local inflammation and mucosal tissue); and (2) evaluate efficacy as determined by correction of the chloride ion transport defect, and wild-type CFTR mRNA and protein expression. (Protocol #9312-066)

 

Appendix D-89.  Dr. Steven M. Albelda of the University of Pennsylvania Medical Center, Philadelphia, Pennsylvania, may conduct gene transfer experiments on 12 subjects with advanced mesothelioma.  The adenovirus vector encoding the Herpes simplex virus thymidine kinase (HSV-TK) gene, H5.020RSVTK, will be administered through a chest tube to the pleural cavity.  Tumor biopsies will be assayed for gene transfer and expression.  Subjects will be monitored for immunological responses to the adenovirus vector.  Ganciclovir will be administered intravenously 14 days following vector administration.  The primary objective of this Phase I study is to evaluate the safety of direct adenovirus vector gene delivery to the pleural cavity of patients with malignant melanoma. (Protocol #9409-090)

 

Appendix D-90.  Drs. Jeffrey Holt and Carlos B. Arteaga of the Vanderbilt University, Nashville, Tennessee, may conduct gene transfer experiments on 10 female patients (over 18 years of age) with metastatic breast cancer. Patient effusions from pleura or peritoneum will be drained and the fluid will be replaced with supernatant containing the retroviral vectors, XM6:antimyc or XM6:antifos, which express c-myc and c-fos antisense sequences, respectively, under the control of a mouse mammary tumor virus promoter.  The objectives of this study are to:  (1)  assess uptake and expression of the vector sequences in breast cancer cells present in pleural and peritoneal fluids, and determine if this expression is tumor specific, (2) assess the safety of localized administration of antisense retroviruses, and (3) monitor subjects for clinical evidence of antitumor response. (Protocol #9409-084)

 

Appendix D-91.  Dr. Jack A. Roth of MD Anderson Cancer Center, Houston, Texas, may conduct gene transfer experiments on 14 non-small cell lung cancer subjects (≥ 18 and ≤ 80 years of age) who have failed conventional therapy and who have bronchial obstruction.  LNSX-based retroviral vectors containing the β-actin promoter will be used to express: (1) the antisense RNA of the K-ras oncogene (LN-K-rasB), and (2) the wildtype p53 tumor suppressor gene (LNp53B).  Tumor biopsies will be obtained to characterized K-ras and p53 mutations.  Relative to their specific mutation, subjects will undergo partial endoscopic resection of the tumor bed followed by bronchoscopic administration of the appropriate retrovirus construct.  The objective of this study is to evaluate the safety and efficacy of intralesional administration of LN-K-rasB and LNp53 retrovirus constructs. (Protocol #9403-031)

 

Appendix D-92.  Drs. Robert E. Sobol and Ivor Royston of the San Diego Regional Cancer Center, San Diego, California, may conduct gene transfer experiments on 12 subjects (≥ 18 years of age) with metastatic colon carcinoma.  The autologous skin fibroblasts will be transduced with the retroviral vector, LNCX/IL-2, which encodes the gene for human interleukin-2 (IL-2).  In this dose-escalation study, subjects will receive subcutaneous injections of lethally irradiated autologous tumor cells.  The objectives of the study are to: (1) evaluate the safety of subcutaneous administration of LNCX/IL-2 transduced fibroblasts, (2) determine in vivo antitumor activity, and (3) monitor cellular and humoral antitumor responses.  (Protocol #9312-060)

 

Appendix D-93.  Dr. Michael Lotze of the University of Pittsburgh, Pittsburgh, Pennsylvania, may conduct gene transfer experiments on 18 subjects (≥ 18 years of age) with advanced melanoma, 6 with T-cell lymphoma, breast cancer, or head and neck cancer.  Subjects should have accessible cutaneous tumors, and have failed standard therapy.  Over 4 weeks, subjects will receive a total of 4 intratumoral injections of autologous fibroblasts transduced with the retrovirus vector, TFG-hIL-12-Neo.  This vector, which consists of the murine MFG backbone, expresses both the p35 and p40 subunits of interleukin-12 (IL-12) and the neoR selection marker.  The objectives of the study are to:  (1) define the local and systemic toxicity associated with peritumoral injections of gene-modified fibroblasts, (2) examine the local and systemic immunomodulatory effects of these injections, and (3) evaluate clinical antitumor efficacy.  (Protocol #9406-081)


 

 

Appendix D-94.  Drs. Evan Hersh, Emmanuel Akporiaye, David Harris, Alison Stopeck, Evan Unger, James Warneke, of the Arizona Cancer Center, Tucson, Arizona, may conduct gene transfer experiments on 25 subjects (≥ 18 years of age) with solid malignant tumors or lymphomas.  A plasmid DNA/lipid complex designated as VCL-1102 (IL-2 Plasmid DNA/DMRIE/DOPE) will be used to transduce the human gene for interleukin-2 (IL-2).  Patients with advanced cancer who have failed conventional therapy will undergo a procedure in which VCL-1102 is injected directly into the tumor mass to induce tumor-specific immunity.  The objectives of the study are to:  (1) determine safety and toxicity associated with escalating doses of VCL-1102; (2) confirm IL-2 expression in target cells; (3) determine biological activity and pharmacokinetics; and (4) determine whether IL-2 expression stimulates tumor regression in subjects with metastatic malignancies.  (Protocol #9412-095)

 

Appendix D-95.  Drs. Richard Morgan and Robert Walker of the National Institutes of Health, Bethesda, Maryland, may conduct gene transfer experiments on 48 human immunodeficiency virus (HIV) seropositive subjects (≥ 18 years of age).  This Phase I/II study involves identical twins (one HIV seropositive and the other HIV seronegative).  CD4(+) T cells will be enriched following apheresis of the HIV seronegative twin, induced to polyclonal proliferation with anti-CD3 and recombinant IL-2, transduced with either the LNL6/NeoR or G1Na/NeoR, and transduced with up to 2 additional retroviral vectors (G1RevTdSN and/or GCRTdSN(TAR)) containing potentially therapeutic genes (antisense TAR and/or transdominant Rev).  These T cell populations will be expanded 10 to 1,000 fold in culture for 1 to 2 weeks and reinfused into the HIV seropositive twin.  Subjects will receive up to 4 cycles of treatment using identical or different combinations of control and anti-HIV retrovirus vectors.  The relative survival of these transduced T cell populations will be monitored by vector-specific polymerase chain reaction, while the subjects' functional immune status is monitored by standard in vitro and in vivo assays.  (Protocol #9503-103)

 

Appendix D-96.  Dr. Harry L. Malech of the National Institutes of Health, Bethesda, Maryland, may conduct gene transfer experiments on 2 subjects ≥ 18 years of age (with or without concurrent serious infection), and 3 subjects ≥ 18 years of age (with or without concurrent serious infection) or minors 13-17 years of age who have concurrent serious infection who have chronic granulomatous disease (CGD).  CGD is an inherited immune deficiency disorder in which blood neutrophils and monocytes fail to produce antimicrobial oxidants (p47phox mutation) resulting in recurrent life-threatening infections.  Subjects will undergo CD34(+) mobilization with granulocyte colony stimulating factor (G-CSF).  These CD34(+) cells will be transduced with the retrovirus vector, MFG-S-p47phox, which encodes the gene for normal p47phox.  The objectives of this study are to:  (1) determine the safety of administering MFG-S-p47phox transduced CD34(+) cells, and (2) demonstrate increased functional oxidase activity in circulating neutrophils.  (Protocol #9503-104)

 

Appendix D-97.  Drs. Chris Evans and Paul Robbins of the University of Pittsburgh, Pittsburgh, Pennsylvania, may conduct gene transfer experiments on 6 subjects (≥ 18 and ≤ 76 years of age) with rheumatoid arthritis.  Rheumatoid arthritis is a chronic, progressive disease thought to be of autoimmune origin.  A gene encoding an interleukin-1 receptor antagonist protein (IRAP) will be delivered to the rheumatoid metacarpal-phalangeal joints to determine the autoimmune reactions can be interrupted.  The vector construct, DFG-IRAP, is based on the MFG murine retrovirus vector backbone, and encodes the human IRAP gene.  Synovial fibroblasts will be generated from the rheumatoid arthritic joint tissue obtained from patients who are scheduled to undergo surgery.  The fibroblasts will be transduced with the DFG-IRAP vector, and the transduced cells injected into the synovial space.  The synovial fluid and joint material will be collected 7 days later to determine the presence and location of the transduced synovial fibroblasts and the level of IRAP in the joint fluid.  (Protocol 9406-074)

 

Appendix D-98.  Dr. R. Scott McIvor of the University of Minnesota, Minneapolis, Minnesota, may conduct gene transfer experiments on 2 children with purine nucleoside phosphorylase (PNP) deficiency.  PNP deficiency results in severe T-cell immunodeficiency, an autosomal recessive inherited disease which is usually fatal in the first decade of life.  Autologous peripheral blood lymphocytes will be cultured in an artificial capillary cartridge in the presence of anti-CD3 monoclonal antibody and interleukin-2 and transduced with the retroviral vector, LPNSN-2, encoding human PNP.   Subjects will undergo bimonthly intravenous administration of transduced T cells for a maximum of 1 year.  The objectives of the study are to determine:  (1) the safety of intravenous administration of transduced T cells in children with PNP deficiency, (2) the efficiency of PNP gene transfer and duration of gene expression in vivo, and (3) the effect of PNP gene transfer on immune function.  (Protocol #9506-110)


 

 

Appendix D-99.  Drs. Nikhil C. Munshi and Bart Barlogie of the University of Arkansas School for Medical Sciences, Little Rock, Arkansas, may conduct gene transfer experiments on 21 subjects (>18 and <65 years of age) with relapsed or persistent multiple myeloma who are undergoing T cell depleted allogeneic bone marrow transplantation.  Donor peripheral blood lymphocytes will be cultured in vitro with interleukin-2 and anti-CD3 monoclonal antibody.  T cell depleted lymphocytes will be transduced with the retroviral construct, G1Tk1SvNa.7, which encodes the Herpes simplex virus thymidine kinase (HSV-TK) gene.  The transduced cells will be reinfused.  In this dose escalation study, 3 subjects will undergo cell-mediated gene transfer per cohort (maximum of 5 cohorts) until Grade III or IV Graft versus Host Disease (GVHD) is observed.  A maximum of 6 additional patients may be entered at that maximum tolerated dose.  The objectives of this study are to determine the:  (1) safety of transduced donor cell infusions, (2) effectiveness of donor cell infusions in decreasing the effects of severe GVHD, (3) effectiveness of donor cell infusions in prolonging multiple myeloma remission, and (4) effectiveness of ganciclovir in eliminating donor cells for the purpose of preventing the depletion of erythrocytes.  (Protocol #9506-107)

 

Appendix D-100.  Dr. Wayne A. Marasco of Dana-Farber Cancer Institute, Boston, Massachusetts, may conduct gene transfer experiments on 6 subjects (≥ 18 and ≤ 65 years of age) with human immunodeficiency virus type-1 (HIV-1).  Autologous lymphocytes from asymptomatic subjects will be transduced ex vivo with a retroviral vector, LNCs105, encoding the sFv105 antibody specific for the HIV-1 envelope protein.  An identical aliquot will be simultaneously transduced with a control retroviral vector lacking the sFv105 cassette.  Transduced cells will be reinfused into patients and the differential survival of both populations of CD4+ lymphocytes compared.  The objective of the study is to determine whether the intracellular expression of a human single chain antibody against HIV-1 envelope glycoprotein gp160 that blocks gp160 processing and the production of infectious virions can safely prolong the survival of CD4(+) lymphocytes in HIV-1-infected subjects.  (Protocol #9506-111)

 

Appendix D-101.  Dr. Henry Dorkin of the New England Medical Center, Boston, Massachusetts, and Dr. Allen Lapey of Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, propose to conduct gene transfer experiments on 16 subjects (≥ 18 years of age).  An E1/partial E4-deleted, replication-deficient, type 2 adenovirus vector, AD2/CFTR-2, will be used to deliver the human cystic fibrosis transmembrane conductance regulator (CFTR) gene by aerosol administration (nebulization) to the lung of CF patients.   Aerosol administration will be initiated only after initial safety data has been obtained from the lobar administration protocol (#9409-091).  This is a single administration dose-escalation study in which subjects will receive between 8 x 106 and 2.5 x 1010 pfu.  Subjects will be assessed for evidence of adverse, systemic, immune, inflammatory, or respiratory effects in response to AD2/CFTR-2.  Subjects will be monitored for virus shedding and transgene expression.  Health care workers present in the facility will be required to sign an Informed Consent document regarding the possibility of virus transmission.  (Protocol #9412-074)

 

Appendix D-102.  Drs. Charles J. Link and Donald Moorman of the Human Gene Therapy Research Institute, Des Moines, Iowa, may conduct gene transfer experiments on 24 female subjects (≥ 18 years of age) with refractory or recurrent ovarian cancer.  Subjects will undergo intraperitoneal delivery (via Tenkhoff catheter) of the vector producing cells (VPC), PA317/LTKOSN.2.  These VPC express the Herpes simplex virus thymidine kinase (HSV-TK) gene which confers sensitivity to killing by the antiviral drug, ganciclovir (GCV).  The LTKOSN.2 retrovirus vector is based on the LXSN backbone.  Two weeks following intraperitoneal delivery of the VPC, subjects will receive 5 mg/kg intravenous GCV twice daily for 14 days.  Subjects will receive between 1 x 105 and 1 x 108 VPC/kg in this dose escalation study.  Subjects will be evaluated by X-ray and peritoneoscopy of the abdomen for evidence of clinical response.  The objectives of this study are to determine the safety of intraperitoneal VPC administration.  (Protocol #9503-100)

 

Appendix D-103.  Dr. David T. Curiel of the University of Alabama, Birmingham, Alabama, may conduct gene transfer experiment of 15 subjects (≥ 18 years of age) with metastatic colorectal cancer.  Subjects will receive intramuscular injection of the polynucleotide vaccine, pGT63, which is a plasmid DNA vector expressing carcinoembryonic antigen (CEA) and hepatitis B surface antigen (HBsAg).  The objectives of the study are to:   (1) characterize the immune response to CEA and HBsAg following a single intramuscular injection and following 3 consecutive intramuscular injections, and (2) determine the safety of intramuscular injection of the plasmid DNA vector at doses ranging between 0.1 to 1.0 milligrams (single dose) and 0.9 to 3.0 milligrams (total multidose). (Protocol #9506-073)


 

 

Appendix D-104.  Dr. Chester B. Whitley of the University of Minnesota, Minneapolis, Minnesota, may conduct gene transfer experiments on two adult subjects (18 years of age or older) with mild Hunter syndrome (Mucopolysaccharidosis Type II).  The autologous peripheral blood lymphocytes will be transduced ex vivo with the retroviral vector, L2SN, encoding the human cDNA for iduronate-2-sulfatase (IDS).  The transduced lymphocytes will be reinfused into the patients on a monthly basis.  The study will determine the frequency of peripheral blood lymphocyte transduction and the half-life of the infused cells.  Evaluation of patients will include measurement of blood levels of IDS enzyme, assessment of metabolic correction by urinary glycosaminoglycan levels, clinical response of the disease, and monitoring for potential toxicity.  This Phase I study is to demonstrate the safety of the L2SN-mediated gene therapy and to provide a preliminary evaluation of clinical efficacy.  (Protocol #9409-087)

 

Appendix D-105.  Drs. James Economou, John Glaspy, and William McBride of the University of California, Los Angeles, California, may conduct gene transfer experiments on 25 subjects (≥ 18 years of age) with metastatic melanoma.  The protocol is an open label, Phase I trial to evaluate the safety and immunological effects of administering lethally irradiated allogeneic and autologous melanoma cells transduced with the retroviral vector, IL-7/HyTK, which encodes the gene for human interleukin-7 (IL-7).  Subjects will receive 1 x 107 irradiated unmodified autologous tumor cells in combination with escalating doses of IL-7/HyTK transduced allogeneic melanoma cells (M24 cell line).  The number of M24 cells administered will be adjusted based on the level of IL-7 expression.  Subjects will receive 3 biweekly subcutaneous injections of M24 cells expressing 10, 100, or 1000 nanograms of IL-7/hour in vivo.  A final cohort of 5 subjects will receive IL-7/HyTK transduced autologous cells.  Subjects will be monitored for antitumor activity by skin tests, biopsy analysis, tumor-specific antibody activity, and cytotoxic T lymphocyte precursor evaluation.  Non-immunologic parameters will also be monitored. (Protocol #9503-101)

 

Appendix D-106.  Dr. Jack A. Roth, MD Anderson Cancer Center, may conduct gene transfer experiments on 42 subjects (≥ 18 years of age) with refractory non-small cell lung cancer (NSCLC).  Subjects will receive direct intratumoral injection of a replication-defective type 5 adenovirus vector, AD5CMV-p53, to deliver the normal human p53 tumor suppressor gene.  The E1 region of AD5CMV-p53 has been replaced with a p53 expression cassette containing the human cytomegalovirus promoter (CMV).  Subjects will be divided into 2 treatment groups:  (1) 21 subjects will receive Ad5CMV-p53 alone, and (2) 21 subjects will receive Ad5CMV-p53 in combination with cisplatin.  Following vector administration, subjects will be isolated for 96 hours during which time, assays will be conducted to demonstrate the lack of shedding of adenovirus vector.  The objectives of this study are determine:  (1) the maximum tolerated dose of AD5CMV-p53, (2) qualitative and quantitative toxicity related to vector administration, and (3) biologic activity.

 

Prior to administration, adenovirus vector stocks will be screened for p53 mutants using the SAOS osteosarcoma cell assay that was submitted by Dr. Roth on June 23, 1995.  This biologic assay compares the activity of a standard stock of Adp53 vector to the activity of newly produced stocks.  The standard stock of Adp53 will be defined as mediating cell death in 100% of SAOS cells (human osteosarcoma cell line with homozygous p53 deletion) at an MOI of 50:1 (titer > 5 x 1010) on day 5 of culture.  The sensitivity of the assay for detecting inactive (presumed mutant) Adp53 vector will be determined by adding increasing amounts of Adluc (control adenovirus vector containing the luciferase gene) to the Adp53 stock to determine the percentage of inactive vector required to decrease growth inhibition of SAOS cells mediated by Adp53.  The test lot of Adp53 will be tested for its ability to inhibit SAOS in a 5 day assay.  Significant loss of inhibitory activity compared with the standard would indicate an unacceptable level of inactive (presumed mutant) vector. (Protocol #9406-079)

 

Appendix D-107A.  Dr. Gary Clayman. M.D. Anderson Cancer Center, Houston, Texas, may conduct gene transfer experiments on 21 subjects (≥ 18 years of age) with refractory squamous cell carcinoma of the head and neck.  Subjects will receive direct intratumoral injection of a replication-defective type 5 adenovirus vector, AD5CMV-p53, to deliver the normal human p53 tumor suppressor gene.  The E1 region of AD5CMV-p53 has been replaced with a p53 expression cassette containing the human cytomegalovirus promoter (CMV).  Subjects will be divided into 2 treatment groups: (1) those with non-resectable tumors, and (2) those with surgically accessible tumors.  Subjects will receive multiple injections of vector in each dose-escalation cohort.  Following vector administration, subjects will be isolated for 48 hours during which time, assays will be conducted to demonstrate the lack of shedding of adenovirus vector.  The objectives of the study are to determine: (1) the maximum tolerated dose of AD5CMV-p53, (2) qualitative and quantitative toxicity related to vector administration, and (3) biologic activity.

 

Prior to administration, adenovirus vector stocks will be screened for p53 mutants using the SAOS osteosarcoma cell assay that was submitted by Dr. Roth on June 23, 1995.  This biologic assay compares the activity of a standard stock of Adp53 vector to the activity of newly produced stocks.  The standard stock of Adp53 will be defined as mediating cell death in 100% of SAOS cells (human osteosarcoma cell line with homozygous p53 deletion) at an MOI of 50:1 (titer > 5x1010) on day 5 of culture.  The sensitivity of the assay for detecting inactive (presumed mutant) Adp53 vector will be determined by adding increasing amounts of Adluc to the Adp53 stock to determine the percentage of inactive vector required to decrease growth inhibition of SAOS cells mediated by Adp53.  The test lot of Adp53 will be tested for its ability to inhibit SAOS in a 5 day assay.  Significant loss of inhibitory activity compared with the standard would indicate an unacceptable level of inactive (presumed mutant) vector.  (Protocol #9412-096)

 

Appendix D-107B.  Drs. Bernard A. Fox and Walter J. Urba of Earle A. Chiles Research Institute, Providence Medical Center, Portland, Oregon, may conduct gene transfer experiments on 18 subjects (≥ 18 years of age) w