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CRISPRa induced expression of native MRI reporter proteins

CRISPRa 诱导天然 MRI 报告蛋白的表达

基本信息

批准号：
10287598
负责人：
Erik Shapiro
金额：
$ 22.3万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10287598
关键词：
Adverse effects Aging BCAR1 gene Binding Proteins CRISPR therapeutics CRISPR/Cas technology Cell Lineage Cell Surface Receptors Cell Survival Cell Therapy Cell Transplantation Cells Chemicals Chromatin Chronic Clustered Regularly Interspaced Short Palindromic Repeats Colorectal Cancer Contrast Media DNA Methylation Detection Disease Engineering Epigenetic Process FDA approved Gene Expression Genes Genome Guide RNA HCT116 Cells Hepatic Human Human Cell Line Image In Vitro Insertional Mutagenesis Investments Lead Link Liver Magnetic Resonance Imaging Malignant Neoplasms Mediating Metabolic Diseases Metals Methods Modification Molecular Abnormality Monitor Mus Nude Mice OATP Transporters Patients Pentetic Acid Performance Promoter Regions Proteins RNA Editing Reporter Reporter Genes Reproducibility Ribonucleoproteins Signal Transduction Site Specificity Stretching System Technology Testing Therapeutic Time Tissues Toxic effect Transcription Initiation Site Transcriptional Activation Transcriptional Activation Domain Transcriptional Regulation Transfection Transplantation Validation base cancer cell cell growth cell type epigenetic regulation experimental study gene therapy genome editing imaging modality immunogenicity in vivo in vivo imaging induced pluripotent stem cell mRNA Expression novel strategies novel therapeutics post-transplant programs protein activation protein expression protein function quadriceps muscle sharing platform tissue regeneration tool tumor

项目摘要

Abstract: Genome editing technologies such as CRISPR-Cas encompass new therapeutic tools for treating and curing a broad set of diseases ranging from cancer to genetic abnormalities to metabolic diseases, even stretching as far as combating aging and regenerating tissue. Extending beyond permanent genome modification, the expanding ensemble of CRISPR technologies based on engineered Cas proteins now possess capabilities for programmable epigenetic regulation, transcriptional control of native genes, and RNA editing; moreover, these systems can be combined for orthogonal control of gene expression. These technologies open up unlimited therapeutic applications but the critical requirement that the targeted or transplanted cells be monitored and tracked in vivo for verifying both intended and unintended consequences, is unmet. Here we propose a new paradigm in imaging reporters based on CRISPR-Cas, well suited for in vivo imaging of cell and gene therapies. Rather than introduce exogenous imaging reporter genes into the genome or cell, we hypothesize that cell and gene therapies can be imaged in vivo by using CRISPR-mediated transcriptional activation (CRISPRa) technology to induce expression of native proteins that function as imaging reporters. We call this paradigm CRISPRa-MRI. The benefits of CRISPRa-MRI are that these proteins would be inherently human, would only be transiently expressed without disrupting the genome and would enable multiplexing either with other reporter proteins or CRISPR therapeutics, simultaneously. As a proof- of-concept, we will focus on the hepatic organic anion transporting polypeptides (OATPs) mouse OATP1A1 (mOATP1A1) and human OATP1B3 (hOATP1B3), recognizing that many other transporters, metal binding proteins and cell surface receptors are also viable endogenous reporter candidates. mOATP1A1 and hOATP1B3 both specifically transport the FDA-approved MRI contrast agent Gd-EOB-DTPA into cells, the accumulation of which results in robust bright MRI signal. Normally, expression of these transporters is limited to the liver, but by using CRISPRa these silent imaging reporter genes can be ‘turned on’ and used to image any cell type or lineage. CRISPRa-MRI can co-opt the technology used for CRISPR editing or therapeutics in the engineered cells or gene therapy, avoiding the potential for insertional mutagenesis or chronic expression of an exogenous gene which may lead to immunogenicity or toxicity, while at the same time eliminating the additional challenge of delivery and expression of conventional reporter genes. Here we propose to develop a CRISPRa strategy for activating endogenous mOATP1A1 and hOATP1B3 in vitro (Specific Aim 1) and then test its performance for in vivo imaging of directly targeted tissues or CRISPRa induction in cells post-transplantation (Specific Aim 2). The promise of imaging reporter genes for cell and gene-based therapies in humans is, as yet, largely unfulfilled. CRISPRa-MRI is a completely novel strategy that can help achieve the potential of imaging reporter genes in humans, especially for monitoring therapies based on CRISPR gene editing or epigenetic modulation.

摘要：CRISPR-Cas等基因组编辑技术为治疗和治疗疾病提供了新的治疗工具