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Development of a mutation-independent approach to treat merosin-deficient congenital muscular dystrophy type 1A (MDC1A)

Arockiaraj, Annie Infancia (2023) Development of a mutation-independent approach to treat merosin-deficient congenital muscular dystrophy type 1A (MDC1A). Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Merosin-deficient congenital muscular dystrophy (MDC1A) is an autosomal recessive disorder caused by mutations in the LAMA2 gene, resulting in a defective form of the extracellular matrix protein laminin-α2 (LAMA2). Individuals diagnosed with MDC1A exhibit progressive muscle wasting and declining neuromuscular functions. No treatments for this disorder are currently available. Our group previously showed that postnatal Lama1 upregulation, achieved through CRISPR activation (CRISPRa), compensates for Lama2 deficiency and prevents neuromuscular pathophysiology in a mouse model of MDC1A. In this study, I assessed the feasibility of upregulating human LAMA1 as a potential therapeutic strategy for individuals with MDC1A, regardless of their mutations. I hypothesized that CRISPRa-mediated upregulation of human LAMA1 would compensate for the lack of LAMA2 and rescue cellular abnormalities in MDC1A fibroblasts. Global transcriptomic and pathway enrichment analyses of fibroblasts collected from individuals carrying pathogenic LAMA2 mutations, compared with healthy controls, indicated higher expression of transcripts encoding proteins that contribute to wound healing, including Transforming Growth Factor-β (TGF-β) and Fibroblast Growth Factor (FGF). These findings were supported by wound-healing assays indicating that MDC1A fibroblasts migrated significantly more rapidly than the controls. Subsequently, the MDC1A fibroblasts were treated with SadCas9-2XVP64 and sgRNAs targeting the LAMA1 promoter. Robust LAMA1 expression was observed, which was accompanied by significant decreases in cell migration and expression of FGFR2, TGF-2, and ACTA2, which are involved in the wound-healing mechanism in MDC1A fibroblasts.
Collectively, our data suggest that CRISPRa-mediated LAMA1 upregulation may be a feasible mutation-independent therapeutic approach for MDC1A. This strategy might be adapted to address other neuromuscular diseases and inherited conditions in which strong compensatory mechanisms have been identified.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Arockiaraj, Annie Infanciaannie.arockiaraj@pitt.eduana100
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairKemaladewi, Dwi U.dwk24@pitt.edudwk24
Committee MemberConley, Yvetteyconley@pitt.eduyconley
Committee MemberPadiath, Quasar S.qpadiath@pitt.eduqpadiath
Committee MemberUrban, Zsolturbanz@pitt.eduurbanz
Date: 9 May 2023
Date Type: Publication
Defense Date: 12 April 2023
Approval Date: 9 May 2023
Submission Date: 23 March 2023
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 159
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: MDC1A, LAMA2, disease modifier, LAMA1, CRISPR activation system, VP64, Migration
Date Deposited: 10 May 2023 01:29
Last Modified: 10 May 2023 01:29

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