scholarly journals Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilia A mice with preexisting FVIII immunity

Blood ◽  
2008 ◽  
Vol 112 (7) ◽  
pp. 2713-2721 ◽  
Author(s):  
Qizhen Shi ◽  
Scot A. Fahs ◽  
David A. Wilcox ◽  
Erin L. Kuether ◽  
Patricia A. Morateck ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgenic Mice ◽  
Hematopoietic Stem Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Genetically Modified ◽  
Hematopoietic Stem ◽  
Inhibitory Antibodies ◽  
Sublethal Irradiation

Abstract Although genetic induction of factor VIII (FVIII) expression in platelets can restore hemostasis in hemophilia A mice, this approach has not been studied in the clinical setting of preexisting FVIII inhibitory antibodies to determine whether such antibodies would affect therapeutic engraftment. We generated a line of transgenic mice (2bF8) that express FVIII only in platelets using the platelet-specific αIIb promoter and bred this 2bF8 transgene into a FVIIInull background. Bone marrow (BM) from heterozygous 2bF8 transgenic (2bF8tg+/−) mice was transplanted into immunized FVIIInull mice after lethal or sublethal irradiation. After BM reconstitution, 85% of recipients survived tail clipping when the 1100-cGy (myeloablative) regimen was used, 85.7% of recipients survived when 660-cGy (nonmyeloablative) regimens were used, and 60% of recipients survived when the recipients were conditioned with 440 cGy. Our further studies showed that transplantation with 1% to 5% 2bF8tg+/− BM cells still improved hemostasis in hemophilia A mice with inhibitors. These results demonstrate that the presence of FVIII-specific immunity in recipients does not negate engraftment of 2bF8 genetically modified hematopoietic stem cells, and transplantation of these hematopoietic stem cells can efficiently restore hemostasis to hemophilic mice with preexisting inhibitory antibodies under either myeloablative or nonmyeloablative regimens.

Functional aspects of factor VIII expression after transplantation of genetically-modified hematopoietic stem cells for hemophilia A

2010 ◽  
Vol 12 (4) ◽  
pp. 333-344 ◽  
Author(s):  
Lucienne M. Ide ◽  
Neal N. Iwakoshi ◽  
Bagirath Gangadharan ◽  
Shawn Jobe ◽  
Robert Moot ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Genetically Modified ◽  
Hematopoietic Stem ◽  
Functional Aspects

scholarly journals CblY371H Transgene Combined with Hematopoietic Deletion of the Endogenous c-Cbl Gene Results in GM-CSF Hypersensitivity and Leukocytosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3672-3672
Author(s):  
Kenneth Lieuw ◽  
Jayasree Krishnamurthy ◽  
Mignon L. Loh
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgenic Mice ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Embryonic Lethality ◽  
Hematopoietic Stem ◽  
Endogenous Gene ◽  
Gm Csf ◽  
Marrow Cells

Abstract Juvenile Myelomonocytic Leukemia (JMML) is a mixed myeloproliferative/myelodysplastic disease that is rapidly fatal with infiltration of myeloid cells into multiple organs. About 15% of JMML patient samples contain a mutation in c-Cbl, and germline mutation results in the predisposition for developing JMML. The c-Cbl gene encodes a multifunctional adaptor protein that contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif that contains E3 ligase activity, and a C-terminal ubiquitin-associated domain. The TKB domain is involved in adaptor functions of the protein, whereas the ubiquitin ligase domain results in mono-ubiquitination of receptors which promotes lysosomal mediated degradation of activated receptors. Interestingly, a hotspot for mutations at residue 371 exists in JMML patients, where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H. This residue belongs in the linker region of the CBL protein, and it was previously observed that Tyr-371 plays key roles in activating the ubiquitin ligase activity of the protein. In vitro, CblY371H mutation does indeed destroy its ligase function, resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene is silenced. How mutant Cbl gives rise to JMML, however, and how it acts in concert with other genes in the pathogenesis of JMML requires further study. To address these questions, we overexpressed the oncogenic CblY371H mutation using transgenic mice. As expected, overexpression of CblY371H by itself in wildtype mice had no apparent phenotype. Therefore, Cbl transgenic mice were bred to Cbl heterozygous knockout mice (Cbl+/-) followed by further breeding in an attempt to generate Cbl transgenic mice with the endogenous Cbl gene inactivated (CblY371H; Cbl-/-). Surprisingly, unlike Cbl null mice, which are viable, overexpression of mutant Cbl allele in Cbl null mice caused embryonic lethality between 11.5 dpc and 12.5 dpc. In order to circumvent the developmental effects of expressing the mutant Cbl protein, we used a conditional Cbl knockout mouse to tissue specifically delete the endogenous Cbl gene. We chose the MMTV-Cre strain, which expresses Cre recombinase in only 10% of hematopoietic stem cells (CD34-; Lin-; Sca-1+; c-Kit+). With subsequent breeding with the CblY371H transgenic mice, we were able to bypass the embryonic lethality and produce mice with the correct genotype (MMTV-Cre;CblY371H;Cblfl/fl). These mice look normal but develop significant leukocytosis and show GM-CSF hypersensitivity even though only 10% of hematopoietic stem cells are affected. These mice, however, appear unaffected by the leukocytosis, and show no obvious difference with its littermates up to one year of age. We conclude that mutant CblY371H by itself is not sufficient for the development of JMML in this model and requires additional cooperating events. Whether further aging of these mice will result in JMML remains to be seen. In conclusion, we have developed a mouse model overexpressing the CblY371H protein ubiquitously, which causes deleterious development when it is the only c-Cbl protein available. This confirms the important role of c-Cbl activity during development. In hematopoietic cells, the overexpression of CblY371H results in leukocytosis and GM-CSF hypersensitivity when the endogenous gene is inactivated. We are currently investigating the cooperating events that are required for the development of JMML in this mouse model. Figure 1. Phenotype of CblY371H Transgenic Mice A and B. Embryonic lethality of Cbl transgenic mice. The embryos look normal on day 10 of development but by day 12.5, no homozygous embryos are found. C and D. There is significant leukocytosis when the CblY371H transgene is combined with inactivation of the endogenous gene only in hematopoietic stem cells using the MMTV Cre. Figure 1. Phenotype of CblY371H Transgenic Mice A and B. Embryonic lethality of Cbl transgenic mice. The embryos look normal on day 10 of development but by day 12.5, no homozygous embryos are found. C and D. There is significant leukocytosis when the CblY371H transgene is combined with inactivation of the endogenous gene only in hematopoietic stem cells using the MMTV Cre. Figure 2. GM-CSF Hypersensitivity of Bone Marrow Cells from Triple Transgenic Mice A. Western Blot of bone marrow cells stimulated with GM-CSF. Panel A shows time course after stimulation of bone marrow cells from conditional Cbl mice (Cblfl/fl) that have the endogenous Cbl gene deleted using the MMTV-Cre transgene. B-D.Quantitation of several blots showing GM-CSF hypersensitivity. When normalized to the nontransgenic mice at time point zero, there is increased activity of downstream signaling pathways with and without GM-CSF. Figure 2. GM-CSF Hypersensitivity of Bone Marrow Cells from Triple Transgenic Mice A. Western Blot of bone marrow cells stimulated with GM-CSF. Panel A shows time course after stimulation of bone marrow cells from conditional Cbl mice (Cblfl/fl) that have the endogenous Cbl gene deleted using the MMTV-Cre transgene. B-D. Quantitation of several blots showing GM-CSF hypersensitivity. When normalized to the nontransgenic mice at time point zero, there is increased activity of downstream signaling pathways with and without GM-CSF. Disclosures No relevant conflicts of interest to declare.

Sustained Phenotypic Correction of Murine Hemophilia A with Pre-Existing Anti-FVIII Immunity Using Lentivirus-Mediated Platelet-Specific FVIII Gene Transfer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 29-29 ◽  
Author(s):  
Qizhen Shi ◽  
Erin L. Kuether ◽  
Brian C. Cooley ◽  
Scot A. Fahs ◽  
Jocelyn A. Schroeder ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Real Time Pcr ◽  
Hemophilia A ◽  
Hematopoietic Stem ◽  
Fviii Activity ◽  
Inhibitory Antibodies ◽  
Tail Clip

Abstract Abstract 29 The development of inhibitory antibodies to exogenous factor VIII (FVIII) is considered a severe and important complication of FVIII infusion in hemophilia A patients. Gene therapy of hemophilia A with inhibitors is especially challenging because functional FVIII activity may be inactivated by circulating inhibitory antibodies if transgene protein is constitutively secreted into the blood circulation. Our previous studies have demonstrated that syngeneic transplantation of hematopoietic stem cells from 2bF8 transgenic mice that express platelet-specific FVIII can efficiently restore hemostasis to hemophilic mice with pre-existing inhibitory antibodies. In the current study, we assessed whether lentivirus-mediated 2bF8 gene transfer could efficiently introduce 2bF8 transgene expression and ameliorate the hemorrhagic phenotype in hemophilic mice with pre-existing immunity. To mimic the clinical situation of an autologous transplant in an inhibitor patient, both donor and recipient FVIIInull mice were immunized with recombinant human B-domain deleted FVIII to induce inhibitory antibody development. Platelet-derived FVIII expression in FVIIInull mice was introduced by 2bF8 lentiviral-mediated bone marrow (BM) transduction and syngeneic transplantation. Following BM reconstitution, mice were analyzed by PCR, quantitative real-time PCR, platelet lysate FVIII activity assay, and inhibitor assay. Phenotypic correction was assessed by tail clip survival test and electrolytic-induced thrombus formation. Expression of the 2bF8 product was detected in all recipients that received 2bF8 lentivirus transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF8 lentivirus transfer vector. Functional platelet-FVIII activity levels in the transduced mice with pre-existing immunity ranged from 0.36 to 6.18 mU/108 platelets (mean 1.56 ± 1.76 mU/108 platelets, n = 10), which was not significantly different from the levels obtained from a parallel non-inhibitor model (1.46 ± 0.87 mU/108 platelets, n = 4). Real-time PCR demonstrated that there was an average of 0.17 ± 0.05 LV DNA copies per cell in peripheral white blood cells from transduced mice. FVIII inhibiter titer gradually declined with the time, indicating that transduced platelet FVIII is well protected from exposure to the immune system, avoiding activation of a memory response. The tail clip survival test showed that 90% of mice survived tail clip challenge. The electrolytic injury model demonstrated that hemostasis was improved in recipients that received 2bF8 lentivirus-transduced BM cells. Furthermore, BM transferred from the primary transplant recipients into immunized FVIIInull secondary recipients demonstrated sustained platelet-FVIII expression, resulting in the correction of the hemophilia A phenotype with pre-existing immunity. This shows that gene transfer has occurred within long-term repopulating hematopoietic stem cells even in the presence of inhibitory antibodies. These results demonstrate that lentivirus-mediated bone marrow transduction/transplantation can provide sustained improvement of hemostasis in hemophilic mice with pre-existing immunity, indicating that this approach may be a promising strategy for gene therapy of hemophilia A with inhibitory antibodies in humans. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Correction of murine hemophilia A following nonmyeloablative transplantation of hematopoietic stem cells engineered to encode an enhanced human factor VIII variant using a safety-augmented retroviral vector

Blood ◽  
2009 ◽  
Vol 114 (3) ◽  
pp. 526-534 ◽  
Author(s):  
Ali Ramezani ◽  
Robert G. Hawley
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Factor Viii ◽  
Insertional Mutagenesis ◽  
Hemophilia A ◽  
Point Mutations ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Human Factor Viii ◽  
A1 Domain

Abstract Insertional mutagenesis by retroviral vectors is a major impediment to the clinical application of hematopoietic stem cell gene transfer for the treatment of hematologic disorders. We recently developed an insulated self-inactivating gammaretroviral vector, RMSinOFB, which uses a novel enhancer-blocking element that significantly decreases genotoxicity of retroviral integration. In this study, we used the RMSinOFB vector to evaluate the efficacy of a newly bioengineered factor VIII (fVIII) variant (efVIII)—containing a combination of A1 domain point mutations (L303E/F309S) and an extended partial B domain for improved secretion plus A2 domain mutations (R484A/R489A/P492A) for reduced immunogenicity—toward successful treatment of murine hemophilia A. In cell lines, efVIII was secreted at up to 6-fold higher levels than an L303E/F309S A1 domain–only fVIII variant (sfVIIIΔB). Most important, when compared with a conventional gammaretroviral vector expressing sfVIIIΔB, lower doses of RMSin-efVIII-OFB–transduced hematopoietic stem cells were needed to generate comparable curative fVIII levels in hemophilia A BALB/c mice after reduced-intensity total body irradiation or nonmyeloablative chemotherapy conditioning regimens. These data suggest that the safety-augmented RMSin-efVIII-OFB platform represents an encouraging step in the development of a clinically appropriate gene addition therapy for hemophilia A.

Reversible Expansion of Hematopoietic Stem Cells (HSC) and CML-Like Disease in Transgenic Mice Expressing BCR-ABL under the Control of the SCL 3′ Enhancer.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 715-715
Author(s):  
Steffen Koschmieder ◽  
Berthold Goettgens ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
Kristin Geary ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgenic Mice ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Molecular Mechanisms ◽  
Leukemic Cells ◽  
Hematopoietic Stem ◽  
Double Transgenic Mice

Abstract Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs. Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by real-time PCR, suggesting that the SCL 3′ enhancer indeed directed BCR-ABL expression to the most primitive hematopoietic cells within the bone marrow. The percentage of HSC in the bone marrow was expanded 7- and 26-fold in double-transgenic as compared to single-transgenic or wild-type control mice within 12 and 21 days, respectively, after BCR-ABL induction. GMP were increased 2- and 3-fold while the number of CMP was decreased 2-fold after 12 days but was increased 1.5-fold after 21 days. MEP were decreased 3-fold at both time points. In keeping with these results, the percentage of Ter-119 positive erythroid cells was decreased while the percentage of Gr-1 positive granulocytic cells was increased in the bone marrow. To assess reversibility of the phenotype, we readministered tetracycline to abrogate BCR-ABL expression. Double-transgenic mice showed rapid clinical improvement, reversion of neutrophilia and leukocytosis, normalization of Gr-1/Mac-1 positive cells in the peripheral blood and spleen, and reversion of splenomegaly. In addition, in mice that had developed lymphoblastic disease, readministration of tetracycline led to disappearance of lymphomas and of B220/BP-1 positive lymphoblastic cells in the peripheral blood. Furthermore, expansion of the HSC compartment in the bone marrow was also reversible, and the percentage of HSC decreased to levels observed in control mice. Repeated induction of BCR-ABL expression by removal of tetracycline led to reappearance of the myeloid and lymphoid phenotype. Again, the disease was reversible, and none of the animals relapsed while on tetracycline, suggesting that the phenotype remained completely dependent on the expression of the oncogene. In conclusion, we present a model of BCR-ABL mediated CML-like disease with expansion of phenotypic hematopoietic stem cells and myeloid progenitor cells in the bone marrow. The target cell population in this model closely resembles the origin of transformation in patients with CML, allowing for in vivo monitoring of early molecular mechanisms of BCR-ABL transformation. We are currently studying the function of the expanded HSC and progenitor cells in transplantation experiments.

scholarly journals Hematopoietic Stem Cells Encoding Porcine Factor VIII Induce Pro-coagulant Activity in Hemophilia A Mice with Pre-existing Factor VIII Immunity

2007 ◽  
Vol 15 (6) ◽  
pp. 1093-1099 ◽  
Author(s):  
Christopher B Doering ◽  
Bagirath Gangadharan ◽  
Hillary Z Dukart ◽  
H Trent Spencer
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Factor Viii ◽  
Hemophilia A ◽  
Hematopoietic Stem ◽  
Coagulant Activity
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