Abstract Glioblastoma multiforme (GBM) is an aggressive primary brain tumor with a median survival of approximately 15 months. Currently, the standard of care therapy includes resection followed by chemotherapy and/or radiotherapy. These treatments are limited by incomplete tumor resection and poor therapeutic delivery due to the blood-tumor and blood-brain barriers. Gene therapy has emerged as a potential therapeutic strategy for the treatment of GBM. In fact, clinical trials have explored the delivery of oncolytic viruses, suicide genes, and the p53 gene, however these therapies are also largely challenged by poor delivery. Nanoparticles (NPs) are typically employed with gene therapies to protect nucleic acids from degradation and improve tissue accumulation and penetration. However, NP gene delivery platforms often rely on passive accumulation in the tumor facilitated by leaky vasculature (i.e., enhanced permeability and retention (EPR) effect) which is intra- and intertumorally heterogeneous and only allows for a small fraction of the NPs to accumulate in the tumor. These proposed studies aim to improve tumoral therapeutic accumulation by developing novel non-viral gene delivery platforms for GBM. Brain penetrating NPs (BPNs) are a class of densely PEGylated polyethyleneimine (PEI) NPs that have demonstrated enhanced tumor targeting as compared to conventionally PEGylated PEI NPs. In our studies, we have modified BPNs (SH-BPNs) to target exofacial thiols overexpressed by tumor cells due to the dysregulated protein synthesis in the tumor microenvironment. First, we will investigate the tumor-tropism and transfection characteristics of SH-BPNs compared to BPNs via two different administration routes: intratumoral injection and systemic delivery. Next, we will further optimize gene delivery by using focused ultrasound (FUS) treatment regimens to enhance therapeutic delivery. To address the challenges of the EPR effect, researchers have employed FUS to enhance the delivery of various therapeutic agents, including NPs, across various biological barriers. We will investigate SH-BPN delivery at various time points relative to FUS treatments. While most FUS-mediated delivery of NPs involves the intravenous administration of microbubbles (MBs) followed by NPs, studies have shown that the coupling of the two to create a NP-MB construct improves transfection efficacy. We will also explore the effects of SH-BPN conjugation to MBs (SH-BPN-MBs). Overall, this study contributes to the development of targeted gene therapy strategies for GBM by addressing delivery challenges and enhancing the therapeutic potential of nucleic acid-based treatments.