Infantile Batten Disease: Effective Therapy and Novel Model

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten) is typically an early onset, neurodegenerative lysosomal storage disorder. INCL is caused by mutations to the gene CLN1 which codes for the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1). PPT1 is a soluble lysosomal enzyme that functions to cleave fatty acyl chains from proteins destined for degradation. Deficiency in PPT1 leads to the accumulation of autofluorescent storage material, a hallmark of the NCLs. The storage material has been implicated in progressive histopathological changes in the brain such as neuronal loss, astrocytosis, microgliosis, and immune cell infiltration. These histopathological changes result in a progression of clinical signs including vision loss, decline in motor function, cognitive deficits, seizures, and premature death. Currently, there are no cures or treatments for INCL. However, a murine model of INCL has been used in pre-clinical therapy studies. The PPT1-/- mouse has been shown to be a reliable model for the human INCL disease. Detailed temporal and spatial histopathological examinations of murine INCL in the brain have led to intracranial gene therapy studies. These pre-clinical studies have resulted in significant improvements in biochemical, histopathological, and functional deficits seen in the untreated PPT1-/- mouse. However, there have only been modest improvements in lifespan. Given the identification and development of improved gene therapy vectors, this was a surprising finding. Therefore, the first section of the dissertation, we pursued a more thorough characterization of the central nervous system to identify potential regions of disease not targeted by intracranial gene therapy. We identified the spinal cord as a significant site of disease that was not previously characterized or corrected. This allowed us to target both the brain and spinal cord with AAV-based gene therapy. We demonstrated that targeting the entirety of the central nervous system was necessary to treat INCL more effectively. From these and historical studies, we identified a multitude of cell types that are involved with INCL pathogenesis. In the central nervous system, INCL has been shown to progress sequentially from astrocytosis, to neuronal loss, to microgliosis and immune cell infiltration. PPT1 is ubiquitously expressed; therefore, its deficiency in INCL could lead to pathology in every cell type. Currently, we are unable to model cellular and metabolic changes in specific cell types in INCL due to ‘cross-correction’. While ‘cross-correction’ is beneficial for the development of therapeutics, it interferes with our ability to understand the role of PPT1 in specific cell types. Therefore, in the second section of the dissertation, we sought to determine the cell-autonomous nature of PPT1. Because PPT1 is a soluble lysosomal hydrolase that can undergo ‘cross-correction’, we developed a chimeric enzyme whereby PPT1 is tethered to the lysosomal membrane. We demonstrated that tethered PPT1 retains its enzymatic function and does not ‘cross-correct’ in vitro and in vivo. We further demonstrated that near-ubiquitous expression of tethered PPT1 could prevent INCL. This lays the groundwork for future studies designed to determine the role of specific cell types in the pathogenesis of INCL

Recent progress and considerations for AAV gene therapies targeting the central nervous system

Abstract Background: Neurodevelopmental disorders, as a class of diseases, have been particularly difficult to treat even when the underlying cause(s), such as genetic alterations, are understood. What treatments do exist are generally not curative and instead seek to improve quality of life for affected individuals. The advent of gene therapy via gene replacement offers the potential for transformative therapies to slow or even stop disease progression for current patients and perhaps minimize or prevent the appearance of symptoms in future patients. Main body: This review focuses on adeno-associated virus (AAV) gene therapies for diseases of the central nervous system. An overview of advances in AAV vector design for therapy is provided, along with a description of current strategies to develop AAV vectors with tailored tropism. Next, progress towards treatment of neurodegenerative diseases is presented at both the pre-clinical and clinical stages, focusing on a few select diseases to highlight broad categories of therapeutic parameters. Special considerations for more challenging cases are then discussed in addition to the immunological aspects of gene therapy. Conclusion: With the promising clinical trial results that have been observed for the latest AAV gene therapies and continued pre-clinical successes, the question is no longer whether a therapy can be developed for certain neurodevelopmental disorders, but rather, how quickly

Synergistic effects of treating the spinal cord and brain in CLN1 disease

Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1(-/-)) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1(-/-) mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1(-/-) mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders.Peer reviewe

Down-Regulation of ZnT8 Expression in INS-1 Rat Pancreatic Beta Cells Reduces Insulin Content and Glucose-Inducible Insulin Secretion

The SLC30A8 gene codes for a pancreatic beta-cell-expressed zinc transporter, ZnT8. A polymorphism in the SLC30A8 gene is associated with susceptibility to type 2 diabetes, although the molecular mechanism through which this phenotype is manifest is incompletely understood. Such polymorphisms may exert their effect via impacting expression level of the gene product. We used an shRNA-mediated approach to reproducibly downregulate ZnT8 mRNA expression by >90% in the INS-1 pancreatic beta cell line. The ZnT8-downregulated cells exhibited diminished uptake of exogenous zinc, as determined using the zinc-sensitive reporter dye, zinquin. ZnT8-downregulated cells showed reduced insulin content and decreased insulin secretion (expressed as percent of total insulin content) in response to hyperglycemic stimulus, as determined by insulin immunoassay. ZnT8-depleted cells also showed fewer dense-core vesicles via electron microscopy. These data indicate that reduced ZnT8 expression in cultured pancreatic beta cells gives rise to a reduced insulin response to hyperglycemia. In addition, although we provide no direct evidence, these data suggest that an SLC30A8 expression-level polymorphism could affect insulin secretion and the glycemic response in vivo

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Recent progress and considerations for AAV gene therapies targeting the central nervous system

Abstract Background Neurodevelopmental disorders, as a class of diseases, have been particularly difficult to treat even when the underlying cause(s), such as genetic alterations, are understood. What treatments do exist are generally not curative and instead seek to improve quality of life for affected individuals. The advent of gene therapy via gene replacement offers the potential for transformative therapies to slow or even stop disease progression for current patients and perhaps minimize or prevent the appearance of symptoms in future patients. Main body This review focuses on adeno-associated virus (AAV) gene therapies for diseases of the central nervous system. An overview of advances in AAV vector design for therapy is provided, along with a description of current strategies to develop AAV vectors with tailored tropism. Next, progress towards treatment of neurodegenerative diseases is presented at both the pre-clinical and clinical stages, focusing on a few select diseases to highlight broad categories of therapeutic parameters. Special considerations for more challenging cases are then discussed in addition to the immunological aspects of gene therapy. Conclusion With the promising clinical trial results that have been observed for the latest AAV gene therapies and continued pre-clinical successes, the question is no longer whether a therapy can be developed for certain neurodevelopmental disorders, but rather, how quickly

Synergistic effects of treating the spinal cord and brain in CLN1 disease.

Infantile neuronal ceroid lipofuscinosis (INCL, or CLN1 disease) is an inherited neurodegenerative storage disorder caused by a deficiency of the lysosomal enzyme palmitoyl protein thioesterase 1 (PPT1). It was widely believed that the pathology associated with INCL was limited to the brain, but we have now found unexpectedly profound pathology in the human INCL spinal cord. Similar pathological changes also occur at every level of the spinal cord of PPT1-deficient (Ppt1-/- ) mice before the onset of neuropathology in the brain. Various forebrain-directed gene therapy approaches have only had limited success in Ppt1-/- mice. Targeting the spinal cord via intrathecal administration of an adeno-associated virus (AAV) gene transfer vector significantly prevented pathology and produced significant improvements in life span and motor function in Ppt1-/- mice. Surprisingly, forebrain-directed gene therapy resulted in essentially no PPT1 activity in the spinal cord, and vice versa. This leads to a reciprocal pattern of histological correction in the respective tissues when comparing intracranial with intrathecal injections. However, the characteristic pathological features of INCL were almost completely absent in both the brain and spinal cord when intracranial and intrathecal injections of the same AAV vector were combined. Targeting both the brain and spinal cord also produced dramatic and synergistic improvements in motor function with an unprecedented increase in life span. These data show that spinal cord pathology significantly contributes to the clinical progression of INCL and can be effectively targeted therapeutically. This has important implications for the delivery of therapies in INCL, and potentially in other similar disorders