Project summary Glioblastoma (GBM) is the most frequent brain tumor with a very poor prognosis. Currently, despite surgery, chemotherapy, and radiotherapy as the standard-of-care treatments, combination of these regimens does not prolong patient survival significantly. Oncolytic viral therapy opens a new era for solid tumor therapy, especially for brain tumors. HSV-1 derived oncolytic viruses (oHSVs) are the most advanced type of oncolytic virus (OV) and the only type of virus that is currently FDA approved in the USA. Very recently, another oHSV (Delytact, marketed by Daiichi) was granted conditional approval for the treatment of GBM patients in Japan. Thus, investigating ways to improve oHSV therapy against GBM is highly significant. However, the anti-tumor efficacy of oncolytic virus (OV) is limited by tumor microenvironment (TME). CD44 signaling regulates GBM TME through interaction with extracellular matrix (ECM) or immune cells. Hyaluronic acid is one of the key extracellular matrix components derived from glucose metabolites in GBM. CD44 is highly expressed in GBM cells and is a marker for glioblastoma stem cells (GSCs). CD44 also bind to other ligands, such as, osteopontin (OPN), chrondroitin and proteoglycan to regulate ECM and immune cell function. Our ongoing experiments show that: 1) CD44 expression in GBM is correlated with poor prognosis of patients, 2) Oncolytic herpes simplex virus type 1 (oHSV) intratumoral injection increases HA secretion in brain tumor xenografts in vivo, 3) oHSV-CD44 significantly downregulates SOX2 and decreases stemness of GSCs, 4) oHSV-CD44 significantly increases anti-tumor efficacy in vivo, and 5) Combination oHSV-CD44 with an AKT inhibitor or irradiation could further decrease SOX2 and Nestin expression in GSCs and reduce GSC stemness. Given these findings, we hypothesize that blocking CD44 signaling in tumor microenvironment (TME) using a CD44 extracellular domain-secreting oncolytic virus (oHSV-CD44) will not only disable CD44-mediated GBM growth and lead to immunosuppression but will also inhibit GSC stemness while sensitizing the cells to chemotherapy and radiotherapy. The hypothesis will be addressed with the following experiments proposed in the Specific Aims: 1) Investigate whether combination oHSV-CD44 with an AKT inhibitor reduces GSC stemness and increases the anti-GBM immune response, and 2) Test whether oHSV-CD44 increases radiotherapy- mediated anti-GBM efficacy. The proposed study is significant and has a broad impact on several research areas. In addition to shedding light on the fundamental understanding of HA-CD44 signaling in GSCs stemness, GBM invasiveness, and standard-of-care treatment sensitivity, this study will uncover the role of CD44 signaling blockade by oHSV-CD44 in enhanced efficacy of GBM treatment, including chemotherapy or radiotherapy. Furthermore, combination of oHSV-CD44 with an AKT inhibition or radiotherapy will provide novel treatment strategies for tumor relapse.