CRISPRa induced expression of native MRI reporter proteins

翻译后摘要：基因组编辑技术，如CRISPR-Cas包括新的治疗工具，治疗和 治愈一系列广泛的疾病，从癌症到遗传异常到代谢疾病，甚至 拉伸到对抗衰老和再生组织的程度。超越永久基因组 修饰，基于工程化Cas蛋白的CRISPR技术的扩展集合现在拥有 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，但关键的要求是，靶向或移植的细胞， 在体内进行监测和跟踪，以验证预期和非预期后果。 在这里，我们提出了一种基于CRISPR-Cas的成像报告子的新范式，非常适合于体内 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中 或者细胞，我们假设细胞和基因疗法可以通过使用CRISPR介导的 转录激活（CRISPRa）技术来诱导天然蛋白质的表达，所述天然蛋白质的功能是 成像报告物。我们称之为CRISPRa-MRI。CRISPRa-MRI的好处是， 蛋白质是人类固有的，只能在不破坏基因组的情况下瞬时表达， 将能够同时与其他报告蛋白或CRISPR治疗剂进行多路复用。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OATP 1A 1 （mOATP 1A 1）和人OATP 1B 3（hOATP 1B 3），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子候选物。mOATP 1A 1和hOATP 1B 3 两者都能特异性地将FDA批准的MRI造影剂Gd-EOB-DTPA转运到细胞中， 这导致稳健的明亮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“打开”并用于成像任何细胞类型， 脉CRISPRa-MRI可以将用于CRISPR编辑或治疗的技术用于工程化的细胞中。 细胞或基因治疗，避免了外源基因的插入诱变或慢性表达的可能性。 可能导致免疫原性或毒性的基因，同时消除额外的挑战 传递和表达常规的报告基因。在这里，我们建议制定CRISPRa策略， 体外激活内源性mOATP 1A 1和hOATP 1B 3（特定目标1），然后检测其性能， 直接靶向组织的体内成像或移植后细胞中的CRISPRa诱导（特异性目标2）。 到目前为止，人类细胞和基因治疗的成像报告基因的前景在很大程度上是 没有实现CRISPRa-MRI是一种全新的策略，可以帮助实现成像报告基因的潜力。 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疗法。

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.