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.
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 (DMDmdx) treated with fordadistrogene movaparvovec, an adeno-associated virus serotype 9 gene therapy containing miniaturised dystrophin. Four treatment groups of ~7–9-week-old DMDmdx rats and two control groups (one wild-type and one DMDmdx group) of ~7–9-week-old DMDmdx 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 DMDmdx 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 DMDmdx rats treated with fordadistrogene movaparvovec. Taken together, this suggests a correlation between mini-dystrophin protein expression and function in the DMDmdx rat model 3 and 6 months after fordadistrogene movaparvovec.
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.
