Multipronged Strategies to Achieve Safe and Efficient Gene Transfer to the Brain

Gene therapy has recently emerged as a powerful treatment alternative for patients suffering from brain tumors and neurodegenerative diseases. Despite the recent advances in understanding underlying mechanisms for disease progression, therapies have not been effective in the clinic due the inability to deliver therapeutics in an effective manner. Non-viral gene vectors that can overcome steric and adhesive barriers in the brain have recently emerged as a promising avenue for treating brain disorders. In this dissertation, I engineered and evaluated the use of polymeric gene vectors that provides safe and efficient transgene expression for the treatment of glioblastoma and neurodegenerative diseases. This thesis work focuses on manipulating the two main components of non-viral gene vectors, the plasmid DNA and the polymer to achieve specific and efficient gene transfer in the brain. The first goal is to achieve widespread distribution with non-viral gene vectors, while allowing for preferential transgene expression in a cell-specific manner. Taking advantage of the upregulation of specific proteins in tumor cells, I selected a promoter that drives expression of these tumor-associated proteins and incorporated that into DNA plasmid to drive expression of a reporter protein. Gene vectors containing these plasmids achieve widespread distribution yet cancer-specific transgene expression. The second goal is to create a polymer delivery system that preferentially degrades in target environments to release the nucleic acid payload. With the addition of a polyethylene glycol (PEG) coating, these gene vectors can penetrate the extracellular matrix of the brain and degrade within the cell cytoplasm to enhance gene transfection efficiency. These engineered gene vectors can achieve effective treatment of diseased cells within the brain by ensuring greater efficiency of transgene expression. The first aim is to tackle the challenge of ensuring widespread distribution of nanoparticles but limiting transgene expression to only cancer cells. Certain proteins have shown an elevated expression profile in brain tumor cells. These associated promoters can be extracted and incorporated into the plasmid DNA constructs. When these promoters are packaged into plasmid constructs and formulated into densely pegylated DNA nanoparticles (DNA-BPN), they can distribute widely through the brain parenchyma. More interestingly, with the use of tumor specific promoters controlling gene expression in the plasmid, they were able to facilitate significantly lower transgene expression in healthy mouse brain compared to cytomegalovirus (CMV), a ubiquitous, high intensity promoter that is commonly used in clinical applications. Finally, these DNA BPN carrying plasmids controlled by tumor specific promoter driven transgene expression were confined to the tumor boundary in an orthotopic brain tumor model. In contrast, transgene expression driven by CMV expanded far past the tumor boundary. The ability to provide widespread distribution yet tumor specific transgene expression using a combination of DNA BPN and tumor specific promoters has a myriad of applications, specifically in infiltrative brain tumors such as glioblastoma. The second aim is to engineer a bio-reducible polymer to enhance gene transfer over commonly used polymer systems for non-viral gene therapy. For example, polyethylenimine (PEI) is considered a gold-standard polymer for its ability to efficiently compact DNA and has been explored in a myriad of clinical trials. However, toxicity concerns limit their effective translation possibly due to its non-biodegradable nature and high density of positive charges. An environmentally sensitive PEI polymer (SSPEI) that will preferentially degrade inside the cell to release the DNA cargo can overcome these limitations. When transfection efficiency was evaluated in vitro, these SSPEI nanoparticles mediated significantly higher transgene expression compared to PEI nanoparticles. In addition, when the surface of these SSPEI nanoparticles is coated with PEG (SSPEI-BPN), the nanoparticles provided widespread distribution into the brain parenchyma and were able to mediate transgene expression at a higher efficiency compared to other conventional systems. Furthermore, these nanoparticles facilitate observably higher transgene expression in the hippocampus compared to clinically used, adeno-associated virus serotype 2 for treating Alzheimer’s disease. Additionally, they efficiently transfect neurons in the substantia nigra pars compacta, the site of neurodegeneration in patients with Parkinson’s disease. And finally, unlike biodegradable nanoparticle systems, the SSPEI BPN retain physiochemical characteristics and transfection efficiency even 2 weeks after shelf-storage at room temperature indicating significant clinical translation potential