CONSTRUCTION AND ANALYSIS OF TRUNCATED MUSCLE-SPECIFIC PROMOTERS *Wang, B; *Zhou, LQ; *Kotchey, N; *Zhu, T; *Tian, MN; ** Jiang, XC; *Li, J; +*Xiao, X Luminometer Read, 40 sec. /mg protein Luciferase Activity + University of Pittsburgh, Pittsburgh, PA. [email protected] INTRODUCTION The tissue-specific promoters are highly CK6) showed significantly higher levels of luciferase expression desirable for the purpose of gene therapy in muscular disorders, but the compared with the original small MCK group (p<0.005). Particularly, full length muscle creatine kinase promoter (MCK) is not well suited for the luciferase activity generated by the tMCK promoter was 18-fold adeno-associated viral (AAV) vectors due to size constraints.1,2 Our aim higher than the original MCK promoter (Fig. 2). is to develop multiple, greatly compact, highly active yet extremely The CMV promoter revealed a high level of luciferase gene tissue-specific promoters, of suitable size to be packaged into AAV viral expression in 293 cells and the liver when compared with MCK vector containing the mini-dystrophin gene. Here we have constructed promoters (data not shown ), indicating that the modified MCK chimeric MCK promoters and compared the activity and tissuepromoters were highly active and also exceedingly tissue-specific. specificity of new promoters in vitro and in vivo. The dMCK-LacZ transgenic mice strongly expressed LacZ in skeletal MATERIALS AND METHODS Construction of MCK Promoters muscles at day 10. The results showed that whole bodies of transgenic and AAV Vector Production: The truncated chimeric promoters LacZ mice all were strongly positive. The high levels of Lac Z gene containing one, two, or three modified MCK enhancers (2R5S) along expressions were observed in the different muscles such as fore limbs with the minimal MCK promoter driving reporter luciferase gene (Luc) (FL), diaphragm muscle (DIA), intercostal muscles (ITM), hind limbs were packaged into AAV vector. The serotype II AAV vectors were (HL), and facial muscles (FAC) (Fig. 3). To determine the activity of made according to a previous protocol.3 the dMCK promoter in mdx mice, a small animal model commonly used Transfection of 293 Cell Line and Myotubes in vitro: Transfection for DMD, the Lac Z transgenic mice bred with either C57/BL10 or mdx of 293 cells was done by the calcium phosphate transfection method.4 mice. Both strain showed dramatic expression of the Lac Z gene in the Transfection of murine C2C12 myoblasts was performed by the incision of the hind limbs at 20 days without difference (Fig. 4). Lipofectamine Plus Reagent Kit. The cells were monitored in day 1, 2, 3, and 4 to observe differentiation. Mice and AAV Vectors Administration: The 2-month-old C57/BL10 mice were treated by AAV2-Luc viruses via IM in thehind leg tibialis anteriors (TA) with 2.5 x 1011 particles of virus. One month post–AAV virus treatment, TA muscle tissues were collected for luciferase activity assay. In addition, 2.5 x 1012 particles of AAV-luciferase vectors were injected into the same age mice via intrasplenic infusion to test tissue– specific expression and livers were collected 15 days after the Fig. 3 Fig. 4 intrasplenic injection. In further studies, GAS TA QUAD Luciferase Assay and Dystrophin Immunostaining: For the in vitro the 6-month-old Dysand in vivo studies, the luciferase activities were determined by a Tg mdx mice Luminometer. Immunostaining against dystrophin was performed demonstrated that the according to a previously described method.5 dMCK promoter has more activity in the Creation of Lac Z and Human Mini-Dystrophin Transgenic Mice: ITM DIA HEART fast skeletal muscles The transgenic mice were generated to test dMCK promoter activity 6 (GAS, TA, QUAD, according to standard procedures. The transgenic mice were identified and ITM), but weak by PCR procedures. The PCR positive founders of Lac Z were bred to or absent in slow C57/BL10 or mdx mice at 3-month-old via a breeding process. The Fig. 5 muscles human-mini-dystrophin transgenic males mated with homozygous mdx (DIA and heart) (Fig. 5), which is in agreement with previous results.7 females to obtain a DISCUSSION A short regulatory sequence with muscle-typehomozygous mdx The Activ ity of Lucifer ase dr iv en by D iffe rent specific activities were found within the 358-bp 5’flanking region.1 The background. Pr omoter s in C 2C12 enhancer activates the MCK promoter only in differentiated muscle RESULTS For the in 1000000 cells. 7 Several of these constructs contain a mutagenized MCK enhancer vitro C2C12 transfection 1 day that significantly increased gene transfer plus a truncated MCK promoter 100000 2 day s experiment, the results region. 1 The CK6 promoter containing 2R5S enhancer and a proximal 3 day s showed that the levels of 10000 4 day s promoter extending from -358 to +7 bp relative to the transcriptional luciferase activity start site was used to drive a mini-dystrophin gene. 8 However, we are 1000 achieved by the chimeric still seeking an optimal small promoter that would satisfy the 100 promoters, especially the Control sMC K- C K6- dC K4- tC K4- C MVrequirement for AAV vector delivery in muscle tissues. In these studies, Luc Luc Luc Luc Luc modified construct Fig. 1 we designed the two or three tandem modified MCK enhancers (2R5S) D ifferent P romoters dMCK (double-modified plus a minimal promoter region (-80 to +7 bp) to drive luciferase, LacZ, enhancers) and tMCK and human mini-dystrophin. The results confirmed that the new (triple-modified enhancers), were significantly higher (> 10 fold) than promoters with small size (500 to 750 bp) are suitable for gene vectors the original truncated MCK promoter (regular enhancer) in such as AAV, and can also benefit the gene therapy for DMD. differentiated C2C12 cells REFERENCES (Fig. 1). ∗ 1 Shield MA et al. Mol Cell Biol 1996; 16: 5058-5068. 100000 ** For in vivo animal 2 Hauser MA et al. Mol Ther 2000; 2: 16-25. ∗ ∗ ∗ studies, both the dMCK 3 Xiao X et al. J Virol 1998; 72: 2224-2232. 2 10000 and the CK6 promoter 4 Qiao C et al. J Virol 2002; 76: 13015-13027. activities demonstrated 5 Wang B et al. PNAS 2000; 97: 13714-13719. 1000 the similar strength to 6 Walsh A et al. J Biol Chem 1989; 264: 6488-6494. the CMV promoter in the 7 Dunant P et al. Mol Ther 2003; 8: 80-89. 100 TA muscle, while the 8 Yue Y et al. Circulation 2003; 108: 1626-1632. tMCK promoter’s AFFILIATED INSTITUTION FOR CO-AUTHORS * activity was 4-fold than Fig. 2 **The State University of New York, Brooklyn, NY ** the CMV promoter (p<0.005). All the modified enhancer MCK groups (dMCK, tMCK, and CMV-Luc, n=4 sMCK-Luc, n=4 CK6-Luc, n=4 dMCK-Luc, n=4 tMCK-Luc, n=4 Comparing with sMCK group, p< 0.005; Comparing with CMV group, p<0.005 52nd Annual Meeting of the Orthopaedic Research Society Paper No: 1000