Human gene therapy最新文献

Canavan disease (CD) is a rare autosomal recessive leukodystrophy caused by biallelic pathogenic variants in the ASPA gene. CD is characterized by developmental delay, macrocephaly, and abnormal muscle tone. The biochemical diagnosis is confirmed by increased N-acetylaspartic acid levels. The phenotypic presentation varies, with 85-90% of individuals exhibiting the severe, typical form, while 10-15% present with a milder, atypical form. Here we report on five patients with a clinical and biochemically proven diagnosis in whom a second pathogenic variant had not yet been identified. Targeted long-read sequencing of the entire ASPA gene revealed an SVA_E retrotransposable element located in intron 4 that had been missed by standard short-read-based diagnostic procedures. Haplotype analysis of all patients showed linkage of the SVA_E element with a noncoding variant in intron 1. Functional characterization of the SVA_E element suggests that transcripts of the affected allele are prone to highly efficient mRNA degradation processes. These findings enhance the precision of genetic diagnostics and enable improved guidance for families as well as facilitating potential access to targeted therapies.

Over the last two decades, adeno-associated viruses (AAVs) have been widely used as viral vectors in gene therapy due to their ability to infect both dividing and nondividing cells, broad tissue specificity, and favorable safety profile. Recombinant AAV (rAAV) production requires a helper virus, typically adenovirus (AdV), which provides essential genes for AAV replication. However, the increasing demand for safer and more efficient rAAV production methods led to the need to develop helper plasmids with minimal AdV components. In this study, we evaluate the impact of AdV E2 and E4 in the productivity and genome packaging of rAAV serotypes 2, 5, 8, and 9, produced by transient transfection. We designed and tested eight novel helper plasmids with different deletions in E2 and E4 genes. Results indicated that deletions in these genes significantly affected rAAV productivity and packaging, particularly for serotypes 8 and 9. Helper plasmids containing minimal essential genes-E2-DBP, E4orf6, and VA RNA-showed near to 10-fold reduction in viral genome packaging compared to the control. However, including E2 L4-22/33K and E4orf3 regions significantly improved viral production, particularly for serotypes 8, and 9. In this study, we also demonstrated that the full E4 gene is crucial to achieving high full-empty ratios, minimizing the production of empty capsids, and enhancing viral release into the culture medium of rAAV8. Accordingly, we created a smaller plasmid, without adenoviral structural proteins that allows a similar rAAV production across all tested serotypes. Overall, these findings provide insights into the genetic requirements for efficient rAAV production and highlight the importance of the E2 and E4 regions for optimizing viral yield and quality. This approach could lead to the development of improved strategies for large-scale rAAV vector production by enabling safer and more cost-effective systems.

Duchenne muscular dystrophy (DMD) is a severe, progressive genetic disorder primarily affecting boys, characterized by muscle degeneration due to mutations in the DMD gene encoding dystrophin, a crucial protein for muscle fiber integrity. The disease leads to significant muscle weakness and eventually to loss of ambulation. Adeno-associated viral (AAV)-microdystrophin (MD) gene therapy shows promise in preclinical and clinical settings. However, muscle fibrosis, a consequence of chronic inflammation and extracellular matrix remodeling, exacerbates disease progression and may hinder therapeutic efficacy. Periostin, a matricellular protein involved in fibrosis, is upregulated in DMD rodent models and correlates with collagen deposition. We previously developed an antisense oligonucleotide strategy to induce exon 17 skipping and so reduce periostin expression and collagen accumulation in the fibrotic D2.mdx mouse model of DMD. Here, we investigated the combined effects of periostin modulation and AAV-MD1 treatment. We found that systemic periostin splicing modulation significantly improved muscle function, assessed by forelimb grip strength and treadmill performance. Importantly, periostin exon skipping increased the MD protein expression. These findings suggest that targeting periostin in conjunction with MD therapy could represent a valid therapeutic strategy for DMD.

Gene therapy has revolutionized modern medicine by offering innovative treatments for genetic disorders, cancers, and immune-related conditions through technologies such as viral vector delivery, genome editing, and genetically modified cell therapies. Despite significant advancements, the classification of gene therapy medicinal products (GTMPs) as genetically modified organisms (GMOs) under EU legislation imposes significant regulatory burdens, hindering early and timely patient access to such therapies. Current GMO regulations, originally designed for agricultural biotechnology, require environmental risk assessments (ERAs) and additional approvals, creating delays and increasing costs-with a particularly negative impact on early academic research. This article examines the scientific and regulatory discrepancies in classifying GTMPs as GMOs, arguing that replication-deficient vectors and non-persistent modified cells may not meet the criteria for GMOs. We highlight the negative impact of GMO requirements on clinical trial feasibility in Europe compared to the U.S., where a categorical exclusion from ERA applies to investigational medicinal products. Proposed solutions include adopting a risk-based regulatory model, harmonizing ERA processes under the revised EU Clinical Trials Regulation, and establishing exemptions for low-risk therapies. By aligning regulatory frameworks with scientific evidence, policymakers can accelerate the translation of gene therapies while maintaining safety standards, ultimately improving patient access to these transformative treatments.

Among solid pediatric tumors, brain tumors are the leading cause of cancer-related mortality. While survival rates have improved for certain pediatric brain tumor subtypes, the overall prognosis remains poor. Consequently, there is an urgent need for novel therapies that are not only effective but also less toxic. Oncolytic viruses have emerged as promising therapeutic agents due to their ability to selectively replicate in tumor cells while sparing healthy tissue and their potential to induce systemic antitumor immune responses. VCN-01 is a replication-competent oncolytic adenovirus whose efficacy has been demonstrated in clinical trials after systemic administration in combination with chemotherapy. Evidence of antitumor activity has also been obtained after intracranial administration in preclinical models of various brain tumors, including high-grade gliomas. However, before progressing to clinical trials for those indications, it is essential to assess the safety of its intracranial administration. In this study, we evaluated the toxicity and biodistribution of VCN-01 following intracranial injection in a Syrian hamster model. Two viral doses were tested: 1.5 × 10⁹ and 1.5 × 10¹⁰ viral particles (vp)/animal, corresponding to 5 and 50 times the starting clinical dose (10¹⁰ vp/patient), respectively. Our toxicity analysis revealed a favorable safety profile, with no adverse effects observed following administration. Biodistribution studies demonstrated that VCN-01 primarily remained confined to the brain, with only minimal presence detected in peripheral tissues. The neutralizing antibody response against the virus was stronger in females than in males, correlating with a lower detection of vp in females compared with males. In conclusion, these findings support the safety of intracranial administration of VCN-01 and provide a strong rationale for its further development as a therapeutic option for patients with brain tumors.

Seven cases of hematological malignancy reported in recipients of Skysona™ (elivaldogene autotemcel) have reignited long-standing concerns about insertional mutagenesis in lentiviral vector (LV)-based gene therapy. Here, we dissect the molecular and clinical evidence underlying these events, place them in the broader context of over 300 patients treated with LV-modified hematopoietic stem and progenitor cells (HSPCs), and review the real-world safety record of LV-engineered chimeric antigen receptor T cells. We show that cancers associated with Skysona are mechanistically linked to the use of a potent viral MNDU3 promoter probably combined with intensive conditioning and growth-factor support, whereas LV products employing weak or physiological promoters continue to display an excellent safety profile. With event rates <0.6/100 patient-years, lower than those after autologous HSCT, the therapeutic index of approved LV-HSPC advanced therapy medicinal products remains favorable. Ongoing optimization of vector design, conditioning, and long-term surveillance, together with emerging genome-editing platforms, is expected to further mitigate residual risk.

The advent of genome editing has kindled the hope to cure previously uncurable, life-threatening genetic diseases. However, whether this promise can be ultimately fulfilled depends on how efficiently gene editing agents can be delivered to therapeutically relevant cells. Over time, viruses have evolved into sophisticated, versatile, and biocompatible nanomachines that can be engineered to shuttle payloads to specific cell types. Despite the advances in safety and selectivity, the long-term expression of gene editing agents sustained by viral vectors remains a cause for concern. Cell-derived vesicles (CDVs) are gaining traction as elegant alternatives. CDVs encompass extracellular vesicles (EVs), a diverse set of intrinsically biocompatible and low-immunogenic membranous nanoparticles, and virus-like particles (VLPs), bioparticles with virus-like scaffold and envelope structures, but devoid of genetic material. Both EVs and VLPs can efficiently deliver ribonucleoprotein cargo to the target cell cytoplasm, ensuring that the editing machinery is only transiently active in the cell and thereby increasing its safety. In this review, we explore the natural diversity of CDVs and their potential as delivery vectors for the clustered regularly interspaced short palindromic repeats (CRISPR) machinery. We illustrate different strategies for the optimization of CDV cargo loading and retargeting, highlighting the versatility and tunability of these vehicles. Nonetheless, the lack of robust and standardized protocols for CDV production, purification, and quality assessment still hinders their widespread adoption to further CRISPR-based therapies as advanced "living drugs." We believe that a collective, multifaceted effort is urgently needed to address these critical issues and unlock the full potential of genome-editing technologies to yield safe, easy-to-manufacture, and pharmacologically well-defined therapies. Finally, we discuss the current clinical landscape of lung-directed gene therapies for cystic fibrosis and explore how CDVs could drive significant breakthroughs in in vivo gene editing for this disease.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While CRISPR-based CFTR editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi^{Cas9(Y66S)eGFP} reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of CFTR functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del CFTR and Y66S eGFP was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP⁺ (Y66S-corrected) cells and eGFP^- (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP^- population exhibited 25.2% of the CFTR-specific transepithelial Cl^- transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP⁺ cells than eGFP^- cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.

Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering and whether these changes can be measured in peripheral biofluids. We previously found that lipid changes that occur in brain with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant HTT allele with expanded cytosine-adenine-guanine (CAG) can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Wild-type or zQ175DN mice were injected with adeno-associated virus 9 (AAV9) bearing a cDNA for a zinc finger protein (ZFP), which preferentially targets mutant HTT (ZFP-HTT) to repress transcription. Proteins from brain tissues were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses of brain tissue and plasma collected from the same mice were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic instability index was assessed using capillary gel electrophoresis of PCR products and was shown to be impeded by ZFP-HTT. Lowering mHTT levels by 43% for 4 months prevented loss of total lipid content including the subclasses sphingomyelin, ceramide, phosphatidylethanolamine and others of caudate-putamen in zQ175DN mice. Moreover, LC-MS analysis of plasma demonstrated total lipid increases and lipid changes in monogalactosyl monoacylglyceride and certain phosphatidylcholine species were reversed with the therapy. In summary, our data demonstrate that analyzing lipid signatures of brain tissue and peripheral biofluids are valuable approaches for evaluating potential therapies in a preclinical model of HD.