Human gene therapy最新文献

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease marked by joint destruction and functional impairment. Tumor necrosis factor (TNF) plays a critical role in RA pathogenesis. Although TNF-targeting drugs are clinically effective, their need for frequent and long-term administration often results in poor patient adherence and suboptimal outcomes. This study developed a gene therapy approach using engineered adeno-associated virus (AAV) vectors to deliver an anti-TNF agent directly into the joint cavity of RA animal models. Animals receiving this therapy demonstrated sustained improvement in clinical scores, inflammatory markers, and joint tissue health. Immunofluorescence staining revealed that AAV vectors could transduce various cell types, including T cells, type A synoviocytes, and dendritic cells. Our results indicate that a single administration of this gene therapy provided long-term efficacy. This suggests that AAV-mediated anti-TNF gene therapy can offer prolonged relief from clinical symptoms and reduce inflammatory damage in a mouse model of RA. This innovative approach presents a promising new therapy with significant clinical prospects to treat patients with RA.

A potential therapeutic approach for cancer treatment is target oxidative phosphorylation and glycolysis simultaneously. The matrix protein of vesicular stomatitis virus (VSV MP) can target the surface of mitochondria, causing morphological changes that may be associated with mitochondrial dysfunction and oxidative phosphorylation inhibition. Previous research has shown that mitochondrial abnormalities can direct glucose metabolism toward glycolysis. Thus, after treatment with VSV MP, glycolysis inhibition is necessary to completely block glucose metabolism and eradicate cancer. Here, to inhibit glycolysis, the 2-deoxy-D-glucose (2-DG), a synthetic glucose analog was used to combine with VSV MP to treat cancer. This study aims to determine how VSV MP affects the glucose bioenergetic metabolism of cancer cells and to evaluate the synergistic effect of 2-DG when combined with VSV. Our results indicated that in U87 and C6 glioblastoma cell lines, VSV MP caused mitochondrial membrane potential loss, cytochrome c release, and glucose bioenergetics metabolism reprogramming. When combined with 2-DG, VSV MP synergistically aggravated cell viability, apoptosis, and G2/M phase arrest. Meanwhile, the combination therapy exacerbated ATP depletion, activated AMPK, and inhibited mammalian target of rapamycin signaling pathways. In addition, 2-DG treatment alone induced autophagy in glioblastoma cells; however, VSV MP inhibited the autophagy induced by 2-DG in combined treatment and finally contributed to the enhanced cytotoxic effect of the combination strategy in U87 and C6 cancer cells. In the orthotopic U87 glioblastoma model and subcutaneous C6 glioblastoma model, the combined treatment led to significant tumor regression and prolonged survival. A potent therapeutic approach for treating glioblastoma may be found in the combination of VSV MP and glycolytic inhibitors.

In the legal context of Chinese law, genetic data are an object of complex rights. At the level of private law, genetic data contain personal information, thus being protected by the Civil Code and the Personal Information Protection Law. At the level of public law, genetic data are important genetic resource that embody both public and national interests, which should also be regulated by public laws such as the Biosecurity Law and the Data Security Law. The recently issued Regulation on the Administration of Human Genetic Resources have refined the approval and record procedure, in order to promote the utilization of genetic data in China. At present, China still lacks sufficient protection for genetic data privacy, and the "informed consent" and "anonymization" system cannot work effectively. On the path of improvement, we should break constraints of individualism and start from the following three levels to strengthen genetic data privacy protection: formulating specialized legislation and leveraging the functions of group organizations and public interest litigation systems.

Choroideremia, an incurable, progressive retinal degeneration primarily affecting young men, leads to sight loss. GEMINI was a multicenter, open-label, prospective, two-period, interventional Phase II study assessing the safety of bilateral sequential administration of timrepigene emparvovec, a gene therapy, in adult males with genetically confirmed choroideremia (NCT03507686, ClinicalTrials.gov). Timrepigene emparvovec is an adeno-associated virus serotype 2 vector encoding the cDNA of Rab escort protein 1, augmented by a downstream woodchuck hepatitis virus post-transcriptional regulatory element. Up to 0.1 mL of timrepigene emparvovec, containing 1 × 10¹¹ vector genomes, was administered by subretinal injection following vitrectomy and retinal detachment. The second eye was treated after an intrasurgery window of <6, 6-12, or >12 months. Each eye was followed at up to nine visits over 12 months. Overall, 66 participants received timrepigene emparvovec, and 53 completed the study. Visual acuity (VA) was generally maintained in both eyes, independent of intrasurgery window duration, even after bilateral retinal detachment and subretinal injection. Bilateral treatment was well tolerated, with predominantly mild or moderate treatment-emergent adverse events (TEAEs) and a low rate of serious surgical complications (7.6%). Retinal inflammation TEAEs were reported in 45.5% of participants, with similar rates in both eyes; post hoc analyses found that these were not associated with clinically significant vision loss at month 12 versus baseline. Two participants (3.0%) reported serious noninfective retinitis. Prior timrepigene emparvovec exposure did not increase the risk of serious TEAEs or serious ocular TEAEs upon injection of the second eye; furthermore, no systemic immune reaction or inoculation effect was observed. Presence of antivector neutralizing antibodies at baseline was potentially associated with a higher percentage of TEAEs related to ocular inflammation or reduced VA after injection of the first eye. The GEMINI study results may inform decisions regarding bilateral sequential administration of other gene therapies for retinal diseases.

Adeno-associated virus-mediated gene therapies for certain muscle disorders require regulatory cassettes that provide high-level, striated muscle-specific activity. However, cardiotoxicity has emerged as a serious concern in clinical trials for Duchenne muscular dystrophy and X-linked myotubular myopathy. While this may be caused by systemic inflammatory effects of the treatment, high transgene expression in the heart may also play a role. Thus, certain muscle disorders may require a modulated level of therapeutic expression in the heart, while others may not require any cardiac expression at all. Additionally, the size of some cargos requires regulatory cassettes to be small enough that large cDNAs and other therapeutic payloads can be accommodated. Thus, we have performed enhancer/promoter optimization to develop highly minimized regulatory cassettes that are active in skeletal muscles, with either low or no detectable activity in cardiac muscle. Our No-heart (NH) cassette is active in most skeletal muscles, but exhibits only very low activity in extensor digitorum longus (EDL), soleus, and diaphragm, and no activity in the heart. By contrast, our Have a Little Heart (HLH) cassette displays high activity in most skeletal muscles, comparable to the ∼800-bp CK8 cassette, with increased activity in EDL, soleus, and diaphragm, and low activity in the heart. Due to their small size, these cassettes can be used in therapeutic strategies with both flexible (e.g., antisense) and stringent (e.g., CRISPR/Cas or bicistronic) size limitations. Thus, our new cassettes may be useful for gene therapies of muscle disorders in which the need for low or almost no expression in cardiac muscle would outweigh the need for high levels of therapeutic product in certain skeletal muscles.

Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.

Double-stranded break (DSB) repair of eukaryotic DNA is mainly accomplished by nonhomologous end joining and homologous recombination (HR). Providing exogenous templates during HR repair can result in the editing of target genes, which is the central mechanism of the well-established clustered regularly interspaced short palindromic repeats (CRISPR) gene editing system. Currently, exogenous templates are mainly DNA molecules, which can provoke a cellular immune response within the cell. In order to verify the feasibility of RNA molecules as repair templates for HR in mammalian cell genome editing, we fused RNA template molecules to the 3'-end of single guide RNA (sgRNA), so that the sgRNA and the homologous template RNA form a single RNA molecule. The results show this construct can be used as a repair template to achieve target gene editing in mammalian cells. In addition, the factors influencing HR mediated by RNA template molecules were investigated, and it was found that increasing the length of homologous arms and inducing an R-loop near the DSBcan effectively promote HR repair. Furthermore, intracellular homologous chromosomes may compete with exogenous RNA templates. The findings in this article provide a reference for the utilization of RNA template molecules to mediate target gene editing in eukaryotic cells, as well as a basis for the study of the mechanism by which RNA molecules mediate the repair of DSBs.

The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in ∼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.