Human gene therapy最新文献

The delivery of a mini-dystrophin gene to skeletal muscles using recombinant adeno-associated virus serotype (AAV) holds great potential as a gene therapy for Duchenne muscular dystrophy (DMD). However, the presence of anti-AAV-neutralizing antibodies (NAbs) may impede the effectiveness of gene transduction. This study aimed to evaluate the prevalence of anti-AAV9 NAbs in Chinese patients with DMD, and to characterize the target population for an AAV gene therapy. A total of one hundred male patients with DMD were included in this study, and demographic and clinical data were collected. A blood specimen was obtained from each participant for the purpose of evaluating the existence of anti-AAV9 NAbs through a cell-based functional assay conducted at a central laboratory. A NAb titer exceeding 1:4 was considered positive. The positivity rates of anti-AAV9 NAb were compared among different subgroups. The median age of this DMD cohort was 8 years old, ranging from 3 to 15 years of age. Forty-two percent of patients tested positive for anti-AAV9 NAb. Notably, all samples from patients under 4 years of age tested negative, and the positivity rates of anti-AAV9 NAb differed significantly across the three age subgroups (<4 years old, ≥4 years old and <12 years old, and ≥12 years old, χ² = 7.221, p = 0.023). Further investigation into the living environment revealed a higher positivity rate of anti-AAV9 NAb in rural patients compared with urban patients (χ² = 3.923, p = 0.048). Moreover, the prevalence in patients from different cities/provinces varied greatly (χ² = 16.550, p = 0.003). There was no statistically significant difference in the positivity rate of NAb among subgroups of patients with different motor functions (ambulatory or nonambulatory) and different treatment strategies (taking or not taking glucocorticoid). In Chinese DMD patients, the prevalence of anti-AAV9 NAb was found to reach 42%. Moreover, the antibody-positive rate in children <4 years of age was low and revealed notable regional discrepancies.

Chronic hypereosinophilia, defined as persistent elevated blood levels of eosinophils ≥1,500/μL, is associated with tissue infiltration of eosinophils and consequent organ damage by eosinophil release of toxic mediators. The current therapies for chronic hypereosinophilia have limited success, require repetitive administration, and are associated with a variety of adverse effects. As a novel approach to treat chronic hypereosinophilia, we hypothesized that adeno-associated virus (AAV)-mediated delivery of an anti-human eosinophil antibody would provide one-time therapy that would mediate persistent suppression of blood eosinophil levels. To assess this hypothesis, we first generated a human monoclonal antibody (mAb) directed against Siglec8, a sialic-acid binding immunoglobulin-like lectin, expressed at high levels on the cell surface of human eosinophils. Transgenic mice with a human immunoglobulin repertoire were immunized with human Siglec8 protein or DNA encoding human Siglec8. Based on target binding assessments, the 08C07 mAb was chosen for further study. The human variable regions of 08C07 were joined to the human Ig constant region, creating H08C07 (hAntiEos), a fully human anti-human eosinophil mAb. Using the gene sequence of hAntiEos, we created AAVrh.10hAntiEos, an AAVrh.10-based vector expressing the heavy and light chains of H08C07. Intravenous administration of AAVrh.10hAntiEos (10¹¹ genome copies or gc) to C57Bl/6 mice resulted in persistent elevated serum levels of hAntiEos. In vivo gene therapy generated hAntiEos bound to recombinant human Siglec8 protein in a dose-dependent manner and to human eosinophils, mediated apoptosis of human eosinophils, and antibody-dependent cellular cytotoxicity activity against human eosinophils. Consistent with these data, administration of AAVrh.10hAntiEos to human CD34⁺ transplanted NSG-SGM3 immunodeficient mice suppressed levels of human eosinophils in vivo. AAVrh.10hAntiEos holds the potential to offer therapeutic benefit to patients with chronic hypereosinophilia.

Oncolytic viruses are able to lyse tumor cells selectively in the liver without killing normal hepatocytes, in addition to activating the immune response. Oncolytic virus therapy is expected to revolutionize the treatment of liver cancer, including hepatocellular carcinoma (HCC), one of the most frequent and fatal malignancies. In this study, reverse genetics techniques were exploited to load NA fragments of the A/PuertoRico/8/34 virus (PR8) with GV1001 peptides derived from human telomerase reverse transcriptase. An in vitro assessment of the therapeutic effect of the recombinant oncolytic virus was followed by an in vivo study in mice with HCC. The recombinant virus was verified by sequencing of the recombinant viral gene sequence, and viral virulence was detected by hemagglutination assays and based on the 50% tissue culture infectious dose (TCID₅₀). The morphological structure of the virus was observed by electron microscopy, and GV1001 peptide was localized by cellular immunofluorescence. The selective cytotoxicity of the recombinant oncolytic virus in vitro was demonstrated in cultured HCC cells and normal hepatocytes, as only the tumor cells were killed; the normal cells were not significantly altered. Consistent with the in vitro results, the recombinant oncolytic influenza virus significantly inhibited liver tumor growth in mice in vivo, in addition to inducing an antitumor immune response, including an increase in the number of CD4⁺ and CD8⁺ T lymphocytes and, in turn, improving survival. Our results suggest that oncolytic influenza virus carrying GV1001 is a promising immunotherapy in patients with HCC.

CLN2 disease (late infantile neuronal ceroid lipofuscinosis) is an autosomal recessive, neurodegenerative lysosomal storage disease that results from loss of function mutations in the CLN2 gene, which encodes tripeptidyl peptidase 1. It affects the central nervous system (CNS) with progressive neurodegeneration and early death, typically at ages from 8 to 12 years. Twenty years ago, our phase I clinical trial treated subjects with CLN2 disease by a catheter-based CNS administration of an adeno-associated virus vector serotype 2 (AAV2) expressing the CLN2 gene. Here we present an analysis of the survival of the 10 treated children and find 2 distinct survival groups with a wide disparity in survival. Group 1 (n = 7) had the typical mean survival of 8.8 ± 0.5 years of age, 3.8 ± 0.6 years post-therapy. Group 2 (n = 3) had a markedly longer mean survival of 23.4 ± 2.4 years of age and 14.9 ± 2.8 years post-therapy (p < 0.00002, survival of group 1 vs. group 2). Long survivors (group 2) at the time of treatment were older (group 1: 5.0 ± 0.6 years; group 2: 8.5 ± 0.9 years; p < 0.02); had similar disease severity (Hamburg clinical score group 1: 4.7 ± 0.5, group 2: 3 ± 0.0, p > 0.05); and had larger CNS ventricular volume (81.1 ± 22.2 cm³ vs. 27.3 ± 7.2 cm³ for group 1; p < 0.02). While the genotype of 3, group 2 subjects, had one allele (509-1G>C) identical to that of three in group 1, the second allele was different. This was unlikely to explain the survival difference, as alleles for both groups were equally predicted deleterious by the Combined Annotation-Dependent Depletion score: 34.9 ± 0.7 and 32.8 ± 0.3 for groups 1 and 2, respectively (a score of >20 is considered deleterious). This represents one of the longest survival studies (up to 20 years) of AAV-treated individuals with hereditary disorders and demonstrates variability of therapeutic efficacy where the genotype on its own has no apparent survival advantage. Protocol registration numbers for the original study: NCT00151216 and NCT00151268; www.clinicaltrials.gov.