Recombinant Adeno-Associated Virus-Mediated Gene Therapy for Treatment of Familial Hypercholesterolemia in Rabbits

Familial hypercholesterolemia (FH) is a genetic disorder characterized by abnormally high concentrations of low-density lipoprotein-cholesterol (LDLcholesterol) in the blood that can contribute to heart disease. FH can result from a defect in the gene for the LDL receptor (LDL-R). FH patients lacking functional LDL-R may benefit from viral-mediated transfer of a functional copy of the open reading frame (ORF) of the LDL-R. Since a recombinant adeno-associated virus (rAAV) is not immunogenic and can be mass-produced, it shows promise for gene therapy applications. AAV6 and AAV8 have been shown to specifically transduce hepatocytes in several species, which normally remove the majority of LDL-cholesterol from the blood via LDL-R-mediated endocytosis. Because of the potential of rAAV to treat FH by delivery of a correct LDL-R ORF to hepatocytes, the liver specificity of these two AAV serotypes was evaluated. Additionally, rabbits were chosen as the animal model for this study because a specific strain of rabbits, Watanabe heritable hyperlipidemic (WHHL), adequately mimics the pathology of FH in humans. Exposure of rabbit liver to rAAV with the marker LacZ and subsequent inspection of liver tissue showed that AAV8 transduced rabbit liver more efficiently than AAV6. To assess the feasibility of producing a rAAV capable of transferring the LDL-R ORF to rabbit hepatocytes in vivo, rAAV8-LDL-R was mass-produced by a baculovirus system in suspension grown insect cells

The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism Chlamydomonas reinhardtii have focused on the length dependence of the intraflagellar transport (IFT) system, which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not been determined. We found that SHF1 encodes a Chlamydomonas orthologue of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as in wild-type cells but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intraflagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses

Primary cilium loss in mammalian cells occurs predominantly by whole-cilium shedding.

The primary cilium is a central signaling hub in cell proliferation and differentiation and is built and disassembled every cell cycle in many animal cells. Disassembly is critically important, as misregulation or delay of cilia loss leads to cell cycle defects. The physical means by which cilia are lost are poorly understood but are thought to involve resorption of ciliary components into the cell body. To investigate cilium loss in mammalian cells, we used live-cell imaging to comprehensively characterize individual events. The predominant mode of cilium loss was rapid deciliation, in which the membrane and axoneme of the cilium was shed from the cell. Gradual resorption was also observed, as well as events in which a period of gradual resorption was followed by rapid deciliation. Deciliation resulted in intact shed cilia that could be recovered from culture medium and contained both membrane and axoneme proteins. We modulated levels of katanin and intracellular calcium, two putative regulators of deciliation, and found that excess katanin promotes cilia loss by deciliation, independently of calcium. Together, these results suggest that mammalian ciliary loss involves a tunable decision between deciliation and resorption