ORCID ID

0000-0002-0961-7299

Publication Date

2020-11-17

Document Type

Doctoral Dissertation

Academic Program

Interdisciplinary Graduate Program

Department

RNA Therapeutics Institute

First Thesis Advisor

Erik Sontheimer

Keywords

CRISPR, AAV, rAAV, gene editing, gene therapy, genome engineering, in vivo, self-deleting, self-inactivating, Nme2Cas9, Cas9, adeno-associated virus, mice

Abstract

One of the major challenges facing medicine and drug discovery is the large number of genetic diseases caused by inherited mutations leading to a toxic gain-of-function, or loss-of-function of the disease protein. Microbiology offered a new glimpse of hope to address those disorders with the adaptation of the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) defense system as a genome editing tool. Cas9 is a unique CRISPR-associated endonuclease protein that can be easily programmed with an RNA [a single-guide RNA (sgRNA)] that is complementary to nearly any DNA locus. Cas9 creates a double-stranded break (DSB) that can be exploited to knock out toxic genes or replenish therapeutic expression levels of essential proteins. In addition to a matching sgRNA sequence, Cas9 requires the presence of a short signature sequence [a protospacer adjacent motif (PAM)] flanking the target locus. Over the past few years, several Cas9-based therapeutic platforms have emerged to correct DNA mutations in a wide range of mammalian cell lines, ex vivo, and in vivo by adapting recombinant adeno-associated virus (rAAV). However, most of the applications of Cas9 in the field have been limited to Streptococcus pyogenes (SpyCas9), which, in its wild-type form, suffers from inaccurate editing at off-target sites. It is also difficult to deliver via an all-in-one (sgRNA+Cas9) rAAV approach due to its large size. In this thesis, I describe other Cas9 nucleases and their development as new AAV-based genome editing platforms for therapeutic editing in vivo in mouse disease models. In the first part of this thesis, I develop the all-in-one AAV strategy to deliver a Neisseria meningitidis Cas9 ortholog (Nme1Cas9) in mice to reduce the level of circulating cholesterol in blood. I also help characterize an enhanced Cas9 from another meningococcus strain (Nme2Cas9) and show that it is effective in performing editing not only in mammalian cell culture, but also in vivo by all-in-one AAV delivery. Additionally, I describe two AAV platforms that enable advanced editing modalities in vivo: 1) segmental DNA deletion by delivering two sgRNAs (along with Nme2Cas9) in one AAV, and 2) precise HDR-based repair by fitting Nme2Cas9, sgRNA and donor DNA within a single AAV capsid. Using these tools, we successfully treat two genetic disorders in mice, underscoring the importance of this powerful duo of AAV and Cas9 in gene therapy to advance novel treatment. Finally, I present preliminary data on how to use these AAV.Nme2Cas9 vectors to treat Alexander Disease, a rare progressive neurological disorder. These findings provide a platform for future application of gene editing in therapeutics.

DOI

10.13028/ey4j-tj18

Rights and Permissions

Copyright is held by the author, with all rights reserved.

Available for download on Wednesday, November 23, 2022

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