CRISPRa induced expression of native MRI reporter proteins

摘要：CRISPR-Cas等基因组编辑技术包含了治疗和治疗癌症的新的治疗工具。 治愈一系列疾病，从癌症到基因异常再到代谢性疾病，甚至 伸展到抗击衰老和再生组织。延伸到永久基因组之外 修饰，基于工程Cas蛋白的CRISPR技术的扩展集成现在拥有 具有可编程表观遗传调控、本地基因转录控制和RNA编辑的能力； 此外，这些系统还可以组合在一起进行基因表达的正交控制。这些技术开放了 无限制的治疗应用，但关键要求靶向或移植的细胞 在活体内监测和跟踪以核实预期和非预期后果的情况未得到满足。 在此，我们提出了一种新的基于CRISPR-CAS的成像记者范式，该范式适用于活体 细胞和基因疗法的成像。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治疗可以通过CRISPR介导的体内成像 转录激活(CRISPRa)技术诱导天然蛋白的表达，这些蛋白的功能是 影像记者。我们称这种范式为CRISPRA-MRI。CRISPRa-MRI的好处是 蛋白质本来就是人类的，只能在不破坏基因组的情况下瞬时表达，而且 将能够同时与其他报告蛋白或CRISPR疗法进行多路复用。作为证据- 在概念上，我们将重点关注肝脏有机阴离子转运多肽(OATPs)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性报告候选。MOATP1A1和hOATP1B3 两者都专门将FDA批准的MRI造影剂Gd-EOB-DTPA转运到细胞内，积累的 这导致了强大的明亮的MRI信号。正常情况下，这些转运蛋白的表达仅限于肝脏，但通过 使用CRISPRa，这些沉默的成像报告基因可以被‘打开’，并用于对任何类型的细胞或 血统。CRISPRa-MRI可以增选用于CRISPR编辑或治疗的技术 细胞或基因治疗，避免插入突变或外源基因的慢性表达 可能导致免疫原性或毒性的基因，同时消除额外的挑战 常规报告基因的传递和表达。在这里，我们建议为以下目标制定CRISPRa战略 体外激活内源性mOATP1A1和hOATP1B3(特异靶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CRISPRa的诱导(特定目标2)。 到目前为止，成像报告基因在人类细胞和基于基因的治疗中的前景还很大 没有实现。CRISPRa-MRI是一种全新的策略，可以帮助实现成像报告的潜力 人类基因，尤其是用于监测基于CRISPR基因编辑或表观遗传调节的治疗。

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.