微孔結構所組成纖維膜具有低質傳效應的優勢，因此在專一性吸附之薄膜層析法中已廣泛使用。在本研究中，由於第二型重組腺病毒之主要受器為heparan sulfate proteoglycans，因此我們運用了肝素化纖維膜直接用來吸附第二型重組腺病毒，藉由此特點發展出一種受質調控型之病毒載體釋放的新穎應用。首先，在我們的研究發現肝素化纖維膜與普遍細胞培養之材料聚乙烯的細胞生長與貼附情況具有類似的效果。因此我們直接將綠色螢光蛋白 (green fluorescent protein GFP)、□-半乳糖分解酶 (□-galactosidase)、冷光蛋白酶 (luciferase)這些報導基因之未純化病毒細胞混合液通過肝素化纖維膜，吸附細胞液中的病毒，再將人類纖維細胞移殖至該膜上，直到細胞貼附於肝素化纖維膜，而原先吸附於膜上病毒將對細胞進行轉導作用。我們證明肝素化纖維膜確實可以用來吸附細胞液中的病毒，吸附在膜上病毒仍然具有活性，可以將基因釋放於細胞中並表現出該報導基因。此研究方法可以避免傳統上利用超高速離心法和層析法製程rAAV2繁雜的缺點。此研究結果證明了肝素化纖維膜之專一性吸附確實可以運用在受質調控型病毒載體傳遞之新穎應用，這對於發展局部性基因傳遞是非常重要工具。

The microporous affinity membrane based on cellulose matrices offers minimal mass-transfer effects in membrane chromatography with low nonspecific adsorption. In this research, we tested a novel application of the microporous, heparinized cellulose matrices (H-CM) for their affinity toward recombinant adeno-associated virus serotype 2 (rAAV2, which uses heparan sulfate proteoglycans as the primary cellular receptor) to develop a controlled, substrate-mediated viral vector delivery. We adsorb rAAV2 from the crude lysate of packaging cells to an epoxy-crosslinked heparin cellulose membrane, which led to vector transduction upon cellular adhesion. When adhered, the human HT-1080 fibroblasts exhibited proliferation kinetics similar to those on the standard polystyrene tissue-culture surface. Using green fluorescent protein and beta-galactosidase as reporters, we showed that the heparin-bound rAAV2 particles remained active and that the rAAV2-heparin binding was reversible and capable of mediating transgene delivery in cell culture. This method avoids the use of conventional ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work explores a new application of affinity cellulose matrices in substrate-mediated viral vector delivery, which can be a useful tool in developing protocols for localized gene transfer.

1. Sevillano G, Rodriguezpuyol M, Martos R, Duque I, Lamas S, Diezmarques ML, et al. Cellulose-Acetate Membrane Improves Some Aspects of Red-Blood-Cell Function in Hemodialysis-Patients. Nephrology Dialysis Transplantation 1990 Jul;5(7):497-499.
2. Burhop KE, Johnson RJ, Simpson J, Chenoweth DE, Borgia J. Biocompatibility of Hemodialysis Membranes - Evaluation in an Ovine Model. Journal of Laboratory and Clinical Medicine 1993 Feb;121(2):276-293.
3. Chang Q, Murtaza Z, Lakowicz JR, Rao G. A fluorescence lifetime-based solid sensor for water. Analytica Chimica Acta 1997 Sep 10;350(1-2):97-104.
4. Kostov Y, Tzonkov S, Yotova L, Krysteva M. Membranes for Optical Ph Sensors. Analytica Chimica Acta 1993 Aug 2;280(1):15-19.
5. Entcheva EG, Yotova LK. Analytical Application of Membranes with Covalently Bound Glucose-Oxidase. Analytica Chimica Acta 1994 Dec 30;299(2):171-177.
6. Desai SD, Blanchard J. In vitro evaluation of pluronic F127-based controlled-release ocular delivery systems for pilocarpine. Journal of Pharmaceutical Sciences 1998 Feb;87(2):226-230.
7. Doheny JG, Jervis EJ, Guarna MM, Humphries RK, Warren RAJ, Kilburn DG. Cellulose as an inert matrix for presenting cytokines to target cells: production and properties of a stem cell factor-cellulose-binding domain fusion protein. Biochemical Journal 1999 Apr 15;339:429-434.
8. Sardonini C, DiBiasio D. Design and operating criteria for hollow fiber bioreactors. Bioprocess Engineering 1996 Nov;15(6):327-330.
9. Ko IK, Iwata H. An approach to constructing three-dimensional tissue. Bioartificial Organs Iii: Tissue Sourcing, Immunoisolation, and Clinical Trials 2001;944:443-455.
10. Katoh R, Urist MR. Surface-Adhesion and Attachment Factors in Bone Morphogenetic Protein-Induced Chondrogenesis in-Vitro. Clinical Orthopaedics and Related Research 1993 Oct(295):295-304.
11. Martson M, Viljanto J, Hurme T, Saukko P. Biocompatibility of cellulose sponge with bone. European Surgical Research 1998 Nov-Dec;30(6):426-432.
12. Takata T, Wang HL, Miyauchi M. Migration of osteoblastic cells on various guided bone regeneration membranes. Clinical Oral Implants Research 2001 Aug;12(4):332-338.
13. De Bartolo L, Morelli S, Bader A, Drioli E. Evaluation of cell behaviour related to physico-chemical properties of polymeric membranes to be used in bioartificial organs. Biomaterials 2002 Jun;23(12):2485-2497.
14. Kino Y, Sawa M, Kasai S, Mito M. Multiporous cellulose microcarrier for the development of a hybrid artificial liver using isolated hepatocytes. Journal of Surgical Research 1998 Sep;79(1):71-76.
15. Yang MB, Vacanti JP, Ingber DE. Hollow Fibers for Hepatocyte Encapsulation and Transplantation - Studies of Survival and Function in Rats. Cell Transplantation 1994 Sep-Oct;3(5):373-385.
16. Risbud MV, Bhonde RR. Suitability of cellulose molecular dialysis membrane for bioartificial pancreas: In vitro biocompatibility studies. Journal of Biomedical Materials Research 2001 Mar 5;54(3):436-444.
17. Cullen B, Watt PW, Lundqvist C, Silcock D, Schmidt RJ, Bogan D, et al. The role of oxidised regenerated cellulose/collagen in chronic wound repair and its potential mechanism of action. International Journal of Biochemistry & Cell Biology 2002 Dec;34(12):1544-1556.
18. Ovington LG. Overview of matrix metalloprotease modulation and growth factor protection in wound healing. Part 1. Ostomy Wound Manage 2002;48(6 Suppl):3-7
19. Miyamoto T, Takahashi S, Ito H, Inagaki H, Noishiki Y. Tissue Biocompatibility of Cellulose and Its Derivatives. Journal of Biomedical Materials Research 1989 Jan;23(1):125-133.
20. Martson M, Viljanto J, Hurme T, Laippala P, Saukko P. Is cellulose sponge degradable or stable as implantation material? An in vivo subcutaneous study in the rat. Biomaterials 1999 Nov;20(21):1989-1995.
21. Moss RB, Rochman D, Spencer LT, Aitken ML, Zeitlhi PL, Waltz D, et al. Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis - A multicenter, double-blind, placebo-controlled trial. Chest 2004 Feb;125(2):509-521.
22. Manno CS, Arruda VR, Pierce GF, Glader B, Ragni M, Rasko J, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response (vol 12, pg 342, 2006). Nature Medicine 2006 May;12(5):592-592.
23. Marshall E. Clinical trials - Gene therapy death prompts review of adenovirus vector. Science 1999 Dec 17;286(5448):2244-2245.
24. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. New England Journal of Medicine 2003 Jan 16;348(3):255-256.
25. Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. Journal of Virology 1998 Feb;72(2):1438-1445.
26. Auricchio A, Hildinger M, O'Connor E, Gao GP, Wilson JM. Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column. Human Gene Therapy 2001 Jan 1;12(1):71-76.
27. Pajusola K, Gruchala M, Joch H, Luscher TF, Yla-Herttuala S, Bueler H. Cell-type-specific characteristics modulate the transduction efficiency of adeno-associated virus type 2 and restrain infection of endothelial cells. Journal of Virology 2002 Nov;76(22):11530-11540.
28. Mizuguchi H, Hayakawa T. Targeted adenovirus vectors. Human Gene Therapy 2004 Nov;15(11):1034-1044.
29. Muzyczka N, Warrington KH. Custom adeno-associated virus capsids: The next generation of recombinant vectors with novel tropism. Human Gene Therapy 2005 Apr;16(4):408-416.
30. Ponnazhagan S, Mahendra G, Kumar S, Thompson JA, Castillas M. Conjugate-based targeting of recombinant adeno-associated virus type 2 vectors by using avidin-linked ligands. Journal of Virology 2002 Dec;76(24):12900-12907.
31. Raty JK, Airenne KJ, Marttila AT, Marjomaki V, Hytonen VP, Lehtolainen P, et al. Enhanced gene delivery by avidin-displaying baculovirus. Molecular Therapy 2004 Feb;9(2):282-291.
32. Yun YH, Goetz DJ, Yellen P, Chen WL. Hyaluronan microspheres for sustained gene delivery and site-specific targeting. Biomaterials 2004 Jan;25(1):147-157.
33. Pandori MW, Hobson DA, Sano T. Adenovirus-microbead conjugates possess enhanced infectivity: A new strategy for localized gene delivery. Virology 2002 Aug 1;299(2):204-212.
34. Scherer F, Anton M, Schillinger U, Henkel J, Bergemann C, Kruger A, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy 2002 Jan;9(2):102-109.
35. Segura T, Chung PH, Shea LD. DNA delivery from hyaluronic acid-collagen hydrogels via a substrate-mediated approach. Biomaterials 2005 May;26(13):1575-1584.
36. Guo T, Zhao JN, Chang JB, Ding Z, Hong H, Chen JN, et al. Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor-beta 1 for chondrocytes proliferation. Biomaterials 2006 Oct;27(7):1095-1103.
37. Huang YC, Riddle K, Rice KG, Mooney DJ. Long-term in vivo gene expression via delivery of PEI-DNA condensates from porous polymer scaffolds. Human Gene Therapy 2005 May;16(5):609-617.
38. De Laporte L, Rea JC, Shea LD. Design of modular non-viral gene therapy vectors. Biomaterials 2006 Oct;27(7):947-954.
39. Stachelek SJ, Song C, Alferiev I, Defelice S, Cui X, Connolly JM, et al. Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants. Gene Therapy 2004 Jan;11(1):15-24.
40. Levy RJ, Song C, Tallafragada S, DeFelice S, Hinson JT, Vyavahare N, et al. Localized adenovirus gene delivery using antiviral IgG complexation. Gene Therapy 2001 May;8(9):659-667.
41. Gu DL, Nguyen T, Gonzalez AM, Printz MA, Pierce GF, Sosnowski BA, et al. Adenovirus encoding human platelet-derived growth factor-B delivered in collagen exhibits safety, biodistribution, and immunogenicity profiles favorable for clinical use. Molecular Therapy 2004 May;9(5):699-711.
42. Bellocq NC, Kang DW, Wang XH, Jensen GS, Pun SH, Schluep T, et al. Synthetic biocompatible cyclodextrin-based constructs for local gene delivery to improve cutaneous wound healing. Bioconjugate Chemistry 2004 Nov-Dec;15(6):1201-1211.
43. Koefoed M, Ito H, Gromov K, Reynolds DG, Awad HA, Rubery PT, et al. Biological effects of rAAV-caAlk2 coating on structural allograft healing. Molecular Therapy 2005 Aug;12(2):212-218.
44. Ito H, Koefoed M, Tiyapatanaputi P, Gromov K, Goater JJ, Carmouche J, et al. Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy. Nature Medicine 2005 Mar;11(3):291-297.
45. Burova E, Loffe E. Chromatooraphic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications. Gene Therapy 2005 Oct;12:S5-S17.
46. Duffy AM, O'Doherty AM, O'Brien T, Strappe PM. Purification of adenovirus and adeno-associated virus: Comparison of novel membrane-based technology to conventional techniques. Gene Therapy 2005 Oct;12:S62-S72.
47. Blouin V, Brument N, Toublanc E, Raimbaud I, Moullier P, Salvetti A. Improving rAAV production and purification: towards the definition of a scaleable process. Journal of Gene Medicine 2004 Feb;6:S223-S228.
48. Brument N, Morenweiser R, Blouin V, Toublanc E, Raimbaud I, Cherel Y, et al. A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and-5. Molecular Therapy 2002 Nov;6(5):678-686.
49. Tsai YH, Wang MY, Suen SY. Purification of hepatocyte growth factor using polyvinyldiene fluoride-based immobilized metal affinity membranes: equilibrium adsorption study. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2002 Jan 5;766(1):133-143.
50. Chen WY, Wu CF, Liu CC. Interactions of imidazole and proteins with immobilized Cu(II) ions: Effects of structure, salt concentration, and pH in affinity and binding capacity. Journal of Colloid and Interface Science 1996 Jun 1;180(1):135-143.
51. Wu CF, Chen WY, Liu HS. Determination of the Binding Constant of Imidazole and Histidine with Immobilized Cu(Ii) by Differential Uv Spectroscopy. Journal of Chemical Engineering of Japan 1995 Aug;28(4):419-424.
52. Belew M, Porath J. Immobilized Metal-Ion Affinity-Chromatography - Effect of Solute Structure, Ligand Density and Salt Concentration on the Retention of Peptides. Journal of Chromatography 1990 Sep 21;516(2):333-354.