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Adenovirus-Mediated Transfer of Type IV Collagen Α5 Chain Cdna

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Adenovirus-Mediated Transfer of Type IV Collagen Α5 Chain Cdna Gene Therapy (2001) 8, 882–890 2001 Nature Publishing Group All rights reserved 0969-7128/01 $15.00 www.nature.com/gt RESEARCH ARTICLE Adenovirus-mediated transfer of type IV collagen ␣5 chain cDNA into swine kidney in vivo: deposition of the protein into the glomerular basement membrane P Heikkila¨1, A Tibell2, T Morita1, Y Chen1,GWu2, Y Sado4, Y Ninomiya5, E Pettersson3 and K Tryggvason1 1Division of Matrix Biology, Department of Medical Biochemistry and Biophysics, Departments of 2Transplantation Surgery, and 3Nephrology, Huddinge Hospital, Karolinska Institutet, Stockholm, Sweden; 4Division of Immunology, Shigei Medical Research Institute, Okayama; 5Department of Molecular Biology and Biochemistry, Okayama University Medical School, Okayama, Japan Gene therapy of Alport syndrome (hereditary nephritis) aims a FLAG epitope in the recombinant ␣5(IV) chain. The results at the transfer of a corrected type IV collagen ␣ chain gene indicate that correction of the molecular defect in Alport syn- into renal glomerular cells responsible for production of the drome is possible. Previously, we had developed an organ glomerular basement membrane (GBM). A GBM network perfusion method for effective in vivo gene transfer into composed of type IV collagen molecules is abnormal in glomerular cells. In vivo perfusion of pig kidneys with the Alport syndrome which leads progressively to kidney failure. recombinant adenovirus resulted in expression of the ␣5(IV) The most common X-linked form of the disease is caused chain in kidney glomeruli as shown by in situ hybridization by mutations in the gene for the ␣5(IV) chain, the ␣5 chain and its deposition into the GBM was shown by immunohisto- of type IV collagen. Full-length human ␣5(IV) cDNA was chemistry. The results strongly suggest future possibilities expressed in HT1080 cells with an adenovirus vector, and for gene therapy of Alport syndrome. Gene Therapy (2001) the recombinant ␣5(IV) chain was shown to assemble into 8, 882–890. heterotrimers consisting of ␣3(IV) and ␣4(IV) chains, utilizing Keywords: Alport syndrome; type IV collagen; basement membrane; glomerulus; gene therapy; adenovirus Introduction Gly-Xaa-Yaa repeat which allows for flexible kinks in the triple helix. In addition to the collagenous domain, the Alport syndrome is an inherited kidney disease charac- type IV collagen molecules have a noncollagenous globu- terized by progressive hematuria, development of renal lar NC1 domain at the carboxyl end, and the aminotermi- 1,2 failure and frequently also hearing loss. The only avail- nal has a noncollagenous 7S domain. Six genetically dis- able treatment is hemodialysis and/or kidney transplan- tinct type IV collagen ␣ chains have been described. The tation. The underlying cause of the disease is a defective ␣1(IV) and ␣2(IV) chains are ubiquitous and are present structure of the type IV collagen framework of the glom- in triple-helical molecules in a 2:1 ratio.9 The other ␣ erular basement membrane (GBM). The disorder results chains have variable and more restricted tissue distri- in deterioration of the GBM. The disease affects about bution. The current understanding of type IV collagen 1 1:5000 males. It has been estimated that 85% of cases are synthesis in the renal glomerulus is illustrated in Figure caused by mutations in the X chromosomal gene enco- 1a and b. In the GBM, ␣1(IV) and ␣2(IV) dominate during ␣ 3–5 ding the 5(IV) collagen chain. The less frequent auto- embryonic development (Figure 1a), but after birth these ␣ somal forms are caused by mutations in the 3(IV) or are replaced by trimers containing ␣3(IV), ␣4(IV) and ␣ 6,7 4(IV) collagen chain genes located on chromosome 2. ␣5(IV) chains. Due to their high cysteine content the Type IV collagen is a basement membrane-specific col- ␣3:␣4:␣5 trimers are thought to be necessary for forming lagen type which is the main structural component of a stronger, more cross-linked GBM collagen network (see 8 these extracellular structures. Similarly to other col- Figure 1b).10–12 In X-linked Alport syndrome caused by a lagens, type IV collagen is a triple-helical protein con- mutation in the ␣5(IV)␣chain gene, the ␣3(IV) and ␣ ␣ sisting of three chains. The collagen chains have (Gly- ␣4(IV)␣chains are usually absent from the GBM, even Xaa-Yaa)n repeats, glycine being the only amino acid though their genes residing on chromosome 2 are intact.13 small enough to fit into the center of the triple helix. The This is presumably due to intracellular degradation of the ␣ type IV collagen chains have many interruptions in the chains in the absence of ␣5(IV) that is essential for the formation of the ␣3:␣4:␣5 trimer. Instead, the Alport GBM contains the embryonic type of collagen IV mol- ␣ ␣ Correspondence: K Tryggvason, Department of Medical Biochemistry and ecules consisting of 1 and 2 chains (Figure 1c). How- Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden ever, since these apparently do not provide sufficient Received 14 April 2000; accepted 14 September 2000 mechanical strength to the GBM and sufficient resistance Type IV collagen gene transfer P Heikkila¨ et al 883 a b c d Figure 1 Illustration of type IV collagen synthesis and incorporation into the GBM network in embryonic, adult and Alport syndrome kidney. (a) In the embryonic glomerulus, ␣1(IV) and ␣2(IV) chains assemble into triple-helical collagen molecules in a 2:1 ratio, and are secreted and deposited into the GBM network (broken lines). (b) Postnatally there is a developmental shift from ␣1(IV) and ␣2(IV) chains to ␣3(IV), ␣4(IV) and ␣5(IV) chains that form trimers in a 1:1:1 ratio.10 These molecules form a more tightly cross-linked meshwork (solid lines), due to a higher content of cysteine residues in the component chains. (c) In X-linked Alport syndrome, absence or abnormal ␣5(IV) chains lead to degradation of the ␣3(IV) and ␣4(IV) chains ␣ ␣ ␣ ␣ ␣ and consequent absence of 3: 4: 5 which are substituted by the embryonic 12: 2 trimers and, thus, a weaker GBM. (d) Gene therapy of X-linked Alport syndrome aims at restoring the situation in a normal adult (b) by introducing a vector synthesizing recombinant ␣5(IV) chains into the glomerular cells. to proteolysis,14 the consequence is deterioration of the gene transfer. The recombinant ␣5(IV) chain was shown structure, hematuria and development of Alport to be incorporated into triple-helical type IV collagen syndrome. molecules containing endogenous ␣3 and ␣4 chains, indi- Alport syndrome is an attractive candidate disease for cating that correction of the molecular defect in Alport gene therapy due to its high kidney specificity and syndrome might be possible. The second objective of this because the isolated blood circulation of the kidneys study was to explore if in vivo perfusion of pig kidneys makes them a good target for organ-specific gene trans- with these viruses would result in expression of the fer. The principle of gene therapy of X-linked Alport syn- recombinant ␣5(IV) chain and deposition of the polypep- drome is depicted in Figure 1d. This requires transfer of tide chain into the GBM. The experiments resulted in the appropriate type IV collagen ␣ chain gene to the efficient gene transfer into glomeruli and expression of endothelial and epithelial cells of the glomerulus, recombinant ␣5(IV) chain, as determined by in situ expression of the protein and intracellular assembly of hybridization and immunolocalization. Importantly, the the exogenous recombinant chain into triple-helical mol- recombinant ␣5 chain was deposited into the pig GBM, ecules together with the endogenous or ␣3, ␣4or␣5 indicating that it was assembled intracellularly into tri- chains, and, finally, secretion of the protein and ple-helical molecules together with endogenous pig type incorporation of the protein into the GBM type IV col- IV collagen ␣ chains and secreted from the cell. The lagen network (see Figure 1d).15 For the development of results strongly suggest the feasibility of gene therapy for gene therapy of Alport syndrome, we have previously Alport syndrome by targeted gene transfer to renal developed an organ perfusion system for adenovirus- glomeruli. mediated gene transfer into renal glomeruli in vivo.16 Using this procedure, we obtained a transfer efficiency of up to 85% of pig glomeruli using an adenovirus contain- Results ing the ␤-galactosidase reporter gene. Surprisingly, the kidney perfusion method resulted in efficient transfer Adenovirus vectors for expression of the type IV only to glomerular cells, while cells in other regions of collagen ␣5 chain the kidney did not take up significant amounts of the For expression of the ␣5 collagen chain two constructs virus. were made (Figure 2). The first adenovirus Ad-A5wt vec- The present study was undertaken to develop further tor contained the full-length human ␣5(IV) chain coding a gene therapy procedure for Alport syndrome. The first sequence,17 and the second one contained the same objective was to obtain expression of full-length human cDNA with an inserted FLAG-tag coding sequence. The ␣5 collagen cDNA with an adenovirus vector in cultured FLAG tag was used to enable easy purification of the cells, and explore whether the recombinant ␣5(IV) chain recombinant ␣5 chain, as well as to distinguish it from is incorporated into triple-helical molecules. Two con- the endogenous one in tissues following in vivo gene structs were made. One construct contained full-length transfer in pigs. The eight-residue FLAG tag sequence normal human ␣5(IV) chain cDNA, while the other one was placed in a 10-residue noncollagenous sequence in had an extra sequence, a FLAG-tag, that enables purifi- the fifth interruption in the collagenous domain of the ␣5 cation of the recombinant protein for functional studies, chain.
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