Transplantation of hematopoietic stem cells encoding a high-expression factor VIII transgene under myeloablative and nonmyeloablative conditioning provides curative factor VIII activity to hemophilia A mice

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.

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408. Correction of the Factor VIII Deficiency in Hemophilia A Mice with Pre-Existing Anti-Factor VIII Inhibitors by Transplantation of Genetically-Modified Hematopoietic Stem Cells Expressing Porcine Factor VIII

Molecular Therapy ◽

10.1016/s1525-0016(16)44614-9 ◽

2007 ◽

Vol 15 ◽

pp. S157

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Factor Viii ◽

Hemophilia A ◽

Genetically Modified ◽

Hematopoietic Stem ◽

Factor Viii Deficiency

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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 ◽

10.1182/blood-2009-01-199653 ◽

2009 ◽

Vol 114 (3) ◽

pp. 526-534 ◽

Cited By ~ 19

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.

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

Molecular Therapy ◽

10.1038/sj.mt.6300146 ◽

2007 ◽

Vol 15 (6) ◽

pp. 1093-1099 ◽

Cited By ~ 43

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

The Journal of Gene Medicine ◽

10.1002/jgm.1442 ◽

2010 ◽

Vol 12 (4) ◽

pp. 333-344 ◽

Cited By ~ 28

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

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Evidence That High-Mobility Group A2 (Hmga2) Expression Contributes to the Ontogeny-Dependent Properties of Fetal Hematopoietic Stem Cells.

Blood ◽

10.1182/blood.v116.21.2634.2634 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2634-2634

Author(s):

Michael R. Copley ◽

David G. Kent ◽

Claudia Benz ◽

Keegan M. Rowe ◽

Stefan H. Woehrer ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Rapid Expansion ◽

High Expression ◽

Primary Host ◽

Self Renewal ◽

Hematopoietic Stem ◽

Renewal Program

Abstract Abstract 2634 Fetal and early neonatal hematopoietic stem cells (HSCs) are distinct from their adult counterparts by their rapid turnover and expansion rates in vivo. However, the mechanisms underlying the regulation of these properties are poorly understood. In previous studies using serial limiting-dilution competitive repopulating transplant assays, our lab has shown that the rapid expansion phenotype of fetal HSCs is at least partially intrinsically determined since significantly more daughter HSCs are produced from fetal as compared to adult HSCs when similar numbers are transplanted into the same type of irradiated adult host. Additionally, we have observed a conversion of fetal HSCs to the adult regeneration phenotype that occurs within six weeks of transplantation in the primary host. To facilitate a comparison of highly-purified subsets of fetal and adult HSCs identified by an identical phenotype, we adopted the use of the CD45+EPCR+CD150+CD48− (E-SLAM) phenotype which we found gave HSC purities of 20–50% for hematopoietic tissues from early fetal to aged adulthood. We then used comparative gene expression analysis to identify candidate regulators of the fetal HSC high self-renewal program. This gave 20 candidate genes whose transcript levels were measured by quantitative real time PCR in E-SLAM cells isolated from E14.5 fetal liver (FL) and adult bone marrow (ABM). Of these genes only Hmga2 and Smarcc1 showed significant differences (p<.05) in expression between fetal and adult HSCs and only Hmga2 maintained this differential expression when the same cells were stimulated to divide for 48 hrs in vitro. To test the hypothesis that high expression of Hmga2 is a necessary and sufficient factor in determining the fetal HSC self-renewal program, purified adult E-SLAM HSCs were transduced with Hmga2-overexpressing or control lentiviruses and the kinetics of transduced vs untransduced hematopoietic cells in a congenic serial-transplantation model were then analyzed. Interestingly, when BM cells from the primary repopulated mice (transplanted 6-weeks earlier) were injected into secondary animals and the peripheral blood was analyzed for donor-type %Y/GFP chimerism, the Hmga2-overexpressing cells were observed to have a competititve advantage and exhibited an ∼6-fold expansion relative to the untransduced cells. In contrast, the control virus-infected BM cells were found to be equally competitive. These findings support the hypothesis that high expression of Hmga2 may be a critical mediator of the high self-renewal phenotype of fetal HSCs. Disclosures: No relevant conflicts of interest to declare.

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High expression of BCL6 inhibits the differentiation and development of hematopoietic stem cells and affects the growth and development of chickens

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-020-00541-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Hongmei Li ◽

Bowen Hu ◽

Shang Hu ◽

Wen Luo ◽

Donglei Sun ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Mitochondrial Function ◽

Growth And Development ◽

Clinical Manifestations ◽

High Expression ◽

Ros Production ◽

Hematopoietic Stem ◽

Atp Concentration

Abstract Background B-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that represses more than 1200 potential target genes. Our previous study showed that a decline in blood production in runting and stunting syndrome (RSS) affected sex-linked dwarf (SLD) chickens compared to SLD chickens. However, the association between BCL6 gene and hematopoietic function remains unknown in chickens. Methods In this study, we used RSS affected SLD (RSS-SLD) chickens, SLD chickens and normal chickens as research object and overexpression of BCL6 in hematopoietic stem cells (HSCs), to investigate the effect of the BCL6 on differentiation and development of HSCs. Results The results showed that comparison of RSS-SLD chickens with SLD chickens, the BCL6 was highly expressed in RSS-SLD chickens bone marrow. The bone marrow of RSS-SLD chickens was exhausted and red bone marrow was largely replaced by yellow bone marrow, bone density was reduced, and the levels of immature erythrocytes in peripheral blood were increased. At the same time, the hematopoietic function of HSCs decreased in RSS-SLD chickens, which was manifested by a decrease in the hematopoietic growth factors (HGFs) EPO, SCF, TPO, and IL-3, as well as hemoglobin α1 and hemoglobin β expression. Moreover, mitochondrial function in the HSCs of RSS-SLD chickens was damaged, including an increase in ROS production, decrease in ATP concentration, and decrease in mitochondrial membrane potential (ΔΨm). The same results were also observed in SLD chickens compared with normal chickens; however, the symptoms were more serious in RSS-SLD chickens. Additionally, after overexpression of the BCL6 in primary HSCs, the secretion of HGFs (EPO, SCF, TPO and IL-3) was inhibited and the expression of hemoglobin α1 and hemoglobin β was decreased. However, cell proliferation was accelerated, apoptosis was inhibited, and the HSCs entered a cancerous state. The function of mitochondria was also abnormal, ROS production was decreased, and ATP concentration and ΔΨm were increased, which was related to the inhibition of apoptosis of stem cells. Conclusions Taken together, we conclude that the high expression of BCL6 inhibits the differentiation and development of HSCs by affecting mitochondrial function, resulting in impaired growth and development of chickens. Moreover, the abnormal expression of BCL6 might be a cause of the clinical manifestations of chicken comb, pale skin, stunted growth and development, and the tendency to appear RSS in SLD chickens.

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