A NOVEL MUSCLE-SPECIFIC PROMOTER

Abstract

The present invention concerns a novel short promoter characterized by a high activity in the skeletal muscles and a low activity in the heart. It then constitutes a valuable candidate especially for driving the expression of transgenes encoding proteins useful for the treatment of muscular dystrophies.

Claims

1. A promoter comprising: the sequence SEQ ID NO: 2; or a sequence having identity greater than or equal to 90% with SEQ ID NO: 2.

2. The promoter according to claim 1, wherein its size does not exceed 350 nucleotides.

3. The promoter according to claim 1, wherein it ensures an expression level in the skeletal muscles higher than in the heart.

4. The promoter according to claim 1, wherein it comprises or consists of the sequence SEQ ID NO: 2.

5. An expression system comprising a promoter comprising: a sequence of SEQ ID NO: 2 or a sequence having identity greater than or equal to 90% with SEQ ID NO: 2; and a transgene placed under the control of said promoter.

6. The expression system according to claim 5, wherein the transgene encodes a protein selected from the group consisting of: dystrophin, HSP-40 homologue B6, calpain 3, dysferlin, sarcoglycan, FKRP (Fukutin-Related Protein) and Anoctamin5.

7. The expression system according to claim 6, wherein the calpain 3 protein has the sequence SEQ ID NO: 8.

8. The expression system according to claim 5, further comprising at least one additional sequence selected from the group consisting of: a sequence for transcript stabilization; a polyadenylation signal; an enhancer sequence; and a target sequence of a microRNA.

9. The expression system according to claim 8, wherein it further comprises at least one target sequence of miR208a.

10. The expression system according to claim 5, wherein it comprises a viral vector.

11. A pharmaceutical composition comprising an expression system comprising a promoter comprising: a sequence of SEQ ID NO: 2 or a sequence having identity greater than or equal to 90% with SEQ ID NO: 2; and a transgene placed under the control of said promoter.

12. (canceled)

13. (canceled)

14. A method of treating a neuromuscular disease, comprising: administering to a subject in need a pharmaceutical composition comprising an expression system comprising a promoter comprising: a sequence of SEQ ID NO: 2 or a sequence having identity greater than or equal to 90% with SEQ ID NO: 2; and a transgene placed under the control of said promoter.

15. The method of claim 14, wherein the composition is administered systemically.

16. The method of claim 14, wherein the disease is selected from the group consisting of muscular dystrophies, congenital muscular dystrophies, congenital myopathies, distal myopathies, myopathies, myotonic syndromes, ion channel muscle diseases, malignant hyperthermia, metabolic myopathies, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), limb-girdle muscular dystrophy 2A (LGMD2A), LGMD2B, LGMD2D, LGMD2I, LGMD1D, and LGMD2L.

17. The expression system of claim 6, wherein the dystrophin is microdystrophin, minidystrophin, or quasidystrophin.

18. The expression system of claim 8, wherein the polyadenylation signal comprises a polyA of the transgene, the polyA of SV40, or the polyA of human β globin (HBB2).

19. The expression system of claim 10, wherein the viral vector is an adeno associated viral vector (AAV).

20. The expression system of claim 19, wherein the AAV comprises a capsid selected from the group consisting of AAV8 capsid, AAV9 capsid, AAV9 rh74 capsid, and AAV9 rh74 P1 capsid.

Description

FIGURES

[0239] FIG. 1: Luciferase activity of GFP-Luc transgene in C57Bl6 albino mice (4 males/15 promoter) injected with 5.sup.e13 vg/kg AAV9-promoter-GFP-Luc (promoter=Desmin or ACTA1)

[0240] A/ Imaging analysis of the injected mice

[0241] B/ Luciferase activity normalized by total protein amount in the different organs (TA: tibialis anterior muscle; diaphragm; heart; liver; kidney; adr glands: adrenal glands)

[0242] FIG. 2: Calpain 3 expression in the TA (tibialis anterior) muscle and in the heart of mice (2 or 3 males/promoter) not injected (NI) or injected with 1.sup.c14 vg/kg AAV9-promoter-hCalpain3-2×target-miR208a

[0243] 2T: 2×target-miR208a

[0244] promoter=Desmin: Des or ACTA1: ACTA

[0245] A/ at RNA level by qRT-PCR after normalization with Rplp0

[0246] B/ at protein level by western blot using an antibody directed against calpain 3.

MATERIALS AND METHODS

[0247] Animal Models

[0248] The animal studies were performed in accordance to the current European legislation on animal care and experimentation (2010/63/EU) and approved by the institutional ethics committee of the Centre d'Exploration et de Recherche Fonctionnelle Expérimentale in Evry, France (protocol APAFIS DAP 2018-024-B#19736). The C57Bl6 albino mice were ordered to the Charles River Facility.

[0249] Expressing Cassette and AAV-Mediated Gene Transfer

[0250] Two different AAV cassettes were designed using the AAV2 ITR sequences, the fusion transgene GFP-Luciferase and the SV40 polyadenylation sequence. The promoter was the only element that differed between the constructs. In this study, the human desmin (Des) promoter (SEQ ID NO: 5) and the truncated ACTA1 promoter of the invention (SEQ ID NO: 2) were compared. The serotype 9 was used for the production of GFP-Luc recombinant adeno-associated virus (AAV9-promoter-GFP-Luc) using the tri-transfection method. The corresponding sequence including the ITR sequences is shown in SEQ ID NO: 6 in relation to the truncated ACTA1 promoter.

[0251] Two other AAV cassettes were also designed using the same promoters but with the human calpain 3 transgene and: [0252] AAV9 ITR sequences; [0253] HBB2 intron inserted between the promoter and the transgene; [0254] two target sequences of mir208a in tandem inserted between the transgene and the HBB2 polyadenylation sequence.

[0255] The serotype 9 was used for the production of recombinant calpain 3 adeno-associated virus (AAV9-promoter-hCalpain3-2×target-miR208a) using the tri-transfection method. The corresponding sequence including the ITR sequences is shown in SEQ ID NO: 7 in relation to the truncated ACTA1 promoter.

[0256] The different vectors were injected by a single systemic administration in the tail vein of male one month-old C57Bl6 Albino mice or C57Bl6 mice in order to express the GFP-Luc transgene or to produce the human calpain 3, respectively. The doses of vector injected were normalized by the body's weight of mice at 5e13vg/kg of AAV9-promoter-GFP-Luc or at 1e14vg/kg of AAV9-promoter-hCalpain3-2×target-miR208a. Two weeks after treatment with AAV9-promoter-GFP-Luc, global body biodistribution was assessed by luciferase imaging in living animals. Three or four weeks after treatment with AAV9-promoter-GFP-Luc and AAV9-promoter-hCalpain3-2×target-miR208a, respectively, mice were sacrificed and tissues collected. The tibialis anterior (TA) muscle was chosen as a representative skeletal muscle.

[0257] Quantification of the luciferase activity by luciferase assay Samples were first homogenized with 500 μL of assay buffer (Tris/Phosphate, 25 mM; Glycerol 15%; DTT, 1 mM; EDTA 1 mM; MgCl2 8 mM) with 0.2% of Triton X-100 and Protease inhibitor cocktail PIC (Roche). Ten μl of lysate were loaded into flat-bottomed wells of a white opaque 96-well plate. The Enspire spectrophotometer was used for quantification of the luminescence. The pumping system delivers D-luciferin (167 μM; Interchim) and assay buffer with ATP (40 nM) (Sigma-Aldrich) to each well of the plate. The signal of Relative Light Unit (RLU) was measured after each dispatching of D-luciferin and ATP, respecting 2 sec delay between each samples. A BCA protein quantification (Thermo Scientific) was performed to normalize the quantity of protein in each sample. The result was expressed as the level of RLU normalized by the protein amount.

[0258] Global Body Biodistribution

[0259] Mice were anesthetized by inhalation of isoflurane and injected intraperitoneally with 50 mg/ml D-luciferin (LifeTechnologies, California, USA). In vivo imaging was performed using IVIS® Lumina Imaging system (PerkinElmer). The software Living Image® (PerkinElmer) was used to analyze the images.

[0260] mRNA Quantification

[0261] Total RNA extraction was performed from frozen tissues following NucleoSpin® RNA Set for NucleoZOL protocol (Macherey Nagel). Extracted RNA was eluted in 60 μl of RNase-free water and treated with Free DNA kit (Ambion) to remove residual DNA. Total RNA was quantified using a Nanodrop spectrophotometer (ND8000 Labtech).

[0262] For quantification of the transgene expression, one μg of RNA was reverse-transcribed using the RevertAid H minus Reverse transcriptase kit (Thermo Fisher Scientific) and a mixture of random oligonucleotides and oligo-dT. Real-time PCR was performed using LightCycler480 (Roche) using specific sets of primers and probes (Thermo Fisher Scientific) for the quantification of human calpain 3:

TABLE-US-00011 (SEQ ID NO: 9) FWD: 5′-CGCCTCCAAGGCCCGT-3′ (SEQ ID NO: 10) REV: 5′-GGCGGAAGCGCTGGCT-3′ and (SEQ ID NO: 11) Probe: 5′-CTACATCAACATGAGAGAGGT-3′.

[0263] For mouse samples, the Rplp0 was used to normalize the data across samples:

TABLE-US-00012 (SEQ ID NO: 12) FWD: 5′-CTCCAAGCAGATGCAGCAGA-3′ (SEQ ID NO: 13) REV: 5′-ATAGCCTTGCGCATCATGGT-3′, and (SEQ ID NO: 14) Probe: 5′-CCGTGGTGCTGATGGGCAAGAA-3′.

[0264] Each experiment was performed in duplicate. Quantification cycle (Cq) values were calculated with the LightCycler® 480 SW 1.5.1 using 2nd Derivative Max method. RT-qPCR results, expressed as raw Cq, were normalized to Rplp0. The relative expression was calculated using the 2.sup.−ΔCt method.

[0265] Western Blot Analysis

[0266] Frozen sections of approximately 1 mm of tissues (Heart and TA muscle) were solubilized in radio immunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail. For calpain 3, 1 mg of tissue was mixed with 40 μl of urea buffer (8M Urea, 2M Thiourea, 3% SDS, 50 mM Tris-HCl pH 6.8, 0.03% Bromophenol Blue pH 6.8, 50% Ultra-pure Glycerol+Protease inhibitor Cocktail (100×) Sigma P8340) and 40 μl of glycerol. Protein extract was quantified by BCA (bicinchoninic acid) protein assay (Pierce). Thirty μg of total protein were processed for western blot analysis, using calpain 3 antibody (CALP-12A2 (Leica Biosystem); COP-COP-080049 (Cosmo Bio)). Fluorescence signal of the secondary antibodies was read on an Odyssey imaging system, and band intensities were measured by the Odyssey application software (LI-COR Biosciences, 2.1 version).

[0267] Statistical Analyses

[0268] Statistical analyses were performed using the GraphPad Prism version 6.04 (GraphPad Software, San Diego, Calif.). Statistical analyses were performed by ANOVA for all experiments. Data were expressed as mean±SD. P values of less than 0.05 were considered statistically significant.

[0269] Results:

[0270] I/ Expression Profile of the New Promoter in Comparison with the Desmin Promoter Using the Reporter Gene GFP-Luc:

[0271] Two weeks after injection in the tail vein of four male one month-old C57Bl6 Albino mice of 5e13vg/kg AAV9-promoter-GFP-Luc, global body biodistribution was assessed by luciferase imaging in living animals using the IVIS system.

[0272] The global body biodistribution was shown to be different between the two promoters (FIG. 1A). Indeed, luciferase imaging revealed a stronger signal in limb muscles for the truncated ACTA1 promoter compared to the Desmin promoter, as well as a lower signal in the thorax.

[0273] At day 21, mice were sacrificed. It is to be noted that one mouse was found dead in the Desmin promoter group. No mortality was observed in the truncated ACTA1 promoter group.

[0274] The luciferase activity was biochemically measured in the sampled muscles and organs, then normalized to the amount of proteins in each sample. The level of luciferase activity is higher in skeletal muscles with the truncated ACTA1 promoter compared to the Desmin promoter (see TA and diaphragm), and lower in the heart (FIG. 1B). No change is observed in the other organs analysed, i.e. liver, kidney and adrenal glands.

[0275] Taken together, these results show that the use of the truncated ACTA1 promoter according to the invention leads to a higher level of transgene expression in skeletal muscles, and a lower level in cardiac muscle, compared to the use of Desmin promoter.

[0276] II/ Validation of the Expression Profile of the New Promoter in Comparison with the Desmin Promoter Using the Calpain 3 Transgene:

[0277] To validate these observations and to confirm that the truncated ACTA1 promoter is adapted for e.g. driving the expression for the human calpain 3 transgene, C57Bl6 male mice (2 or 3 per group) were intravenously injected with the rAAVs (AAV9-promoter-hCalpain3-2×target-miR208a) at the dose of 1e14 vg/kg. Four weeks after injection, the animals were euthanized. Muscles and heart were sampled for molecular analyses.

[0278] The calpain 3 expression was measured at mRNA levels (FIG. 2A). The level of Calpain 3 mRNA is higher in Tibialis anterior (TA) and lower in the heart for the truncated ACTA1 promoter compared to the Desmin promoter.

[0279] The levels of Calpain 3 protein was also measured (FIG. 2B). The level of Calpain 3 is higher in the TA muscle for the truncated ACTA1 promoter compared to the Desmin promoter. In an expected manner, there is no expression of Calpain 3 protein in the heart with both promoters, because of the presence in the cassettes of two target sequences for miR208a.

CONCLUSIONS

[0280] All these results show that, compared to the Desmin promoter, the small-sized truncated ACTA1 promoter according to the invention leads to a higher level of transgene expression in skeletal muscles, and a lower level in heart.