Molecular Therapy Methods & Clinical Development

A major challenge in gene therapy for GJB2-related hearing loss (DFNB1)--the most common form of hereditary deafness--is achieving efficient and precise connexin 26 delivery. Herein, we engineered two cell type-specific promoters (GJB2-1 and WFS1-2274) and developed an AAV capsid, AAV-MAS012, with enhanced transduction efficiency in mature cochlear cells. Our AAV-mediated gene therapy systems restored hearing of low-to-mid-frequencies in newborn Gjb2 cKO mice to wild-type levels and maintained for 45 weeks. Additionally, our therapeutic systems restored low-to-mid-frequencies hearing function to wild-type levels in adult Gjb2 cKO mice. A humanized version of the therapy, AAV-MAS012-WFS1-2274-hGJB2, rescued hearing function in two distinct Gjb2-deficient mouse models, and demonstrated a favorable safety profile in nonhuman primates. This study represents the first successful hearing restoration in adult Gjb2-deficient mice. The significant therapeutic efficacy of the humanized gene therapy system shows great potential for clinical translation in DFNB1 patients.

Reprogramming of Muller glial (MG) cells into retinal neurons has the potential to treat vision loss by regenerating the retina. Development of efficient gene delivery systems to target the MG cells is critical. Adeno-associated virus (AAV) serotypes and promoter specificity are important factors that influence AAV transduction profile in the retina. However, studies that optimize these parameters to specifically target MG cells are limited, in particular in rats which are commonly used for eye research. Here we tested 4 AAV serotypes and 14 promoters to optimize gene delivery to human MG cells in vitro and/or rat MG cells in vivo. We showed that the combinatorial use of MG-specific serotypes and promoters achieved high specificity for MG cell targeting, with ShH10Y serotype and the GFAP (gfaABC1D) promoter as the best performing tool to target rat MG cells in vivo. We developed new AAV vectors using known and novel MG-specific promoters and engineered short promoter variants to improve the cargo capacity of AAV delivery. Our results highlighted a number of promoters that can target MG cells in vitro or in vivo. This study further expands the AAV toolbox to target MG cells, which has important implications for retinal gene therapy development.

Recombinant adeno-associated virus (rAAV) vectors are pivotal for gene therapy; however, the encapsidation of residual DNA, particularly plasmid backbone sequences, pose significant safety risks. Recent studies have identified the p5 promoter, which contains a Rep-binding element and a terminal resolution site (TRS), as a cryptic origin of replication that facilitates packaging of upstream sequences. In this study, we investigated the effect of p5 TRS modifications on impurity DNA levels in a single-plasmid All-in-One (AiO) AAV production system. Wild-type p5 (p5wt) promoted significant packaging of upstream plasmid backbone DNA, especially when the backbone was positioned between p5wt and the inverted terminal repeat. Introducing mutations or deletions in the p5 TRS significantly reduced encapsidation of plasmid-derived sequences, including kanamycin resistance genes, and improved the ratio of full to partial particles, as seen with the p5{Delta}loop variant. Furthermore, the p5{Delta}loop-AiO system showed higher rAAV yields than both conventional triple-transfection methods and previously reported p5-spacer variants. Thus, our findings suggest a robust vector design strategy for minimizing DNA impurities, thereby enhancing the safety and efficacy of AAV-based gene therapy.

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.

Gene therapy may provide a way to restore inner ear function to deaf and dizzy patients. The mouse is a crucial model system for functional genomics because of the numerous genetic models for hearing loss and inner ear dysfunction. Using an advance generation, E1-/E3-/E2b-(preterminal protein-/polymerase-) Type 5 Adenovirus, we investigated several routes of virus microinjection to determine which were most reproducible in targeting the endolymphatic fluid compartment of the cochlea. We found that when adenovirus is injected via the round window, transduced cells are found only adjacent to the scala tympani and not in the organ of Corti, suggesting that Adenovirus is unable to penetrate the basilar membrane or bony wall of the modiolus. Delivery to the cochlea via the semicircular canals is also inefficient. In contrast, our new method, via a stylomastoid foramen cochleostomy, increases the likelihood of adenovirus gene transfer to the scala media including cells in the organ of Corti and stria vascularis while preserving some hearing. The ability to target delivery of virus and other therapeutic reagents to specific inner ear fluid compartments will facilitate in vivo testing of candidate molecules implicated in multiple aspects of inner ear physiology and regeneration.

Inherited retinal diseases (IRDs) are a heterogeneous group of genetic disorders that cause progressive vision loss. A subset of IRDs is associated with ubiquitously expressed genes involved in fundamental cellular processes, often resulting in multisystem disease. Among these is Zellweger spectrum disorder (ZSD), caused by pathogenic variants in PEX genes required for peroxisome biogenesis and function. There are no proven targeted disease-modifying treatments for ZSD, and it is unclear whether localized restoration of peroxisome function is sufficient to mitigate retinal degeneration. We previously demonstrated that HsPEX1 retinal gene augmentation therapy in a mouse model of mild ZSD homozygous for the murine equivalent (PEX1-p.[Gly844Asp]) of the most common deleterious allele in patients (PEX1-c.[2528G>A], PEX1-p.[Gly843Asp]), improved retinal electrophysiological response. Here, we present a comprehensive, dose-range evaluation of a re-designed, clinically relevant AAV8-delivered HsPEX1 subretinal gene therapy, employing expanded outcome measures. We observed a marked improvement in functional vision, retinal response, photoreceptor structure, retinal pigment epithelium integrity, subretinal inflammation, and peroxisomal metabolites, durable to the endpoint of 6 months post single subretinal injection. These studies provide preclinical proof-of-concept that localized retinal gene replacement can mitigate vision loss in peroxisome-mediated IRD.

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.

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.

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.

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.

PurposeCompare the effect of MEK inhibition on iPSC-derived retinal pigmental epithelial (RPE) cells generated from a patient who developed MEK inhibitor-Associated Retinopathy (MEKAR) versus a patient who did not develop retinopathy. DesignCase-control SubjectsTwo female patients with Neurofibromatosis Type 1 who were treated with MEK inhibitors. One patient developed MEKAR, the other did not. MethodsRPE were generated from human induced pluripotent stem cells (hiPSCs) from these two patients. These hiPSC-derived RPE were treated with selumetinib for 10 days. Main Outcome MeasuresPhagocytic activity and changes in gene expression ResultsAs previously reported, there was a significant increase in internalized rhodopsin in phagocytosis assays, yet this was only found in hiPSC-derived RPE from the patient who developed MEKAR. Selumetinib decreased expression of genes related to fluid transport and cell volume, including aquaporins and solute transporters. At baseline, cells from the patients without MEKAR had higher expression of these genes. Interestingly, selumetinib-induced changes in gene expression only reached statistical significance in cells from the patient who did not develop MEKAR, suggesting these changes may be a compensatory protective mechanism. Patients susceptible to forming MEKAR may have increased phagocytosis without a compensatory change in expression of genes related to fluid flux, thereby inhibiting their ability to transport fluid out of the subretinal space. ConclusionsMEK inhibitor-Associated Retinopathy may only affect susceptible patients whose retinal pigment epithelium cannot sufficiently regulate expression of genes related to fluid transport and cell volume, altering the ability of these cells to properly function.

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.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

BackgroundThe optic nerve serves as a vital conduit for visual signaling, and its degeneration in optic neuropathy results in irreversible vision loss. It is also a widely used model for studying central nervous system (CNS) injury and repair. Although adeno-associated virus (AAV) and lentivirus are extensively applied in CNS research, their transduction efficiency and cell-type specificity within the optic nerve remain poorly characterized. This study aimed to identify the most effective viral vector, serotype, and promoter for direct gene delivery to the adult rat optic nerve. MethodsSprague-Dawley rats (7-10 weeks) received intra-optic nerve injections of lentiviral or AAV vectors encoding GFP under different promoters (CAG, CMV, or GFAP). Two to three weeks post-injection, optic nerves were collected for immunohistochemistry with markers of oligodendrocytes (Olig2), astrocytes (GFAP, Sox9), and microglia (IBA1). Transduction efficiency and cell-type specificity were assessed using confocal microscopy. ResultsAAV2, AAV5, and lentivirus showed minimal transduction, with only sparse GFP-positive cells observed near injection sites. In contrast, AAV-PHP.eB carrying the CAG promoter yielded robust and widespread GFP expression near the injection site. Quantitative analysis revealed that approximately 90% of transduced cells were Olig2-positive oligodendrocytes, indicating strong tropism for this glial population. ConclusionAAV-PHP.eB driven by the CAG promoter enables efficient gene delivery to the optic nerve, with a predominant tropism for oligodendrocytes. This targeted intra-optic nerve injection approach offers a reliable platform for manipulating oligodendrocytes and investigating mechanisms of CNS development, injury, and repair relevant to both optic neuropathies and other CNS diseases.

{gamma}-secretase is a multi-subunit enzyme complex responsible for cleaving hundreds of substrates in diverse cellular contexts. Variation in subunit composition - including the use of alternate catalytic subunits Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2) - results in diverse {gamma}-secretase complexes. Point mutations in PSEN1 and PSEN2 cause familial forms of Alzheimers disease, while loss-of-function mutations in the {gamma}-secretase subunits PSEN1, PSENEN and NCSTN cause acne inversa. To advance therapeutic strategies targeting {gamma}-secretase in Alzheimers disease, a better understanding of individual {gamma}-secretase complexes is required. In this study, we used CRISPR-Cas9 genome engineering to generate PSEN2-knockout iPSCs in order to compare the consequence of PSEN2 knockout versus PSEN1 knockout in iPSC-derived brain cells. In contrast to PSEN1-knockout, PSEN2-knockout did not alter APP cleavage or A{beta} generation in iPSC-neurons, nor did it disrupt Nicastrin maturation. Similarly, PSEN2-knockout had little impact on TREM2 processing in iPSC-microglia. Instead, our data indicate that loss of PSEN2 primarily impacts the endo-lysosomal system in iPSC-neurons, causing an accumulation of early endosome markers and a reduction in lysosomal markers - phenotypes not observed in PSEN1-knockout neurons. Taken together, these findings highlight distinct and non-redundant functions of PSEN1 and PSEN2 in human brain cells, reinforcing findings in animal models and subcellular localisation studies. This work advances our understanding of distinct {gamma}-secretase complex functions and provides insights that will support future therapeutic efforts to inhibit, modulate or stabilise {gamma}-secretase.

The need for safe, allogeneic cell therapies for cancer is driving a growing interest in CAR-NK-based therapies, which, unlike CAR-T cell therapies, offer the potential for off-the-shelf administration. Lentiviruses pseudotyped with vesicular stomatitis virus glycoprotein G (VSV-G) are commonly used for genetic modification of cell therapy products. Their use in NK cells, however, is limited by low transduction efficiency. This study explores the complexities of NK cell transduction using lentiviral vectors pseudotyped with VSV-G. We demonstrate that efficient transduction depends on multiple factors such as NK cell activation, construct design, lentivirus pseudotype selection, and the use of transduction enhancers. By optimizing these elements, we achieved effective transduction, facilitating the use of VSV-G-pseudotyped LVs for therapeutic NK cell production. Our optimized workflow comprises NK cell activation with interleukins, followed by transduction with a NK cell-specific CAR construct using VSV-G-pseudotyped LVs in the presence of BX795 and Retronectin, resulting in excellent transduction efficiency without compromising NK cell phenotype or growth. This allows for the use of a widely used gene transfer vector with an excellent safety record for producing therapeutic NK cell products.

Among mammals, spiny mice (Acomys spp.) exhibit the unique capacity to regenerate parts of their nervous system. Studying this phenomenon has the potential to reveal new targets that can slow or halt human neurodegenerative disorders. Unfortunately, research tools (e.g., transgenic lines, gene delivery vehicles) are lacking compared to those available for other rodent models. Here, we tested systemic adeno-associated viral vectors (AAVs) in Acomys dimidiatus and identified three promising candidates: X1.1, CAP-Mac, and MaCPNS1. Characterizing their tropism following intravenous delivery, we found that in the brain, MaCPNS1 and X1.1 primarily transduced astrocytes. In the peripheral nervous system, MaCPNS1 efficiently transduced dorsal root ganglia, axon bundles of the ear pinnae, and enteric neurons throughout the gastrointestinal tract. As a proof-of-concept, we used MaCPNS1 to chemogenetically modulate the activity of enteric neurons, successfully decreasing gastric motility in vivo and increasing colonic motility ex vivo. We expect these findings to enable functional studies of the uniquely regenerative nervous system of Acomys, which may in turn help advance neuroregenerative therapeutics for humans. Summary StatementIdentification of an AAV tool to efficiently deliver transgenes to the central and peripheral nervous systems of spiny mice enables functional studies of the nervous system in a mammalian model of regeneration.