Lipodystrophies are a group of disorders featuring reduced adipose tissue mass or function, which often leads to significant metabolic disease, reduced lifespan and impaired quality of life. Individuals with congenital generalised lipodystrophy (CGL) have severely reduced adipose tissue mass. The loss of healthy systemic lipid storage typically causes hepatic steatosis and lipoatrophic diabetes. In addition, adipocyte-secreted hormones including leptin and adiponectin are dramatically reduced. Leptin has critical roles regulating appetite and broader effects on lipid and glucose metabolism. Daily injection with recombinant leptin is currently the only specific, approved treatment for CGL. The consequences of adiponectin loss in these patients are not fully understood. Likewise, the potential therapeutic benefit of adiponectin delivery is unclear. Here we examine the effect of delivering leptin or adiponectin by adeno-associated virus (AAV) as a potential gene therapy treatment for metabolic disease in CGL using a well-characterised murine model of the condition. AAV-mediated leptin delivery significantly improved hepatic steatosis and hyperinsulinemia. However, adiponectin delivery did not lead to any observed beneficial effects. This demonstrates that gene therapy approaches may have the potential to offer long-term delivery of leptin in individuals with lipodystrophy, without the need for continuous supply of perishable therapeutics and painful daily injections.

Adeno-associated virus (AAV) is widely used for liver-directed gene therapy, with AAV serotype 3B (AAV3B) showing particularly high tropism for human hepatocytes. However, pre-existing neutralizing antibodies (NAbs) against AAV represent a major barrier to efficient gene transfer and patient eligibility. We have recently demonstrated that subjects with intermediate NAb levels (1:5-1:45), that would be excluded from clinical trials, can be converted in eligible patients by the administration of IgG-cleaving enzymes. It is thus crucial to assess not only the seroprevalence but also titer distribution. Here, we analyzed anti-AAV3B NAb titers, using a validated cell-based transduction inhibition assay, in healthy adults and patients with Wilson's disease, a hepatic metabolic disorder amenable to AAV3B-mediated gene therapy. Two independent cohorts of healthy adults (n = 200 and n = 580) were analyzed; 33-36% of individuals were NAb positive, and prevalence increased with age. Differences were observed across ethnicities. Importantly, a substantial proportion of seropositive individuals (19%) exhibited intermediate NAb titers. In contrast, only 17.4% of Wilson's disease patients (n = 23) were positive, all with high NAb titers. These findings provide insights into target population eligibility, and titer distribution data support the potential of NAb-reduction approaches to expand access to systemic AAV3B-based gene therapies.

Genetic therapies offer a promising strategy for treating cystic fibrosis (CF) lung disease, particularly for individuals with modulator-ineligible genotypes. However, clinical translation has been slower than anticipated. While CF gene therapy candidates typically demonstrate high efficacy in preclinical studies, all products trialled to date have failed to progress beyond Phase I/II, raising questions about the predictive capacity of current preclinical models. One contributing factor may be the absence of severe muco-obstructive lung disease in commonly used CF rodent models, highlighting the need to evaluate gene vectors under conditions that more closely recapitulate the CF lung environment. In this study, we evaluated a vesicular stomatitis virus glycoprotein G (VSV-G) pseudotyped lentiviral (LV) vector carrying the luciferase reporter gene under conditions of Pseudomonas aeruginosa lung infection in wild-type and CF rats. Bioluminescence imaging was conducted on days 7 and 14 post-LV vector administration to assess lung transgene expression under infected and non-infected conditions. At day 7, there was no significant difference in bioluminescence between P. aeruginosa-infected and non-infected animals (p = 0.537). From day 7 to day 14, bioluminescence decreased 136-fold in non-infected animals (p < 0.0001), compared with a 7-fold decrease in infected animals (p = 0.043), with a significant infection × time interaction indicating that the magnitude of decline was greater in non-infected controls (p = 0.024). These findings demonstrate that lung disease state can modulate transgene expression and persistence, highlighting the importance of evaluating gene therapy vectors under conditions that more closely mimic the CF lung environment.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from loss-of-function mutations in the dystrophin gene, leading to progressive muscle wasting and premature death. We present the mini-dystrophin protein expression-function relationship from a post-hoc analysis of mini-dystrophin quantification and function (strength) data at 3 and 6 months in biceps femoris and pectoralis muscles from a DMD rat model (DMD[mdx]) treated with fordadistrogene movaparvovec, an adeno-associated virus serotype 9 gene therapy containing miniaturised dystrophin. Four treatment groups of ~7-9-week-old DMD[mdx] rats and two control groups (one wild-type and one DMD[mdx] group) of ~7-9-week-old DMD[mdx] rats received fordadistrogene movaparvovec (1E + 13, 3E + 13, 1E + 14 or 3E + 14 vg/kg) or formulation buffer, respectively. Muscle samples were harvested at 3 or 6 months post-administration. Previously reported treatment of DMD[mdx] rats with fordadistrogene movaparvovec increased body weight, improved clinical chemistry parameters, ameliorated skeletal and cardiac muscle histopathology and improved muscle and cardiac function, all in a dose-dependent manner. Data from this post-hoc analysis indicate that expression of the mini-dystrophin protein levels is sustained or increased (at lower doses) from 3 to 6 months post-dose in DMD[mdx] rats treated with fordadistrogene movaparvovec. Taken together, this suggests a correlation between mini-dystrophin protein expression and function in the DMD[mdx] rat model 3 and 6 months after fordadistrogene movaparvovec.

Cortical spreading depolarization (SD) underlies migraine, frequently co-occurs with seizures, and is associated with poor prognosis after stroke. In rodent models, most genetic therapies for epilepsy suppress seizures by enhancing neuronal K[+] conductance. Although seizures and SD often co-occur and SD causes temporary neuronal silencing, it remains unknown whether K[+]-conductance-enhancing gene therapy affects SD. Here, we present a genetic method for targeted downregulation of SD using adeno-associated virus to express KCNN4, which encodes the Ca[2+]-gated KCa3.1 channels. We tested the effect of KCNN4 expression in rodent neocortex using patch-clamp electrophysiology, functional imaging in brain slices, and field potential recording in vivo. KCNN4 expression reduced acute seizure-like activity, lowered SD amplitude several-fold both in vivo and in vitro, and slowed SD propagation in brain slices, where epileptiform activity paradoxically reappeared faster after SD. Moreover, SD triggered a Ca[2+] rebound upon restoration of epileptiform activity, which was counterbalanced by KCa3.1-mediated afterhyperpolarization. Targeted suppression of SD amplitude using KCNN4 expression provides a proof-of-principle for potential gene therapy models for disorders complicated by SD. Reducing SD via increased neuronal K[+] conductance may have a side effect worth considering in gene therapy for epilepsy, as some literature suggest that SD itself may terminate seizures.

Adenine base editors (ABEs), composed of Cas9 (i.e., SpCas9) nickase, adenosine deaminase, and sgRNA, can convert A•T base pairs to G•C base pairs in the genome without causing DNA double-strand breaks, thereby offering broad application prospects. The strict protospacer adjacent motif requirement and off-target effects of wild-type SpCas9 notably restricted its application until the identification of two SpCas9 variants, SpCas9-NG and SpRY, which have overcome these limitations. However, how to select appropriate ABEs from various SpCas9 variants remains unclear. In this study, we evaluated NGG-ABE8e, NG-ABE8e, and SpRY-ABE8e in human cells and explored the impact of sgRNA design on their performance. The results indicated that NGG-ABE8e showed higher editing activity at most tested NGG-PAM sites, whereas SpRY-ABE8e showed stronger mismatch discrimination and lower detectable A-to-G editing at the selected predicted off-target sites with NGG-PAM sites. Regarding sgRNA design, we found that spacer length and 5' guanine (G) addition affected editing outcomes in a site- and editor-dependent manner. Notably, adding an extra 5' G was not uniformly beneficial and could reduce editing efficiency, especially when the native spacer already began with G. In conclusion, this study provides insights into selecting optimal ABEs and designing effective sgRNAs for the treatment of human genetic diseases.

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.