Gene Therapy最新文献

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 (R² = 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.

Recombinant adeno-associated viruses (AAVs) have become increasingly popular as gene therapy vectors in recent years. Like all viruses, AAVs undergo dynamic structural changes in response to varying temperature and pH conditions. However, the specific capsid regions involved in these processes remain unknown. In this study, we employed Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to investigate the impact of pH and temperature on the structure and conformational dynamics of AAV capsids. Our analysis identified specific regions of the capsid that are sensitive to these environmental changes. Additionally, our data elucidated the structural basis for DNA uncoating or leakage triggered by low pH or high temperature. Detailed structural characterization of AAVs by HDX-MS in this study deepens our understanding of viral capsid conformational dynamics and stability in AAV transduction and manufacturing and storage conditions, paving the way for formulation development and next-generation capsid engineering.

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.

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.

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 ^hInGeTox 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 ^hInGeTox to cells infected with LV at the pre-clinical stage of development, we hope that ^hInGeTox 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.

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.

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.

Onasemnogene abeparvovec (OA) is the first gene replacement therapy (GT) approved for 5q spinal muscular atrophy (SMA). While effective, it can cause severe side effects, including thrombotic microangiopathy (TMA). The pathophysiology, risk factors, and management of viral-vector-related TMA remain unclear. This study aimed to evaluate TMA frequency among Brazilian patients treated with OA and characterize their clinical and laboratory profiles. This retrospective, multicenter study analyzed 294 Brazilian patients with 5q SMA treated with OA between October 2020 and September 2024, of whom seven (2.4%) developed TMA. The average age at OA administration was 20.4 months, and the average weight was 11.5 kg. Three patients had documented infections before OA administration. TMA symptoms appeared within 6-10 days post-infusion. All patients showed hemolytic anemia, thrombocytopenia, and at least one organ dysfunction. Treatment included plasmapheresis in two cases and increased corticosteroid doses in four cases. One patient died from TMA complications. Whole exome sequencing in five patients identified no pathogenic variants linked to TMA. TMA is a rare but severe complication of OA therapy for SMA. Prompt recognition and management, often with corticosteroids, are crucial for improving outcomes.