CRISPR-based in vivo attenuation of HSV to improve immune control of genital herpes infection

About This Grant

SUMMARY Genital herpes simplex virus (HSV-1 and HSV-2) infections are painful and can result in transmission to sexual partners and newborns; the latter can be highly susceptible to severe disease with detrimental outcomes. While the host immune response mostly ensures that HSV remains in a latent state in dorsal root ganglions (DRG), fluctuations in immunity can allow the virus to escape effective immune surveillance. This can lead to infectious viral shedding from mucosal or skin sites where lesions may also develop. Strategies that fortify the natural immune response has the potential to limit frequency, degree and duration of HSV reactivation. This project will utilize CRISPR-Cas9 technology to attenuate HSV, while maintaining immune stimulation. The herpes protein pUL56 is encoded by late HSV-1/HSV-2 gene UL56. We and others have shown that pUL56 targets host cell protein degradation pathways, antagonizes innate immune sensing, and affects virulence in vivo. Recently, we demonstrated that a UL56 deletion HSV-1 mutant fails to cause secondary skin lesions and death in a mouse model. Interestingly, it still induces long-lasting protective immunity against infection with the wildtype virus. Importantly, as shown in new preliminary data, the deletion mutant is also attenuated in a vaginal infection mouse model. Thus, UL56 may represent a promising new therapeutic target. This MPI application leverages Dr. Jensen’s experience working with HSV infection models and the UL56 deletion HSV mutant virus and Dr. Kaminski's hands-on competence in developing and validating CRISPR-Cas9- based antiviral therapeutic to test the feasibility of CRISPR-based targeting of the UL56 gene. Specifically, we hypothesize that genetic ablation of UL56 expression will create an attenuated virus that will cause less viral shedding and milder disease. In the R21 proof-of-concept phase, we will develop tools for CRISPR-mediated disabling of the HSV-1/HSV- 2 UL56 gene. These will be tested in a vaginal infection mouse model. In the R33 further-validation phase, we will utilize the clinically relevant guinea pig model of genital herpes infection. We will further develop and test the efficacy of HSV-vector-based delivery of CRISPR gRNA targeting the UL56 gene. The effects on viral reservoir, shedding, and disease severity and frequency will be evaluated. The overall goal of these studies is to determine if CRISPR technology can be used to attenuate HSV in vivo and thereby prevent or reduce recurrent disease and viral shedding. This study will provide a basis for future development of potential therapeutical approaches focused on targeting UL56 for genital herpes infections.