Antagonists of bacterial sequences

Abstract

The present invention refers to antagonist molecules, particularly nucleic acid effector molecules directed against a bacterial RNA.

Claims

1. An isolated antagonist directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA is homologous or complementary to RNA or DNA host sequences and/or innate bacterial sequences, coupled with or enclosed by a pharmaceutical carrier which increases the efficacy of the antagonist to enter target cells, wherein said antagonist is a nucleic acid effector molecule selected from (i) antisense molecules, ribozymes, siRNA molecules, and shRNA molecules (ii) precursors of the molecules from (i) and (iii) DNA molecules encoding the molecules from (i) and (ii).

2. The isolated antagonist of claim 1 directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA is a contiguous bacterial RNA stretch that has a length of at least 10 nucleotides.

3. The isolated antagonist of claim 1 directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA that is homologous or complementary to RNA or DNA host sequences and/or innate bacterial sequences has a length of at least 15 nucleotides, and wherein the degree of homology or complementarity is at least 75%.

4. The isolated antagonist of claim 1 directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA has a stem-loop secondary structure.

5. The isolated antagonist of claim 1 directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA is from mycobacteria.

6. The isolated antagonist of claim 1 directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA is from the mycobacterial tuf gene.

7. The isolated antagonist of claim 1, wherein said carrier is suitable for the treatment of bacterial infections.

8. The isolated antagonist of claim 1 which is present as: (i) a naked nucleic acid, (ii) a non-viral vector including liposomes, cationic lipids, polyethylenimine, poly-L-lysine or other non-viral compounds suitable for gene delivery, (iii) a viral vector suitable for gene delivery, or (iv) a bacterial vector including invasive or intracellular bacterial vectors.

9. The isolated antagonist of claim 1, wherein said antagonist is coupled to ferromagnetic nanoparticles.

10. The isolated antagonist of claim 1 which is covalently or non-covalently linked to a targeting moiety.

11. A pharmaceutical composition comprising more than one antagonist of claim 1 together with said pharmaceutically acceptable carrier which increases the efficacy of the antagonist to enter target cells, wherein said carrier is selected from the group consisting of lipids, liposomes, ferromagnetic nano-particles, viral vectors, bacterial vectors and gene carriers.

12. The isolated antagonist according to claim 1, where said antagonist is a nucleic acid molecule with at least one sugar, backbone or nucleobase modified ribonucleotide.

13. The isolated antagonist according to claim 1, where said antagonist is a nucleic acid effector molecule which is modified with lipid residues, cholesterol residues or florophors.

14. The isolated antagonist according to claim 1, wherein said pharmaceutical carrier is selected from the group consisting of lipids, liposomes, ferromagnetic nano-particles, viral vectors, bacterial vectors and gene carriers.

15. The isolated antagonist of claim 1, wherein said antagonist is directed against bacterial mRNA from a portion of the mycobacterial tuf gene comprising the sequence according to SEQ ID NO: 3.

16. The isolated antagonist according to claim 2, wherein the bacterial RNA is from M. tuberculosis.

17. The isolated antagonist according to claim 10, wherein said targeting moiety is a bacterial delivery peptide.

18. The isolated antagonist of claim 7, wherein the bacterial infections are caused by or associated with intracellular bacteria.

19. The isolated antagonist of claim 7 wherein said carrier is suitable for the treatment of mycobacterial infections.

20. The isolated antagonist of claim 19, wherein the mycobacterial infections are caused by and/or associated with M. tuberculosis.

21. The isolated antagonist of claim 8, wherein said bacterial vector is based on a bacteria selected from the group consisting of E. coli, Salmonella species, Listeria monocytogenes, and Mycobacteria.

22. The isolated antagonist of claim 4, wherein the stem has a length of at least 15 base pairs.

23. The isolated antagonist of claim 22, wherein the stem has a length of at least 20 base pairs.

24. The isolated antagonist of claim 4 wherein the stem has a degree of self-complementarity within the stem of at least 70%.

25. The isolated antagonist of claim 24 wherein the degree of self-complementarity within the stem is at least 80%.

26. The isolated antagonist of claim 25 wherein the degree of self-complementarity within the stem is at least 90%.

27. The isolated antagonist of claim 26 wherein the degree of self-complementarity within the stem is 100%.

28. The isolated antagonist of claim 4 wherein the stem has a length of at least 10 base pairs.

29. The isolated antagonist of claim 28 wherein the stem has a degree of self-complementarity within the stem of at least 70%.

30. An isolated antagonist directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA has a stem-loop secondary structure, the stem-loop secondary structure is homologous to RNA or DNA host sequences and/or innate bacterial sequences, the bacterial RNA is from mycobacteria, and wherein the degree of identity within the homologous regions of the stem sequences and the innate or bacterial sequences is at least 70% for the strand with the highest homology, coupled to or enclosed by a pharmaceutical carrier which increases the efficacy of the antagonist to enter target cells, wherein said carrier is selected from the group consisting of lipids, liposomes, ferromagnetic nano-particles, viral vectors, bacterial vectors and gene carriers.

31. The isolated antagonist according to claim 30, wherein the degree of identity within the homologous regions of the stem sequences and the innate or bacterial sequences is at least 75% for the strand with the highest homology.

32. An isolated antagonist directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA has a stem-loop secondary structure, the stem-loop secondary structure is homologous to RNA or DNA host sequences and/or innate bacterial sequences, and the antagonist is selected from the group consisting of nucleic acid effector molecules, aptamers, and combinations thereof, coupled with or enclosed by a pharmaceutical carrier which increases the efficacy of the antagonist to enter target cells, wherein said carrier is selected from the group consisting of lipids, liposomes, ferromagnetic nano-particles, viral vectors, bacterial vectors and gene carriers.

33. The isolated antagonist according to claim 8, wherein said viral vector is selected from the group consisting of AAV vectors, adenoviral vectors, lentiviral vectors, retroviral vectors, and herpes viral vectors.

34. An isolated antagonist directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA has a stem-loop secondary structure, the stem-loop secondary structure is homologous to RNA or DNA host sequences and/or innate bacterial sequences, and the antagonist is selected from the group consisting of nucleic acid effector molecules, aptamers and combinations thereof, wherein said nucleic acid effector molecules and aptamers comprise (a) a non-naturally occurring sugar, backbone or nucleobase modified ribonucleotide or deoxyribonucleotide building blocks, (b) lipid residues, (c) cholesterol residues, and/or (d) fluorophors.

35. An isolated antagonist directed against a bacterial RNA or a precursor thereof, wherein the bacterial RNA is homologous or complementary to RNA or DNA host sequences and/or innate bacterial sequences, said antagonist is a modified nucleic acid effector molecule or aptamer, and wherein the modification is selected from the group consisting of (a) a non-naturally occurring sugar, backbone or nucleobase modified ribonucleotide or deoxyribonucleotide building blocks, (b) lipid residues, (c) cholesterol residues, and (d) fluorophors.

36. The isolated antagonist of claim 10 which is covalently linked to a targeting moiety.

37. The isolated antagonist of claim 30, wherein said antagonist is covalently linked to a targeting moiety.

38. The isolated antagonist of claim 32, wherein said antagonist is covalently linked to a targeting moiety.

Description

FIGURES

(1) FIG. 1: Computer-based identification algorithm (RNA base pair matrix, shift width 1 nt) of miRNA analogous structures having a pre-determined length of 140 nt by self-alignment. The intramolecular self-complementarity is expressed by continuous lines consisting of individual dots.

(2) FIG. 2: Examples of miRNA analogous structures derived from M. tuberculosis sequences (SEQ ID NO: 1, 2, 3 & 4).

(3) FIG. 3: Sequence conservation within the miRNA analogous structures from M. tuberculosis. a) Alignment tree of all miRNA candidates of M. tuberculosis. b) Portion of the sequence alignment. *This structure differs from other structure sequences by being homologous to two human sequences (SEQ ID NO: 5, 6, 7, 8, 9 & 10).

(4) FIG. 4: Sequence conservation within miRNA analogous structures from different Bacteria. *miRNA analogous structure with homology to human MHC-II and Myelin P0 is absolutely conserved among the tuf genes of M. tuberculosis and M. bovis and partly conserved in M. smegmatis. It does not occur in any of the other investigated organisms.

(5) FIG. 5: Predicted structure of the miRNA analogous structure having homology to human sequences as shown in FIGS. 3 and 4, and a simplified schematic depiction of this structure (SEQ ID NO: 3).

(6) FIG. 6: Sequences, structures (dot-bracket depiction) and folding energies of potential mature miRNA molecules (ranging from 23-mere to 16-mere) which may be obtained by processing from the stem of the precursor structure shown in FIG. 5. a) 23-mere (residues 11-33, 12-34, 13-35, and 14-36 of SEQ ID NO: 3), 22-mere (residues 11-32, 12-33, 13-34, 14-35, and 15-36 of SEQ ID NO: 3), 21-mere (residues 11-31, 12-32, 13-33, 14-34, 15-35, and 16-36 of SEQ ID NO: 3), 20-mere (residues 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, and 17-36 of SEQ ID NO: 3), 19-mere (residues 11-29, 12-30, 13-31, 14-32, 15-33, 16-34, 17-35, and 18-36 of SEQ ID NO: 3), 18-mere (residues 11-28, 12-29, 13-30, 14-31, 15-32, 16-33, 17-34, 18-35, and 19-36 of SEQ ID NO: 3), 17-mere (residues 11-27, 12-28, 13-29, 14-30, 15-31, 16-32, 17-33, 18-34, 19-35, and 20-36 of SEQ ID NO: 3), and 16-mere (residues 11-26, 12-27, 13-28, 14-29, 15-30, 16-31, 17-32, 18-33, 19-34, 20-35, and 21-36 of SEQ ID NO: 3) processed from 5′ terminal stem. b) 23-mere (residues 41-63, 42-64, 43-65, and 44-66 of SEQ ID NO: 3) 22-mere (residues 41-62, 42-63, 43-64, 44-65, and 45-66 of SEQ ID NO: 3), 21-mere (residues 41-61, 42-62, 43-63, 44-64, 45-65, and 46-66 of SEQ ID NO: 3), 20-mere (residues 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, and 47-66 of SEQ ID NO: 3), 19-mere (residues 41-59, 42-60, 43-61, 44-62, 45-63, 46-64, 47-65, and 48-66 of SEQ ID NO: 3), 18-mere (residues 41-58, 42-59, 43-60, 44-61, 45-62, 46-63, 47-64, 48-65, and 49-66 of SEQ ID NO: 3), 17-mere (residues 41-57, 42-58, 43-59, 44-60, 45-61, 46-62, 47-63, 48-64, 49-65, and 50-66 of SEQ ID NO: 3), and 16-mere (residues 41-56, 42-57, 43-58, 44-59, 45-60, 46-61, 47-62, 48-63, 49-64, 50-65, and 51-66 of SEQ ID NO: 3) processed from 3′ terminal stem.

(7) FIG. 7: Homology between the bacterial miRNA target sequence (SEQ ID NO: 3) and potential human host sequences. The sequence homology to the myelin P0 mRNA (SEQ ID NO: 17 and residues 32-68 of SEQ ID NO: 3) and to the plus strand of the myelin P0 gene (SEQ ID NO: 16 and residues 32-68 of SEQ ID NO: 3) is shown.

(8) FIG. 8: Homology between the bacterial mRNA target sequence (SEQ ID NO: 3) and human host sequences. Homology to the minus strand of the myelin P0 gene (SEQ ID NO: 11 and residues 12-34 of SEQ ID NO: 3).

(9) FIG. 9: Homology between the bacterial target miRNA sequence (SEQ ID NO: 3) and human host sequences. Homology to the plus strand DNA (SEQ ID NO: 13 and residues 8-38 of SEQ ID NO: 3) and minus strand DNA (SEQ ID NO: 12 and residues 40-68 of SEQ ID NO: 3) of the MHC-II cluster between the two MHC-II subunits HLA-DRB7 and HLA-DRB8.

(10) FIG. 10: Specific MHC-II inhibition in primary human monocytes by an miRNA candidate-derived siRNA. Monocytes were isolated from a buffy coat, transfected with chemically synthesized siRNAs using nucleofection prior to IFN-γ stimulation. MHC-II expression was detected by FACS analysis 24 h after transfection. Upper panel: GFP-directed control siRNA; lower panel: miRNA candidate-derived MHC-II-specific siRNA (SEQ ID NO: 3, 14 & 15).

(11) FIG. 11: Examples for the positioning of nucleic acid based antagonists with regard to the sequence of the identified bacterial target RNA (a). b) positioning against unpaired regions, e.g. the 5′ terminal sequence, the loop and the 3′ terminal sequence, or positioning against portions of the paired stem region. c) Positioning against two, three or several of the regions described under b). d) Positioning against the whole sequence from a). e) Positioning against discontinuous sequences, i.e. both free ends excluding the hairpin (stem loop) structure. f) Positioning against discontinuous sequences from the hairpin structure (SEQ ID NO: 3).

(12) FIG. 12: Examples for the positioning of nucleic based antagonists relative to the structure of an identified bacterial target RNA. Positioning against unpaired areas such as the 5′ terminus, the loop and the 3′ terminus of the sequence or against portions of the paired stem regions. Further positionings are directed against two, three or more of these structural elements or against the whole sequence. Further antagonists may be directed against discontinuous portions of the target structure, e.g. against both free ends or the hairpin region.

(13) FIG. 13: Specific examples of nucleic acid based antagonists directed against the identified bacterial mycobacterial target RNA (a). b) Oligodesoxyribonucleotide which is preferably directed with its 3′ end against an unpaired of the portion of the target sequence. c) Ribozyme directed against a target sequence GUH (wherein H is any base except G). d) Antisense RNA which preferably has a length between 70 and 120 nucleotides and a structure with free 5′ and 3′ termini. e) siRNA molecule having a sense and an antisense strand and two nt 3′ overhangs. Preferably, the guide strand of the siRNA is not capable of intramolecular folding or has a positive free Gibbs folding energy. f) shRNA molecules including the guide structure from e) (SEQ ID NO: 3, 18, 19, 20, 21, 22, 23 & 24).

EXAMPLE

1. Identification of miRNA Analogous Structures in Bacteria

(14) By means of bioinformatic analysis on the basis of self-complementarity and length (FIG. 1) miRNA-analogous RNA secondary structures were identified in the transcriptome of M. tuberculosis (FIG. 2) (SEQ ID NO: 1, 2, 3 & 4). Next, the conservation of the identified RNA structures was analysed (FIG. 3). It was found that the sequences exhibit a very high homology within the stem region of the hairpin structure and have a variability in the loop region. Noteworthy is a sequence, which does not show any homology in the middle sequence portion including the loop region to the other sequences (FIG. 3b) (SEQ ID NO: 5, 6, 7, 8, 9 & 10).

(15) This sequence is a transcribed sequence from the tuf gene of M. tuberculosis and M. bovis including M. bovis BCG and exhibits some homology to a sequence from M. smegmatis (FIGS. 3 and 4) (SEQ ID NO: 5, 6, 7, 8, 9 & 10). It exhibits at its 3′ terminus a hairpin structure characteristic for miRNA molecules (FIG. 5) (SEQ ID NO: 3). Further, the hairpin structure exhibits a high degree of homology to human host sequences sufficient for RNA interference (FIGS. 7-9) (SEQ ID NO: 11, 12, 13, 16 & 17 and residues 12-34, 8-38, 40-68 and 32-68 of SEQ ID NO: 3).

(16) The sequence portions homologous to human host sequences fulfil the criteria of preferred active guide sequences in antisense sRNA or mature miRNA molecules (FIG. 6) (residues 11-33, 12-34, 13-35, 14-36, 11-32, 12-33, 13-34, 14-35, 15-36, 11-31, 12-32, 13-33, 14-34, 15-35, 16-36, 11-30, 12-31, 13-32, 14-33, 15-34, 16-35, 17-36, 11-29, 12-30, 13-31, 14-32, 15-33, 16-34, 17-35, 18-36, 11-28, 12-29, 13-30, 14-31, 15-32, 16-33, 17-34, 18-35, 19-36, 11-27, 12-28, 13-29, 14-30, 15-31, 16-32, 17-33, 18-34, 19-35, 20-36, 11-26, 12-27, 13-28, 14-29, 15-30, 16-31, 17-32, 18-33, 19-34, 20-35, 21-36, 41-63, 42-64, 43-65, 44-66, 41-62, 42-63, 43-64, 44-65, 45-66, 41-61, 42-62, 43-63, 44-64, 45-65, 46-66, 41-60, 42-61, 43-62, 44-63, 45-64, 46-65, 47-66, 41-59, 42-60, 43-61, 44-62, 45-63, 46-64, 47-65, 48-66, 41-58, 42-59, 43-60, 44-61, 45-62, 46-63, 47-64, 48-65, 49-66, 41-57, 42-58, 43-59, 44-60, 45-61, 46-62, 47-63, 48-64, 49-65, 50-66, 41-56, 42-57, 43-58, 44-59, 45-60, 46-61, 47-62, 48-63, 49-64, 50-65, and 51-66 of SEQ ID NO: 3). This means that these sequence portions substantially have positive or only slightly negative folding energies and/or comprise long free termini (cf. Ref. 1). This criterion is particularly fulfilled by the shorter hypothetic guide sequences and preferably by those derived from the 5′ terminal portion of the stem (FIG. 6a).

(17) The expression of the tuf mRNA is known (cf. Ref. 2), it is differentially expressed following bacterial phagocytosis (cf. Ref. 3), and the 3′-terminal hairpin structure is known to function as atypical transcriptional terminator (cf. Ref. 4). However, unknown is the high homology of the stem region of the 3′-terminal hairpin structure (SEQ ID NO: 3) to human sequences. These host sequences are the 3′UTR of the myelin P0 mRNA (SEQ ID NO: 17) or the corresponding sequences of the myelin P0 gene (SEQ ID NO: 11 & 16) and a non-transcribed region in the MHC-II cluster between both MHC-II subunits HLA-DRB7 and HLA-DRB8 (SEQ ID NO: 12 & 13).

(18) The homologous region to the myelin P0 mRNA (SEQ ID NO: 17) and to the plus strand of the myelin P0 gene (SEQ ID NO: 16) comprises 32 bases in a 37 base long homologous region having a maximum of 15 contiguous complementary bases (32/37/15) (FIG. 7) (residues 32-68 of SEQ ID NO: 3). The homology to the minus strand of the myelin P0 gene (SEQ ID NO: 11) comprises 20/23/15 bases (FIG. 8) (residues 12-34 SEQ ID NO:3). The homology to the plus strand of the DNA of the MHC-II cluster comprises 24/31/16 bases (SEQ ID NO: 12 and residues 8-38 of SEQ ID NO: 3) and to the minus strand DNA 26/29/16 bases (SEQ ID NO: 13 and residues 40-68 of SEQ ID NO: 3) (FIG. 9).

(19) MHC-II expression is down-regulated in human cells in the course of infection with M. tuberculosis (Refs. 5 and 6). The molecular mechanisms of this down-regulation is unknown, although, the lipoprotein LprG of M. tuberculosis is known to be able to inhibit the MHC-II dependent antigen presentation in human macrophages (Ref. 7). It is assumed, that the identified bacterial target RNAs alone or together with LprG might play a role in MHC-II down-regulation. A sRNA derived from the miRNA candidate with high homology to the intergenic region between MHC-II subunits HLA-DRB7 and HLA-DRB8 was able to efficiently inhibit MHC-II expression in IFN-γ stimulated primary human monocytes as shown in FIG. 10. (SEQ ID NO: 3, 21 & 22). Further, 20-30% of all tuberculosis incidents also inflict the central nervous system (CNS). In this process, the interference of a bacterial mRNA with the myelin P0 gene might play a role. (SEQ ID NO: 3, 11, 16 & 17).

2. Development of Nucleic Acid Based Antagonists

(20) The interference of any bacterial target sequence, e.g. a tuf mRNA derived miRNA analogous structure, but not limited thereto, with corresponding host sequences can be reduced and/or inhibited by nucleic acid effector molecules. These nucleic effector molecules may be directed against the identified bacterial miRNA analogous sequences or precursors thereof, e.g. the complete tuf mRNA which comprises the hairpin structure at its 3′ terminus within or without the miRNA analogous sequence regions.

(21) The therapeutic efficacy of the novel nucleic acid based antagonists is achieved by functional blocking and/or cleavage of the bacterial target sequences or precursors thereof. Further, the antagonists are suitable for diagnostic applications, particularly in order to identify the bacterial target sequences as biomarkers for the stage and the course of a bacterial infection.

(22) The antagonists may be directed against any region of the bacterial target sequence, preferably a region having a length of at least 15 nucleotides (FIG. 11). (SEQ ID NO: 3). The target regions comprise the 3′ or 5′ termini, the stem region, the hairpin, and the regions between these structural elements. Further, the antagonists may be directed against specific structural element, e.g. against both free termini without the hairpin structure or the 5′ and 3′ terminal loop bases adjacent to the hairpin without the bases in the centre of the loop structure and portions of the stem region (FIG. 12).

(23) In FIG. 13, specific embodiments are shown:

(24) An antisense oligodesoxyribonucleotide preferably directed with its 3′ end against an accessible (unpaired) region of the target sequence (SEQ ID NO: 18), a hammerhead ribozyme directed against a GUH sequence (wherein H is any base except G) (SEQ ID NO: 19), an antisense RNA molecule which preferably has a length between 70 and 120 nucleotides and which comprises free (accessible) 5′ and 3′ ends (SEQ ID NO: 20), or an siRNA molecule, the guide strand of which preferably is not capable of intramolecular folding (SEQ ID NO: 21 & 22) or two shRNA molecules derived therefrom in which the antisense strand can be upstream or downstream of the sense strand. (SEQ ID NO: 23 & 24).

(25) It should be noted that that the nucleic acid based antagonists shown in FIG. 13 are only exemplary embodiments of the present invention without limiting its scope. For example, the nucleic acid based antagonists may also be directed to portions of the tuf mRNA (or any other bacterial RNA), which are located outside the miRNA analogous structure at the 3′ terminus of the tuf mRNA.

REFERENCES

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