Clinically Translatable MRI Reporter Genes and Imaging Methods with Ultra-High Specificity and Sensitivity

Massachusetts General Hospital, Boston MA

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Abstract

PROJECT SUMMARY Magnetic resonance (MR) reporter genes have the potential to monitor transgene expression non- invasively in real time at high resolution. These genes can be applied to interrogate the efficacy of gene therapy, monitor oncolytic virotherapy, and assess cellular differentiation, cell trafficking, and specific metabolic activity. Despite initial efforts toward developing MRI reporter genes, there are numerous complications, caused by the low sensitivity and specificity of detection, the requirement for substrates with undesirable pharmacokinetics, and the difficult interpretation of often delayed signal changes. We have previously demonstrated that many of these challenges can be overcome with the use of a lysine rich protein (LRP) reporter gene, that is detectable by chemical exchange saturation transfer (CEST) MRI, in which a frequency-selective radio-frequency pulse is used to saturate the amide exchangeable proton signal of the protein reporter. Our previous research efforts (supported by R01-EB031008) were focused on optimizing the amide proton-based reporter gene stability, maximizing the reporter protein expression, and optimizing the detection sensitivity by maximizing the amide proton exchange rate (kex). While we succeeded on all three counts, we found that the small chemical shift dispersion (Dw) of amide protons ultimately limits the sensitivity and specificity, while the very high RF powers required to efficiently saturate these fast-exchanging amide protons limits the clinical translation potential. Taking the reporter gene technology to the next level will require both (1) designing reporter proteins with exchangeable protons with significantly increased Dw, allowing for much higher specificity and sensitivity; and (2) developing MRI methods for detecting fast exchanging protons that do not require high saturation powers and can be implemented on clinical MRI scanners. In our previous research efforts to optimize amide-based reporter proteins, we discovered peptides and proteins containing tyrosine (Tyr) and tryptophan (Trp) residues that demonstrated CEST contrast from highly shifted (4-10 ppm) phenol OH and indole NH protons, with optimal kex of 2000-6000 s-1. In addition, we discovered that a water resonant chemical exchange-sensitive spin-lock (CESL) magnetic resonance fingerprinting (MRF) method can provide high detection sensitivity for fast exchanging protons without requiring high RF powers, making it suitable for clinical translation. We hypothesize that chemical exchange sensitive reporter proteins can be detected with ultra-high sensitivity and specificity under clinically relevant conditions. To test this hypothesis, and establish the clinical potential of MRI reporter genes, we will capitalize on two transformative technologies developed in our labs; (Aim 1) the discovery of Tyr- and Trp-based reporters with large Dw and fast kex; (Aim 2) the development of a CESL-MRF method for quantification of the reporter protein concentration and kex. Finally, the new reporter genes and CESL-MRF will be tested for imaging oncolytic virotherapy in mouse tumor models (Aim 3).