Tolerization of a Type I Allergic Immune Response through Transplantation of Genetically Modified Hematopoietic Stem Cells

The curative potential of Type I interferons for patients suffering from Myeloproliferative Neoplasms (MPNs) has been reported and these are the only class of drugs that can lead to reduction of the mutant allelic burden in patients. However, modelling IFN treatment in mice has been challenging. Here, we report the use of murine pegylated IFNα (murine ropeginterferon-a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) to model IFN treatment in transgenic MPN mouse models. We started treating JAK2V617Ff/+;vavCre and control vavCre mice (n=6-8) with PBS or mRopeg (600 ng/mouse/week), by subcutaneous injections from the time they were 4 weeks old. The mice were bled every 2 weeks from the facial vein and the blood parameters were monitored. We observed significant normalization of platelet and WBC counts in Jak2-V617F fl/+ vavCre mice to wild type levels. No effect on hematocrit and hemoglobin level was observed in the Jak2-V617F fl/+ vavCre mice. VavCre control animals showed no sign of negative effect such as cytopenia during the entire treatment course. We observed a highly significant prolongation of the survival of mRopeg treated JAK2V617Ff/+;vavCre mice over a duration of 80 days of treatment. While all the PBS treated JAK2V617Ff/+;vavCre mice died within 60 days, all the mRopeg treated mice were still alive till the end of the treatment duration. We also generated a novel transgenic mouse model that conditionally expresses hybrid mutant CALR protein (murine exons 1-8 and human CALR del52 exon9) from the endogenous murine Calr locus. We bred them into vavCre background (in both heterozyhous and homozygous states) to induce expression of CALR-del52 in hematopoietic cells. Upon Cre recombinase expression, the endogenous murine exon 9 is replaced by the human del52 exon 9 and the expression of the humanized Calr-del52 oncoprotein is detectable by Western blot analysis using mutant CALR specific antibodies. Calr-del52 animals develop an essential thrombocythemia (ET) like phenotype when expressed in a heterozygous state with elevated number of hematopoietic stem cells and megakaryocytes in the bone marrow. In the homozygous state, the thrombocythemia is more severe with splenomegaly and older animals show anemia with increased WBC. Bone marrow histology shows megakaryocytic hyperplasia with no sign of fibrosis up to age of one year. We treated a cohort of animals with 600 ng mRopeg/PBS once a week for 4 weeks. Peripheral blood counts were determined at baseline and at regular intervals during treatment. At the end of treatment, mice were sacrificed, and splenic and bone marrow cells were immunophenotyped and quantified by FACS. We observed correction of thrombocythemia in the homozygous Calr-del52 mice but no unspecific decrease of platelet count in the vavCre mRopeg treated animals. We observed significant specific reduction of the long-term hematopoietic stem cells (LT-HSCs/fraction A) in homozygous CALR-del52 mice. In conclusion, Type I IFN treatment significantly reduces platelet counts to normal levels in both JAK2 and CALR mutant driven MPN mouse models. The prolongation of survival of JAK2V617F transgenic mice upon Type I IFN treatment is particularly remarkable; as no survival data is reported until now in any clinical trials or other animal models. Further experiments are required to understand the mechanism of action of this phenomenon. Disclosures No relevant conflicts of interest to declare.

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466. Modeling and Immune Monitoring of Human Adaptive T and B Antigen-Specific Responses in Humanized Mice Upon Immunizations With Lentivirus-Induced Dendritic Cells: Potential Application for Functional Evaluation of Genetically Modified Human Hematopoietic Stem Cells

Molecular Therapy ◽

10.1016/s1525-0016(16)35479-x ◽

2014 ◽

Vol 22 ◽

pp. S179

Keyword(s):

Stem Cells ◽

Dendritic Cells ◽

Hematopoietic Stem Cells ◽

Potential Application ◽

Genetically Modified ◽

Immune Monitoring ◽

Humanized Mice ◽

Functional Evaluation ◽

Hematopoietic Stem

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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 ◽

10.1182/blood-2008-02-138214 ◽

2008 ◽

Vol 112 (7) ◽

pp. 2713-2721 ◽

Cited By ~ 70

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.

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Copy Number Determination of Genetically-Modified Hematopoietic Stem Cells

Genetic Modification of Hematopoietic Stem Cells - Methods in Molecular Biology ◽

10.1007/978-1-59745-409-4_19 ◽

2009 ◽

pp. 281-298 ◽

Cited By ~ 3

Author(s):

Todd Schuesler ◽

Lilith Reeves ◽

Christof von Kalle ◽

Elke Grassman

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Copy Number ◽

Genetically Modified ◽

Hematopoietic Stem ◽

Copy Number Determination

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Genetically Modified Hematopoietic Stem Cells for Gene Therapy in Beta-Thalassemia: Power of Viral Vector Towards Clinical Application

10.2174/9789815036756121010011 ◽

2021 ◽

pp. 133-144

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Hematopoietic Stem Cells ◽

Clinical Application ◽

Viral Vector ◽

Genetically Modified ◽

Beta Thalassemia ◽

Hematopoietic Stem

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Mucopolysaccharidosis Type I Phenotypically Corrected with Edited Hematopoietic Stem Cells: Instead of altering the IDUA gene, a protein was inserted in a repurposable place in the genome known as a “safe harbor locus”

American Journal of Medical Genetics Part A ◽

10.1002/ajmg.a.61223 ◽

2020 ◽

Vol 182 (2) ◽

pp. 275-276

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Type I ◽

Safe Harbor ◽

Mucopolysaccharidosis Type ◽

Mucopolysaccharidosis Type I ◽

Hematopoietic Stem ◽

Idua Gene

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Bone Morphogenetic Proteins Regulate the Developmental Program of Human Hematopoietic Stem Cells

Journal of Experimental Medicine ◽

10.1084/jem.189.7.1139 ◽

1999 ◽

Vol 189 (7) ◽

pp. 1139-1148 ◽

Cited By ~ 270

Author(s):

Mickie Bhatia ◽

Dominique Bonnet ◽

Dongmei Wu ◽

Barbara Murdoch ◽

Jeff Wrana ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Bone Morphogenetic Proteins ◽

Transforming Growth Factor ◽

Critical Role ◽

Type I ◽

Hematopoietic Tissue ◽

Hematopoietic Stem ◽

Proliferation And Differentiation

The identification of molecules that regulate human hematopoietic stem cells has focused mainly on cytokines, of which very few are known to act directly on stem cells. Recent studies in lower organisms and the mouse have suggested that bone morphogenetic proteins (BMPs) may play a critical role in the specification of hematopoietic tissue from the mesodermal germ layer. Here we report that BMPs regulate the proliferation and differentiation of highly purified primitive human hematopoietic cells from adult and neonatal sources. Populations of rare CD34+CD38−Lin− stem cells were isolated from human hematopoietic tissue and were found to express the BMP type I receptors activin-like kinase (ALK)-3 and ALK-6, and their downstream transducers SMAD-1, -4, and -5. Treatment of isolated stem cell populations with soluble BMP-2, -4, and -7 induced dose-dependent changes in proliferation, clonogenicity, cell surface phenotype, and multilineage repopulation capacity after transplantation in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Similar to transforming growth factor β, treatment of purified cells with BMP-2 or -7 at high concentrations inhibited proliferation yet maintained the primitive CD34+CD38− phenotype and repopulation capacity. In contrast, low concentrations of BMP-4 induced proliferation and differentiation of CD34+ CD38−Lin− cells, whereas at higher concentrations BMP-4 extended the length of time that repopulation capacity could be maintained in ex vivo culture, indicating a direct effect on stem cell survival. The discovery that BMPs are capable of regulating repopulating cells provides a new pathway for controlling human stem cell development and a powerful model system for studying the biological mechanism of BMP action using primary human cells.

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