Lecture VI. Viral vectors continued... 27th November

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1 Lecture VI Viral vectors continued... 27th November

2 Vectors Non-viral/plasmids Viral naked DNA Lipoplexes RNA Retroviruses (including lentiviruses) DNA Adenoviral AAV Herpes complexes with Viroplexes other chemicals (lipoplexes enriched In specific viral proteins)

3 Viral vectors Integrating Lentiviral -retroviral -AAV (limited) Non-integrating Adenoviral HSV Integration depends on: -LTR sequences and integrase (retroviruses) - ITR seqeuences and rep proteins (AAV)

4 Kay et al., Nature Medicine 7, (2001) Generic strategy for engineering a virus into a vector.

5 Transduction of a target cell Kay et al., Nature Medicine 7, (2001)

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7 AAV vectors adeno-associated associated viral vectors

8 AAV adenovirus

9 Infectious cycle of AAV

10 Cell infection through heparan sulphate binding co-receptors: α V β 5 and FGF-2 receptor

11 Adeno-associated viruses 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 are 145 nt-long ITR (inverted terminal repeats) ITR necessary in cis initiation of replication - packaging signal - integration into genom

12 AAVs insigth AAV genome is a linear single stranded DNA flanked by inverted terminal repeats ITR(145nt); The genome has 2 genes: -cap (encodes viral capsid protein) -rep (encodes 4 overlapping Rep proteins)

13 AAV genome organization

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15 Site-specific integration AAV integrates usually stably into a specific site on chromosome 19q13.3 (AAVS1) Integration region- AAVS1 (RBS,TRS) Rep78 and Rep68 bind to a 109 bp DNA fragment near AAVS1 and can mediate complex formation (DNA of chromosome 19 and AAV harpin DNA) Viral DNA replication within AAVS1 are likely involved in sitespecific integration;

16 AAV serotypes 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

17 AAV vector trafficking

18 AAV vectors removal of rap and cap genes transgene insertion ITR ITR

19 Ways of production of AAV vectors - dependent on helper vector - helper-vectors independent

20 Construction of AAV vectors system with helper adenoviral co-infection with adenovirus packaging cells adenovirus recombinant AAV

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

22 AAV Helper-Free System For production of AAV vectors only three sets of AAV vectors are required: E1, E2A, E4 & VA

23 Vectors in AAV helper-free system

24 Helper-free production of AAV vectors (2)

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26 AAV vectors features - due to the lack of Rep68 and Rep78 the specific integration into chromosom 19 is lost - unspecific integration (low efficacy, about 5-10%) - episomal expression - because of non-immunogenic nature the episomal expression in non-dividing cells can be long-term

27 AAV-2 vectors tropism to skeletal muscles

28 AAV vectors, in contrast to adenoviral, can provide long-term expression serce myszy Champion et al., Circulation 2003

29 Adeno-Associated Virus Vectors Targeting Courtesy of Andrzej Rutkowski

30 AAV cell entry via heparin sulfate proteoglicans (HSPG) direct membrane co-receptors: HSPG a v ß 5 integrin FGFR1 Cell entry is independent on adenovirus presence. after B. J. Carter

31 Different transduction efficiency of AAV-2 viral vectors

32 Different transduction efficiency of AAV-2 viral vectors Endothelial Bronchial epithelial Vascular smooth muscle Skeletal muscle

33 Targeting of AAV vectors to endothelial cells GFP 587-SIGYPLP pxx6 AAVsig Nicklin et al. Mol. Ther vol. 4 (2)

34 Targeting of AAV vectors to endothelial cells AAVsig HUVEC HSVEC 10,000 vector particles/cell wtaav 5.9-fold 28.2-fold HUVEC HSVEC Mol. Ther vol. 4 (2)

35 Methods enhancing the effectiveness of AAV vectors Jazwa et al.,, Curr Gene Therapy, 2006, in press A) transcapsidation B) bi-specific antibody ITR 5 AAV2 transgene 3 ITR AAV9 Rep Cap AAV2/9 cell-surface receptor (for e.g. endothelial cell) C) mosaic capsid D) chimeric capsid Rep1 Cap1 Rep2 Cap2 ITR 5 SIGYPLP 3 ITR Rep Cap 19/1 3/1 1/1 1/3 1/19 Different ratios of plasmids AAVsig

36 Targeting of AAV vectors usage of different serotypes

37 Features of AAV vectors 1. Long term expression Advantages 2. High efficiency of transduction of many cell types 3. Non-pathogenic viruses. Low risk of cellular immune response, which is additionaly limited by removal of viral sequences Limitations 1. Unspecific integration 2. Small capacity max. 4 kbp 3. Low efficiency of transduction of certain cell types targeting might berequired 4. Difficulty of production in sufficient titer for in vivo work 5. Risk of humoral immunity: antibodies detect capsid proteins

38 AAV- concatamerisation

39 Application of AAV in clinical gene therapy 1. Nervous system diseases Canvan disease 2. Cystic fibrosis 3. Haemophilia transfer of factor IX 4. Muscular dystrophy

40 Other vectors

41 HSV Herpes simplex viruses

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43 Other delivery systems - alpha-viruses - baculoviruses

44 Other viral vectors 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

45 Viral vectors in clinical gene therapy

46 Verma & Weitzmann, Ann Rev Biochem 2005

47 Phases of clinical trials Pre-clinical Clinical Revision Post marketing

48 Phases of clinical trials

49 Registered drugs are only a portion of all tested

50 Clinical trials of gene therapy

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54 Construction of 1st generation of adenoviral vectors

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56 Decreasing of recombination risk

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58 Adenoviral vectors in cardiovascular gene therapy

59 Construction of helper-dependent viruses ψ loxp loxp molekularne nozyczki Wirus pomocniczy ψ Packaging cells Wektor gutless Leczniczy gen Sygnal pakowania DNA do kapsydu zostal wyciety NIE pakuje sie do kapsydow Produkuje bialka kapsydow, Pakuje sie do gotowych kapsydow

60 Transfer of helper-dependent gutless vector harboring apoe gene protects from atherosclerosis apoe-/- control HdAd-gen gen-apoe Tętnica myszy chorej Tętnica myszy zdrowej

61 Transfer of leptin gene to ob/ob mice reduces obesity Myszy ob/ob mysz kontrolna Morsy et al Proc Natl Acad Sci USA 95: mysz leczona

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64 Wirus opryszczki (HSV-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

65 Zastosowanie wektorów w HSV-1 Badania eksperymentalne: 1. Nowotwory, w tym nowotwory układu nerwowego 2. Choroby obwodowegi układu nerwowego 3. Chorby centralnego układu nerwowego 4. Uszkodzenie rdzenia kręgowego 5. Leczenie bólu transfer do nerwów czuciowych wydaje się być najbardziej obiecujący