(593) A murine reporter system to assess delivery and duration of genome edited or transplanted cells

Background: Cell and molecular therapies require methods to assess efficacy of delivery in vivo and duration of effect of delivered cells or therapeutics. For pre-clinical studies in animal models, reporter systems provide opportunities to assess delivery and duration in ways not practical or possible in humans, but can reveal insights into efficacy as well as barriers and toxicities one might encounter in humans for the delivery vehicle and pathway. The plethora of fluorescent proteins available and evolved bioluminescent systems allow one to construct reporter systems that can provide precise localization and in vivo detection, respectively, facilitating longitudinal studies for duration as well.

Methods: As a test cargo, the bacterial recombinase Cre was chosen as it can be delivered as a protein, an mRNA or a DNA transgene. For a reporter, a fusion protein was developed that consists of the monomeric fluorescent protein mVenus (excitation max l = 515nm , emission max l = 527nm), and the firefly luciferase variant akaluciferase that emits in the near infrared range (lmax = 677 nm) with the substrate akalumine. A construct in which transcriptional terminators are flanked by LoxP sites, followed by the reporter coding sequence was generated and under control of the CAG promoter. Homology arms to the mouse Rosa26 locus were included and the construct evaluated for expression in the presence and absence of Cre. Having performed as designed, founder mouse lines were generated. The founders were crossed with 2 different lines of Cre-expressing mice. One in which Cre is expressed during gametogenesis for systemic activation of the reporter as a positive control and the other using villin-Cre to restrict Cre expression to intestinal epithelia. To assess sensitivity, cells from the positive control line were delivered to the lungs of C57BL/6J mice through an endoscope. Reporter signal was evaluated by luminescence on a Perkin-Elmer IVIS in live mice and by whole body fluorescence microscopy and immunofluorescence of specific tissues by microscopy.

Results: The positive control mice showed whole body luminescence ~1 million times above background (Figure 1). Fluorescence microscopy of tissues from these animals showed mVenus expression throughout, but different organs had very different levels of fluorescence, with skeletal muscle showing the strongest and liver the weakest. Animals from the villin-Cre cross showed intestinal expression by fluorescence and luminescence in the abdominal region in live animals (Figure 1). The sensitivity experiments in which fibroblasts from the positive control mice were instilled into the lungs of C57BL/6J mice showed that the threshold for luminescent detection of these cells was approximately 1 million cells.

Conclusions: The Cre-sensitive reporter can be used to assess delivery systems in live animals with a large dynamic range. Additionally, the positive control line developed provides a detectable cell donor source for transplant and cellular delivery strategies. The incorporation of bioluminescence allows longitudinal studies in individual mice to determine the duration of edited or transplanted cells.

Acknowledgements: This research was supported by the Carlsberg Foundation Internationalisation fellowship grant [CF17-0157, CF19-0020] and the Research Institute for Children’s Health.

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