Peptide-Modified Hybrid Recombinant Adeno-Associated Virus Serotype Between AAV9 and AAVrh74 with Reduced Liver Tropism and Increased Muscle Transduction

Abstract

The invention relates to a recombinant adeno-associated virus (AAV) capsid protein, which is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins comprising at least one copy of a peptide comprising the RGD motif, wherein said recombinant peptide-modified hybrid AAV capsid protein has a further reduced liver tropism and an increased muscle transduction compared to the recombinant hybrid AAV capsid protein not having said peptide. The invention relates also to the derived peptide-modified hybrid AAV serotype vector particles packaging a gene of interest and their use in gene therapy, in particular for treating neuromuscular genetic diseases, in particular muscular genetic diseases.

Claims

1. A recombinant adeno-associated virus (AAV) capsid protein, which is a peptide-modified hybrid between AAV serotype 9 (AAV9) and AAV serotype 74 (AAVrh74) capsid proteins comprising at least one copy of a peptide comprising the RGD motif, wherein said recombinant peptide-modified hybrid AAV capsid protein has a further reduced liver tropism and an increased muscle transduction compared to the recombinant hybrid AAV capsid protein not having said peptide.

2. The recombinant hybrid AAV capsid protein according to claim 1, which results from the replacement of a variable region in the AAV9 or AAVrh74 capsid sequence with the corresponding variable region of the other AAV serotype capsid sequence, wherein the variable region of AAV9 capsid corresponds to the sequence situated from any one of positions 331 to 493 to any one of positions 556 to 736 in AAV9 capsid of SEQ ID NO: 1 or a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the sequence situated from positions 493 to 556 in AAV9 capsid of SEQ ID NO: 1, and the variable region of AAVrh74 capsid corresponds to the sequence situated from any one of positions 332 to 495 to any one of positions 558 to 738 in AAVrh74 capsid of SEQ ID NO: 2 or a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 consecutive amino acids of the sequence situated from positions 495 to 558 in AAVrh74 capsid of SEQ ID NO: 2.

3. The recombinant hybrid AAV capsid protein according to claim 2, which results from the replacement of the variable region corresponding to the sequence situated from positions 449 to 609 in AAV9 capsid of SEQ ID NO: 1 or from positions 450 to 611 in AAVrh74 capsid of SEQ ID NO: 2 with the corresponding variable region of the other AAV serotype capsid sequence.

4. The recombinant hybrid AAV capsid protein according to claim 1, which is derived from a hybrid AAV capsid protein comprising a sequence selected from the group consisting of the sequences SEQ ID NO: 3 and SEQ ID NO: 4 and the sequences having at least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequences.

5-6. (canceled)

7. The recombinant hybrid AAV capsid protein according to claim 1, wherein the peptide comprising the RGD motif is of up to 30 amino acids, and comprises or consists of any one of: RGDLGLS (SEQ ID NO: 8), LRGDGLS (SEQ ID NO: 14), LGRGDLS (SEQ ID NO: 15), LGLRGDS (SEQ ID NO: 16), LGLSRGD (SEQ ID NO: 17) and RGDMSRE (SEQ ID NO: 18).

8-10. (canceled)

11. The recombinant hybrid AAV capsid protein according to claim 7, wherein the sequences SEQ ID NO: 8 and 14 to 18 are flanked by GQSG (SEQ ID NO: 9) and AQAA (SEQ ID NO: 10), respectively at the N- and C-terminal end of the peptide.

12. The recombinant hybrid AAV capsid protein according to claim 7, wherein the peptide comprises or consists of SEQ ID NO: 13.

13. (canceled)

14. The recombinant hybrid AAV capsid protein according to claim 1, wherein the at least one copy of the peptide comprising the RGD motif is inserted into a site exposed on the AAV capsid surface chosen from a site around any of positions 261, 383, 449, 575 or 590 according to the numbering in SEQ ID NO: 3.

15. (canceled)

16. The recombinant hybrid AAV capsid protein according to claim 14, wherein the at least one copy of the peptide comprising the RGD motif is inserted around position 449 or 590 according to the numbering in SEQ ID NO: 3.

17. (canceled)

18. The recombinant hybrid AAV capsid protein according to claim 16, wherein the insertion site of the at least one copy of the peptide comprising the RGD motif is from positions 587 to 592 according to the numbering in SEQ ID NO: 3, and wherein the peptide comprising the RGD motif replaces all the residues from positions 587 to 592 of the AAV capsid protein according to the numbering in SEQ ID NO: 3.

19. (canceled)

20. The recombinant hybrid AAV capsid protein according to claim 1, which comprises a sequence selected from the group consisting of SEQ ID NO: 5 and the sequences having at least 85%, 90%, 95%, 97%, 98% or 99% identity with said sequence.

21. The recombinant hybrid AAV capsid protein according to claim 1, which is a hybrid VP1, VP2 or VP3 protein.

22. A recombinant chimeric AAV capsid protein, which is selected from the group consisting of: a chimeric VP1 protein comprising: (i) a VP1-specific N-terminal region having a sequence from natural or artificial AAV serotype other than AAV9 and AAVrh74, (ii) a VP2-specific N-terminal region having a sequence from AAV9, AAVrh74 or natural or artificial AAV serotype other than AAV9 and AAVrh74, and (iii) a VP3 C-terminal region having the sequence of a hybrid VP3 protein according to claim 21, and a chimeric VP2 protein comprising: (i) a VP2-specific N-terminal region having a sequence from natural or artificial AAV serotype other than AAV9 and AAVrh74, and (ii) a VP3 C-terminal region having the sequence of a hybrid VP3 protein according to claim 21.

23. A polynucleotide encoding the recombinant hybrid AAV capsid protein according to claim 1, in expressible form, and eventually further encoding AAV Replicase protein in expressible form.

24. (canceled)

25. An AAV vector particle packaging a gene of interest, which comprises the hybrid recombinant AAV capsid protein according to claim 1, and eventually also at least one AAV capsid protein from natural or artificial AAV serotype other than AAV9 and AAVrh74.

26. The AAV vector particle according to claim 25, wherein the gene of interest 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. A pharmaceutical composition comprising a therapeutically effective amount of AAV vector particles according to claim 26.

28. A method of treating a disease by gene therapy, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising the recombinant hybrid AAV capsid protein according to claim 1.

29. The method according to claim 28, wherein the gene therapy targets a gene responsible for a neuromuscular genetic disorders selected from the group consisting of Duchenne muscular dystrophy, Limb-girdle muscular dystrophies, Spinal muscular atrophy, Myotubular myopathy, Pompe disease and Glycogen storage disease III.

30. The method according to claim 29, wherein the target gene is selected from the group consisting of DMD, CAPN3, DYSF, FKRP, ANO5, SMN1, MTM1, GAA and AGL genes.

Description

FIGURE LEGENDS

[0134] FIG. 1: Design of peptide insertion in the hybrid AAV serotype between AAV9 and AAVrh74.

[0135] A. Schematic representation of the Cap genes (VP1) of AAV9, AAVrh74 and the AAV9-rh74 hybrid capsid highlighting the sequences of the variable region. B. The VP1 protein of the AAV-MT capsid derives from the insertion of 15 amino acid (15-mer) between amino acid 586 and 593 of the VP1 of the AAV9-rh74 capsid.

[0136] FIG. 2: Comparative biodistribution of AAV9-RH74 and AAV-MT in GSDIII mice. GSDIII mice were tail vein injected with 2×10.sup.12 vector genome/mouse of the indicated AAV vectors. Three months after injection, tissues were collected. PBS-injected mice were used as controls. (A-F) Vector genome copy number per cell (VGCN/cell) measured in liver (A); heart (B); diaphragm (C); quadriceps (D); extensor digitorum longus (EDL) (E) and gastrocnemius (F). Data were presented as mean ±standard deviation. Statistical analyses were performed by ANOVA (*=p<0.05 vs. PBS; §=p<0.05 vs. ALL; n=4-5 mice per group).

EXAMPLE

Peptide-Modified Hybrid AAV9-rh74 Serotype Vector

1. Material and Methods

Plasmid Construction for New Serotypes

[0137] To construct the plasmid containing AAV2 Rep sequence and Hybrid Cap 9-rh74, a fragment of 1029 nt, containing the highly variable part of AAV-rh74 Cap flanked with AAV9 Cap sequence fragments and restriction sites BsiWI in 5′ and Eco47III in 3′, was synthesized (GENEWIZ). This fragment was then inserted using the mentioned restriction sites in the plasmid pAAV2-9, which contains AAV2 Rep and AAV9 Cap, to replace the AAV9 Cap corresponding sequence. Peptide engraftment was performed by replacing the QQNAAP hexapeptide (SEQ ID NO: 11) in the AAV9-rh74 capsid with the GQSGRGDLGLSAQAA (SEQ ID NO: 13) amino acid sequence.

AAV Production

[0138] HEK293T cells were grown in suspension in 250 mL of serum-free medium. The cells were transfected with 3 plasmids: i) a transgene plasmid, containing AAV2 ITRs flanking an expression cassette ii) the helper plasmid pXX6, containing adenoviral sequences necessary for AAV production, and iii) a plasmid containing AAV Rep and Cap genes, defining the serotype of AAV. Two days after transfection, the cells were lysed to release the AAV particles.

[0139] The viral lysate was purified by affinity chromatography. Viral genomes were quantified by a TaqMan real-time PCR assay using primers and probes corresponding to the ITRs of the AAV vector genome (Rohr et al., J. Virol. Methods, 2002, 106, 81-88).

In Vivo Studies

[0140] All mouse studies were performed according to the French and European legislation on animal care and experimentation (2010/63/EU) and approved by the local institutional ethical committee (protocol no. 2016-002C). AAV vectors were administered intravenously via the tail vein to three month-old male GDE knockout mice. PBS-injected littermates were used as controls. Three months after vector injections, tissues were harvested and homogenized in DNAse/RNAse free water using Fastprep tubes (6.5 m/s; 60 seconds).

Vector Genome Copy Number (VGCN) Quantification

[0141] For vector genome copy number (VGCN) quantification in samples, DNA was extracted from samples using MagNA Pure 96 Instrument (Roche). Real-time PCR was performed on 1μL of DNA, using the protocol for AAV vectors titration described above. Exon Mex5 of titin gene was used as genomic DNA loading control.

2. Results

[0142] The AAV9-rh74 hybrid capsid was engineered by inserting a peptide in the common region between VP1, VP2 and VP3 between Q at positions 586 and I at position 593 of SEQ ID NO: 3 (FIG. 1). AAV capsid modification was performed according to Kienle EC (Dissertation for the degree of Doctor of natural Sciences, Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany, 2014) using a peptide as disclosed in Michelfelder et al. (PLoS ONE, 2009, 4, e5122). Briefly, the hexapeptide QQNAAP (SEQ ID NO: 11) present in the common region between VP1, VP2 and VP3 of the hybrid Cap9-rh74 (positions 587 to 592 of SEQ ID NO: 3) is mutated to the octapeptide GQSGAQAA (SEQ ID NO: 12) and peptide P1 (RGDLGLS; SEQ ID NO: 8) is inserted between glycine at position 4 and alanine at position 5. The peptide-modified hybrid Cap9-rh74 having the insertion of the peptide P1 has the amino acid sequence SEQ ID NO: 5 and the corresponding coding sequence is SEQ ID NO: 6. Vectors are produced by triple transfection in HEK293 cells grown in suspension and purified by affinity chromatography as described in example 1. The resulting AAV Vector called AAV-Muscle Transducer (AAV-MT) was injected in GDE knockout mice, an animal model of GSDIII, in parallel with AAV9-rh74 at the dose of 2×10.sup.12 vg/mouse. Tissues were obtained three months post-injection and vector genome copy number (VGCN) was measured by quantitative PCR to assess tissue targeting (FIG. 2). In the liver, injection of AAV9-rh74 resulted in significantly increased vector genome copies compared to PBS injected mice (p<0.05; FIG. 2A) while liver transduced with AAV-MT did not showed a significant increase in VGCN compared to the PBS group. In general, AAV-MT showed better transduction efficacy in muscles when compared to the parental capsid AAV9-rh74 (FIG. 2B-F) Importantly, in diaphragm, quadriceps and gastrocnemius, the AAV-MT outperformed the parental capsid (p<0.05 vs. AAV9-rh74 group). Finally, in heart and extensor digitorum longus (EDL) a significantly higher VGCN were measured after injection with the AAV-MT capsid but not with the AAV9-rh74 when compared with PBS injected mice. These data indicate that the AAV-MT capsid outperforms the AAV9-rh74 in muscle transduction and maintains or even ameliorates the pronounced liver detargeting observed for the parental capsid.