EDC/NHS-mediated heparinization of small intestinal submucosa for recombinant adeno-associated virus serotype 2 binding and transduction.
A major challenge in the use of gene transfer vectors as therapeutic tools is controlling vector administration at a desired tissue site. One potential solution is implanting tissue-engineering constructs loaded with gene transfer vectors such as viruses for localized transgene delivery. In this work, we conjugated recombinant adeno-associated virus serotype 2 (rAAV2) to a heparinized small intestinal submucosa (H-SIS) matrix, which resulted in vector transduction upon cellular adhesion. H-SIS was prepared by incorporating heparin, the rAAV2 receptor, into SIS through N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) mediated crosslinking. Incorporated heparin adsorbed rAAV2 onto the H-SIS matrix for conjugation. Using green fluorescent protein and beta-galactosidase as reporters, we showed that conjugated rAAV2 was active and capable of mediating transgene delivery in cell culture. Additionally, we applied H-SIS to adsorb unpurified rAAV2 from the crude lysate of packaging cells for conjugation, avoiding the use of ultracentrifugation or chromatography in preparation of infectious rAAV2 for transduction. Our work provides a unique, modified tissue substrate H-SIS for rAAV2 binding and transduction, which can be a useful tool in developing localized gene transfer.
Yue
Tse-Wei
TW
Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang Fu Road, Hsinchu 300, Taiwan.
Chien
Wei-Che
WC
Tseng
S-Ja
SJ
Tang
Shiue-Cheng
SC
D002234:Carbodiimides / Q000494:pharmacology*; D002448:Cell Adhesion; D002460:Cell Line; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D005354:Fibrosarcoma / Q000473:pathology; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D008156:Luciferases / Q000378:metabolism; D012703:Serotyping; D013388:Succinimides / Q000494:pharmacology*; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C518491:1-ethyl-3-(3-(diethylamino)propyl)carbodiimide; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D013388:Succinimides; D049452:Green Fluorescent Proteins; D006493:Heparin; D008156:Luciferases; D001616:beta-Galactosidase; C001426:N-hydroxysuccinimide