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

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.; 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 Nanomedicin

Site Specific Knock-In Genome Editing in Human HSCs Using Baboon Envelope gp Pseudotypedviral Derived “Nanoblades” Loaded with Cas9/sgRNA Combined with Donor Encoding AAV-6

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. Here, we have designed ?Nanoblades?, a new technology that will deliver a genomic cleaving agent into cells. These are genetically modified Murine Leukemia Virus (MLV) or HIV derived virus like particle (VLP), in which the viral structural protein Gag has been fused to the Cas9. These VLPs are thus loaded with Cas9 protein together with the guide RNAs. Thus, nanoblades are devoid of any viral-derived genetic material. Highly efficient gene editing was obtained in cell lines, IPS cells and primary mouse and human cells (Mangeot et al. Nature Communication, 2019). However, their delivery into target cells can be technically challenging when working with primary immune cells. Now we showed that nanoblades were remarkably efficient for entry into human T, B and hematopoietic stem cells thanks to their surface co-pseudotyping with baboon retroviral and VSVG envelope glycoproteins. We were able to induce efficient, transient and very rapidlygenome-editing in human induced pluripotent stem cells reaching up to 70% in the empty spiracles homeobox 1 (EMX1) and muscular dystrophy (MD) gene locus. A brief nanoblade incubation of primary human T and B cells resulted in 40% and 20% editing of the Wiskott-Aldrich syndrome (WAS) gene locus, while hematopoietic stem cells treated for 18 h with nanoblades allowed 30-40% gene editing in the WAS gene locus and up to 80% for the Myd88 genomic target. Moreover, for the HIV- and MLV-derived nanoblades no cell toxicity and low to undetectable off-target effects were demonstrated.Finally, we also treated hHSCs with nanoblades in combination with an AAV-6 donor encoding vector resulting in over 20% of stable expression cassette knock-in into the WAS gene locus. Currently, we are evaluating these gene modified HSCs for their long-term reconstitution of NOD/SCIDgC-/- mice.Summarizing, this new technology is simple to implement in any laboratory, shows high flexibility for different targets including primary immune cells of murine and human origin, is relatively inexpensive and therefore have important prospects for basic and clinical translation in the area of gene therapy.Fil: Gutierrez, Alejandra. Inserm; FranciaFil: 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. Inserm; FranciaFil: Mangeot, Philippe E.. Inserm; FranciaFil: Costa, Caroline. Inserm; FranciaFil: Bernandin, Ornellie. Inserm; FranciaFil: Fusil, Floriane. Inserm; FranciaFil: Froment, Gisèle. Inserm; FranciaFil: Martin, Francisco. Inserm; FranciaFil: Bellabdelah, Karim. Universidad de Granada; EspañaFil: Ricci, Emiliano P.. Inserm; FranciaFil: Ayuso, Eduard. Universite de Nantes; FranciaFil: Cosset, François loic. Inserm; FranciaFil: Verhoeyen, Els. Inserm; FranciaAmerican Society of Cell and Gene Therapy 22nd Annual MettingWashingtonEstados UnidosAmerican Society of Cell and Gene Therap