Search Results (408)

At present, there is no cure for the human genetic disorder ataxia-telangiectasia (A-T), which is managed by supportive care. This disorder arises due to mutations in the ATM (ataxia-telangiectasia mutated) gene and is characterised by a defect in the response to DNA damage, oxidative stress, mitochondrial dysfunction and immune deficiency. The ATM protein is activated by DNA damage, reactive oxygen species (ROS), and a variety of other stimuli, which leads to the phosphorylation or altered cellular localisation of multiple protein substrates that participate in cellular defence pathways. ATM plays a central role in orchestrating cellular defence against stress, which forms a focal point for approaches to treating the symptoms in this disorder. These strategies involve boosting mitochondrial function and dampening the inflammatory response. A more direct approach to treatment is gene therapy, yet the leading method using Adeno-Associated Virus (AAV) is hampered by the large size of the ATM gene itself. The use of antisense oligonucleotides (ASO) as an alternative gene therapeutic approach to treat patients has been increasingly utilised. However, only certain mutations fit the criteria for ASO-based intervention, which encourages rescue through readthrough of premature truncation mutations. For this reason, small-molecule-based therapies addressing the multi-system nature of the disease are urgently required. We address these different approaches to therapy and the outcome of numerous recent clinical trials with A-T patients, as well as ongoing research that has potential to lead to therapy. Full article

Background: Process analytical technology (PAT) enhances product quality by monitoring and controlling critical quality attributes (CQAs), offering better insights into process performance. Raman spectroscopy is a promising PAT tool to support the development of control systems for continuous and automated processes. In this study, Raman spectroscopy was used to monitor in real-time ultra- and diafiltration (UF/DF) of adeno-associated virus (AAV) and lentiviral vector (LV). Method: The incorporation of a Raman probe in a tangential flow filtration (TFF) system, when operated in open loop, enabled spectral acquisition during a five-fold concentration of clarified bulk followed by a buffer exchange to PBS with five diafiltration volumes. Results: Principal Component Analysis reduced the dimensionality of the training set, and a Partial Least Squares model was developed for each parameter in each phase. After validation, Raman monitoring platforms showed good performance in predicting CQA of both viral vectors. Batch-to-batch variability was identified as the principal source of spectral variation, and its impact on UF/DF operation was observed. Discussion: This study emphasises the potential of Raman spectroscopy as a PAT for monitoring and control strategies in the downstream processing of viral vectors. Conclusions: These monitoring platforms enhance understanding of TFF’s operation and enable timely decision making by providing real-time information on multiple parameters. Full article

Existing vectored immunoprophylaxis (VIP) approaches have primarily focused on IgG, which provides systemic protection but is less specialized in mucosal immunity. In contrast, secretory IgA (sIgA) plays a central role at epithelial surfaces, promoting pathogen neutralization while limiting inflammation. Although monoclonal IgA therapies are effective, their short half-life requires repeated dosing. Thus, VIP strategies enabling sustained sIgA expression at mucosal sites could transform mucosal infection prevention and treatment. This review outlines the key challenges associated with in vivo IgA expression and discusses critical considerations for VIP-mediated IgA delivery at mucosal surfaces, with emphasis on its potential for clinical translation. We provide a detailed overview of platforms for targeted IgA expression, including adeno-associated virus (AAV), adenoviral and lentiviral vectors, and lipid nanoparticle-based systems, alongside relevant routes of administration. Additionally, we examine emerging strategies to enhance the robustness, durability, and localization of IgA expression in vivo. Overall, VIP-enabled IgA expression represents an emerging strategy for enhancing mucosal immunity, with continued advances required to establish its role in the prevention and treatment of mucosal infections. Full article

The adeno-associated virus (AAV) has become the vector of choice for gene therapy and experimental gene delivery, owing to its non-pathogenic nature and ability to achieve persistent gene expression across diverse tissues. AAV has emerged as a key platform in cellular reprogramming and regenerative medicine, with applications spanning transcription factor delivery for in vivo lineage conversion and tissue repair across the CNS, heart, and musculoskeletal systems. However, significant limitations remain, particularly in the context of induced pluripotent stem cell (iPSC) engineering. We assess barriers to efficient iPSC transduction including receptor-dependent entry deficits and activation of p53-dependent DNA damage responses. Although AAV is widely described as non-integrating, evidence indicates that integration events occur in rapidly proliferating and actively reprogramming cells. Critically, we synthesize evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads, a cross-tissue problem not addressed in existing AAV reviews, and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing. These aspects, underrepresented in recent platform-level reviews, are specifically emphasized here as a resource for researchers designing rigorous AAV-based reprogramming and gene therapy strategies. Full article

Background/Objectives: PTEN Hamartoma Tumour Syndrome (PHTS) is a rare inherited disorder caused by germline PTEN mutations, presenting with cancer predisposition and neurodevelopmental abnormalities, including macrocephaly. PTEN functions as a tumour suppressor by regulating the PI3K/AKT/mTOR pathway and contributes to cellular adhesion, migration, and genomic stability. As no curative therapy exists, gene therapy represents a promising avenue to correct the underlying genetic defect. Methods: In this study we evaluated the therapeutic potential of AAV9-mediated PTEN gene delivery in a Pten^Δ5/+ mouse model, which is predominantly characterized by progressive lymphoid hyperplasia leading to lymph node tumour development, as well as hepatic focal lesions and macrocephaly. Results: Systemic vector delivery did not improve lymph node enlargement which represents the predominant disease phenotype in the Pten^Δ5/+ mice at the doses tested, due to restricted vector biodistribution, but it did effectively rescue the hepatic lesions, including steatosis and steatohepatitis-like changes. Mechanistically, AAV-mediated PTEN expression significantly reduced elevated p-AKT levels, indicating attenuated hyperactivation of the PI3K/AKT/mTOR pathway. Additionally, intracranial delivery of the same vector in Pten^Δ5/+ mouse neonates ameliorated macrocephaly without apparent adverse effects. Conclusions: These findings highlight the therapeutic potential of AAV9-mediated PTEN gene therapy in PHTS, particularly for hepatic and neuroanatomical manifestations. Optimization of vector biodistribution and transduction efficiency will be critical for clinical translation. Full article

Background: Duchenne muscular dystrophy (DMD) has, for the first time, several mutation-specific drugs in clinical use. Four exon-skipping antisense oligonucleotides and one adeno-associated virus (AAV) micro-dystrophin gene therapy carry US FDA approval, and one nonsense-readthrough agent (ataluren) holds a conditional European authorisation that has since been placed under review; ataluren has never been approved by the FDA. Regulatory approval, however, has not translated cleanly into demonstrable clinical benefit, and in resource-limited settings it has not translated into access at all. Methods: We compiled published efficacy estimates for each approved therapy: the EMBARK randomised trial for delandistrogene moxeparvovec (Elevidys), the STRIDE registry for ataluren, and the long-term extension studies of eteplirsen, golodirsen, viltolarsen and casimersen. We read these alongside mutation-spectrum and clinical data from a Kazakh DMD cohort (n = 34), one of the first Central Asian populations to be characterised systematically. Because these sources differ in design, population, endpoint and follow-up, the comparison is indirect and descriptive rather than pooled or statistical. On that basis we assembled three descriptive maps: biological versus clinical efficacy, eligibility versus access, and an order-of-magnitude estimate of cost per year of preserved ambulation. Results: Across the approved therapies, larger biological dystrophin restoration did not correspond to longer preservation of walking. We describe this as the absence of the expected positive association rather than as evidence of a negative one: the comparison rests on a small number of drugs whose values come from heterogeneous studies. We did not—and with these data could not—test it as a formal correlation. Ataluren, with roughly 2% dystrophin restoration, was associated with a longer reported delay in loss of ambulation than Elevidys, which restores 34–51% micro-dystrophin. In the Kazakh cohort, 26.5% of patients were biologically eligible for exon-skipping and 11.8% for ataluren, yet realistic access to any mutation-specific therapy was effectively zero as of April 2026. Our order-of-magnitude cost estimates ran from about $12,000 per additional year of ambulation for standard of care to about $1.3 million for AAV gene therapy, a hundredfold range that did not track clinical effect. Conclusions: We argue that the conventional priority ordering (gene therapy first, exon-skipping second, standard care as background) does not hold up when weighed against patient-relevant outcomes and cost, and may reasonably be inverted for resource-limited systems. This is our interpretation of an indirect comparison, not an evidence-based clinical recommendation. On that reading, the highest-value investments for Central Asia are early molecular diagnosis, universal access to glucocorticoids and specialised physiotherapy, and individual-import pathways for ataluren, while AAV gene therapy is, in our view, a lower near-term priority until its durability and safety data improve. Full article

Background: Adeno-associated virus (AAV) vector-mediated gene transfer is an emerging treatment strategy for severe cardiac disorders with genetic etiology. We refined the AAV toolkit to achieve efficient and selective expression of the therapeutic transgene in mouse hearts. Methods: Using vectors with a reporter transgene, we evaluated AAV administration routes, AAV serotype tropism to the myocardium, and cardiospecific promoters. Results: We showed that systemic AAV administration provides potent delivery and uniform transduction of cardiac tissue, outperforming localized injection techniques. The MyoAAV 2A capsid variant enabled an improved heart-to-liver transduction ratio compared to the parental AAV9 serotype. Screening a panel of cardiac and pan-muscular promoters in vitro and in vivo verified the superiority of the cardiac troponin T (cTnT) promoter for robust heart-specific transgene expression. Finally, we demonstrated that the cumulative properties of systemic AAV delivery, the MyoAAV 2A serotype, and the cTnT promoter allowed for efficient cardiac synthesis of the therapeutic transgene—an artificial miRNA designed for the gene suppression strategy of FLNC-related cardiomyopathy. Conclusions: Our findings establish an effective AAV approach for transgene transfer into the mouse heart and promote the development of gene therapy for cardiac disorders. Full article

Adeno-associated virus (AAV) is a small ssDNA satellite virus that receives wide attention due to its popularity as a safe and effective gene therapy vector. The AAV cell entry receptor (AAVR) for most serotypes is a glycoprotein containing five polycystic kidney disease (PKD) domains with which AAV interacts. AAV serotypes can be classified into three groups: those that interact primarily with PKD1, those whose interactions with PKD2 are stronger, and AAV4-like serotypes whose transduction is AAVR-independent. A phylogenetic analysis of AAVR and paralog KIAA0319 revealed AAVR amino acid variability in the region of PKD1 that is bound by AAV. We hypothesized that the substitution, in all cat-like animals, of a glutamate at a site that is an arginine (R353) in human AAVR may interfere with the binding of clade H AAVs that interact exclusively with PKD1. Analysis of PKD1 mutations, including ELISA, shows that an R353E substitution of glutamate for arginine affects the binding of the clade H AAVs that interact primarily with PKD1. Full article

Subretinal and intravitreal injection of retinal gene therapy is associated with serious adverse events and poor efficacy. We sought to improve retinal gene therapy delivery by developing fibrin hydrogel encapsulated adeno-associated virus (FE-AAV). Here we investigate conditions for storage and stability for FE-AAV. FE-AAV containing 1.9 × 10⁹ genome copies of AAV2/2-CMV-GFP was manufactured using fibrinogen reconstituted in 0.01 M sodium citrate, pH 7 (NaC) or phosphate-buffered saline containing 0.001% (v/v) Pluronic F68 (F68). Samples were stored at either −80 °C or 4 °C for up to 16 weeks. Changes in transduction efficiency, and mechanical and physical properties were evaluated. In vitro transduction was significantly (p < 0.05) reduced for FE-AAV manufactured with NaC. In contrast, we observed no change in transduction through 16 weeks for FE-AAV made with F68. Physical changes occurred in FE-AAV stored at −80 °C. In contrast to FE-AAV formulated with NaC, FE-AAV formulated with F68 and stored at 4 °C for 16 weeks was essentially equivalent to freshly made FE-AAV and retained the ability to transduce retinal pigment epithelial (RPE) cells in the pig eye. We conclude that FE-AAV formulated with F68 and stored at 4 °C is stable and shows potential for retinal gene therapy for at least 4 months following manufacture. Full article

The role of gluconeogenesis in kidney disease has increasingly drawn attention. Fructose-1,6-bisphosphatase 1 (FBP1) is a key rate-limiting enzyme in gluconeogenesis that suppresses glycolysis and reduces lactate production. In this study, we first analyzed public transcriptomic datasets of diabetic kidney disease (DKD) and validated the findings in 24-week-old BKS-db mice and in high-glucose-induced human renal tubular epithelial (HK-2) cells. We further constructed tubular-specific FBP1 overexpression/knockdown mouse models via adeno-associated virus serotype 9 (AAV-9) and combined pharmacological inhibition of lactate dehydrogenase B (LDHB) to dissect the underlying mechanism. Analysis of public clinical transcriptomic datasets showed that renal tubular FBP1 expression was positively correlated with estimated glomerular filtration rate (eGFR). In vivo, tubular-specific FBP1 overexpression in BKS-db mice reduced 24-h urinary protein and decreased renal lactate accumulation (p < 0.05) compared with diabetic controls. In vitro, high glucose-induced lactate elevation in HK-2 cells was reversed by FBP1 overexpression, while co-treatment with an LDHB inhibitor abolished this protective effect. Our findings suggest that FBP1 represents a potential experimental therapeutic target associated with alleviation of renal lactic acid accumulation and mitochondrial injury in preclinical DKD models. Full article

Background: Scalability and cost remain major manufacturing barriers limiting broad patient access to adeno-associated virus (AAV) gene therapies. While capsid engineering has advanced vector biology, comparatively fewer innovations have addressed fundamental upstream productivity constraints. Transient triple-plasmid transfection is still the dominant AAV production platform and relies on large bacterial backbone plasmids that impose DNA burden and contribute significantly to the cost-of-goods. Methods: In this study, we evaluated a compact Nanoplasmid^TM DNA system (Aldevron) as a structural redesign of the transfection substrate to enhance upstream productivity. Conventional pUC-based triple-plasmid systems were compared to fully substituted Nanoplasmid^TM equivalents across suspension HEK293 production platforms optimised via response surface Design of Experiments. Hybrid plasmid configurations were also constructed to assess component-level contributions. Results: Complete substitution with Nanoplasmid^TM resulted in up to a 10-fold increase in vector genome titre relative to conventional plasmids under matched conditions. Hybrid systems failed to recapitulate this improvement, demonstrating that full-system backbone minimisation is required to ensure high yield. Productivity gains were preserved across transfection reagents and suspension media. Nanoplasmid^TM deployment represents a scalable, capsid-independent upstream intensification approach that improves yield without altering capsid biology. Conclusions: Integration of this approach within a design-for-manufacturability framework offers a practical route to reducing bacterial plasmid elements increasing safety, enhancing process robustness, and improving economic feasibility of AAV therapeutics. Full article

Background: Selecting the most efficient and specific adeno-associated virus (AAV) capsids for gene delivery to the nervous system via minimally invasive routes is critical to gene therapy advancement. While AAV9, rAAV2-retro, AAV-PHP.eB, and AAV-MacpnS1 have demonstrated significant central nervous system (CNS) transduction ability after systemic delivery, their tropism, efficiency, and safety profiles in a developmentally relevant model have yet to be systematically compared. This study comparatively evaluated four capsids after intravenous administration in neonatal C57BL/6 mice. Methods: Transgene expression was quantitatively assessed across multiple CNS regions, as well as in the heart and liver. Associated biochemical indicators of hepatic stress were also evaluated. Results: The resulting transduction profiles were distinct and capsid-specific. Both AAV9 and AAV-MacpnS1 induced widespread CNS transduction and robust peripheral organ expression. However, AAV-MacpnS1-neuronal tropism in the thalamus was superior, and it was also associated with the most prominent biochemical indicators of hepatic stress. In contrast, rAAV2-retro was remarkably specific to the medulla and spinal motor neurons, demonstrating a valuable safety profile. AAV-PHP.eB achieved broad cellular transduction in the spinal cord, but it was the least specific towards cholinergic motor neurons. Furthermore, transduction in DRG neurons using AAV9 and AAV-MacpnS1 was efficient, but that using rAAV2-retro or AAV-PHP.eB was not. Conclusions: These findings provide an “atlas-like” comparative framework that clearly outlines the strengths and limitations of each vector. They also offer valuable guidance on selecting the most suitable AAV capsid for fundamental neuroscience applications and for developing targeted gene therapies, particularly for neurodevelopmental and motor neuron disorders, where intravenous administration in the early stages of life is a promising strategy. Full article

Rare genetic disorders of the central nervous system (CNS) remain some of the most complex and challenging diseases to treat for several reasons. Targeting the CNS, especially the brain, presents one of the greatest obstacles in gene therapy using adeno-associated virus (AAV) vectors. Although various AAVs have been identified for their ability to transduce different cells in the CNS, their effectiveness and efficiency are significantly limited by the presence of neutralising antibodies (NAbs) and restricted cargo capacity. Despite these challenges, our understanding of AAV structure and technological advances continue to enable researchers to develop innovative strategies that have resulted in groundbreaking, FDA-approved therapeutic products now available for Leber congenital amaurosis (LCA) (Luxturna^®), spinal muscular atrophy (SMA) (Zolgensma^®), and the two recent gene therapy products for aromatic L-amino acid decarboxylase (AADC) deficiency, Kebilidi^® and Upstaza^®, which currently hold FDA and EMA approval, respectively. This review aims to highlight recent advances in the field of AAV gene therapy for neurological disorders, identify research gaps, and suggest areas for future investigation to enable potential breakthroughs particularly in neurodegenerative, neurodevelopmental, and neuromuscular disorders. We foresee that more tissue- and cell-specific AAV vectors designed using AI-powered platforms will emerge to precisely and efficiently target specific brain regions, transforming how CNS disorders are treated. Full article

Glutaric aciduria type 1 (GA1) is a rare neurometabolic disorder caused by glutaryl-CoA dehydrogenase (GCDH) deficiency, leading to the accumulation of neurotoxic metabolites that can cause both acute encephalopathic crises and progressive, insidious brain injury. Current management primarily relies on a protein-restricted diet, which remains therapeutically insufficient and burdensome for patients, highlighting the need for disease-modifying therapies. In this study, we established a novel GA1 mouse model using CRISPR/Cas9 technology and evaluated the preclinical efficacy of systemic recombinant adeno-associated virus (rAAV)-mediated gene therapy. Under standard dietary conditions without high-lysine challenge, our GA1 model exhibited sustained cerebral and hepatic glutaric acid (GA) accumulation and distinct chronic vacuolation in the hippocampus and cerebellum, mirroring the insidious-onset GA1 phenotype. Five-week-old mice received a single intravenous injection of rAAV-hGCDH using either rAAV2/8 or rAAV2/9 serotypes. Systemic rAAV-mediated gene therapy significantly reduced GA accumulation and attenuated chronic neuropathological changes in this GA1 mouse model for both serotypes. Our findings support the hypothesis that peripheral metabolic correction may play an important role in preventing the chronic neuropathological changes associated with GCDH deficiency. However, further investigation using tissue-specific expression systems is required to definitively delineate the relative contributions of hepatic versus central GCDH restoration to the observed neuroprotection. Full article

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches. Full article