Molecular Therapy

Heart disease affects millions of individuals and prime editing (PE) may enable curative therapies that address the underlying drivers of heart disease. Here we describe the establishment and optimization of an in vivo cardiac PE platform which mediates efficient editing in the heart with no detectable editing in the liver. We performed a proof-of-concept test on RNA binding motif protein 20 (RBM20), which if mutated, can cause dilated cardiomyopathy (DCM) in humans. Our dual-AAV based PE therapeutic rescued cardiomyopathy phenotypes in the heterozygous Rbm20R636Q mouse model. To further develop PE targeting human RBM20, we introduced a novel humanized mouse model carrying human RBM20 wildtype (WT) or R634Q mutant sequences and displaying RBM20 cardiomyopathy phenotypes. Our human RBM20 PE therapeutic efficiently corrected the pathogenic mutation and rescued phenotypes in the humanized RBM20 mouse model. Our findings demonstrate the potential of in vivo cardiac PE in treating heart disease, offer a valuable humanized DCM mouse model for developing various therapies, and present an optimized in vivo PE platform that can be adopted for targeting other organs and tissues.

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.

Brain vascular aging is increasingly recognized as a critical therapeutic target for age-related cognitive decline. Oxidative stress, bioenergetic dysfunction, and molecular damage play central roles in the progression of vascular aging, contributing to cerebrovascular dysfunction and impaired cognitive function. While naturally occurring polyphenols such as resveratrol (RSV) have demonstrated potential in mitigating aging-related pathologies, their poor bioavailability and limited brain targeting efficiency significantly constrain their therapeutic impact. As a result, high doses or advanced drug delivery strategies are necessary to achieve meaningful physiological effects. We introduce a novel nanocarrier system designed to enhance RSV delivery to the cerebral endothelium by leveraging the natural formation of an apolipoprotein E (ApoE)-enriched protein corona around fusogenic liposomes (FL) in vivo. These nanoparticles directly fuse with cytoplasmic cell membranes and thus evade endocytosis. We found that once in the circulation FL spontaneously acquire a protein corona, which is highly enriched in ApoE, a key ligand for brain endothelial low-density lipoprotein receptors (LDLR). Based on this observation, we engineered an ApoE-functionalized protein corona around FL (ApoE-FL) to systematically evaluate whether this mechanism could be exploited for targeted brain delivery. Following optimization and physicochemical characterization, the RSV-loaded liposomes were evaluated in vitro using human cerebral microvascular endothelial cells and in vivo C57BL/6 aged mice to assess their therapeutic potential. Both FL and engineered ApoE-FL liposomal delivery systems exhibited a strong affinity for endothelial cell membranes in vitro. The knockdown of the ApoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), significantly reduced liposomal docking. Microscopy analysis revealed that both ApoE-FL and non-functionalized FL directly fused with endothelial plasma membranes, thus bypassing intracellular organelles and minimizing lysosomal degradation. This suggests that the naturally formed ApoE corona in vivo may contribute to efficient cerebrovascular targeting, a property successfully replicated by the engineered ApoE corona strategy. In vivo biodistribution and kinetic studies demonstrated that especially ApoE-FL achieved enhanced brain-targeting efficiency, prolonged cerebrovascular retention, and extended targeting distance along the arteriovenous axis. This emphasizes that fusogenic liposomes effectively engage almost the entire microvascular network, including capillaries and post-capillary venules. Functionally, fusogenic liposome-delivered RSV improved blood-brain barrier (BBB) integrity, enhanced neurovascular coupling (NVC) responses, and promoted brain vascularization in aged mice. Single-cell RNA sequencing (scRNA-seq) revealed enhanced endothelial angiogenesis and barrier protective transcriptional profiles in cerebrovascular cells treated with ApoE-FL/RSV, suggesting a molecular basis for the observed vascular benefits. Liposomal RSV delivery achieved near-complete cerebrovascular and cognitive rejuvenation in aged mice applying a 2000-fold lower RSV dose than oral administration used as control sample. Thus, ApoE-FL liposomes exhibited exceptionally high delivery efficiency in deeper brain regions, further expanding their therapeutic potential. These findings underscore the importance of targeted drug delivery in optimizing therapeutic outcomes and establish ApoE-functionalized fusogenic liposomes as a promising strategy for mitigating brain vascular aging and cognitive decline. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/709925v1_ufig1.gif" ALT="Figure 1000"> View larger version (52K): org.highwire.dtl.DTLVardef@f7966dorg.highwire.dtl.DTLVardef@b4ea4corg.highwire.dtl.DTLVardef@18240a9org.highwire.dtl.DTLVardef@634f6a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Neuronal intranuclear inclusion disease (NIID) is a polyglycine disease that primarily affects the neuronal and neuromuscular systems. Here, we developed a novel transgenic mouse model that faithfully recapitulates the multisystemic impairments associated with polyG intranuclear inclusions. Our findings demonstrate that polyG expression induces neurodegeneration, behavioral deficits, and age-dependent accumulation of uN2CpolyG aggregates across multiple tissues.

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

The blood-brain barrier (BBB) severely restricts the delivery of systemically administered adeno-associated virus (AAV) vectors for central nervous system (CNS) gene therapy. To overcome this limitation, we engineered a library of AAV9 capsid variants through rational design focused on the low-density lipoprotein receptor-related protein 6 (LRP6), a conserved mediator of transcytosis. A multi-tiered screening strategy, encompassing human BBB endothelial cells followed by neuronal and glial target cells in vitro, identified three lead variants (QL9-21, QL9-22, and QL9-25) with markedly enhanced transduction potential. In mice, these variants achieved a 5-28 fold increase in brain-wide gene delivery compared to AAV9, without elevating hepatic tropism. Crucially, evaluation in non-human primates (NHPs) revealed that the lead variant, QL9-21, mediated a striking 3-40 fold enhancement in viral genome delivery across all examined brain regions versus AAV9, while concurrently reducing liver accumulation by 2.6 fold. Our study establishes an LRP6-guided engineering platform that yields novel AAV9 vectors capable of efficient, species-conserved BBB penetration coupled with a favorable safety profile, representing a significant advance toward clinically translatable CNS gene therapies.

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.

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

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

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

Methylmalonic acidemia (MMA) is a recessive genetic disease caused by variants in the MMUT (mitochondrial enzyme methylmalonyl-CoA mutase) gene or by defects in transport or metabolism of MMUT cofactor (5 deoxyadenosylcobalamin), including variants in the MMAB gene. For the most recurrent pathogenic MMAB variant, c.556C>T (R186W), we identified a corrective editing strategy using adenine base editing. Deploying an adenine base editor mRNA and optimized hybrid guide RNA with lipid nanoparticles, we observed efficient in vitro corrective editing of the variant to wild-type, with minimized bystander editing and off-target editing in hepatocytes. These observations lay the groundwork for a gene editing therapy for patients with MMA resulting from at least one copy of the MMAB c.556C>T (R186W) variant, as well as a platform of similar therapies for patients with MMA caused by other variants amenable to adenine base editing.

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

Owing to pivotal roles in CNS debris clearance and homeostasis, microglia are central targets for the therapy of neurodegenerative diseases. Intricate proximity to neurons, the inherent danger of neuroimmune toxicity, and intrinsically high plasticity and adaptability, impose high hurdles on microglia modulation. Attenuated viruses are being tested extensively against CNS malignancies (i.e., cancer virotherapy); yet, aside from viral vector-mediated payload delivery, virotherapy for non-neoplastic CNS disease remains unexplored. Here we report disseminated targeting of microglia with the highly attenuated polio:rhinovirus chimera, PVSRIPO, that culminated in profound, durable microglia reprogramming. This phenotype, rooted in extended cytoplasmic viral (v)RNA replication, was non-cytopathogenic and did not yield virus progeny or dissemination. vRNA replication in microglia triggered selective interferon (IFN) regulatory factor (IRF) 3/IRF7 transcriptional programs in the relative absence of NF{kappa}B-driven proinflammatory cytokine responses and elicited robust phagocytosis of both tumor cells and amyloid-beta. Targeting of microglia with PVSRIPO mediated immunotherapy in a mouse glioma model and the clearance of oligomeric amyloid-beta deposits in an injectable model of neurotoxic amyloid accumulation. This work identifies attenuated virotherapy as an approach to safely and effectively invigorate microglia function in immune surveillance and neurotoxic debris clearance.

siRNA delivery platforms capable of accessing both central and peripheral tissues are critically needed to expand the therapeutic potential of oligonucleotides. To address this, we developed a novel siRNA-antibody conjugate by attaching an Hprt-targeting siRNA to an engineered antibody shuttle. This shuttle targets the insulin-like growth factor 1 receptor (IGF1R) using a fused antibody fragment (Clone F) and utilizes an antibody backbone with no tissue-relevant binding in this study. The resulting conjugate, designated Clone F-Hprt, demonstrated robust in vivo knockdown across multiple tissues. Clone F-Hprt demonstrated enhanced penetration into central nervous system (CNS) tissues compared to unconjugated siRNA following intracerebroventricular (ICV) and intravenous (IV) administration. In peripheral tissues, Clone F-Hprt achieved widespread knockdown in muscle, heart, and lung, consistent with IGF1R expression. The conjugate was well tolerated across all routes, including with repeated dosing. Although several receptor-mediated approaches for CNS delivery are progressing to the clinic (e.g., targeting the transferrin receptor), clinical validation remains to be demonstrated. Our findings highlight IGF1R as an alternative receptor capable of supporting delivery across both central and peripheral tissues, offering a complementary strategy for expanding the therapeutic landscape of oligonucleotide delivery.

Acute ischemic stroke (AIS) remains a leading cause of disability worldwide, and effective treatments are urgently needed beyond reperfusion therapy. Translating preclinical success to clinical impact has been hindered by variability in animal models and the lack of translational biomarkers that predict outcomes across species. To overcome these barriers, we developed a robust rat AIS model optimized for consistency and severity, enabling rigorous therapeutic testing. Additionally, we tested a panel of common clinical serum biomarkers to improve translation from rodents to humans. We demonstrated that serum neurofilament light chain (NfL) -a biomarker widely used in clinical stroke studies-strongly correlated with functional outcomes, establishing a translational link that has not been previously reported in rats. Notably, NfLs predictive capabilities outperformed infarct volume, a key prognostic factor in moderate and severe strokes, as well as traditional serum biomarkers intercellular adhesion molecule-1 (ICAM-1) and S100 calcium binding protein (S100B). Using this platform, we evaluated the therapeutic impact of neural stem cell-derived extracellular vesicles (NSC EVs), a novel biologic therapy poised for clinical trials, on stroke outcome in our rat AIS model. A three-dose regimen of NSC EV over 48 hours produced the best outcomes in stroked animals evidenced by smaller infarct volume, improved neurologic score, and reduced serum NfL, although single-dose and two-dose regimens were both effective at some endpoints. These findings not only validate NfL as a cross-species biomarker but also provide critical dosing insights for NSC EV therapy, accelerating the path from bench to bedside for AIS treatment.

Sickle cell disease (SCD) is caused by the production of an abnormal adult hemoglobin that generates sickle-shaped red blood cells (RBCs). Transplantation of autologous genetically corrected hematopoietic stem/progenitor cells (HSPCs) represents a promising therapy. Persistent fetal hemoglobin expression improves SCD. Here, we engineered the fetal HBG1/2 promoters by replacing the BCL11A repressor binding site (BS) with a TAL1:GATA1 motif recognized by transcriptional activators. We exploited the prime editing nuclease (PEn) that efficiently installed the TAL1:GATA1 motif in K562 cells, outperforming the original PE. Non-homologous end joining (NHEJ) and/or alternative-end joining (alt-EJ) pathway inhibition enhanced precise editing. However, this strategy was poorly efficient in patients HSPCs. Alternatively, we used CRISPR/Cas9 nuclease to either disrupt the BCL11A BS via NHEJ and/or alt-EJ or to replace it with the TAL1:GATA1 motif via homology-directed repair (HDR) using a donor ssODN template. NHEJ and alt-EJ inhibition improved product purity, reducing InDels and achieving superior precise editing efficiency compared to PEn in K562 and HSPCs. HDR-edited HSPCs preserved clonogenic capacity and differentiated into RBCs showing elevated HBG expression and correction of the sickling phenotype. These results demonstrate that replacing the BCL11A BS with a TAL1:GATA1 motif is a potent strategy for reactivating HBG1/2 to treat SCD.

Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR- and -{beta} chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell-derived islet grafts (SC-islets) in vivo. This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.

The dy3K/dy3K Lama2-/- mouse is a model for the severe form of LAMA2-related dystrophy and peripheral neuropathy (LAMA2-RD). In the dystrophic mice, a compensating laminin subunit, Lm4, that lacks polymerization and -dystroglycan-binding activity, replaces the missing Lm2 subunit. It was previously found that an 4-laminin can be modified with two small laminin-binding linker proteins, i.e. LNNd{Delta}G2 and miniagrin to facilitate polymerization and -dystroglycan binding respectively, to enable the key missing functions. Adeno-associated virus serotype 9 (AAV9) was used to deliver minigenes coding for the two proteins in dystrophic mice. AAV9-LNNd{Delta}G2 utilized a universal CBh promoter while AAV9-miniagrin utilized either the CBh promoter or muscle-specific SPc5-12 promoter. The phenotype in the dy3K/dy3K mice was evaluated following i.v. postnatal injection with either AAV9 -LNNd{Delta}G2 alone or in combination with AAV9- LNNd{Delta}G2 + AAV9- miniagrin. Double AAV treatment was found to substantially increase survival and ambulation, as well as increase forelimb grip-strength and improve muscle histology. Of note, the sciatic nerve amyelination characteristic of laminin 2-deficiency was prevented. While single treatment with LNNd{Delta}G2 was inferior to double treatment for muscle strength and survival, it corrected the radial sorting deficit equally, revealing that enablement of laminin polymerization is a sufficient requirement for myelination. HighlightsO_LIThe dy3K/dy3K (Lama2-/-) mouse, a model for severe LAMA2-related dystrophy, expresses laminin-411 that is unable to polymerize or bind to -dystroglycan (DG). C_LIO_LILNNd{Delta}G2 and miniagrin are laminin-411-binding proteins that enable polymerization and DG binding. C_LIO_LIAAV9 delivery of genes coding for LNNd{Delta}G2 and miniagrin ameliorated the dystrophic phenotype in muscle and nerve (survival, growth, mobility, and grip-strength, muscle and nerve histopathology). C_LIO_LISciatic nerve amyelination was prevented by LNNd{Delta}G2 alone. C_LI

Profound degeneration of dopamine (DA) neurons and reduced DA levels in the brain is recognized as an underlying cause of Parkinsons Disease (PD). The standard treatment for PD is levodopa (L-DOPA), but its effectiveness wanes over time and prolonged usage can lead to L-DOPA-induced-dyskinesia (LID). An adeno-associated virus (AAV)-based strategy to overexpress aromatic l-amino acid decarboxylase (AADC) in the striatum combined with L-DOPA therapy shows promise for symptomatic improvement but requires an invasive delivery approach. Here, we generated enhancer AAVs to drive AADC expression in key cell types and paired them with a blood-brain barrier (BBB)-penetrant capsid. We characterized the AAVs in mouse following multiple routes of administration and found that cell-type specific viral treatment ameliorated motor deficits and LID in PD disease models. This cell type-specific viral rescue strategy showed similar or better phenotypic rescue compared to a ubiquitous targeting approach and improved mortality. Additionally, we characterized the expression of an AAV-AADC vector capable of mouse phenotypic rescue in non-human primate (NHP) following two routes of administration. This novel therapeutic strategy in combination with L-DOPA may enable a less invasive and better tolerated approach to treat motor deficits in PD patients.