Novel cell and gene therapies hold great potential to alter the clinical course of a range of chronic diseases, yet uncertainties remain regarding their long-term efficacy and impact. These uncertainties pose challenges for Health Technology Assessment (HTA) committees, which systematically assess health interventions seeking public subsidy. Haematological conditions are a common target for gene therapies; however, the perspectives of those both directly and indirectly impacted by funding decisions for therapies in this disease area have not been investigated. To support future HTA considerations of gene therapies, we explored the perspectives of individuals living with haematological conditions who may become eligible for such treatments, alongside their caregivers and members of the general population in Australia. We undertook interviews and focus groups with 55 participants with lived experience and 32 general population members to elicit responses on what might influence decision making on gene therapies; the potential benefits or harms of gene therapies; any perceived barriers to gene therapy access; and/or uncertainties associated with the respective gene therapy. Here, we describe the parallel perspectives of health consumers and the general population, providing five key areas of consideration for HTA decision-making: individual patient and disease factors; geographical, financial and clinical access; balancing disease and quality of life benefits with adverse effects; individual and health system costs; and remaining treatment uncertainties. These will require resolution before widespread acceptability and uptake of gene therapy.

Direct intratumoral administration of immune-stimulating agents can generate systemic immunity following a tumor-localized immune response. Interleukin 12 (IL-12) activates T cells and reverses immunosuppression, generating a localized patient-antigen-specific immune response. Changes in the tumor microenvironment in response to this cytokine facilitate the function of checkpoint inhibitors. In this study, a plasmid (p)-based approach was used to generate intratumor expression of IL-12 protein and of a polypeptide designed to inhibit PD-1/PD-L1 binding, potentially to function as a checkpoint inhibitor. An updated technology, heat-and-impedance gene electrotransfer (HIGET), was used for tumor transfection to increase the reproducibility of protein expression. Efficacy was evaluated in a difficult mouse model combining subcutaneous and intraperitoneal melanomas. While each monotherapy, pIL-12 HIGET or pPD1ex HIGET, significantly increased mouse survival, the combination therapy produced significantly longer survival times than either monotherapy alone.

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.

Hearing loss poses substantial global healthcare challenges, and conventional treatments remain inadequate for genetic etiologies, necessitating innovative approaches like gene therapy. This study presents the first comprehensive bibliometric analysis of hearing loss gene therapy to systematically map global trends, key contributors, and technological breakthroughs. We analyzed 2913 publications (1993-2025) from Web of Science, PubMed, and Scopus using advanced tools like CiteSpace, VOSviewer, and Bibliometrix. Metrics encompassed publication trends, country and institutional contributions, journal influence, author productivity, and keyword networks, with a focus on adeno-associated virus vectors and CRISPR/Cas9 technologies. Results indicate an exponential growth in publications, driven primarily by the USA and China, with the USA maintaining the highest citation impact. Harvard University, the University of Michigan, and Fudan University emerged as the top contributing institutions. Keyword analysis identified AAV-driven hair cell regeneration and CRISPR/Cas9-mediated mutation correction as the dominant research frontiers. While AAV-based studies currently dominate highly cited works, CRISPR/Cas9 applications have gained significant momentum since 2015. Furthermore, clinical trials targeting genes like OTOF reflect rapid translational progress, with round-window membrane injection emerging as the most prevalent and promising surgical delivery approach. Ultimately, this study highlights the dynamic evolution of auditory gene therapy, providing a roadmap that bridges foundational discoveries with clinical applications while underscoring the necessity of global collaboration.

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.

Vitamin D receptor (VDR) regulates musculoskeletal biology, but its adult, tissue-specific roles are difficult to resolve with germline or conventional conditional knockouts. We developed recombinant adeno-associated viral vectors (rAAVs) to drive Cre recombinase selectively in bone or muscle and used them to delete Vdr postnatally in Vdr[fl/fl] mice. To engineer a muscle-selective vector, we screened AAV9 constructs carrying candidate muscle promoters and identified tMCKΔ63 as the most selective promoter. Packaging this cassette in the myotropic AAVMYO capsid further reduced off-target skeletal expression while preserving strong muscle transduction. Local intramuscular delivery of AAVMYO-tMCKΔ63 enabled unilateral targeting with minimal systemic spread. In parallel, a bone-selective AAV8-Sp7 vector supported skeletal delivery. These vectors produced tissue-restricted Vdr deletion in Vdrmuscle[AAV] and Vdrbone[AAV] mice. Muscle-targeted VDR loss reduced grip strength (-9.27%, p < 0.01) and endurance (-16.58%, p < 0.05). Bone-targeted deletion caused modest but significant skeletal changes, including increased cortical thickness (+ 7%, p < 0.05) and higher vertebral stiffness (+ 27%, p < 0.001), without effects on body weight or tibial strength. This scalable, crossbreeding-independent strategy enables compartment-specific functional studies in floxed models, including genes with embryonic lethality or complex tissue interactions. It also provides a general framework for iterative capsid-promoter optimization to maximize specificity in vivo across diverse tissues.

Tumorigenicity is a key safety concern for CRISPR/Cas9-based gene therapies, yet its assessment remains challenging due to the lack of relevant and sensitive in vivo models. This HESI Global multi-site study evaluated the Soft Agar Colony Formation (SACF) assay and the Growth in Low Attachment (GILA) assay for in vitro transformation testing of CRISPR/Cas9-edited cells as part of tumorigenicity risk assessment. Across four laboratories, MCF10A cells were spiked with varying concentrations of cells carrying a CRISPR/Cas9-mediated knockout of the known tumor suppressor PTPN12 to determine the assays' limit of detection (LOD), robustness and reproducibility. Both assays demonstrated a consistent LOD of 0.8% at most sites, with SACF showing a broader dynamic range and slightly stronger correlation across laboratories. GILA exhibited weaker correlation due to plateauing at high spike-in concentrations (>12.5%), and excluding these conditions improved inter-laboratory correlations. Additionally, the study identified key determinants of assay success, including the selection of appropriate cell lines and lots, and ensuring sufficient recovery time post-thaw. Together, these data establish multi-site reproducibility of SACF and GILA in the MCF10A-PTPN12 model evaluated here and support their use as animal-free, development-stage characterization tools contributing to tumorigenicity evidence generation.

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 × 10[12] 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.

Recombinant adeno-associated virus (rAAV) mediated gene therapy is an effective approach for targeting therapeutic genes to retinal photoreceptors. Complete congenital stationary night blindness (cCSNB) is a genetically heterogeneous inherited retinal disease caused by mutations in one of several genes that are part of a large, interdependent depolarizing bipolar cell (DBC) signalplex required for normal synaptic signaling with photoreceptors. These genes include NYX, GRM6, TRPM1, GPR179, and LRIT3, and the resulting cCSNB phenotype is characterized by abnormally low-light vision, myopia, and nystagmus, but does not include retinal degeneration. Because of the non-progressive and recessive nature of cCSNB, we investigated the potential of a gene augmentation approach in the mature retina to improve retinal function and cortical visual acuity. We used a mouse model of cCSNB caused by LRIT3 loss to evaluate the efficacy of a single subretinal injection of an rAAV expressing LRIT3 in either rods or cones, and the extent of restoration of retinal function and visual acuity. We show that gene augmentation by expressing LRIT3 in Lrit3[-/-] retinas of mature mice restores the DBC synaptic signaling complex and is sufficient to rescue retinal function and improve visual acuity.

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.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability. It is caused by a trinucleotide expansion in the 5' UTR of the Fragile X messenger ribonucleoprotein 1 (FMR1) gene leading to loss of expression of Fragile X messenger ribonucleoprotein (FMRP). There is currently no cure for FXS. We developed an FMR1 gene therapy based on an adeno-associated viral vector designed with strong translational potential for future clinical testing. The viral vector was tested in Fmr1 knockout mice using two translationally relevant delivery routes and ages corresponding to in utero, toddler, and adolescent ages in humans. Functional studies showed that the FMR1 gene therapy improved select translational FXS phenotypes spanning three critical domains: sensory hyperexcitability, adaptation to change, and altered brain activity. Expression after intracerebroventricular injection was most prominent in the forebrain, whereas intravenous delivery predominantly led to expression across midbrain and brainstem, suggesting that a dual route may be needed to achieve full brain coverage. Biodistribution analyses further suggested that FMRP expression must be titrated carefully for optimal rescue. In summary, we show that FMR1 gene therapy using delivery routes and vehicles approved for clinical use improves core phenotypes in a mouse model for FXS.

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.