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Recombinant adeno-associated virus virus-derived vectors (rAAVs) are among the most used viral delivery system for in vivo gene therapies with a good safety profile. However, rAAV production methods often lead to a heterogeneous vector population, in particular with the presence of undesired empty particles. Analytical ultracentrifugation sedimentation velocity (AUC-SV) is considered as the gold analytical technique allowing the measurement of relative amounts of each vector subpopulation and components like particle aggregates, based on their sedimentation coefficients. This letter presents the principle and practice of AUC experiments for rAAVs characterization. We discuss our results in the framework of previously published works. In addition to classical detection at 260 nm, using interference optics in the ultracentrifuge can provide an independent estimate of weight percentages of the different populations of capsids, and of the genome size incorporated in rAAV particles.

In the past two decades, adeno-associated virus (AAV) vector manufacturing has made remarkable advancements to meet large-scale production demands for preclinical and clinical trials. In addition, AAV vectors have been extensively studied for their safety and efficacy. In particular, the presence of empty AAV capsids and particles containing “inaccurate” vector genomes in preparations has been a subject of concern. Several methods exist to separate empty capsids from full particles; but thus far, no single technique can produce vectors that are free of empty or partial (non-unit length) capsids. Unfortunately, the exact genome compositions of full, intermediate, and empty capsids remain largely unknown. In this work, we used AAV-genome population sequencing to explore the compositions of DNase-resistant, encapsidated vector genomes produced by two common production pipelines: plasmid transfection in human embryonic kidney cells (pTx/HEK293) and baculovirus expression vectors in Spodoptera frugiperda insect cells (rBV/Sf9). Intriguingly, our results show that vectors originating from the same construct design that were manufactured by the rBV/Sf9 system produced a higher degree of truncated and unresolved species than those generated by pTx/HEK293 production. We also demonstrate that empty particles purified by cesium chloride gradient ultracentrifugation are not truly empty but are instead packaged with genomes composed of a single truncated and/or unresolved inverted terminal repeat (ITR). Our data suggest that the frequency of these “mutated” ITRs correlates with the abundance of inaccurate genomes in all fractions. These surprising findings shed new light on vector efficacy, safety, and how clinical vectors should be quantified and evaluated.

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long-term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy.

International audience; Recombinant adeno-associated virus (AAV) vectors have evolved as the most promising technology for gene therapy due to their good safety profile, high transduction efficacy, and long-term gene expression in non-dividing cells. AAV-based gene therapy holds great promise for treating genetic disorders like inherited blindness, muscular atrophy, or bleeding disorders. Multiple naturally occurring and engineered AAV serotypes exist, which differ in capsid sequence and as a consequence in cellular tropism. Individual AAV capsids differ in thermal stability and have a characteristic melting temperature (Tm), which enables serotype-specific discrimination of AAV vectors. Differential scanning fluorimetry (DSF) combined with a dye-like SYPRO Orange (SO-DSF), which binds to hydrophobic regions of unfolded proteins, has been successfully applied to determine the Tm of AAV capsids. Here, we present DSF measurement of intrinsic fluorescence signal (iDSF) as a simple alternative method for determination of AAV capsid Tm. The study demonstrates that DSF measurement of intrinsic fluorescence signal is a simple, accurate, and rapid alternative to SO-DSF, which enables characterization of AAV capsid stability with excellent precision and without the need of SO or any other dye.

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells and sustained maintenance of the viral genome. However, the conclusion from clinical data using these vectors is that there is a need to develop new AAVs with a higher transduction efficiency and specificity for relevant target tissues. To overcome these limitations, we chemically modified the surface of the capsid of AAV vectors. These modifications were achieved by chemical coupling of a ligand by the formation of a thiourea functionality between the amino group of the capsid proteins and the reactive isothiocyanate motif incorporated into the ligand. This strategy does not require genetic engineering of the capsid sequence. The proof of concept was first evidenced using a fluorophore (FITC). Next, we coupled the N-acetylgalactosamine ligand onto the surface of the AAV capsid for asialoglycoprotein receptor-mediated hepatocyte-targeted delivery. Chemically-modified capsids also showed reduced interactions with neutralizing antibodies. Taken together, our findings reveal the possibility of creating a specific engineered platform for targeting AAVs via chemical coupling., Bioconjugated AAV vectors, achieved by coupling of ligands on amino groups of the capsid, are of great interest for gene delivery. Chemical modifications can be used to enhance cell tropism and to decrease interactions with neutralizing antibodies.

International audience; Viral vectors have a great potential for gene delivery, but manufacturing is a big challenge for the industry. The baculovirus-insect cell is one of the most scalable platforms to produce recombinant adeno-associated virus (rAAV) vectors. The standard procedure to generate recombinant baculovirus is based on Tn7 transposition which is time-consuming and suffers technical constraints. Moreover, baculoviral sequences adjacent to the AAV ITRs are preferentially encapsidated into the rAAV vector particles. This observation raises concerns about safety due to the presence of bacterial and antibiotic resistance coding sequences with a Tn7-mediated system for the construction of baculoviruses reagents. Here, a faster and safer method based on homologous recombination (HR) is investigated. First, the functionality of the inserted cassette and the absence of undesirable genes into HR-derived baculoviral genomes are confirmed. Strikingly, it is found that the exogenous cassette showed increased stability over passages when using the HR system. Finally, both materials generated high rAAV vector genome titers, with the advantage of the HR system being exempted from undesirable bacterial genes which provides an additional level of safety for its manufacturing. Overall, this study highlights the importance of the upstream process and starting biologic materials to generate safer rAAV biotherapeutic products.

With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

With the success of clinical trials using recombinant adeno-associated viral vectors (rAAV), regulatory agencies ask for a more comprehensive characterization of process- and product- related impurities found in rAAV stocks in order to assess the potential risks for patients. During production, rAAV capsids are known to internalize illegitimate DNA fragments in addition to their recombinant genome. These contaminants can come from plasmid or helper virus DNA as well as from the producer host cell. Here, we describe a method based on high-throughput sequencing to identify and quantify residual DNA in rAAV vector lots. Contrary to qPCR, SSV-Seq (Single-Stranded DNA Virus Sequencing) offers a nonselective approach to determine the percentage of each DNA contaminant and analyze rAAV vector genome identity.

International audience; Adeno-associated virus (AAV) vectors are extensively used for gene therapy clinical trials. Accurate and standardized titration methods are essential for characterizing and dosing AAV-based drugs and thus to assess their safety and efficacy. To this end, the Reference Standard Materials (RSM) working group generated standards for AAV serotype 2 and serotype 8. The AAV8RSM (ATCC® VR-1816™) was deposited to the American Type Culture Collection in 2014 and is available to the scientific community. Here, three independent laboratories of the RSM working group provide stability data of the AAV8RSM 2 years after the initial characterization and after container relabeling performed at the ATCC. The AAV8RSM showed constant titers across experimental conditions: 1.48 ± 0.62 × 1012 vector genome (vg)/ml, 9.38 ± 11.4 × 108 infectious units (IU)/ml and 5.76 ± 2.39 × 1011 total particles (p)/ml as determined by qPCR, TCID50 and ELISA, respectively. Additionally, the AAV8RSM capsid protein integrity assessed by SDS-PAGE was equivalent to the original analyses. In conclusion, the AAV8RSM titers remained stable for two years under appropriate storage conditions (

International audience; Adeno-associated viral vectors (AAV) are efficient engineered tools for delivering genetic material into host cells. The commercialization of AAV-based drugs must be accompanied by the development of appropriate quality control (QC) assays. Given the potential risk of co-transfer of oncogenic or immunogenic sequences with therapeutic vectors, accurate methods to assess the level of residual DNA in AAV vector stocks are particularly important. An assay based on high-throughput sequencing (HTS) to identify and quantify DNA species in recombinant AAV batches is developed. Here, it is shown that PCR amplification of regions that have a local GC content >90% and include successive mononucleotide stretches, such as the CAG promoter, can introduce bias during DNA library preparation, leading to drops in sequencing coverage. To circumvent this problem, SSV-Seq 2.0, a PCR-free protocol for sequencing AAV vector genomes containing such sequences, is developed. The PCR-free protocol improves the evenness of the rAAV genome coverage and consequently leads to a more accurate relative quantification of residual DNA. HTS-based assays provide a more comprehensive assessment of DNA impurities and AAV vector genome integrity than conventional QC tests based on real-time PCR and are useful methods to improve the safety and efficacy of these viral vectors.

Although the clinical use of recombinant adeno-associated virus (rAAV) vectors is constantly increasing, the development of suitable quality control methods is still needed for accurate vector characterization. Among the quality criteria, the titration of infectious particles is critical to determine vector efficacy. Different methods have been developed for the measurement of rAAV infectivity in vitro, based on detection of vector genome replication in trans-complementing cells infected with adenovirus, detection of transgene expression in permissive cells, or simply detection of intracellular vector genomes following the infection of indicator cells. In the present study, we have compared these methods for the titration of infectious rAAV8 vector particles, and, to assess their ability to discriminate infectious and non-infectious rAAV serotype 8 particles, we have generated a VP1-defective AAV8-GFP vector. Since VP1 is required to enter the cell nucleus, the lack of VP1 should drastically reduce the infectivity of rAAV particles. The AAV8 reference standard material was used as a positive control. Our results demonstrated that methods based on measurement of rAAV biological activity (i.e., vector genome replication or transgene expression) were able to accurately discriminate infectious versus non-infectious particles, whereas methods simply measuring intracellular vector genomes were not. Several cell fractionation protocols were tested in an attempt to specifically measure vector genomes that had reached the nucleus, but genomes from wild-type and VP1-defective AAV8 particles were equally detected in the nuclear fraction by qPCR. These data highlight the importance of using suitable controls, including a negative control, for the development of biological assays such as infectious unit titration. Keywords: AAV vectors, gene therapy, quality control, titration, infectivity

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans -complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production.

Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (

International audience; Adeno-associated virus (AAV) vectors are promising clinical candidates for therapeutic gene transfer, and a number of AAV-based drugs may emerge on the market over the coming years. To insure the consistency in efficacy and safety of any drug vial that reaches the patient, regulatory agencies require extensive characterization of the final product. Identity is a key characteristic of a therapeutic product, as it ensures its proper labeling and batch-to-batch consistency. Currently, there is no facile, fast, and robust characterization assay enabling to probe the identity of AAV products at the protein level. Here, we investigated whether the thermostability of AAV particles could inform us on the composition of vector preparations. AAV-ID, an assay based on differential scanning fluorimetry (DSF), was evaluated in two AAV research laboratories for specificity, sensitivity, and reproducibility, for six different serotypes (AAV1, 2, 5, 6.2, 8, and 9), using 67 randomly selected AAV preparations. In addition to enabling discrimination of AAV serotypes based on their melting temperatures, the obtained fluorescent fingerprints also provided information on sample homogeneity, particle concentration, and buffer composition. Our data support the use of AAV-ID as a reproducible, fast, and low-cost method to ensure batch-to-batch consistency in manufacturing facilities and academic laboratories.

International audience; Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new “Free-ITR” qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.

Adeno-associated virus (AAV) vectors have emerged as key clinical candidates for gene therapy. Yet, the efficiency and safety of these 20-25 nm biological nanoparticles remain difficult to harmonize across pre-clinical studies due to the limitations of current analytical tools. The presence of residual DNA, protein contaminants, empty particles and VP subunits resulting from incomplete capsid assembly are variables that can strongly modulate the reliability of in vivo data and that, therefore, need to be closely monitored in AAV research laboratories. In this work, 70 AAV preparations, obtained with various production (baculo/Sf9 and triple transfection system) and purification (iodixanol gradient and double cesium-chloride gradient) techniques were analyzed using a thermal shift assay based on the fluorescent dye Sypro® Orange. The fluorescence fingerprint obtained did not only allow to discriminate various AAV serotypes based on their capsid melting temperatures, but also enabled to probe the homogeneity and purity of AAV vector preparations, investigated in parallel using dynamic light scattering (DLS) and polyacrylamide gel electrophoresis. In particular, a double fluorescence transition indicated the presence of capsid-associated protein contaminants whereas a high initial fluorescence background correlated with the presence of free protein contaminants and capsid subunits, possibly resulting from capsid degradation during vector purification or storage. The variability, sensitivity and precision of this assay were further investigated in two different AAV research laboratories. This simple, fast (analysis of 94 preps in ~6 hrs) and low-cost assay emerges as a relevant tool for characterization of AAV vector preparations and will help to increase the reliability of in vivo gene transfer studies.

A 2010 report that Adeno-associated virus 2 (AAV2) encodes an additional protein in the second open reading frame of the cap(sid) gene significantly changed our perception of the biology of this virus. This so-called assembly-activating protein or AAP appears to interact with the major viral capsid protein 3 (VP3) and to chaperone its assembly into virus-like particles, via mechanisms that remain unknown. Moreover, it was shown that deliberate mutation of the AAP gene diminishes AAV particle production.As the AAP open reading frame fully overlaps with that of the AAV capsid proteins VP1/VP2 (and partially VP3), it is likely that the AAP gene is inadvertently disrupted or mutated during molecular AAV evolution protocols that involve capsid gene shuffling (homology-directed recombination of cap gene fragments). To study this possibility comprehensively and to provide solutions, if needed, we first investigated the role of AAP for 9 further AAV serotypes other than AAV2. Therefore, we knocked out AAP expression in serotypes 1, 3-9 and rh10 while leaving the rep and cap genes intact. Indeed, all mutants were markedly impaired in particle production, confirming that functional AAP is critically required for at least 10 different AAV serotypes.Notably, all vectors were fully restored upon co-delivery of the corresponding AAP via an extra plasmid during particle production. Furthermore, when testing the cross-reactivity of all AAPs with the AAV2-AAP mutant (AAV2mut), we observed a broad interchangeability except for AAP4 and 5 which predominantly rescued their cognate serotype.These results were confirmed independently in a baculovirus AAV2 vector production system where we also observed a dramatic reduction in particle yields upon AAP mutation. Interestingly, in both, mammalian and insect cells, we consistently noted that absence of AAP reduced the steady-state levels of VP proteins, suggesting a similar biological function of AAP in the two heterologous cellular systems. As we could rule out an effect on the transcriptional level, we postulate that AAP-mediated chaperoning of AAV virion assembly indirectly stabilizes VP proteins in cells of different species.To explicitly dissect the role of AAP in the context of molecular AAV evolution, we produced five different AAV capsid libraries with increasing complexities, subcloned 46 randomly selected shuffled AAPs from these and tested them with the AAV-AAP mutants. Out of these 46 shuffled AAPs, 37 could rescue AAV2mut, and two more efficiently complemented an AAV5-AAP mutant. In total, 84.8 % of all tested AAPs were functional.Of note, the highest proportion of defect AAPs was found in libraries that contained the diverse capsids of AAV4 and 5. We thus shuffled serotypes 2, 4, 5, 8 and 9 and produced viral libraries with or without a cocktail of excess AAP from all five serotypes. Strikingly, albeit up to 40% of all AAP genes were predicted to be non-functional as a result of inadvertent shuffling, AAP trans-complementation had no significant effect on particle yields.Collectively, our data suggest that albeit AAP is disrupted to some extent during AAV capsid gene shuffling, this is likely not a major restriction for the efficiency and success of this technology.

Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression in a wide variety of animals, including primates. The rAAV genome is packaged into the virion as single-stranded DNA devoid of any viral genes. A proportion of the single-stranded genomes are converted into transcriptionally active double-stranded DNA (dsDNA) early after nuclear entry by second-strand synthesis mediated by host repair DNA polymerases or/and by annealing of the rAAV (-) and (+) strands. Second-generation, self-complementary vectors are packaged as single-strand hairpins and rapidly assume a dsDNA conformation independent of the action of polymerases. In both cases, linear dsDNA vector genomes circularize and can undergo concatemerization into higher order forms (McCarty et al. Annu Rev Genet 38: 819-845, 2004; Schultz and Chamberlain Mol Ther 16: 1189-1199, 2008; Duan et al. J Virol 72: 8568-8577, 1998). As a result, rAAV vector genomes are maintained mainly as circular monomeric and concatemeric episomal forms in skeletal muscle and liver (Schnepp et al. J Virol 77: 3495-3504, 2003; Penaud-Budloo et al. J Virol 82: 7875-7885, 2008; Nakai et al. J Virol 75: 6969-6976, 2001). Moreover, in nonhuman primate skeletal muscle, it has been shown that rAAV episomes assimilate into chromatin with a typical nucleosomal pattern that presumably is important for persistence and gene expression in quiescent tissues over a period of several years (Penaud-Budloo et al. J Virol 82: 7875-7885, 2008). Conversely, although rAAV is not considered as an integrative vector per se, introduction of exogenous DNA into the nuclear compartment can result in low-level vector assimilation into the host genome. One mechanism appears to involve vector insertion at sites of double-strand DNA breaks using cellular DNA repair enzymes. As rAAV gene transfer technology and applications mature, a better characterization of the genetic fate of the rAAV genome is critical to accurately evaluate the risk/benefit ratio for a particular disease indication. In this chapter, two complementary methods are detailed to enable characterization of rAAV molecular structure in a particular target tissue and estimation of its integration frequency.

Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression in a wide variety of animals, including primates. The rAAV genome is packaged into the virion as single-stranded DNA devoid of any viral genes. A proportion of the single-stranded genomes are converted into transcriptionally active double-stranded DNA (dsDNA) early after nuclear entry by second-strand synthesis mediated by host repair DNA polymerases or/and by annealing of the rAAV (-) and (+) strands. Second-generation, self-complementary vectors are packaged as single-strand hairpins and rapidly assume a dsDNA conformation independent of the action of polymerases. In both cases, linear dsDNA vector genomes circularize and can undergo concatemerization into higher order forms (McCarty et al. Annu Rev Genet 38: 819-845, 2004; Schultz and Chamberlain Mol Ther 16: 1189-1199, 2008; Duan et al. J Virol 72: 8568-8577, 1998). As a result, rAAV vector genomes are maintained mainly as circular monomeric and concatemeric episomal forms in skeletal muscle and liver (Schnepp et al. J Virol 77: 3495-3504, 2003; Penaud-Budloo et al. J Virol 82: 7875-7885, 2008; Nakai et al. J Virol 75: 6969-6976, 2001). Moreover, in nonhuman primate skeletal muscle, it has been shown that rAAV episomes assimilate into chromatin with a typical nucleosomal pattern that presumably is important for persistence and gene expression in quiescent tissues over a period of several years (Penaud-Budloo et al. J Virol 82: 7875-7885, 2008). Conversely, although rAAV is not considered as an integrative vector per se, introduction of exogenous DNA into the nuclear compartment can result in low-level vector assimilation into the host genome. One mechanism appears to involve vector insertion at sites of double-strand DNA breaks using cellular DNA repair enzymes. As rAAV gene transfer technology and applications mature, a better characterization of the genetic fate of the rAAV genome is critical to accurately evaluate the risk/benefit ratio for a particular disease indication. In this chapter, two complementary methods are detailed to enable characterization of rAAV molecular structure in a particular target tissue and estimation of its integration frequency.

Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (i.m.) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2-3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the i.m. or the intravenous (i.v.) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.

Recombinant adeno-associated virus (rAAV) vectors are capable of mediating long-term gene expression following administration to skeletal muscle. In rodent muscle, the vector genomes persist in the nucleus in concatemeric episomal forms. Here, we demonstrate with nonhuman primates that rAAV vectors integrate inefficiently into the chromosomes of myocytes and reside predominantly as episomal monomeric and concatemeric circles. The episomal rAAV genomes assimilate into chromatin with a typical nucleosomal pattern. The persistence of the vector genomes and gene expression for years in quiescent tissues suggests that a bona fide chromatin structure is important for episomal maintenance and transgene expression. These findings were obtained from primate muscles transduced with rAAV1 and rAAV8 vectors for up to 22 months after intramuscular delivery of 5 × 10 12 viral genomes/kg. Because of this unique context, our data, which provide important insight into in situ vector biology, are highly relevant from a clinical standpoint.

International audience; We developed a drug-free regional intravenous (RI) delivery protocol of recombinant adeno-associated virus (rAAV) 1 and 8 to an entire limb in the nonhuman primate (NHP), and compared the results with those produced by intramuscular (IM) delivery of the same dose of vector. We show that RI delivery of both serotypes was remarkably well tolerated with no adverse side-effects. After IM, muscle transduction was restricted to the site of injection with a high number of vector copies per cell for rAAV1. In contrast, although RI delivery resulted in a lower vector copy per cell, it was detectable in the vast majority of muscles of the injected limb. The amounts of circulating infectious rAAV were similar for both serotypes and modes of delivery. At autopsy at up to 34 months after vector administration, similar biodistribution patterns were found for both vectors and for both modes of delivery, with numerous organs found to be positive for vector sequence when assayed using PCR and Southern blot. Altogether, we demonstrated that RI is a simple and efficient transduction protocol in NHPs, resulting in higher expression of the transgene with a lower number of vector genomes per cell. However, regardless of the mode of delivery, concerns continue to be raised by the presence of vector sequences detected at distant sites.