Approaches to enhancing the retroviral transduction of human synoviocytes.
This report concerns a clinical trial for rheumatoid arthritis (RA), approved by the US National Institutes of Health and the Food and Drug Administration. An amphotropic retrovirus (MFG-IRAP) was used ex vivo to transfer a cDNA encoding human interleukin-1 receptor antagonist (IL-1Ra) to synovium. The protocol required the transduced cells to secrete at least 30 ng IL-1Ra/10(6) cells per 48 h before reimplantation. Here we have evaluated various protocols for their efficiency in transducing cultures of human rheumatoid synoviocytes. The most reliably efficient methods used high titer retrovirus (approximately 10(8) infectious particles/ml). Transduction efficiency was increased further by exposing the cells to virus under flow-through conditions. The use of dioctadecylamidoglycylspermine (DOGS) as a polycation instead of Polybrene (hexadimethrine bromide) provided an additional small increment in efficiency. Under normal conditions of static transduction, standard titer, clinical grade retrovirus (approximately 5 x 10(5) infectious particles/ml) failed to achieve the expression levels required by the clinical trial. However, the shortfall could be remedied by increasing the time of transduction under static conditions, transducing under flow-through conditions, or transducing during centrifugation.
Del Vecchio
M A
MA
Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, PA, USA. cevans@rics.bwh.harvard.edu
Georgescu
H I
HI
McCormack
J E
JE
Robbins
P D
PD
Evans
C H
CH
D001172:Arthritis, Rheumatoid / Q000235:genetics / Q000601:surgery / Q000628:therapy; D002478:Cells, Cultured; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D012190:Retroviridae / Q000235:genetics*; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics / Q000627:therapeutic use; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*
C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D012795:Sialoglycoproteins