1. ADENOVIRUS AS A VECTOR IN GENE THERAPY

2. Methods of gene delivery

3. Methods of gene delivery
Viral Vectors:
Adenovirus
Retrovirus
Lentivirus
Adeno-associated virus (AAV)
Herpes simplex virus (HSV)
Non-viral vector based
Naked DNA (plasmid DNA): injection or genegun
Liposomes (cationic lipids): mix with genes
Ex-vivo
In vivo

4. Why use viral vectors Virus at transferring viral DNA into host cells
Specific target cells: depending on the viral attachment proteins (capsid or glycoproteins)
Gene replacement: non-essential genes of virus are deleted and exogenous genes are inserted
Very efficient

5. The Ideal Vector for Gene Transfer
High concentration of virus allowing many cells to be infected or transduced
Convenience and reproducibility of production
Ability to transduce dividing and non-dividing cells
Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episome
A transcriptional unit that can respond to manipulation of its regulatory elements
Ability to target the desired type of cell
No components that elicit an immune response

6. Adenovirus Structure Size: 70-90nm Non-enveloped icosahedral virus
Capsid comprised of 3 surface coat proteins
Fibers
Pentons
Hexons

7. Electron Micrograph of Purified Adenoviruses Particles

8. Replication Cycle Absorption and Penetration
Bind to cell surface receptor
Enters cell by endocytosis
Transcription
Early transcription- Codes for non-structural, regulatory proteins
Late transcription- Codes for replication substrates and machinery
Assembly
Exit- Cell lysis

9. Adenovirus use in gene therapy

10. Gene structure and organization
-2 origins of replication -ITR
-Transcription Units
5 “early” (E1A, E1B, E2, E3, E4)
2 “delayed early” (IVa2 and IX)
1 major late -> (L1-L5)

11. Transcription Units

12. Transcription Units E1, E2, E4
Encode essential regulatory proteins involved in:
Transactivation of viral and cellular promoters
DNA replication
Cell cycle
Apoptosis
Essential for replication in cell culture

13. Transcription Units E3 Escaping host immune defenses
Preventing T cell cytotoxicity
Tumor necrosis factor (TNF) action
Dispensable for replication in cell culture

14. Transcription Units Late genes
Encodes proteins required for packaging the viral genome
Essential for replication in cell culture

15. Adenovirus Attachment

16. Adenovirus Internalization

17. Why adenoviruses are good vectors for gene therapy
Gene therapy works by manipulating viruses to contain “good genes” in which they can transport to the cells to code for needed protein/hormone/enzyme/etc
They do not incorporate their genes into the host genome
The Adenovirus is ubiquitous- it has been isolated from a large number of different species with over 100 known serotypes

18. Why adenoviruses are good vectors for gene therapy
Can rapidly infect a large range of human cells
Low pathogenicity in humans
Can hold large segments of DNA
Genome does not undergo rearrangement at high rates
DNA is easy to manipulate with current recombinant DNA techniques

19. Adenovirus for Gene Therapy
Evolution of Adenovectors
1st generation: E1- and E3 +/-
2nd generation: E1-, E2- or E4-, E3 +/-
Helper dependent: deletion of all viral genes

20. 1st and 2nd generation

21. Construction of a Recombinant Adenovirus
Deletion of E1 and E3 regions
-Renders the virus replication defective: E1 being essential, it is complemented in a specially designed packaging cell line (293)
-Makes room for gene of interest
Gene of interest is cloned into a transfer vector
Gene of interest is transferred into the viral genome by homologous recombination

22. Construction of a Recombinant Adenovirus

24. 2nd Vector generations?
O’Neal, W.K. et al. Toxicological comparison of E2a-deleted and first-generation adenoviral vectors expressing a1-antitrypsin after systemic delivery. Human Gene Therapy, July 1998

25. Adenovirus for Gene Therapy
Adenoviruses can be converted into efficient gene transfer vehicles
Adenoviral vectors are not inherently dangerous
Not all adenoviral vectors have equivalent toxicity profiles
The dose of vector delivered is related to the toxicity observed
Standardization of dose specification is necessary

26. Advantages of using a recombinant Adenovirus
1. Broad host range
Can infect a broad range of mammalian cells
Allows for the expression of recombinant proteins in most mammalian cell lines and tissues
Have been used extensively to express human as well as non-human proteins

27. Advantages of using a recombinant Adenovirus
2. Infection and expression of genes in replicative and non-replicative cells
Retroviruses can only infect replicative cells
Transfection cannot be done in non-replicative cells
Best system to study gene expression in primary non-replicative cells
Allows for a direct comparison of results obtained with transformed cell lines and primary cells

28. Advantages of using a recombinant Adenovirus
3. Replicates efficiently to high titers
Production of 1012 to 1013 VP/mL

29. Advantages of using a recombinant Adenovirus
4. Helper independent Ad can accommodate up to 7.5 kb of foreign DNA
Ad can normally encapsidate a viral DNA molecule slightly bigger than the normal DNA (105%)
To provide additional cloning space, the E1 and E3 early regions of Ad have been deleted

30. Advantages of using a recombinant Adenovirus
5. No insertional mutagenesis; remains epichromosomal
Retroviruses integrate randomly into the host chromosome and can inactivate genes or activate oncogenes
Ad remains epichromosomal and therefore does not interfere with other host genes.

31. Advantages of using a recombinant Adenovirus
6. Simultaneous expression of multiple genes
Insertion of two genes in a double expression cassette
Co-infection using different recombinant viruses each expressing a different protein
Modulation of the expression by changing the MOI

32. Disvantages of using a recombinant Adenovirus
High toxicity
Low transgene expression
Replicant Competent Adenovirus (RCA)

33. Helper-dependent adenoviral vectors: construction

34. Helper-dependent adenoviral vectors: production

35. Helper-dependent adenoviral vectors:
advantages
High-efficiency in vivo transduction
High- level and long-term transgene expression
Large cloning capacity ~ 37 kb
Low toxicity profile

36. Summary of Adv capacity
Designation/ Name of the Vectors
Capacity to carry transgene
Adenovirus (full genome intact)
1.8kb
First generation Adenovirus vectors
(E1-E3 deleted Adenovirus)
4-5 kb
Second generation Adenovirus vectors
(E1-E3 and E2-E4 deleted Adenovirus)
6-7 kb
Gutless Adenovirus (All genes deleted except ITR and packaging signals)
30-35 kb

37. Helper-dependent adenoviral vectors:
amplification
Viral prep
CsCl
Triple flasks
(293 cells )

38. Helper-dependent adenoviral vectors: production
Helper virus
Helper virus AdLC8cluc
Helper virus AdNG163R-2; HDΔ28E4LacZ
Donna Palmer and Philip Ng, Molecular Therapy, November 2003

39. Helper-dependent adenoviral vectors: production
Cell line: 116
C. pNG159 plasmid
D. Southern blot for ψ; Western for Cre
Donna Palmer and Philip Ng, Molecular Therapy, November 2003

40. Helper-dependent adenoviral vectors:
amplification

41. Toxicity after adenovirus administration
Immediate toxicity (Acute)
Inflammatory/innate immunity
Early toxicity
Hepatitis, coagulopathy; CTL vs. viral
products
Late toxicity (Chronic)
CTL/Ab response to virus/transgene;
accumulation of viral proteins

42. Toxicity after adenovirus administration

43. Immediate toxicity
Time: 1-96 hours after treatment
Characterized by: elevation of several cytokines (IL-6, IL-12, TNFα) and signs of thrombocytopenia.
Involved cells: Macrophage, Kupffer cells
Effect: elimination of ~90% vector from liver within first 24h
The immediate toxicity is indipendent of viral gene expression and it is due to an innate response to viral capsid proteins

44. Toll-like receptor 9 44

45. 45

46. Strategies to prevent adenovirus-induced immune response
Circumventing the adaptive immune response by adaptations of the patients:
Immunomodulation or immunosuppression
Circumventing the adaptive immune response by adaptating adenoviral vectors:
Altering native Ad vector and cell surface receptor interactions
Vector microencapsulation
Serotype switching
Helper-virus dependent vectors
Use of nonhuman Ad vectors

47. PEGylation of therapeutic vectors
PEG (polyethyleneglycol):
Low toxicity profile
Not have immunogenic properties
Has been approved by FDA
Activated PEG makes a covalent bond with ε-amino group of lysine on the capsid

48. Levels of pro-inflammatory cytokines
PEGylation of HD-Ad significantly reduce cytokines (IL-6, IL-12, TNF-α) production, 6h after administration of vectors in C57BL/6 mice
Croyle M. et al. Gene Therapy 2005

49. Platelet Count PEGylation of helper-dependent adenovirus prevents
vector-induced thrombocytopenia after systemic delivery

50. Transduction efficiency and vector readministration of PEGylated Ad
A: Control
B: HD-Ad-BGeo
C: PEG- HD-Ad-BGeo
D: HD-Ad-BGeo
after immunizzation with HD-AdSTK109
E: PEG-HD-Ad-BGeo
PEGylation does not compromise transduction efficiency in vitro and in vivo could achieve significant gene expression upon readministration
Croyle M. et al. Gene Therapy 2005

51. Species-specific differences in the biodistribution of and response to PEG-modified vectors 

52. PEGylation of HD-Ad: Reduces vector-induced tissue damage
Decreases prolongation of clotting time
Prevents vector-induced cytokine release
Does not change transduction efficiency of vectors

53. Using a Human Adenovirus Vector in Vaccination
Using viral vectors to deliver antigens:
Recombinant antigen DNA is inserted into the adenoviral vector.
The vector binds to the cell membrane, is packaged in a vesicle, and then injects the antigen DNA into the nucleus.
Cell then makes the antigen using the recombinant antigen DNA, while the cell's own DNA remains unaltered.

54. Using a Human Adenovirus Vector in Vaccination
Primary criticism is the issue of pre-existing immunity
35-55% of the Ad population has neutralizing Abs, in particular against Ad5
These Abs might limit Ad based vaccines vector’s efficacy
Use of alternate serotypes that are antigenically different and thus cannot be neutralized by these Abs is an option in this respect
However still conflicting data exists in this regard
Incase of Merck Ad-5 based HIV vaccine, the effect of neutralizing Abs can be overcome by increasing the dose of the vaccine in clinical trials

55. Using a Human Adenovirus Vector in Vaccination
Evidence also exist that alternative route of administration (oral or intranasal) instead of injection can overcome pre-existing vector immunity
The issue of pre-existing Ad vector immunity is a source of frequent debate and experimental data exist for support on both sides of the argument.

56. Future work using adenovirus as a vector
The majority of clinical trials done using the adenovirus in gene therapy have been phase 1
Phase 1 trials are used to determine safety, feasibility, and toxicity of the process
The Phase 1 trials have set the stage for future work with the next generation of Adeno vectors that will show less stimulation of the host immune system and can be selectively targeted to specific tissues.
Live recombinant adenovirus vaccine are being developed
Provides hope for practitioners in using a more economical vaccine that provides a longer lasting immunity .
Ongoing research is aimed at determining effectiveness of recombinant vaccines

57. References
Palmer, D.; Ng, P. Improved system for helper-dependent adenoviral vector production.
Mol. Ther. 2003, 8, 846–852
Lasaro, M.O.; Ertl, H.C. New insights on adenovirus as vaccine vectors. Mol. Ther. 2009, 17,
1333–1339.
Brunetti-Pierri, N.; Ng, P. Progress towards the clinical application of helper-dependent adenoviral vectors for liver and lung gene therapy. Curr. Opin. Mol. Ther. 2006, 8, 446–454.
Wonganan P, Clemens CC, Brasky K, Pastore L, Croyle MA
Species differences in the pharmacology and toxicology of PEGylated helper-dependent adenovirus. Mol Pharm Feb 7;8(1):78-92.