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

项目摘要 基因治疗代表了一种潜在的机制，可以恢复衰竭器官中的基因表达模式， 特别是在心力衰竭中，其中多种蛋白质的表达下调导致舒张衰竭。 用于体内基因编辑的方法已经经历了显著的发展，特别是新的工程化的基因编辑方法。 用于递送转基因或作为CRISPR/Cas9的载体的腺相关病毒（AAV）载体的变体。 然而，验证成功的基因编辑和时空表达的纵向监测 模式仍然需要侵入性活检。活检遭受空间采样误差，造成疼痛， 心脏衰竭会增加猝死的风险。连续非侵入性和多器官定量 基因编辑货物的递送和转导以及随后的转基因表达将提供关键的 数据用于进一步开发基因疗法，并可能用于全面的患者监测。磁 共振成像（MRI）在美国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。一种称为化学的新兴MRI方法 交换饱和转移（CEST）利用磁化强度的内源交换， 大分子和水用于体内分子成像。我们以前开发了CEST-MRI方法 用于组织纤维化、代谢功能障碍、细胞追踪的非侵入性心脏成像，最近， 定量基因编码的50-赖氨酸报告肽的表达。在本建议中，我们力求 开发CEST-MRI方法，利用AAV 2病毒衣壳蛋白3（VP 3）的表面赖氨酸残基 用于细胞AAV 2转导和内体逃逸的内源性CEST-MRI。接下来，我们寻求将联合收割机 用CEST-MRI评估心脏和肝脏中转基因表达的时空模式 以及心脏结构/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MRI扫描仪上实现，并提供用于跟踪基因的内源性机制。 无需活检即可编辑货物和随后的多尺度结果。

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.