1. ¹Scott-Ritchey Research Center/College Vet Med, Auburn University, Auburn, AL
2. ²Gene Therapy Center, University of North Carolina, Chapel Hill, NC

Abstract

Deficiency of lysosomal |[beta]|-galactosidase causes G_M1 gangliosidosis, an inherited, progressive neurological disorder in which G_M1 ganglioside accumulates in all tissues, including thymus, liver and brain. In addition to abnormal neuronal histology and function, gangliosidosis pathology involves a significant inflammatory component and therefore is similar to other neurodegenerative disorders such as Alzheimer and Parkinson disease. G_M1 gangliosidosis occurs in humans, mice and domesticated animals, including cats, and the feline gangliosidosis model has been used previously by our laboratory to test several therapeutic strategies. In this study, stem cell transplantation and gene therapy have been evaluated separately and in combination as therapeutic approaches for feline G_M1 gangliosidosis.

A lentiviral vector (TK291) was constructed in which a human |[beta]|-galactosidase cDNA is driven by a modified long terminal repeat promoter. Pseudotyped with the vesicular stomatitis virus G protein, TK291 transduced at least 75% of fibroblasts and mesenchymal stem cells (MSC) isolated from normal and G_M1 gangliosidosis-affected cats after one round of infection. Both wild-type and diseased cells expressed above normal levels of |[beta]|-galactosidase activity after transduction and were evaluated for therapeutic potential in cross-correction assays in vitro. Also, TK291 was injected directly into the cerebral cortex of diseased cats, which subsequently expressed elevated levels of |[beta]|-galactosidase.

In addition, bone marrow MSC were evaluated for therapeutic potential from at least 2 perspectives. First, mesenchymal stem cells were tested as |[beta]|-galactosidase delivery vehicles by assessing enzyme transfer to recipient cells in vitro. MSC were evaluated both before and after transduction with lentiviral vector TK291 and found to deliver |[beta]|-galactosidase to recipient cells through conditioned media. Additionally, mesenchymal stem cells were tested by in vitro differentiation assays for their potential to replace diseased neurons in G_M1 gangliosidosis cats. After exposure to a neuronal induction cocktail for 24 hours, feline mesenchymal stem cells increased by up to 20-fold the expression of several neuronal markers, including neurofilament M, TrkA, tau and GAP43. Expression of the stem cell marker nestin decreased substantially after induction, as did non-neuronal markers such as osteopontin and glial fibrillary acidic protein. When transplanted into organotypic brain slice cultures from fetal cats, MSC migrated broadly throughout the slices and assumed morphologies typical of neuronal or glial cells.

Finally, a combination of stem cell and gene therapy was tested for therapeutic potential by intracortical injection of mesenchymal stem cells transduced by TK291. Transplanted cells survived for at least 6 weeks in immunosuppressed cats, where they migrated from the injection site and expressed |[beta]|-galactosidase activity for the duration of the experiment. These encouraging results provide the impetus for continued studies of stem cell and gene therapy for G_M1 gangliosidosis.