Squaramide-modified adeno-associated virus vectors

Abstract

The present invention relates to adeno-associated virus (AAV) vectors modified by the covalent coupling of at least one compound comprising a squaramide moiety to at least one amino group of an amino acid residue of the capsid of the AAV vectors. The AAV vectors are useful in transducing a cell, especially for gene therapy.

Description

This invention relates to adeno-associated virus (AAV) vectors modified by the covalent coupling of at least one compound comprising a squaramide moiety to at least one amino group of an amino acid residue of the capsid of the AAV vectors. The AAV vectors are useful in transducing cells, especially for gene therapy.

Claims

CLAIMS 1. An adeno-associated virus (AAV) vector particle comprising a moiety of formulawherein N* is a nitrogen atom of a primary amino group from a surface-exposed amino acid residue of a capsid polypeptide from the AAV vector; ---- represents the point of attachment to the AAV vector’s capsid; and RL-NH- is a functional moiety including a nitrogen containing group -NH- and a group RL, and wherein said functional moiety RL-NH- comprises a steric shielding agent, a labelling agent, a cell-type specific ligand, a drug moiety or a combination thereof.

2. The AAV vector particle according to claim 1, wherein the functional moiety RL- NH- comprises a group Z and one or more spacers L, and the adeno-associated virus (AAV) vector particle is represented by formula (IIa):wherein Z is H or a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins, or functionally active fragments thereof, membrane receptors or functionally activefragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties, and L comprises one or more groups selected from the group consisting of an arylene or a heteroarylene group, an optionally substituted group comprising saturated or unsaturated, linear or branched, C1-C40hydrocarbon chains, preferably one or more groups C1-6alkyl, C1-6alkylamine or C1-6acyl, a polyethylene glycol (PEG), a polypropylene glycol (PPG), an alkylene amine; an acyl group, an amino acid moiety, a polyether of a branched polyol, a β-alanine polymer, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.

3. The AAV vector particle according to claim 2, wherein L comprises more than one spacers groups selected from the group consisting of L1, L2and L3, and said moiety of formula (IIa) is selected from the group consisting of formula (IIa1), (IIa2), (IIa3), (IIa4), (IIa5), (IIa6), (IIa7), (IIa8), (IIa9), (IIa10), (IIa11), (IIa12), (IIa13), (IIa14), (IIa15), (IIa16), (IIa17), (IIa18), (IIa19) and (IIa20):wherein L1is one or more groups selected from the group consisting of a polyethylene glycol (PEG), comprising 1 to 40, preferably 1 to 20, ethylene glycol monomers, a polyether of a branched C3-12polyol, preferably a branched C3-6polyol, and a β-alanine polymer comprising 1 to 40, preferably 1 to10, β-alanine monomers, or a mixture thereof; L2comprises one or more arylene or a heteroarylene groups; L3is one or more groups selected from an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, a C1-6alkylene group, being said C1-6alkylene a linear C1-6alkylene group or a branched C3-6alkylene group; a C1-6alkylenamine group and C1-6acyl group; wherein L3is covalently linked to L2by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and wherein, L1and L2, or L1and L3are covalently linked by an amide moiety or a bioisostere moiety thereof; or wherein L1and L3covalently linked by an ether bond.

4. The AAV vector particle according to any of claims 2 or 3, or a pharmaceutically acceptable salt thereof, wherein Z comprises a linear or a cyclic peptide or a saccharide, preferably wherein said peptide features biological activity, and beingthe saccharide selected from the group consisting of monosaccharides, oligosaccharides and polysaccharides.

5. The AAV vector particle according to any of claims 2 to 4, wherein the peptide is a blood brain barrier (BBB) shuttle peptide, preferably a BBB shuttle peptide selected from the group consisting of a peptide THR or a peptide with a RGD motif, including a cyclic RGD peptide; and wherein the saccharide is selected from the group consisting of mannose, galactose, N-acetylglucosamine, fucose, fructose, glucose, xylose trehalose, desosamine, glucuronic acid, S6-galactose, S6-N- acetylgalactosamine, P6-mannose, P6-glucose, sialic acid, S1-fructose and P1- fructose.

6. The AAV vector particle according to any of claims 1 to 5, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12; or pseudotypes, chimeras, and variants thereof.

7. The AAV vector particle according to any of claims 1 to 6, wherein the moiety of formula (II) is selected from the group consisting of formula (IIb), (IIc), (IId), (IIe), (IIf), (IIg), (IIh), (IIj), (IIk), (IIm), (IIn), (IIp), (IIq), (IIr), (IIs), (IIt), (IIv), (IIw) and (IIx):(IIc),being at least one of Ra, Rband Rca group R’, and wherein Z is a peptide or a saccharide, Ar is arylene group or a heteroarylene group comprising one or more heteroatoms selected from the group consisting of N, O, S and Se; R1is selected from the group consisting of H, C1-6alkyl, C1-6haloalkyl and Z-(OCH2-CH2)n-, Z-C(O)NH-(CH2)q-(OCH2-CH2)n- and Z-NHC(O)-(CH2)q-(OCH2-CH2)n-; preferably R1 is H; m1and m2are each independently 0, 1 or 2, m3, m4, m5and m6are each independently selected from 1 to 6, preferably 1 or 2, and n and n’ are each independently selected from 1 to 40, preferably 1 to 20, and q is selected from 1 to 3.

8. A pharmaceutical composition comprising an AAV vector particle according to any of claims 1 to 7, and at least one pharmaceutically acceptable vehicle.

9. An AAV vector particle according to any of claims 1 to 7, or a pharmaceutical composition according to claim 8, for use as a medicament.

10. An AAV vector particle according to any of claims 1 to 7, or a pharmaceutical composition according to claim 8, for use in gene therapy.

11. A method of delivering a nucleic acid to a cell, the method comprising contacting a cell with an AAV vector particle according to any one of claims 1 to 7 and a nucleic acid to be expressed in the contacted cell.

12. A compound of formula (Illa):whereinZ is H or a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, glycosylated peptides, proteins, glycoproteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acids or peptide aptamers, vitamins, and drugs moieties, andL consists of one or more groups selected from the group consisting of an arylene or a heteroarylene group, an optionally substituted group comprising saturated orunsaturated, linear or branched C2-C40hydrocarbon chains, a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers, a polypropylene glycol (PPG) comprising 1 to 40 propylene glycol monomers, a polyether of a branched C3-12polyol, an arginine derivative, a β-alanine polymer comprising 1 to 40 β-alanine monomers, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof; and wherein L comprises at least one or more groups selected from the group consisting of an arylene or a heteroarylene group, a polyethylene glycol (PEG) comprising 1 to 40 ethylene glycol monomers and a β-alanine polymer comprising 1 to 40 β-alanine monomers.

13. The compound of claim 12, wherein L comprises more than one spacers groups selected from the group consisting of L1, L2, and L3, and said compound of formula (III) is selected from the group consisting of formula (Illa1), (IIIa2), (Illa3), (IIIa4), (Illa5), (IIIa6), (IIIa7), (IIIa8), (IIIa9), (Illa10), (Illa11), (IIIa12), (IIIa13), (IIIa14), (Illa15), (IIIa16), (IIIa17), (IIIa18), (IIIa19) and (IIIa20):wherein L1is one or more groups selected from the group consisting of a polyethylene glycol (PEG), comprising 1 to 40, preferably 1 to 20, ethylene glycol monomers, a poly ether of a branched C3-12polyol, preferably a branched C3-6polyol, and a β-alanine polymer comprising 1 to 40, preferably 1 to10, β-alanine monomers, or a mixture thereof; L2comprises one or more arylene or a heteroarylene groups; L3is one or more groups selected from the group consisting of an amino acid moiety, preferably an arginine moiety or a β-alanine moiety, a C1-6alkylene group, being said C1-6alkylene a linear C1-6alkylene group or a branched C3-6alkylene group; a C1-6alkylenamine group and C1-6acyl group, being said C1-6alkylene a linear C1-6alkylene group or a branched C3-6alkylene group; wherein L3is covalently linked to L2by one carbon atom of the arylene group or by one carbon atom or one heteroatom of the heteroarylene group; and wherein L1and L2or L1and L3are covalently linked by an amide moiety or a bioisostere moiety thereof, or wherein L1and L3covalently linked by an ether bond.

14. A method for manufacturing an AAV vector particle according to any one of claims 1 to 7, wherein said method comprises reacting a compound of formula (III) according to any of claims 12 or 13 with an amino group present within the capsid of the AAV vector.

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