Viral Viral vectors vectors
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