Introduction:

For those suffering from hereditary diseases caused by gene mutations, viral gene delivery, specifically using recombinant adeno-associated viruses (rAAV), is the most advanced option available. Gene addition therapy holds great promise; however, one significant obstacle in rAAV-based therapy is the in vivo assessment of transgene expression. An effective way to monitor the success of viral transduction within the body is by tagging it with a cis-linked reporter. The reporter gene should be as small as possible, and allow direct imaging through magnetic resonance imaging (MRI). We have incorporated superCESTide, a reporter gene evolved to provide chemical exchange saturation transfer (CEST) MRI contrast, into an rAAV transgene and evaluated the resulting vector to monitor gene expression in the liver non-invasively.

Objective: To evaluate rAAV carrying the superCESTide reporter and a novel steady-state CEST acquisition for their capabilities in monitoring gene therapy in the liver.

Methods: We configured a rAAVs to express the superCESTide reporter gene or control tdTomato under the control of the enhanced chicken beta-actin promoter (Figure 1A) and pseudoserotyped with an AAV8 capsid and delivered 0.9×10^13 vg/kg into the tail vein of juvenile C57Bl/6 mice. The images were acquired after 5 weeks of injection.

MRI studies were performed on a Bruker Biospec 11.7 T scanner. In vivo CEST MRI was acquired on two groups of mice: one group expressing tdTomato (control, Ns=5) and another expressing superCESTide+tdTomato (superCESTide, Ns=4). Animals were anesthetized with 0.5%–2% isoflurane during scanning. We optimized a steady-state saturation buildup with interleaved FLASH readouts of the magnetization and radial k-space sampling for rapid and motion-robust CEST imaging. Each TR included a Proton Resonance Enhancement for CEST imaging and Shift Exchange (PRECISE) saturation pulse (100 ms) with B_1,peak = 2.4 mT, followed by a 7.5-degree flip angle pulse for detection (TR/TE = 110 ms/1.22 ms). A Z-spectrum was recorded from -5 ppm to +5 ppm in 0.2 ppm steps, with S0 obtained by setting the saturation offset = 200 ppm. The acquisition time was approximately 10 minutes. Fluorescence images were acquired on an IVIS Spectrum CT with an excitation wavelength of 554 nm and an emission wavelength of 581 nm. The fluorescence exposure time was 6 seconds, and the field of view was set to 13 cm.

Results: Figure 1A shows the transgene maps for tdTomato (control) and superCESTide/tdTomato (CEST) viruses. Fluorescence images in Figure 1B clearly demonstrate that fluorescence primarily occurs in the liver region for both groups of rAAV-injected mice and is absent in the uninjected mouse indicating a successful viral transduction. Figure 1C-D show T2-weighted images and 1E-F show MTR_asym maps overlayed over respective CEST M₀ MR images at week 5 post-injection. The higher CEST contrast in the mouse injected with rAAV encodes to superCESTide (Figure 1E) in contrast to the mouse injected with strongly suggests that the reporter gene can track viral transduction in vivo (Supporting Figure S1). Figure 1G presents a correlation of the ratio of superCESTide to beta-actin determined via RT-PCR versus CEST MRI contrast. Clear CEST contrast in the liver above that of controls for the superCESTide mice is evident, with the contrast showing correlation (, P value = 0.0458) between liver contrast and superCESTide expression as quantified by PCR.

Conclusions: We have successfully detected the expression of superCESTide in the livers of mice following rAAV administration. There is a strong correlation between CEST contrast and superCESTide expression, indicating the potential for using an MRI reporter gene to monitor gene expression in vivo.

Image/Figure:

Image/Figure Caption:

Figure 1 A. Transgene maps for tdTomato (control) and superCESTide/tdTomato (CEST) viruses. B. Fluorescence images of uninjected mouse, tdtomato and superCESTide+tdTomato mice. C-D. T2-weighted images and, E-F. MTR_asym maps overlayed over respective CEST M₀ MR images at week 5 post-injection of tdtomato and superCESTide+tdTomato mice respectively. G. Correlation of the ratio of superCESTide to beta-actin determined via RT-PCR versus CEST MRI contrast.

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