Plerixafor Single Agent for Autologous Stem Cells Mobilization and Collection in Adult Thalassemic Patients: Towards the Assessment of the Suitable Hematopoietic Stem Cell Source for Gene Therapy of Beta-Thalassemia.

Abstract 586

Gene therapy of inherited blood diseases requires harvest of hematopoietic stem cells (HSCs) from patients and autologous transplantation of genetically modified cells. In order to achieve correction of the disease, high number of HSCs and previous conditioning of the host bone marrow (BM) are necessary. In the clinical application of gene therapy for thalassemic patients the choice of the HSC source is a crucial issue. On one side, the minimal target dose poses a challenge for the use of steady state BM since reinfusion of high numbers of beta globin gene modified CD34⁺ cells is probably necessary to gain sufficient correction of the genetic defect in order to achieve transfusion independency; on the other side, the disease related features and complications of thalassemic patients (i.e. splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilizing agent.

In April 2011 a clinical protocol exploring the use of Plerixafor (AMD3100) as single agent was started (“Plerixafor mobilized stem cells as source for gene therapy of beta-thalassemia”, acronym AMD-THAL, EudraCT2011-000973-30). Aims of the trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization in adult patients affected by beta-thalassemia, to characterize stem/progenitor cells mobilized from the BM and peripheral blood of treated subjects and to achieve gene transfer efficiency of mobilized CD34⁺ cells at a level comparable to that obtained using steady state BM.

Four patients (01, 02, 03 and 04) were enrolled and already mobilized to date (August 2012). All patients are affected by transfusion dependent beta-thalassemia and aged 28 (01), 41 (02), 39 (03), 33 (04). Two are splenectomized (02 and 03); all subjects are regularly iron chelated with adequate organ function. Administration of Plerixafor subcutaneously as single agent and at the single dose of 0.24 mg/kg resulted in mobilization of CD34⁺ cells/mcl with a peak of 78 cells at 9 hrs (01), 70 cells at 7 hrs (02) and 69 cells at 8 hrs (03); suboptimal mobilization was observed in patient 04 (peak 18 at 8 hrs). Patient 03 received a second dose at 0.40 mg/kg 24 hrs after the first dose and underwent a second leucoapheretic procedure. Harvest by leukoapharesis resulted in procurement of the following CD34⁺ cells/kg: 1.84 × 10⁶ (01) and 4.43 × 10⁶ (02) with a unique leukoapheretic procedure, and 3.57 × 10⁶ (03) with two leukoapheresis. No apheresis was performed for patient 04 because the minimum target of 20 CD34⁺ cells/mcl in peripheral blood was not reached. CD34⁺ cells selection through Clinimacs Miltenyi resulted in the following yield: 1.2 × 10⁶ CD34⁺ cells/Kg, 65% recovery (01), 2.66 × 10⁶ CD34⁺ cells/Kg, 60% recovery (02), 1.78 × 10⁶ CD34⁺ cells/Kg, 50% recovery (03). No severe adverse event occurred. Recorded side effects were: grade 3 hypotension related to the apheretic procedure (01), mild grade 1 facial disestesia (02 and 04) and hyperleukocytosis (02: WBC from 13.6 to 42.6 × 10³/mcl). In addition, steady state and Plerixafor primed BM aspirates were performed to analyze any modification in CD34⁺ concentration in the BM following Plerixafor administration. In fact, Plerixafor administration resulted in enrichment of CD34+ cells concentration in the BM.

Purified CD34⁺ cells from leukoapheresis of the 4 treated patients were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation of CD34⁺ cells were tested following transplantation in NSG mice. Experiments are ongoing but preliminary results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high reconstitution potential and are efficiently transduced with a lentiviral based vector, named GLOBE, encoding for the human beta-globin (Roselli et al., 2010), thus being a suitable source of target cells for gene therapy.