Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency
Gene therapies are being developed for many rare genetic diseases and provide hope for patients and their families who currently have few treatment options available. Lung surfactant deficiencies comprise a group of rare genetic lung diseases, where babies present at birth with severe respiratory di...
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| 格式: | Thesis |
| 语言: | English |
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2024
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| _version_ | 1826317716228669440 |
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| author | Ruffle, AR |
| author2 | Hyde, SC |
| author_facet | Hyde, SC Ruffle, AR |
| author_sort | Ruffle, AR |
| collection | OXFORD |
| description | Gene therapies are being developed for many rare genetic diseases and provide hope for patients and their families who currently have few treatment options available. Lung surfactant deficiencies comprise a group of rare genetic lung diseases, where babies present at birth with severe respiratory distress requiring mechanical ventilation for survival. Severe surfactant deficiency can result from mutations in the <em>ABCA3</em> gene which encodes the ABCA3 lipid transporter. The ABCA3 transporter is present in alveolar type II (ATII) cells, specifically, the limiting membrane of intracellular lamellar bodies (LBs) that assemble and store surfactant prior to secretion onto the lung surface. A recombinant lentiviral (rLV) vector based on Simian Immunodeficiency Virus (SIV) and pseudotyped with the F and HN glycoproteins from Sendai virus (rSIV.F/HN) was selected to accommodate the large ABCA3 cDNA. Candidate rLV genome plasmids were constructed for ABCA3 expression under control of the synthetic CG dinucleotide-free hCEF promoter and confirmed to express ABCA3 protein in a human lung cell line by western blotting. Alternative promoter sequences based on the endogenous promoter of the human <em>SFTPB</em> gene were also investigated, using rLVs expressing Green Fluorescent Protein as a reporter transgene. To evaluate the functional activity of ABCA3 expressed from the rLV vector, <em>in vitro</em> models of ATII cells were explored under both submerged and ‘air-lifted’ air-liquid interface culture conditions and gene editing was performed to ablate the <em>ABCA3</em> gene. Using transmission electron microscopy on cells cultured under submerged conditions, LBs could be observed in the human A549 cell line but were not present in human H441 cells; although air-lifting H441 cells resulted in the emergence of some LB structures. An <em>in vitro</em> cell detoxification assay was established which showed reduced viability of cells expressing pathogenic variants of ABCA3 compared with wild-type ABCA3, after exposure to the cytotoxic drug doxorubicin. To explore potential to treat ABCA3-surfactant deficiency, highly purified and concentrated rSIV.F/HN.hCEF.ABCA3 vector was produced at large (5l) scale, resulting in vector titres suitable for testing <em>in vivo</em>. Intranasal instillation was used to deliver rSIV.F/HN expressing ABCA3 to the lungs of wild-type (BALB/c) mice, confirming expression of vector-derived <em>ABCA3</em> and no evidence of acute toxicity. A conditional <em>Abca3</em> knockout murine model was also developed where tamoxifen administration resulted in knockout of the <em>Abca3</em> gene to induce a respiratory disease phenotype. Overall, rLV vectors expressing ABCA3 are a promising strategy for the treatment of ABCA3-mediated surfactant dysfunction. |
| first_indexed | 2025-03-11T16:58:19Z |
| format | Thesis |
| id | oxford-uuid:9403933a-5318-4c36-bf93-b6f0ff1ccc64 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2025-03-11T16:58:19Z |
| publishDate | 2024 |
| record_format | dspace |
| spelling | oxford-uuid:9403933a-5318-4c36-bf93-b6f0ff1ccc642025-03-03T16:37:08ZLentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiencyThesishttp://purl.org/coar/resource_type/c_db06uuid:9403933a-5318-4c36-bf93-b6f0ff1ccc64Gene therapyPulmonary surfactantRespiratory distress syndromeSurfactant deficiencyLentivirusesEnglishHyrax Deposit2024Ruffle, ARHyde, SCGill, DRRoberts, TCMilne, THart, SGene therapies are being developed for many rare genetic diseases and provide hope for patients and their families who currently have few treatment options available. Lung surfactant deficiencies comprise a group of rare genetic lung diseases, where babies present at birth with severe respiratory distress requiring mechanical ventilation for survival. Severe surfactant deficiency can result from mutations in the <em>ABCA3</em> gene which encodes the ABCA3 lipid transporter. The ABCA3 transporter is present in alveolar type II (ATII) cells, specifically, the limiting membrane of intracellular lamellar bodies (LBs) that assemble and store surfactant prior to secretion onto the lung surface. A recombinant lentiviral (rLV) vector based on Simian Immunodeficiency Virus (SIV) and pseudotyped with the F and HN glycoproteins from Sendai virus (rSIV.F/HN) was selected to accommodate the large ABCA3 cDNA. Candidate rLV genome plasmids were constructed for ABCA3 expression under control of the synthetic CG dinucleotide-free hCEF promoter and confirmed to express ABCA3 protein in a human lung cell line by western blotting. Alternative promoter sequences based on the endogenous promoter of the human <em>SFTPB</em> gene were also investigated, using rLVs expressing Green Fluorescent Protein as a reporter transgene. To evaluate the functional activity of ABCA3 expressed from the rLV vector, <em>in vitro</em> models of ATII cells were explored under both submerged and ‘air-lifted’ air-liquid interface culture conditions and gene editing was performed to ablate the <em>ABCA3</em> gene. Using transmission electron microscopy on cells cultured under submerged conditions, LBs could be observed in the human A549 cell line but were not present in human H441 cells; although air-lifting H441 cells resulted in the emergence of some LB structures. An <em>in vitro</em> cell detoxification assay was established which showed reduced viability of cells expressing pathogenic variants of ABCA3 compared with wild-type ABCA3, after exposure to the cytotoxic drug doxorubicin. To explore potential to treat ABCA3-surfactant deficiency, highly purified and concentrated rSIV.F/HN.hCEF.ABCA3 vector was produced at large (5l) scale, resulting in vector titres suitable for testing <em>in vivo</em>. Intranasal instillation was used to deliver rSIV.F/HN expressing ABCA3 to the lungs of wild-type (BALB/c) mice, confirming expression of vector-derived <em>ABCA3</em> and no evidence of acute toxicity. A conditional <em>Abca3</em> knockout murine model was also developed where tamoxifen administration resulted in knockout of the <em>Abca3</em> gene to induce a respiratory disease phenotype. Overall, rLV vectors expressing ABCA3 are a promising strategy for the treatment of ABCA3-mediated surfactant dysfunction. |
| spellingShingle | Gene therapy Pulmonary surfactant Respiratory distress syndrome Surfactant deficiency Lentiviruses Ruffle, AR Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency |
| title | Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency |
| title_full | Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency |
| title_fullStr | Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency |
| title_full_unstemmed | Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency |
| title_short | Lentiviral gene therapy for the treatment of ABCA3-mediated surfactant deficiency |
| title_sort | lentiviral gene therapy for the treatment of abca3 mediated surfactant deficiency |
| topic | Gene therapy Pulmonary surfactant Respiratory distress syndrome Surfactant deficiency Lentiviruses |
| work_keys_str_mv | AT rufflear lentiviralgenetherapyforthetreatmentofabca3mediatedsurfactantdeficiency |