Ligand-directed therapeutic CRISPR/Cas gene editing in tumor cells

ABSTRACT The human body is composed of at least 400 different cell types, each distinguishable by distinct cellular markers. Cancer cells also express distinct cellular markers, differentiating them from their precursor cell-type prior to malignant transformation. Accordingly, the broad goal of oncology is targeted, tumor-specific therapies designed to maximize therapeutic efficacy while avoiding undesired off-target effects. This is recognized as a major obstacle for the application of current gene editing technologies. Proposed here is a strategy for tumor-specific gene editing involving combined use of the chimeric adeno-associated virus / phage (AAVP) vector together with the clustered regularly-interspaced short palindromic repeat (CRISPR) / CRISPR-associated protein (Cas) gene editing system – collectively referred to as AAVP-CRISPR. The tissue-specific targeting aspect is carried out by the phage capsid of AAVP, which can be modified to display a targeting peptide for ligand-directed delivery of a desired transgene. AAVP, first introduced in 2006, is a well-characterized gene delivery tool that has been explored in numerous tumor models. Here we plan to introduce two separate CRISPR/Cas systems with previously demonstrated gene editing efficacy into the AAVP vector. The first construct will contain the conventional CRISPR/Cas9 system, while the second construct will contain the more recently discovered hypercompact CRISPR/CasΦ system, referred to as AAVP-CRISPR/Cas9 and AAVP-CRISPR/CasΦ, respectively. The two constructs will be engineered to display RGD4C, a well-known, clinically viable tumor- targeting peptide, and carry various gene editing guide RNA (gRNA) combinations. Gene editing using the two constructs will first be performed in an in vitro setting, to confirm activity of the elements of each CRISPR/Cas system, including receptor binding, cell internalization, Cas gene expression, and gene editing activity. Several gRNA configurations will be tested in vitro and AAVP-CRISPR constructs containing the leading gRNA candidates will be evaluated for tumor targeting, Cas expression, and gene editing activity in vivo. Successful completion of the proposed studies will provide insight into the efficacy of AAVP-CRISPR for tumor-specific gene editing and contribute to the development of much needed novel therapeutic strategies for cancer.