PRODUCTION OF LARGE-SIZED QUASIDYSTROPHINS USING OVERLAPPING AAV VECTORS

Abstract

The present invention concerns a quasidystrophin (QD) having the structure CH1CH2H1R1R2R3H2R8R9 in its N-terminal part and advantageously further comprising the R16 and R17 rod domains, as well as the dual AAV vector system which allows producing it.

Claims

1-15. (canceled)

16. A quasidystrophin (QD) having the structure CH1CH2H1R1R2R3H2R8R9 in its N-terminal part.

17. The quasidystrophin according to claim 16, wherein the QD comprises rod domains R16 and R17.

18. The quasidystrophin according to claim 17 deprived of rod domains R4, R5, R6, and R7 (ΔR4-R7), rod domains R10, R11, R12, R13, R14, and R15 (ΔR10-R15), and rod domains R18 and R19 (ΔR18-R19).

19. The quasidystrophin according to claim 18 having the sequence SEQ ID NO: 2 or a sequence having at least 90% identity thereto.

20. The quasidystrophin according to claim 18 having the sequence as set forth in SEQ ID NO: 20.

21. The quasidystrophin according to claim 17 deprived of rod domains R4, R5, R6, and R7 (ΔR4-R7), rod domains R10, R11, R12, and R13 (ΔR10-R13), and rod domains R18, R19, R20 and R21 (ΔR18-R21).

22. The quasidystrophin according to claim 21 having the sequence SEQ ID NO: 3 or a sequence having at least 90% identity thereto.

23. A nucleic acid sequence encoding the quasidystrophin according to claim 17.

24. The nucleic acid sequence according to claim 23 comprising or consisting of SEQ ID NO: 4 or a sequence having at least 70% identity thereto.

25. The nucleic acid sequence according to claim 23 comprising or consisting of a sequence as set forth in SEQ ID NO: 5 or SEQ ID NO: 22.

26. A dual AAV vector system comprising two AAV vectors, wherein a first AAV vector comprises, between 5′ and 3′ AAV ITRs, a first nucleic acid sequence that encodes a N-terminal part of a quasidystrophin, and a second AAV vector comprises, between 5′ and 3′ AAV ITRs, a second nucleic acid sequence that encodes a C-terminal part of a quasidystrophin, wherein the first and second nucleic acid sequences comprise an overlapping region that permits the production by recombination of the quasidystrophin according to claim 16.

27. The dual AAV vector system according to claim 26, wherein the first nucleic acid sequence has the sequence SEQ ID NO: 6 or SEQ ID NO: 23 and the second nucleic acid sequence has the sequence SEQ ID NO: 7.

28. The dual AAV vector system according to claim 26, wherein the first nucleic acid sequence has the sequence SEQ ID NO: 8 and the second nucleic acid sequence has the sequence SEQ ID NO: 9.

29. A cell transduced with the dual AAV vector system according to claim 26.

30. The cell of claim 29, wherein the cell is a muscle cell.

31. A composition comprising, in a pharmaceutically acceptable carrier, the dual AAV vector system according to claim 26.

32. The composition according to claim 31, wherein the dual AAV vector system is present in a cell into which it has been transduced.

33. An AAV vector which is the first AAV vector or the second AAV vector of the dual AAV vector system according to claim 26.

34. A method for treating muscular dystrophy in a subject in need thereof, comprising administering a composition comprising the dual AAV vector system according to claim 26 to the subject.

35. The method of claim 34, wherein the muscular dystrophy is Duchenne muscular dystrophy (DMD).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0217] FIG. 1: Scheme of the different forms of dystrophins:

[0218] A/ Scheme of the full-length dystrophin;

[0219] B/ Scheme of the MD1 microdystrophin as disclosed by Yue et al.;

[0220] C/ Scheme of the ΔH2-R15 quasidystrophin (SEQ ID NO: 1) as disclosed by Kodippili et al. (DD);

[0221] D/ Scheme of the ΔR4-R7ΔR10-R15ΔR18-R19 quasidystrophin (SEQ ID NO: 2) according to the invention (WL1);

[0222] E/ Scheme of the ΔR4-R7ΔR10-R13ΔR18-R21 quasidystrophin (SEQ ID NO: 3) according to the invention (WL2).

[0223] FIG. 2: Analysis of the production level of the different forms of dystrophins:

[0224] A/ Western blotting with anti-dystrophin antibody

[0225] The TA muscles of 3-month old mdx mice were injected with 1×10.sup.10 vg of AAVs. TA muscles were recovered 30 days after injection and processed for western blotting with an antibody (DysB) against dystrophin. Panels 6 to 8 (Opt) show the level of protein WL1 (280 kDa) obtained with a dual AAV vector system according to the invention in comparison to that obtained with AAV2/9-hMD1 (panel 9: 138 kDa), human control sample (FL: panel 2: 427 kDa) and mdx (KO: panels 3 and 4), respectively. Control muscles from age-matched mdx and C57BL/10 were injected with saline only.

[0226] B and C/ The TA muscles of 1-2 month old WT mice were injected with 1-3×10.sup.10 vg of AAVs. TA muscles were collected 30 days after injection and processed for western blotting with an antibody against dystrophin (DysB and Dys2) and against α-actinin level (α-actinin level was used as a normalizer). Comparison of protein level expressed from different constructs: [0227] WL1 wt (expressed from SEQ ID NO: 8+9) panels 1 to 3 in upper blot; [0228] WL2 wt (expressed from SEQ ID NO: 13+14: panels 4 to 6 in upper blot; [0229] WL1 Opt (expressed from SEQ ID NO: 6+7): panels 7 to 9 in upper blot; [0230] PBS: panel 10 in upper blot; [0231] DD (expressed from SEQ ID NO: 18+19): panels 1 to 3 in lower blot; [0232] WL1 Opt (expressed from SEQ ID NO: 6+7: panels 4 to 6) in lower blot; [0233] PBS: panel 7 in lower blot.

[0234] FIG. 3: Evaluation of the dystrophin expression and distribution in TA muscles

[0235] 1 month old Dba2_Mdx mice were intravenously injected with either: [0236] 5′AAV vector harboring SEQ ID NO: 6 and 3′AAV vector harboring SEQ ID NO: 7 (7 mice injected; n=3 representative ones on WB); [0237] 5′AAV vector harboring SEQ ID NO: 6 only (2 mice injected; n=1 representative one on WB); [0238] 3′AAV vector harboring SEQ ID NO: 7 only (2 mice injected; n=1 representative one on WB); [0239] No vector, i.e. PBS (5 mice injected; n=2 representative ones on WB)

[0240] TA muscles were recovered 70 days after injection and processed for western blotting or cryosectioning.

[0241] A/ Western blotting (WB) with anti-dystrophin antibodies (Dys N-ter and Dys C-ter) and α-actinin antibody. Expected size=280 kDa; Full-length dystrophin=427 kDa.

[0242] B/ Immunolabelling in cryosections with Dys N-ter antibody. The percentage of positive fibers is mentioned on each picture.

[0243] FIG. 4: Evaluation of the dystrophin therapeutic effect in TA muscles treated as in FIG. 3

[0244] A/ Staining of sections with Hematoxylin, Phloxine, Saffron (HPS)

[0245] B/ Expression level of TMEM8C (top left), CD11b (top right) and fibronectin (bottom)

[0246] FIG. 5: Evaluation of the dystrophin expression in the heart of mice treated as in FIG. 3 by western blotting with anti-dystrophin antibodies (Dys N-ter and Dys C-ter) and α-actinin antibody

[0247] FIG. 6: Evaluation of the dystrophin expression and distribution in TA (Tibialis Anterior) and DIA (diaphragm)

[0248] Dba2_Mdx mice were intravenously injected with either: [0249] 5′AAV vector harboring SEQ ID NO: 6 and 3′AAV vector harboring SEQ ID NO: 7 (5′mut+3′ or Dual Dys mut); 5 mice injected; [0250] 5′AAV vector harboring SEQ ID NO: 23 and 3′AAV vector harboring SEQ ID NO: 7 (5′cor+3′ or Dual Dys cor); 5 mice injected;

[0251] For comparison, non injected (NI) Dba2_Mdx mice (n=3) and Dba2 WT mice were included in the experiments.

[0252] TA and DA muscles were recovered 3 weeks after injection and processed for western blotting or cryosectioning.

[0253] A/ Western blotting (WB) with anti-dystrophin antibody (Dys2) and α-actinin antibody (used to normalize the sample). Expected size=280 kDa; Full-length dystrophin=427 kDa; L=ladder; HL=high molecular weight ladder, T+=positive control.

[0254] B/ Immunolabelling in cryosections with Dys2 antibody. The percentage of positive fibers is mentioned on each picture.

[0255] FIG. 7: Evaluation of the dystrophin therapeutic effect in muscles treated as in FIG. 6:

[0256] Expression level of TMEM8C (top left), CD11 b (top right) and fibronectin (bottom).

MATERIAL AND METHODS

[0257] In vivo gene transfer. Different mouse models were used in this study: wild-type C57BL/10 and C57BL/6J; mdx and Dba_2Mdx (D2.B10-mdx/J). All mouse procedures were done according to protocol approved by the Ethic Committee at the CERFE of Evry animal facility and under appropriate biological containment. Adeno-associated virus vectors were produced using three-plasmid constructs protocol. After sacrifice, tissues (TA muscles and heart) were collected, snap-frozen in liquid nitrogen-cooled isopentane and stored at −80° C.

[0258] Western blot. Total proteins were extracted from tissue samples. Protein extracts were separated on gels NuPAGE™, then transferred on a nitrocellulose membrane. Membranes were then blocked with Odyssey Blocking Buffer and PBS, then hybridized with the adequate antibody (antiDystrophin DysB or Dys2 antibody, rabbit alpha-actinin (Life Technologies)) and with secondary anti-mouse or anti-rabbit-conjugated (680 or 800) antibodies.

[0259] Immunohistochemistry TA muscle cryo-sections were stained using Mouse on Mouse (M.O.M) kit (Vector Labs). Primary antibodies were incubated overnight at 4° C. followed by 3 washes with PBS-0.1% tween, and incubated with goat anti-mouse or goat anti-rabbit secondary antibodies Alexa 594 (Life Technologies). Antibodies against dystrophin (Dys2, 1:100, mouse monoclonal, Novocastra) were used.

[0260] HPS staining. TA muscle cryo-sections were stained using Hematoxylin (nuclear staining), Phloxine (cytoplasmic staining) and Saffron (collagen).

[0261] PCR quantification of dystrophic profile. Expression of TMEM8C (for measuring fiber regeneration/TGE Mm00481256_ml, ThermoFisher), CD11b (for measuring inflammation/TGE Mm00434455_ml) and fibronectin (for measuring fibrosis/TGE Mm01256744_ml) was quantified on cDNA collected from cryo-sections by RT-quantitative PCR (RTqPCR).

Results:

[0262] 1) Construction of AAV Vectors

[0263] Different recombinant AAV2/8 or AAV2/9 vectors were constructed to evaluate their relative efficiency for dystrophin production:

TABLE-US-00001 TABLE 1 List of tested dystrophins Dystrophin Regulatory Name (MW) Encoding sequences sequences FL Full length — — (FIG. 1A) dystrophin (427 kDa) MD1 Microdystrophin See Yue et al. See Yue et al. (FIG. 1B) (138 kDa) DD Minidystrophin 5′AAV: SEQ ID NO: 18 See Kodippili (FIG. 1C) (280 kDa) 3′AAV: SEQ ID NO: 19 et al. (SEQ ID NO: 1) WL1 wt quasidystrophin Overlapping AAV spC5-12 (FIG. 1D) (280 kDa) vectors: promoter, (SEQ ID NO: 2) 5′AAV: SEQ ID NO: 8 chimeric intron 3′AAV: SEQ ID NO: 9 and SV40 pA WL1 Opt quasidystrophin Overlapping AAV spC5-12 or (280 kDa) vectors: promoter, hDysOpt (SEQ ID NO: 20) 5′AAV (5′mut): chimeric intron or SEQ ID NO: 6 and SV40 pA T+ 3′AAV: SEQ ID NO: 7 signal (FIG. 1D) WL1 Cor quasidystrophin Overlapping AAV spC5-12 (FIG. 1D) (280 kDa) vectors: promoter, (SEQ ID NO: 2) 5′AAV (5′cor): chimeric intron SEQ ID NO: 23 and SV40 pA 3′AAV: SEQ ID NO: 7 signal WL2 wt quasidystrophin Overlapping AAV spC5-12 (FIG. 1E) (280 kDa) vectors: promoter, (SEQ ID NO: 3) 5′AAV: SEQ ID NO: 13 chimeric intron 3′AAV: SEQ ID NO: 14 and SV40 pA signal

[0264] 2) Analysis of Dystrophin Profile after Intramuscular (IM) Delivery

[0265] To evaluate the muscle transduction and level of expression of the protein produced from the dual vector system according the invention, an in vivo analysis of the vectors was performed. One-month-old dystrophin deficient (mdx) mice were intramuscularly injected with 1e10 vg of AAV vectors. Tibialis anterior (TA) muscles were recovered 30 days after injection and processed for western blotting with an antibody (DysB) against dystrophin (FIG. 2A): Panels 6 to 8 (Opt) show the level of protein WL1 OPT (280 kDa) obtained with a dual AAV vector system according to the invention in comparison to that obtained with AAV2/9-hMD1 (panel 9: 138 kDa), human dystrophin (FL: panel 2: 427 kDa) and mdx (KO: panels 3 and 4), respectively. Control muscles from age-matched mdx and C57BL/10 were injected with saline only. As shown, these data confirm that a truncated dystrophin according to the invention can be efficiently produced based on a dual AAV vector system.

[0266] Further experiments were performed to evaluate different dual AAV vector systems. 1-month-old wt mice were intramuscularly injected with 1-3e10 vg of AAV vectors. Tibialis anterior (TA) muscles were recovered 30 days after injection and processed for western blotting with antibodies against dystrophin (Dys2 and DysB) and α-actinin. FIGS. 2B and C show the quantification of protein level obtained after normalization with α-actinin using StudioLight software.

[0267] FIG. 2B shows the results revealing that the quasidystrophin according to the invention (WL1 Opt) is produced at a significant level, higher than WL1 wt and WL2 wt and even better than the one obtained with the construct according to the prior art (DD).

[0268] Therefore, further experiments were performed using said construct identified as the most promising for functional evaluation.

[0269] 3) Analysis of Dystrophin Profile after Intravascular (IV) Injection

A/ Using WL1 Opt

[0270] Following the IM analysis, WL1 Opt was selected for intravascular (IV) injection. Four week old Dba_2Mdx mice received the preparations (n=7 for 5′+3′; n=2 for 5′ alone; n=2 for 3′ alone; n=5 for PBS) by intravascular injection (5e11vg/mouse). Endpoint analyses were performed 70 days post gene transfer.

3-1 in the Muscles

[0271] Quasidystrophin production based on the dual AAV vector system according to the invention was confirmed after intravenous injection (FIG. 3A).

[0272] Immunohistochemical analysis of TA muscle sections (FIG. 3B) shows a labelling of dystrophin at the membrane and confirms the proper expression of the quasidystrophin according to the invention, despite a certain heterogeneity in the expression level between mice.

[0273] In order to evaluate the therapeutic effect of the quasidystrophin according to the invention administered by intravenous injection, TA sections were stained with HPS which reveals the general state of a tissue, especially inflammation, fiber regeneration, fibrosis. When compared to wt and mdx mice, the deficient mice expressing the quasidystrophin of the invention show an intermediate profile (FIG. 4A). This observation was corroborated by the measurement of relevant markers (see FIG. 4B).An improvement can be observed with a level in treated animals between the level observed in wt and mdx mice for each criteria.

3-2 in the Heart

[0274] Besides its beneficial effects on muscles as reported above, the transgenic quasidystrophin according to the invention is also expressed in the heart of the injected mice (see FIG. 5).

B/ Using WL1 Cor

[0275] It has been noticed that codon optimization of WL1 resulted in two mutations in WL1 ORF (R49S and F748S). Therefore, the corresponding sequence in the 5′AAV vector (SEQ ID NO: 6) have been corrected (AGC at position 790 converted into AGG and TCT at position 2887 converted into TTC).

[0276] The experiments have then be repeated using both constructs: the new (cor) Dual Dys vector system (mutations corrected; 5′cor+3′) corresponding to WL1 Cor (SEQ ID NO: 22) and the old (mut) Dual Dys vector system (5′mut+3′, n=5) corresponding to WL1 Opt (SEQ ID NO: 5).

[0277] As shown in FIG. 6A/6B and even at an early stage after intravenous injection (3 weeks), proper quasidystrophin production was observed with both dual AAV vector systems in the tibialis anterior and diaphragm muscles.

[0278] With respect to the level of expression, it appears that the new (cor) Dual Dys vector system (mutations corrected; 5′cor+3′), i.e. the optimized version encoding the native quasidystrophin, gives promising results.

[0279] With respect to the therapeutic effect of the quasidystrophins so produced, FIG. 7 confirms that both constructs give rise to similar profiles of the treated mice, i.e. an amelioration in relevant markers for regeneration, inflammation and fibrosis.