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.

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.

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.

Serotonergic psychedelics have attracted considerable interest as promising therapeutic agents. However, the molecular mechanisms linking their acute hallucinogenic-like effects to longer-lasting neuroplastic responses remain incompletely understood, partly because of the scarcity of native neural models suitable for mechanistic studies. Here, we developed a neural stem cell-derived in vitro model capable of differentiating into neuronal and glial lineages and, after characterization, used it to investigate the molecular pharmacology of serotonergic psychedelics. A panel comprising tryptamines, phenethylamines and ergolines, including psychedelic compounds and selected non-psychedelic analogues, was evaluated alongside ketamine and TrkB agonists. Endpoints included dendritogenesis, synaptogenesis, immediate-early gene induction, BDNF expression and lactate production. TrkB silencing abolished dendritogenic responses to serotonergic psychedelics, ketamine and TrkB agonists, whereas 5-HT2A receptor silencing selectively impaired serotonergic psychedelic-induced plasticity and altered TrkB-dependent responses. Most serotonergic compounds also increased synaptogenesis and induced c-Fos and Egr-2 expression, although ligand-specific differences were evident, particularly for psilocin and the phenethylamines DOI and Ariadne. Uncoupling of Gq/11 or Gi/o protein-dependent signaling differentially modified neuroplastic and transcriptional responses, indicating a ligand and endpoint dependent contribution of both pathways. Serotonergic psychedelics further induced a 5-HT2A receptor dependent lactate response that was generally sensitive to disruption of either Gq/11 or Gi/o protein coupling. Taken together, these findings support a model in which serotonergic psychedelics recruit an integrated 5-HT2A-TrkB signaling network with distinct structural, transcriptional and metabolic outputs, and establish this neural stem cell-derived system as a valuable platform for screening and dissecting the signaling basis of psychedelic action.

Delivery of cells or vectors in advanced therapies is probably the major challenge for genetic disorders that affect a large part of the body such as Duchenne Muscular Dystrophy (DMD). Here, we describe a novel approach for systemic cell delivery based upon an implantable bio-scaffold composed of aligned polycaprolactone nanofibers coated with laminin, able to support adhesion and extensive proliferation of mesoderm cells both in vitro and when implanted subcutaneously in a DMD mouse model. The scaffold is rapidly vascularised leading to cell entering the circulation and colonising multiple distal organs, including distant skeletal muscles and heart. Cells survive in colonized muscles and differentiate into muscle fibres that produce well detectable levels of dystrophin and -sarcoglycan. These results are game changing for cell therapy, as they allow colonization of life essential but "difficult to reach" muscles such as diaphragm and heart while avoiding invasive catheterization. Once optimised, this approach will rapidly enter clinical experimentation for DMD, other muscular dystrophies, and possibly other genetic disorders of the mesoderm. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/715524v1_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@11dfd34org.highwire.dtl.DTLVardef@1da6599org.highwire.dtl.DTLVardef@14427f0org.highwire.dtl.DTLVardef@19a242a_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Study design and therapeutic outcome. Muscle biopsies were obtained from Duchenne muscular dystrophy (DMD) patients to isolate human DMD mesangioblasts (DMD-hMabs). Cells were genetically corrected using a lentivirus carrying a snRNA able to induce exon skipping (U7snRNA), generating U7-hMabs (1). U7-hMabs were seeded onto laminin-coated polycaprolactone (Lam-PCL) nanofiber scaffolds and implanted into the back muscle of DMD-NSG mice. This platform enabled systemic distribution of hMabs cells through circulation, resulting in engraftment across multiple muscle groups, including tibialis anterior, triceps, diaphragm and heart. C_FIG

CAR-T immunotherapy has achieved remarkable efficacy in hematologic malignancies. However, the widespread clinical adoption of autologous CAR-T products remains constrained by high costs, lengthy manufacturing process, and limited accessibility. Universal or off the shelf CAR-T (UCAR-T) cells derived from healthy donors offer a promising alternative, enabling immediate treatment at a lower cost. However, the allogeneic nature of UCAR-T cells triggers immune rejection by the host immune system after infusion, thereby compromising their persistence and therapeutic efficacy. Current strategies to circumvent this rejection focus on disrupting HLA class I expression. Although this modification allows UCAR-T cells to successfully evade T cell mediated elimination, the loss of HLA class I molecules renders them vulnerable to attack by host natural killer (NK) cells. In contrast to previous approaches that attempt to retain certain non-classical HLA molecules (such as HLA-E or HLA-G) to inhibit NK cells, we directly focused on editing the ligands that mediate NK cell rejection. Through transcriptomic and in vitro validation analyses, we found that UL16 binding proteins (ULBP) 2/5/6 were substantially upregulated in UCAR-T cells compared with nontransduced donor T cells. Elevated ULBP expression effectively activates the NKG2D receptor on allogeneic NK cells and leads to killing of UCAR-T cells, thereby impairing UCAR-T function. To test whether abrogating this NK activating signal could improve UCAR-T persistence and antitumor efficacy, we generated ULBP knockout UCAR-T cells using CRISPR-Cas9 editing. Deletion of ULBP2/5/6 significantly reduced NK cell mediated killing in vitro without affecting CAR expression or T cell effector function. Compared with wild type UCAR-T cells, ULBP deficient UCAR-T cells exhibited enhanced tumor killing efficacy in the presence of NK cells. Collectively, our findings identify ULBP upregulation as one of the mechanisms underlying NK cell mediated rejection of HLA deficient UCAR-T cells. Targeted ablation of ULBP molecules provides a novel strategy to confer resistance to host NK cells, thereby improving the therapeutic potential of universal CAR T products.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare autosomal recessive metabolic disorder due to loss-of-function ALDH5A1 mutations impairing the catabolism of {gamma}-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the brain. In SSADHD, pathologic accumulation of GABA and its metabolic by-product {gamma}-hydroxybutyrate (GHB) corresponds to a clinical syndrome dominated by developmental delay and epilepsy in half of patients with risk of sudden death in adolescence and adulthood. Brain-wide ALDH5A1 gene replacement for SSADHD is unavailable, and whether such treatment will reverse the SSADHD phenotype is unknown. We developed an inducible mouse SSADHD model, Aldh5a1lox-STOP, enabling Cre-dependent Aldh5a1 restoration to evaluate gene therapy feasibility. In the absence of SSADH, Aldh5a1lox-STOP mice exhibit hyperactivity and excessive serum GHB levels, culminating in death by [~]postnatal day 22, recapitulating the severe SSADHD condition. Systemic delivery of a blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV) carrying a Cre gene to Aldh5a1lox-STOP mice leads to brain-wide SSADH restoration, serum GHB level reduction, normalization of hyperactivity, and substantial increase in survival. As a step toward clinical translation, we further assessed an AAV encompassing a functional native promoter (FLnP) of ALDH5A1 tethered to its human coding sequence, namely AAV-FLnP-hALDH5A1. Aldh5a1lox-STOP mice were effectively rescued when treated with AAV-FLnP-hALDH5A1 packaged in the blood-brain barrier (BBB)-penetrating capsid PHP.eB. These findings provide preclinical proof that SSADH gene replacement therapy is feasible and potentially effective.

Genetically engineered human induced pluripotent stem cells (hiPSCs) represent a promising platform for regenerative medicine and next-generation immunotherapies. While recent advances enable stroma-free differentiation of hiPSCs into mature CD3TCR{beta} cytotoxic T lymphocytes (CTLs), overall efficiency remains limited. Here, we identify small-molecule modulators that enhance T cell output, particularly at the ProT cell stage. Targeted and stage-specific inhibition of AHR, DOT1L, or GSK3 drives robust maturation from ProT to CD4 immature single-positive (ISP) cells, markedly increasing CD4CD8 populations and augmenting CTL production of up to 2000 fold. hiPSC-derived T (iT) cells matured under these conditions display superior activity in cytotoxicity assays using AMG-701 (BCMAxCD3) or Blinatumomab (CD19xCD3). These effects were reproducible across independent hiPSC lines, diverse hematopoietic progenitor generation methods, and multiple stroma-free differentiation platforms, and were further validated in cord blood CD34 cells. Notably, AHR inhibition enhanced T cell development and promoted B lymphopoiesis, revealing shared regulatory pathways in lymphoid lineage specification. We also demonstrate that the Oct4-activating compound OAC1 functions as a weak AHR inhibitor, partially recapitulating the effects of canonical AHR blockers in both cellular and zebrafish AHR reporter systems. Collectively, our findings define key molecular circuits governing human lymphoid differentiation and establish practical strategies to optimize the yield and function of hiPSC-derived cytotoxic T cells. This work advances the development of both universal and autologous hiPSC-derived T cell therapies, offering a path forward even for patient-specific hiPSC lines with suboptimal T cell differentiation potential.

Transforming growth factor-beta (TGF-{beta}), a potent promoter of extracellular matrix deposition and suppressor of infiltrating immunity, has arisen as an attractive target for improving outcomes in tissue fibrosis and cancer immune therapy. Despite the promise of TGF-{beta} inhibitors for attenuating the progression of fibrotic disorders or as adjuncts for cancer immunotherapy, current systemically administered inhibitors that target the ligand or receptors have significant on-target liabilities, including cardiotoxicity and development of pre-malignant cutaneous squamous lesions. Recently, an engineered mini monomer of TGF-{beta} (mmTGF-{beta}), which potently and specifically inhibits TGF-{beta} activity, was shown to strongly synergize with checkpoint inhibitors to suppress cancer progression in an aggressive model of melanoma when genetically delivered using an engineered form of vaccinia virus that preferentially infects cancer cells. Despite these promising results, however, a significant fraction of the mmTGF-{beta} was found to misfold, likely due to mispairing of the cysteines that comprise its cystine knot. Here, we demonstrate that inclusion of a modified form of the TGF-{beta} pro-domain that lacks its dimerization motif, the bowtie knot, dramatically improves both the folding and inhibitory activity upon secretion by mammalian cells, thus overcoming one of the major limitations of genetically delivering mmTGF-{beta}. Furthermore, we show that fusion of mmTGF-{beta} to a CD44 binding domain enhances the inhibitory potential of mmTGF-{beta} on immune cells, and on other cell types which express CD44, by more than 30-fold compared to cells negative for CD44. Together, these modifications provide a framework for further enhancing the efficacy and safety of mmTGF-{beta} for cancer immune therapy, and possibly also tissue fibrosis, when delivered genetically using vaccinia, or other related approaches.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual loss in elderly individuals for which no effective treatments are currently available. The photoreceptor loss in dry AMD is secondary to the demise of the retinal pigment epithelium (RPE) cells. The accumulation of extracellular deposits, known as drusen, resulting in part from deficient lysosomal and autophagosomal degradation, is a key feature of dry AMD pathogenesis. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway that maintains proteostasis by targeting specific cytosolic proteins for lysosomal translocation and degradation. LAMP2A (lysosome-associated membrane protein 2A) functions as the key lysosomal receptor required for CMA. Using Lamp2a knockout mouse, we show that selective CMA dysfunction recapitulates AMD-like pathologies, including sub-RPE lipid and protein deposits, RPE atrophy, Bruchs membrane thickening, and impaired autophagic activity. Furthermore, we identify large-conductance Ca{superscript 2}-activated K (BK) channels as a therapeutic target for restoring autophagic activity. Mechanistically, pharmacological activation of BK channels with the small-molecule agonist GLA-1-1 enhances macroautophagy and stimulates autophagic flux by promoting autophagosome-lysosome fusion. Importantly, oral administration of GLA-1-1 in markedly attenuates structural, functional, and molecular retinal abnormalities in Lamp2a-deficient mice, suggesting that pharmacological activation of macroautophagy through facilitating autophagosome-lysosome fusion can partially compensate for CMA deficiency. Together, these findings demonstrate that pharmacological activation of macroautophagy can ameliorate the retinal phenotype resulting from CMA dysfunction and support BK channel activation by GLA-1-1 as a promising therapeutic strategy for dry AMD.

BackgroundPrimary astrocyte cultures derived from neonatal rodent cortices provide a controlled system for investigating astrocyte-specific mechanisms. However, mixed glial preparations frequently contain contaminating microglia and oligodendrocyte progenitor cells, and most existing protocols require pooling tissue from multiple mouse pups to obtain sufficient astrocyte yields. This approach is impractical as it obscures sex and genotype, limits investigations of sex dependent astrocyte phenotypes, and precludes studies in certain transgenic models. To address this gap, our protocol achieves a high astrocyte yield from a single neonatal mouse brain, enabling sex- and genotype-specific cultures without the need for pooling. Mechanical removal of oligodendrocyte progenitors combined with pharmacological depletion of microglia using a Colony Stimulating Factor 1 Receptor (CSF1R) inhibitor produces highly enriched astrocytes suitable for functional assays, including those focused on sex-specific biology. MethodsCortical tissue was isolated from a single mouse pup is mechanically dissociated in astrocyte media. Cell suspensions are transferred to poly-D-lysine-coated flasks in astrocyte media. After 10-15 days in culture, OPCs are mechanically removed by horizontal shaking and microglia are selectively depleted by incubating cultures with CSF1R inhibitor PLX5622 for 24, 48, 72 and 96 hours. After PLX treatment, media is replaced and enriched astrocytes were maintained or passaged for experimentation. The sex of the pups is determined by PCR performed on DNA extracted from tail biopsies. ResultsImmunocytochemical analysis for astrocyte and microglia markers (GFAP and Iba1, respectively) showed that 24 hours of PLX5622 treatment did not fully eliminate microglia from mixed glial cultures. Extending treatment to 48 hours effectively depleted microglia while minimizing cytotoxicity and astrocyte loss and produced a pure, high-yield, sex-specific primary astrocyte culture. PCR reliably enabled the sex identification of pups used in culture using DNA extracted from tail biopsies. DiscussionThis protocol provides an efficient and reproducible method for generating high-purity, sex-specific primary astrocyte cultures from a single mouse brain. It improves consistency and purity while eliminating the need to pool tissue, preserving sex and genotype and enabling studies in transgenic mouse lines of both sexes.

BackgroundChoroid plexus (ChP) produces cerebrospinal fluid (CSF), and regulates brain development and adult subventricular zone (SVZ) neurogenesis, but its role in hippocampal subgranular zone (SGZ) neurogenesis in adulthood and early postnatal stages is not well understood. Current tools to directly manipulate neonatal ChP/CSF volume are very limited, representing an urgent need in the field. MethodsWe first discovered the specific "leaky" expression of DTR gene in the ChP of adult ROSA26-iDTR mice which can be used to specifically ablate ChP in adult brain that generated robust and long-lasting ablation of ChP and reduction of CSF volume. In this study, we the effectiveness of ROSA26-iDTR allele in ablating neonatal ChP. We also developed a novel AAV2/5-CMV-DTR vector with validated ChP tropism in both neonatal and adult mice, which induces substantial CSF loss in both neonates and adult mice. With both the ROSA26-iDTR genetic and AAV2/5-DTR viral-mediated ChP ablation in young adults and at defined postnatal ages, we quantified ventricular CSF volume by MRI and characterized postnatal neurogenesis. Doublecortin-positive (DCX+) neuroblasts, Ki67+ proliferating cells, and TUNEL+ apoptotic cells were quantified in SVZ and SGZ using confocal microscopy and machine learning-assisted cell counting. ResultsWe show that ROSA26-iDTR-mediated ChP ablation is inefficient before postnatal day 10, suggesting that this line may be of limited utility for CSF reduction in the early neonatal period before P10. P3-5 Dtx treatment of a previously used dosage of 20ng/g dosage did not lead to a reduction in CSF volume. Higher dosage of 40ng/gX3 Dtx dosage at p3-5 generated only moderate partial reduction of CSF in third ventricle and total CSF volume, with indication of toxicity associated with high Dtx dosage in general. In contrast, p10-12 injection of 20ng/gX3 Dtx led to robust CSF reduction. To target early neonatal days, AAV2/5 CMV-DTR virus shows high tropism for ChP epithelial cells and leads to near-complete ablation of CSF in neonatal brains. ChP/CSF loss in neonates or young adult mice leads to a substantial reduction of DCX+ cells at the SVZ but a moderate but significant reduction of SGZ DCX+ neuroblasts, without changes in Ki67+ or TUNEL+ cells. ConclusionsThis study reports a novel role of the ChP/CSF in maintaining the neuroblast pool in the neurogenic niches in both early postnatal and adult stages. Moreover, we expand the available tools to target the ChP and CSF production in the neonate, with potential uses in treating conditions such as neonatal hydrocephalus.

Systemic delivery of adeno-associated virus (AAV) for gene therapy of central nervous system (CNS) disorders is limited by inefficient blood-brain barrier (BBB) penetration and dose-limiting toxicity in peripheral organs, notably the liver and dorsal root ganglia (DRG)1-5. Here, we report the development of novel AAV variants via a proprietary capsid engineering platform (REACH). In non-human primates (NHPs), intravenous administration of lead variants resulted in transgene expression levels in the brain that were 600-2000 fold higher than AAV9 at the RNA level, concomitant with a 10-50 fold reduction in liver tropism and minimal off-target exposure in the heart and DRG. These engineered capsids achieve unprecedented, pan-CNS transduction with a markedly improved safety profile, representing a transformative platform for treating a broad spectrum of neurological diseases.

Alzheimers disease (AD), the leading cause of dementia, affects over 33 million people worldwide, with pathogenesis tied to amyloid-{beta} (A{beta}) accumulation. Although anti-A{beta} monoclonal antibodies have shown clinical benefits, they often cause side effects including amyloid-related imaging abnormalities and brain microhemorrhage, especially in APOE E4 allele carriers. Here we used PHP.eB serotype adeno-associated virus (AAV), a vector with enhanced central nervous system (CNS) tropism, to deliver an A{beta} antibody expression vector (AAV-LEC) into the CNS of APP/PS1 and 5xFAD mice intravenously. The AAV-LEC-mediated expression of anti-A{beta} antibodies in the CNS significantly reduced the number and size of A{beta} plaques at various stages in both APP/PS1 and 5xFAD mice, alongside improved spatial learning and memory. It also reversed abnormal glial activation with reduced disease-associated microglia and astrocytes, and restored oligodendrocyte differentiation and myelin formation. No brain microhemorrhage or liver damage was detected following the AAV-antibody treatment. Thus, this AAV-mediated strategy offers a promising, convenient and safe AD therapeutic approach in the future.

The establishment of mixed hematopoietic chimerism is a promising way to induce immune tolerance for islet replacement therapy and to treat the underlying autoimmunity in Type 1 diabetes (T1D). Mixed chimerism not only promotes effective thymic negative selection of autoreactive cells but also restores regulatory T cell (Treg) function and peripheral tolerance. In the current study, we determined that a novel class of donor-derived CD8+CD44+CD122+ Tregs (d-CD8+CD122+ Tregs) plays a crucial role in controlling autoimmunity in non-obese diabetic (NOD) mice with induced mixed chimerism. Using adoptive T cell transfer experiments, we showed that d-CD8+CD122+ Tregs abrogate autoimmunity by selectively depleting the exogenously injected diabetogenic T cells in Recombination-Activating Gene deficient NOD mice. These d-CD8+CD122+ Tregs from NOD chimeras show upregulation of Helios, Programmed cell death protein 1, perforin, granzyme-B, CD39, Folate receptor 4, and downregulation of proinflammatory markers like Scart1 and Scart2. Using in vitro assays, we show that d-CD8+CD122+ Tregs respond specifically to a Complementarity-Determining Region-3 peptide sequence derived from T cell receptors of islet antigen-specific autoreactive T cells. Thus, mixed chimerism might be a method to revitalize CD8+CD122+ Tregs which are decreased in number and functionality in NOD mice. Similarly, we found that individuals with T1D have a deficiency in CD8+CD122+ Tregs, suggesting a potential loss of regulatory function accompanies disease onset. Revitalizing CD8+CD122+ Tregs may offer a new therapeutic strategy of restoring immune tolerance in autoimmune diabetes. One sentence summary Inducing mixed donor chimerism in NOD mice generates donor-derived CD8+CD122+ Tregs that suppress autoimmunity and restore immune tolerance by selectively eliminating autoreactive T cells.

Spiral ganglion neurons (SGNs) are the primary auditory afferents in the inner ear. These neurons degenerate in response to a number of conditions, including auditory neuropathies, concussions, and aging. Research to assess the extent of degeneration and to test the efficacy of protective or rehabilitative strategies requires quantification of SGNs from tissue sections. However, manual counting of SGNs can be arduous and time-consuming due to dense crowding and the lack of reliable nuclear-specific labels. SGNs receive afferent input via GluA2-containing AMPA receptors. As the Gria2 transcripts that code for GluA2 must undergo RNA editing to ensure calcium impermeability, we hypothesized that SGNs would express high levels of the adenosine deaminase acting on RNA (ADAR) enzyme ADARB1. Here we confirm enriched expression of Adarb1 in SGNs via in situ hybridization and show that anti-ADARB1 antibodies robustly label the nuclei of both type I and type II SGNs in cochlear sections from young and aged mice. Neuronal specificity was confirmed using antibodies against neurofilament heavy chain (NFH), human antigen D (HuD), GATA binding protein 3 (GATA3), and SRY-box 2 (SOX2). A blinded investigator manually counted SGNs via NFH staining, and these were compared to automated counts of ADARB1-positive nuclei using the analyze particles function in ImageJ. A concordance correlation coefficient and Bland-Altman analysis demonstrated strong agreement between the manual and automated counts. Additionally, immunolabeling of ADARB1 in macaque and human temporal bone sections confirm robust labeling of SGN nuclei, suggesting broad utility of ADARB1 immunolabeling for automated counts of SGNs across species.

Immune mobilizing monoclonal TCR against cancer (ImmTAC) are cancer therapeutics that activate T cells through recognition of a tumor-associated antigenic MHC/peptide complex. A first-in-class ImmTAC, Tebentafusp, is approved for the treatment of metastatic uveal melanoma. While clinical efficacy is thus established, the cellular mechanisms underpinning ImmTAC action are not fully resolved. Using a recently established experimental strategy to generate suppressed human primary cytotoxic T lymphocytes (CTL), we have investigated an ImmTAC that recognizes a peptide derived from the tumor associated antigen NY-ESO-1 in comparison to direct engagement of a TCR recognizing the same MHC/peptide complex. In response to endogenous antigen presentation, ImmTACs could elicit tumor cell cytolysis by suppressed CTL, but not IFN{gamma} secretion, in a manner dependent on the engager affinity for CD3{varepsilon}. ImmTACs enhanced the efficient execution of subcellular CTL polarization steps required for effective cytolysis and could trigger calcium signaling. These data establish that ImmTACs activate CTL similarly to direct engagement of a TCR by MHC/peptide and are likely to retain this capability under suppressive conditions such as in the tumor microenvironment.