1. ¹Department of Experimental Hematology, Hannover Medical School, Hannover, Germany
2. ²Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany
3. ³Department of Hematology, Erasmus MC, The Netherlands
4. ⁴Eufets AG, Idar-Oberstein, Germany
5. ⁵Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA

Correspondence: Dr J Bohne, Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strae 1, D-30625 Hannover, Germany. E-mail: bohne.jens@mh-hannover.de

⁶Address vector requests to schambach.axel@mh-hannover.de

Received 13 January 2006; Revised 11 April 2006; Accepted 13 April 2006; Published online 8 June 2006.

Abstract

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 4 10⁷ 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.