Publication
Gene-targeted strategies to eliminate HIV latent cells through synthetic activators and suicide lentivectors
| datacite.subject.fos | Ciências Médicas::Medicina Básica | pt_PT |
| dc.contributor.advisor | Gonçalves, João, 1967- | |
| dc.contributor.advisor | Marta, Mariana Santa, 1978- | |
| dc.contributor.author | Perdigão, PRL | |
| dc.date.accessioned | 2017-11-27T17:42:49Z | |
| dc.date.available | 2017-11-27T17:42:49Z | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017 | |
| dc.description | Tese de doutoramento, Farmácia (Biotecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2017 | pt_PT |
| dc.description.abstract | Despite the success of antiretroviral therapy, a cure for HIV-1 infection remains elusive. The persistence of cellular reservoirs harboring transcriptionally silent (latent) HIV provirus is responsible for the viremia rebound observed following treatment withdrawal. Stimulation of latent viral expression is considered critical to target HIV reservoirs for elimination through a “shock and kill” approach. Pharmacological drugs have systematically proven ineffective to drastically reduce the reservoir size and may cause severe side effects owing to their indiscriminate mode of action. In the present thesis, gene-targeted strategies were explored to stimulate and eliminate HIV latent cells. To stimulate latent virus expression, we designed synthetic activators based on transcription activator-like effector (TALE) proteins that recognize conserved regions on HIV 5’LTR promoter. Four TALE activators strongly induced HIV transcription, acting in cooperation to specifically enhance viral expression from cell line models of HIV-1 latency. Moreover, we show that histone deacetylase inhibitors can further enhance the effect of TALE-mediated activation in highly repressed latent cells. To further potentiate the elimination of stimulated latent cells, we conjugated an HIV-responsive suicide lentivector to our TALE activator technology. For this purpose, we incorporated a modified 5’LTR promoter into the suicidal lentivector as a safety mechanism to dissociate TALE-driven activation, restricting the responsiveness of this plasmid to the HIV regulatory proteins. The therapeutic plasmid was capable of specifically eliminate latently infected cells stimulated by TALE activators through a Tat/Rev-dependent expression of the diphtheria toxin. Finally, we presented a “gene-free” approach to specifically activate latent HIV expression through protein delivery of cell-penetrating zinc-finger activators (CPP-ZFA). A single activator based on Cys2His2 zinc-finger domains proved effective at inducing viral expression from the primer binding site downstream of 5’LTR promoter. When conjugated with positively charged nuclear localization signal repeats, this synthetic activator efficiently translocated across cell membrane without the need of carriers. Short-term presence of CPP-ZFA following protein delivery was sufficient to stimulate gene expression in HIV-1 latent cells, offering a safer alternative to avoid off-target effects from prolonged exposure to these synthetic activators. In resume, this work provides proof-of-concept that synthetic activators and suicide lentivectors constitute promising candidates for the eradication of HIV-1 reservoirs through gene-targeted strategies. | pt_PT |
| dc.description.provenance | Submitted by Amelia Janeiro (ajaneiro@reitoria.ul.pt) on 2017-10-27T14:40:32Z No. of bitstreams: 1 ulsd731054_td_Pedro_Perdigao.pdf: 6176325 bytes, checksum: 69d0aaac454a14b71fb360c6f4753021 (MD5) | en |
| dc.description.provenance | Made available in DSpace on 2017-11-27T17:42:49Z (GMT). No. of bitstreams: 1 ulsd731054_td_Pedro_Perdigao.pdf: 6176325 bytes, checksum: 69d0aaac454a14b71fb360c6f4753021 (MD5) Previous issue date: 2017 | en |
| dc.description.sponsorship | Institute for Chemical Biology and the National Institutes of Health, DP1CA174426; Bill and Melinda Gates Foundation, Grand Challenges Explorations Round | pt_PT |
| dc.identifier.tid | 101385455 | pt_PT |
| dc.identifier.uri | http://hdl.handle.net/10451/29864 | |
| dc.language.iso | eng | pt_PT |
| dc.relation | ERADICATION OF HIV-1 INFECTED CELLS BY TARGETING NANOPARTICLES CARRYING THERAPEUTIC PLASMIDS | |
| dc.relation | Artificial Zinc-Finger transcription factors libraries: Identification of HIV-1 cellular restriction factors and Therapeutic application in HIV-1 infection. | |
| dc.subject | Teses de doutoramento - 2017 | pt_PT |
| dc.title | Gene-targeted strategies to eliminate HIV latent cells through synthetic activators and suicide lentivectors | pt_PT |
| dc.type | doctoral thesis | |
| dspace.entity.type | Publication | |
| oaire.awardTitle | ERADICATION OF HIV-1 INFECTED CELLS BY TARGETING NANOPARTICLES CARRYING THERAPEUTIC PLASMIDS | |
| oaire.awardTitle | Artificial Zinc-Finger transcription factors libraries: Identification of HIV-1 cellular restriction factors and Therapeutic application in HIV-1 infection. | |
| oaire.awardURI | info:eu-repo/grantAgreement/FCT//SFRH%2FBD%2F81941%2F2011/PT | |
| oaire.awardURI | info:eu-repo/grantAgreement/FCT/3599-PPCDT/VIH%2FSAU%2F0013%2F2011/PT | |
| oaire.awardURI | info:eu-repo/grantAgreement/FCT/3599-PPCDT/VIH%2FSAU%2F0020%2F2011/PT | |
| oaire.awardURI | info:eu-repo/grantAgreement/FCT/3599-PPCDT/HIVERA%2F0002%2F2013/PT | |
| oaire.fundingStream | 3599-PPCDT | |
| oaire.fundingStream | 3599-PPCDT | |
| oaire.fundingStream | 3599-PPCDT | |
| person.familyName | Perdigão | |
| person.givenName | Pedro Ricardo Lucas | |
| person.identifier.ciencia-id | 0F1E-EFD6-AF01 | |
| person.identifier.orcid | 0000-0003-2943-3515 | |
| project.funder.identifier | http://doi.org/10.13039/501100001871 | |
| project.funder.identifier | http://doi.org/10.13039/501100001871 | |
| project.funder.identifier | http://doi.org/10.13039/501100001871 | |
| project.funder.identifier | http://doi.org/10.13039/501100001871 | |
| project.funder.name | Fundação para a Ciência e a Tecnologia | |
| project.funder.name | Fundação para a Ciência e a Tecnologia | |
| project.funder.name | Fundação para a Ciência e a Tecnologia | |
| project.funder.name | Fundação para a Ciência e a Tecnologia | |
| rcaap.rights | openAccess | pt_PT |
| rcaap.type | doctoralThesis | pt_PT |
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| thesis.degree.name | Doutoramento em Farmácia | pt_PT |