Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34+/CD38− cells, comprising ~1%–3% of all CD34+ cells, were isolated from healthy cord blood CD34+ cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-βAS3-FB). Isolated CD34+/CD38− cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34+ cells and required up to 40-fold less vector for transduction compared to bulk CD34+ preparations containing an equivalent number of CD34+/CD38− cells. Transduction of isolated CD34+/CD38− cells was comparable to CD34+ cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34+/38− cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34+/CD38− cells or unfractionated CD34+ cells. In vivo studies showed equivalent engraftment of transduced CD34+/CD38− cells when transplanted in competition with 100-fold more CD34+/CD38+ cells. This work provides initial evidence for the beneficial effects from isolating human CD34+/CD38− cells to use significantly less vector and potentially improve transduction for HSC gene therapy.

Lentiviral vectors designed for the treatment of the hemoglobinopathies require the inclusion of regulatory and strong enhancer elements to achieve sufficient expression of the β-globin transgene. Despite the inclusion of these elements, the efficacy of these vectors may be limited by transgene silencing due to the genomic environment surrounding the integration site. Barrier insulators can be used to give more consistent expression and resist silencing even with lower vector copies. Here, the barrier activity of an insulator element from the human ankyrin-1 gene was analyzed in a lentiviral vector carrying an antisickling human β-globin gene. Inclusion of a single copy of the Ankyrin insulator did not affect viral titer, and improved the consistency of expression from the vector in murine erythroleukemia cells. The presence of the Ankyrin insulator element did not change transgene expression in human hematopoietic cells in short-term erythroid culture or in vivo in primary murine transplants. However, analysis in secondary recipients showed that the lentiviral vector with the Ankyrin element preserved transgene expression, whereas expression from the vector lacking the Ankyrin insulator decreased in secondary recipients. These studies demonstrate that the Ankyrin insulator may improve long-term β-globin expression in hematopoietic stem cells for gene therapy of hemoglobinopathies.

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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.