Transgene Expression in Dogs After Liver-Directed Hydrodynamic Delivery of Sleeping Beauty Transposons Using Balloon Catheters.

The Sleeping Beauty transposon system has been extensively tested for integration of reporter and therapeutic genes in vitro and in vivo in mice. Dogs were used as a large animal model for human therapy and minimally invasive infusion of DNA solutions. DNA solutions were delivered into the entire liver or the left side of the liver using balloon catheters for temporary occlusion of venous outflow.

Role of transgene regulation in ex vivo lentiviral correction of artemis deficiency.

Artemis is a single-stranded endonuclease, deficiency of which results in a radiation-sensitive form of severe combined immunodeficiency (SCID-A) most effectively treated by allogeneic hematopoietic stem cell (HSC) transplantation and potentially treatable by administration of genetically corrected autologous HSCs. We previously reported cytotoxicity associated with Artemis overexpression and subsequently characterized the human Artemis promoter with the intention to provide Artemis expression that is nontoxic yet sufficient to support immunodevelopment. Here we compare the human Artemis promoter (APro) with the moderate-strength human phosphoglycerate kinase (PGK) promoter and the strong human elongation factor-1α (EF1α) promoter to regulate expression of Artemis after ex vivo lentiviral transduction of HSCs in a murine model of SCID-A.

High-dose enzyme replacement therapy in murine Hurler syndrome.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive disease that is systemic, including progressive neurodegeneration, mental retardation and death before the age of 10 years. MPS I results from deficiency of α-L-iduronidase (IDUA) in lysosomes and subsequent accumulation of glycosaminoglycans (GAG). Clinical enzyme replacement therapy (ERT) with intravenous laronidase reverses some aspects of MPS I disease (e.

Left ventricular failure produces profound lung remodeling and pulmonary hypertension in mice: heart failure causes severe lung disease.

Chronic left ventricular failure causes pulmonary congestion with increased lung weight and type 2 pulmonary hypertension. Understanding the molecular mechanisms for type 2 pulmonary hypertension and the development of novel treatments for this condition requires a robust experimental animal model and a good understanding of the nature of the resultant pulmonary remodeling. Here we demonstrate that chronic transverse aortic constriction causes massive pulmonary fibrosis and remodeling, as well as type 2 pulmonary hypertension, in mice.

Systemic correction of storage disease in MPS I NOD/SCID mice using the sleeping beauty transposon system.

The Sleeping Beauty (SB) transposon system is a nonviral vector that directs transgene integration into vertebrate genomes. We hydrodynamically delivered SB transposon plasmids encoding human alpha-L-iduronidase (hIDUA) at two DNA doses, with and without an SB transposase gene, to NOD.129(B6)-Prkdc(scid) IDUA(tm1Clk)/J mice.

Protection of mice from methotrexate toxicity by ex vivo transduction using lentivirus vectors expressing drug-resistant dihydrofolate reductase.

Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs).

Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses.

Background: The Sleeping Beauty (SB) transposon system is a non-viral vector system that can integrate precise sequences into chromosomes. We evaluated the SB transposon system as a tool for gene therapy of mucopolysaccharidosis (MPS) types I and VII.

Methods: We constructed SB transposon plasmids for high-level expression of human beta-glucuronidase (hGUSB) or alpha-L-iduronidase (hIDUA).

Methotrexate preconditioning allows sufficient engraftment to confer drug resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity.

Methotrexate (MTX) is an effective antitumor agent that has been demonstrated to be particularly useful in the treatment of hematopoietic neoplasms but causes substantial hematologic and gastrointestinal toxicity. We previously demonstrated that transplantation with transgenic marrow expressing drug-resistant dihydrofolate reductase (DHFR) into animals preconditioned by irradiation substantially protected recipient mice from the toxic side effects of methotrexate administration. Here we test the use of methotrexate itself as a preconditioning agent for engraftment of drug-resistant transgenic marrow, subsequently conferring drug resistance upon recipient animals.

Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human alpha-L-iduronidase gene.

Murine models of lysosomal storage diseases provide an opportunity to evaluate the potential for gene therapy to prevent systemic manifestations of the disease. To determine the potential for treatment of mucopolysaccharidosis type I using a gene delivery approach, a recombinant adeno-associated virus (AAV) vector, vTRCA1, transducing the human iduronidase (IDUA) gene was constructed and 1 x 10(10) particles were injected intravenously into 1-day-old Idua(-/-) mice. High levels of IDUA activity were present in the plasma of vTRCA1-treated animals that persisted for the 5-month duration of the study, with heart and lung of this group demonstrating the highest tissue levels of gene transfer and enzyme activity overall.

Biodistribution and toxicity studies of VSVG-pseudotyped lentiviral vector after intravenous administration in mice with the observation of in vivo transduction of bone marrow.

Lentiviral vectors can confer high levels of gene transfer and transgene expression in a variety of cell types. However, the biodistribution and toxicity after intravenous administration have not been reported. To address these issues of biodistribution and toxicity, an HIV-1-based vector, HR'cmvGFP, was administered to normal BALB/c mice by tail-vein injection.

Methotrexate exacerbates tumor progression in a murine model of chronic myeloid leukemia.

Expression of drug-resistant forms of dihydrofolate reductase (DHFR) in hematopoietic cells confers substantial resistance of animals to antifolate administration. In this study, we tested whether the chemoprotection conferred by expression of the tyrosine-22 variant DHFR could be used for more effective therapy of the 32Dp210 murine model of chronic myeloid leukemia (CML). 32Dp210 tumor cells were found to be sensitive to methotrexate (MTX) in vitro, whereas cells expressing the tyrosine-22 DHFR gene were protected from MTX at up to micromolar concentrations.