Gene therapy for surfactant protein B deficiency using recombinant AAV vectors
Gene therapies are being developed for rare genetic diseases and offer hope for conditions with few treatment options. Surfactant protein B (SP-B) deficiency is a rare genetic disease of the lung causing severe respiratory distress in neonates and is fatal within the first months of life. Recombinan...
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| Format: | Thesis |
| Language: | English |
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2021
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| _version_ | 1826315540736507904 |
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| author | Meyer-Berg, H |
| author2 | Gill, D |
| author_facet | Gill, D Meyer-Berg, H |
| author_sort | Meyer-Berg, H |
| collection | OXFORD |
| description | Gene therapies are being developed for rare genetic diseases and offer hope for conditions with few treatment options. Surfactant protein B (SP-B) deficiency is a rare genetic disease of the lung causing severe respiratory distress in neonates and is fatal within the first months of life. Recombinant adeno-associated virus (rAAV) is being investigated for delivery of functional SP-B to the lungs of SP-B deficient neonates, to produce surfactant for secretion onto the lung surface to increase chances of survival.
To screen AAV serotypes for efficient targeting of alveolar type II (ATII) cells of the lung parenchyma, rAAV5, rAAV6.2 and rAAV9 expressing reporter transgenes were delivered to murine lungs via intranasal instillation. Serotype 5 mediated the highest transduction levels in the target ATII cells (19.4 %, p< 0.05 versus naive). The in situ hybridisation method RNAscope was adapted to include a DNase digestion step to allow for detection of transgene mRNA expression after rAAV delivery and showed that the hCEFI promoter mediated the highest expression levels in ATII cells (11.1 transgene mRNA copies per cell, p< 0.05 versus rAAV5.SP-B MG and p< 0.0001 versus all other groups). Therefore, rAAV5.hCEFI.SP-B was the rAAV vector selected for use in the murine lung. To validate the vector for human lung delivery, eight AAV capsids were screened in human models of the lung parenchyma: precision cut lung slices (PCLS) generated from lung resections and lung bud organoids (LBO) generated from human embryonic stem cells. The rAAV5 vector did not transduce the PCLS model, or the LBO after microinjection to the lumen; but vectors rAAV6, rAAV6.2 and rAAV6.2FF showed distinct levels of transduction in both models (p< 0.005 versus naive), indicating their suitability for gene delivery to the human lung parenchyma and highlighting the limitations of the murine model for AAV serotype selection in lung. Reporter gene expression using the CMV promoter was more robust than the hCEFI promoter in both the PCLS (hCEFI expression not detected) and LBO model (about 5-fold), but hCEFI mediated distinct levels of expression long-term. To test the capability of rAAV6.2 to mediate therapeutic protein expression in a human model of the SP-B deficient lung, embryonic stem cells were gene edited to incorporate the most common disease-causing 121ins2 mutation in SP-B deficiency. Subsequently, LBOs were generated from this edited cell line by sequential differentiation and delivery of rAAV6.2.CMV.SP-B resulted in expression of mature SP-B protein, highlighting the translational potential of the selected rAAV vector. |
| first_indexed | 2024-03-07T07:26:47Z |
| format | Thesis |
| id | oxford-uuid:088b6ca7-0b1e-4bc2-ad37-a5798e3e9f75 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:27:56Z |
| publishDate | 2021 |
| record_format | dspace |
| spelling | oxford-uuid:088b6ca7-0b1e-4bc2-ad37-a5798e3e9f752024-12-01T10:51:08ZGene therapy for surfactant protein B deficiency using recombinant AAV vectorsThesishttp://purl.org/coar/resource_type/c_db06uuid:088b6ca7-0b1e-4bc2-ad37-a5798e3e9f75Gene therapyRare diseasesAdeno-associated virus 2LungsEnglishHyrax Deposit2021Meyer-Berg, HGill, DHyde, SCarlisle, RClark, HGene therapies are being developed for rare genetic diseases and offer hope for conditions with few treatment options. Surfactant protein B (SP-B) deficiency is a rare genetic disease of the lung causing severe respiratory distress in neonates and is fatal within the first months of life. Recombinant adeno-associated virus (rAAV) is being investigated for delivery of functional SP-B to the lungs of SP-B deficient neonates, to produce surfactant for secretion onto the lung surface to increase chances of survival. To screen AAV serotypes for efficient targeting of alveolar type II (ATII) cells of the lung parenchyma, rAAV5, rAAV6.2 and rAAV9 expressing reporter transgenes were delivered to murine lungs via intranasal instillation. Serotype 5 mediated the highest transduction levels in the target ATII cells (19.4 %, p< 0.05 versus naive). The in situ hybridisation method RNAscope was adapted to include a DNase digestion step to allow for detection of transgene mRNA expression after rAAV delivery and showed that the hCEFI promoter mediated the highest expression levels in ATII cells (11.1 transgene mRNA copies per cell, p< 0.05 versus rAAV5.SP-B MG and p< 0.0001 versus all other groups). Therefore, rAAV5.hCEFI.SP-B was the rAAV vector selected for use in the murine lung. To validate the vector for human lung delivery, eight AAV capsids were screened in human models of the lung parenchyma: precision cut lung slices (PCLS) generated from lung resections and lung bud organoids (LBO) generated from human embryonic stem cells. The rAAV5 vector did not transduce the PCLS model, or the LBO after microinjection to the lumen; but vectors rAAV6, rAAV6.2 and rAAV6.2FF showed distinct levels of transduction in both models (p< 0.005 versus naive), indicating their suitability for gene delivery to the human lung parenchyma and highlighting the limitations of the murine model for AAV serotype selection in lung. Reporter gene expression using the CMV promoter was more robust than the hCEFI promoter in both the PCLS (hCEFI expression not detected) and LBO model (about 5-fold), but hCEFI mediated distinct levels of expression long-term. To test the capability of rAAV6.2 to mediate therapeutic protein expression in a human model of the SP-B deficient lung, embryonic stem cells were gene edited to incorporate the most common disease-causing 121ins2 mutation in SP-B deficiency. Subsequently, LBOs were generated from this edited cell line by sequential differentiation and delivery of rAAV6.2.CMV.SP-B resulted in expression of mature SP-B protein, highlighting the translational potential of the selected rAAV vector. |
| spellingShingle | Gene therapy Rare diseases Adeno-associated virus 2 Lungs Meyer-Berg, H Gene therapy for surfactant protein B deficiency using recombinant AAV vectors |
| title | Gene therapy for surfactant protein B deficiency using recombinant AAV vectors |
| title_full | Gene therapy for surfactant protein B deficiency using recombinant AAV vectors |
| title_fullStr | Gene therapy for surfactant protein B deficiency using recombinant AAV vectors |
| title_full_unstemmed | Gene therapy for surfactant protein B deficiency using recombinant AAV vectors |
| title_short | Gene therapy for surfactant protein B deficiency using recombinant AAV vectors |
| title_sort | gene therapy for surfactant protein b deficiency using recombinant aav vectors |
| topic | Gene therapy Rare diseases Adeno-associated virus 2 Lungs |
| work_keys_str_mv | AT meyerbergh genetherapyforsurfactantproteinbdeficiencyusingrecombinantaavvectors |