Stem cell therapy for treatment of central nervous system pathology in α-mannosidosis guinea pigs.

Abstract

Lysosomal storage disorders (LSD) are a group of heritable genetic diseases resulting from a deficiency of one or more lysosomal enzpe activities, with broad pathological consequences. One of the most difficult aspects of these diseases to treat is central nervous system (CNS) pathology. Numerous strategies are being pursued in order to develop effective therapies for CNS pathology in LSD. One of these strategies involves the implantation of stem cells for the in vivo secretion of deficient enzyme in the brain, to be taken up by host cells, to therapeutic effect. α-Mannosidosis is a LSD resulting from a functional deficiency of lysosomal α-mannosidase. This deficiency results in the accumulation of various oligosaccharides in the lysosomes of affected individuals, to cause progressive neurological degeneration and other somatic pathology. We have a guinea pig model of this disease that closely models human α-mannosidosis. Although enzyme replacement therapy has shown great promise for treatment of somatic pathology in α-mannosidosis guinea pigs, it is not effective for treatment of brain pathology. Thus, this disease model was chosen as an appropriate disorder for the evaluation of intra-cranial stem cell implantation as a therapeutic approach. α-Mannosidosis guinea pigs display significant neurological abnormalities as part of the course of their disease. We postulated that the development of tests to quantitate the loss of neurological function underlying these characteristics would be useful for evaluation of therapies in this model. The first aim of this study was thus to establish such tests. The Morris water maze has been used to evaluate therapies for neurological disease in mouse models of LSD, and its use in guinea pigs has more recently been described for analysis of memory and learning difficulties arising from prenatal ethanol exposure. A pilot study was first carried out to detetmine the feasibility of using this test to investigate cognitive deficits in α-mannosidosis guinea pigs. Following the observation that neurological pathology develops to a readily quantifiable extent by three months of age, a larger study was undertaken using naïve (not previously tested) three month old α-mannosidosis and normal guinea pigs, to provide a background against which cell implantation could later be evaluated. Previously, gait changes have been observed in α-mannosidosis guinea pigs (relative to normal animals) at two and three months of age (Dr. Kim Hemsley, unpublished observation). This thesis included further investigation of these changes. A previously developed general neurological examination protocol (originally established by Dr. Allison Crawley) was also further modified (in collaboration with Dr. Allison Crawley) and used to test all of the animals in this study, in order to chart the progression of pathology observed in α-mannosidosis. The establishment the Morris water maze along with further characterisation of gait changes and neurological abnormalalities further expands the existing battery of histological and biochemical tests available for the analysis of pathology in the α-mannosidosis guinea pig. The availability of such tests thus adds to the potential utility of this animal model to evaluate CNS treatment options in LSD. Another aim of this study was to construct an embryonic stem (ES) cell line for the over-expression of recombinant human lysosomal α-mannosidase (rhαM), and to evaluate the potential of this cell line for therapeutic, supra-physiological expression of α-mannosidase in the α-mannosidosis brain. With a view to achieving sustained over-expression of rhαM in mouse ES cells and their differentiated progeny, expression vectors were constructed before generating transfected mouse ES cell clones, and isolating and characterising these clones for rhaM expression and differentiation potential. Further characterisation of the highest expressing clone included a cross-correction experiment, which showed that enzyme produced by transfected cells was able to be endocytosed by α-mannosidase-deficient human skin fibroblasts and mediate a reduction in stored oligosaccharides. These results suggest possible therapeutic utility of mouse ES cell clones expressing rhaM for ES cell therapy in the α-mannosidosis guinea pig brain. Additionally, constructing this cell line using pluripotent mouse ES cells allows the flexibility to take advantage of future development of techniques for the manipulation and implantation of embryonic stem cells and their differentiated progeny. Future developments in this area should thus open up further avenues of investigation for stem cell therapies in the guinea pig model and other animal models of α-mannosidosis. The implantation of pluripotent ES cells into sites of neonatal or adult animals carries the risk of teratorna formation. In order to facilitate site-specific differentiation of implanted pluripotent cells based on local environment (in contrast to continued division and teratoma formation), implantation of low cell numbers has previously been utilised. Whilst characterisation of the transfected cell lines was ongoing, preliminary studies using this approach were carried out in order to develop the methods required for this therapy. This involved the implantation of untransfected differentiated mouse ES cells into the dentate gyrus of neonatal α-mannosidosis and normal (or heterozygous) guinea pig brains. Animals were sacrificed at various time points to determine survival and engraftment of implanted cells, as well as a group being taken through to 13 weeks post-implantation and tested with various behavioural tests. Surviving graft derived cells were detected only out to eight weeks post-surgery. No graft-derived cells were observed, however, in any of the animals at 13 weeks post implantation. Further studies suggested that the lack of cell survival may be due to an immune response against the implanted cells despite the immunosuppression of implanted animals, or perhaps be due to a sub-optimal phenotype of implanted cells. There was no effect on behavioural pathology in α-mannosidosis guinea pigs as a result of this treatment. Histological analysis confirmed that these animals had no surviving cells, and no apparent reduction in lysosomal storage in the hippocampus. Thus, further refinement of differentiation and implantation protocols would be required for the development of a potentially effective therapy in this model. The following review introduces the general field of LSD and specifically α-mannosidosis, beginning with the biology of the lysosome and the deficiencies that result in LSD. This review also discusses historical, current and future therapeutic approaches to treatment of LSD, including stem cell therapies.