Human gene therapy最新文献

Recombinant adeno-associated viruses (rAAVs) are widely used viral vectors in human gene therapy. However, DNA impurities, such as plasmid DNA and host cell DNA, remain a significant quality control concern for final products. Our study examined purified rAAV1-ZsGreen1, rAAV2-ZsGreen1, rAAV5-ZsGreen1, and rAAV6-ZsGreen1 samples and found that they contained 0.69-3.27% DNA impurities derived from three plasmids, as detected by droplet digital PCR. These plasmid-derived impurities primarily consisted of those derived from the pAAV plasmid (≥98.88%), with small amounts of pRC1, pRC2mi342, pRC5, or pRC6 (≤0.91%), and pHelper (≤0.21%) plasmids. To determine the DNA strand form of these impurities within the capsids, we used two different DNases with distinct substrate specificities. The extracted DNA impurities from the rAAV samples exhibited high sensitivity to nuclease P1 but not to lambda exonuclease. Similarly, host cell DNA encapsulated within the capsids revealed similar sensitivities to the nucleases. These findings indicate that DNA impurities derived from the plasmids and host cell DNA are encapsulated into rAAV capsids as single-stranded DNA, likely through a mechanism similar to that of the rAAV genome.

Human embryonic kidney 293 (HEK 293) cells are the main producer cell line for recombinant adeno-associated virus (rAAV) production. However, AAV vector yields among 293 clones vary considerably. To elucidate the biological basis for these differences, whole genomes of an adherent and a suspension 293 cell clone with high-yield rAAV were sequenced using nanopore technology. All 293 cell derivative lines showed a twofold copy number gain at the adenoviral integration site across, suggesting a genome duplication event. To our surprise, the two high-producer clones, despite having been separately developed, are biologically closely grouped together as compared to other commonly used 293 clones. Their genomes contain a similar adenoviral gene integration region, which likely leads to high expression of proteins that facilitate AAV replication and packaging. Thus, genome duplication in the adenovirus integration locus may be a key factor affecting AAV production yield.

The objective of this article is to summarize the research progress and discuss the current difficulties of gene-based therapeutic angiogenesis in lower limb ischemic diseases, so as to provide new research directions for the non-invasive treatment of lower limb ischemia. The basic and clinical trials of gene-based therapeutic angiogenesis in lower limb ischemia in recent years were read and reviewed. Growth factors such as vascular endothelial growth factor, hepatocyte growth factor, and fibroblast growth factor have been extensively studied for their application in lower limb ischemic diseases. However, clinical studies across various phases have shown inconsistent efficacy endpoints. The efficacy of gene therapy remains questionable. Before exploring efficient methods of delivering pro-angiogenic genes to ischemic tissues, clarification is needed regarding whether the goal of gene therapy is to simply promote collateral circulation or create a conducive tissue microenvironment for angiogenesis. In conclusion, pre-clinical and clinical studies have demonstrated the potential of therapeutic angiogenesis, but more systematic and comprehensive research is needed to explore safer, more effective, and cost-effective treatment methods.

Adeno-associated virus (AAV)-based therapeutics have the potential to transform the lives of patients by delivering one-time treatments for a variety of diseases. However, a critical challenge to their widespread adoption and distribution is the high cost of goods. Reducing manufacturing costs by developing AAV capsids with improved yield, or fitness, is key to making gene therapies more affordable. AAV fitness is largely determined by the amino acid sequence of the capsid, however, engineered AAVs are rarely optimized for manufacturability. Here, we report a state-of-the art machine learning (ML) model that predicts the fitness of AAV2 capsid mutants based on the amino acid sequence of the capsid monomer. By combining a protein language model (PLM) and classical ML techniques, our model achieved a significantly high prediction accuracy (Pearson correlation = 0.818) for capsid fitness. Importantly, tests on completely independent datasets showed robustness and generalizability of our model, even for multimutant AAV capsids. Our accurate ML-based model can be used as a surrogate for laborious in vitro experiments, thus saving time and resources, and can be deployed to increase the fitness of clinical AAV capsids to make gene therapies economically viable for patients.

Endothelial activation and dysfunction are key early steps in atherogenesis. Vascular gene therapy targeting endothelial inflammation and cholesterol accumulation could decrease atherosclerosis progression. ATP-binding cassette subfamily A member 1 (ABCA1) exhibits anti-inflammatory properties and promotes cholesterol efflux. A mouse model showed that systemic endothelial overexpression of ABCA1 decreased diet-induced atherosclerosis. To test if local ABCA1 endothelial overexpression protects against atherosclerosis, we used helper-dependent adenoviral vectors (HDAd) to express ABCA1 or a "Null" control in the carotid endothelium of hyperlipidemic rabbits. Both ABCA1 mRNA and endothelial protein were increased 3 days after vector infusion. After 24 weeks on a high-fat diet, laser-microdissected endothelium showed increased ABCA1 mRNA expression, but whole-vessel ABCA1 mRNA was decreased with HDAdABCA1. Endothelial ABCA1 protein could not be measured at 24 weeks, so its overexpression may be transient. CD68 expression was decreased (-23%, p < 0.001), but ITGAM (-15%, p = 0.3) was unchanged. Macrophage markers for both M1-like macrophages (IL1B: -44% [p = 0.02]; IL6: -40% [p = 0.02]; CCL2: -25% [p = 0.02]) and M2-like macrophages (ARG1: -27% [p = 0.03]; IL10: -23% [p = 0.09]; TGFB1: -13% [p < 0.001]) were also decreased. The inflammatory cytokines IL6 (-100%; p < 0.001) and TNF (p < 0.05) were significantly decreased in the laser-microdissected endothelium, but VCAM1 (+5%, p = 1.0) was unchanged and ICAM1 (+101%; p = 0.03) increased. Lesion size, intimal lipid, and intimal macrophage content were all unchanged (p > 0.5 for all), and vascular cholesterol measured by mass spectrometry (-11%; p = 0.9) also showed no difference. There was a small decrease in the intimal/medial ratio. scRNAseq revealed that vector transcripts were not restricted to endothelial cells after 24+ weeks but were detected in most cell types. The exception was modulated smooth muscle cells, which were found in substantial numbers in larger lesions. Overall, transient overexpression of ABCA1 in the vascular endothelium subtly alters the expression of inflammatory markers, providing only a modest atheroprotection.

β-thalassemia and sickle cell disease (SCD) are global monogenic blood system disorders, and reactivated δ-globin is expected to replace missing or abnormal β-globin. With the development of gene editing technology, activating γ-globin for treating β-thalassemia and SCD has been highly successful. However, δ-globin, as another important potential therapeutic target, has few related studies. Gene editing technology introduced cis-acting elements, including NF-Y, KLF1, GATA1, and TAL1, into the regulatory region of HBD, successfully activating the expression of δ-globin. It was confirmed that the activation effect of δ-globin was closely related to the location of the introduced cis-acting elements. In this study, the mutation creates a de novo binding site for KLF1 at -85∼93 bp upstream of the transcription start site of the HBD gene, as well as the site for TAL1 and GATA1 cobinding motifs at -59 to ∼-78 bp, which could effectively activate δ-globin.

Niemann-Pick disease, type C1 (NPC1), is a rare, fatal neurodegenerative disorder caused by pathological variations in NPC1. We and others have previously demonstrated the efficacy of systemic adeno-associated virus (AAV) gene therapy with AAV9 in murine models of NPC1. The presence of neutralizing antibodies (NAbs) caused by natural exposure to wildtype AAVs may impair AAV transduction efficacy and reduce or negate the benefit of gene therapy. In addition, there remains the question of whether individuals seroconvert with age and whether seroconversion limits the window of therapeutic efficacy. Thus, we assessed the prevalence of anti-AAV9 and anti-AAV2 NAbs in serum samples from 22 individuals with NPC1 at two different time points: one closer to diagnosis (0.9-17 years old) and another collected between 4 and 15 years later during follow-up (6-28 years old). At a titer of <1:5, we found that more than half of the cohort lacked NAbs against either AAV2 (68.2%) or AAV9 (59.1% at time 1, 63.6% at time 2). Notably, only 3 out of 22 individuals showed a transition from undetectable to detectable NAb titers, and most participants maintained stable titers over time, unaffected by age. These data support the feasibility of systemic or direct CNS AAV9 gene therapy in this patient population.