Recombinant adeno-associated virus (AAV) vectors are predominantly nonintegrating, but rare genomic integration events have been associated with oncogenesis in neonatal murine models. Here we report a case of a neuroepithelial tumor that developed in a 5-year-old boy with severe mucopolysaccharidosis type I (MPSI, Hurler subtype) 4 years after intracisternal magna administration of AAV serotype 9 gene therapy. The patient underwent successful resection of the primary tumor. Postoperatively, he has continued to have advanced cognitive function for his age, a finding that indicates mitigation of MPSI. Molecular analysis of the tumor showed clonal integration of rearranged AAV vector elements into the gene PLAG1 and expression of a chimeric AAV-PLAG1 transcript.

Gene therapies have demonstrated transformative potential for a range of genetic disorders, including immunodeficiencies, hematopoietic conditions, and neuromuscular diseases. However, the application of these approaches to cystic fibrosis (CF) and other airway diseases remains constrained by the challenge of efficient gene delivery to target epithelial cells. Adeno-associated virus (AAV) vectors are widely used for in vivo gene delivery due to their favorable safety profile and capacity for long-term transgene expression in non-dividing cells. Nonetheless, current AAV capsids require high doses to achieve therapeutic efficacy in the airways, raising safety concerns. Here we report the development of novel AAV capsid variants with markedly enhanced transduction efficiency of airway epithelial cells. Using unbiased peptide-modified AAV libraries and round-over-round screening in well-differentiated primary cultures of human airway epithelia (HAE), we identified 20 novel capsids that efficiently transduced cells at doses 10- to 100-fold lower than those required by existing vectors (termed AAV-AE). These variants demonstrated high transgene expression in HAE, primary human basal cells, tracheal explants from nonhuman primates, and murine airways in vivo. These optimized AAV capsids represent a significant advancement in pulmonary gene therapy, offering a versatile platform for the delivery of gene addition and editing reagents to treat CF and other respiratory diseases.

Huntington's disease involves progressive corticostriatal dysfunction; however, the timing of region-specific transcriptional changes remains unresolved at cellular resolution. Here, we provide a temporal single-nucleus transcriptomic atlas of striatum and motor cortex from zQ175 knock-in mice at 6 and 18 months. This full-length Huntingtin model enables staging of progressive circuit dysfunction. Striatal projection neurons show extensive early dysregulation with progressive striosomal identity erosion, whereas cortical pyramidal neuron dysfunction was layer-specific and coincided with motor symptom onset. By modeling genotype-dependent effects, we distinguish region- and cell type-specific signatures of core disease mechanisms from age-related changes and compensatory adaptations. Integrating gene co-expression and transcription factor regulatory networks, we predict candidate regulators of stage-specific dysfunction. These findings, validated across human HD and mouse model datasets, reveal temporal dynamics of disease pathogenesis in regionally distinct and interconnected neuronal populations, establishing a framework for understanding cell type-selective vulnerability.

Abstract Background Huntington’s disease (HD) involves progressive corticostriatal dysfunction, yet the temporal dynamics and cell type-specific vulnerability patterns remain incompletely understood. While recent single-cell studies in rapidly progressing models have revealed early developmental and regional changes, temporal profiling distinguishing pathogenic mechanisms from normal aging in full-length HTT models remains lacking. Resolving stage-specific temporal dynamics across interconnected striatal and cortical neuronal populations over protracted time is essential for identifying drivers of cellular dysfunction. Methods A temporal single-nucleus transcriptomic atlas was generated from striatum and motor cortex from heterozygous zQ175 knock-in mice at early symptomatic (6 months) and late symptomatic (18 months) stages. This full-length huntingtin model enables staging of progressive circuit dysfunction alongside physiological aging. The high inherited CAG repeat length of the zQ175 model places cells beyond the somatic expansion threshold associated with transcriptional dysregulation and identity erosion in vulnerable human neuronal populations, yet prior to the de-repression crisis and cell loss observed at the most extreme expansions in HD, providing a tractable window into the progressive molecular pathogenic cascade. Genotype-dependent effects were modeled to distinguish cell type-specific signatures of disease mechanisms from age-related and compensatory changes. Integration of weighted gene co-expression and transcription factor regulatory networks with protein-protein interaction databases predicted candidate regulators of stage-specific programs. Findings were validated across human HD datasets and the rapidly progressive R6/2 mouse model. Results Temporal gene and network analysis revealed diverging, converging and biphasic patterns of transcriptional changes, distinguishing progressive disease and neuronal identity loss from aging. 21 cell type-specific gene co-expression modules were validated in human HD and R6/2 mice datasets, revealing stage-specific shifts in cellular stress, proteostasis, and synaptic programs. Disease modules enriched for CAG repeat length-dependent genes resolved their temporal progression. Shared vulnerability across cortical and striatal projection neurons implicated epigenetic regulator Zswim6 and splicing factors Rbfox1 and Celf2 in corticostriatal dysfunction. Integrative network analysis identified Foxo1, Neurod2, and Npas2 as stage-specific transcriptional regulators. Cross-species validation established conserved gene regulatory modules in human HD, establishing generalizable cell type-specific gene modules of translational relevance. Conclusions This temporally resolved atlas reveals stage-specific transcriptional dynamics of disease-relevant gene expression programs and physiological trajectories in vulnerable neuronal populations. distinguished from aging alone. This work establishes an important framework for understanding the temporal and regional coordination of pathogenic mechanisms, providing molecular insights into stage-specific therapeutic intervention.

In February 2026, the US Food and Drug Administration (FDA) published a draft guidance on a new plausible mechanism framework for the development and approval of individualized therapies for genetic conditions. Here, we report initial proof-of-concept studies supporting a customizable prime editing platform geared to the treatment of 7 urea cycle disorders (UCDs) and other liver-centered disorders, as well as the outcome of a formal meeting with the FDA to discuss the use of the platform in an “umbrella-of-umbrellas” clinical trial including subjects with any of the 7 UCDs. We anticipate our findings will be of interest to academic investigators and industry sponsors who wish to pursue expeditious FDA approvals of therapies for ultra-rare diseases using the plausible mechanism framework.

Huntington's disease (HD) is caused by an expanded CAG trinucleotide repeat within the huntingtin (HTT) gene. Genetic modifiers of disease onset and progression in HD implicate somatic instability (SI) of the expanded CAG repeat as a key pathogenic driver, with MSH3 emerging as a leading therapeutic target. Reducing SI, particularly in the most affected neuronal cell type, medium spiny neurons (MSNs) of the striatum, is thus a rational therapeutic strategy for HD. To inform the development of an SI-targeted therapy, we generated a computational model simulating SI in MSNs to infer therapeutic effects on MSN survival resulting from an intervention that reduces SI. The model takes advantage of HD patient data to predict therapeutic benefit across a range of inherited CAG lengths and ages of intervention, considering the degree of target engagement regionally and per cell. To target SI experimentally, we designed an artificial microRNA to lower MSH3 mRNA (miMSH3) after delivery with AAV-DB-3, a previously described MSN-targeting AAV capsid variant. AAV-DB-3.miMSH3 achieved from 48 to 94% MSH3 mRNA reduction in MSNs of nonhuman primates (NHPs), which, when modeled, would reduce the composite Unified Huntington Disease Rating Scale change over baseline from 50 to over 120% as well as delay motor symptom onset by many years. AAV-DB-3.miMSH3 also showed robust target engagement in vivo with up to 46% reduction in SI in HdhQ111 mice. The integration of preclinical experimental data and the computational model support the translational potential of AAV-DB-3.miMSH3 as a disease modifying therapy applicable for HD patients with a broad range of inherited repeat lengths. ### Competing Interest Statement PTR, DEL and BLD are founders of Latus Bio. BPS, PTR, DEL, ARH, NS, CPC, CMF, JB, JCS, GJY, JC, PPG, and JJC are employees of Latus Bio. BLD has sponsored research and or serves an advisory role for Carbon Bio, Seamless Ther, and Latus Bio. PPG is a Latus Bio board member. All other authors declare that they have no competing interests. Hereditary Disease Foundation, https://ror.org/02a3w7r50, NA CHOP RI, NA Latus Bio, NA

Heterozygous variants in SYNGAP1 and STXBP1 cause distinct neurodevelopmental disorders due to haploinsufficiency of essential synaptic proteins. As gene targeted approaches to correct these disorders often target non-conserved genomic regions, thus limiting their clinical translation, we generated humanized mouse models wherein the entire Syngap1 or Stxbp1 loci were replaced with their human counterparts. Stxbp1 humanized mice exhibited impaired viability, while Stxbp1 hybrid mice (Stxbp1Hu/+) were viable and suitable for evaluating target engagement of human-specific therapeutics. Syngap1 humanized mice were viable and successfully crossed with Syngap1 heterozygous mice to produce a Syngap1 humanized-haploinsufficient model (Syngap1Hu/-). Syngap1Hu/- mice displayed haploinsufficient levels of human SYNGAP1, disease-relevant behaviors, and EEG abnormalities including epileptiform activity and generalized slowing. Importantly, parallel analysis in a cohort of patients with SYNGAP1-disorder revealed similar electrophysiological signatures. Finally, we showed that human gene-targeted antisense oligonucleotides modulate human SYNGAP1 expression in Syngap1Hu/- neurons. Together, we describe new models to support pre-clinical therapeutic development for SYNGAP1 and STXBP1 disorders and identify translational biomarkers of SYNGAP1-disorder in mice and humans to benchmark therapeutic testing.

Huntington’s disease (HD) is caused by a CAG repeat expansion in the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here we identify TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Disrupted nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 occurs in HD mouse and human brains, with TDP-43 also co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum of HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD. Nguyen et al. identify TDP-43 and METTL3 as key regulators of disrupted RNA splicing in Huntington’s disease, offering insight into how TDP-43 mislocalization and aberrant m6A RNA modification and localization relate to disease pathogenesis.

Gene therapy development, re-engineering, and application to patients hold promise to revolutionize medicine, including therapies for disorders of the brain. Advances in delivery modalities, expression regulation, and improving safety profiles are of critical importance. Additionally, each inherited disorder has its own unique characteristics as to regions and cell types impacted and the temporal dynamics of that impact that are essential for the design of therapeutic design strategies. Here, we review the current state of the art in gene therapies for inherited brain disorders, summarizing key considerations for vector delivery, gene addition, gene silencing, gene editing, and epigenetic editing. We provide examples from animal models, human cell lines, and, where possible, clinical trials. This review also highlights the various tools available to researchers for basic research questions and discusses our views on the current limitations in the field.

In the past 10 years, CRISPR-Cas9 has revolutionized the gene-editing field due to its modularity, simplicity, and efficacy. It has been applied for the creation of in vivo models, to further understand human biology, and toward the curing of genetic diseases. However, there remain significant delivery barriers for CRISPR-Cas9 application in the clinic, especially for in vivo and extrahepatic applications. In this work, high-throughput molecular barcoding techniques were used alongside traditional screening methodologies to simultaneously evaluate LNP formulations encapsulating ribonucleoproteins (RNPs) for in vitro gene-editing efficiency and in vivo biodistribution. This resulted in the identification of a lung-tropic LNP formulation, which shows efficient gene editing in endothelial and epithelial cells within the lung, targeting both model reporter and clinically relevant genomic targets. Further, this LNP shows no off-target indel formation in the liver, making it a highly specific extrahepatic delivery system for lung-editing applications.

Amyotrophic lateral sclerosis (ALS) involves motor neuron death due to mislocalized TDP-43. Pathologic TDP-43 associates with stress granules (SGs), and lowering the SG-associated protein ataxin-2 (ATXN2) using Atxn2-targeting antisense oligonucleotides prolongs survival in TAR4/4 sporadic ALS mice but failed in clinical trials likely due to poor target engagement. Here we show that an AAV with potent motor neuron transduction delivering Atxn2-targeting miRNAs reduces Atxn2 throughout the central nervous system at doses 40x lower than published work. In TAR4/4 mice, miAtxn2 increased survival (50%) and strength, and reduced motor neuron death, inflammation, and phosphorylated TDP-43. TAR4/4 transcriptomic dysregulation recapitulated ALS gene signatures that were rescued by miAtxn2, identifying potential therapeutic mechanisms and biomarkers. In slow progressing hemizygous mice, miAtxn2 slowed disease progression, and in ALS patient-derived lower motor neurons, our AAV vector transduced >95% of cells and potently reduced ATXN2 at MOI 4 logs lower than previously reported. These data support AAV-RNAi targeting ATXN2 as a translatable therapy for sporadic ALS.

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by a CTG repeat expansion that accumulate as toxic CUG repeat RNA. Functional sequestration of muscleblind-like (MBNL) proteins by CUG repeat RNA leads to deleterious, yet predictable changes in alternative splicing in DM. Genetic medicines for DM that reduce CUG repeat RNA or increase MBNL are advancing, but application of a viral-based approach must contend with high phenotypic and molecular variability. To address this, we have repurposed well-described cassette exons that are excluded by MBNL action to tune translational output of a therapeutic protein. We show that these splicing events can be taken out of genetic context and respond to changes in MBNL concentration or accumulation of toxic CUG repeat RNA, can deliver therapeutic MBNL1 protein to improve skeletal muscle myotonia or prevent cardiac toxicity associated with MBNL1 overexpression in mice, and distinguish DM patient-derived skeletal muscle myotubes from isogenic controls. Further work to fine-tune events to specific tissue targets and therapeutic cargos is needed, but these events can increase the therapeutic window for viral-based approaches for DM1. ### Competing Interest Statement The authors have declared no competing interest.

Cytoplasmic aggregation and concomitant dysfunction of the prion-like, RNA-binding protein TDP-43 underpin several fatal neurodegenerative diseases, including amyotrophic lateral sclerosis. To elucidate endogenous defenses, we systematically scoured the entire human Hsp70 network for buffers of TDP-43 toxicity. We identify 30 J-domain proteins (2 DNAJAs, 10 DNAJBs, 18 DNAJCs), 6 Hsp70s, and 5 nucleotide-exchange factors that mitigate TDP-43 toxicity. Specific chaperones reduce TDP-43 aggregate burden and detoxify diverse synthetic or disease-linked TDP-43 variants. Sequence-activity mapping unveiled unexpected, modular mechanisms of chaperone-mediated protection. Typically, DNAJBs collaborate with Hsp70 to suppress TDP-43 toxicity, whereas DNAJCs act independently. In human cells, specific chaperones increase TDP-43 solubility and enhance viability under proteotoxic stress. Strikingly, spliceosome-associated DNAJC8 and DNAJC17 retain TDP-43 in the nucleus and promote liquid-phase behavior. Thus, we disambiguate a diverse chaperone arsenal embedded in the human proteostasis network that counters TDP-43 toxicity and illuminate mechanistic gateways for therapeutic intervention in TDP-43 proteinopathies.

Tandem repeat (TR) size variation is implicated in ~50 neurological disorders, yet its impact on gene regulation in the human brain remains largely unknown. In the present study, we quantified the impact of TR size variation on brain gene regulation across distinct molecular phenotypes, based on 4,412 multi-omics samples from 1,597 donors, including 1,586 newly sequenced ones. We identified ~2.2 million TR molecular quantitative trait loci (TR-xQTLs), linking ~139,000 unique TRs to nearby molecular phenotypes, including many known disease-risk TRs, such as the G2C4 expansion in C9orf72 associated with amyotrophic lateral sclerosis. Fine-mapping revealed ~18,700 TRs as potential causal variants. Our in vitro experiments further confirmed the causal and independent regulatory effects of three TRs. Additional colocalization analysis indicated the potential causal role of TR variation in brain-related phenotypes, highlighted by a 3ʹ-UTR TR in NUDT14 linked to cortical surface area and a TG repeat in PLEKHA1, associated with Alzheimer’s disease. Mapping of multi-omic molecular quantitative trait loci associated with tandem repeat size variation in up to 4,412 human brain samples from 1,597 donors offers insights into how these variants affect gene regulation and mediate disease risk.

Recent work has shown that prolonged expression of recombinant proteins after adeno-associated virus (AAV)–mediated delivery of gene therapy to long-lived, ventricle-lining ependymal cells can profoundly affect disease phenotypes in animal models of neurodegenerative diseases. Here, we performed in vivo screens of millions of peptide-modified capsid variants of AAV1, AAV2, and AAV9 parental serotypes in adult nonhuman primates (NHPs) to identify capsids with potent transduction of key brain tissues, including ependyma, after intracerebroventricular injection. Through these screens, we identified an AAV capsid, AAV-Ep + , with markedly increased potency in transducing ependymal cells and cerebral neurons in NHPs. AAV-Ep + ’s potency was conserved in three species of NHP, two mouse strains, and human neurons derived from induced pluripotent stem cells. To apply AAV-Ep + to the treatment of ceroid lipofuscinosis type 2 disease, a lysosomal storage disorder caused by loss-of-function mutations in tripeptidyl-peptidase 1 ( TPP1 ), we used the capsid to package the human TPP1 transgene (AAV-Ep + .hTPP1) and delivered the construct by intracerebroventricular injection into mice lacking TPP1 activity. AAV-Ep + provided robust and therapeutically relevant TPP1 protein concentrations in these mice, significantly improving tremor and life span. In NHPs, high cerebrospinal fluid (CSF) TPP1 concentrations were achieved after intracerebroventricular delivery of AAV-Ep + .hTPP1 at a total dose of 1 × 10 12 viral genomes, which was more than 30× lower than previously reported doses in NHPs. These results suggest that AAV-Ep + may be a potent vector for gene therapy applications where CSF protein expression is required.

Pathogenic variants in over 1700 genes can cause neurogenetic disorders. Monogenetic diseases are ideal targets for genetic therapies; however, the blood-brain barrier (BBB), post-mitotic neurons, and inefficient delivery platforms make gene therapies for neurogenetic diseases challenging. Following nusinersen's 2016 approval, the development of gene therapies for neurogenetic disorders has advanced rapidly, with new delivery vehicles [e.g., BBB-crossing capsids, engineered viral-like proteins, lipid nanoparticles (LNPs)] and novel therapeutic strategies (e.g., regulatory elements, novel RNA therapeutics, tRNA therapies, epigenetic and gene editing). Patient-led disease foundations have accelerated treatment development by addressing trial readiness and supporting translational research. We review the current landscape and future directions in developing gene therapies for neurogenetic disorders.