Treatment of adult MPSI mouse brains with IDUA-expressing mesenchymal stem cells decreases GAG deposition and improves exploratory behavior.

BACKGROUND: Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity. METHODS: MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses. RESULTS: After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months. CONCLUSIONS: These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice.

Granulocyte-macrophage colony-stimulating factor gene based therapy for acute limb ischemia in a mouse model.

BACKGROUND: Granulocyte-colony-stimulating factor (GM-CSF) is a pleiotropic factor for hematopoiesis that stimulates myeloblasts, monoblasts and mobilization of bone marrow stem cells. Therefore, the GM-CSF gene is a potential candidate for vessel formation and tissue remodeling in the treatment of ischemic diseases. METHODS: A new mouse limb ischemia was established by surgery and gene transfer was performed by injection of 100 microg of a plasmid carrying GM-CSF. Muscle force and weight, histology, capillary density, circulating stem cells and monocytes were determined after 3-4 weeks. RESULTS: More than 60% of nontreated ischemic animals showed gangrene below the heel after 4 weeks, whereas the GM-CSF gene-treated animals showed only darkening of nails or toes. These animals demonstrated a full recovery of the affected muscles in terms of weight, force and muscle fiber structure, but the muscles of nontreated ischemic animals lost approximately 50% weight, 86% force and their regular structure. When the GM-CSF gene was injected into the contralateral limb, only partial loss was observed, demonstrating a distant effect of GM-CSF. The capillary density in the GM-CSF-treated group was 52% higher in relation to the nontreated group. Blood analysis by flow cytometry showed that the GM-CSF-treated group had 10-20% higher levels of circulating monocytes and Sca-1(+). CONCLUSIONS: We conclude that the direct administration of GM-CSF gene in limb ischemia had a strong therapeutic effect because it promoted the recovery of muscle mass, force and structure by mobilizing therapeutic cells and augmenting the number of vessels.

Hydrodynamics- and ultrasound-based transfection of heart with naked plasmid DNA.

A novel, efficient transfection method, based on ultrasound and hydrodynamics, has been developed to transfect heart tissue with plasmid DNA. An ultrasound probe was aimed at the heart of anesthetized rats for 30 sec, at an intensity of 1 MHz and 2 W/cm2. The aorta was clamped and a phosphate-buffered saline (PBS) solution containing pSV-LacZ was quickly injected into the left ventricle. Each animal was maintained in this condition for 20 sec, and then the clamp was opened and the needle was removed. Electrocardiography, performed after 4 weeks, showed mild or no sign of ischemia in all groups. Visual evaluation of heart tissue samples from rats that received 100 microg of pSV-LacZ in 100 microl had only a few blue cells, indicating transfection, and those that received only PBS had no blue cells. However, all heart tissue samples from rats transfected with 100 to 500 microg of pSV-LacZ in 200 microl, or with 200 to 500 microg of pSV-LacZ in 100 micro had many blue cells. The base and epicardium of the heart tissue samples had many more blue cells than did the rest of the samples. Histological results, based on staining with hematoxylin and eosin, showed similar results between control and transfected groups. Therefore, we concluded that gene delivery by plasmid vector in association with ultrasound and hydrodynamics was highly effective in transfecting rat heart.