Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

<p>Abstract</p> <p>Background</p> <p>Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of <it>in vivo </it>expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells.</p> <p>Results</p> <p>We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus.</p> <p>Conclusions</p> <p>These findings indicate that the p53-responsive pCLPG retrovirus did offer expression <it>in vivo </it>and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.</p

A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites

Insertional mutagens such as viruses and transposons are a useful tool for performing forward genetic screens in mice to discover cancer genes. These screens are most effective when performed using hundreds of mice, however until recently a major limitation to performing screens on this scale has been the cost effective isolation and sequencing of insertion sites. Here we present a method for the high-throughput isolation of insertion sites using a highly efficient splinkerette-PCR method coupled with capillary or 454 sequencing. This protocol includes a description of the procedure for DNA isolation, DNA digestion, linker or splinkerette ligation, primary and secondary PCR amplification, and sequencing. This method, which takes about 1 week to perform, has allowed us to isolate hundreds of thousands of insertion sites from mouse tumours and, unlike other methods, has been specifically optimised for the isolation of insertion sites generated with the murine leukaemia virus (MuLV), and can easily be performed in 96 well plate format for the efficient multiplex isolation of insertion sites

Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells

Background: Hematopoietic stem cells (HSC), in particular mobilized peripheral blood stem cells, represent an attractive target for cell and gene therapy. Efficient gene delivery into these target cells without compromising self-renewal and multipotency is crucial for the success of gene therapy. We investigated factors involved in the ex vivo transduction of CD34 + HSCs in order to develop a clinically relevant transduction protocol for gene delivery. Specifically sought was a protocol that allows for efficient transduction with minimal ex vivo manipulation without serum or other reagents of animal origin. Methodology/Principal Findings: Using commercially available G-CSF mobilized peripheral blood (PB) CD34 + cells as the most clinically relevant target, we systematically examined factors including the use of serum, cytokine combinations, prestimulation time, multiplicity of infection (MOI), transduction duration and the use of spinoculation and/or retronectin. A self-inactivating lentiviral vector (SIN-LV) carrying enhanced green fluorescent protein (GFP) was used as the gene delivery vehicle. HSCs were monitored for transduction efficiency, surface marker expression and cellular function. We were able to demonstrate that efficient gene transduction can be achieved with minimal ex vivo manipulation while maintaining the cellular function of transduced HSCs without serum or other reagents of animal origin. Conclusions/Significance: This study helps to better define factors relevant towards developing a standard clinical protocol for the delivery of SIN-LV into CD34 + cells

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. Keywords: AML; RUNX1; CKMT1; cyclocreatine; arginine metabolismNational Cancer Institute (U.S.) (NIH 1R35 CA210030-01)Stand Up To CancerBridge ProjectNational Cancer Institute (U.S.) (David H. Koch Institute for Integrative Cancer Research at MIT. Grant P30-CA14051