Human Protamine-1 as an MRI Reporter Gene Based on Chemical Exchange

Abstract

Genetically engineered reporters have revolutionized the understanding of many biological processes. MRI-based reporter genes can dramatically improve our ability to monitor dynamic gene expression and allow coregistration of subcellular genetic information with high-resolution anatomical images. We have developed a biocompatible MRI reporter gene based on a human gene, the human protamine-1 (hPRM1). The arginine-rich hPRM1 (47% arginine residues) generates high MRI contrast based on the chemical exchange saturation transfer (CEST) contrast mechanism. The 51 amino acid-long hPRM1 protein was fully synthesized using microwave-assisted technology, and the CEST characteristics of this protein were compared to other CEST-based contrast agents. Both bacterial and human cells were engineered to express an optimized hPRM1 gene and showed higher CEST contrast compared to controls. Live cells expressing the hPRM1 reporter gene, and embedded in three-dimensional culture, also generated higher CEST contrast compared to wild-type live cells.

Cardiac Chemical Exchange Saturation Transfer MR Imaging Tracking of Cell Survival or Rejection in Mouse Models of Cell Therapy

“…Mice were anesthetized and maintained with 2% isoflurane in oxygen, the pectoral muscles were dissected at the fourth intercostal space, mosquito oxide nanoparticle-labeled cells (4-7) has raised concerns of altered gene expression, tissue retention of particles after cell death, and difficulty in differentiating between labeled cells and tissue necrosis (2,(8)(9)(10)(11)(12)(13). Chemical exchange saturation transfer (CEST) MR imaging is an emerging molecular imaging technique wherein the contrast (change in signal intensity) of either paramagnetic CEST agents (14,15) or CEST-active reporter genes (16)(17)(18) is selectively activated by using radiofrequency irradiation at target specific offset frequencies (19). Subsequently, paramagnetic CEST-labeled cells or CEST MR imaging reporter gene-expressing cells can be visualized without disruption of underlying MR image integrity.…”

“…Subsequently, paramagnetic CEST-labeled cells or CEST MR imaging reporter gene-expressing cells can be visualized without disruption of underlying MR image integrity. Prior CEST MR imaging studies have been conducted to track paramagnetic CEST-labeled cells in subcutaneous tumor models in mice (14,15) and image CEST reporter gene-expressing cells implanted in the rodent brain (16)(17)(18). While CEST MR imaging is suited for cell tracking in cardiac cell therapy, conventional CEST approaches are unsuitable for preclinical cardiac imaging and have been limited to stationary organs and the lungs (20).…”

“…Since CEST contrast is selectively activated, the combined use of CESTactive biomaterials (33), CEST MR imaging reporter genes (16,18,32), and multiagent paramagnetic CEST labeling of different cell populations (15) has the potential to demonstrate multiple processes that drive cell fate decisions in regenerative therapy. borders on teratoma formation (C3H) and complete rejection of implanted cells (C57BL/6J).…”

“…Further study is warranted as to whether labeling of cells with paramagnetic CEST agents induces changes in gene expression similar to those observed with iron oxide-based agents. However, emerging CEST MR imaging labels, including CEST-active reporter genes (16,18,32) and engineered nonmetallic CEST macromolecules (33,34), could be used in place of lanthanide metal-based labels. A secondary limitation to our study is the use of high radiofrequency saturation power to generate CEST contrast that would not be possible in clinical settings.…”

NMR Hyperpolarization Techniques of Gases

“…Examples include enzymes or transporters that can act on synthetic contrast agents, [178] proteins that naturally contain paramagnetic metals, including ferritin, [179] MagA, [180] MntR, [181] tyrosinase, [182] and cytochrome P450, [183] and diamagnetic CEST agents such as lysine rich-protein, [184] human protamine sulfate [185] and proteins that alter water diffusivity in tissue. [186] Comparatively fewer contrast agents have been designed for heteronuclear MRI, all of them based on enzymatic or transporter interactions with 19 F compounds, [187] HP 13 C compounds, [187c, 188] or 31 P substrates.…”