ANTISENSE OLIGOMERS FOR CONTROLLING CANDIDA ALBICANS INFECTIONS

Abstract

The present disclosure relates to the use of antisense oligomers in the treatment or therapy of Candida albicans infections. The present disclosure further describes the use of antisense oligomers in antisense therapy to inhibit the morphological transition of Candida albicans from yeast to filamentous form.

Claims

1. An isolated oligomer comprising: one or more sequences at least 95% identical to SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO: 9.

2. (canceled)

3. The isolated oligomer of claim 1, for use in the treatment or therapy of C. albicans related infections.

4. The isolated oligomer of claim 1, for use in the treatment or therapy of vaginal infection and/-or oral infections.

5. The isolated oligomer of claim 1, wherein the one or more sequences are at least 96% identical to SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO: 9.

6. A composition comprising at least two isolated oligomers, each isolated oligomer comprising: a sequence at least 95% identical to SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO: 9.

7. The composition of claim 6, wherein the sequence is at least 96% identical to SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO: 9.

8. The composition according to of claim 6, comprising two or three isolated oligomers having the following sequences respectively: SEQ ID NO: 7 and SEQ ID NO: 8; SEQ ID NO: 7 and SEQ ID NO: 9; SEQ ID NO: 8 and SEQ ID NO: 9; or SEQ ID NO: 7; SEQ ID NO: 8 and SEQ ID NO: 9.

9. The composition according to of claim 6, wherein the composition is a coating composition.

10. An article comprising the composition of claim 6.

11. The article of claim 10, wherein the article is a medical device.

12. The article of claim 10, wherein the article is an intravaginal tampon, a sanitary napkin, or a panty liners.

13. The isolated oligomer of claim 1, comprising a sequence of SEQ ID NO: 7; SEQ ID NO: 8; or SEQ ID NO: 9.

14. The article of claim 10, wherein the article is a patch, a catheter, a stent, a contact lens or a pacemaker.

15. A method of inhibiting C. albicans filamentation, the method comprising contacting C. albicans with the isolated oligomer of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

[0042] FIG. 1 shows the antisense oligomers sequences according to the description.

[0043] FIG. 2 shows the anti-EFG1 and anti-HWP1 oligomers sensitivity and specificity against Candida species determined by fluorescence in situ hybridization assays.

[0044] FIG. 3 shows the cytotoxicity effect of 40 nM of anti-EFG1, anti-HWP1 and anti-HYR1 oligomers against 3T3 cell line (Fibroblast cells, Embryonic tissue, Mouse from CCL3, American Type Culture Collection).

[0045] FIG. 4 shows the effects of anti-EFG1, anti-HWP1 and anti-HYR1 oligomers on C. albicans filamentation.

[0046] FIG. 5 shows the effect of 40 nM of anti-EFG1 on C. albicans filamentation, on EFG1 gene expression and on efg1p translation at 24 h of incubation.

[0047] FIG. 6 shows the results related to different combinations with 40 nM of each oligomer (anti-EFG1, anti-HWP1 and anti-HYR1) on C. albicans filamentation during 24 h of incubation.

[0048] FIG. 7 shows the performance of anti-EFG1 oligomer on different human body fluids: artificial saliva (AS) and urine (AU) during 24 h and blood at 48 h of incubation.

DETAILED DESCRIPTION

[0049] The present disclosure relates to the use of ASOs to target specific genes involved in the morphological transition of C. albicans from yeast to filamentous form.

[0050] The present disclosure further describes the use of ASO in AST to inhibit the morphological transition of C. albicans from yeast to filamentous form.

[0051] In one embodiment, ASOs targeting the three different genes were designed and synthetized to ensure the total blockade of C. albicans filamentation. The target regions were selected from each gene taking into account its high specificity against C. albicans genome and lower specificity against Homo sapiens genome. The regions selected were (5′-ACAATAACGGTATGCC-3′), (5′ CGCTTATTACATGTTATCA 3′) and (5′ GCTTACTCTCAACC 3′) for EFG1, HWP1 and HYR1, respectively.

[0052] In an embodiment, the target regions selected for methylation were:

TABLE-US-00001 SEQ ID No. 1-for EFG1: 5′-.sup.47ACAATAACGGTATGCC.sup.62-3′; SEQ ID No. 2-for HWP1: 5′ .sup.33CGCTTATTACATGTTATCA.sup.51 3′; SEQ ID No. 3-for HYR1: 5′ .sup.36GCTTACTCTCAACC.sup.49 3′.

[0053] In one embodiment, for each sequence of the target regions selected the reverse complement was determined in order to design the respective ASOs. The sequences determined were (5′ GGCATACCGTTATTGT 3′), (5′TGATAACATGTAATAAGCG3′) and (5′GGTTGAGAGTAAGC 3′) for EFG1, HWP1 and HYR1, respectively.

[0054] The reverse complement sequences determined for methylation were:

TABLE-US-00002 SEQ ID No. 4-for EFG1: 5′ GGCATACCGTTATTGT 3′; SEQ ID No. 5-for HWP1: 5′TGATAACATGTAATAAGCG3′; SEQ ID No. 6-for HYR1: 5′GGTTGAGAGTAAGC 3′.

[0055] In one embodiment, in order to increase the ASOs hit-rate, part of the oligonucleotides belonging to each selected sequence were chemically modified based on second generation nucleic acid mimics design (2′-O-methyl).

[0056] In another embodiment, once it has been demonstrated that the inclusion of the two or more modifications in each end of the nucleic acid mimics increase its stability in human serum, antisense oligomers were designed and synthesized.

[0057] In one embodiment, anti-EFG1 oligomer was designed and synthetized with four 2′-O-methyl chemical modifications (5′-mG mG mC nnA TACCGTTA mU mU mG mU-3′). Anti-HWP1 oligomer was designed and synthetized with two chemical modifications (5′ mUmGATAACATGTAATAAGmCmG 3′). Anti-HYR1 oligomer was designed and synthetized with three chemical modifications (5′ mGmGmU TGA GAG TAmA mGmC 3′).

[0058] In an embodiment, the methylated sequences were:

TABLE-US-00003 SEQ ID No. 7-for EFG1: 5′-mG mG mC mA TACCGTTA mU mU mG mU-3′; SEQ ID No. 8-for HWP1: 5′ mUmGATAACATGTAATAAGmCmG 3′; SEQ ID No. 9-for HYR1: 5′ mGmGmU TGA GAG TAmA mGmC 3′.

[0059] Methods for the alignment of sequences for comparison are well known in the art, such methods include BLAST and FASTA. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). The sequence identity values, which are indicated in the present subject matter as a percentage were determined over the entire amino acid sequence, using BLAST with the default parameters.

[0060] In an embodiment, FIG. 1 shows the ASOs sequences for EFG1 (anti-EFG1), HWP1 (anti-HWP1) and HYR1 (anti-HYR1) genes of C. albicans and the respective 2′-O-methyl chemical modifications insertions. FIG. 1B summarizes the characteristics of the three ASOs sequences in terms of its size, melting temperature and percentage of guanine and cytosine (GC).

[0061] In an embodiment, FIG. 2 shows Anti-EFG1 and anti-HWP1 oligomers sensitivity and specificity against Candida species determined by fluorescence in situ hybridization assays. FIG. 2A summarizes the intensity of fluorescence obtained after 2 h of hybridization at 37° C. for different Candida species tested. FIG. 2B shows the fluorescence images of Candida species hybridization with anti-EFG1 and anti-HWP1 labelled with red fluorescein (56-FAM) and green fluorescein (TYE 563).

[0062] In an embodiment, FIG. 3 shows the cytotoxicity effects of 40 nM of Anti-EFG1, anti-HWP1 and anti-HYR1 oligomers against 3T3 cell line (Fibroblast cells, Embryonic tissue, Mouse from CCL3, American Type Culture Collection). These were measured using MTS kit (CellTiter 96® Aqueous One Solution Cell Proliferation Assay, Promega). The error bars represent standard deviation.

[0063] In an embodiment, FIG. 4 shows the effects of anti-EFG1, anti-HWP1 and anti-HYR1 oligomers on C. albicans filamentation. FIG. 4A shows the effect of 40 nM of each oligomer in terms of percentage of C. albicans filamentation inhibition. FIG. 4B shows the fluorescence images of C. albicans filamentation reduction when treated with the ASOs for 24 h. The control is related to C. albicans cultured in same conditions in absence of the oligomers. The error bars represent standard deviation. Statistical differences among the different time point tested (P<0.05) are marked with *.

[0064] In an embodiment, FIG. 5A shows the effect of 40 nM of anti-EFG1 on C. albicans filamentation ability after 24 h of incubation. The effect on EFG1 gene expression is measured by qRT-PCR (as shown in FIG. 5B) and on efg1p translation obtained by nanoLC-MS/MS analysis (as shown in FIG. 5C). The error bars represent standard deviation. Statistical differences between C. albicans treated with anti-EFG1 oligomer and untreated (P<0.05) are marked with *.

[0065] In an embodiment, FIG. 6 shows the results related with different combinations with 40 nM of each oligomer (anti-EFG1, anti-HWP1 and anti-HYR1). FIG. 6A shows the cytotoxicity effect against 3T3 cell line. This is measured using MTS kit. FIG. 6B shows the effect on C. albicans filamentation (% of inhibition). FIG. 6C shows the fluorescence images of C. albicans filamentation reduction at 8 h and 24 h of incubation. The error bars represent standard derivation. Statistical differences between C. albicans treated with mixed ASOs and untreated (P<0.05) are marked with *.

[0066] In an embodiment, FIG. 7 shows the performance of anti-EFG1 oligomer on human body fluids (AS-artificial saliva; AU-artificial urine and blood). FIG. 7A shows anti-EFG1 oligomer effect against C. albicans filamentation. FIG. 7B shows anti-EFG1 oligomer effect against EFG1 gene expression.

[0067] The above described embodiments are combinable.

[0068] The term “comprising” whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0069] The following claims further set out particular embodiments of the disclosure.

REFERENCES

[0070] Huang, G. (2012) Regulation of phenotypic transitions in the fungal pathogen Candida albicans. Virulence 3, 251-261 [0071] Silva, S. et al. (2017) Candida Species Biofilms' Antifungal Resistance. J. Fungi 3, 8 DeVos, S. L. and Miller, T. M. (2013) Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA. Neurotherapeutics DOI: 10.1007/s13311-013-0194-5 [0072] Potaczek, D. P. et al. (2016) Antisense molecules: A new class of drugs. J. Allergy Clin. Immunol. 137, 1334-1346 [0073] Ecker et al., (1995). OLigonucleotides inhibiting Candida germ tube formation. U.S. Ser. No. 00/569,141A