Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.
Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.
Schambach
A
A
Department of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany. schambach.axel@mh-hannover.de
Mueller
D
D
Galla
M
M
Verstegen
M M A
MM
Wagemaker
G
G
Loew
R
R
Baum
C
C
Bohne
J
J
D000818:Animals; D018952:Antigens, CD34; D002460:Cell Line; D005434:Flow Cytometry; D017988:Gammaretrovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D008253:Macaca mulatta; D008297:Male; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000821:virology; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection; D038301:Virus Inactivation
D018952:Antigens, CD34