CRISPR Mediated Precision Nanomedicine for Patient Derived Malignant Glioma Stem Cells

Glioma stem cells (GSCs, cancer stem cells in high grade glioma) are recently recognized as the driving force behind the malignancy, invasiveness, chemo/radio therapy resistance, and aggressive recurrence in one of the most lethal brain tumors known to man, glioblastoma multiforme (GBM). Large scale and comprehensive genome (complete gene sets) analyses based on patient GBM clinical tissue samples, such as The Cancer Genome Atlas (TCGA) project, have identified genetic traits that further separate clinically encountered GBMs into distinct subclasses (Classical, Neural, Proneural, and Mesenchymal) that correlate with individual patient response to therapy, offering hope that Precision Medicine tailored for individual tumor profiles is on the horizon to offer improved therapeutic benefits. Genetic stratification of GBM patients reveals gene signatures underlying their respective unique GSC subtypes, such as the recently identified four master transcription factors (TFs) that control the GSC fate by my collaborators at Harvard Medical School, namely Sox2, Olig2, Sall2, and Pou3F2, as the core GSC drivers of the Proneural subclass of GBM. [Scope & Significance]: However, patient genome-specific Precision Medicine remained a talking point, until the recent development of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing technologies (technologies that insert, delete, or change gene sequences using flexible gene targeting strategies), spearheaded by collaborators at my host institution, MIT. The CRISPR technology utilizes engineered microbial Cas9 endonucleases and single-guide RNAs (sgRNAs), oligo nucleotides that recruit Cas9 to eliminate or activate the target gene. It is a highly innovative engineering feat tailoring naturally occurring microbial immune systems for mammalian applications, and allow for efficient and precise targeting of almost any gene in the mammalian genomes for editing. This project aims to use GSC cells in a patient derived xenograft mouse model to validate the therapeutic potential of CRISPR mediated Precision Medicine against the malignant GSCs with patient-specific genetic signatures. [Hypothesis]: By leveraging a novel non-viral nanoparticle system developed by my host, Dr. Robert Langer at MIT/Koch Institute for Integrative Cancer Research for efficient in vivo co-delivery of Cas9 mRNA and sgRNAs targeting GSCs, we can build on our expertise and leadership in Neuro-Oncology translational research to achieve efficacious elimination of the GSC populations in a patient specific manner, and concoct personalized Precision Nanomedicine based on CRISPR principles established at MIT to help extend lives. [Specific Aims]: Aim 1, Collaborative visits to Dr. Langer's Institute at MIT allow for optimization of non-viral lipopolymeric nanoparticle mediated nanomedicine, custom made to use CRISPR technology to eliminate GSC signature genes in order to inhibit the malignant growth of GSCs. Aim 2, Visits to MIT allow for up-to-date optimization of the in vivo delivery strategies of this unique nanomedicine, in synchrony with breakthroughs made in the CRISPR technologies at MIT. Aim 3, broadened collaboration with leading scientists at Harvard/MIT/Broad Institute will enhance the translational values of my integrative research approach. [Career Development Potential]: This is the blueprint for a first in class direct, precise, and multiplex (simultaneous targeting of multi-genes), non-viral gene therapy against patient-specific GSCs in the brain, which can be translated to clinical s