Prediction of second neurological attack in patients with clinically isolated syndrome using support vector machines

The aim of this study is to predict the conversion from clinically isolated syndrome to clinically definite multiple sclerosis using support vector machines. The two groups of converters and non-converters are classified using features that were calculated from baseline data of 73 patients. The data consists of standard magnetic resonance images, binary lesion masks, and clinical and demographic information. 15 features were calculated and all combinations of them were iteratively tested for their predictive capacity using polynomial kernels and radial basis functions with leave-one-out cross-validation. The accuracy of this prediction is up to 86.4% with a sensitivity and specificity in the same range indicating that this is a feasible approach for the prediction of a second clinical attack in patients with clinically isolated syndromes, and that the chosen features are appropriate. The two features gender and location of onset lesions have been used in all feature combinations leading to a high accuracy suggesting that they are highly predictive. However, it is necessary to add supporting features to maximise the accuracy. © 2013 IEEE

Thermostabilité des Virus Adéno-Associés (AAV)

Les virus adéno-associés (AAVs) sont des virus à ADN simple brin, nonenveloppés, considérés comme des candidats de choix pour la thérapie génique. Pour augmenter les chances de succès des thérapies géniques basées sur l’AAV, des efforts sont actuellement faits pour développer de nouvelles capsides virales, qui seraient plus résistantes à l’immunité préexistante, plus spécifiques de certains tissus, et compatibles avec une production à grande échelle. L’un des défis posés par le développement de nouveaux vecteurs consiste à comprendre comment conférer de nouvelles fonctions biologiques aux capsides d’AAVs, sans compromettre leur intégrité structurale. Pour ce faire, il est nécessaire d’améliorer notre compréhension des mécanismes gouvernant la métastabilité des capsides d’AAVs. L’objectif de cette thèse était d’étudier la thermostabilité des AAVs, ses liens avec leurs propriétés biologiques, ainsi que ses applications dans le domaine du contrôle qualité des préparations d’AAVs recombinants Dans un premier temps, nous étendons les travaux existants à l’étude de virus AAVs ancestraux (AncAAVs), reconstruits in silico. Nous montrons que Anc80, l’ancêtre commun prédit d’AAV1, 2, 8 et 9, est plus thermostable que ses descendants (ΔT = 15-20°C). Nous identifions ensuite, par une analyse de type phénotype-phylogénie, 12 acides aminés jouant potentiellement un rôle important dans la stabilisation des capsides virales. Nous montrons ensuite que la thermostabilité des capsides d’AAVs, mesurée par fluorimétrie différentielle à balayage (DSF), est utile pour déterminer, à l’échelle protéique, l’identité des préparations de vecteurs viraux, une opération requise par les agences réglementaires. Pour finir, nous appliquons ce test d’identité à l’étude de l’homogénéité structurale des librairies d’AAVs. Ces travaux de thèse pourraient s’avérer utiles pour développement et le manufacturing de nouveaux AAVs recombinants pour la thérapie génique.Adeno-associated virus (AAV) vectors have emerged as promising gene delivery vehicles for gene therapy. To improve the probability of success of AAV-based therapeutic strategies, efforts are currently being made to engineer novel capsids able to produce and purify well, escape pre-existing immunity, and target specific cell populations more efficiently. One challenge in AAV vector engineering is to understand how to confer new functions to the viral capsid without altering its structural integrity. To do so, there is a critical need to gain further knowledge on the mechanisms steering AAV capsid metastability. The objective of this thesis is to investigate the thermal stability of AAVs, its impact on AAV biology, and applications to quality control of AAV preparations. First, we extend existing thermal stability studies to in silico reconstructed ancestral AAV particles (AncAAVs), and show that, Anc80, the common putative ancestor of AAV1, 2, 8 and 9, is 15-20°C more thermostable than its contemporary homologs. Using phenotype-tophylogeny mapping, we also identify a set of 12 residues potentially playing a key role in capsid metastability. Second, we demonstrate that capsid thermal stability, as measured by Differential Scanning Fluorimetry (DSF), can be used for identification of AAV preparations at the protein level, a requirement of regulatory agencies. Last, we apply this identity assay to the study of capsid mosaic formation in AAV library preparations. This work will help guide the engineering and manufacturing of improved AAV vectors for gene therapy

The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

International audienceThe adeno-associated virus (AAV) vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral cofactor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid’s dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

International audienceGeneration and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multi-plexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombi-nant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina

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

International audienceAdeno-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

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

International audienceGene 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