LACTAM-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 lactam moiety (e.g., β-lactam) 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.

Claims

1. An adeno-associated virus (AAV) vector comprising a moiety according to formula (II): ##STR00067## or a pharmaceutically acceptable salt thereof, wherein N* is a nitrogen atom of an amino group of an amino acid residue of the AAV vector's capsid; ----- represents the point of attachment to the AAV vector's capsid; Z is a functional moiety comprising a cell-type specific ligand, a labelling agent, a steric shielding agent, a drug moiety or combinations thereof; L is a linker; R.sup.1, R.sup.1′, R.sup.2 and R.sup.2′ are each independently hydrogen, halogen, or an optionally substituted C.sub.1-6 alkyl; or R.sup.1 and R.sup.1′ or R.sup.2 and R.sup.2′, together with their intervening atoms, may come together of form an optionally substituted spiro-fused ring; or R.sup.1 and R.sup.2, together with their intervening atoms, may form an optionally substituted 3- to 7-membered saturated, partially unsaturated, or aryl or heteroaryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

2. The AAV vector according to claim 1, wherein at least one N* is a nitrogen atom of an amino group of a lysine residue of the AAV vector's capsid.

3. The AAV vector according to claim 1, wherein Z comprises or consists of a cell-type specific ligand selected from the group consisting of saccharides, hormones, peptides, proteins or functionally active fragments thereof, membrane receptors or functionally active fragments thereof, antibodies or functionally active fragments thereof, spiegelmers, nucleic acid or peptide aptamers, vitamins, and drugs.

4. (canceled)

5. The AAV vector according to claim 3, wherein Z is selected from the group consisting of mannose, galactose, fucose, desosamine, N-acetylglucosamine, N-acetylgalactosamine, S6-galactose, S6-N-acetylgalactosamine, glucuronic acid, P6-galactose and P1-galactose.

6. The AAV vector according to claim 1, wherein Z comprises or consists of a steric shielding agent selected from the group consisting of polyethylene glycol, pHPMA, and polysaccharides.

7. The AAV vector according to claim 1, wherein L comprises an optionally substituted group comprising saturated or unsaturated, linear or branched C.sub.2-C.sub.40 hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkylene diamines, and combinations thereof.

8. The AAV vector according to claim 7, wherein L comprises a polyethylene glycol (PEG), comprising 2 to 40 ethylene glycol monomers.

9-11. (canceled)

12. The AAV vector according to claim 1, comprising a moiety according to formula (IIa) ##STR00068## or a pharmaceutically acceptable salt thereof, wherein L.sup.1 is an optionally substituted aryl or heteroaryl group; L.sup.2 is an optionally substituted heteroaryl group; and L.sup.3 is a linker.

13-15. (canceled)

16. The AAV vector according to claim 1, comprising a moiety according to formula (IIc): ##STR00069## or a pharmaceutically acceptable salt thereof, wherein L.sup.3 is a linker; R.sup.3, R.sup.3′, R.sup.4 and R.sup.4′ are each independently hydrogen, halogen, —OR, —NR.sub.2, —CN, —SR or an optionally substituted group selected from C.sub.1-6 alkyl or a 3- to 7-membered saturated, partially unsaturated, aryl, or heteroaryl ring having 0-3 heteroatoms independently selected form nitrogen, oxygen, or sulfur; or R.sup.3 and R.sup.4, or R.sup.3′ and R.sup.4′, together with their intervening atoms, may form an optionally substituted 3- to 7-membered saturated, partially unsaturated, aryl or heteroaryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R is independently selected from hydrogen or C.sub.1-6 alkyl; and each is independently a single or double bond.

17. The AAV vector according to claim 16, wherein L.sup.3 is an optionally substituted group selected from the group consisting of saturated or unsaturated, linear or branched C.sub.2-C.sub.40 hydrocarbon chains, polyethylene glycol, polypropylene glycol, pHPMA, PLGA, polymers of alkyl diamines and combinations thereof; preferably L.sup.3 is polyethylene glycol.

18. (canceled)

19. (canceled)

20. The AAV vector according to claim 1, comprising a moiety according to formula (IIe) ##STR00070## or a pharmaceutically acceptable salt thereof, wherein R.sup.3, R.sup.3′, R.sup.4 and R.sup.4′ are each independently hydrogen, halogen, —OR, —NR.sub.2, —CN, —SR or an optionally substituted group selected from C.sub.1-6 alkyl or a 3- to 7-membered saturated, partially unsaturated, aryl, or heteroaryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur; or R.sup.3 and R.sup.4, or R.sup.3′ and R.sup.4′, together with their intervening atoms, may form an optionally substituted 3- to 7-membered saturated, partially unsaturated, aryl or heteroaryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur; each R is independently selected from hydrogen or C.sub.1-6 alkyl; and n is an integer ranging from 1 to 20.

21. The AAV vector according to claim 1, comprising: ##STR00071## or a pharmaceutically acceptable salt thereof.

22. The AAV vector according to claim 1, wherein said AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, pseudotypes, chimeras, and variants thereof.

23. The AAV vector according to claim 1, wherein said AAV vector comprises at least one transgene, and wherein the transgene is optionally under control of a promoter.

24. The AAV vector according to claim 23, wherein said AAV vector comprises at least one transgene comprising the cDNA from a GBA gene.

25. A pharmaceutical composition comprising an AAV vector according to claim 1 and at least one pharmaceutically acceptable vehicle.

26. (canceled)

27. A method of manufacturing an AAV vector of claim 1 comprising the step of incubating an AAV with a compound of formula (I) ##STR00072## or a salt thereof, under conditions suitable to obtain the AAV vector according to claim 1.

28. A compound of formula (Ia): ##STR00073## or a salt thereof, wherein Z is a functional moiety comprising a cell-type specific ligand, a labelling agent, a steric shielding agent, a drug moiety or combinations thereof; L.sup.1 is an optionally substituted aryl or heteroaryl group; L.sup.2 is an optionally substituted heteroaryl group; and L.sup.3 is a linker; and R.sup.1, R.sup.1′, R.sup.2 and R.sup.2′ are each independently hydrogen, halogen, or an optionally substituted C.sub.1-6 alkyl; or R.sup.1 and R.sup.1′ or R.sup.2 and R.sup.2′, together with their intervening atoms, may come together to form an optionally substituted spiro-fused ring; or R.sup.1 and R.sup.2, together with their intervening atoms, may form an optionally substituted 3- to 7-membered saturated, partially unsaturated, or aryl or heteroaryl ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

29-39. (canceled)

40. The AAV vector according to claim 1, wherein R.sup.1, R.sup.1′, R.sup.2 and R.sup.2′ are hydrogen.

41. The AAV vector according to claim 12, wherein L.sup.2 is an optionally substituted 5- or 6-membered heteroaryl group comprising 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

42. A method for transducing a cell of a subject, comprising administering to said subject an AAV vector according to claim 1.

43. A method of gene therapy in a subject in need thereof, comprising administering to said subject an AAV vector according to claim 1.

44. A method of preventing and/or treating a CNS disease in a subject in need thereof, comprising administering to said subject an AAV vector according to claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0433] FIG. 1 is a schematic representation illustrating the coupling between a compound comprising a lactam (e.g., β-lactam) according to the invention and a AAV surface-exposed primary amine.

[0434] FIG. 2A, FIG. 3A, FIG. 4A, FIG. 5, FIG. 6A, FIG. 7, FIG. 8, FIG. 9A, FIG. 10A, FIG. 11, FIG. 12 and FIG. 13 are SDS-PAGE gels with silver staining evidencing AAV capsid integrity. 10.sup.12 vg of AAV2-eGFP, AAV5-eGFP, AAV8-eGFP, or AAV9-eGFP were added to a solution of lactam linkers (1), (2), (3), (4), (5), (7), (8) or (9) (3E6 eq) in TBS buffer (pH 9.3) and incubated for 4 h at 20° C. 10.sup.10 vg of each coupling was analyzed by SDS-PAGE and silver staining. VP1, VP2 and VP3 are the three proteins constituting the AAV capsid. Capsid protein molecular weight is indicated at the right of the images according to a protein ladder. FIG. 2A: AAV2 incubated with linker (1); FIG. 3A: AAV2 incubated with linker (2); FIG. 4A: AAV2 incubated with linker (3); FIG. 5: AAV2 incubated with linker (4); FIG. 6A: AAV8 incubated with linker (1); FIG. 7: AAV8 incubated with linker (3); FIG. 8: AAV2 incubated with linker (5); FIG. 9A: AAV5 incubated with linker (1); and FIG. 10A: AAV9 incubated with linker (1). FIG. 11: AAV2 incubated with linker (7). FIG. 12 AAV2 incubated with linker (8). FIG. 13: AAV2 incubated with linker (9).

[0435] FIG. 2B, FIG. 3B, FIG. 4B, FIG. 6B, FIG. 9B, and FIG. 10B are western blot analysis. 10.sup.12 vg of AAV2-eGFP, AAV5-eGFP, AAV8-eGFP, or AAV9-eGFP were added to a solution of lactam linkers (1), (2) or (3) (3E6 eq) in TBS buffer (pH 9.3) and incubated for 4 h at 20° C. 10.sup.10 vg of each coupling was analyzed by Western blot using Concanavalin-HRP (ConA) or biotinylated lectins RCA1 and UEA1 to detect VP proteins coupled to their linker. FIG. 2B: AAV2 incubated with linker (1), ConA staining; FIG. 3B: AAV2 incubated with linker (2), RCA1 staining; FIG. 4B: AAV2 incubated with linker (3), UEA1 staining; FIG. 6B: AAV8 incubated with linker (1), ConA staining; FIG. 9B: AAV5 incubated with linker (1), ConA staining; FIG. 10B: AAV9 incubated with linker (1), ConA staining.

[0436] FIG. 14A and FIG. 14B are graphs showing quantitative analyses of the striatal and nigral volumes in the mouse right brain hemisphere that are positive for GFP 48 days after a single, unilateral intrastriatal injection of 5.5E8 vg of either AAV2, (1)-AAV2, or (7)-AAV2 vectors. Bars represent the group mean; circles, triangle, and square dots represent the individual animal values. FIG. 14A: Percent volume of striatum expressing GFP. FIG. 14B: Percent volume of substantia nigra expressing GFP.

[0437] FIG. 15A, FIG. 15B, and FIG. 15C are two immunohistochemistry photographs, showing the GFP staining of mouse brain slices at the striatal, thalamic, and nigral levels, 48 days after a single injection of AAV2 or (1)-AAV2 in the right striatum. FIG. 15A: immunohistochemistry photograph of mouse brain slices at the striatal level. FIG. 15B: immunohistochemistry photograph of mouse brain slices at the thalamic level. FIG. 15C: immunohistochemistry photograph of mouse brain slices at the nigral level.

[0438] FIG. 16 is a graph showing fluorimetric determination of glucocerebrosidase (GCase) enzymatic activity following transduction of HEK293 cells with either AAV2.GBA1, (1)-AAV2.GBA1, or Man-NCS-AAV2.GBA1.

EXAMPLES

[0439] The present invention is further illustrated by the following examples.

[0440] The following abbreviations are used:

[0441] ACN: Acetonitrile;

[0442] AgOTf: Silver (I) trifluoromethanesulfonate;

[0443] DBU: 1,8-Diazabicyclo[5.4.0]undec-7-ene;

[0444] DCM: Dichloromethane;

[0445] DIAD: Diisopropyl azodicarboxylate;

[0446] DMF: Dimethylformamide;

[0447] DPBS: Dulbecco's phosphate buffered saline;

[0448] EtOAc: Ethyl acetate;

[0449] EtOH: Ethanol;

[0450] MeOH: Methanol;

[0451] n-BuLi: n-Butyllithium;

[0452] TBS: Tris buffered saline;

[0453] THF: Tetrahydrofuran;

[0454] TMSOTf: Trimethylsilyl trifluoromethanesulfonate.

I. Synthesis of Compounds of Formula (I)

I.1. Material and Analytical Methods

Materials

[0455] Solvents, reagents and starting material were purchased and used as received from commercial sources, unless otherwise specified.

[0456] The intermediates and compounds described below were named using ChemBioDraw® Ultra version 12.0 (Perkin Elmer).

Analytical Methods

[0457] HPLC

Apparatus: HPLC—Shimadzu Nexera-i LC-2040C 3D with DAD detector;
Column: Gemini-NX 3μ C18 (4.6×50 mm), 110 Å, column no. OOB-4453-EO;
HPLC-1 conditions:
Wavelength: 200.0 nm±4.0 nm; 283.0 nm±4.0 nm; Flow: 0.5 ml/min; Column temperature: 30° C.; Injection volume: 1.0 μl;
Elution: gradient with mobile phase A, water, and mobile phase B, acetonitrile.

TABLE-US-00005 TABLE 3 HPLC-1 conditions Time [min] Mobile phase A [%] Mobile phase B [%] 0.0 95 5 2.0 95 5 9.5 20 80 10.5 20 80 12.0 95 5 14.0 95 5
Sample preparation: Dissolve sample in ACN to obtain final concentration 0.5 mg/ml.
HPLC-2 conditions:
Wavelength: 200.0 nm±4.0 nm; 283.0 nm±4.0 nm; Flow: 0.5 ml/min; Column temperature: 45° C.; Injection volume: 1.5 μl;
Elution: gradient with mobile phase A, water with 0.1% formic acid, and mobile phase B, acetonitrile with 0.1% formic acid.

TABLE-US-00006 TABLE 4 HPLC-2 conditions Time [min] Mobile phase A [%] Mobile phase B [%] 0.0 90 10 9.5 20 80 10.5 20 80 12.0 90 10 14.0 90 10
Sample preparation: Dissolve sample in ACN to obtain final concentration 0.5 mg/ml.
HPLC-3 conditions:
Wavelength: 205.0 nm±4.0 nm; 283.0 nm±4.0 nm; Flow: 0.5 ml/min; Column temperature: 30° C.; Injection volume: 1.0 μl;
Elution: gradient with mobile phase A, water, and mobile phase B, acetonitrile.

TABLE-US-00007 TABLE 4a HPLC-3 conditions Time [min] Mobile phase A [%] Mobile phase B [%] 0.0 95 5 4.0 95 5 19.0 20 80 21.0 20 80 24.0 95 5 28.0 95 5
Sample preparation: Dissolve sample in H.sub.2O/ACN (1:1) to obtain final concentration 0.25 mg/ml.

[0458] LC/MS

Apparatus: Shimadzu LCMS-2020 Single Quadrupole Liquid Chromatograph Mass Spectrometer;

[0459] Column: Acquity UPLC 1.8 μm C18 (2.1×50 mm), 100 Å, column no. 186003532;
UHPLC conditions: Wavelength: 220.0 nm±4.0 nm; 254.0 nm±4.0 nm;
Flow: 0.5 ml/min; Column temperature: 25° C.; Autosampler temperature: 20° C.;
Injection volume: 1 μl;
Elution: gradient with mobile phase A, water with 0.10% formic acid, and mobile phase B, acetonitrile with 0.1% formic acid.

TABLE-US-00008 TABLE 5 LC/MS conditions Time [min] Time [min] (6 min) (12 min) Mobile phase A [%] Mobile phase B [%] 0.00 0.00 95 5 4.00 10.00 5 95 5.00 10.50 5 95 5.20 11.00 95 5 6.00 12.00 95 5
MS conditions: Mass range: 100-1500 m/z; Ionization: alternate; Scan speed: 15 000 u/sec.
Sample preparation: Dissolve sample in ACN to obtain final concentration 0.25 mg/ml.

[0460] Flash Chromatography

Conditions-1:

Apparatus: Pure C-850 FlashPrep, BUCHI;

Column: PF-15-C18-F0080, Puriflash, 15 μm;

[0461] Conditions: Wavelength: 200 nm; 254 nm and ELSD detector; Flow: 30 ml/min;
Elution: gradient with mobile phase A, water, and mobile phase B, acetonitrile.

TABLE-US-00009 TABLE 6 Chromatography conditions-1 Time [min] Mobile phase A [%] Mobile phase B [%] 00.00 100 0 7.60 95 5 21.20 89 11 30.00 81 19 34.90 79 21 52.00 79 21 74.90 0 100
Sample preparation: The crude material was dissolved in H.sub.2O/ACN.

Conditions-2:

Apparatus: Puriflash XS 420, Interchim;

Column: PF-RP-HP-F0040, Puriflash, 15 μm;

[0462] Conditions: Wavelength: 220 nm; 254 nm; Flow: 30 ml/min;
Elution: gradient with mobile phase A, water, and mobile phase B, acetonitrile.

TABLE-US-00010 TABLE 6a Chromatography conditions-2 Time [min] Mobile phase A [%] Mobile phase B [%] 00.00 95 5 45.00 60 40 55.00 0 100 70.00 0 100
Sample preparation: The crude material was dissolved in H.sub.2O/ACN (1:1).

I.2. Synthesis of Building Blocks

Building Block 1 (BB1): 2-[2-(2-azidoethoxy)ethoxy]ethan-1-ol

[0463] ##STR00056##

[0464] A mixture of 2-[2-(2-chloroethoxy)ethoxy]ethanol (1 eq., 5.0 g) and sodium azide (1 eq., 1.9 g) in dry DMF (30 mL) was stirred at 90° C. overnight. The reaction mixture was cooled down to room temperature, diluted with THF (10 mL) and stirred for 15 min. The resulting suspension was filtered, the cake was washed with THF, and the filtrate was concentrated under reduced pressure to deliver the product as a yellow oil (5.1 g, yield 98%). LC/MS (6 min): RT=1.79 min, found [M+H].sup.+ 176.00; .sup.1H NMR (300 MHz, Chloroform-d) δ 3.73-3.60 (m, 8H), 3.57 (dd, J=5.5, 3.7 Hz, 2H), 3.36 (t, J=5.0 Hz, 2H), 2.78 (s, 1H).

Building Block 3 (BB3): 1-(4-ethynylbenzoyl)azetidin-2-one

[0465] ##STR00057##

[0466] A mixture of 4-ethynylbenzoic acid (1 eq., 1.0 g) and SOCl.sub.2 (3 eq., 12.2 g/7.4 mL) was kept under reflux overnight, next was concentrated under vacuum to give an orange solid. 2-Azetidinone (1 eq., 0.43 g) was dissolved in THF (20 mL), the solution was cooled down to −78° C., and 2M solution of nBuLi in hexane (1 eq., 3.04 mL) was dropwise added. The resulting suspension was stirred at −78° C. for 15 minutes, and next treated dropwise with a solution of 4-ethynylbenzoyl chloride in THF (5 mL). The reaction mixture was allowed to stir at room temperature overnight, and was quenched with an aqueous NH.sub.4Cl solution. The product was extracted with DCM (2×50 mL), the organic layers were combined, dried over MgSO.sub.4, filtered, and evaporated under reduced pressure to dryness. The crude material was purified by silica gel column chromatography using hexane/EtOAc as an eluent (100:0->80:20->50:50) to deliver 0.45 g (yield: 37%) of the product as a yellow solid. LC/MS (6 min): RT=2.76 min, found [M+H].sup.+ 199.70; .sup.1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, 2H), 7.60 (d, 2H), 4.46 (s, 1H), 3.66 (t, J=5.5 Hz, 2H), 3.11 (t, J=5.5 Hz, 2H).

I.3. Preparation of Compounds of Formula (I)

Compound (1): 1-(4-(1-(2-(2-(2-(((3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)benzoyl)azetidin-2-one

[0467] ##STR00058##

[0468] Step 1: [3,4,5,6-tetrakis(acetyloxy)oxan-2-yl]methyl acetate (1 eq., 5.0 g) was dissolved in dry DCM (50 mL) under argon atmosphere, morpholine (4 eq., 1.6 g) was added, and the solution was stirred at room temperature overnight. The reaction mixture was then washed twice with 2M HCl (2×50 mL), water (50 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure. The resulting yellow oil was dissolved in dry DCM (50 mL) under argon atmosphere, the solution was cooled down to 0° C., and treated with trichloroacetonitrile (10 eq., 16.7 g/11.6 mL). After being stirred for 1h at 0° C., DBU (0.2 eq., 0.35 g/0.35/mL) was added, the reaction mixture was stirred at 0° C. for 1h, and next at room temperature for 1h. The solvents were removed under reduced pressure, and the brown oily residue was purified by silica gel column chromatography using hexane/EtOAc (1:1) as eluent to yield the product (4.3 g, yield 68.8%) as an yellowish oil. .sup.1H NMR (300 MHz, DMSO-d6) δ 10.15 (s, 1H), 6.22 (s, 1H), 5.33-5.16 (m, 3H), 4.26-3.96 (m, 3H), 2.15 (s, 3H), 2.12-1.86 (m, 9H).

[0469] Step 2: To a suspension of [3,4,5-tris(acetyloxy)-6-[(2,2,2-trichloroethanimidoyl)oxy]oxan-2-yl]methyl acetate (1 eq., 4.34 g) and molecular sieves 4 Å (to keep the reaction mixture anhydrous) in dry DCM (40 mL) under argon atmosphere was added BB1 (1.5 eq., 0.53 g) at room temperature. The mixture was cooled down to −25° C., and TMSOTf (1.1 eq., 2.15 h/1.75 mL) was added. The reaction mixture was stirred at −25° C. for 1 h, and then was allowed to stir at room temperature overnight. The reaction was quenched with saturated NaHCO.sub.3 solution (60 mL), DCM (100 mL) was added, the phases were separated, and the organic phase was washed with water (100 mL), brine (50 mL), and dried over MgSO.sub.4. The resulting suspension was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using hexane/EtOAc (1:1) as eluent to deliver 1.8 g of the product (yield 59%) as an colorless oil. .sup.1H NMR (300 MHz, Chloroform-d) δ 5.82-5.64 (m, 3H), 5.28 (d, J=1.8 Hz, 1H), 4.78-4.64 (m, 1H), 4.55-4.42 (m, 2H), 4.25-4.16 (m, 1H), 4.08 (dd, J=5.1, 2.2 Hz, 9H), 3.85-3.75 (m, 2H), 2.48 (ddd, J=34.4, 15.5, 1.6 Hz, 12H).

[0470] Step 3: [3,4,5-Tris(acetyloxy)-6-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}oxan-2-yl]methyl acetate (1 eq., 0.3 g) was dissolved in 7N NH.sub.3 in MeOH (5 mL) and stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness, and the crude product was used for the next step without further purification (200 mg, yield quantitative).

[0471] Step 4: To a solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-6-(hydroxymethyl)oxane-3,4,5-triol (1 eq., 0.2 g) in THF/MeOH (3 mL/1 mL) were added BB3 (1.1 eq., 0.130 g), copper (I) acetate (0.25 eq., 0.018 g), and L-ascorbic acid (1.1 eq., 0.126 g). The reaction mixture was stirred at 60° C. for 1h. Then, the solvents were removed under reduced pressure, and the residue was purified by reversed phase flash column chromatography (Column FP-ID-C18, H.sub.2O:ACN, starting from H.sub.2O 100% to ACN 100%) to deliver 94.2 mg (yield: 27.8%) of the product (1) as a white solid foam. LC/MS (6 min): RT=2.11 min, found [M+H].sup.+ 537.15; LC/MS (12 min): RT=4.59 min, found [M+H].sup.+ 537.3; HPLC-1 purity: 99.14% (200 nm), 99.25% (283 nm); .sup.1H NMR (300 MHz, DMSO-d6) δ 8.70 (s, 1H), 8.05-7.91 (m, 4H), 4.74 (dd, J=8.9, 4.5 Hz, 2H), 4.60 (dd, J=6.6, 3.4 Hz, 4H), 4.45 (t, J=6.0 Hz, 1H), 3.88 (t, J=5.1 Hz, 2H), 3.73-3.38 (m, 15H), 3.12 (t, J=5.4 Hz, 2H).

Compound (2): 1-(4-(1-(2-(2-(2-(((3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)benzoyl)azetidin-2-one

[0472] ##STR00059##

[0473] Step 1: To a suspension of [3,4,5-tris(acetyloxy)-6-bromooxan-2-yl]methyl acetate (1 eq., 5.0 g) and molecular sieves 4A (to keep the reaction mixture anhydrous) in dry DCM (100 mL) under argon atmosphere was added BB1 (1.5 eq., 3.1 g) and AgOTf (1.4 eq., 1.75 g) at room temperature. The reaction mixture was stirred at room temperature overnight. The resulting suspension was filtered through a pad of Celite, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using hexane/EtOAc (1:1) as eluent to deliver 2.5 g of the product (yield 40%) as a yellow oil. .sup.1H NMR (300 MHz, DMSO-d6) δ 5.25 (dd, J=3.5, 1.1 Hz, 1H), 5.14 (dd, J=10.4, 3.5 Hz, 1H), 4.93 (dd, J=10.4, 7.9 Hz, 1H), 4.72 (d, J=8.0 Hz, 1H), 4.22-4.13 (m, 1H), 4.04 (dd, J=6.3, 1.6 Hz, 2H), 3.86-3.74 (m, 1H), 3.67-3.47 (m, 9H), 3.39 (dd, J=5.6, 4.2 Hz, 2H), 2.11 (s, 3H), 2.00 (d, J=3.9 Hz, 6H), 1.91 (s, 3H).

[0474] Step 2: [3,4,5-tris(acetyloxy)-6-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}oxan-2-yl]methyl acetate (1 eq., 2.0 g) was dissolved in 7N NH.sub.3 in MeOH (15 mL) and stirred at room temperature for 72h. The solvent was removed under reduced pressure to dryness, and the crude product was used for the next step without further purification (1.33 g, yield quantitative).

[0475] Step 3: To a solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-6-(hydroxymethyl)oxane-3,4,5-triol (1 eq., 0.344 g) in THF/MeOH (3 mL/1 mL) were added BB3 (1.1 eq., 0.217 g), copper (I) acetate (0.05 eq., 0.006 g), and L-ascorbic acid (1.1 eq., 0.192 g). The reaction mixture was stirred at 60° C. for 1h. Then, the solvents were removed under reduced pressure, and the residue was purified by reversed phase flash column chromatography (Column FP-ID-C18, H.sub.2O:ACN, starting from H.sub.2O 100% to ACN 100%) to deliver 29 mg (Y: 5.5%) of the product as a white solid foam. LC/MS (6 min): RT=2.06 min, found [M+H].sup.+ 537.25; LC/MS (12 min): RT=3.123 min, found [M+H].sup.+ 537.3; HPLC-1 purity: 98.51% (200 nm), 98.80% (283 nm); .sup.1H NMR (300 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.01-7.93 (m, 4H), 4.85 (d, J=4.2 Hz, 1H), 4.72 (d, J=4.7 Hz, 1H), 4.58 (dt, J=11.1, 5.4 Hz, 3H), 4.37 (d, J=4.5 Hz, 1H), 4.07 (d, J=6.7 Hz, 1H), 3.88 (t, J=5.1 Hz, 2H), 3.68 (t, J=5.4 Hz, 2H), 3.61 (t, J=3.5 Hz, 1H), 3.50 (pd, J=7.8, 6.5, 4.2 Hz, 9H), 3.27 (d, J=4.7 Hz, 2H), 3.12 (t, J=5.4 Hz, 2H).

Compound (3): 1-(4-(1-(2-(2-(2-(((3S,4R,5S,6S)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)benzoyl)azetidin-2-one

[0476] ##STR00060##

[0477] Step 1: A suspension of anhydrous sodium acetate (1 eq., 2.5 g) and acetic anhydride (60 mL) was refluxed for 5 min, next L-(−)-fucose (1 eq., 5.0 g) was added and the reaction mixture was refluxed overnight. The hot solution was poured on an ice water, and the mixture was extracted with DCM (200 mL). The organic phase was washed with water (300 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to deliver the product as a brown oil (8.5 g, yield 83.7%). .sup.1H NMR (300 MHz, Chloroform-d) δ 5.62 (d, J=8.3 Hz, 1H), 5.27 (dd, J=2.8, 1.4 Hz, 1H), 5.23-5.19 (m, 1H), 5.01 (dd, J=10.4, 3.4 Hz, 1H), 3.90 (dd, J=6.4, 1.2 Hz, 1H), 2.15-1.91 (m, 20H), 1.16 (d, J=6.4 Hz, 3H).

[0478] Step 2: 2,3,5-tris(acetyloxy)-6-methyloxan-4-yl acetate (1 eq., 8.5 g) was dissolved in dry DCM (90 mL) under argon atmosphere, morpholine (4 eq., 8.9 g) was added, and the solution was stirred at room temperature overnight. The reaction mixture was then washed with 2M HCl (2×90 ml), water (100 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure. The resulting yellow oil was dissolved in dry DCM (60 mL) under argon atmosphere, the solution was cooled down to 0° C., and treated with trichloroacetonitrile (10 eq., 24.4 g/16.9 mL). After being stirred for 1h at 0° C., DBU (0.2 eq., 0.51 g/0.51 mL) was added, the reaction mixture was stirred at 0° C. for 1h, and next at room temperature for 1 hour. The solvents were removed under reduced pressure, and the brown oily residue was purified by silica gel column chromatography using hexane/EtOAc (1:1) as eluent to yield the product (5.42 g, yield 65.2%) as a yellowish oil.

[0479] Step 3: To a suspension of 3,5-bis(acetyloxy)-2-methyl-6-[(2,2,2-trichloroethanimidoyl)oxy]oxan-4-yl acetate (1 eq., 5.42 g) and molecular sieves 4A (to keep the reaction mixture anhydrous) in dry DCM (60 mL) under argon atmosphere was added BB1 (1.5 eq., 3.28 g) at room temperature. The mixture was cooled down to −25° C., and TMSOTf (1.1 eq., 3.05 g/2.48 mL) was added. The reaction mixture was stirred at −25° C. for 1h, and then was allowed to stir at room temperature overnight. The reaction was quenched with saturated NaHCO.sub.3 solution (80 mL), next DCM (100 mL) was added, the phases were separated, and the organic phase was washed with water (100 mL), brine (50 mL), and dried over MgSO.sub.4. The solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using hexane/EtOAc (1:1) as eluent to deliver 0.87 g of the product (yield 15.0%) as an colorless oil. .sup.1H NMR (300 MHz, Chloroform-d) δ 5.27-5.15 (m, 2H), 5.09-4.98 (m, 1H), 4.53 (d, J=7.9 Hz, 1H), 4.03-3.94 (m, 1H), 3.82-3.74 (m, 1H), 3.74-3.62 (m, 9H), 3.41 (t, J=5.0 Hz, 2H), 2.18 (s, 3H), 2.06 (s, 3H), 1.99 (s, 3H), 1.23 (d, J=6.4 Hz, 3H).

[0480] Step 4: 3,5-bis(acetyloxy)-2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-6-methyloxan-4-yl acetate (1 eq., 0.87 g) was dissolved in 7N NH.sub.3 in MeOH (12 mL) and stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness, and the crude product was used for the next step without further purification (700 mg, yield quantitative).

[0481] Step 5: To a solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-6-methyloxane-3,4,5-triol (1 eq., 0.200 g) in THF/MeOH (3 mL/1 mL) were added BB3 (1.1 eq., 0.136 g), copper (I) acetate (0.05 eq., 0.004 g), and L-ascorbic acid (1.1 eq., 0.121 g). The reaction mixture was stirred at 60° C. for 1h. Then, the solvents were removed under reduced pressure, and the residue was purified by reversed phase flash column chromatography (Column FP-ID-C18, H.sub.2O:ACN, starting from H.sub.2O 100% to ACN 100%) to deliver 51 mg (Y: 15.7%) of the product as a white solid foam. LC/MS (6 min): RT=2.16 min, found [M+H].sup.+ 521.25; LC/MS (12 min): RT 3.271 min, found [M+H].sup.+ 521.4; HPLC-1 purity: 99.08% (200 nm), 99.01% (283 nm); .sup.1H NMR (300 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.01-7.92 (m, 4H), 4.63-4.56 (m, 3H), 4.52-4.48 (m, 1H), 4.42-4.38 (m, 1H), 3.88 (t, J=5.1 Hz, 2H), 3.77 (q, J=6.5 Hz, 1H), 3.68 (t, J=5.4 Hz, 2H), 3.64-3.38 (m, 12H), 3.13 (t, J=5.4 Hz, 2H), 1.04 (d, J=6.5 Hz, 3H).

Compound (4): 1-(4-(1-(2-(2-(2-(((3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)benzoyl)azetidin-2-one

[0482] ##STR00061##

[0483] Step 1: A solution of erythromycin (1 eq., 46 g) in EtOH (2500 mL)-6N HCl (16 vol, 740 mL) was refluxed for 4 hours. The reaction mixture was cooled to room temperature, and the liquid was decanted from a dark insoluble material. The solution was washed with chloroform (6×2000 mL). The aqueous ethanolic layer was concentrated under reduced pressure to remove ethyl alcohol. 1-Butanol (1000 mL) was added followed by water (500 mL). The phases were separated, the aqueous layer was washed with 1-butanol (4×1000 mL), and the combined organic layers were washed with water (1000 mL). All aqueous layers were combined, and concentrated under reduced pressure to dryness. The oily residue was dissolved in EtOH (10 mL), and diethyl ether was added (200 mL) to form a precipitate. The solid was filtered, washed with diethyl ether, and dried to give desosamine hydrochloride (6.6 g, Y=60%) as a white solid. LC/MS (6 min): No UV absorbance detected (lack of chromophore), the mass detected on the positive ionization, RT=0.45 min, found [M+H].sup.+ 175.75 (a free base); .sup.1H NMR (300 MHz, DMSO-d6) δ 9.73 (d, J=41.8 Hz, 1H), 7.01 (s, 0.5H), 6.70 (d, J=4.5 Hz, 0.5H), 6.01 (s, 0.5H), 5.62 (s, 0.5H), 5.00 (d, J=3.4 Hz, 0.5H), 4.38 (dd, J=7.3, 3.0 Hz, 0.5H), 4.04 (ddd, J=11.4, 6.2, 2.2 Hz, 0.5H), 3.60 (qd, J=6.0, 1.9 Hz, 1H), 3.23 (d, J=10.2 Hz, 0.5H), 2.77-2.64 (m, 6H), 2.05-1.93 (m, 1H), 1.40 (qd, J=11.9, 8.0 Hz, 1H), 1.15 (dd, J=17.0, 6.2 Hz, 3H).

[0484] Step 2: To a solution of Bu.sub.3P (1 eq., 8.2 mL) in toluene (20 vol., 150 mL) at −30° C. was dropwise added DIAD (1 eq., 6.7 g) under argon atmosphere The resulting solution was stirred for 20 min., and desosamine hydrochloride (1 eq., 7.0 g) was added. The mixture was stirred at −30° C. for 45 min, next 2-mercaptopyridine (1 eq., 3.7 g) was added, and the reaction mixture was allowed to stir at room temperature for 16h. The reaction was filtered through a pad of Celite, the pad was washed with DCM, and the filtrate was concentrated under reduced pressure to dryness. The residue was treated with toluene (100 mL), the mixture was stirred for 20 min at room temperature, and the solid was filtered off, washed with toluene (3×100 mL), and dried to deliver the product (4.29 g, yield=48%) as an orange solid. LC/MS (6 min): RT=1.48 min, found [M+H].sup.+ 269.65; .sup.1H NMR (300 MHz, Methanol-d4) δ 8.61 (d, J=4.9 Hz, 2H), 7.20 (t, J=4.9 Hz, 1H), 5.61 (d, J=9.8 Hz, 1H), 3.87-3.75 (m, 1H), 3.66 (t, J=9.8 Hz, 1H), 3.15 (d, J=13.3 Hz, 1H), 2.61 (s, 6H), 2.00 (d, J=10.5 Hz, 1H), 1.51 (q, J=12.4 Hz, 2H), 1.25 (d, J=6.2 Hz, 3H).

[0485] Step 3: A suspension of anhydrous sodium acetate (1 eq., 0.82 g) in acetic anhydride (12 vol., 32 mL) was refluxed for 5 min. To this suspension was added 4-(dimethylamino)-6-methyl-2-(pyrimidin-2-ylsulfanyl)oxan-3-ol (1 eq., 2.69 g) and the reaction was kept under reflux for 1 h. The hot solution was poured on ice water (100 mL), basified with solid NaHCO.sub.3, and the product was extracted with DCM (3×100 mL). The organic layers were combined, dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (DCM: MeOH, 0-10% MeOH) to give the product (1.21 g, yield=39%) as a yellow solid. LC/MS (6 min): RT=1.84 min, found [M+H].sup.+ 312.00; .sup.1H NMR (300 MHz, DMSO-d6) δ 8.66 (dd, J=8.1, 4.9 Hz, 2H), 7.27 (dt, J=6.3, 4.9 Hz, 1H), 6.55 (d, J=5.1 Hz, 0.35H), 5.62 (d, J=10.1 Hz, 0.65H), 5.06 (dd, J=11.2, 5.2 Hz, 0.35H), 4.83 (t, J=10.1 Hz, 0.65H), 4.00 (d, J=4.0 Hz, 0.35H), 3.77-3.65 (m, 0.65H), 3.01-2.84 (m, 1H), 2.20 (d, J=5.5 Hz, 6H), 1.92 (d, J=10.8 Hz, 3H), 1.82 (dd, J=13.5, 4.1 Hz, 1H), 1.36 (q, J=12.3 Hz, 1H), 1.13 (dd, J=6.1, 4.7 Hz, 3H).

[0486] Step 4: A suspension of silver trifluoromethanesulfonate (3 eq., 2.60 g) and molecular sieves (4A, 0.16 g) in dry DCM was cooled to at 0° C. in darkness (wrapping with aluminum foil). A solution of 4-(dimethylamino)-6-methyl-2-(pyrimidin-2-ylsulfanyl)oxan-3-yl acetate (1 eq., 1.05 g) and BB1 (2 eq., 1.18 g) in dry DCM was next added (total DCM: 40 vol., 42 mL). The reaction mixture was stirred for 2h at 0° C., and then was allowed to stir at room temperature overnight. The reaction was quenched with saturated NaHCO.sub.3 solution (50 mL) till pH 8, the mixture was filtered through a pad of Celite, and the phases were separated. The aqueous layer was extracted with DCM (3×50 mL). The organic layers were combined, dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography (DCM: MeOH, 0-5% MeOH) to give the product as a yellow solid (498 mg, yield=33%). LC/MS (6 min): No UV absorbance detected (lack of chromophore), the mass detected on the positive ionization, RT=1.74 min, found [M+H].sup.+ 333.10; .sup.1H NMR (300 MHz, DMSO-d6) δ 4.24 (d, J=7.2 Hz, 1H), 3.90-3.79 (m, 1H), 3.69-3.50 (m, 10H), 3.40 (dd, J=5.6, 4.1 Hz, 2H), 3.16 (d, J=5.2 Hz, 2H), 2.58 (s, 6H), 1.86 (d, J=13.0 Hz, 1H), 1.36 (q, J=11.8 Hz, 1H), 1.19 (d, J=6.1 Hz, 3H).

[0487] Step 5: To a solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-4-(dimethylamino)-6-methyloxan-3-ol (1 eq., 0.170 g) in THF/DMF (6 mL/1 mL) were added BB3 (1.1 eq., 0.112 g), L-ascorbic acid (1.1 eq., 0.109 g), and copper (I) acetate (0.25 eq., 0.016 g). The reaction mixture was stirred at 60° C. for 1 h. Then, the solvents were evaporated under reduced pressure, and the residue was purified by reversed phase column chromatography (Column FP-ID-C18, H.sub.2O:ACN, starting from H.sub.2O 100% to ACN 100%) to give the product (53 mg, yield=19.5%) as a white solid foam. LC/MS (6 min): RT=1.89 and 2.08 min (two peaks), found [M+H].sup.+ 532.40; LC/MS (12 min): RT=3.15 min, found [M+H].sup.+ 532.4; HPLC-2 purity: 89.52% (200 nm), 93.02% (283 nm); .sup.1H NMR (300 MHz, Acetonitrile-d3) δ 8.31 (s, 1H), 7.99 (s, 4H), 4.63-4.58 (m, 2H), 4.23 (d, J=7.1 Hz, 1H), 3.93 (t, J=5.0 Hz, 2H), 3.89-3.83 (m, 1H), 3.71 (t, J=5.5 Hz, 2H), 3.58 (ddt, J=11.8, 6.1, 2.9 Hz, 8H), 3.41-3.34 (m, 1H), 3.32-3.23 (m, 1H), 3.09 (t, J=5.5 Hz, 2H), 2.77 (s, 6H), 1.46 (q, J=12.0 Hz, 2H), 1.23 (d, J=6.2 Hz, 3H).

Compound (5): 1-(4-(1-(2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)benzoyl)azetidin-2-one

[0488] ##STR00062##

[0489] Step 1: To a solution of BB1 (1 eq., 0.125 g) in THF/DMF (4.4 ml/0.6 ml) were added BB3 (1.1 eq., 0.156 g), L-ascorbic acid (1.1 eq., 0.152 g), and copper (I) acetate (0.25 eq., 0.022 g). The reaction mixture was stirred at 60° C. for 2h. Then, the solvents were evaporated under reduced pressure, and the residue was purified by reversed phase column chromatography (Column PF-RP-HP-F0040, H2O:ACN, starting from ACN 5% to 100%) to give the product (141 mg, Y: 53%) as a white solid foam. LC/MS (6 min): RT=2.25, found [M+H].sup.+ 374.75; LC/MS (12 min): RT=3.33 min, found [M+H].sup.+ 375.2; HPLC-3 purity: 97.93% (205 nm), 98.91% (283 nm); .sup.1H NMR (300 MHz, Acetonitrile-d3) δ 8.31 (s, 1H), 7.99 (s, 4H), 4.59 (dd, J=5.6, 4.6 Hz, 2H), 3.92 (dd, J=5.6, 4.6 Hz, 2H), 3.71 (t, J=5.5 Hz, 2H), 3.64-3.53 (m, 6H), 3.50-3.44 (m, 2H), 3.09 (t, J=5.5 Hz, 2H), 2.63 (s, 1H).

Compound (6): N-[4,5-dihydroxy-6-(hydroxymethyl)-2-{2-[2-(2-{4-[4-(2-oxoazetidine-1-carbonyl)phenyl]-1H-1,2,3-triazol-1-yl}ethoxy)ethoxy]ethoxy}oxan-3-yl]acetamide

[0490] ##STR00063##

[0491] Step 1: β-D-Glucosamine pentaacetate (10 g, 1.0 eq.) was dissolved in DCE (75 vol, 750 mL) at room temperature. TMSOTf (4.6 mL, 1.0 eq.) was added, the mixture was stirred at 50° C. for 30 min, cooled down and treated with triethylamine (2.7eq., 9.7 mL). The resulting solution was stirred at room temperature for 15 min, then filtered through a short plug of silica gel, and washed with ethyl acetate. The crude material was purified by silica gel column chromatography using DCM/MeOH as an eluent (100:0->90:10) to deliver 5 g of product as a colorless oil (Yield: 59%). .sup.1H NMR (300 MHz, Chloroform-d) δ 5.94 (d, J=7.4 Hz, 1H), 5.22 (t, J=2.5 Hz, 1H), 4.89 (ddd, J=9.2, 2.1, 1.3 Hz, 1H), 4.20-4.08 (m, 3H), 3.57 (dt, J=8.9, 4.3 Hz, 1H), 2.08 (s, 3H), 2.07-2.02 (m, 9H).

[0492] Step 2: To a suspension of [6,7-bis(acetyloxy)-2-methyl-3aH,5H,6H,7H,7aH-pyrano[3,2-d][1,3]oxazol-5-yl]methyl acetate (4.0 g, 1.0 eq.) and molecular sieves 4A (to keep the reaction mixture anhydrous) in dry DCM under argon atmosphere was added BB1 (6.4 g, 1.5 eq.) at room temperature. The reaction mixture was stirred for 30 min, then H.sub.2SO.sub.4 (4 drops, 1.0 eq.) was added dropwise and the solution was stirred at room temperature overnight. The reaction was quenched with saturated NaHCO.sub.3 solution, DCM (50 mL) was added, the phases were separated, and the organic phase was washed with water (100 mL), brine (100 mL), and dried over MgSO.sub.4. The solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using Hex/EtOAc as an eluent (1:1) to deliver 3 g of the product as a yellow solid (yield: 62%). LC/MS (6 min) RT=2.55 min, found [M+H]+ 505.15; .sup.1H NMR (300 MHz, DMSO-d6) δ 7.91 (d, J=9.1 Hz, 1H), 5.08 (dd, J=10.5, 9.4 Hz, 1H), 4.82 (t, J=9.7 Hz, 1H), 4.65 (d, J=8.5 Hz, 1H), 4.18 (dd, J=12.3, 4.8 Hz, 1H), 4.09-3.98 (m, 1H), 3.88-3.62 (m, 3H), 3.53 (dt, J=4.3, 2.2 Hz, 8H), 3.39 (dd, J=5.6, 4.2 Hz, 2H), 2.02 (s, 3H), 1.97 (s, 3H), 1.91 (s, 3H), 1.76 (s, 3H).

[0493] Step 3: [3,4-Bis(acetyloxy)-6-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-5-acetamidooxan-2-yl]methyl acetate (2.5 g, 1.0 eq.) was dissolved in 7N NH.sub.3 in MeOH (33 mL, 15 vol.) and stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness, and the crude product was used for the next step without further purification (1.6 g, yield: 95%). .sup.1H NMR (300 MHz, DMSO-d6) δ 7.65 (d, J=8.8 Hz, 1H), 4.31 (d, J=8.3 Hz, 1H), 3.85-3.75 (m, 1H), 3.67 (d, J=11.6 Hz, 1H), 3.59 (dd, J=5.7, 4.3 Hz, 2H), 3.56-3.45 (m, 8H), 3.44-3.35 (m, 4H), 3.09-3.03 (m, 2H), 1.75 (s, 3H).

[0494] Step 4: To a solution of N-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl)acetamide (0.592 g, 1.0 eq.) in MeOH (2 mL)/THF (6 mL) were added BB3 (0.343 g, 1.0 eq.), L-ascorbic acid (0.333 g, 1.1 g), and copper (I) acetate (0.024 g, 0.25 g). The reaction mixture was stirred at 60° C. for 3 h. Then, the solvents were evaporated under reduced pressure and the residue was purified by reversed phase column chromatography (Column PF-15-C18-F0080, H2O:ACN, starting from H.sub.2O 100% to ACN 100%) to give 450 mg of the product as a white solid foam (yield: 44%). LC/MS (6 min) RT=2.04 min, found [M+H].sup.+ 578.30; RT=2.04 min, found [M+H].sup.+ 578.30; HPLC-1 purity: 98.76% (200 nm), 98.71% (284 nm). RT=6.37 min, found [M+H].sup.+ 578.2; .sup.1H NMR (300 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.97 (d, J=2.7 Hz, 4H), 7.65 (d, J=8.9 Hz, 1H), 4.98 (d, J=4.7 Hz, 1H), 4.90 (d, J=5.2 Hz, 1H), 4.60 (t, J=5.1 Hz, 2H), 4.53 (s, 1H), 4.30 (d, J=8.3 Hz, 1H), 3.88 (t, J=5.2 Hz, 2H), 3.77 (dt, J=9.6, 3.6 Hz, 1H), 3.67 (q, J=5.5 Hz, 3H), 3.56-3.40 (m, 9H), 3.27 (ddd, J=10.1, 8.1, 5.3 Hz, 1H), 3.12 (t, J=5.4 Hz, 2H), 3.06 (q, J=4.8, 3.5 Hz, 2H), 1.77 (s, 3H).

Compound (7): sodium (3,4,5-trihydroxy-6-{2-[2-(2-{4-[4-(2-oxoazetidine-1-carbonyl)phenyl]-1H-1,2,3-triazol-1-yl}ethoxy)ethoxy]ethoxy}oxan-2-yl)methyl sulfate

[0495] ##STR00064##

[0496] Step 1 & 2: as described for compound (2)

[0497] Step 3: To a solution of 2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-6-(hydroxymethyl)oxane-3,4,5-triol (1 eq., 0.309 g) in THF/MeOH (3 mL/1 mL) were added BB3 (1.1 eq., 0.182 g), copper (I) acetate (0.05 eq., 0.006 g), and L-ascorbic acid (1.1 eq., 0.161 g). The reaction mixture was stirred at 60° C. for 1h. Then, the solvents were removed under reduced pressure, and the residue was purified by reversed phase flash column chromatography (Column FP-ID-C18, H.sub.2O:ACN, starting from H.sub.2O 100% to ACN 100%) to deliver 283 mg (Y: 57.5%) of the product as a white solid foam. LC/MS (6 min) RT=1.99 min, found [M+H].sup.+ 537.35.

[0498] Step 4: To a solution of 1-[4-(1-{2-[2-(2-{[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}ethoxy)-ethoxy]ethyl}-1H-1,2,3-triazol-4-yl)benzoyl]azetidin-2-one (1 eq., 0.283 g) in dry DMF was added SO3Py (0.097 g). The reaction mixture was stirred at room temperature overnight. Then, the solvent was removed under reduced pressure, and the residue was purified by prep-HPLC (Gemini-NX 5 μm C18, H2O:ACN:TFA, starting from H.sub.2O 85% to ACN 95%). The appropriate fractions were combined, neutralized by TEA, evaporated under reduced pressure, dissolved in water, and passed through Dowex 50WX4 Na+-form to deliver 50 mg (Y: 14%) of the product as a white solid foam; LC/MS (6 min) RT=1.94 min, found [M−Na]− 615.30; LC/MS (12 min) RT=2.803 min, found [M−Na]− 615.3; HPLC purity: 93.54% (200 nm), 94.56% (283 nm); 1H NMR (300 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.04-7.90 (m, 4H), 4.83 (d, J=4.3 Hz, 1H), 4.69 (d, J=5.0 Hz, 1H), 4.60 (t, J=5.2 Hz, 2H), 4.52 (d, J=4.8 Hz, 1H), 4.08 (d, J=7.1 Hz, 1H), 3.89 (t, J=5.2 Hz, 2H), 3.85-3.73 (m, 3H), 3.68 (t, J=5.4 Hz, 2H), 3.62-3.46 (m, 9H), 3.25 (d, J=4.1 Hz, 2H), 3.12 (t, J=5.4 Hz, 2H).

Compound (8): [(5-acetamido-3,4-dihydroxy-6-{2-[2-(2-{4-[4-(2-oxoazetidine-1-carbonyl)phenyl]-1H-1,2,3-triazol-1-yl}ethoxy)ethoxy]ethoxy}oxan-2-yl)methoxy]sulfonic acid

[0499] ##STR00065##

[0500] Step 1: [3,4,6-Tris(acetyloxy)-5-acetamidooxan-2-yl]methyl acetate (1 eq., 10 g) was dissolved in DCE (75 vol., 750 mL) at room temperature. Then, TMSOTf (1 eq., 4.6 mL) was added, the mixture was stirred at 50° C. for 30 min, cooled down, and treated with triethylamine (2.7 eq., 9.7 mL). The resulting solution was stirred at room temperature for 15 min, then filtered through a short plug of silica gel, and washed with ethyl acetate. The crude material was purified by silica gel column chromatography using DCM/MeOH as an eluent (100:0->90:10) to deliver 2.6 g of the product as a colorless oil (yield 31%). 1H NMR (300 MHz, Chloroform-d) δ 5.99 (d, J=6.8 Hz, 1H), 5.46 (t, J=2.9 Hz, 1H), 4.90 (dd, J=7.5, 3.3 Hz, 1H), 4.28-4.15 (m, 2H), 4.10 (dd, J=10.7, 5.6 Hz, 1H), 4.03-3.96 (m, 1H), 2.12 (s, 3H), 2.06 (dd, J=5.5, 1.1 Hz, 9H).

[0501] Step 2: To a suspension of [6,7-bis(acetyloxy)-2-methyl-3aH,5H,6H,7H,7aH-pyrano[3,2-d][1,3]oxazol-5-yl]methyl acetate (1 eq., 2.6 g) and molecular sieves 4A (to keep the reaction mixture anhydrous) in dry DCM under argon atmosphere was added BB1 (1.5 eq., 2.1 g) at room temperature. The reaction mixture was stirred for 30 min, then H.sub.2SO.sub.4 was added dropwise and the solution was stirred at room temperature overnight. The reaction was quenched with saturated NaHCO.sub.3 solution, DCM (50 mL) was added, the phases were separated, and the organic phase was washed with water (50 mL), brine (50 mL), and dried over MgSO.sub.4. The solution was filtered, the filtrate was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography using DCM/MeOH as an eluent (100:0->95:5) to deliver 2.2 g of the product as a yellow solid (yield 55%). LC/MS (6 min) The mass detected on the positive ionization, RT=2.5 min, found [M+H].sup.+ 505.25; 1H NMR (300 MHz, DMSO-d6) δ 7.79 (d, J=9.2 Hz, 1H), 5.21 (d, J=3.4 Hz, 1H), 4.97 (dd, J=11.2, 3.5 Hz, 1H), 4.55 (d, J=8.5 Hz, 1H), 4.03 (s, 3H), 3.94-3.74 (m, 2H), 3.63-3.50 (m, 8H), 3.40 (dd, J=5.6, 4.2 Hz, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.89 (s, 3H), 1.77 (s, 3H).

[0502] Step 3: [3,4-Bis(acetyloxy)-6-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-5-acetamidooxan-2-yl]methyl acetate (1 eq., 2.2 g) was dissolved in 7N NH.sub.3 in MeOH (15 vol., 33 ml) and stirred at room temperature overnight. The solvent was removed under reduced pressure to dryness, and 1.62 g of the crude product was used for the next step without further purification (yield: 98%). LC/MS 6 min) The mass detected on the positive ionization, RT=1.54 min, found [M+H].sup.+ 378.70, [M−H]− 376.50. 1H NMR (300 MHz, DMSO-d6) δ 7.60 (d, J=8.9 Hz, 1H), 4.60-4.53 (m, 2H), 4.48 (d, J=4.3 Hz, 1H), 4.28 (d, J=8.4 Hz, 1H), 3.83-3.67 (m, 2H), 3.65-3.55 (m, 4H), 3.51-3.36 (m, 6H), 3.31 (d, J=10.0 Hz, 3H), 1.80 (s, 3H).

[0503] Step 4: To a solution of N-(2-{2-[2-(2-azidoethoxy)ethoxy]ethoxy}-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl)acetamide (1 eq., 0.300 g) in THF (35 vol., 0.5 mL)/DMF (5 vol., 1.5 mL) were added BB3 (1 Eq., 0.174 g), L-ascorbic acid (1.1 Eq., 0.169 g), and copper (I) acetate (0.25 Eq., 0.024 g). The reaction mixture was stirred at 60° C. for 2h. Then, the solvents were evaporated under reduced pressure, and the residue was purified by reversed phase column chromatography (Column FP-ID-C18, H.sub.2O:ACN, starting from H.sub.2O 100% to ACN 100%) to give 342 mg of the product as a white solid foam (yield: 74%). LC/MS (6 min) RT=2.0 min, found [M+H].sup.+ 578.35. 1H NMR (300 MHz, DMSO-d6) δ 8.71 (s, 1H), 8.03-7.88 (m, 4H), 7.61 (d, J=9.0 Hz, 1H), 4.60 (dd, J=5.9, 3.8 Hz, 4H), 4.51 (d, J=4.3 Hz, 1H), 4.25 (d, J=8.4 Hz, 1H), 3.88 (t, J=5.2 Hz, 2H), 3.80-3.61 (m, 5H), 3.57-3.37 (m, 10H), 3.28 (t, J=6.2 Hz, 1H), 3.12 (t, J=5.4 Hz, 2H), 1.76 (s, 3H).

[0504] Step 5: To a solution of N-[4,5-dihydroxy-6-(hydroxymethyl)-2-{2-[2-(2-{4-[4-(2-oxoazetidine-1-carbonyl)phenyl]-1H-1,2,3-triazol-1yl}ethoxy)ethoxy]ethoxy} oxan-3-yl]acetamide (1 eq., 0.340 g) in dry DMF was added SO3Py (1.15 Eq, 0.108 g) at room temperature. The reaction mixture was stirred at room temperature overnight, the solvents were removed under reduced pressure, and the residue was purified by prep-HPLC (PF-RP-AQ-F0120, H.sub.2O:ACN, starting from H.sub.2O 95% to ACN 100%). The appropriate fractions were combined, evaporated under reduced pressure, dissolved in water, and passed through Dowex 50WX4 Na+-form to deliver 23 mg of the product as a white solid foam (Yield 6%). LC/MS (6 min) RT=1.88, found [M+H].sup.+ 657.50, [M−H]− 655.45. LC/MS (12 min) RT=2.85 min, found [M−H]− 656.5. HPLC purity-1: 96.91% (200 nm), 97.46% (283 nm). 1H NMR (300 MHz, DMSO-d6) δ 8.72 (d, J=1.4 Hz, 1H), 8.06-7.87 (m, 4H), 7.59 (d, J=9.0 Hz, 1H), 4.69-4.46 (m, 4H), 4.25 (d, J=8.5 Hz, 1H), 3.94-3.65 (m, 8H), 3.64-3.40 (m, 10H), 3.12 (t, J=5.6 Hz, 2H), 1.76 (d, J=1.3 Hz, 3H).

Compound (9): Sodium 3,4,5-trihydroxy-6-{2-[2-(2-{4-[4-(2-oxoazetidine-1-carbonyl)phenyl]-1H-1,2,3-triazol-1-yl}ethoxy)ethoxy]ethoxy} oxane-2-carboxylate

[0505] ##STR00066##

[0506] Step 1: To a suspension of methyl 3,4,5-tris(acetyloxy)-6-bromooxane-2-carboxylate (1 eq., 4.0 g) and molecular sieves 4A (to keep the reaction mixture anhydrous) in dry DCM under argon atmosphere was added BB1 (1.5 eq., 2.65 g) and AgOTf (1.4 Eq, 3.62 g) at room temperature. The reaction mixture was stirred at room temperature overnight. The resulting suspension was filtered through a pad of Celite, the filtrate was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using hexane/EtOAc (1/1) as eluent to deliver 2.0 g of the product as a yellow oil (yield 40.4%). 1H NMR (300 MHz, DMSO-d6) δ 5.34 (t, J=9.6 Hz, 1H), 5.01-4.86 (m, 2H), 4.79 (dd, J=9.6, 8.0 Hz, 1H), 4.44 (d, J=9.9 Hz, 1H), 3.86-3.74 (m, 1H), 3.64-3.48 (m, 12H), 3.39 (dd, J=5.6, 4.2 Hz, 2H), 2.00-1.95 (m, 9H).

[0507] Step 2: To a solution of 3,4,5-Tris(acetyloxy)-6-{2-[2-(2-azidoethoxy) ethoxy]ethoxy}oxan-2-yl]methyl acetate (1 eq., 1.0 g) in MeOH (15 ml) was added about 2M NaOH solution (10 mL). The reaction mixture was stirred at room temperature overnight. The solvents were removed under reduced pressure to dryness, the residue was dissolved in H2O and acidified with 1N HCl solution to pH=4. Water was removed under reduced pressure, and the crude product was used for the next step without further purification (calculated on the azide: 700 mg, yield quantitative).

[0508] Step 3: To a solution of 6-{2-[2-(2-Azidoethoxy)ethoxy]ethoxy}-3,4,5 trihydroxyoxane-2-carboxylic acid (1 eq., 0.921 g) in THF (9 mL)/MeOH (3 mL) were added BB3, copper (I) acetate (0.8 eq., 0.262 g), and L-ascorbic acid (1.7 eq., 0.804 g). The reaction mixture was stirred at 60° C. for overnight. The solvents were removed under reduced pressure, and the residue was purified by prep-HPLC (Gemini-NX 5 μm C18, H2O:ACN:TFA, starting from ACN 15% to ACN 95%). The appropriate fractions were combined, neutralized with TEA, evaporated under reduced pressure, dissolved in water, and passed through Dowex 50WX4 Na+-form to deliver 45 mg of the product as a white solid foam (yield 3.1%). LC/MS (6 min) RT=1.78 min, found [M+H].sup.+ 551.30 (a free acid). LC/MS (12 min) RT=3.123 min, found [M+H].sup.+ 551.4 (a free acid). HPLC purity: 96.51% (200 nm), 97.32% (283 nm). 1H NMR (300 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.04-7.88 (m, 4H), 4.98-4.87 (m, 2H), 4.61 (t, J=5.2 Hz, 2H), 4.14 (d, J=7.7 Hz, 1H), 3.85 (dt, J=25.8, 5.2 Hz, 4H), 3.68 (t, J=5.5 Hz, 2H), 3.54 (dt, J=9.1, 5.3 Hz, 9H), 3.13 (t, J=5.7 Hz, 3H), 2.95 (s, 1H).

II. Synthesis of AAVs

[0509] AAVs were generated by coupling the lactam linkers of the invention to at least one AAV surface-exposed primary amine, as schematically represented on FIG. 1.

II.1. Production and Purification of AAVs

Production and Purification

[0510] AAVs were produced and purified according to well-known techniques in the art.

Quality Control

[0511] Quality of the AAV2, AAV5, AAV8, and AAV9 batches was assessed by titration (qPCR using the LightCycler 480 from Roche, ddPCR using the QX200 from Bio-Rad, and ELISA).

[0512] A purity check was performed on a 10% SDS PAGE gel using silver staining: only VP1, VP2 and VP3 proteins in the correct stoichiometry of 1:1:10 were detectable, indicating a purity of the AAV preparation of over 95%.

[0513] Integrity of the packaged vector genome was also assessed on a 0.8% agarose gel stained with gel red: only a single sharp band about 3.2 kb was found, which corresponds to the size of the packaged vector genome, hence demonstrating its integrity in the AAV particles.

[0514] Endotoxin levels in the final products were determined using the Endosafe®-nexgen-PTS™ spectrometer (Charles River Laboratories). All samples passed the test with a detection threshold of 0.5 EU/mL.

[0515] Finally, functional tests were carried out in U87-MG cells, transduced with the AAV vectors (AAV2-eGFP; AAV5-eGFP; AAV8-eGFP; AAV9-eGFP) with MOI 1 000, 10 000 and 100 000. Total gDNA was isolated from each well and Cp values as direct measure of the vector copies (vg)/cell were determined by qPCR using eGFP-specific PCR primers.

II.2. Coupling of Compounds Comprising β-Lactam to AAVs

Materials

[0516] Compounds (1)-(5), (7)-(9) were obtained as detailed above in example 1.3.

[0517] The following AAVs, obtained as detailed in example II.1, were used: [0518] AAV2-eGFP: 1.0×10.sup.13 vg/mL in DPBS+Ca.sup.2+, Mg.sup.2+, 0.001% Pluronic F68 at pH 7.4; [0519] AAV5-eGFP: 1.0×10.sup.13 vg/mL in DPBS+Ca.sup.2+, Mg.sup.2+, 0.001% Pluronic F68 at pH 7.4; [0520] AAV8-eGFP: 1.0×10.sup.13 vg/mL in DPBS+Ca.sup.2+, Mg.sup.2+, 0.001% Pluronic F68 at pH 7.4; [0521] AAV9-eGFP: 1.0×10.sup.13 vg/mL in DPBS+Ca.sup.2+, Mg.sup.2+, at pH 7.4.

TABLE-US-00011 TABLE 7 Other material and reagents: Description Specification TBS, pH 9.3 50 mM Tris, 150 mM NaCl, pH: 9.3 Formulation buffer DPBS + Ca.sup.2+, Mg.sup.2+, 0.001% Pluronic F68 Concanavalin A-HRP SIGMA (L6397) Biotin-RCA1 Vector Lab (ZG1009) Biotin-UEA1 Vector Lab (ZG0901)

Methods

[0522] The coupling of the lactam linkers on AAVs capsids was carried out with a 3.0E6 fold molar excess of the linker in a total reaction volume of 1 mL. The final concentration of AAVs was 1.0E12 vg/mL.

[0523] The Lactam Compounds were brought to room temperature before being weighed into 1.5 mL reaction tube and dissolved in the suitable volume of buffer TBS, pH 9.3.

[0524] The AAVs were thawed at 20° C. 1 h before the coupling reactions are set up.

[0525] The coupling reactions were set up in 2.0 ml polypropylene reaction tubes The reaction tubes were gently shaken on an orbital shaker in horizontal position for 4h at 20° C.

[0526] Formulation/filtration: removal of free linkers. For each coupling mixture one PD MidiTrap G-25 column is required. The columns are equilibrated 5 times with 4 ml of formulation buffer (DPBS, Ca2+, Mg2+, 0.001% F68). The coupling reactions mixtures are loaded onto the columns and the samples are allowed to enter the bed. Elution of the rAAVs occurs with 1.5 ml formulation buffer. 5 fractions at 0.3 ml are collected dropwise in 1.5 ml PP-tubes. Fractions 2-5 are pooled and qPCR titer is determined for each pooled fraction. Pooled fractions are then sterile filtrated using Acrodisc PP, PES, 0.2 μM 1 cm.sup.2. The filtrated fractions were frozen at −80° C. in aliquots of 50 μL.

II.3. Characterization of AAVs

[0527] II.3.a. Titration of Vector Genones (vg)

[0528] For all coupling reactions quantitative real time PCT (qPCR) titers were determined using a LightCycler 480 (Roche) for samples taken after the formulation/filtration step, see Table 8. The high values of % input in the last column of Table 8 show that very few losses were sustained during the coupling.

TABLE-US-00012 TABLE 8 qPCR titers Coupling VG/ml Vol (ml) Total VG % Input 1 (1) + AAV2 8.64 × 10.sup.11 1.2  1.04 × 10.sup.12 104% 2 (2) + AAV2 9.47 × 10.sup.11 1.2  1.14 × 10.sup.12 114% 3 (3) + AAV2 8.69 × 10.sup.12 1.2  1.04 × 10.sup.12 104% 4 (4) + AAV2 8.05 × 10.sup.11 1.2  9.66 × 10.sup.12  97% 5 (1) + AAV8 9.34 × 10.sup.11 1.2  1.12 × 10.sup.12 112% 6 (3) + AAV8 1.04 × 10.sup.11 1.2  1.25 × 10.sup.12 125% 7 (3) + AAV5 6.62 × 10.sup.11 1.2  7.95 × 10.sup.12  80% 8 (5) + AAV2 7.23 × 10.sup.11 1.2  8.68 × 10.sup.11  87% 9 (1) + AAV5 6.91 × 10.sup.11 1.2  8.29 × 10.sup.11  83% 10 (1) + AAV9 8.45 × 10.sup.11 1.2 1.014 × 10.sup.12 101% 11 (7) + AAV2 9.25 × 10.sup.11 1.2  1.11 × 10.sup.12 111% 12 (8) + AAV2 8.49 × 10.sup.11 1.2  1.02 × 10.sup.12 102% 13 (9) + AAV2 7.91 × 10.sup.11 1.2  9.50 × 10.sup.11  95%
II.3.b. Analysis of coupling by SDS-PAGE and Lectin WB

Purpose

[0529] The purity and integrity of the obtained AAV vectors was evaluated by silver staining of SDS-PAGE gels. The efficacy of the coupling of the saccharide moieties on the AAVs was further studied by western blot analysis using various lectin stainings which bind selectively to the respective coupled saccharides.

Method

[0530] VP proteins corresponding to 1.0×10.sup.10 vg of rAAV after coupling were compared to VP proteins of the original AAV as negative control by SDS-PAGE (10% PAA gel) and subsequent silver staining. Gel was migrated up to MW 32 kDa.

[0531] Additional identical gels were used for Western Blotting (WB) and subsequent detection of linkers coupled to VP proteins using lectin staining: Concanavalin-HRP (ConA) or biotinylated lectins RCA1 and UEA1.

[0532] The analysis of the couplings by silver stain and WB was performed on frozen samples.

[0533] Successful coupling should result in a shift of the VP proteins towards higher molecular mass, and specific lectin staining (when applicable).

Results

[0534] Results are presented on FIGS. 2-13 and summarized in Table 9 below.

TABLE-US-00013 TABLE 9 Mobility shift Lectin Coupling on silver gel staining 1 (1) + AAV2 yes yes (ConA) 2 (2) + AAV2 yes yes (RCA1) 3 (3) + AAV2 yes yes (UEA1) 4 (4) + AAV2 yes ND 5 (1) + AAV8 yes yes (ConA) 6 (3) + AAV8 yes yes (UEA1) 7 (3) + AAV5 inconclusive yes (UEA1) 8 (5) + AAV2 yes ND 9 (1) + AAV5 yes yes (ConA) 10 (1) + AAV9 yes yes (ConA) 11 (7) + AAV2 yes ND 12 (8) + AAV2 yes ND 13 (9) + AAV2 yes ND ND: not determined

Conclusion

[0535] Mobility shifts and/or specific lectin staining were observed for all modified AAVs, evidencing effective coupling of Lactam compounds on AAVs.

II.3.c. Infectivity Assay (U87-MG Glioblastoma Cells)

[0536] In case SDS-PAGE show the expected mobility shift of the VP proteins, it was tested if infectivity of the AAVs can be observed. For this purpose U87-MG cells were transduced at MOIs 10.000 and 100.000. Transduced cells were analyzed 72h after transduction via monitoring of transduced (eGFP-positive) and non-transduced cell population by fluorescence microscopy.

[0537] Representative bright field and fluorescence pictures were taken from the transduced cells, showing that U87-MG cells were efficiently transduced by all the AAVs (data not shown).

III. In Vivo Evaluation of AAVs

III.1 Evaluation of the Transduction Properties of Three AAV Vectors in the Mouse Brain

[0538] The objective of the study was to investigate the transduction properties of three recombinant AAV2 vectors expressing GFP (AAV2, (1)-AAV2, and (7)-AAV2) in the mouse brain following a single, unilateral intrastriatal injection.

Materials

Animals

[0539] Eight (8) adult male C.sub.57BL/6 mice (Mus musculus), purchased from Janvier Labs.

Test Items

[0540] “AAV2” is a recombinant AAV2 vector comprising an unmodified capsid and carrying a CAG-eGFP expression cassette.

[0541] “(1)-AAV2” is a recombinant AAV2 vector comprising a modified capsid with surface-bound mannose linkers and carrying a CAG-eGFP expression cassette.

[0542] “(7)-AAV2” is a recombinant AAV2 vector comprising a modified capsid with surface-bound 6-O-sulfated galactose linkers and carrying a CAG-eGFP expression cassette.

Methods

Test Items

[0543] (1)- and (7)-AAV2 were produced as described in section II above. Briefly, mannose and 6-O-sulfated galactose linkers were covalently attached to the primary amines of lysine residues exposed at the surface of the AAV2 capsid after a 4-hour co-incubation of the linkers with the AAV2 vectors in Tris buffer pH 9.3 at 20° C., and a dialysis of the mix against buffered saline sterile solution (BSSS)+0.001% Pluronic® F68 to remove free molecules that did not bind to the AAV capsid.

Study Design

[0544] Eight (8) mice underwent stereotactic surgery and were randomly injected with the test items into the right striatum, according to Table 10 below.

TABLE-US-00014 TABLE 10 Treatment schedule. Group n Test item Target G1 2 AAV2 Right striatum G2 3 (1)-AAV2 Right striatum G3 3 (7)-AAV2 Right striatum

Surgical Procedures

[0545] Buprenorphine (0.1 mg/kg; 10 mL/kg, s.c.) was given as an analgesic before and after surgery. The animals were placed individually in an anesthetic chamber supplied with a continuous flow of oxygen (1.5 μL/min) and 3% isoflurane, and upon loss of consciousness, were stabilized in a stereotactic frame (Kopf) with the head fixed into position with ear bars. The skin of the skull was incised to allow for the unilateral injection of one of the test items using a glass pipette, at the coordinates described in Table 11. For an atlas of the mouse brain, see Paxinos & Franklin, 2019. The mouse brain in stereotaxic coordinates (5th ed.). San Diego, Calif.: Elsevier Science Publishing Co Inc.

TABLE-US-00015 TABLE 11 Injection coordinates. AP: anterior-posterior; ML: medial-lateral; DV: dorsal-ventral. AP ML DV Volume/target Titer/target Target (mm) (mm) (mm) (μL) (vg/mL) Right striatum +1.0 2.1 −2.6 0.5 5.5 × 10.sup.11 +0.3 2.3 −2.6 0.5 5.5 × 10.sup.11

[0546] Animals were allowed to recover for 48 days before euthanasia was carried out.

Ex Vivo Analysis

[0547] Euthanasia and Tissue Processing

[0548] At the end of the in vivo phase, animals were euthanized, and tissue were collected. Euthanasia was performed in accordance with European Veterinary Medical Association guidelines.

[0549] At termination, the brain of each animal was quickly removed and fixed in paraformaldehyde (PFA; 4%). After 3 days, the tissues were cryoprotected in 20% sucrose solution (in 0.1 M PBS) at 4° C. overnight, then frozen for sectioning into 50 μm thick coronal sections using a cryostat. Free-floating sections were placed in PBS azide and stored at 4° C.

[0550] GFP Immunohistochemistry

[0551] Definition of the percentage of transduced brain volume in the regions of interest was made based upon GFP immunohistochemistry. One in every four sections was used for immunohistochemistry.

[0552] Tissue sections were taken from the refrigerator and left to adjust to room temperature. After thorough rinsing with PBS, endogenous peroxidase activity and antigenic sites were blocked by a 10-min incubation in peroxidase-blocking solution (Dako, S2023) followed by a 30-min incubation in PBS, 2% BSA, 0.3% Triton X-100 and 0.01% thimerosal, respectively.

[0553] Sections were then exposed first to a polyclonal anti-GFP antibody (Ab3080, Merck), diluted 1:1000 in PBS containing 0.2% BSA, 0.3% Triton X-100 and 0.01% thimerosal, and next to the Envision+anti-rabbit HRP-tagged secondary antibody (Dako, K4011) diluted in the same buffer. After thorough rinsing with PBS, HRP was reacted with the DAB+ substrate (Dako) for approximately 30 seconds. The chromogenic reaction was stopped by several washes with PBS.

[0554] Sections were then mounted onto slides and counterstained with a Nissl stain. The slides were digitized using a PannoramicScan II (3DHISTECH, Hungary) at a ×20 magnification with an extended mode in which 5-layer focus is automatically acquired and then flatten.

[0555] The regions of the right hemisphere corresponding to the striatum and the substantia nigra were digitally drawn using the MERCATOR software (Mercator, Explora Nova, La Rochelle, France) on 10 and 6 sections, respectively.

[0556] The total and GFP-positive volumes of these 2 brain structures were determined based on a threshold detection method, using the formula

V=ΣStd

where “ΣS” is the sum of surface areas; “t” is the average section thickness; and “d” is the number of slices between two consecutives analyzed sections measured.

[0557] The percentage of transduced brain volume in each region of interest was then calculated.

Results

[0558] Immunohistochemically-stained brain slices of G1 and G2 groups are shown in FIG. 14. The percentages of right striatum and right substantia nigra volumes with a positive GFP-staining are provided for each vector in Table 12 below and in FIGS. 14A and 14B.

TABLE-US-00016 TABLE 12 Mean percentages of GFP-positive striatal and nigral volumes (right hemisphere only) 48 days post intrastriatal injection. Brain structure AAV2 (1)-AAV2 (7)-AAV2 Striatum 39% 70% 56% Substantia nigra 39% 52% 48%

Conclusions

[0559] The objective of the study was to investigate the transduction properties of three recombinant AAV2 vectors expressing GFP (AAV2, (1)-AAV2, and (7)-AAV2) in the mouse brain following a single, unilateral intrastriatal injection in the right hemisphere.

[0560] All three AAV vectors drove the expression of GFP and the extent of GFP staining in specific brain structures (volumes) was used as an indicator of the ability of each vector to transduce brain cells.

[0561] Both (1)-AAV2 and (7)-AAV2 vectors outperformed the AAV2 vector in the striatum and the substantia nigra of the right hemisphere when delivered in the parenchyma (striatum), covering 70% and 56% of the right striatum and 52% and 48% of the right substantia nigra, respectively, when AAV2 only transduced 39% of the same regions. (1)-AAV2 achieved the largest coverage of both the striatal and nigral structures (striatum: 70% for (1)-AAV2 vs 56% for (7)-AAV2 vs 39% for AAV2; substantia nigra: 52% for (1)-AAV2 vs 48% for (7)-AAV2 vs 39% for AAV2).

[0562] Besides the measurements in striatal and nigral regions, the (1)-AAV2 vector showed the best transduction properties, both in terms of distribution and transgene expression levels, considering the whole brain. After a single administration in the right striatum, many brain structures of both the right and left hemisphere, which were not or poorly transduced by the AAV2 vector, showed a positive signal for (1)-AAV2-driven GFP expression. In particular, large portions of the cortex and the hippocampus were intensely stained (FIG. 15).

IV. In Vitro Evaluation of a Test Candidate: AAV2-GBA1

[0563] The objective of the study was to determine the glucocerebrosidase (GCase) activity in HEK293 cells transduced with recombinant AAV2 vectors expressing the GBA1 gene (AAV2.GBA1, (1)-AAV2.GBA1 or Man-NCS-AAV2.GBA1).

[0564] GBA1 codes for GCase, a lysosomal enzyme that converts glucosylceramide into glucose and ceramide. Mutations in the GBA1 gene are the most common genetic risk factor for Parkinson's disease and Gaucher disease.

Materials

Test Items

[0565] “AAV2.GBA1” (also referred to as “AAV2” in FIG. 16) is a recombinant AAV2 vector comprising an unmodified capsid and carrying a CAG-GBA expression cassette.

[0566] “(1)-AAV2.GBA1” (also referred to as “(1)-AAV2” in FIG. 16) is a recombinant AAV2 vector comprising a modified capsid with surface-bound mannose beta-lactam linkers (more particularly with the compound (1) as described in Table 1 or 2) and carrying a CAG-GBA expression cassette.

[0567] “Man-NCS-AAV2.GBA1” (also referred to as “Man-NCS-AAV2” in FIG. 16) is a recombinant AAV2 vector comprising a modified capsid with mannose isothiocyanate linkers and carrying a CAG-GBA expression cassette.

Methods

Test Items

[0568] AAV2.GBA1, (1)-AAV2.GBA1 and Man-NCS-AAV2.GBA1 were produced as described above in section “II. SYNTHESIS OF AAVs” or in WO2017/212019.

[0569] Briefly, mannose with either beta-lactam or isothiocyanate linkers were covalently attached to the primary amines of lysine residues exposed at the surface of the AAV2.GBA1 capsids after a 4-hour co-incubation of the linkers with the AAV2.GBA1 vectors in Tris buffer pH 9.3 at 20° C. Coupled vectors (1)-AAV2.GBA1 and Man-NCS-AAV2.GBA1 were formulated in buffered saline sterile solution (BSSS)+0.001% Pluronic® F68 using a PD MidiTrap G-25 desalting column to remove free molecules that did not bind to the AAV capsid. AAV2.GBA1 was formulated in D-PBS+Ca.sup.2++Mg.sup.2++0.001% Pluronic® F68.

Gcase Activity Assay

[0570] HEK293 cells were seeded. On day 2, they were transduced with vectors carrying the GBA1 gene (AAV2.GBA1, (1)-AAV2.GBA1 or Man-NCS-AAV2.GBA1) at a MOI of 10.sup.6 (one well/condition). 96 hours post-transduction, for each well, supernatant was collected (1 mL) and dry cell pellet were prepared. Both were stored at −80° C. until GCase activity assay was performed.

[0571] Dry cell pellets were homogenized in NaCl 0.9% and sonicated. Then, protein concentration was determined using a Pierce BCA Protein Assay in cell pellets and supernatants. 4-methylumbelliferyl (4-MU) served as a calibrator and a dilution range was prepared in stop solution (glycine buffer, pH 10.5). GCase activity was measured in samples using 4-methylumbelliferyl β-D-glucopyranoside substrate in sodium phosphate buffer (pH 5.5) supplemented with sodium taurocholate hydrate at 37° C. for 30 minutes. The reaction was stopped by adding stop solution, and substrate fluorescence was measured (excitation wavelength=355 nm; emission wavelength=460 nm) with a fluorimeter (Mithras LB 940 BERTHOLD Technologies). All samples and calibrators were run in triplicates.

Results

[0572] For each well, GCase activity was measured in the supernatant (secreted GCase) and in the cellular pellet (non-secreted GCase). The total GCase activity per condition, being the sum of activities in both the supernatant and the pellet, is also reported. The total GCase activity was 624.3 nmol/h/well vs 384.6 nmol/h/well vs 276.1 nmol/h/well for (1)-AAV2.GBA1, AAV2.GBA1 and Man-NCS-AAV2.GBA1 vectors respectively. In the supernatant, enzymatic activity was 517.4 nmol/h/well vs 315.5 nmol/h/well vs 218.1 nmol/h/well; in the pellet enzymatic activity was 106.9 nmol/h/well vs 69.1 nmol/h/well vs 58.0 nmol/h/well for the three vectors respectively.

Conclusion

[0573] (1)-AAV2.GBA1 outperformed AAV2.GBA1 and Man-NCS-AAV2.GBA1 vectors with higher GCase activities measured in both the supernatant and the pellet.

[0574] These results are promising and demonstrate the unexpected superiority of AAV vectors comprising a lactam-linked moiety.