Mucopolysaccharidosis (MPS) IIIB is a neurodegenerative lysosomal storage disease (LSD) caused by deficient degradation of heparan sulfate. Clinically this manifests as cognitive decline, developmental regression, impaired mobility and ultimately premature death. There are currently no effective therapies. Due to the neurodegenerative nature of this disease, optimal CNS transduction is necessary for human trials. Several groups have demonstrated improvement of the mouse model using different adeno-associated viral (AAV) vectors. We have recently demonstrated that AAV8 has better brain gene delivery in MPS IIIB than wild type mice. A similar finding of altered brain delivery in Sly Syndrome compared to wild type mice has been published for AAV9. However, for translation to human trials, it is essential to identify a highly effective AAV capsid serotype which will deliver to cells in the requisite brain regions. More generally, for any treatment of human neurologic disease in which the central nervous system (CNS) is of substantially larger volume and is phylogenetically distant compared to our current mouse models, we will need to identify an optimal vector and delivery method for CNS approaches. To this end, we have developed a novel two-step bar code AAV vector system that allows assessment of multiple AAV vector serotypes within the same animal, greatly reducing the number of animals needed for statistical comparisons of brain delivery. This system has a genetic bar code that identifies each vector and a second bar code that is incorporated during PCR amplification of each brain region isolated. The bar code system allows determination of distribution and the expression levels of each serotype in anatomical areas of interest. We will use this novel two-step barcoded AAV vector system to simultaneously identify brain delivery of 40 AAV serotypes and capsid variants in wild type and MPS IIIB mice as well as in non-human primates - the closest to human model available to us. We will identify whether injections into the body of the brain or the less invasive injection into the fluid around the brain method provides a better vector distribution. We will identify which wild- type AAV serotypes or capsid mutants provide the best delivery by region, are altered by presence of the disease, and are similar between primate and mouse models. The results will inform clinical trial vector selection across the spectrum of central neurologic disorders, including MPS III. Subsequently, our MPS IIIB gene construct will be packaged into the optimal vector to assess treatment effect in MPS IIIB mice. We hypothesize that CNS transduction and distribution will differ by serotype and species and that some serotypes will transduce differently between wild type and Sanfilippo Syndrome mice.
Our specific aims are therefore: 1. We will determine the brain delivery of AAV serotypes in non-human primates (NHP) and in wild type and MPS IIIB affected mice. We will use a novel two-step bar-coded AAV vector system to allow simultaneous delivery and assessment of 40 serotypes with capsid variants in each animal via injections into the brain or surrounding fluid. Brain distribution for each serotype will be assessed by quantitative next generation RNA sequencing of the various brain regions. The top three vectors for brain delivery by this method will be used individually to identify the cell types treated and pattern of gene expression in mice and NHP. 2. Assess the effect of the AAV serotype with the best distribution in the thought processing and motor coordination regions of the brain carrying the MPS IIIB gene to treat the MPS IIIB mouse. We will use day/night activity, hearing, coordination, lifespan, lysosomal storage and enzyme assays to determine preclinical benefit in the mouse model. Overall, these studies will determine the effects of species, delivery site and disease state on brain delivery from a multitude of AAV serotypes. Through this study, we will identify the most promising vector(s) for clinical trial development in MPS IIIB and other neurodegenerative disorders. If this project is successful, we will be in a position to quickly move towards such clinical trials.
The goals of this research are to improve on current CNS-directed gene therapy approaches for Sanfilippo Syndrome and neurodegenerative diseases in general by comparing 40 promising vectors directly and confirming the cell type and expression levels of the best vectors in murine and primate models. The efficacy of the best vector will then be assessed in the murine model of Mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo Syndrome type B). This disease has a devastating impact on the children and families affected and this proposal will identify an optimal therapy to relieve this disease burden.