京都大学 / 新制・課程博士 / 博士(医学) / 甲第25159号 / 医博第5045号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 森田 智視, 教授 永井 洋士 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

In Canada alone, there were an estimated 800,000 visually impaired people in 2007, costing the federal government an annual amount of $15.8 billion in services, treatments and lost revenue. These costs are estimated to double by the year 2032, as the population ages. The leading causes of visual impairment and blindness is retinal degeneration, characterized by the progressive death of retinal cells. The research presented in this PhD thesis aimed to prevent retinal degeneration by over-expressing the X-linked Inhibitor of Apoptosis (XIAP) in retinal cells using plasmid and adeno-associated viral vectors. The work is divided into four sequential chapters targeted at developing an anti-apoptotic gene therapy strategy to prevent retinal cell death. The first chapter examines XIAP gene therapy in the treatment of Leber’s Hereditary Optic Neuropathy (LHON). In vitro studies using the 661W cone-photoreceptor cell line showed that XIAP over-expression significantly lowers cell death when 661W cells are exposed to a number of apoptotic stimuli. In a mouse model of Leber’s Hereditary Optic Neuropathy (LHON), XIAP expression in retinal ganglion cells (RGCs) protected the ultrastructure of the RGC axons within the optic nerve, in addition to providing evidence of functional protection. The second and third chapters further examine the potential for XIAP gene therapy in the treatment of retinal disease by developing an in vivo model of retinal detachment in cats, followed by evaluating the efficacy of XIAP gene therapy intervention. When XIAP was over-expressed in the photoreceptor cells, there was significant structural protection and trends in preservation of function in this model of degeneration. Finally, the fourth chapter explores an alternate method to viral gene therapy by evaluating the efficacy and toxicity of chitosan microparticles as a protein delivery system to the retina. Results show that chitosan microparticles are mucosal-adhesive and are non-toxic at low concentrations in vitro in 661W cells and in vivo in rats. This thesis work provides strong evidence that XIAP gene therapy is an effective method for preventing retinal degeneration, and works as a broad spectrum gene therapy strategy that can be applied to different forms of retinal degeneration.

Deafblindness is a combined impairment of vision and hearing with an incidence of about 1: 8000 children and 1: 5500 adults. The most common genetic causes are the Stickler (STL) and Usher (USH) syndromes. The main goal of this work is to provide an up-to-date overview of STL and USH in the Czech and Slovak Republic (CR and SR), to determine the correlations between the genotype and phenotype in our population and the associated diagnostic criteria. Using sequencing and MLPA we examined 45 patients from 28 families for suspected STL. We found potentially causal variants of STL genes in 39 patients from 22 families. Fifteen different COL2A1 variants (8 being novel) were found in 28 patients from 18 families and 4 novel COL11A1 variants were found in 11 patients from 4 families. We identified the cause of the disease in 79 % of the families. The USH study involved 30 patients from 27 families. The most frequent cause was USH2A pathogenic variants, i.e. 19 variants in 14 families, 9 being novel. Less common were pathogenic variants in MYO7A (6 variants in 3 families, 5 being novel), USH1C and CDH23 (3 variants, 2 being novel, in 2 families both) genes. In 2 families, compound heterozygosity was found for variants in two different USH genes. The deafblindness etiology was clarified for 24 patients from...

The paper introduces briefly topics of retinal detachment, vitrectomy and applicable postoperative care. A system for increasing patient comfort is described, which continuously records and signalizes head tilt. The software part of aforementioned system is implemented, using C++ Builder programming environment and helper libraries supplied by system's hardware component vendors. This software application allows user to analyze recorded data and manage the system. Application's user interface and usage is described in detail, as well as implementation details concerning internal functions and organization.