Human gene therapy最新文献

Adeno-associated viral (AAV) vectors are increasingly used for preclinical and clinical cardiac gene therapy approaches. However, gene transfer to cardiomyocytes poses a challenge due to differences between AAV serotypes in terms of expression efficiency in vitro and in vivo. For example, AAV9 vectors work well in rodent heart muscle cells in vivo but not in cultivated neonatal rat ventricular cardiomyocytes (NRVCMs), necessitating the use of AAV6 vectors for in vitro studies. Therefore, we aimed to develop an AAV that could efficiently express genes in NRVCMs, human engineered heart tissue (hEHT), and mammalian hearts. The production of AAV6 vectors results in lower yields compared with AAV9. Hence, we used random AAV9 peptide libraries and selected variants on NRVCMs at the vector genome and RNA levels in parallel. The enriched library variants were characterized using high-throughput analysis of barcoded variants, followed by individual validation of the most promising candidates. Interestingly, we found striking differences in NRVCM transduction and gene expression patterns of the AAV capsid variants depending on the selection strategy. AAV variants selected based on the vector genome level enabled the highest transduction but were outperformed by AAVs selected on the RNA level in terms of expression efficiency. In addition, we identified a new AAV9 capsid variant that not only allowed significantly higher gene expression in NRVCMs compared with AAV6 but also enabled similar gene expression in murine hearts as AAV9 wild-type vectors after being intravenously injected into mice. Moreover, the novel variant facilitated significantly higher gene expression in hEHT compared with AAV9. Therefore, this AAV variant could streamline preclinical gene therapy studies of myocardial diseases by eliminating the need for using different AAVs for NRVCMs, hEHT, and mice.

Complement-mediated thrombotic microangiopathy (TMA) in the form of atypical hemolytic uremic syndrome (aHUS) has emerged as an immune complication of systemic adeno-associated virus (AAV) gene transfer that was unforeseen based on nonclinical studies. Understanding this phenomenon in the clinical setting has been limited by incomplete data and a lack of uniform diagnostic and reporting criteria. While apparently rare based on available information, AAV-associated TMA/aHUS can pose a substantial risk to patients including one published fatality. Reported cases were originally limited to pediatric Duchenne muscular dystrophy patients receiving micro- or mini-dystrophin transgenes via AAV9 but have subsequently been reported in both pediatric and adult patients across a range of disorders, transgenes, promoters, and AAV capsid types. This article provides an introduction to the complement system, TMA and aHUS, and anticomplement therapies, then presents clinical reviews of AAV-associated TMA/aHUS cases that have been reported publicly. Finally, exploration of risk factors and current and future mitigation approaches are discussed.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). While gene therapy holds promise as a cure, the cell-type-specific heterogeneity of CFTR expression in the lung presents significant challenges. Current CF ferret models closely replicate the human disease phenotype but have limitations in studying functional complementation through cell-type-specific CFTR restoration. To address this, we developed a new transgenic ferret line, CFTR^{int1-eGFP(lsl)}, in which a Cre-recombinase (Cre)-excisable enhanced fluorescent protein (eGFP) reporter cassette is knocked in (KI) to intron 1 of the CFTR locus. Breeding this reporter line with CFTR^G551D CF ferret resulted in a novel CF model, CFTR^{int1-eGFP(lsl)/G551D}, with disease onset manageable via the administration of CFTR modulator VX770. In this study, we confirmed two key properties of the CFTR^{int1-eGFP(lsl)/G551D} CF ferrets: (1) cell-type-specific expression of the CFTR(N-24)-eGFP fusion protein, driven by the intrinsic CFTR promoter, in polarized epithelial cultures and selected tissues, and (2) functional reversion of the KI allele via Cre-mediated excision of the reporter cassette. This model provides a valuable tool for studying the effects of targeted CFTR reactivation in a cell-type-specific manner, which is crucial for enhancing our understanding of CFTR's roles in modulating airway clearance and innate immunity, and for identifying relevant cellular targets for CF gene therapy.

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.

Worldwide, thousands of male patients who carry ATP Binding Cassette Subfamily D Member 1 (ABCD1) mutations develop adrenomyeloneuropathy (AMN) in mid-adulthood, a debilitating axonopathy of the spinal cord. Today AAV gene therapy brings the most hope for this orphan disease. We previously reported that an AAV9-MAG-hABCD1 vector injected intravenously in the neonatal period prevented the disease in 2-year-old Abcd1-/- mice, the AMN mouse model. In the current study, the same vector was injected intracisternally at 18 months of age, when about half of Abcd1-/- mice start losing balance and motricity. As soon as 1-3 months after vector injection, motor tests have evolved differently in treated and untreated (UT) mice. Six months after vector, treated mice (n = 24) had near-normal motor performances, whereas neurological state had deteriorated in UT mice (n = 34). In five white matter regions of the cervical spinal cord, hABCD1 expression at 24 months of age was present in 22% (18-27) of oligodendrocytes (OLs) and 22% (17-26) of astrocytes and not detected in neurons or microglia. Abundant hABCD1 expression was also observed in OLs and astrocytes in the cerebellum and brainstem and, to a lesser level, in the lower spinal cord, not in the dorsal root ganglia or brain cortex. In conclusion, the effect of the AAV9-MAG-hABCD1 vector at an early symptomatic stage of the Abcd1-/- mouse model paves a new oligotropic way for the gene therapy of AMN.

Adeno-associated virus (AAV) vectors have demonstrated safety and efficacy for gene transfer to hepatocytes in preclinical models, in various clinical trials and from a clinical experience with a growing number of approved gene therapy products. Although the exact duration is unknown, the expression of therapeutic genes in hepatocytes remains stable for several years after a single administration of the vector at clinically relevant doses in adult patients with hemophilia and other inherited metabolic disorders. However, clinical applications, especially for diseases requiring high AAV vector doses by intravenous administrations, have raised several concerns. These include the high prevalence of pre-existing immunity against the vector capsid, activation of the complement and the innate immunity with serious life-threatening complications, elevation of liver transaminases, liver growth associated with loss of transgene expression, underlying conditions negatively affecting AAV vector safety and efficacy. Despite these issues, the field is rapidly advancing with a better understanding of vector-host interactions and the development of new strategies to improve liver-directed gene therapy. This review provides an overview of the current and emerging challenges for AAV-mediated liver-directed gene therapy.

Mucopolysaccharidosis type IVA (MPS IVA) is an autosomal congenital metabolic lysosomal disease caused by a deficiency of the N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) gene, leading to severe skeletal dysplasia. The available therapeutics for patients with MPS IVA, enzyme replacement therapy and hematopoietic stem cell transplantation, revealed limitations in the impact of skeletal lesions. Our previous study, a significant leap forward in MPS IVA research, showed that liver-targeted adeno-associated virus (AAV) gene transfer of human GALNS (hGALNS) restored GALNS enzymatic activity in blood and multiple tissues and partially improved the aberrant accumulation of storage materials. This promising approach was further validated in our current study, where we delivered AAV8 vectors expressing hGALNS, under the control of a liver-specific or ubiquitous promoter, into MPS IVA murine disease models. The results were highly encouraging, with both AAV8 vectors leading to supraphysiological enzymatic activity in plasma and improved cytoplasmic vacuolization of chondrocytes in bone lesions of MPS IVA mice. Notably, the ubiquitous promoter constructs, a potential game-changer, resulted in significantly greater enzyme activity levels in bone and improved pathological findings of cartilage lesions in these mice than in a liver-specific one during the 12-week monitoring period, reinforcing the positive outcomes of our research in MPS IVA treatment.

Systemic delivery of adeno-associated virus (AAV) vectors targeting the central nervous system has the potential to solve many neurodevelopmental disorders, yet it is made difficult by the filtering effect of the blood-brain barrier and systemic complications. To overcome this limitation, we attempted to inject a Venus-expressing, oligodendrocyte-selective AAV9 viral vector in the ventricles together with lipid microbubbles and subjected them to focused ultrasound (FUS); the resulting mechanical stimulation on the brain ventricles is able to open small, temporary gaps from which vector particles can leak and spread. Our findings indicate that FUS can increase viral vector diffusion across both the anteroposterior and left-right axes without influencing cell tropism; significant effects were found with 60 and 90 s exposure time, but no effects were observed with longer intervals. Taken together, these results highlight a new strategy for the safe and effective delivery of viral vectors and offer new perspectives for the development and application of gene therapies for central nervous system diseases.

Delandistrogene moxeparvovec is a gene transfer therapy for Duchenne muscular dystrophy (DMD) that uses an adeno-associated viral vector to deliver a micro-dystrophin transgene to skeletal and cardiac muscle. This study evaluated the long-term survival and cardiac efficacy of delandistrogene moxeparvovec in a DMD-mutated (DMD^MDX) rat model of DMD-related cardiomyopathy. DMD^MDX male rats, aged 21-42 days, were injected with 1.33 × 10¹⁴ viral genomes/kilogram (vg/kg) delandistrogene moxeparvovec and followed for 12, 24, and 52 weeks. Ambulation was recorded via the Photobeam Activity System, whereas echocardiograms, cardiomyocyte contractility, calcium handling, and histological analysis of fibrosis were used to evaluate cardiac disease at 12-, 24-, and 52-weeks post-treatment. A separate cohort of rats was used to assess the impact of delandistrogene moxeparvovec on survival. Treatment with delandistrogene moxeparvovec extended median survival in DMD^MDX rats to >25 months versus the 13-month median survival in saline-control-treated DMD^MDX rats. Compared with saline control, delandistrogene moxeparvovec therapy elicited statistically significant improvements across cardiac parameters approaching wild-type values with additional benefits in mobility, histopathology, and fibrosis observed. Transgene expression was maintained up to >25 months and micro-dystrophin expression was broadly distributed across skeletal and cardiac muscle. Taken together, these findings demonstrate long-term cardiac efficacy and improved survival following delandistrogene moxeparvovec treatment in DMD^MDX rats.