A Muscle Hybrid Promoter as a Novel Tool for Gene Therapy

Abstract

Gene therapy is a promising strategy to cure rare diseases. The lack of regulatory sequences ensuring specific and robust expression in skeletal and cardiac muscle is a substantial limitation of gene therapy efficiency targeting the muscle tissue. Here we describe a novel muscle hybrid (MH) promoter that is highly active in both skeletal and cardiac muscle cells. It has an easily exchangeable modular structure, including an intronic module that highly enhances the expression of the gene driven by it. In cultured myoblasts, myotubes, and cardiomyocytes, the MH promoter gives relatively stable expression as well as higher activity and protein levels than the standard CMV and desmin gene promoters or the previously developed synthetic or CKM-based promoters. Combined with AAV2/9, the MH promoter also provides a high in vivo expression level in skeletal muscle and the heart after both intramuscular and systemic delivery. It is much more efficient than the desmin-encoding gene promoter, and it maintains the same specificity. This novel promoter has potential for gene therapy in muscle cells. It can provide stable transgene expression, ensuring high levels of therapeutic protein, and limited side effects because of its specificity. This constitutes an improvement in the efficiency of genetic disease therapy.

Keywords: AAV; C2C12; expression cassette; heart; muscle promoter; myotubes; skeletal muscles; vector.

Figure 1

The MH Promoter Is Composed of 4 Main Modules that Together Are Predicted to Efficiently Bind Transcription Factors (A) Analysis of the Des gene (encoding desmin) promoter, from 2,500 bp before the TSS to the first intron inclusive, revealed predicted cluster-binding TFs (enhancer) in positions −970 bp to −826 bp according to the TSS and the second cluster in the final part of intron 1. (B) Analysis of the Ckm promoter from 2,500 bp before the TSS to the start codon (including the first intron) revealed 3 predicted main cluster-binding TFs. The strongest cluster corresponded to an enhancer identified from −1,256 bp to −1,050 bp, the second one was identified within intron 1, and the third cluster corresponded to the core promoter (from −358 bp to +7 bp). (C) Functional elements of the promoter were chosen using in silico analysis and then optimized and combined to give a modular structure. The MH promoter is composed of the following linked modules: (1) the Des gene enhancer (enh1); (2) the Ckm gene enhancer (enh2); (3) the Ckm gene core promoter (with modifications within the proximal promoter [pp]); and (4) a designed intron consisting of a SIE derived from the Ckm gene. Moreover, the 5′ UTR derived from the Ckm gene, including potential sequences enhancing translation, was inserted after the designed intron. Around the TSS, the following sequences occur: TATA box, Inr, and DPE. (D) Analysis of the MH promoter revealed predicted, very strong cluster-binding TFs localized through almost the entire 1,030-bp sequence. (A), (B), and (D) were prepared using Cister software.

Figure 2

The MH Promoter Is Highly Active in Muscle Cells (A–C) Proliferating C2C12 cells (A), C2C12 cells differentiated to myotubes (B), and H9C2 cells (C) were transfected with constructs encoding secretory luciferase under the control of different promoters. Luciferase and enzyme activity was measured based on luminescence 48 h after transfection. The MH promoter was more active than the DES or CMV promoters but less active than the constitutive promoter of EF1a. During C2C12 differentiation, the activity of the DES promoter increased, whereas the activity of the CMV promoter decreased. However, the activity of the MH promoter was higher than that of these two promoters. 4 biological replicates were performed, with 4 technical replicates each. Boxes represent 25/75 percentiles, with the median value and whiskers representing minimum and maximum values. RLU, relative luminescence units normalized to transfection efficiency and total protein amount. Values are presented using a log₁₀ scale.

Figure 3

The Key Element of the MH Promoter Is an Intron-Containing SIE (A) A set of MH promoter variants lacking particular module(s) was prepared. (B–F) Cells were transfected, and luciferase activity was measured using luminescence in the following cell lines: (B) C2C12 myoblasts, (C) C2C12 myotubes, (D) H9C2 cardiomyocytes, (E) HEK293 cells, and (F) HepG2 cells. Deletion of any element decreased MH promoter activity at least by half in all muscle cells but not in HEK293 or HepG2 cells. Deletion of the intron module decreased MH promoter activity most significantly, to a much greater degree than deletion of the core/pp. When the pp was deleted, there was no difference in whether the TATA box remained or was deleted, suggesting that this regulatory cassette has low significance when other cassettes (Inr or DPE) are in proximity. 3 biological replicates were performed, with 4 technical replicates each. Boxes represent 25/75 percentiles, with the median value and whiskers representing minimum and maximum values (*p < 0.01; **p < 0.05; ***p < 0.001; ****p < 0.0001; n/s, not significant). Values are presented using a log₁₀ scale.

Figure 4

MH Promoter Activity and Specificity (A) The MH promoter is specific for muscle cells. The MH promoter was more active than the CMV promoter in muscle cells (67-fold difference in myotubes). The activity of the MH promoter was at least 5-fold lower than the activity of the CMV promoter in all control non-muscle cell lines (HEK293, NHDF, HepG2, HeLa, and HDMEC cells). The MH promoter was more active than the DES promoter in all the examined cell lines (data not shown). Bars represent mean values with SD. 3 biological replicates were performed, each in 4 technical replicates normalized to transfection efficiency and total protein amount. Values are presented using a log₁₀ scale. (B) The MH promoter has favorable kinetics activity in C2C12 cells. C2C12 cells were transfected with constructs encoding secretory luciferase under control of various promoters, and luciferase activity was measured based on luminescence in the days following transfection. MH promoter-controlled expression starts later than that of the CMV promoter, but its expression lasts longer. The luminescence intensity decrease after 72 h is due to plasmid loss in cells after division because C2C12 cells proliferate very rapidly. 3 biological replicates were performed, with 4 technical replicates each. Bars represent mean values with SDs (*p < 0.01, **p < 0.05, ***p < 0.001). Values are presented using a log₁₀ scale.

Figure 5

The MH Promoter Is Highly Active in Both Skeletal and Heart Muscle In Vivo after Intramuscular Delivery Newborn wild-type mice were injected into TA muscle with AAV2/9 encoding EGFP under control of the MH (n = 3), DES (n = 3), or CMV (n = 4) promoters. Tissues were analyzed 6 weeks thereafter. The MH promoter provided the highest expression level in muscle tissue and lower expression compared with the CMV promoter in the liver and lungs. The DES promoter always led to the lowest expression in all tissues. (A) qPCR analysis showed that the MH promoter provides the highest expression levels in comparison with CMV or DES promoters in muscle tissues. The opposite effect was observed in the lungs and liver, where CMV promoter-driven expression was higher than for the MH promoter. DES promoter-controlled expression was the lowest in all tissues and comparable with MH in the lung. Each point represents the mean expression level in particular tissue for each mouse in 4 technical replicates. The horizontal line represents the mean value for all mice (*p < 0.01, **p < 0.05, ***p < 0.001, ****p < 0.0001). Values are presented using a log₁₀ scale. (B) For gastrocnemius muscle, the Egfp expression level was normalized to both the Rplp0 expression level and to the vector copy number, showing that the MH promoter provides an expression level 2.7 times higher than the CMV promoter and 150 times higher than the DES promoter in cells transduced with the AAV. (C) Analysis of representative injected TA muscles from mice treated with different vectors. Nuclei were stained with DAPI (blue). When EGFP (green) was detected, it was homogeneously distributed throughout the muscle section.

Figure 6

Systemic Delivery of the AAV with the MH Promoter Provides a High Level of Expression in Muscles and a Low Level of Expression in Other Analyzed Tissues (A–D) Newborn wild-type mice were injected systemically via the temporal vein with AAV2/9 vectors encoding EGFP under the control of the CMV, MH, or DES promoters. Tissues were analyzed 8 weeks thereafter via qPCR (A and B) and western blotting (C and D). The MH promoter provided high expression and protein levels in muscle tissues, comparable with those for the CMV promoter, and a lower level in other tested tissues, comparable with that for the DES promoter. (A) qPCR analysis of muscle tissues showed comparable expression levels driven by the CMV and MH promoters in the heart and slightly higher levels for CMV than MH in skeletal muscle. (B) qPCR analysis of selected control non-muscle tissues showed the highest expression levels for the CMV promoter. The MH and DES promoters showed comparable expression levels in these tissues. In (A) and (B), each point represents the mean expression level (4 technical replicates) in that particular tissue for each mouse (n = 5). The horizontal line represents the mean value (*p < 0.01, **p < 0.05, ***p < 0.001, ****p < 0.0001). Values are presented using a log₁₀ scale. (C) Western blot analysis of the heart, quadriceps and gastrocnemius muscles, and liver were performed. For the DES promoter, three mice with the highest expression level quantified with qPCR were analyzed, but EGFP was detectable only in the heart. In the heart, the highest protein level was observed for the MH promoter. In skeletal muscle, the EGFP protein levels were slightly higher for the MH than for the CMV promoter. In the liver, the highest protein level was observed for the CMV promoter, with a barely detectable level for the MH promoter. For the lungs, brain, small intestine, and kidneys, EGFP detection signals were low for all promoters (data not shown). (D) Densitometry analysis of the western blot staining presented in (C). Bars represent the mean EGFP band intensity normalized to loading control bands. Whiskers represent SDs.