Manufacturing and Characterization of a Recombinant Adeno-Associated Virus Type 8 Reference Standard Material

Ayuso, Eduard; Blouin, Véronique; Lock, Martin; McGorray, Susan P.; León, Xavier; Alvira, Mauricio R.; Auricchio, Alberto; Bucher, Stéphanie; Chtarto, Abdelwahed; Clark, K. Reed; Darmon, Christophe; Doria, Monica; Fountain, Will; Gao, Guangping; Gao, Kai; Giacca, Mauro; Kleinschmidt, J A; Leuchs, Barbara; Melas, Catherine; Mizukami, Hiroaki; Müller, Marcus; Noordman, Yvet; Bockstael, Olivier; Ozawa, Keiya; Pythoud, Catherine; Sumaroka, Marina; Surosky, Richard; Tenenbaum, Liliane; Linden, Inge Van der; Weins, Brigitte; Wright, J. Fraser; Zhang, Xinhua; Zentilin, Lorena; Bosch, Fátima; Snyder, Richard O.; Moullier, Philippe

doi:10.1089/hum.2014.057

Cited by 82 publications

(85 citation statements)

References 32 publications

(61 reference statements)

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“…Sample with titers between 3 Â 10 11 and 3 Â 10 12 particles/mL (pt/mL), as titrated by ELISA and calibrated using the rAAV8 Reference Standard Materials (RSM8), 23 were subjected to AAV-ID ( Figure 5D). Results show linearity of particle dose and DSF amplitude for one order of magnitude, yet there was not an indication of a loss of linearity at the highest concentration tested ( Figure 5E).…”

Section: Aav-id Signal Amplitude Is a Function Of Virion Concentrationmentioning

confidence: 99%

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

Pacouret

Bouzelha

Shelke³

et al. 2017

Molecular Therapy

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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.

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Section: Aav-id Signal Amplitude Is a Function Of Virion Concentrationmentioning

confidence: 99%

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

Pacouret

Bouzelha

Shelke³

et al. 2017

Molecular Therapy

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“…25 Viral genome particle titers were determined by a quantitative real-time polymerase chain reaction (PCR) method. 26 …”

Section: Gene Therapy Vector Productionmentioning

confidence: 99%

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia

Bär

Povedano

Serrano

et al. 2016

Blood

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Key Points• Telomerase gene therapy in a mouse model of aplastic anemia targets the bone marrow and provides increased and stable telomerase expression.• Telomerase expression leads to telomere elongation and subsequently to the reversal of aplastic anemia phenotypes.Aplastic anemia is a fatal bone marrow disorder characterized by peripheral pancytopenia and marrow hypoplasia. The disease can be hereditary or acquired and develops at any stage of life. A subgroup of the inherited form is caused by replicative impairment of hematopoietic stem and progenitor cells due to very short telomeres as a result of mutations in telomerase and other telomere components. Abnormal telomere shortening is also described in cases of acquired aplastic anemia, most likely secondary to increased turnover of bone marrow stem and progenitor cells. Here, we test the therapeutic efficacy of telomerase activation by using adeno-associated virus (AAV)9 gene therapy vectors carrying the telomerase Tert gene in 2 independent mouse models of aplastic anemia due to short telomeres (Trf1-and Tert-deficient mice). We find that a high dose of AAV9-Tert targets the bone marrow compartment, including hematopoietic stem cells. AAV9-Tert treatment after telomere attrition in bone marrow cells rescues aplastic anemia and mouse survival compared with mice treated with the empty vector. Improved survival is associated with a significant increase in telomere length in peripheral blood and bone marrow cells, as well as improved blood counts. These findings indicate that telomerase gene therapy represents a novel therapeutic strategy to treat aplastic anemia provoked or associated with short telomeres. (Blood.

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“…Continuing improvement in the manufacturing process using adherent mammalian cells with transient transfection, suspension culture and invertebrate cells, aimed at large-scale production of recombinant AAV vectors, will be critical for the success of vector commercialization [97-99]. Similarly, better characterization of vector preparations, such as the recent multicenter studies using reference standard material [100], will provide critical information to allow for direct comparison between vector parameters, and preclinical and clinical study outcomes. Notably, most of these clinical successes are primarily focused on orphan diseases, but the progression to more common diseases, including vaccines, is anticipated.…”

Section: Aav Manufacturing Issuesmentioning

confidence: 99%

Obstacles and future of gene therapy for hemophilia

Arruda

Samelson‐Jones

2015

Expert Opinion on Orphan Drugs

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Introduction The recent success of early-phase clinical trials for adeno-associated viral (AAV) liver-directed gene therapy for hemophilia B (HB) demonstrates the potential for gene therapy, in the future, to succeed protein-based prophylaxis therapy for HB. Significant obstacles, however, need to be overcome prior to widespread adoption. The largest obstacles include immune responses to the AAV capsid including preexisting neutralizing antibodies (NAbs) and a delayed cellular immune response. Emerging evidence suggests that the latter is vector-dose dependent. Furthermore, the development and eradication of inhibitors remains a significant safety concern. Similarly, biological differences between Factor VIII and Factor IX (FIX) impose challenges to direct adoption of the successes for HB to hemophilia A (HA). Areas covered The advantages and limitations of the current strategies addressing these obstacles for gene therapy for HB and HA are discussed, as well as vector manufacturing issues relevant to widespread adoption. Alternative strategies including both ex-vivo and in-vivo lentiviral-based methods are discussed, though we focus on AAV-based approaches because of their recent clinical success and potential. Expert opinion Our opinion is that these obstacles can be overcome with current approaches, and AAV-based gene therapy for HB will likely translate into future clinical care. Innovative approaches are, however, likely needed to solve the current problems obstructing HA gene therapy.

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Manufacturing and Characterization of a Recombinant Adeno-Associated Virus Type 8 Reference Standard Material

Cited by 82 publications

References 32 publications

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

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

Telomerase gene therapy rescues telomere length, bone marrow aplasia, and survival in mice with aplastic anemia

Obstacles and future of gene therapy for hemophilia

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