The EHA Research Roadmap: Hematopoietic Stem Cell Gene Therapy.

Gene therapy for Wiskott-Aldrich syndrome: History, new vectors, future directions

Efficient Nanoparticle-Mediated Delivery of Gene Editing Reagents into Human Hematopoietic Stem and Progenitor Cells

Safety and Clinical Benefit of Lentiviral Hematopoietic Stem Cell Gene Therapy for Wiskott-Aldrich Syndrome

Evaluation of: Aiuti A, Biasco L, Scaramuzza S et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science 341(6148), 1233151 (2013).Wiskott–Aldrich syndrome (WAS), an X-linked primary immunodeficiency disease (PID) with unique and characteristic features, had been considered to be a good candidate for gene therapy. In 2010, hematopoietic stem cell (HSC) gene therapy, using a retroviral vector, was performed for WAS patients; however, concerns remain regarding the long-term safety of this therapy as several patients with PID developed myeloproliferative diseases due to insertional mutagenesis related to HSC gene therapy using retroviral vectors. Aiuti et al. first reported HSC gene therapy for WAS using a lentiviral vector and compared the safety and efficacy of the two therapies in the context of the same disease background. They undertook a detailed study of the vector integration sites and concluded that lentiviral HSC gene therapy was safer than retroviral gene therapy.

Autologous hematopoietic stem cell (HSC) gene therapy has gone through remarkable advancements in recent years, especially for the treatment of sickle cell disease (SCD). However, the collection of HSCs from SCD patients requires unique considerations, as granulocyte colony-stimulating factor (G-CSF)-mediated mobilization is contraindicated, and plerixafor-only mobilization is highly variable. Consequently, alternative mobilization regimens that are safe for SCD patients and generate better cell yields are desirable for SCD HSC gene therapy. Here, we evaluated a combination of plerixafor (AMD3100, a CXCR4 antagonist) with GroβT (MGTA-145/GroβT, a CXCR2 agonist) against the current gold-standard G-CSF for HSC gene therapy in nonhuman primates (NHPs) for HSC mobilization, leukapheresis, ex vivo gene editing to reactivate fetal hemoglobin, and transplantation. AMD3100/GroβT rapidly and reliably mobilized phenotypically primitive HSCs within hours even in a G-CSF non-responder. Average CD34/CD90 frequency in the blood and yields after enrichment were comparable in both mobilization regimens. Rapid recovery and robust multilineage long-term engraftment of gene-modified HSCs was achieved in the bone marrow and blood of animals. In summary, AMD3100/GroβT allows highly efficient and reliable mobilization of HSCs, providing a G-CSF-free regimen specifically for SCD but also any other hematological disease or disorder treatable with HSC gene therapy.

Safety of Autologous Hematopoietic Stem Cell Transplantation with Gene Addition Therapy for Transfusion-Dependent β-Thalassemia, Sickle Cell Disease, and Cerebral Adrenoleukodystrophy

Current and future treatments for sickle cell disease: From hematopoietic stem cell transplantation to invivo gene therapy.

Hematopoietic stem cell (HSC) gene therapy has effectively become a therapeutic option largely due to the resounding clinical successes in patients with primary immunodeficiencies (PIDs) such as X-linked severe immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), Wiskott-Aldrich syndrome (WAS), and chronic granulomatous disease (CGD). HSC gene therapy is also being investigated in patients with metabolic diseases such as X-linked adrenoleukodystrophy (ALD) and metachromatic leukodystrophy (MLD) and inherited blood disorders such as β-thalassemia and sickle cell disease (SCD) where some therapeutic benefits have been reported more recently. Safer and more efficient self-inactivating (SIN) γ-retroviral and lentiviral vectors have been developed to overcome the genotoxicity imparted by γ-retroviral vectors with intact long terminal repeat (LTR). The discovery and maturation of gene-editing platforms, including zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, offer exciting prospective strategies for further improving gene therapy by targeting the repair of diseased genes.

Virus-based vectors are widely used in hematopoietic stem cell (HSC) gene therapy, and have the ability to integrate permanently into genomic DNA, thus driving long-term expression of corrective genes in all hematopoietic lineages. To date, HSC gene therapy has been successfully employed in the clinic for improving clinical outcomes in small numbers of patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy (ALD), thalassemia, chronic granulomatous disease (CGD), and Wiskott-Aldrich syndrome (WAS). However, adverse events were observed during some of these HSC gene therapy clinical trials, linked to insertional activation of proto-oncogenes by integrated proviral vectors leading to clonal expansion and eventual development of leukemia. Numerous studies have been performed to understand the molecular basis of vector-mediated genotoxicity, with the aim of developing safer vectors and lower-risk gene therapy protocols. This review will summarize current information on the mechanisms of insertional mutagenesis in hematopoietic stem and progenitor cells due to integrating gene transfer vectors, discuss the available assays for predicting genotoxicity and mapping vector integration sites, and introduce newly-developed approaches for minimizing genotoxicity as a way to further move HSC gene therapy forward into broader clinical application.

Hematopoietic stem cells (HSCs) reconstitute blood cells throughout life. DNA-level correction of HSCs allows for a one-time cure of genetic diseases, including sickle cell disease (SCD). Sickle cell disease is one of the most common single-gene disorders; therefore, SCD is a prime candidate for gene therapy. Several drug therapies are available for SCD, including hydroxyurea, which is the first-line choice despite requiring lifelong administration. Allogeneic HSC transplantation is a one-time, curative treatment for SCD with limited availability of histocompatible donors. Therefore, autologous HSC gene therapy was developed using patients' own HSCs with lentiviral gene addition/silencing and clustered regularly interspaced short palindromic repeats gene editing, making gene therapy applicable to most patients. However, the established method of HSC gene therapy requires costly and complex ex vivo HSC culture. Therefore, in vivo HSC gene therapy is being developed to treat SCD, envisioning a single-injection HSC-targeted gene delivery system. This review discusses various therapeutic methods to treat SCD, the development of HSC gene therapy, and clinical gene therapy trials in SCD, ranging from FDA-approved to novel in vivo gene therapy.

Primary immunodeficiencies (PIDs) are rare hereditary disorders that affect the immune system. PIDs have a devastating prognosis and thus require definitive treatment to prevent morbidity and mortality. The primary treatment provided to individuals suffering from PID is allogeneic hematopoietic stem cell transplantation (alloHSCT), and it carries the risk of graft-versus-host disease (GvHD) or graft failure.Inherited mutations across hematopoietic lineages can be corrected by autologous hematopoietic stem cell gene therapy (HSC-GT). Strimvelis is the first approved HSC-GT for the treatment of adenosine deaminase-deficient SCID (ADA-SCID). Newer gene-editing technologies can circumvent insertional mutagenesis caused by the viral vectors; thus, HSC-GT is likely to be successful in the treatment of PIDs. Chances of insertional mutagenesis caused by the gamma-retroviral vector and toxicities related to erroneous genetic modification of non-targeted site by the techniques used for gene editing can pose risks to the success of HSC-GT.Anti-retroviral treatment has made remarkable progress over the years, with a positive impact on the lives of patients. The shortcomings, like treatment-associated toxicity, need for lifelong therapy, adherence to medication, and drug resistance, have been challenging. Innovative cell and gene therapies are being developed, like chimeric antigen receptor T-cells that specifically target cells infected with HIV, genome editing strategies, and gene therapies that contribute in making the patients immune to HIV infection, but they have failed to achieve positive results. The chapter discusses the development of HSC-GT for PIDs and AIDs.

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

British Society for Gene and Cell Therapy Annual Conference Glasgow9–11th June 2015Conference Abstracts

330. Lentiviral Vector Mediated Hematopoietic Stem Cell Gene Therapy Combined with Non-Lethal Conditioning Restores T Cell Function in the Murine Model of Wiskott-Aldrich Syndrome

Hematopoietic stem cell (HSC) gene therapy has become in the recent years an attractive therapeutic strategy for primary immunodeficiencies and other inherited disorders, offering several potential advantages over allogeneic HSC transplantation. Early-generation gammaretroviral vectors have shown important limitations and risks, with the exception of adenosine deaminase-deficient SCID (ADA-SCID), for which the cumulative experience has established the long-term efficacy and safety. Gene therapy for ADA-SCID has now become the first ex vivo HSC gene therapy approved in the European Union. Currently, self-inactivating vectors, and particularly HIV-derived lentiviral vectors, are the most used platform for genetic correction of HSC. Clinical trials for SCID-X1, Wiskott-Aldrich syndrome, and recently ADA-SCID showed sustained engraftment of gene-corrected cells, restored immune function, and general improvement of clinical condition, with a positive safety profile. Continuous monitoring will be important to confirm long-term safety and efficacy. Preclinical proof of concept has been obtained for several other primary immunodeficiencies (e.g., from deficiencies in gp91 phox , Artemis, RAG1, RAG2, perforin, Munc 13-4, and FOXP3 deficiencies), with important hurdles due to requirement of highly controlled transgene expression. Recent advances in gene-editing technology may allow to further expand the applications of gene therapy to other primary immunodeficiencies.