Synthetic Cell State Engineering for Primary Human Cellular Therapies

PROJECT SUMMARY/ABSTRACT The human genome encodes the unique functional properties of hundreds of different cell types and states across human development, in health and disease. Complex germline genetic changes over evolutionary time have given rise to new cell types, and more rapidly similarly complex somatic genetic changes underly the cell state transformation of cancers. I propose the scientific challenge of whether new therapeutically useful human cell states can similarly be created through complex synthetic genetic changes of living primary human cells. To reach the complexity of genetic perturbation required to access potential new synthetic human cell states, I propose the development and application of two key technical innovations, 1) CRISPR-All, an arbitrarily combinatorial genetic perturbation platform across perturbation type and scale in primary human cells, and 2) the combinatorial construction of new, “non-evolved” human genes from existing functional human protein domains, initially focusing on building new human transcription factors. I will apply these systems in primary human T cells due to their abundance and thus suitability for large scale screening efforts for the discovery of synthetic cell states, and the immediate therapeutic relevance of human T cell genetic manipulations for cancer immunotherapy. Using a tiered screening approach, I will first use CRISPR-All to discover complex combinations of genetic perturbations and non-evolved, synthetic transcription factors that functionally enhance human T cell function. Then, using CRISPR-All’s ability to pair arbitrarily complex genetic perturbations with single cell RNA and ATAC sequencing, I will perform small scale pooled screens with single cell readouts of 100s of functional candidate complex perturbations and synthetic transcription factors to determine the degree of variance in each construct’s resulting cell states compared to cell atlases of natural human T cell states. Finally, top candidates with divergent transcriptomes and chromatin profiles will be characterized through detailed genomic, mechanistic, computational, and functional analysis to assess whether a stable, therapeutically useful, non- natural cell state has been accessed. This study is expected to generate new technical platforms for human cellular engineering, provide greatly expanded insights into the stability of natural human cell states, and suggest whether the human genome is truly optimal in every cell type in every disease setting, or whether new cell states can be genetically accessed to tailor the genome of a specific human cell for a specific desired therapeutic function.