Autologous Ex Vivo Lentiviral Gene Therapy for Adenosine Deaminase Deficiency.

BACKGROUND: Severe combined immunodeficiency due to adenosine deaminase (ADA) deficiency (ADA-SCID) is a rare and life-threatening primary immunodeficiency. METHODS: We treated 50 patients with ADA-SCID (30 in the United States and 20 in the United Kingdom) with an investigational gene therapy composed of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) transduced ex vivo with a self-inactivating lentiviral vector encoding human ADA. Data from the two U.S. studies (in which fresh and cryopreserved formulations were used) at 24 months of follow-up were analyzed alongside data from the U.K. study (in which a fresh formulation was used) at 36 months of follow-up. RESULTS: Overall survival was 100% in all studies up to 24 and 36 months. Event-free survival (in the absence of reinitiation of enzyme-replacement therapy or rescue allogeneic hematopoietic stem-cell transplantation) was 97% (U.S. studies) and 100% (U.K. study) at 12 months; 97% and 95%, respectively, at 24 months; and 95% (U.K. study) at 36 months. Engraftment of genetically modified HSPCs persisted in 29 of 30 patients in the U.S. studies and in 19 of 20 patients in the U.K. study. Patients had sustained metabolic detoxification and normalization of ADA activity levels. Immune reconstitution was robust, with 90% of the patients in the U.S. studies and 100% of those in the U.K. study discontinuing immunoglobulin-replacement therapy by 24 months and 36 months, respectively. No evidence of monoclonal expansion, leukoproliferative complications, or emergence of replication-competent lentivirus was noted, and no events of autoimmunity or graft-versus-host disease occurred. Most adverse events were of low grade. CONCLUSIONS: Treatment of ADA-SCID with ex vivo lentiviral HSPC gene therapy resulted in high overall and event-free survival with sustained ADA expression, metabolic correction, and functional immune reconstitution. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01852071, NCT02999984, and NCT01380990.).

BCR-JAK2 drives a myeloproliferative neoplasm in transplanted mice.

BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.

Brief report: reduced expression of CD18 leads to the in vivo expansion of hematopoietic stem cells in mouse bone marrow.

Leukocyte adhesion deficiency type-I is a primary immunodeficiency caused by mutations in the ITGB2 gene (CD18 leukocyte integrin) which lead to defects in leukocyte extravasation. To investigate the role of CD18 in hematopoietic stem cell (HSC) biology, we have thoroughly characterized the HSCs of CD18 Itgb2(tm1bay) hypomorphic mice (CD18(HYP) ) both by flow cytometry and using in vitro and in vivo transplantation assays. Flow cytometry analyses and cultures in methyl cellulose revealed that bone marrow (BM) from CD18(HYP) mice was enriched in hematopoietic precursors, mainly early quiescent short-term and long-term Hematopoietic progenitors cells. Strikingly, BM competition assays showed a progressive expansion of CD18(HYP) -derived hematopoiesis in recipient mice. Additionally, we provide evidence that this HSC expansion was not caused by an increased homing capacity of CD18(HYP) HSCs or by alterations in the hematopoietic environment of CD18(HYP) mice due to defects in neutrophils clearance. On the contrary, our data demonstrated that the reduced expression of CD18 causes a cell-autonomous expansion in the HSC compartment, thus revealing unexpected regulatory functions for CD18 in mouse HSCs.

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.

The downregulated membrane expression of CD18 in CD34+ cells defines a primitive population of human hematopoietic stem cells.

BACKGROUND: CD18 is the common beta subunit of ß2 integrins, which are expressed on hematopoietic cells. ß2 integrins are essential for cell adhesion and leukocyte trafficking. METHODS: Here we have analyzed the expression of CD18 in different subsets of human hematopoietic stem and progenitor cells (HSPCs) from cord blood (CB), bone marrow (BM), and mobilized peripheral blood (mPB) samples. CD34+ cells were classified into CD18high and CD18low/neg, and each of these populations was analyzed for the expression of HSPC markers, as well as for their clonogenity, quiescence state, and repopulating ability in immunodeficient mice. RESULTS: A downregulated membrane expression of CD18 was associated with a primitive hematopoietic stem cells (HSC) phenotype, as well as with a higher content of quiescent cells and multipotent colony-forming cells (CFCs). Although no differences in the short-term repopulating potential of CD18low/neg CD34+ and CD18high CD34+ cells were observed, CD18low/neg CD34+ cells were characterized by an enhanced long-term repopulating ability in NSG mice. CONCLUSIONS: Overall, our results indicate that the downregulated membrane expression of CD18 characterizes a primitive population of human hematopoietic repopulating cells.

Lentiviral Hematopoietic Stem Cell Gene Therapy Rescues Clinical Phenotypes in a Murine Model of Pompe Disease.

Pompe disease is a lysosomal storage disorder caused by malfunctions of the acid alpha-glucosidase (GAA) enzyme with a consequent toxic accumulation of glycogen in cells. Muscle wasting and hypertrophic cardiomyopathy are the most common clinical signs that can lead to cardiac and respiratory failure within the first year of age in the more severe infantile forms. Currently available treatments have significant limitations and are not curative, highlighting a need for the development of alternative therapies. In this study, we investigated the use of a clinically relevant lentiviral vector to deliver systemically GAA through genetic modification of hematopoietic stem and progenitor cells (HSPCs). The overexpression of GAA in human HSPCs did not exert any toxic effect on this cell population, which conserved its stem cell capacity in xenograft experiments. In a murine model of Pompe disease treated at young age, we observed phenotypic correction of heart and muscle function with a significant reduction of glycogen accumulation in tissues after 6 months of treatment. These findings suggest that lentiviral-mediated HSPC gene therapy can be a safe alternative therapy for Pompe disease.

Lentiviral gene therapy for X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of phagocytic cells1,2. We report the initial results of nine severely affected X-linked CGD (X-CGD) patients who received ex vivo autologous CD34+ hematopoietic stem and progenitor cell-based lentiviral gene therapy following myeloablative conditioning in first-in-human studies (trial registry nos. NCT02234934 and NCT01855685). The primary objectives were to assess the safety and evaluate the efficacy and stability of biochemical and functional reconstitution in the progeny of engrafted cells at 12 months. The secondary objectives included the evaluation of augmented immunity against bacterial and fungal infection, as well as assessment of hematopoietic stem cell transduction and engraftment. Two enrolled patients died within 3 months of treatment from pre-existing comorbidities. At 12 months, six of the seven surviving patients demonstrated stable vector copy numbers (0.4-1.8 copies per neutrophil) and the persistence of 16-46% oxidase-positive neutrophils. There was no molecular evidence of either clonal dysregulation or transgene silencing. Surviving patients have had no new CGD-related infections, and six have been able to discontinue CGD-related antibiotic prophylaxis. The primary objective was met in six of the nine patients at 12 months follow-up, suggesting that autologous gene therapy is a promising approach for CGD patients.

Enhancing Lentiviral and Alpharetroviral Transduction of Human Hematopoietic Stem Cells for Clinical Application.

Ex vivo retroviral gene transfer into CD34+ hematopoietic stem and progenitor cells (HSPCs) has demonstrated remarkable clinical success in gene therapy for monogenic hematopoietic disorders. However, little attention has been paid to enhancement of culture and transduction conditions to achieve reliable effects across patient and disease contexts and to maximize potential vector usage and reduce treatment cost. We systematically tested three HSPC culture media manufactured to cGMP and eight previously described transduction enhancers (TEs) to develop a state-of-the-art clinically applicable protocol. Six TEs enhanced lentiviral (LV) and five TEs facilitated alpharetroviral (ARV) CD34+ HSPC transduction when used alone. Combinatorial TE application tested with LV vectors yielded more potent effects, with up to a 5.6-fold increase in total expression of a reporter gene and up to a 3.8-fold increase in VCN. Application of one of the most promising combinations, the poloxamer LentiBOOST and protamine sulfate, for GMP-compliant manufacturing of a clinical-grade advanced therapy medicinal product (ATMP) increased total VCN by over 6-fold, with no major changes in global gene expression profiles or inadvertent loss of CD34+CD90+ HSPC populations. Application of these defined culture and transduction conditions is likely to significantly improve ex vivo gene therapy manufacturing protocols for HSPCs and downstream clinical efficacy.

Lentiviral Vector-Mediated Correction of a Mouse Model of Leukocyte Adhesion Deficiency Type I.

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene and is characterized by recurrent and life-threatening bacterial infections. These mutations lead to defective or absent expression of ß2 integrins on the leukocyte surface, compromising adhesion and extravasation at sites of infection. Three different lentiviral vectors (LVs) conferring ubiquitous or preferential expression of CD18 in myeloid cells were constructed and tested in human and mouse LAD-I cells. All three hCD18-LVs restored CD18 and CD11a membrane expression in LAD-I patient-derived lymphoblastoid cells. Corrected cells recovered the ability to aggregate and bind to sICAM-1 after stimulation. All vectors induced stable hCD18 expression in hematopoietic cells from mice with a hypomorphic Itgb2 mutation (CD18(HYP)), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18(HYP) recipients. hCD18(+) hematopoietic cells from transplanted CD18(HYP) mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18(HYP) mice subjected to two different inflammation models demonstrated that the LV-mediated gene therapy completely restored neutrophil extravasation in response to inflammatory stimuli. Finally, these vectors were able to correct the phenotype of human myeloid cells derived from CD34(+) progenitors defective in ITGB2 expression. These results support for the first time the use of hCD18-LVs for the treatment of LAD-I patients in clinical trials.