The packaging capacity of adeno-associated virus (AAV) and the potential forwild-type-plusAAV gene therapy vectors

Autosomal recessive genetic forms (DFNB) account for most cases of profound congenital deafness. Adeno-associated virus (AAV)-based gene therapy is a promising therapeutic option, but is limited by a potentially short therapeutic window and the constrained packaging capacity of the vector. We focus here on the otoferlin gene underlying DFNB9, one of the most frequent genetic forms of congenital deafness. We adopted a dual AAV approach using two different recombinant vectors, one containing the 5′ and the other the 3′ portions of otoferlin cDNA, which exceed the packaging capacity of the AAV when combined. A single delivery of the vector pair into the mature cochlea ofOtof−/−mutant mice reconstituted the otoferlin cDNA coding sequence through recombination of the 5′ and 3′ cDNAs, leading to the durable restoration of otoferlin expression in transduced cells and a reversal of the deafness phenotype, raising hopes for future gene therapy trials in DFNB9 patients.

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Immune Response Against Gene Therapy Vectors: Influence of Synovial Fluid on Adeno-Associated Virus Mediated Gene Transfer to Chondrocytes

Journal of Clinical Immunology ◽

10.1023/b:joci.0000019781.64421.5c ◽

2004 ◽

Vol 24 (2) ◽

pp. 162-169 ◽

Cited By ~ 44

Author(s):

Virginie Cottard ◽

Chiara Valvason ◽

Géraldine Falgarone ◽

Didier Lutomski ◽

Marie-Christophe Boissier ◽

...

Keyword(s):

Gene Therapy ◽

Immune Response ◽

Gene Transfer ◽

Synovial Fluid ◽

Adeno Associated Virus ◽

Gene Therapy Vectors

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The Structure of an AAV5-AAVR Complex at 2.5 Å Resolution: Implications for Cellular Entry and Immune Neutralization of AAV Gene Therapy Vectors

Viruses ◽

10.3390/v12111326 ◽

2020 ◽

Vol 12 (11) ◽

pp. 1326

Author(s):

Mark A. Silveria ◽

Edward E. Large ◽

Grant M. Zane ◽

Tommi A. White ◽

Michael S. Chapman

Keyword(s):

Gene Therapy ◽

Binding Sites ◽

Polycystic Kidney ◽

Adeno Associated Virus ◽

Gene Therapies ◽

The Us ◽

Gene Therapy Vectors ◽

Cellular Entry ◽

Resolution Structure

Adeno-Associated Virus is the leading vector for gene therapy. Although it is the vector for all in vivo gene therapies approved for clinical use by the US Food and Drug Administration, its biology is still not yet fully understood. It has been shown that different serotypes of AAV bind to their cellular receptor, AAVR, in different ways. Previously we have reported a 2.4Å structure of AAV2 bound to AAVR that shows ordered structure for only one of the two AAVR domains with which AAV2 interacts. In this study we present a 2.5Å resolution structure of AAV5 bound to AAVR. AAV5 binds to the first polycystic kidney disease (PKD) domain of AAVR that was not ordered in the AAV2 structure. Interactions of AAV5 with AAVR are analyzed in detail, and the implications for AAV2 binding are explored through molecular modeling. Moreover, we find that binding sites for the antibodies ADK5a, ADK5b, and 3C5 on AAV5 overlap with the binding site of AAVR. These insights provide a structural foundation for development of gene therapy agents to better evade immune neutralization without disrupting cellular entry.

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The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice

Journal of Clinical Investigation ◽

10.1172/jci37607 ◽

2009 ◽

Vol 119 (8) ◽

pp. 2388-2398 ◽

Cited By ~ 148

Author(s):

Jiangao Zhu ◽

Xiaopei Huang ◽

Yiping Yang

Keyword(s):

Gene Therapy ◽

Immune Responses ◽

Adaptive Immune Responses ◽

Adeno Associated Virus ◽

Virus Gene ◽

Adaptive Immune ◽

Gene Therapy Vectors ◽

Myd88 Pathway

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Stability of Recombinant Mosaic Adeno-Associated Virus Vector rAAV/DJ/CAG at Different Temperature Conditions

Journal of Biomedical Nanotechnology ◽

10.1166/jbn.2021.3183 ◽

2021 ◽

Vol 17 (11) ◽

pp. 2114-2124

Author(s):

Alicja Bie´nkowska-Tokarczyk ◽

Maciej Małecki

Keyword(s):

Gene Therapy ◽

Uv Light ◽

Temperature Fluctuations ◽

Effect Of Temperature ◽

Transduction Efficiency ◽

Microscopy Imaging ◽

Adeno Associated Virus ◽

Targeted Gene Delivery ◽

Gene Therapy Vectors ◽

The Stability

The nanometer size and biological characteristics of recombinant adeno-associated virus vectors (rAAV) make them particularly useful as gene therapy vectors and they have been successfully used in this role. Our latest research revealed that the rAAV/DJ/CAG mosaic vector offers highly efficient targeted gene delivery to melanoma cells metastasized to the lungs and that the transduction is temperature dependent. In order to further explore the ability of the rAAV/DJ/CAG vector to deliver highly selective transduction, this study was designed to identify the transduction stability of rAAV/DJ/CAG under various conditions. The temperatures used in this study ranged from −196 ° (liquid nitrogen) to 90 °, and the effect of temperature fluctuations (freeze-thaw, cooling-heating cycles) was also studied. This research also investigated the effects of UV radiation (ultraviolet) on the rAAV/DJ/CAG activity. Changes in the transduction efficiency were assessed via fluorescence microscopy imaging and the qPCR method. Under the test conditions, the transduction efficiency was reduced by approx. 35%, on average. High temperatures (70 °/90 °) and UV light proved to have the most detrimental impact. Changes in the stability of the rAAV/DJ/CAG structure are manifested by variations in the number of genome copies (gc) and GFP+ cells. Temperature fluctuations resulted in differences in the number of gc while maintaining a similar number of GFP+ cells, which may indicate specific changes in the rAAV/DJ/CAG structure, triggering disorders or degradation in the vector entry. This study provides interesting insights into rAAV/DJ/CAG, and the implications of these findings provide a basis for developing new protocols in cancer gene therapy.

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