BioSafety ConsiderationsBioSafety Considerations

ForFor

Viral VectorsViral Vectors

Biosafety Considerations of Biosafety Considerations of Viral VectorsViral Vectors

Lifestyles of the Small & InfectiousLifestyles of the Small & Infectious Terminology of Gene ExpressionTerminology of Gene Expression Viruses to VectorsViruses to Vectors Vectors and More VectorsVectors and More Vectors

Lifestyles of the Small and Lifestyles of the Small and InfectiousInfectious

Very Basic VirologyVery Basic Virology

What Are Viruses?What Are Viruses? Small, infectious obgligate intracellular Small, infectious obgligate intracellular

parasites whose genome is either DNA or parasites whose genome is either DNA or RNA.RNA.

The viral genome is replicated within a The viral genome is replicated within a host cell and uses cellular systems to host cell and uses cellular systems to direct the synthesis of other virion direct the synthesis of other virion components.components.

These newly synthesized components are These newly synthesized components are used to assemble progeny virions which used to assemble progeny virions which are responsible for transmission of the are responsible for transmission of the viral genome to the next host cell.viral genome to the next host cell.

Common Viral StrategiesCommon Viral Strategies All viruses package their genome All viruses package their genome

inside a particle that mediates inside a particle that mediates transmission of the viral genome transmission of the viral genome from host to host.from host to host.

The viral genome contains the The viral genome contains the information for initiating and information for initiating and completing an infectious cycle.completing an infectious cycle.

All viruses are able to establish All viruses are able to establish themselves in a host population.themselves in a host population.

Terminology of Gene Terminology of Gene ExpressionExpression

What is…???What is…???

cDNA cDNA : a DNA copy of an mRNA: a DNA copy of an mRNA– Only contains the protein coding Only contains the protein coding

domain (not introns)domain (not introns)– When transcribed the RNA When transcribed the RNA

requires no processing requires no processing – Can be translated directlyCan be translated directly

TransgeneTransgene– A gene that is put into some sort A gene that is put into some sort

of expression vector to be of expression vector to be delivered into a celldelivered into a cell

Expression CassetteExpression Cassette

Piece of DNA that contains all the Piece of DNA that contains all the elements necessary for a gene to elements necessary for a gene to be expressed in a cell.be expressed in a cell.

Elements of an Expression Elements of an Expression VectorVector

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.

CisCis && TransTrans Acting Acting ElementsElements

CisCis – present on the same piece of – present on the same piece of DNA/RNA being acted on; cannot DNA/RNA being acted on; cannot function separately.function separately.– Promoters, signaling sitesPromoters, signaling sites

TransTrans – elements that act on a – elements that act on a different piece of DNA/RNA than different piece of DNA/RNA than the one they are present on.the one they are present on.– Viral replication proteins, RNA Viral replication proteins, RNA

polymerasepolymerase

Complementing Cell LinesComplementing Cell Lines Complementation: Providing trans-acting Complementation: Providing trans-acting

functions to rescue a nucleic acid that is functions to rescue a nucleic acid that is missing, or mutant in, those functions.missing, or mutant in, those functions.

By stably transfecting cells to express a By stably transfecting cells to express a gene product necessary for viral gene product necessary for viral replication, can then grow viruses deleted replication, can then grow viruses deleted for that gene.for that gene.– HEK293 cells express Ad E1; will complement HEK293 cells express Ad E1; will complement

E1 deleted adenoviruses.E1 deleted adenoviruses.

Viruses to VectorsViruses to Vectors

Biosafety ConsiderationsBiosafety Considerations

Why Use Viruses?Why Use Viruses?

Viruses have evolved to efficiently Viruses have evolved to efficiently condense, package, & deliver nucleic condense, package, & deliver nucleic acids to cells.acids to cells.

Relatively easy to generate & renew.Relatively easy to generate & renew. Infect a wide variety of cell types.Infect a wide variety of cell types. Production of proteins w/ authentic post Production of proteins w/ authentic post

translational modifications.translational modifications. Potential for regulated production.Potential for regulated production. Potential for Potential for in vivoin vivo gene delivery. gene delivery.

Concerns About Concerns About Recombinant VirusesRecombinant Viruses

Pathogenicity of parental virus.Pathogenicity of parental virus.– Can engineer to be repl. incompetantCan engineer to be repl. incompetant

Cytopathogenicity of vector.Cytopathogenicity of vector.– eg. Spike proteins on Adenoviruseg. Spike proteins on Adenovirus

Requirements for specialized Requirements for specialized facilities.facilities.

Scale-up considerations.Scale-up considerations. Training requirements.Training requirements.

Replication Incompetent Replication Incompetent VirusesViruses

To avoid potential pathogenicity of viral To avoid potential pathogenicity of viral vectors – disable them so they cannot vectors – disable them so they cannot replicate in target cells.replicate in target cells.

Usually accomplished by deleting genes Usually accomplished by deleting genes that provide necessary that provide necessary transtrans-acting -acting functions from the vector genome.functions from the vector genome.

Introduction of genes & defective vector Introduction of genes & defective vector into cells results in synthesis of vector into cells results in synthesis of vector genomes & packaging of the defective genomes & packaging of the defective genomes into virus particles. genomes into virus particles.

Reconstitution of Replication Reconstitution of Replication Competent Virus (RCV)Competent Virus (RCV)

During amplification & packaging During amplification & packaging of defective viral vector genomes of defective viral vector genomes they can, at low frequency, re-they can, at low frequency, re-acquire the genes necessary for acquire the genes necessary for autonomous growth, making the autonomous growth, making the virus replication competent again. virus replication competent again.

Occurs by recombination.Occurs by recombination.

Strategies to Avoid Strategies to Avoid RCV ReconstitutionRCV Reconstitution

Split genomes: putting replication Split genomes: putting replication genes on different DNA constructsgenes on different DNA constructs

Remove viral regulatory regionsRemove viral regulatory regions Produce as a transient single batch Produce as a transient single batch

rather than continuous culturerather than continuous culture Use non-host cell linesUse non-host cell lines

– reduces “rescue”reduces “rescue”

Why Do Split Genomes Why Do Split Genomes Decrease RCV Risk?Decrease RCV Risk?

Pseudotyping of VirusesPseudotyping of Viruses The use of different viral surface proteins to The use of different viral surface proteins to

affect the host range of the virus and/or to affect the host range of the virus and/or to change physical properties of the viral change physical properties of the viral particles.particles.

Some viruses are surrounded by cell-derived Some viruses are surrounded by cell-derived membranes & foreign surface proteins can membranes & foreign surface proteins can be included in these envelopes.be included in these envelopes.– G glycoprotein of vesticular stomatitis virusG glycoprotein of vesticular stomatitis virus– Eliminates ability to regenerate original host Eliminates ability to regenerate original host

rangerange

VectorsVectors

……and more Vectorsand more Vectors

Typical Viruses UsedTypical Viruses Used

Retrovirus/LentivirusRetrovirus/Lentivirus AdenovirusAdenovirus Baculovirus (insect cells)Baculovirus (insect cells) Poxvirus (vaccinia, fowlpox)Poxvirus (vaccinia, fowlpox) HerpesvirusHerpesvirus Alpha virus (SFV, sindbis, VEE)Alpha virus (SFV, sindbis, VEE) Adeno-Associated VirusAdeno-Associated Virus

Common Viral Vectors Common Viral Vectors

BSL 1 BSL 2 BSL 2/3

Baculovirus(insect cells)

AdenovirusRetrovirus/Lentivirus

(HIV, SIV, HTLV)

Adeno-AssociatedVirus

Poxvirus(vaccinia, fowlpox)

Alphavirus(semliki forest,sindbis, VEE)

Herpesvirus(Epstein-Barr,

Herpes viruses)Flavivirus

BSL 1 BSL 2 BSL 2/3

Baculovirus(insect cells)

AdenovirusRetrovirus/Lentivirus

(HIV, SIV, HTLV)

Adeno-AssociatedVirus

Poxvirus(vaccinia, fowlpox)

Alphavirus(semliki forest,sindbis, VEE)

Herpesvirus(Epstein-Barr,

Herpes viruses)Flavivirus

RetrovirusesRetroviruses

Based on murine leukemia virus Based on murine leukemia virus – Risk Group 1 and 2Risk Group 1 and 2

Simple genomic structure: all the Simple genomic structure: all the retroviral genes can be removed & retroviral genes can be removed & supplied extraneouslysupplied extraneously– Three genes: Three genes: gag, pol, envgag, pol, env

Biosafety ConcernsBiosafety Concerns

Amphotropic viruses are capable of Amphotropic viruses are capable of infecting human cells, therefore infecting human cells, therefore biosafety concern is on effects of biosafety concern is on effects of the expressed genethe expressed gene

Replication competent retroviral Replication competent retroviral breakthroughsbreakthroughs

LentivirusesLentiviruses Complex retrovirus, based on HIV Complex retrovirus, based on HIV

genomegenome

LentivirusLentivirus

Capable of infecting non-dividing Capable of infecting non-dividing cellscells– Provirus DNA must enter nucleus for Provirus DNA must enter nucleus for

integration of DNA to occurintegration of DNA to occur– Simple retroviruses rely on dissolution Simple retroviruses rely on dissolution

of nuclear membrane during mitosisof nuclear membrane during mitosis– Lentiviruses encode nuclear Lentiviruses encode nuclear

localization signals which transport localization signals which transport complex into nucleuscomplex into nucleus

Biosafety Concerns for Biosafety Concerns for LentivirusLentivirus

Generation of replication Generation of replication competent viruscompetent virus

Infection of non-target cellsInfection of non-target cells Inappropriate expression of gene Inappropriate expression of gene

product in non-intended cell typeproduct in non-intended cell type Insertional mutagenesisInsertional mutagenesis ““Rescue” by other human Rescue” by other human

pathogenic virusespathogenic viruses

Insertional MutagenegisInsertional Mutagenegis

Lentivirus integrates into the host Lentivirus integrates into the host chromosome at random.chromosome at random.

Possible to integrate in area such Possible to integrate in area such that downstream LTR could that downstream LTR could function as a promoter for growth function as a promoter for growth regulation.regulation.

““Rescue” Rescue”

Host genome may contain Host genome may contain endogenous retrovirusendogenous retrovirus

Recombination event could lead to Recombination event could lead to reactivation or “rescue” of reactivation or “rescue” of replication competent virusreplication competent virus

How Is Safety Engineered How Is Safety Engineered Into Lentiviral Vectors?Into Lentiviral Vectors?

Lentivirus- 3rd generation Lentivirus Lentivirus- 3rd generation Lentivirus System. Significantly modified for System. Significantly modified for biosafety.biosafety.

– Packaging vector lacks both LTRs and Packaging vector lacks both LTRs and expresses only gag and pol.expresses only gag and pol.

– Rev is supplied in Rev is supplied in transtrans on a separate on a separate vector.vector.

– The vector expressing the packaged viral The vector expressing the packaged viral genome has a self-inactivating LTR and genome has a self-inactivating LTR and expresses no viral gene productsexpresses no viral gene products

– The envelope protein is VSV-G and is also The envelope protein is VSV-G and is also expressed on a separate vectorexpressed on a separate vector

– Packaged virus expresses no viral gene Packaged virus expresses no viral gene productsproducts

AdenovirusesAdenoviruses

Risk Group 2Risk Group 2 Deletions of the E1 region render Deletions of the E1 region render

the virus replication incompetent; the virus replication incompetent; can be propagated in a can be propagated in a complementing cell line (293 cells)complementing cell line (293 cells)

Airborne Transmitted; infects Airborne Transmitted; infects broad range of cellsbroad range of cells

Adenovirus VectorAdenovirus VectorAdvantagesAdvantages

High titersHigh titers Infects wide ranges Infects wide ranges

of cellsof cells Infects dividing and Infects dividing and

quiescent cellsquiescent cells Virion stabilityVirion stability

DisadvantagesDisadvantages

Have had some Have had some adverse eventsadverse events

Transient expressionTransient expression Problems w/ Problems w/

subsequent subsequent administration in administration in gene therapygene therapy

Based on human Based on human pathogenpathogen

RCA breakthroughsRCA breakthroughs

BaculovirusBaculovirus Risk group 1 agentRisk group 1 agent Enveloped virus w. double stranded Enveloped virus w. double stranded

DNA genomeDNA genome Insect pathogen that does not Insect pathogen that does not

propagate in mammalian cellspropagate in mammalian cells Used for many years to express Used for many years to express

proteins; will also deliver genes and proteins; will also deliver genes and mediate expression in mammalian mediate expression in mammalian cellscells

Baculoviruses as Gene Delivery Baculoviruses as Gene Delivery Vehicles for Mammalian CellsVehicles for Mammalian Cells

Virus will deliver its DNA into Virus will deliver its DNA into mammalian cellsmammalian cells

Viral promoters are not activated in Viral promoters are not activated in mammalian cells but if expression mammalian cells but if expression cassette is included with a mammalian cassette is included with a mammalian promoter, it will be expressedpromoter, it will be expressed

No overt deleterious effects on No overt deleterious effects on mammalian cells have been shownmammalian cells have been shown

Baculovirus VectorsBaculovirus Vectors

AdvantagesAdvantages Ease of UseEase of Use Broad host rangeBroad host range Efficiency & lack of Efficiency & lack of

toxicitytoxicity Non-replicative in Non-replicative in

mammalian cellsmammalian cells Inactivated by Inactivated by

human complementhuman complement

LimitationsLimitations Relatively Relatively

uncharacterizeduncharacterized Potential for Potential for

maintenance of viral maintenance of viral DNADNA

No evidence of No evidence of in vivo in vivo deliverydelivery

Potential expression Potential expression of viral gene productsof viral gene products

Vaccinia VirusVaccinia Virus Risk Group 2Risk Group 2 Enveloped virus w/ double stranded Enveloped virus w/ double stranded

DNA genomeDNA genome Extensive use as recombinant protein Extensive use as recombinant protein

expression vectorexpression vector Wide host rangeWide host range Clone large DNA fragments (> 20 Kb)Clone large DNA fragments (> 20 Kb) Used as a live vaccine against Used as a live vaccine against

smallpoxsmallpox

Vaccina Virus VectorVaccina Virus Vector

AdvantagesAdvantages

Broad host rangeBroad host range Easy to generate Easy to generate

virusesviruses Accepts large insertsAccepts large inserts High expression levelHigh expression level Molecular virology Molecular virology

well understoodwell understood

LimitationsLimitations

Lytic infectionsLytic infections Readily Readily

transmisible agenttransmisible agent Vaccination Vaccination

requirementrequirement Scale-up Scale-up

considerationsconsiderations

Avipox VectorsAvipox Vectors Poxviral vectors are replication Poxviral vectors are replication

competentcompetent Poxviruses that infect avian species Poxviruses that infect avian species

(fowlpox and canarypox) are (fowlpox and canarypox) are replication defective in mammalian replication defective in mammalian cellscells

Vectors based on these viruses might Vectors based on these viruses might provide a safer alternative to vaccinia provide a safer alternative to vaccinia based vectors for based vectors for in vivoin vivo use use

Alpha VirusesAlpha Viruses Risk Group 2 and 3 agentsRisk Group 2 and 3 agents Enveloped virus w/ single stranded RNA Enveloped virus w/ single stranded RNA

genomegenome Arthropod-borne viruses; replicate in Arthropod-borne viruses; replicate in

cells from vertebrates & invertebratescells from vertebrates & invertebrates Consequence of human alphavirus Consequence of human alphavirus

infection can be mild or significant infection can be mild or significant disease.disease.

Main Main in vivoin vivo application is to expression application is to expression of antigen to elicit an immune responseof antigen to elicit an immune response

Recombinant AlphavirusesRecombinant Alphaviruses Vector systems have been developed Vector systems have been developed

from three alpha viruses: from three alpha viruses: – Sindbis virus (SIN, risk group 2); Sindbis virus (SIN, risk group 2); – Semliki Forest Virus (SFV, risk group 2/3); Semliki Forest Virus (SFV, risk group 2/3); – Venezuelan Equine Encephalitis (VEE, Venezuelan Equine Encephalitis (VEE,

risk group 3)risk group 3) SIN and SFV systems are SIN and SFV systems are

commercially availablecommercially available

Alphaviral VectorsAlphaviral Vectors

AdvantagesAdvantages

High expression High expression levellevel

Broad host rangeBroad host range High titersHigh titers Ease of useEase of use

LimitationsLimitations

CytopathicCytopathic Replication Replication

competent viruscompetent virus Based on human Based on human

pathogenpathogen Large Scale Large Scale

productionproduction

Herpes Virus Herpes Virus

Risk Group 2Risk Group 2 HSV1 & HSV2: Enveloped virus w/ HSV1 & HSV2: Enveloped virus w/

double stranded DNA genomedouble stranded DNA genome Replicates in the nucleusReplicates in the nucleus Dual life cycle: lytic growth in Dual life cycle: lytic growth in

epithelial cells; latent infection in epithelial cells; latent infection in neuronal cellsneuronal cells

Herpesviral VectorsHerpesviral Vectors

AdvantagesAdvantages

Accepts large Accepts large insertsinserts

Broad host rangeBroad host range High titerHigh titer Latency in neuronal Latency in neuronal

cells persistence of cells persistence of expressionexpression

DisadvantagesDisadvantages

Virus spread by Virus spread by direct contactdirect contact

Complex, extensive Complex, extensive engineering of engineering of vectorsvectors

Latency not well-Latency not well-understoodunderstood

Adeno-Associated Virus Adeno-Associated Virus (AAV)(AAV)

Risk group 1 agentRisk group 1 agent Human parvovirus-not associated with Human parvovirus-not associated with

any diseaseany disease Majority of population is seropositiveMajority of population is seropositive Requires a helper virus (adeno or Requires a helper virus (adeno or

herpes coinfection for replication)herpes coinfection for replication) AAV integrates into the host cell AAV integrates into the host cell

chromosome (19)chromosome (19) Lack of initiation of immune responseLack of initiation of immune response

AVV VectorsAVV VectorsAdvantagesAdvantages

Infects multiple cell Infects multiple cell typestypes

No viral genes in No viral genes in vectorvector

Long term gene Long term gene expression expression (persistence of (persistence of genome)genome)

No immune responseNo immune response

LimitationsLimitations

Limited insert sizeLimited insert size Helper virus Helper virus

contaminationcontamination Large Scale Large Scale

productionproduction Genome persistence Genome persistence

not understoodnot understood

Emerging VectorsEmerging Vectors Simian Virus 40Simian Virus 40 RhabdovirusRhabdovirus Influenza VirusInfluenza Virus PoliovirusPoliovirus Hepatis B VirusHepatis B Virus Epstein Barr VirusEpstein Barr Virus ParvovirusParvovirus

Chimeric virusesChimeric viruses– Adeno-retroviralAdeno-retroviral– Adeno-AAVAdeno-AAV– Alpha-retroviralAlpha-retroviral– Alpha-rhabdoviralAlpha-rhabdoviral

Challenges of Risk Challenges of Risk AssessmentAssessment

A= viral vector systemA= viral vector system B= expression constructB= expression construct Quite often…Quite often… A + B = A*A + B = A* But sometimes…But sometimes… A + B = CA + B = C

Need realistic risk assessment for Need realistic risk assessment for – Protection of personnelProtection of personnel– Guidance for containment & work practicesGuidance for containment & work practices

Thanks To:Thanks To:

Patrick Condreay, PhDPatrick Condreay, PhD– GloxoSmithKline Discovery ResearchGloxoSmithKline Discovery Research

Flint, Enquist, Krug, Racaniello, Flint, Enquist, Krug, Racaniello, SkalakaSkalaka– Authors: Authors: Principles of VirologyPrinciples of Virology

Bristol-Myers Squibb Central NJ IBCBristol-Myers Squibb Central NJ IBC