ViralViral vectorsvectors

Vectors

Non-viral/plasmids Viral

RNA DNA Retroviruses(includinglentiviruses)

AdenoviralAAV Herpes

„naked” DNA

Lipoplexes

Viroplexes(lipoplexes enrichedIn specific viral proteins)

complexes withother chemicals

Characteristics of commonly used vectorsVector Genome Insert capacity Integration Duration Ease

of manufacture

Retrovirus ssRNA 8-10 kb yes extended easy

Lentivirus ssRNA 9 kb yes extended difficult

Adenovirus dsDNA 7-8 kb No transient very easy

Guttlesadenovirus dsDNA 35 kb No extended difficult

AAV ssDNA 4-5 kb unclear extended difficult at scale

lipid basedplasmid dsDNA unlimited No transient easy

naked DNA dsDNA unlimited No variable very easy

ViralViral vectorsvectors

IntegratingIntegrating NonNon--integratingintegratingLentiviral-retroviral-AAV (limited)

AdenoviralHSV

Integration depends on:-LTR sequences and integrase (retroviruses) - ITR seqeuences and rep proteins (AAV)

Integrated transgene

Advantage: - perpetual- may provide a stable expression and a cure

Disadvantage: - random insertion may lead to silencing of a transgene

or inactivation or dysregulated activation ofhost genes

- unknown, long-term effects of the transgene

EpisomalEpisomal transgenetransgene

Advantage- no risk of insertional mutagenesis

Disadvantage- transient expression- repeated treatments may be necessary

ViralViral vectorsvectors

Viruses have evolved to deliver nucleic acids to the cells

RM Twyman, Gene transfer to animal cells. 2005

StrategiesStrategies for for viralviral vectorsvectors constructionsconstructions

RM Twyman, Gene transfer to animal cells. 2005

Helper-independent

required genesare delivered in trans

Viral vectors used in clinical trials of gene therapy

1. Adenoviruses

2. Retrovirusesa) oncoretrovirusesb) lentiviruses

3. Adeno-associated viruses

4. Herpes simplex virus

5. Poxviruses (including vaccinia)

6. Semliki Forest Virus

7. Measles virus

Transgene insertion into viral vectors

In vitro-restriction enzymesand ligase are needed

In vivo-Homologousrecombination

RetroviralRetroviral expressionexpression systemsystem

Gag – core proteins, matric, nucleocapsidPol – reverse transcriptase and integraseEnv – envelope glycoproteins

All retrovirus genomes consist of two molecules of RNA, which are s/s, (+)sense and have 5' cap and 3' poly-(A) (equivalent to mRNA). These vary in size from ~8-11kb. Retrovirus genomes have 4 unique features:

1.They are the only viruses which are truly diploid. 2.They are the only RNA viruses whose genome is produced by cellular transcriptional machinery (without any participation by a virus-encoded polymerase). 3.They are the only viruses whose genome requires a specific cellular RNA (tRNA) for replication. 4.They are the only (+)sense RNA viruses whose genome does not serve directly as mRNA immediately after infection.

Two RNA molecules are physically linked as a dimer by hydrogen bonds (co-sediment). In addition, there is a 3rd type of nucleic acid present in all particles, a specific type of tRNA (usually trp, pro or lys) - required for replication

FeaturesFeatures ofof retrovirusesretroviruses

Retroviral vectors

1. Oncoretroviruses: a) Alpharetrovirus – Avian leucosis virus (RSV) b) Betaretrovirus – Mouse mammary tumor virus (MMTV)c) Gammaretrovirus – Murine leukemia virus (Mo-MLV)d) Deltaretrovirus – Bovine leukemia viruse) Epsilonretrovirus – Walleye dermal sarcoma virus

2. Lentivirus – HIV, HTLV, BLV

3. Spumavirus – human spumavirus(human foamy virus – HMV)

Family Retroviridae

RetroviralRetroviral infectiousinfectious cyclecycle

How to make a vector from a virus?

-replace the coding region of the virus with the therapeutic gene/reporter gene

-cist-acting sequences remain intact

- the construct is intrioduced into the packaging cell line, providing the structuralviral proteins in trans

Construction of retrovirus vector

1. Construction of retrovirus vector as a recombinant plasmid in E.coli

2. Introduction of a plasmid into a packaging cell line

3. Incorporation of vector transcripts into transmissible virus particles

4. Conversion of the transcripts into double-stranded DNA by reversetranscriptase

Important components of retroviral cloning strategy1. Separation of genes and sequences acting in-cis into different plasmid

vectors in order to avoid viral reconstruction through recombinantion

- coding sequences, required for formation of infectious particle act in-trans- in-trans sequences are delivered in separate plasmids and are present

in genome of packaging cells

2. Construction of a plasmid containing in-cis required sequences plus a therapeutic gene

Cis-acting elements required for efficientgene transduction and integration

- a promoter and a polyadenylation signal

- a packaging signal

- a primer-binding site and a polypurine tract for initiation

- sequences at the termini of the viral LTR for integration

StructureStructure ofof a a retroviralretroviral vectorvector

U3 R U5

Ψ

Structure of a plasmid used for production of retroviral vectors

therapeutic genereporter gene

StagesStages ofof retroviralretroviral constructionconstruction (1) (1)

StagesStages ofof retroviralretroviral constructionconstruction (2) (2)

Consequences of such modifications

Due to destruction of a natural, well-functioning gene composition, required for theformation of infectious virus, the resulting system of retroviral vector synthesissuffers from:

- low efficiency of packaging of vectors in comparison to wild type viruses

- formation of a large number of defective particles, which inhibit the transductionefficiency

BindingBinding ofof retroviralretroviral vectorvector to a to a cellcell surfacesurface

Pseudotyping

-one species of retrovirus is capable of incoporating the envelope protein or another species or a different virus

-the endogenous or heterologous envelope protein can be provided in trans to a replication-defective vector that lack the envelope coding sequence

- can be used to alter the tropism or increase the titer of the vectors

1. Ecotropic - infect only rodent cells (Eco-Rreceptor)

2. Xenotropic - infect most mammalianexcept rodent

3. Amphotropic - infect all mammalian (receptor Ram-1 and Glvr-1)

4. Pantropic - infect various species - VSV glicoprotein

TypesTypes ofof retroviralretroviral vectorsvectors

RetroviralRetroviral vectorsvectors cancan be be usedused for for transductiontransduction inin many many speciesspecies

Titer (miano)

Concentration of viral particles and/or virionswhich are able to transduce the cells

The titer represents only a small fractionof a total number of viral particles

LentiviralLentiviral vectorsvectorsTransfect non-dividing cells

Naturally infect cells expressing CD4 – change to a VSVG – resultsin a broad range of transfectable cell types

Lentiviral vectors are based on:HIV-1, HIV-2

SIV FIV

Self-inactivating lentiviruses: deletion of 299 bp in 3’ LTR causes aftertransduction inactivation of 5’LTR, decreasing the risk of recombination andvector mobilisation

ComparisonComparison ofof oncoretrovirusesoncoretroviruses andand lentiviruseslentiviruses

Self-inactivating vectors have deletion in 3’ LTR

Nuclear transport possible: proteins involved: integrase, one of Gag protein, Vpr

TargetingTargeting ofof lentivirallentiviral vectorsvectors to to hematopoietichematopoietic stemstem cells cells

Blood, November 2005

Retroviral vectors – properties

Capacity 4-5 kb – oncoretroviruses8-9 kb - lentiviruses

Ability to integrate yesTissue specificity yesAbility to transfect non-dividing cells no – oncoretroviruses

yes – lentiviruses

Duration of expression long-termLevel of expression moderateSafety risk of insertional

mutagenesis

GETTING INTEGRATED:may affect a retroviral vector's pre-integration

Different host factors

complex (PIC). The factors influencing the integrations are unknown.

IntegrationIntegration ofof retroviralretroviral vectorsvectors

-MMLV – integrate at transcriptional start site- HIV-1 – integrate to the transcribed region of a gene

ApplicationApplication ofof retroviralretroviral vectorsvectors inin genegene therapytherapy

1. Inherited diseases: a) metabolic disorders – eg. Hypercholesterolemiab) haemophiliac) immunodeficiency diseases

2. Transfer of a suicide gene – thymidine kinase – prevention of graft versushost reaction GvH (przeszczep przeciw gospodarzowi) in patients afterallogeneic bone marrow transplantation

3. Transfer of genes to stem cells

Adenoviral vectors

AdenovirusesMore than 50 serotypes – type 2 & 5 are used

Genom: 36 kbp, more than 50 proteinsE1 region– contains genes regulating the expression of genes necessary for viral replication

E2 i E4 regions – together with E1 are required for viral replication

E3 region– is not required for replication, modulates response of cell to infections

AdenovirusesMore than 50 serotypes – type 2 & 5 are used

Genom: 36 kbp, more than 50 proteinsE1 region– contains genes regulating the expression of genes necessary for viral replication

E2 i E4 regions – together with E1 are required for viral replication

E3 region– is not required for replication, modulates response of cell to infections

AdenoviralAdenoviral serotypesserotypes andand disordersdisorders causedcaused by by themthem

BindingBinding andand internalizationinternalization ofof adenovirusadenovirus

StagesStages ofof adenoviraladenoviral infectioninfection

StagesStages ofof adenoviraladenoviral infectioninfection

Adenoviruses and adenoviral vectors

•• ~40 ~40 serotypesserotypes ofof adenovirusesadenoviruses (for (for genegene therapytherapy typetype 2 2 andand 5 5 werewere usedused), ), causingcausing usuallyusually mildmild illnessillness inin humanshumans

•• GenomeGenome consistsconsists a 36 a 36 kbkb doubledouble--strandedstranded linearlinear DNA DNA withwith ITR ITR sequencessequences atat eacheachendend, , withwith::

EarlyEarly genesgenes ((responsibleresponsible for for viralviral genegene transcriptiontranscription, DNA , DNA replicationreplication, , hosthost immuneimmunesuppressionsuppression andand hosthost cellcell apoptosisapoptosis

LateLate genesgenes ((codingcoding proteinsproteins requiredrequired for for virusvirus assemblyassembly))

•• E1 E1 earlyearly genegene isis essentialessential for for thethe subsequentsubsequent adenoviraladenoviral genegene expressionexpression

leczniczy gen

region E1Adenoviral DNA

DNA of 1st generation vector

ConstructionConstruction ofofadenoviraladenoviralvectorsvectors ofof 1st 1st generationgeneration by by homologoushomologousrecombinationrecombination

Homologic recombination inHEK 293 cells

PackagingCell

HEK 293

Shuttlevector

transgene

DecreasingDecreasing ofof recombinationrecombination riskrisk

ProductionProduction ofof adenoviraladenoviral vectorsvectors withoutwithout homologoushomologous recombinationrecombination (1) (1)

E1 genes are deleted from adenoviral genomeHEK 293 cells provide in trans the required E1 genes

ProductionProduction ofof adenoviraladenoviral vectorsvectors withoutwithout homologoushomologous recombinationrecombination (2) (2)

ProductionProduction ofof adenoviraladenoviral vectorsvectors withoutwithout homologoushomologous recombinationrecombination (3) (3)

TypesTypes ofof adenoviraladenoviral vectorsvectors

Adenoviral vectors

Benihoud K. et al. 1999. Current Opinion in Biotechnology 10: 440.

ΔE1 E1 (and E3) E1 complementing cells 7.5 kb Viral protein neosynthesis, viral replicationdespite lack of E1

ΔE1E4 E1,E4 (and E3) E1 and E4complementing cells

10 kb Reduced viral proteinneosynthesis,

ΔE1E2 E1,E2A or E2B(and E3)

E1 and E2complementing cells

9 kb Block of viral replicationsever inhibition of viralprotein synthesis

Vector Deletions Production Capacity Features

E1 E3

E2 E4

ApplicationApplication ofof adenoviraladenoviral vectorsvectors inin genegene therapytherapy

1. Gene therapy of inborn errors in metabolism – lack of OCT 2. Gene therapy of monogenic diseases – cystic fibrosis3. Gene therapy of cardiovascular diseases – transfer of angiogenic genes4. Gene therapy of cancer – it is possible that toxicity and immongenicity

will enhance the therapeutic effectiveness

TypesTypes ofof adenoviraladenoviral vectorsvectors

vectorvectorcapacitycapacity

1st1stgenerationgeneration

Short expression of transgene after adenoviral gene transfer Serotype change – does not help much

Mechanisms of adenoviral-induced inflammation

Adenoviral vectors of the first generation GreatGreat::•• VeryVery high high transductiontransduction efficiencyefficiency•• BroadBroad hosthost andand cellcell typetype rangesranges•• CanCan be be preparedprepared inin high high titerstiters•• CanCan transducetransduce mitoticmitotic andand postpost--mitoticmitotic cellscells•• Do not Do not integrateintegrate withwith genomegenome•• CanCan harborharbor ~ 7 ~ 7 kbkb ofof transgenetransgene

But:But:•• StrongStrong immuneimmune responseresponse to to viralviral proteinsproteins eliminateeliminate virallyvirally transducedtransduced cells cells withinwithin 30 30

daysdays•• NeutralizingNeutralizing antibodyantibody responseresponse preventsprevents readministrationreadministration ofof adenovirusadenovirus vectorvector ofof thethe

same same serotypeserotype

ThusThus:: AdenoviralAdenoviral vectorsvectors provideprovide thethe high but high but transienttransient (<4 (<4 weeksweeks) )

transgenetransgene expressionexpression

Sekwencja ITRNecessary for production of a transgene ITRITRTherapeuticTherapeutic genegene

Ψ sequence

Stuffer (intron)Or the whole transgene

HelperHelper--dependentdependent adenoviraladenoviral vectorsvectors

HelperHelper--dependentdependent adenoviraladenoviral vectorsvectors

Adenoviral vectorsAdenoviral vectors

No viral protein neosynthesis, No viral replication, Helpervirus contaminants (<0.1%)

ΔE1 E1 (and E3) E1 complementing cells 7.5 kb Viral protein neosynthesis, viral replicationdespite lack of E1

ΔE1E4 E1,E4 (and E3) E1 and E4complementing cells

10 kb Reduced viral proteinneosynthesis,

ΔE1E2 E1,E2A or E2B(and E3)

E1 and E2complementing cells

9 kb Block of viral replicationsever inhibition of viralprotein synthesis

Gutless All viral genes E1 complementing cells,helper virus

36 kb

Benihoud K. et al. 1999. Current Opinion in Biotechnology 10: 440.

Vector Deletions Production Capacity Features

E1 E3

E2 E4

•• Very high transduction efficiencyVery high transduction efficiency

•• Broad host species and cell type rangeBroad host species and cell type range

•• Can Can transducetransduce mitotic and postmitotic and post--mitotic cellsmitotic cells

•• Can harbor ~ 35 kb (!) of Can harbor ~ 35 kb (!) of transgenetransgene

•• Do not integrate with genomeDo not integrate with genome

•• Do not produce any viral proteinsDo not produce any viral proteins

•• Show significantly reduced Show significantly reduced immunogenicityimmunogenicity in vivoin vivo

Drawback:Drawback:•• Difficult for producing in high titersDifficult for producing in high titers

Helper-dependent adenoviralvectors