We are developing the first genetically engineered (GE) reporter system for cell tracking using hyperpolarized (HP) magnetic resonance spectroscopic imaging (MRSI) to address an urgent need for the quantitative tracking of cellular therapies. This GE probe would allow for longitudinal studies by identifying the presence and viability of cellular therapies in vivo with high sensitivity. The advent of HP-MRSI holds the potential to overcome traditional limiting challenges of MR by increasing the signal 10⁶ over traditional MRI methods while enabling the longitudinal detection of enzyme function¹. 

            Here, we report an MR-compatible, highly specific enzyme-substrate pair incorporating porcine liver esterase (PLE) and a 1-methyl-cyclopropylester protecting group². We have adapted a highly specific enzyme-substrate pair from an established fluorescence imaging system using porcine liver esterase (PLE) and a 1-methyl-cyclopropylester protecting group and derivatives that have very low background hydrolysis by endogenous esterases. Substrates were identified with favorable characteristics for enabling dynamic nuclear polarization magnetic resonance spectroscopic imaging (DNP-MRSI) including long T₁ relaxation times, sufficient metabolism, and low toxicity.

The hydrolysis of these substrates is visualizable in vitro by HP-MRI without the need for isotopic labeling for evaluation experiments using purified PLE. By doping with isotopically-labeled glassing solvent, such as ¹³C pyruvate, we can polarize our substrate by DNP through polarization transfer. HP spectroscopy experiments using natural abundance substrates have allowed for the characterization of PLE hydrolysis rates, T₁ relaxation rates of substrate and product 1-cyclopropyl ring and the carbonyl carbons, and the chemical shift of both. Even without deuteration of the ring, substrates show T₁ relaxation rates greater than 40s, and viable chemical shifts on both quaternary carbons.

Cell lines were subsequently developed for in cellulo and in vivo use that stably express only PLE and PLE as a P2A with dTomato, for fluorescence confirmation of expression, under a constitutive EF-1α promoter using a sleeping beauty transposase transfection system. A control cellular line of HEK293t as well as two primary hepatocellular carcinoma (HCC) cell lines, SNU-449 and Huh7, expressing these constructs were successfully generated. These GE cell lines were used to generate a PLE-flank tumor model of HCC. HCC and control cell lines also did not show any toxicity up to 10mM substrate concentration in media.

Ongoing studies include in vitro efficacy testing using DNP-MR spectroscopy of PLE-expressing cells with HP ¹³C isotopically-labelled substrate in a previously published cellular perfusion system³.  The ability of this construct to enable cell tracking in vivo will then be determined through DNP-MRSI of HP ¹³C substrates through tail vein injection to mice with flank tumors grown using the stable PLE-expressing primary HCC lines.

            In summary, here a HP gene reporter system is described with the potential to enable functional tracking of diverse cell types.

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