1. Adeno-associated virus (AAV) vectors

2. AAV virus
AAV is a small, non-enveloped virus of ~22 nm in size, belonging to Parvoviridae family and Dependovirus genus
Adeno-associated virus-based vectors have gained prominence in gene transfer studies due to their non-pathogenic nature and low immunogenicity compared to other viral vectors such as adenovirus

3. AAV vectors
In pre-clinical studies, AAV vectors have shown an ability to transduce a wide variety of tissues, provide stable transgene expression and exhibit relatively low immunogenicity. AAV serotype 2 is the prototype vector and has been extensively tested for its safety in pre-clinical and clinical studies. Other alternate serotypes [AAV1, AAV5, AAV8 and AAV9] have also generated promising pre-clinical data for treatment of a variety of disease states such as muscular dystrophy, hemophilia and α 1-anti trypsin deficiency. These vectors, when compared to AAV2, show remarkably diverse tissue tropism and low immune activation.

4. AAV vectors
It has a single stranded (ss) DNA genome of ~4.7 kb which contains two open reading frames encoding the rep and cap genes flanked by 145 base pair long ITR sequence.
Productive infection of the virus requires the presence of other helper viruses such as adenovirus or herpes simplex virus
During the production of rAAV vectors, the transgene cassette is incorporated between the ITR containing plasmid whereas the rep-cap is supplied in trans along with the helper function genes in a triple plasmid transfection protocol. This generates replication defective vectors which exist as episomes in hostcells.
Currently 12 different AAV serotypes (AAV1-12) have been utilised as gene therapy vectors while several other variants are also known to exist

5. PARVOVIRUS ● Were identified in 1960 ● Are very small and simple
● Parvoviridae family contains 6 genus divided into two subfamilies

6. PARVOVIRUS formed by particles icosahedral, without envelop,
constituted by only proteins (50%) and DNA (50%)

7. Genomic organization of the wt-AAV-2
The inverted terminal repeats (ITRs) flank the two open reading frames (ORFs).
The four-nonstructural proteins encoded from the rep gene are driven by the p5 and p19 promoters, whereas the structural Cap proteins are regulated by the p40 promoter. Additionally, the Assembly Activating Protein (AAP) was found recently within the cap gene.

8. The two larger proteins Rep78/68 play an essential role in viral genome integration and regulation of AAV gene expression, whereas the smaller Rep proteins are involved in viral genome encapsidation.
Rep proteins act both as repressors and activators of AAV transcription in respect to the absence and presence of helper viruses such as adenoviruses (Ad) or herpes simplex viruses (HSV) by interacting with several cellular proteins.
In the absence of Rep proteins, as it is the case in recombinant AAVs, integration of the viral genome into the human genome is rare and random.
There are several hotspots for integration of wtAAV genomes such as the human chromosome 19q13.42, known as the AAVSI site, but as well some other accessible chromatin regions for preferred integration have been found (5p13.3 and 3p24.3).

9. VP proteins
The AAV capsid consists of 60 capsid protein subunits composed of the three cap proteins VP1, VP2, and VP3, which are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry.
The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products.
VP1, VP2 and VP3 share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (NLSs).
These elements are conserved in almost all parvoviruses.

10. Natural Tropism and HSPG motif
The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor. Its binding motif consists of five amino-acids located on the capsid surface

11. the initial contact with HSPG, FGFR-1, HGFR and/or laminin is followed by an interaction with αVβ5 and/or αVβ1 which propably leads to an intracellular activation of enzymes involved in the rearrangement of cytoskeletal proteins like actin, via PI3K-pathway . In general, the receptor-mediated endocytosis (RME) is a complex process proteins and co-factors form clathrin coated pits

12. The AAV cycle

14. Assembly-activating protein
A gene encoding for the assembly-activating protein (AAP) was recently (in 2010) discovered in AAV serotype 2 genome. Its gene product is conserved among all AAV serotypes, illustrating its essential role in virus life cycle. Its functions comprise transport of the viral structural proteins to the nucleolus and involvement in following capsid assembly.
The AAP gene, located in the Cap coding region, is translated from an alternative open reading frame (ORF) with unconventional start codon.

15. After several contacts with HSPG the viral capsid proteins get rearranged.
Clathrin-mediated endocytosis and cellular trafficking into the cell’s center follows. After acidification and following endosomal escape, the viral genome is transferred into the nucleus and replicated (lytic phase) or integrated into the host genome (latent phase).

16. The rearrangment of the capsid structure is probably essential for interaction with other cofactors, which leads to endocytosis. The factors respectively co-receptors of the cellular surface are known to enhance the initial binding affinity of HSPG: Fibroblast growth factor receptor 1 (FGFR-1), hepatocyte growth factor receptor (HGFR) and laminin receptor. It is known that AAVs affect both αVβ5 and αVβ1integrin. The αVβ1 -binding site is an asparagine-glycine-arginine motif. These integrins interact with intracellular molecules like Rho, Rac and Cdc42 GTPases.

17. The first viral particles in the nuclear area can be detected after 15 minutes and an accumulation of virions takes place after 30 minutes post transfection.
After arrival, the viral genomes are transported into the nucleus. It is not entirely clear in which way the transport is accomplished. The viral particles seem to use different pathways to enter the nucleus, either via the nuclear pore complexes with their maximal pore size of 23 nm. In this case, the viral capsid (25 nm diameter) has to be remodeled.

18. ● The ITRs serve as primers for the host cells’ DNA polymerase, which converts the single-stranded virus genome into double-stranded DNA (ds DNA) as a part of the viruses’ replicative cycle. They also play important roles in viral genome integration into and rescue from the hosts genome, the formation of concatamers in the host cell nucleus and encapsidation of the viral genome into preformed capsids. ● Due to these essential functions, the ITR structures cannot be deleted from a viral vector and need to be delivered in cis.

20. Transduction of dividing and non-dividing cells
RECOMBINANT AAV
deletion of the entire parent genome replaced with the therapeutic transgene with the exception of the 145 nucleotides of the ITRs
Transduction of dividing and non-dividing cells
lung, neurons, eye, liver, muscle, hematopoietic progenitors, joint synovium, endothelial cells and gut.
ITR p5
p19
p40
ITR
Cis plasmid
ITR 2
ITR 2
gene of interest

21. Strategy For Recombinant AAV Production
Cis plasmid
Trans plasmid
Virion
ITR 2
ITR 2 + =
AAV2/2
gene of interest
Rep 2
Cap 2
4.7 Kb

22. Recombinant AAV Production
● A producton protocol of AAV vectors in the absence of a helper virus is widely employed for triple plasmid transduction of human embryonic kidney 293 cells.
● The adeno‐virus regions that mediate AAV vector replication (namely, the VA, E2A and E4 regions) were assembled into a helper plasmid. When this helper plasmid is co-transfected into 293 cells along with plasmids encoding the AAV vector genome and rep cap genes, the AAV vector is produced as efficiently as when using adenovirus infection.

23. Generation of rAAV vectors requires 4 key components:
1) a plasmid containing the AAV Rep and Cap genes required for capsid formation and replication
2) a plasmid containing the necessary adenovirus helper genes  
3) a cassette containing the transgene enclosed by two inverted terminal repeats (ITR)
4) a viral packaging cell line

25. rAAV Production Harvest Trans Cis Adeno Helper Triple Transfection
rep
cap
Trans
ITR
ITR
Pro
Transgene pA
E2A E4
VAI
Cis
Adeno Helper
Triple Transfection
HEK 293 cells
(E1)
3 days
Harvest

26. Improvements on rAAV2 Purification by SSCP
●Single step gravity-flow heparin based affinity column
● Increased purity
● Increased potency (5 ~10-fold)
● Highly reproducible
● No expensive or special equipment required
● Shortened purification process from 4 days to 1 day

27. Single-step Column Purification (SSCP) of rAAV2 Vectors
Cell pellet
Freeze/thaw x 3
Benzonase & DOC treatment
Cell lysate
1 Day
Filtration through 5 mm & 0.8 mm Millex filter
Load onto Heparin Agarose column
Wash with 0.1M NaCl PBS
Elute with 0.4M NaCl PBS
Desalting & concentration
Quality control assays

28. Improved Quality of SSCP Purified rAAV2 Vectors
Purity
(SDS-PAGE)
CsCl
Heparin
Activity in vivo
15.3
Heparin
VP1
CsCl
VP2
VP3
Relative b-gal expression
3.3
Method
CsCl
Heparin
Yield (GC)
7.3 x 1012
5.7 x 1012

31. The AAV vector genome can be pseudotyped by packaging with capsids from different AAV serotypes. A combination of the capsid and the route of administration will determine whether gene delivery is local or systemic. AAV1 capsids produce the most efficient transduction after direct intramuscular injection, whereas AAV8 capsids achieve global gene delivery after intravenous injection. In this example, gene delivery is detected by expression of a transduced gene expressing green fluorescent protein.

32. AAV Vectors with Different Capsids
Have Different Transduction Characteristics
AAV genome plasmid
Packaging plasmid
Virion
Target Tissue
Rep 2
Cap 1
AAV2/1
Muscle
Rep 2
Cap 2
AAV2/2
Muscle-Liver-Retina
Rep 2
Cap 3
AAV2/3
Inner ear
ITR 2
ITR 2
Rep 2
Cap 4 =
AAV2/4
SNC, Retina
gene of interest
Rep 2
Cap 5
AAV2/5
Lung
Rep 2
Cap 7
AAV2/7
Muscle
Rep 2
Cap 8
AAV2/8
Liver
Rep 2
Cap 9
AAV2/9
Lung

33. Capsid shuffling
a | Various capsid DNA sequences are derived from adeno-associated viruses (AAVs) with different transduction properties (hexagons of different colours). b | The capsid DNA sequences are randomly digested and then PCR ligated back into a 'wild-type' AAV plasmid (capS, shuffled cap gene). The AAV capsid library can contain between 106 and 107 unique sequences

34. e | Selected capsid sequences that survive the selection are then cloned into a vector production system and used to pseudotype standard AAV vector genomes (containing a reporter or therapeutic expression cassette) and tested for transduction properties in cells, animals or humans.
c | The recombinant AAV wild-type viruses are expanded (with the addition of a replication helper virus, not shown) without any selection in cells. d | The AAV viral library is expanded under selective pressure, allowing viruses that survive the selection to be further propagated. With stronger selective pressure, the diversity of the capsid library is reduced and select clones are enriched

35. Dozens of different naturally occurring AAV capsids, as well as genetically engineered ones, have been isolated for study, from humans and from other species. The capsid sequences are highly conserved, from 60% to 99%, but studies with naturally occurring serotypes and purpose-engineered capsids have shown that even small differences in capsid sequence may affect tissue tropism of a vector and can be exploited to improve therapeutic outcomes.

36. Transcapsidation
process that involves the packaging of the ITR of one serotype of AAV into the capsid of a different serotype. Most examples of this involve using the heavily studied AAV2 genome being packaged into other AAV serotypes to examine the efficacy. The drawback to using transcapsidation in modifying AAV tropism is a potentially lower titer yield, depending on which two serotypes are used. In addition, due to the interaction between the C-terminus of the protein coat of the capsid and the viral genome, lower yields of AAV particles can be expected during production

37. Plasmid mixing approach used to generate transcapsidated rAAV.
Plasmid mixing approach used to generate transcapsidated rAAV. Helper plasmid DNA containing the capsid gene from any two AAV serotypes (one is represented by a red capsid gene, and the other is represented by a blue capsid gene) was cotransfected at different ratios (19:1, 3:1, 1:1, 1:3, and 1:19) during the standard production scheme to generate rAAV particles. The topology maps of potential viral products are shown and colored according to the proportion of subunits from the ratio of helper plasmids in the transfection mixtures.
Plasmid mixing approach used to generate transcapsidated rAAV. Helper plasmid DNA containing the capsid gene from any two AAV serotypes (one is represented by a red capsid gene, and the other is represented by a blue capsid gene) was cotransfected at different ratios (19:1, 3:1, 1:1, 1:3, and 1:19) during the standard production scheme to generate rAAV particles.

38. Adsorption of Receptor Ligands
Adsorption of Receptor Ligands to the AAV Capsid Surface is the addition of foreign peptides to the surface of the capsid. The main goal is to be able to specifically target cells that no AAV serotype currently has a tropism towards, and this greatly expands the uses of AAV as a gene therapy tool. Early approaches involved using AAV-specific antibodies that are linked to a second antibody that specifically links to cell receptors on the target cells. These modifications proved to be successful in altering the vector tropism towards the target cells

39. Mosaic Capsid
●Mosaic Capsid modification involves the packaging of the AAV genome/rAAV transgene into an AAV capsid made up of a mixture of unmodified capsid proteins from two separate serotypes.
●T o do so, two separate plasmids each with genes from a separate serotype are transfected together during packaging, which theoretically leads to a packaged virus with roughly 50% of the viral proteins from one serotype and 50% from the other.
● Results from experimentation show that even though the original serotypes may poorly transfect a certain tissue type individually, the modified mosaic capsids may be able to transfect more efficiently leading to speculation that mosaic capsids trafficking mechanism is different

40. Chimeric Capsid
Chimeric capsids are packaged capsid which has a foreign protein sequence inserted into the Open Reading Frame of the capsid gene. The most commonly used chimeric modifications are:
● The use of epitope coding sequences fused to either the N or C termini of the capsid coding sequences to attempt to expose new peptides on the surface of the viral capsid without affecting gene function.

41. ● The use of epitope sequences inserted into specific positions in the capsid coding sequence for the same reason as above, but using a different approach of tagging the epitope into the coding sequences itself.
The main goal of using chimeric capsids in rAAV vectors is to expand the range of cell types that can be transfected and to increase the efficiency of transduction.

42. ●AAV are currently the most favoured vectors for gene therapy of retina given their ability to efficiently target various retinal layers, most likely due to their relatively small size and ability to spread after subretinal delivery.
● In addition, AAV have an excellent safety profile and low immunogenicity which allows for long-term expression of the therapeutic gene after a single administration, a desirable feature when treating a chronic condition like IR and re-administration of AAV to the subretinal space
● Each AAV serotype has unique transduction characteristics (i.e target cells, kinetic of transgene expression) because different AAV capsids interact with different receptors on target cells and/or impact the AAV post-entry transduction. This allows the user to select the most appropriate AAV serotype to transduce the retinal cell layer of interest

43. ● Following subretinal delivery, virtually all AAV serotypes tested efficiently transduce RPE, with AAV2/1, AAV2/ 4 and AAV2/6 being the most specific in various animal models.
This may result from the RPE accessibility from the subretinal space into which vectors are injected, an inherent permissiveness of the RPE to AAV infection (specifically, the presence of AAV receptors and co-receptors at the RPE cell membrane), and/or lastly, the phagocytic properties of the RPE that may facilitate entry of AAV particles .

44. ● Conversely, the levels of PR transduction vary significantly among different sero-types. AAV2/5, 2/7, 2/8 and 2/9 have all been demonstrated to efficiently transduce PR, in addition to RPE with AAV2/8 being the most efficient serotype in mice, pigs, dogs and non-human primates .
● Particular attention has been recently directed towards the identification of AAV vectors that efficiently transduce cone PR in addition to rods, which make up the majority of PR in the retina. This is required for gene therapy of cone and cone-rod dystrophies, such as Stargardt disease, achromatopsia and age-related macular degeneration.

45. The AAV serotypes’ ability to transduce cones has been evaluated in the pig and non-human primate retinas, which present cone density and distribution more similar to humans than mice.
Among these, serotypes AAV2/5,AAV2/8 and AAV2/9 were found to present the highest cone transduction properties.

47. novel AAV capsid variants with enhanced gene transfer efficiency and altered tropism have been generated in recent years by either rational design or directed evolution

48. Overview image of a retinal slice from a CNGA3 knockout mouse after AAV-mediated gene therapy stained for cGMP (green) and CNGA3 (red). The untreated (left) part with missing CNGA3 expression shows high levels of cGMP in cone photoreceptors. The treatment-border area (dashed rectangle) is also shown in higher magnification. In the treated part (right) AAV-mediated expression of CNGA3 lowered cGMP to levels that cannot be detected by cGMP-immunohistochemistry

50. Subretinal Delivery Of Pseudotyped AAV Vectors Encoding EGFP: Histology At Day 28
rpe
onl
inl

51. Intravitreal Delivery Of AAV2/2 Vectors Encoding EGFP: Histology At Day 28
Inner retina
Ciliary Body
Optic nerve

52. Use of Tissue Specific Promoters to Restrict Gene Expression in the Eye
AAV2/5pCMVeGFP
AAV2/5p-563OA1eGFP
AAV2/5p-600hRHOeGFP
ONL
RPE

53. Appropriate Viral Vectors and Promoters To Treat Murine Models Of Retinal Disorders
Disease
Locus
Cell
AAV RP
Rho PR
2/5
MERTK
RPE
2/1
LCA
RPE65
Stargardt
ABCR
OA1
Promoter
Rhodopsin
RPE65-OA1
RPE65-OA1
Rhodopsin
RPE65-OA1

54. Retrograde transduction of spinal motor neurons by AAV vectors.
Expression of β-gal activity in the skeletal muscle (A) and spinal cord (B) 4 weeks after injection of different AAV-LacZ vector serotypes in the gastrocnemius muscle. (A) AAV2/1 shows remarkable transduction of skeletal muscle, followed by AAV2/7 and AAV2/5. Conversely, the muscle transduction level for AAV2/2 and AAV2/8 is low. (B) Longitudinal sections of lumbar spinal cord to visualize targeting of motor neurons by retrograde transport of AAV (left side injected with AAV-LacZ). β-Gal activity restricted to motor neurons is visible only with AAV2/2 and AAV2/1

55. Gene transfer approaches to treat LGMDs
Gene transfer approaches to treat LGMDs. Disease-causing genes or muscle regulator genes are encapsidated into AAV vectors to be delivered intramuscularly or systemically

56. The figure illustrates the concept of muscle-directed systemic cancer gene therapy. First, an AAV vector encoding anti-angiogenic agents is injected into a muscle. After a single injection, secreted anti-angiogenic agents are circulating throughout the entire body. These circulating factors suppress both the primary tumor and undetectable metastatic lesions through inhibition of tumor angiogenesis. If the gene therapy alone is not sufficient to suppress all the tumors, other therapies such as radiation, chemotherapy or immunotherapy can be added to enhance the effect.

57. Systemic anti-angiogenic cancer gene therapy
(A) Construction of recombinant AAV vector plasmids expressing murine endostatin (AAV/mEnd) and control GFP expressing vector (AAV/GFP). (B) Serum levels of endostatin after intramuscular injection of AAV/End (42 days after vector injection). Intramuscular injection of AAV/End increased the serum endostatin level. (C) Effects of AAV-mediated endostatin expression on pancreatic tumor growth and liver metastasis

58. Expression of transgenes following intramuscular administration of AAVs. Following injection of the respective AAV serotypes of the AAV/mEnd vectors, which express murine endostatin together with GFP, into quadriceps muscles, expression of GFP and plasma concentrations of mEnd were analyzed (A). Four AAV serotypes of AAV/Luc, which express luciferase, were injected into the quadriceps muscles of DDY mice. Four weeks after injection, these mice were analyzed by in vivo imaging system

59. Inhibition of tumor growth after administration of AAV/IL24
Inhibition of tumor growth after administration of AAV/IL24. One week after subcutaneous injection of Ehrlich ascites tumor cells, mice were treated with AAV/IL24 or control AAV/GFP. The mean tumor volumes 56 days after injection were significantly smaller in animals that received AAV/IL24 than in those that received AAV/GFP (P < 0.001)