Parkin-independent mitophagy controls chemotherapeutic response in cancer cells

Mitophagy is an evolutionarily conserved process that selectively targets impaired mitochondria for degradation. Defects in mitophagy are often associated with diverse pathologies, including cancer. Because the main known regulators of mitophagy are frequently inactivated in cancer cells, the mechanisms that regulate mitophagy in cancer cells are not fully understood. Here, we identified an E3 ubiquitin ligase (ARIH1/HHARI) that triggers mitophagy in cancer cells in a PINK1-dependent manner. We found that ARIH1/HHARI polyubiquitinates damaged mitochondria, leading to their removal via autophagy. Importantly, ARIH1 is widely expressed in cancer cells, notably in breast and lung adenocarcinomas; ARIH1 expression protects against chemotherapy-induced death. These data challenge the view that the main regulators of mitophagy are tumor suppressors, arguing instead that ARIH1-mediated mitophagy promotes therapeutic resistance

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

CD20 and CD19 targeted vectors induce minimal activation of resting B lymphocytes

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction

Nanoblades allow high-level genome editing in murine and human organoids

Genome engineering has become more accessible thanks to the CRISPR-Cas9 gene-editing system. However, using this technology in synthetic organs called “organoids” is still very inefficient. This is due to the delivery methods for the CRISPR-Cas9 machinery, which include electroporation of CRISPR-Cas9 DNA, mRNA, or ribonucleoproteins containing the Cas9-gRNA complex. However, these procedures are quite toxic for the organoids. Here, we describe the use of the “nanoblade (NB)” technology, which outperformed by far gene-editing levels achieved to date for murine- and human tissue-derived organoids. We reached up to 75% of reporter gene knockout in organoids after treatment with NBs. Indeed, high-level NB-mediated knockout for the androgen receptor encoding gene and the cystic fibrosis transmembrane conductance regulator gene was achieved with single gRNA or dual gRNA containing NBs in murine prostate and colon organoids. Likewise, NBs achieved 20%–50% gene editing in human organoids. Most importantly, in contrast to other gene-editing methods, this was obtained without toxicity for the organoids. Only 4 weeks are required to obtain stable gene knockout in organoids and NBs simplify and allow rapid genome editing in organoids with little to no side effects including unwanted insertion/deletions in off-target sites thanks to transient Cas9/RNP expression

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus

Editorial: Gene Therapy for Fanconi Anemia Enters a New Clinical Era

International audienc

Humanized Mice Are Precious Tools for Preclinical Evaluation of CAR T and CAR NK Cell Therapies

Chimeric antigen receptor (CAR) T-cell therapy represents a revolutionary treatment for hematological malignancies. However, improvements in CAR T-cell therapies are urgently needed since CAR T cell application is associated with toxicities, exhaustion, immune suppression, lack of long-term persistence, and low CAR T-cell tumor infiltration. Major efforts to overcome these hurdles are currently on the way. Incrementally improved xenograft mouse models, supporting the engraftment and development of a human hemato-lymphoid system and tumor tissue, represent an important fundamental and preclinical research tool. We will focus here on several CAR T and CAR NK therapies that have benefited from evaluation in humanized mice. These models are of great value for the cancer therapy field as they provide a more reliable understanding of sometimes complicated therapeutic interventions. Additionally, they are considered the gold standard with regard to assessment of new CAR technologies in vivo for safety, efficacy, immune response, design, combination therapies, exhaustion, persistence, and mechanism of action prior to starting a clinical trial. They help to expedite the critical translation from proof-of-concept to clinical CAR T-cell application. In this review, we discuss innovative developments in the CAR T-cell therapy field that benefited from evaluation in humanized mice, illustrated by multiple examples