Genetic/genomic manipulation techniques (gene transfer/delivery, gene editing, etc.) have become more and more mature, and the illegal use as gene doping in sports has drawn attentions. World Anti-Doping Agency (WADA) st...Genetic/genomic manipulation techniques (gene transfer/delivery, gene editing, etc.) have become more and more mature, and the illegal use as gene doping in sports has drawn attentions. World Anti-Doping Agency (WADA) strictly prohibits gene doping, and has issued guideline on quantitative real-time PCR (qPCR) detections. However, the technical feature of qPCR makes it difficult to detect new doping targets, and codon changes on targets may also affect detection efficiency. Here, we prepare standard materials for genomic and transgenic versions of human EPO (hEPO) gene, and design qPCR primers to check the consequences of codon changes on gene doping detection. We confirm that carefully designed qPCR assays could indeed capture transgene signal, but codon changes on the transgene could severely undermine detection efficiency. We have also mimicked real world gene doping scenario by mixing genomic and transgenic versions of hEPO, and qPCR could detect wild-type but not codon-changed transgenes. As a method validation for such a challenge, we also use Sanger sequencing to confirm that sequencing could easily capture gene doping even for codon-changed transgenes. Our study confirms that codon changes will challenge qPCR-based gene doping detection, and calls for un-biased detection tools based on high-throughput sequencing in the future.
Beyond its well-known role in orofacial recurrent infections, HSV-1 has garnered significant attention in neuroscience for contrasting reasons. On one hand, it has been found to be involved in neurodegenerative processes...Beyond its well-known role in orofacial recurrent infections, HSV-1 has garnered significant attention in neuroscience for contrasting reasons. On one hand, it has been found to be involved in neurodegenerative processes; on the other, it may represent a versatile platform for gene therapy of brain diseases, due to its large genome that enables the delivery of sizable or multiple genes. These opposite features underscore the importance of understanding HSV-1 interactions with neural tissues in view of its employment as a gene therapy platform. We recently developed a new generation of highly defective backbones that proved very efficient and safe after direct injection in the brain parenchyma. Here we aimed at probing in depth the safety of viral batches that lack obvious unwanted (specifically, fusogenic) activities during production and, therefore, may escape negative selection. We employed whole-genome sequencing, electrophysiology, and viral engineering to compare different viral batches. We identified mutations (in particular A to I at position 549 in the UL27 gene) that confer fusogenic capacity to the envelop glycoprotein gB, inducing a hyperexcitable phenotype in transduced neurons. Such syncytial variants should be identified and avoided for any application of HSV-1 vectors implicating their direct injection in the nervous system.
Sensitive quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is essential for gene therapy success. Conventional cell-based transduction inhibition assays often encounter matrix-induced artifac...Sensitive quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is essential for gene therapy success. Conventional cell-based transduction inhibition assays often encounter matrix-induced artifacts resulting from variable serum content across dilutions, which artificially inflate transduction baselines and mask partial neutralization. To address this challenge, we developed the constant serum concentration (CSC) assay, which maintains constant serum levels across dilutions to stabilize assay baselines and enhance NAb detection sensitivity. Using human sera across multiple AAV serotypes, we demonstrated that CSC reclassified up to 21.7% of samples classified as non-neutralizing by a conventional variable serum concentration (VSC) assay format. This improved sensitivity was validated using monoclonal antibody and multi-species serum test benchmarks and enhanced the reliability of seronegative control pool selection. Importantly, CSC detected persistent seropositivity in preclinical seroreversion models up to one year longer than a conventional VSC assay. Since even low-level neutralizing antibodies can significantly impact gene therapy efficacy, these findings have direct implications for optimizing AAV redosing strategies and refining patient stratification. By addressing fundamental limitations in NAb quantification while maintaining procedural simplicity, the CSC assay provides crucial insights into antibody persistence with translational relevance across species and clinical settings.
The main structural components of mucus produced in the lung are mucin 5B (MUC5B) and mucin 5AC (MUC5AC) where a relatively higher expression of MUC5B is typical in health. In the lungs of individuals with asthma, there...The main structural components of mucus produced in the lung are mucin 5B (MUC5B) and mucin 5AC (MUC5AC) where a relatively higher expression of MUC5B is typical in health. In the lungs of individuals with asthma, there is a shift from MUC5B to MUC5AC as the predominantly secreted mucin which has been shown to impair mucociliary clearance (MCC) and increase airway mucus plug formation. Given its role in asthmatic lung disease, MUC5AC represents a potential therapeutic target where a gene delivery approach could be leveraged to modulate its expression. For these purposes, we explored adeno-associated virus serotype 6 (AAV6), as a viral gene vector to transduce airway epithelial cells and reduce MUC5AC expression via siRNA delivery. We confirmed that AAV6 was able to transduce epithelial cells in vitro as well as in the airways of healthy mice in vivo with high transgene expression in mucus-secreting goblet cells. Using multiple particle tracking analysis, we observed that AAV6 was capable of penetrating both normal and MUC5AC-enriched mucus barriers. AAV6 carrying MUC5AC-targeting siRNA was evaluated as a prophylactic treatment in HAE cell cultures before IL-13 challenge. IL-13 stimulated HAE cultures treated with AAV6-MUC5AC siRNA had significantly reduced MUC5AC mRNA and protein expression compared to untreated controls. Mucociliary transport in IL-13 stimulated HAE cultures was also maintained and comparable to healthy controls following AAV6-MUC5AC siRNA treatment. Together, these findings support that AAV6 may be used as an inhaled gene therapy to suppress MUC5AC overexpression and restore normal airway clearance function in asthma.
The development of targeted vector systems for gene therapy has made impressive progress during the last decade. Promising vector candidates were identified by screening large pools of adeno-associated virus (AAV) mutant...The development of targeted vector systems for gene therapy has made impressive progress during the last decade. Promising vector candidates were identified by screening large pools of adeno-associated virus (AAV) mutants in small animal models. However, it became apparent that targeted AAV mutants isolated from rodents may not function in humans as the tropism of individual AAV mutants can differ between species. To identify novel vascular-targeted AAV capsid mutants suitable for treating human patients, we generated a set of AAV2 display peptide libraries and screened them in the common marmoset, a non-human primate. To evaluate the impact of different AAV library production methods, progress of the screening process was monitored by next generation sequencing. Particle distribution and enrichment was compared between different AAV libraries and selection rounds. We observed enrichment of AAV variants in the brain and other well-perfused organs (lung, heart, kidney) potentially mediated by high capsid affinity for the vascular endothelium in general. In vitro experiments on primary human microvascular endothelial cells isolated from a set of different organs (brain, heart, lung, liver, kidney and spleen) confirmed superior transduction of a selected AAV variant displaying the "DWP" amino acid sequence motif compared to natural AAV serotypes 1-9.
Preexisting anti-AAV antibodies pose a significant challenge to the success of Adeno-associated Virus (AAV) mediated gene therapies, as they can diminish therapeutic effectiveness, restrict patient eligibility for treatm...Preexisting anti-AAV antibodies pose a significant challenge to the success of Adeno-associated Virus (AAV) mediated gene therapies, as they can diminish therapeutic effectiveness, restrict patient eligibility for treatment, and cause serious health issues during treatment. This study introduces the first point-of-care (POC) test for the rapid, quantitative detection of AAV8 binding antibodies in patients' plasma, serum, and blood, leveraging Chembio's Dual Path Platform (DPP) technology. The DPP AAV8 Total Antibody (TAb) assay delivers results within 20 min from sample addition, with a dynamic range of 0-32 µg/ml when evaluated with purified human polyclonal antibodies that bind to AAV8, with reasonable specificity and sensitivity relative to Chembio's AAV8 TAb ELISA (R² = 0.90). Moreover, the assay demonstrated strong correlations with Chembio's AAV8 neutralizing antibody (NAb) ELISA and cell-based NAb assays (R² = 0.97 in plasma) (Cell-based assay adapted from BioAgilytix EU protocol). This rapid and reliable test can facilitate the screening of potential gene therapy patients for pre-existing antibodies that bind to AAV8 and assess their suitability for AAV8-mediated gene therapy.
Vesicular glutamate transporter 3 (VGLUT3) is prominently expressed in the inner hair cells of the cochlea, playing a vital role in auditory signal transmission to the brain. Previous studies have shown that Vglut3 gene...Vesicular glutamate transporter 3 (VGLUT3) is prominently expressed in the inner hair cells of the cochlea, playing a vital role in auditory signal transmission to the brain. Previous studies have shown that Vglut3 gene knockout in mice causes severe sensorineural hearing loss without affecting hair cell integrity. However, the cochlear structure of the aged Vglut3 remains inadequately explored. In this study, we analyzed the cochlear structure of aged Vglut3 mice, revealing significant degeneration of inner hair cells, synapses, and stereocilia. To explore the potential of gene therapy to restore cochlear structure, we employed AAV8 vectors to express Vglut3 in the cochleae of 5-week-old Vglut3 mice. Twenty-seven weeks post-injection, we conducted a series of experiments to evaluate the efficacy of our gene therapy approach. Auditory brainstem response (ABR) testing demonstrated restoration of auditory function following gene therapy. Immunohistochemical staining and scanning electron microscopy (SEM) analysis revealed substantial recovery of inner hair cells and stereocilia post-injection. Our findings provide important insights into the development of novel therapeutic strategies for age-related hearing loss.
Duchenne muscular dystrophy (DMD) is caused by pathogenic sequence variants occurring in the DMD gene which lead to the loss of the dystrophin protein, a molecular 'shock absorber' that protects muscle from contraction-i...Duchenne muscular dystrophy (DMD) is caused by pathogenic sequence variants occurring in the DMD gene which lead to the loss of the dystrophin protein, a molecular 'shock absorber' that protects muscle from contraction-induced injury. The large size of the dystrophin open reading frame precludes delivery of the full-length protein using a single adeno-associated virus (AAV) vector, which led to the development of internally-deleted dystrophin minigenes encoding partially-functional dystrophin. Indeed, five such microdystrophin therapies have been assessed in various clinical programmes. In 2023, Elevidys (Sarepta Therapeutics) received accelerated approval based on levels of dystrophin as a surrogate biomarker. In 2024, it received full approval despite unclear efficacy (i.e. not meeting primary or secondary outcomes in a phase 3 trial). Additionally, in 2025, two DMD individuals treated with Elevidys died after acute liver failure. A separate microdystrophin therapy, PF-06939926 (Pfizer) was discontinued for both efficacy and safety reasons (including the deaths of two clinical trial participants). Solid Biosciences, Genethon, REGENXBIO, and Insmed continue to develop microdystrophin therapies differing in transgene structure, promoter sequences, and AAV serotype. Here we describe recent progress in AAV-microdystrophin therapeutics development, and discuss the challenges facing such approaches, including pre-existing anti-capsid immunity, anti-transgene immunity, the unknown functionality of microdystrophin transgenes, transduction of muscle stem cells, and long-term transgene persistence.
Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from locali...Collagen disorders encompass a wide range of genetic conditions caused by pathogenic variants in collagen genes for which there is an unmet need for treatments. They present various clinical features, ranging from localised tissue abnormalities to severe systemic complications. Symptoms differ significantly and depend on the pathogenic variant, which can affect various systems, including the musculoskeletal, cardiovascular, and respiratory systems, highlighting the complex implications of collagen gene pathogenic variants and the wide range of expression patterns among different collagen types. Gene-editing technologies, particularly Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas systems, have emerged as promising therapeutic options for these disorders, representing a putative one-for-all treatment strategy. This review provides an overview of current gene-editing strategies aimed at collagen-related diseases, including osteogenesis imperfecta, Alport syndrome, and dystrophic epidermolysis bullosa. We explore the application of CRISPR-Cas9, which facilitates targeted DNA modifications, base editing (BE), and prime editing (PE), enabling precise single-nucleotide alterations without double-strand breaks (DSB). Preclinical and clinical studies have shown the potential of gene therapy to enhance collagen production, restore tissue integrity, and alleviate symptoms. However, challenges persist, including the lack of recurring mutations, the need for improved delivery methods, the reduction of off-target effects, and the development of novel therapies. Despite these challenges, advancements in gene editing techniques appear promising in enhancing editing efficiency while minimising unintended mutations, paving the way for more precise and safer genetic interventions for collagen disorders. Gene editing is fundamentally transforming medicine and biotechnology. Its applications encompass advanced diagnostics, tailored therapeutic strategies, and solutions for rare genetic disorders. By enabling precise genetic modifications, gene editing is paving the way for treatments of previously untreatable diseases, including those linked to collagen pathogenic variants. This review discusses the latest advancements in gene therapy techniques targeting collagen-related disorders. It explores innovative approaches like CRISPR-Cas9-mediated gene editing and highlights emerging strategies, such as allele-specific inactivation and base editing (BE). By examining these cutting-edge therapies and their potential clinical applications, this review highlights the transformative impact of gene editing in treating collagen-related conditions, while also identifying critical challenges and future directions for research.
Inherited retinal diseases are a devasting and incurable cause of blindness which frequently affect patients at a young age, and developing effective treatments has been an important research priority in recent decades....Inherited retinal diseases are a devasting and incurable cause of blindness which frequently affect patients at a young age, and developing effective treatments has been an important research priority in recent decades. Treatments must be validated in randomised-control trials, which involve measuring benefit according to prospectively defined endpoints. A wide variety of conventional clinical endpoints and emerging anatomical, physiological, and functional biomarkers may be selected. Different options may be better or worse at capturing clinically significant differences and identifying real differences between experimental groups. This review provides an overview of some proven and potential endpoints for randomised-control trials involving inherited retinal disease patients. Clinical endpoints and biomarkers are discussed, and the work required to validate biomarkers for use in trials is outlined. Unlike in general medicine, ophthalmological clinical endpoints may all be conceptualised as surrogates for maintained vision. Selecting optimal endpoints is essential to ensure that treatments are assessed fairly, such that resources are directed towards interventions that stand to truly benefit patients with inherited retinal diseases.
Rod-cone dystrophies (RCD) are caused by mutations in over 100 genes associated with photoreceptor function, leading to progressive and sequential loss of rod and cone photoreceptors. These mutations generally disrupt re...Rod-cone dystrophies (RCD) are caused by mutations in over 100 genes associated with photoreceptor function, leading to progressive and sequential loss of rod and cone photoreceptors. These mutations generally disrupt retinal metabolism and oxidative stress response accelerating disease progression and vision loss. SPVN06 is an adeno-associated virus (AAV)-based gene- and mutation-agnostic investigational therapy designed to slow cone degeneration by delivering long-term expression of rod-derived cone viability factor (RdCVF) and its full-length isoform, thioredoxin RdCVFL, following a single subretinal administration. These proteins support cone survival by promoting glucose metabolism and reducing oxidative damage, respectively, providing a gene and mutation independent therapeutic approach for RCD. SPVN06 IND-enabling program included pharmacology evaluation in the rd10/rd10 mouse model of RCD (1.0 × 10 vector genomes (vg)/eye up to 1 month) along with systemic and ocular safety and biodistribution evaluation in non-human primates (NHPs, 6.0 × 10 to 3.0 × 10 vg/eye up to 3 months). In the rd10/rd10 mice, SPVN06 showed preserved vision, as assessed by optokinetic tracking. In NHPs, SPVN06 was well-tolerated up to 6.0 × 10 vg/eye, with high and stable RdCVF and RdCVFL mRNA expression levels in the retina and retinal pigment epithelium. These results supported the initiation of the ongoing Phase I/II PRODYGY trial with RCD (NCT05748873).
Adeno-associated virus (AAV)-based gene replacement therapies in Duchenne muscular dystrophy (DMD) aim to restore dystrophin function via the introduction of micro- or mini-dystrophins. We report dystrophin and mini-dyst...Adeno-associated virus (AAV)-based gene replacement therapies in Duchenne muscular dystrophy (DMD) aim to restore dystrophin function via the introduction of micro- or mini-dystrophins. We report dystrophin and mini-dystrophin concentrations generated by immunoaffinity liquid chromatography-tandem mass spectrometry (IA-LC-MS/MS) in skeletal muscle biopsies from ambulatory participants with DMD in a phase 1b study of fordadistrogene movaparvovec, an AAV9-based gene replacement construct. The assay performed robustly for 26 months, as demonstrated by limited variability in calibration standards for peptides LLQV (dystrophin and mini-dystrophin) and LEMP (mini-dystrophin only), quality control samples consisting of spiked mini-dystrophin in DMD skeletal muscle lysate, as well as unspiked, pooled, non-dystrophic skeletal muscle lysate (normal pool). Average values for LLQV in the normal pool tested as part of clinical sample and long-term stability runs were similar to validated values. Biopsy samples showed minor or absent LLQV and absent LEMP signals pre-treatment with fordadistrogene movaparvovec infusion, but signals substantially increased at Days 60 and 360, on average. There was strong concordance in LEMP and LLQV expression change between Days 60 and 360 (R = 0.91; p < 0.001). IA-LC-MS/MS enables reproducible, stable, and reliable quantification of dystrophin/mini-dystrophin following fordadistrogene movaparvovec infusion. ClinicalTrials.gov identifier: NCT03362502.
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocereb...Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.
This study explores the potential of adeno-associated virus serotype 9 (AAV9) to deliver therapeutic genes directly into the memory circuit throughout the olfactory bulb (OB), a critical memory and sensory processing reg...This study explores the potential of adeno-associated virus serotype 9 (AAV9) to deliver therapeutic genes directly into the memory circuit throughout the olfactory bulb (OB), a critical memory and sensory processing region. Using convection-enhanced delivery (CED) of AAV9 encoding green fluorescent protein (GFP), we mapped the extensive neural connectivity from the OB to key memory-related brain regions, including the entorhinal cortex (EC) and hippocampus. Our findings reveal significant transduction of neural pathways and underscore the potential of targeting the OB connectome for therapeutic interventions in progressive neurodegenerative disorders such as Alzheimer's disease or mild cognitive impairment. Targeting the OB connectome will pave the way for new therapeutic strategies to preserve neuronal function and slow the progression, offering a promising avenue beyond symptomatic relief to address the underlying mechanisms of the disease.
The selection of an appropriate promoter is important to the design and optimisation of adeno-associated viral (AAV) vector-based cardiac gene therapies. The expression cassette design can impact efficacy and safety of t...The selection of an appropriate promoter is important to the design and optimisation of adeno-associated viral (AAV) vector-based cardiac gene therapies. The expression cassette design can impact efficacy and safety of the vector. This study is the first to use a novel AAV barcode-seq method for the simultaneous evaluation of a panel of cardiac-specific promoters in a high-throughput manner. Functional analyses of our cardiac promoter kit packaged in three different capsids were performed using neonatal rat ventricular myocytes (NRVM), human iPSC-derived cardiomyocytes (hiPSC-CMs), HuH7 hepatocellular carcinoma cells, as well as mouse, rat, sheep and pig models. The cardiac troponin T (cTnT) promoter showed the most promise overall as a cardiac-specific promoter across all cardiac models tested. The results validate the barcode-seq technique as a powerful and versatile approach that enables high-throughput, quantitative analysis of various expression cassettes in commonly used models of cardiac gene therapy.
Suleman S, Alhaque S, Guo A
… +18 more, Zhang H, Payne A, Zahn M, Fawaz S, Khalifa MS, Jobling S, Hay D, Franco M, Fronza R, Wang W, Strobel-Freidekind O, Deichmann A, Takeuchi Y, Gil-Farina I, Klapwijk J, Perera S, Schmidt M, Themis M
Lentivirus vectors are effective for treatment of genetic disease. However, safety associated with vector related genotoxicity is of concern and currently available models are not reliably predictive of safety in humans....Lentivirus vectors are effective for treatment of genetic disease. However, safety associated with vector related genotoxicity is of concern and currently available models are not reliably predictive of safety in humans. We have developed InGeTox as the first human in vitro platform that uses induced pluripotent stem cells and their hepatocyte like cell derivatives to better understand vector-host interactions that relate vectors to their potential genotoxicity. Using lentiviral vectors carrying the eGFP expression cassette under SFFV promoter activity, that only differ by their LTR and SIN configuration, we characterised vector host interactions potentially implicated in genotoxicity. To do this, lentiviral infected cells were subjected to an array of assays and data from these was used for multi-omics analyses of vector effects on cells at early and late harvest time points. Data on the integration sites of lentiviral vectors in cancer genes and differential expression levels of these genes, showed that both vector configurations are capable of activating cancer genes. Through IS tracking in bulk infected cell populations, we also saw an increase in the viral sequence count in cancer genes present over time which were differentially regulated. RNASeq also showed each vector had potential to generate fusion transcripts with the human genome suggestive of gene splicing or vector mediated readthrough from the internal SFFV promoter. Initially, after infection, both vector configurations were associated with differential expression of genes associated cytokine production, however, after culturing over time there were differences in differential expression in cells infected by each LV. This was marked in particular by the expression of genes involved in the response to DNA damage in cells transduced by the SIN vector, suggesting effects likely to prevent tumour development, in contrast to the expression of genes involved in methylation, characteristic of tumour development, in cells transduced by the LTR vector. Both sets of lentiviral infected cells were also found associated with differential expression of MECOM and LMO2 genes known to be associated with clonal dominance, supporting their potential genotoxicity. Alignment of transcriptomic signatures from iPSC and HLC infected cultures with known cancer gene signatures showed the LTR vector with a higher cancer score than the SIN vector over time in iPSC and also in HLC, which further suggests higher genotoxic potential by the LTR configuration lentivirus. By application of InGeTox to cells infected with LV at the pre-clinical stage of development, we hope that InGeTox can act as a useful pre-clinical tool to identify lentivirus-host interactions that may be considered contributory to genotoxicity to improve safer lentiviral vector design for gene therapy.
Di Donfrancesco A, Adelizzi A, Giri A
… +13 more, Duchi R, Boito S, Barandalla M, Massaro G, Santanatoglia C, Cappellozza E, Perota A, Di Meo I, Tiranti V, Bottani E, Galli C, Persico N, Brunetti D
In utero fetal gene therapy (IUFGT) has the potential to correct severe monogenic disorders before irreversible damage occurs. Despite promising results in small and large animal models, its translation to clinical pract...In utero fetal gene therapy (IUFGT) has the potential to correct severe monogenic disorders before irreversible damage occurs. Despite promising results in small and large animal models, its translation to clinical practice remains limited by technical challenges, safety concerns, and the lack of standardized protocols in relevant disease models species. We established and validated a minimally invasive, ultrasound-guided approach for systemic gene delivery in fetal pigs using a self-complementary AAV9 vector encoding GFP under a CAG promoter. Injections were performed at different gestational ages (GA 80 and GA 108) via intracardiac or umbilical venous routes. Postnatal outcomes were monitored, and transgene biodistribution and expression were assessed by qPCR, ddPCR, immunofluorescence, and Western blotting. Inflammatory response, toxicity, and maternal safety were evaluated through cytokine profiling and histological analyses. The procedure was well tolerated, with no significant maternal morbidity or adverse obstetric outcomes beyond one preterm delivery. Biodistribution analysis revealed widespread vector presence in peripheral tissues, with robust GFP expression in liver and heart. Importantly, there was no evidence of significant tissue toxicity, necrosis, or fibrosis in any of the organs analyzed. Mild increases in pro-inflammatory cytokines (GM-CSF, GRO-α, IFN-γ) were observed but were not associated with histopathological changes. No anti-AAV9 capsid antibodies were detected in sera from piglets or sows, suggesting a minimal immune response to the vector. These findings demonstrate the safety, feasibility, and efficacy of ultrasound-guided IUFGT in pigs, supporting its potential as a translational platform for therapeutic gene delivery in fetuses affected by severe congenital diseases. This model offers a valuable framework for further preclinical development of prenatal interventions, particularly for disorders with early onset, such as mitochondrial diseases.