Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Here, we use engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

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.

Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B‐cell malignancies. Notwithstanding, CAR T‐cell manufacturing requires complex procedures impeding the broad supply chain. Here, we provide evidence that human CD19‐CAR T cells can be generated directly in vivo using the lentiviral vector CD8‐LV specifically targeting human CD8⁺ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8⁺ T cells and efficacious elimination of CD19⁺ B cells. Further, upon injection of CD8‐LV into mice transplanted with human CD34⁺ cells, induction of CAR T cells and CD19⁺ B‐cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue‐invading CAR T cells and complete elimination of the B‐lymphocyte‐rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8⁺CAR T cells active against CD19⁺ cells, yet with similar adverse effects currently notorious in the clinical practice. Synopsis T cells were reprogrammed with chimeric antigen receptors (CARs) in situ upon systemic injection of CD8‐targeted lentiviral vectors, thus providing proof‐of‐principle for converting CAR T cell therapy from an autologous, individualized to an off‐the‐shelf product. In situ CAR T cell generation was evidenced in mice transplanted with human PBMC or CD34⁺ stem cells. In situ generated CAR T cells expanded upon antigen recognition and eliminated CD19⁺ cells. Some mice developed side‐effects resembling the cytokine release syndrome observed in patients treated with CAR T cells.

Hematopoietic stem cell (HSC)-based gene therapy holds promise for the cure of many diseases. The field is now moving toward the use of lentiviral vectors (LVs) as evidenced by 4 successful clinical trials. These trials used vesicular-stomatitis-virus-G protein (VSV-G)-LVs at high doses combined with strong cytokine-cocktail stimulation to obtain therapeutically relevant transduction levels; however, they might compromise the HSC character. Summarizing all these disadvantages, alternatives to VSV-G-LVs are urgently needed. We generated here high-titer LVs pseudotyped with a baboon retroviral envelope glycoprotein (BaEV-LVs), resistant to human complement. Under mild cytokine prestimulation to preserve the HSC characteristics, a single BaEV-LV application at a low dose, resulted in up to 90% of hCD34⁺ cell transduction. Even more striking was that these new BaEV-LVs allowed, at low doses, efficient transduction of up to 30% of quiescent hCD34⁺ cells, whereas high-dose VSV-G-LVs were insufficient. Importantly, reconstitution of NOD/Lt-SCID/γc^−/− (NSG) mice with BaEV-LV-transduced hCD34⁺ cells maintained these high transduction levels in all myeloid and lymphoid lineages, including early progenitors. This transduction pattern was confirmed or even increased in secondary NSG recipient mice. This suggests that BaEV-LVs efficiently transduce true HSCs and could improve HSC-based gene therapy, for which high-level HSC correction is needed for life-long cure.