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Novel AAV engineering technology: identification of improved AAV variants for gene addition and genome engineering in primary human cells

Cabanes Creus, Martí; (2019) Novel AAV engineering technology: identification of improved AAV variants for gene addition and genome engineering in primary human cells. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Vectors based on Adeno-associated virus (AAV) are currently the most used gene transfer tools in the human gene therapy field. In order to overcome some of the intrinsic limitations of AAVs, lots of efforts have been put around AAV capsid engineering, with the ultimate goal of developing new recombinant serotypes with novel properties such as enhanced tropism for specific human cell types, increased manufacturability and decreased human seroreactivity. The work described in this thesis focus in an exhaustive revision of the process of new vector development. Firstly, we adapted new cloning strategies and bacterial selection on the AAV library construction field, allowing the generation of larger libraries, and reducing the time and effort needed for assembly. Secondly, we describe a new method based on codon optimization that when applied to AAV DNA-family shuffling overcomes the limitation encountered by phylogenetically distant serotypes to contribute on chimeric clones due to lack of DNA homology to other parental serotypes. This allowed to increase the complexity of the generated libraries. Thirdly, we generated and describe here a new high-throughput AAV vector selection platform to perform selection based on functional transduction (transgene expression).Briefly, we substituted the rep gene present on the AAV genome for a GFP reporter cassette under the control of a promoter of choice, and kept the cap library. This allows to transduce the cells of interest, FACS-sort the GFP positive cells and recover the capsid genes that encoded capsids that led to efficient functional transduction using PCR. Finally, we applied those three new AAV engineering technologies to develop novel capsids with improved tropism for primary human CD34+ cells, T-cells and hepatocytes.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Novel AAV engineering technology: identification of improved AAV variants for gene addition and genome engineering in primary human cells
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) Licence (https://creativecommons.org/licenses/by-nc-nd/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. - Some third party copyright material has been removed from this e-thesis.
UCL classification: UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10071599
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