Protocols involving the use of rDNA/sNA for gene transfer into humans, whether done directly in the subject or in vitro and subsequently put into the subject, must be submitted to both the APB and the Stanford University Institutional Review Board (IRB) for Medical Human Subjects. Federal regulations require the local IBC (at Stanford, the APB), upon receiving submission of a Human Gene Transfer protocol, to review the following aspects to determine if NIH Recombinant Advisory Committee (RAC) review is required:

  • The protocol uses a new vector, genetic material, or delivery methodology that represents a first-in- human experience, thus presenting an unknown risk.
  • The protocol relies on preclinical safety data that were obtained using a new preclinical model system of unknown and unconfirmed value.
  • 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 bodies to evaluate the protocol rigorously.

Dependent upon the above findings the protocol will be either be submitted for RAC review or the APB will state that RAC review is not required.

For additional information concerning Stanford University’s IRB panels, please access the panel’s web site at https://researchcompliance.stanford.edu/.

Table 1. Recombinant and Synthetic Nucleic Acid Molecules (NIH guidelines).

If your experiment is in an exempt category, APB approval is not necessary. If your experiment does not fall within the exempt categories, you must have current APB approval (also see Table 2).

Is your synthetic nucleic acid designed to: (1) neither replicate nor generate nucleic acids that can replicate in any living cell, and (2) not integrate into DNA, and (3) not produce a toxin that is lethal for vertebrates at an LD50 of less than 100 nanograms per kilogram body weight? Yes Exempt (III-F-1)
Is your recombinant or synthetic nucleic acid molecule not in an organism, cell or virus and not been modified or manipulated to render it capable of penetrating cellular membranes? Yes Exempt (III-F-2)
Is your recombinant or synthetic nucleic acid molecule solely from a single source that exists contemporaneously in nature? Yes Exempt (III-F-3)
Is your recombinant or synthetic nucleic acid molecule solely from a prokaryotic host and propagated in the same host or transferred to another host by naturally occurring means? Yes Exempt (III-F-4)
Is your recombinant or synthetic nucleic acid molecule from a eukaryotic host and propagated in the same host? Yes Exempt (III-F-5)
Is your recombinant or synthetic nucleic acid molecule from species that naturally exchange DNA? Yes Exempt (III-F-6)
Does your genomic DNA contains a transposable element that does not contain any recombinant and/or synthetic nucleic acids? Yes Exempt (III-F-7)
Recombinant or synthetic nucleic acid molecule which does not present a significant risk to health or the environment, as determined by the NIH* Yes Exempt (III-F-8)
*The NIH has determined that rDNA/sNA from infectious agents of BL-2 (see Appendix A) or above is not exempt and must receive Biosafety approval. Additionally, certain cloning vectors, i.e. Adeno- or Sindbis-based vectors, or amphotropic MMLV based vectors, are some examples of rDNA that are non-exempt.

Table 2. Experiments requiring APB approval.

Approval required for experiments involving:
(Specific NIH Guideline Section)		 Further Information and Examples:
Deliberate transfer of a drug resistance trait to microorganisms that are not known to acquire the trait naturally if such acquisition could compromise the ability to control disease agents in humans, veterinary medicine or agriculture. (III-A-1-a)
  • Transferring a drug resistance trait that is used, had previously been used, may be used (including outside the U.S.), or that is related to other drugs that are used to treat or control disease agents.
  • Examples include transfer of: erythromycin resistance into Borrelia burgdorferi; pyrimethamine resistance into Toxoplasma gondii; chloramphenicol resistance into Rickettsia conorii; tetracycline resistance into Porphyromonas gingivalis.
Cloning of DNA, RNA or synthetic nucleic acid molecules encoding toxins lethal to vertebrates at an LD50 of <100 ng/kg body weight. (III-B-1)
  • Cloning toxins (or using plasmids that express toxins with low LD50s).
  • Examples include: botulinum, tetrodotoxin, ricin, T-2, saxitoxin, abrin, tetanus, Shigella dysenteriae neurotoxin, pertussis, Staph aureus Beta, shigatoxin, and conotoxins.
Transfer of recombinant or synthetic nucleic acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules into human research participants. (III-C-1)
  • Use of recombinant or synthetic nucleic acid molecules, or DNA or RNA derived from recombinant or synthetic nucleic acid molecules, that meet ANY of the following four criteria:
    • Contain >100nt, or
    • Possess biological properties that enable genome integration, or
    • Have the potential to replicate in a cell, or
    • Can be translated or transcribed.
    • Examples include: use of a defective adenoviral vector to deliver the CFTR gene intranasally to patients with Cystic Fibrosis; introduction of an HSV-TK transduced cell line into patients with epithelial ovarian carcinoma; introduction of a shRNA delivered in a plasmid, bacterial or viral vector.
Risk Group 2, Risk Group 3, Risk Group 4 or Restricted Agents used as Host-Vector Systems. (III-D-1)
  • The introduction of recombinant or synthetic nucleic acid molecules into Risk Group 2, 3, 4, or Restricted Agents that meet ANY of the following criteria:
    • Have the potential to replicate in a cell, or
    • Possess biological properties that enable genome integration, or
    • Produce a toxin lethal to vertebrates at an LD50 of <100ug/kg body weight
  • Examples include: Adenovirus, Herpes virus, Lentivirus, Amphotropic or VSV-g pseudotyped Murine Retrovirus, Human Retrovirus, Vaccinia virus Vesicular Stomatitis virus, and Adeno-Associated virus with helper virus.
DNA from Risk Group 2, Risk Group 3, Risk Group 4 or Restricted Agents cloned into nonpathogenic prokaryotic or lower eukaryotic host-vector systems. (III-D-2)
  •  Transfer of DNA from Risk Group 2, 3, 4, or Restricted Agents into
    nonpathogenic prokaryotes or lower eukaryotes.
  • Use of pathogens or defective pathogens as vectors.
  • Examples include: Adenovirus, Herpes virus, Lentivirus, Amphotropic or VSV-g pseudotyped Murine Retrovirus, Human Retrovirus, Vaccinia virus Vesicular Stomatitis virus, and Adeno-Associated virus with helper virus.
Infectious DNA or RNA viruses or defective DNA or RNA viruses in the presence of helper virus in tissue culture systems. (III-D-3)
  •  rDNA experiments involving Risk Group 2, 3, or 4 pathogens.
  • rDNA experiments involving ≤ 2/3 of the genome from eukaryotic viruses in the presence of a helper virus.
  • Examples include: HIV, HTLV-I & II, West Nile Virus, and Lymphocytic
    Choriomeningitis Virus.
Whole animals, including transgenic animals. (III-D-4)
  • Experiments utilizing any of the following that may lead to transmissible infection either directly or indirectly as a result of complementation or recombination in the animal:
    • > 2/3 of eukaryotic viral genome, or
    • Animals containing sequences from viral vectors, or
    • Stable integration of recombinant or synthetic nucleic acid molecules, or
      nucleic acids derived therefrom, into the germline.
  • Use of viable recombinant or synthetic nucleic acid molecule-modified Risk
    Group 2, 3, 4 or Restricted Agent microorganisms tested on whole animals.
Whole plants. (III-D-5)
  • Experiments involving exotic infectious agents when recombinant or synthetic nucleic acid molecule techniques are associated with whole plants.
  • Experiments with plants involving cloned genomes of readily transmissible exotic infectious agents.
  • Experiments with plants involving readily transmissible exotic infectious agents (i.e. soybean rust fungus Phakopsora pachyrhizi, maize streak or other viruses) in the presence of their specific arthropod vectors.
  • Experiments involving plants or their associated organisms and the introduction of sequences encoding potent vertebrate toxins.
  • Experiments involving microbial pathogens of insects, arthropods or small animals associated with plants if the recombinant or synthetic nucleic acid molecule-modified organism can detrimentally impact the ecosystem.
Large-scale DNA work. (III-D-6)
  •  ≥ 10 liters of culture combined.
  • Examples include: Use of ≥10 L fermentor; growing up to five 2 L flasks of rDNA culture (i.e. E. coli K-12).
Influenza virus. (III-D-7)
  • Experiments with Influenza virus shall be conducted at the BSL containment corresponding to the Risk Group of the virus that was the source of the majority of segments.
  • Experiments that alter antiviral susceptibility may increase containment level requirements.
  • Examples of BSL3 influenza work: 1957-1968 Human H2N2, Highly pathogenic avian influenza H5N1 strains within the Goose/Guangdong/06-like H5 lineage (HPAI H5N1), 1918 H1N1.
a Refers to the parental or wild-type virus and some of the common deletions used in viral vectors. MMLV, Moloney murine leukemia virus; SIV, simian immunodeficiency virus.
b Refers to ability of vector to infect cells from a range of species. Ecotropic generally means able to infect only cells of the species originally isolated from or identified in. Please note that the ecotropic host for HIV and HSV would be human cells, but the ecotropic host for MMLV would be murine cells. Amphotropic and VSV-G-pseudotyped virus host range includes human cells.
c Shown are general categories of cellular genes and functions. Please note that there are differences in the containment level for the same class depending on whether the viral vector integrates into the recipient genome at a high rate. The general categories are as follows: S, structural proteins (actin, myosin, etc.); E, enzymatic proteins (serum proteases, transferases, oxidases, phosphatases, etc.); M, metabolic enzymes (amino acid metabolism, nucleotide synthesis, etc.); G, cell growth, housekeeping; CC, cell cycle, cell division; DR, DNA replication, chromosome segregation, mitosis and meiosis; MP, membrane proteins, ion channels, G-coupled protein receptors, transporters, etc.; T, tracking genes such as those for green fluorescent proteins and luciferases and photoreactive genes; TX, active subunit genes for toxins such as ricin, botulinum toxin and Shiga and Shiga-like toxins; R, regulatory genes for transcription and cell activators such as cytokines, lymphokines and tumor suppressors; Ov and Oc, oncogenes identified via transforming potential of viral and cellular analogs, or mutations in tumor suppressor genes resulting in a protein that inhibits/moderates the normal cellular wild- type proteins. This does not include SV40 T antigen. SV40 T-antigen-containing cells should not be considered more hazardous than the intact virus. SV40 is considered a risk level 1 agent (the lowest level) according to the NIH Guidelines. The prevalence of SV40 infection in the U.S. population due to contaminated polio vaccine does not seem to have caused a statistically significant increase in the rate
of cancers. However, the data from the various studies on SV40 association with cancer are equivocal (Strickler et al. 1998; Butel and Lednicky, 1999; Dang-Tan et al., 2004).
d This is a general assessment of containment levels for laboratory construction and use of these vectors for nonproduction quantities only based on the 4th edition of BMBL. This table cannot cover every potential use within a research or laboratory settings; as information is gained, risk assessments and containment levels may be changed. Local IBCs should use all available information and their best judgment to determine appropriate containment levels. BSL – 1* refers to the containment level based on parent virus risk group. However, most procedures involving the handling and manipulation of the viral vectors are done at BSL – 2 to protect cell cultures and viral stocks from contamination.
e Certain specific strains of poxviruses, such as MVA, NYVAC, ALVAC and TROVAC, are considered low-risk agents and can be handled at BSL – 1 in certain cases.

From Biological Safety Principles and Practices, 4th ed., pg. 524, D.O. Fleming and D.L. Hunt, Ed, ASM Press, 2006.

Figure 1. Genome Editing and Gene Drives
Image credit: Kevin Esvelt

Important Information

Human Gene Transfer

Conducting Gene Transfer experiments into human subjects requires both an IRB and an APB protocol.