Initiation of a Pre-Clinical Gene Therapy Study in Non-Human Primates Using Lentivectors Targeting Hematopoietic Cells for Correction of Fabry Disease.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5542-5542 ◽  
Author(s):  
Jagdeep S. Walia ◽  
Makoto Yoshimitsu ◽  
Josh D. Silvertown ◽  
Armando Poeppl ◽  
Vanessa I. Rasaiah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fabry Disease ◽  
Peripheral Blood ◽  
Rhesus Macaques ◽  
Marker Gene ◽  
Safety Information ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Abstract Fabry disease is a lysosomal storage disorder (LSD) due to low or absent levels of α-galactosidase A (α-gal A). This results in accumulation of substrate with terminal galactosyl residues such as globotriaosylceramide (Gb3) in lysosomes causing pathology in different organs. Previously, we have demonstrated correction of the deficiency in Fabry mice in numerous gene therapy studies targeting hematopoietic cells. Here we have initiated a pre-clinical gene therapy study in non-human primates (NHPs) targeting Fabry disease. Three rhesus macaques are housed in our animal facility and implantation of telemetric devices and vascular access ports have occurred. We have mobilized hematopoietic stem/progenitor cells from all three animals independently by treatment for 5 days with 10 μg/kg/day of recombinant human granulocyte colony stimulating factor (rhuG-CSF) and 200 μg/kg/day recombinant human stem cell factor (rhuSCF). On the 5th day of mobilization, all animals underwent leukapheresis. To be more clinically relevant, we are using a protocol with an unmodified, commercially available apheresis machine for the rhesus macaques, which can be used for humans of an equivalent weight. From each successful apheresis, we collected approximately 1 x109/kg mobilized peripheral blood mononuclear cells (MoPBMNCs). After collection of MoPBMNCs, we isolated CD34+ cells using an anti-human CD34 antibody (clone 12.8) with a recovery of approximately 15–20 x 106 CD34+ cells per kg body weight of the animal with >80% purity. Collected CD34+ cells are stored in liquid nitrogen. These cells will be prestimulated for 24 hours with huSCF, huFlt3L, huIL-6 and huTPO (kindly provided by Amgen) and will be transduced with a concentrated bicistronic lentivector (LV) that engineers co-expression of huα-gal A and huCD25, a cell surface marker for transduced cells. Our lab has recently shown overexpression of a rhesus form of CD25 in >80% of transduced rhesus BM CD34+ cells mediated by a LV, validating its candidacy as a marker gene. Transduced cells will then be transplanted autologously in the NHPs after myeloablation by irradiation (10Gy) or mild chemotherapy (fludarabine and cyclophosphamide). The irradiation protocol has been optimized and a special plexiglass chamber, with the capacity for inhalational and intravenous anesthesia as well as a space for a HEPA filter, has been prepared for the animal procedures. The transplanted animals will be followed for at least one year and outcomes will be assessed by full measurement of safety parameters, α-gal A activity in plasma and relevant organs along with the real-time PCR and LAM PCR on BM and peripheral blood cells for the persistence of LV. Gb3 levels will also be examined in different organs compared to pre-transplant levels in tissue biopsies. We expect that this preclinical study in NHPs will serve as a roadmap to clinical gene therapy of Fabry disease using LV and provide important safety information for the use of this promising gene delivery system.

Retroviral transduction efficiency of G-CSF+SCF–mobilized peripheral blood CD34+ cells is superior to G-CSF or G-CSF+Flt3-L–mobilized cells in nonhuman primates

Blood ◽  
2003 ◽  
Vol 101 (6) ◽  
pp. 2199-2205 ◽  
Author(s):  
Peiman Hematti ◽  
Stephanie E. Sellers ◽  
Brian A. Agricola ◽  
Mark E. Metzger ◽  
Robert E. Donahue ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Peripheral Blood ◽  
Nonhuman Primates ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Retroviral Transduction ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Gene transfer experiments in nonhuman primates have been shown to be predictive of success in human clinical gene therapy trials. In most nonhuman primate studies, hematopoietic stem cells (HSCs) collected from the peripheral blood or bone marrow after administration of granulocyte colony-stimulating factor (G-CSF) + stem cell factor (SCF) have been used as targets, but this cytokine combination is not generally available for clinical use, and the optimum target cell population has not been systematically studied. In our current study we tested the retroviral transduction efficiency of rhesus macaque peripheral blood CD34+ cells collected after administration of different cytokine mobilization regimens, directly comparing G-CSF+SCF versus G-CSF alone or G-CSF+Flt3-L in competitive repopulation assays. Vector supernatant was added daily for 96 hours in the presence of stimulatory cytokines. The transduction efficiency of HSCs as assessed by in vitro colony-forming assays was equivalent in all 5 animals tested, but the in vivo levels of mononuclear cell and granulocyte marking was higher at all time points derived from target CD34+ cells collected after G-CSF+SCF mobilization compared with target cells collected after G-CSF (n = 3) or G-CSF+Flt3-L (n = 2) mobilization. In 3 of the animals long-term marking levels of 5% to 25% were achieved, but originating only from the G-CSF+SCF–mobilized target cells. Transduction efficiency of HSCs collected by different mobilization regimens can vary significantly and is superior with G-CSF+SCF administration. The difference in transduction efficiency of HSCs collected from different sources should be considered whenever planning clinical gene therapy trials and should preferably be tested directly in comparative studies.

Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 203-203
Author(s):  
Theo Gomes ◽  
Stephanie Sellers ◽  
Robert E. Donahue ◽  
Rima Adler ◽  
Andre La Rochelle ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Insertional Mutagenesis ◽  
Ex Vivo ◽  
Safety Issue ◽  
Retroviral Vectors ◽  
Ex Vivo Expansion ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Abstract There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

Mobilization as a Preparative Regimen for Hematopoietic Stem Cell (HSC) Transplantation in Rhesus Macaques.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1709-1709
Author(s):  
Andre Larochelle ◽  
Cynthia L. Perez ◽  
Allen Krouse ◽  
Mark Metzger ◽  
Simon Fricker ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Dose ◽  
Peripheral Blood ◽  
Rhesus Macaques ◽  
Myeloablative Conditioning ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Preparative Regimen ◽  
Hsc Transplantation

Abstract The myeloablative conditioning regimens currently used for hematopoietic stem cell (HSC) transplantation are associated with significant morbidity and mortality. Alternative strategies to promote engraftment of infused HSCs with increased safety warrant investigation. In a murine model, we previously demonstrated that, in absence of irradiation, mobilization with AMD3100 (a CXCR4 antagonist) before marrow transplantation vacated microenvironmental niches and resulted in higher levels of engraftment of transplanted HSCs compared to controls (no AMD3100 treatment before transplantation) (Abkowitz JL et al., Blood (ASH Annual Meeting Abstracts)104 (11): 1187, 2004). In this study, we hypothesized that AMD3100 mobilization before transplantation could also promote HSC engraftment in a large animal model, eliminating the need for toxic myeloablative conditioning. Peripheral blood cells from two rhesus macaques were collected by apheresis 3 hours after administration of a single dose of AMD3100 1mg/Kg. CD34+ cells were enriched and transduced for four days in the presence of cytokines and fibronectin with non-expression Moloney murine leukemia virus-derived retroviral vectors (G1PLI) that carry a bacterial neomycin phosphotransferase resistance gene (neoR). The neoR-marked CD34+ cells were reinfused in the non-myeloablated animals, immediately after AMD3100 mobilization and apheresis repeated on the day of transplantation. NeoR-marking levels of approximately 0.1% were detected in both peripheral blood MNC and granulocytes at two months (animal 2RC102) and four months (animal RQ4791) after transplantation. Previous transplantation studies performed without prior myeloablative conditioning or mobilization preparative regimen resulted in no long-term in vivo gene marking. We mathematically confirmed that this observed level of gene marking is what can be expected when AMD3100 mobilization is used as a conditioning regimen. Previous studies have estimated the number of long-term repopulating HSCs at 6 per 105 CD34+ cells (Abkowitz JL et al, Blood96: 3399, 2000). In animal RQ4791, approximately 4.5X107 CD34+ cells, and therefore 2700 HSCs, were mobilized after AMD3100 administration. The total number of HSCs per animal is thought to be conserved in mammals and has been estimated at 11,000 to 22,000 (Abkowitz JL et al, Blood100: 2665, 2002). Hence, 12–24% of HSCs were mobilized after a single dose of AMD3100, consequently opening 12–24% of microenvironmental niches for engraftment. If 1% of engrafted HSCs are marked, 0.12–0.24% long-term marking levels are expected, correlating well with the observed marking level of 0.1%. These results imply that the number of available niches in large animals, as in murine models, regulates the number of HSCs that engraft. As importantly, mobilization with AMD3100 could provide a non-toxic preparative approach in large mammals, including humans, to improve HSC engraftment in transplantation for genetic and other nonmalignant disorders.

Methylguanine Methyltransferase-Based In Vivo Selection Results in Only Transient Improvement in Long-Term Marking after Autologous Transplantation of Transduced Hematopoietic Stem Cells in Rhesus Macaques.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3272-3272
Author(s):  
Andre Larochelle ◽  
Uimook Choi ◽  
Nora Naumann ◽  
Josh R. Clevenger ◽  
Harry L. Malech ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Steady State ◽  
Ex Vivo ◽  
Rhesus Macaques ◽  
Autologous Transplantation ◽  
Survival Advantage ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Selective Survival

Abstract In vivo selective survival advantage of transduced cells contributed to clinically beneficial levels of genetic correction of lymphocytes following X-SCID gene therapy. For most blood disorders there will be no constitutive selective advantage of the gene-corrected cells. Alternatively, a selectable gene incorporated into the vector may provide selective survival advantage. The P140K mutant of human O6-methylguanine-DNA methyltransferase (MGMT*) is a candidate mammalian selectable gene for hematopoietic stem cell (HSC) gene therapy. AMD3100-mobilized CD34+ cells from 5 rhesus macaques were transduced daily from day 2 to 4 of culture using oncoretroviral (n=2 animals) or lentiviral (n=3 animals) vectors encoding the gp91phox-IRES-MGMT* cassette or the GFP-MGMT* fusion protein, respectively. Transduced CD34+ cells were selected after (in vivo, n=4) or before (ex vivo, n=1) autologous transplantation in rhesus macaques using the BG (120mg/m2)/TMZ 400 mg/m2 combination for in vivo selection and the BG (5uM)/BCNU (7.5uM) combination for ex vivo selection. Marking of peripheral blood (PB) cells was evaluated by FACS and/or real-time PCR. Bulk CD34+ cells were marked at 27–58% after transduction with oncoretroviral or lentiviral vectors. Four animals were transplanted with transduced non-selected CD34+ cells. Small fractions of cultured cells not transplanted were exposed to BG/BCNU resulting in an increase of marking to 88–97% in each case, confirming the in vitro survival advantage. Cells from animals #1 and #2 were transduced with oncoretroviral vectors and steady-state marking of 3.5% was obtained in PB. Animal #1 received BG/TMZ infusions at 3 and 6 months post-transplant. Marking declined to 3.3% and 1.1% after BG/TMZ treatment 1 and 2, respectively. Animal #2 received one cycle of BG/TMZ at 4 months post-transplant. Full hematopoietic recovery was not achieved and the animal died of infectious complications one month after treatment. Marking of 2% was detected in the PB at the time of death. Cells from animals #3 and #4 were transduced with lentiviral vectors. Animal #3 received 4 monthly infusions of BG/TMZ starting 5 months after transplantation. Marking increased from 0.1% at steady-state to 1.8% in PB after the first cycle but rapidly declined to 0.2%. Despite significant myelosuppression, additional cycles of BG/TMZ resulted in no significant improvement in marking. Animal #4 received 4 monthly infusions of BG/TMZ starting 3 months after transplantation. Marking increased from 3.3% at steady-state to 29.2% after the first cycle but rapidly declined to 6.2%. Each additional cycle of BG/TMZ resulted in a transient increase in marking with a peak increase gradually declining with each cycle. Animal #5 was transplanted with CD34+ cells transduced with lentiviral vector expressing GFP-MGMT* and exposed to BG/BCNU ex vivo before transplantation. At the time of reinfusion, 55% of the cells were vector positive. Stable hematopoietic recovery required one month, compared to an average recovery of 2 weeks in animals transplanted with transduced cells without ex vivo selection. Steady state marking in PB of only 0.7% was detected. These data combined with the theoretic concern that the use of cytotoxic drugs could increase the risk of leukemogenesis in the setting of drug-resistance gene therapy, raise concerns for the clinical applicability of this approach.

Suicide Gene (HSVtk) Ablation of Hematopoietic Stem Cells and Their Progeny In the Rhesus Macaque Model: An Approach to Deletion of Dangerous Clones.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3759-3759
Author(s):  
Cecilia Barese ◽  
Connor King ◽  
Stephanie Sellers ◽  
Allen E Krouse ◽  
Mark E Metzger ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Hematopoietic Stem Cells ◽  
Rhesus Macaque ◽  
Conflicts Of Interest ◽  
Marker Gene ◽  
Suicide Gene ◽  
Marker Genes ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3759 For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Incremental Innovation of Ex Vivo Hematopoietic Stem Cell Engineering to Expand Clinical Gene Therapy Applications

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4707-4707
Author(s):  
Erika Zonari ◽  
Giacomo Desantis ◽  
Carolina Petrillo ◽  
Oriana Meo ◽  
Samantha Scaramuzza ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Genetically Engineered ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Clinical Gene Therapy ◽  
Ex Vivo Culture

Abstract Transplantation of genetically engineered, autologous hematopoietic stem and progenitor cells (HSPC) is becoming a promising alternative to allogeneic stem cell transplantation for curing genetic diseases, avoiding the risks of graft versus host disease and prolonged immunosuppression. Most clinical gene therapy protocols are based on CD34+ HSPC engineered during >2 days of ex vivo culture. By xenotransplanting mobilized peripheral blood (mPB) CD34+ HSPC, which were lentivirally (LV) marked with different fluorescent proteins according to CD38/CD90 expression levels allowing quantitative assessment of the contribution of CD38/CD90 subpopulations to hematopoietic reconstitution (n=48 NSG mice, 3 experiments), we identified 2 distinct waves of reconstitution: (1) short term repopulation (up to 2 months) mostly driven by CD34+CD38intCD90+/- cells and (2) long-term repopulation driven by CD34+CD38-CD90+ (70%) and CD34+CD38-CD90- cells (30%). Notably, an intermediate wave extending from 2 to 4 months driven by CD34+CD38low cells was selectively eliminated by prolonged ex vivo culture and could be rescued when culture time was reduced to 1 day. We therefore developed a novel LV transduction protocol able to provide curative levels of gene transfer during a single day of ex vivo culture. Stimulating CD34+ cells or CD34+CD38- cells with Prostaglandin E2 (PGE2) increased gene transfer with VSVg-pseudotyped LVs by 1.5-2 fold acting on early steps of transduction, an effect that was further potentiated by the late-acting compound Cyclosporin A. Using large-scale vector preparations for gene therapy of mucopolysaccharidosis type 1, chronic granulomatous disease or beta-thalassemia, we show by in vitro and xenotransplantation assays that a 1-day PGE2 protocol achieved similar transduction efficiencies into BM or MPB HSPC from healthy donors and patients as our 62h benchmark protocol. PGE2 treatment did not result in toxicity or skewed multi-lineage differentiation. However, shortening ex vivo culture increased engraftment levels in the NSG mouse model. To entirely avoid culturing progenitor cells, we explored the feasibility to limit ex vivo manipulation to HSC-enriched CD34+CD38- cells that may be co-transplanted with unmanipulated CD34+ progenitor cells devoid of long-term engraftment potential. This could further improve hematopoietic reconstitution, increase safety by reducing the LV integration load infused into the patient and downscale ex vivo manipulation making the process more efficient and economically sustainable. To this end, we optimized a sequential bead-based, GMP-compatible selection procedure to separate mPB into a CD34+CD38- stem and CD38+ progenitor cell fraction. We reached high purity (87+/-6.6% CD34+) and recovery of CD34+CD38- cells (37.3+/-8.7%), making their isolation clinically viable. Bead-selected CD34+CD38- cells showed higher engraftment potential than equivalent numbers of FACS-sorted cells. Co-infusion of unmanipulated (culture-sensitive) CD38+ supporter cells with genetically-engineered CD34+CD38- cells into NSG mice resulted in rapid engraftment followed by near-complete replacement of untransduced short-term repopulating progenitors by gene-marked HSPC deriving from CD34+CD38- cells after the 3rd month post-transplant. Finally, we explored ex vivo expansion of mPB CD34+CD38- cells with arylhydrocarbon receptor antagonists and/or pyrimido-indole-derivatives. These cells expanded 3-10 fold in a 7-14 d time-window, far less than seen for total CD34+ cells, thereby facilitating culture handling and reducing cost. Unlike CD34+ cells, expanded mPB CD34+CD38- cells largely maintained their SCID-repopulating potential providing proof-of-concept for the expansion of gene-modified HSC. This clinically applicable platform will improve the efficacy, safety and sustainability of ex vivo gene addition and open up new opportunities in the field of gene editing. Disclosures Ciceri: MolMed SpA: Consultancy.

Gene Transfer to CD34+ Cells From Peripheral Blood Restores Osteoclast Function in Infantile Malignant Osteopetrosis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2058-2058
Author(s):  
Ilana Moscatelli ◽  
Christian Thudium ◽  
Maria Askmyr ◽  
Ansgar S Schulz ◽  
Oscar Porras ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Transcriptional Level ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Malignant Osteopetrosis ◽  
Infantile Malignant Osteopetrosis ◽  
Hsc Transplantation

Abstract Abstract 2058 PURPOSE: Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by nonfunctional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for the a3 subunit of a proton pump used by the osteoclast to acidify the resorption area. As a consequence of the lack of resorption, remodeling of bone is severely hampered, which results in dense and fragile bone. This, in turn, causes bone marrow failure followed by anemia and hepatosplenomegaly. The only curative treatment for IMO is HSC transplantation, but this form of therapy is associated with high mortality, especially when an HLA-identical donor is not available. IMO is thus a candidate disease for development of gene therapy because of its fatal outcome early in life if treatment with HSC transplantation is not possible. We have previously shown that the murine oc/oc disease model of osteopetrosis can be rescued by gene therapy targeting hematopoietic stem cells (Johansson et al, Blood 2007). The aim of the present study was to rescue the phenotype of human IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. METHODS AND RESULTS: CD34+ cells from peripheral blood of three IMO patients were isolated without need for mobilization as they have high levels (around 3%) of circulating blood progenitors (Steward et al, Biol Blood Marrow Transplant. 2005). These cells were cultured in SFEM medium with 50 ng/ml M-CSF, 30 ng/ml GM-CSF, 10 ng/ml IL-6, 200 ng/ml SCF and 50 ng/ml Flt3L for 2 weeks. During culture the cells expanded 500 fold and gradually lost CD34 expression while 50% became positive for CD14, a marker for osteoclast precursors. The cells were transduced with SIN lentiviral vectors expressing either endogenous or codon optimized TCIRG1, plus GFP, under a SFFV promotor. The transduction efficiency was approximately 40% at 2 weeks. Cells were then differentiated to mature osteoclasts by culturing for 10 days on bone slices with α-MEM containing 10% serum, 50 ng/ml M-CSF and 50 ng/ml RANKL. Expression of GFP was retained throughout differentiation. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1 compared to controls. Interestingly the protein appeared only at the end of the differentiation protocol suggesting regulation at the post-transcriptional level, a phenomenon that is under further investigation. Vector-corrected IMO osteoclasts generated increased Ca2+ release and bone degradation products such as C-telopeptide of type 1 collagen (CTX-1) into the media, while non-corrected IMO osteoclasts failed to resorb bone. Resorption per osteoclast (CTX-1/TRAP ratio) was 20–50% of that of osteoclasts derived from normal CD34+ cord blood cells and about 2–6 fold higher than that of osteoclasts derived from non-transduced IMO CD34+ cells. CONCLUSION: In conclusion we provide the first in vitro evidence of lentiviral-mediated correction of a genetic disease involving the osteoclast lineage, supporting further development of gene therapy of IMO and other diseases affecting these cells. Disclosures: Richter: Novartis: Honoraria; Bristol-Myers Squibb: Honoraria.

Integration-Mediated Activation of PRDM16 and HMGA2 in Multiple Clones without Adverse Hematopoietic Consequences Following Transplant of Autologous MGMTP140K Gene-Modified CD34+ Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2053-2053
Author(s):  
Jennifer E Adair ◽  
Brian C Beard ◽  
Grant Trobridge ◽  
Frederic Bushman ◽  
Maciej M Mrugala ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Frequency Analysis ◽  
Peripheral Blood ◽  
Hematopoietic Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Retrovirus Integration ◽  
Time Point

Abstract Abstract 2053 Drug resistance gene therapy with mutant MGMTP140K gene-modified hematopoietic CD34+ cells has been proposed to circumvent myelosuppression associated with alkylating agent chemotherapy; however, the safety of this approach has not yet been demonstrated. We have achieved successful engraftment of MGMTP140K gene-modified hematopoietic progenitor cells in 3 glioblastoma patients following BCNU (600mg/m2) conditioning, as part of a Phase I/II clinical study which includes post-transplant combination O6-benzylguanine (O6BG) and temozolomide (TMZ) chemotherapy. Two of these patients, one of whom remains alive with no evidence for disease progression at more than 2 years since diagnosis, received a total of 9 and 4 cycles of O6BG/TMZ chemotherapy, respectively. We observed transient increases in the number of circulating gene-modified white blood cells (WBCs) and granulocytes in these patients following each cycle. Analysis of CD34+ colony forming cells (CFCs) in peripheral blood revealed increases in the number of gene-modified CFCs over time and with multiple cycles of chemotherapy. Given this, we have conducted a longitudinal retroviral integration site (RIS) analysis in an attempt to identify any selective advantage for gene-modified clones following repeated O6BG/TMZ regimens. In the first patient, at day 200 following transplant, 2 separate RISs were identified, each mapping to the intronic region between exons 1 and 2 of the human PRDM16 gene, previously identified as being associated with insertional activation and clonal expansion in a gene therapy trial for X-linked chronic granulomatous disease (Ott et al 2006). Semi-quantitative capture site frequency analysis revealed that these 2 clones with PRDM16 integrations constituted >20% of the gene-modified cell pool, which included hundreds of RISs at this time point in this patient. Overall gene marking at this same time point was 39.9% of peripheral blood WBCs, indicating that as many as 8% of circulating WBCs arose from these two clones. In the second patient, 3 distinct RISs were identified mapping to the 3'untranslated region (UTR) of the HMGA2 gene, within a cluster of let-7 microRNA binding domains known to be responsible for post-transcriptional regulation of this gene product. While HMGA2-associated RISs have been reported in 2 other gene therapy trials for β-thalassemia (Cavazzana-Calvo et al 2010) and X-linked severe combined immunodeficiency syndrome (Wang et al 2010), the genomic loci of these RISs mapped to the third intron of this genomic sequence. Capture frequency analysis indicated that overall contribution of the most abundant HMGA2-associated clone identified in this study reached 5.2% of gene-modified peripheral blood WBCs at day 300 after transplant. Analysis of PRDM16 and HMGA2 transcripts in each patient's cells by reverse-transcriptase real-time PCR indicated increased transcription of these genes in peripheral blood and bone marrow WBCs. Analysis of WBC subsets in the first patient found PRDM16 RISs present in granulocyte and CD3+ blood cell lineages, suggesting these clones originated from multipotential gene-modified hematopoietic stem cells capable of differentiating despite PRDM16 transactivation. Furthermore, since discontinuation of chemotherapy, we have observed a steady decline in levels of both PRDM16 clones, with complete disappearance of one clone. Extended analysis of HMGA2 expression and clone tracking in WBC subsets of the second patient is currently under way. Importantly, analysis of both patients bone marrow demonstrated normal cytogenetics, with no evidence for MDS or leukemia by hematopathology and flow cytometry. We believe that these data, in combination with other clinical gene therapy studies identifying similar clones, indicate the PRDM16 and HMGA2 gene sequences as “hot-spots” for either initial retrovirus integration in CD34+ hematopoietic progenitor and stem cells, or as genomic loci where aberrant gene expression, as a result of retrovirus integration, is associated with increased cell proliferation. At this time, these observations suggest that in the absence of underlying genomic disease, in vivo fluctuations in PRDM16- and HMGA2-associated clones following transplant may be a common, clinically inconsequential phenomenon in retrovirus-mediated gene therapy targeting CD34+ cell populations for modification. Disclosures: No relevant conflicts of interest to declare.

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.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 586-586
Author(s):  
Marta Claudia Frittoli ◽  
Bernhard Gentner ◽  
Maria Rosa Lidonnici ◽  
Annamaria Aprile ◽  
Laura Bellio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Steady State ◽  
Peripheral Blood ◽  
Single Agent ◽  
Beta Thalassemia ◽  
Target Cells ◽  
Beta Globin ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract 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. Disclosures: No relevant conflicts of interest to declare.

The Challenge Of HSCs Procurement For Gene Therapy: Exploring Plerixafor As Mobilization Agent

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2901-2901
Author(s):  
Maria Rosa Lidonnici ◽  
Annamaria Aprile ◽  
Marta Claudia Frittoli ◽  
Giacomo Mandelli ◽  
Bernhard Gentner ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Transduction Efficiency ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Nsg Mice ◽  
Molecular Features ◽  
Cd34 Cells

Abstract Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of autologous genetically engineered hematopoietic stem/progenitor cells (HSPCs) in myeloablated patients. Hematopoietic reconstitution and clinical benefit are related to cell dose, although single disease features might play a role favoring selection of relevant progenitor populations. Gene therapy trials in young pediatric patients are performed isolating CD34+ cells from bone marrow (BM), while in adults mobilized peripheral blood stem cells (PBSC) should represent the favorite target. In the context of gene therapy for thalassemia, the choice of HSPC source is crucial since intrinsic characteristics of patients (splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilization agent and prompt investigation of new agents. Moreover, adult thalassemic patients may possibly have a decreased BM stem cell reservoir, due to the BM suppression in response to multiple transfusions. A phase II clinical protocol exploring the use of Plerixafor as a single mobilizing agent in adult patients affected by transfusion dependent beta-thalassemia (EudraCT 2011-000973-30) started in 2012 at our hospital. Plerixafor selectively and reversibly antagonizes the binding of SDF-1 to its receptor CXCR4 with subsequent egress of HSCs to the peripheral blood. The availability of a new source of HSPCs, potentially superior in terms of CD34+ cell yield, transduction efficiency and biological features to steady-state BM, would have a significant impact on the feasibility and efficacy of gene therapy. 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 7 to 9 hrs. Three out of four patients achieved the minimal target cell dose (2 x 106 cells/kg) and no severe adverse event occurred. To the aim of engineering Plerixafor-mobilized CD34+ cells for gene therapy, we performed a comprehensive characterization of their biological, molecular and functional properties. In vivo reconstitution potential and lympho-myeloid differentiation were tested following transplantation in NSG mice and compared to those of PBSCs mobilized by G-CSF. Percentages of engrafted human cells in NSG mice transplanted with Plerixafor -PBSCs were about 2- to 5-fold higher than those found in mice transplanted with G-CSF PBSCs. On the same line, the SRC frequency, obtained by pooled engraftment data, was significantly higher (1 SRC out of 47.875 CD34+ cells vs.1 SRC out of 141.203 CD34+ cells). The phenotypic analysis of the frequency of primitive hematopoietic sub-populations revealed that Plerixafor mobilizes preferentially HSPCs and LT-HSPCs, with a percentage of CD34+ CD38-/low CD90+ CD45RA- CD49f+ cells higher than that found in G-CSF PBSCs. This result mirrors the enhanced number of SRCs found in the CD34+ cell population mobilized by Plerixafor. In order to further define the molecular features of HSPCs from different sources, we are studying signalling networks in response to specific cytokines by phospho-proteins analysis and gene expression by microarrays analysis. Our studies are focused on self-renewal, homing, engraftment and multilineage differentiation processes and bioinformatic analysis will reveal the molecular machinery underlying 'stemness' properties of Plerixafor mobilized cells. Disclosures: No relevant conflicts of interest to declare.

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