Transcript AUTO BSL-1 - Institutional Biosafety Committee

Town Hall Discussion: AAV
Institution Biosafety Committee
February 23, 2016
11:00am
S120 BST
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Who Are We?
Position
Research Interests/Experience
Nathan Urban, PhD
Vice Provost, Special
Events;
Prof of Neuroscience
Neural Circuits research
Bill Yates, PhD
Co-Director, RCCO:
Vestibular System research
Prof of Otolaryngology &
Neuroscience
Kelly Cole, PhD
Chair, IBC;
Assoc. Prof of
Immunology
Influenza Virus Vaccine research
Beverly Harding, MSL
Office Director, IBC
DOD research/safety; 15+ lab
Molly Stitt-Fischer, PhD
Biosafety Officer
Environmental and Occ health
What is an IBC?
NIH Guidelines for Research Involving
Recombinant or Synthetic Nucleic Acid
Molecules (NIH Guidelines); Section I-E-2
An Institutional Biosafety Committee (IBC) is a
committee that:
1) Is comprised of Faculty, Technical Members,
Community Members (Section IV-B-2),
and
2) Reviews, Approves, and Oversees research
projects in accordance with the responsibilities
defined in the NIH Guidelines
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Why is IBC review required?
 Federal funding - the mandate for
institutional compliance
 Public Transparency - to increase
public trust of science and research;
see: Freedom of Information Act FOIA 1967
 Public meeting
 Providing IBC meeting minutes upon request
 Based on Investigator integrity
established at Alisomar, CA in 1975
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What are IBCs required to review?
 All research involving recombinant or
synthetic nucleic acid molecules for
conformity with the NIH Guidelines
 Potential risk to environment and public
health
 Containment level and practices according to
the NIH Guidelines
 Adequacy of facilities, applicable SOPs, and
investigator and laboratory staff training
 Institutional and Investigator compliance
(e.g. Adverse events, incident reporting, etc.)
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IBC review includes the following
considerations (Section IV-B-2):
Safety Assessment
• Risk Assessment (Section II-A-3)
• Biosafety Containment (Section II-B)
• Classification of risk (Appendix B)
Assessment of research facilities
• EHS cooperative assessment
Procedures
Safety practices
Training of personnel
• Investigator, Research staff
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Risk assessment involves the
following factors:
1) Virulence
2) Pathogenicity
3) Infectious dose
4) Environmental stability
5) Route of spread
6) Communicability
7) Operations (SOPs, procedures)
8) Quantity
9) Vaccine or treatment availability
10) Gene Product effects
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Appendix B-I: Risk Group 1 (RG1)
• RG1 agents are not associated with
disease in healthy adult humans
– Examples of RG1 agents include adenoassociated virus (AAV – all serotypes); and
recombinant or synthetic AAV constructs
produced in the absence of a helper virus
and that do not contain oncogenes.
NOTE: 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
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Appendix B-I: Risk Group 2 (RG2)
• RG2 agents are associated with human
disease which is rarely serious and for
which preventive or therapeutic
interventions are often available with
disease in healthy adult humans
– Examples of RG2 agents include many
bacteria including S. aureus and B. pertussis;
fungal agents including Cryptococcos
neoformans; parasitic agents including T.
gondii and Giardii; and viruses including
adenovirus, some influenza , hepatitis and
lentiviruses.
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Why Does IBC Recommend Use of
AAV/AAV Vectors at BSL-2/ABSL-2?
• OSHA Bloodborne Pathogen Standard,
29 CFR 1910.1030 requires institutions
to minimize exposure of personnel to
bloodborne pathogens
– “Pathogenic microorganisms that are
present in human blood, human body fluids,
human tissues or other potentially
infectious material, including recombinant
or synthetic nucleic acid molecules.”
– University of Pittsburgh Bloodborne
Pathogens Exposure Control Plan
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Other Potentially Infectious Material (OPIM)
Include but are not limited to:
– All human body fluids;
– Any unfixed tissue or organ other than intact skin from a
human (living or dead);
– Human cell lines or cultures, human tissue cultures, human
organ cultures;
– Non‐human primate blood, body fluids or other tissues;
– Blood, body fluids or other tissues from experimental
animals infected with bloodborne pathogens;
– Liquid or solid culture medium or other materials containing
biological agents capable of causing disease in healthy adults
(i.e. equivalent to agents handled at Biosafety level 2 or
above, visit
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm);
– Liquid or solid culture medium or other materials containing
or potentially contaminated with recombinant or synthetic
nucleic acids or samples from animals experimentally
exposed to recombinant or synthetic nucleic acid molecules.
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Other Potentially Infectious Material (OPIM)
Include but are not limited to:
– All human body fluids;
– Any unfixed tissue or organ other than intact skin from a
human (living or dead);
– Human cell lines or cultures, human tissue cultures, human
organ cultures;
– Non‐human primate blood, body fluids or other tissues;
– Blood, body fluids or other tissues from experimental
animals infected with bloodborne pathogens;
– Liquid or solid culture medium or other materials containing
biological agents capable of causing disease in healthy adults
(i.e. equivalent to agents handled at Biosafety level 2 or
above, visit
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm);
– Liquid or solid culture medium or other materials containing
or potentially contaminated with recombinant or synthetic
nucleic acids or samples from animals experimentally
exposed to recombinant or synthetic nucleic acid molecules.
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Why Are Human Cell Lines Considered
Potentially Infectious?
•
•
It is not possible to test every cell line for
every possible pathogen
There are unknown pathogens that may be
harbored in human cell lines
– OSHA letter of interpretation to the American
Biological Safety Association
– Keene, J., Ask the Experts – Biosafety
Requirements for Human Cell Lines. Journal of
the American Biological Safety Association, 8(3),
p 132-134, 2003.
– CDC/NIH Biosafety in Microbiological and
Biomedical Laboratories, 5th edition, 2009.
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Human Cell Lines – Potential to Harbor
Adventitious Infectious Agents
Laboratory-Acquired Infections in Clinical Patients
Year
1995
Infectious Agent
Acute Hepatitis B Virus in 6
Patients1,2
Source of Infectious Agent
Viral contamination of liquid nitrogen storage
tank from compromised cryopreservation bag
Contamination of Biological Products with Adventitious Agents
Year
2013
2011
Adventitious Agent
Porcine circovirus type 13
Mouse minute virus4-6
Product/Company
RotarixTM Vaccine Glaxo Smith Kline
Genetech, Amgen, Merrimack
Pharmaceuticals
Identification of Adventitious Agents in Established Cell Cultures
Year
=2014
2013
Adventitious Agent or
Contamination Identified
Cell Line or Source of
Contamination
BK Polyoma virus7,8
Coccidioides posadasii9
SVGp12 (ATCC)
Commercial Rhesus
monkey kidney cells
1.
2.
3.
4.
5.
6.
7.
8.
9.
Lancet 1995; 346: 137-40.
Journal of Virolological Methods 1996; 60: 81-88.
Human Vaccines and Immunotherapeutics. 2013; 9(11):
PDA Journal of Pharmaceutical Science and Technology.
PDA Journal of Pharmaceutical Science and Technology.
PDA Journal of Pharmaceutical Science and Technology.
Journal of Virology. 2014; 88(13): 7556-7568.
Journal of General Virology. 1992; 73: 2871-78.
Journal of Clinical Microbiology. 2013; 51(4): 1288-90.
2398-2408.
2011; 65(6): 715-29.
2011; 65(6): 599-611.
2011; 65: 580-88.
Productive
Infection/Pot
Transmissible
Yes
Yes
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Historical Precedent for AAV
Timeline for AAV
2001
IBC reorganization under RCCO
2001-2010
All AAV handled under BSL-2/ABSL-2
June 2011
AAV downgrade guidance
Nov. 2014
AAV downgrade guidance revision
Dec. 2015
AAV downgrade guidance revision
March 2016
AAV downgrade guidance revision
AAV is BSL-1 BUT it is grown in Human
Cell Lines which are BSL-2…
In 2011 IBC identified specific criteria to
allow BSL-1 downgrades for AAV vectors 15
Vector Core Facilities
• Vector Core Facilities make make
obtaining viral vectors easy
• Production methods vary by Core
– How is vector produced?
– Is it purified?
– Does Core do quality assurance to
check for purity?
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AAV Purification
1. AAV grown in human cells
2. Supernatants collected
3. Iodoxinal Gradient
Centrifugation/Column Purification
4. Buffer Exchange
5. Titer determined by PCR or Other
Infectivity Assay
BUT HOW WELL DID PURIFICATION WORK?
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AAV-Purification by Iodoxinol Gradient
Human Gene Therapy 2010; 21: 1259-1271.
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AAV-Purification by Iodoxinol Gradient
J. Gene Med 2004; 6: S223-S228.
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AAV Purification
1. AAV grown in human cells
2. Supernatants collected
3. Iodoxinal Gradient
Centrifugation/Column Purification
4. Buffer Exchange
5. NEED TO HAVE CORE PERFORM QC SDS-PAGE to CHECK PURITY OF PREP!
6. Titer determined by PCR or Other
Infectivity Assay
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Commonly Used Vector Cores
CORE
PURIFICATION PROCEDURE
BSL
UNC
Iodoxinal Gradient + Column Purification; QC AUTO BSL-1
check via SDS-PAGE/Silver Stain per vector
per lot; provide purity and titer per lot
Salk (U Pitt)
Iodoxinal Gradient + Column Purification; QC AUTO BSL-1
check via SDS-PAGE/Silver Stain per vector
per lot; provide purity and titer per lot
U Penn
Iodoxinal Gradient + Column Purification; QC BSL-2; can
check via SDS-PAGE available upon request
request downfor $120 per vector per lot; provide purity
grade with QC
and titer per lot
Stanford
Provides unpurified AAV unless otherwise
requested; Core recommends use at BSL-2
BSL-2 unless
proof of purity
with docs
Using AAV at BSL-1
1. Consult IBC website for updates AAV
SOP / listing of current Vector Cores
– Use one that is auto downgraded
2. Consult database of AAV vectors to
see if anyone else has obtained
same vector/lot of interest and
order same
– Planned support for the development
of master AAV database
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Using AAV at BSL-1
3. Place order for vector; be sure to
request:
•
•
Purification of vector
Quality Control check of purity (i.e., SDSPAGE) even if additional cost
4. Provide IBC proof of vector and QC if
obtained from a Core that is not
identified for automatic BSL-1
5. Maintain lab documentation on each
vector/each lot for life of vector
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AAV downgrade criteria
The downgrade criteria includes a description
of purification steps, and Quality Control data
to verify purification success
No
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Where to find resources
The guidance documents are posted on the
IBC website under the Resources menu block
www.ibc.pitt.edu
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Current AAV use by BSL
The data shown below is from the IBC
database on 18 February 2016
Active protocols using AAV
80
70
60
50
40
30
20
10
0
1
2 -downgraded
2
2+
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What if I am out of Compliance?
• Contact the IBC Immediately
• Provide information
– Vector(s) and source
– Are animals involved
– IBC will evaluate on case-by-case basis
• Some options:
– Move studies to BSL-2 if compromising
safety or integrity of other studies
– See if QC is obtainable quickly to resolve
compliance issue
– Halt work as last resort
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http://grants.nih.gov/reproducibility/index.htm
http://nexus.od.nih.gov/all/2016/01/29/authentication-of-key-biological-andor-chemical-resources-in-nih-grant-applications/
More resources
For guidance, training, meeting dates, and
news: www.ibc.pitt.edu or call 412-383-1768
or by email [email protected]
NEW!
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Our Goal
Our goal is to better understand your needs
and
streamline
the
process
where
possible…without compromising safety!
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