Type IV collagen : regulation of COL4A1 and COL4A2 genes and gene therapy of Alport syndrome

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Medical Biochemistry and Biophysics

Sammanfattning: Type IV collagen is an important structural protein in basement membranes, which am thin sheet-like structures located between organized tissues and the connective tissue. Type IV collagen is composed of six genetically different [alpha] chains. The COL4A1 and COL4A2 genes that code for the widespread [alpha]1(IV) and [alpha]2(IV) chains are located on the same chromosome head-to-head and share a common promoter. The COL4A3 and COL4A4, as well as COL4A5 and COL4A6 have similar pairwise localization. Transcriptional regulation of the unique bidirectional promoter of COL4A1 and COL4A2 was studied using a minigene construct which enabled the detection of transcriptional activity to Ionic directions simultaneously. The role of several promoter elements in the control of bidirectional was characterized by mutational analysis. The promoter was shown to contain both bidirectionally functioning promoter elements, such as a GC box and a CTC element, as well as gene specific regulatory elements like a CAAT box. Type IV collagen has a particular role in the glomerular basement membrane (GBM) of the kidney. The [alpha]3(IV), [alpha]4(IV) and [alpha]5(IV) chains are specific to this extracellular structure of the kidney filtration barrier. Mutations in the genes encoding any of these three chains lead to Alport syndrome (AS), a progressive hereditary kidney disease. Characteristic for the disease is abnormal structure of the GBM. The X-linked AS, which affects one in 5000 males, is caused by mutations in the gene for the [alpha]5(IV) chain leading to abnormalities in the type IV collagen network of the GBM with abnormal or absent type IV collagen molecules composed of [alpha]3, [alpha]4 and [alpha]5 chains. Gene therapy of Alport syndrome aims at the transfer of a corrected type IV collagen [alpha] chair gene into renal glomerular cells responsible for production of the GBM. In an attempt to transfer genes into kidneys for treatment of AS, an organ perfusion method was developed using pig kidneys. The method developed in this study, provides highly effective and targeted adenovirus-mediated gene transfer into glomerular cells in vivo. The approach utilizes a closed-circuit system for recirculating the adenovirus in the kidney which is attached to the system via renal artery, vein, and ureter. To study the expression of the recombinant [alpha]5 chain of type IV collagen, full-length human and canine [alpha]5(IV) cDNAs were constructed and expressed in 293 and HT1080 cells. The recombinant proteins were secreted to the medium mainly as a monomeric form. However, by immunoprecipitations using a FLAG epitope in the recombinant [alpha]5(IV) chain the recombinant human [alpha]5(IV) chain was shown to assemble with the [alpha]3(IV) and [alpha]4(IV) chains in HT1080 cells, indicating that correction of the molecular defect in AS might be feasible. Using in vivo perfusion of pig kidneys with the recombinant adenovirus, expression of the [alpha]5(IV) chain in kidney glomeruli was obtained, as shown by in situ hybridization. In addition using immunohistochemistry, the recombinant [alpha]5(IV) chain was shown to deposit into the GBM. The results of this study demonstrate that three prerequisites for gene therapy of AS can be achieved. First, an efficient method for targeted gene transfer into kidney glomeruli was developers. Second, the recombinant [alpha]5(IV) chain of type IV collagen was shown to assemble with the [alpha]3(IV) and [alpha]4(IV) chains. Third, the recombinant [alpha]5(IV) chain introduced into renal glomerular cells in vivo was shown to deposit into the GBM. These results strongly imply that Alport syndrome can be treated by gene therapy.

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