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Miniaturized CRISPR activation for a single AAV delivery method to treat LAMA2-Related Muscular Dystrophies

Cheng Zhang, Jia Qi (2023) Miniaturized CRISPR activation for a single AAV delivery method to treat LAMA2-Related Muscular Dystrophies. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Programmable recruitment of transcription factors to activate genes in a targeted and specific manner, commonly known as CRISPR activation (CRISPRa), offers unprecedented opportunities for therapeutic interventions. For example, the upregulation of the compensatory gene LAMA1 using CRISPRa to treat LAMA2-related deficient congenital muscular dystrophies (LAMA2-RD) has emerged as a mutation-independent therapeutic approach for this condition.

However, the viral delivery of CRISPRa components has been complicated by their size and the limited packaging capacity of adeno-associated viruses (AAVs), necessitating the use of dual AAV approaches that contribute to a common bottleneck in gene therapy, including high dose, potential toxicity, and production cost.

Here, I present a miniaturized CRISPRa system that can be packaged into a single AAV and apply this approach to upregulate a compensatory gene Lama1 in a mouse model representing a severe form of LAMA2-RD. Single-AAV9 carrying S. aureus dCas9, driven by novel mini promoter 4XNRF1, combined with tripartite activators VP64-delp65-delRTA and a single guide RNA targeting the mouse Lama1 promoter resulted in systemic LAMA1 expression, including critical target tissues for LAMA2-RD such as the skeletal muscles and peripheral nerves. Importantly, the single AAV-mediated Lama1 upregulation significantly improved neuromuscular functions and extended the lifespan of the severe LAMA2-RD mouse model.

Compared to the current state of CRISPRa therapeutic in LAMA2-RD, here, I successfully rescued the disease phenotype in a much more severe mouse model with only half the viral load. These results are crucial to advance the therapeutic development of upregulating the disease modifier gene for LAMA2-RD. Moreover, the components and engineered cassettes presented are adaptable for AAV-based gene therapy for different genetic diseases, such as other neuromuscular disorders and haploinsufficiency-related diseases.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Cheng Zhang, Jia Qijic147@pitt.edujic147
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKemaladewi, Dwi Udwi.kemaladewi@chp.edudwk24
Committee MemberQuasar, Padiath Saleemqpadiath@pitt.eduqpadiath
Committee MemberRoman, Beth Lromanb@pitt.eduromanb
Committee MemberAmbrosio,
Date: 24 August 2023
Date Type: Publication
Defense Date: 31 July 2023
Approval Date: 24 August 2023
Submission Date: 4 August 2023
Access Restriction: 1 year -- Restrict access to University of Pittsburgh for a period of 1 year.
Number of Pages: 139
Institution: University of Pittsburgh
Schools and Programs: School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Gene therapy Neuromuscular diseases
Date Deposited: 24 Aug 2023 13:53
Last Modified: 24 Aug 2023 13:53


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