Publications by authors named "Beverly L Davidson"

162 Publications

Mis-splicing in Huntington's disease: harnessing the power of comparative transcriptomics.

Trends Neurosci 2021 Nov 6. Epub 2021 Nov 6.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, PA 19104, USA. Electronic address:

A recent paper by Elorza et al. describes an 'intersect-RNA-seq' analysis of Huntington's disease (HD) by parallel RNA sequencing (RNA-seq) profiling of HD brain tissues from humans and mice. This work illustrates a broadly applicable strategy to elucidate splicing alterations in neurological diseases by integrating the transcriptome profiles of human patient tissues and animal models.
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http://dx.doi.org/10.1016/j.tins.2021.10.009DOI Listing
November 2021

Toxicity after AAV delivery of RNAi expression constructs into nonhuman primate brain.

Nat Med 2021 Nov 18;27(11):1982-1989. Epub 2021 Oct 18.

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia Research Institute, Philadelphia, PA, USA.

RNA interference (RNAi) for spinocerebellar ataxia type 1 can prevent and reverse behavioral deficits and neuropathological readouts in mouse models, with safety and benefit lasting over many months. The RNAi trigger, expressed from adeno-associated virus vectors (AAV.miS1), also corrected misregulated microRNAs (miRNA) such as miR150. Subsequently, we showed that the delivery method was scalable, and that AAV.miS1 was safe in short-term pilot nonhuman primate (NHP) studies. To advance the technology to patients, investigational new drug (IND)-enabling studies in NHPs were initiated. After AAV.miS1 delivery to deep cerebellar nuclei, we unexpectedly observed cerebellar toxicity. Both small-RNA-seq and studies using AAVs devoid of miRNAs showed that this was not a result of saturation of the endogenous miRNA processing machinery. RNA-seq together with sequencing of the AAV product showed that, despite limited amounts of cross-packaged material, there was substantial inverted terminal repeat (ITR) promoter activity that correlated with neuropathologies. ITR promoter activity was reduced by altering the miS1 expression context. The surprising contrast between our rodent and NHP findings highlight the need for extended safety studies in multiple species when assessing new therapeutics for human application.
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http://dx.doi.org/10.1038/s41591-021-01522-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8605996PMC
November 2021

Next-generation strategies for gene-targeted therapies of central nervous system disorders: A workshop summary.

Mol Ther 2021 Dec 20;29(12):3332-3344. Epub 2021 Sep 20.

Children's Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

The National Institute of Neurological Disorders and Stroke (NINDS) held a workshop titled "Next generation strategies for gene-targeted therapies of central nervous system (CNS) disorders" in September 2019 in Bethesda, MD, USA. The meeting brought together a multi-disciplinary group of experts in the field of CNS-directed gene-targeted therapy delivery from academia, industry, advocacy, and the government. The group was charged with identifying the key challenges and gaps in this evolving field, as well as suggesting potential solutions. The workshop was divided into four sessions: (1) control of level and location, (2) improving delivery and distribution, (3) enhancing models and manufacturing, and (4) impacting patients. Prior to the workshop, NINDS established working groups of key opinion leaders (KOLs) for each session. In pre-meeting teleconferences, KOLs were tasked with identifying the research gaps and key obstacles that delay and/or prevent gene-targeted therapies to move into the clinic. This approach allowed for the workshop to begin with problem-solving discussions and strategy development, as the key issues had been established. The overall purpose of the workshop was to consider knowledge gaps and potential strategies to inform the community around CNS gene-targeted therapies, including but not limited to researchers and funders.
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http://dx.doi.org/10.1016/j.ymthe.2021.09.010DOI Listing
December 2021

Regulated control of gene therapies by drug-induced splicing.

Nature 2021 08 28;596(7871):291-295. Epub 2021 Jul 28.

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

So far, gene therapies have relied on complex constructs that cannot be finely controlled. Here we report a universal switch element that enables precise control of gene replacement or gene editing after exposure to a small molecule. The small-molecule inducers are currently in human use, are orally bioavailable when given to animals or humans and can reach both peripheral tissues and the brain. Moreover, the switch system, which we denote X, does not require the co-expression of any regulatory proteins. Using X, the translation of the desired elements for controlled gene replacement or gene editing machinery occurs after a single oral dose of the inducer, and the robustness of expression can be controlled by the drug dose, protein stability and redosing. The ability of X to provide temporal control of protein expression can be adapted for cell-biology applications and animal studies. Additionally, owing to the oral bioavailability and safety of the drugs used, the X switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.
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http://dx.doi.org/10.1038/s41586-021-03770-2DOI Listing
August 2021

Immortalized striatal precursor neurons from Huntington's disease patient-derived iPS cells as a platform for target identification and screening for experimental therapeutics.

Hum Mol Genet 2021 Nov;30(24):2469-2487

Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA.

We have previously established induced pluripotent stem cell (iPSC) models of Huntington's disease (HD), demonstrating CAG-repeat-expansion-dependent cell biological changes and toxicity. However, the current differentiation protocols are cumbersome and time consuming, making preparation of large quantities of cells for biochemical or screening assays difficult. Here, we report the generation of immortalized striatal precursor neurons (ISPNs) with normal (33) and expanded (180) CAG repeats from HD iPSCs, differentiated to a phenotype resembling medium spiny neurons (MSN), as a proof of principle for a more tractable patient-derived cell model. For immortalization, we used co-expression of the enzymatic component of telomerase hTERT and conditional expression of c-Myc. ISPNs can be propagated as stable adherent cell lines, and rapidly differentiated into highly homogeneous MSN-like cultures within 2 weeks, as demonstrated by immunocytochemical criteria. Differentiated ISPNs recapitulate major HD-related phenotypes of the parental iPSC model, including brain-derived neurotrophic factor (BDNF)-withdrawal-induced cell death that can be rescued by small molecules previously validated in the parental iPSC model. Proteome and RNA-seq analyses demonstrate separation of HD versus control samples by principal component analysis. We identified several networks, pathways, and upstream regulators, also found altered in HD iPSCs, other HD models, and HD patient samples. HD ISPN lines may be useful for studying HD-related cellular pathogenesis, and for use as a platform for HD target identification and screening experimental therapeutics. The described approach for generation of ISPNs from differentiated patient-derived iPSCs could be applied to a larger allelic series of HD cell lines, and to comparable modeling of other genetic disorders.
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http://dx.doi.org/10.1093/hmg/ddab200DOI Listing
November 2021

Mavis Agbandje-McKenna's Lifelong Commitment to Teaching and Research.

Hum Gene Ther 2021 Apr;32(7-8):319-320

UNC Gene Therapy Center and the Department of Pharmacology, Chapel Hill, NC, USA.

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http://dx.doi.org/10.1089/hum.2021.29157.almDOI Listing
April 2021

Gene therapy for ALS: A review.

Mol Ther 2021 Dec 9;29(12):3345-3358. Epub 2021 Apr 9.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Electronic address:

Amyotrophic lateral sclerosis (ALS) has historically posed unique challenges for gene-therapy-based approaches, due to a paucity of therapeutic targets as well as the difficulty of accessing both the brain and spinal cord. Recent advances in our understanding of disease mechanism and ALS genetics, however, have combined with tremendous strides in CNS targeting, gene delivery, and gene editing and knockdown techniques to open new horizons of therapeutic possibility. Gene therapy clinical trials are currently underway for ALS patients with SOD1 mutations, C9orf72 hexanucleotide repeat expansions, ATXN2 trinucleotide expansions, and FUS mutations, as well as sporadic disease without known genetic cause. In this review, we provide an in-depth exploration of the state of ALS-directed gene therapy, including antisense oligonucleotides, RNA interference, CRISPR, adeno-associated virus (AAV)-mediated trophic support, and antibody-based methods. We discuss how each of these approaches has been implemented across known genetic causes as well as sporadic ALS, reviewing preclinical studies as well as completed and ongoing human clinical trials. We highlight the transformative potential of these evolving technologies as the gene therapy field advances toward a true disease-modifying treatment for this devastating illness.
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http://dx.doi.org/10.1016/j.ymthe.2021.04.008DOI Listing
December 2021

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington's disease.

Proc Natl Acad Sci U S A 2021 01;118(4)

Department of Neurobiology and Behavior, University of California, Irvine, CA 92697;

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.
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http://dx.doi.org/10.1073/pnas.2021836118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848703PMC
January 2021

Author Correction: Striatal neurons directly converted from Huntington's disease patient fibroblasts recapitulate age-associated disease phenotypes.

Nat Neurosci 2020 Oct;23(10):1307

Department of Developmental Biology, Center for Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41593-020-00714-3DOI Listing
October 2020

Regional Variation of Splicing QTLs in Human Brain.

Am J Hum Genet 2020 08 25;107(2):196-210. Epub 2020 Jun 25.

Bioinformatics Interdepartmental Graduate Program, University of California, Los Angeles, Los Angeles, CA 90095, USA; Center for Computational and Genomic Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

A major question in human genetics is how sequence variants of broadly expressed genes produce tissue- and cell type-specific molecular phenotypes. Genetic variation of alternative splicing is a prevalent source of transcriptomic and proteomic diversity in human populations. We investigated splicing quantitative trait loci (sQTLs) in 1,209 samples from 13 human brain regions, using RNA sequencing (RNA-seq) and genotype data from the Genotype-Tissue Expression (GTEx) project. Hundreds of sQTLs were identified in each brain region. Some sQTLs were shared across brain regions, whereas others displayed regional specificity. These "regionally ubiquitous" and "regionally specific" sQTLs showed distinct positional distributions of single-nucleotide polymorphisms (SNPs) within and outside essential splice sites, respectively, suggesting their regulation by distinct molecular mechanisms. Integrating the binding motifs and expression patterns of RNA binding proteins with exon splicing profiles, we uncovered likely causal variants underlying brain region-specific sQTLs. Notably, SNP rs17651213 created a putative binding site for the splicing factor RBFOX2 and was associated with increased splicing of MAPT exon 3 in cerebellar tissues, where RBFOX2 was highly expressed. Overall, our study reveals a more comprehensive spectrum and regional variation of sQTLs in human brain and demonstrates that such regional variation can be used to fine map potential causal variants of sQTLs and their associated neurological diseases.
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http://dx.doi.org/10.1016/j.ajhg.2020.06.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413857PMC
August 2020

Gene therapy matures to medicines.

Hum Mol Genet 2019 10;28(R1):R1-R2

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, the Children's Hospital of Philadelphia.

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http://dx.doi.org/10.1093/hmg/ddz182DOI Listing
October 2019

Neuronal network dysfunction precedes storage and neurodegeneration in a lysosomal storage disorder.

JCI Insight 2019 11 1;4(21). Epub 2019 Nov 1.

Division of Child Neurology, Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Accumulation of lysosomal storage material and late-stage neurodegeneration are hallmarks of lysosomal storage disorders (LSDs) affecting the brain. Yet, for most LSDs, including CLN3 disease, the most common form of childhood dementia, it is unclear what mechanisms drive neurologic symptoms. Do deficits arise from loss of function of the mutated protein or toxicity from storage accumulation? Here, using in vitro voltage-sensitive dye imaging and in vivo electrophysiology, we find progressive hippocampal dysfunction occurs before notable lysosomal storage and neuronal loss in 2 CLN3 disease mouse models. Pharmacologic reversal of lysosomal storage deposition in young mice does not rescue this circuit dysfunction. Additionally, we find that CLN3 disease mice lose an electrophysiologic marker of new memory encoding - hippocampal sharp-wave ripples. This discovery, which is also seen in Alzheimer's disease, suggests the possibility of a shared electrophysiologic signature of dementia. Overall, our data describe new insights into previously unknown network-level changes occurring in LSDs affecting the central nervous system and highlight the need for new therapeutic interventions targeting early circuit defects.
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http://dx.doi.org/10.1172/jci.insight.131961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6948765PMC
November 2019

Doubling down on siRNAs in the brain.

Nat Biotechnol 2019 08;37(8):865-866

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, the Children's Hospital of Philadelphia, and the Department of Pathology and Laboratory Medicine, The University of Pennsylvania, Philadelphia, PA, USA.

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http://dx.doi.org/10.1038/s41587-019-0204-1DOI Listing
August 2019

Standard screening methods underreport AAV-mediated transduction and gene editing.

Nat Commun 2019 07 30;10(1):3415. Epub 2019 Jul 30.

The Children's Hospital of Philadelphia, Philadelphia, PA, 19104, USA.

Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.
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http://dx.doi.org/10.1038/s41467-019-11321-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667494PMC
July 2019

Publisher Correction: Lysosomal storage diseases.

Nat Rev Dis Primers 2019 May 17;5(1):34. Epub 2019 May 17.

Office of the Clinical Director and Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA.

In the originally published version of Figure 3, APP was incorrectly linked to CMA. In addition, the label for NCP2 was omitted, and GlcSph was incorrectly labelled as GlcCer. This figure has now been corrected.
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http://dx.doi.org/10.1038/s41572-019-0089-9DOI Listing
May 2019

CRISPR to the Rescue: Advances in Gene Editing for the Gene.

Brain Sci 2019 Jan 21;9(1). Epub 2019 Jan 21.

The Raymond G. Perelman Center of Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.

Gene-editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is promising as a potential therapeutic strategy for many genetic disorders. CRISPR-based therapies are already being assessed in clinical trials, and evaluation of this technology in Fragile X syndrome has been performed by a number of groups. The findings from these studies and the advancement of CRISPR-based technologies are insightful as the field continues towards treatments and cures of Fragile X-Associated Disorders (FXADs). In this review, we summarize reports using CRISPR-editing strategies to target Fragile X syndrome (FXS) molecular dysregulation, and highlight how differences in FXS and Fragile X-associated Tremor/Ataxia Syndrome (FXTAS) might alter treatment strategies for each syndrome. We discuss the various modifications and evolutions of the CRISPR toolkit that expand its therapeutic potential, and other considerations for moving these strategies from bench to bedside. The rapidly growing field of CRISPR therapeutics is providing a myriad of approaches to target a gene, pathway, or transcript for modification. As cures for FXADs have remained elusive, CRISPR opens new avenues to pursue.
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http://dx.doi.org/10.3390/brainsci9010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357057PMC
January 2019

Generation of Spinocerebellar Ataxia Type 2 induced pluripotent stem cell lines, CHOPi002-A and CHOPi003-A, from patients with abnormal CAG repeats in the coding region of the ATXN2 gene.

Stem Cell Res 2019 01 10;34:101361. Epub 2018 Dec 10.

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, USA; Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and The University of Pennsylvania, USA. Electronic address:

Spinocerebellar Ataxia Type 2 (SCA2) is an autosomal dominant disease characterized by progressive degeneration of the cerebellum, brain stem, and spinal cord. SCA2 is caused by spontaneous misfolding and aggregate formation from abnormal CAG trinucleotide repeat expansion in the coding region of the ATXN2 gene. Here we describe the generation of two distinct iPSC lines from patients with SCA2.
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http://dx.doi.org/10.1016/j.scr.2018.101361DOI Listing
January 2019

AAV-Mediated Progranulin Delivery to a Mouse Model of Progranulin Deficiency Causes T Cell-Mediated Toxicity.

Mol Ther 2019 02 17;27(2):465-478. Epub 2018 Nov 17.

Department of Neurology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Adeno-associated virus-mediated gene replacement is emerging as a safe and effective means of correcting single-gene mutations affecting the CNS. AAV-mediated progranulin gene (GRN) delivery has been proposed as a treatment for GRN-deficient frontotemporal dementia and neuronal ceroid lipofuscinosis, and recent studies using intraparenchymal AAV-Grn delivery to brain have shown moderate success in histopathologic and behavioral rescue in mouse models. Here, we used AAV9 to deliver GRN to the lateral ventricle to achieve widespread expression in the Grn null mouse brain. We found that, despite a global increase in progranulin, overexpression resulted in dramatic and selective hippocampal toxicity and degeneration affecting neurons and glia. Hippocampal degeneration was preceded by T cell infiltration and perivascular cuffing. GRN delivery with an ependymal-targeting AAV for selective secretion of progranulin into the cerebrospinal fluid similarly resulted in T cell infiltration, as well as ependymal hypertrophy. Interestingly, overexpression of GRN in wild-type animals also provoked T cell infiltration. These results call into question the safety of GRN overexpression in the CNS, with evidence for both a region-selective immune response and cellular proliferative response. Our results highlight the importance of careful consideration of target gene biology and cellular response to overexpression prior to progressing to the clinic.
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http://dx.doi.org/10.1016/j.ymthe.2018.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6369714PMC
February 2019

Adeno-Associated Virus Production, Purification, and Titering.

Curr Protoc Mouse Biol 2018 Dec 29;8(4):e56. Epub 2018 Nov 29.

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Adeno-associated virus (AAV) vectors are exemplary tools for studying gene function in vivo and are particularly favorable for transferring genes of interest into brain tissues. They have shown great promise as a gene therapy vector for preclinical and clinical applications. However, the ability to use this tool is often hampered because the viruses themselves are not readily available. Many methods have been developed for AAV production. Here, we describe a simple method for small- to medium-scale (10 -10 viral particles) production of AAV based on Polyethylenimine Max (PEI Max)-mediated triple transfection of HEK 293 cells and purification with iodixanol gradient ultracentrifugation. These methods will provide users with ample material of sufficient quality for performing in vivo gene transfer. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.56DOI Listing
December 2018

Viral Vectors for Gene Transfer.

Curr Protoc Mouse Biol 2018 Dec 28;8(4):e58. Epub 2018 Nov 28.

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Viral vectors are a promising tool for effective delivery of genetic material into cells. They take advantage of the natural ability of a virus to deliver a genetic payload into cells while being genetically modified such that their ability to replicate is crippled or removed. Here, an updated overview of routinely used viral vectors, including adeno-associated viruses (AAV), retroviruses/lentiviruses, and adenoviruses (Ads), is provided, as well as perspectives on their advantages and disadvantages in research and gene therapy. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.58DOI Listing
December 2018

Techniques for Intracranial Stereotaxic Injections of Adeno-Associated Viral Vectors in Adult Mice.

Curr Protoc Mouse Biol 2018 Dec 5;8(4):e57. Epub 2018 Nov 5.

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Stereotaxic intracranial injection of viral vectors is a valuable technique to directly deliver genetic material to a specific population of cells in the central nervous system of a mouse model. This enables scientists to test candidate gene therapies or disease modulators that can then provide insight into the pathological mechanisms of disease. In this article, we present a standardized method of conducting intracranial stereotaxic injection of adeno-associated virus into a specific brain region in a mouse model. Support protocols are provided for virus dialysis and testing new stereotaxic coordinates with dye. © 2018 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.57DOI Listing
December 2018

Author Correction: Lysosomal storage diseases.

Nat Rev Dis Primers 2018 Oct 18;4(1):36. Epub 2018 Oct 18.

Office of the Clinical Director and Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA.

In the version of the article originally published, in Figure 2 and the accompanying legend, LIMP 2 was incorrectly referred to as LIMP 1. The article has now been corrected.
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http://dx.doi.org/10.1038/s41572-018-0037-0DOI Listing
October 2018

Lysosomal storage diseases.

Nat Rev Dis Primers 2018 10 1;4(1):27. Epub 2018 Oct 1.

Office of the Clinical Director and Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA.

Lysosomal storage diseases (LSDs) are a group of over 70 diseases that are characterized by lysosomal dysfunction, most of which are inherited as autosomal recessive traits. These disorders are individually rare but collectively affect 1 in 5,000 live births. LSDs typically present in infancy and childhood, although adult-onset forms also occur. Most LSDs have a progressive neurodegenerative clinical course, although symptoms in other organ systems are frequent. LSD-associated genes encode different lysosomal proteins, including lysosomal enzymes and lysosomal membrane proteins. The lysosome is the key cellular hub for macromolecule catabolism, recycling and signalling, and defects that impair any of these functions cause the accumulation of undigested or partially digested macromolecules in lysosomes (that is, 'storage') or impair the transport of molecules, which can result in cellular damage. Consequently, the cellular pathogenesis of these diseases is complex and is currently incompletely understood. Several LSDs can be treated with approved, disease-specific therapies that are mostly based on enzyme replacement. However, small-molecule therapies, including substrate reduction and chaperone therapies, have also been developed and are approved for some LSDs, whereas gene therapy and genome editing are at advanced preclinical stages and, for a few disorders, have already progressed to the clinic.
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http://dx.doi.org/10.1038/s41572-018-0025-4DOI Listing
October 2018

The long non-coding RNA NEAT1 is elevated in polyglutamine repeat expansion diseases and protects from disease gene-dependent toxicities.

Hum Mol Genet 2018 12;27(24):4303-4314

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG-repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock-in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA-protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reducing it enhanced mHTT-dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG-repeat expansion diseases.
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http://dx.doi.org/10.1093/hmg/ddy331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276831PMC
December 2018

Disease-Associated Short Tandem Repeats Co-localize with Chromatin Domain Boundaries.

Cell 2018 09 30;175(1):224-238.e15. Epub 2018 Aug 30.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

More than 25 inherited human disorders are caused by the unstable expansion of repetitive DNA sequences termed short tandem repeats (STRs). A fundamental unresolved question is why some STRs are susceptible to pathologic expansion, whereas thousands of repeat tracts across the human genome are relatively stable. Here, we discover that nearly all disease-associated STRs (daSTRs) are located at boundaries demarcating 3D chromatin domains. We identify a subset of boundaries with markedly higher CpG island density compared to the rest of the genome. daSTRs specifically localize to ultra-high-density CpG island boundaries, suggesting they might be hotspots for epigenetic misregulation or topological disruption linked to STR expansion. Fragile X syndrome patients exhibit severe boundary disruption in a manner that correlates with local loss of CTCF occupancy and the degree of FMR1 silencing. Our data uncover higher-order chromatin architecture as a new dimension in understanding repeat expansion disorders.
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http://dx.doi.org/10.1016/j.cell.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175607PMC
September 2018

Cardiac mTORC1 Dysregulation Impacts Stress Adaptation and Survival in Huntington's Disease.

Cell Rep 2018 04;23(4):1020-1033

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA; The Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Huntington's disease (HD) is a dominantly inherited neurological disorder caused by CAG-repeat expansion in exon 1 of Huntingtin (HTT). But in addition to the neurological disease, mutant HTT (mHTT), which is ubiquitously expressed, impairs other organ systems. Indeed, epidemiological and animal model studies suggest higher incidence of and mortality from heart disease in HD. Here, we show that the protein complex mTORC1 is dysregulated in two HD mouse models through a mechanism that requires intrinsic mHTT expression. Moreover, restoring cardiac mTORC1 activity with constitutively active Rheb prevents mortality and relieves the mHTT-induced block to hypertrophic adaptation to cardiac stress. Finally, we show that chronic mTORC1 dysregulation is due in part to mislocalization of endogenous Rheb. These data provide insight into the increased cardiac-related mortality of HD patients, with cardiac mHTT expression inhibiting mTORC1 activity, limiting heart growth, and decreasing the heart's ability to compensate to chronic stress.
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http://dx.doi.org/10.1016/j.celrep.2018.03.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967646PMC
April 2018

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo.

Neurobiol Dis 2018 07 13;115:182-193. Epub 2018 Apr 13.

The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States; Department of Pathology & Laboratory Medicine, Philadelphia, PA 19104, United States. Electronic address:

The neuronal ceroid lipofuscinoses are a class of inherited neurodegenerative diseases characterized by the accumulation of autofluorescent storage material. The most common neuronal ceroid lipofuscinosis has juvenile onset with rapid onset blindness and progressive degeneration of cognitive processes. The juvenile form is caused by mutations in the CLN3 gene, which encodes the protein CLN3. While mouse models of Cln3 deficiency show mild disease phenotypes, it is apparent from patient tissue- and cell-based studies that its loss impacts many cellular processes. Using Cln3 deficient mice, we previously described defects in mouse brain endothelial cells and blood-brain barrier (BBB) permeability. Here we expand on this to other components of the BBB and show that Cln3 deficient mice have increased astrocyte endfeet area. Interestingly, this phenotype is corrected by treatment with a commonly used GAP junction inhibitor, carbenoxolone (CBX). In addition to its action on GAP junctions, CBX has also been proposed to alter lipid microdomains. In this work, we show that CBX modifies lipid microdomains and corrects membrane fluidity alterations in Cln3 deficient endothelial cells, which in turn improves defects in endocytosis, caveolin-1 distribution at the plasma membrane, and Cdc42 activity. In further work using the NIH Library of Integrated Network-based Cellular Signatures (LINCS), we discovered other small molecules whose impact was similar to CBX in that they improved Cln3-deficient cell phenotypes. Moreover, Cln3 deficient mice treated orally with CBX exhibited recovery of impaired BBB responses and reduced autofluorescence. CBX and the compounds identified by LINCS, many of which have been used in humans or approved for other indications, may find therapeutic benefit in children suffering from CLN3 deficiency through mechanisms independent of their original intended use.
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http://dx.doi.org/10.1016/j.nbd.2018.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969532PMC
July 2018

Overcoming Limitations Inherent in Sulfamidase to Improve Mucopolysaccharidosis IIIA Gene Therapy.

Mol Ther 2018 04 31;26(4):1118-1126. Epub 2018 Jan 31.

Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Sulfamidase (SGSH) deficiency causes mucopolysaccharidosis type IIIA (MPS IIIA), a lysosomal storage disease (LSD) that affects the CNS. In earlier work in LSD mice and dog models, we exploited the utility of adeno-associated viruses (AAVs) to transduce brain ventricular lining cells (ependyma) for secretion of lysosomal hydrolases into the cerebrospinal fluid (CSF), with subsequent distribution of enzyme throughout the brain resulting in improved cognition and extending lifespan. A critical feature of this approach is efficient secretion of the expressed enzyme from transduced cells, for delivery by CSF to nontransduced cells. Surprisingly, we found that SGSH was poorly secreted from cells, resulting in retention of the expressed product. Using site-directed mutagenesis of native SGSH, we identified an improved secretion variant that also displayed enhanced uptake properties that were mannose-6-phosphate receptor independent. In studies in MPS IIIA-deficient mice, ependymal transduction with AAVs expressing variant SGSH improved spatial learning and reduced memory deficits, substrate accumulation, and astrogliosis. Secondary lysosomal enzyme elevations in the CSF and brain parenchyma were also resolved. In contrast, ependymal transduction with AAVs expressing wild-type SGSH had significantly lower CSF SGSH levels and limited impacts on behavior. These results demonstrate the utility of a previously undescribed SGSH variant for improved MPS IIIA brain gene therapy.
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http://dx.doi.org/10.1016/j.ymthe.2018.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079371PMC
April 2018

Striatal neurons directly converted from Huntington's disease patient fibroblasts recapitulate age-associated disease phenotypes.

Nat Neurosci 2018 03 5;21(3):341-352. Epub 2018 Feb 5.

Department of Developmental Biology, Center for Regenerative Medicine, Washington University School of Medicine, St. Louis, MO, USA.

In Huntington's disease (HD), expansion of CAG codons in the huntingtin gene (HTT) leads to the aberrant formation of protein aggregates and the differential degeneration of striatal medium spiny neurons (MSNs). Modeling HD using patient-specific MSNs has been challenging, as neurons differentiated from induced pluripotent stem cells are free of aggregates and lack an overt cell death phenotype. Here we generated MSNs from HD patient fibroblasts through microRNA-based direct neuronal conversion, bypassing the induction of pluripotency and retaining age signatures of the original fibroblasts. We found that patient MSNs consistently exhibited mutant HTT (mHTT) aggregates, mHTT-dependent DNA damage, mitochondrial dysfunction and spontaneous degeneration in culture over time. We further provide evidence that erasure of age stored in starting fibroblasts or neuronal conversion of presymptomatic HD patient fibroblasts results in differential manifestation of cellular phenotypes associated with HD, highlighting the importance of age in modeling late-onset neurological disorders.
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http://dx.doi.org/10.1038/s41593-018-0075-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857213PMC
March 2018

Unravelling Endogenous MicroRNA System Dysfunction as a New Pathophysiological Mechanism in Machado-Joseph Disease.

Mol Ther 2017 04 22;25(4):1038-1055. Epub 2017 Feb 22.

CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, Coimbra 3004-504, Portugal; Faculty of Pharmacy, University of Coimbra, Coimbra 3000-548, Portugal. Electronic address:

Machado-Joseph disease (MJD) is a genetic neurodegenerative disease caused by an expanded polyglutamine tract within the protein ataxin-3 (ATXN3). Despite current efforts, MJD's mechanism of pathogenesis remains unclear and no disease-modifying treatment is available. Therefore, in this study, we investigated (1) the role of the 3' UTR of ATXN3, a putative microRNA (miRNA) target, (2) whether miRNA biogenesis and machinery are dysfunctional in MJD, and (3) which specific miRNAs target ATXN3-3' UTR and whether they can alleviate MJD neuropathology in vivo. Our results demonstrate that endogenous miRNAs, by targeting sequences in the 3' UTR, robustly reduce ATXN3 expression and aggregation in vitro and neurodegeneration and neuroinflammation in vivo. Importantly, we found an abnormal MJD-associated downregulation of genes involved in miRNA biogenesis and silencing activity. Finally, we identified three miRNAs-mir-9, mir-181a, and mir-494-that interact with the ATXN3-3' UTR and whose expression is dysregulated in human MJD neurons and in other MJD cell and animal models. Furthermore, overexpression of these miRNAs in mice resulted in reduction of mutATXN3 levels, aggregate counts, and neuronal dysfunction. Altogether, these findings indicate that endogenous miRNAs and the 3' UTR of ATXN3 play a crucial role in MJD pathogenesis and provide a promising opportunity for MJD treatment.
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http://dx.doi.org/10.1016/j.ymthe.2017.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383579PMC
April 2017
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