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 (p=0.0275, n=10).

Protein Optimization Evolving Tool (POET) based on Genetic Programming

“…S-2) good CESTides can deviate from the poly-L-lysine like sequence. This is especially important for designing a new version of genetically encoded CEST based reporters 19 . Less charged reporters reduce intracellular interactions with other proteins while the use of more varied amino acids increases the intracellular expression level of the reporter as it is not dependent on the supply of a single amino acid.…”

“…Moreover, POET evolved five new CESTides that produce CEST contrast at 5 ppm that is between 0.6-0.8 of the contrast that K12 produces at 3.6 ppm. Considering the low CEST background that cells and tissue produce at 5 ppm 30,34 , it is anticipated that assembling these peptides will result in a reporter gene that is more sensitive than the 3.6 ppm based genetically encoded CEST reporters [15][16][17][18][19] . These findings indicate that POET can assist in mining the fitness landscape to identify peptides with a new function that cannot evolve otherwise.…”

“…While this contrast was demonstrated to be most efficient for poly-L-lysine by van Zijl and colleagues 12 and later on was optimized for several other peptides 13,14 , contrast improvement using rational protein design remained a challenge. Moreover, when the peptides were genetically encoded and expressed in cells or rodents, the background contrast that was generated by cellular metabolites was very high [15][16][17][18][19] . Therefore, there was a need to generate peptides that provide MRI contrast at a frequency where the background CEST contrast is low.…”

Using Genetic Programming to Predict and Optimize Protein Function

“…CEST [10] is a Magnetic Resonance Imaging (MRI) contrast approach in which peptides with exchangeable protons or molecules are saturated and detected indirectly through enhanced water signals after transfer. The main advantage of CEST based proteins is that they can be encoded into DNA and expressed in live cells and tissue, thus allowing tracking non-invasively with MRI [11,12,13,14]. POET here aims to aid DE by predicting better functioning CEST contrast proteins and replacing the rigorous and costly task of screening and mutagenesis.…”

An end‐to‐end AI‐based framework for automated discovery of rapid CEST/MT MRI acquisition protocols and molecular parameter quantification (AutoCEST)

“…The potential benefit of using the CEST contrast mechanism was demonstrated in a variety of clinical applications, including cancer detection and grading, 4 stroke characterization, 5 characterization of neurodegenerative disorders, 6 kidney disease monitoring, 7 , 8 cartilage and intervertebral disc imaging, 9 , 10 cell tracking, 11 , 12 , 13 and cardiac disease assessment. 14 …”