Molecular Therapy

Autosomal dominant retinitis pigmentosa (adRP) caused by RHO mutations is a leading form of inherited retinal degeneration. Extensive allelic heterogeneity of RHO pathogenic variants limits the translational applicability of mutation-specific gene therapies. To address this, we developed SNARE (SNP-guided Silencing of Aberrant RHO Expression), a mutation-independent, allele-specific antisense oligonucleotide (ASO) strategy. SNARE selectively suppresses mutant RHO transcripts by targeting the common, benign c.-26A/G single-nucleotide polymorphism (SNP) as an allelic discriminator. Candidate gapmer ASOs were screened in engineered reporter lines and validated in patient-derived retinal organoids, identifying RHOligo-A as the lead c.-26A-targeting candidate. In vitro, RHOligo-A achieved robust, preferential knockdown of the target allele, improving RHO localization in retinal organoids, and demonstrated a favorable safety profile with minimal transcriptomic off-target effects and no detectable immunostimulatory activity. Subsequent validation in a novel, humanized RHOP347L/WT mouse model, achieved sustained c.-26A-linked allele-selective suppression, retinal structure preservation, and significantly restored visual function, upon a single intravitreal administration. These findings establish RHOligo-A and SNARE as a scalable, mutation-independent therapeutic platform with strong translational potential and substantial clinical reach for RHO-associated adRP.

Parkinson's disease (PD) is characterized by progressive DAergic neurodegeneration and the accumulation of aggregated -Synuclein (Syn), which drives chronic neuroinflammation through sustained activation of innate and adaptive immune responses. Regulatory T cells (Tregs) exert potent immunosuppressive functions and have shown neuroprotective effects in preclinical PD models; however, clinical translation of polyclonal Treg therapies has been limited by poor tissue specificity and insufficient therapeutic efficacy. To overcome these limitations, we engineered induced human Tregs (iTregs) expressing chimeric antigen receptors (CARs) directed against pathological Syn aggregates. Among the CAR designs tested, only a nanobody-based construct incorporating NbSyn87 displayed selective antigen-dependent activation in response to Syn preformed fibrils (PFFs). Intriguingly, despite the ability of the parental NbSyn87 nanobody to bind both monomeric and aggregated Syn, incorporation into the CAR architecture conferred functional selectivity for aggregated conformers. This feature enabled discrimination between pathological extracellular aggregates and physiological monomeric Syn, providing an important safety advantage. To evaluate therapeutic activity in vivo, we established an immunodeficient mouse model of synucleinopathy permissive to human cell engraftment. iTregs preferentially accumulated within Syn-rich brain regions and, in the presence of astrocyte-derived human IL-2 with antigen-independent mechanism. Conversely, only CAR iTregs directed against Syn significantly reduced microglial and astrocytic activation, decreased pro-inflammatory cytokine expression, and attenuated Syn pathology. Collectively, these findings demonstrate that Syn-specific CAR iTregs can selectively exert potent local immunomodulatory effects, establishing a promising antigen-specific cellular immunotherapy platform for PD and other synucleinopathies.

Proteostasis impairment at the level of the endoplasmic reticulum (ER) is a salient feature of Alzheimers disease (AD). The unfolded protein response (UPR) is the main pathway to cope with ER stress, where the expression of the transcription factor X-Box binding protein 1 (XBP1) is central to establish repair programs. To artificially enforce the adaptive capacity of the UPR in the AD brain, we recently reported the protective effects of overexpressing active XBP1 in the brain using adeno-associated vectors (AAVs) of AD mice, in addition to aged animals. Here we have generated a next generation vector suitable for clinical testing by (i) expressing codon-optimized human XBP1s without artificial tags, (ii) the use of the synapsin promoter to restrict expression to neurons, and (iii) incorporating a novel variant of AAV2 (AAV-TT) with greater biodistribution (here termed Proteostaser-1). Treatment of 5xFAD mice with Proteostaser-1 improved spatial learning and synaptic plasticity, and reduced the deposition of amyloid plaques in the brain. Proteostaser-1 administration also improved cognition in a model of sporadic AD based on the intracerebral injection of amyloid {beta} oligomers. Our results further support the therapeutic potential of the UPR as a strategy to ameliorate AD features and sustain synaptic function.

Hemoglobinopathies, including sickle cell disease (SCD) and thalassemia syndromes, affect millions of individuals worldwide who have limited access to curative therapies. Autologous hematopoietic stem cell transplant following ex vivo CRISPR editing of the BCL11A erythroid enhancer reactivates fetal hemoglobin (HbF) and achieves an effective cure, but the resource constraints of clinically approved procedures for editing by electroporation (EP) severely limit widespread implementation. We directly compared the functional outcomes of EP delivery of Cas9 ribonucleoprotein with lipid nanoparticle (LNP) delivery of Cas9 mRNA in primary human HSPCs obtained from healthy HbAA donors and from patients with SCD. While higher editing rates are achieved with EP, LNP-treated HSPCs exhibited greater viability and cell yields that persisted throughout a multi-stage in vitro erythroid differentiation protocol. By day 20, the yield of mature red blood cells (CD71lowCD235ahigh) was lowest in the EP cohorts. Across treatment groups, we observed HbF induction proportional to indel frequency. LNP editing of SCD patient-derived HSPCs as low as 25% modified alleles still caused HbF production and reduced the propensity for sickling of in vitro differentiated RBCs. These findings highlight the critical trade-offs among manufacturing ease, delivery-associated toxicity, and functional performance across two modalities of therapeutic genome editing for hemoglobinopathies.

Despite substantial advances in diagnostic testing, 10-15% of Usher syndrome patients remain without a genetic diagnosis, having significant implications for genetic counseling and potential future therapeutic interventions. In this study, genome sequencing data from probands clinically presenting with Usher syndrome were analyzed. Two novel deep-intronic variants were identified in PCDH15, c.3983+3635A>G and c.3123-1728A>G, in two independent patients. Both deep-intronic variants were classified as likely pathogenic and predicted to alter PCDH15 pre-mRNA splicing. Using a minigene splice assay and iPSC-derived photoreceptor precursor cells from patients, we confirmed that both variants lead to the inclusion of a pseudoexon in the PCDH15 transcript introducing a stop codon and subsequent premature termination of protein translation. We designed and evaluated antisense oligonucleotides (ASOs) with the purpose of redirecting aberrant pre-mRNA splicing caused by both deep-intronic variants. For both variants, designed ASOs were successful in restoring normal splicing patterns, highlighting their potential as a future therapeutic intervention strategy to halt the progression of retinitis pigmentosa caused by these novel variants. Overall, these findings contribute to the understanding of Usher syndrome caused by deep-intronic pathogenic variants in PCDH15 and describe for the first time the use of an ASO-mediated splice correction strategy for individuals diagnosed with these variants.

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-{beta} (A{beta}) plaques, neurodegeneration, and cognitive decline. {beta}-Site amyloid precursor protein cleaving enzyme 1 (BACE1) catalyzes the rate-limiting step in A{beta} production and remains a therapeutic target for AD. However, effective delivery of RNA therapeutics to the brain remains challenging due to the blood-brain barrier (BBB). Here, we evaluated the feasibility of using clinical-grade mesenchymal stem cell-derived extracellular vesicles (EVs) as systemic carriers for Bace1-targeting small interfering RNA (siRNA) in the 5xFAD mouse model of AD. Engineered EVs crossed the BBB and delivered siRNA cargo to the brain, with uptake observed in both neurons and astrocytes. Systemic therapy with EVs engineered to encapsulate Bace1 siRNA resulted in reduced brain Bace1 protein levels and a decrease in amyloid plaque burden compared with control EVs carrying scrambled siRNA. The reduction was most pronounced in larger, high-intensity plaques, suggesting that Bace1 suppression may preferentially limit plaque growth and maturation. Repeated systemic administration was well tolerated, with no evidence of treatment-associated toxicity. These findings establish a proof-of-concept feasibility for EV-mediated delivery of Bace1-targeting siRNA to the brain and support further development of engineered EVs as a therapeutic platform for neurodegenerative diseases. Future studies incorporating behavioral, molecular, and mechanistic analyses will be required to determine the extent to which Bace1 suppression delivered through EVs can modify disease progression and improve functional outcomes in AD.

Neurodegenerative diseases are increasingly associated with vascular dysfunction beyond progressive neuronal degeneration, yet how vascular pathology contributes to disease progression remains poorly understood, largely due to the lack of a neurodegenerative disease model capable of capturing neuronal pathology alongside associated vascular dysfunction. Here, we developed a microengineered 3D vascularized brain tissue model that integrates neurospheroids with a self-assembled, perfusable vascular network to recapitulate key features of the neurovascular interface. Using this model, we investigated the vascular contribution to Parkinson's disease pathology by introducing -synuclein preformed fibrils into the engineered vasculature. Intravascular -syn fibril exposure induced endothelial barrier disruption, vascular leakage, inflammation, and vascular regression. Notably, this vascular insult was accompanied by intraneuronal -synuclein aggregation within neurospheroids, suggesting that vascular dysfunction may facilitate the exposure of neural tissue to pathogenic -synuclein. Our neurodegenerative disease modeling approach establishes a versatile and tractable platform for investigating vascular contributions to neurodegenerative disease progression.

Cytomegalovirus (CMV) has been implicated in glioblastoma (GBM) progression. Ongoing clinical trials are assessing therapeutic approaches targeting CMV in GBM but to date no new therapy has been approved outside the standard of care. Previous preclinical studies have highlighted the potential of the antiviral drug Cidofovir (CDV) in GBM; however, its clinical use is limited by dose-dependent nephrotoxicity and poor cellular uptake, necessitating high intravenous doses to achieve therapeutic activity. Brincidofovir (BCV), a lipid conjugate of CDV has been developed, which does not induce nephrotoxicity and has significantly greater cellular bioavailability. Here we examined the effects of BCV in a newly established CMV-driven GBM model (SB28) and in patient-derived tumor neurospheres. We show that BCV prolongs survival in vivo and exerts both CMV-dependent and independent antitumor effects. Mechanistically, BCV induces DNA damage and cell cycle dysregulation in GBM cells and inhibits proliferation of patient-derived neurospheres in a dose-dependent manner. These data identify BCV as a dual-action therapeutic that suppresses viral oncomodulation while directly targeting tumor cell viability.

Scalable human models that capture interactions between distinct tissues remain limited, constraining mechanistic insight and therapeutic prediction. Here, we established a scalable, automation-compatible human neuromuscular organoid (NMO) platform that enables integrated analysis of neuronal and muscle lineages in spinal muscular atrophy (SMA). Patient-derived NMOs reproducibly self-organise into spinal cord and skeletal muscle compartments and form functional neuromuscular circuits. SMA NMOs recapitulate early disease features, including reduced survival motor neuron (SMN) protein levels and impaired neuromuscular junction (NMJ) maturation. Single-nucleus RNA sequencing identifies lineage-specific transcriptional changes across neuronal and muscle compartments preceding functional deficits. Using this platform, we compared two clinically relevant SMN2 splicing modulators and observed distinct, cell-type-dependent responses. While both compounds increased SMN levels and NMJ number, only one enhanced myofiber growth and improved contractile function. These findings highlight muscle maturation, rather than NMJ number alone, as a key determinant of functional recovery and establish NMOs as a scalable system for studying cell-type-specific therapeutic responses.

Echovirus 18 (E18) is a predominant pathogen causing aseptic meningitis in children, and post-E18 infection frequently triggers myasthenia gravis-like autoimmune neurological damage. This pathological process relies on neonatal Fc receptor (FcRn)-mediated IgG transcytosis across mucosal barriers, and FcRn also acts as an essential functional receptor required for E18 attachment and uncoating during host cell invasion. At present, no E18-specific prophylactic vaccine has been clinically approved, and anti-FcRn monoclonal antibodies are the available therapeutics to alleviate autoantibody-mediated tissue injury. We constructed an integrated automated phylogenetic pipeline named evolution_conservation, which enables rapid tracing of the evolutionary position and genetic relatedness of clinical isolates to identify closely related strains from previous outbreaks. Serving as an in silico alternative to animal experiments, this pipeline supports reference-guided vaccine design and longitudinal comparative assessment of vaccine safety and efficacy, facilitates identification of patient populations presenting rare post-viral sequelae, and accelerates clinical trial progression. In this study, we inserted the pre-screened linear epitope E18-3 into a truncated hepatitis B core (HBc) scaffold to generate chimeric virus-like particles (VLPs). A non-invasive floss-based gingival mucosal immunization mouse model was established, with subcutaneous Freunds adjuvant immunization set as the control group. ELISA results confirmed that gingival mucosal delivery of particulate HBc-E18-3 VLPs alone could induce sustained high levels of antigen-specific intestinal IgG in vivo. Drawing on research paradigms of therapeutic neoantigen vaccines for tumor recurrence prevention, the evolution_conservation bioinformatic pipeline and mucosal VLP platform described herein establish an innovative framework for developing antigen-competitive prophylactic and therapeutic vaccines targeting FcRn for myasthenia gravis and autoimmune encephalitis.

Compact type II-C Cas9 nucleases are attractive for therapeutic genome editing because their small size enables packaging into adeno-associated viral (AAV) vectors, and their extended protospacer-adjacent motifs (PAMs) reduce off-target cleavage while expanding targeting scope. Yet characterized type II-C orthologs have edited mammalian cells far less efficiently than the canonical SpCas9. Here, we used embedding-based metagenomic mining of >4.7 x 10 proteins, combined with AlphaFold3 structure prediction and locus-context analysis, to identify three previously uncharacterized compact type II-C Cas9 orthologs, NsuCas9 (1,092 aa), PsuCas9 (1,084 aa), and GfoCas9 (1,074 aa), and benchmarked them in vitro and in human HEK293T cells. All three are robust RNA-guided nucleases with distinct PAM specificities (N CC, N NYAA, and N RHAA, respectively), divergent thermal profiles, and asymmetric sgRNA cross-compatibility. In human cells, PsuCas9 with an N ATAA PAM reaches 78.4% indels and matches or exceeds SpCas9 at multiple loci, representing the first natural compact type II-C ortholog reported to do so, while GfoCas9 and NsuCas9 add complementary coverage. All three show a strong deletion-biased repair signature and no detectable editing across 33 predicted off-target sites. These compact, high-fidelity nucleases expand the CRISPR targeting space for AAV-deliverable therapeutic editing.

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

The blood-brain barrier (BBB) is a highly selective cell layer that restricts the diffusion of diverse chemical entities into the central nervous system (CNS) from systemic circulation. Macromolecular therapeutics including oligonucleotides, peptides, and monoclonal antibodies exhibit only minimal brain distribution after systemic dosing due to exclusion by the BBB. Receptor-mediated transcytosis (RMT) has evolved to transport vital cargo across the BBB through a specialized vesicular transport pathway. Transferrin receptor 1 (TfR1) shuttles transferrin, its natural ligand, across the BBB, as well as TfR1-binding IgG antibodies and conjugates. Here, we describe a novel monovalent TfR1-binding VHH-Fc for the delivery of oligonucleotide cargo, including antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) across the BBB in rodents and non-human primates (NHPs), supporting the translational potential of the VHH-antisense RMT platform for the treatment of neurological disorders. We explore the role of binding affinity, conjugation site, drug-antibody ratio (DAR), and conjugation chemistry, and determine that binding affinity, DAR and conjugation site are major determinants of RMT capacity and brain activity of siRNAs delivered across the BBB. Graphical Abstract / Highlights O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/744307v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@d1d648org.highwire.dtl.DTLVardef@4b22d3org.highwire.dtl.DTLVardef@db8b6borg.highwire.dtl.DTLVardef@19e5ac3_HPS_FORMAT_FIGEXP M_FIG C_FIG - Anti-TfR1 (-TfR1) VHH ligands formatted as heterodimeric, 2-chain monovalent VHH-Fc were engineered for conjugation to siRNA and ASO. - Systematic in vivo evaluation of VHH clones spanning a range of TfR1 binding affinities revealed a relationship between TfR1 binding affinity and the CNS activity of intravenously dosed VHH-Fc-siRNA conjugates. - By optimizing TfR1 binding affinity, conjugation site, and conjugation chemistry, we identified VHH-Fc-siRNA molecules that efficiently cross the BBB via receptor-mediated transcytosis and reduce target mRNA across CNS tissues, including deeper brain regions, after intravenous (IV) or subcutaneous (SC) dosing in mice and non-human primates (NHPs).

SummaryA catch-all intronic guide RNA pair excises the KIAA1549--BRAF oncofusion across its major variants, with productive junction excision confirmed by gain-of-function PCR in patient-derived glioma cells. An allele-specific guide selectively disrupts BRAF V600E, in patient-derived pediatric low-grade glioma cells. Pediatric low-grade glioma (pLGG) is the most common brain tumor of childhood, accounting for 30--50% of all pediatric central nervous system malignancies1. The disease is almost universally driven by activating mutations in the BRAF serine/threonine kinase: a chromosomal tandem duplication generating the KIAA1549--BRAF oncofusion in approximately 70% of cases, or the BRAF V600E gain-of-function point mutation in approximately 15%2. Current targeted pharmacotherapies, including the RAF inhibitor tovorafenib, require continuous dosing, are not allele-specific, and carry risks of long-term toxicity in children. A one-time genomic intervention that permanently disables the oncogenic BRAF alteration while preserving wild-type BRAF signaling represents a compelling therapeutic alternative. In this study, we describe the design and experimental validation of allele-specific CRISPR guide RNAs targeting both the KIAA1549--BRAF oncofusion and the BRAF V600E point mutation. For the oncofusion, we developed a double-cut intronic excision strategy in which a guide RNA targeting KIAA1549 intron 14 is paired with a guide RNA targeting BRAF intron 11. Because the genomic breakpoints of all four major fusion variants (KB 16:9, 15:9, 16:11, and 15:11) fall within these introns, a single guide pair can address the full landscape of fusion heterogeneity in a single intervention. For BRAF V600E, we exploited a unique PAM sequence created by the pathogenic TBA transversion at codon 600, enabling allele-specific SpCas9 and AsCas12a guide designs that distinguish the mutant from the wild-type allele at single-nucleotide resolution. We screened guide RNA candidates by ribonucleoprotein (RNP) nucleofection in A375 human melanoma cells (BRAF V600E homozygous) and in patient-derived 3635 PXA glioma cells (BRAF V600E heterozygous). The top KIAA1549 intron 14 guide, K9_i14_A_Cas9, achieved 66% indel frequency in A375 cells. The top BRAF intron 11 guides, B_i11_A_Cas9 and B_i11_D_Cas9, achieved 84% and 85% indel frequency, respectively. For BRAF V600E, the best allele-specific SpCas9 guide achieved l57% editing in A375 cells and l74% editing in 3635 PXA patient-derived glioma cells. Dual-cut excision of the KIAA1549--BRAF junction was confirmed by a gain-of-function PCR assay designed to detect the excision junction amplicon ([~]191 bp) produced by NHEJ-mediated rejoining of the KIAA1549 intron 14 and BRAF intron 11 cut ends.

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.

Pancreatic beta cells exhibit marked resistance to proliferation, posing a barrier to therapeutic strategies aimed at restoring beta-cell mass in diabetes. Here, we present a transient, non-integrative approach to stimulate beta-cell proliferation using in vitro transcribed (IVT) mRNAs encoding cell cycle regulators. In rodent beta cells and human-beta cell derived EndoC-BH5 cells, chemically modified IVT mRNAs activated cell cycle entry and subsequent mitosis. A single dose of cyclin D1 and CDK4 IVT mRNAs nearly doubled the number of rat beta cells. However, achieving cell division in human beta cells required co-delivery of MYC IVT mRNA. The mitogenic response of beta cells peaked within 36-60 hours, and declined thereafter, reflecting the transient nature of IVT mRNA. Transcriptomic profiling revealed temporary activation of proliferative pathways and reversible downregulation of beta-cell maturation markers. Importantly, we detected no evidence of sustained proliferation. Our findings demonstrate that mRNA-based delivery of cell cycle regulators can overcome the intrinsic cell cycle block in beta cells and may provide a controllable approach for beta-cell regeneration.

Glioblastoma studies employ syngeneic orthotopic models to preserve tumor-immune interactions, but intracranial tumor burden is challenging to monitor longitudinally. Bioluminescence imaging enables non-invasive assessment, although reporter immunogenicity may compromise model fidelity. We engineered murine GL261 glioma cells to stably express nanoluciferase (NLuc) and compared them with parental GL261 (WT) and GL261 cells expressing red-shifted firefly luciferase (Red-FLuc). In vitro, GL261-NLuc retained growth kinetics and morphology comparable to GL261-WT and produced >100-fold stronger bioluminescence than GL261-Red-FLuc. In immunocompetent mice, GL261-NLuc formed lethal brain tumors with survival and tumor histopathology, immune profile, and response patterns to experimental oncolytic virus therapy broadly resembling GL261-WT. In contrast, GL261-Red-FLuc tumors regressed and exhibited heightened inflammation and increased infiltration of activated CD8+ T-cells. Longitudinal imaging of GL261-NLuc tumors detected treatment-associated changes in growth kinetics not captured by survival alone. These establish GL261-NLuc as a practical reporter for longitudinal immunocompetent glioblastoma studies amenable to immunotherapy evaluations.

Nonarteritic anterior ischemic optic neuropathy (NAION) is the leading cause of acute optic nerve related vision loss in older adults, yet no disease modifying therapy exists. Although ischemia is a defining feature of NAION, prior therapeutic efforts targeting vascular insufficiency or nonspecific oxidative stress have failed to prevent irreversible retinal ganglion cell (RGC) degeneration, underscoring an unresolved mechanistic gap between ischemic insult and permanent axonal failure. In this endeavour, we identify lipid peroxidation as an important driver of neurodegeneration in NAION. Analyses of human NAION retina, together with a rigorously validated mouse model, demonstrated a remarkable activation of phospholipid peroxidation within the retina following ischemic injury. RGC-specific overexpression of glutathione peroxidase 4 (GPX4), the only known enzyme capable of directly detoxifying phospholipid hydroperoxides within biological membranes, confers striking protection of RGC survival, axonal integrity, and visual function. We further demonstrate that mitochondrial-targeted GPX4 provides superior protection, suggesting mitochondria as a critical locus of lipid peroxidation-driven vulnerability in NAION. Leveraging real-time multiparametric in vivo imaging to directly interrogate axonal metabolism and function, we demonstrate that RGC-specific GPX4 overexpression robustly restores axonal and retinal mitochondrial abundance, improves ATP bioenergetics, and suppresses superoxide stress following optic nerve ischemia. Mitochondria-targeted GPX4 expression further restores axonal transport and retinofugal projections to central visual targets, thereby stabilizing visual pathway connectivity. Notably, these neuroprotective effects are recapitulated by Ebselen, a clinically tested GPX mimetic, identifying lipid peroxide detoxification as a translatable and imaging-validated therapeutic strategy. Collectively, this work establishes ischemia-induced lipid peroxidation as an essential driver of neurodegeneration in NAION and identifies GPX4 as a key molecular determinant of retinal ganglion cell resilience.

Astrocyte border formation is a conserved neuroprotective response to neural tissue disruption, yet astrocyte border states at implanted biomaterials remain less well characterized than injury responses. Here, we developed the Astrocyte Border Characterization (ABC) Tool, which leverages a shear-thinning, injectable biomaterial to locally deliver astrocyte-specific RiboTag AAVs and small molecule regulators in the mouse striatum, enabling molecular profiling and phenotypic modulation of astrocyte border (AB) cells. Spatially precise delivery of AAV using the ABC Tool yielded enhanced specificity and robust RiboTag expression in AB cells from 7-70 days post injection. Temporal transcriptomic profiling of AB cells revealed predominantly acute, transient changes in genes governing dedifferentiation, proliferation, metabolic reprogramming, and inflammation regulation. Persistent changes accounted for only 14% of regulated genes but involved critical gain of functions in immune regulation and host defense that mirrored astrocyte border responses at chronic CNS injuries. Local delivery of indiscriminate or astrocyte-selective ablation molecules delayed, rather than prevented, border formation, ultimately yielding thicker astrocytes borders with increased inflammation and fibrosis at the biomaterial-tissue interface. Conversely, local delivery of {beta}-hydroxybutyrate (BHB) from the ABC Tool altered key aspects of the transcriptional reprogramming to attenuate chronic astrocyte reactivity and prevent biomaterial contraction without exacerbating inflammation or fibrosis. Our findings establish the ABC Tool as a bioassay for studying and manipulating astrocyte borders at implanted biomaterials and identify focal metabolic regulation as a strategy to modulate AB cell phenotypes and enhance the CNS biocompatibility of biomaterials.

More than two million deaths annually are attributed to liver-related conditions, making primary human hepatocytes (PHH) an invaluable in vitro model for studying liver pathophysiology and the molecular mechanisms underlying hepatic diseases. However, because PHH do not proliferate in culture, CRISPR gene editing has been highly inefficient. Here, we report lipofection- and lentivirus-mediated protocols for CRISPR-Cas9 delivery in mouse-passaged primary human hepatocytes (mpPHH), a system that enables PHH expansion in liver-humanized mice. We achieve robust gene editing efficiencies exceeding 90% in mpPHH while maintaining cell viability. We demonstrate the utility of these protocols by disrupting CYP3A4 to impair xenobiotic metabolism and by showing that edited mpPHH efficiently engraft and expand in mice, generating liver-humanized animals. We establish the feasibility of arrayed CRISPR screening in mpPHH using an 85-gene screen to identify host factors influencing hepatitis B virus (HBV) infection, and validate key findings in humanized mice by targeting the HBV entry receptor SLC10A1 (NTCP), which reduced viral infection in vivo. Our methodology enables scalable genetic manipulation of mpPHH, opening new avenues for HBV research and liver disease modeling.