Dipg-32. preclinical testing of the efficacy and safety of crispr/cas9 gene edited car-t cell therapy for diffuse midline glioma (dmg)

Abstract DMGs are highly aggressive and difficult-to-treat brain tumors in children. Recent advances in Chimeric Antigen Receptor (CAR) expressing T cell therapy suggest application for brain tumors. Recent studies also demonstrate feasibility of GD2, Her2 and B7H3 CAR-T infusions in childhood brain tumors. We identified and developed a novel CAR-T therapy, targeting the CD99 antigen, which is homogenously expressed at high levels on DMG tumor cells. These CAR-T cells showed initial clearance of tumor, but the long-term persistence and anti-tumor efficacy of these CAR-T cells in DIPG xenografts were limited due to fratricide, as T cells also express CD99. To overcome this obstacle, we have further optimized a protocol in which we first knock-out CD99 from the human donor T cells using CRISPR-cas9 gene-editing and subsequently transducing with our CD99 virus in the pure population of CD99KO T cells. Systemic delivery of these gene-edited CAR-T cells showed enhanced in vivo persistence with complete clearance of tumor in DMG mouse models, and no tumor recurrence was seen well-beyond the time frame of expected tumor recurrence after treatment with un-edited CD99 CAR-T cells. In addition, loco-regional delivery of these gene-edited CD99 CAR-T cells when administered directly to the 4th ventricle, even at a low dose, showed similar anti-tumor efficacy with significantly increased animal survival. These data suggest a regional approach analogous to Omaya reservoir administered drug in humans may be a promising alternative approach to limit any toxicity.