Garyfalia Karponi

Hematopoietic Stem Cell Mobilization for Gene Therapy of Adult Patients With Severe β-Thalassemia: Results of Clinical Trials Using G-CSF or Plerixafor in Splenectomized and Nonsplenectomized Subjects

The safety and efficacy of hematopoietic stem cell (HSC) mobilization was investigated in adult splenectomized (SPL) and non-SPL patients with thalassemia major, in two clinical trials, using different mobilization modes: granulocyte-colony-stimulating factor (G-CSF)-alone, G-CSF following pretreatment with hydroxyurea (HU), plerixafor-alone. G-CSF-mobilization was both safe and effective in non-SPL patients. However, in SPL patients the procedure resulted in excessive response to G-CSF, expressed as early hyperleukocytosis necessitating significant dose reduction, and suboptimal CD34(+) cells yields. One-month HU-pretreatment prevented hyperleukocytosis and allowed successful CD34(+) cell collections when an optimal washout period was maintained, but it significantly prolonged the mobilization procedure. Plerixafor resulted in rapid and effective mobilization in both SPL and non-SPL patients and was well-tolerated. For gene therapy of thalassemia, G-CSF or Plerixafor could be used as mobilization agents in non-SPL patients whereas Plerixafor appears to be the mobilization agent of choice in SPL adult thalassemics in terms of safety and efficacy.

Safe mobilization of CD34+ cells in adults with β-thalassemia and validation of effective globin gene transfer for clinical investigation

We conducted a pilot trial to investigate the safety and effectiveness of mobilizing CD34(+) hematopoietic progenitor cells (HPCs) in adults with β-thalassemia major. We further assessed whether thalassemia patient CD34(+) HPCs could be transduced with a globin lentiviral vector under clinical conditions at levels sufficient for therapeutic implementation. All patients tolerated granulocyte colony-stimulating factor well with minimal side effects. All cell collections exceeded 8 × 10(6) CD34(+) cells/kg. Using clinical grade TNS9.3.55 vector, we demonstrated globin gene transfer averaging 0.53 in 3 validation runs performed under current good manufacturing practice conditions. Normalized to vector copy, the vector-encoded β-chain was expressed at a level approximating normal hemizygous protein output. Importantly, stable vector copy number (0.2-0.6) and undiminished vector expression were obtained in NSG mice 6 months posttransplant. Thus, we validated a safe and effective procedure for β-globin gene transfer in thalassemia patient CD34(+) HPCs, which we will implement in the first US trial in patients with severe inherited globin disorders. This trial is registered at www.clinicaltrials.gov as #NCT01639690.

Optimizing autologous cell grafts to improve stem cell gene therapy

Over the past decade, stem cell gene therapy has achieved unprecedented curative outcomes for several genetic disorders. Despite the unequivocal success, clinical gene therapy still faces challenges. Genetically engineered hematopoietic stem cells are particularly vulnerable to attenuation of their repopulating capacity once exposed to culture conditions, ultimately leading to low engraftment levels posttransplant. This becomes of particular importance when transduction rates are low or/and competitive transplant conditions are generated by reduced-intensity conditioning in the absence of a selective advantage of the transduced over the unmodified cells. These limitations could partially be overcome by introducing megadoses of genetically modified CD34(+) cells into conditioned patients or by transplanting hematopoietic stem cells hematopoietic stem cells with high engrafting and repopulating potential. On the basis of the lessons gained from cord blood transplantation, we summarize the most promising approaches to date of increasing either the numbers of hematopoietic stem cells for transplantation or/and their engraftability, as a platform toward the optimization of engineered stem cell grafts.

Poor stem cell harvest may not always be related to poor mobilization: lessons gained from a mobilization study in patients with β-thalassemia major: POOR STEM CELL HARVEST IN OPTIMAL MOBILIZATION

Hematopoietic stem cell mobilization and leukapheresis in adult patients with β‐thalassemia have recently been optimized in the context of clinical trials for obtaining hematopoietic stem cells for thalassemia gene therapy. In some patients, however, the yield of cluster of differentiation 34‐positive (CD34+) cells was poor despite successful mobilization, and a modification of apheresis settings was mandatory for harvest rescue.

The Functional Effect of Repeated Cryopreservation on Transduced CD34+ Cells from Patients with Thalassemia

Stable gene marking and effective engraftment of gene-modified CD34+ hematopoietic stem cells is a prerequisite for gene therapy success but may be challenged by the inevitable cryopreservation of the final product prior to extensive quality assurance testing. We investigated the β-globin gene transfer potency in fresh and cryopreserved CD34+ cells from mobilized patients with β-thalassemia, as well as the qualitative impact of repeated freeze/thaw cycles on the functionality of cultured and unmanipulated CD34+ cells in terms of engrafting capacity in a xenotransplantation model, under partial myeloablation. Cells transduced fresh or after one freeze-thaw cycle yielded similar clonogenic and gene transfer frequencies. Repeated cryopreservation cycles did not affect the transduction rates whereas either one or two freeze-thaw cycles of cultured-but not of unmanipulated-cells significantly reduced their clonogenicity. No differences in the engrafting potential of gene-corrected cells subjected to either none or up to two cryopreservation cycles, were encountered post xenotransplantation. Overall, we assessed the gene transfer efficiency, clonogenicity and engrafting capacity of cryopreserved CD34+ cells and the impact of repeated freeze/thaw cycles in their performance. These observations may prove essential in the design of gene therapy trials, considerably facilitating their logistics.

291. CD26 Inhibition Enhances Chimerism of Lentivirally Transduced Hematopoietic Stem Cells in a Non-Myeloablative Setting

Despite the progress of gene therapy for monogenic diseases over the last years, the engrafting capacity of gene-modified cells, as well as constant and high-level gene expression in vivo, require further optimization, especially when a reduced intensity conditioning is mostly preferred. Culture conditions applied for effective gene transfer lead to significant loss of long-term repopulating cells and ultimately, impaired engraftment. The ex vivo inhibition of CD26 peptidase activity with Diprotin A was shown to improve homing and engraftment of unmanipulated hematopoietic stem cells (HSCs). We here, sought to assess the homing and engrafting capacity of lentivirally GFP-transduced murine bone marrow (BM) cells under competitive niche settings generated by a non-myeloablative conditioning. Following CD26 inhibition with 5mM Diprotin A of GFP-transduced murine HSCs, we assessed migration towards SDF-1 in transwell systems and the engraftment capacity after partial myeloablation (Busulfan 100mg/kg, equivalent to 8mg/kg in humans) in a syngeneic mouse model, allowing for donor chimera detection (C57Bl6-CD45.2+ donors/PepBoy-CD45.1+ recipients). We also investigated the possible effects of Diprotin A on gene transfer efficiency and transgene expression in bulk and clonogenic cultures of HSCs. In vitro, Diprotin A significantly increased the expression of the CXCR4 receptor (25.6±0.75 vs 16.4±1.02, P=0.02), as well as the %migration of gene-modified HSCs towards SDF-1 over untreated transduced cells (71.78±3.79% vs 55.71±5.34%, P=0.03), implying a potentially enhanced engrafting dynamic in the BM. Indeed, in vivo, the Diprotin A-treated transduced HSCs exhibited faster hematologic reconstitution by, at least, one week (P=0.03) and both superior long-term engraftment and GFP expression in all hematopoietic tissues (peripheral blood, BM, spleen) of the recipients, over non-Diprotin A-treated transduced cells (blood 4th month post-transplant, %CD45.2+: 77.26±5.26 vs 13.11±11.69, P=0.0002; %GFP+: 30.21±6.86 vs 3.9±2.33, P=0.03). The increased GFP expression observed in vivo in the Diprotin A cohort reflected the enhanced engraftment rates, since Diprotin A per se did not affect gene transfer efficiency or transgene expression prior to transplantation (%transduction with Diprotin A: 93±3.05 vs without Diprotin A: 89.9±7.47, P=0.73; %GFP in pools of colonies with Diprotin A: 74.8±7.14 vs without Diprotin A: 72.1±8.45, P=0.81). Upon sacrifice, the Diprotin A animals displayed sustained gene marking with ~1 vector copy in all hematopoietic tissues, as opposed to almost undetectable vector copy number (VCN) in the non-Diprotin A cohort. Likewise, individual colonies from the chimeric BM, demonstrated significantly higher VCN in the Diprotin A mice (2.8±0.48 vs 0.37±0.37, P=0.01). Overall, the ex vivo treatment with Diprotin A seems to abrogate the negative impact of culture conditions, allowing for enhanced donor chimerism under partial myeloablation and consequently, increased gene marking in vivo. This ex vivo, easily applicable approach may serve to overcome major constraints for the clinical implementation of gene therapy should the data be confirmed with human CD34+ cells.