Download rDNA Risk Assessment Worksheet

rDNA Risk Assessment Worksheet
From the PHAC e-learning module: ‘For recombinant organisms. The parental and modified pathogens
should be considered together when examining each of the risk factors. Risk group assessments for
recombinant organisms require the researcher to consider the effect of the modification in the context of all
the risk factors discussed in the course.’ In other works, rDNA risk assessment includes the assessment of
the combined host, insert (donor nucleic acid segment) or deletion and vector (if microorganism is used) in
the context of the procedures being used and the facility and personnel involved.
A. Host/ Parent
1. What is the name of the agent you have modified using DNA recombination?
2. Check all that apply.
Virus
Bacteria
Human
Animal
Cell line
Plant
Fungus/Yeast
Insect
Other __________________
3. What is the risk group of the biological agent before rDNA modification? See CBSG for risk
group definitions.
RG-1
RG-2
RG-3
RG-4
4. How have the changes been made? Or What method is used to make the modifications?
5. How has the agent been changed? Briefly describe insertions, deletions and any other relevant
rDNA changes.
6.
Do the changes that have been made:
Cause viral DNA to integrate into the host genome?
Attenuate the agent, if yes explain how or describe the attenuation. Describe
how extensively the attenuated strain has been utilized without incident (or
has the attenuation been proven in animal models?)
Enhance pathogenicity or virulence? – if yes please describe
Confer the ability to produce a new toxin or encodes a toxin molecule with a
LD 50 of <100nanogram/kg of body weight.
Alter the host range or tropism? If yes describe.
Decrease the infectious dose?
Increase the agent’s survival time outside the host?
Alter pharmacological activity? E.g. Increase resistance to antibiotics or
drugs used to treat infection or render a useful vaccine ineffective?
Make the agent transmissible through a route not previously able? Indicate
all that apply:
inhalation
ingestion
injection
mucosal membranes/skin exposure
Allow a pathogen to evade diagnostic or detection modalities?
Include oncogenes ?
Increase or decrease replication competence?
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yes
yes
no
no
unk
unk
yes
yes
no
no
unk
unk
yes
yes
yes
yes
no
no
no
no
unk
unk
unk
unk
yes
yes
yes
yes
yes
yes
yes
yes
no
no
no
no
no
no
no
no
unk
unk
unk
unk
unk
unk
unk
unk
B. Inserts (donor nucleic acid segments)
1. If sequences were inserted, what is the source of any inserted DNA (e.g. genomic, cDNA,
synthetic, coding/non-coding sequences)
2. If sequences were inserted, what was the Risk Group of the agent the sequences were derived
from?
RG-1
RG-2
RG-3
RG-4
3. Do the changes that have been made
the parent organism?
increase,
decrease,
not affect the Risk Group of
4. Describe the properties of the donor nucleic acid segment. e.g. foreign gene expression &
function? Is there potential for altering the cell cycle? Examples below from Kost etal. (4th
Edition Biological Safety: Principles and Practices)
a. Structural protein, Enzymatic proteins
b. DNA replication
c. Chromosome segregation
d. Membrane proteins
e. Tracking genes (GFP)
f. Cell growth, housekeeping
g. Cell cycle, cell division
h. Toxins
i. Regulatory genes for transcription and cell activators such as cytokines, lymphokines and
tumour suppressors
j. Oncogene potential; insertional mutagenesis
C. Vector
1. Vector/vector backbone is _________________ (bacterial plasmid, phage, virus, YAC, etc.)
2. Vector /vector backbone risk group?
RG-1
RG-2
RG-3
RG-4
3. Vectors will be:
[ ] Constructed in the lab
[ ]purchased from a vendor (name and cat.# or url
[ ] obtained elsewhere (specify)___________________________
5. Have any safety features been incorporated into a viral vector if used?
)
yes
no
Describe (e.g. Is a helper virus or packaging cell line required, replication defective, etc.)?
6. If HIV backbone (refer also to Working with Lentiviral Vectors Guideline):
a. Generation of vector? ( e.g. 2nd, 3rd, 4th)
b. How many gene-bearing plasmids?
c. Is there a self-inactivating (SIN) mechanism (e.g. 3’LTR) making the virus replication
incompetent? Describe the SIN mechansim and include a discussion of the probability of
generating a replication-competent virus?
d. How many/which accessory genes are absent?
e. What is the virus envelope?
f. Vector titer and volume in use?
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D. Overall Risk Assessment
1. What Risk Group would you assign to this modified agent?
RG-1
RG-2
RG-3
RG-4
2. At what Containment Level (CL) should this agent be handled? See CBSG for CL definitions.
CL-1
CL-2
CL-3
CL-4 (cannot be done at the U of M)
3. Do the changes,( even if they do not change the original Risk Group/Containment Level),
require any additional safety work practices or facility design requirements?
yes
no
4. What is the scale of the work?
Research
Production scale?( ~ >10liter)
5. List any special precautions that you think are necessary for work with the modified agent.
E.g. aerosol producing procedures, work with sharps, centrifugation, work in BSC, medical
surveillance, post exposure , PPE, etc..
Ref: Containment level definitions from CBSG
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