Molecular Therapy Methods & Clinical Development

Recombinant Adeno-associated viruses (AAVs) are widely used for gene delivery in the central nervous system and have become central tools in both gene therapy and basic neuroscience research. However, although AAV serotypes have been extensively characterized in rodent models, their performance in human neurons, particularly those derived from induced pluripotent stem cells (iPSCs), remains poorly characterized. While human iPSC-derived neurons are increasingly used for disease modeling and drug screening, their susceptibility to viral transduction varies and remains difficult to predict. In this study, we systematically evaluated the transduction efficiency and toxicity profiles of 18 wild-type and engineered AAV serotypes across three distinct types of iPSC-derived neurons, relevant to disease modeling and drug discovery: cortical projection neurons, NGN2- induced forebrain-like neurons, and dopaminergic neurons and four doses (1E3, 1E4, 1E5 and 2E5 genome copies per cell). Using automated high-throughput confocal imaging and quantification of reporter gene expression, we identified several serotypes with robust and efficient transduction across all neuronal subtypes. Among these, three serotypes AAV6, AAV6.2 and AAV2.7m8 showed consistently high performance. To assess safety, we quantified cell number and neurite morphology, finding that while high transduction and gene expression correlate with toxicity, sensitivity varied across neuronal subtypes, with NGN2 neurons being most vulnerable and dopaminergic neurons most resilient. Finally, we validated our findings in a more complex 3D model by testing one of the best-performing serotypes, AAV2.7m8, in both whole and dissociated human cerebellar organoids. Together, our results establish a benchmark dataset for AAV performance in human iPSC- derived neurons and provide practical guidance for AAV based gene delivery in human in vitro neural models. This resource will be valuable for both basic research and preclinical applications aiming to manipulate gene expression in human neurons and understanding AAV tropism in disease-relevant cell types.

The efficacy of adeno-associated virus (AAV)-mediated systemic gene therapy for central nervous system (CNS) diseases is often limited by the blood-brain barrier (BBB). This study systematically evaluated the tissue distribution of three BBB-crossing AAV capsid variants (PHP.eB, CNSRCV300, and BI-hTFR1) following intravenous injection in mice, using either a constitutive promoter (CAG) or a neuron-specific promoter (hSyn) to drive EGFP reporter expression. Compared with AAV9, both PHP.eB and CNSRCV300 demonstrated significantly enhanced BBB penetration and brain transduction efficiency. While the use of the hSyn promoter led to reduced transgene expression in the brain compared with the CAG promoter, and substantially decreased visible reporter expression in peripheral organs, viral deposition in the liver could still be detected via immunohistochemistry. Overall, CNSRCV300 exhibited the most favorable balance between brain-targeting efficiency and biosafety, highlighting its potential as a promising delivery vector. In summary, both the capsid and promoter jointly influence AAV-mediated expression in vivo, and although cell type-specific promoters can reduce off-target expression, residual viral deposition in non-target tissues remains a potential safety concern.

PurposeTo characterize the cellular tropism and temporal dynamics of adeno-associated virus 2 (AAV2)-retro-mediated gene delivery in the adult mouse retina following intravitreal injection. MethodsAdult C57BL/6J mice received single or sequential intravitreal injections of AAV2-retro carrying the mGreenLantern (mGL) reporter gene. Retinas were collected at 1-, 3-, and 14-days post-injection (dpi) and processed for immunofluorescence analysis. Transduced cell types were identified using cell-type markers, including cone arrestin, RBPMS, and AP-2. The number and distribution of mGL-positive cells were quantified on whole retinas or retinal cross-sections to assess transduction efficiency, specificity, and spatial coverage. ResultsReporter expression was detected in the outer retina at 1 dpi and increased markedly at 3 and 14 dpi. AAV2-retro demonstrated strong tropism for photoreceptors and retinal pigment epithelium (RPE), with robust labeling of both rods and cones. In contrast to the robust outer retinal expression, transduction in the inner nuclear layers was limited to a few retinal ganglion and amacrine cells, reflecting strong cell-type specificity. Reporter expression was distributed widely across the retina, exceeding the localized pattern typically observed following subretinal delivery with conventional AAV2 vectors. Sequential injections further increased reporter expression and spatial coverage compared with single injections. ConclusionsAAV2-retro enables efficient, outer retina-specific gene delivery following intravitreal administration. This approach overcomes the limitations of traditional intravitreal gene transfer and provides a minimally invasive alternative to subretinal injection. AAV2-retro- mediated transduction may facilitate preclinical studies of retinal degeneration and support the development of gene therapies aimed at preserving photoreceptors and RPE function.

BackgroundAdeno-associated virus (AAV) vectors are foundational to gene therapy but remain difficult to manufacture at high quality. Vector preparations frequently contain empty capsids and truncated genomes, diminishing potency and increasing immunogenic and production burdens. Conventional assays such as qPCR and ddPCR quantify only short regions, overestimating functional genomes and failing to resolve truncation patterns. MethodsWe applied the NanoMosaic Tessie nanoneedle platform to quantify AAV9 capsid and genome titers, directly distinguishing full-length (>4 kb) and truncated genomes. A 4.5 kb CAG- Luciferase-WPRE-bGH transgene packaged in AAV9 was analyzed using (i) nanoneedle "Probe Walk" assays to map truncations, (ii) PacBio SMRT long-read sequencing for orthogonal validation, and (iii) sedimentation velocity analytical ultracentrifugation (SV-AUC) to assess particle heterogeneity. ResultsProbe-walk mapping revealed asymmetric packaging with a [~]570 bp truncation hotspot 0.44-1.01 kb from the left inverted terminal repeat (ITR). PacBio sequencing confirmed positional concordance, identifying left partial reads clustering within the same region. SV-AUC resolved four major populations--empty (1.8%), partial (4.6%), full-length (70.4%), and high-molecular-weight (HMW) species (18.5%)--suggesting dimeric or multimeric capsids co-sedimenting with full-genome particles. Discussion and ConclusionsThe nanoneedle platform provided quantitative, region-specific insights into genome integrity that aligned with sequencing data while requiring minimal sample and processing time. The disproportion between molecular and AUC estimates indicates that apparent "full" species may contain long partial genomes or multimeric capsids bearing partial genomes. Together, these results establish the NanoMosaic Tessie system as a critical quality attribute (CQA) tool for assessing genome integrity and guiding process optimization. Integrating nanoneedle-based analytics early in development enables detection of truncation hotspots, improvement of vector fidelity, and acceleration of scalable, high-quality AAV manufacturing.

Niemann-Pick disease, type C1 (NPC1) is a rare, fatal neurodegenerative disorder caused by pathological variants in NPC1, which encodes a lysosomal cholesterol transporter. FDA-approved treatments are limited and do not target the underlying genetic defect. Both systemic and central nervous system (CNS) delivery of AAV9-hNPC1 have shown significant disease amelioration in NPC1 murine models. To assess the impact of dose in null Npc1m1N/m1N mice, we systemically administered three different doses of AAV9-hNPC1 at 4 weeks old. Then, to assess the impact of age, we administered the medium dose before phenotypic onset or at early- or late-stage of disease progression (4, 6 or 8 weeks old, respectively). Higher vector doses and earlier treatment were associated with significantly increased lifespan, slower disease progression, and enhanced CNS transduction. In Npc1I1061T/I1061T mice, a model recapitulating a common human hypomorphic variant, similar benefits ensued. Our findings help define dose ranges, treatment ages, and efficacy in hypomorphic models of NPC1 deficiency and suggest that higher doses of AAV9-hNPC1 in pre-symptomatic disease states are likely to yield better outcomes in NPC1 individuals. Summary BlurbSystemic AAV9-hNPC1 gene therapy in null Npc1m1N mice at higher doses or with earlier administration and treatment of hypomorphic Npc1I1061T mice delays disease progression and increases lifespan.

PurposeRetinal ganglion cells (RGCs) are essential for visual signal transmission, yet they are vulnerable to injury and degeneration. Gene modulation in RGCs using adeno-associated virus (AAV) offers a promising avenue for neuroprotection and regeneration, but promoters lack sufficient RGC specificity, limiting precision needed for preclinical studies. This study aims to identify novel promoter-enhancer combinations (PECs) to achieve gene expression preferentially in RGCs. MethodsWe evaluated existing transcriptomic data to identify Neuritin 1(Nrn1) as a gene with highly restricted RGC expression in the retina. Synthetic PECs derived from human and mouse Nrn1 loci were incorporated into AAV2 vectors driving expression of a nuclear-targeted reporter GreenLantern. AAVs were delivered via intravitreal injection into C57BL6/J mice, and transduction efficiency and RGC specificity were evaluated in both young and aged retinas and those subjected to intraorbital optic nerve crush (ONC), using immunohistochemistry and quantitative analysis of RBPMS+ cells. ResultsWe found that AAV2 with a human Nrn1 PEC drives gene expression in RGCs. Quantitative analysis revealed that over 83% of transduced cells were RBPMS-positive, indicating robust RGC selectivity and significantly outperforming ubiquitous promoters. Notably, the Nrn1 PEC retained strong and selective transgene expression in RGCs in aged mice and following ONC, demonstrating its resilience under aged and injury conditions. ConclusionThe Nrn1 PEC enables efficient and injury-resilient gene expression in RGCs, addressing a key limitation in cell-specific targeting. This AAV-incorporated PEC offers a robust platform for evaluating neuroprotective interventions and accelerates translational development of gene therapies for glaucoma and other optic neuropathies.

Recombinant adeno-associated virus (rAAV) vectors show therapeutic potential, but their biomanufacturing is limited by low yields and high costs. Host cell-cycle modulation is emerging as a promising strategy to enhance rAAV production. Two G2/M phase-arresting small molecules, ABT-751, a microtubule inhibitor, and helenalin, a thiol-reactive sesquiterpene lactone, were applied post-transfection in HEK293 cells to evaluate how cell-cycle arrest and stress pathways influence rAAV yields. ABT-751 induced G2/M arrest with minimal cytotoxicity, leading to a near five-fold increase in rAAV vector genomes across multiple serotypes and production platforms. Helenalin caused G2/M arrest, yet suppressed rAAV production. Comparative transcriptomic profiling (RNA-Seq) revealed that helenalin altered expression of a widespread set of genes (4,579) compared to control, characterized by rampant p53, ferroptosis, and endoplasmic reticulum dysregulation that overflowed into unfolded protein response with CHOP induction and apoptosis. ABT-751 elicited a more moderate, targeted response (1,895 differentially expressed genes) in a similar subset of pathways, including compensatory mechanisms mitigating oxidative stress. Together, these findings indicate that cell-cycle arrest alone is insufficient to improve rAAV yield. Indeed, tailored cell-cycle modulation, coupled with balanced activation of cellular stress pathways, can enhance rAAV manufacturing efficiency, facilitating more scalable and cost-effective gene therapy production strategies for the future.

Ectrodactyly-Ectodermal Dysplasia-Cleft Lip/Palate (EEC) syndrome is a rare disorder caused by dominant-negative mutations in the TP63 gene, frequently leading to limbal stem cell deficiency (LSCD) and progressive corneal degeneration. Current therapeutic strategies are limited, primarily due to impaired epithelial renewal and poor proliferative capacity of patient-derived cells. We have recently shown that decreasing the expression of the mutated allele by means of siRNA-mediated silencing can restore epithelial cell proliferation. However, the clinical utility of this approach is hindered by the presence of different TP63 mutations causing EEC syndrome, and the need for continuous siRNA administration to achieve sustained gene silencing. To address these challenges, we employed a CRISPR/Cas9-based genome editing strategy to disrupt mutant TP63 alleles in human induced pluripotent stem cells (hiPSCs) derived from EEC patients carrying R279H and R304Q mutations. Targeted editing of exon 6 induced frameshift mutations that activated nonsense-mediated mRNA decay, leading to a significant reduction in mutant transcript levels. Edited hiPSC-derived corneal epithelial cells exhibited improved cell proliferation compared to unedited isogenic controls. These findings demonstrate the feasibility and therapeutic potential of allele-specific genome editing to correct TP63-associated epithelial defects in EEC syndrome paving the way toward future regenerative therapies for TP63-related corneal diseases.

Instability of the inverted terminal repeats (ITRs) in AAV transfer plasmids has long hindered consistent and efficient production of therapeutic AAV vectors. The palindromic, GC-rich ITR sequence readily forms secondary structures, making them highly mutable in transfer plasmids. Indeed, a recent survey observed mutated ITRs in [~]40% of AAV transfer plasmids from labs around the world. Conventional strategies to mitigate this issue - such as using specialized E. coli strains, suboptimal culture conditions, or modified ITR sequences - have limited effect and often compromise plasmid and AAV yield. Here, by combinatorial optimization of the plasmid backbone structure and ITR flanking sequences, we established MuteFree, an AAV transfer plasmid system that eliminated ITR mutations for both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV). Specifically, MuteFree reduced ITR mutation rates from a range of 32-100% in various transfer plasmids tested to 0% after serial passage of host E. coli for >160 population doublings. Moreover, in three GMP-grade AAV plasmid manufacturing projects initially cancelled due to severe and incurable ITR mutations, replacing conventional backbone with MuteFree completely solved the problem, reducing mutation occurrence to zero under standard GMP manufacturing conditions. Notably, MuteFree supports the packaging of potent AAV virus. The MuteFree system thus presents a robust solution to ITR instability, enabling high-fidelity and high-yield AAV production of AAV-based gene therapy vectors that is fully compatible with existing GMP manufacturing workflows.

Background & AimsAchieving functional cure for chronic hepatitis B (CHB) will likely require a combinatorial approach targeting multiple aspects of the complex hepatitis B virus (HBV) life cycle and host adaptive immune responses. Here, we developed a therapeutic vaccination strategy targeting the PreS1 region of L-HBsAg, required for cellular entry of both hepatitis B and D viruses. An established potent T-cell inducing platform, recombinant adenovirus (Ad), was used as a nanoparticle scaffold for PreS1 attachment, to generate antibodies that neutralize virus entry and to establish T-cell mediated immune control. Approach & ResultsScreening a cohort of 61 patients diagnosed with CHB revealed minimal evidence of natural anti-PreS1 responses. Thus, Ad particles encoding multiple HBV antigens were decorated with PreS1 peptide using DogTag/DogCatcher protein superglue. Mice vaccinated with PreS1-decorated Ad induced robust anti-PreS1 antibody responses that neutralized HBV and HDV infection. In contrast, an undecorated Ad encoding L-HBsAg failed to neutralize HBV, demonstrating that PreS1 decoration was required for potent HBV neutralization. Strong CD8+ and CD4+ T-cell responses were induced against HBV antigens encoded in the Ad genome. ConclusionsPreS1-decorated Ad combines immunological HBV and HDV entry inhibition with potent anti-HBV T-cell induction in a single platform, providing a promising addition to current therapeutic strategies against CHB, with particular utility in HBV/HDV co-infection.

Promoters and vectors are critical components of gene therapy, enabling the delivery and expression of therapeutic genes to correct both loss- and gain-of-function mutations. Adeno-associated virus (AAV) vectors are the leading platform for in vivo gene delivery; however, the widely used Streptococcus pyogenes Cas9 (SpCas9, 4.1 kb) approaches the AAV packaging limit of 4.7 kb. This constraint often necessitates dual-vector systems, which reduce therapeutic efficiency, or the use of smaller nucleases such as SaCas9 (3.2 kb) and AacCas12b (3.4 kb), which have lower PAM site frequencies. To enhance promoter selection for gene therapy applications, we developed a strategy to identify compact, cell-preferred RNA polymerase II (Pol II) promoters. Analysis of approximately 300 compact Pol II promoters revealed that exogenous expression levels in one cell type correlate more strongly with those in other cell types than with endogenous expression, underscoring the importance of exogenous expression efficiency in promoter selection. Using this approach, we identified a compact Pol II promoter #2 (Pro2, 133 bp) that drives robust transgene expression in human retinal ganglion cells (RGCs). To enable single-AAV delivery of SpCas9, we analyzed three commonly used Pol III promoters (H1, 7SK and U6) and determined their minimal functional lengths using a CRISPR/Cas9 reporter assay. We further engineered three compact hybrid Pol II/III promoters which combined pro2 with minimal H1, 7SK and U6 (276, 294, and 323 bp) capable of co-expressing SpCas9 and gRNA, enabling efficient genome editing in both transfected HEK293 cells (approaching 100%) and human RGCs (up to 55.9%) from human stem cell-derived retinal ganglion cells (RGCs). Together, these findings establish a framework for developing single-AAV CRISPR-based gene therapy strategies. Authors contributionsPWZ and DJZ conceived the study, designed the experiments, performed data analysis and interpretation, and were the primary contributors to manuscript writing. STZ played a key role in data collection and correlation analysis. YYC, SL, LF, CJK, YD, CAB, JC, and DW contributed to the execution of essential experiments and subsequent data analysis. All authors have read and approved the final manuscript. Declaration of interestsThe authors declare no conflicts of interest.

Pigs and chickens are not only the most important livestock species for global food production but also serve as key model organisms in various research disciplines. The pig is widely used in translational research due to its anatomical and physiological similarity to humans, providing valuable insights into immunology, metabolism, and disease mechanisms. In contrast, the chicken has become an essential model for studies related to poultry health, animal welfare, and developmental biology. Its externally developing embryo offers exceptional accessibility for experimental manipulation. Recent advances in genome editing technologies, particularly CRISPR/Cas9, have further expanded the potential of these species for functional genomic studies, although the efficient delivery of such tools remains a major challenge. By using virus-like particles (VLPs), we have been able to overcome this limitation. Here, we evaluated VLPs as delivery vehicles for genome engineering tools in pigs and chickens, two key livestock species at the human-animal interface. VLP-mediated delivery enabled efficient Cre recombination and high CRISPR/Cas9 editing rates in porcine cells, organoids, and oocytes, particularly when multiplexed. In chickens, VLPs supported robust Cre recombination and Cas9-mediated editing in cell culture, tracheal organ cultures, and in ovo. Reporter VLPs and dCas9 VLPs further demonstrated the versatility of this platform across porcine and avian systems. Together, these findings establish VLPs as an efficient and time-saving strategy for gene editing in livestock, with relevance for animal health, agricultural productivity, and translational One Health research.

Efficient and cell-specific gene delivery to cochlear inner hair cells (IHCs) remains a major challenge for inner ear gene therapy. Here, we identify and characterize a novel AAV2-derived capsid, AAV-WM04, that enables highly efficient and selective IHC transduction at low doses. Using an in vivo-directed evolution strategy, we generated a randomized AAV2 capsid library with 9-amino acid insertions and performed iterative selection in the adult mouse cochlea. Next-generation sequencing revealed enrichment of several variants, among which AAV-WM04 exhibited superior packaging efficiency and pronounced IHC tropism. AAV-WM04 achieved near-complete IHC transduction throughout the cochlear axis in adult mice, outperforming clinically relevant vectors with minimal off-target expression and no detectable ototoxicity. Robust and exclusive IHC transduction was further validated in non-human primates following round window membrane delivery, underscoring translational potential. Therapeutically, AAV-WM04 enabled efficient dual-AAV trans-splicing delivery of the large OTOF gene, resulting in uniform full-length otoferlin expression in IHCs. In a humanized Otof Q829X/Q829X mouse model, AAV-WM04 restored auditory function across a broad frequency range at relatively low doses and achieved durable hearing recovery. Collectively, these findings establish AAV-WM04 as a next-generation IHC-targeted vector with high efficiency, safety, and cross-species applicability for precision gene therapy of hereditary hearing loss.

Background and AimsPropionic acidemia (PA) is a rare autosomal recessive disorder caused by mutations in PCCA or PCCB, which encode the two subunits of propionyl-CoA carboxylase (PCC). PCC deficiency causes toxic metabolite accumulation and multi-organ damage. Current management, including dietary restriction, pharmacological support, and liver transplantation, does not restore enzymatic activity. We developed a dual-gene adeno-associated virus (AAV) therapy that delivers both PCC subunits to treat both PA subtypes. MethodsWe generated a clinically relevant PCCA-R73W knock-in mouse model and administered AAV8 vectors encoding native human PCCA and PCCB under the control of a liver-specific thyroxine-binding globulin promoter (AAV8-TBG-hPCCA-P2A-hPCCB). Metabolite levels and organ safety were longitudinally assessed. ResultsDual-gene therapy produced dose-dependent reductions in plasma C3/C2 ratio, 3-hydroxypropionic acid, 2-methylcitric acid, and propionylglycine, and significantly outperformed single-gene (PCCA-only) therapy. Neonatal facial-vein injection achieved metabolic correction comparable to or better than adult treatment. The longitudinal follow-up revealed sustained efficacy over a 16-week period, with no signs of hepatotoxicity or adverse effects. ConclusionsSingle-dose, dual-gene AAV therapy achieves sustained metabolic correction and demonstrates long-term safety in a clinically relevant PA model, supporting its translational potential for both type I and type II propionic acidemia.

Cone-rod dystrophies (CoRD) are inherited retinal diseases (IRDs) with variable ages of onset, characterized by the progressive loss of cones, followed by secondary degeneration of rods. Cone-rod homeobox (CRX) is a transcription factor that regulates gene expression essential for photoreceptor development and maintenance. Mutations in CRX gene, including CRXE168d2 and CRXE80A, are implicated in autosomal dominant CoRDs. Although these mutations show distinct pathogenic mechanisms, published studies in knock-in mouse models have suggested a common treatment strategy: increasing WT CRX expression to reduce the detrimental activities of mutant proteins. This study employs two independent strategies of CRX augmentation to evaluate their therapeutic potential in CrxE168d2/+ and CrxE80A/+ mouse models. The Tet-On-hCRX transgenic system, a platform of proof-of-principle gene therapy, induces consistent and pan-photoreceptor expression of CRX augmentation in diseased retinae, allowing for the faithful assessment of functional and behavioral recovery. AAV-mediated CRX augmentation confirms the biosafety, delivery efficiency and efficacy of viral transduction in diseased retinae. Both strategies have achieved significant treatment outcomes in cone photoreceptor survival and overall photoreceptor functions in young adulthood. Treated cones survive past the age point of complete cone loss in untreated controls of both models. Treated rods show functional improvement and long-term survival through later adulthood. This preclinical study establishes valuable treatment regimens and benchmarks for CRX augmentation in the treatment of CRX-associated IRDs, and offers new insights into the mechanisms for photoreceptor development and survival.

Matrix metalloproteinases (MMPs) are rapidly expressed and activated in response to oxidative stress and contribute to various pathological conditions. Cisplatin is a highly effective chemotherapeutic agent; however, its clinical use is limited by its associated permanent hearing loss (ototoxicity). While cispwlatin-induced oxidative stress and inner ear cell death are well-established, the contribution of MMPs remains unclear. In this study, we demonstrate that cisplatin exposure triggers activation of MMP-2 and MMP-9 and expression of an intracellular N-terminal-truncated isoform of MMP-2 in mouse inner ear hair cells. Pharmacological inhibition of MMP-2 and genetic knockdown of Mmp-9 enhanced hair cell survival and attenuated cisplatin-induced inflammation and cytotoxicity. Furthermore, proteomic analysis revealed that proteins involved in intracellular trafficking, including RAB proteins, may serve as potential substrates of intracellular MMP-2 upon cisplatin exposure, pointing to a previously unrecognized mechanism of cisplatin-induced hair cell injury. In vitro analysis confirmed that MMP-2 cleaves RAB9A in response to cisplatin, and in silico analyses predicted MMP-2-preferred cleavage sites on RAB9A. Collectively, our findings identify MMP-2 as a promising therapeutic target for mitigating cisplatin-induced ototoxicity.

We have developed a vector platform for delivery of foreign peptides by genetic modification of the temperate lambda ({lambda}) bacteriophage. This delivery platform is capable of displaying peptides or proteins on either terminus of the structural {lambda} head protein D, present in [~]420 copies per phage particle, and {lambda} side tail fiber (Stf), present at 12 copies per phage particle. Proteins and peptides can be easily fused for display through the low-cost and high-efficiency methods of recombineering and {lambda} prophage induction for recombinant phage preparation described here. To improve this vector technology for use in antigen selection and immunotherapy, we introduced several mutations in the bacterial host and resident prophage {lambda} that improve engineering, induction, phage stability, yield, fusion protein accommodation capacity, and longevity in animal systems. We tested the ability of this {lambda} display system to identify useful antigens and generate antibodies in a mouse model. We report its success as a new technology for both applications: the selection and delivery of therapeutic peptides and proteins.

Objectives: Patients with Cystic fibrosis (CF) often receive aminoglycosides (AGs) to manage recurrent pulmonary infections, placing them at risk for ototoxicity. Chronic AG use can lead to complex cochlear damage affecting inner and outer hair cells, the stria vascularis, and spiral ganglion neurons. The greatest damage is typically in the basal cochlear region, which encodes high-frequency hearing, with additional involvement of more apical regions. While extended-high-frequency (EHF) hearing loss (EHFHL; 9-16 kHz) is often the earliest sign of AG ototoxicity, speech in noise (SiN) effects are rarely studied. Our overall hypothesis is that SiN perception difficulties in individuals with CF, treated with AGs, are related to combined cochlear and neural damage, primarily in the EHF range but also in the standard frequency (SF; 0.25-8 kHz) range. Three mechanisms that contribute to SiN perception were evaluated in children and young adults: 1) a primary effect of reduced EHF sensitivity, measured by pure-tone audiometry (PTA) and transient-evoked otoacoustic emissions (TEOAEs); 2) a secondary effect of subclinical damage in the SF range, measured by PTA and TEOAEs; and 3) additional neural effects, measured by middle ear muscle reflex (MEMR) threshold (afferent) and growth functions (efferent).Design:A total of 185 participants were enrolled; 101 individuals with CF treated with intravenous AGs and 84 age and sex-matched Controls without hearing concerns or CF. Assessments included EHF and SF PTA; the Bamford-Kowal-Bench (BKB)-SIN test for SiN perception; double-evoked TEOAEs with chirp stimuli from 0.71 to 14.7 kHz; and ipsilateral and contralateral wideband MEMR thresholds and growth functions using broadband stimuli. Results: Reduced sensitivity at EHFs (PTA, TEOAEs) was not associated with impaired SiN perception in the CF group. SF hearing, regardless of EHF status, was the primary predictor of SiN performance in the CF group. Increased MEMR growth was also significantly associated with poorer SiN in the CF group. Conclusions: In CF, impaired SiN perception was primarily predicted by SF hearing impairment, with additional involvement of the efferent auditory pathway through increased MEMR growth. These results build on prior evidence for efferent neural effects due to ototoxic exposures, supporting both sensory (afferent) and neural (efferent) mechanisms that contribute to listening difficulties in CF. Thus, preventive and intervention strategies should consider these combined mechanisms in people with AG ototoxicity to address their SiN problems.

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

Mucopolysaccharidosis III (MPS III or Sanfilippo disease) is a spectrum of 4 genetic disorders (MPS IIIA-D), caused by defects in the genes SGSH, NAGLU, HGSNAT and GNS encoding enzymes involved in degradation of heparan sulfate (HS). HS accumulates in brain tissues and causes neuronal dysfunction and neurodegeneration leading to neuropsychiatric problems, developmental delays, childhood dementia, blindness and death during the second decade of life. Previously, we demonstrated that pathophysiological mechanisms, underlying MPS IIIC in mouse models, involves functional pathological changes, affecting synaptogenesis and synaptic transmission and leading to learning and memory deficits. These results suggested that a treatment for MPS III could be developed by using compounds inducing synaptogenesis. In the current study, we tested the efficacy of a synthetic peptide ACTH(4-7)PGP, an analog of adrenocorticotropic hormone fragment, previously used as a neuroprotective and anti-inflammatory medication for treatment of acute neurological conditions, including stroke. We show that intranasal administration of ACTH(4-7)PGP restores defective synaptic transmission in CA1 pyramidal neurons of MPS IIIA and MPS IIIC mouse models and rescues the decrease in synaptic proteins in cultured MPS IIIC mouse hippocampal neurons and iPSC-derived neurons of human MPS IIIA, MPS IIIB and MPS IIIC patients. Furthermore, daily intranasal administration of ACTH(4-7)PGP to MPS IIIC and MPS IIIA mice reduces hyperactivity and rescues defects in working and spatial memory, delays progression of CNS pathology including neuroinflammation and axonal demyelination, and increases the lifespan. Together with the absence of any adverse reactions to ACTH(4-7)PGP in the MPS III and WT mice, our results justify testing the drugs efficacy in clinical settings.