Developing Gene Therapy for Mitigating Multisystemic Pathology in Fabry Disease: Proof of Concept in an Aggravated Mouse Model.

Fabry disease (FD) is a multisystemic lysosomal storage disorder caused by the loss of α-galactosidase A (α-Gal) function. The current standard of care, enzyme replacement therapies, while effective in reducing kidney pathology when treated early, do not fully ameliorate cardiac issues, neuropathic manifestations, and risk of cerebrovascular events. Adeno-associated virus (AAV)-based gene therapies (AAV-GT) can provide superior efficacy across multiple tissues owing to continuous, endogenous production of the therapeutic enzyme and lower treatment burden. We set out to develop a robust AAV-GT to achieve optimal efficacy with the lowest feasible dose to minimize any safety risks that are associated with high-dose AAV-GTs. In this proof-of-concept study, we evaluated the effectiveness of an rAAV9 vector expressing human GLA transgene under a strong ubiquitous promoter, combined with woodchuck hepatitis virus posttranscriptional regulatory element (rAAV9-hGLA). We tested our GT at three different doses, 5e10 vg/kg, 2.5e11 vg/kg, and 6.25e12 vg/kg in the G3Stg/GLAko Fabry mouse model that has tissue Gb3 substrate levels comparable with patients with FD and develops several early FD pathologies. After intravenous injections of rAAV9-hGLA at 11 weeks of age, we observed dose-dependent increases in α-Gal activity in the key target tissues, reaching as high as 393-fold of WT in the kidneys and 6156-fold in the heart at the highest dose. Complete or near-complete substrate clearance was observed in animals treated with the two higher dose levels tested in all tissues except for the brain. We also found dose-dependent improvements in several pathological biomarkers, as well as prevention of structural and functional organ pathology. Taken together, these results indicate that an AAV-GT under a strong ubiquitous promoter has the potential to address the unmet therapeutic needs in patients with FD at relatively low doses.

Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells.

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1-2M). The cryoprotectants used included 2M 1,2-propanediol (PROH, cell membrane permeable) and 0.5M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

Homogeneous differentiation of hepatocyte-like cells from embryonic stem cells: applications for the treatment of liver failure.

One of the major hurdles of cellular therapies for the treatment of liver failure is the low availability of functional human hepatocytes. While embryonic stem (ES) cells represent a potential cell source for therapy, current methods for differentiation result in mixed cell populations or low yields of the cells of interest. Here we describe a rapid, direct differentiation method that yields a homogeneous population of endoderm-like cells with 95% purity. Mouse ES cells cultured on top of collagen-sandwiched hepatocytes differentiated and proliferated into a uniform and homogeneous cell population of endoderm-like cells. The endoderm-like cell population was positive for Foxa2, Sox17, and AFP and could be further differentiated into hepatocyte-like cells, demonstrating hepatic morphology, functionality, and gene and protein expression. Incorporating the hepatocyte-like cells into a bioartificial liver device to treat fulminant hepatic failure improved animal survival, thereby underscoring the therapeutic potential of these cells.

Green's function methods for analysis of oxygen delivery to tissue by microvascular networks.

Delivery of oxygen to tissue is an essential function of the circulatory system. The distance that oxygen can diffuse into oxygen-consuming tissue is small, and so tissue oxygenation is critically dependent on the spatial arrangement of microvessels in tissue. Theoretical methods have been developed to simulate the spatial distribution of oxygen levels in tissue surrounding a network of microvessels. Here, numerical methods based on a Green's function approach are presented, for realistic three-dimensional network geometries derived from observations of skeletal muscle, brain, and tumor tissues. Relative to finite-difference methods, the Green's function approach reduces the number of unknowns in the numerical formulation and allows rapid computations even for complex vascular geometries. Generally, the boundary conditions on the exterior of the computational domain are not known. Imposition of a no-flux boundary condition can lead to exaggerated estimates of the extent of hypoxia in the tissue region. A new version of the method is described that avoids this problem and can be applied to arbitrarily shaped tissue domains.

Comparison of PSGL-1 microbead and neutrophil rolling: microvillus elongation stabilizes P-selectin bond clusters.

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophil's surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.