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https://hdl.handle.net/1822/77903
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Campo DC | Valor | Idioma |
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dc.contributor.author | Sousa, Diana Andrade | por |
dc.contributor.author | Gaspar, Ricardo | por |
dc.contributor.author | Ferreira, Celso J. O. | por |
dc.contributor.author | Baltazar, Fátima | por |
dc.contributor.author | Rodrigues, L. R. | por |
dc.contributor.author | Silva, Bruno F. B. | por |
dc.date.accessioned | 2022-05-25T11:56:42Z | - |
dc.date.available | 2022-05-25T11:56:42Z | - |
dc.date.issued | 2022-05-19 | - |
dc.identifier.citation | Sousa, Diana; Gaspar, Ricardo; Ferreira, Celso J. O.; Baltazar, Fátima; Rodrigues, Lígia R.; Silva, Bruno F. B., In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes. Pharmaceutics, 14(5), 1087, 2022 | por |
dc.identifier.uri | https://hdl.handle.net/1822/77903 | - |
dc.description.abstract | Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems. | por |
dc.description.sponsorship | This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and the Project FCOMP-01– 0124-FEDER-021053 (PTDC/SAU-BMA/121028/2010). This research was also supported by the Microfluidic Layer-by-layer Assembly of Cationic Liposome—Nucleic Acid Nanoparticles for Gene Delivery project (032520) co-funded by FCT and the ERDF through COMPETE2020. Diana A. Sousa (D.A.S) and Celso J.O. Ferreira (C.J.O.F) acknowledge FCT for the grants PD/BD/139083/2018 and SFRH/BD/149199/2019, respectively. | por |
dc.language.iso | eng | por |
dc.publisher | Multidisciplinary Digital Publishing Institute (MDPI) | por |
dc.relation | info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F04469%2F2020/PT | por |
dc.relation | info:eu-repo/grantAgreement/FCT/5876-PPCDTI/PTDC%2FSAU-BMA%2F121028%2F2010/PT | por |
dc.relation | info:eu-repo/grantAgreement/FCT/POR_NORTE/PD%2FBD%2F139083%2F2018/PT | por |
dc.relation | info:eu-repo/grantAgreement/FCT/POR_NORTE/SFRH%2FBD%2F149199%2F2019/PT | por |
dc.rights | openAccess | por |
dc.rights.uri | http://creativecommons.org/licenses/by/4.0/ | por |
dc.subject | CRISPR | por |
dc.subject | Cas9 | por |
dc.subject | gene knockout | por |
dc.subject | CL-DNA | por |
dc.subject | lipoplex | por |
dc.subject | plasmid | por |
dc.subject | gene delivery | por |
dc.subject | multivalent cationic lipids | por |
dc.subject | MVL5 | por |
dc.title | In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic LiposomeDNA Complexes | por |
dc.type | article | - |
dc.peerreviewed | yes | por |
dc.relation.publisherversion | https://www.mdpi.com/1999-4923/14/5/1087 | por |
dc.comments | CEB55561 | por |
oaire.citationStartPage | 1087 | por |
oaire.citationIssue | 5 | por |
oaire.citationConferencePlace | Switzerland | - |
oaire.citationVolume | 14 | por |
dc.date.updated | 2022-05-24T21:41:34Z | - |
dc.identifier.eissn | 1999-4923 | por |
dc.identifier.doi | 10.3390/pharmaceutics14051087 | por |
dc.description.publicationversion | info:eu-repo/semantics/publishedVersion | - |
dc.subject.wos | Science & Technology | por |
sdum.journal | Pharmaceutics | por |
oaire.version | VoR | por |
Aparece nas coleções: | CEB - Publicações em Revistas/Séries Internacionais / Publications in International Journals/Series |
Ficheiros deste registo:
Ficheiro | Descrição | Tamanho | Formato | |
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document_55561_1.pdf | 2,55 MB | Adobe PDF | Ver/Abrir |
Este trabalho está licenciado sob uma Licença Creative Commons