Clonal Dominance With Retroviral Vector Insertions Near the ANGPT1 and ANGPT2 Genes in a Human Xenotransplant Mouse Model.

Insertional leukemogenesis represents the major risk factor of hematopoietic stem cell (HSC) based gene therapy utilizing integrating viral vectors. To develop a pre-clinical model for the evaluation of vector-related genotoxicity directly in the relevant human target cells, cord blood CD34(+) HSCs were transplanted into immunodeficient NOD.SCID.IL2rg(-/-) (NSG) mice after transduction with an LTR-driven gammaretroviral vector (GV). Furthermore, we specifically investigated the effect of prolonged in vitro culture in the presence of cytokines recently described to promote HSC expansion or maintenance. Clonality of human hematopoiesis in NSG mice was assessed by high throughput insertion site analyses and validated by insertion site-specific PCR depicting a GV typical integration profile with insertion sites resembling to 25% those of clinical studies. No overrepresentation of integrations in the vicinity of cancer-related genes was observed, however, several dominant clones were identified including two clones harboring integrations in the ANGPT1 and near the ANGPT2 genes associated with deregulated ANGPT1- and ANGPT2-mRNA levels. While these data underscore the potential value of the NSG model, our studies also identified short-comings such as overall low numbers of engrafted HSCs, limited in vivo observation time, and the challenges of in-depth insertion site analyses by low contribution of gene modified hematopoiesis.

Lentiviral MGMT(P140K)-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter.

Notwithstanding recent successes, insertional mutagenesis as well as silencing and variegation of transgene expression still represent considerable obstacles to hematopoietic gene therapy. This also applies to O(6)-methylguanine DNA methyltransferase (MGMT)-mediated myeloprotection, a concept recently proven clinically effective in the context of glioblastoma therapy. To improve on this situation we here evaluate a SIN-lentiviral vector expressing the MGMT(P140K)-cDNA from a combined A2UCOE/PGK-promoter. In a murine in vivo chemoselection model the A2UCOE.PGK.MGMT construct allowed for significant myeloprotection as well as robust and stable selection of transgenic hematopoietic cells. In contrast, only transient enrichment and severe myelotoxicity was observed for a PGK.MGMT control vector. Selection of A2UCOE.PGK.MGMT-transduced myeloid and lymphoid mature and progenitor cells was demonstrated in the peripheral blood, bone marrow, spleen, and thymus. Unlike the PGK and SFFV promoters used as controls, the A2UCOE.PGK promoter allowed for sustained vector copy number-related transgene expression throughout the experiment indicating an increased resistance to silencing, which was further confirmed by CpG methylation studies of the PGK promoter. Thus, our data support a potential role of the A2UCOE.PGK.MGMT-vector in future MGMT-based myeloprotection and chemoselection strategies, and underlines the suitability of the A2UCOE element to stabilize lentiviral transgene expression in hematopoietic gene therapy.

Efficiency and safety of O6-methylguanine DNA methyltransferase (MGMT(P140K))-mediated in vivo selection in a humanized mouse model.

Efficient O6-methylguanine DNA methyltransferase (MGMT(P140K))-mediated myeloprotection and in vivo selection have been demonstrated in numerous animal models and most recently in a phase I clinical study in glioblastoma patients. However, this strategy may augment the genotoxic risk of integrating vectors because of chemotherapy-induced DNA damage and the proliferative stress exerted during the in vivo selection. Thus, to improve the safety of the procedure, we evaluated a self-inactivating lentiviral MGMT(P140K) vector for transduction of human cord blood-derived CD34⁺ cells followed by transplantation of the cells into NOD/LtSz-scid/Il2rγ⁻/⁻ mice. These experiments demonstrated significant and stable enrichment of MGMT(P140K) transgenic human cells in the murine peripheral blood and bone marrow. Clonal inventory analysis utilizing linear amplification-mediated polymerase chain reaction and high-throughput sequencing revealed a characteristic lentiviral integration profile. Among the bone marrow insertions retrieved, we observed considerable overlap to previous MGMT(P140K) preclinical models or the clinical study. However, no significant differences between our chemotherapy-treated and nontreated cohorts were observed. This also hold true when specific cancer gene databases and a functional annotation of hit genes by the Panther Database with respect to molecular function, biological process, or cellular component were assessed. Thus, in summary, our data demonstrate efficient and long-term in vivo selection without overt hematological abnormalities using the lentiviral MGMT(P140K) vector. Furthermore, the study introduces humanized mouse models as a novel tool for the pre-clinical assessment of human gene therapy related toxicity.

Myeloprotection by cytidine deaminase gene transfer in antileukemic therapy.

Gene transfer of drug resistance (CTX-R) genes can be used to protect the hematopoietic system from the toxicity of anticancer chemotherapy and this concept recently has been proven by overexpression of a mutant O(6)-methylguaninemethyltransferase in the hematopoietic system of glioblastoma patients treated with temozolomide. Given its protection capacity against such relevant drugs as cytosine arabinoside (ara-C), gemcitabine, decitabine, or azacytidine and the highly hematopoiesis-specific toxicity profile of several of these agents, cytidine deaminase (CDD) represents another interesting candidate CTX-R gene and our group recently has established the myeloprotective capacity of CDD gene transfer in a number of murine transplant studies. Clinically, CDD overexpression appears particularly suited to optimize treatment strategies for acute leukemias and myelodysplasias given the efficacy of ara-C (and to a lesser degree decitabine and azacytidine) in these disease entities. This article will review the current state of the art with regard to CDD gene transfer and point out potential scenarios for a clinical application of this strategy. In addition, risks and potential side effects associated with this approach as well as strategies to overcome these problems will be highlighted.