Cell cycle status of CD34(+) hemopoietic stem cells determines lentiviral integration in actively transcribed and development-related genes.

Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory toward safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34(+) cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that ~10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis.

Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation.

Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudotyped lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6×10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy.

The new self-inactivating lentiviral vector for thalassemia gene therapy combining two HPFH activating elements corrects human thalassemic hematopoietic stem cells.

To address how low titer, variable expression, and gene silencing affect gene therapy vectors for hemoglobinopathies, in a previous study we successfully used the HPFH (hereditary persistence of fetal hemoglobin)-2 enhancer in a series of oncoretroviral vectors. On the basis of these data, we generated a novel insulated self-inactivating (SIN) lentiviral vector, termed GGHI, carrying the (A)γ-globin gene with the -117 HPFH point mutation and the HPFH-2 enhancer and exhibiting a pancellular pattern of (A)γ-globin gene expression in MEL-585 clones. To assess the eventual clinical feasibility of this vector, GGHI was tested on CD34(+) hematopoietic stem cells from nonmobilized peripheral blood or bone marrow from 20 patients with ß-thalassemia. Our results show that GGHI increased the production of γ-globin by 32.9% as measured by high-performance liquid chromatography (p=0.001), with a mean vector copy number per cell of 1.1 and a mean transduction efficiency of 40.3%. Transduced populations also exhibited a lower rate of apoptosis and resulted in improvement of erythropoiesis with a higher percentage of orthochromatic erythroblasts. This is the first report of a locus control region (LCR)-free SIN insulated lentiviral vector that can be used to efficiently produce the anticipated therapeutic levels of γ-globin protein in the erythroid progeny of primary human thalassemic hematopoietic stem cells in vitro.

The essential requirement for Runx1 in the development of the sternum.

Runx1 is highly expressed in chondroprogenitor and osteoprogenitor cells and in vitro experiments suggest that Runx1 is important in the early stages of osteoblast and chondrocyte differentiation. However, because Runx1 knockout mice are early embryonic lethal due to failure of hematopoiesis, the role of Runx1 in skeletogenesis remains unclear. We studied the role of Runx1 in skeletal development using a Runx1 reversible knockout mouse model. By crossing with Tie2-Cre deletor mice, Runx1 expression was selectively rescued in the endothelial and hematopoietic systems but not in the skeleton. Although Runx1(Re/Re) embryos survived until birth and had a generally normal skeleton, the development of mineralization in the sternum and some skull elements was significantly disrupted. In contrast to wild-type embryos, the sternum of E17.5 Runx1(Re/Re) embryos showed high levels of Sox-9 and collagen type II expression and lack of development of hypertrophic chondrocytes. In situ hybridization analysis demonstrated that, in contrast to the vertebrae and long bones, the sternum of wild-type embryos expresses high levels of Runx1, but not Runx2, the master regulator of skeletogenesis. Thus, although Runx1 is not essential for major skeletal development, it does play an essential role in the development of the sternum and some skull elements.

Restoration of Runx1 expression in the Tie2 cell compartment rescues definitive hematopoietic stem cells and extends life of Runx1 knockout animals until birth.

Mice deficient in the runt homology domain transcription factor Runx1/AML1 fail to generate functional clonogenic hematopoietic cells and die in utero by embryonic day 12.5. We previously generated Runx1 reversible knockout mice, in which the Runx1 locus can be restored by Cre-mediated recombination. We show here that selective restoration of the Runx1 locus in the Tie2 cell compartment rescues clonogenic hematopoietic progenitors in early Runx1-null embryos and rescues lymphoid and myeloid lineages during fetal development. Furthermore, fetal liver cells isolated from reactivated Runx1 embryos are capable of long-term multilineage lymphomyeloid reconstitution of adult irradiated recipients, demonstrating the rescue of definitive hematopoietic stem cells. However, this rescue of the definitive hematopoietic hierarchy is not sufficient to rescue the viability of animals beyond birth, pointing to an essential role for Runx1 in other vital developmental processes.