Establishing Methods To Improve CAR T-cell Therapy and Genome-scale CRISPR-Cas9 Knockouts
The Banovich lab uses cutting-edge genomic technologies to better understand treatment outcomes in cancer. Use of these technologies requires extensive optimization. Here I will discuss how we are working to optimize two such technologies: (1) Single-cell RNA-seq (scRNA-seq), a technique used to distinguish the expression of different genes in a group of cells, applied to patients undergoing chimeric antigen receptor T-cell therapy (CAR-T cell therapy); (2) Genome-scale CRISPR-Cas9 knockout (GeCKO), a technique used to inhibit the function of every gene in the genome, applied to in vitro Multiple Myeloma (MM) samples.
Glioblastoma Multiforme (GBM) is a type of brain cancer that forms from glial cells of the brain and spinal cord, which affects an individual’s central nervous system. In order to treat patients with GBM, CAR T-cell therapy can be used to eliminate the progressive brain tumors. In CAR T-cell therapy, T-cells, a type of white blood cell, are extracted from a patient’s blood and altered in the lab to produce a receptor. When these altered T-cells are re-infused into the patient through injection into the spinal fluid, these receptors can bind to the antigens of the cancer cells, enabling the patient’s cancer cells to be targeted and destroyed by these altered T-cells. We hope to use scRNA-seq to better understand the immune responses surrounding CAR-T cell therapy. However, samples from patients oftentimes contain very few cells. Thus, a minimum concentration of cells needs to be found to determine exactly how many cells are required for scRNA-seq. To accomplish this, we utilized an immortalized T-cell system amenable to in vitro culture, Jurkat cells. These cells were grown, frozen at varying cell count (thousands) and volume (µL) pairings, centrifuged and counted to determine cell viability. The results from the counting provided information on the degree of recovery of the Jurkat cells after cryostorage.
Multiple Myeloma (MM) is a type of bone marrow cancer which originates from plasma cells, a type of white blood cell. To identify novel genomic vulnerabilities in MM, we are hoping to perform in vitro screens of MM cells by knocking out every gene in the genome. This will be accomplished using a genome-scale CRISPR-Cas9 knockout. A lentivirus “delivery system” was constructed to deliver a plasmid library (Library A), which produces guide RNAs, to the genes in MM cells. The guide RNAs, by connecting with the CRISPR-Cas9 knockout, will silence the genes in the MM cells by creating indels and thus disrupting the coding sequencing of genes in the MM cells. The results will help optimize the packaging, selection, and delivery of the GeCKO lentiviral library.