The Cancer Genome as a Novel Target for Malignant Osteosarcoma Immunotherapy

Osteosarcoma (OS) is a rare but devastating form of bone cancer that often affects pediatric populations. Patient survival is highly dependent on the stage of OS diagnosis. If identified early, when cancer remains localized, the five-year survival is 74%; however, this drops to just 27% when diagnosed with metastatic disease. Traditionally, targeted therapies attempt to preferentially kill cancer cells by exploiting overexpressed cell surface proteins, differences in metabolic activity, or changes in the tumor microenvironment; however, these features are not unique to cancer cells, resulting in damage to off-target tissues, and are not uniformly present in all patients. We propose the use of clustered regularly interspaced short palindromic repeats (CRISPR) to kill OS cells with unprecedented specificity based on the presence of patient-specific mutations. After identifying the primary mutation(s) driving a patient’s disease, we will formulate guide RNA and Cas9 to specifically cut OS cell DNA at the site of the mutation. By co-delivering these components with exogenous donor DNA encoding diphtheria toxin subunit A (DT-A), a cytotoxic protein, this strategy will kill OS cells while leaving normal cells unedited and unharmed. Further, because DT-A induces immunogenic cell death, it will both directly kill cancer cells and recruit immune cells to the site of OS cell death where they will detect neoantigens and promote a systemic anti-cancer response that eradicates tumors from the body. To demonstrate the efficacy of this approach, we will use the Campylobacter jejuni Cas9 variant to target the R172H hot spot mutation in Trp3, a gene encoding a key tumor suppressor gene whose homologous human gene, TP53, is mutated in 90% of OS cases. We will first use CRISPR to introduce the R172H mutation into an existing murine OS cell line. We will then create a custom genome editing formulation that specifically target cells harboring the R172H mutation while being inert in cells that contain wild-type Trp53. After selectively inserting DT-A, we will evaluate the specificity and efficiency of our genome-targeted therapy. We will then show the therapeutic efficacy of genome-targeted immunotherapy using a syngeneic mouse model of OS. By both directly killing OS cells and engaging the immune system to destroy OS cells systemically—including metastases—this approach will eradicate tumors and prolong survival. If successful, this approach would represent a dramatic shift in OS treatment paradigms, dramatically improve metastatic OS survival and reduce or eliminate side effects. Ultimately, we envision a clinical pipeline for target personalization in which biopsies are collected, sequenced, and used to develop formulations customized to that individual’s mutation(s). This process would be fast, inexpensive, and require minimal modification to current clinical practices, thereby minimizing barriers to adoption.