Publicação
In vitro transcribed mRNA optimization for gene therapy in the eye
| Resumo: | Inherited retinal diseases (IRDs) represent a heterogeneous group of degenerative diseases, caused by around 300 gene mutations, without an effective treatment. IRDs constitute the leading cause of blindness worldwide, with a prevalence of 1 in 2000 individuals. Gene therapy emerges as a promis- ing therapeutic option for the possible treatment of IRDs. Recent advancements in mRNA therapeutics motivated the exploration of in vitro transcribed mRNA for therapeutic applications including the COVID- 19 vaccines. Despite the success of mRNA-based vaccination, little research has been done on the application of mRNA technology for ocular diseases. Delivering mRNA to the retina holds substantial advantages, as mRNA is completely functional in the cytoplasm and does not require nuclear entry, hence does not integrate with the genome. Furthermore, it presents a transient nature, low-cost produc- tion, and low immunogenicity. This project aims to explore mRNA efficiency in two retinal pigment epi- thelium (RPE) cell models: ARPE-19, human induced Pluripotent Stem Cells-derived RPE (hiPSCs- RPE) and also retinal organoids. In this work, the delivery of a mRNA (GFPmRNA) using commercial lipid-based nanoparticles (LNPs) was explored. Results demonstrated that mRNA is efficiently delivered to RPE in a short time (1h-3h). Moreover, mRNA complexed with LNPs, successfully transfect both ARPE-19 and hiPSCs- RPE, that lasted up to 30 days without toxicity. Notably, complexed mRNA demonstrated superior transfection efficiency compared to pDNA and AAV2 strategies. On the other hand, AAV6 exhibited similar efficiency compared to mRNA delivery in hiPSCs-RPE and higher in ARPE-19. Despite this, the increased expression of MCP1 was only observed in viral delivery compared to mRNA transfection, suggesting potential immunogenicity. Furthermore, in retinal organoids only few Müller glia cells were transfected. In summary, this study showed a clear advantage of mRNA delivery compared to other strate- gies in RPE cells and highlights the potential for developing mRNA-based gene therapies for the treatment of IRDs. |
|---|---|
| Autores principais: | Castro, Mariana Parreiras Dias Urbano de |
| Assunto: | lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso embargado |
| Instituição associada: | Universidade Nova de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório Institucional da UNL |
| _version_ | 1868983221808529408 |
|---|---|
| author | Castro, Mariana Parreiras Dias Urbano de |
| author_facet | Castro, Mariana Parreiras Dias Urbano de |
| author_role | author |
| contributor_name_str_mv | Lemos, Luisa Braga, Margarida RUN |
| country_str | PT |
| creators_json_txt | [{\"Person.name\":\"Castro, Mariana Parreiras Dias Urbano de\"}] |
| datacite.contributors.contributor.contributorName.fl_str_mv | Lemos, Luisa Braga, Margarida RUN |
| datacite.creators.creator.creatorName.fl_str_mv | Castro, Mariana Parreiras Dias Urbano de |
| datacite.date.Accepted.fl_str_mv | 2023-11-28T00:00:00Z |
| datacite.date.available.fl_str_mv | 2026-09-30T00:00:00Z |
| datacite.date.embargoed.fl_str_mv | 2026-09-30T00:00:00Z |
| datacite.rights.fl_str_mv | http://purl.org/coar/access_right/c_f1cf |
| datacite.subjects.subject.fl_str_mv | lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| datacite.titles.title.fl_str_mv | In vitro transcribed mRNA optimization for gene therapy in the eye |
| dc.contributor.none.fl_str_mv | Lemos, Luisa Braga, Margarida RUN |
| dc.creator.none.fl_str_mv | Castro, Mariana Parreiras Dias Urbano de |
| dc.date.Accepted.fl_str_mv | 2023-11-28T00:00:00Z |
| dc.date.available.fl_str_mv | 2026-09-30T00:00:00Z |
| dc.date.embargoed.fl_str_mv | 2026-09-30T00:00:00Z |
| dc.format.none.fl_str_mv | application/pdf |
| dc.identifier.none.fl_str_mv | http://hdl.handle.net/10362/161846 |
| dc.language.none.fl_str_mv | eng |
| dc.rights.none.fl_str_mv | http://purl.org/coar/access_right/c_f1cf |
| dc.subject.none.fl_str_mv | lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| dc.title.fl_str_mv | In vitro transcribed mRNA optimization for gene therapy in the eye |
| dc.type.none.fl_str_mv | http://purl.org/coar/resource_type/c_bdcc |
| description | Inherited retinal diseases (IRDs) represent a heterogeneous group of degenerative diseases, caused by around 300 gene mutations, without an effective treatment. IRDs constitute the leading cause of blindness worldwide, with a prevalence of 1 in 2000 individuals. Gene therapy emerges as a promis- ing therapeutic option for the possible treatment of IRDs. Recent advancements in mRNA therapeutics motivated the exploration of in vitro transcribed mRNA for therapeutic applications including the COVID- 19 vaccines. Despite the success of mRNA-based vaccination, little research has been done on the application of mRNA technology for ocular diseases. Delivering mRNA to the retina holds substantial advantages, as mRNA is completely functional in the cytoplasm and does not require nuclear entry, hence does not integrate with the genome. Furthermore, it presents a transient nature, low-cost produc- tion, and low immunogenicity. This project aims to explore mRNA efficiency in two retinal pigment epi- thelium (RPE) cell models: ARPE-19, human induced Pluripotent Stem Cells-derived RPE (hiPSCs- RPE) and also retinal organoids. In this work, the delivery of a mRNA (GFPmRNA) using commercial lipid-based nanoparticles (LNPs) was explored. Results demonstrated that mRNA is efficiently delivered to RPE in a short time (1h-3h). Moreover, mRNA complexed with LNPs, successfully transfect both ARPE-19 and hiPSCs- RPE, that lasted up to 30 days without toxicity. Notably, complexed mRNA demonstrated superior transfection efficiency compared to pDNA and AAV2 strategies. On the other hand, AAV6 exhibited similar efficiency compared to mRNA delivery in hiPSCs-RPE and higher in ARPE-19. Despite this, the increased expression of MCP1 was only observed in viral delivery compared to mRNA transfection, suggesting potential immunogenicity. Furthermore, in retinal organoids only few Müller glia cells were transfected. In summary, this study showed a clear advantage of mRNA delivery compared to other strate- gies in RPE cells and highlights the potential for developing mRNA-based gene therapies for the treatment of IRDs. |
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| format | masterThesis |
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| identifier.url.fl_str_mv | http://hdl.handle.net/10362/161846 |
| inst_facet_str | urn:organizationAcronym:unl{{{_:::_}}}Universidade Nova de Lisboa |
| instacron_str | unl |
| institution | Universidade Nova de Lisboa |
| instname_str | Universidade Nova de Lisboa |
| language | eng |
| network_acronym_str | run |
| network_name_str | Repositório Institucional da UNL |
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| person_str_mv | Castro, Mariana Parreiras Dias Urbano de |
| publishDate | 2023 |
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| reponame_str | Repositório Institucional da UNL |
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| spelling | engpt_PTInherited retinal diseases (IRDs) represent a heterogeneous group of degenerative diseases, caused by around 300 gene mutations, without an effective treatment. IRDs constitute the leading cause of blindness worldwide, with a prevalence of 1 in 2000 individuals. Gene therapy emerges as a promis- ing therapeutic option for the possible treatment of IRDs. Recent advancements in mRNA therapeutics motivated the exploration of in vitro transcribed mRNA for therapeutic applications including the COVID- 19 vaccines. Despite the success of mRNA-based vaccination, little research has been done on the application of mRNA technology for ocular diseases. Delivering mRNA to the retina holds substantial advantages, as mRNA is completely functional in the cytoplasm and does not require nuclear entry, hence does not integrate with the genome. Furthermore, it presents a transient nature, low-cost produc- tion, and low immunogenicity. This project aims to explore mRNA efficiency in two retinal pigment epi- thelium (RPE) cell models: ARPE-19, human induced Pluripotent Stem Cells-derived RPE (hiPSCs- RPE) and also retinal organoids. In this work, the delivery of a mRNA (GFPmRNA) using commercial lipid-based nanoparticles (LNPs) was explored. Results demonstrated that mRNA is efficiently delivered to RPE in a short time (1h-3h). Moreover, mRNA complexed with LNPs, successfully transfect both ARPE-19 and hiPSCs- RPE, that lasted up to 30 days without toxicity. Notably, complexed mRNA demonstrated superior transfection efficiency compared to pDNA and AAV2 strategies. On the other hand, AAV6 exhibited similar efficiency compared to mRNA delivery in hiPSCs-RPE and higher in ARPE-19. Despite this, the increased expression of MCP1 was only observed in viral delivery compared to mRNA transfection, suggesting potential immunogenicity. Furthermore, in retinal organoids only few Müller glia cells were transfected. In summary, this study showed a clear advantage of mRNA delivery compared to other strate- gies in RPE cells and highlights the potential for developing mRNA-based gene therapies for the treatment of IRDs.application/pdfpt_PTIn vitro transcribed mRNA optimization for gene therapy in the eyeCastro, Mariana Parreiras Dias Urbano deLemos, LuisaBraga, MargaridaHostingInstitutionOrganizationalRUNe-mailmailto:run@unl.ptrun@unl.pt2023-11-282026-09-30T00:00:00Z2023-11-28T00:00:00ZHandlehttp://hdl.handle.net/10362/161846http://purl.org/coar/access_right/c_f1cfembargoed accesslARPE-19Gene therapyhuman induced Pluripotent Stem CellsmRNALipid NanoparticlesRetinal Organoids44587575 bytesliteraturehttp://purl.org/coar/resource_type/c_bdccmaster thesishttp://purl.org/coar/access_right/c_f1cfapplication/pdffulltexthttps://run.unl.pt/bitstreams/c8cfc440-48cb-411c-8ea3-c608ec910d3f/download |
| spellingShingle | In vitro transcribed mRNA optimization for gene therapy in the eye Castro, Mariana Parreiras Dias Urbano de lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| status | SINGLETON |
| subject.fl_str_mv | lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| title | In vitro transcribed mRNA optimization for gene therapy in the eye |
| title_full | In vitro transcribed mRNA optimization for gene therapy in the eye |
| title_fullStr | In vitro transcribed mRNA optimization for gene therapy in the eye |
| title_full_unstemmed | In vitro transcribed mRNA optimization for gene therapy in the eye |
| title_short | In vitro transcribed mRNA optimization for gene therapy in the eye |
| title_sort | In vitro transcribed mRNA optimization for gene therapy in the eye |
| topic | lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| topic_facet | lARPE-19 Gene therapy human induced Pluripotent Stem Cells mRNA Lipid Nanoparticles Retinal Organoids |
| url | http://hdl.handle.net/10362/161846 |
| visible | 1 |