Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa
<p>Dominant mutations in RHO (rhodopsin) are the most common cause of autosomal dominant retinitis pigmentosa (RHO-adRP). RHO-adRP causes progressive loss of rod cells, followed by cone cells, leading to blindness. This disease is a candidate for CRISPR gene editing, as reduction of mutant rho...
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| Médium: | Diplomová práce |
| Jazyk: | English |
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2021
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| Témata: |
| _version_ | 1826315789863485440 |
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| author | Peddle, CF |
| author2 | MacLaren, RE |
| author_facet | MacLaren, RE Peddle, CF |
| author_sort | Peddle, CF |
| collection | OXFORD |
| description | <p>Dominant mutations in RHO (rhodopsin) are the most common cause of autosomal dominant retinitis pigmentosa (RHO-adRP). RHO-adRP causes progressive loss of rod cells, followed by cone cells, leading to blindness. This disease is a candidate for CRISPR gene editing, as reduction of mutant rhodopsin is associated with phenotypic rescue. This thesis optimises all-in-one CRISPR/Cas9 and CRISPRi adeno-associated viruses (AAVs) and explores their potential as RHO-adRP gene therapy vectors.</p>
<p>Following construct optimisation in vitro, a CRISPR/Cas9 and CRISPRi plasmid carrying SaCas9 and dSaCas9.KRAB, respectively, were generated. Both demonstrated knock down of endogenously expressed EGFP in a transgenic cell line. For allele-specific knock down of RHO, the CRISPR constructs must target a region unique to the mutant allele. Bioinformatic screening of RHO identified seven non-pathogenic single nucleotide polymorphisms (SNPs) that were targetable with SaCas9. Two of these SNPs could be targeted allele-specifically, with high CRISPR/Cas9 gene disruption and CRISPRi gene repression rates of 38.5-73.2 % and 43.1-65.8 %, respectively. In heterozygous patients, targeting the SNP on the mutant RHO offers a mutation-independent allele-specific targeting strategy.</p>
<p>As a proof-of-concept for targeting rod cells, the AAV were subretinally injected into Nrl-EGFP mice, in whom enhanced green fluorescent protein (EGFP) expression is limited to rod photoreceptors. To restrict Cas9 expression to rods cells only, a novel 154 bp rod cell-specific promoter was identified. This shortened promoter from the PDE6B gene was active in human retinal explants, the human retinoblastoma-derived cell line Y79, and drove strong rod cell-specific expression in mice. The CRISPRi AAV was unable to repress EGFP, despite high transgene expression. The CRISPR/Cas9 AAV however, drove strong EGFP disruption. It produced 4.1 % EGFP indels, resulting in a reduction of EGFP mRNA and fluorescence of 55.3% and 36.5 %, respectively. The all-in-one CRISPR/Cas9 AAV is therefore able to drive strong gene disruption in rod cells in vivo, making it a useful vector for the treatment of RHO-adRP.</p>
<p>This thesis describes in detail the development of these all-in-one CRISPR/Cas9 and CRISPRi AAV vectors from proof-of-concept to in vivo study and advances the potential for using such therapies in future treatments of RHO-adRP.</p> |
| first_indexed | 2024-03-06T19:20:37Z |
| format | Thesis |
| id | oxford-uuid:19f44d06-9c2f-44e5-907c-fc38736c36eb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:32:35Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:19f44d06-9c2f-44e5-907c-fc38736c36eb2024-12-01T15:47:21ZDevelopment of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosaThesishttp://purl.org/coar/resource_type/c_db06uuid:19f44d06-9c2f-44e5-907c-fc38736c36ebRetinal degenerationGene therapyCRISPR (Genetics)Retinitis pigmentosaEnglishHyrax Deposit2021Peddle, CFMacLaren, REMcClements, ME<p>Dominant mutations in RHO (rhodopsin) are the most common cause of autosomal dominant retinitis pigmentosa (RHO-adRP). RHO-adRP causes progressive loss of rod cells, followed by cone cells, leading to blindness. This disease is a candidate for CRISPR gene editing, as reduction of mutant rhodopsin is associated with phenotypic rescue. This thesis optimises all-in-one CRISPR/Cas9 and CRISPRi adeno-associated viruses (AAVs) and explores their potential as RHO-adRP gene therapy vectors.</p> <p>Following construct optimisation in vitro, a CRISPR/Cas9 and CRISPRi plasmid carrying SaCas9 and dSaCas9.KRAB, respectively, were generated. Both demonstrated knock down of endogenously expressed EGFP in a transgenic cell line. For allele-specific knock down of RHO, the CRISPR constructs must target a region unique to the mutant allele. Bioinformatic screening of RHO identified seven non-pathogenic single nucleotide polymorphisms (SNPs) that were targetable with SaCas9. Two of these SNPs could be targeted allele-specifically, with high CRISPR/Cas9 gene disruption and CRISPRi gene repression rates of 38.5-73.2 % and 43.1-65.8 %, respectively. In heterozygous patients, targeting the SNP on the mutant RHO offers a mutation-independent allele-specific targeting strategy.</p> <p>As a proof-of-concept for targeting rod cells, the AAV were subretinally injected into Nrl-EGFP mice, in whom enhanced green fluorescent protein (EGFP) expression is limited to rod photoreceptors. To restrict Cas9 expression to rods cells only, a novel 154 bp rod cell-specific promoter was identified. This shortened promoter from the PDE6B gene was active in human retinal explants, the human retinoblastoma-derived cell line Y79, and drove strong rod cell-specific expression in mice. The CRISPRi AAV was unable to repress EGFP, despite high transgene expression. The CRISPR/Cas9 AAV however, drove strong EGFP disruption. It produced 4.1 % EGFP indels, resulting in a reduction of EGFP mRNA and fluorescence of 55.3% and 36.5 %, respectively. The all-in-one CRISPR/Cas9 AAV is therefore able to drive strong gene disruption in rod cells in vivo, making it a useful vector for the treatment of RHO-adRP.</p> <p>This thesis describes in detail the development of these all-in-one CRISPR/Cas9 and CRISPRi AAV vectors from proof-of-concept to in vivo study and advances the potential for using such therapies in future treatments of RHO-adRP.</p> |
| spellingShingle | Retinal degeneration Gene therapy CRISPR (Genetics) Retinitis pigmentosa Peddle, CF Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa |
| title | Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa |
| title_full | Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa |
| title_fullStr | Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa |
| title_full_unstemmed | Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa |
| title_short | Development of all-in-one CRISPR/Cas9 and CRISPRi AAV constructs to treat autosomal dominant retinitis pigmentosa |
| title_sort | development of all in one crispr cas9 and crispri aav constructs to treat autosomal dominant retinitis pigmentosa |
| topic | Retinal degeneration Gene therapy CRISPR (Genetics) Retinitis pigmentosa |
| work_keys_str_mv | AT peddlecf developmentofallinonecrisprcas9andcrispriaavconstructstotreatautosomaldominantretinitispigmentosa |