Human iPSC-derived retinal organoids offer a human-relevant platform for inherited retinal disease (IRD) gene therapy, yet robust AAV transduction in vitro remains challenging. Here we show that culture in BrainPhys™ (BP) medium markedly enhances AAV-mediated gene delivery. Brief BP exposure during the transduction window improves uptake, whereas continuous BP culture yields the strongest effects, indicating contributions from acute entry processes and longer-term cellular remodelling. Quantitative proteomics reveal coordinated upregulation of viral entry and trafficking mediators, including integrins that form αVβ5, and the trans-Golgi entry factor GPR108, together with enrichment of endosomal/ER-Golgi transport proteins, providing a mechanistic basis for enhanced vector processing. BP concurrently promotes neuronal maturation and metabolism, with pronounced increases in neurotrophic and synaptic proteins and broad enhancement of oxidative phosphorylation components. Functional calcium imaging demonstrates comparable neuronal dynamics across conditions but reveals robust, recurrent network-level bursts under BP, consistent with strengthened connectivity. BP also preserves retinal ganglion cells and maintains expression of canonical markers, aligning with a BDNF-centred interactome and improved circuit integration. Collectively, these findings identify the culture environment as a critical determinant of AAV efficacy in human retinal tissue models and position BP as a simple, scalable strategy to reduce vector requirements, enhance retinal cells targeting, and increase the fidelity of organoid-based non-animal testing for IRD gene therapy development.
Optogenetic gene therapy has emerged as a mutation-agnostic therapeutic modality for vision restoration in advanced retinal degenerative diseases, where conventional gene replacement strategies are no longer applicable. By introducing light-sensitive opsins into surviving retinal neurons using viral vectors, this approach restores photosensitivity after photoreceptor loss. Advances in opsin engineering, vector design, and cell-specific targeting have accelerated the translation of optogenetics from proof-of-concept studies to an expanding clinical pipeline. This review summarizes recent progress in optogenetic vision restoration, focusing on the evolution of opsin technologies, retinal target cell selection—including retinal ganglion cells, bipolar cells, and residual cone photoreceptors—and viral vector platforms for retinal gene delivery. We also discuss key translational and drug development challenges, including insufficient light sensitivity, interspecies differences, immune responses to viral vectors and microbial proteins, and the lack of standardized clinical endpoints for patients with profound visual impairment. Addressing these barriers will be essential to establishing optogenetics as a clinically viable therapeutic modality and to enabling next-generation vision restoration therapies.
Helper plasmids that depend on native adenovirus gene expression have long been the standard for transient adeno-associated virus (AAV) production. Here, we demonstrate that engineering the required helper gene expression can greatly increase AAV production relative to the use of native adenovirus gene regulation. Two different engineered helper plasmid designs improved AAV vector genome (VG) titers up to 5-fold compared to a standard helper plasmid. A substantial decrease in adenovirus E4orf6 and an increase in L4 22K and 33K gene expression were associated with the improved engineered helper plasmids. VG titer improvement across capsid serotypes, plasmid transfection platforms and genome designs suggest that engineering helper gene expression is widely suited to improving AAV manufacturing yields while also maintaining consistent vector quality attributes.
The incidence of esophageal disease ranging from gastroesophageal reflux disease (GERD) to cancer has dramatically risen over the last 10 years. However, there have been limited descriptions of effective gene delivery methods for the esophagus. We developed a simple and effective electroporation-based gene delivery procedure to target the distal esophagus in living animals. We have used this approach to prevent formation of dilated intercellular space (DIS), a pathological feature of GERD, which is characterized by disruption of tight junctions and loss of epithelial barrier function. We found that tight junction proteins and the Na+,K+-ATPase β1 subunit were decreased in biopsies from patients with GERD. Transfection of esophageal epithelial cells with plasmids expressing the Na+,K+-ATPase β1 subunit increased levels of tight junction proteins and transepithelial electrical resistance. Electroporation-mediated gene delivery to the distal esophagus resulted in no injury in either rat or rabbit models. Gene expression was detected within 2 days in the esophageal mucosa and persisted for at least 12 weeks. DIS developed by 12 weeks in a rabbit GERD model and was reduced by 70% when the Na+,K+-ATPase β1 subunit was delivered. These results are encouraging for future development of gene-based treatments for GERD and its downstream consequences.
Hereditary hearing loss, predominantly attributed to mutations in the GJB2 gene, constitutes a significant global health issue, with the c.235delC mutation being the most prevalent pathogenic allele in East Asian populations. Existing interventions, such as hearing aids and cochlear implants, mitigate symptoms but fail to correct the underlying genetic defects, highlighting the need for precise therapeutic approaches. In this study, we developed a cellular disease model (293T-GJB2mut-EGFP) incorporating the c.235delC mutation and employed prime editing (PE) to restore the GJB2 functionality. We systematically optimized the pegRNA architecture and identified that the optimal pegRNA (PBS 9 bp, RTT 16 bp) achieved 37.15% correction with PE2. Incorporating Csy4 or evopreQ1motifs at the pegRNA 3' end resulted in comparable efficiencies (~37%). The introduction of two synonymous mutations (pegRNAMMD2) increased the efficiency to 48.94%. Among the seven prime editor variants, PEmax showed the highest intrinsic activity (49.14%). The PE3b nicking strategy with a + 1 nick sgRNA further improved the efficiency to 58.05%. To address adeno-associated virus (AAV) packaging limitations, a split-intein dual-AAV system for PEmax (bisected between residues 1153-1154) was developed. After nicking optimization, it showed comparable correction efficiency to a previously reported split site (residues 1024-1025), with no statistically significant difference. Off-target deep sequencing of the top four predicted loci revealed no significant editing above the background. These findings establish a comprehensively optimized prime editing toolkit for GJB2 c.235delC correction and provide a foundation for future AAV-mediated in vivo gene therapy for hereditary hearing loss.
Recombinant adeno-associated virus (rAAV) vectors underpin many approved and late-stage gene therapies, yet manufacturing costs remain a major driver of therapy price. Here, we present the first comprehensive, platform-resolved cost analysis of rAAV production across three industrially relevant suspension platforms: transient transfection, baculovirus infection, and producer cell line (PCL). Using a bottom-up model, we decompose total cost-of-goods (COGs) into upstream, downstream, drug product, and quality control contributions at bioreactor scales from 50 L to 2000 L. We show that, on a per-batch basis, baculovirus infection is the most cost-efficient platform, followed by transient transfection and PCLs however, when costs are normalized to vector titer (cost per 1 × 10¹² vg), the ranking shifts, with transient transfection becoming the lowest-cost platform under the modeled assumptions, followed by baculovirus infection and PCL platforms, underscoring the dominant influence of productivity on unit cost. We identify platform-specific cost drivers—plasmids, transfection reagent, and media in transient transfection; media and perfusion consumables in PCL platforms; and affinity capture chromatography in baculovirus processes—with buffer preparation consistently representing the largest downstream material cost across platforms. We further quantify the impact of process development levers: transfection optimization and perfusion-based intensification reduce cost per dose by up to an order of magnitude, whereas affinity resin reuse and capsid enrichment strategies provide modest, incremental savings. Incorporating indication-specific annual viral genome demand reveals how process optimization and scale-up together reduce batch burden and lower dose cost by up to two orders of magnitude, suggesting the potential for improved supply feasibility, even for high-dose neuromuscular indications, under the modeled assumptions of productivity improvement and process optimization. Together, these results provide a quantitative framework linking platform choice, process scale, and unit operations to rAAV manufacturing COGs.
Treatment effectiveness is hindered by the phenotypic plasticity of cancer and the genetic complexity of tumors. However, CRISPR-Cas-based medicines face challenges with specificity, off-target effects, and tumor heterogeneity adaptability. This work investigates the possible combination of quantum biological processes, artificial intelligence, and nanomaterials to improve CRISPR gene editing and modulate or reverse selected malignant phenotypes. Quantum machine learning (QML) can be used to simulate quantum processes like electron tunneling in DNA repair and spin-dependent enzyme activity. To enable exact tumor phenotypic reversal, these models will be combined with optimization approaches powered by AI to direct CRISPR editing in oncogenic signaling networks. Graphene, gold nanoparticles, and lipid-based vectors are some of the nanomaterials that will be used as carriers to effectively and deliver CRISPR systems in a biocompatible manner to the cancer microenvironment. We hypothesize that selected homeostatic gene-expression states may be partially restored in experimental cancer models through the integration of quantum-informed AI, CRISPR gene alteration, and nanomaterial delivery. This integrated strategy could support future cancer therapies that move beyond tumor suppression toward controlled modulation of malignant cell states, although substantial preclinical and clinical validation remains necessary.
Recombinant adeno-associated virus (rAAV) platforms have achieved significant success in clinical gene therapy; however, many still rely on ubiquitous promoters. This robust and widespread transgene expression can cause off-target effects, immune activation, and systemic toxicity, limiting their suitability for diseases requiring tissue-specific expression, such as surfactant protein B (SP-B) deficiency. Here, we aimed to improve the precision of AAV-lung gene therapy by evaluating computationally predicted lung-specific promoters with AAV6.2FF, a capsid with strong lung tropism. Promoter strength and specificity were assessed following administration of AAV6.2FF encoding the human placental alkaline phosphatase (AP) reporter gene in mice. Cross-species activity was evaluated in precision-cut lung slices (PCLS) from ferrets and pigs. We identified promoter 5979 as lung-specific in mice, outperforming the ubiquitous CASI promoter (comprised of the cytomegalovirus enhancer, chicken β-actin promoter, and ubiquitin C regulatory elements) in transgene expression and specificity, independent of AAV capsid or route of administration. In a conditional SP-B knockout model, AAV6.2FF-5979-hSPB extended survival in SP-B-deficient mice and outperformed its CASI-driven counterpart. Promoter 5979 exhibited the highest activity among the four synthetic promoters in ferret PCLS, but demonstrated limited activity in pig PCLS, underscoring species-specific differences. This study demonstrates the therapeutic value of tissue-specific promoters in targeted gene therapy while emphasizing the importance of refining algorithmic prediction platforms to ensure reliable cross-species and clinical performance.
Adeno-associated virus (AAV) is widely accepted as a delivery vector for in vivo gene therapy due to its relatively low immunogenicity, minimal toxicity, sustained efficacy, and broad tropism. However, its unpredictable cross-species applicability remains a troublesome hurdle for broader clinical applications. Thus, designing novel AAV capsids with enhanced cross-species applicability is urgently needed. In this review, we present AAV bioengineering methods, including rational design, directed evolution, and artificial intelligence-based design, with the goal of creating novel AAV variants that are translatable to humans. Using representative examples, we also evaluate how each method addresses key species-dependent barriers-receptor usage, intracellular trafficking, immune recognition, and toxicity-that critically determine cross-species translatability.
In pursuit of a gene transfer agent with efficient pulmonary transduction, the UK Respiratory Gene Therapy Consortium has developed a lentiviral vector pseudotyped with the envelope proteins, F and HN from Sendai virus (rSIV.F/HN). In contrast to other viral vectors, pulmonary rSIV.F/HN delivery achieves sustained gene expression ( ~ 2 years in mice) in the lungs and systemic circulation following a single dose. Here, we investigate the application of the rSIV.F/HN vector-platform for wider indications, including systemic disorders that require serum expression of therapeutic proteins. To assess the potential for rSIV.F/HN to produce systemic proteins, intravenous vector delivery was characterised and compared against intrapulmonary administration, achieved via 'nasal sniffing'. Both delivery routes achieved sustained (at least 1 year) systemic expression of the secreted reporter protein Gaussia luciferase. Systemic rSIV.F/HN delivery resulted in widespread protein expression across multiple organs, accompanied by the generation of significant anti-vector neutralising antibodies limiting vector readministration. Conversely, localised airway transduction was observed following pulmonary administration, which we have previously shown is not an impediment to efficient vector readministration. These data support intrapulmonary rSIV.F/HN delivery for systemic protein production, with sustained high-level transgene expression and feasible readministration.
Genome editing has progressed from a laboratory capability for targeted DNA manipulation to a clinically relevant strategy for correcting, silencing, or regulating genes implicated in human disease. In this Review, we synthesize the mechanisms, capabilities, and constraints of the principal programmable platforms-zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas systems-and highlight how base editors, prime editors, and epigenetic editors expand the range of achievable outcomes beyond double-strand break-dependent repair to precise nucleotide substitutions, small insertions/deletions, and transcriptional modulation. We compare genome-editing cargo formats, including plasmid DNA, viral-vector DNA, mRNA, guide RNA, and ribonucleoprotein complexes, together with the delivery modalities used to transport them, including AAV, adenoviral and herpesviral vectors, lipid nanoparticles (LNPs), electroporation, and virus-like particles. We then consolidate key biomedical applications enabled by these technologies, spanning endogenous gene tagging, high-throughput functional variant screening, molecular recording, and the generation of genetically faithful disease models. Across oncology, respiratory, hematologic, cardiovascular, metabolic, neurodegenerative, viral, ocular, and immune disorders, genome editing is advancing both ex vivo and in vivo interventions, including engineered cellular immunotherapies, hematopoietic stem and progenitor cell editing for hemoglobinopathies, and emerging liver-directed programs for lipid and coagulation targets. Finally, we discuss priorities for broad clinical implementation: improving editing fidelity and PAM flexibility, increasing performance in non-dividing cells, enabling tissue-selective delivery to difficult organs (for example, lung and central nervous system), and addressing manufacturing scalability, long-term monitoring, and equitable global access.
Recombinant Adeno-Associated Virus (rAAV) is the leading viral vector platform for gene therapy. A persistent challenge in rAAV manufacturing and quality control is accurate assessment of the DNA content of purified viral capsids. Empty particles, as well as ‘partial’ and over-filled species commonly contaminate even highly purified rAAV preparations. These impurities contribute to high production costs and reduced therapeutic efficacy, resulting in higher required doses and increased risks to patient health. Here we present high-resolution electrospray differential mobility analysis (ES-DMA) as a novel benchtop analytical modality for rAAV vector characterization. Sampling microliter volumes of analyte, this method achieves angstrom-scale particle sizing which we demonstrate is sufficient to resolve DNA encapsidation by rAAVs via differential electrical mobility. We benchmark this approach against charge detection mass spectrometry (CD-MS), an emerging gold standard for rAAV analytics, and identify a robust near-linear correlation between electrical mobility and mass of aerosolized rAAV particles. This relationship enables rapid identification and relative quantification of empty, full, and partial / over-filled rAAV capsids by ES-DMA.
Hereditary protein C (PC) deficiency, which is caused by PROC gene mutations, increases the risk of venous thromboembolism and offers limited treatment options. In this study, we developed adeno-associated virus serotype 8 (AAV8) vectors carrying either murine PC (AAV8-mPROC) or human PC (AAV8-hPROC) transgenes. These vectors were delivered through tail vein injection into PROC knockout mice. The highest dose of AAV8-mPROC (6.00E + 12 vg/kg) resulted in PC activity and antigen levels reaching 200.7% and 190.1%, respectively, which were maintained at 171.8% and 165.2%, respectively, by week 48. Similarly, the highest dose of AAV8-hPROC (8.00E + 12 vg/kg) resulted in 295.4% PC activity and 3.72 μg/ml human protein C antigen, which were maintained at 195.1% and 1.13 μg/ml, respectively, by week 48. In the vein thrombosis model, AAV8-mPROC and AAV8-hPROC significantly reduced the thrombus weight from 12.11 ± 3.39 mg to 7.19 ± 2.28 mg and 6.81 ± 2.28 mg, respectively. In the pulmonary embolism model, the proportion of embolized vessels decreased from 88.53% to approximately 60.62% in the AAV8-mPROC group and 62.33% in the AAV8-hPROC group. Our study has established a preclinical foundation for the safe and effective application of AAV vector-based gene therapy in treating inherited PC deficiency.
Use of adeno-associated virus (AAV) vectors has revolutionized in vivo gene therapy, but the presence of pre-existing neutralizing antibodies remains a major barrier that can hinder clinical application. While large-animal models such as non-human primates have been used to study anti-AAV immunity, their high cost and limited accessibility present challenges for studying the impact of AAV immunity on AAV-based therapies. Here, we evaluate pigs as an immunologically relevant large-animal model for investigating humoral barriers to AAV-based gene therapy. Using ELISA-based profiling across 11 AAV serotypes, we detected immunoglobulin G (IgG) antibodies against AAV capsids in pigs as early as two weeks of age, with titers increasing with age and displaying serotype-specific dynamics. Animals maintained in standard housing displayed greater inter-individual variations and broader serotype-specific reactivities. Functional assays demonstrated that these antibodies neutralized AAV particles in a dose- and serotype-dependent manner, with IgG depletion restoring transduction in vitro. Sequence analysis indicated that capsid identity can partially predict cross-reactive binding, but only under controlled conditions. These findings establish pigs as a tunable model capable of recapitulating age- and environment-dependent features of anti-AAV immunity, providing a platform for studying humoral barriers and evaluating immune evasion strategies in AAV-based gene therapy.
Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in the treatment of hematologic cancers; however, their use is often accompanied by inflammatory toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). These toxicities, ranging from mild to life-threatening, are partly driven by bystander myeloid cell activation (BMCA) and the subsequent release of pro-inflammatory cytokines such as IL-6 and IL-1β. Although previous studies have described the individual contributions of GM-CSF, IFN-γ, and TNFα secreted by CAR-T cells, a comprehensive characterization of CAR-T-derived inflammatory factors has been lacking. In this study, we characterized the soluble factors secreted by activated CAR-T cells derived from human peripheral blood and assessed their role in BMCA. Comparative cytokine analyses across human T cell subsets, including CAR-T cells, identified multiple candidates involved in BMCA. Antibody-mediated neutralization confirmed that four factors, GM-CSF, IFN-γ, TNFα, and GPIbα, play dominant roles in driving BMCA. Furthermore, siRNA-mediated knockdown of these factors in CAR-T cells significantly reduced BMCA without impairing their anti-tumor activity. These findings are consistent with prior reports on the inflammatory roles of GM-CSF, IFN-γ, and TNFα during CAR-T cell therapy and, importantly, identify GPIbα as a previously unrecognized contributor to CAR-T-associated inflammatory toxicities. Targeting these factors through antibody blockade or genetic modification may represent a promising strategy to mitigate inflammatory toxicities and improve the safety of CAR-T cell therapies.
The inhibition of estrogen receptor (ER)-mediated genomic signaling in ER-positive cancer cells has long been a primary focus of therapeutic strategies. Here, we introduce a switchable competitive inhibition system for ERα-mediated transcriptional regulation, termed DOCTER (Drug-induced On-Off Competitor for Transcription mediated by ERα). DOCTER integrates the Tet-On induced Cre-loxP recombination system to enable precise, reversible on/off switching. We demonstrate that DOCTER effectively inhibits ERα-mediated transcriptional regulation in breast cancer cells, modulating both exogenous and endogenous gene expression, and remains effective in cells harboring ERα ligand-binding domain (LBD) mutations. Upon drug induction, DOCTER exhibits controllable and reversible inhibition. To visualize these dynamic switching events in real time, we developed a multi-color fluorescent reporting system that enables monitoring of complete DOCTER switch-off within 24 hours. Our study provides a novel approach for time-specific transcriptional regulation and offers broad potential for applications in genetic research and therapeutic development.
Antibody-oligonucleotide conjugates (AOCs) effectively integrate the delivery capability of antibodies with the specific gene regulatory function of oligonucleotides, offering a novel strategy for extrahepatic delivery. Unlike antibody-drug conjugates (ADCs), AOCs employ nucleic acid payloads, enabling specific gene modulation with reduced off-target effects, thus providing new avenues for treating genetic disorders. While AOC candidates for conditions such as Duchenne muscular dystrophy (DMD) have progressed to Phase III trials, their development remains constrained by limited targets like TfR1, and their activity is highly dependent on antibody selection, linker design, and modification strategies. This study delves into the fundamental principles of target selection and antibody engineering, emphasizing that the endocytic efficiency of the target receptor is a critical factor for the successful delivery of AOC therapeutics. Taking TfR1 as an example, it analyzes the advantages and disadvantages of different antibody formats, examines the structure-activity relationships between linker chemistry and pharmacokinetics/pharmacodynamics, and further explores nucleic acid modification strategies aimed at enhancing delivery efficiency. Finally, the study outlines future directions for AOC development, including advances in bispecific antibodies, peptide conjugation, gene editing, and artificial intelligence-driven approaches, aiming to provide forward-looking perspectives and a theoretical foundation for the design of AOC therapeutics.
Hemophilia A (HA), an X-linked bleeding disorder caused by factor VIII (FVIII) deficiency, is primarily managed with exogenous therapeutic agents; however, this treatment approach remains burdensome and fails to provide durable hemostatic control. Adeno-associated viral (AAV) vectors enabling endogenous FVIII expression have emerged as promising alternatives to address these limitations, but existing vectors show limited transduction efficiency and declining activity over time. Here, we report the development and preclinical evaluation of two bioengineered AAV8 vectors, HMR-001 and its codon-optimized variant HMR-001z, designed to enhance genome integrity, hepatocellular delivery, and translational efficiency. In hemophilia A mice, intravenous administration of HMR-001 induced dose-dependent and sustained FVIII expression, achieving substantial hemostatic improvement at the highest investigated dose (2 × 10¹³ vg/kg), with blood loss reduced to levels comparable to Xyntha prophylaxis. High-order triple-linkage ddPCR quantification revealed dose-dependent increases in full-length vector genome abundance, reaching 3.74, 21.55, and 48.52 copies per diploid genome at doses of 2 × 10¹², 8 × 10¹², and 2 × 10¹³ vg/kg, respectively. Building on these markedly improved genome delivery and preservation profiles, HMR-001z achieved complete hemostatic correction at a dose of 1 × 10¹³ vg/kg, exhibiting approximately 30-fold higher FVIII expression than HMR-001 and reaching ~400 IU/dL. Collectively, these findings demonstrate that genome-level optimization combined with codon-usage refinement synergistically enhances AAV8-mediated FVIII expression, establishing HMR-001z as a durable and translationally advanced gene therapy candidate for hemophilia A.
Charcot-Marie-Tooth disease type 4C is a demyelinating neuropathy caused by loss of function mutations in the SH3TC2 gene, that is highly expressed in myelinating Schwann cells. We generated and tested a clinical stage vector with a minimal human MPZ promoter driving expression of SH3TC2. Groups of 1-month old Sh3tc2-/- mice were treated with 3 different doses of AAV9-hMPZmini.SH3TC2.SV40pA or the formulation buffer by lumbar intrathecal injection. Outcomes were compared 8 weeks post injection by behavioral, electrophysiological, proteomics, morphological analysis and evaluation of tissue integrity and inflammatory responses. Vector biodistribution to the peripheral nerves and high rates of cell-specific therapeutic gene expression in Schwann cells resulted in significant therapeutic benefits in the CMT4C model. Treated mice showed improved motor performance in grip strength and motor nerve conduction velocities. Morphological analysis revealed significant improvement in g-ratios, myelin thickness and ratios of demyelinated fibers in lumbar roots and femoral nerves of treated mice. Proteomic profiles showed correction of muscle denervation associated pathobiochemical processes in treated mice. Not observed tissue toxicity or immune reactions in neural tissues or peripheral organs. This study provides proof of principle for dose-dependent effectiveness and safety of intrathecal AAV9-mediated gene replacement paving the way for clinical translation.
