Transfer of lacZ marker gene to the meniscus.
BACKGROUND
Lesions in the avascular two-thirds of the meniscus do not heal well and are of concern clinically. Various growth factors promote the synthesis of matrix by meniscal cells and thus have the potential to augment healing. However, their clinical application is severely hindered by problems with delivery. An attractive approach to overcoming such problems is to transfer genes that encode the growth factors in question to the site of the injury. As a prelude to this, we evaluated methods for delivering genes to the meniscus.


METHODS
Gene transfer was evaluated in vitro and in vivo with a lacZ marker gene, which expresses the enzyme beta-galactosidase. Two types of vectors were tested: an adenovirus and a retrovirus. Monolayers of lapine, canine, and human meniscal cells, as well as intact lapine and human menisci, were used for the in vitro studies. Lesions were created in the menisci of rabbits and dogs for the in vivo studies. Gene transfer to the sites of the experimental meniscal lesions in vivo was accomplished in two ways. In the lapine model, a suspension of adenovirus carrying the lacZ marker gene was mixed with whole blood and the clot was inserted into the lesion. In the canine model, retrovirally transduced allogenic meniscal cells carrying the lacZ marker gene were embedded in collagen gels and transferred to the defects. The animals were killed at various time-points, and gene expression was evaluated by histological examination of sections stained with 5-bromo-4-chloro-indolyl-beta-D-galactose (X-gal), from which a blue chromagen is released in the presence of beta-galactosidase.


RESULTS
Monolayer cultures of lapine, canine, and human meniscal cells were susceptible to genetic transduction by both adenoviral and retroviral vectors. In vitro gene transfer to intact human and lapine menisci proved possible both by direct, adenoviral, delivery and indirect, retroviral, delivery. Gene expression persisted for at least twenty weeks under in vitro conditions. With regard to the in vivo studies, gene expression persisted within the clot and in some of the adjacent meniscal cells for at least three weeks in the lapine defect model. In the canine defect model, gene expression persisted within the transplanted, transduced meniscal cells for at least six weeks.


CONCLUSIONS
It is possible to transfer genes to sites of meniscal damage and to express them locally within the lesion for several weeks.
Goto
H
H
Ferguson Laboratory, University of Pittsburgh School of Medicine, Pennsylvania 15213, USA.
Shuler
F D
FD
Lamsam
C
C
Moller
H D
HD
Niyibizi
C
C
Fu
F H
FH
Robbins
P D
PD
Evans
C H
CH
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D018014:Gene Transfer Techniques; D005819:Genetic Markers / Q000235:genetics*; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007763:Lac Operon / Q000235:genetics*; D008592:Menisci, Tibial / Q000473:pathology*; D011817:Rabbits; D014945:Wound Healing / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics
D005819:Genetic Markers; D001616:beta-Galactosidase