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.

Mitochondrial DNA depletion syndromes (MDS) are inherited conditions caused by pathogenic variants in mitochondrial DNA maintenance genes. Most MDS are severe, fatal and incurable conditions. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an MDS resulting from loss-of-function mutations in the TYMP gene, encoding Thymidine Phosphorylase (TP). Systemic nucleoside accumulation caused by TP deficiency disrupts mitochondrial nucleotide homeostasis, which underlies disease progression and mortality. Current treatments, including liver and hematopoietic stem cell transplantation, partially restore TP activity but are invasive, carry substantial risk and are limited by donor availability. Here, we establish TYMP-mRNA in lipid nanoparticles (hTYMP-mRNA-LNPs) as a safe non-viral protein-replacement therapy for MNGIE. Intravenous administration of hTYMP-mRNA-LNPs induced robust hepatic TP expression in a mouse model of MNGIE, was well tolerated and restored circulating nucleosides to wild-type levels within hours, lasting up to three weeks, at a preclinical minimally effective dose of 0.25mg/kg. To facilitate repeat administration and patient access, we demonstrate enhanced efficiency of subcutaneous mRNA-LNP delivery by co-administration of recombinant or mRNA-encoded (SPAM1-mRNA-LNPs) hyaluronidase, achieving effective hepatic TP expression and systemic nucleoside clearance. These findings establish mRNA-LNP-mediated protein replacement as a therapeutic strategy for a primary mitochondrial disease where transient liver-targeted expression is sufficient to correct a systemic metabolic defect. More broadly, our results support the development of mRNA-LNP therapeutics and their subcutaneous delivery as a generalizable platform for treating monogenic diseases, through repeatable, non-viral protein replacement.

Clinical applications of gene editors including CRISPR-Cas9 are limited by the cell-type specificity and tissue penetrance of current in vivo delivery options. Cell-mediated gene editor delivery could overcome these obstacles. To date, systems utilizing immortalized cell lines have demonstrated gene editor delivery in vitro. Because T cells traffic throughout the organism and engage in intercellular communication with individual target cells, we wondered whether they could be engineered to deliver gene editing machinery. Here, we demonstrate that primary human T cells can produce retroviral-based enveloped delivery vehicles (EDVs) packaging functional Cas9 ribonucleoproteins. When paired with fusogenic VSV-G, T cell-generated Cas9-EDVs edited a diverse array of target cells. We developed a miniaturized Moloney murine leukemia virus-based system that proved superior to existing architectures in this context and optimized both lentiviral and site-specific knock-in approaches for T cell engineering. These Enveloped Particle Information Courier- (EPIC-) T cells offer key advantages as delivery systems.

Mitochondrial DNA (mtDNA) base editors (mtBEs) are promising tools for studying and correcting pathogenic mtDNA mutations. However, the application of mtBEs is often hindered by substantial mtDNA-wide off-target editing that accumulates with prolonged mtBE expression resulting from plasmid DNA or viral delivery. To overcome this limitation, we developed mtBE-VLPs, a virus-like-particle (VLP) platform for transiently delivering mtBEs in vitro and in vivo with minimal off-target effects. While canonical protein-packaging VLPs yielded undetectable mtDNA base editing, engineered mRNA-packaging VLPs enabled efficient but transient mtBE cargo delivery. By iteratively engineering mRNA-packaging VLP compositions, we improved mtBE-VLP-mediated base editing efficiencies from less than 1% to greater than 50% across multiple target mtDNA loci in bulk populations of treated cultured cells. To further improve the packaging and delivery of certain mtBEs, we developed a strategy to selectively reduce mtBE cargo expression in VLP producer cells but not transduced cells, which increased VLP-mediated base editing efficiencies for certain mtBEs by 7.1-fold. Compared to plasmid DNA delivery in vitro, mtBE-VLPs exhibited equivalent on-target editing but 23- to 41-fold lower mtDNA-wide off-target editing, highlighting the improved editing specificity offered by transient mtBE delivery. mtBE-VLPs efficiently installed a pathogenic mtDNA mutation in mouse embryonic fibroblasts (88% efficiency) and corrected a pathogenic mtDNA mutation in a human patient-derived cell line (41% efficiency). Additionally, a single subretinal injection of mtBE-VLPs yielded 6-20% (average 12%) mtDNA base editing in the mouse retinal pigment epithelium with minimal mtDNA-wide off-target editing. These findings establish mRNA-packaging mtBE-VLPs as promising vehicles for efficient and precise mitochondrial base editing via transient mtBE delivery.

Antisense oligonucleotides (ASOs) inducing exon-skipping during pre-mRNA splicing offer promising treatment for diseases such as Duchenne muscular dystrophy. Recently, dual-targeting ASOs that simultaneously bind two splicing regulatory sequences have been shown to improve exon-skipping efficiency. Current ASOs are designed to interact with target RNAs solely based on Watson-Crick base pairing. Here we report the development of dual-targeting three-dimensional bridge (3D-bridge) ASOs in which tertiary interactions are engineered to increase target-binding affinity and the number of target residues occupied. We designed a 3D-bridge ASO for skipping exon 44 of dystrophin mRNA and biochemically validated its enhanced binding affinity for the target RNA over a similar ASO with the tertiary interactions disrupted. We subsequently optimized the 3D-bridge ASO by varying its ends and ribose chemical modifications using patient-derived induced directly reprogrammed myotubes and identified an LNA/DNA mixmer as the most potent. We further demonstrate enhanced exon-skipping efficacy and potency of the 3D-bridge ASO compared to previously reported ASOs in myotubes differentiated from patient-derived primary myoblasts and MyoD-reprogrammed fibroblasts harboring mutations amenable to reframing through exon 44 skipping. In conclusion, the 3D-bridge ASO technology improves the current dual-targeting design, demonstrating the utility and power of structure-based ASO design methods.

Adeno-associated virus (AAV) mediated gene therapies continue to show promise for the treatment of genetic diseases. However, delivery and safety still represent significant prohibitive barriers restricting translation of these delivery modalities to humans. We describe a novel AAV9 variant, AAV9fyhr or FYHR, identified from screening a controlled randomization point mutation AAV9 library in mice and nonhuman primates followed by directed evolution of in vivo CNS-derived libraries screened in human iPSC differentiated cortical neurons. Capsid variants of interest were identified by coupling long read sequencing and computational structure modeling. Structure-function analysis of our lead candidate reveal that two targeted amino acid changes are sufficient to result in beneficial pleiotropic effects across viral stability, cell surface attachment, and gene delivery. We developed a novel multiplexed barcoding reporter system, enabling a time and cost-effective option to gather spatially resolved single cell capsid validation data in mice and nonhuman primates. When administered intrathecally, AAV9fyhr exhibits 2-to-6-fold higher gene delivery across several tissues in the CNS while minimally altering the tropism to peripheral tissues in comparison to AAV9. These findings demonstrate the potential of whole capsid engineering for the rational development of translational next generation AAV capsids.

Duchenne muscular dystrophy (DMD) is a fatal disorder caused by dystrophin mutations, leading to progressive muscle degeneration and cardiac failure. Although AAV-based DMD therapies with innovative designs to overcome the cargo limits of the virus are effective in animal models, these current systems may face clinical translation challenges related to efficiency, off-target effects, and immunogenicity. We developed a multi-vector RNA End Joining (REJ) system to split large genes into multiple co-delivered AAVs that engage the cell's intrinsic spliceosome to precisely reassemble RNA segments that encode very large scar-free proteins. We show the system is efficient and has negligible off-target interactions. In vivo, REJ vectors expressing the adenine base editor Abe8e, mini-dystrophin Dp253, or native full-length dystrophin Dp427 each prevented muscle degeneration. Machine-learning histopathology of more than 120,000 myofibers revealed robust reductions in nuclear infiltration, improved centronucleation, and normalized hypertrophy, supported by functional, transcriptomic, and behavioral analyses. These findings establish REJ as a clinically viable AAV-based strategy for DMD that enables expression of large therapeutic proteins while avoiding potentially antigenic bacterial protein- or DNA-recombinases.

Safe, efficient gene delivery throughout the CNS remains a central obstacle to treating genetic diseases of the brain and spinal cord. We describe BI-TfR1 CapX, a human transferrin receptor (TfR1)-binding AAV capsid that, after a low intravenous dose in adult humanized TFRC mice, transduced more than 80% of cortical and spinal neurons and ~60% of cortical astrocytes, with reduced distribution and expression in several peripheral tissues relative to AAV9. Unlike other engineered BBB-crossing capsids, CapX bound the CNS vasculature within minutes and a large fraction of its genomes reached parenchymal nuclei within 24 hours. Delivering a Prnp-targeted epigenetic silencer (Prnp-CHARM), CapX reduced brain Prnp mRNA by over 95% requiring 18-fold fewer vector genomes per brain cell than AAV-PHP.eB for 50% lowering. A CapX delivered dual-vector cytosine base editor installed a protein-truncating PRNP stop codon lowering brain prion protein by 80%. These properties make CapX an attractive vehicle for human CNS gene therapy.

Mislocalization and aggregation of the RNA-binding proteins (RBPs) FUS and TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), contributing to combined loss- and gain-of-function toxicity. Here, we present a protein engineering strategy that appends optimized anti-aggregation motifs to FUS and TDP-43, thereby enhancing their nuclear localization and solubility, and enabling functional replacement of disease-associated proteins. We show that these engineered chimeric RBPs reduce levels of aggregation-prone endogenous proteins through autoregulatory mechanisms. In addition, chimeric FUS and TDP-43 recapitulate key nuclear functions, including DNA repair and mRNA processing. Notably, chimeric FUS restores neurite outgrowth in diseased iPSC-derived neurons, while chimeric TDP-43 suppresses cryptic exon inclusion in patient-derived fibroblasts. Collectively, these results demonstrate that engineered chimeric RBPs can rebalance RBP homeostasis by simultaneously addressing protein aggregation and mislocalization, thus mitigating RBP-associated loss- and gain-of-function toxicity, highlighting a generalizable strategy for therapeutic protein replacement in neurodegenerative disease.

Development of gene therapies for USH2A-associated diseases has been limited by the large size of the USH2A coding sequence, which exceeds the packaging capacity of adeno-associated viral vectors. Here, we evaluated high-capacity adenoviral vectors (HC-AdVs) as vehicles for a mutation-independent USH2A gene supplementation strategy in USH2A human retinal organoids. First, we generated human induced pluripotent stem cell (hiPSC) lines carrying a nonsense mutation in USH2A exon-61 (61KO). Similarly to our previously established exon-13 knockout model (13KO), 61KO hiPSC-derived retinal organoids showed reduced levels of usherin and its interacting proteins, adhesion G protein-coupled receptor V1 (ADGRV1), and whirlin, at the photoreceptor connecting cilium. Delivery of a HC-AdV5 encoding full-length USH2A under the control of the photoreceptor-specific human rhodopsin kinase (hGRK1) promoter restored usherin expression and promoted re-localization of ADGRV1 and whirlin to their physiological sites at the connecting cilium in 13KO and 61KO photoreceptors. Unexpectedly, usherin expression was also observed within mutant Muller glial cells. These findings provide proof-of-concept that HC-AdV5-mediated delivery of full-length USH2A can successfully promote the rescue of usherin and relocate its interacting partners in USH2A-defective retinal organoids, demonstrating its potential as a variant-independent therapeutic approach for inherited retinal disorders caused by USH2A mutations.

Stargardt disease is a juvenile-onset retinal dystrophy characterized by the buildup of cytotoxic lipofuscin deposits in the retinal pigment epithelium (RPE), leading to photoreceptor degeneration and eventual blindness. Currently, there are no FDA-approved treatments for Stargardt disease. Bisretinoids, byproducts of the visual cycle, are the major cytotoxic components of the lipofuscin deposits, and bisretinoid synthesis relies on the traffic of retinol from the bloodstream to the retina. Selective targeting of the key retinol transporter, Retinol-Binding Protein 4 (RBP4), offers an appealing strategy for halting the buildup of lipofuscin in the RPE and arresting the progression of Stargardt disease. Retinol delivery depends on RBP4 interaction with another serum protein, Transthyretin (TTR). We previously reported several libraries of RBP4 antagonists that effectively blocked the association of the TTR-RBP4-retinol tertiary complex, thereby lowering the overall retinol load in the retina; however, some chemotypes displayed off-target activity that warranted further optimization. Here, we report the pharmacological characterization of AKR-XI-85 and its analogs as promising non-retinoid small-molecule RBP4 antagonists. AKR-XI-85 displayed excellent in vitro and in vivo efficacy and desirable pharmacokinetic properties without any limiting off-target activity. In Abca4-/- mice, chronic dosing of the compound induced a prolonged reduction in serum RBP4 levels and achieved a dramatic, 70 % reduction in the accumulation of A2E, a critical component of toxic lipofuscin. As such, AKR-XI-85 may be an attractive drug candidate for the treatment of Stargardt disease and other lipofuscin-dependent retinopathies.

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.

In vivo hematopoietic stem/progenitor cell (HSPC) gene therapy remains limited by low gene-editing efficiency and a lack of clinically applicable selection strategies to enrich therapeutically corrected progeny. We used in vivo base and prime editing to introduce a nonpathogenic EPOR variant into HSPCs, conferring erythropoietin hypersensitivity and promoting preferential expansion of gene-corrected erythroid cells. EPOR editing was combined with three therapeutic approaches for the correction of hemoglobinopathies: {gamma}-globin gene addition, {gamma}-globin reactivation, or correction of the sickle cell disease mutation. Tropism-modified helper-dependent adenoviral vectors (HDAd6/3+) targeting HSPCs were used to simultaneously deliver the EPOR-editing machinery and the corresponding therapeutic components. In vitro studies in an erythroid progenitor cell line and primary CD34+ cells demonstrated that the EPORW439* variant conferred a strong proliferative advantage to therapeutically modified erythroid progenitors. Mice humanized with CD34+ cells from a {beta}/{beta}-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic {gamma}-globin transgene which resulted in >70% HbF-positive erythroid cells and substantial reversion of the disease-associated phenotype, including reduced oxidative stress, near-complete elimination of splenic iron deposition, and reduced splenomegaly. Importantly, these effects were achieved after simple intravenous administration of the vectors following HSPC mobilization and cytokine prophylaxis, without subsequent pharmacologic selection. Together, these findings establish a strategy to amplify the therapeutic benefit of otherwise limited in vivo HSPC gene editing for hemoglobinopathies.

A challenge of "once-and-done" adeno associated virus (AAV)-based gene therapy is the inability to modulate the level of therapeutic protein expression post-administration. Herein, we demonstrate the utility of an adenosine deaminase acting on RNA (ADAR) - mediated gene switch to control AAV-delivered gene expression. Using a premature termination codon (PTC) in the human Factor IX (hFIX) transgene, we established an ON switch, where expression of hFIX is contingent on rescuing the PTC mutation via RNA editing. In vitro and in vivo studies demonstrated silencing of the hFIX transgene by the PTC mutation and induction of protein expression by administration of an ADAR-recruiting trigger RNA. Mice transduced with a hepatotropic AAV capsid encoding an ApoE-hAAT hFIX-PTC transgene expression cassette showed a dose-dependent response between the levels of LNP-delivered trigger RNA and the amount of plasma hFIX expression achieved. We observed predictable and reproducible levels of hFIX expression upon multiple rounds of RNA editing and demonstrated that this system can achieve clinically relevant levels of hFIX. This work suggests that ADAR-mediated RNA editing may be a valuable tool for tunable expression of therapeutic transgenes in applied gene therapies.

Microglia play key roles in brain development, homeostasis, and neurodegeneration. Although multiple strategies for viral gene delivery to microglia have been reported, they have not been directly compared. Here, we developed microglia-targeting AAV capsids and benchmarked them against existing approaches. The novel capsids exhibit improved transduction efficiency in cultured mouse and human microglia, as well as neurons and astrocytes. However, when we compared microglial transduction efficiency of the novel capsids with published engineered and naturally occurring capsids after intracranial injection, all capsids achieved efficient and specific transduction when paired with a genome incorporating IBA1 promoter and miR-124 target sites. In contrast, CAG promoter did not support efficient microglial transduction. Moreover, blood-brain barrier- crossing capsids carrying IBA1 promoter and miR-124 target sites efficiently transduced microglia at high doses but exhibited off-target expression. Together, our work provides improved capsids for in vitro manipulation of microglia and establishes viral genome design, not capsid identity, as the principal determinant of efficient in vivo microglial targeting.

Adeno-associated virus (AAV) vectors are widely used for gene delivery to the central nervous system, but natural capsid tropism is broad and cell-type restriction is typically imposed transcriptionally using promoters and enhancers that consume packaging capacity and often drive weak expression. Here, we engineer neuronal targeting directly into the AAV-DJ capsid by ablating its endogenous heparan sulfate proteoglycan (HSPG) affinity and genetically displaying a nanobody against the Group 1 metabotropic glutamate receptor mGluR5 within the VP1 subunit at a permissive capsid loop (T456), generating AAV-m5. Western blot confirmed incorporation of the nanobody-VP1 fusion into assembled capsids. In primary hippocampal neuron cultures, ablating HSPG binding abolished infectivity and nanobody display rescued transduction while restricting GFP expression almost exclusively to mGluR5-positive neurons. Heparin competition assays showed that, unlike wild-type AAV-DJ, AAV-m5 transduction was unaffected by exogenous heparin, confirming that entry occurs independently of HSPG binding. Packaged with a strong constitutive promoter (CAG), AAV-m5 achieved neuron-restricted expression comparable to or exceeding that of wild-type AAV-DJ driven by a neuron-specific promoter (hSyn) and produced negligible expression under an astrocyte-specific promoter (GFAP) despite promoter activity in glia, demonstrating that capsid-level targeting can substitute, or complement, transcriptional restriction. Following stereotactic injection into the mouse hippocampus, AAV-m5 achieved an eight-fold higher proportion of transduced neurons than wild-type AAV-DJ at equivalent titers. Delivery to Grm5-null hippocampus reduced both the intensity and the spatial extent of transduction, confirming that the broad hippocampal transduction achieved by AAV-m5 is mGluR5-dependent. Together, these results establish nanobody-functionalized AAV-DJ as a modular, single-component platform for precision CNS gene delivery that circumvents the packaging and expression trade-offs of promoter-based cell-type restriction, with potential for retargeting to additional CNS cell types and disease-relevant receptors.

Adeno-associated viral (AAV) vectors possess immense potential for gene therapy applications. However, microglia can mount an immune response against AAV vectors. Yet, microglia are therapeutic targets since they play a driving role in the progression of neurodegenerative diseases. Thus, it is critical to understand the cellular processes driving the microglia-mediated immune response against AAV vectors. To this end, we modelled AAV6 induced inflammation upon retinal gene therapy in minipigs. Upon AAV6 administration, the IBA1+ cells demonstrated a morphological transition to the amoeboid form, indicating microglial reactivity. We also observed the invasion of peripheral immune cells in the retina. To elucidate the underlying molecular mechanisms, we transduced human iPSC derived microglia-like cells (iMGLs) in vitro with AAV6 and AAV8 respectively. Only AAV6 transduced iMGLs exhibited morphological features and a transcriptomic signature of microglial reactivity. The magnitude of intracellular AAV6 viral genomes was significantly higher as compared to the AAV8 condition. The elevated levels of intracellular AAV6 genomes induced a DNA damage response in AAV6-transduced iMGLs, which is a key feature of neuroinflammation-linked microglial reactivity. Thus, our work highlights the impact of intracellular viral genome accumulation on microglial reactivity and its implications for retinal gene therapy associated inflammation.

Spinocerebellar ataxia type 3 (SCA3) is most common subtype of autosomal dominant spinocerebellar ataxia worldwide and the most prevalent CAG expansion disorder in the east Asian population, but disease-modifying therapies remain unavailable. SCA3 is caused by the accumulation of polyglutamine- (polyQ-) expanded mutant ataxin-3 (mAtxn3), while the wild-type ataxin-3 (wtAtxn3) plays an important cellular role. Thus, allele-selective clearance of mAtxn3 without affecting wtAtxn3 is highly desired for SCA3 treatment. To achieve this goal, we tested previously reported compounds that have been shown to selectively degrade the Huntingtons disease causing mutant HTT protein by tethering the expanded polyQ to the autophagosome protein LC3. Here, we demonstrated that two of these compounds, AN2 and GW5074, selectively reduced both soluble and aggregated mAtxn3 with expanded polyQ (84Q), but not wtAtxn3, in both cellular and mouse models of SCA3. Mechanistically, both compounds lowered mAtxn3 by enhancing its autophagic degradation without obvious activation of global autophagy. At the molecular level, both compounds increased mAtxn3-LC3 interaction and colocalization to enhance the allele-selectively autophagic degradation of mAtxn3. Phenotypically, both compounds rescued cell viability and ATP content of SCA3 cellular models. In SCA3 patient pluripotent stem cells (iPSCs) derived cortical neurons, both compounds allele-selectively reduced mAtxn3 and partially rescued neurite shortening. Importantly, in a well-established SCA3 transgenic mouse model (MJD84.2), intracerebroventricular, intraperitoneal, and oral administration allele-selectively reduced mAtxn3 in multiple brain regions and rescued motor function deficits. Oral dosing achieved nanomolar cerebellar exposure and showed no overt liver, kidney, or glial toxicity over 28 days. Analyses of 1303 Chinese patients with SCA3 and ATXN3 constructs of different polyglutamine lengths further supported the compounds polyQ length-dependent activity with relative sparing of normal-length ataxin-3. Together, these findings provide preclinical proof of concept that utilizing orally available small molecule compounds to allele-selectively enhance mAtxn3 autophagic degradation for SCA3 treatment. One-sentence summarySmall-molecule autophagosome-tethering compounds selectively lower mutant ataxin-3 through an LC3- and autophagy-dependent mechanism and improve disease-related phenotypes in preclinical models of SCA3.

Antisense oligonucleotides (ASOs) are a powerful therapeutic modality, but their full potential is hindered by pharmacokinetic properties that affect tissue and cellular delivery. Lipid conjugation is increasingly used to modulate ASO's biodistribution and promote extrahepatic activity, yet lipid dependent effects on in vivo functional delivery, particularly in the central nervous system (CNS), remain less explored. Here, we performed a side by side in vivo comparison of cholesterol, palmitic acid (C16:0), docosanoic acid (C22:0), and eicosapentaenoic acid (C20:5) conjugated to a fully phosphorothioated 3 10 3 LNA gapmer ASO targeting the Malat1 long non coding RNA. Lipid-ASO conjugates were administered systemically or locally in the brain of mice and evaluated for tissue level and cellular level distribution by imaging, qPCR and single-cell RNA sequencing, simultaneously annotating cell origin and global transcriptional changes within the cell. Following systemic administration in mice, lipid conjugation improved overall multi organ efficacy compared to unconjugated ASO, but with pronounced tissue specific differences. Single cell sequencing of liver and heart transcriptomes revealed lipid dependent cellular uptake patterns and transcriptional responses distinct from administration of unconjugated ASO. After intracerebroventricular administration, selected fatty acid conjugates enhanced silencing in deep brain regions such as the striatum, whereas cholesterol conjugation impaired functional delivery despite increased CNS retention. Light-sheet microscopy showed restricted parenchymal penetration of cholesterol ASOs compared with broader but heterogeneous distribution of palmitic acid conjugate. Together, these findings demonstrate that lipid identity critically determines ASO efficacy, productive cellular uptake, and regional CNS engagement, emphasizing the need for context specific lipid design in ASO therapeutic development.

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.