POLY (BETA-AMINO ESTER) MEDIATED GENE DELIVERY TO HUMAN ADIPOSE DERIVED MESENCHYMAL STEM CELLS (hAMSCs) & ROUTES OF hAMSCs ADMINISTRATION FOR BRAIN TUMOR TREATMENT

Glioblastoma (GB) is a severe type of brain tumor with 26,000 adult, 5,000 pediatric cases diagnosed every year. In addition to primary tumors, ~170,000 new metastatic cancers to brain are diagnosed annually1,2. The median patient survival remains a mere 14.6 months despite an expensive treatment combining surgical resection, chemotherapy and radiation. There is an immediate need to develop alternative therapies for GB that can be easily administered at an affordable cost. Stem cells have provided an attractive platform to develop therapies in this regard due to their inherent tropism towards inflammation or site of injury and their proliferation capacity. Having the ability to non-virally modify stem cells to deliver therapeutic gene(s) of interest will change the way treatment is strategized and administered for GB. The first step towards developing this treatment involves efficiently engineering patient-derived human adipose derived mesenchymal stem cells (hAMSCs) to express gene(s) of interest and characterizing the modified hAMSCs. This thesis involves identifying an optimal poly-beta-amino-ester (PBAE) polymer formulation to transfect patient-derived hAMSCs and characterizing the migration, proliferation and stemness of hAMSCs modified to express single or multiple genes of interest. The thesis consists of two parts. The first half comprises of identification of a polymer formulation for optimal transfection of patient-derived hAMSCs and characterization of nanoparticle modified hAMSCs. We were able to screen for optimal gene expression using a library of PBAE polymers previously created in our laboratory. We showed that primary patient-derived hAMSCs can be transfected using PBAE nanoparticles and the gene expression can be seen even five days after transfection. Though there is not just one single polymer formulation to obtain optimal expression, a combination of polymer structure, polymer to DNA weights ratio and DNA dosage resulted in transfection efficacy of up to 60%. We also observed variability in transfection efficacy among hAMSCs obtained from individual patients, suggesting that every hAMSC culture does not result in similar gene expression for a given formulation. However, we observed a transfection efficacy of at least 30-40% for any given patient-derived hAMSC culture, with some cultures having transfection efficacy as high as 60%. Multiple cultures of primary hAMSCs obtained from patients have been transfected to express individual gene or a combination of genes. We observed that hAMSCs modified to express receptor protein GFP before and after transfection maintained their migration ability, proliferation capacity and stemness. In addition, hAMSCs expressing two genes maintained their stemness as well. These results suggest that we have the ability to transfect multiple plasmids within a single PBAE nanoparticle resulting in an effective and efficient multiple gene delivery, without significant cytotoxicity. The latter half of the thesis evaluates the routes of administration of hAMSCs in a mouse model of glioblastoma in vivo. Virally transduced bioluminescent hAMSCs have been administered to mice through intracardiac, nasal, intrathecal or intravenous delivery. The distribution of hAMSCs was studied over a period of 5 days. The total flux of the fluorescence signal for each mouse (for days 0-4) was qualitatively compared for different routes. We observed the most diffused distribution of hAMSCs in for intracardiac administration. These findings indicate the PBAE polymers can be used to effectively transfect primary patient-derived hAMSCs to express gene(s) of interest. Further, changes to polymer structure, polymer to DNA weights ratio and DNA dosage influence the transfection efficacy. Moreover, hAMSCs can be used as delivery vehicles in a brain tumor model, with different routes of administration resulting in varied distribution of the injected cells. In addition, patient-derived hAMSCs non-virally engineered to express gene(s) of interest maintain their migration ability, proliferative capacity and stemness, thus making hAMSCs an attractive gene delivery vehicle for brain tumor patients