Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery

Autores
Abrey Recalde, Maria Jimena; Gutierrez Guerrero, Alejandra; Mangeot, Phillipe; Costa, Caroline; Bernandin, Ornelie; Froment, Giselle; Molina, Francisco Martin; Karim, Bellabdelah; Frecha, Cecilia Ariana; Ricci, Emiliano; Cosset, Francose; Verhoeyen, Els
Año de publicación
2018
Idioma
inglés
Tipo de recurso
documento de conferencia
Estado
versión publicada
Descripción
Programmable nucleases have enabled rapid and accessible genome engineering in cells andliving organisms. However, their delivery into target cells can be challenging, specially intoprimary cells. Here, we have designed Nanoblades, a new technology to deliver a genomiccleaving agent into cells. These are murine leukemia virus-derived virus like particle (VLP),which are loaded with Cas9 protein through fusion with the gag viral structural protein, andwith guide RNAs. Cas9 together with gRNAs introduces site specifics double strand break(DSBs) in target gene which can be repaired by non-homologous end-joining (NHEJ) or byhomology-directed repair (HDR) introducing a new sequence from an exogenous templateDNA bearing homology to the sequences flanking the DSBs (donor-DNA).Previously, we demonstrated that Nanoblades were extremely efficient in delivery of theirCas9/sgRNA cargo into K562 human cell line and human T, B , HSCs and HSCs derivedprogenitors T cells (pro-T cells ), thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelopes.The objective of this work was to edit Wiscott Aldrich Syndrome (WAS) gene locus by HDRusing Nanoblades and AAV6 carrying a donor-DNA, which consists in GFP reporter geneflanking by homologous arms of the WAS gene.AAV6 were added to K562 cells at different times points with respect to Nanobladesaddition, in order to find optimal time that maximizes HDR. Different multiplicities ofinfection (MOI) of AAV were tested. HDR-mediated gene editing was determined by PCRand GFP expression by FACS, 7 days after Nanoblades addition.Our results shows that HDR-mediated edition of WAS gene occurred in a 50% of cells, whennanoblades and AAV6 (MOI 100000 vg/cell) were added at same time. We are currentlytesting this protocol of AAV6 and Nanoblades in HSCs and pro-T cells.In summary, Nanoblades in combination with AAV6 carrying donor-DNA are efficienttools for gene editing and have important prospects for basic and clinical translation forgene therapy.
Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina
Fil: Gutierrez Guerrero, Alejandra. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina
Fil: Mangeot, Phillipe. Inserm; Francia
Fil: Costa, Caroline. Inserm; Francia
Fil: Bernandin, Ornelie. Inserm; Francia
Fil: Froment, Giselle. Inserm; Francia
Fil: Molina, Francisco Martin. No especifíca;
Fil: Karim, Bellabdelah. No especifíca;
Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina
Fil: Ricci, Emiliano. Inserm; Francia
Fil: Cosset, Francose. Inserm; Francia
Fil: Verhoeyen, Els. Inserm; Francia
LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de Fisiología
Mar del Plata
Argentina
Sociedad Argentina de Investigación Clínica
Sociedad Argentina de Inmunologia
Sociedad Argentina de Fisiología
Sociedad Argentina de Virología
Asociación Argentina de Nanomedicina
Materia
NANOBLADES
GENE EDITING
CRISPR
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:11336/183376
Acceder
id CONICETDig_b5626ea3d534c9bf4c63121df0e71a50
oai_identifier_str oai:ri.conicet.gov.ar:11336/183376
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette deliveryAbrey Recalde, Maria JimenaGutierrez Guerrero, AlejandraMangeot, PhillipeCosta, CarolineBernandin, OrnelieFroment, GiselleMolina, Francisco MartinKarim, BellabdelahFrecha, Cecilia ArianaRicci, EmilianoCosset, FrancoseVerhoeyen, ElsNANOBLADESGENE EDITINGCRISPRhttps://purl.org/becyt/ford/3.4https://purl.org/becyt/ford/3Programmable nucleases have enabled rapid and accessible genome engineering in cells andliving organisms. However, their delivery into target cells can be challenging, specially intoprimary cells. Here, we have designed Nanoblades, a new technology to deliver a genomiccleaving agent into cells. These are murine leukemia virus-derived virus like particle (VLP),which are loaded with Cas9 protein through fusion with the gag viral structural protein, andwith guide RNAs. Cas9 together with gRNAs introduces site specifics double strand break(DSBs) in target gene which can be repaired by non-homologous end-joining (NHEJ) or byhomology-directed repair (HDR) introducing a new sequence from an exogenous templateDNA bearing homology to the sequences flanking the DSBs (donor-DNA).Previously, we demonstrated that Nanoblades were extremely efficient in delivery of theirCas9/sgRNA cargo into K562 human cell line and human T, B , HSCs and HSCs derivedprogenitors T cells (pro-T cells ), thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelopes.The objective of this work was to edit Wiscott Aldrich Syndrome (WAS) gene locus by HDRusing Nanoblades and AAV6 carrying a donor-DNA, which consists in GFP reporter geneflanking by homologous arms of the WAS gene.AAV6 were added to K562 cells at different times points with respect to Nanobladesaddition, in order to find optimal time that maximizes HDR. Different multiplicities ofinfection (MOI) of AAV were tested. HDR-mediated gene editing was determined by PCRand GFP expression by FACS, 7 days after Nanoblades addition.Our results shows that HDR-mediated edition of WAS gene occurred in a 50% of cells, whennanoblades and AAV6 (MOI 100000 vg/cell) were added at same time. We are currentlytesting this protocol of AAV6 and Nanoblades in HSCs and pro-T cells.In summary, Nanoblades in combination with AAV6 carrying donor-DNA are efficienttools for gene editing and have important prospects for basic and clinical translation forgene therapy.Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Gutierrez Guerrero, Alejandra. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Mangeot, Phillipe. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornelie. Inserm; FranciaFil: Froment, Giselle. Inserm; FranciaFil: Molina, Francisco Martin. No especifíca;Fil: Karim, Bellabdelah. No especifíca;Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; ArgentinaFil: Ricci, Emiliano. Inserm; FranciaFil: Cosset, Francose. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaLXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de FisiologíaMar del PlataArgentinaSociedad Argentina de Investigación ClínicaSociedad Argentina de InmunologiaSociedad Argentina de FisiologíaSociedad Argentina de VirologíaAsociación Argentina de NanomedicinaFundación Revista Medicina2018info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/conferenceObjectReuniónJournalhttp://purl.org/coar/resource_type/c_5794info:ar-repo/semantics/documentoDeConferenciaapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/183376Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de Fisiología; Mar del Plata; Argentina; 2018; 1-1CONICET 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 Técnicas2025-09-29T10:00:12Zoai:ri.conicet.gov.ar:11336/183376instacron: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 10:00:12.524CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
title Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
spellingShingle Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
Abrey Recalde, Maria Jimena
NANOBLADES
GENE EDITING
CRISPR
title_short Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
title_full Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
title_fullStr Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
title_full_unstemmed Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
title_sort Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery
dc.creator.none.fl_str_mv Abrey Recalde, Maria Jimena
Gutierrez Guerrero, Alejandra
Mangeot, Phillipe
Costa, Caroline
Bernandin, Ornelie
Froment, Giselle
Molina, Francisco Martin
Karim, Bellabdelah
Frecha, Cecilia Ariana
Ricci, Emiliano
Cosset, Francose
Verhoeyen, Els
author Abrey Recalde, Maria Jimena
author_facet Abrey Recalde, Maria Jimena
Gutierrez Guerrero, Alejandra
Mangeot, Phillipe
Costa, Caroline
Bernandin, Ornelie
Froment, Giselle
Molina, Francisco Martin
Karim, Bellabdelah
Frecha, Cecilia Ariana
Ricci, Emiliano
Cosset, Francose
Verhoeyen, Els
author_role author
author2 Gutierrez Guerrero, Alejandra
Mangeot, Phillipe
Costa, Caroline
Bernandin, Ornelie
Froment, Giselle
Molina, Francisco Martin
Karim, Bellabdelah
Frecha, Cecilia Ariana
Ricci, Emiliano
Cosset, Francose
Verhoeyen, Els
author2_role author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv NANOBLADES
GENE EDITING
CRISPR
topic NANOBLADES
GENE EDITING
CRISPR
purl_subject.fl_str_mv https://purl.org/becyt/ford/3.4
https://purl.org/becyt/ford/3
dc.description.none.fl_txt_mv Programmable nucleases have enabled rapid and accessible genome engineering in cells andliving organisms. However, their delivery into target cells can be challenging, specially intoprimary cells. Here, we have designed Nanoblades, a new technology to deliver a genomiccleaving agent into cells. These are murine leukemia virus-derived virus like particle (VLP),which are loaded with Cas9 protein through fusion with the gag viral structural protein, andwith guide RNAs. Cas9 together with gRNAs introduces site specifics double strand break(DSBs) in target gene which can be repaired by non-homologous end-joining (NHEJ) or byhomology-directed repair (HDR) introducing a new sequence from an exogenous templateDNA bearing homology to the sequences flanking the DSBs (donor-DNA).Previously, we demonstrated that Nanoblades were extremely efficient in delivery of theirCas9/sgRNA cargo into K562 human cell line and human T, B , HSCs and HSCs derivedprogenitors T cells (pro-T cells ), thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelopes.The objective of this work was to edit Wiscott Aldrich Syndrome (WAS) gene locus by HDRusing Nanoblades and AAV6 carrying a donor-DNA, which consists in GFP reporter geneflanking by homologous arms of the WAS gene.AAV6 were added to K562 cells at different times points with respect to Nanobladesaddition, in order to find optimal time that maximizes HDR. Different multiplicities ofinfection (MOI) of AAV were tested. HDR-mediated gene editing was determined by PCRand GFP expression by FACS, 7 days after Nanoblades addition.Our results shows that HDR-mediated edition of WAS gene occurred in a 50% of cells, whennanoblades and AAV6 (MOI 100000 vg/cell) were added at same time. We are currentlytesting this protocol of AAV6 and Nanoblades in HSCs and pro-T cells.In summary, Nanoblades in combination with AAV6 carrying donor-DNA are efficienttools for gene editing and have important prospects for basic and clinical translation forgene therapy.
Fil: Abrey Recalde, Maria Jimena. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina
Fil: Gutierrez Guerrero, Alejandra. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina
Fil: Mangeot, Phillipe. Inserm; Francia
Fil: Costa, Caroline. Inserm; Francia
Fil: Bernandin, Ornelie. Inserm; Francia
Fil: Froment, Giselle. Inserm; Francia
Fil: Molina, Francisco Martin. No especifíca;
Fil: Karim, Bellabdelah. No especifíca;
Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Hospital Italiano. Instituto de Medicina Traslacional E Ingenieria Biomedica. - Instituto Universitario Hospital Italiano de Buenos Aires. Instituto de Medicina Traslacional E Ingenieria Biomedica.; Argentina
Fil: Ricci, Emiliano. Inserm; Francia
Fil: Cosset, Francose. Inserm; Francia
Fil: Verhoeyen, Els. Inserm; Francia
LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de Fisiología
Mar del Plata
Argentina
Sociedad Argentina de Investigación Clínica
Sociedad Argentina de Inmunologia
Sociedad Argentina de Fisiología
Sociedad Argentina de Virología
Asociación Argentina de Nanomedicina
description Programmable nucleases have enabled rapid and accessible genome engineering in cells andliving organisms. However, their delivery into target cells can be challenging, specially intoprimary cells. Here, we have designed Nanoblades, a new technology to deliver a genomiccleaving agent into cells. These are murine leukemia virus-derived virus like particle (VLP),which are loaded with Cas9 protein through fusion with the gag viral structural protein, andwith guide RNAs. Cas9 together with gRNAs introduces site specifics double strand break(DSBs) in target gene which can be repaired by non-homologous end-joining (NHEJ) or byhomology-directed repair (HDR) introducing a new sequence from an exogenous templateDNA bearing homology to the sequences flanking the DSBs (donor-DNA).Previously, we demonstrated that Nanoblades were extremely efficient in delivery of theirCas9/sgRNA cargo into K562 human cell line and human T, B , HSCs and HSCs derivedprogenitors T cells (pro-T cells ), thanks to their surface co-pseudotyping with baboonretroviral and VSV-G envelopes.The objective of this work was to edit Wiscott Aldrich Syndrome (WAS) gene locus by HDRusing Nanoblades and AAV6 carrying a donor-DNA, which consists in GFP reporter geneflanking by homologous arms of the WAS gene.AAV6 were added to K562 cells at different times points with respect to Nanobladesaddition, in order to find optimal time that maximizes HDR. Different multiplicities ofinfection (MOI) of AAV were tested. HDR-mediated gene editing was determined by PCRand GFP expression by FACS, 7 days after Nanoblades addition.Our results shows that HDR-mediated edition of WAS gene occurred in a 50% of cells, whennanoblades and AAV6 (MOI 100000 vg/cell) were added at same time. We are currentlytesting this protocol of AAV6 and Nanoblades in HSCs and pro-T cells.In summary, Nanoblades in combination with AAV6 carrying donor-DNA are efficienttools for gene editing and have important prospects for basic and clinical translation forgene therapy.
publishDate 2018
dc.date.none.fl_str_mv 2018
dc.type.none.fl_str_mv info:eu-repo/semantics/publishedVersion
info:eu-repo/semantics/conferenceObject
Reunión
Journal
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/11336/183376
Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de Fisiología; Mar del Plata; Argentina; 2018; 1-1
CONICET Digital
CONICET
url http://hdl.handle.net/11336/183376
identifier_str_mv Viral derived “nanoblades” loaded with cas9/ sgrna ribonucleoproteins and aav6 for donor dna cassette delivery; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica;LXVI Reunión Anual de la Sociedad Argentina de Inmunologia y Reunión anual de la Sociedad Argentina de Fisiología; Mar del Plata; Argentina; 2018; 1-1
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/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.coverage.none.fl_str_mv Nacional
dc.publisher.none.fl_str_mv Fundación Revista Medicina
publisher.none.fl_str_mv Fundación Revista Medicina
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
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
_version_ 1844613780178206720
score 13.070432

Publicaciones similares