Increasing Transduction Efficiency in Human Hematopoietic Stem Cells for Gene Therapy

Abstract Abstract 2052 Background: Sickle cell disease (SCD) is a multisystem disease, associated with severe episodes of acute illness and progressive organ damage. Currently, the only curative treatment is allogeneic hematopoietic stem cell transplant (HSCT); however, this is limited by availability of HLA compatible donors and by immunological complications of graft rejection or graft-versus-host disease. Autologous stem cell gene therapy for SCD has the potential to treat this illness without the immune suppression needed for current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levasseuer Blood 102:4312–9, 2003). Initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with the βAS3lentiviral (LV) vector for gene therapy of sickle cell disease has been demonstrated in our lab. However, this complex lentiviral vector is produced at a sub-optimal titer and large production batches would be needed to supply clinical trials. Hypothesis: Although, it has been proven that the βAS3 gene can be transduced into CD34+ hematopoietic stem/progenitor cells (HSPC), the transduction efficiency is still not optimal. The CD34+ cell population includes rare long-lived stem cells but also more abundant progenitors, which would be short-lived after transplant. We hypothesize that isolating the more primitive HSPC population (CD34+/CD38− cells approximately 1% of all CD34+ cells) and transducing them with the βAS3 lentiviral vector will increase transduction efficiency and greatly reduce vector needs. Methods: CD34+/CD38− cells were isolated from cord blood (CB) CD34+ cells obtained from healthy donors by fluorescence activated cell sorting (FACS) and transduced with the CCL.βAS3.FB LV vector. After 14 days in culture, vector copy number (VCN) was determined by qPCR. Isolation of a more primitive cell was confirmed via long term culture (LTC) assay for 90 days. At 2–3 weeks intervals, non-adherent cell number was obtained, VCN was analyzed and CFU assays were performed to assess their capability to fully maintain their hematopoietic potential after transduction. Results: CD34+/CD38− cells were effectively isolated using FACS (n=7; 6,329–33,742 cells; 34–99% theoretical yield). The isolated CD34+/CD38- cells were able to generate progeny over an extended period of LTC compared to the CD34+ cells whose cell expansion declined ∼60 days in culture. CFU assays demonstrated that βAS3 gene-modified CB CD34+/CD38- cells were fully capable of maintaining their hematopoietic potential. The isolated CD34+/CD38- cells required 3–40 fold less vector for transduction compared to an equivalent number of these cells contained within the larger, non-fractionated CD34+ preparations. Transduction of CD34+/CD38- cells measured at day 14, by qPCR, was improved relative to CD34+ cells, mean VCN 2.5, +/− SEM 0.33 (range 2–3.5) vs. VCN 1.3, +/− 0.40 (range 0.5–2), respectively (p=0.03). In LTC, VCN remained higher over time in the CD34+/CD38- cells compared to the CD34+ cells, mean VCN 2.0, +/− SEM 0.13 (range 1.6–2.7) vs. VCN 0.5, +/− 0.09 (range 0.2–0.9) respectively. In vivo studies are ongoing to investigate the transduction efficiency of stem/progenitor cells engrafting from CD34+ and CD34+/CD38- cells transplanted in the NSG mouse model. Immunomagnetic isolation of CD34+/CD38- cells using columns is underway in anticipation of potential use in future clinical trials. Further investigations into the mechanisms for increased transduction in the CD34+/CD38- cells are ongoing. Conclusions: This work provides initial evidence for the beneficial effects from isolating human CB CD34+/CD38− cells to improve transduction with the βAS3LV vector for gene therapy of sickle cell disease. Disclosures: No relevant conflicts of interest to declare.