Molecular MRI for in vivo tracking of gene editing and gene edited cells

项目摘要 基因疗法代表了一种潜在的机制，可以恢复衰竭器官的基因表达模式， 尤其是在心力衰竭中，多种蛋白质表达下调导致舒张性心衰。 体内基因编辑的方法已经有了长足的发展，特别是在新型工程技术的帮助下 腺相关病毒(AAV)载体的变种，用于运送转基因或作为CRISPR/CAS9的载体。 然而，成功的基因编辑和时空表达的纵向监测的验证 模式仍然需要侵入性活组织检查。活组织检查存在空间抽样错误、造成疼痛和患者 心力衰竭会增加猝死的风险。连续无创性多脏器定量检查 基因编辑货物的运送和转导以及随后的转基因表达将提供关键的 为进一步开发基因疗法提供数据，并有可能用于全面的患者监测。磁性 磁共振成像(MRI)在全美拥有庞大的安装基础，被用作 心脏结构和功能的常规临床评估。一种名为化学的新兴磁共振成像方法 交换饱和转移(CEST)利用内部的磁化交换 用于活体分子成像的大分子和水。我们之前已经开发了CEST-MRI方法 用于组织纤维化、代谢功能障碍、细胞跟踪的无创心脏成像，以及最近的 量化基因编码的50-赖氨酸报告肽的表达。在这项建议中，我们寻求 建立利用AAV2病毒衣壳蛋白3(VP3)表面赖氨酸残基的CEST-MRI方法 用于细胞AAV2转导和内体逃逸的内源性CEST-MRI。接下来，我们寻求将 用CEST-MRI对转基因在心脏和肝脏中表达的时空模式进行评估 伴随着心脏结构/功能的相应变化。如果成功，这些方法可以很容易地 在现有的临床核磁共振扫描仪上实现，并提供了一种跟踪基因的内生机制 编辑货物和随后的多尺度结果，而不需要活检。

Project Summary Gene therapy represents a potential mechanism to restore gene expression patterns in failing organs, particularly in heart failure where downregulated expression of multiple proteins contributes to diastolic failure. Methods for in vivo gene editing have undergone significant development, particularly with novel engineered variants of adeno-associated viral (AAV) vectors for delivery of transgenes or as vehicles for CRISPR/Cas9. However, verification of successful gene editing and longitudinal monitoring of spatiotemporal expression patterns still require invasive biopsies. Biopsies suffer from spatial sampling error, inflict pain, and in patients with heart failure increase the risk of sudden death. Serial non-invasive and multi-organ quantification of the delivery and transduction of gene editing cargo and subsequent transgene expression would provide critical data for further development of gene therapies, and potentially for comprehensive patient monitoring. Magnetic resonance imaging (MRI), which has a large install base throughout the United States, is used as part of routine clinical assessment of cardiac structure and function. An emerging MRI approach termed chemical exchange saturation transfer (CEST) utilizes the endogenous exchange of magnetization between macromolecules and water for in vivo molecular imaging. We have previously developed CEST-MRI methods for non-invasive cardiac imaging of tissue fibrosis, metabolic dysfunction, cell tracking, and most recently to quantify the expression of a genetically encoded 50-Lysine reporter peptide. In this proposal, we seek to develop CEST-MRI methods that exploit the surface Lysine residues of the AAV2 viral capsid protein 3 (VP3) for endogenous CEST-MRI of cellular AAV2 transduction and endosomal escape. Next, we seek to combine such assessment with CEST-MRI of spatiotemporal patterns of transgene expression in the heart and liver alongside corresponding changes in cardiac structure/function. If successful, these methods can be easily implemented on existing clinical MRI scanners, and provide an endogenous mechanism for tracking of gene editing cargo and subsequent multi-scale outcomes without the need for biopsy.