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Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice
Mariacarmela Allocca, … , Jean Bennett, Alberto Auricchio
Mariacarmela Allocca, … , Jean Bennett, Alberto Auricchio
Published April 15, 2008
Citation Information: J Clin Invest. 2008;118(5):1955-1964. https://doi.org/10.1172/JCI34316.
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Technical Advance Genetics Article has an altmetric score of 13

Serotype-dependent packaging of large genes in adeno-associated viral vectors results in effective gene delivery in mice

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Abstract

Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4–/– mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.

Authors

Mariacarmela Allocca, Monica Doria, Marco Petrillo, Pasqualina Colella, Maria Garcia-Hoyos, Daniel Gibbs, So Ra Kim, Albert Maguire, Tonia S. Rex, Umberto Di Vicino, Luisa Cutillo, Janet R. Sparrow, David S. Williams, Jean Bennett, Alberto Auricchio

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Figure 2

Genome integrity of rAAV2/5-CMV-Abca4, -MYO7A, -CEP290, and -EGFP-8.9.

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Packaging capacity of rAAV serotypes.
(A) Average titers of rAAV2/1, 2, ...
(A) Southern blot analysis of vector DNA isolated directly from rAAV large preparations (2.5 × 1010 GC/lane) and separated on alkaline agarose gels. Lane 1 contains a marker DNA fragment obtained by restriction digestion from the pAAV2.1-CMV-Abca4 plasmid; lane 2 contains the same DNA fragment as in lane 1 digested with DNaseI, as control of DNaseI activity; lanes 3 and 4: genomes isolated from rAAV2/5-CMV-Abca4; lanes 5 and 6: genomes isolated from rAAV2/5-CMV-MYO7A; lanes 7 and 8: genomes isolated from rAAV2/5-CMV-CEP290; lanes 9 and 10: genomes isolated from rAAV2/5-CMV-EGFP-8.9 (lanes 1–10 belong to the same gel but were noncontiguous); lanes 11 and 12: genomes isolated from rAAV2/5-CMV-EGFP (2.6 kb). Samples in lanes 3, 5, 7, 9, and 11 were treated with DNaseI. (B) Assessment of rAAV2/5-CMV-Abca4 genome length following in vivo delivery. Top panel: Schematic representation of the rAAV2/5-CMV-Abca4 genome with the 2 probes used for the Southern blot analysis. Bottom panel, upper blots: Southern blot analysis of genomic DNA from uninjected muscles (lanes 1 and 3) and an equivalent amount of genomic DNA from murine muscle injected with rAAV2/5-CMV-Abca4 (lanes 2 and 4) digested with NcoI and NotI (lanes 1 and 2) or NcoI alone (lanes 3 and 4). Lanes belong to the same gel but were noncontiguous. The arrows indicate the bands of the expected size. Lower blots: Southern blot analysis with a probe specific for the PDE6B gene used as loading control. Molecular weights are indicated on the left. (C) Western blot analysis with anti-ABCA4 (upper blot) or anti–α-tubulin (lower blot) antibodies of lysates from Cos cells transduced with rAAV2/5. Lane 1: retina from wild-type mouse; lane 2: samples transduced with rAAV2/5-CMV-Abca4; lane 3: samples transduced with rAAV2/5-CMV-EGFP. Anti–α-tubulin was used as loading control. The amounts (μg) of protein loaded are indicated under the respective lanes.

Copyright © 2025 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)

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