[CCoE Notice] CHE PhD Defense: Engineering Fate and Function of Human Cells by Expression of Heterologous Proteins

[Knudsen, Rachel W]

NAME: Fatemeh Sadeghi

DATE: Tuesday, December 3, 2019

TIME: 2:00PM

PLACE: Mechanical Engineering Large Conference Room

CHAIR/ADVISOR: Dr. Navin Varadarajan
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TITLE:

Engineering Fate and Function of Human Cells by Expression of Heterologous Proteins

Cell-based therapies hold great potential toward novel treatment approaches for a wide range of diseases including cancer. Cell therapies can be engineered to repurpose their cellular functions and enhance their therapeutic potentials by the aid of heterologous proteins of nonhuman origins. In this dissertation, I have engineered mammalian cells with 1) a small molecule inducible, plant-derived, nucleocytoplasmic protein shuttling platform, and 2) an insect-derived trehalose transporter that can enhance the function and phenotype of chimeric antigen receptor (CAR)-T cells after cryopreservation.

First, we have designed an inducible protein translocation system using the hormone receptor involved in the immune response mechanism of the plant upon pathogen infection. The receptor, Non-Expresser of Pathogenesis Related 1 (NPR1) is translocated to the nucleus in the presence of its hormone ligand, salicylic acid (SA) in plant cells. To our knowledge there are no studies reporting the use of this system in mammalian cells. In this study, we have shown that nucleocytoplasmic shuttling of a reporter protein fused to the transactivation domain of this receptor can be regulated through treatment with the SA hormone in human embryonic kidney (HEK293) cells. We have demonstrated the SA-dependent protein localization at the single-cell level in a time-and concentration-dependent manner.

In the second part of this dissertation, we have genetically modified CD19-targeting CAR-T cells to facilitate the intracellular uptake of the trehalose sugar. Trehalose is a non-toxic natural cryoprotectant that is accumulated in organisms including yeast, bacteria, and invertebrates upon dehydration or desiccation stress.  Here, we have shown that the proliferation and the chemokine expression levels of the central memory and effector memory T cells are impaired upon cryopreservation. To address these issues, we have expressed TRET1, a facilitated transporter of trehalose, on the surface of CAR-T cells. We observed an improvement in the proliferation and upregulation of CD62L expression of effector memory population in TRET1-expressing CAR-T cells when they were incubated with trehalose prior to cryopreservation.
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