scholarly journals Correcting Rare Blood Disorders Using Coagulation Factors Produced In Vivo By Shielded Living TherapeuticsTM Products

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2065-2065 ◽  
Author(s):  
Guillaume Carmona ◽  
Lauren Barney ◽  
Jared Sewell ◽  
Ryan Newman ◽  
Christine Carroll ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cell Line ◽  
Immune Cells ◽  
Hemophilia A ◽  
Genetically Modified ◽  
Human Cells ◽  
Blood Disorders ◽  
Cell Therapies

Hemophilia A arises from mutations in the F8 gene, affecting ~ 1/5000 males. Treatment options include frequent intravenous factor and subcutaneous non-factor therapies. While these approaches have been widely used, they have significant limitations, such as breakthrough bleeds and joint disease due to suboptimal adherence, non-ideal factor kinetics, inhibitor generation, (Weyand, Blood 2018) as well as risk of thrombotic events and coagulation test interference with newer non-factor therapies. (Peters, Nat Rev Drug Discov 2018) Alternative modalities such as cell therapies with genetically modified, ready-made human cells are being investigated. To avoid a cytotoxic immune response by the host, allogeneic cells either need to be physically shielded and/or the host immunosuppressed. Various biomaterials, e.g. hydrogels, could serve as the physical barrier that prevents host immune cells from accessing the allogeneic cells, avoiding the need for immunosuppression altogether. However, the host can still activate a foreign body response (FBR), targeting the biomaterial, which significantly limits cell survival and durability of cell therapies. (Anderson, Semin Immunol 2008) We have successfully identified a library of proprietary small molecules, which when conjugated to alginate used to create encapsulating spheres, limit the FBR (Bochenek, Nat Biomed Eng 2018). In addition, we further reduced the FBR using two-compartment design, 1.5 mm diameter spheres, in which the cells are encapsulated in an inner compartment surrounded by an outer, acellular compartment. Using this innovative technology, we aimed to create a novel product that will deliver long-term, sustained human coagulation factor VIII (hFVIII) in vivo. First, we selected a human epithelial cell line with optimal properties for encapsulation within the spheres; considerations included safety, contact inhibition and longevity. We genetically modified this cell line using a non-viral vector and an optimized the coding sequence for a B-domain deleted hFVIII to create a proprietary engineered cell line that constitutively expresses high levels of this protein. Second, we optimized the inner compartment matrix by modulating cell density/sphere and by the addition of a novel modified alginate; these changes maximized cell viability and protein production in vivo. Finally, we further optimized the acellular outer compartment with a proprietary mixture of small-molecule-modified and unmodified alginates. The resulting SIG-001 product candidate consists of two-compartment, 1.5 mm spheres encapsulating hFVIII-expressing human cells. The spheres are sufficiently porous to allow gasses, nutrients, and secreted proteins to freely diffuse, while limiting FBR and prohibiting cell contact with the host's tissues including immune cells. Our in vitro studies demonstrated similar secretion of hFVIII protein by non-encapsulated and encapsulated engineered cells, along with viability of the same cell line after encapsulation. Several doses of SIG-001 were administered intraperitoneally to mice. Stable hFVIII production and good cell viability was shown for spheres retrieved after long-term placement in immunocompromised mice (up to 6 months). Furthermore, our data showed dose-responsive hFVIII activity and efficacious correction of the bleeding phenotype in immunocompetent Hemophilia A mice (Carmona, ISTH 2019). In conclusion, SIG-001 can deliver sustained therapeutic plasma levels of hFVIII in vivo. Our technology could eliminate the need for regular factor injections, lowering the patient burden and providing consistent factor levels without the peaks and troughs observed with factor and non-factor therapies. It also has the potential for expanded use in pediatric patients, and allows for re-dosing if needed. Additionally, there is no concern about the pre-existing antibodies to viral capsids which limit eligibility for gene therapies. We aim to use our technology platform to develop a new category of medicines for severe chronic diseases including rare blood disorders such as Hemophilia A, and to advance their development into clinical testing. Disclosures Carmona: Sigilon Therapeutics: Employment. Barney:Sigilon Therapeutics: Employment. Sewell:Sigilon Therapeutics: Employment. Newman:Sigilon Therapeutics: Employment. Carroll:Sigilon Therapeutics: Employment. Beauregard:Sigilon Therapeutics: Employment. Huang:Sigilon Therapeutics: Employment. Heidebrecht:Sigilon Therapeutics: Employment. Corzo:Sigilon Therapeutics: Employment. Moller:Sigilon Therapeutics: Employment. Smith:Sigilon Therapeutics: Employment. Peritt:Sigilon Therapeutics: Employment. Vivaldi:Sigilon Therapeutics: Employment.

102 Cord-blood derived NK cells, and CAR-T cells, an attractive improved immunotherapy treatment to be considered for hematological malignancies

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A113-A113
Author(s):  
Mireia Bachiller García ◽  
Lorena Pérez-Amill ◽  
Anthony Battram ◽  
Alvaro Urbano-Ispizua ◽  
Beatriz Martín-Antonio
Keyword(s):  
T Cells ◽  
Tumor Cell ◽  
Cord Blood ◽  
Immune Cells ◽  
In Vivo Models ◽  
Cell Clusters ◽  
Cytotoxicity Assays ◽  
Anti Tumor Activity

BackgroundMultiple myeloma (MM) remains an incurable hematological malignancy where a proportion of patients relapse or become refractory to current treatments. Administration of autologous T cells modified with a chimeric antigen receptor (CAR) against B cell maturation antigen (BCMA) has achieved high percentages of complete responses. Unfortunately, the lack of persistence of CART-BCMA cells in the patient leads to relapses. On the other side, cord-blood derived natural killer cells (CB-NK) is an off-the-shelf cellular immunotherapy option to treat cancer patients with high potential due to their anti-tumor activity. However, clinical results in patients up to date have been sub-optimal. Whereas CB-NK are innate immune cells and their anti-tumor activity is developed in a few hours, CART cells are adaptive immune cells and their activity develops at later time points. Moreover, we previously described that CB-NK secrete inflammatory proteins that promote the early formation of tumor-immune cell clusters bringing cells into close contact and thus, facilitating the anti-tumor activity of T cells. Therefore, we hypothesized that the addition of a small number of CB-NK to CART cells would improve the anti-tumor activity and increase the persistence of CART cells.MethodsT cells transduced with a humanized CAR against BCMA and CB-NK were employed at 1:0.5 (CART:CB-NK) ratio. Cytotoxicity assays, activation markers and immune-tumor cell cluster formation were evaluated by flow cytometry and fluorescence microscopy. In vivo models were performed in NSG mice.ResultsThe addition of CB-NK to CART cells demonstrated higher anti-MM efficacy at low E:T ratios during the first 24h and in long-term cytotoxicity assays, where the addition of CB-NK to CART cells achieved complete removal of tumor cells. Analysis of activation marker CD69 and CD107a degranulation from 4h to 24h of co-culturing proved differences only at 4h, where CD69 and CD107a in CART cells were increased when CB-NK were present. Moreover, CB-NK accelerated an increased formation of CART-tumor cell clusters facilitating the removal of MM cells. Of note, CB-NK addition did not increase total TNFα and IFNγ production. Finally, an in vivo model of advanced MM with consecutive challenge to MM cells evidenced that the addition of CB-NK achieved the highest efficacy of the treatment.ConclusionsOur results suggest that the addition of ‘off-the-shelf’ CB-NK to CART cells leads to a faster and earlier immune response of CART cells with higher long-term maintenance of the anti-tumor response, suggesting this combinatorial therapy as an attractive immunotherapy option for MM patients.

scholarly journals Libidibia ferreaFruit Crude Extract and Fractions Show Anti-Inflammatory, Antioxidant, and Antinociceptive EffectIn Vivoand Increase Cell ViabilityIn Vitro

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Tamires Rocha Falcão ◽  
Aurigena Antunes de Araújo ◽  
Luiz Alberto Lira Soares ◽  
Iuri Brilhante de Farias ◽  
Wliana Alves Viturino da Silva ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Cell Viability ◽  
Gallic Acid ◽  
Cell Line ◽  
Crude Extract ◽  
Leukocyte Migration ◽  
Total Glutathione ◽  
Anti Inflammatory

Background.Libidibia ferrea(L. ferrea)is found throughout the northeastern region of Brazil, where it has been used in folk medicine with beneficial effects on many inflammatory disorders.Purpose. This study investigated the phytochemical composition of the crude extract and fractions ofL. ferreafruit and evaluated its anti-inflammatory and antinociceptive activitiesin vivoand effect on cell viabilityin vitro.Methods. Characterization of polyphenols present in crude extract (CE), hydroalcoholic fractions of 20-80% ethanol (CE20, CE40, CE60, and CE80), aqueous fraction (AqF), and ethyl acetate (EAF) fractions ofL. ferreafruit was performed by chromatographic analysis.Anti-inflammatory activity was evaluated by using a carrageenan-induced peritonitis model submitted to a leukocyte migration assay and myeloperoxidase activity (MPO) analysis. Total glutathione and malondialdehyde (MDA) levels were assessed to evaluate the oxidative stress level. Antinociceptive activity was evaluated by acetic acid-induced abdominal writhing and hot plate test.In vitrocell viability was determined by using MTT assay in a mouse embryonic fibroblast cell line (3T3 cells).Results. Chromatography revealed the presence of ellagic acid content in EAF (3.06), CE (2.96), and CE40 (2.89). Gallic acid was found in EAF (12.03), CE 20 (4.43), and CE (3.99).L. ferreacrude extract and all fractions significantly reduced leukocyte migration and MPO activity (p<0.001).L. ferreaantioxidant effect was observed through high levels of total glutathione and reduction of MDA levels (p<0.001). Acetic acid-induced nociception was significantly inhibited after administration ofL. ferreacrude extract and all fractions (p<0.001). Crude extract and all fractions significantly increased the viability of the 3T3 cell line (p<0.05).Conclusions. The appropriate extraction procedure preserves the chemical components ofL. ferreafruit, such as gallic acid and ellargic acid. Crude extract and fractions ofL. ferreafruit exhibited anti-inflammatory, antioxidant, antinociceptive activitiesin vivoand enhanced cell viabilityin vitro.

scholarly journals Subcutaneous Immunization of Leishmania HSP70-II Null Mutant Line Reduces the Severity of the Experimental Visceral Leishmaniasis in BALB/c Mice

Vaccines ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 141
Author(s):  
José Carlos Solana ◽  
Laura Ramírez ◽  
Emma CL Cook ◽  
Elena Hernández-García ◽  
Silvia Sacristán ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Cell Line ◽  
Subcutaneous Administration ◽  
Chronic Phase ◽  
Severe Form ◽  
Vaccine Potential ◽  
Dilution Experiments ◽  
Subcutaneous Immunization

Leishmania infantum parasites cause a severe form of visceral leishmaniasis in human and viscerocutaneous leishmaniasis in dogs. Recently, we reported that immunization with an attenuated L. infantum cell line, lacking the hsp70-II gene, protects against the development of murine cutaneous leishmaniasis. In this work, we analyzed the vaccine potential of this cell line towards the long-term protection against murine visceral leishmaniasis. This model shows an organ-dependent evolution of the disease. The infection can resolve in the liver but chronically affect spleen and bone marrow. Twelve weeks after subcutaneous administration of attenuated L. infantum, Bagg Albino (BALB/c) mice were challenged with infective L. infantum parasites expressing the luciferase-encoding gene. Combining in vivo bioimaging techniques with limiting dilution experiments, we report that, in the initial phase of the disease, vaccinated animals presented lower parasite loads than unvaccinated animals. A reduction of the severity of liver damage was also detected. Protection was associated with the induction of rapid parasite-specific IFN-γ production by CD4+ and CD8+ T cells. However, the vaccine was unable to control the chronic phase of the disease, since we did not find differences in the parasite burdens nor in the immune response at that time point.

scholarly journals The severe combined immunodeficient-human peripheral blood stem cell (SCID-huPBSC) mouse: a xenotransplant model for huPBSC-initiated hematopoiesis

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 89-100 ◽  
Author(s):  
SR Goan ◽  
I Fichtner ◽  
U Just ◽  
L Karawajew ◽  
W Schultze ◽  
...  
Keyword(s):  
Cell Line ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Dose Range ◽  
Human Cells ◽  
Colony Stimulating Factor ◽  
Immunodeficient Mice ◽  
Stimulating Factor

Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose-intensified anticancer chemotherapy and were transplanted into unconditioned “nonleaky” young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL-3), IL-6, and granulocyte- macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment.

The Kinetics and Extent of Engraftment of Chronic Myelogenous Leukemia Cells in Non-Obese Diabetic/Severe Combined Immunodeficiency Mice Reflect the Phase of the Donor’s Disease: An In Vivo Model of Chronic Myelogenous Leukemia Biology

Blood ◽  
1998 ◽  
Vol 92 (4) ◽  
pp. 1390-1396 ◽  
Author(s):  
Francesco Dazzi ◽  
Debora Capelli ◽  
Robert Hasserjian ◽  
Finbarr Cotter ◽  
Margherita Corbo ◽  
...  
Keyword(s):  
Cell Line ◽  
Chronic Myelogenous Leukemia ◽  
Chronic Phase ◽  
Human Cells ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
In Vivo Model ◽  
Fish Analysis ◽  
Rate Of Increase

Abstract In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML). Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model. A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice. Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene. One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site. Similar short-term kinetics were observed using51Cr-labeled cells. The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks. At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173. CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks. The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0.5%/week). FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal. We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor’s disease, thus providing an in vivo model of CML biology. © 1998 by The American Society of Hematology.

Genetically Modified Pc 12 Brain Grafts: Survivability and Inducible Nerve Growth Factor Expression

1996 ◽  
Vol 5 (1) ◽  
pp. 57-68
Author(s):  
Daniel C. Rohrer ◽  
Gajanan Nilaver ◽  
Valerie Nipper ◽  
Curtis A. Machida
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Cell Line ◽  
Neurodegenerative Disorders ◽  
Genetically Modified ◽  
Rat Striatum ◽  
Nerve Growth

Neural transplantation of genetically modified cells has been successfully employed to reverse functional deficits in animal models of neurodegenerative disorders, including Parkinson's disease. While implanted PC12 cells secrete dopamine in vivo and can ameliorate dopamine deficiency in parkinsonian rat model systems, these cells either degenerate within 2-3 wk postimplantation (presumably due to the lack of neural trophic factor support at the site of implantation), or in some cases, form a tumor mass leading to the death of the host animal. To address these limitations, we have developed a genetically modified PC12 cell line that can synthesize nerve growth factor (NGF) under the control of a zinc-inducible metallothionein promoter. When implanted in the rat striatum and under in vivo zinc stimulation, these cells will neurodifferentiate, express tyrosine hydroxylase, and will undergo survival through potential autocrine trophic support. This regulatable cell line and general approach may provide additional insight on the potential utilization of cell transplants for treatment of Parkinson's disease and other neurodegenerative disorders.

In vivo GABA release and kinetics of transgene loss in a GABAergic cell line after long-term transplantation into the rat brain

Neuroscience ◽  
2012 ◽  
Vol 203 ◽  
pp. 244-254 ◽  
Author(s):  
J. Mejía-Toiber ◽  
J.H. Limón-Pacheco ◽  
A. Gonzalez-Gallardo ◽  
M. Giordano
Keyword(s):  
Rat Brain ◽  
Cell Line ◽  
Gaba Release ◽  
Kinetics Of

Rational Combinations Including a Novel Syk Inhibitor, Fostamatinib Disodium (FosD) in Diffuse Large B Cell Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 283-283
Author(s):  
Randall M Rossi ◽  
Valerie Grose ◽  
Polly Pine ◽  
Richard I Fisher ◽  
Craig T. Jordan ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
B Cell Lymphoma ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
In Vivo Studies

Abstract Abstract 283 Certain malignant B-cells rely upon B-cell receptor-mediated survival signals. Spleen tyrosine kinase (Syk) initiates and amplifies the B-cell receptor-mediated signal. We and others have demonstrated that fostamatinib disodium (FosD: a prodrug of R406, a potent and specific inhibitor of Syk) induces apoptosis in lymphoma cell lines and primary tumors. A recent clinical trial has demonstrated significant clinical activity of FosD in relapsed/refractory B-cell non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia, and minimal overlap in toxicities with conventional agents. Given this background, future development in B-cell NHL will include rational combinations of FosD and currently available therapies. Therefore, we conducted in vitro and in vivo studies of rational combinations including FosD, in anticipation of clinical trial development. First, using a human DLBCL cell line of GCB genotype, (OCI-Ly19), we analyzed in vitro the combination of R406 with the following agents: fludarabine, rapamycin, rituximab, bendamustine and bortezomib. Increased cytotoxicity was observed using in vitro culture assays with the addition of fludarabine, rapamycin, or rituximab to R406. Cell viability at 72 hours was 25% with R406 alone, 27% for fludarabine alone, and only 9% for the fludarabine/R406. At 48 hours, cell viability was 49% using R406 alone, 31% using rituximab alone, and 21% for rituximab/R406. At 120 hours using primary lymphoma cells (DLCL27), there were no viable cells treated with the rapamycin/FosD combination, compared with rapamycin alone (7%) or FosD alone (25%) The addition of bortezomib or bendamustine to FosD resulted in only a minimal additive increase in cytotoxicity. Results with all combinations were similar with the OCI-Ly10 human DLBCL line of ABC genotype. We then performed in vivo studies by subcutaneous transplantation of the DLBCL cell line OCI-Ly19, (engineered to express luciferase allowing for real time in vivo imaging) into immune deficient NOD/SCID mice which reproducibly formed tumors. Recipient animals were separated into uniform cohorts when the tumors were less than or equal to 500 mm3 in size. The animals were then simultaneously treated with FosD (n=7; 3 gm/kg ad. lib.; translates into 2-5 micromolar R406 systemically throughout the 24h period) and either bortezomib, (n=6; 0.4 mg/kg weekly IP), or rituximab, (n=13; 3 mg/kg, 2x weekly IP). Analysis of the OCI-Ly19 tumor volumes at day 46 showed a median of 2364 mm3 with bortezomib alone compared with 1823 mm3 with bortezomib and FosD. When FosD was combined with rituximab the most significant cytotoxicity was observed: (p=0.01; median tumor volume of 497 mm3 following the combination) in comparison to either FosD alone (3150 mm3) or rituximab alone (1764 mm3). We conclude that the addition of FosD appears to increase activity against NHL of several drugs, including fludarabine and rapamycin. These agents have significant activity in indolent and mantle cell NHL as well as CLL. Moreover, there is no evidence that FosD impedes rituximab responses in vitro or in vivo; in fact we have suggested possible synergy with the combination of rituximab and FosD. Based upon the documented single agent activity of FosD in humans, and this data, clinical trials are now indicated using these promising combinations in NHL and CLL. Disclosures: Pine: Rigel: Employment. Friedberg:Rigel: Research Funding.

In Vivo Selection Of Genetically Manipulated Hematopoietic Stem Cells For Platelet Gene Therapy Of Hemophilia A

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2329-2329
Author(s):  
Qizhen Shi ◽  
Jocelyn A. Schroeder ◽  
David A. Wilcox ◽  
Robert R. Montgomery ◽  
Yingyu Chen
Keyword(s):  
Gene Therapy ◽  
Hemophilia A ◽  
Genetically Modified ◽  
Conditioning Regimen ◽  
Selective Treatment ◽  
Hematopoietic Stem ◽  
In Vivo Selection ◽  
Fviii Activity ◽  
Selection Of

Abstract Our previous studies have demonstrated that targeting FVIII expression to platelets (2bF8) by lentiviral (LV) gene delivery to hematopoietic stem cells (HSCs) corrects bleeding diathesis in hemophilia A mice with or without inhibitors. Although the bleeding diathesis is improved in transduced recipients, the transduction efficiency using our current 2bF8 LV, is only about 10%, resulting a median level of platelet-FVIII (Plt-F8) of 1.5 mU/108 platelets even thought a myeloablative conditioning regimen was employed. It has been shown in clinical trials that efficient stem cell gene transfer and myeloablation is not required when there is a powerful selective advantage to the genetically modified cells. We hypothesize that incorporating a drug-resistance gene into the 2bF8 LV construct will allow for in vivo selection of 2bF8 LV-transduced cells which will result in the increase of therapeutic levels of Plt-F8 for hemophilia A gene therapy and reduce the potential for genotoxicity. To address our hypothesis, we constructed a new lentiviral vector, pWPT-2bF8/MGMT, which harbors dual genes, the 2bF8 gene and a drug-resistance gene, the MGMTP140K cassette. To explore the feasibility of the MGMT-based in vivo selection system, HSCs from FVIIInull mice were transduced with 2bF8/MGMT LV at an MOI (multiplicity of infectious) of 1, which is 1/10 of the MOI used for our regular 2bF8 LV transduction, and transplanted into littermates pre-conditioned with a non-myeloablative regimen, 660 cGy total body irradiation (TBI). We chose a low MOI because one of the goals of using the MGMT selection system is to reduce the potential for genotoxicity. After bone marrow (BM) reconstitution, the recipients were treated with O6-benzylguanine (BG) followed by 1, 3-bis-2 chloroethyl-1-nitrosourea (BCNU) monthly for 3 or 4 times. As determined by a chromogenic assay on platelet lysates, functional Plt-F8 expression in recipients was only 0.22 ± 0.15 mU/108 platelets before the drug treatment, but remarkably increased to 4.33 ± 5.48 mU/108 platelets (n = 16) after BG/BCNU drug-selective treatments. The levels of Plt-F8 in the untreated transduced control group remained low over the study period. FVIII activity was not detected in the plasma in any of the recipients even with Plt-F8 as high as 22 mU/108 platelets. The average copy number of 2bF8/MGMT proviral DNA per cell was determined by quantitative real-time PCR. 2bF8 proviral DNA was barely detectable (0.01 ± 0.02 copies/cell) in recipients before drug-selective treatment, but it increased to 0.42 ± 0.15 copies/cell after BG/BCNU treatments, confirming that 2bF8/MGMT genetically modified cells were effectively enriched in vivo after drug-selective treatment. When the tail clip survival test was used to assess phenotypic correction of the FVIIInull coagulation defect, 15 of 16 treated animals survived the tail clip challenge; in contrast, none of the untransduced FVIIInull control mice survived. When ROTEM analysis was used to determine the whole blood clotting time (CT), the CT was shortened from 3043 ± 728 seconds (n = 7) to 931± 273 seconds (n = 6) (P < 0.0001) in treated transduced recipients when compared to FVIIInull mice. There was no significant difference between wild type (722 ± 270 seconds, n = 7) and treated recipients. To ensure sustained Plt-F8 expression in BG/BCNU treated transduced recipients, some primary recipients were sacrificed 9 months after transplantation and BM mononuclear cells were transplanted into secondary recipients. Platelet lysate FVIII activity assays showed that the levels of Plt-F8 in secondary recipients were similar to those in primary recipients, confirming that long-term repopulating HSCs were successfully genetically modified by 2bF8/MGMT LV. When a low intensity pre-conditioning regimen of 440 cGy TBI was used, the levels of Plt-F8 increased from 0.06 ± 0.12 mU/108 platelets to 1.86 ± 2.06 mU/108 platelets after BG/BCNU drug-selective treatment. It is notable that no anti-FVIII inhibitory antibodies were detected in the treated recipients even after rhFVIII challenge, indicating that immune tolerance was induced in the treated animals. In contrast, all FVIIInull mice under the same challenge developed various levels of inhibitors. Taken together, we have established a powerful in vivo selective system that allows us to enrich 2bF8 LV-transduced cells and to enhance platelet-FVIII expression for hemophilia A gene therapy. Disclosures: No relevant conflicts of interest to declare.

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