Human gene therapy最新文献

Adeno-associated viral vectors (AAVs) are promising tools for gene therapy. However, scaling up the production of AAVs to produce high-quality vectors at high yields for clinical purposes has proven to be challenging. In the present study, we optimized the production process of AAV in a fixed-bed bioreactor using transient transfection in adherent HEK-293T cells. We systematically optimized the key process parameters, namely cell seeding density, cell density at transfection, and DNA-to-cell ratio, based on the yield obtained, starting from a prototype batch, followed by ten batch runs. Here, we packaged a reporter gene (enhanced green fluorescent protein) and a therapeutic gene (lysyl oxidase) into AAV9 capsids as part of our process development program to be applied for future current Good Manufacturing Practices production and clinical trial application. Throughout the experiments, media conditions, transfection processes, and mechanical parameters were kept identical, while monitoring pH, dissolved oxygen, and media glucose concentration during a production process of approximately 10 days. We demonstrate that by optimizing these parameters, the fixed-bed bioreactor was able to support as many as 1.6-2.8 × 10⁶ cells/carrier strip, up to 3 × 10⁹ cells/m² bioreactor. Through this multivariate optimization process, we increased viral yield by about 7.6-fold (range of 5.7-10.4-fold for the optimized process runs) over the prototype batch. The total AAV vector yield average was 2.3 × 10¹⁴ vg (range 1.1 × 10¹⁴ vg to 4.95 × 10¹⁴ vg), corresponding to an average per cell yield of 1.4 × 10⁵ vg/cell (range 0.85 × 10⁵-2.46 × 10⁵vg/cell). In conclusion, our findings highlight that optimizing process parameters in a fixed-bed bioreactor presents a promising strategy for scalable and cost-effective AAV vector production.

Recombinant adeno-associated virus (rAAV) vectors have emerged as a leading viral vector for in vivo gene therapy because they offer significant advantages over other viral vectors, such as stable physicochemical properties, low pathogenicity, low integration risk, and long-term expression of the transgene. In recent years, the number of rAAV products that have received approval for commercial marketing and clinical trials has grown rapidly, bringing hope for the treatment of refractory and rare diseases. However, rAAV products are highly innovative and complex, and the manufacturing processes to produce them are diverse and rapidly improving. Concurrently, the quality control methodologies and technologies are rapidly advancing and evolving. As biotechnology rapidly advances, there is a heightened need for communication between regulatory authorities and entities applying for rAAV products to be used for in-human trials or commercial marketing. Here, we focus on the discussion of chemistry, manufacturing, and control issues such as the control of adventitious viruses in different manufacturing processes and quality control during the biologics license application of rAAV products. It is expected to provide references and suggestions for the manufacturing and quality control of rAAV vectors, thereby accelerating the high-quality development of gene therapy products.

Myocardial fibrosis represents a maladaptive response to some pathological stimulus affecting the heart's functions, predisposing to arrhythmia and ultimately heart failure (HF). Stathmin 1 (Stmn1) is a core protein in regulating the formation of the mitotic spindle. Although the dysregulation of Stmn1 has been confirmed to be related to the occurrence of kidney or liver fibrosis, the role of Stmn1 in HF remains obscure. In this study, bioinformatic analysis (GSE150736) of myocardial tissues from HF patients suggested that Stmn1 was significantly upregulated compared with healthy controls. The consistent results were also observed in the heart tissues of the rat model of HF. Furthermore, we demonstrated that Adenovirus-mediated overexpression of Stmn1 in the peri-infarct border zone area significantly improved cardiac fibrosis and collagen deposition, as evidenced by the decreased expression of transforming growth factor-beta1 (TGF-β1), collagen IV, and alpha-smooth muscle actin in heart tissues. In vitro, overexpression of Stmn1 reduced the production and deposition of collagen in cardiac fibroblasts (CFs) induced by TGF-β1, and thus inhibited the activation of CFs into myofibroblasts. Mechanistically, upregulation of Stmn1 significantly suppressed the phosphorylation of p38 both in vivo and in vitro. Moreover, we demonstrated that Stmn1 was transcriptionally regulated by the cohesin and CCCTC-binding factor (Ctcf) and functionally mediated the cardioprotective effects of Ctcf. Collectively, this work established Stmn1 as a promising therapeutic target for myocardial fibrosis in HF. [Figure: see text].

Dozens of gene therapies (GTs) have received regulatory approval, and hundreds more are in various research phases. To characterize the ethical, legal, and social implications (ELSI) associated with the clinical development of this relatively new therapeutic class, we conducted a scoping review of the literature. Articles were eligible if they were written in English and discussed ELSI in the context of human somatic GT clinical research. 273 articles published from 2013 to 2024 met the eligibility criteria. To characterize and synthesize the ELSI associated with human somatic GT clinical research, thematic analysis was performed on extracted ELSI-relevant text from 179 articles published between 2019 and early 2024, including reviews, empirical research articles, opinions/commentaries, reports, news articles, and blog posts. Twenty-four themes were identified, leading to the generation of five high-level themes: (1) assessment of the risks and benefits of GTs is scientifically and ethically challenging, (2) communication and engagement with the patient community is crucial, (3) access and justice issues are heightened, (4) ethical GT trial design requires thoughtful consideration, and (5) strategic decision-making about GT research has ethical implications and is impacted by financial considerations and the regulatory context. Potential approaches to the identified ELSI are explored and discussed.

The development of efficient and safe muscle-directed gene therapy is an unmet medical need. One of the bottlenecks in muscle-directed gene therapy is the high levels of muscle-targeted transcription required in these afflicted target tissues. To circumvent this problem, novel transcriptional cis-regulatory elements (CREs) were identified by transcriptome-wide data-mining that led to a significant increase of transgene expression in skeletal muscle, heart, and diaphragm after adeno-associated viral vector 9 (AAV9) gene transfer. The expression achieved with this CRE arrays outperformed that obtained with several quintessential muscle-targeted promoters, such as the synthetic SPc5-12 and MHCK7 promoters, used in various muscle-targeted gene therapy clinical trials. Incorporation of these CRE arrays led up to a robust 20- to 30-fold increase in luciferase reporter gene expression when compared with the SPc5-12 and MHCK7 promoters. To validate their therapeutic efficacy, AAV9 vectors containing CREs and encoding α-glucosidase (GAA) were administered to GAA-/- knockout mice that mimic Pompe disease (glycogen storage disease type II) in human subjects. The CRE arrays resulted in a significant 25-fold increase in GAA protein and GAA mRNA expression in different skeletal muscles, leading to GAA activity levels comparable with those of wild-type mice. Subsequently, this led to a significant decrease in glycogen accumulation and a restoration of centronuclear localization similar to those of wild-type levels. Most importantly, long-term correction of skeletal muscle, diaphragm, and cardiac function was achieved in GAA-/- knockout mice treated with the CRE-containing AAV9 vectors yielding normal phenotypes indistinguishable from wild type. This robust phenotypic correction was demonstrated based on grip and hanging tests, cardiac conductance assays as reflected by PR interval prolongation, and diaphragm contractility function tests. The current study has broad implications for improving outcomes of future clinical trials in Pompe patients and other genetic disorders that affect skeletal muscle, heart, and diaphragm.

Several unexpected fatalities in patients who received adeno-associated virus (AAV)-based gene therapies have recently occurred. These tragic events have cast a pall over the entire sector with some stakeholders suggesting that AAV is patently unsafe as a gene delivery platform and ought not to be pursued. This conclusion is not warranted.

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.