Imaging of adeno-associated viral capsids for purposes of gene editing using CEST NMR/MRI.

PURPOSE: Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. METHODS: NMR experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800-MHz vertical scanner. In vitro experiments were performed for testing of CEST-NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase-polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. RESULTS: CEST-NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. CONCLUSION: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

Individually tailored spatial-spectral pulsed CEST MRI for ratiometric mapping of myocardial energetic species at 3T.

PURPOSE: CEST MRI has been used to probe changes in cardiac metabolism via assessment of CEST contrast from Cr. However, B1 variation across the myocardium leads to spatially variable Cr CEST contrast in healthy myocardium. METHODS: We developed a spatial-spectral (SPSP) saturation pulsed CEST protocol to compensate for B1 variation. Flip angle maps were used to individually tailor SPSP pulses comprised of a train of one-dimensional spatially selective subpulses selective along the principal B1 gradient dimension. Complete Z-spectra in the hearts of (n = 10) healthy individuals were acquired using conventional Gaussian saturation and SPSP schemes and supported by phantom studies. RESULTS: In simulations, the use of SPSP pulses reduced the average SD of the effective saturation B1 values within the myocardium (n = 10) from 0.12 ± 0.02 µT to 0.05 ± 0.01 µT (p < 0.01) and reduced the average SD of Cr CEST contrast in vivo from 10.0 ± 4.3% to 6.1 ± 3.5% (p < 0.05). Results from the hearts of human subjects showed a significant reduction of CEST contrast distribution at 2 ppm, as well as amplitude, when using SPSP saturation. Corresponding phantom experiments revealed PCr-specific contrast generation at body temperature when SPSP saturation was used but combined PCr and Cr contrast generation when Gaussian saturation was used. CONCLUSION: The use of SPSP saturation pulsed CEST resulted in PCr-specific contrast generation and enabled ratiometric mapping of PCr to total Cr CEST contrast in the human heart at 3T.

CEST MRI reveals a correlation between visceral fat mass and reduced myocardial creatine in obese individuals despite preserved ventricular structure and function.

Systolic cardiac function is typically preserved in obese adults, potentially masking underlying declines in cardiomyocyte metabolism that may contribute to heart failure. We used chemical exchange saturation transfer (CEST) MRI, a sensitive method for measurement of myocardial creatine, to examine whether myocardial creatine levels correlate with cardiac structure, contractile function, or visceral fat mass in obese adults. In this study, obese (body mass index, BMI > 30, n = 20) and healthy (BMI < 25, n = 11) adults were examined with dual-energy x-ray absorptiometry to quantify fat masses. Cine MRI and myocardial tagging were performed at 1.5 T to measure ventricular structure and global function. CEST imaging with offsets in the range of ±10 parts per million (ppm) were performed in one mid-ventricular slice, where creatine CEST contrast was calculated at 1.8 ppm following field homogeneity correction. Ventricular structure, global function (ejection fraction 69.4 ± 4.3% healthy versus 69.6 ± 9.3% obese, NS), and circumferential strain (-17.0 ± 2.3% healthy versus -16.5 ± 1.5% obese, NS) and strain rate were preserved in obese adults. However, creatine CEST contrast was significantly reduced in obese adults (6.8 ± 1.3% healthy versus 4.1 ± 2.7% obese, p = 0.001). Creatine CEST contrast was inversely correlated with total body fat% (ρ = -0.45, p = 0.011), visceral fat mass (ρ = -0.58, p = 0.001), and septal wall thickness (ρ = -0.44, p = 0.013), but uncorrelated to ventricular function or contractile function. In conclusion, creatine CEST-MRI reveals a strong correlation between heightened body and visceral fat masses and reduced myocardial metabolic function that is independent of ventricular structure and global function in obese adults.

Quantitative Gadolinium-Free Cardiac Fibrosis Imaging in End Stage Renal Disease Patients Reveals A Longitudinal Correlation with Structural and Functional Decline.

Patients with end stage renal disease (ESRD) suffer high mortality from arrhythmias linked to fibrosis, but are contraindicated to late gadolinium enhancement magnetic resonance imaging (MRI). We present a quantitative method for gadolinium-free cardiac fibrosis imaging using magnetization transfer (MT) weighted MRI, and probe correlations with widely used surrogate markers including cardiac structure and contractile function in patients with ESRD. In a sub-group of patients who returned for follow-up imaging after one year, we examine the correlation between changes in fibrosis and ventricular structure/function. Quantification of changes in MT revealed significantly greater fibrotic burden in patients with ESRD compared to a healthy age matched control cohort. Ventricular mechanics, including circumferential strain and diastolic strain rate were unchanged in patients with ESRD. No correlation was observed between fibrotic burden and concomitant measures of either circumferential or longitudinal strains or strain rates. However, among patients who returned for follow up examination a strong correlation existed between initial fibrotic burden and subsequent loss of contractile function. Gadolinium-free myocardial fibrosis imaging in patients with ESRD revealed a complex and longitudinal, not contemporary, association between fibrosis and ventricular contractile function.