Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease (CRISPR/Cas) genome editing has enabled scientists to design and build next-generation cell-based therapies. T cell-based immunotherapies are particularly suitable CRISPR/Cas tools as after editing they can be expanded to sufficient numbers for use in humans. Recent efforts have shown that precise CRISPR/Cas knock-in at the T cell receptor a constant (TRAC) locus with a chimeric antigen receptor (CAR) targeted towards the B cell antigen CD19 is more effective over randomly integrating viral-based engineered CAR-T cells in mouse models of human B cell leukemia. Therefore, while CRISPR/Cas cellular therapies may improve patient outcomes, like traditional CAR- T cell therapy, it is not known if every patient will respond equivalently or if some patients will be prone to off- target, life-threatening side effects. What is needed are translationally-relevant technologies that can monitor the fate of CRISPR/Cas-edited CAR-T cells in vivo to provide the information needed to understand patient treatment response/non-response, relapse and/or toxicity. The long-term objective of this application is to develop molecular imaging reporter gene technologies for non-invasive visualization of TRAC-targeted CAR-T cells in patients. Specifically, we propose to develop novel translationally-relevant magnetic resonance imaging (MRI) and positron emission tomography (PET) reporter gene assays to noninvasively track the fate of TRAC-edited CAR-T cells over time and to validate these technologies in preclinical mouse models.
The specific aims of this new application are: to compare minicircle and non-integrating lentiviral CRISPR/Cas donor vectors for their ability to efficiently edit T cells at the TRAC loci with a CD19-targeted CAR (Aim 1.1); to develop donor vectors co-encoding our CAR, a bioluminescence reporter gene, and either the human MRI reporter organic anion transporter polypeptide 1B3 (OATP1B3) or the PET reporter sodium iodide symporter (NIS), and to validate TRAC-editing and functional reporter expression in vitro (Aim 1.2); to evaluate the sensitivity of OATP1B3-based MRI and NIS-based PET for visualizing CAR-T cells in vivo, as well as perform longitudinal PET and MRI of CAR-T cells in mouse models of B cell malignancies (Aim 2.1); and finally, to develop a dual-reporter translationally-relevant CRISPR/Cas system and evaluate the ability to noninvasively monitor CAR-T cells in mice with both PET and MRI (Aim 2.2). The significance of this work will be to provide powerful cell tracking technologies to allow the fate of CRISPR/Cas-edited CAR-T cells, or other CRISPR/Cas-edited cellular therapies, to be monitored noninvasively in preclinical models and patients. This information will allow more precise monitoring of these transformative therapies in individual patients to better assess safety as well as to relate cell biodistribution to patient outcomes.

Public Health Relevance

This project addresses the lack of clinically-relevant tools to noninvasively visualize next-generation chimeric antigen receptor (CAR) T cell cancer immunotherapies being built using clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease (CRISPR/Cas) genome editing technologies. To solve this need, we will build and validate new CRISPR/Cas systems that can efficiently engineer human T cells with a CAR at a defined DNA location that has been shown to improve the effectiveness of the therapy in animal models, in addition to co-engineering the cells with reporter genes that will make imageable proteins to permit the cells to be visible with magnetic resonance imaging (MRI) and/or positron emission tomography (PET). In the future, we predict these new imaging tools will allow doctors to better assess the long-term distribution and survival of the CRISPR/Cas-edited T cells once injected into patients, to provide critical information on their overall safety as well as their therapeutic effectiveness in individual patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Exploratory/Developmental Cooperative Agreement Phase I (UH2)
Project #
1UH2EB028907-01
Application #
9810816
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Atanasijevic, Tatjana
Project Start
2019-09-05
Project End
2021-07-31
Budget Start
2019-09-05
Budget End
2020-07-31
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Western Ontario
Department
Type
DUNS #
208469452
City
London
State
ON
Country
Canada
Zip Code
N6 3K7