Targeted exon skipping of NF1 exon 17 as a therapeutic for neurofibromatosis type I
We investigated the feasibility of utilizing an exon-skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin protein expression and GTPase activating protein (GAP)-related domain (GRD) fu...
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Published in | Molecular therapy. Nucleic acids Vol. 28; pp. 261 - 278 |
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Main Authors | , , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Elsevier Inc
14.06.2022
American Society of Gene & Cell Therapy Elsevier |
Subjects | |
Online Access | Get full text |
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Summary: | We investigated the feasibility of utilizing an exon-skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin protein expression and GTPase activating protein (GAP)-related domain (GRD) function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function, which was confirmed by in vitro assessments utilizing an Nf1 cDNA-based functional screening system. Skipping of exons 17 or 52 fit our criteria, as minimal effects on protein expression and GRD activity were noted. Antisense phosphorodiamidate morpholino oligomers (PMOs) were utilized to skip exon 17 in human cell lines with patient-specific pathogenic variants in exon 17, c.1885G>A, and c.1929delG. PMOs restored functional neurofibromin expression. To determine the in vivo significance of exon 17 skipping, we generated a homozygous deletion of exon 17 in a novel mouse model. Mice were viable and exhibited a normal lifespan. Initial studies did not reveal the presence of tumor development; however, altered nesting behavior and systemic lymphoid hyperplasia was noted in peripheral lymphoid organs. Alterations in T and B cell frequencies in the thymus and spleen were identified. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with pathogenic variants in exon 17, as homozygous deletion of exon 17 is consistent with at least partial function of neurofibromin.
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We evaluate neurofibromin sequence in silico; utilize an in vitro cDNA model system; design and test antisense oligos; and utilize them in vitro in human cell lines with exon-specific mutations. Additionally we model exon loss in vivo and demonstrate at least partial function of NF1, providing proof of concept. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 These authors contributed equally |
ISSN: | 2162-2531 2162-2531 |
DOI: | 10.1016/j.omtn.2022.03.011 |