Redesigned reporter gene for improved proton exchange-based molecular MRI contrast

Abstract

Reporter gene imaging allows for non-invasive monitoring of molecular processes in living cells, providing insights on the mechanisms underlying pathology and therapy. A lysine-rich protein (LRP) chemical exchange saturation transfer (CEST) MRI reporter gene has previously been developed and used to image tumor cells, cardiac viral gene transfer, and oncolytic virotherapy. However, the highly repetitive nature of the LRP reporter gene sequence leads to DNA recombination events and the expression of a range of truncated LRP protein fragments, thereby greatly limiting the CEST sensitivity. Here we report the use of a redesigned LRP reporter (rdLRP), aimed to provide excellent stability and CEST sensitivity. The rdLRP contains no DNA repeats or GC rich regions and 30% less positively charged amino-acids. RT-PCR of cell lysates transfected with rdLRP demonstrated a stable reporter gene with a single distinct band corresponding to full-length DNA. A distinct increase in CEST-MRI contrast was obtained in cell lysates of rdLRP transfected cells and in in vivo LRP expressing mouse brain tumors ([Formula: see text], n = 10).

Figure 1

Screening various polypeptides in search of an optimal LRP-based reporter gene backbone. (a) Z-spectra for a variety of polypeptides with different amino acid motifs. (b) The corresponding CEST contrast observed as a function of saturation frequency offset (MTR${}_{\mathit{asym}}$). (c) Polypeptide sequences and their respective CEST contrast at the amide exchangeable proton chemical shift of 3.6 ppm. The amide proton CEST contrast for the RHGP polypeptide (containing the arginine, histidine, glycine and proline amino acid motif) is similar in magnitude to that of PLL. A large CEST contrast is also observed at 2 ppm for the guanidyl amine exchangeable proton of the RHGP polypeptide. A: Alanine, PLL: poly-L-lysine.

Figure 2

RNA characterization of the rdLRP reporter. (a) Amino acid sequence of the rdLRP consisting of RHGP units with different lysine spacers with a total of 100 amino acid residues. (b,c) RT-PCR of total mRNA from HEK293T cell lysates transfected with rdLRP, evaluating the BGH reverse tag (b) and the v5 epitope tag (c) sites. A single well-defined band is observed from cell lysates transfected with rdLRP, but not from control cell lysates. The base-pair lengths are consistent with expression of full-length rdLRP mRNA (b, 557 bp; c, 491 bp). Raw gel images are shown in Supplementary Information Fig. S2.

Figure 3

In vitro CEST-MRI contrast evaluation of the rdLRP reporter. (a,b) Z-spectra of control (blue trace) and rdLRP (red trace) in transfected HEK293T (a) and GL261N4 (b) cell lysates acquired at 4.7T. (c,d) The corresponding MTR${}_{\mathit{asym}}$ spectra. Increased CEST contrast is observed at $\sim$ 2 ppm corresponding to the guanidyl amine exchangeable protons of arginine amino acid residues and at $\sim$ 3.6 ppm corresponding to the amide exchangeable protons of the peptide bond generated by coupling two amino acids.

Figure 4

In vivo imaging of the rdLRP. (a,b) Amide proton CEST amplitude maps of representative mice implanted with either GL261N4 glioma cells (a) or with rdLRP transfected GL261N4 cells (b). (c) T${}_2$ relaxation time analysis for the two imaged groups, demonstrating the evident edema at the tumor region. Notably, no statistically significant differences were calculated at the tumor region between the rdLRP and control group mice (two-tailed t-test, $p=0.961$, t = 0.0491, df = 18, n = 10 imaging data points per group). (d,e) T${}_2$-weighted images for the mice imaged at (a,b), respectively. (f,g) The amide proton CEST contrast, obtained using a frequency selective saturation pulse (a,b) is overlaid atop the tumor region in the T${}_2$-weighted images presented in (d) and (e), respectively. (h) Quantitative group comparison for the amide proton CEST amplitude. Statistically significant CEST signal increase was obtained in the rdLRP group (two-tailed t-test, $p=0.0275$, n = 10 imaging data points per group, t = 2.399, df = 18, difference between means: $1.542\pm 0.643$% (mean ± SEM)). The box plots represent the median, interquartile range, Tukey style whiskers, outliers (yellow) and individual data points.