Advisor(s)
Deeann Wallis
Committee Member(s)
Andre Leier
Kim Keeling
School
Joint Health Sciences (Interdisciplinary)
Document Type
Thesis
Department (new version)
Genetics
Date of Award
1-6-2025
Abstract
Modification of pre-mRNA splicing using antisense oligonucleotides (ASOs) can be used to skip one or more exons carrying pathogenic DNA sequence variants. Our previ-ously published data indicates NF1 exon 52 is a good target for exon skipping as a cDNA screen indicates an NF1 isoform lacking exon 52 maintains both high neurofi-bromin expression and the ability to suppress Ras activity. To develop an exon skipping approach, we designed antisense phosphorodiamidate morpholino oligomers (PMOs) to skip exon 52. Our lead PMO has a low IC50 of 45nM and is able to restore NF1 expres-sion and Ras-suppression in a cell line with pathogenic variation in exon 52. To deter-mine the essentiality of exon 52 in vivo, we’ve developed a novel mouse model with deletion of exon 52 (DelE52). Mice null for NF1 exon 52 show no gross abnormalities or histological lesions. We have established an aging cohort of DelE52 mice that are currently between 6-20 months and show no evidence of tumor development. This pro-vides further proof-of-concept that exon 52 is not essential for neurofibromin function. Further, we’ve created a novel humanized exon 52 mouse model containing pathogenic variant R2550X in exon 52 (hR2550X) for pre-clinical ASO testing. We have devel-oped and optimized a digital droplet PCR (ddPCR) protocol to assess skipping efficien-cy. Preliminary testing of the hR2550X model in conjunction with a floxed allele and CAGG-Cre inducible NF1 loss with VIVO-PMOs shows RNA skipping efficiency in multiple tissues. Hence, exon skipping holds therapeutic potential for intragenic NF1 exon 52 mutations.
ProQuest ID
Recommended Citation
Church, Cameron Lee, "Targeted Exon Skipping Of Nf1 Exon 52 As A Mutation Specific Therapeutic For Neurofibromatosis Type 1" (2025). All ETDs from UAB. 7265.
https://digitalcommons.library.uab.edu/etd-collection/7265
