1357791 - Quantitative imaging of gene therapy delivery vehicles using CEST-NMR/MRI

Over the last decade gene therapy has rapidly expanded to include in vivo somatic cell gene editing, particularly the use of adeno-associated virus (AAV) vectors as delivery vehicles for gene editing machinery. AAV vector-mediated gene therapy has been explored as a promising approach for treating different cardiovascular and muscular conditions, including Pompe disease, atherosclerosis, and Barth syndrome. However, the reliance upon invasive biopsies to verify gene editing is a boundary to successful translation. AAV2 capsids contain over 1,000 surface Lysine residues that can potentially generate CEST contrast. We tested this using NMR CEST under varying pH, density, biological transduction stage, serotypes, and in mixed biological media. Subsequent experiments optimized CEST saturation schemes for AAV contrast detection at 7T.

Methods:

NMR-CEST experiments were performed using an 800MHz scanner on AAV2 phantoms (pH 7, 37°C). CEST contrast was assessed on AAV2 samples with varying capsid densities (5×10³-5×10⁸ vg/μL), pH values (4-7.5), and later multiple AAV serotypes (1, 5, 6, 7, 9) across a range of saturation powers (3-9μT). To test whether AAV2 generates CEST contrast during endosomal transport, HEK293T cells were transduced with AAV2 and then harvested at multiple timepoints following removal of viral media. Afterwards, endosomes were lysed, isolated, and buffered in solution for NMR-CEST experiments.

AAV2 phantoms were then imaged at 7T with Z-spectra acquired following varying values for saturation B₁, saturation pulse duration, and saturation duty cycle across different combinations. To probe the impact of dilution in background tissue protein content, additional experiments were carried out on a phantom containing cell lysate and AAV2 using different saturation schemes for optimization of contrast detection.

Results:

Water-suppressed NMR of AAV2 identified 3 potential groups of high-density protons at 0.6, 3.0, and 3.6ppm (Figure 1A) of which the exchangeable protons at 0.6ppm generated significant CEST contrast (Figure 1B). CEST contrast of AAV2 at 0.6ppm correlated with capsid density and decreased with acidification. Similar CEST contrast was observed across other AAV serotypes. Quantification of viral titer and subsequent regression analysis revealed a positive linear correlation between CEST contrast and viral density. CEST contrast of AAV2 at 7T demonstrated a dependence upon saturation scheme parameters, but when optimized produced CEST contrast of 9-12% in phantoms (Figure 2). Dilution of AAV2 capsids in cell lysate reduced CEST contrast of AAV2 to approximately 3.3%.

Conclusion:

AAV2 viral capsids generates robust CEST contrast in vitro across a variety of chemical environments, concentrations, and saturation schemes. Additional experiments to explore the effectiveness of AAV2 viral capsids as an in vivo endogenous CEST contrast agent for gene therapy tracking is needed.