SMRT Sequencing Enables High-Throughput Identification of Novel AAVs from Capsid Shuffling and Directed Evolution.

The use of AAV capsid libraries coupled with various selection strategies has proven to be a remarkable approach for generating novel AAVs with enhanced and desired features. The inability to reliably sequence the complete capsid gene in a high-throughput manner has been the bottleneck of capsid engineering. As a result, many library strategies are confined to localized and modest alterations in the capsid, such as peptide insertions or single variable region (VR) alterations. The caveat of short reads by means of next-generation sequencing (NGS) hinders the diversity of capsid library construction, shifting the field away from whole-capsid modifications. We generated AAV capsid shuffled libraries of naturally occurring AAVs and applied directed evolution in both mice and non-human primates (NHPs), with the goal of yielding AAVs that are compatible across both species for translational applications. We recovered DNA from the tissues of injected animal and used single molecule real-time (SMRT) sequencing to identify variants enriched in the central nervous system (CNS). We provide insights and considerations for variant identification by comparing bulk tissue sequencing to that of isolated nuclei. Our work highlights the potential advantages of whole-capsid engineering, as well as indispensable methodological improvements for the analysis of recovered capsids, including the nuclei-enrichment step and SMRT sequencing.

Dual-isoform hUBE3A gene transfer improves behavioral and seizure outcomes in Angelman syndrome model mice.

Loss of the maternal UBE3A allele causes Angelman syndrome (AS), a debilitating neurodevelopmental disorder. Here, we devised an AS treatment strategy based on reinstating dual-isoform expression of human UBE3A (hUBE3A) in the developing brain. Kozak sequence engineering of our codon-optimized vector (hUBE3Aopt) enabled translation of both short and long hUBE3A protein isoforms at a near-endogenous 3:1 (short/long) ratio, a feature that could help to support optimal therapeutic outcomes. To model widespread brain delivery and early postnatal onset of hUBE3A expression, we packaged the hUBE3Aopt vector into PHP.B capsids and performed intracerebroventricular injections in neonates. This treatment significantly improved motor learning and innate behaviors in AS mice, and it rendered them resilient to epileptogenesis and associated hippocampal neuropathologies induced by seizure kindling. hUBE3A overexpression occurred frequently in the hippocampus but was uncommon in the neocortex and other major brain structures; furthermore, it did not correlate with behavioral performance. Our results demonstrate the feasibility, tolerability, and therapeutic potential for dual-isoform hUBE3A gene transfer in the treatment of AS.

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

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.

Widespread Expression of a Membrane-Tethered Version of the Soluble Lysosomal Enzyme Palmitoyl Protein Thioesterase-1.

"Cross-correction," the transfer of soluble lysosomal enzymes between neighboring cells, forms the foundation for therapeutics of lysosomal storage disorders (LSDs). However, "cross-correction" poses a significant barrier to studying the role of specific cell types in LSD pathogenesis. By expressing the native enzyme in only one cell type, neighboring cell types are invariably corrected. In this study, we present a strategy to limit "cross-correction" of palmitoyl-protein thioesterase-1(PPT1), a lysosomal hydrolase deficient in Infantile Neuronal Ceroid Lipofuscinosis (INCL, Infantile Batten disease) to the lysosomal membrane via the C-terminus of lysosomal associated membrane protein-1 (LAMP1). Tethering PPT1 to the lysosomal membrane prevented "cross-correction" while allowing PPT1 to retain its enzymatic function and localization in vitro. A transgenic line harboring the lysosomal membrane-tethered PPT1 was then generated. We show that expression of lysosome-restricted PPT1 in vivo largely rescues the INCL biochemical, histological, and functional phenotype. These data suggest that lysosomal tethering of PPT1 via the C-terminus of LAMP1 is a viable strategy and that this general approach can be used to study the role of specific cell types in INCL pathogenesis, as well as other LSDs. Ultimately, understanding the role of specific cell types in the disease progression of LSDs will help guide the development of more targeted therapeutics. One Sentence Synopsis: Tethering PPT1 to the lysosomal membrane is a viable strategy to prevent "cross-correction" and will allow for the study of specific cellular contributions in INCL pathogenesis.

Histochemical localization of palmitoyl protein thioesterase-1 activity.

Infantile neuronal ceroid lipofuscinosis (INCL, Infantile Batten disease) is an invariably fatal neurodegenerative pediatric disorder caused by an inherited mutation in the PPT1 gene. Patients with INCL lack the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1, EC 3.1.2.22), resulting in intracellular accumulation of autofluorescent storage material and subsequent neuropathology. The Ppt1(-/-) mouse is deficient in PPT1 activity and represents a useful animal model of INCL that recapitulates most of the clinical and pathological aspects of the disease. Preclinical therapeutic experiments performed in the INCL mouse include CNS-directed gene therapy and recombinant enzyme replacement therapy; both seek to re-establish therapeutic levels of the deficient enzyme. We present a novel method for the histochemical localization of PPT1 activity in the Ppt1(-/-) mouse. By utilizing the substrate CUS-9235, tissues known to be positive for PPT1 activity turn varying intensities of blue. Presented here are histochemistry data showing the staining pattern in Ppt1(-/-), wild type, and Ppt1(-/-) mice treated with enzyme replacement therapy or AAV2/9-PPT1-mediated gene therapy. Results are paired with quantitative biochemistry data that confirm the ability of CUS-9235 to detect and localize PPT1 activity. This new method complements the current tools for the study of INCL and evaluation of effective therapies.

An anti-neuroinflammatory that targets dysregulated glia enhances the efficacy of CNS-directed gene therapy in murine infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is an inherited neurodegenerative lysosomal storage disease (LSD) caused by a deficiency in palmitoyl protein thioesterase-1 (PPT1). Studies in Ppt1(-/-) mice demonstrate that glial activation is central to the pathogenesis of INCL. Astrocyte activation precedes neuronal loss, while cytokine upregulation associated with microglial reactivity occurs before and concurrent with neurodegeneration. Therefore, we hypothesized that cytokine cascades associated with neuroinflammation are important therapeutic targets for the treatment of INCL. MW01-2-151SRM (MW151) is a blood-brain barrier penetrant, small-molecule anti-neuroinflammatory that attenuates glial cytokine upregulation in models of neuroinflammation such as traumatic brain injury, Alzheimer's disease, and kainic acid toxicity. Thus, we used MW151, alone and in combination with CNS-directed, AAV-mediated gene therapy, as a possible treatment for INCL. MW151 alone decreased seizure susceptibility. When combined with AAV-mediated gene therapy, treated INCL mice had increased life spans, improved motor performance, and eradication of seizures. Combination-treated INCL mice also had decreased brain atrophy, astrocytosis, and microglial activation, as well as intermediary effects on cytokine upregulation. These data suggest that MW151 can attenuate seizure susceptibility but is most effective when used in conjunction with a therapy that targets the primary genetic defect.

Astrocytosis in infantile neuronal ceroid lipofuscinosis: friend or foe?

Infantile neuronal ceroid lipofuscinosis (INCL; infantile Batten disease) is an inherited paediatric neurodegenerative disease. INCL is caused by a deficiency in the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1) and is thus classified as a lysosomal storage disease. Pathological examination of both human and murine INCL brains reveals progressive, widespread neuroinflammation. In fact, astrocyte activation appears to be the first histological sign of disease. However, the role of astrocytosis in INCL was poorly understood. The hallmark of astrocyte activation is the up-regulation of intermediate filaments, such as glial fibrillary acidic protein (GFAP) and vimentin. The role of astrocytosis in INCL was studied in a murine model lacking PPT1 and the intermediate filaments GFAP and vimentin (triple-knockout). This murine model of INCL with attenuated astrocytosis had an exacerbated pathological and clinical phenotype. The triple-knockout mouse had a significantly shortened lifespan, and accelerated cellular and humoural neuroinflammatory response compared with the parental PPT1(-/-) mouse. The data obtained from the triple-knockout mouse strongly suggest that astrocyte activation plays a beneficial role in early INCL disease progression. A more thorough understanding of the glial responses to lysosomal enzyme deficiencies and the accumulation of undergraded substrates will be crucial to developing effective therapeutics.