Human gene therapy最新文献

As impact of adeno-associated virus (AAV) empty capsids on drug product safety and quality remains inconclusive, downstream purification strategy has been focusing on empty capsid removal. Anion-exchange chromatography (AEX) has made significant progress in separating empty from full capsids in recent years. Still, achieving baseline resolution between different AAV subpopulations remains challenging due to subtle charge differences. With a certain AAV construct design, this difficulty is compounded when upstream packaging efficiency is low or when empty and full capsids of a particular serotype have similar electrostatic charge profiles. To improve separation and product purity, secondary interaction mechanisms using multimodal (mix-mode) chromatography are often introduced. In this study, we present a case study on developing a polishing chromatography step to remove empty capsids from AAV7 and AAV8 preparations. To create a challenging feed material for the polishing step, we used small gene-of-interest (GOI) and poorly packaged starting materials. We investigated multiple critical process parameters, including buffer matrix, salt concentration, pH, peak fractionation strategies, and column chemistry (strong AEX vs. mix-mode). Mass photometry (MP) and charge detection mass spectrometry (CDMS) were used to characterize capsid populations. Optimized AEX conditions for AAV8 achieved 80% full capsids by MP and 90% GOI-containing capsids by CDMS. For AAV7, the mix-mode column demonstrated improved resolution compared with the standard AEX gradient method. These results demonstrate that mix-mode chromatography provides an alternative polishing option for serotypes where traditional AEX fails to achieve the desired separation.

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by defective ciliary motility, leading to recurrent respiratory infections and chronic airway damage. Gene therapy holds promise for treating PCD, but its effectiveness in patient-derived models remains uncertain. This study aimed to evaluate the therapeutic potential of lentiviral gene delivery in restoring ciliary function in patient-derived nasal apical-out airway organoids. Using nasal epithelial cells from both healthy individuals and PCD patients with mutations in DNAAF1, DNAAF3, or DNAAF6, we established organoid models to assess gene therapy efficacy. Lentiviral vectors successfully restored the expression and proper localization of DNAAF proteins in mutant organoids, significantly improving ciliary beating frequency and the proportion of organoids with functional cilia. These findings provide proof-of-concept evidence supporting gene therapy as a viable approach to correct ciliary defects in PCD, paving the way for targeted treatments.

The natural tissue tropism of adeno-associated viruses (AAVs) is being widely exploited in their use as vectors for transgene delivery for gene therapy of human diseases. A major limitation of this approach is the prevalence of antibodies to AAV capsid antigens, which can neutralize the infused vector and thus affect expression of the transgene. There are limited data on the prevalence of such antibodies, total (TAb) and neutralizing (NAb), to determine the eligibility of patients for gene therapy using specific AAV vectors. Using whole capsid ELISA for TAb and transduction inhibition assay (mCherry-based flow cytometry method for AAV5 and luciferase-based assay for AAV8) for NAb, in this study, we have evaluated the seroprevalence of anti-AAV5 and anti-AAV8 antibodies in three groups: healthy individuals (AAV5 n = 130, AAV8 n = 75), individuals with hemophilia A (AAV5 n = 62, AAV8 n = 88), and individuals with hemophilia B (AAV5 n = 42, AAV8 n = 55). The TAb prevalence for AAV5 in the three groups was 77.7%, 90.3%, and 95.2%, respectively, and that for AAV8 was 89.3%, 93.2%, and 92.7%, respectively. The AAV5 NAb seropositivity in the three groups was 89.2%, 100%, and 100% and that against AAV8 was 45.3%, 46.6%, and 45.5%, respectively. To check endpoint titer for AAV5 NAb, 21 out of the 42 hemophilia B samples were screened, using a dilution of 1 in 80 and 1 in 160. Among the AAV5 hemophilia B samples with higher dilutions, 81% of the individuals had a titer of ≤80. Age-stratified AAV5 and 8 TAb and NAb seroprevalence showed high prevalence across all age groups in all three groups of samples screened. High positivity among AAV5 NAb at lower dilutions should be further evaluated for cross-reactivity.

The study of β-hemoglobinopathies and associated β-globin genes has revealed that genetic elements, such as the Locus Control Region (LCR) or the replication Initiation Region (IR) of the β-globin gene locus, are essential for the regulation of β-globin genes replication and expression. The LCR at 5' of the β-globin genes plays major role in the intricate regulation of transcription of the "β-like globin genes" expression in situ and in gene therapy protocols by viral gene transfer, ensuring globin gene expression independent from integration site and exerting a critical role in chromatin organization and boundary formation. The IR element, located at the 5' site of the HBB gene promoter, functions as the initiation point for physiological, bidirectional DNA replication, both in situ and within an episomal vector, and induces replication in positions that do not possess such capacity. It enhances plasmid replication, establishment, and transgene expression in the descendants of transfected human CD34+ cells during colony-forming cell assays. A third required genetic element is the promoter of the transgene(s). This is either the HBB gene native promoter or the CD34+ cell-functional ubiquitous promoter spleen focus-forming virus. Both promoters, in in vitro studies, can direct accurate, efficient transcription from episomal, S/MAR-based vectors. Mutations in the HBB gene native promoter as well as in LCR and IR lead to β-thalassemia. Another genetic element, the S/MAR, deriving from the 5' of the human β-interferon gene, ensures plasmid nonintegration and long-term nuclear retention in the prototype episomal vector pEPI-1 and derivative episomal vectors. Such S/MAR-based episomal vectors form the basis from which the genetic elements collectively- HBB gene promoter, LCR, and IR-represent a comprehensive model for the design of efficient episomal vectors with efficient transcription, replication, and long-term nuclear retention of vector for gene therapy applications for the β-hemoglobinopathies within the context of gene addition strategy.

In gene therapy using adeno-associated virus (AAV) vectors, treatment-induced anti-AAV antibodies pose barriers for re-administration of the same or different AAV serotype vectors. We aimed to investigate whether the administration of AAV5, AAV8, or AAV9 in Cynomolgus monkeys resulted in the formation of cross-reactive antibodies. To achieve this, we developed a Biacore SPR-based total binding antibody (TAb) assay to identify anti-AAV antibodies in monkey plasma and assess the cross-reactivity of these antibodies against AAV5, AAV8, or AAV9 vectors on a sensor chip. AAV5, AAV8, and AAV9 vectors were immobilized onto the surface of a CM5 sensor chip on Fc2, Fc3, and Fc4 flow cells, respectively, using amine coupling, while Fc1 served as a reference. Plasma samples flowed through four channels, followed by injecting anti-monkey IgG and IgM antibodies to determine the immunoglobulin (Ig) isotypes. We analyzed TAb against the AAV serotypes in the plasma using a Biacore-based TAb assay 29 days after administration to evaluate the anti-AAV antibody responses. The TAb detected by the Biacore-based assay showed cross-reactivity between antibodies against AAV8 and AAV9; however, there was minimal cross-reactivity between antibodies against AAV5 and those against AAV8 or AAV9. Both IgG and IgM TAb were detected at 29 days post-dosing, and the antibody profiles determined by both the Biacore and ELISA platforms were comparable. The Biacore assessment confirmed the absence of cross-reactivity of anti-AAV5 antibodies against AAV8 and AAV9 vectors, and vice versa. This absence of cross-reactive antibodies against a specific AAV serotype indicated the possibility of re-administering a different AAV serotype.

Adeno-associated virus (AAV) vectors have emerged as versatile and promising tools in gene therapy due to their favorable safety profile, broad tissue tropism, and long-term gene expression. However, pre-existing immunity, especially in the form of neutralizing antibodies (NAbs) remains a significant barrier, reducing vector efficacy and restricting patient eligibility. This review provides a comprehensive overview of the immunological landscape affecting AAV gene therapy, including global seroprevalence, environmental influences, and antibody cross-reactivity stemming from natural parvovirus exposure or vaccination of animal research models.We detail the mechanisms underlying immune detection and vector clearance, covering innate pattern recognition receptors, complement activation, and adaptive immune effector functions such as antibody-dependent complement deposition, cytotoxicity, and phagocytosis.We further analyze how species, age, serotype, administration route, and target tissue contribute to immune susceptibility and variable transduction outcomes. To overcome these challenges, we propose a three-pronged classification of mitigation strategies: (1) immune-focused strategies, such as plasmapheresis, immunoadsorption, enzymatic antibody cleavage, corticosteroids, and B cell depletion; (2) delivery-focused strategies, which include targeting immune-privileged sites, localized or intrathecal delivery, and timing of vector administration; and (3) capsid-focused strategies, comprising rational capsid engineering and the use of decoy particles or empty capsids.We also discuss promising advances such as AAV-specific regulatory T cells and re-dosable AAV platforms. This strategic framework offers a roadmap for tailoring gene therapy approaches to individual immune profiles and improving the safety, efficacy, and accessibility of AAVbased therapeutics.

Adeno-associated viral vectors (AAVs) are promising tools for gene therapy. However, scaling up the production of AAVs to produce high-quality vectors at high yields for clinical purposes has proven to be challenging. In the present study, we optimized the production process of AAV in a fixed-bed bioreactor using transient transfection in adherent HEK-293T cells. We systematically optimized the key process parameters, namely cell seeding density, cell density at transfection, and DNA-to-cell ratio, based on the yield obtained, starting from a prototype batch, followed by ten batch runs. Here, we packaged a reporter gene (enhanced green fluorescent protein) and a therapeutic gene (lysyl oxidase) into AAV9 capsids as part of our process development program to be applied for future current Good Manufacturing Practices production and clinical trial application. Throughout the experiments, media conditions, transfection processes, and mechanical parameters were kept identical, while monitoring pH, dissolved oxygen, and media glucose concentration during a production process of approximately 10 days. We demonstrate that by optimizing these parameters, the fixed-bed bioreactor was able to support as many as 1.6-2.8 × 10⁶ cells/carrier strip, up to 3 × 10⁹ cells/m² bioreactor. Through this multivariate optimization process, we increased viral yield by about 7.6-fold (range of 5.7-10.4-fold for the optimized process runs) over the prototype batch. The total AAV vector yield average was 2.3 × 10¹⁴ vg (range 1.1 × 10¹⁴ vg to 4.95 × 10¹⁴ vg), corresponding to an average per cell yield of 1.4 × 10⁵ vg/cell (range 0.85 × 10⁵-2.46 × 10⁵vg/cell). In conclusion, our findings highlight that optimizing process parameters in a fixed-bed bioreactor presents a promising strategy for scalable and cost-effective AAV vector production.