Restoration of T and B Cell Differentiation after RAG1 Gene Transfer in Human RAG1 Defective Hematopoietic Stem Cells.

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34+ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID.

Utilizing epigenetic regulators to improve HSC-based lentiviral gene therapy.

ABSTRACT: The curative benefits of autologous and allogeneic transplantation of hematopoietic stem cells (HSCs) have been proven in various diseases. However, the low number of true HSCs that can be collected from patients and the subsequent in vitro maintenance and expansion of true HSCs for genetic correction remains challenging. Addressing this issue, we here focused on optimizing culture conditions to improve ex vivo expansion of true HSCs for gene therapy purposes. In particular, we explored the use of epigenetic regulators to enhance the effectiveness of HSC-based lentiviral (LV) gene therapy. The histone deacetylase inhibitor quisinostat and bromodomain inhibitor CPI203 each promoted ex vivo expansion of functional HSCs, as validated by xenotransplantation assays and single-cell RNA sequencing analysis. We confirmed the stealth effect of LV transduction on the loss of HSC numbers in commonly used culture protocols, whereas the addition of quisinostat or CPI203 improved the expansion of HSCs in transduction protocols. Notably, we demonstrated that the addition of quisinostat improved the LV transduction efficiency of HSCs and early progenitors. Our suggested culture conditions highlight the potential therapeutic effects of epigenetic regulators in HSC biology and their clinical applications to advance HSC-based gene correction.

IL3 Has a Detrimental Effect on Hematopoietic Stem Cell Self-Renewal in Transplantation Settings.

The ex vivo expansion and maintenance of long-term hematopoietic stem cells (LT-HSC) is crucial for stem cell-based gene therapy. A combination of stem cell factor (SCF), thrombopoietin (TPO), FLT3 ligand (FLT3) and interleukin 3 (IL3) cytokines has been commonly used in clinical settings for the expansion of CD34+ from different sources, prior to transplantation. To assess the effect of IL3 on repopulating capacity of cultured CD34+ cells, we employed the commonly used combination of STF, TPO and FILT3 with or without IL3. Expanded cells were transplanted into NSG mice, followed by secondary transplantation. Overall, this study shows that IL3 leads to lower human cell engraftment and repopulating capacity in NSG mice, suggesting a negative effect of IL3 on HSC self-renewal. We, therefore, recommend omitting IL3 from HSC-based gene therapy protocols.

Ex Vivo Expansion of Hematopoietic Stem Cells for Therapeutic Purposes: Lessons from Development and the Niche.

Expansion of hematopoietic stem cells (HSCs) for therapeutic purposes has been a "holy grail" in the field for many years. Ex vivo expansion of HSCs can help to overcome material shortage for transplantation purposes and genetic modification protocols. In this review, we summarize improved understanding in blood development, the effect of niche and conservative signaling pathways on HSCs in mice and humans, and also advances in ex vivo culturing protocols of human HSCs with cytokines or small molecule compounds. Different expansion protocols have been tested in clinical trials. However, an optimal condition for ex vivo expansion of human HSCs still has not been found yet. Translating and implementing new findings from basic research (for instance by using genetic modification of human HSCs) into clinical protocols is crucial to improve ex vivo expansion and eventually boost stem cell gene therapy.