Epigenetically optimized replication-defective Herpes Simplex Virus vectors for multigenic gene therapy in Alzheimer’s disease.

. Alzheimer's disease (AD) is the most common neurodegenerative disorder, significantly impacting older adults and placing a substantial economic burden on healthcare systems. Despite extensive research, including numerous clinical trials and drug development efforts, current treatments for AD have shown limited success. Recently approved monoclonal antibodies, while offering disease-modifying potential by clearing Aβ plaques, still face challenges like amyloid-related imaging abnormalities (ARIA) and limited effectiveness due to the complex and multifactorial nature of AD. AD arises from a combination of genetic, environmental, and lifestyle factors, involving multiple pathological processes such as Aβ plaques, tau tangles, neuroinflammation, and neurodegeneration. The biochemical mechanisms of AD are deeply interconnected and dynamic, evolving as the condition progresses, making it difficult for monotherapies to effectively address the disease. Recognizing these complexities, combination therapies are increasingly valued for their comprehensive approach. Gene therapy platforms that target multiple pathways, tackling the diverse factors driving AD, are emerging as promising strategies. Here we propose to develop and refine novel replication-defective (rd) Herpes simplex virus (HSV) vector as gene therapy platform aimed at treating AD. The rdHSV platform is a promising neurotrophic vector with the ability to deliver multiple therapeutic genes, due to its large payload capacity (~35 kb) and ensuring long-term transgene expression through viral and cellular insulators that prevent host silencing. Safety was achieved by removing all immediate early (IE) genes, including those encoding infected cell proteins (ICP) 4, ICP27, and ICP0. These high-capacity vectors, exclusively generated by our laboratory, are supported by preliminary data showing durable (up to 1 year), non-toxic multi-gene expression in the brain positioning it as a potential strategy to address the multifactorial nature of AD. In Aim 1, we will explore the epigenetic mechanisms underlying the interaction between viral and cellular insulators, focusing on how they affect chromatin structure, DNA methylation, and histone modifications. This is crucial for ensuring stable transgene expression in neurons, as controlling the epigenetic environment is key to the long-term success of gene therapy. Enhancing the epigenetic compatibility of the transgene cassette in neurons could also be adapted for use in other CNS cell types in the future. In Aim 2, we will test the therapeutic efficacy of the rdHSV vectors in an in vivo AD model (3×Tg-AD mice), testing the ability of vectors expressing genes targeting Aβ clearance (NEP) and tau degradation (TRIM11). Both genes are downregulated in AD. We will test weather their stable expression will reduce pathology, improve cognitive function, and alleviate neuroinflammation in both preventing and therapeutic settings. We will test the vectors carrying these therapeutic genes, both individually and in combination. By addressing the complex causes of AD, this approach seeks to provide a more effective treatment than the current monogenic therapies that have proven ineffective. Project Number: 1R21AG098360-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Selene Ingusci | Institution: UNIVERSITY OF PITTSBURGH AT PITTSBURGH, PITTSBURGH, PA | Award Amount: $433,710 | Activity Code: R21 | Study Section: Chronic Dysfunction and Integrative Neurodegeneration Study Section[CDIN] View on NIH RePORTER: https://reporter.nih.gov/project-details/11285560