Encapsulated cells producing retroviral vectors for in vivo gene transfer.
BACKGROUND
Because gene therapy of the future will primarily take an in vivo approach, a number of problems associated with its current implementation exist. Currently, repeated delivery of a vector in vivo is necessary to ensure adequate transfer of the therapeutic gene. This may lead to the development of an immune response against the vector, thus interfering with gene delivery. To circumvent this problem, retroviral vector packaging cells that permanently produce recombinant retroviral vector particles have been encapsulated.


METHODS
Vector (pBAG)-producing amphotropic cells were encapsulated in beads composed of polymerized cellulose sulphate. These capsules were analysed in vitro for expression of the vector construct using X-gal staining, as well as for the release of particles by performing RT-PCR from culture supernatant. Infectivity studies were performed in vitro and in vivo. The latter was assayed using histological sections of the microcapsule and the surrounding area stained for beta-galactosidase activity and by RT-PCR.


RESULTS
In culture, the virus-producing cells inside the capsules remained viable and released virus into the culture medium for at least 6 weeks. To test whether these capsules, upon implantation into mice, also release vector virions that infect the surrounding cells, two different models were used. In the first, capsules were implanted in the fat pad of the mammary gland of Balb/c mice. The capsules were well tolerated for at least 6 weeks and a self-limiting inflammatory reaction without any other gross immune response was observed during this period. Furthermore, the virus-producing cells remained viable. In the second model, SCID mice were immunologically reconstituted by subcutaneous implantation of thymus lobes from MHC-identical Balb/c newborn mice and gene transfer into lymphoid cells was achieved by retroviral vectors released by co-implanted capsules.


CONCLUSIONS
The implantation of such capsules containing cells that continually produce retroviral vector particles may be of use for in vivo gene therapy strategies. The data presented demonstrate the feasibility of the concept.
Saller
Robert M
RM
Institute of Molecular Virology, GSF-Research Centre for Environment and Health, Oberschleissheim, Germany. saller@bavarian.nordic.deE
Indraccolo
Stefano
S
Coppola
Vincenzo
V
Esposito
Giovanni
G
Stange
Jan
J
Mitzner
Steffen
S
Amadori
Alberto
A
Salmons
Brian
B
Günzburg
Walter H
WH
D016475:3T3 Cells; D000818:Animals; D000831:Animals, Newborn; D001940:Breast / Q000378:metabolism; D002469:Cell Separation; D018920:Coculture Techniques; D004339:Drug Compounding; D005260:Female; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D051379:Mice; D008807:Mice, Inbred BALB C; D016513:Mice, SCID; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors