The Diversity of Parvovirus Telomeres

Parvoviridae are small viruses composed of a 4–6 kb linear single-stranded DNA protected by an icosahedral capsid. The viral genes coding non-structural (NS), capsid, and accessory proteins are flanked by intriguing sequences, namely the telomeres. Telomeres are essential for parvovirus genome replication, encapsidation, and integration. Similar (homotelomeric) or different (heterotelomeric) at the two ends, they all contain imperfect palindromes that fold into hairpin structures. Up to 550 nucleotides in length, they harbor a wide variety of motifs and structures known to be recognized by host cell factors. Our study aims to comprehensively analyze parvovirus ends to better understand the role of these particular sequences in the virus life cycle. Forty Parvoviridae terminal repeats (TR) were publicly available in databases. The folding and specific DNA secondary structures, such as G4 and triplex, were systematically analyzed. A principal component analysis was carried out from the prediction data to determine variables signing parvovirus groups. A special focus will be put on adeno-associated virus (AAV) inverted terminal repeats (ITR), a member of the genus Dependoparvovirus used as vectors for gene therapy. This chapter highlights the diversity of the Parvoviridae telomeres regarding shape and secondary structures, providing information that could be relevant for virus-host interactions studies

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 β1-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

Unraveling the Complex Story of Immune Responses to AAV Vectors Trial After Trial

International audienceOver the past decade, vectors derived from adeno-associated virus (AAV) have established themselves as apowerful tool for in vivo gene transfer, allowing long-lasting and safe transgene expression in a variety of humantissues. Nevertheless, clinical trials demonstrated how B and T cell immune responses directed against the AAVcapsid, likely arising after natural infection with wild-type AAV, might potentially impact gene transfer safetyand efficacy in patients. Seroprevalence studies have evidenced that most individuals carry anti-AAV neutralizingantibodies that can inhibit recombinant AAV transduction of target cells following in vivo administration ofvector particles. Likewise, liver- and muscle-directed clinical trials have shown that capsid-reactive memoryCD8+T cells could be reactivated and expanded upon presentation of capsid-derived antigens on transduced cells,potentially leading to loss of transgene expression and immune-mediated toxicities. In celebration of the 25thanniversary of the European Society of Gene and Cell Therapy, this review article summarizes progress madeduring the past decade in understanding and modulating AAV vector immunogenicity. While the knowledgegenerated has contributed to yield impressive clinical results, several important questions remain unanswered,making the study of immune responses to AAV a priority for the field of in vivo transfer

Lentiviral transduction of immune cells.

International audienceGene transfer into mammalian cells has been of crucial importance for studies determining the role of specific genes in the differentiation and cell fate of various hematopoietic lineages. Until recently, the majority of these studies were performed in transformed cell lines due to difficulties in achieving levels of transfection of greater than 1-3% in primary hematopoietic cells. Vectors based on retrovirus and lentivirus backbones have revolutionized our ability to transfer genes into primary hematopoietic cells. These vectors have allowed extensive ex vivo and in vivo studies following introduction of a gene of interest and have been used clinically in individuals suffering from cancers, infections, and genetic diseases. Ex vivo lentiviral gene transfer can result in efficient transduction of progenitor cells (>80%) that can then be further differentiated into immune lineage cells including T, B, dendritic, or natural killer cells. Alternatively, differentiated immune cells can themselves be transduced ex vivo with lentiviral vectors. Here, we discuss optimization of technologies for human immunodeficiency virus (HIV)-based gene transfer into murine and human progenitor and immune cell lineages

Risk Mitigation of Immunogenicity: A Key to Personalized Retinal Gene Therapy

Gene therapy (GT) for ocular disorders has advanced the most among adeno-associated virus (AAV)-mediated therapies, with one product already approved in the market. The bank of retinal gene mutations carefully compiled over 30 years, the small retinal surface that does not require high clinical vector stocks, and the relatively immune-privileged environment of the eye explain such success. However, adverse effects due to AAV-delivery, though rare in the retina have led to the interruption of clinical trials. Risk mitigation, as the key to safe and efficient GT, has become the focus of ‘bedside-back-to-bench’ studies. Herein, we overview the inflammatory adverse events described in retinal GT trials and analyze which components of the retinal immunological environment might be the most involved in these immune responses, with a focus on the innate immune system composed of microglial surveillance. We consider the factors that can influence inflammation in the retina after GT such as viral sensors in the retinal tissue and CpG content in promoters or transgene sequences. Finally, we consider options to reduce the immunological risk, including dose, modified capsids or exclusion criteria for clinical trials. A better understanding and mitigation of immune risk factors inducing host immunity in AAV-mediated retinal GT is the key to achieving safe and efficient GT

La thérapie génique des rétinites pigmentaires héréditaires

Les rétinites pigmentaires, ou dystrophies rétiniennes héréditaires, sont des maladies dégénératives cécitantes d’origine génétique. La thérapie génique est une approche révolutionnaire en plein essor qui ouvre la voie au traitement de maladies jusqu’ici incurables. Une thérapie génique, le Luxturna®, a obtenu une autorisation de mise sur le marché par la FDA (Food and Drug Administration) fin 2017 et l’EMA (European Medicines Agency) fin 2018. Ce traitement, à l’efficacité démontrée, destiné aux patients porteurs d’une amaurose congénitale de Leber ou d’une rétinopathie pigmentaire en lien avec une mutation bi-allélique du gène RPE65, apporte beaucoup plus de questions que de réponses. Nous présentons, dans cette revue, les avancées actuelles, puis les défis technologiques, économiques et éthiques à surmonter pour que la thérapie génique améliore nos pratiques médicales

Chem-AAV: Chemically Modified AAV for Targeting Gene Delivery

21st Annual Meeting of the American-Society-of-Gene-and-Cell-Therapy (ASGCT), Chicago, IL, MAY 16-19, 2018International audienc

The SSV‐Seq 2.0 PCR‐Free Method Improves the Sequencing of Adeno‐Associated Viral Vector Genomes Containing GC‐Rich Regions and Homopolymers

International audienceAdeno-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

Analytical ultracentrifugation sedimentation velocity for the characterization of recombinant adeno-associated virus vectors sub-populations

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

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. Keywords: AAV vectors, gene therapy, quality control, titration, infectivit