Engineered immune cells to treat enzymopathies and cancers

Abstract

The ability to edit the genome of human cells has potentially countless applications in advanced medicine. There are currently two major genome engineering methods: non-targeted genome engineering and targeted genome engineering. Non-targeted genome engineering comprises of viral and non-viral tools. Targeted genome engineering comprises of Zinc-Finger Nucleases, TALENs, and CRISPR-based tools. Inarguably, these tools provide new avenues to create therapies to treat diseases such as enzymopathies and cancers.We have developed methods for editing the genome of human B cells and T cells. Specifically, we precisely inserted a therapeutic transgene into these cells by developing methods for CRISPR-Cas9 along with recombinant adeno-associated virus (rAAV) to mediate insertion of α-L-iduronidase (IDUA). Furthermore, we tested the efficacy of the engineered B and T cells to express IDUA enzyme to treat Hurler syndrome, a form of enzymopathy. We demonstrate that successful engraftment of the engineered cells in a mouse model of Hurler syndrome shows direct positive outcomes and is superior to current standard treatments. Importantly, this platform has potential use not only for Hurler syndrome or enzymopathies, but also other diseases. We also developed a method for editing the genome of human monocytes. Specifically, we developed a highly efficient method that allowed CRISPR-Cas-based in the form of mRNA to edit the genome of monocytes by deploying a pan-RNase inhibitor along with CRISPR-based mRNA with gRNA. We have also created a platform for delivery of CRISPR-Cas9 in combination with rAAV to insert a transgene into monocytes. Importantly, we targeted SIRPA gene for knockout and showed enhanced functional properties in the engineered monocytes against CD47-expressing cancer cells in vitro. The focus of this work was to develop methods for engineering primary human immune cells with the goal of creating clinical products to treat enzymopathies and cancers. Future work will focus on further optimizations to improve efficacy of the engineered methods and treatments.