Gene Therapy最新文献

Adeno-associated virus (AAV)-based gene therapies have garnered significant attention and investment due to their clinical success and potential to address underlying causes of many diseases. AAV vectors provide effective delivery of therapeutic genetic material to disease-relevant tissues. When evaluating safety and efficacy of recombinant AAV vectors, biodistribution profiles play a critical role in novel therapy development. Herein, a biodistribution metadata analysis was performed on ten studies involving 51 cynomolgus macaques (Macaca fascicularis). The macaques received a self-complementary or single-stranded AAV9 vector containing chicken ß-actin (CBA) or cytomegalovirus (CMV173) promoters expressing fluorescent reporters or a human SMN1 gene. These studies covered various routes of administration (ROA) including intravenous (IV), intracisternal magna (ICM), and lumbar puncture intrathecal (IT) injection. Metadata analysis of AAV9 biodistribution showed relatively uniform vector genome delivery throughout spinal cord tissues over multiple timepoints and ROAs. Moreover, decreased expression efficiency of viral DNA in liver was observed regardless of the ROA, macaque age, or viral construct used. To understand this trend, epigenetic profiling of tissue-localized AAV9 vector genome DNA was performed. Experimental evidence supports partial silencing and repression of transgene expression in macaque liver. These findings point to plausible strategies to consider in preclinical development of AAV9 mediated gene therapies.

This article provides a regulatory perspective on secondary malignancy of T-cell origin as a rare adverse reaction to the currently marketed CD19- or BCMA-directed chimeric antigen receptor (CAR) T-cell therapies. To assess the risk, causality between reported suspected adverse reactions and CAR T-cell therapy was assessed applying the principles of the World Health Organization-Uppsala Monitoring Centre causality categories, alongside a review of scientific publications and data from registries/ databases. By 11 April 2024, 38 cases of T-cell malignancy after CAR T-cell therapy were reported in patients aged 29-80 years. In 19 patients, tumour samples were tested for the presence of CAR transgene, which was detected in seven cases. Most of the T-cell malignancies were diagnosed within 12 months of treatment (22/33; 67%). The reporting rate is approximately one case per 1000 patients treated. An overall causal relationship was established with at least a reasonable possibility. Regulatory measures included updates to the product information, risk management plan, and educational materials. An additional pharmacovigilance activity was requested from the marketing authorisation holders (MAHs) to strengthen the process of genetic testing of residual tumour samples. To further characterise this risk and understand underlying mechanisms, continued efforts from healthcare professionals, MAHs and regulators are essential. Well-documented case reports, including information on genetic testing of tumour samples, are considered crucial elements.

Retinitis pigmentosa (RP) associated with mutations in the rhodopsin gene (RHO) is a significant cause of blindness. Here we report on the application of adenine base editing of the c.1030C>T (p.Q344X) RHO mutation linked to RP. Using a fluorescence reporter cell system, we optimized editing by exploring base editors, sgRNA, and delivery methods. Flow cytometry, western blotting, and immunofluorescence microscopy confirmed the restoration of full-length rhodopsin after editing. DNA sequencing verified editing at the target nucleotide and the absence of bystander edits within the editing window. Polyethylenimine cationic polymer transfection of cells with a plasmid containing the NG-ABE8e adenine base editor and A6 guide RNA that placed the targeted adenine in position 6 of the editing window resulted in 31.0% gDNA sequence correction and 26.3% rhodopsin protein correction as determined by flow cytometry. Purified NG-ABE8e protein complexed with A6-sgRNA showed 32.2% gDNA editing and 44.5% rhodopsin correction. Plasmid NG-ABE8e and A6-sgRNA co-encapsulated into lipid nanoparticles (LNPs) and transfected into the reporter cell system resulted in the highest editing (42.6% gDNA editing and 65.9% rhodopsin correction). These results demonstrate the successful correction of the c.1030C>T RHO mutation and provide the foundation for base editing as a treatment for RP.

Adeno-associated viruses (AAVs) hold significant promise for gene therapy targeting the central nervous system (CNS). However, current delivery methods are either invasive or cause significant systemic exposure. Intranasal (IN) delivery presents a promising noninvasive alternative for direct CNS targeting, though its efficacy in delivering AAVs to the brain has seldom been explored. Here, we quantitatively assessed AAV transduction in the brain and peripheral organs of Swiss, BALB/c, and C57BL/6 J mice following IN administration, using intravenous (IV) injection as a benchmark for comparison. Our findings revealed that IN administration of the AAV9 vector achieved approximately 15% of the transduction efficiency and 9% of the gene expression levels observed with IV delivery. Importantly, IN delivery significantly reduced systemic exposure to most major peripheral organs by up to 1.34 × 10⁴-fold compared to IV injection. The ratios of gene transduction between the brain and various peripheral tissues were calculated, revealing that for key organs such as the liver, stomach, kidney, and spleen, IN delivery achieved higher brain-to-peripheral transduction ratios than IV delivery. These findings underscore the potential of IN delivery for noninvasive brain-targeted gene delivery with significant reductions in peripheral exposure.

Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.

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.

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.

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.

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.