Mucopolysaccharidosis type IIIB and GalNAc Transferase double knockout mice and additional studies in gene therapy and animal models to assess pathogenesis and therapy

摘要

The Mucopolysaccharidoses (MPSs) are a class of lysosomal storage diseases characterized by lysosomal accumulation of glycosaminoglycan (GAG). Understanding the pathogenesis of the neuropathic forms of MPS and improving treatment options for these disorders are major areas of research in the laboratory of Dr. Ellinwood. The neuropathic MPSs include MPS I, II, III (A-D), and VII, all of which have primary storage of heparan sulfate (HS), and a secondary accumulation of GM2 and GM3 gangliosides. Clinically patients with the severe forms of these disorders have central nervous system defects. Mucopolysaccharidosis III, also known as Sanfilippo syndrome, has four subtypes designated as A-D. These subtypes are due to deficiency of four different and distinct lysosomal enzymes. Mucopolysaccharidosis type IIIB (MPS IIIB) results from an inherited deficiency of N-acetyl-α-D-glucosaminidase (Naglu) activity which leads to lysosomal accumulation of HS as a primary storage substrate, and secondary accumulation GM2 and GM3 gangliosides. Mucopolysaccharidosis type IIIC is characterized by deficiency of the HSGNAT gene product, a lysosomal membrane enzyme. No treatment exists for MPS IIIB, but potential treatments could involve a combination of gene therapy (GT) and hematopoietic stem cell therapy (HSCT). To this end we construction a lentiviral vector containing hNaglu-cDNA under the control of the hPGK promoter. This vector will be used in the future for ex vivo transduction of HSCT to be followed by transplantation into lethally irradiated MPS IIIB mice. While work proceeds on development of therapy there is still a critical need for a better understanding of the disease process in MPS III. Use of mouse models could aid this process very effectively. To this end we used a double knockout (DKO) approach to test a theory on an approach to improve treatment of MPS IIIB. This approach had as its goal a better understanding of the pathogenesis of MPS IIIB, by targeting, through substrate reduction, ganglioside accumulation at the genetic level. This DKO approach entailed breeding mice deficient in both Naglu (the causative gene in MPS IIIB) and GalNAcT activity. The latter is the enzyme required for synthesis of GM2 and other complex gangliosides. If the DKO mice showed improvement relative to the single KO MPS IIIB this would provide strong proof or principle to move forward with assessment of substrate deprivation therapy (SDT) targeting gangliosides, for which drugs are available. Contrary to our expectation and to double knockout (DKO) studies where GalNAcT was knocked out in combination with other LSDs, our DKO mice showed a drastically shortened lifespan (24.5 ± 1.4 weeks, versus 50.5 ± 0.9 wks (MPS IIIB), and 38.6 ± 1.2wks (GalNAcT)). To confirm that HS storage was the primary element resulting in the accelerated disease in our DKO mice, and not a locus tightly linked to the Naglu gene in the embryonic cell like used to generate these mice, we replicated our study with MPS IIIA mice, and found a virtually identical result (27.5 ± 1.8 weeks, versus 53.8 ± 1.6 wks.). All DKO mice showed motor signs of hind limb ataxia and hyper-extension, which were not seen in single KO or normal mice. At approximately 5 months of age the MPS IIIB DKO showed a unique pattern of vacuolization and nerve fiber degeneration in the corpus callosum as well as the mild intracytoplasmic vacuolation of neurons and glia typical of MPS IIIB affected mice at this age. We analyzed motor performance on a rocking Rotor-rod beginning at 3 months of age. The DKO IIIA and DKO IIIB mice showed impaired performance and were statistically different from all parental lines. In particular, the DKO MPS IIIB mice were significantly different from the parent MPS IIIB strains at 3, 5, and 6 months (p≤0.0245). In conclusion we identified an accelerated form of MPS IIIB within a DKO model system which showed white matter changes, with attendant performance deficits and a drastically shortened lifespan. This was in stark contrast to our expectations of a salutary response to the elimination of GM2. While contrary to our expectations, the accelerated pathology and clinical signs seen in these mice represent an improved system with which to study the MPS IIIB mice and their response to therapies. It may also be a useful model to investigate white matter pathogenesis as well as the role of complex gangliosides in animals with MPS III. Another KO technology which could accelerate MPS III disease research involves a conditional knockout strategy applied to MPS IIIC. The HSGNAT gene product which is deficient in MPS IIIC is a membrane bound protein which is not subject to cross-correction from other cells. By using a Cre-loxP recombination system to conditionally knockout HSGNAT gene in MPS IIIC mice, studies could focus on the specific aspects of the disease, both temporally, and by tissue and cell type. To this end we also constructed a knockout plasmid vector, pBY49 under the control of the PGK promoter, which targeted the HSGNAT mouse locus. Together these studies have potential to contribute important knowledge and resources to MPS targeted research.