Repopulation of B-lymphocytes with restricted gene expression using haematopoietic stem cells engineered with lentiviral vectors

Abstract The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder caused by mutations in the WASP gene. WASP is an effector protein in the actin polymerization pathway. Characterized by immunodeficiency, micro-thrombocytopenia and eczema, untreated WAS patients may also suffer hemorrhages, recurring infections and potential malignancies. WAS can be cured by bone marrow (BM) transplant but because many patients lack a suitable donor, stem cell-targeted gene transfer is being developed as an alternative therapeutic approach. We have demonstrated correction of the T-cell proliferation defect in Wasp− mice using MSCV oncoretroviral vectors (Blood102:3108, 2003). However, our competitive repopulation studies in mice with both wild-type (WT) and gene-corrected Wasp− BM, demonstrated only a modest selective advantage for gene modified lymphocytes. Correction of the lymphocytopenia was observed only in animals having high proportions of transduced cells. In addition, variability in the level of gene expression among gene-corrected cells was associated with only partial correction of the T-cell cytokine secretory defects. Thus, the efficiency of stem cell-targeted gene transfer as well as the level and consistency of gene expression are likely to be key factors that determine success in any clinical application of gene transfer for the treatment of WAS. In attempting to improve vector design, we have used lentiviral vectors because of their greater efficiency in transducing repopulating stem cells as we recently demonstrated in a non-human primate model (Blood103:4062, 2004). The WASP gene is regulated by two promoter regions. The proximal promoter lies immediately adjacent to the translation start site with the distal promoter found 6 kb upstream, followed by an alternate first exon. We have developed a series of third generation, self-inactivating lentiviral vectors containing the MSCV, proximal or distal WASP promoters driving GFP in the reverse transcriptional orientation. The WASP promoters were active in lymphocytes but not HeLa cells in vitro. However, expression was low in lymphocytes and granulocytes in mice transplanted with genetically modified stem cells. In an effort to abrogate any position-effect variegation and enhance expression, we generated a new series of vectors with the transcriptional unit in the forward orientation that also contained the woodchuck post-transcriptional regulatory element, the chicken beta-globin 5′ DNase I hypersensitive site 4 (I) and human beta-interferon scaffold attachment region (S) insulator elements. Either GFP or murine Wasp cDNA were included for expression analysis. Despite the complexity of the SI-containing vectors, titers of concentrated preparations ranged from 9x106 TU/ml to 5x107 TU/ml and enabled transduction of both cell lines and murine hematopoietic stem cells. Low-level GFP expression from the proximal promoter was detected in HeLa cells with higher expression found in lymphocytes (NALM6 and Jurkat cells). Expression of GFP under the control of the proximal WASP promoter was detected in vivo in multiple hematopoietic lineages in mice transplanted with transduced stem cells. Future efforts will focus on further characterization and optimization of vector design with the goal of achieving consistent, high level expression.

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Identification of the Regulatory Elements and Target Genes of Megakaryopoietic Transcription Factor MEF2C

Thrombosis and Haemostasis ◽

10.1055/s-0039-1678694 ◽

2019 ◽

Vol 119 (05) ◽

pp. 716-725 ◽

Cited By ~ 2

Author(s):

Xianguo Kong ◽

Lin Ma ◽

Edward Chen ◽

Chad Shaw ◽

Leonard Edelstein

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Genetic Variation ◽

Transcription Factors ◽

Cell Fate ◽

Target Genes ◽

Regulatory Elements ◽

Haematopoietic Stem Cells ◽

Platelet Production ◽

Induced Pluripotent

AbstractMegakaryopoiesis produces specialized haematopoietic stem cells in the bone marrow that give rise to megakaryocytes which ultimately produce platelets. Defects in megakaryopoiesis can result in altered platelet counts and physiology, leading to dysfunctional haemostasis and thrombosis. Additionally, dysregulated megakaryopoiesis is also associated with myeloid pathologies. Transcription factors play critical roles in cell differentiation by regulating the temporal and spatial patterns of gene expression which ultimately decide cell fate. Several transcription factors have been described as regulating megakaryopoiesis including myocyte enhancer factor 2C (MEF2C); however, the genes regulated by MEF2C that influence megakaryopoiesis have not been reported. Using chromatin immunoprecipitation-sequencing and Gene Ontology data we identified five candidate genes that are bound by MEF2C and regulate megakaryopoiesis: MOV10, AGO3, HDAC1, RBBP5 and WASF2. To study expression of these genes, we silenced MEF2C gene expression in the Meg01 megakaryocytic cell line and in induced pluripotent stem cells by CRISPR/Cas9 editing. We also knocked down MEF2C expression in cord blood-derived haematopoietic stem cells by siRNA. We found that absent or reduced MEF2C expression resulted in defects in megakaryocytic differentiation and reduced levels of the candidate target genes. Luciferase assays confirmed that genomic sequences within the target genes are regulated by MEF2C levels. Finally, we demonstrate that small deletions linked to a platelet count-associated single nucleotide polymorphism alter transcriptional activity, suggesting a mechanism by which genetic variation in MEF2C alters platelet production. These data help elucidate the mechanism behind MEF2C regulation of megakaryopoiesis and genetic variation driving platelet production.

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MicroRNA Expression Profiling in Sorted AML Subpopulations: A Possible Role for miR-155/BIC in Stem Cell Maintenance and Leukemogenesis.

Blood ◽

10.1182/blood.v106.11.466.466 ◽

2005 ◽

Vol 106 (11) ◽

pp. 466-466 ◽

Cited By ~ 2

Author(s):

Eric R. Lechman ◽

Kristin J. Hope ◽

Fernando J. Suarez Saiz ◽

Katsuto Takenaka ◽

Carlo M. Croce ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Stem Cell ◽

B Cell ◽

Lentiviral Vectors ◽

Array Analysis ◽

Hematopoietic Stem ◽

Cell State ◽

Mirna Array ◽

Stem Cell State

Abstract MicroRNAs (miRNAs) are a new class of non-coding small RNAs that negatively regulate the expression of protein-encoding genes. Mature miRNAs are excised sequentially from primary miRNA (pri-miRNA) foldback precursor transcripts, and regulate gene expression at the post-transcriptional level. miRNAs functionally suppress gene expression by either inhibition of protein synthesis or by direct cleavage of the target mRNA. miRNA expression is tissue and developmental stage restricted, suggesting important roles in tissue specification and/or cell lineage determination. miRNAs are implicated in the regulation of diverse processes including cell growth control, apoptosis, fat metabolism and insulin secretion, and may be involved in the maintenance of the embryonic stem cell state. Several recent lines of evidence suggest a role for miRNAs in hematological malignancies. Many characterized miRNAs are located at fragile sites, minimal loss of heterozygosity regions, minimal regions of amplification or common breakpoint regions in human cancers. For example, chromosomal translocation t(8;17) in an aggressive B-cell leukemia results in fusion of miR-142 precursor and a truncated MYC gene. Furthermore, both miR-15 and miR-16 are located within a 30 kb deletion in CLL, and in most cases of this cancer both genes are deleted or underexpressed. In addition, mice transplanted with hematopoietic stem cells (HSC) overexpressing both c-Myc and the miR-17–92 polycistron developed cancers earlier with a more aggressive nature when compared to lymphomas generated by c-myc alone. To address the role of miRNAs in the regulation and maintenance of the hematopoietic stem cell state and leukemogenesis, we sorted 6 primary AML patient samples into 4 populations based on the expression of CD34/CD38 and performed miRNA array analysis. We identified a subset of miRNAs whose expression profile could discriminate the CD34+/CD38- fractions from more mature populations. In particular, BIC/miR-155 was found to be over-expressed in leukemic stem cells (LSC). Validation by qRT-PCR revealed this expression pattern in 5 of the 6 sorted AML samples. Furthermore, within umbilical cord blood (CB) cells, BIC/miR-155 is more highly expressed in the primitive CD34+38- fraction as compared to mature sub-fractions as assessed by Affymetrix microarray. miRNA array analysis also revealed elevated levels of miR-155 in bulk primary AMLs as compared to normal BM. Intriguingly, BIC/miR-155 was first identified as a common retroviral insertion site in avian leucosis virus induced B cell lymphomas, and BIC/miR-155 overexpression has been observed in all subtypes of Hodgkin’s lymphoma. To test the hypothesis that miR-155 is important in LSC/HSC function, we designed lentiviral vectors for RNAi mediated knockdown of BIC/miR-155. Knockdown of BIC/miR-155 within a novel CD34+ leukemic cell line resulted in a loss of CD34 expression and reduced proliferative potential. Additionally, knockdown within CB led to alterations in colony forming capacity. Additionally, we have recently generated lentiviral vectors for the enforced overexpression of BIC/miR-155. In vivo studies to investigate the effects of BIC/miR-155 over-expression and knockdown are ongoing and will be discussed.

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