420 the challenge of HSCs procurement for gene therapy of beta thalassemia: exploring plerixafor as mobilization agent

420 The Challenge of HSCs Procurement for Gene Therapy of beta Thalassemia Exploring Plerixafor as Mobilization Agent Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Socie[.]

HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I through both the TPO and TGF-beta pathways, with significantly increased levels of phosphorylation of MAPK and Smad proteins respectively Biotinylation studies revealed increased levels of cell surface receptors following mTOR inhibition Pharmacologic disruption of endocytosis reversed the effects of rapamycin treatment, with equivalent cytokine signaling in treated and untreated cells In utero HSC transplantation studies revealed significant enhancement of HSC engraftment levels in rapamycin-treated cells compared to fresh whole bone marrow controls (Figure 1) Conclusions: These results support a role for mTOR in modulating HSC signal transduction by regulation of cytokine receptor endocytosis, resulting in optimized engraftment following transplantation These studies will ultimately improve understanding of how the manipulation of multiple signaling circuits may be optimized to control self-renewal and quiescence of HSCs to best support in vitro expansion and transplantation for CID responsiveness, with the strongest response achieved by deleting an AP2 binding site involved in lysosomal targeting Studies with CB CD34+ cells demonstrated this delta-AP2 variant increased expansion of total cells nearly 3-fold, to an average of over 80 times the starting number by day 21 of culture (P=0.03) This included a 4.5-fold increase in the expansion of CD41a+/CD42b+ megakaryocytes by day 14 (P=0.01) and a 3.1-fold increase in the expansion of CD235a+/CD41a- erythroid cells by day 21 (P=0.03) We also noticed a nearly 6-fold increase in an atypical CD235a+/ CD41a+ cell population (P=0.05), peaking at 14+/-3% of all cells at day 14 of culture for the delta-AP2 vector Studies from others in mice reported the emergence of a similar cell population under conditions of hematopoietic stress that exhibited an erythroid/ megakaryocyte bi-potential precursor phenotype In our studies, sorting and colony assays demonstrated a commensurate increase in the number of clonogenic progenitor colonies, restricted largely to the CD235a-/CD41a- population, although two of the colonies grown from the CD235a+/CD41a+ population in CID exhibited a CFU-Mix phenotype with both erythroid and megakaryocytic cells In conclusion, these hyperactive variants appear to improve the overall potency of the Mpl-based cell growth switch, with the greatest expansion seen with an apparently novel erythroid/megakaryocyte bi-potential cell population 420 The Challenge of HSCs Procurement for Gene Therapy of beta-Thalassemia: Exploring Plerixafor as Mobilization Agent 419 Improved Expansion of Primitive Erythroid/Megakaryocyte Precursors from Cord Blood CD34+ Cells with Hyperactive Variants of Pharmacologically-Regulated Mpl Maria Rosa Lidonnici,1,5 Annamaria Aprile,1 Marta Frittoli,1,5 Giacomo Mandelli,1 Bernhard Gentner,1,2,5 Laura Bellio,3 Elena Cassinerio,4 Laura Zanaboni,4 Silvano Rossini,3 Maria Domenica Cappellini,4 Fabio Ciceri,2 Sarah Marktel,2 Giuliana Ferrari.1,5 H San Raffaele-Telethon Institute for Gene Therapy (HSRTIGET), Milan, Italy; 2Hematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy; 3Immunohematology and Transfusion Medicine Unit, San Raffaele Scientific Institute, Milan, Italy; 4Università di Milano, Ca Granda Foundation IRCCS, Milan, Italy; 5Università Vita-Salute San Raffaele, Milan, Italy The clinical utility of allogeneic umbilical cord blood (CB) transplantation is limited by poor engraftment secondary to the inadequate number of hematopoietic stem/progenitor cells per cord and the delayed differentiation of these cells post-transplant We have been investigating a means of expanding and activating these hematopoietic stem/progenitor cells based on a pharmacologically regulated Mpl cell growth switch This involves fusing the signaling domain of the thrombopoietin receptor Mpl to an artificial dimerization domain that is, in turn, specific for a small drug molecule called a chemical inducer of dimerization (CID) We previously reported that viral vector transduction of CB CD34+ cells with a wild-type version of this Mpl cell growth switch allowed for a 20-fold, CID-mediated expansion of total cells in culture (Richard  Blau, 2003) However, in contrast to similar studies with mouse bone marrow which allowed for the virtually limitless expansion of a primitive undifferentiated population in culture, these CB CD34+ cultures underwent terminal differentiation after a few weeks, with a predominantly erythroid phenotype In order to determine whether this performance could be improved by augmenting Mpl signaling, we modified the conventional hybrid Mpl cassette by deleting known target sites involved in the degradation of Mpl These hyperactive variants of Mpl in factor-dependent BaF/3 cells reduced the threshold Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of a significant dose of autologous genetically engineered hematopoietic stem cells (HSCs) Gene therapy trials in young pediatric patients are performed by transplanting CD34+ cells from steady state BM, while in adults G-CSF mobilized peripheral blood cells are the preferred targets In the context of gene therapy for thalassemia, the choice of HSC source is crucial since intrinsic characteristics of patients (splenomegaly and thrombophilia) dictate caution in the use of G-CSF and prompt investigation of new agents A phase II clinical protocol exploring the use of Plerixafor as a single mobilizing agent in adult patients affected by transfusion dependent beta-thalassemia was approved (EudraCT 2011-000973-30) and started in 2012 Plerixafor selectively and reversibly antagonizes the binding of SDF-1 to its receptor CXCR4 with subsequent egress of HSCs to the peripheral blood Aims of our trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization, to characterize mobilized stem/ progenitor cells in the BM and peripheral blood and to achieve gene transfer efficiency in mobilized CD34+ cells at a level comparable to that obtained using steady state BM cells Four subjects were enrolled and treated by subcutaneously administration of Plerixafor at the single dose of 0.24 mg/kg followed by leukoapheresis Mobilization of CD34+ cells occurred very rapidly with a peak between to hrs Only one patient received a second dose (0.40 mg/kg) at 24 hrs after the first one and underwent a second leukoapheretic procedure Three out of four patients achieved the minimal target cell dose (2 x 106 cells/kg) and no severe adverse events occurred Purified CD34+ cells Eyayu Belay,1 Christophre P Miller,1 Amanda Kortum,1 Beverly Torok-Storb,2 C Anthony Blau,1 David W Emery.1 Institute for Stem Cell & Regenerative Medicine, Univeristy of Washington, Seattle, WA; 2Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S161 HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY I from leukoaphereses were analyzed for their biological and functional properties, subpopulations composition and expression profile In vivo reconstitution potential and lymphomyeloid differentiation were tested following transplantation in NSG mice We also compared Plerixafor-mobilized peripheral blood cells with CD34+ cells derived from BM pre- and post-Plerixafor treatment Cells were transduced with GLOBE lentiviral vector, carrying the beta-globin gene, to assess gene transfer efficiency and transgene expression The results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high in vivo hematopoietic reconstitution potential and are efficiently transduced, thus representing a suitable source of target cells for gene therapy 421 Leukocyte Telomere Maintenance after Transplant and In Vivo Chemoselection of Mutant MGMTP140K Gene-Modified Hematopoietic Cells Christopher R Burtner,1 Devikha Chandrasekaran,1 Brian C Beard,1 Hans-Peter Kiem,1 Jennifer E Adair.1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Engraftment of a sufficient number of gene-modified hematopoietic stem cells (HSCs) to elicit a therapeutic effect remains a significant hurdle to the success of gene therapy for certain diseases where genemodified cells not have an intrinsic in vivo selective advantage over non-modified cells While drug resistance gene therapy in HSCs may provide a strategy to overcome this limitation, the biological effects of repeated chemoselective pressure on gene-modified cells is unknown It has been established that leukocyte telomere attrition correlates with increasing age in patients, and HSC transplant has been demonstrated to accelerate human telomere attrition in both the autologous and allogeneic transplant setting Thus, we applied a quantitative PCR method of leukocyte telomere length (LTL) measurement to determine whether repetitive chemoselection after autologous transplant of O6benzylguanine (O6BG)-resistant methylguanine methyltransferase (MGMTP140K) gene-modified CD34+ cells caused accelerated telomere shortening in both the pigtailed macaque (Macaca nemestrina) and in three brain tumor patients enrolled in a clinical trial (NCT #00669669) We did not observe telomere attrition with age in the pigtailed macaque, consistent with telomere dynamics in the rhesus macaque and likely due to variation among animals in baseline telomere length Interestingly, in an analysis of 28 pigtailed macaques receiving high-dose total body irradiation followed by autologous MGMTP140K gene-modified HSCs, we did not see a decrease in LTL at time points immediately following and up to one year post-transplantation In 16 animals that received subsequent rounds of non-myeloablative O6BG and the alkylating agent bischloroethylnitrosourea BCNU for chemoselection, a trend in LTL shortening was observed that did not reach statistical significance In 11 animals for which longitudinal data is available, individual telomere trends varied, but interestingly, animals exhibited a positive correlation between gene marking levels and telomere length as a result of chemoselection These data suggest that expression of the MGMTP140K transgene in modified cells may exert a role in telomere maintenance to a degree that corresponds with the level of gene marking Analyses of LTL in gene modified cells compared to non-gene-modified cells of different lineages are ongoing In of patients who received MGMTP140K-modified mobilized CD34+ cells after non-myeloablative conditioning with single-agent BCNU, the administration of 3-4 rounds of O6BG and temozolomide chemotherapy had no significant effect on telomere length, although LTL for all three patients at the beginning of study was lower than that expected due to age alone However, in the third patient, a significant decline in gene marking was observed after chemotherapy cycles that corresponded with a >40% decline in LTL This is consistent with the hypothesis that expression of MGMTP140K, measured as S162 the number of gene-modified cells, positively regulates telomere maintenance in a way that counters the loss expected from the replicative demand of transplant and chemotherapy 422 Assessing Clonal Composition of Human Hematopoiesis in an NSG Transplantation Model after In Vitro Expansion of Transduced Cord Blood CD34 Cells Reinhard Haemmerle,1 Ruhi Phaltane,2 Michael Rothe,1 Thomas Moritz,2 Ute Modlich.1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2REBIRTH Research Group Reprogramming and Gene Therapy, Hannover Medical School, Hannover, Germany Transplantation of genetically modified hematopoietic stem cells (HSC) can cause severe adverse reactions in patients due to insertional deregulation of endogenous genes Recently, great efforts were made to achieve expansion of transplantable HSCs by the usage of novel cytokines and small molecules during extended culture periods As in vitro culture required for cell modification may actually contribute to adverse reactions, prolongation of ex vivo culture time and the choice of cytokines may further affect the clonal repertoire of retrovirally transduced HSCs following transplantation To address this question we transduced and expanded human cord-blood CD34+ (CB-CD34+) cells under four different cytokine conditions: SCF, TPO, and FLT3L alone (1), plus GCSF (2)1, or plus StemReginin-1 (3)2; and SCF, TPO, FGF-1, IGFBP-2, Angptl5 (4)3 After 10 days of expansion, the progenies of 5x104 CB-CD34+ cells were transplanted into NOD SCID.IL2Rgc-/- (NSG) mice (4 groups, n=6 each) Transductions were performed on days and (2xMOI20) with the mutagenic gammaretroviral vector RSF91.eGFP.pre with efficiencies of 90%99% irrespective of the cytokine conditions Cells expanded in medium containing StemReginin-1 formed 1.2-3.8-fold more colonies in methylcellulose assay than cells cultured in the other cytokine conditions Moreover, clearly more cells retained expression of the CD34 marker (34% vs 9.4%-19.9%) FACS analysis of blood and bone-marrow 24 weeks after transplantation showed no statistically significant differences between the conditions in terms of engraftment (39.4% (1), 17.6% (2), 25.1% (3), 27.0% (4)) or mean GFP expression (24.7% (1), 63.5% (2), 52.7% (3), 25.3% (4)) due to high variations within all groups Blood lineage contributions of transduced cells did not indicate vector-induced clonal outgrowth or lineage skewing and histopathology remained normal Insertion site analysis was performed by LAM-PCR and deep sequencing on 24 mice and four pre-transplant samples A total of 3198 unique integration sites clustering around transcription start sites were retrieved From each mouse on average 72 ± 21 integrations were recovered, a clear reduction compared to the pre-transplant samples (305 ± 118) In six mice integrations with high read counts (>50% of all reads) potentially representing dominant clones were detected Furthermore, preliminary analysis by nrLAM-PCR in three mice revealed additional unique integrations that were not found by LAM-PCR One of these integrations with a high read count (84% of total reads) was located in the fourth intron of ANGPT1 Although comparison between the four groups was difficult due to high variations within the groups, deep sequencing in individual humanized NSG mice revealed integrations near relevant cancer-related genes and apparently dominant integrations Next, the influence of the vector insertion on the outgrowth of potential dominant clones will be further addressed 1Neff et al., Blood, 2005; Boitano et al., Science, 2010; 3Zhang et al., Blood 2008 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... gene- modified hematopoietic stem cells (HSCs) to elicit a therapeutic effect remains a significant hurdle to the success of gene therapy for certain diseases where genemodified cells not have an intrinsic... with the hypothesis that expression of MGMTP140K, measured as S162 the number of gene- modified cells, positively regulates telomere maintenance in a way that counters the loss expected from the. .. single -agent BCNU, the administration of 3-4 rounds of O6BG and temozolomide chemotherapy had no significant effect on telomere length, although LTL for all three patients at the beginning of study