ORIGINAL ARTICLE Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors A Schambach 1,2,6 , D Mueller 1 , M Galla 1 , MMA Verstegen 3 , G Wagemaker 3 , R Loew 4 , C Baum 1,5 and J Bohne 1 1 Department of Experimental Hematology, Hannover Medical School, Hannover, Germany; 2 Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany; 3 Department of Hematology, Erasmus MC, The Netherlands; 4 Eufets AG, Idar-Oberstein, Germany and 5 Division of Experimental Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA 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, gammaretro- viral 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 tran- scripts, we obtained a strong enhancement in titer (up to 4  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. Gene Therapy (2006) 13, 1524–1533. doi:10.1038/ sj.gt.3302807; published online 8 June 2006 Keywords: mouse leukemia virus; RNA processing; hematopoiesis Introduction Self-inactivating (SIN) retroviral vectors lack enhancer– promoter sequences in the U3 region of their long- terminal repeats (LTRs) and use internal cis-regulatory sequences to initiate transcription of a gene of interest. 1 The SIN design has several important advantages: it reduces the risk of recombination to replication-compe- tent retroviruses (RCR), impedes the mobilization of vector RNA in case of RCR superinfection, increases the autonomy of the internal promoter 1 and theoretically reduces the risk of insertional upregulation of neighbor- ing alleles depending on the choice of the internal enhancer/promoter. These features can be achieved without compromising the potency of the integrated transgene allele. Although the deletion of enhancer sequences from the LTR impairs overall transcript levels and increases 3 0 read-through, 2 improved RNA proces- sing of the internal transcript still allows the generation of SIN vectors that mediate comparable transgene expression levels as their LTR counterparts; 3 SIN vectors are thus of interest for a variety of applications in human gene therapy. On the basis of foamyvirus (FV) or lentiviruses such as the human immunodeficiency virus (HIV), SIN vectors can be produced from transiently transfected packaging cells, without substantial loss of titers compared to constructs containing intact LTR sequences. 4,5 In contrast, the first generations of gammaretroviral SIN vectors based on murine leukemia virus (MLV) suffered from strongly reduced titers. 1 While MLV vectors cannot transduce non-dividing cells, they still represent impor- tant tools for human gene therapy, because they do not require the incorporation of any sequences overlapping with coding sequences of gag, pol, env or accessory genes, 6 in contrast to the most common forms of vectors based on HIV or FV. 4,5 In addition, MLV SIN vectors are likely to increase the safety of human gene therapy protocols when used under conditions where MLV-based LTR vectors already show therapeutic efficiency. 7–10 Before the present study, the mechanisms responsible for the severe titer reduction of early generations of gammaretroviral SIN vectors were unclear. When we introduced the post-transcriptional element (PRE) from Woodchuck hepatitis virus into the 3 0 -untranslated region of SIN vectors, we were able to increase infectious titers above 10 6 transducing units per ml of unconcen- trated cell-free culture supernatant. 3,11 Owing to the Received 13 January 2006; revised 11 April 2006; accepted 13 April 2006; published online 8 June 2006 Correspondence: Dr J Bohne, Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strae 1, D-30625 Hannover, Germany. E-mail: bohne.jens@mh-hannover.de 6 Address vector requests to schambach.axel@mh-hannover.de Gene Therapy (2006) 13, 1524–1533 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt