Gene Therapy最新文献

The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorders. rAAV vectors have emerged as promising vehicles for in vivo gene therapy due to their favorable safety profile, high tissue specificity, and ability to induce sustained transgene expression. However, their limited packaging capacity has been a significant challenge for delivering large CRISPR molecules. To overcome this limitation, innovative strategies have been developed, including the use of compact Cas orthologs, dual rAAV vector systems, and trans-splicing rAAV vectors. These approaches have significantly improved the efficiency of genome editing for therapeutic applications. This review presents recent advancements in rAAV-CRISPR-mediated in vivo gene therapy, highlighting key technological innovations, current challenges, and the therapeutic potential of these strategies in the development of next-generation gene therapies.

Hypercholesterolemia, defined by high low-density lipoprotein cholesterol levels, critically increases the risk of atherosclerotic cardiovascular disease, which represents the foremost cause of worldwide morbidity and mortality. While established lipid-lowering therapies, primarily statins, are effective for many patients, a significant proportion either fail to achieve optimal LDL-C targets, experience dose-limiting side effects, or face challenges with the long-term adherence required for sustained cardiovascular benefit. The recent emergence and rapid advancement of precise gene editing technologies most notably CRISPR-Cas9 and its advanced variants like base editing and prime editing offer a revolutionary therapeutic paradigm. These tools have the potential to achieve durable modification of the expression or function of genes fundamentally involved in cholesterol metabolism. This comprehensive overview integrates the current knowledge of critical cholesterol regulatory pathways and the main protein targets that are suitable for gene editing. The fundamental mechanisms, relative advantages, and inherent limitations of gene editing platforms and delivery systems for clinical translation are examined. The expanding preclinical data and groundbreaking clinical evidence highlighting the transformative potential of gene editing to achieve significant and lasting reductions in LDL-C, especially through promising therapies like VERVE base editors targeting PCSK9 and ANGPTL3 are critically evaluated. The challenges including off-target effects, delivery efficiency and specificity, long-term safety and durability, complex ethical considerations, and evolving regulatory landscapes that must be rigorously navigated for these therapies to become mainstream clinical practice are thoroughly addressed. Successfully overcoming these challenges could mark the beginning of a new era of personalized, one-time treatments for hypercholesterolemia.

Antibody-drug conjugates (ADCs) are a promising class of targeted cancer therapeutics, but their efficacy is often limited by off-target toxicity caused by payload leakage after internalization into tumor cells. To overcome this, we developed an ADC alternative using an antibody-guided adeno-associated virus (AAV) vector system that delivers suicide genes specifically to cancer cells. By displaying Protein A on the AAV VP2 capsid, we enabled IgG binding and stable complex formation on the capsid, leading to efficient antibody-guided transduction under our assay conditions. This modular platform allows flexible retargeting specific tumor-associated antigens simply by changing the antibody, without genetic re-engineering of the capsid. Using an AAV2 heparan sulfate binding knockout (HBKO) background to minimize nonspecific infection, we achieved antigen-specific transduction for multiple targets including CD20, EGFR, PSMA, CEA, and CD5 (with varying levels of enhancement depending on the target). In vitro, the system successfully directed EGFP expression and, upon delivery of the pro-apoptotic gene BAX, induced selective apoptosis in target-positive cells. Unlike conventional ADCs, this strategy is designed to minimize the risk of extracellular payload leakage and to confine cytotoxicity primarily to transduced cells, thereby supporting more selective tumor targeting. Our approach may offer a versatile alternative for targeted cancer therapy, with the potential for further development into customizable and precision-guided gene-based treatments.

Guidance from the U.S. Food and Drug Administration and other regulatory agencies recommends long-term follow-up (LTFU) studies of gene therapy recipients. The primary objective of LTFU studies is to understand the long-term safety of gene therapy products; evaluation of efficacy outcomes may be a secondary goal. To learn more about LTFU study design and conduct, we conducted a descriptive study of key characteristics of LTFU gene therapy study protocols registered in ClinicalTrials.gov, including data about status and duration, funding source, enrollment, number of clinical trial sites per study, eligibility criteria, geographic location, and intent to monitor and report adverse events. This analysis enabled a better understanding of how registered LTFU studies are currently designed and stimulated ideas for improvement, which are discussed. Most notably, our results suggest that there is a lack of harmonization in how safety outcomes are monitored and reported across LTFU studies. Standardization and/or harmonization of outcome reporting for LTFU studies of GTs may increase their scientific value. The development of better guidance and innovative approaches for LTFU study design and conduct would help support best practices and the fulfillment of LTFU commitments to understand the overall long-term benefit-risk profile of GT products.

Sensitive quantification of adeno-associated virus (AAV) neutralizing antibodies (NAbs) is essential for gene therapy success. Conventional cell-based transduction inhibition assays often encounter matrix-induced artifacts resulting from variable serum content across dilutions, which artificially inflate transduction baselines and mask partial neutralization. To address this challenge, we developed the constant serum concentration (CSC) assay, which maintains constant serum levels across dilutions to stabilize assay baselines and enhance NAb detection sensitivity. Using human sera across multiple AAV serotypes, we demonstrated that CSC reclassified up to 21.7% of samples classified as non-neutralizing by a conventional variable serum concentration (VSC) assay format. This improved sensitivity was validated using monoclonal antibody and multi-species serum test benchmarks and enhanced the reliability of seronegative control pool selection. Importantly, CSC detected persistent seropositivity in preclinical seroreversion models up to one year longer than a conventional VSC assay. Since even low-level neutralizing antibodies can significantly impact gene therapy efficacy, these findings have direct implications for optimizing AAV redosing strategies and refining patient stratification. By addressing fundamental limitations in NAb quantification while maintaining procedural simplicity, the CSC assay provides crucial insights into antibody persistence with translational relevance across species and clinical settings.