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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.

The tumor microenvironment hinders CAR T-cell access, activation, and persistence at the tumor site, thereby impacting on the therapeutic efficacy. To tackle these obstacles, ongoing efforts are focusing on further engineering CAR T-cells to enhance their homing, fitness, long-term persistence, and antitumor activity. Advances in genetic modification have prompted the development of armored CAR T-cells equipped with a combination of synergistic elements strengthening their function. These include cytokine release, chemokine receptor expression, immune checkpoint inhibition, gene-editing of inhibitory molecules, or metabolic reprogramming, among others. Multiarmored CAR T-cells may allow addressing the unmet clinical needs of patients with solid tumors or hard-to-treat hematological malignancies who do not benefit from conventional CAR T-cell therapy. Accordingly, several clinical trials are currently assessing the safety and efficacy of these novel CAR constructs.

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.

Until a few years ago, the onset of acute myeloid leukemia (AML) was entirely ascribed to genetic lesions in hematopoietic stem cells. These mutations generate leukemic stem cells, which are known to be the main ones responsible for chemoresistance and relapse. However, in the last years, increasing evidence demonstrated that dynamic interplay between leukemic cells and bone marrow (BM) niche is of paramount relevance in the pathogenesis of myeloid malignancies, including AML. Specifically, BM stromal niche components, such as mesenchymal stromal cells (MSCs) and their osteoblastic cell derivatives, play a key role not only in supporting normal hematopoiesis but also in the manifestation and progression of myeloid malignancies. Here, we reviewed recent clinical and experimental findings about how genetic and functional alterations in MSCs and osteolineage progeny can contribute to leukemogenesis and how leukemic cells in turn generate a corrupted niche able to support myeloid neoplasms. Moreover, we discussed how the newest single-cell technologies may help dissect the interactions between BM stromal cells and malignant hematopoiesis. The deep comprehension of the tangled relationship between stroma and AML blasts and their modulation during disease progression may have a valuable impact on the development of new microenvironment-directed therapeutic strategies, potentially useful for a wide cohort of patients.

Acute myeloid leukemia (AML) still represents an unmet clinical need for adult and pediatric patients. Adoptive cell therapy by chimeric antigen receptor (CAR)-engineered T cells demonstrated a high therapeutic potential, but further development is required to ensure a safe and durable disease remission in AML, especially in elderly patients. To date, translation of CAR T-cell therapy in AML is limited by the absence of an ideal tumor-specific antigen. CD123 and CD33 are the 2 most widely overexpressed leukemic stem cell biomarkers but their shared expression with endothelial and hematopoietic stem and progenitor cells increases the risk of undesired vascular and hematologic toxicities. To counteract this issue, we established a balanced dual-CAR strategy aimed at reducing off-target toxicities while retaining full functionality against AML. Cytokine-induced killer (CIK) cells, coexpressing a first-generation low affinity anti-CD123 interleukin-3–zetakine (IL-3z) and an anti-CD33 as costimulatory receptor without activation signaling domains (CD33.CCR), demonstrated a powerful antitumor efficacy against AML targets without any relevant toxicity on hematopoietic stem and progenitor cells and endothelial cells. The proposed optimized dual-CAR cytokine-induced killer cell strategy could offer the opportunity to unleash the potential of specifically targeting CD123+/CD33+ leukemic cells while minimizing toxicity against healthy cells.

La leucemia mieloide acuta (LMA) è una neoplasia del sangue caratterizzata dalla proliferazione clonale anomala di blasti indifferenziati nella nicchia midollare. Diversi lavori hanno suggerito che le cellule di LMA siano in grado di rimodellare il microambiente midollare generando una "nicchia leucemica" in grado di favorire la malattia, sopprimendo al contempo la normale emopoiesi. Infatti, esse alterano il differenziamento osteogenico delle cellule mesenchimali stromali derivate dal midollo osseo (BMSC), favorendo l’accumulo di osteoprogenitori. Ulteriori studi sono necessari per comprendere gli effetti che le alterazioni della componente osteogenica del midollo osseo hanno sulla malattia. In questo lavoro abbiamo studiato i cambiamenti indotti dalle cellule leucemiche nella capacità delle BMSCs di formare osso e una nicchia midollare funzionale. Per farlo abbiamo utilizzato due sistemi in vivo, specifici per valutare in un ambiente fisiologico il potenziale osteogenico e la capacità di stabilire una nicchia stromale completa. Abbiamo riscontrato che le BMSC derivanti da paziente (L-BMSC) e le BMSC derivate da donatore sano (N-BMSC) presentano morfologia, immunofenotipo, potenziale proliferativo e differenziativo simili in vitro ma le L-BMSC mostrano una diminuzione significativa dei livelli degli mRNA di Kit-ligand, un fattore che regola l'emopoiesi, e di Osterix, un gene che regola l'osteogenesi. Inoltre, gli impianti ottenuti da L-BMSC hanno mostrato una ridotta capacità di formazione ossea matura e hanno sviluppato una nicchia ricca di osteoprogenitori con un aumento di cellule osteoprogenitrici esprimenti Osterix senza però mostrare variazione nel numero di osteoclasti. Questi dati suggeriscono che le cellule leucemiche possono modulare irreversibilmente la differenziazione delle BMSC verso i preosteoblasti, con una riduzione della quantità di osso. È interessante notare che il nostro secondo modello in vivo ha mostrato che gli impianti derivati da L, Acute myeloid leukemia (AML) is a blood neoplasm characterized by the abnormal clonal proliferation of undifferentiated blasts which progressively occupy and transform the bone marrow (BM) niche. In the last decades, several evidences suggested that AML cells remodel the BM microenvironment generating a “leukemia niche” able to favor leukemia cell survival, spreading, and chemoresistance, while suppressing normal hematopoiesis. Several murine models have shown that AML cells skew the bone marrow-derived mesenchymal stromal cells (BMSC) toward an ineffective osteolineage differentiation with an accumulation of osteoprogenitors in the AML BM. However, further studies are needed to understand the effects that alterations in the osteogenic component of BM have on the disease. We investigated if AML derived-BMSC (AML-BMSC) have undergone significant changes in their capability to form bone and a BM niche after exposure to patient leukemia in the BM using two in vivo systems specific to assess in a physiologic environment the osteogenic potential and the capacity to establish a complete stromal niche. We found that AML-BMSC and healthy donor-derived BMSCs (HD-BMSC) displayed a similar in vitro morphology, immunophenotype, proliferative, and differentiation potential. However AML-BMSC exhibit a significantly diminished mRNA levels of Kit-ligand/KITLG, a factor regulating hematopoiesis within the niche, and of Osterix/SP7, a gene regulating osteogenesis. Histological analysis of the transplants harvested from the first in vivo model revealed new bone formation in both AML- and HD-BMSC groups. However, AML-BMSCs showed a reduced mature bone formation capacity and developed an osteoprogenitor-rich niche with the presence of Osterix-expressing osteoprogenitor cells. Comparative analysis of osteoclast formation failed to show significant differences. These data suggest that leukemia cells can irreversibly modulate BMSC differentiation toward pre-osteoblasts, with a reduction in

Objective: To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection. Methods: Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow. Results: An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell. Conclusion: Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.