Hematopoietic stem cell gene editing rescues B-cell development in X-linked agammaglobulinemia.

BACKGROUND: X-linked agammaglobulinemia (XLA) is an inborn error of immunity that renders boys susceptible to life-threatening infections due to loss of mature B cells and circulating immunoglobulins. It is caused by defects in the gene encoding the Bruton tyrosine kinase (BTK) that mediates the maturation of B cells in the bone marrow and their activation in the periphery. This paper reports on a gene editing protocol to achieve "knock-in" of a therapeutic BTK cassette in hematopoietic stem and progenitor cells (HSPCs) as a treatment for XLA. METHODS: To rescue BTK expression, this study employed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system that creates a DNA double-strand break in an early exon of the BTK locus and an adeno-associated virus 6 virus that carries the donor template for homology-directed repair. The investigators evaluated the efficacy of the gene editing approach in HSPCs from patients with XLA that were cultured in vitro under B-cell differentiation conditions or that were transplanted in immunodeficient mice to study B-cell output in vivo. RESULTS: A (feeder-free) B-cell differentiation protocol was successfully applied to blood-mobilized HSPCs to reproduce in vitro the defects in B-cell maturation observed in patients with XLA. Using this system, the investigators could show the rescue of B-cell maturation by gene editing. Transplantation of edited XLA HSPCs into immunodeficient mice led to restoration of the human B-cell lineage compartment in the bone marrow and immunoglobulin production in the periphery. CONCLUSIONS: Gene editing efficiencies above 30% could be consistently achieved in human HSPCs. Given the potential selective advantage of corrected cells, as suggested by skewed X-linked inactivation in carrier females and by competitive repopulating experiments in mouse models, this work demonstrates the potential of this strategy as a future definitive therapy for XLA.

Anti-CD45 PBD-based antibody-drug conjugates are effective targeted conditioning agents for gene therapy and stem cell transplant.

Stem cell gene therapy and hematopoietic stem cell transplantation (SCT) require conditioning to ablate the recipient's hematopoietic stem cells (HSCs) and create a niche for gene-corrected/donor HSCs. Conventional conditioning agents are non-specific, leading to off-target toxicities and resulting in significant morbidity and mortality. We developed tissue-specific anti-human CD45 antibody-drug conjugates (ADCs), using rat IgG2b anti-human CD45 antibody clones YTH24.5 and YTH54.12, conjugated to cytotoxic pyrrolobenzodiazepine (PBD) dimer payloads with cleavable (SG3249) or non-cleavable (SG3376) linkers. In vitro, these ADCs internalized to lysosomes for drug release, resulting in potent and specific killing of human CD45+ cells. In humanized NSG mice, the ADCs completely ablated human HSCs without toxicity to non-hematopoietic tissues, enabling successful engraftment of gene-modified autologous and allogeneic human HSCs. The ADCs also delayed leukemia onset and improved survival in CD45+ tumor models. These data provide proof of concept that conditioning with anti-human CD45-PBD ADCs allows engraftment of donor/gene-corrected HSCs with minimal toxicity to non-hematopoietic tissues. Our anti-CD45-PBDs or similar agents could potentially shift the paradigm in transplantation medicine that intensive chemo/radiotherapy is required for HSC engraftment after gene therapy and allogeneic SCT. Targeted conditioning both improve the safety and minimize late effects of these procedures, which would greatly increase their applicability.

An improved medium formulation for efficient ex vivo gene editing, expansion and engraftment of hematopoietic stem and progenitor cells.

Gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR-Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat hematological disorders, and clinical trials using these tools are now under way. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and their in vitro expansion for clinical application. In this study, we assessed the effect of different culture medium compositions on the ability of HSPCs to proliferate and undergo homology-directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex vivo culture. We demonstrated that by supplementing the culture medium with stem cell agonists and by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion. Overall, the implementation of this optimized ex vivo HSPC culture protocol can improve the efficacy, feasibility, and applicability of gene editing as a key step to unlocking the full therapeutic potential of this powerful technology.

Preclinical Optimization and Safety Studies of a New Lentiviral Gene Therapy for p47phox-Deficient Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is an inherited blood disorder of phagocytic cells that renders patients susceptible to infections and inflammation. A recent clinical trial of lentiviral gene therapy for the most frequent form of CGD, X-linked, has demonstrated stable correction over time, with no adverse events related to the gene therapy procedure. We have recently developed a parallel lentiviral vector for p47phox-deficient CGD (p47phoxCGD), the second most common form of this disease. Using this vector, we have observed biochemical correction of CGD in a mouse model of the disease. In preparation for clinical trial approval, we have performed standardized preclinical studies following Good Laboratory Practice (GLP) principles, to assess the safety of the gene therapy procedure. We report no evidence of adverse events, including mutagenesis and tumorigenesis, in human hematopoietic stem cells transduced with the lentiviral vector. Biodistribution studies of transduced human CD34+ cells indicate that the homing properties or engraftment ability of the stem cells is not negatively affected. CD34+ cells derived from a p47phoxCGD patient were subjected to an optimized transduction protocol and transplanted into immunocompromised mice. After the procedure, patient-derived neutrophils resumed their function, suggesting that gene correction was successful. These studies pave the way to a first-in-man clinical trial of lentiviral gene therapy for the treatment of p47phoxCGD.

Targeted gene correction of human hematopoietic stem cells for the treatment of Wiskott - Aldrich Syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.

Enhanced CAR T cell expansion and prolonged persistence in pediatric patients with ALL treated with a low-affinity CD19 CAR.

Chimeric antigen receptor (CAR)-modified T cells targeting CD19 demonstrate unparalleled responses in relapsed/refractory acute lymphoblastic leukemia (ALL)1-5, but toxicity, including cytokine-release syndrome (CRS) and neurotoxicity, limits broader application. Moreover, 40-60% of patients relapse owing to poor CAR T cell persistence or emergence of CD19- clones. Some factors, including the choice of single-chain spacer6 and extracellular7 and costimulatory domains8, have a profound effect on CAR T cell function and persistence. However, little is known about the impact of CAR binding affinity. There is evidence of a ceiling above which increased immunoreceptor affinity may adversely affect T cell responses9-11. We generated a novel CD19 CAR (CAT) with a lower affinity than FMC63, the high-affinity binder used in many clinical studies1-4. CAT CAR T cells showed increased proliferation and cytotoxicity in vitro and had enhanced proliferative and in vivo antitumor activity compared with FMC63 CAR T cells. In a clinical study (CARPALL, NCT02443831 ), 12/14 patients with relapsed/refractory pediatric B cell acute lymphoblastic leukemia treated with CAT CAR T cells achieved molecular remission. Persistence was demonstrated in 11 of 14 patients at last follow-up, with enhanced CAR T cell expansion compared with published data. Toxicity was low, with no severe CRS. One-year overall and event-free survival were 63% and 46%, respectively.

The ALK(F1174L) mutation potentiates the oncogenic activity of MYCN in neuroblastoma.

The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.