Mannose-coupled AAV2: A second-generation AAV vector for increased retinal gene therapy efficiency.

Inherited retinal diseases are a leading and untreatable cause of blindness and are therefore candidate diseases for gene therapy. Recombinant vectors derived from adeno-associated virus (rAAV) are currently the most promising vehicles for in vivo therapeutic gene delivery to the retina. However, there is a need for novel AAV-based vectors with greater efficacy for ophthalmic applications, as underscored by recent reports of dose-related inflammatory responses in clinical trials of rAAV-based ocular gene therapies. Improved therapeutic efficacy of vectors would allow for decreases in the dose delivered, with consequent reductions in inflammatory reactions. Here, we describe the development of new rAAV vectors using bioconjugation chemistry to modify the rAAV capsid, thereby improving the therapeutic index. Covalent coupling of a mannose ligand, via the formation of a thiourea bond, to the amino groups of the rAAV capsid significantly increases vector transduction efficiency of both rat and nonhuman primate retinas. These optimized rAAV vectors have important implications for the treatment of a wide range of retinal diseases.

Evaluation of an AAV9-mini-dystrophin gene therapy candidate in a rat model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by loss-of-function mutations in the dystrophin gene and is characterized by muscle wasting and early mortality. Adeno-associated virus-mediated gene therapy is being investigated as a treatment for DMD. In the nonclinical study documented here, we determined the effective dose of fordadistrogene movaparvovec, a clinical candidate adeno-associated virus serotype 9 vector carrying a human mini-dystrophin transgene, after single intravenous injection in a dystrophin-deficient (DMDmdx) rat model of DMD. Overall, we found that transduction efficiency, number of muscle fibers expressing the human mini-dystrophin polypeptide, improvement of the skeletal and cardiac muscle tissue architecture, correction of muscle strength and fatigability, and improvement of diastolic and systolic cardiac function were directly correlated with the amount of vector administered. The effective dose was then tested in older DMDmdx rats with a more dystrophic phenotype similar to the pathology observed in older patients with DMD. Except for a less complete rescue of muscle function in the oldest cohort, fordadistrogene movaparvovec was also found to be therapeutically effective in older DMDmdx rats, suggesting that this product may be appropriate for evaluation in patients with DMD at all stages of disease.

Cellular and Structural Characterization of VP1 and VP2 Knockout Mutants of AAV3B Serotype and Implications for AAV Manufacturing.

AAV virion biology is still lacking a complete understanding of the role that the various structural subunits (VP1, 2, and 3) play in virus assembly, infectivity, and therapeutic delivery for clinical indications. In this study, we focus on the less studied adeno-associated virus AAV3B and generate a collection of AAV plasmid substrates that assemble virion particles deficient specifically in VP1, VP2, or VP1 and 2 structural subunits. Using a collection of biological and structural assays, we observed that virions devoid of VP1, VP2, or VP1 and 2 efficiently assembled virion particles, indistinguishable by cryoelectron microscopy (cryo-EM) from that of wild type (WT), but unique in virion transduction (WT > VP2 > VP1 > VP1 and 2 mutants). We also observed that the missing structural subunit was mostly compensated by additional VP3 protomers in the formed virion particle. Using cryo-EM analysis, virions fell into three classes, namely full, empty, and partially filled, based on comparison of density values within the capsid. Further, we characterize virions described as "broken" or "disassembled" particles, and provide structural information that supports the particle dissolution occurring through the two-fold symmetry sites. Finally, we highlight the unique value of employing cryo-EM as an essential tool for release criteria with respect to AAV manufacturing.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

AAV8 locoregional delivery induces long-term expression of an immunogenic transgene in macaques despite persisting local inflammation.

Adeno-associated virus (AAV) vectors are considered efficient vectors for gene transfer, as illustrated by recent successful clinical trials targeting retinal or neurodegenerative disorders. However, limitations as host immune responses to AAV capsid or transduction of limited regions must still be overcome. Here, we focused on locoregional (LR) intravenous perfusion vector delivery that allows transduction of large muscular areas and is considered to be less immunogenic than intramuscular (IM) injection. To confirm this hypothesis, we injected 6 cynomolgus monkeys with an AAV serotype 8 (AAV8) vector encoding for the highly immunogenic GFP driven by either a muscle-specific promoter (n = 3) or a cytomegalovirus (CMV) promoter (n = 3). We report that LR delivery allows long-term GFP expression in the perfused limb (up to 1 year) despite the initiation of a peripheral transgene-specific immune response. The analysis of the immune status of the perfused limb shows that LR delivery induces persisting inflammation. However, this inflammation is not sufficient to result in transgene clearance and is balanced by resident regulatory T cells. Overall, our results suggest that LR delivery promotes persisting transgene expression by induction of Treg cells in situ and might be a safe alternative to IM route to target large muscle territories for the expression of secreted therapeutic factors.

Intra-CSF AAV9 and AAVrh10 Administration in Nonhuman Primates: Promising Routes and Vectors for Which Neurological Diseases?

The identification of the most efficient method for whole central nervous system targeting that is translatable to humans and the safest route of adeno-associated virus (AAV) administration is a major concern for future applications in clinics. Additionally, as many AAV serotypes were identified for gene introduction into the brain and the spinal cord, another key to human gene-therapy success is to determine the most efficient serotype. In this study, we compared lumbar intrathecal administration through catheter implantation and intracerebroventricular administration in the cynomolgus macaque. We also evaluated and compared two AAV serotypes that are currently used in clinical trials: AAV9 and AAVrh10. We demonstrated that AAV9 lumbar intrathecal delivery using a catheter achieved consistent transgene expression in the motor neurons of the spinal cord and in the neurons/glial cells of several brain regions, whereas AAV9 intracerebroventricular delivery led to a consistent transgene expression in the brain. In contrast, AAVrh10 lumbar intrathecal delivery led to rare motor neuron targeting. Finally, we found that AAV9 efficiently targets respiratory and skeletal muscles after injection into the cerebrospinal fluid (CSF), which represents an outstanding new property that can be useful for the treatment of diseases affecting both the central nervous system and muscle.

Capsid-specific removal of circulating antibodies to adeno-associated virus vectors.

Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.

Five Years of Successful Inducible Transgene Expression Following Locoregional Adeno-Associated Virus Delivery in Nonhuman Primates with No Detectable Immunity.

Anti-transgene immune responses elicited after intramuscular (i.m.) delivery of recombinant adeno-associated virus (rAAV) have been shown to hamper long-term transgene expression in large-animal models of rAAV-mediated gene transfer. To overcome this hurdle, an alternative mode of delivery of rAAV vectors in nonhuman primate muscles has been described: the locoregional (LR) intravenous route of administration. Using this injection mode, persistent inducible transgene expression for at least 1 year under the control of the tetracycline-inducible Tet-On system was previously reported in cynomolgus monkeys, with no immunity against the rtTA transgene product. The present study shows the long-term follow-up of these animals. It is reported that LR delivery of a rAAV2/1 vector allows long-term inducible expression up to at least 5 years post gene transfer, with no any detectable host immune response against the transactivator rtTA, despite its immunogenicity following i.m. gene transfer. This study shows for the first time a long-term regulation of muscle gene expression using a Tet-On-inducible system in a large-animal model. Moreover, these findings further confirm that the rAAV LR delivery route is efficient and immunologically safe, allowing long-term skeletal muscle gene transfer.

Tetramer-Based Enrichment of Preexisting Anti-AAV8 CD8+ T Cells in Human Donors Allows the Detection of a TEMRA Subpopulation.

Pre-existing immunity to AAV capsid may compromise the safety and efficiency of rAAV-mediated gene transfer in patients. Anti-capsid cytotoxic immune responses have proven to be a challenge to characterize because of the scarcity of circulating AAV-specific CD8+ T lymphocytes which can seldom be detected with conventional flow cytometry or ELISpot assays. Here, we used fluorescent MHC class I tetramers combined with magnetic enrichment to detect and phenotype AAV8-specific CD8+ T cells in human PBMCs without prior amplification. We showed that all healthy individuals tested carried a pool of AAV8-specific CD8+ T cells with a CD45RA+ CCR7- terminally-differentiated effector memory cell (TEMRA) fraction. Ex vivo frequencies of total AAV-specific CD8+ T cells were not predictive of IFNγ ELISpot responses but interestingly we evidenced a correlation between the proportion of TEMRA cells and IFNγ ELISpot positive responses. TEMRA cells may then play a role in recombinant AAV-mediated cytotoxicity in patients with preexisting immunity. Overall, our results encourage the development of new methods combining increased detection sensitivity of AAV-specific T cells and their poly-functional assessment to better characterize and monitor AAV capsid-specific cellular immune responses in the perspective of rAAV-mediated clinical trials.

Chemical modification of the adeno-associated virus capsid to improve gene delivery.

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.

Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls.

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.

RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect.

Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.

Safety and Long-Term Efficacy of AAV4 Gene Therapy in Patients with RPE65 Leber Congenital Amaurosis.

The aim of this study was the evaluation of the safety and efficacy of unilateral subretinal injection of the adeno-associated vector (AAV) serotypes 2 and 4 (AAV2/4) RPE65-RPE65 vector in patients with Leber congenital amaurosis (LCA) associated with RPE65 gene deficiency. We evaluated ocular and general tolerance and visual function up to 1 year after vector administration in the most severely affected eye in nine patients with retinal degeneration associated with mutations in the RPE65 gene. Patients received either low (1.22 × 1010 to 2 × 1010 vector genomes [vg]) or high (between 3.27 × 1010 and 4.8 × 1010 vg) vector doses. An ancillary study, in which six of the original nine patients participated, extended the follow-up period to 2-3.5 years. All patients showed good ophthalmological and general tolerance to the rAAV2/4-RPE65-RPE65 vector. We observed a trend toward improved visual acuity in patients with nystagmus, stabilization and improvement of the visual field, and cortical activation along visual pathways during fMRI analysis. OCT analysis after vector administration revealed no retinal thinning, except in cases of macular detachment. Our findings show that the rAAV2/4.RPE65.RPE65 vector was well tolerated in nine patients with RPE65-associated LCA. Efficacy parameters varied between patients during follow-up.

Long-term microdystrophin gene therapy is effective in a canine model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV-microdystrophin gene therapy in DMD patients.

Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells.

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 (≤0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.

AAV-ID: A Rapid and Robust Assay for Batch-to-Batch Consistency Evaluation of AAV Preparations.

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.

Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons.

Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

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.

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6ß-deficient Dogs.

We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing.

Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.