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Adeno-associated virus (AAV vector) -Introduction

Landscape, protocol and guidelines of Adeno-associated virus (AAV) vector system, AAV production,AAV transduction and AAV gene therapy are described below. The detail information is mentioned about AAV vector plasmids cloning, AAV packaging, purification, AAV caspsid evolution and insigt of AAV gene therapy.

Adeno Associated Virus (AAV)

Custom AAV Production
AAV Vector System
CRISPR/Cas9 AAV Production
Optogenetics AAV Production
Pre-made AAV Production
AAV Control Virus Production
AAV-LC3 Autophagy Flux Detection

Adeno Associated Virus (AAV) Packaging System

AAV Vector System
AAV Rep-Cap Plasmids
AAV Expression Vector

Adeno-associated virus (AAV vector) -Index


Adeno-associated virus (AAV vector) and AAV gene therapy-Knowledge Base

1. What is Adeno-associated virus (AAV) vector?

Adeno-associated virus (AAV) is a small single strand DNA virus, member of human parvovirus, originally described in 1960s by Bob Atchison at Pittsburgh and Wallace Rowe at NIH as a contaminant in preparation of adenovirus [1,2]. Shortly after its discovery, AAV was then isolated from humans, but serological studies suggested that AAV itself did not cause any disease despite being present in people infected with helper viruses such as adenovirus or herpes virus [3]. With the development of recombinant AAV vectors (rAAV), the adenovirus helper genes required for AAV replication have been identified, which can be cloned into plasmids for AAV production to liberate the need of helper virus. Currently, AAV production adopts the 3-plasmid co-transfection system (AAV plasmid with gene of interest, AAV packaging plasmid AAV-RC (AAV replication and AAV capsid) and pHelper (AAV helper plasmid)) in AAV-293 cells, which significantly simplifies the AAV purification process.

2. AAV Gene Therapy Review

AAV has been proved as the most excellent gene therapy vector. To date, more than 204 clinical trials have been carried out using AAV vectors for gene delivery [4], and promising gene therapy outcomes have been achieved from Phase 1, Phase 2 and Phase 3 trials for a great number of diseases (Table 1), including lipoprotein lipase deficiency (LPLD) [5], spinal muscular atrophy (SMA) [6], retinal dystrophy [7,8], cystic fibrosis [9,10], Duchenne Muscular Dystrophy [11], Hemophilia [12], congestive heart failure [13], Parkinson's disease [14] and Rheumatoid Arthritis [15,16]. Among them, Luxturna from Spark has been approved by FDA to treat patients with retinal dystrophy, AVXS-101 by AveXis has shown outstanding therapeutic effects in patients with spinal muscular atrophy, while AAVs with micro-dystrophin and coagulation factor IX have displayed great potential in the treatment of Duchenne Muscular Dystrophy and Hemophilia respectively.

a)Luxturna for treatment of retinal dystrophy
The FDA has approved LUXTURNA™ (voretigene neparvovec-rzyl), a one-time gene therapy product designed for the treatment of the LCA ( Leber's congenital amaurosis) patients with confirmed biallelic RPE65 mutation-associated retinal dystrophy in December 2017. LUXTURNA is the first and only pharmacologic treatment for an inherited retinal disease (IRD) and the first AAV vector gene therapy for a genetic disease approved in the U.S. LUXTURNA uses AAV2 to carry out functional copies of the RPE65 gene delivery to the retinal pigment epithelial (RPE) cells to compensate for the RPE65 mutation [17]. With the functional RPE65 gene delivery, retinal pigment epithelial cells begin producing the RPE65 protein [18], letting 11-cis-retinal (a critical visual pigment component) regenerate to restore the visual cycle [4,19]. To date, Phase 1 studies have shown potential benefits of gene replacement in RPE65-mediated inherited retinal dystrophy, and the Phase 3 studies also demonstrate great efficacy and safety of Luxturna gene therapy in participants inherited retinal dystrophy [20].

Table1. Selected clinical trials using AAV vectors.
Indication Gene AAV serotype Route of AAV delivery Phase Sponsor
Lipoprotein lipase deficiency LPL AAV1 Intramuscular II–Ⅲ Amsterdam Molecular Therapeutics
LPL AAV1 Intramuscular I UniQure Biopharma B.V.
Spinal muscular atrophy 1 SMN AAV9 Intravenous I AveXis
SMN AAV9 Intravenous AveXis
SMN AAV9 Intravenous AveXis
Spinal muscular atrophy SMN AAV9 Intravenous I AveXis
SMN AAV9 Intravenous AveXis
SMN AAV9 Intravenous AveXis
Retinal dystrophy RPE65 AAV2 Subretinal I–II University College, London
PDE6B AAV5 Subretinal I–II Horama S.A
Cystic fibrosis CFTR AAV5 Lung, via aerosol I NIDDK
Hemophilia A HLP-FVIII-V3 AAV8 Intravenous I University College, London
Hemophilia B FIX AAV2 Intravenous I Spark Therapeutics
FIX16 AAVrh10 Intravenous I–II Ultragenyx Pharmaceutical Inc
Arthritis TNFR:Fc AAV2 Intraarticular I Targeted Genetics Corporation
TNFR:Fc AAV2 Intraarticular I–II Targeted Genetics Corporation
hIFN-b AAV5 Intraarticular I Arthrogen
hIFN-b AAV5 Intraarticular I Arthrogen
Congestive heart failure SERCA2a AAV1 Intracoronary I–II Celladon Corporation
SERCA2a AAV1 Intracoronary II Celladon Corporation
SERCA2a AAV1 Intracoronary II Imperial College London
SERCA2a AAV1 Intracoronary II Assistance Publique - Hôpitaux de Paris
SERCA2a AAV1 Intracoronary I–II Celladon Corporation
Parkinson's disease GAD AAV2 Surgical infusion I Neurologix, Inc.
GAD AAV2 Intrastriatal I Genzyme, a Sanofi Company
NTN AAV2 Intrastriatal I Genzyme
NTN AAV2 Bilateral Intraputaminal (IPu) II Genzyme
NTN AAV2 Bilateral Intraputaminal and Intranigral I–II Sangamo Therapeutics
Alzheimer's disease NGF AAV5 Bilateral, stereotactic I Ceregene
APOE2 AAVrh.10 Intracisternal I Weill Medical College of Cornell University

b)AVXS-101 for treatment of spinal muscular atrophy (SMA)
AVXS-101 is being developed by AveXis (Owned by Novartis) to carry out gene delivery of a functional copy of the SMN1 gene by using AAV9 vector to motor neuron cells in SMA patients [21]. The SMN1 transgene in AVXS-101 contains double-stranded DNA, meaning it consists of the genetic instructions and takes the same form as natural genes, so that it can be activated more quickly to produce continuous and sustainable SMN protein, resulting in more efficient gene therapy in vivo. The safety, tolerability and impressive improvements in motor function of AVXS-101 in all patients with SMA Type 1 has been guaranteed during Phase 1 clinical trials [51]. Based on the preliminary top-line results, the FDA has granted AVXS-101 Orphan Drug Designation for the treatment of all types of SMA and Breakthrough Therapy Designation, as well as Fast Track Designation, for the treatment of SMA Type 1. Likewise, the European Medicines Agency (EMA) also granted AveXis access into its PRIority Medicines (PRIME) program for AVXS-101 for the treatment of SMA Type 1 in January 2017. Then in December 2017, AveXis also announced the start of an open-label, dose-comparison, multi-center Phase 1 trial, also known as STRONG, to evaluate the safety, optimal dosing and proof of concept for efficacy of AVXS-101 gene therapy in two distinct age groups of patients with SMA Type II. Nowadays, AveXis is running a Phase 3 trial, called STR1VE (NCT03306277) in infants with SMA type 1 to further understanding of both the safety of AVXS-101 gene therapy, and how well AVXS-101 may work in SMA patients, which may bring new hope for pediatric and adult patients with other types of motor neuron diseases.

c)AAV with micro-dystrophin for treatment of Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD), resulting from dystrophin gene mutation, is a severe genetic disease resulting in body-wide muscle degeneration and necrosis in boys and young men [22]. In gene therapy theory, replacing or correcting the mutated dystrophin gene with a functional one would cure this disease [23]. However, the enormous size of dystrophin cDNA and the distribution of muscle throughout the body presented great challenges to this arduous task. To address these obstacles, researchers have developed the highly abbreviated micro-dystrophin gene and designed systemic gene delivery with AAV2.5, which was a translational optimized AAV variant, inducing stronger transduction in skeletal muscles than AAV2 and displaying lower crossreactivity to AAV2 neutralizing antibodies [11]. To date, preclinical data suggests that intravascular AAV micro-dystrophin delivery can significantly ameliorate muscle pathology, enhance muscle force, and attenuate dystrophic cardiomyopathy in animals, such as mice and canines [24,25]. Phase 1 clinical trials of AAV2.5 in DMD patients have shown that rationally designed AAV vector (AAV2.5) was safe and well tolerated, setting the foundation of customizing AAV vectors best suiting the clinical objectives [11]. Meanwhile, more clinical trials are required to estimate the clinical efficacy of AAV2.5 with micro-dystrophin gene, and the findings display a great prospects of body-wide DMD therapy with a synthetic micro-dystrophin AAV vector [26].

d)AAV with coagulation factor IX for treatment of Hemophilia B
Hemophilia B (HB) is an X-linked disease, caused by a deficiency in functional coagulation factor IX protein (FIX) [27]. Currently, preclinical data suggests AAV vector-mediated gene transfer of coagulation factor IX to the skeletal muscle or to liver have shown sustained correction of hemophilia B in mice and dogs [28,29]. The two initial phase I/II clinical trials with FIX cDNA gene delivery by AAV vectors for treatment of hemophilia B displayed no serious adverse effects, but demonstrated contrary results, no therapeutic level of FIX in muscle trial while some therapeutic effects closely with that of hemophilic dogs [30]. To date, gene therapy for Hemophilia B via systemic administration of AAV vectors containing an optimized coagulation factor IX construct has achieved considerable progress, ameliorating the bleeding phenotype numerous patients [31]. However, challenges remain for sustained gene therapy in patients, and efforts are still required to improve long term expression of factor IX, via optimisations to the AAV vector, transgene cassette and correction strategy.

3. Advantages and drawbacks of AAV vector-mediated gene transfer

a) Advantages of AAV-mediated gene transfer
AAV has been developed into a very attractive candidate for creating viral vectors for gene therapy and for the creation of isogenic human disease models due to various advantages. 1) Superior biosafety rating. The wild type AAV has not currently been known to cause disease in vivo, and further security of recombinant AAV gene delivery in vivo is ensured after removal of most AAV genome elements. 2) Low immunogenicity. AAV causes a very mild immune response in vivo, lending further support to its apparent lack of pathogenicity during gene delivery. 3) Broad range of infectivity. AAV can infect both dividing and quiescent cells in vivo, allowing gene delivery to a highly diverse range of cell types. 4) Stable expression. Long term gene delivery in vivo can be mediated by AAV.

b) Drawbacks of AAV-mediated gene transfer
Although adenovirus benefits a great deal of disease therapies, it does present some drawbacks.
1) The major drawback is its limited cloning capacity (less than 4.7kb) of the vector, which restricts its use in gene delivery of large genes.(Table 3) [32]. 2) Generation of neutralizing antibodies against AAV in the Non-Human Primates (NHP) and human, may attenuate the cure effect of AAV-mediated gene therapy [33].

Table 2. Comparison between Retrovirus, Lentivirus, Adenovirus and Adeno-associated virus (AAV) vectors.
Comparison Retrovirus Lentivirus Adenovirus AAV
Genome ss RNA ss RNA ds DNA ss DNA
Integration Yes Yes No No
Packaging Capacity 3kb 4kb 5.5kb 2kb
Time to peak expression 72h 72h 3h-72h cell: 7 days; animals: 2 weeks
Sustainable time about 3 weeks stable expression transient expression > 6 months
Cell Type most dividing/non-dividing Cells most dividing/non-dividing Cells most dividing/non-dividing Cells most dividing/non-dividing Cells
Titer 10^7 TU/ml 10^8 TU/ml 10^11 PFU/ml 10^12 vg/ml
Animal experiment suitable low efficiency lowest efficiency most suitable
Immune Response high medium medium mild

4. AAV Genome Structure (ITR,Rep,Cap,VP1,VP2,VP3) and AAV virus assembly

The AAV shell is approximately 25nm in diameter and encapsidates a single-stranded DNA genome of 4.7 kilobases. The genome consists of two large open reading frames (ORFs) flanked by 145bp inverted terminal repeats (ITR), which are the only cis-acting elements required for AAV genome replication and AAV packaging. The left ORF encodes four replication proteins, Rep40, Rep52, Rep68, and Rep78, in charge of site-specific integration, as well as regulation of AAV capsid formation initiation within the AAV genome, while the right ORF encodes the viral structural proteins, VP1, VP2, and VP3, which interact together to assemble into icosahedral virion shells comprising 60 subunits each (Figure1).

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5. Life Cycle of AAV Virus-From Infection To Regeneration

AAV transduces cells through several stages: (1) viral binding to cell surface receptor/coreceptor, (2) endocytosis of the virus, (3) intracellular trafficking of the virus through the endosomal compartment, (4) endosomal escape of the virus, (5) intracellular trafficking of the virus to the nucleus and nuclear import, (6) virion uncoating, (7) viral genome conversion from a single-stranded to a double-stranded genome capable of expressing an encoded gene [34-36]. Since AAV has no ability to encode polymerases, AAV is dependent upon cellular polymerase activity to replicate its own genome [37]. The presence of a helper virus such as adenovirus is indispensable for wild-type AAV to facilitate gene expression and replication. (Figure 2A) Without helper virus, expression of Rep68/Rep78 would be restricted owing to Ying Yang 1 (YY1) repression of the P5 promoter, leading to inhibition of AAV genome replication and gene expression, and initiation of AAV chromosome integration (Figure 2B) [38]. AAV establishes latency by undergoing specifically integration into a genome site, termed as the adeno-associated virus integration site 1 (AAVS1), a 4kb region on chromosome 19 (q13.4).

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So far, no known disease has been demonstrated to be related to AAV though AAV has been detected in many tissues of animal species. Serological studies show that most people are exposed to wild-type AAV during childhood and adolescence and prevalence rates for antibody titers exceed 60% among adults, while no detrimental effects or lytic infections have been observed [39,40].

6. AAV serotypes and AAV Tissue-specific Tropism

Over the past decades, numerous AAV serotypes have been identified with variable tropism. To date, 12 AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human/nonhuman primate tissues. Among them, AAV2, AAV3, AAV5, AAV6 were discovered in human cells, while AAV1, AAV4, AAV7, AAV8, AAV9, AAV10 (AAVrh10), AAV11, AAV12 in nonhuman primate samples [41]. Genome divergence among different serotypes is most concentrated on hypervariable regions (HVRs) of virus capsid, which might determine their tissue tropism[42]. Besides virus capsid, tissue tropisms of AAV vectors are also influenced by cell surface receptors, cellular uptake, intracellular processing, nuclear delivery of the vector genome, uncoating, and second strand DNA conversion [3]. To effectively enable the transduction of tissues or limit AAV tropism to specific tissues in vivo, researchers have developed a number of mosaic AAV vectors or chimeric AAV vectors by engineering custom-designed AAV capsids to carry out gene therapy [43,44].

a)AAV Serotype 2 (AAV2)
AAV Serotype 2 (AAV2) is the first isolate of AAVs and also the best studied AAV serotype, which stands for natural tropism towards skeletal muscles [45], neurons [46], vascular smooth muscle cells [47], and hepatocytes [48]. AAV2 possesses three receptors, among which heparan sulfate protoglycan (HSPG) functions as a primary receptor to scavenge AAV particles and impair the infection efficiency [49], while aVβ5 integrin and fibroblast growth factor receptor 1 (FGFR-1) enable AAV to enter the cell by receptor-mediated endocytosis as co-receptors [50-52]. In possession of great gene therapy potentials, AAV2 has been used for cancer treatments, such as ovarian carcinoma (OvCa) without any effect on healthy cells currently [53].

b)AAV Serotype 8 (AAV8) and AAV Serotype 9 (AAV9)
In addition to AAV2, the most widely used AAV serotypes are AAV Serotype 8 (AAV8) and AAV Serotype 9 (AAV9). AAV8 is a robust vector for gene delivery to liver and a promising research tool for delivering genes to various target organs with higher efficiency than most other serotypes. As demonstrated, efficient transduction of liver, skeletal muscle and heart can be detected after systemic injection of AAV8 [54]. More importantly, a single tail vein injection of AAV8 was as efficient as portal vein injection at any dose and a high dose of AAV8 transduced skeletal muscle throughout the body, including the diaphragm, the entire cardiac muscle, and the pancreas, smooth muscle, and brain at substantial levels [55]. Compared with AAV2, AAV9 displays a similar profile with widely disseminated transduction, just with much more efficient [56]. Additionally, the ability of AAV9 to bypass the blood-brain barrier (BBB) enables intravascular administration as a non-invasive delivery route to achieve widespread CNS gene expression. What’s more, AAV9 intravenous gene delivery has already shown promising results for several diseases in animal models, including lysosomal storage disorders and motor neuron diseases, opening the way to the first clinical trial in the field [57].

c)Other AAV serotypes
Today, 12 AAV serotypes have been designed as gene delivery vectors. It might be difficult to interpret AAV serotype tissue tropism due to interstudy variations in vector titers and doses, promoters, and transgenes. A general hierarchy of transduction efficiency in major tissues and cell types has been established at table 1 and table 2. For instance, AAV2 can moderately transduce several tissue types, including central nervous system, liver, muscle, and lung. Similarly within the CNS, AAV1 and AAV5 show higher transduction frequencies than AAV2 in all injected regions [58,59], while AAV4 just appears to transduce specific cell types, such as the astrocytes and ependyma in the subventricular zone [60]. In skeletal muscle cells, AAV1, AAV6 and AAV7 is reported to present very high transduction rate [56,61-63], while AAV8 exhibits transduction ability with super efficiency not only in hepatocytes but also in other organs [44,61,64]. In addition to skeletal muscle cells, AAV6 has been also reported to mediate more efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 [64], which may supply AAV6 with more significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis considering the lower immunogenicity of AAV6 than of AAV2 [44,61].

Table 3. AAV serotypes and their respective Tropism.
AAV Serotype Tissue tropism
CNS Retina Lung Liver Pancreas Kidney Heart Muscle
AAV1
AAV2
AAV3
AAV4
AAV5
AAV6
AAV7
AAV8
AAV9
AAV-DJ
AAV-DJ/8
AAV-Rh10
AAV-retro
AAV-PHP.B
AAV8-PHP.eB
AAV-PHP.S
Table 4. Transduction efficiency comparison between different AAV serotypes.
Cell Line AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV8 AAV9 AAV-DJ AAV-DJ/8
Huh-7 13 100 2.5 0 0.1 10 0.7 0 500 0.2
HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 500 0.3
Hela 3 100 2 0.1 6.7 1 0.2 0.1 667 0.2
HepG2 3 100 16.7 0.3 1.7 5 0.3 ND 1250 0.5
Hep1A 20 100 0.2 1 0.1 1 0.2 0 400 0.1
911 17 100 11 0.2 0.1 17 0.1 ND 500 0
CHO 100 100 14 1.4 333 50 10 1 25000 5
COS 33 100 33 3.3 5 14 2 0.5 500 0.3
MeWo 10 100 20 0.3 6.7 10 1 0.2 2857 1
NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND 500 0.1
A549 14 100 20 ND 0.5 10 0.5 0.1 1000 0.1
HT1180 20 100 10 0.1 0.3 33 0.5 0.1 333 0.2
Monocytes 1111 100 ND ND 125 1429 ND ND 100 ND
Immature DC 2500 100 ND ND 222 2857 ND ND 200 ND
Mature DC 2222 100 ND ND 333 3333 ND ND 100 ND

7. AAV Vector System (AAV Vector System)

a)Introduction of rAAV vector system

Though wild-type AAV is not associated with human disease, it is naturally defective and requiring helper adenovirus or herpes simplex virus (HSV) coinfection for AAV replication, so recombinant AAV (rAAV) has been an attractive vector for gene therapy. In 1984, rAAV was used for the first time by replacing the viral genome with a transgene, which was then transfected into cells that were infected with adenovirus for production of rAAV.

Traditionally, rAAV vectors used in clinical trials were prepared with a plasmid containing the therapeutic gene flanked by AAV-inverted terminal repeats (ITRs), co-transfected with AAV packaging plasmid AAV-RC (AAV replication and AAV capsid) and pHelper (AAV helper plasmid). The adenovirus helper factors, such as E1A, E1B, E2A, E4ORF6 and VA RNAs, would be provided by the third helper plasmid. Given that HEK293 cells, a commonly used AAV production cells, already contain the E1A/E1b gene, only E2A, E4ORF6 and VA RNAs need to be provided by the helper plasmid.

b)Helper-free rAAV system

The current method of the rAAV production is based on the adenovirus-free transient transfection of all elements that are required for AAV production in host cells, such as HEK293 cells. It involves the co-transfection of 3 plasmids into AAV production cells as shown in Figure 3.

1. AAV-GOI: an AAV ITR-containing plasmid carrying the gene of interest (GOI);

2. AAV-RC: an AAV serotype plasmid that carries Rep and Capsid genes;

3. pHelper: an AAV helper plasmid that provides the helper genes isolated from adenovirus.

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8. AAV engineering (AAV caspid evolution)

Although more than 100 AAV serotype variants have been found in the nature, significant progress has been made for rAAV production, the transduction efficiency and specificity for some tissues and cell types in animal and primates is low. The non-targeted delivery by AAV requires high doses and potentially resulting in toxicities, problematic manufacturing burdens and suboptimal efficacy. Therefore, an important area in the development of AAV as a vector concerns the engineering of altered cell tropisms to increase rAAV mediated gene delivery efficiency and specificity. Among various AAV serotypes, the variations in transducing efficiencies could be caused by differences in their respective receptor contents on target cells.

A great number of strategies for improving AAV delivery efficiency and specificity have been developed. Methodologically, the methods to improve AAV tropism and specificity can be divided into two categories. One is the assembly and recombination of proteins between different viruses, which can produce hybrid rAAVs. The other is to recombine, redesign, or introduce random mutations into the capsid protein of AAV by different methods to artificially increase the variance of AAV serotypes, and then screen the appropriate AAV serotypes.

a)Hybrid rAAV Vectors

The commonly used approach to generate hybrid rAAV vectors is transcapsidation, which is a process involving the packaging of the ITR from one AAV serotype into the capsid of another serotype, which may determine the tissue tropism of hybrids. For instance, if it is rAAV2/5 that is to be produced based on the helper-free system discussed above, the AAV packaging plasmid AAV-RC should be pRep2/Cap5, that is, the Rep gene is derived from AAV2, while the Capsid gene from AAV5. The packaging scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is named as rAAV2/5, in which the genome is based on recombinant AAV2, while the capsid based on AAV5. The tissue tropism displayed by this rAAV2/5 hybrid virus is assumed to be the same with AAV5.

b)AAV revolution

There are two main ways to evolve AAV, one is rational design, the other is direct evolution (Figure4).

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Rational Design

Some common method of rational design AAV capsid, based on knowledge about delivery mechanisms coupled with AAV structural analysis [65], are listed as follows.

(1) Phosphorylation of tyrosine residues in AAV capsid results in ubiquitylation and promotes proteasomal degradation of AAV virions, leading to the creation of vectors in which tyrosines were mutated to phenylalanines by site-directed mutagenesis [66].

(2) Analysis of potential docking sites for a murine IgG2a antibody with the AAV2 surface to determine sterically accessible candidate positions, which were then subjected to extensive site-directed mutagenesis to develop variants with reduced neutralization by antibodies in vitro [67].

(3) Incorporation of high-affinity ligands into the AAV capsid can confer binding to alternative cell surface receptors and thereby restrict or redirect viral tropism [68].

AAV Directed Evolution

The conventional process of directed evolution is generating a library by error-prone PCR, peptide display or DNA family shuffling at firs. Then, this AAV viral library was packaged as a pool and purified, and selected under certain pressure in vitro or in vivo. After infection of the target cell or tissue, the viruses would recover from adenovirus-mediated replication or PCR amplification. Three or more rounds selection later, the highly infectious and specific AAV capsid species would be enriched (Figure 5) [69].

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Point Mutation (Error-Prone PCR)

The simplest strategy for AAV diversification is mutagenesis of individual residues in the AAV viral capsid, either in a site-directed or in a randomized manner, expecting the introduced amino acid changes may alter essential AAV vector properties. Randomized mutations can be introduced to AAV capsid-encoding sequence by error-prone PCR (Figure 6).

Although point mutations are easily introduced to AAV capsid-encoding sequence, there still exist problems. 1) Only 0.01–1% of AAV mutations are typically beneficial; 2) By far, number of clones to cover all combinations of multiple mutations within a single AAV capsid exceeds technical possibilities. It would be more useful to focus on a specific region of the AAV capsid, especially the surface-exposed areas and AAV capsid protrusions which are more likely to interact with cellular receptors and antibodies [70].

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Peptide Display

Peptide insertion of novel receptor-binding elements on the AAV capsid surface can provide affinity for the receptor specifically expressed in the target cell type. To maximize success of this AAV peptide display strategy, it is critical to capitalize on sites in the AAV capsid that mediate surface exposure of the peptide.

In the case of AAV2, it can be achieved by insertion before or after arginine 588 (R588), one of the two vital residues required for AAV2 binding to HSPG. In the AAV2-7m8 variant, 7m8 disrupts the HSPG-binding motif, taking the arginines apart (Figure 7) [71].

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AAV family shuffling (DNA Shuffling of AAV capsids)

A chimeric capsid–encoding library can be generated by AAV family shuffling (Figure 8). Fragments of various AAV capsid genes are re-assembled based on partial homologies (typically >80% for most AAV serotypes). This library contains chimeras with broad diversity in cell tropism and neutralizing antibody resistance.

This approach can also harness and blend functions from newly identified AAV serotypes, or even rationally designed AAV capsid mutants, to potentially create a combinatorially limitless number of novel variants with new properties [72].

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9. AAV Vector Transduction-AAV vector Gene Delivery in vivo

For normal tissues or organs, such as heart, liver, kidney, breast, pancreas, ovary, brain, eye, skeleton muscle, adipose tissue, etc., Genemedi systematically organizes the corresponding optimal AAV serotype, gene delivery method and injection volume for mouse and rat tissue infection. Recommended AAV gene delivery methods for different organs is shown in Table5.

Table5. AAV gene delivery methods in specific organs.
Infection organ Recommended serotype Injection route Animal Injection volume (μl)
Heart AAV9 Multiple points in situ Rat 10-15/point, 3-5 points
Mouse 10-15/point, 3-5 points
Tail vein Rat 250 (200g body weight)
Mouse 100
Liver AAV8 or AAV9 Tail vein Rat 200 (200g body weight)
Mouse 100
Whole brain AAV-PHP.eB AAV-PHP.B Tail vein Rat 200 (200g body weight)
Mouse 100
Lateral ventricle AAV9 Stereotactic Rat 1-5
Mouse 1-5
Brain tissue AAV9 Stereotactic Rat 2-3
Mouse 1-2
Fat AAV9 Intraperitoneal injection for intraperitoneal fat Rat 300
Mouse 150-200
in situ injection for subcutaneous fat Rat 10-15/point, 3-5 points
Mouse 10-15/point, 3-5 points
Skeletal muscle AAV1 or AAV9 In situ injection Rat 10-15/point, 3-5 points
Mouse 10-15/point, 3-5 points
Eyes AAV2, AAV10 or AAV-DJ vitreous chamber injection Rat 3-5
Mouse 1-3
subretinal space injection Rat 1-2
Mouse 1-2
Lung AAV6 Intratracheal injection Rat 100 (200g weight)
Mouse 50-75
Kidney AAV2 or AAV9 Renal pelvis injection Rat 10-15/point, 3-5 points
Mouse 10-15/point, 3-5 points
Intestine AAV1 or AAV5 Enema Rat 200 (200g weight)
Mouse 100

Detailed information of AAV in vivo animal injection protocol can be seen in AAV User Manual.

10. Adeno-associated virus (AAV vector) -Production Protocol,Guidelines And References

AAV Production Protocol--Genemedi

pdf下载AAV protocol Download

1. AAV plasmid construction

The gene of interest is cloned into one of the ITR/MCS-containing AAV vectors to generate AAV-GOI. The purity and RNAse contaminants of AAV viral plasmid should be taken into consideration.

2. AAV packaging

The recombinant AAV viral plasmid AAV-GOI is co-transfected into the AAV-293 cells with AAV helper plasmid pHelper (carrying adenovirus-derived genes) and AAV-RC (carrying AAV-2 replication and capsid genes), which together supply all the trans-acting factors required for AAV replication and packaging in the AAV-293 cells.

3. Harvesting AAV particles

Collecting the cells and freeze/thaw the cell pellet to release the AAV virus.

4. AAV purification

Purify the AAV virus with gradient ultracentrifugation to separate out contaminants from the impure AAV preparations.

5.AAV titer detection

Determine AAV titer by real-time quantitative PCR (QPCR) using primers targeting the AAV ITR. Titer values are then identified by comparison to a standard curve of a plasmid sample of known concentration.

6.Quality control of AAV

After AAV virus titer detection, the infection activity needs to be evaluated before animal experiments by infecting cell such as 293T, CHO to test the gene expression. MOI for AAV mediated gene transduction in cell will be controlled ranging from 10^4 to 10^5.

Adeno-associated virus (AAV vector) - Guidelines

pdf下载NIH Guidelines for Adeno-Associated (AAV) Lentivirus and Adenovirus pdf下载Guidance for Gene Therapy Clinical trials-FDA pdf下载Human Gene Therapy for Hemophilia
pdf下载Human Gene Therapy For Rare Diseases pdf下载Human Gene Therapy for Retinal Disorders

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2. Hoggan MD, NR Blacklow and WP Rowe. (1966). Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics. Proc Natl Acad Sci U S A 55:1467-74.
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