Antagonistic roles of canonical and Alternative-RPA in disease-associated tandem CAG repeat instability.

Expansions of repeat DNA tracts cause >70 diseases, and ongoing expansions in brains exacerbate disease. During expansion mutations, single-stranded DNAs (ssDNAs) form slipped-DNAs. We find the ssDNA-binding complexes canonical replication protein A (RPA1, RPA2, and RPA3) and Alternative-RPA (RPA1, RPA3, and primate-specific RPA4) are upregulated in Huntington disease and spinocerebellar ataxia type 1 (SCA1) patient brains. Protein interactomes of RPA and Alt-RPA reveal unique and shared partners, including modifiers of CAG instability and disease presentation. RPA enhances in vitro melting, FAN1 excision, and repair of slipped-CAGs and protects against CAG expansions in human cells. RPA overexpression in SCA1 mouse brains ablates expansions, coincident with decreased ATXN1 aggregation, reduced brain DNA damage, improved neuron morphology, and rescued motor phenotypes. In contrast, Alt-RPA inhibits melting, FAN1 excision, and repair of slipped-CAGs and promotes CAG expansions. These findings suggest a functional interplay between the two RPAs where Alt-RPA may antagonistically offset RPA's suppression of disease-associated repeat expansions, which may extend to other DNA processes.

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

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.

Singles engage the RNA interference pathway.

Single-stranded RNAs interact with components of the RNA interference pathway to reduce the expression of target mRNAs. Now, Lima et al. and Yu et al. show that, with extensive chemical modifications, small single-stranded RNAs can robustly induce gene silencing with efficacy similar to their double-stranded counterparts.

Regulated control of gene therapies by drug-induced splicing.

So far, gene therapies have relied on complex constructs that cannot be finely controlled1,2. 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 Xon, does not require the co-expression of any regulatory proteins. Using Xon, 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 Xon 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 Xon switch system provides an unprecedented opportunity to refine and tailor the application of gene therapies in humans.

APOE2 gene therapy reduces amyloid deposition and improves markers of neuroinflammation and neurodegeneration in a mouse model of Alzheimer disease.

Epidemiological studies show that individuals who carry the relatively uncommon APOE ε2 allele rarely develop Alzheimer disease, and if they do, they have a later age of onset, milder clinical course, and less severe neuropathological findings than people without this allele. The contrast is especially stark when compared with the major genetic risk factor for Alzheimer disease, APOE ε4, which has an age of onset several decades earlier, a more aggressive clinical course and more severe neuropathological findings, especially in terms of the amount of amyloid deposition. Here, we demonstrate that brain exposure to APOE ε2 via a gene therapy approach, which bathes the entire cortical mantle in the gene product after transduction of the ependyma, reduces Aß plaque deposition, neurodegenerative synaptic loss, and, remarkably, reduces microglial activation in an APP/PS1 mouse model despite continued expression of human APOE ε4. This result suggests a promising protective effect of exogenous APOE ε2 and reveals a cell nonautonomous effect of the protein on microglial activation, which we show is similar to plaque-associated microglia in the brain of Alzheimer disease patients who inherit APOE ε2. These data increase the potential that an APOE ε2 therapeutic could be effective in Alzheimer disease, even in individuals born with the risky ε4 allele.

A novel iPSC model reveals selective vulnerability of neurons in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is an ultra-rare, inherited lysosomal storage disease caused by mutations in the gene sulfatase modifying factor 1 (SUMF1). MSD is characterized by the functional deficiency of all sulfatase enzymes, leading to the storage of sulfated substrates including glycosaminoglycans (GAGs), sulfolipids, and steroid sulfates. Patients with MSD experience severe neurological impairment, hearing loss, organomegaly, corneal clouding, cardiac valve disease, dysostosis multiplex, contractures, and ichthyosis. Here, we generated a novel human model of MSD by reprogramming patient peripheral blood mononuclear cells to establish an MSD induced pluripotent stem cell (iPSC) line (SUMF1 p.A279V). We also generated an isogenic control iPSC line by correcting the pathogenic variant with CRISPR/Cas9 gene editing. We successfully differentiated these iPSC lines into neural progenitor cells (NPCs) and NGN2-induced neurons (NGN2-iN) to model the neuropathology of MSD. Mature neuronal cells exhibited decreased SUMF1 gene expression, increased lysosomal stress, impaired neurite outgrowth and maturation, reduced sulfatase activities, and GAG accumulation. Interestingly, MSD iPSCs and NPCs did not exhibit as severe of phenotypes, suggesting that as neurons differentiate and mature, they become more vulnerable to loss of SUMF1. In summary, we demonstrate that this human iPSC-derived neuronal model recapitulates the cellular and biochemical features of MSD. These cell models can be used as tools to further elucidate the mechanisms of MSD pathology and for the development of therapeutics.

Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define in vivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.

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

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.

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

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.

Regional Variation of Splicing QTLs in Human Brain.

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.

Neuronal genetic rescue normalizes brain network dynamics in a lysosomal storage disorder despite persistent storage accumulation.

Although neurologic symptoms occur in two-thirds of lysosomal storage disorders (LSDs), for most we do not understand the mechanisms underlying brain dysfunction. A major unanswered question is if the pathogenic hallmark of LSDs, storage accumulation, induces functional defects directly or is a disease bystander. Also, for most LSDs we do not know the impact of loss of function in individual cell types. Understanding these critical questions are essential to therapy development. Here, we determine the impact of genetic rescue in distinct cell types on neural circuit dysfunction in CLN3 disease, the most common pediatric dementia and a paradigmatic neurodegenerative LSD. We restored Cln3 expression via AAV-mediated gene delivery and conditional genetic rescue in a CLN3 disease mouse model. Surprisingly, we found that low-level rescue of Cln3 expression in neurons alone normalized clinically relevant electrophysiologic markers of network dysfunction, despite the presence of substantial residual histopathology, in contrast to restoring expression in astrocytes. Thus, loss of CLN3 function in neurons, not storage accumulation, underlies neurologic dysfunction in CLN3 disease. This impliesies that storage clearance may be an inappropriate target for therapy development and an ineffectual biomarker.

Evaluating the state of the science for adeno-associated virus integration: An integrated perspective.

On August 18, 2021, the American Society of Gene and Cell Therapy (ASGCT) hosted a virtual roundtable on adeno-associated virus (AAV) integration, featuring leading experts in preclinical and clinical AAV gene therapy, to further contextualize and understand this phenomenon. Recombinant AAV (rAAV) vectors are used to develop therapies for many conditions given their ability to transduce multiple cell types, resulting in long-term expression of transgenes. Although most rAAV DNA typically remains episomal, some rAAV DNA becomes integrated into genomic DNA at a low frequency, and rAAV insertional mutagenesis has been shown to lead to tumorigenesis in neonatal mice. Currently, the risk of rAAV-mediated oncogenesis in humans is theoretical because no confirmed genotoxic events have been reported to date. However, because insertional mutagenesis has been reported in a small number of murine studies, there is a need to characterize this genotoxicity to inform research, regulatory needs, and patient care. The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance, and bioethics are discussed.

Gene-based therapeutics for rare genetic neurodevelopmental psychiatric disorders.

We are in an emerging era of gene-based therapeutics with significant promise for rare genetic disorders. The potential is particularly significant for genetic central nervous system disorders that have begun to achieve Food and Drug Administration approval for select patient populations. This review summarizes the discussions and presentations of the National Institute of Mental Health-sponsored workshop "Gene-Based Therapeutics for Rare Genetic Neurodevelopmental Psychiatric Disorders," which was held in January 2021. Here, we distill the points raised regarding various precision medicine approaches related to neurodevelopmental and psychiatric disorders that may be amenable to gene-based therapies.

Gene therapy for ALS: A review.

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.

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

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.

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

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.

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

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.

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

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.