Regenerative Therapies for Inherited Blood Disorders

Gene and stem cell-based regenerative therapies hold the promise of restoring lost, damaged, or aging cells and tissues in the human body. This laboratory is investigating novel strategies and stem cell concepts to advance the translational field of regenerative medicine, with a focus on inherited disorders affecting blood-forming hematopoietic stem cells (HSCs), including: 1) Gene therapy: Genetic correction of adult HSCs using CRISPR/Cas and viral vector technologies, and ex vivo expansion of genetically corrected HSCs; 2) Cell Therapy: Derivation of engraftable HSCs from genetically corrected induced pluripotent stem cells (iPSCs); 3) Drug Therapy: Investigations of the mechanisms of action of eltrombopag and clinical applications in patients with inherited bone marrow failure (Fanconi anemia, Diamond Blackfan Anemia).

Genetic correction and expansion of HSCs

Autologous transplantation of genetically modified HSCs offers a potentially curative treatment for inherited blood disorders, such as bone marrow failure syndromes and immuno-deficiencies. This approach has been successfully employed in recent years using modified viral vectors to express functional copies of the affected gene in HSCs. A clinical trial using this approach, known as gene addition therapy, is under development in Dr. Larochelle’s group for the treatment of leukocyte adhesion deficiency (LAD). In recent years, transformative advances have emerged to precisely manipulate cellular genomes and correct mutations. These approaches, known as genome editing therapies, include a range of nuclease systems that can target, cleave, and repair specific genomic sequences at sites of inherited disease-generating mutations. The CRISPR/Cas9 system potentially offers the greatest flexibility for genome editing because of its striking efficiency and ease of use. Dr. Larochelle’s group is developing and optimizing this technology for the genetic correction of human HSCs and pluripotent stem cells. Due to low efficiency of gene correction of current genome editing protocols, clinical application of this technology depends on our ability to efficiently expand HSCs prior to and/or following genetic correction to allow sufficient numbers of HSCs for transplantation. Various approaches are being investigated, including activation of Notch and hypoxic pathways, regulation of the Unfolded Protein Response (UPR) in expanding HSCs, and CRISPRa/CRISPRi-based activation/inhibition of epigenetic modifiers and HSC regulators.

Derivation of HSCs from induced pluripotent stem cells (iPSCs)

With the development of iPSC technologies via transient expression of embryonic stem cell (ESC) transcription factors in somatic cells emerged the concept of generating iPSCs from an individual patient, correcting the genetic defect using genome editing approaches (e.g. CRISPR/Cas9), and differentiating the disease-free iPSCs into transplantable HSCs. The ability to generate an endless supply of matched, tailor-made HSCs for autologous transplantation would be a revolution in clinical hematology. However, despite significant advances toward clinical applications, protocols to derive HSCs from iPSCs are inefficient at producing functional HSCs required for clinical applications. With a primary focus on epigenome editing targeting regulatory sequences defining HSC-identity, Dr. Larochelle’s group is optimizing a scalable culture system for hematopoietic differentiation of human iPSCs derived from normal individuals and subjects with inherited blood disorders. Single cell transcriptomic (scRNAseq) and chromatin accessibility (ATAC-seq) comparison of bona fide HSCs and hematopoietic cells generated using this culture system will allow the identification of essential genetic programs that current differentiation protocols fail to activate. Ultimately, these studies will lead to optimized methodologies for the generation of definitive human HSCs with high-level, multilineage, long-term, hematopoietic reconstitution for therapeutic applications.

Eltrombopag for the treatment of inherited bone marrow failure

Eltrombopag is a synthetic small molecule mimetic of thrombopoietin (TPO), a primary positive regulator of HSC survival. In recent clinical trials, Eltrombopag was found to improve trilineage hematopoiesis in patients with acquired bone marrow failure, indicating that this drug can stimulate the small number of residual HSCs in the bone marrow. Dr. Larochelle’s group has identified general mechanisms of action by which eltrombopag promotes HSC function, including DNA double strand break repair activity and a unique ability to maintain HSCs under inflammatory conditions mediated by interferon-γ. Based on these findings, a clinical trial testing the impact of eltrombopag in Fanconi anemia, an inherited bone marrow failure disorder associated with DNA repair defects, is in progress in Dr. Larochelle’s laboratory. Interim analysis suggest safety and efficacy of eltrombopag in these patients.