Mediated cellular delivery of LNA oligonucleotides

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1 MEDIATED CELLULAR DELIVERY OF LNA OLIGONUCLEOTIDES

Cross-reference to Related Applications

This application is the U.S. National Stage of International Application No. PCT/DK2008/000019, filed Jan. 18, 2008, which, in turn, claims the benefit of Danish Patent Application Number PA200700087, filed Jan. 19, 2007.

The present invention relates to novel modified oligomeric compounds and to methods of making and using such compounds. The invention fiirther relates to methods of enhancing the cellular uptake of oligomeric compounds comprising conjugating a metal chelator to those.

SUMMARY OF THE INVENTION

The present invention relates to oligomeric compounds having a metal chelator covalently conjugated thereto. The metal chelator enhances functional cellular uptake of the oligomeric compounds compared to its native counterpart. The present invention can be used in combination with other known moieties that enhances cellular uptake. The oligomeric compounds can be used to direct specific inhibition, degradation or thethering of other functional groups to their target RNAs and DNAs, both coding and non-coding.

BACKGROUND OF THE INVENTION

A general object of some current therapeutic approaches is to interfere with, or otherwise modulate, gene expression.

One method for inhibiting the expression of specific genes involves the use of oligonucleotides, particularly oligonucleotides that are complementary to a specific target messenger RNA (mRNA) sequence, known as antisense oligonucleotides. Several oligonucleotides are currently undergoing clinical trials for such use.

Oligonucleotides and their analogs can be designed to have particular properties. A number of chemical modifications have been introduced into oligomeric compounds to increase their usefiilness as therapeutic agents. Such modifications include those designed to increase binding affinity to a target strand, to increase cell penetration, to stabilize against nucleases and other enzymes that degrade or interfere with the structure or activity of the oligonucleotide, to provide a mode of disruption (terminating event) once the oligonucleotide is bound to a target, and to improve the pharmacokinetic properties of the oligonucleotide. Despite such modifications, the cellular uptake of oligomeric compounds remains poor

Oligonucleotides have been formulated with various with transfection agents, including anionic and cationic lipids and polyarnines, in an attempt to improve their ability to permeate biological membranes. Of the transfection agents used, polyethylenimines (PEI) are generally the most eflicient and least expensive delivery vehicles. Kren, B. T.; Parashar, B.; Bandyopadhyay, P.; Chowdhury, N. R.; Chowdhury, J . R.; Steer, C. J. Proc. Natl.Acad. Sci. U.S.A. 1999,96, 10349-10354. It was observed, however, that, although complexes of excess PEI and oligonucleotide phosphorothioates were efficiently taken up by cells, the oligonucleotides failed to dissociate in the cytoplasm, resulting in no appreciable enhancement in the antisense activity of the oligonucleotides. Dheur, S.; Dias, N.; Van Aerschot, A.; Herdewijn, P.; Bettinger, T.; Remy, J .-S.; Helene, C.; Saison-Behmoaras, E. T. Antisense Nucleic Acid Drug Dev. 1999, 9, 515-525.

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Another approach has been attempts to enhance the cellular uptake of oligomeric compounds comprising conjugating the compounds to amphipathic moieties such as amphipathic peptides. (PCT Publication WO 2004/016274).

Conjugation of terpyridine to PNA nucleotides have previously been shown to mediate cellular uptake of a PNA oligonucleotide as monitored by fluorescence. This process was suggested to be dependent on Zn“ chelating. These experiments were carried out in HeLa cells using elevated concentrations (1-2.5 p.M) of fluorophor labeled oligonucleotides added directly to the cell culture media. Addition of high concentrations of oligonucleotides usually leads to some uptake, probably through so called phase fluid endocytosis, as can be observed as a punctuated intracellular distribution, mostly located to endosomes. Interestingly conjugation of terpyridine to PNA led to increased nuclear localization of the oligo, indicating that oligonucleotide may be free to base pair with a potential target sequence, however, no such data was presented (Andreas Fuss, Andreas Schleifenbaum, Mareike Goritz, Andrew Riddel, Carsten Schultz, Roland Kramer, J . Am. Chem. Soc. 2006, 128(8), 5986-5987.)

A need therefore exists in the art for the development of means to improve the cellular uptake and cellular distribution of oligomeric compounds.

RNA-mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co-suppression, transgene silencing, imprinting, methylation, and possibly position-effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signalling (Mattick 2001; EMBO reports 2, 11: 986-991). Recent studies indicate that antisense transcription is a very common phenomenon in the mouse and human genomes (Okazaki et al. 2002; Nature 420: 563-573; Yelin et al. 2003, Nature Biotechnol.). Thus, antisense modulation of gene expression in eukaryotic cells, e.g. human cells appear to be a common regulatory mechanism. In light of this, the present invention provides a method for detection and fiinctional analysis of non-coding antisense RNAs, as well as a method for detecting the overlapping regions between sense-antisense transcriptional units.

SUMMARY OF THE INVENTION

The present invention relates to metal chelating agents (such as 2,2':6',2'-terpyridine (terpyridine)) facilitating the delivery of a wide variety of modified oligonucleotides both ex vivo and in vivo.

In certain embodiments the conjugation of metal chelating agents is used for the modification of LNA-modified oligonucleotides, particularly when the oligonucleotides are used for delivery of miRNA knockdown oligonucleotides, in particular for enabling knockdown studies in hard-to-transfect cell lines and primary culture.

The invention provides for an oligonucleotide composition optionally substituted with one or more metal chelators. In a preferred embodiment, at least one non-natural monomer of the oligonucleotide composition is substituted with one or more metal chelators.

The present invention in one aspect provides for a compound of formula I:

OLIGO-L-CHEL (1)

wherein “OLIGO” designates an oligonucleotide composition, “CHEL” designates a metal chelator and “L” is a linking moiety consisting of from 1 to 6 consecutive groups of the formula —(CR1R2 )WYZ—, wherein R1 and R2 are separately hydrogen or a heteroatom such as O, S, Se, O, Si, N, P; or a

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Ci-C20 alkyl group optionally substituted with one or more heteroatoms such as oxygen atoms, nitrogen atoms, and/or sulphur atoms, optionally containing aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as poly-[3-alanine, polyglycine, polylysine, peptides, oligo/polysaccharides; or

R1 and R2 are separately forming a double bond to the carbon;

W is an integer of from 0 and up;

Y is a heteroatom such as O, S, Se, O, Si, N, P, to which hydrogen, alkyl, allyl, aromates, carbocycles or other heteroatoms are attached;

Z is an integer from 0 and up;

and wherein the consecutive groups can be the same or dif

ferent.

Examples of a C i-C20 alkyl group include a straight chain, branched chain or cyclic alkyl group such as a methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-decyl, cyclopropyl, 2,2-dimethylcyclopropyl, cyclopentyl, cyclohexyl and menthyl group.

In a further aspect the present invention relates to compounds as described above with an antisense, silencing or knock-down eflicacy.

The present invention furthermore provides for a method of synthesising a compound of formula I, comprising the following steps:

a) coupling a linker moiety with a suitable solid phase protecting group to the 5'-end of an oligonucleotide composition by a conventionally used process,

b) cleaving off said protecting group using a suitable reagent,

c) conjugating to said linker moiety a methal chelator, and

d) deprotecting said oligonucleotide composition.

In an additional embodiment, the invention provides a method of synthesising a compound of formula I, comprising the following steps:

a) attaching a linker moiety to the solid phase by conventionally used processes, thereby attaching the linker to the 3'-end of an oligonucleotide,

b) synthesising an oligonucleotide composition using conventionally used processes,

c) deprotecting said oligonucleotide composition, and

d) conjugating to said linker moiety a metal chelator.

The invention further provides for kits comprising a compound of formula I for use in diagnostics.

A further aspect of the present invention is the use of any of the above mentioned compounds for the treatment of a disease.

FIGURES

FIG. 1 shows the effect on MCF7 cells of co-transfection with pMIR-21 (hsa-miR-21 firefly luciferase reporter construct) and the indicated miR-21 inhibiting oligonucleotides with or without terpyridine conjugates. The diagram shows fold up regulation; FL (RLU)/RL (RLU). Relative light units (RLU); firefly luminescence (FL); Renilla luminescence (RL).

FIG. 2 shows the effect of terpyridine conjugation in absence of transfection reagent. The diagram shows fold up regulation; FL (RLU)/RL (RLU). Relative light units (RLU); firefly luminescence (FL); Renilla luminescence (RL). Relative light units (RLU); firefly luminescence (FL); Renilla luminescence (RL).

FIG. 3 shows the effect of terpyridine conjugation in absence of transfection reagent in HeLa, HeLa 3S, and MCF7

4 cells. The diagram shows fold up regulation of FL (RLU)/RL (RLU). Relative light units (RLU); firefly luminescence (FL); Renilla luminescence (RL).

FIG. 4 shows the effect of terpyridine-CPP conjugation in absence of transfection reagent in HeLa cells. The diagram shows fold up regulation of FL (RLU)/RL (RLU). Relative light units (RLU); firefly luminescence (FL); Renilla luminescence (RL).

DETAILED DESCRIPTION OF THE INVENTION

In a particular embodiment of the invention oligonucleotides are referred to as “oligonucleotide compositions”.

“Oligonucleotide compositions” are oligonucleotides wherein at least one monomer is a non-natural nucleotide also designated a “modified monomer unit”, which preferably is a LNA monomer as defined below and the remaining monomers are natural nucleotides. Preferred LNA monomers are oxy-LNA, alpha-LNA and arnino-LNA as defined below.

A “reference oligonucleotide composition” is a “oligonucleotide composition” as defined above wherein one or more non-natural nucleotide, preferably a LNA monomer is replaced with a natural nucleotide.

An example of an oligonucleotide composition of the invention and the corresponding reference oligonucleotide composition are shown in Table 1

Oligonucleotide compositions and reference oligonucleotide compositions shown in Table 1 is based on the reverse complementary sequence of the microRNA hsa-miR-21 (miRBase accession number MIMAT0000076).

The present invention also provides a kit for detection of nucleic acids. The present invention also provides a kit for sequence specific inactivation of intracellular nucleic acids. The present invention also provides a kit for introduction of nucleic acids to a cell.

For the kits according to the invention, the reaction body is preferably a solid support material, e.g. selected from borosilicate glass, soda-lime glass, polystyrene, polycarbonate, polypropylene, polyethylene, polyethyleneglycol terephthalate, polyvinylacetate, polyvinylpyrrolidinone, polymethylmethacrylate and polyvinylchloride, preferably polystyrene and polycarbonate. The reaction body may be in the form of a specimen tube, a vial, a slide, a sheet, a film, a bead, a pellet, a disc, a plate, a ring, a rod, a net, a filter, a tray, a microtitre plate, a stick, or a multi-bladed stick.

A written instruction sheet stating the optimal conditions for use of the kit typically accompanies the kits.

LNA substituted oligomers are preferably chemically synthesized using commercially available methods and equipment as described in the art (Koshkin et al., Tetrahedron 541