Insertional Oncogenesis after Retroviral Gene Transfer in Hematopoietic Stem Cells

__Abstract__ This thesis is focuses on the insertional oncogenesis brought about by gamma-retroviral vector insertions. In hematopoietic gene therapy, gamma-retroviral vectors can be used to deliver therapeutic transgenes into target cells of patients with monogenic disorders, which has been successfully shown in three human diseases. The addition of the therapeutic gene to the host cell genome has the opportunity to cure the disorder. The mechanism that allows insertion of the transgene in the host cell genome can unfortunately also introduce deregulation of the genes surrounding the insertion site, sometimes with leukemia as a result. In the studies described here, the insertion profiles in mouse or human hematopoietic cells were analyzed and the frequency of oncogenic mutations was determined. In addition, software that allows automated determination and annotation of retroviral insertion sites was developed

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and I healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+)T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation

Avoiding cytotoxicity of transposases by dose-controlled mRNA delivery

The Sleeping Beauty (SB) transposase and its newly developed hyperactive variant, SB100X, are of increasing interest for genome modification in experimental models and gene therapy. The potential cytotoxicity of transposases requires careful assessment, considering that residual integration events of transposase expression vectors delivered by physicochemical transfection or episomal retroviral vectors may lead to permanent transposase expression and resulting uncontrollable transposition. Comparing retrovirus-based approaches for delivery of mRNA, episomal DNA or integrating DNA, we found that conventional SB transposase, SB100X and a newly developed codon-optimized SB100Xo may trigger premitotic arrest and apoptosis. Cell stress induced by continued SB overexpression was self-limiting due to the induction of cell death, which occurred even in the absence of a co-transfected transposable element. The cytotoxic effects of SB transposase were strictly dose dependent and heralded by induction of p53 and c-Jun. Inactivating mutations in SB’s catalytic domain could not abrogate cytotoxicity, suggesting a mechanism independent of DNA cleavage activity. An improved approach of retrovirus particle-mediated mRNA transfer allowed transient and dose-controlled expression of SB100X, supported efficient transposition and prevented cytotoxicity. Transposase-mediated gene transfer can thus be tuned to maintain high efficiency in the absence of overt cell damage

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wildtype genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model

Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development

A strict balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is required in orde

Comprehensive Investigation of Parameter Choice in Viral Integration Site Analysis and Its Effects on the Gene Annotations Produced

Introducing therapeutic genes into hematopoietic stem cells using retroviral vector-mediated gene transfer is an effective treatment for monogenic diseases. The risks of therapeutic gene integration include aberrant expression of a neighboring gene, resulting in oncogenesis at low frequencies (10(-7)-10(-6)/transduced cell). Mechanisms governing insertional mutagenesis are the subject of intensive ongoing studies that produce large amounts of sequencing data representing genomic regions flanking viral integration sites (IS). Validating and analyzing these data require automated bioinformatics applications. The exact methods used vary between applications, based on the requirements and preferences of the designer. The parameters used to analyze sequence data are capable of shaping the resulting integration site annotations, but a comprehensive examination of these effects is lacking. Here we present a web-based tool for integration site analysis, called Methods for Analyzing ViRal Integration Collections (MAVRIC), and use its highly customizable interface to look at how IS annotations can vary based on the analysis parameters. We used the integration data of the previously published adenosine deaminase severe combined immunodeficiency (ADA-SCID) gene therapy trials for evaluation of MAVRIC. The output illustrates how MAVRIC allows for direct multiparameter comparison of integration patterns. Careful analysis of the SCID data and reanalyses using different parameters for trimming, alignment, and repeat masking revealed the degree of variation that can be expected to arise due to changes in these parameters. We observed mainly small differences in annotation, with the largest effects caused by masking repeat sequences and by changing the size of the window around the IS

Multipotent Stromal Cells Induce Human Regulatory T Cells Through a Novel Pathway Involving Skewing of Monocytes Toward Anti-inflammatory Macrophages

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4(+)CD25(hi)FoxP3(+) T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-1 mediates induction of Tregs. TGF-1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC. Stem Cells2013;31:1980-199

Stimulation of id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells.

Background— Bone morphogenetic proteins (BMPs) are multifunctional proteins that regulate the proliferation, differentiation, and migration of a large variety of cell types. Like other members of the transforming growth factor-ß family, BMPs elicit their cellular effects through activating specific combinations of type I and type II serine/threonine kinase receptors and their downstream effector proteins, which are termed Smads. In the present study, we investigated BMP receptor/Smad expression and signaling in endothelial cells (ECs) and examined the effects of BMP on EC behavior. Methods and Results— Immunohistochemical analysis of tissue sections of human colon and mouse heart and aorta showed that BMP receptors are expressed in ECs in vivo. Bovine aortic ECs and mouse embryonic ECs were found to express BMP receptors and their Smads. BMP receptor activation induced the phosphorylation of specific Smad proteins and promoted EC migration and tube formation. Id1 was identified as a BMP/Smad target in ECs. Ectopic expression of Id1 mimicked BMP-induced effects. Importantly, specific interference with Id1 expression blocked BMP-induced EC migration. Conclusions— The BMP/Smad pathway can potently activate the endothelium. Id1 expression is strongly induced by BMP in ECs. Ectopic expression of Id1 induces EC migration and tube formation. Moreover, Id1 played a critical role in mediating BMP-induced EC migration

一般社団法人神緑会事業報告

It is a longstanding question which bone marrow-derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34+CD1a- and CD34+CD1a+ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34+CD1a- thymocytes but not in CD34+CD1a+ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34+CD1a- subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34+CD1a- thymocytes toward the T-cell lineage, as shown by T-cell receptor δ gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34+CD1a+ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34+CD1a- and CD34+CD1a+ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity