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Viral Vectors In The Research Laboratory: Just How Safe Are They?
Dawn P. Wooley, Ph.D., SM(NRM), RBP, CBSP
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Learning Objectives
Recognize hazards associated with viral vectors in research and animal testing laboratories.
Interpret viral vector modifications pertinent to risk assessment.
Understand the difference between gene delivery vectors and viral research vectors.
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Outline
Introduction to Viral Vectors
Lentiviral Vectors (+RNA virus)
Adenovirus Vectors (DNA virus)
Novel (-)RNA virus vectors
Conclusions
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Increased Use of Viral Vectors in Research
Difficulties in DNA delivery to mammalian cells<50% with traditional transfection methodsUp to ~90% with viral vectors
Increased knowledge about viral systems
Commercialization has made viral vectors more accessible
Many new genes identified and cloned (transgenes)
Gene therapy
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What is a Viral Vector?
Viral Vector: A viral genome with deletions in some or all essential genes and possibly insertion of a transgene
Plasmid: Small (~2-20 kbp) circular DNA molecules that replicates in bacterial cells independently of the host cell chromosome
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Molecular Biology Essentials
Flow of genetic informationNucleic acid polarityUnderstanding cDNAcis and trans-acting sequences
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Genetic flow & nucleic acid polarity
mRNA (+)Noncoding DNA Strand (-)
Coding DNA Strand (+)
cDNA(-)(Copy DNA aka complementary DNA)
RT
5' 3'5'3'
5' 3'
5' 3'
5'3'
5'3'3'5'5'3'
mRNA (+)
Proteins
ds DNA in plasmid
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Viral Vector Design and Production
HelperCell
Vector+
VectorHelper Constructs
+
Vector++
Helper Constructs
1
2
3
Note: These viruses are replication-defective but still infectious.
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The Marketing of Lentiviral Vectors
ViraPower™ Lentiviral Expression Systems (Invitrogen)
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Features of Lentiviral Vectors
Naturally integrate into chromosomeLong term persistence
Infect resting as well as dividing cells
Do not harm target cells as they enter
Up to ~8 kb of foreign gene sequences can be packaged
Relatively convenient packaging systems
Can be manufactured in large quantities
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Lentivirus is a genus, not a species
Family: RetroviridaeGenus: Lentivirus
Human immunodeficiency virus 1 (human)Human immunodeficiency virus 2 (human)Simian immunodeficiency virus (monkey)Bovine immunodeficiency virus (cow)Feline immunodeficiency virus (cat)Caprine arthritis encephalitis virus (goat)Visna/maedi virus (sheep)
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Generations of HIV vectors
First Generation VectorsΨ minus helper constructs and split genome (gag/pol & env); three plasmids with pseudotyping
Second Generation VectorsDeletion of HIV accessory genes vpr, vif, vpu and nef
HIVHIV
tat
LTR LTR
polvif nef
vpuvprenvrev
gagψ
10 kbp
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Generations of HIV vectors
Third Generation VectorsFour plasmids, tat eliminated, rev supplied in trans
Fourth Generation VectorsSelf Inactivating Vectors (SIN); 3 of 9 HIV genes left
HIVHIV
tat
LTR LTR
polvif nef
vpuvprenvrev
gagψ
10 kbp
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HIV vector hazards
Integration
Transgene
High mutation rate
High transduction efficiencies
Amphotropic envelope (broad tropism)
Recombination
Seroconversion
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The marketing of Adenovirus vectors
AdEasy™ Adenoviral Vector System (Stratagene)
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Features of Adenovirus Vectors
Relatively Safe (millions of recruits vaccinated)Ad2 and Ad5 do not cause cancerRarely integrate into the chromosomeTransient expression onlyInfects many cell types and resting cellsAerosol deliveryCan accept large foreign DNA insertsStable and amenable to purification (1011)Generate strong immune response
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Generations of Adenovirus vectorsFirst Generation Vectors
Two early genes deleted (E1 or E1/E3)
Second Generation VectorsThree early genes deleted (E1, E3, E4)Commercialized as AdEasy (He et al., 1998)
Third Generation Vectors“Gutless vectors”All viral genes deleted; only essential cis-acting sequences retained
AdenoAdenoITR ITR
ΔE3 ΔE4ΔE1
36 kbp
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Adenovirus vector hazards
Recombination with wild type strainsRecombination during virus productionContamination with helper virus (gutless)TransgeneImmune responseDifficulties in detecting exposure (prevalence)Altered tropism – capsid and fiber proteins
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(-) RNA Viruses
Nonsegmented
Filoviridae (Ebola)
Rhabdoviridae (Rabies)
Paramyxoviridae (Hendra& Nipah)
Segmented
Orthomyxoviridae (Flu)
Bunyaviridae (Hanta)
Arenaviridae (Lassa)
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Minus-Strand RNA Virus Vectors
Generation of biologically contained Ebola viruses
Peter Halfmann*, Jin Hyun Kim*, Hideki Ebihara†‡, Takeshi Noda†, Gabriele Neumann*, Heinz Feldmann‡,and Yoshihiro Kawaoka*†§¶
*Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706; §Division of Virology, Department of Microbiology and Immunology, and †International Research Center for Infectious Diseases, Institute of Medical Science,University of Tokyo, Tokyo 113-0033, Japan; and ‡Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada and Department of Medical Microbiology, University of Manitoba, Winnipeg, MB, Canada R3E 3R2
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved December 12, 2007 (received for review August 25, 2007)
PNAS January 29, 2008 vol. 105 no. 4 1129–1133
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Nucleic Acid Polarity for (–)RNA Viruses
Anti-genome / mRNA (+)
(-) RNA virus genome
cDNA (+)
Viral VectorWild Type Virus
5' 3'
3' 5'
(-) RNA virus genome3' 5'
5'
Proteins
(-) RNA virus genome3' 5'
3'
(-) RNA virus genome3' 5'
Viral Polymerase
Viral Polymerase
RT
T7 Polymerase
(not encapsidated)
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Viral Vector Design and Production forthe Ebola Virus Vector
HelperCell supplies
one gene
Vector (missing one gene)
cDNA codes for(-) RNA genome
+Helper Constructs (5)
cDNAs encode essentialproteins for: replication &
transcription
VirusNaïveCell
Virus
Amplification
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(-) RNA Vector Hazards
Includes RG3 and RG4 agentsLittle known about recombination for (-) RNA virusesLittle known about the replication cycles for these virusesSuch unknowns create difficulties with regard to accurate risk assessments
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Animal Studies
Push to move animals to lower containment levelsExcretions and secretionsIssues of mixed waste (bio and chemical)Proper equipment for containmentRecombination with endogenous sequences or wild type species
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ConclusionsViral vectors can be handled safelyRealize that modern vector systems are becoming increasingly complexUnderstanding all hazards → accurate risk assessmentsKnow the purpose and potential uses of the vector with respect to its designPay attention to the transgene, especially when function not fully understoodBe cautious in prematurely lowering containment levels for novel vector types
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Questions?
“The difficulty lies, not in the new ideas, but in escaping from the old ones, which ramify, for those brought up as most of us have been, into every corner of our minds.”
John Maynard Keynes (1936)