Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease

Autores
Abrey Recalde, Maria Jimena; Gonzalez Hermida, Paula; Baez, Veronica; Jerusalinsky, Diana Alicia; Frecha, Cecilia Ariana
Año de publicación
2018
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by a progressive loss of cognitive functions. One of the hallmarks is the formation of amyloid plaques, composed mainly by Aß peptide oligomers (AβOs). Neprilysin (NEP) is the most important endopeptidase and crucial for the degradation of Aß in the brain, avoiding amyloid plaques formation. Because NEP is decreased in brains of patients with AD, we aim to develop a lentiviral vector (LV) capable of specifically expressing in hippocampal neurons, to study its role in AD and therefore its possible therapeutic function.First, a construct containing the complete cDNA of NEP downstream of the hippocampal-specific promoter human synapsin (SYN-1) was performed. NEP cDNA was obtained from pBOB-NEP plasmid and was cloned under the SYN-1 promoter to obtain SYN-NEP plasmid. SYN-NEP also contains the ubiquitous CMV promoter, located outside the sequence to be packaged in LV. The correct cloning was checked by BamHI/KpnI digestion followed by 1% agarose gel electrophoresis and was verified by sequencing. To test NEP expression under the CMV promoter, 293T cells were transfected with SYN-NEP. SYN-RFP plasmid expressing the reporter red fluorescent protein (RFP) and pBOB-NEP were used as transfection and positive controls, respectively. After 48 hours NEP expression was evaluated by western blot (WB) and RFP by fluorescence microscopy.Electrophoresis of SYN-NEP digestion resulted in two bands of 3392pb and 7644pb, which coincided with the molecular weights of the insert containing NEP and backbone, respectively. This in turn was validated by sequencing. Transfection efficiency calculated by expression of RFP was 80%. WB showed a 85kDa band only in those lanes corresponding to transfection with pBOB-NEP and SYN-NEP.In conclusion, NEP was successfully cloned downstream of SYN-1 promoter and is expressed correctly under a ubiquitous promoter. This construction is the first step for the production of LV.
Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; Argentina
Fil: Gonzalez Hermida, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; Argentina
Fil: Baez, Veronica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina
Fil: Jerusalinsky, Diana Alicia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; Argentina
LXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio
Argentina
Sociedad Argentina de Investigación Clinica
Sociedad Argentina de Inmunología
Sociedad Argentina de Farmacología Experimental
Sociedad Argentina de Nanomedicina
Asociación Argentina de ciencia y tecnología de animales de Laboratorio
Materia
Alzheimer's disease
NEPYLISIN
LENTIVIRAL VECTORS
SYNAPSIN PROMOTER
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/198440
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/198440
network_acronym_str CONICETDig
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network_name_str CONICET Digital (CONICET)
spelling Design of a lentiviral vector as a therapeutic strategy against alzheimer´s diseaseAbrey Recalde, Maria JimenaGonzalez Hermida, PaulaBaez, VeronicaJerusalinsky, Diana AliciaFrecha, Cecilia ArianaAlzheimer's diseaseNEPYLISINLENTIVIRAL VECTORSSYNAPSIN PROMOTERhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by a progressive loss of cognitive functions. One of the hallmarks is the formation of amyloid plaques, composed mainly by Aß peptide oligomers (AβOs). Neprilysin (NEP) is the most important endopeptidase and crucial for the degradation of Aß in the brain, avoiding amyloid plaques formation. Because NEP is decreased in brains of patients with AD, we aim to develop a lentiviral vector (LV) capable of specifically expressing in hippocampal neurons, to study its role in AD and therefore its possible therapeutic function.First, a construct containing the complete cDNA of NEP downstream of the hippocampal-specific promoter human synapsin (SYN-1) was performed. NEP cDNA was obtained from pBOB-NEP plasmid and was cloned under the SYN-1 promoter to obtain SYN-NEP plasmid. SYN-NEP also contains the ubiquitous CMV promoter, located outside the sequence to be packaged in LV. The correct cloning was checked by BamHI/KpnI digestion followed by 1% agarose gel electrophoresis and was verified by sequencing. To test NEP expression under the CMV promoter, 293T cells were transfected with SYN-NEP. SYN-RFP plasmid expressing the reporter red fluorescent protein (RFP) and pBOB-NEP were used as transfection and positive controls, respectively. After 48 hours NEP expression was evaluated by western blot (WB) and RFP by fluorescence microscopy.Electrophoresis of SYN-NEP digestion resulted in two bands of 3392pb and 7644pb, which coincided with the molecular weights of the insert containing NEP and backbone, respectively. This in turn was validated by sequencing. Transfection efficiency calculated by expression of RFP was 80%. WB showed a 85kDa band only in those lanes corresponding to transfection with pBOB-NEP and SYN-NEP.In conclusion, NEP was successfully cloned downstream of SYN-1 promoter and is expressed correctly under a ubiquitous promoter. This construction is the first step for the production of LV.Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaFil: Gonzalez Hermida, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaFil: Baez, Veronica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; ArgentinaFil: Jerusalinsky, Diana Alicia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; ArgentinaLXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de LaboratorioArgentinaSociedad Argentina de Investigación ClinicaSociedad Argentina de InmunologíaSociedad Argentina de Farmacología ExperimentalSociedad Argentina de NanomedicinaAsociación Argentina de ciencia y tecnología de animales de LaboratorioFundacion Revista Medicina2018info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/198440Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease; LXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio; Argentina; 2016; 110-111CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.saic.org.ar/reunion-anualNacionalinfo:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T09:38:22Zoai:ri.conicet.gov.ar:11336/198440instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 09:38:22.506CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
title Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
spellingShingle Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
Abrey Recalde, Maria Jimena
Alzheimer's disease
NEPYLISIN
LENTIVIRAL VECTORS
SYNAPSIN PROMOTER
title_short Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
title_full Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
title_fullStr Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
title_full_unstemmed Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
title_sort Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease
dc.creator.none.fl_str_mv Abrey Recalde, Maria Jimena
Gonzalez Hermida, Paula
Baez, Veronica
Jerusalinsky, Diana Alicia
Frecha, Cecilia Ariana
author Abrey Recalde, Maria Jimena
author_facet Abrey Recalde, Maria Jimena
Gonzalez Hermida, Paula
Baez, Veronica
Jerusalinsky, Diana Alicia
Frecha, Cecilia Ariana
author_role author
author2 Gonzalez Hermida, Paula
Baez, Veronica
Jerusalinsky, Diana Alicia
Frecha, Cecilia Ariana
author2_role author
author
author
author
dc.subject.none.fl_str_mv Alzheimer's disease
NEPYLISIN
LENTIVIRAL VECTORS
SYNAPSIN PROMOTER
topic Alzheimer's disease
NEPYLISIN
LENTIVIRAL VECTORS
SYNAPSIN PROMOTER
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by a progressive loss of cognitive functions. One of the hallmarks is the formation of amyloid plaques, composed mainly by Aß peptide oligomers (AβOs). Neprilysin (NEP) is the most important endopeptidase and crucial for the degradation of Aß in the brain, avoiding amyloid plaques formation. Because NEP is decreased in brains of patients with AD, we aim to develop a lentiviral vector (LV) capable of specifically expressing in hippocampal neurons, to study its role in AD and therefore its possible therapeutic function.First, a construct containing the complete cDNA of NEP downstream of the hippocampal-specific promoter human synapsin (SYN-1) was performed. NEP cDNA was obtained from pBOB-NEP plasmid and was cloned under the SYN-1 promoter to obtain SYN-NEP plasmid. SYN-NEP also contains the ubiquitous CMV promoter, located outside the sequence to be packaged in LV. The correct cloning was checked by BamHI/KpnI digestion followed by 1% agarose gel electrophoresis and was verified by sequencing. To test NEP expression under the CMV promoter, 293T cells were transfected with SYN-NEP. SYN-RFP plasmid expressing the reporter red fluorescent protein (RFP) and pBOB-NEP were used as transfection and positive controls, respectively. After 48 hours NEP expression was evaluated by western blot (WB) and RFP by fluorescence microscopy.Electrophoresis of SYN-NEP digestion resulted in two bands of 3392pb and 7644pb, which coincided with the molecular weights of the insert containing NEP and backbone, respectively. This in turn was validated by sequencing. Transfection efficiency calculated by expression of RFP was 80%. WB showed a 85kDa band only in those lanes corresponding to transfection with pBOB-NEP and SYN-NEP.In conclusion, NEP was successfully cloned downstream of SYN-1 promoter and is expressed correctly under a ubiquitous promoter. This construction is the first step for the production of LV.
Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; Argentina
Fil: Gonzalez Hermida, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; Argentina
Fil: Baez, Veronica. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina
Fil: Jerusalinsky, Diana Alicia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina
Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional e Ingeniería Biomédica - Hospital Italiano. Instituto de Medicina Traslacional e Ingeniería Biomédica.- Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional e Ingeniería Biomédica; Argentina
LXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio
Argentina
Sociedad Argentina de Investigación Clinica
Sociedad Argentina de Inmunología
Sociedad Argentina de Farmacología Experimental
Sociedad Argentina de Nanomedicina
Asociación Argentina de ciencia y tecnología de animales de Laboratorio
description Alzheimer’s disease (AD) is a chronic neurodegenerative disorder characterized by a progressive loss of cognitive functions. One of the hallmarks is the formation of amyloid plaques, composed mainly by Aß peptide oligomers (AβOs). Neprilysin (NEP) is the most important endopeptidase and crucial for the degradation of Aß in the brain, avoiding amyloid plaques formation. Because NEP is decreased in brains of patients with AD, we aim to develop a lentiviral vector (LV) capable of specifically expressing in hippocampal neurons, to study its role in AD and therefore its possible therapeutic function.First, a construct containing the complete cDNA of NEP downstream of the hippocampal-specific promoter human synapsin (SYN-1) was performed. NEP cDNA was obtained from pBOB-NEP plasmid and was cloned under the SYN-1 promoter to obtain SYN-NEP plasmid. SYN-NEP also contains the ubiquitous CMV promoter, located outside the sequence to be packaged in LV. The correct cloning was checked by BamHI/KpnI digestion followed by 1% agarose gel electrophoresis and was verified by sequencing. To test NEP expression under the CMV promoter, 293T cells were transfected with SYN-NEP. SYN-RFP plasmid expressing the reporter red fluorescent protein (RFP) and pBOB-NEP were used as transfection and positive controls, respectively. After 48 hours NEP expression was evaluated by western blot (WB) and RFP by fluorescence microscopy.Electrophoresis of SYN-NEP digestion resulted in two bands of 3392pb and 7644pb, which coincided with the molecular weights of the insert containing NEP and backbone, respectively. This in turn was validated by sequencing. Transfection efficiency calculated by expression of RFP was 80%. WB showed a 85kDa band only in those lanes corresponding to transfection with pBOB-NEP and SYN-NEP.In conclusion, NEP was successfully cloned downstream of SYN-1 promoter and is expressed correctly under a ubiquitous promoter. This construction is the first step for the production of LV.
publishDate 2018
dc.date.none.fl_str_mv 2018
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
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http://purl.org/coar/resource_type/c_5794
info:ar-repo/semantics/documentoDeConferencia
status_str publishedVersion
format conferenceObject
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/198440
Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease; LXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio; Argentina; 2016; 110-111
CONICET Digital
CONICET
url http://hdl.handle.net/11336/198440
identifier_str_mv Design of a lentiviral vector as a therapeutic strategy against alzheimer´s disease; LXI Reunión anual de la Sociedad Argentina de Investigación Clinica; LXVI, Reunión anual de la Sociedad Argentina de Inmunología; XLVIII Reunión Anual de la Sociedad Argentina de Farmacología Experimental; VII Reunión Anual de la Sociedad Argentina de Nanomedicina y V Congreso Nacional de la Asociación Argentina de Ciencia y Tecnología de Animales de Laboratorio; Argentina; 2016; 110-111
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.saic.org.ar/reunion-anual
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
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dc.format.none.fl_str_mv application/pdf
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dc.coverage.none.fl_str_mv Nacional
dc.publisher.none.fl_str_mv Fundacion Revista Medicina
publisher.none.fl_str_mv Fundacion Revista Medicina
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instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
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