The Risk Assessment and Biosafety Program for Recombinant DNA Research Peili Zhu, Jonathan Koolpe, Mei-Chuan Huang, Biosafety Officer

Office of Environmental Health and Safety, University of California, San Francisco

Introduction • To achieve biological and medical research goals, recombinant DNA (rDNA) materials and

techniques are often used in various animal, human, and basic research which raises health and safety concerns for research personnel and animal facility staff.

• Information identified by risk assessment when working with rDNA materials will provide

guidance for:

o Selecting appropriate biosafety levels

o Implementing appropriate safety practices

o Identifying appropriate safety equipment

o Establishing facility safeguards

• Risk assessments using these criteria will assist in preventing exposures to or laboratory

acquired infections from rDNA materials.

Challenge How to conduct risk assessments for rDNA research?

Example 1. Viral vectors: What will be risk assessment differences between using ecotropic

retrovirus (MMLV), replication incompetent lentivirus, and replication competent HIV as

vectors?

Example 2. Genes: What will be risk assessment differences between rDNA manipulations of

genes such as GFP, oncogene (Ras), and prion genes?

Example 3. Hosts: What will be risk assessment differences between commonly used hosts

such as E. coli K12, human cells, and polio virus?

Example 4. Human Gene Transfer Studies: How to conduct risk assessment for using

genetically modified Listeria monocytogenes as vector in human gene transfer studies?

Additional Challenges • rDNA research is being used in increasing numbers of laboratory, animal, and clinical

research studies

• rDNA technology is rapidly developing/changing

• Regulations change frequently

Goal • Implementation of appropriate and effective biosafety programs to cover all aspects of rDNA

research (human, animal, basic)

• Program will assist universities when overseeing rDNA research to ensure both researcher

safety and compliance with regulations

I. Online BUA Application System

Conclusion

Developing and implementing an effective biosafety program for rDNA research continues to be vitally important for biomedical research to ensure both safety and compliance at UCSF. Implementing similar programs will assist other universities and research institutions to oversee animal, human, and basic rDNA research and to ensure that these research activities also remain safe and are in compliance with all applicable regulations.

Result

The online BUA application system, the BUA review and risk assessment for rDNA research

conducted by the IBC, the online Biosafety and laboratory specific trainings, the collaboration

among IBC, IRB and IACUC, and Occupational Health Program constitute a successful

biosafety program at UCSF.

Such a program oversees the animal, human, and basic rDNA research at UCSF and ensures

that these research activities are in compliance with various regulations.

II. Institutional Biosafety Committee Review

Recombinant DNA materials or technology

Infectious Agents (IA)

Bloodborne Pathogens (BBP)

Biological Toxins

Method Design and implement a campus biosafety program for rDNA research by using an online

Biological Use Authorization (BUA) application system, Institutional Biosafety Committee (IBC)

review, and online training program. The program will oversee rDNA research at all campuses and

to ensure that these research activities are in compliance with various regulations.

I. Online BUA Application System

Online BUA application indicates if a study involving:

Select Agents

Animals

Generate Genetically Modified Animals

Shipping of biological materials

Online BUA application system ensures that detailed information regarding vectors, genes, hosts

and constructs used for rDNA research has been provided by Principal Investigators.

Vector Information

Vector Name: Lentivirus

Risk Group: 2

Vector Type: Viral

Replication incompetent: Yes

Causes human disease: Yes

Ecotropic: No

Amphotropic: Yes

Used in Animals: Yes

Describe the method by which you will verify replication incompetence, and specify the frequency of testing. For

example, the UCSF Sandler Lentiviral RNAi Core’s Lentivirus Replication Safety Assay (e.g. every third batch):

UCSF Sandler Lentiviral RNAi Core's Lentivirus Replication Safety Assay.

Every third batch of the virus will be tested.

Provide an explanation of the hazards to humans and describe the possible modes of transmission:

During infection, there is a possibility that the replication-incompetent lentivirus may convert to a replication competent state.

If the researcher is accidentally infected, it could result in expression of the inserted gene. Possible mode of transmission is

via skin (accidental puncture or contact with open wound).

Provide a detailed description of this vector and safety features of its design (such as which generation of viral vector,

gene deletions, etc.):

Lentiviruses can infect both dividing and nondividing cells and can integrate into the genome resulting in long-term, heritable

expression of the inserted gene. The lenti vectors chosen to be used in the lab are second and third generation tripartite

systems in which viral replication genes are deleted and packaging components are on separate vectors so that the infectious

viral particle cannot replicate in target host cell. Nevertheless the viral supernatant produced by lentivirus packaging cell line

has the potential to infect humans and therefore will be handled with caution under strict BSL2 conditions.

Gene Information

Gene or Gene Family Name: Ras family (K, H and N-Ras) and Ras pathway genes

Gene Biological Sources: Human cells and mouse tissue

Highest Risk Group of Sources: 2

Expressed as RNA/Protein: Yes

Indicate which RNA or protein will be produced: Ras (K, H and N); Raf, MEK, MAPK, PI3K, p85

Adverse Immune Reaction: No

(Proto) Oncogenic: Yes

Toxigenic: No

Enhances Pathogenicity: No

Pathogenic to Humans: No

Teratogenic: No

Not sure: No

None: No

Associated vectors: Adenovirus/Viral, Lentivirus/Viral, pcDNA/Plasmid,

Retrovirus/Viral

Describe the nature of cellular activity that will result if this gene is expressed or knocked down.

Cell signaling

Provide an assessment of the hazardous potential of cloning, expressing or inhibiting the target of these DNA/RNA

segments that encode substances that are immunogenic, (proto)oncogenic, teratogenic, toxigenic or enhance

pathogenicity:

Recombinant materials we are using do not encode pathogens or toxins known to affect humans or animals. Expression of

oncogenes or inhibition of tumor suppressor genes is potentially hazardous to the researcher. Therefore, we perform these

experiments in a certified biosafety cabinet and follow UCSF guidelines such as using personal protective equipment (lab

coat and gloves) and proper handling of hazardous waste including solid waste in red bags placed in hard sided plastic

containers, decontamination of surfaces with 10%bleach and treating liquid waste with 10% bleach for 30 minutes.

Host Information

Host Name: human established cell lines

Risk Group: 2

Infectious Agent: No

Bloodborne Pathogen: Yes

Host details (if applicable):

Vector-Gene Construct(s) for human established cell lines

Vectors \ Genes: Ras family (K, H and N-Ras) and Ras pathway genes

Adenovirus Yes

Lentivirus Yes

pcDNA Yes

Retrovirus Yes

rDNA Research Safety

rDNA Safety

Confirm that, in case of accidental exposure to recombinant materials, you will follow this SOP:

Personnel who are exposed to recombinant materials will immediately wash the site of exposure (wound, eye,

mucosa, etc.) with copious amounts of water for 15 minutes and then immediately call the Needlestick and

Exposure Hotline at 415-353-7842 for all UCSF campuses and affiliated institutions. Important: Report

exposure to Biosafety Officer (415-514-2824) within 72 hours.

If the SOP cannot be followed, describe how exposure will be managed:

This SOP will be followed as described.

Describe methods by which experiment(s) will be conducted safely:

rDNA experiments are carried out at the bench which is covered with bench paper to prevent contaminating the

bench and wiped clean with 10% bleach periodically. Growth media is decontaminated with 10% bleach before it is

discarded. When working with lentiviral vector, we use 2nd and 3rd generation vectors components of which are

present on separate plasmids and cannot package an infectious viral particle until all components are mixed properly

which we perform in a biosafety cabinet in a BSL2 designated room for the safety of personnel. Similarly,

adenoviral vector is missing essential genes and requires helper cell line for virus packaging. We package

replication-deficient viruses only in a biosafety cabinet under strict BSL2 conditions and follow UCSF guidelines.

All transfection and infection work is also performed in a biosafety cabinet in a BSL2 room using proper PPE

(labcoat and gloves). Solid waste is discarded in properly labeled hard plastic containers lined with red bags. Liquid

waste is inactivated with 10% bleach for 30 minutes before discarding. Sharps are collected in a sharps container

and disposed as biohazardous solid waste.

If these constructs were accidentally released outside of the laboratory, will there be significant implications

to the environment? If 'Yes', please describe:

Constructs we are using do not pose significant implication to the environment.

Animal Use for rDNA Research

Approved Protocols

IACUC Protocol

AN085050-01A

Approved Animal Species

Species

Mouse

• Provide a concise narrative describing your animal use in this study.

• If you use hazardous materials in animals, provide details for the experiment procedures involved. Step-by-step

descriptions of procedures are not necessary, but focus should be placed on procedural steps where laboratory or

LARC personnel are vulnerable to exposure to potentially hazardous materials.

• Provide a risk assessment of using hazardous materials in animals for personnel handling and caring for animals.

The mice used in our studies will grow tumors either 1) of de novo origin through activation of oncogenic transgenes; or 2) of

human/mouse origin through xenograft implantation of cancer cell lines. We will deliver RNAi-based therapies to these mice

via intravenous injection with the objective of inducing tumor regression. Activation of oncogenic transgenes will be achieved

through intranasal inhalation of adenovirus (up to 2.5x10^7 pfu) or intratracheal installation of lentivirus (up to 1x10^6 pfu).

Avertin will be used as an anesthetic. Newly infected animals will be treated in accordance with ABSL2 guidelines including

proper PPE (lab coat and gloves), biohazard labeling on animal cages, investigator cage handling for 48h, and proper hazardous

waste handling and disinfection procedures (solid waste in red bags in hard plastic containers, sharps waste in sharps container,

liquid waste treated with 10% bleach for 30 minutes). There should be no risk to LARC personnel for experiments with lenti

and adenovirus as they will not be handling these animals for the first 48 hours post infection. There is a small risk of exposure

to researchers: 1. we will use replication deficient lenti and adenovirus. 2. Accidental exposure to adenovirus is mild respiratory

disease. However, its expression is transient. Lenitvirus on the other hand can integrate into the genome resulting in long-term

heritable expression of the inserted gene. Although replication deficient lentivirus that we are using mitigates this risk, we will

strictly follow ABSL2 guidelines and biosafety procedures outlined above and on the website http://or.ucsf.edu/ehs/7240-

DSY/9696 to further lower the risk.

Hazardous Materials

Type Materials Risk Group

BBP A primary cell line or cells derived directly from human tissue 2

Vector Adenovirus 2

Vector Lentivirus 2

Genetically Modified Animals

Research project summary

BAC Huntingtin S421 mice: We have used the Gladstone Transgenic Core to create lines of mice expressing the

huntingtin, the protein that causes Huntington's disease. We are generating specific site mutants, to study the role that a

specific phosphorylation site plays in the toxicity of the mutant version of huntingtin.

Arc transgenic mice: We have used the Gladstone Transgenic Core to generate a transgenic mouse with the protein Arc

tagged with His and Flag tags, expressed at levels similar to endogenous Arc levels. This will allow for efficient

purification of Arc and its binding partners in vivo. The mice were made by the Gladstone transgenic facility and are in

the C57B6 background.

Dendra2-LC3 mice: We have used the Gladstone Transgenic Core to generate transgenic mice expressing a fluorescent

protein Dendra2 fused to LC3, which serves as a reporter for autophagosomes. This transgenic mouse model will be a

useful tool to study mammalian autophagy.

PKD1 knockout mice: We have used the Gladstone Transgenic Core to make PKD1 knockout mice to study the novel

role of (PKD1) in the trafficking of synaptic proteins and learning and memory.

Risk assessment.

There is very minimal to zero risk if these mice were introduced into wild populations or other colonies. The BAC

Huntingtin S421 mice develop a slow-onset neurodegenerative condition that has not been shown to be transmissible

and only can be inherited. The Arc transgenic mice and Dendra-LC3 mice express protein at levels comparable to

endogenous and the proteins are not transmissible. Although the PKD1 knockout mice have severe learning deficits,

this phenotype is not transmissible into wild populations.

Risk minimization.

All mice are kept in secure cages in a barrier facility in Gladstone Institute. During maintenance, cages are left closed

unless mice are being watched in close proximity. If animals are to be moved out of their holding room, primary

containment (cage or transport box) and secondary containment (closed paper bag) are used to prevent escape. Mice

are tracked by ear markings and a record is made of which cage the mouse resides. Colony population is recorded in

spreadsheet software. They will only be removed from the animal facility when they will be sacrificed. After being

sacrificed, the carcasses are disposed as pathological waste.

rDNA Research Information

III. Biosafety Training