Use of a Synthetic AAV Capsid for Gene Therapy of Muscle and Central Nervous System Disorders

Abstract

The invention relates to the use of a recombinant porcine adeno-associated virus (AAV) vector comprising a peptide-modified porcine AAV serotype 1 (AAVpol) capsid in gene therapy of muscle and/or central nervous system (CNS) disorders, in particular neuromuscular diseases such as genetic neuromuscular diseases.

Claims

1-16. (canceled)

17. A method of treating nervous system disorders and neuromuscular disorders affecting the nervous system by gene therapy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a recombinant porcine adeno-associated virus (AAV) vector comprising a peptide-modified capsid protein, wherein the peptide-modified capsid protein comprises at least one peptide comprising the sequence MPLGAAG (SEQ ID NO: 2) or a variant comprising only one or two amino acid mutations in said sequence, and wherein said at least one peptide is inserted into a capsid from a porcine AAV serotype 1.

18. The method according to claim 17, wherein the recombinant porcine AAV vector is characterized by the combination of liver detargeting and transgene expression levels in different muscle groups, and in the brain and spinal cord that are at least equivalent if not superior to that of AAV9 vector, after systemic administration, in particular intravenous administration.

19. The method according to claim 17, wherein the peptide comprises the sequence GMPLGAAGA (SEQ ID NO: 3), or a variant comprising one or two amino acid deletions or substitutions in said sequence.

20. The method according to claim 19, wherein the peptide comprises or consists of the sequence GQRGMPLGAAGAQAA (SEQ ID NO: 4).

21. The method according to claim 17, wherein the peptide is inserted between residues N567 and S568 or between residues N569 and T570 of the capsid protein; said positions being determined by alignment with SEQ ID NO: 1.

22. The method according to claim 21, wherein the peptide replaces all the residues from positions 565-567 and 568-570 or all the residues from positions from positions 567-569 and 570-572; said positions being determined by alignment with SEQ ID NO: 1.

23. The method according to claim 17, wherein said peptide-modified capsid protein comprises a sequence selected from the group consisting of the sequence SEQ ID NO: 5, and the sequences having at least 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 5 which comprise said peptide, and the fragment thereof corresponding to VP2 or VP3 capsid protein.

24. The method according to claim 17, wherein the recombinant porcine AAV vector is a vector particle packaging a gene of interest for therapy.

25. The method according to claim 24, wherein the gene of interest for therapy is operably linked to a promoter functional in neurons and/or glial cells.

26. The method according to claim 24, wherein the gene of interest for therapy is selected from the group consisting of: (i) therapeutic genes; (ii) genes encoding therapeutic proteins or peptides such as therapeutic antibodies or antibody fragments and genome editing enzymes; and (iii) genes encoding therapeutic RNAs such as interfering RNAs, guide RNAs for genome editing and antisense RNAs capable of exon skipping.

27. The method according to claim 17, which is for treating a neuromuscular disease affecting the nervous system,.

28. The method according to claim 17, wherein the disease is a genetic neuromuscular disease affecting the nervous system.

29. The method according to claim 17, , wherein the disease is a genetic neuromuscular disease affecting the nervous system selected from the group comprising : (i) myopathies; (ii) spinal muscular atrophies and motor neuron diseases; (iii) Myotonic syndrome; (iv) Hereditary motor and sensory neuropathies; (v) Hereditary paraplegia and Hereditary ataxia; and (vi) Congenital myasthenic syndromes.

30. The method according to claim 29, wherein the myopathies are muscular dystrophies including congenital muscular dystrophies and/or the Myotonic syndrome is myotonic dystrophy type 1 or type 2.

31. The method according to claim 17, wherein the recombinant porcine AAV vector is a vector particle packaging a functional version of a gene responsible for a genetic neuromuscular disorder affecting the nervous system or a therapeutic RNA targeting said gene responsible for the disease.

32. The method according to claim 31, wherein the genetic neuromuscular disorder affecting the nervous system and the gene responsible for said disease are selected from the group comprising: Duchenne muscular dystrophy and Becker muscular dystrophy (DMD gene); Limb-girdle muscular dystrophies (DYSF, FKRP genes); Myotonic dystrophy type 1 (DMPK gene) and type 2 (CNBP/ZNF9 gene); Centronuclear myopathies (DNM2, BIN1 genes); Pompe disease (GAA gene); Glycogen storage disease III (AGL gene); Spinal muscular atrophy (SMN1, ASAH1 genes); Amyotrophic lateral sclerosis (SOD1, ALS2, SETX, FUS, ANG, TARDBP, FIG4, OPTN and others); Hereditary paraplegia (SPAST, SPG7); Charcot-Marie-Tooth, Type 4B1 (MTMR2), and Congenital myasthenic syndrome (CHAT, AGRN).

33. The method according to claim 31, wherein said gene responsible for the genetic neuromuscular disorder affecting the nervous system is selected from the group comprising: DMD, DYSF, FKRP, DNM2, BIN1, GAA, AGL, SMN1 and ASAH1 genes.

34. The method according to claim 31, wherein said gene responsible for the genetic neuromuscular disorder affecting the nervous system is selected from the group comprising: FKTN, POMT1, POMT2, POMGNT1, POMGNT2, LMNA, ISPD, GMPPB, LARGE, LAMA2, TRIM32, and B3GALNT2.

35. The method according to claim 17, which is for the gene therapy of Spinal muscular atrophy, wherein said vector comprises a peptide-modified capsid protein comprising the sequence SEQ ID NO: 5 or a sequence having at least 95%, 96%, 97%, 98% or 99% identity with said sequence which comprises the peptide of any one of SEQ ID NO: 2 to 4, and said vector further packaging a human SMN1 gene operably linked to a promoter functional in neurons and/or glial cells.

36. The method according to claim 17, wherein the recombinant porcine AAV vector is administered by systematic route, by intracerebral, intracerebroventricular, intracisternal, and/or intrathecal routes, or by a combination thereof.

37. The method according to claim 36, wherein the systemic route is an intravascular route.

Description

FIGURE LEGENDS

[0133] FIG. 1: Body weight over time of Mtml-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO-AAVpo1), AAVpo1A1 (KO-AAVpo1A1), AAV8 (KO-AAV8), AAV9 (KO-AAV9), AAVrh10 (KO-AAVrh10). Untreated wild-type (WT-PBS) and Mtml-KO (KO-PBS) mice are used as controls.

[0134] FIG. 2: Muscles weight of Mtml-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Untreated wild-type (WT + PBS) and Mtml-KO (KO + PBS) mice are used as controls. TA: Tibialis Anterior EDL: Extensor Digitorum Longus. Qua: Quadriceps. Ga: Gastrocnemius. Sol: Soleus. Triceps. Biceps. Diaphragm. Heart. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison post-test (* P<0.05 vs. KO + AAV8; ** P<0.01 vs. KO + AAV; *** P<0.001 vs. KO + AAV8; $ P<0.05 vs. KO + AAV9; $$ P<0.01 vs. KO + AAV9; $$$ P<0.001 vs. KO + AAV9).

[0135] FIG. 3: Vector Copy Number (VCN) in muscles of Mtml-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Untreated wild-type mice (WT-PBS) are used as control. TA: Tibialis Anterior EDL: Extensor Digitorum Longus. Qua: Quadriceps. Ga: Gastrocnemius. Sol: Soleus. Triceps. Biceps. Diaphragm. Heart. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison post-test (* P<0.05 vs. KO + AAV8; ** P<0.01 vs. KO + AAV; *** P<0.001 vs. KO + AAV8; $ P<0.05 vs. KO + AAV9; $$ P<0.01 vs. KO + AAV9).

[0136] FIG. 4: Vector Copy Number (VCN) in organs of Mtml-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison post-test (* P<0.05 vs. KO + AAV8; ** P<0.01 vs. KO + AAV; *** P<0.001 vs. KO + AAV8; $ P<0.05 vs. KO + AAV9; $$ P<0.01 vs. KO + AAV9; $$$ P<0.001 vs. KO + AAV9).

[0137] FIG. 5: hMTM1 mRNA level in muscles of Mtml-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). MTM1 mRNA levels are expressed relative to expression in KO + AAV8. TA: Tibialis EDL: Extensor Digitorum Anterior Longus. Qua: Quadriceps. Ga: Gastrocnemius. Sol: Soleus. Triceps. Biceps. Diaphragm. Heart. Statistical analyses were performed by using one-way ANOVA followed by Tukey’s multiple comparison post-test (* P<0.05 vs. KO + AAV8; ** P<0.01 vs. KO + AAV; *** P<0.001 vs. KO + AAV8; $ P<0.05 vs. KO + AAV9; $$ P<0.01 vs. KO + AAV9; $$$ P<0.001 vs. KO + AAV9).

[0138] FIG. 6: hMTM1 mRNA level in organs of Mtml-KO mice treated with various AAV vectors expressing MTM1. AAVpo1 (KO + AAVpo1), AAVpo1A1 (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). hMTM1 mRNA levels are expressed relative to expression in KO + AAV8. Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison post-test (* P<0.05 vs. KO + AAV8; ** P<0.01 vs. KO + AAV; *** P<0.001 vs. KO + AAV8; $ P<0.05 vs. KO + AAV9; $$$ P<0.001 vs. KO + AAV9).

[0139] FIG. 7: hMTM1 protein level in muscles of Mtml-KO mice treated with various AAV vectors expressing hMTM1. AAVpo1 (KO + AAVpo1), AAVpolAl (KO + AAVpo1A1), AAV8 (KO + AAV8), AAV9 (KO + AAV9), AAVrh10 (KO + AAVrh10). Untreated wild-type (WT + PBS) and Mtml-KO (KO + PBS) mice are used as controls. GAPDH is used as internal control.

[0140] FIG. 8: Immunolocalization of SMN protein in neurons of spinal cord of C57BL/6 mice injected with AAVpo1A1-SMN1 vector at 5x10.sup.13 vg/kg. The SMN protein is fused to an HA-tag and detected with an anti-HA antibody. Neurons were labelled with an anti-NeuN antibody. Arrows show motoneurons expressing HA-SMN. Scale bars = 200 .Math.m (left) or 50 .Math.m (right).

EXAMPLES

Materials and Methods

[0141] The AAVpo1A1 capsid of porcine origin (nucleotide sequence SEQ ID NO: 13 encoding the protein of SEQ ID NO: 5 comprising the peptide of SEQ ID NO: 4 replacing all the residues from positions 567-569 and 570-572 of AAVpo1 capsid protein of SEQ ID NO: 1) was compared with the serotypes 8, 9, rh10 and po1 (Bello et al., Gene Therapy, 2009, 16, 1320-1328. doi: 10.1038/gt.2009.821), in a constitutive knockout of the myotubularin gene (Mtm1 KO mouse line) described previously (Buj-Bello et al., PNAS, 2002, 99, 15060-5. doi:10.1073/pnas.212498399; Al-Qusairi, et al., PNAS, 2009, 106, 18763-8. doi:10.1073/pnas.0900705106). The vectors were all produced by a triple transfection method using HEK 293 cells and carried a cassette expressing human MTM1 under the control of the human desmin promoter (1 kb) and a target sequence of miR208a (Raguz et al., Dev. Biol., 1998, 201, 26-42; Paulin D & Li Z, Exp. Cell. Res., 2004, Nov 15;301(1):1-7; Roudault et al., Circulation, 2013, 128, 1094-104. doi: 10.1161/CIRCULATIONAHA.113.001340). The AAVpo1A1 and AAV9 capsids were also assessed in C57BL/6 mice, with a cassette expressing human SMN fused to an HA tag sequence under the control of the ubiquitous CAG promoter (Meyer et al., Molecular Therapy, 2015, 23. doi: 10.1038/mt.2014.210).

[0142] A single dose of 2x10.sup.13 vg/kg of each vector expressing MTM1 was administrated intravenously in 3-week-old mutant mice and tissues were harvested and frozen in nitrogen 4 weeks post-injection. As control, PBS was injected in Mtm1-KO and wild-type littermate males. C57BL/6 mice received a dose of 8x10.sup.12 vg/kg of either AAV9 or AAVpo1A1 vectors at the age of 4 weeks, and tissues were collected 3 weeks later.

[0143] The number of vector genomes per diploid genome was quantified from 32 ng of total DNA by Taqman real-time PCR using a LightCycler480 thermocycler (Roche). The titin gene was used for standardization with primers and probe: 5′-AAAACGAGCAGTGACGTGAGC-3′ (forward; SEQ ID NO: 6), 5′-TTCAGTCATGCTGCTAGCGC-3′ (reverse; SEQ ID NO: 7) and 5′-TGCACGGAAGCGTCTCGTCTCAGTC-3′ (probe; SEQ ID NO: 8). Primers used for vector genome (MTMI) amplification were: 5′-TTGGTTGTCCAGTTTGGAGTCTACT-3′ (forward; SEQ ID NO: 9), 5′-CCGTCACTGCAATGCACAAG-3′ (reverse; SEQ ID NO: 10) and 5′-ATATCAAGCTCGTTTTGAC-3′ (probe; SEQ ID NO: 11). Primers used for vector genome (SMN1) amplification were: 5′-CAGTGCAGGCTGCCTATCAG-3′ (forward; SEQ ID NO: 15), 5′-TGTGGGCCAGGGCATTAG-3′ (reverse; SEQ ID NO: 16), 5′-AAGTGGTGGCTGGTGTG-3′ (probe; SEQ ID NO: 17). Other primers used for vector genome (SMN1) amplification were: 5′-GCTGCCTCCATTTCCTTCTG-3′ (forward; SEQ ID NO: 18), 5′-ACATACTTCCCAAAGCATCAGCAT-3′ (reverse; SEQ ID NO: 19), 5′-CACCACCTCCCATATGTCCAGATTCTCTTG-3′ (probe; SEQ ID NO: 20).

[0144] The level of MTM1 transcripts was quantified from 350 ng of total RNA subjected to reverse transcription using RevertAid H Minus Reverse Transcriptase kit (Thermo Scientific). Next, a cDNA amount was amplified by qPCR using a LightCycler480 thermocycler (Roche). The RPLP0 gene was used for standardization with primers and probe: 5′-CTCTGGAGAAACTGCTGCCT-3′ (forward; SEQ ID NO: 21), 5′-CTGCACATCACTCAGAATTTCAA-3′ (reverse; SEQ ID NO: 22) and 5′-AGGACCTCACTGAGATTCGGGATATGC -3′ (probe; SEQ ID NO: 23).

[0145] Proteins were extracted and analyzed by NuPAGE 4-12% Bis-Tris gel electrophoresis and western blotting. Membranes were probed with a polyclonal antibody against human myotubularin (Abnova). A mouse monoclonal antibody specific for GAPDH (Merck Millopore) was used as internal control. Detection was performed with a secondary antibody (Donkey anti-Goat 800 or Goat anti-Mouse 680 (Invitrogen) and the Odyssey infrared imaging system (LI-COR Biotechnology Inc.).

[0146] For immunostaining of the vector-derived HA-SMN, C57BL/6 mice were injected with a single dose of 5x10.sup.13 vg/kg of AAVpo1A1 vector and euthanized 4 weeks later by intraperitoneal anesthetic injection (10 mg/kg xylazine, 100 mg/kg ketamine), followed by intracardiac perfusion with PBS and then 4% paraformaldehyde (PFA). Tissues were isolated and postfixed by incubation in 4% PFA. Spinal cord was then incubated in a PBS-sucrose solution (30%). Serial coronal cryostat sections of lumbar spinal cord were processed for mouse IgG blocking with a Mouse-on-Mouse IgG Blocking Solution (Invitrogen), then anti-HA tag (hSMN) staining with a rabbit anti-HA primary antibody (Sigma-Aldrich) and for anti-NeuN staining with a mouse anti-NeuN primary antibody (Sigma-Aldrich). Detection was performed with fluorescent-conjugated secondary antibodies (Goat anti-rabbit Alexa Fluor 488 and Goat anti-mouse Alexa Fluor 594 (Invitrogen)). Sections were mounted with FluoroMount-G medium + DAPI, and the images were captured using the axioscan Z1 (Zeiss).

Results

[0147] AAV vectors (AAVpo1, AAVpo1A1, AAV8, AAV9, AAVrh10) expressing MTM1 were injected intravenously at 2x10.sup.13 vg/kg in Mtm1-KO mice at 3 weeks of age. From two weeks post-injection, the body weight of treated KO and WT mice was similar, whereas untreated KO mice started to lose weight after 6 weeks of age (FIG. 1). Skeletal muscles of mutant mice from AAVpo1- and AAVpo1A1- treated groups, such as Tibialis Anterior (TA), quadriceps (Qua), gastrocnemius (Ga) and triceps (Tri), were heavier than in the AAV8-treated group mice (FIG. 2).

[0148] According to vector genome quantification in skeletal muscles (FIGS. 3 and 4), the AAVpo1A1 vector transduced most skeletal muscles as efficiently as the AAV8 vector. Interestingly, the AAVpo1A1 vector resulted in low transduction levels of organs such as heart, liver, spleen, kidney, lungs and brain.

[0149] The expression of the MTM1 transgene was analyzed by RT-qPCR in various muscles and organs (FIGS. 5 and 6). The AAVpo1A1 vector led to higher MTM1 transcript levels in all skeletal muscles compared to AAV8, despite similar transduction levels, reaching transgene expression levels comparable to the AAV9 vector. Moreover, administration of the AAVpo1A1 vector detargeted transgene expression in organs such as liver and spleen, with much lower MTM1 transcript levels than those observed after AAV8 vector delivery. Transgene expression levels were higher in regions of the central nervous system, such as cortex and spinal cord, of AAVpo1A1-treated mice compared to the AAV8 group, and even higher than in AAV9-treated mice in the spinal cord.

[0150] MTM1 protein expression was analyzed in various muscles (gastrocnemius, triceps, and diaphragm) by immunoblotting (FIG. 7). The AAVpo1A1 vector administration resulted in higher MTM1 protein levels in skeletal muscles of mutant mice compared to the AAV8 and AAVpo1 vectors.

[0151] The AAVpo1A1 and AAV9 vectors expressing SMN1 were injected intravenously at 8x10.sup.12 vg/kg in C57BL/6 mice at 4 weeks of age. Several muscles and organs were collected 3 weeks later. The AAVpolAl vector transduced at similar levels all skeletal muscles and heart in WT mice. As previously observed in Mtm1-KO mice, administration of the AAVpo1A1 vector resulted in low transduction of the liver.

[0152] Transgene expression was analyzed by RT-qPCR, and results show that SMN1 transcript levels were similar in skeletal muscles after AAVpo1A1 and AAV9 vector transduction.. The levels of AAVpo1A1-derived SMN1 mRNA were lower in heart, liver, spleen and kidney compared to AAV9. In the central nervous system, AAVpo1A1-derived SMN1 transcripts were present in all analyzed regions (cortex, cerebellum and spinal cord), with levels slightly higher in spinal cord.

[0153] To assess the cellular localization of SMN in spinal cord, immunofluorescence stainings using anti-HA antibodies and anti-NeuN antibodies were performed 4 weeks after injection of AAVpo1A1-SMNI vector at 5x10.sup.13 vg/kg. As shown in FIG. 8, HA-SMN was expressed in neurons, and in particular in the large motoneurons located in the ventral horns of the spinal cord. The majority of motoneurons (mean 80%, range 72% to 94%, n=4 mice) were transduced with AAVpo1A1 and expressed the transgene.

[0154] Altogether this demonstrates the improved potency and tissue specificity of AAVpo1A1 vector for muscle- and/or CNS-directed gene transfer since it advantageously combines high transgene expression levels in skeletal muscle, brain and spinal cord comparable to the AAV9 vector and vector detargeted transgene expression in other organs such as liver and spleen.