Generation of stable retrovirus packaging cell lines after transduction with herpes simplex virus hybrid amplicon vectors.
BACKGROUND
A number of properties have relegated the use of Moloney murine leukemia virus (Mo-MLV)-based retrovirus vectors primarily to ex vivo protocols. Direct implantation of retrovirus producer cells can bypass some of the limitations, and in situ vector production may result in a large number of gene transfer events. However, the fibroblast nature of most retrovirus packaging cells does not provide for an effective distribution of vector producing foci in vivo, especially in the brain. Effective development of new retrovirus producer cells with enhanced biologic properties may require the testing of a large number of different cell types, and a quick and efficient method to generate them is needed.


METHODS
Moloney murine leukemia virus (Mo-MLV) gag-pol and env genes and retrovirus vector sequences carrying lacZ were cloned into different minimal HSV/AAV hybrid amplicons. Helper virus-free amplicon vectors were used to co-infect glioma cells in culture. Titers and stability of retrovirus vector production were assessed.


RESULTS
Simultaneous infection of two glioma lines, Gli-36 (human) and J3T (dog), with both types of amplicon vectors, generated stable packaging populations that produced retrovirus titers of 0.5-1.2 x 10(5) and 3.1-7.1 x 10(3) tu/ml, respectively. Alternatively, when cells were first infected with retrovirus vectors followed by infection with HyRMOVAmpho amplicon vector, stable retrovirus packaging populations were obtained from Gli-36 and J3T cells producing retrovirus titers comparable to those obtained with a traditional retrovirus packaging cell line, Psi CRIPlacZ.


CONCLUSIONS
This amplicon vector system should facilitate generation of new types of retrovirus producer cells. Conversion of cells with migratory or tumor/tissue homing properties could result in expansion of the spatial distribution or targeting capacity, respectively, of gene delivery by retrovirus vectors in vivo.
Molecular Neurogenetics Unit and Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
Miguel
M
Sena-Esteves
Jürgen A
JA
Hampl
Sara M
SM
Camp
Xandra O
XO
Breakefield
D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D005784:Gene Amplification; D015316:Genetic Therapy; D005822:Genetic Vectors; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008979:Moloney murine leukemia virus / Q000502:physiology*; D012367:RNA, Viral / Q000235:genetics; D018139:Simplexvirus / Q000235:genetics*; D014161:Transduction, Genetic; D019065:Virus Assembly
D012367:RNA, Viral