GENE THERAPY EXPRESSION SYSTEM ALLOWING AN ADEQUATE EXPRESSION IN THE MUSCLES AND IN THE HEART OF SGCG

Abstract

The present invention concerns an expression system for systemic administration comprising a sequence encoding gamma-sarcoglycan (SGCG) placed under the control of a promoter allowing an adequate expression of SGCG in the skeletal muscles and in the heart, and its use for the treatment of Limb-Girdle Muscular Dystrophy type C.

Claims

1-14. (canceled)

15. An expression system for systemic administration comprising a sequence encoding gamma-sarcoglycan (SGCG) placed under the control of a promoter allowing expression of SGCG in the skeletal muscles and in the heart, wherein the ratio between the SGCG expression in the skeletal muscles and the SGCG expression in the heart is superior or equal to 0.9.

16. The expression system according to claim 15, wherein the system is configured to express SGCG in the skeletal muscles in a quantity superior or equal to 0.3 times a quantity expressed endogenous.

17. The expression system according to claim 15, wherein the expression system is configured to express SGCG in the heart in a quantity inferior or equal to 8 times a quantity expressed endogenous.

18. The expression system according to claim 15, wherein the promoter is a tMCK promoter.

19. The expression system according to claim 18, wherein the tMCK promoter has a sequence as set forth in SEQ ID NO: 4.

20. The expression system according to claim 15, wherein the SGCG protein has the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

21. The expression system according to claim 15, wherein the sequence encoding the SGCG protein has the sequence SEQ ID NO: 3.

22. The expression system according to claim 15, wherein the expression system comprises SEQ ID NO: 5 or SEQ ID NO: 6.

23. The expression system according to claim 15, wherein the expression system comprises a viral vector.

24. The expression system according to claim 23, wherein the viral vector is an adeno-associated viral (AAV) vector.

25. The expression system according to claim 24, wherein the AAV vector is of serotype 8 or 9.

26. The expression system according to claim 25, wherein the expression system comprises an adeno-associated viral (AAV)2/8 or an AAV2/9 vector.

27. A pharmaceutical composition comprising the expression system according to claim 15.

28. The pharmaceutical composition according to claim 27, wherein the composition is a gene therapy composition.

29. A method of treating a pathology caused by a SGCG deficiency, comprising administering the pharmaceutical composition according to claim 27.

30. The method of claim 29, wherein the pathology caused by a SGCG deficiency is Limb-Girdle Muscular Dystrophy type C (LGMD2C or LGMD R5).

31. The method of claim 29, wherein the pharmaceutical composition is administered systemically.

32. The method of claim 31, wherein the pharmaceutical composition is administered by intravenous injection.

Description

FIGURES

[0189] FIG. 1: A/ Western blot detection of γ-sarcoglycan (SGCG) expression in the tibialis anterior (TA) muscle and the heart of a mouse or a macaca using a γ-sarcoglycan antibody (Ab203113-Abcam) B/ Graphical presentation of SGCG expression in each tissue based on the signals detected in (A). Statistical ANOVA test: (*) indicates a P value of less than 0.05 (statistically significant). ns: non significant

[0190] FIG. 2: Luciferase activity of GFP-Luc transgene normalized by total protein amount in TA muscle and heart from C57B16 albino mice injected with AAV9-prom-GFP-Luc (Des, CK8 and tMCK).

[0191] FIG. 3: Vector genome copy number (VGCN) per diploid genome measured by QPCR in tissues (TA, heart and liver) from 3 groups of Sgcg-/- mice intravenously injected with an AAV8 vector harboring SGCG under the control of the desmin promoter (AAV8-Des-SGCG) or the CK8 promoter (AAV8-CK8-SGCG) or the tMCK promoter (AAV8-tMCK-SGCG).

[0192] FIG. 4: A/SGCG mRNA normalized by P0 endogenous level measured by RT-QPCR in tissues (TA, heart and liver) from the 3 groups of Sgcg-/- mice intravenously injected with an AAV8 vector harboring SGCG under the control of the desmin promoter (AAV8-Des-SGCG) or the CK8 promoter (AAV8-CK8-SGCG) or the tMCK promoter (AAV8-tMCK-SGCG). B/ Ratio between the relative abundance of SGCG/P0 mRNA and the VGCN in each tissue. C/ Ratio of the relative abundance of SGCG mRNA in heart versus TA muscle. The dotted line corresponds to a ratio of 1 (same expression level in heart and TA muscle). Statistical ANOVA test: (*) indicates a P value of less than 0.05 (statistically significant).

[0193] FIG. 5: A/ Western blot detection of human γ-sarcoglycan expression in the TA muscle and the heart of the 5 mice of each group (Sgcg-/- mice intravenously injected with an AAV8 vector harboring SGCG under the control of the desmin promoter (AAV8-Des-SGCG) or the CK8 promoter (AAV8-CK8-SGCG) or the tMCK promoter (AAV8-tMCK-SGCG)), using a human-specific γ-sarcoglycan antibody (Ab203112-Abcam). B/ Graphical presentation of SGCG expression in each tissue (Ht: heart; TA: tibialis anterior) based on the signals detected in (A). Statistical ANOVA test: (*) indicates a P value of less than 0.05 (statistically significant). ns: non significant

[0194] FIG. 6: Immunostaining anti-SGCG performed in TA and heart of Sgcg-/- mice intravenously injected with an AAV8 vector harboring SGCG under the control of the desmin promoter (AAV8-Des-SGCG) or the CK8 promoter (AAV8-CK8-SGCG) or the tMCK promoter (AAV8-tMCK-SGCG). Scale bar = 100 .Math.m.

[0195] FIG. 7: A/ Graphic correlation between the percentage of SGCG expression and the percentage of centronucleated fibers. The black dots correspond to muscle from WT mice and the white ones from KO-Sgcg mice. The grey dots correspond to muscle from KO-Sgcg injected with different level of AAV transduction efficiency (5e12 vg/kg, 1e13 vg/kg and 5e13 vg/kg of AAV8-Des-SGCG) B/ Western blot detection of γ-sarcoglycan expression in the TA muscle and the heart of WT mice intravenously injected with PBS or AAV8-Des-SGCG (3.sup.e14 vg/kg) using a γ-sarcoglycan antibody (Ab203113-Abcam) C/ Graphical presentation of SGCG expression in each tissue (Ht: heart; TA: tibialis anterior) based on the signals detected in (B).

[0196] FIG. 8: A/ Western blot detection of human γ-sarcoglycan expression in the TA muscle and the heart of rats of each group (Sprague dawley intravenously injected with an AAV8 vector harboring SGCG under the control of the tMCK promoter (AAV8 tMCK), the desmin promoter (AAV8 Desmin) and the MHCK7 promoter (AAV8 MHCK7), using a human-specific γ-sarcoglycan antibody (Ab203112-Abcam). B/ Graphical presentation of SGCG expression in each tissue (Heart; TA: tibialis anterior) based on the signals detected in (A). Statistical Student test: (*) indicates a P value of less than 0.05, (***) indicates a P value of less than 0.001 (statistically significant) ns: non significant

[0197] FIG. 9: Molecular ratio rMyh6 / rMyh7 measured by RT-QPCR transcripts in heart from the 3 groups of Sprague Dawley rat intravenously injected with PBS or, with an AAV8 vector harboring SGCG under the control of the tMCK promoter (AAV8-tMCK-SGCG),the desmin promoter (AAV8-Desmin-SGCG) and the MHCK7 promoter (AAV8-MHCK7-SGCG). Statistical ANOVA test: (**) indicates a P value of less than 0.001.

MATERIALS AND METHODS

Animal Models

[0198] 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).

[0199] The Sgcg.sup.-/- mouse strain (Hack et al., J. Cell. Biol. 1998;142:1279-87) was used in this study. These mice were bred in a pure C57BL/6J background by crossing 10 times onto the C57BL/6J background. The C57B⅙J and C57B16 albino mice were ordered to the Charles River Facility. Samples from macaca were provided by Inserm UMR 1089, Atlantic Gene Therapies, Institut de Recherche Thérapeutique (IRT 1) Université de Nantes (France) and Silabe (67207 Niederhausbergen, France). One-month-old male Sprague Dawley rats were also used in this study.

Expressing Cassette and AAV-mediated Gene Transfer

[0200] Three different AAV cassettes were designed using the same ITR sequences, transgene GFP-Luc and polyA HBB2. The promoter was the only element that differs between the constructs. In this study, the human desmin (Des) promoter (SEQ ID NO: 13), the CK8 promoter (Goncalves et al., Mol Ther. 2011;19(7): 1331-41; SEQ ID NO: 14) and the tMCK promoter (Wang et al., Gene Therapy 2008;15:1489-99; SEQ ID NO: 4) were compared. The serotype 9 was used for the production of GFP-Luc recombinant adeno-associated virus (AAV9-prom-GFP-Luc).

[0201] Three other AAV cassettes were also designed using the same promoters but with the SGCG transgene (see SEQ ID NO: 6 in relation to the tMCK promoter). Moreover, the MHCK7 promoter as disclosed in WO2019/152474 (SEQ ID NO: 15) was further tested in this context. The serotype 8 was used for the production of recombinant SGCG adeno-associated virus (AAV8-prom-SGCG).

[0202] Viral genomes were quantified by a TaqMan™ real-time PCR assay using the primer pairs and TaqMan™ probes specific for the polyA HBB2 sequence:

TABLE-US-00001 FWD: 5′-CCAGGCGAGGAGAAACCA-3′ (SEQ ID NO: 7),

TABLE-US-00002 REV: 5′-CTTGACTCCACTCAGTTCTCTTGCT-3′ (SEQ ID NO: 8 ), and

TABLE-US-00003 Probe: 5′-CTCGCCGTAAAACATGGAAGGAACACTTC-3′ (SEQ ID  NO: 9).

[0203] The different vectors were injected by a single systemic administration in the tail vein in order to express the GFP-Luc transgene in male one month-old C57B16 Albino mice or to restore γ-sarcoglycan expression in muscle of female five week-old Sgcg-/- mice. The doses of vector injected were normalized by the body’s weight of mice at 5el3vg/kg of AAV9-prom-GFP-Luc or at 5e12 vg/kg, 1e13 vg/kg 5e13 vg/kg or 3e14 vg/kg of AAV8-prom-SGCG. Three or two weeks after treatment, respectively, mice were sacrificed and tissues collected. The tibialis anterior (TA) muscle was chosen as a representative skeletal muscle.

[0204] Besides, one month old male Sprague Dawley rats were injected intravenously into the tail vein with the three AAV8 vectors MHCK7-hSGCG, Desmin-hSGCG and tMCK-hSCGC at a dose of 3e14vg/kg. Another rat group injected with PBS was also included as a control. One month after the injection, the rats were sacrified. The heart and the tibialis anterior (TA) muscles were collected.

Quantification of the Luciferase by Luciferase Assay

[0205] Samples were first homogenized with 500 .Math.L of assay buffer (Tris/Phosphate, 25 mM; Glycerol 15%; DTT, 1 mM; EDTA 1 mM; MgC12 8 mM) with 0.2% of Triton X-100 and Protease inhibitor cocktail PIC (Roche). Ten .Math.l 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 .Math.M; 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.

Histological and Immunohistochemistry Analyses

[0206] Eight micrometers transversal cryosections were cut from liquid nitrogen-cooled isopentane frozen TA muscles or hearts. The transverse cryosections were then blocked with PBS containing 20% Fetal calf serum (FCS) for 1 h and incubated overnight at 4° C. with a rabbit monoclonal primary antibody directed against the human γ-sarcoglycan protein (Abcam - ab203112). After washing with PBS, sections were incubated with a goat anti-rabbit secondary antibody conjugated with AlexaFluor 594 dyes (Thermo Fisher Scientific) for 1h at room temperature.

[0207] After washing with PBS, sections were mounted with Fluoromount-G and DAPI (SouthernBiotech), and visualized on a fluorescence microscope (Zeiss - Zeiss Axiophot 2). A complete image acquisition of all sections was finally carried out using the AXIOSCAN microscope (Zeiss).

[0208] For determining the number of centronucleated fibers, the sections were labelled with a rabbit anti-laminin antibody (DAKO-Z0097), using a goat anti-rabbit antibody conjugated with AlexaFluor 488 dyes (Thermo Fisher Scientific) as secondary antibody and mounted with Fluoromount-G and DAPI (SouthernBiotech). Image acquisition of all sections was finally carried out using the AXIOSCAN microscope (Zeiss). The morphometric analyses of the skeletal muscles to define the number of centronuclear fibres (CNF/mm.sup.2) were performed as followed:

[0209] Scanned RGB images containing 8bits channels of the laminin Immunofluorescence and DAPI staining captured at l0x magnification are processed using the FIJI software for nuclei and fibers segmentation. Nuclei segmentation is performed based on the DAPI intensity using global thresholding (IsoData) and particles analysis. Fibers are segmented based on the laminin staining using the MorphoLib plugin ‘morphological segmentation’ tool (border image option) and ImageJ particles analysis tool (object circularity > .2, object size filter depending on muscle type and species).

[0210] Nuclei and fibers Regions of Interest (ROI) are converted to spatial objects using the R software (RImageJROI, spatstat and sp libraries) and intra-fiber nuclei identified by intersection of nuclei and fibers objects. For intra-fiber nuclei, their distance to the fiber center of gravity and closest membrane point is calculated.

[0211] Size, shape, fluorescence intensity filtering are performed to exclude artefacts (nerves identified as fiber, spited or merged fibers ... ).

[0212] Centro nucleated fibers are identified based on the distance between the nucleus and the closest membrane (relative to fiber Feret diameter or absolute distance, user’s choice).

Viral Genome Copy Numbers (VGCN) Measurement in Tissues

[0213] Genomic DNA was extracted from frozen tissues using the NucleoMag Pathogen kit (Macherey Nagel) with the KingFisher robot (Thermo Fisher Scientific) according to manufacturer instructions. Vector genome copy number was determined using qPCR from 20 ng of genomic DNA. A serial dilution of a DNA sample of a plasmid harboring one copy of each amplicon was used as standard curve. Real-time PCR was performed using LightCycler480 (Roche Roche) with 0.2 .Math.M of each primer and 0.1 .Math.M of the probe according to the protocol of Absolute QPCR Rox Mix (Thermo Fisher Scientific). A sequence located in the polyA HBB2 of the cassette was used for the quantification of viral genome. The primer pairs and Taqman™ probes specific for the polyA HBB2 sequence were the same as disclosed above (SEQ ID NO: 7 to 9).

[0214] The ubiquitous acidic ribosomal phosphoprotein (P0) was used for genomic DNA quantification. Primer pairs and Taqman™ probe used for P0 amplification were:

TABLE-US-00004 FWD: 5′-CTCCAAGCAGATGCAGCAGA-3′ (SEQ ID NO: 10),

TABLE-US-00005 REV: 5′-ATAGCCTTGCGCATCATGGT-3′ (SEQ ID NO: 11), a nd

TABLE-US-00006 Probe: 5′-CCGTGGTGCTGATGGGCAAGAA-3′ (SEQ ID NO: 12 ).

[0215] The number of diploid genomes is half of the number of copies of P0 gene. The level of transduction of the tissue is determined by the VGCN per diploid genome.

mRNA Quantification

[0216] Total RNA extraction was performed from frozen tissues following NucleoSpin® RNA Set for NucleoZOL protocol (Macherey Nagel). Extracted RNA was eluted in 60.Math.l of RNase-free water and treated with TURBO™ DNase kit (Ambion) to remove residual DNA. Total RNA was quantified using a Nanodrop spectrophotometer (ND8000 Labtech).

[0217] For quantification of the transgene expression, one .Math.g 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 commercial sets of primers and probes for the quantification of human γ-sarcoglycan (Hs00165089_ml; Thermo Fisher Scientific). For mouse samples, the ubiquitous acidic ribosomal phosphoprotein (P0) was used to normalize the data across samples as well as the VGCN quantification described previously.

[0218] 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 P0. The relative expression was calculated using the 2.sup.-ΔCt Livak method.

Measurement of the Transcript Ratio Myh6/Myh7

[0219] The transcripts of Myh6 and Myh7 were quantified by RT-QPCR using commercial sets of primers and probes for the quantification of rMyh6 (Rn00691721_g1; Thermo Fisher Scientific) and rMyh7 (Rn01488777_g1; Thermo Fisher Scientific). The result is expressed as a molecular ratio of the transcripts Myh6 versus Myh7.

Western Blot Analysis

[0220] Frozen sections of approximately 1 mm of tissues (Liver, Heart or TA muscle) were solubilized in radio immunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail. Protein extract was quantified by BCA (bicinchoninic acid) protein assay (Pierce). Thirty .Math.g of total protein were processed for western blot analysis, using an anti-γ-sarcoglycan antibody (human-specific: Ab203112 and for common recognition of mouse human and macaca form :Abcam; Ab203113).

[0221] 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).

Statistical Analyses

[0222] Statistical analyses were performed using the GraphPad Prism version 6.04 (GraphPad Software, San Diego, CA). Statistical analyses were performed using the statistical ANOVA or Sudent test as indicated. Data were expressed as mean ± SD. P values of less than 0.05 were considered statistically significant (*).

RESULTS

I/ Endogenous SGCG Expression Profile in Mouse and Macaca

[0223] In order to define the relative proportion of the endogenous SGCG between heart and skeletal muscle in different species, the relative abundance of the SGCG protein was investigated in different tissues (TA muscle as a representative of the skeletal muscles and the heart) of wild type mice or macaca.

[0224] FIG. 1 reveals that in mice, SGCG is produced at a similar level in the TA muscle and in the heart. In the macaca, which is a mammalian model for humans, it is observed that the quantity of SGCG in the heart is drastically inferior to the SGCG quantity in the TA muscle.

II/ Evaluation of Different Promoters in C57BL6 Mice

[0225] A study was performed to identify an expression construct displaying an expression profile in heart and TA muscle, similar as much as possible to that observed with the endogenous gene, i.e. with an expression at a similar level or even higher in the TA muscle than in the heart.

[0226] For this purpose, different promoters known to have a muscular activity have been tested using the reporter gene GFP-Luc.

[0227] Experiments were performed to compare the desmin promoter, the CK8 promoter and the tMCK promoter. The desmin promoter was chosen because it corresponds to the one tested by Israeli et al. (Mol Ther Methods Clin Dev. 2019; 13:494-502) who have reported its efficiency for restoring muscular activity.

[0228] FIG. 2 reveals that: [0229] The AAV9-CK8-GFP-Luc vector is the more efficient to transduce both heart and TA muscle; [0230] The AAV9-tMCK-GFP-Luc appear to be weaker in terms of promoter strength but more equilibrated between heart and skeletal muscle expression.

[0231] It clearly appears that the tMCK promoter is a promising candidate, ensuring an adequate expression in in heart and TA muscle, as observed with the endogenous gene in the mouse and macaca. On the contrary, the desmin and CK8 promoters give rise to a very high expression in heart, superior to that observed in the TA muscle, with a possible associated cardiac toxicity.

III/ Validation of the tMCK Promoter in Sgcg-/- Mice

[0232] To validate these observations, further studies were performed to compare 3 different SGCG AAV8 vectors intravenously injected in SGCG deficient mice. The promising tMCK promoter was compared to the two other promoters as tested above, i.e. the Desmin promoter and the CK8 promoter.

[0233] First, the efficacy of transduction was compared between the 3 constructs.

[0234] As shown by FIG. 3, there is no bias regarding the infectiosity of the 3 vectors since they transduced at the same level the same tissues. The liver was clearly the organ the most transduced (~1 VGCN / diploid genome). The similar transduction of heart and TA muscle reached around 0.01 VGCN / diploid genome. With this low level of infection, there was no risk to reach a saturation effect that could interfere with the following analyses.

[0235] Then, the transcriptional activity of the 3 promoters was compared.

[0236] The level of SGCG mRNA in TA muscle was not clearly different between the 3 groups of mice. On the contrary, the activity of the tMCK promoter appeared much lower in the heart compared to the 2 other groups of mice, with a statistically significant difference at least with the CK8 promoter. As the number of tranduced cells in the liver is very high, the level of SGCG mRNA is also high (FIG. 4A).

[0237] The normalization of the mRNA SGCG abundance by the VGCN confirmed that the tMCK promoter activity is significantly different from both the Des and CK8 promoter activity (FIG. 4B).

[0238] Finally the SGCG mRNA ratio heart versus TA muscle obtained with the tMCK promoter (about 0.6) appeared to be more in adequation with the endogenous conditions (FIG. 4C), i.e. a higher expression in the TA muscle that in the heart.

[0239] These observations were confirmed by investigating SGCG protein expression in these different tissues:

[0240] As revealed by FIG. 5, whereas the amount of transgene protein is significantly higher in heart than in TA muscle in the groups of mice injected with the AAV8-CK8-SGCG vector and with the AAV8-Des-SGCG vector, this is not the case for mice injected with the AAV8-tMCK-SGCG vector: the quantity of SGCCG is not significantly different between the heart and the TA muscle. Moreover, it is to be noted that the level of Sgcg protein in TA muscle is similar whatever the promoter used. Based on these results, the tMCK is confirmed to have an adequate expression profile, i.e.: [0241] a high activity in the TA muscle similar to the desmin and CK8 promoters; [0242] a lower activity in the heart than the desmin and CK8 promoters. Direct observation on TA and cardiac tissues (FIG. 6) confirmed that the expression of SGCG in TA muscle was not clearly different between the 3 groups of mice. On the contrary, the heart from mice injected with the AAV8-tMCK-SGCG vector displayed fewer positive fibers compared with the 2 other groups of mice.

IV/ Determination of Critical Amounts of SCGC in the Muscles and in the Heart

[0243] In order to determine the minimal therapeutically effective amount of SGCG in the muscles and the maximal not toxic amount of SGCG in the heart, further experiments were performed using the AAV8-Des-SGCG vector which has been shown above to lead to an adequate level of expression in the TA muscle but an excessive level of expression in the heart, possibly toxic.

[0244] It can be concluded from FIG. 7A that in order to reach an acceptable centronucleation level (comparable or even slightly superior to the one observed in muscles of WT mice, i.e. up to 20%), the expression system should allow expressing at least 30% of the normal level of SGCG in the skeletal muscles.

[0245] On another hand, FIGS. 7B and 7C reveal that said system, potentially toxic in the heart, leads to a SGCG level in the heart 8 times greater than the one observed in the heart of WT mice.

V/ Evaluation of Different Promoters in Rats

V-1/Protein SGCG Expression Profile

[0246] The experiments disclosed above in mice were further performed in rats, adding as a new tested promoter, the MHCK7 promoter (AAV8-MHCK7-SGCG vector).

[0247] FIG. 8 reveals that in rats, the amount of transgene protein was significantly higher in heart than in TA muscles in the group of rats injected with the AAV8 Desmin-SGCG vector and with the AAV8 MHCK7-SGCG vector.

[0248] On the contrary, the transgene protein was equally expressed in the TA muscle and in the heart with the AAV8 tMCK-SGCG vector.

[0249] It is to be noted that the expression profile ratio obtained with the AAV8 Desmin-SGCG vector and the AAV8 tMCK-SGCG vector is similar in mice and in rats.

V-2/ Impact on the Heart

[0250] The measurement of the transcript ratio Myh6/Myh7 is a good indicator to detect modification of the heart tissue that accompanies stress induced pathological conditions in heart (Scheuermann et al., EMBO J. 2013; 32(13): 1805-16).

[0251] FIG. 9 shows that this ratio was not significantly modified in the group of rats injected with the vector AAV8 tMCK-SGCG (8.3) compared to the PBS control group (10.2). It further reveals that even if not statistically different, the ratio was strongly reduced in the heart of rats injected with the AAV8 Desmin-SGCG vector (1.8). Finally, the ratio was significantly lower in the heart of rats injected with AAV8 MHCK7-SGCG vector (0.8) in comparison with the PBS control and the AAV8-tMCK groups of rats.

[0252] Overall, the tMCK promoter is driving an equal expression between heart and skeletal muscle whereas with the two other promoters Desmin and MHCK7, SGCG is more expressed in the heart than in skeletal muscle as observed both in rat and mice. In addition, only the tMCK promoter conserves the correct ratio Myh6/Myh7 while this ratio is modified with the two other promoters, indicating cellular stress in the heart.

CONCLUSIONS

[0253] As known in the art, the two most important organs that need to be targeted for the treatment of LGMD2C patients are the skeletal muscles and heart.

[0254] Based on the measurement of the endogenous SGCG protein in Wild Type mouse and macaca, it was concluded that the expression in the heart is preferably at the same level or even lower than in the skeletal muscles.

[0255] Regarding these different aspects, the AAV8-tMCK-SGCG vector was confirmed to be a very promising candidate. The level of expression is significantly reduced in heart compared to the 3 other promoters. Besides and in the TA muscle, the expression of the transgene is near to what obtained with the AAV8-Des-SGCG vector, a vector widely described as efficient to transduce the skeletal muscle and restore muscular activity (see e.g. Israeli et al., Mol Ther Methods Clin Dev. 2019; 13:494-502).