Transcript BioSafety Considerations

BioSafety Considerations
For
Viral Vectors
Biosafety Considerations of Viral
Vectors
Lifestyles of the Small & Infectious
 Terminology of Gene Expression
 Viruses to Vectors
 Vectors and More Vectors
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Lifestyles of the Small and
Infectious
Very Basic Virology
What Are Viruses?
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Small, infectious obgligate intracellular
parasites whose genome is either DNA or
RNA.
The viral genome is replicated within a host
cell and uses cellular systems to direct the
synthesis of other virion components.
These newly synthesized components are
used to assemble progeny virions which are
responsible for transmission of the viral
genome to the next host cell.
Common Viral Strategies
All viruses package their genome inside
a particle that mediates transmission of
the viral genome from host to host.
 The viral genome contains the
information for initiating and completing
an infectious cycle.
 All viruses are able to establish
themselves in a host population.
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Terminology of Gene Expression
What is…???
 cDNA
: a DNA copy of an mRNA
– Only contains the protein coding
domain (not introns)
– When transcribed the RNA requires
no processing
– Can be translated directly
 Transgene
– A gene that is put into some sort of
expression vector to be delivered into
a cell
Expression Cassette
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Piece of DNA that contains all the
elements necessary for a gene to be
expressed in a cell.
Elements of an Expression Vector
Promoters
are tailored to
type of cell
system:
bacteria,
mammalian,
insect, etc.
Termination
signals are
tailored to
prokaryotic or
eukaryotic
systems:
eukaryotic
cells require
polyA signal.
Cis & Trans Acting Elements
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Cis – present on the same piece of
DNA/RNA being acted on; cannot
function separately.
– Promoters, signaling sites
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Trans – elements that act on a different
piece of DNA/RNA than the one they
are present on.
– Viral replication proteins, RNA polymerase
Complementing Cell Lines
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Complementation: Providing trans-acting
functions to rescue a nucleic acid that is
missing, or mutant in, those functions.
By stably transfecting cells to express a gene
product necessary for viral replication, can
then grow viruses deleted for that gene.
– HEK293 cells express Ad E1; will complement E1
deleted adenoviruses.
Viruses to Vectors
Biosafety Considerations
Why Use Viruses?
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Viruses have evolved to efficiently condense,
package, & deliver nucleic acids to cells.
Relatively easy to generate & renew.
Infect a wide variety of cell types.
Production of proteins w/ authentic post
translational modifications.
Potential for regulated production.
Potential for in vivo gene delivery.
Concerns About
Recombinant Viruses
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Pathogenicity of parental virus.
– Can engineer to be repl. incompetant
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Cytopathogenicity of vector.
– eg. Spike proteins on Adenovirus
Requirements for specialized facilities.
 Scale-up considerations.
 Training requirements.
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Replication Incompetent Viruses
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To avoid potential pathogenicity of viral
vectors – disable them so they cannot
replicate in target cells.
Usually accomplished by deleting genes that
provide necessary trans-acting functions from
the vector genome.
Introduction of genes & defective vector into
cells results in synthesis of vector genomes &
packaging of the defective genomes into virus
particles.
Reconstitution of Replication
Competent Virus (RCV)
During amplification & packaging of
defective viral vector genomes they
can, at low frequency, re-acquire the
genes necessary for autonomous
growth, making the virus replication
competent again.
 Occurs by recombination.
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Strategies to Avoid
RCV Reconstitution
Split genomes: putting replication genes
on different DNA constructs
 Remove viral regulatory regions
 Produce as a transient single batch
rather than continuous culture
 Use non-host cell lines
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– reduces “rescue”
Why Do Split Genomes
Decrease RCV Risk?
Pseudotyping of Viruses
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The use of different viral surface proteins to
affect the host range of the virus and/or to
change physical properties of the viral
particles.
Some viruses are surrounded by cell-derived
membranes & foreign surface proteins can be
included in these envelopes.
– G glycoprotein of vesticular stomatitis virus
– Eliminates ability to regenerate original host range
Vectors
…and more Vectors
Typical Viruses Used
Retrovirus/Lentivirus
 Adenovirus
 Baculovirus (insect cells)
 Poxvirus (vaccinia, fowlpox)
 Herpesvirus
 Alpha virus (SFV, sindbis, VEE)
 Adeno-Associated Virus
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Common Viral Vectors
BSL 1
Baculovirus
(insect cells)
AdenoAssociated
Virus
BSL 2
BSL 2/3
Adenovirus
Retrovirus/
Lentivirus
(HIV, SIV, HTLV)
Poxvirus
(vaccinia, fowlpox)
Alphavirus
(semliki forest,
sindbis, VEE)
Herpesvirus
(Epstein-Barr,
Herpes viruses)
Flavivirus
Retroviruses
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Based on murine leukemia virus
– Risk Group 1 and 2
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Simple genomic structure: all the
retroviral genes can be removed &
supplied extraneously
– Three genes: gag, pol, env
Biosafety Concerns
Amphotropic viruses are capable of
infecting human cells, therefore
biosafety concern is on effects of the
expressed gene
 Replication competent retroviral
breakthroughs
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Lentiviruses
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Complex retrovirus, based on HIV genome
Lentivirus
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Capable of infecting non-dividing cells
– Provirus DNA must enter nucleus for
integration of DNA to occur
– Simple retroviruses rely on dissolution of
nuclear membrane during mitosis
– Lentiviruses encode nuclear localization
signals which transport complex into
nucleus
Biosafety Concerns for Lentivirus
Generation of replication competent
virus
 Infection of non-target cells
 Inappropriate expression of gene
product in non-intended cell type
 Insertional mutagenesis
 “Rescue” by other human pathogenic
viruses
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Insertional Mutagenegis
Lentivirus integrates into the host
chromosome at random.
 Possible to integrate in area such that
downstream LTR could function as a
promoter for growth regulation.
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“Rescue”
Host genome may contain endogenous
retrovirus
 Recombination event could lead to
reactivation or “rescue” of replication
competent virus
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How Is Safety Engineered Into
Lentiviral Vectors?
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Lentivirus- 3rd generation Lentivirus
System. Significantly modified for biosafety.
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Packaging vector lacks both LTRs and expresses
only gag and pol.
Rev is supplied in trans on a separate vector.
The vector expressing the packaged viral
genome has a self-inactivating LTR and
expresses no viral gene products
The envelope protein is VSV-G and is also
expressed on a separate vector
Packaged virus expresses no viral gene products
Adenoviruses
Risk Group 2
 Deletions of the E1 region render the
virus replication incompetent; can be
propagated in a complementing cell line
(293 cells)
 Airborne Transmitted; infects broad
range of cells
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Adenovirus Vector
Disadvantages
Advantages
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High titers
Infects wide ranges of
cells
Infects dividing and
quiescent cells
Virion stability
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Have had some adverse
events
Transient expression
Problems w/
subsequent
administration in gene
therapy
Based on human
pathogen
RCA breakthroughs
Baculovirus
Risk group 1 agent
 Enveloped virus w. double stranded
DNA genome
 Insect pathogen that does not
propagate in mammalian cells
 Used for many years to express
proteins; will also deliver genes and
mediate expression in mammalian cells
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Baculoviruses as Gene Delivery
Vehicles for Mammalian Cells
Virus will deliver its DNA into
mammalian cells
 Viral promoters are not activated in
mammalian cells but if expression
cassette is included with a mammalian
promoter, it will be expressed
 No overt deleterious effects on
mammalian cells have been shown
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Baculovirus Vectors
Advantages
 Ease of Use
 Broad host range
 Efficiency & lack of
toxicity
 Non-replicative in
mammalian cells
 Inactivated by human
complement
Limitations
 Relatively
uncharacterized
 Potential for
maintenance of viral
DNA
 No evidence of in vivo
delivery
 Potential expression of
viral gene products
Vaccinia Virus
Risk Group 2
 Enveloped virus w/ double stranded
DNA genome
 Extensive use as recombinant protein
expression vector
 Wide host range
 Clone large DNA fragments (> 20 Kb)
 Used as a live vaccine against smallpox
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Vaccina Virus Vector
Limitations
Advantages
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Broad host range
Easy to generate
viruses
Accepts large inserts
High expression level
Molecular virology well
understood
Lytic infections
 Readily
transmisible agent
 Vaccination
requirement
 Scale-up
considerations
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Avipox Vectors
Poxviral vectors are replication
competent
 Poxviruses that infect avian species
(fowlpox and canarypox) are replication
defective in mammalian cells
 Vectors based on these viruses might
provide a safer alternative to vaccinia
based vectors for in vivo use
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Alpha Viruses
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Risk Group 2 and 3 agents
Enveloped virus w/ single stranded RNA
genome
Arthropod-borne viruses; replicate in cells
from vertebrates & invertebrates
Consequence of human alphavirus infection
can be mild or significant disease.
Main in vivo application is to expression of
antigen to elicit an immune response
Recombinant Alphaviruses
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Vector systems have been developed
from three alpha viruses:
– Sindbis virus (SIN, risk group 2);
– Semliki Forest Virus (SFV, risk group 2/3);
– Venezuelan Equine Encephalitis (VEE, risk
group 3)
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SIN and SFV systems are commercially
available
Alphaviral Vectors
Advantages
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High expression
level
Broad host range
High titers
Ease of use
Limitations
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Cytopathic
Replication
competent virus
Based on human
pathogen
Large Scale
production
Herpes Virus
Risk Group 2
 HSV1 & HSV2: Enveloped virus w/
double stranded DNA genome
 Replicates in the nucleus
 Dual life cycle: lytic growth in epithelial
cells; latent infection in neuronal cells
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Herpesviral Vectors
Advantages
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Accepts large inserts
Broad host range
High titer
Latency in neuronal
cells persistence of
expression
Disadvantages
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Virus spread by
direct contact
Complex, extensive
engineering of
vectors
Latency not wellunderstood
Adeno-Associated Virus (AAV)
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Risk group 1 agent
Human parvovirus-not associated with any
disease
Majority of population is seropositive
Requires a helper virus (adeno or herpes
coinfection for replication)
AAV integrates into the host cell chromosome
(19)
Lack of initiation of immune response
AVV Vectors
Limitations
Advantages
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Infects multiple cell
types
No viral genes in vector
Long term gene
expression (persistence
of genome)
No immune response
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Limited insert size
Helper virus
contamination
Large Scale
production
Genome persistence
not understood
Emerging Vectors
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Simian Virus 40
Rhabdovirus
Influenza Virus
Poliovirus
Hepatis B Virus
Epstein Barr Virus
Parvovirus
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Chimeric viruses
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Adeno-retroviral
Adeno-AAV
Alpha-retroviral
Alpha-rhabdoviral
Challenges of Risk Assessment
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A= viral vector system
B= expression construct
Quite often…
A + B = A*
But sometimes…
A+B=C
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Need realistic risk assessment for
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– Protection of personnel
– Guidance for containment & work practices
Thanks To:
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Patrick Condreay, PhD
– GloxoSmithKline Discovery Research
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Flint, Enquist, Krug, Racaniello, Skalaka
– Authors: Principles of Virology
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Bristol-Myers Squibb Central NJ IBC