Search

Decades of biological and clinical research have led to important advances in recombinant adeno-associated viruses rAAV-based gene therapy gene therapy. However, several challenges must be overcome to fully exploit the potential of rAAV vectors. Innovative approaches to modify viral genome and capsid elements have been used to overcome issues such as unwanted immune responses and off-targeting. While often successful, genetic modification of capsids can drastically reduce vector yield and often fails to produce vectors with properties that translate across species. Here, we describe a chemical bioconjugation strategy to modify tyrosine residues on AAV capsids using specific ligands, thereby circumventing the need to genetically engineer the capsid sequence. Aromatic electrophilic substitution of the phenol ring of tyrosine residues on AAV capsids improved the in vivo transduction efficiency of rAAV2 vectors in both liver and retinal targets. This tyrosine bioconjugation strategy represents an innovative technology for the engineering of rAAV vectors for human gene therapy.

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 forin vivotherapeutic 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 immune 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,viathe 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.

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.

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; The development of new bio-orthogonal ligation methods for the conjugation of native proteins is of particular importance in the field of chemical biology and biotherapies. In this work, we developed a traceless electrochemical method for protein bioconjugation. The electrochemically promoted tyrosine-click (e-Y-CLICK) allowed the chemoselective Y-modification of peptides and proteins with labeled urazoles. A low potential is applied in an electrochemical cell to activate urazole anchors in situ and on demand, without affecting the electroactive amino acids from the protein. The versatility of the electrosynthetic approach was shown on biologically relevant peptides and proteins such as oxytocin, angiotensin 2, serum bovine albumin, and epratuzumab. The fully conserved enzymatic activity of a glucose oxidase observed after e-Y-CLICK further highlights the softness of the method. The e-Y-CLICK protocols were successfully performed in pure aqueous buffers, without the need for co-solvents, scavenger or oxidizing chemicals, and should therefore significantly broaden the scope of bioconjugation.

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

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.

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.