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Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1(-/- )mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

I Geni Attivatori di Ricombinazione (RAG) sono strettamente regolati durante la differenziazione linfocitaria e le loro mutazioni causano una serie di gravi disturbi immunologici. Il trapianto di cellule staminali/progenitrici ematopoietiche (HSPC) è il trattamento di elezione, ma è limitato dalla disponibilità dei donatori e dalla tossicità. Per superare questi problemi, abbiamo sviluppato strategie di modifica genetica mirate all'inserimento di una sequenza correttiva nel gene RAG1 umano mediante riparazione diretta dell'omologia (HDR) e le abbiamo convalidate attraverso piattaforme su misura in 2D, 3D e in vivo per valutare rigorosamente il ripristino dell'espressione e della funzione. Mentre l'integrazione nell'introne 1 ha raggiunto livelli di correzione subottimali, l'inserimento in fase nell'esone 2 ha consentito una fedele riproduzione dell'espressione e dell'attività fisiologica di RAG1, permettendo contemporaneamente di interrompere gli effetti dominanti-negativi degli alleli ipomorfici non riparati. La nostra strategia di editing mediante HDR ha reso altamente efficiente la ricostituzione di RAG1 nelle HSPC da donatori sani e pazienti con mutazioni ipomorfiche di RAG1, sostenendo la differenziazione delle cellule T e superando il blocco della differenziazione delle cellule B nei pazienti. L'efficienza di correzione genetica ha superato la proporzione minima di HSPC funzionali richiesta per salvare l'immunodeficienza nei topi Rag1-/-, supportando fortemente la traduzione clinica della strategia di editing delle HSPC per il trattamento della deficienza causata da mutazioni di RAG1., Recombination Activating Genes (RAG) are tightly regulated during lymphoid differentiation and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem/progenitor cell (HSPC) transplantation is the treatment of choice but limited by donor availability and toxicity. To overcome these issues, we developed gene editing (GE) strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored 2D, 3D and in vivo platforms to stringently assess rescue of expression and function. Whereas integration into intron 1 achieved suboptimal levels of correction, in-frame insertion into exon 2 drove faithful recapitulation of physiologic hRAG1 expression and activity, allowing to concomitantly disrupt dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated GE enabled highly efficient hRAG1 reconstitution in HSPCs from healthy donors and hypomorphic RAG1 patients, supporting T-cell differentiation and overcoming the patients’ B-cell differentiation block. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, strongly supporting the clinical translation of HSPC GE for treating RAG1 deficiency.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.