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Contents The intent of this contribution is to provide an update of the progress we have made towards developing a method/treatment to permanently sterilize cats. Our approach employs two complementary methodologies: RNA interference ( RNAi) to silence genes involved in the central control of reproduction and a virus-based gene therapy system intended to deliver RNAi selectively to the hypothalamus (where these genes are expressed) via the systemic administration of modified viruses. We selected the hypothalamus because it contains neurons expressing Kiss1 and Tac3, two genes essential for reproduction and fertility. We chose the non-pathogenic adeno-associated virus ( AAV) as a vector whose tropism could be modified to target the hypothalamus. The issues that must be overcome to utilize this vector as a delivery vehicle to induce sterility include modification of the wild-type AAV to target the hypothalamic region of the brain with a simultaneous reduction in targeting of peripheral tissues and non-hypothalamic brain regions, identification of RNAi targets that will effectively reduce the expression of Kiss1 and Tac3 without off-target effects, and determination if neutralizing antibodies to the AAV serotype of choice are present in cats. Successful resolution of these issues will pave the way for the development of a powerful tool to induce the permanent sterility in cats. [ABSTRACT FROM AUTHOR]

Background: Inferior vena cava (IVC) filter placement may be life-saving, but after contraindications to anticoagulation remit, patient management is uncertain.Methods: We followed patients who had venous thromboembolism, followed by treatment with permanent IVC filter placement, and were anticoagulated long-term as soon as safety allowed. We conducted annual physical examinations and ultrasound surveillance of the lower extremity deep veins and of the IVC filter site. Clot detected at the filter site was treated with graded intensities of anticoagulation, depending on the clot burden.Results: Symptomatic DVT occurred in 24 of 121 patients (20%; 95% CI, 14%-28%); symptomatic pulmonary embolism (one fatal) was diagnosed in six patients (5%; 95% CI, 2%-10%). There were 45 episodes of filter clot in 36 patients (30%; 95% CI, 22%-38%). The rate of major bleeding (6.6%) was similar to that of a concurrent persistently anticoagulated cohort without IVC filters (5.8%).Conclusions: If therapeutic anticoagulation can be safely begun in patients with IVC filters inserted after venous thromboembolism, further management with clinical surveillance, including ultrasound examination of the IVC filter and graded degrees of anticoagulation therapy if filter clot is detected, has a favorable prognosis. This approach appears valid for patients with current IVC filter and can serve as a comparison standard in subsequent clinical trials to optimize clinical management of these patients. [ABSTRACT FROM AUTHOR]

Background: The current standard initial therapies for deep venous thrombosis are low-molecular-weight heparin and unfractionated heparin. In a dose-ranging study of patients with symptomatic deep venous thrombosis, fondaparinux had efficacy and a safety profile similar to those of low-molecular-weight heparin (dalteparin).Objective: To evaluate whether fondaparinux has efficacy and safety similar to those of enoxaparin in patients with deep venous thrombosis.Design: Randomized, double-blind study.Setting: 154 centers worldwide.Patients: 2205 patients with acute symptomatic deep venous thrombosis.Intervention: Fondaparinux, 7.5 mg (5.0 mg in patients weighing <50 kg and 10.0 mg in patients weighing >100 kg) subcutaneously once daily, or enoxaparin, 1 mg/kg of body weight, subcutaneously twice daily for at least 5 days and until vitamin K antagonists induced an international normalized ratio greater than 2.0.Measurements: The primary efficacy outcome was the 3-month incidence of symptomatic recurrent venous thromboembolic complications. The main safety outcomes were major bleeding during initial treatment and death. An independent, blinded committee adjudicated all outcomes.Results: 43 (3.9%) of 1098 patients randomly assigned to fondaparinux had recurrent thromboembolic events compared with 45 (4.1%) of 1107 patients randomly assigned to enoxaparin (absolute difference, -0.15 percentage point [95% CI, -1.8 to 1.5 percentage points]). Major bleeding occurred in 1.1% of patients receiving fondaparinux and 1.2% of patients receiving enoxaparin. Mortality rates were 3.8% and 3.0%, respectively.Limitations: Follow-up was incomplete in 0.4% of fondaparinux-treated patients and 1.0% of enoxaparin-treated patients.Conclusions: Once-daily subcutaneous fondaparinux was at least as effective (not inferior) and safe as twice-daily, body weight-adjusted enoxaparin in the initial treatment of patients with symptomatic deep venous thrombosis. [ABSTRACT FROM AUTHOR]

The diagnosis of venous thromboembolic disease, and pulmonary embolism in particular, remains problematic. Physicians should strongly consider empiric anticoagulation if the best available diagnostic tests are inconclusive, because treatment is usually safe and successful. Twice-daily subcutaneous low-molecular-weight heparin, dosed without monitoring, may eventually replace standard heparin for most treatment of venous thromboembolism, but it is not yet labeled for the treatment of pulmonary embolism. Deep venous thromboembolism and pulmonary embolism should be treated with anticoagulants rather than inferior vena cava filters, even in oncology patients, unless anticoagulation is contraindicated; if so, when the contraindication remits, anticoagulation should be employed. The most effective prophylaxis of venous thromboembolism in at-risk patients should be used, with prolonged duration if evidence from clinical trials supports efficacy and safety. Low-dose warfarin should be used to prevent venous thrombosis and indwelling central venous catheter thrombosis in patients with cancer. [ABSTRACT FROM AUTHOR]

Background: Heparin-induced thrombocytopenia (HIT) is an antibody-mediated adverse drug reaction that can lead to devastating thromboembolic complications, including pulmonary embolism, ischemic limb necrosis necessitating limb amputation, acute myocardial infarction, and stroke.Methods: The methods of this guideline follow the Methodology for the Development of Antithrombotic Therapy and Prevention of Thrombosis Guidelines: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines in this supplement.Results: Among the key recommendations for this article are the following: For patients receiving heparin in whom clinicians consider the risk of HIT to be > 1%, we suggest that platelet count monitoring be performed every 2 or 3 days from day 4 to day 14 (or until heparin is stopped, whichever occurs first) (Grade 2C). For patients receiving heparin in whom clinicians consider the risk of HIT to be < 1%, we suggest that platelet counts not be monitored (Grade 2C). In patients with HIT with thrombosis (HITT) or isolated HIT who have normal renal function, we suggest the use of argatroban or lepirudin or danaparoid over other nonheparin anticoagulants (Grade 2C). In patients with HITT and renal insufficiency, we suggest the use of argatroban over other nonheparin anticoagulants (Grade 2C). In patients with acute HIT or subacute HIT who require urgent cardiac surgery, we suggest the use of bivalirudin over other nonheparin anticoagulants or heparin plus antiplatelet agents (Grade 2C).Conclusions: Further studies evaluating the role of fondaparinux and the new oral anticoagulants in the treatment of HIT are needed. [ABSTRACT FROM AUTHOR]

Cost-effectiveness information can help health-system participants make decisions about diagnostic or therapeutic innovations that are more expensive but incrementally safe and effective. However, these analyses cannot help decide whether a less expensive approach is "sufficient" and funds ought to be allocated to other medical issues entirely. At present, formulary committees are commonly determining that medications are "mostly equivalent" in efficacy and safety and choosing individual medications or classes of medications based on cost. Clinicians may not agree with these decisions and will need to understand their rationale. For prophylaxis of venous thromboembolism after hip and knee replacement and hip fracture surgery, many preventive modalities are available with different safety and efficacy profiles and different costs. It is possible to list the important safety and efficacy outcomes of prophylaxis, estimate their incidences and costs, and "model" comparisons of one modality vs another, in order to help decide whether one is preferable. [ABSTRACT FROM AUTHOR]

Background: A fixed-dose regimen of rivaroxaban, an oral factor Xa inhibitor, has been shown to be as effective as standard anticoagulant therapy for the treatment of deep-vein thrombosis, without the need for laboratory monitoring. This approach may also simplify the treatment of pulmonary embolism.Methods: In a randomized, open-label, event-driven, noninferiority trial involving 4832 patients who had acute symptomatic pulmonary embolism with or without deep-vein thrombosis, we compared rivaroxaban (15 mg twice daily for 3 weeks, followed by 20 mg once daily) with standard therapy with enoxaparin followed by an adjusted-dose vitamin K antagonist for 3, 6, or 12 months. The primary efficacy outcome was symptomatic recurrent venous thromboembolism. The principal safety outcome was major or clinically relevant nonmajor bleeding.Results: Rivaroxaban was noninferior to standard therapy (noninferiority margin, 2.0; P=0.003) for the primary efficacy outcome, with 50 events in the rivaroxaban group (2.1%) versus 44 events in the standard-therapy group (1.8%) (hazard ratio, 1.12; 95% confidence interval [CI], 0.75 to 1.68). The principal safety outcome occurred in 10.3% of patients in the rivaroxaban group and 11.4% of those in the standard-therapy group (hazard ratio, 0.90; 95% CI, 0.76 to 1.07; P=0.23). Major bleeding was observed in 26 patients (1.1%) in the rivaroxaban group and 52 patients (2.2%) in the standard-therapy group (hazard ratio, 0.49; 95% CI, 0.31 to 0.79; P=0.003). Rates of other adverse events were similar in the two groups.Conclusions: A fixed-dose regimen of rivaroxaban alone was noninferior to standard therapy for the initial and long-term treatment of pulmonary embolism and had a potentially improved benefit-risk profile. (Funded by Bayer HealthCare and Janssen Pharmaceuticals; EINSTEIN-PE ClinicalTrials.gov number, NCT00439777.). [ABSTRACT FROM AUTHOR]

Molecular-level interactions between glucosamine and glycerol have shed light on the specific binding motif required for the plasticization of chitosan with glycerol via the gel theory mechanism. Here, we describe a spectroscopic study of the intermolecular interactions between the monomeric repeat unit of chitosan, glucosamine, and simplified 1,2- and 1,3-diol units of glycerol (i.e, 1,3-propanediol and ethylene glycol). The material properties of chitosan films containing these diols at varying concentrations were characterized using ATR-IR, DMA, TGA, and SEM. The combined results indicate that these diols plasticize chitosan via the lubricity theory mechanism, which differs from glycerol that plasticizes via the gel theory mechanism. At low concentrations, this difference in mechanism has a minimal impact on the material properties. However, at high concentrations of the diols, the necessity of a secondary hydrogen bonding interaction for the retention of chitosan plasticization is observed with a significant increase in the Young's modulus of the materials. The impact of hydrophobicity within the diols was also investigated in chitosan films using 1,2-propanediol and 2-methyl-1,3-propanediol. The combined analyses provide strong evidence that both primary and secondary interactions are responsible for determining the mechanism of chitosan plasticization., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Recombinant adeno-associated virus (rAAV) vectors are an effective and well-established tool in the growing gene therapy field, with five FDA-approved AAV-mediated gene therapies already on the market and numerous more in clinical trials. However, manufacturing rAAV vectors is an expensive, timely, and labor-intensive process that limits the commercial use of AAV-mediated gene therapies. To address this limitation, we screened producer cells for genes that could be targeted to increase rAAV yield. Specifically, we performed a CRISPR-based genome-wide knockout screen in HEK 293 cells using an antibody specific to intact AAV2 capsids coupled with flow cytometry to identify genes that modulate rAAV production. We discovered that the knockout of a group of heparan sulfate biosynthesis genes previously implicated in rAAV infectivity decreased rAAV production. Additionally, we identified several vesicular trafficking proteins for which knockout in HEK 293 cells increased rAAV yields. Our findings provide evidence that host proteins associated with viral infection may have also been co-opted for viral assembly and that the genetic makeup of viral producer cells can be manipulated to increase particle yield., Competing Interests: DECLARATION OF INTERESTS: B.L.D. serves on the advisory board of Latus Biosciences, Patch Bio, Spirovant Biosciences, Resilience, and Carbon Biosciences and has sponsored research unrelated to this work from Roche, Latus, and Spirovant. Authors have filed a patent related to this manuscript through the Children’s Hospital of Philadelphia.

Spinocerebellar ataxia type 1 (SCA1) is an adult-onset neurodegenerative disease caused by an expansion of the CAG repeat region of the ATXN1 gene. Currently there are no disease-modifying treatments; however, previous work has shown the potential of gene therapy, specifically RNAi, as a potential modality. Cas9 editing offers potential for these patients but has yet to be evaluated in SCA1 models. To test this, we first characterized the number of transgenes harbored in the common B05 mouse model of SCA1. Despite having five copies of the human mutant transgene, a 20% reduction of ATXN1 improved behavior deficits without increases in inflammatory markers. Importantly, the editing approach was confirmed in induced pluripotent stem cell (iPSC) neurons derived from patients with SCA1, promoting the translatability of the approach to patients., Competing Interests: B.L.D. serves on the advisory board of Latus Biosciences, Spirovant Biosciences, Resilience, and Carbon Biosciences. She has sponsored research from Roche, Latus, and Saliogen., (Crown Copyright © 2024 Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.)

Heterozygous variants in SYNGAP1 and STXBP1 lead to distinct neurodevelopmental disorders caused by haploinsufficient levels of post-synaptic SYNGAP1 and pre-synaptic STXBP1, which are critical for normal synaptic function. While several gene-targeted therapeutic approaches have proven efficacious in vitro , these often target regions of the human gene that are not conserved in rodents, hindering the pre-clinical development of these compounds and their transition to the clinic. To overcome this limitation, here we generate and characterize Syngap1 and Stxbp1 humanized mouse models in which we replaced the mouse Syngap1 and Stxbp1 gene, respectively, with the human counterpart, including regulatory and non-coding regions. Fully humanized Syngap1 mice present normal viability and can be successfully crossed with currently available Syngap1 haploinsufficiency mouse models to generate Syngap1 humanized haploinsufficient mice. Stxbp1 mice were successfully humanized, yet exhibit impaired viability (particularly males) and reduced STXBP1 protein abundance. Mouse viability could be improved by outcrossing this model to other mouse strains, while Stxbp1 humanized females and hybrid mice can be used to evaluate target engagement of human-specific therapeutics. Overall, these humanized mouse models represent a broadly available tool to further pre-clinical therapeutic development for SYNGAP1 and STXBP1 disorders., Competing Interests: Conflicts of interest BLP and BLD are inventors on two patents relevant to therapeutic development for neurodevelopmental disorders, PCT/US2020/031672 and UPN-22–9943. AJF is an inventor on UPN-22–9943.

Recombinant adeno-associated virus (rAAV)-based gene therapies are expanding in their application. Despite progress in manufacturing, current analytical methods for product quantification and characterization remain largely unchanged. Although critical for product and process development, in-process testing, and batch release, current analytical methods are labor-intensive, costly, and hampered by extended turnaround times and low throughput. The field requires more efficient, cost-effective analytical techniques capable of handling large sample quantities to accelerate product and process development. Here, we evaluated Stunner from Unchained Labs for quantifying and characterizing rAAVs and compared it with established analytical methods. Stunner is a combinatorial analytic technology platform that interpolates ultraviolet-visible (UV-Vis) absorption with static and dynamic light scattering (SLS/DLS) analysis to determine capsid and genomic titer, empty and full capsid ratio, and assess vector size and polydispersity. The platform offers empirical measurements with minimal sample requirements. Upon testing hundreds of rAAV vectors, comprising various serotypes and transgenes, the data show a strong correlation with established analytical methods and exhibit high reproducibility and repeatability. Some analyses can be applied to in-process samples from different purification stages and processes, fulfilling the demand for rapid, high-throughput analysis during development. In sum, the pipeline presented streamlines small- and large-batch analytics., Competing Interests: B.L.D. serves an advisory role and/or receives sponsored research support for her laboratory from Roche, Resilience, and Latus Bio. R.J.-G. and K.L. are employees of Unchained Labs. J.C.M.v.d.L. serves as consultant and/or scientific advisor for 4BIO Capital, Avantor, Vibalogics, and Vyriad., (© 2024 The Authors.)

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 Aβ 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., Competing Interests: Declaration of interests D.M.H. is on the scientific advisory board of C2N Diagnostics and has equity. D.M.H. is on the scientific advisory board of Denali Therapeutics, Genentech, and Cajal Therapeutics and consults for Asteroid. B.L.D. serves an advisory role with equity in Latus Biosciences, Patch Bio, Voyager Therapeutics, Carbon Biosciences, Spirovant Biosciences, Resilience, Panorama Medicines, Saliogen, and Homology Medicines. She has sponsored research from Novartis, Roche, Latus, Homology Medicines, Saliogen, and Spirovant. B.T.H. is on the scientific advisory board of Latus Bio and has an equity interest. B.T.H. has a family member who works at Novartis, and owns stock in Novartis; he serves on the scientific advisory board of Dewpoint and owns stock. He serves on a scientific advisory board of or is a consultant for AbbVie, Aprinoia Therapeutics, Arvinas, Avrobio, Axial, Biogen, BMS, Cure Alzheimer’s Fund, Cell Signaling, Dewpoint, Eisai, Genentech, Ionis, Latus, Novartis, Sangamo, Sanofi, Seer, Takeda, the US Department of Justice, Vigil, and Voyager. M.S.K., L.T., Y.C., and P.T.R. are founders of and shareholders in Latus Biosciences., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both in vitro and in vivo . We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency in vivo , supporting the use of the X on cassette over traditional constitutively expressing AAV approaches. These data depicting improvements to deletions and insight into allelic heterogeneity after CRISPR-del will inform therapeutic approaches for phenotypes that require either large mono- or biallelic deletions, such as autosomal recessive diseases or where mutant allele-specific gRNAs are not readily available, or in situations where the targeted sequence for excision is located multiple times in a genome., Competing Interests: B.L.D. serves on the advisory board of Latus Bio, Patch Bio, Spirovant Biosciences, Resilience, and Carbon Biosciences and has sponsored research unrelated to this work from Roche, Latus, and Spirovant., (© 2024 The Authors.)

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., Competing Interests: Declaration of Competing Interest RAN is an advisor for LatusBio and Orchard Therpeutics. All other authors declare no conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Recombinant adeno-associated viral vectors (rAAVs) are a promising strategy to treat neurodegenerative diseases because of their ability to infect non-dividing cells and confer long-term transgene expression. Despite an ever-growing library of capsid variants, widespread delivery of AAVs in the adult central nervous system remains a challenge. We have previously demonstrated successful distribution of secreted proteins by infection of the ependyma, a layer of post-mitotic epithelial cells lining the ventricles of the brain and central column of the spinal cord, and subsequent protein delivery via the cerebrospinal fluid (CSF). Here we define a functional ependyma promoter to enhance expression from this cell type. Using RNA sequencing on human autopsy samples, we identified disease- and age-independent ependyma gene signatures. Associated promoters were cloned and screened as libraries in mouse and rhesus macaque to reveal cross-species function of a human DNA-derived von Willebrand factor domain containing 3A ( VWA3A ) promoter. When tested in mice, our VWA3A promoter drove strong, ependyma-localized expression of eGFP and increased secreted ApoE protein levels in the CSF by 2-12× over the ubiquitous iCAG promoter., Competing Interests: B.L.D. serves an advisory role with equity in Latus Biosciences, Patch Bio, Voyager Therapeutics, Carbon Biosciences, Spirovant Biosciences, Resilience, Panorama Medicines, Saliogen, and Homology Medicines. She has sponsored research from Novartis, Roche, Latus, Homology Medicines, Saliogen, and Spirovant. B.T.H. is on the scientific advisory board of Latus Bio and has an equity interest. He has a family member who works at Novartis and owns stock in Novartis; he serves on the SAB of Dewpoint and owns stock. He serves on a scientific advisory board or is a consultant for AbbVie, Aprinoia Therapeutics, Arvinas, Avrobio, Axial, Biogen, BMS, Cure Alz Fund, Cell Signaling, Dewpoint, Eisai, Genentech, Ionis, Latus, Novartis, Sangamo, Sanofi, Seer, Takeda, the US Department of Justice, Vigil, and Voyager., (© 2023 The Authors.)

Alternative splicing of neuronal genes is controlled partly by the coordinated action of polypyrimidine tract binding proteins (PTBPs). While PTBP1 is ubiquitously expressed, PTBP2 is predominantly neuronal. Here, we define the PTBP2 footprint in the human transcriptome using brain tissue and human induced pluripotent stem cell-derived neurons (iPSC-neurons). We map PTBP2 binding sites, characterize PTBP2-dependent alternative splicing events, and identify novel PTBP2 targets including SYNGAP1, a synaptic gene whose loss-of-function leads to a complex neurodevelopmental disorder. We find that PTBP2 binding to SYNGAP1 mRNA promotes alternative splicing and nonsense-mediated decay, and that antisense oligonucleotides (ASOs) that disrupt PTBP binding redirect splicing and increase SYNGAP1 mRNA and protein expression. In SYNGAP1 haploinsufficient iPSC-neurons generated from two patients, we show that PTBP2-targeting ASOs partially restore SYNGAP1 expression. Our data comprehensively map PTBP2-dependent alternative splicing in human neurons and cerebral cortex, guiding development of novel therapeutic tools to benefit neurodevelopmental disorders., (© 2023. The Author(s).)

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., Competing Interests: Declaration of interests B.L.D. is a founder of Spark Therapeutics, Spirovant Sciences and Latus Biosciences. She serves an advisory role and/or receives sponsored research support for her laboratory from Roche, NBIR, Homology Medicines, Resilience, Spirovant Sciences, Patch Biosciences, Saliogen therapeutics, Panorama Medicines, and Voyager Therapeutics. B.P.S., C.M.Y., and A.I. have no competing interests. B.P.S and B.L.D. are co-inventors on U.S. Patent Application No. 17/594,651 entitled: CRISPR/Cas9 Gene Editing of ATXN2 for the Treatment of Spinocerebellar Ataxia Type 2., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)