T cells are a highly significant cell type to target for gene therapy. The 12 T cell receptor (TCR) complex is used by most circulating T cells to recognize antigen and initiate immune responses. T cells can be engineered to express specific, cloned TCR genes encoding receptors enabling them to initiate a desired immune response. In principle, this approach would yield direct immunotherapy against a wide variety of pathogen and tumors. Gene therapy can also address incurable genetic and acquired diseases of T cells, several of which disproportionately impact minority communities in the U.S., including those caused by HIV and HTLV-1. Embryonic and adult stem cells and lentiviral vectors are promising vehicles for introducing therapeutic gene products into T cells. But this approach will require major advances in knowledge of DNA sequences regulating transcription during T cell development in vivo. Viral vectors currently used to transduce stem cells cannot generate T lineage-specific or developmentally controlled transcription. Furthermore, they are frequently silenced upon chromosomal incorporation. The properties of the T cell receptor (TCR)-1 Locus Control Region (LCR) DNA can be directly applied towards overcoming these significant limitations. However, stem cell-transducing viral vectors carrying therapeutic genes have very limited space for additional exogenous gene regulatory DNA. Thus, the application of TCR1 LCR activity to gene therapy vector development will require localization and characterization of the DNA sub-sequences that support its powerful function. This laboratory has been studying the in vivo activity of the TCR1 LCR. The goal of this project is to enable the creation of a "miniaturized" version of the TCR1 LCR that supports maximal LCR activity using the minimum amount of DNA. A hurdle to achieving this goal is the lack of essential molecular knowledge of two key LCR elements (named HS1'and HS4). We hypothesize that the HS1'and HS4 region contain multiple, localized functional sub-sequence elements critical to TCR1 LCR activity. Using an array of molecular, biochemical, cellular and in vivo functional assays well established in our laboratory, we propose here to map these functional sub-elements of the HS1'and HS4 regions. With this information, we further hypothesize that construction of a minimally sized, but fully active, TCR1 mini-LCR is achievable. In this project, we will construct and test TCR1 mini-LCR versions based on the knowledge obtained here from the analyses of HS1'and HS4, and prior knowledge of the molecular components of two other critical LCR sub-regions. The proposed work is essential to applying the functional molecular components of TCR1 LCR DNA towards designing safe, effective vectors producing cell type-restricted therapeutic gene expression in T cells.

Public Health Relevance

Stem cells and lentiviral vectors are promising vehicles for introducing therapeutic gene products into T cells to correct genetic deficiencies, or enable them to initiate specific immune responses against a wide variety of pathogens and cancers. In addition, HIV and HTLV-1 cause devastating and currently incurable diseases of T cells (AIDS and Adult T cell leukemia) that disproportionately affect members of minority and underserved populations. This project will reveal DNA elements of the powerful gene regulatory activity of the T cell receptor-1 gene locus control region that can be used to direct safe, effective therapeutic gene expression specifically to T cells to treat these conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Enhancement Award (SC1)
Project #
5SC1GM095402-04
Application #
8607965
Study Section
Special Emphasis Panel (ZGM1-MBRS-X (GC))
Program Officer
Dunsmore, Sarah
Project Start
2011-02-09
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
4
Fiscal Year
2014
Total Cost
$275,400
Indirect Cost
$95,400
Name
Hunter College
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
620127915
City
New York
State
NY
Country
United States
Zip Code
10065
Ku?erová-Levisohn, Martina; Lovett, Jordana; Lahiji, Armin et al. (2014) Derivation of T cells in vitro from mouse embryonic stem cells. J Vis Exp :e52119
Lahiji, Armin; Ku?erová-Levisohn, Martina; Holmes, Roxanne et al. (2014) Adapting in vitro embryonic stem cell differentiation to the study of locus control regions. J Immunol Methods 407:135-45
Lahiji, Armin; Kucerová-Levisohn, Martina; Lovett, Jordana et al. (2013) Complete TCR-? gene locus control region activity in T cells derived in vitro from embryonic stem cells. J Immunol 191:472-9