Highlights d NHEJ-mediated gene editing enables highly efficient editing in human long-term HSCs d NHEJ-mediated editing restores mutant coding frames across FA complementation groups d Corrected FA-HSCs have a marked proliferative advantage in vitro and in vivo

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34 + cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34 + cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34 + cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.

Background:Chimeric antigen receptors (CARs) have been used in the past several years in cancer therapy to redirect immune effector cells. Despite impressive preliminary efficacy of CAR‐T cells in multiple myeloma (MM), NK cell engineering has emerged as a competitive and safer approach. NK‐92 is a universal, cheap and fast “off‐the‐shelf” cellular therapeutic previously used in clinical trials. Although modest responses with these cells have been reported in MM, their oncolytic potential can be enhanced by genetic modification. So far, two preclinical studies have been performed with CAR NK‐92 against MM, targeting CD138 or CS1 (SLAMF7). However, there are still reasonable doubts about its clinical outcomes due to on‐target off‐tumor effect or fratricide, respectively.Aims:Thus, the aim of our study is to generate and compare two novel CAR NK‐92 products for MM treatment.Methods:NK‐92MI cells were lentivirally transduced with the full‐length ectodomain sequence of the human native NKG2D receptor or with an anti‐BCMA scFv, both containing identical 4–1BB costimulatory and CD3‐ζ signaling domains (Figure 1A). To compare the efficacy between these 2nd generation NKG2D‐CAR NK‐92MI and BCMA‐CAR NK‐92MI cells, the same MOI 10 was used to transduce them; both populations were then purified by FACS sorting to obtain stable modified cell lines and vector copy number was measured by qPCR to ensure similar CAR expression. Cytotoxicity assays were performed by 3‐hours Calcein‐AM analysis. We used MM cell lines with different expression of target ligands: U266, BCMAhigh and NKG2DLhigh; XG‐1, a NK resistant cell line, BCMAhigh and NKG2DLlow; NCI H929 R20, a bortezomib resistant cell line with NKG2DLlow. K562, a leukemia cell line with BCMAnegative and NKG2DLhigh expression, was used as negative control.Results:NKG2D‐CAR NK‐92MI cells consistently showed much higher in vitro antitumor activity than the parental line NK‐92MI against U266 (84 ± 2% vs 40.7 ± 4% at a 1:1 E:T ratio), XG‐1 (67.9 ± 9% vs 18.5 ± 4% at a 16:1 E:T ratio) and NCI H929 #R20 (50.9 ± 6% vs 23.7 ± 2% at a 16:1 E:T ratio) cell lines (Figure 1B). NKG2D‐CAR NK‐92MI proved specificity in a blocking assay with an anti‐NKG2D antibody (82% decrease compares to baseline lysis). Furthermore, the oncolytic potential of NKG2D‐CAR NK‐92MI was not altered by physiological levels of soluble MICA, described in MM patients, or mandatory 10Gy irradiation prior to clinical use. Next, we compared cytotoxicity between NKG2D‐CAR NK‐92MI and BCMA‐CAR NK‐92MI against U‐266 (84 ± 2% vs 91.9 ± 3% at a 1:1 E:T ratio), XG‐1 (67.9 ± 9% vs 89.9 ± 2% at a 16:1 E:T ratio) and K562 (94 ± 3% vs 25.74 ± 4% at a 1:1 E:T ratio) cell lines (Figure 1C). Strikingly, there were no significant differences between NKG2D‐CAR NK and the gold standard BCMA‐CAR against MM cell lines with high and similar BCMA and NKG2DL expression. In addition, correlation between target ligands expression on the tumor and efficacy and specificity of both CARs was also shown. None of the CAR NK‐92MI studied populations showed toxicity against PBMCs from healthy donors and in vivo MM orthotopic xenograft mouse model experiments are ongoing.Summary/Conclusion:We have generated two novel and stable CAR NK‐92 immunoproducts that improve the oncolytic efficacy of the parental cell line. Indeed, NKG2D‐CAR NK‐92MI cells are as equally efficient as BCMA‐CAR NK‐92MI cells to eradicate diverse MM cells. To summarize, all these data show the feasibility to use this NK ‘off‐the‐shelf’ approach as immunotherapy for MM.image

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Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused by deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions leads to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from FA patients was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

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