期刊
MOLECULAR THERAPY
卷 22, 期 11, 页码 1900-1909出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/mt.2014.139
关键词
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资金
- NIH NHLBI [ROI HL097088]
- NIH [R01 GM082946]
- ECIA grant from the Bayer Hemophilia Awards Program
Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of virtual family shuffling to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of similar to 8 x 10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.
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