Recombinant Nucleic Acids, Pathogens and Toxins

Any research on pathogens that may, inadvertently or purposefully, increase its virulence or pathogenicity must be reviewed by the BSO and the IBC for potential inclusion in the US government’s Potential Pandemic Pathogen Care and Oversight (P3CO) program.

A potential pandemic pathogen (P3) is one that satisfies both of the following criteria:

1. It is likely highly transmissible and likely capable of wide and uncontrollable spread in human populations, and 
2. It is likely highly virulent and likely to cause significant morbidity and/or mortality in humans.

For more information on potential pandemic pathogen care and oversite visit the Recommended Policy Guidance for Departmental Development of Review Mechanisms for Potential Pandemic Pathogen Care and Oversight page.

The introduction of CRISPR based gene editing tools has made precise, targeted genomic modification a simple procedure available to virtually anyone.  Additionally, CRISPR based genomic editing has ushered in the era of gene drives, genomic alterations that no longer obey Mendelian inheritance rules.  Rather, genes drives are passed to every offspring in each successive generation.

All projects utilizing CRISPR and/or Gene drives are required to be submitted to the IBC for review.  In addition, stringent biosecurity measures may be required to prevent transgenic organisms from accidental release.

Lentiviral vectors are typically HIV derived, replication incompetent viral particles used for the stable genomic integration and expression of transgenes or shRNA in both dividing and non-dividing cells.  Lentiviral vectors are often used in tissue and cell culture studies and in animal (mouse) studies.

Research using lentiviral vectors is inherently dangerous due to the vector’s origination and the stable integration of exogenous DNA into the chromosome of the host.  Therefore a number of risks to human safety have been identified.

These risks are:

• The inadvertent generation of Replication Competent Lentivirus (RCL)
• The potential for oncogenesis due to accidental exposure
• Dependent on the nature of the introduced transgene

Due to the risks associated with lentiviral vectors, efforts have been made to make them safer.  These efforts have been successful in the development of third generation vectors consisting of a four (4) plasmid systems.  The separation of lentiviral vector generation factors onto multiple plasmids has reduced the risk of generating RCL to virtually zero (with two caveats, see below).  However the risks of oncogenesis through integration and that of expression of the exogenous transgene are still present.  Third generation systems still possess a risk for RCL generation in HIV positive individuals as discovered in clinical trials using lentiviral vectors for gene therapy and when transducing primary human cell lines that have not been screened for HIV.   

Investigators who intend to develop their own Lentiviral vector production systems must contact the biosafety office for further guidance (716-829-3301).  

The risks of exposure to laboratory personnel lie mainly in accidental auto-inoculation followed by mucosal exposure and aerosol inhalation.  The health risks inherent to lentiviral work warrant a basal level of safety precautions, described below, when handling the vectors or inoculated animals.  The nature of the transgene may require increased safety precautions beyond the basal level as determined by the risk assessments performed by the PI, the BSO and the University’s IBC.

The NIH’s Recombinant DNA Advisory Committee (RAC) recommends that all lentiviral vectors be generated and used under BSL-2 conditions.  In addition, dependent on the nature of the transgene, the RAC advises increased biological safety considerations on a case-by-case basis.  The University at Buffalo IBC recommends the following precautions are used when generating and working with lentiviral vectors:

• Generation III lentiviral vectors are utilized
• Vector manipulation is performed in certified Class II biological safety cabinet (BSC)
• Base level PPE be worn at all times (gloves, lab coat, eye-protection)
• The use of sharps and needles be minimized and that engineered safety needles be considered
• Only the tissue/cell culture to be inoculated should be present in the BSC (no other cultures or media should be in the BSC)
• Filter cap cell culture plates are utilized, particularly if non-infected cell cultures are in the same incubator at the same time
• Centrifugation must be performed using sealed tubes or aerosol tight rotors which are to be closed and opened in the BSC.
• 10% Household bleach or 70% ethanol must be readily available
• All surfaces are to be decontaminated on a regular basis while working and after work is complete
• All waste be treated as regulated medical waste (RMW) and disposed of according to University RMW policies (call or email UB EH&S or the BSO for questions regarding the University at Buffalo’s RMW policies).
• A laboratory specific Exposure Response Plan (ERP) must be developed and approved by the IBC.  A template for the ERP can be found at (website).

The use of lentiviral vectors in mice is well documented.  The University at Buffalo’s Laboratory Animal Facility (LAF) has developed a Standard Operating Procedure for Investigators. This SOP outlines the precautions, PPE, decontamination, and waste disposal procedures specific to the LAF and lentiviral use in mice.  As with laboratory use, all lentiviral vector use must be reviewed and approved by the IBC on a case-by-case basis as well as by the University at Buffalo’s IACUC committee.

A case-by-case risk assessment must be performed for all lentiviral vector work due to the nature of the transgene or shRNA to be transduced.  Investigators must submit their detailed protocol to the IBC using the CLICK submission portal. Increased biosafety precautions may be required including:

• Double gloves
• The use of a surgical mask with face guard

Laboratory personnel who are HIV positive or engage in behaviors shown to increase the risk of acquiring HIV should consider self-reporting and removing themselves from working with lentiviral vectors.  Clinical studies have shown that HIV co-infection can mobilize the integrated provirus and generate RCL.

In the event of an accidental exposure, individuals should follow their laboratory specific Exposure Response Plan.  For an inoculation, individuals are to immediately wash the affected area with soap and water for 10 minutes.  For all other exposures, the affected area is to be rinsed with water for 10 minutes.

Individuals may be provided anti-retroviral drugs following consultation with their physician.  

Employees who accidently expose themselves through inoculation or other injury should also fill out a worker’s compensation form.

The U.S. Department of Health and Human Services (HHS) and U.S. Department of Agriculture (USDA) have published the regulations that govern the possession, use, and transfer of Select Agents and Toxins (42 CFR part 73, 7 FR part 331, and 9 CFR part 121).

Any researcher desiring to obtain, possess or work with Select Agents must be approved by the Department of Health and Human Services or the United States Department of Agriculture (USDA) . Please contact the University at Buffalo’s Responsible Official (829-3301) to begin the application process.

For your reference, a list of agents and toxins is available from the CDC.

Researchers desiring to work with regulated Select Toxins of biological origin should be aware that possession of small quantities not exceeding a maximum threshold may be allowed under the Select Agent regulations. The list of toxins and exempt quantities is also available from the CDC. Note the use of select agent toxins of biological origin in any quantity may require approval of the University's Institutional Biosafety Committee.

Coding for these toxins may be restricted, even if the amount of toxin generated or possessed remains under the maximum threshold. See Restricted Experiments below. 

An individual or entity may not conduct, or possess products (i.e., select agents that are not known to acquire a drug resistance trait naturally, if such acquisition could compromise the control of disease agents in humans, veterinary medicine, or agriculture, or recombinant and/or synthetic nucleic acids containing genes for the biosynthesis of select toxins lethal for vertebrates at an LD[50] < 100 ng/kg body weight) resulting from, the following experiments unless approved by and conducted in accordance with the conditions prescribed by the Administrator:

Restricted experiments:

1. Experiments that involve the deliberate transfer of, or selection for, a drug resistance trait to select agents that are not known to acquire the trait naturally, if such acquisition could compromise the control of disease agents in humans, veterinary medicine, or agriculture.
2. Experiments involving the deliberate formation of synthetic or recombinant nucleic acids containing genes for the biosynthesis of select toxins lethal for vertebrates at an LD[50]<100 ng/kg body weight.

NOTE: These types of experiments are NOT prohibited, but permission must be obtained from the lead agency, which in our case is the CDC.

A specific subclass of Select Agents fall under the NIH’s DURC Policy.  Dual Use Research of Concern (DURC) is life sciences research that, based on current understanding, can be reasonably anticipated to provide knowledge, information, products, or technologies that could be directly misapplied to pose a significant threat with broad potential consequences to public health and safety, agricultural crops and other plants, animals, the environment, materiel, or national security.