Human gene therapy最新文献

In recent decades, medical and scientific advances have led to the development of new therapeutic approaches for Duchenne muscular dystrophy (DMD), including gene therapy (GT), which is currently being evaluated. Recruiting enough children in clinical trials remains a challenge, depending on parental decisions. Numerous studies have already been carried out to understand these decision-making factors. To date, no study in Europe has been conducted among the various stakeholders lived experience in a DMD GT trial. Our qualitative study explored participants' perceptions using a social representation method and compared them. We recruited 42 participants, divided into 2 groups comprising 21 parents and 21 professionals participating in GNT-014, a DMD natural history study. Each participant was interviewed on four questions about clinical trials, GT, and the facilitators and barriers of the clinical trial pathway. A prototypical and categorical analysis was carried out using "Pointe-au-Sel" software to analyze the data quantitatively. This method highlights which perceptions are shared within the same group and brings out the most important and most frequently evoked terms. We exported the data as a superimposed scatterplot of the representations of both groups for each question. We obtained a total of 453 evocations for the parents' group and a total of 611 evocations for the professionals' group. For clinical trial and GT, hope and scientific progress are common to the core of both groups but are not at the same level of representation. Parents evoked human contact as the main facilitator and what their child may undergo and become for barriers. For professionals, the facilitators and barriers are centered on the terms that can influence the proper conduct of the trial. These comparative results imply that the vision of the different stakeholders is not totally shared in trial participation. On the contrary, the term GT may also have an influence on professionals, including caregivers.

The development of potency assays for Advanced Therapy Medicinal Products (ATMPs) presents significant challenges due to the variability of starting materials and the complex mechanisms of action involved. This article aims to address the following key question: How can we design robust and reliable potency assays for ATMPs that accommodate product-specific challenges and align with evolving regulatory standards? To answer this, we employed a mixed-methods approach, synthesizing data from scientific literature, industry reports, and regulatory guidelines to identify current limitations and innovative solutions for potency assay development. Our methodology integrates a systematic review of academic publications (2018-2024) to capture recent advancements in biotechnology and their applicability to potency testing. We complemented this with an analysis of industry perspectives, drawn from webinars and white papers, as well as a detailed comparison of global regulatory frameworks, including the FDA's new guidance on potency assurance for Cellular and Gene Therapy Products (CGTs/ATMPs). Additionally, we developed a comprehensive database to analyze potency assays used in approved, rejected, and withdrawn CGT/ATMP products, focusing on technical and regulatory challenges. Based on this multilevel analysis, we propose a product-specific framework for designing, developing, and validating potency assays for different ATMP categories, taking into account their unique technical and regulatory constraints. We also highlight emerging technologies, such as droplet digital polymerase chain reaction and reporter gene assays, as innovative tools for improving the precision and reliability of potency testing. Our findings underscore the need for flexible, risk-based strategies in potency assay development that evolve throughout product development and clinical trial phases. Future recommendations emphasize assay standardization, the definition of acceptable variability, and stronger correlations between in vitro potency data and clinical outcomes.

The first marketed gene therapy medicinal products based on adeno-associated virus (AAV-GTMP) show promise for the treatment of various diseases, including rare diseases with unmet medical needs. AAV is traditionally considered nonpathogenic to humans, is incapable of self-replication, and, after introduction into various cell types, remains primarily episomal. Several reports have examined the risks of AAV-GTMP, including the risks associated with unintended integration events of elements from the recombinant (r) AAV vector into the host genome. Such events can be one of the steps in the multistep process of tumor formation. To date, rAAV-gene therapy (GT) vectors have not been shown to induce tumors in humans or non-rodent species, and the potential for rAAV-mediated carcinogenicity in humans is still considered theoretical. Nevertheless, a critical review of publicly available scientific data on rAAV-related integration events and a contextualization of the numbers of AAV-GT vector DNA integrations with the absolute burdens of environmental, lifestyle and background tumorigenic genotoxicities is warranted. From a regulatory perspective, it is advisable to implement a long-term safety follow-up for patients who have undergone treatment with high doses of AAV-GT, in accordance with the risk-based approach that has been established for advanced therapy medicinal products.

Limb-girdle muscular dystrophy type 2E/R4 (LGMD2E/R4) is an ultra-rare autosomal recessive disorder caused by mutations in SGCB, the gene that encodes for β-sarcoglycan (SGCB), a component of the dystrophin-associated protein complex that stabilizes muscle fibers during contractions. Bidridistrogene xeboparvovec is an investigational adeno-associated virus-mediated gene transfer therapy designed to deliver a codon-optimized, full-length human SGCB and induce targeted expression of functional human SGCB protein. Interim safety and efficacy data from a clinical trial in patients with LGDM2E/R4 aged 4-15 years (NCT03652259) support further clinical development of bidridistrogene xeboparvovec. However, less is known about the effects of this agent in patients with more advanced LGMD2E/R4, who on average are older and heavier, which prompted their inclusion in studies VOYAGENE (NCT05876780, phase 1) and EMERGENE (NCT06246513, phase 3). In the preclinical study presented here, we delivered bidridistrogene xeboparvovec (0.185 × 10¹³ vg/kg, 0.37 × 10¹³ vg/kg, 0.74 × 10¹³ vg/kg, 1.85 × 10¹³ vg/kg, or 7.4 × 10¹³ vg/kg) to Sgcb-/- mice aged 27-42 weeks (n = 4 per dose) with age-matched saline-treated Sgcb-/- and C57BL/6J mice used as controls. Approximately 12 weeks after administration, we observed SGCB expression and found evidence of reduction in muscle fibrosis, reduction in muscle damage, and restoration of muscle force. Overall, a dose-dependent increase in vector exposure across tissue types was observed, with a nonlinear, exposure-dependent increase in both SGCB expression and functional improvement that reached saturation at 7.4 × 10¹³ vg/kg. Pharmacokinetic and pharmacodynamic analyses demonstrated a robust relationship between vector biodistribution, SGCB expression, and muscle force, further supporting clinical development of bidridistrogene xeboparvovec at the highest dose (7.4 × 10¹³ vg/kg), across a broad LGMD2E/R4 population and regardless of disease progression.

Significant progress has been made in gene therapy for Duchenne muscular dystrophy (DMD), a severe genetic disorder primarily affecting pediatric patients. However, the immune responses triggered by high-dose systemic delivery of adeno-associated virus (AAV) vectors remain a major challenge. These responses include the generation of long-lasting anti-capsid antibodies and potential immunity against the therapeutic transgene, rendering gene therapy ineffective. In addition, pre-existing anti-AAV antibodies exclude patients from eligibility for treatment. To address these limitations, we have developed an immunosuppression (IMS) strategy aimed at mitigating immune responses to the AAV capsid while enhancing microdystrophin expression. Using an optimized expression cassette (AAV9-UFµDys1) for sustained microdystrophin expression in striated muscle and heart, we observed a 40% improvement in muscle force compared with animals receiving a GFP-encoding control AAV9 vector. In mdx mice, a single-dose IMS regimen significantly increased microdystrophin expression in cardiac and skeletal tissues and repeat dosing further enhanced expression, an effect not observed in non-IMS-treated mdx mice. To model pre-existing immunity, we immune-challenged wild-type mice with empty AAV9 capsids and tracked antibody responses over time. The IMS regimen effectively reduced total anti-AAV antibody levels and increased microdystrophin expression in UFµDys1-treated mice. These findings highlight the potential of IMS to minimize immune barriers, facilitate repeat AAV administration, and expand the therapeutic window for DMD gene therapy. Our results support the further development of AAV-mediated approaches using either microdystrophin-expressing vectors or next-generation systems delivering full-length or near-full-length dystrophin.

Recombinant adeno-associated virus (rAAV) vectors are among the most effective for gene therapy. A significant advancement in rAAV vector production is developing the triple-plasmid transfection method, which remains the most widely used technique. In this study, we used Expi293F^TM (Expi293F) and Viral Production Cells 2.0 (VPC2.0 cells) to evaluate various transfection reagents, comparing transgene protein expression levels and intracellular plasmid copy numbers to optimize rAAV production. Our findings indicated that the effectiveness of transfection reagents in promoting higher rAAV production was cell-dependent and that rAAV productivity correlated more with plasmid levels in the cell nucleus than with transgene protein expression levels. Confocal laser microscopy revealed that in cells transfected with the high-yield transfection reagent, a large amount of free plasmid DNA entered the nucleus, whereas the transfection reagents themselves did not. These results provide new insights into the intracellular mechanisms underlying efficient rAAV vector production. Furthermore, identifying transfection reagents that facilitate nuclear plasmid delivery will aid in the selection of optimal reagents for high-yield AAV production.

Mutations in human COLQ, which encodes the collagen-like tail subunit (ColQ) of asymmetrical acetylcholinesterase (AChE), cause congenital myasthenic syndrome (CMS) with deficiency of end plate AChE. A valuable animal model of COLQ-CMS is the Colq-deficient (Colq^-/-) mouse, which lacks asymmetrical AChE in skeletal and cardiac muscles. Mutant Colq^-/- mice fail to thrive, and many die before reaching maturity. With the aim of developing a treatment for COLQ-CMS, Colq^-/- mice were injected at postnatal day 26-29 with three doses of an adeno-associated virus type rh74 carrying full-length human COLQ (AAVrh74-COLQ): 5 × 10¹³ viral genomes per kilogram (vg/kg) (intravenously [IV]), 1 × 10¹⁴ vg/kg (IV), and 2 × 10¹⁴ vg/kg (1 × 10¹⁴ vg/kg IV + 1 × 10¹⁴ vg/kg intraperitoneally). Motor performance was evaluated using rotarod, grip strength, and wire hang tests weekly for 12 weeks. Voluntary ambulation and repetitive nerve stimulation (RNS) were assessed once before euthanasia. Protein and RNA expression of COLQ was measured via immunohistochemistry (IHC) and reverse transcriptase quantitative PCR (RT-qPCR), respectively. Mice treated with AAVrh74-COLQ at 1 × 10¹⁴ and 2 × 10¹⁴ vg/kg doses showed 100% survival and no adverse side effects. Mice injected with 2 × 10¹⁴ vg/kg showed almost full recovery and similar scores to wild type that were significantly higher than vehicle-injected mutants for grip strength (p value <0.0001), rotarod (p value <0.0001), and RNS (p value <0.0001). Similar improvements were observed in mice injected with 1 × 10¹⁴ vg/kg, although the recovery of grip strength was incomplete. Mice injected with 5 × 10¹³ vg/kg showed incomplete recovery. IHC demonstrated full recovery of protein expression in 1 × 10¹⁴ and 2 × 10¹⁴ vg/kg mice, and RT-qPCR unambiguously demonstrated that the source of the ColQ was human COLQ. In summary, a single treatment of AAVrh74-COLQ (1 × 10¹⁴ to 2 × 10¹⁴ vg/kg) was effective and safe for Colq^-/-mice, which reproduce many of the clinical features of the human COLQ-CMS phenotype. Thus, these results support a similar therapy for patients affected with COLQ-CMS.

Corneal neovascularization (CoNV) is both a sight-threatening condition in and of itself and a major risk factor associated with corneal graft failure. Here, we determine the effectiveness of an adeno-associated viral vector (AAV)-based gene therapy targeting both hematic and lymphatic neovascularization in a murine model of severe CoNV. We first assessed the profile of transgene expression mediated by intrastromal injection of AAV2/8[Y733F] via longitudinal visualization of an enhanced Green Fluorescent Protein (eGFP) transgene and found that this serotype mediates a temporary (∼18 day) transduction of the corneal epithelium and sustained (≥148 day) transduction within the stroma. Constitutively expressed sFlt1 or sFlt4 were prophylactically delivered via intrastromal injection of AAV2/8[Y733F] vector at various intervals prior to aggressive induction of CoNV in a murine model. The extent of CoNV induced was quantified by fluorescein angiography and immunohistochemistry 17 days after induction. AAV2/8[Y733F]-CMV-sFlt1 was highly effective in the prevention of hemangiogenesis (HA) induced at 3, 28, and 210 days after intrastromal injection, but ineffective in the prevention of lymphangiogenesis. Two variants of AAV2/8[Y733F]-CMV-sFlt4 were ineffective in the prevention of angiogenesis when delivered alone, but combined delivery of AAV2/8[Y733F]-CMV-sFlt1 and AAV2/8[Y733F]-CMV-sFlt4 suggested a synergistic effect. Our results show that a single intrastromal injection of AAV2/8[Y733F]-CMV-sFlt1 is sufficient to protect against a robust stimulus for corneal HA over the long term. This technique could also be applied ex vivo to reduce the risk of failure in cases of "high-risk" corneal transplantation.

Adeno-associated virus (AAV) vectors have emerged as the leading gene therapy vehicle due to their favorable safety profile and sustained payload expression. Approved therapies such as voretigene neparvovec (Luxturna) and omnasemnogene abeparvovec (Zolgensma) rely on the tropism of natural AAV variants. The majority of discovered natural AAVs and engineered AAV capsids have not been comprehensively profiled for their biodistribution, especially at single-cell resolution. Recent advances in single nuclei sequencing can enable further refinement of AAV cell-type specificity and reduce off-target effects. However, low levels of transduction and muted sensitivity of current single-cell detection methods make screening pooled capsids at single-cell resolution challenging. Here, we develop SNAC (Single-Nuclei Atlas of Capsid distribution), an improved method for single-nuclei profiling of AAV transduction at multiplex scale. We provide proof of concept using the nonhuman primate eye as a model system, showing that we can accurately identify and quantify vector expression in all major retinal cell types. Furthermore, the ranking of capsids by SNAC agrees with that from pre-established tissue sampling protocols. Our method promises to reduce the time, effort, and cost of accurate cell-type-specific profiling of AAV capsids.