SARS-CoV-2 spreads across the globe, infecting more than 128 million people and claiming over 2.7 million lives with an absence of definitive treatment up to date. Therefore, there is an immediate need to develop treatments fighting against the COVID-19 global pandemic. The goal of my project is to generate and assess an innovative treatment for the SARS-COV-2 virus infection. Our treatment formulates computationally designed proteins, and we want to evaluate its therapeutic effects using human induced pluripotent stem cell (h-iPSC) derived cell lines and organoids. The designed protein is a combinatorial cage (mosaic cage) containing spike binders previously shown to significantly inhibit SARS-CoV-2 viral infection and F-domains that were shown to activate the Tie2 pathway. The Tie2 pathway is a key regulator of vascular stability, where active Tie2 can strengthen cell-cell junctions and enhances endothelial cell survival, thus enhancing blood vessel stability. We hypothesize that the designed protein would neutralize the spike protein to block viral entry and activate the Tie2 pathway to alleviate sepsis in COVID-19 infected patients. We will test spike-binding activity and determine the activation level of the Tie2 pathway of this mosaic cage in iPSC-derived spike-overexpressing endothelial cells. We expect to measure a strong spike-binding affinity of designed proteins and strong downstream pathway signals pAKT, pERK, pFAK in designed protein-treated iPSC-derived endothelial cells. We also plan to test the mosaic cage’s activities using Kidney Organoids. If our hypothesis is correct, we will apply the findings clinically for their potential intranasal administration as a COVID-19 therapeutic.

Over the past fifty years, survival rates for most cancers have risen as innovative treatments have been developed. However, Diffuse Intrinsic Pontine Glioma (DIPG), a rare pediatric brainstem tumor, has seen no such improvement and remains one of the deadliest cancers. DIPGs are genetically distinguished from adult gliomas by a lysine-to-methionine mutation in a variant of histone H3 called H3.3 (H3.3K27M), found in 80% of DIPG tumors. This mutation is absent in canonical H3.1 and H3.2, yet it triggers a global reduction in the levels of polycomb repressive complex 2 (PRC2) mediated H3K27me3 (tri-methylation) marks, which is traditionally considered a driving event in tumorigenesis. However, genome-wide studies of H3K27me3 marks in DIPG cells have revealed that this global loss of H3.3K27me3 is accompanied by a sharp increase in H3K27me3 repressive marks at certain genes. These trimethylation spikes represent regions with high transcriptional repression and may be key in understanding and treating DIPG. Our lab has previously described the inhibition of PRC2 using a computationally designed protein EBdCas9. Using a complementary guide-RNA tiled to the promoter region of a gene, I can target EBdCas9 to that specific region on the genome, remove existing H3K27me3 marks, and increase gene transcription. Identification of candidate genes which are repressed in DIPG cells under these H3K27me3 spikes remains an ongoing project that I am working on. Results of one target, tumor-suppressor gene p16, demonstrate that I can transfect primary DIPG cells with EBdCas9 plasmid and a p16-specific gRNA to trigger a 20-fold increase in p16 expression. I hypothesize that restoration of critical cell-cycle genes like p16 will reduce DIPG viability and offer potential therapeutic targets. I’m currently working to improve transfection efficiency in order to increase p16 expression, and future steps include in-vivo testing of this system, either via mouse model or a brainstem organoid.