CRISPRa induced expression of native MRI reporter proteins

CRISPRa 诱导天然 MRI 报告蛋白的表达

• 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
• 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

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 等基因组编辑技术涵盖了用于治疗和治疗的新治疗工具。 治疗从癌症到遗传异常到代谢疾病的广泛疾病，甚至 伸展以对抗衰老和再生组织。超越永久基因组 修饰，基于工程化 Cas 蛋白的 CRISPR 技术的扩展组合现在拥有 可编程表观遗传调控、天然基因转录控制和 RNA 编辑的能力； 此外，这些系统可以组合起来用于基因表达的正交控制。这些技术开放 无限的治疗应用，但关键要求是靶向或移植细胞 体内监测和跟踪以验证预期和非预期后果尚未得到满足。 在这里，我们提出了一种基于 CRISPR-Cas 的成像报告基因的新范例，非常适合体内 细胞和基因治疗的成像。而不是将外源成像报告基因引入基因组 或细胞，我们假设细胞和基因疗法可以通过使用 CRISPR 介导的体内成像 转录激活（CRISPRa）技术诱导天然蛋白质的表达，其功能 影像记者。我们将这种范例称为 CRISPRa-MRI。 CRISPRa-MRI 的好处是这些 蛋白质本质上是人类的，只会在不破坏基因组的情况下短暂表达，并且 将能够同时与其他报告蛋白或 CRISPR 疗法进行多重分析。作为证明—— 概念上，我们将重点关注肝脏有机阴离子转运多肽 (OATP) 小鼠 OATP1A1 (mOATP1A1) 和人 OATP1B3 (hOATP1B3)，认识到许多其他转运蛋白、金属结合 蛋白质和细胞表面受体也是可行的内源报告基因候选者。 mOATP1A1 和 hOATP1B3 两者都特异性地将 FDA 批准的 MRI 造影剂 Gd-EOB-DTPA 转运到细胞中， 这会产生强大的明亮 MRI 信号。通常，这些转运蛋白的表达仅限于肝脏，但通过 使用 CRISPRa，这些沉默成像报告基因可以“打开”并用于对任何细胞类型或 血统。 CRISPRa-MRI 可以在工程改造中采用用于 CRISPR 编辑或治疗的技术 细胞或基因治疗，避免插入诱变或外源性慢性表达的可能性 可能导致免疫原性或毒性的基因，同时消除额外的挑战 常规报告基因的递送和表达。在这里，我们建议开发一种 CRISPRa 策略 体外激活内源性 mOATP1A1 和 hOATP1B3（具体目标 1），然后测试其性能 直接靶向组织的体内成像或移植后细胞中的 CRISPRa 诱导（具体目标 2）。 迄今为止，成像报告基因在人类细胞和基因治疗中的前景在很大程度上是未知的。 未实现的。 CRISPRa-MRI 是一种全新的策略，可以帮助发挥成像记者的潜力 人类基因，特别是用于监测基于 CRISPR 基因编辑或表观遗传调节的治疗。