Siobhan Williams

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a ‘differential multiple cleavage-site’ model

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a ‘differential multiple cleavage site’ model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.

Assessment of eye length changes in accommodation using dynamic extended-depth OCT

It has been suggested that accommodation induces increases in axial eye length which could contribute to the development of myopia. However, it is debated whether changes in eye length occur during accommodation as the degree of change varies widely across literature. In this study, an extended-depth optical coherence tomography (OCT) system that provides dynamic whole eye biometry was utilized to assess changes in lens thickness (LT) and axial eye length (AEL) in young subjects responding to step disaccommodation stimuli of amplitude 2D, 4D, and 6D. The decrease in lens thickness with disaccommodation was strongly correlated with stimulus amplitude. No statistically significant changes in AEL during accommodation were observed.

Objective dynamic changes in lens curvature during accommodation using optical coherence tomography images (Conference Presentation)

Purpose: To objectively quantify dynamic changes in lens shape during accommodation using two-dimensional OCT images Methods: In-vivo responses to an accommodative step stimulus of three subjects (aged 22, 39, and 45) were captured using a custom-made extended-depth SD-OCT system operating at 840 nm following an IRB-approved protocol (Ruggeri et al. 2012). Subjects focused on a visual accommodation target designed to produce an adjustable step stimulus of accommodation. Accommodative responses to 2-D and 4-D stimuli were captured ~1.5s before and ~4.5s after stimulation. Lens thickness, anterior curvature, and posterior curvature were measured using a newly-developed algorithm (validated using a calibration sphere). Dynamic changes in lens thickness and curvature were then fitted with an exponential model to produce time dependent constants. Results: All calibration OCT images were automatically analyzed in under 2 seconds. A radius of 7.793mm ± 0.051mm was calculated resulting in a difference of 2.4μm from the reported nominal value of the calibration sphere. Anterior lens radius decreased over time in all subjects. Radius of the posterior lens experienced a slight increase for all subjects. Conclusion: This study demonstrates the feasibility of quantifying the dynamic changes in lens curvature and thickness during accommodation using extended-depth OCT combined with a step accommodation stimulus and an automated segmentation algorithm.

System for on- and off-axis volumetric OCT imaging and ray tracing aberrometry of the crystalline lens

We present a new in vitro instrument for measuring shape and wavefront aberrations of the primate crystalline lens, both on- and off-axis, while simulating accommodation with a motorized lens stretching system. The instrument merges spectral domain optical coherence tomography (SD-OCT) imaging and ray tracing aberrometry using an approach that senses wavefront aberrations of the lens with the OCT probing beam. Accuracy and repeatability of aberration measurements were quantified. Preliminary experiments on two human and four cynomolgus monkey lenses demonstrate the ability of the system to measure the lens shape, spherical aberration and peripheral defocus, and their changes during simulated accommodation.

Simultaneous refraction measurement and OCT axial biometry of the eye during accommodation(Conference Presentation)

The purpose of this project is to design and evaluate a system that will enable objective assessment of the optical accommodative response in real-time while acquiring axial biometric information. The system combines three sub-systems which were integrated and mounted on a joystick x-y-z adjustable modified slit-lamp base to facilitate alignment and data acquisition: (1) a Shack-Hartmann wavefront sensor for dynamic refraction measurement, provided software calculates sphere, cylinder and axis values, (2) an extended-depth Optical Coherence Tomography (OCT) system using an optical switch records high-resolution cross-sectional images across the length of the eye, from which, dynamic axial biometry (corneal thickness, anterior chamber depth, crystalline lens thickness and vitreous depth) can be extracted, and (3) a modified dual-channel accommodation stimulus unit based on the Badal optometer for providing a step change in accommodative stimulus. The prototypal system is capable of taking simultaneous measurements of both the optical and the mechanical response of lens accommodation. These measurements can provide insight into correlating changes in lens shape with changes in lens power and ocular refraction and ultimately provide a more comprehensive understanding of accommodation, presbyopia and an objective assessment of presbyopia correction techniques.