AAV-delivered meganucleases for durable control of genital HSV disease

The National Institute of Allergy and Infectious Diseases awarded Fred Hutchinson Cancer Center $465,116 on April 13, 2026, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to develop and evaluate AAV-delivered meganucleases as a therapeutic approach for durable control of genital herpes simplex virus infection.

The R21 phase funds research extending prior work targeting HSV-1 to HSV-2, and determining whether intravenous or intrathecal administration is the optimal delivery route for AAV encoding anti-HSV meganucleases. Previous mouse studies demonstrated that IV-delivered AAV/meganuclease therapy eliminated up to 97% of latent HSV DNA from dorsal root ganglia and reduced viral shedding. R21 specific aims include evaluating intrathecal versus intravenous delivery to reduce AAV dose, minimize systemic exposure, and avoid pre-existing or induced anti-AAV immunity; assessing AAV biodistribution, antiviral efficacy, and dose response; and determining whether intrathecal administration can avoid neutralizing antibodies present in serum and allow re-dosing of AAV. A planned R33 phase will address safety, efficacy, and clinical translation issues.

Performance occurs in Seattle, Washington from April 13, 2026 through March 31, 2028. University of Washington, acting through its Office of Sponsored Programs, is a sub-recipient performing viral genome analysis and quantification. UW Virology will conduct digital PCR, quantitative PCR, and next-generation sequencing assays to measure gene editing of viral genomes and quantify virus shedding in mouse infection models, including HSV DNA viral load testing on tissue samples and ocular and vaginal swabs, and NGS sequence analysis for on-target and genomic off-target gene editing quantitation. The sub-award was obligated on July 7, 2026.

Abstract

Herpes simplex virus (HSV) establishes latency in ganglionic neurons of the peripheral nervous system. Latent HSV can later reactivate, causing recurrent disease and possible transmission to new hosts. Current anti-HSV therapy is inadequate, in that IT does not eliminate latent HSV, and thus is only suppressive rather than curative. We developed a therapeutic approach based on gene editing using HSV-specific meganucleases. We showed that intravenous (IV) administration of adeno-associated virus (AAV) encoding anti-HSV-1 meganucleases can eliminate up to 97% of latent HSV DNA from dorsal root ganglia in mouse models of latent HSV-1 genital infection. We also demonstrated that this reduction in ganglionic viral load led to a corresponding reduction of viral shedding from treated VS. Control mice. This approach offers the potential for a durable means of controlling latent HSV infection and subsequent reactivations, or even achieving a functional cure. In the R21 phase, we propose to extend our work to target HSV-2, and to determine whether IV or localized intrathecal (IT) administration is the optimal route. In the specific aims of the R33 phase we plan to address simultaneously several outstanding issues regarding both HSV biology and the safety and efficacy of in vivo gene therapy that are critical for clinical translation of our work. R21 phase: specific aim 1. Evaluate IT VS. IV delivery of AAV/meganuclease therapy as a means to reduce AAV dose, minimize systemic exposure, and avoid pre-existing/induced anti-AAV immunity. We will evaluate the AAV biodistribution, antiviral efficacy, and dose response after IT VS. IV administration, and the doses at which toxicity occurs. We will also evaluate the ability of IT-delivered AAV to avoid neutralizing antibody present in serum, and whether IT administration will allow RE-dosing of AAV. R33 phase: specific aim 2. Compare the natural history of ganglionic HSV load and peripheral viral shedding in latently infected mice after IT or IV meganuclease therapy VS. Untreated control animals. We will perform long-term studies of the ganglionic viral load and the frequency and quantity of peripheral shedding in control VS. AAV-meganuclease treated animals. These studies will shed light on the stability of reservoirs, the relationship of ganglionic viral load reductions with the frequency and quantity of viral shedding, and provide new insights into possible HSV RE-seeding of ganglionic reservoirs after viral reactivations. R33 phase: specific aim 3. Determine the host genomic consequences of AAV/meganuclease therapy, and compare to the effects after crispr/CAS9 exposure. We will perform an unbiased evaluation of genomic disruptions after our meganuclease therapy, and directly compare with crispr/CAS9 approaches, using the state-of-the-art techniques for the detection of off-target events, including guide-SEQ, improved discover-SEQ+, and analyses for the insertion of AAV vector (ITR-SEQ) and HSV DNA (hybridization capture coupled with next generation sequencing) into the host genome.