Lecture VI

Viral vectors – continued...

27th November

Vectors

Non-viral/plasmids Viral

RNA DNA Retroviruses(includinglentiviruses)

AdenoviralAAV Herpes

„naked” DNA

Lipoplexes

Viroplexes(lipoplexes enrichedIn specific viral proteins)

complexes withother chemicals

ViralViral vectorsvectors

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

AdenoviralHSV

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

GenericGeneric strategystrategy for for engineeringengineering a a virusvirusintointo a a vectorvector. .

Kay et al., Nature Medicine 7, 33 - 40 (2001)

TransductionTransduction ofof a a targettarget cellcell

Kay et al., Nature Medicine 7, 33 - 40 (2001)

AAV AAV vectorsvectors

adenoadeno--associatedassociated viralviral vectorsvectors

AAV

adenovirus

InfectiousInfectious cyclecycle ofof AAV AAV

Cell infection – through heparan sulphate bindingco-receptors: αVβ5 and FGF-2 receptor

AdenoAdeno--associatedassociated virusesviruses –– AAV AAV

Small, non-pathogenic single stranded DNA viruses

For replication require additional genes delivered by other viruses(adenoviruses or herpes simplex viruses)

Genom AAV – 4681 nucleotides, at both ends there are145 nt-long ITR (inverted terminal repeats)

ITR – necessary in cis – initiation of replication- packaging signal- integration into genom

AAVsAAVs insigthinsigth• AAV genome is a linear single stranded

DNA flanked by inverted terminal repeatsITR(145nt);

• The genome has 2 genes:-cap (encodes viral capsid protein)-rep (encodes 4 overlapping Rep proteins)

AAV AAV genomegenome organizationorganization

SiteSite--specificspecific integrationintegration• AAV integrates usually stably into

a specific site on chromosome19q13.3 (AAVS1)

• Integration region- AAVS1 (RBS,TRS)

• Rep78 and Rep68 bind to a 109 bp DNA fragment near AAVS1 andcan mediate complex formation(DNA of chromosome 19 andAAV harpin DNA)

• Viral DNA replication withinAAVS1 are likely involved in site-specific integration;

AAV AAV serotypesserotypes

11 serotypes are known

AAV-2 serotype is the most commonly used

Different serotypes can employ various receptors to enter the cells

- AAV-2: heparan sulphate

- AAV-1 & AAV-5 – sialic acid

- AAV-5 co-receptor: PDGF-B receptor

AAV AAV vectorvector traffickingtrafficking

removal of rap and cap genestransgene insertion

AAV vectors

ITRITR

WaysWays ofof productionproduction ofof AAV AAV vectorsvectors

- dependent on helper vector

- helper-vectors independent

ConstructionConstruction ofof AAV AAV vectorsvectors –– system system withwith helperhelper adenoviraladenoviral

packagingcells

co-infectionwith adenovirus

recombinantAAV

adenovirus

Production of AAV vectors – it is safer to omit helper adenovirus

AAV AAV HelperHelper--FreeFree SystemSystem

For production of AAV vectors only three sets of AAV vectors are required: E1, E2A, E4 & VA

VectorsVectors inin AAV AAV helperhelper--freefree systemsystem

HelperHelper--freefree productionproduction ofof AAV AAV vectorsvectors (2) (2)

AAV AAV vectorsvectors featuresfeatures

- due to the lack of Rep68 and Rep78 the specific integration intochromosom 19 is lost

- unspecific integration (low efficacy, about 5-10%)

- episomal expression

- because of non-immunogenic nature the episomal expression innon-dividing cells can be long-term

AAVAAV--2 2 vectorsvectors tropismtropism to to skeletalskeletal musclesmuscles

AAV AAV vectorsvectors, , inin contrastcontrast to to adenoviraladenoviral, , cancan provideprovidelonglong--termterm expressionexpression

Champion et al., Circulation 2003

serce myszy

Adeno-AssociatedVirus VectorsTargeting

Courtesy of Andrzej Rutkowski

AAV cell entryvia heparin sulfate

proteoglicans (HSPG)direct membraneco-receptors:

avß5 integrinFGFR1

Cell entry is independent on adenovirus presence. after B. J. Carter

HSPG

DifferentDifferent transductiontransduction efficiencyefficiency ofof AAVAAV--2 2 viralviral vectorsvectors

Endothelial

Bronchial epithelial

Vascular smooth muscle

Skeletal muscle

DifferentDifferent transductiontransduction efficiencyefficiency ofof AAVAAV--2 2 viralviral vectorsvectors

GFP587-SIGYPLP

pXX6

AAVsigNicklinNicklin et al. Mol. et al. Mol. TherTher. 2001 vol. 4 (2). 2001 vol. 4 (2)

TargetingTargeting ofof AAV AAV vectorsvectors to to endothelialendothelial cellscells

HUVECHSVEC

10,000 vector particles/cell

5.9-fold HUVEC28.2-fold HSVEC

AAVsig wtAAV

Mol. Ther. 2001 vol. 4 (2)

TargetingTargeting ofof AAV AAV vectorsvectors to to endothelialendothelial cellscells

C) mosaic capsid

19/1 3/1 1/1 1/3 1/19

Different ratios of plasmids

Rep1 Cap1 Rep2 Cap2

B) bi-specific antibody

cell-surfacereceptor (for e.g.endothelial cell)

A) transcapsidation

AAV95’

transgene

3’AAV2

ITR

ITR Rep Cap

AAV2/9

D) chimeric capsid

5’

Rep Cap

3’

ITR

ITR

SIGYPLP

AAVsig

MethodsMethods enhancingenhancing thethe effectivenesseffectiveness ofof AAV AAV vectorsvectors

Jazwa et al.,, Curr Gene Therapy, 2006, in press

Targeting of AAV vectors – usage of different serotypes

FeaturesFeatures ofof AAV AAV vectorsvectorsAdvantages

1. Long term expression

2. High efficiency of transduction of many cell types

3. Non-pathogenic viruses. Low risk of cellular immune response, which isadditionaly limited by removal of viral sequences

Limitations

1. Unspecific integration2. Small capacity – max. 4 kbp3. Low efficiency of transduction of certain cell types – targeting might

berequired4. Difficulty of production in sufficient titer for in vivo work5. Risk of humoral immunity: antibodies detect capsid proteins

AAVAAV-- concatamerisationconcatamerisation

ApplicationApplication ofof AAV AAV inin clinicalclinical genegene therapytherapy

1. Nervous system diseases – Canvan disease2. Cystic fibrosis3. Haemophilia – transfer of factor IX 4. Muscular dystrophy

OtherOther vectorsvectors

HSV

HerpesHerpes simplexsimplex virusesviruses

Other delivery systems

- alpha-viruses

- baculoviruses

OtherOther viralviral vectorsvectors

1.retro-adenoviral vectors: contains LTR of retrovirus, capsid and other genome features of adenovirus

2. Polio virus, hepatatis A virus

3. Ebola virus

lentiwiral vectors containing proteins of Ebola virus capsid- infects cells of respiratory epithelium

ViralViral vectorsvectors inin clinicalclinical genegene therapytherapy

Verma & Weitzmann, Ann Rev Biochem 2005

Pre-clinical Clinical Revision Postmarketing

PhasesPhases ofof clinicalclinical trialstrials

PhasesPhases ofof clinicalclinical trialstrials

RegisteredRegistered drugsdrugs areare onlyonly a a portionportion ofof allall testedtested

ClinicalClinical trialstrials ofof genegene therapytherapy 19891989--20042004

ConstructionConstruction ofof 1st 1st generationgeneration ofof adenoviraladenoviral vectorsvectors

DecreasingDecreasing ofof recombinationrecombination riskrisk

AdenoviralAdenoviral vectorsvectors inin cardiovascularcardiovascular genegene therapytherapy

ψ

loxP loxPmolekularne nozyczki

Leczniczy gen

Packagingcells

Wirus pomocniczy

Produkujebialka kapsydow,

Sygnal pakowaniaDNA do kapsydu

zostal wyciety

Wektor gutless

NIE pakuje siedo kapsydow

Pakuje sie do gotowych kapsydow

ConstructionConstruction ofof helperhelper--dependentdependent virusesviruses

ψ

Transfer Transfer ofof helperhelper--dependentdependent gutlessgutless vectorvector harboringharboringapoEapoE genegene protectsprotects fromfrom atherosclerosisatherosclerosis

apoEapoE--//-- controlcontrol HdAdHdAd--gengen--apoEapoE

Tętnica myszy chorej Tętnica myszy zdrowej

Transfer Transfer ofof leptinleptin genegene to to obob//obob mice mice reducesreducesobesityobesity

Morsy et al. 1998. Proc Natl Acad Sci USA 95:7866-7871.

Myszy Myszy obob//obob

mysz kontrolnamysz kontrolna mysz leczonamysz leczona

Wirus opryszczki (HSVWirus opryszczki (HSV--1)1)

Zalety bardzo duży genom – 152 kbp – liniowy, dwuniciowy DNA, zawiera przynajmniej 84 geny (ciągłe) – około połowa tych genów jest zbędna dla replikacji wirusa.

Możliwość wprowadzania transgenów o długości przynajmniej 30 kb

Możliwość uzyskiwania dużej liczby kopii cząstek wirusa

Nieotksyczne, mogą przebywać w stanie latencji przez długi okres czasu w komórkach

Transdukują liczne typy komórek

Ograniczenia

Brak doświadczenia z zastosowaniem rekombinowanych herpeswirusów u pacjentów

Problemy z nacelowaniem transdukcji na określony typ komórek

Zastosowanie wektorZastosowanie wektoróów HSVw HSV--1 1

Badania eksperymentalne:

1. Nowotwory, w tym nowotwory układu nerwowego2. Choroby obwodowegi układu nerwowego 3. Chorby centralnego układu nerwowego 4. Uszkodzenie rdzenia kręgowego5. Leczenie bólu – transfer do nerwów czuciowych wydaje się być najbardziej obiecujący