Showing papers by "Beverly L. Davidson published in 2022"

An orally available, brain penetrant, small molecule lowers huntingtin levels by enhancing pseudoexon inclusion

Abstract: Huntington's Disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the huntingtin (HTT) gene. The mutant HTT (mHTT) protein causes neuronal dysfunction, causing progressive motor, cognitive and behavioral abnormalities. Current treatments for HD only alleviate symptoms, but cerebral spinal fluid (CSF) or central nervous system (CNS) delivery of antisense oligonucleotides (ASOs) or virus vectors expressing RNA-induced silencing (RNAi) moieties designed to induce mHTT mRNA lowering have progressed to clinical trials. Here, we present an alternative disease modifying therapy the orally available, brain penetrant small molecule branaplam. By promoting inclusion of a pseudoexon in the primary transcript, branaplam lowers mHTT protein levels in HD patient cells, in an HD mouse model and in blood samples from Spinal Muscular Atrophy (SMA) Type I patients dosed orally for SMA (NCT02268552). Our work paves the way for evaluating branaplam's utility as an HD therapy, leveraging small molecule splicing modulators to reduce expression of dominant disease genes by driving pseudoexon inclusion.

DeepRepeat: direct quantification of short tandem repeats on signal data from nanopore sequencing

Abstract: Abstract Despite recent improvements in basecalling accuracy, nanopore sequencing still has higher error rates on short-tandem repeats (STRs). Instead of using basecalled reads, we developed DeepRepeat which converts ionic current signals into red-green-blue channels, thus transforming the repeat detection problem into an image recognition problem. DeepRepeat identifies and accurately quantifies telomeric repeats in the CHM13 cell line and achieves higher accuracy in quantifying repeats in long STRs than competing methods. We also evaluate DeepRepeat on genome-wide or candidate region datasets from seven different sources. In summary, DeepRepeat enables accurate quantification of long STRs and complements existing methods relying on basecalled reads.

DeepRepeat: direct quantification of short tandem repeats on signal data from nanopore sequencing

Abstract: Abstract Despite recent improvements in basecalling accuracy, nanopore sequencing still has higher error rates on short-tandem repeats (STRs). Instead of using basecalled reads, we developed DeepRepeat which converts ionic current signals into red-green-blue channels, thus transforming the repeat detection problem into an image recognition problem. DeepRepeat identifies and accurately quantifies telomeric repeats in the CHM13 cell line and achieves higher accuracy in quantifying repeats in long STRs than competing methods. We also evaluate DeepRepeat on genome-wide or candidate region datasets from seven different sources. In summary, DeepRepeat enables accurate quantification of long STRs and complements existing methods relying on basecalled reads.

Haplotyping SNPs for allele-specific gene editing of the expanded huntingtin allele using long-read sequencing

Abstract: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by CAG trinucleotide repeat expansions in exon-1 of huntingtin (HTT). Currently, there is no cure for HD, and the clinical care of individuals with HD is focused on symptom management. Previously, we showed allele-specific deletion of the expanded HTT allele (mHTT) using CRISPR-Cas9 by targeting nearby (<10 kb) SNPs that created or eliminated a protospacer adjacent motif (PAM) near exon-1. Here, we comprehensively analyzed all potential PAM sites within a 10.4-kb genomic region flanking exon-1 of HTT in 983 individuals with HD using a multiplex targeted long-read sequencing approach on the Oxford Nanopore platform. We developed computational tools (NanoBinner and NanoRepeat) to de-multiplex the data, detect repeats, and phase the reads on the expanded or the wild-type HTT allele. One SNP common to 30% of individuals with HD of European ancestry emerged through this analysis, which was confirmed as a strong candidate for allele-specific deletion of the mHTT in human HD cell lines. In addition, up to 57% HD individuals may be candidates for allele-specific editing through combinatorial SNP targeting. Cumulatively, we provide a haplotype map of the region surrounding exon-1 of HTT in individuals affected with HD. Our workflow can be applied to other repeat expansion diseases to facilitate the design of guide RNAs for allele-specific gene editing.

Combined overexpression of ATXN1L and mutant ATXN1 knockdown by AAV rescue motor phenotypes and gene signatures in SCA1 mice

Abstract: Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by a (CAG) repeat expansion in the coding sequence of ATXN1. The primary mechanism of disease in SCA1 is toxic gain of function by polyglutamine-expanded mutant ATXN1 and is compounded by partial loss of wild-type function. Addressing both disease mechanisms, we have shown that virally expressed RNA interference targeting ATXN1 can both prevent and reverse disease phenotypes in SCA1 mice, and that overexpression of the ATXN1 homolog, ataxin 1-like (ATXN1L), improves disease readouts when delivered pre-symptomatically. Here, we combined these therapeutic approaches into two, dual component recombinant adeno-associated virus (rAAV) vectors and tested their ability to reverse disease in symptomatic SCA1 mice using behavior, pathological, and next-generation sequencing assays. Mice treated with vectors expressing human ATXN1L (hATXN1L) alone showed motor improvements and changes in gene expression that reflected increases in pro-development pathways. When hATN1L was combined with miS1, a previously validated microRNA targeting hATXN1, there was added normalization of disease allele-induced changes in gene expression along with motor improvements. Our data show the additive nature of this two-component approach for a more effective SCA1 therapy.

Antagonistic roles of canonical and alternative RPA in tandem CAG repeat diseases

Abstract: Tandem CAG repeat expansion mutations cause >15 neurodegenerative diseases, where ongoing expansions in patients’ brains are thought to drive disease onset and progression. Repeat length mutations will involve single-stranded DNAs prone to form mutagenic DNA structures. However, the involvement of single-stranded DNA binding proteins (SSBs) in the prevention or formation of repeat instability is poorly understood. Here, we assessed the role of two SSBs, canonical RPA (RPA1-RPA2-RPA3) and the related Alternative-RPA (Alt-RPA, RPA1-RPA4-RPA3), where the primate-specific RPA4 replaces RPA2. RPA is essential for all forms of DNA metabolism, while Alt-RPA has undefined functions. RPA and Alt-RPA are upregulated 2- and 10-fold, respectively, in brains of Huntington disease (HD) and spinocerebellar ataxia type 1 (SCA1) patients. Correct repair of slipped-CAG DNA structures, intermediates of expansion mutations, is enhanced by RPA, but blocked by Alt-RPA. Slipped-DNAs are bound and melted more efficiently by RPA than by Alt-RPA. Removal of excess slipped-DNAs by FAN1 nuclease is enhanced by RPA, but blocked by Alt-RPA. Protein-protein interactomes (BioID) reveal unique and shared partners of RPA and Alt-RPA, including proteins involved in CAG instability and known modifiers of HD and SCA1 disease. RPA overexpression inhibits rampant CAG expansions in SCA1 mouse brains, coinciding with improved neuron morphology and rescued motor phenotypes. Thus, SSBs are involved in repeat length mutations, where Alt-RPA antagonistically blocks RPA from suppressing CAG expansions and hence pathogenesis. The processing of repeat length mutations is one example by which an Alt-RPA↔RPA antagonistic interaction can affect outcomes, illuminating questions as to which of the many processes mediated by canonical RPA may also be modulated by Alt-RPA.

Temporal Phenotypic Changes in Huntington’s Disease Models for Preclinical Studies

Abstract: Background: Mouse models bearing genetic disease mutations are instrumental in the development of therapies for genetic disorders. Huntington’s disease (HD) is a late-onset lethal dominant genetic disorder due to a CAG repeat within exon 1 of the Huntingtin (Htt) gene. Several mice were developed to model HD through the expression of a transgenic fragment (exon 1 of the human HTT), the knock-in mutation of the CAG repeat in the context of the mouse Htt gene, or the full-length HTT human gene. The different mouse models present distinct onset, symptoms, and progression of the disease. Objective: The objective of this study is to advise on the best behavioral tests to assess disease progression in three HD mouse models. Methods: We tested N171-82Q transgenic mice, zQ175 knock-in mice, and BACHD full-length mice in a comprehensive behavior test battery in early, mid-, and late disease stages. Results: We contrast and compare the models and the emerging phenotypes with the available literature. These results suggest the most effective behavioral tests and appropriate sample sizes to detect treatment efficacy in each model at the different ages. We provide options for early detection of motor deficits while minimizing testing time and training. Conclusion: This information will inform researchers in the HD field as to which mouse model, tests and sample sizes can accurately and sensitively detect treatment efficacy in preclinical HD research.

Huntington’s disease phenotypes are improved via mTORC1 modulation by small molecule therapy

Abstract: Huntington’s Disease (HD) is a dominantly inherited neurodegenerative disease for which the major causes of mortality are neurodegeneration-associated aspiration pneumonia followed by cardiac failure. mTORC1 pathway perturbations are present in HD models and human tissues. Amelioration of mTORC1 deficits by genetic modulation improves disease phenotypes in HD models, is not a viable therapeutic strategy. Here, we assessed a novel small molecule mTORC1 pathway activator, NV-5297, for its improvement of the disease phenotypes in the N171-82Q HD mouse model. Oral dosing of NV-5297 over 6 weeks activated mTORC1, increased striatal volume, improved motor learning and heart contractility. Further, the heart contractility, heart fibrosis, and survival were improved in response to the cardiac stressor isoprenaline when compared to vehicle-treated mice. Cummulatively, these data support mTORC1 activation as a therapeutic target in HD and consolidates NV-5297 as a promising drug candidate for treating central and peripheral HD phenotypes and, more generally, mTORC1-deficit related diseases.

Mapping PTBP splicing in human brain identifies targets for therapeutic splice switching including SYNGAP1

Abstract: Alternative splicing of neuronal genes is controlled in part by the coordinated action of the polypyrimidine tract binding proteins (PTBP1 and PTBP2). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal, controlling the expression of such targets as DLG4, which encodes PSD95, a protein important in synaptic function whose deficiency causes neurodevelopmental disorders. Here, we fully define the PTBP2 footprint in the human transcriptome using both human brain tissue and neurons derived from human induced pluripotent stem cells (iPSC-neurons). We identify direct PTBP2 binding sites and define PTBP2-dependent alternative splicing events, finding novel targets such as STXBP1 and SYNGAP1, which are synaptic genes also associated with neurodevelopmental disorders. The resultant PTBP2 binding and splicing maps were used to test if PTBP2 binding could be manipulated to increase gene expression in PTBP-targeted genes that cause disease when haploinsufficient. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and non-sense mediated decay. Antisense oligonucleotides that disrupt PTBP binding sites on SYNGAP1 redirect splicing and increase gene and protein expression. Collectively, our data provide a comprehensive view of PTBP2-dependent alternative splicing in human neurons and human cerebral cortex, guiding the development of novel therapeutic tools that may benefit a range of neurodevelopmental disorders.