WO2015052630A1

WO2015052630A1 - Antisense oligonucleotides for prevention of atherosclerosis and cardiovascular infections

Info

Publication number: WO2015052630A1
Application number: PCT/IB2014/065084
Authority: WO
Inventors: Dan Ericson; Tomas KAČERGIUS
Original assignee: Uab Bioseka
Priority date: 2013-10-07
Filing date: 2014-10-06
Publication date: 2015-04-16
Also published as: LT6214B; LT2013109A

Abstract

The present invention provides isolated antisense oligonucleotides (ASOs) and method for reducing or inhibiting biofilm formation such as biofilm in human cardiovascular system and on the surfaces contacting with blood and/or blood serum in order to prevent atherosclerosis and cardiovascular infections. The present invention further provides compositions such as biopharmaceutical compositions comprising said ASOs.

Description

Antisense Oligonucleotides for Prevention of Atherosclerosis and Cardiovascular Infections

All patent and non-patent references cited in the present application, are thereby incorporated by reference in their entirety.

Technical Field

This invention is from the field of microbiology and is related to the bacterial biofilm

development in human cardiovascular system as well as on the surfaces contacting with blood and/or blood serum. The present invention is specifically related with the prevention and treatment of atherosclerosis and cardiovascular infections caused by the bacterial biofilm using antisense oligonucleotides and their biopharmaceutical compositions.

Background of the Invention

Cardiovascular diseases continue to be one of the major reasons of mortality worldwide (Mendis et al in Global Atlas on cardiovascular disease prevention and control (World Health

Organization, Geneva (2011), pg 8-14)). Spectrum of these diseases is very broad and involves such dangerous life-threatening conditions as coronary heart disease, myocardial infarction and stroke. Fundamental reason for that is atherosclerosis when arterial blood vessels are narrowed due to the formation of atheromatous plaques. In turn, the causes of atherogenesis are complex, including hyperlipidemia, hypercoagulability, oxidative stress, endothelial dysfunction, infection and inflammation which injuries the wall of blood vessels.

On the basis of known research studies, it can be highlighted that the infectious factor is very significant in the atherogenesis, especially taking into consideration the microorganisms which compose the normal human microbiota (Koren et al in Human oral, gut and plaque microbiota in patients with atherosclerosis (Proc Nat Acad Sci USA 108 [Suppl 1] (2011), pg 4592-4598)).

Streptococci comprising normal human microbiota are one of the infectious sources for human cardiovascular infections. Streptococcus mutans and Streptococcus sobrinus species are among them that usually present in human mouth. These streptococci are capable to form biofilm on the surface of oral tissues. Structural matrix of this biofilm consists of the water-insoluble polymer - glucan which is synthesized using glucose molecules from the hydrolyzed sucrose by several isoforms of glucosyltransferase (Gtf) enzyme, i.e. S. mutans GtfB and GtfC as well as S.

sobrinus Gtfl (Bowen and Koo in Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms (Caries Res 45 (2011), pg 69-86); Koo et al in The exopolysaccharide matrix: a virulence determinant of cariogenic biofilm (J Dent Res 92 (2013), pg 1065-1073)). S. mutans GtfB and GtfC are encoded by gtfB and gtfC genes for the synthesis of water-insoluble and partly water-soluble glucans, respectively, whereas S.

sobrinus Gtfl is encoded by gtfl gene for the production of water-insoluble glucan. The glucosyltransferases are localized not only on the bacterial cell wall but they are also secreted into the environment in a free form. Due to activity of these enzymes, S. mutans, S. sobrinus and other oral bacteria can adhere virtually to any type of natural and artificial surfaces - dental enamel, mucous membrane and other human tissues as well as plastic, glass, because the synthesized glucan is a very sticky material by its physical characteristics. Reasonably, the Gtf enzymes have become important targets for the development of various pharmaceutically active substances in order to inhibit the adhesion of S. mutans and S. sobrinus.

S. mutans and S. sobrinus constantly invade into the bloodstream and cardiovascular system through the injuries in oral tissues due to daily oral care practices, professional dental treatment and/or pathological conditions, thereby causing a significant bacteremia (Nakano et al in

Streptococcus mutans and cardiovascular diseases (J Dent Sci Rev 44 (2008), pg 29-37)).

Human blood contains abundantly glucose, which S. mutans and S. sobrinus bacteria and their secreted glucosyltransferases can use for the synthesis of glucans. The bacterial

glucosyltransferases can synthesize this sticky polymer adhesive to the blood vessel walls as well as becoming the adherence sites for platelets and their aggregation in cardiovascular system. The known research studies demonstrate a very high identification rate of S. mutans within human heart valve tissues and atheromatous plaques - 65% and 75% of cases, respectively (Nakano et al in Detection of cariogenic Streptococcus mutans in extirpated heart valve and atheromatous plaque specimens (J Clin Microbiol 44 (2006), pg 3313-3317); Nakano et al in Detection of oral bacteria in cardiovascular specimens (Oral Microbiol Immunol 24 (2009), pg 64-68)). S. sobrinus is also found in atheromatous plaques, however at less frequency. That proves a direct link between these oral streptococci and pathogenesis of atherosclerosis. Moreover, it can be also supported by the results from animal experiments, as reported by Kesavalu et al in Increased atherogenesis during Streptococcus mutans infection in ApoE-null mice (J Dent Res 91 (2012), pg 255-260). The implication of S. mutans and S. sobrinus to the development of atherosclerosis may be related to the enzymatic activity of their secreted glucosyltransferases, that is, the synthesis of glucans. The experimental studies show that S. mutans strains with defects in the gtfB and gtfC genes exhibit considerably lower capabilities to cause platelet aggregation in the blood as compared to normal S. mutans strains (Taniguchi et al in Defect of glucosyltransferases reduces platelet aggregation activity of Streptococcus mutans: Analysis of clinical strains isolated from oral cavities (Arch Oral Biol 55 (2010), pg 410-416)). Thus, inhibiting the production of streptococcal glucosyltransferases is practically important not only for the prophylaxis of dental caries but also for the prevention of cardiovascular diseases. Herein described invention provides a method how to inhibit and/or reduce S. mutans and S. sobrinus biofilm formation in tissues of human cardiovascular system as well as on the surfaces contacting with blood and/or blood serum. This method is based on the use of antisense oligonucleotides (ASOs) which inhibit selectively the expression of S. mutans gtfB, gtfC and S. sobrinus gtfl genes, thereby suppressing the synthesis of glucosyltransferases and glucans. The inhibition of glucan production prevents development of bacterial biofilm that in turn can be used for the prevention of atherosclerosis and cardiovascular infections (e.g., bacterial endocarditis) caused by S. mutans and S. sobrinus bacteria as well as for suppression and reduction of the atheromatous plaques' formation. Moreover, the ASOs and method of bacterial biofilm inhibition provided in this invention is used to treat various surfaces that are in contact with human tissues and blood and/or blood serum such as e.g., tubes, catheters, dialysis membranes, probes, prosthetic heart valves, prostheses, implants, etc., in order to achieve the antibiofilm effect against S. mutans and S. sobrinus present on these surfaces. Importantly, the bacteria attach firmly to these surfaces e.g., catheters, implants and prostheses, because of the produced glucan polymers, and form biofilms which limit the abilities of immune system to eliminate the bacteria. The bacterial biofilms formed on tubes, catheters, dialysis membranes, probes, prosthetic heart valves and bone or joint prostheses cause the inflammatory processes, and consequently rejection of the prostheses, thereby inducing particularly complicated and life-threatening pathological conditions. Accordingly, ASOs and method of this invention solve complexically the problems of prevention as well as treatment of atherosclerosis, cardiovascular and related infections.

Summary of the Invention As discussed herein above, there is thus a need to solve the problems associated with prevention and treatment of atherosclerosis, cardiovascular and related infections which are caused by the bacteria invading into blood circulation system, cardiovascular tissues and other objects that are in contact with tissues of cardiovascular system and blood and/or blood serum. The present invention provides substances and method that solve these problems, in particular, the development of atherosclerosis and infections of cardiovascular system caused by bacterial biofilms and glucans.

The main patented object of this invention is the substance - isolated antisense oligonucleotides (ASOs) corresponding to these features:

a) ASOs comprising of nucleotide (nt) sequences according to SEQ ID NO: 1, 12, 18;

b) ASOs comprising of nucleotide sequences, which are the fragments SEQ ID NO: 1, 12, 18; or c) ASOs having at least 85% sequence identity to SEQ ID NO: 1, 12, 18.

Also, object of this invention is the ASOs which nucleotide sequences differ from the indicated SEQ ID NO: 1 , 12, 18 by one, two, three or four nucleotides, and among them the ASO fragments that nucleotide sequences selected from the group consisting of: SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11 , 19, 20, 21, 22, 23.

The ASOs any of the indicated above forms can be conjugated with peptide facilitating penetration through the bacterial cell wall. Also, object of this invention is the ASOs any of the indicated above forms that are used for prevention, inhibition and reduction of the atheromatous plaques' formation in human cardiovascular tissues and/or blood, which component is serum. Also, object of this invention is the biopharmaceutical composition comprised of the ASOs any of the indicated above forms, and other parts for the biopharmaceutical and medical use.

Also, object of this invention is the biopharmaceutical composition comprised of the ASOs any of the indicated above forms, and other parts, among them adjuvants and/or carriers used in biopharmacy.

Also, object of this invention is the biopharmaceutical composition comprised of the ASOs any of the indicated above forms, and other parts, in which a cationic polymer is used as the carrier. Also, object of this invention is the biopharmaceutical composition comprised of the ASOs any of the indicated above forms, and other parts, used for the prevention and treatment of atherosclerosis.

Also, object of this invention is the biopharmaceutical composition comprised of the ASOs any of the indicated above forms, and other parts, used for the prevention and treatment of bacterial endocarditis.

Also, object of this invention is the biopharmaceutical composition comprised of the ASOs any of the indicated above forms, and other parts, used for the treatment of surfaces that are in contact with human tissues and blood and/or blood serum, e.g., tubes, catheters, dialysis membranes, probes, prosthetic heart valves, prostheses, implants, etc., in order to achieve the antibiofilm effect against S. mutans and S. sobrinus present on these surfaces.

Finally, this invention includes the method for control of S. mutans and S. sobrinus culture growth, that covers the use of ASOs for suppression of the ability of these bacteria to synthesize biofilm matrix composed of exopolysaccharides, administration of ASOs for inhibiting specifically and simultaneously the expression of S. mutans gtfB, gtfC and S. sobrinus gtfl mRNAs, thereby at the same time suppressing the ability of these bacteria to produce water- insoluble and partly water-soluble glucan polymers. Description of the Drawings

Fig. 1 shows the fragment of nucleotide sequence SEQ ID NO: 13, i.e. 5'- UCUGUUAAGAUUAAGCAAUGGUCUGCCAAGUACUUUAAUGGGACAAAUAUUUUA GGGCGCGGAGCAGGCUAUGUCUUAAAAGA-3', in the original output of Mfold program on predicting S. mutans gtfB mRNA secondary structure model with the delineated binding site. As seen in this model, the location contains bulge loop which consists of unpaired nucleotides (5'-UAAGCA-3'), pointing out that the selected ASO can be a strong competitor in the formation of heteroduplex. Symbols -- - - - - -" and "\" mean connection between appropriate nucleotides, and they are used for better representation of bulge and hairpin loops, respectively. The region of gtfB mRNA shown in fig. 1 begins from 3049 nt, and the delineated binding site begins from 3056 nt, that is, the same as in gtfB gene of S. mutans (see Example 1).

Fig. 2 shows a selection of the multiple sequence alignment of the glucosyltransferase genes between various Streptococcus species and strains produced employing the MAFFT online server at the Max Planck Institute for Development Biology. The conserved target region (consisting of 26 nucleotides:

5'- GTTAAGATTAAGCAATGGTCTGCCAA-3 ' with SEQ ID NO: 15) is delineated in the large rectangular, and the mismatches are delineated in the small rectangles. The indicated Streptococcus species and strains:

ENAIAAN58705IAAN5870_0/1-4431 - STREPTOCOCCUS MUTANS UA159

GLUCOSYLTRANSFERASE-I;

ENAIAAN58706IAAN5870_l/l-4368 - STREPTOCOCCUS MUTANS UA159

GLUCOSYLTRANSFERASE-SI;

ENAID88654ID88654.1_2/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PYT216;

ENAID88660ID88660.1_3/l-5684 - STREPTOCOCCUS MUTANS GENE FOR GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PYT223;

ENAID89977ID89977.1_4/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PTHl;

ENAID88657ID88657.1_5/l-5686 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PYT239;

ENAIM17361IM17361.1_6/1-10029 - STREPTOCOCCUS MUTANS

GLUCOSYLTRANSFERASE (GTFB AND GTFC) GENES, COMPLETE CDS;

ENAID88651ID88651.1_7/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PSK6;

ENAIAB299801IAB29980_8/1-4410 - STREPTOCOCCUS CRICETI GTFI GENE FOR GLUCOSYLTRANSFERASE, COMPLETE CDS;

ENAIAB273728IAB27372_9/l-4930 - STREPTOCOCCUS CRICETI GTFI GENE FOR GLUCOSYLTRANSFERASE, COMPLETE CDS, STRAIN: GTC242;

ENAIAB355819IAB35581_10/l-4602 - STREPTOCOCCUS DENTIROUSETTI GTFI GENE FOR GLUCOSYLTRANSFERASE-I, COMPLETE CDS; ENAIAB275384IAB27538_11/1- 5423 - STREPTOCOCCUS DENTISUIS GTFI GENE FOR GLUCOSYLTRANSFERASE, COMPLETE CDS;

ENAIAB272987IAB27298_12/l-4984 - STREPTOCOCCUS ORISUIS GTF GENE FOR GLUCOSYLTRANSFERASE, COMPLETE CDS;

ENAID63570ID63570.1_13/l-6838 - STREPTOCOCCUS SOBRINUS GENE FOR

GLUCOSYLTRANSFERASE GTF-I, COMPLETE CDS;

ENAIM17391IM17391.1_14/l-4995 - STREPTOCOCCUS DOWNEI

GLUCOSYLTRANSFERASE PRECURSOR, GENE, COMPLETE CDS. Fig. 3 shows a selection of the multiple sequence alignment of the glucosyltransferase genes between various Streptococcus species and strains, which are found in human body, produced employing the MAFFT online server at the Max Planck Institute for Development Biology. The conserved target region (consisting of 26 nucleotides: 5'-

CGCGTCATGTTTGAAGGTTTCTCTAA-3 ' with SEQ ID NO: 17) is delineated in the large rectangular. The indicated Streptococcus species and strains: EN AIAAN587051 A AN5870_0/ 1-4431 - STREPTOCOCCUS MUTANS UA159 GLUCOSYLTRANSFERASE-I;

ENAIAAN58706IAAN5870_l/l-4368 - STREPTOCOCCUS MUTANS UA159

GLUCOSYLTRANSFERASE-SI;

ENAID88654ID88654.1_2/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PYT216;

ENAID88660ID88660.1_3/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PYT223;

ENAID89977ID89977.1_4/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PTHl ;

ENAID88657ID88657.1_5/l-5686 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PYT239;

ENAIM17361 IM17361.1_6/1-10029 - STREPTOCOCCUS MUTANS

GLUCOSYLTRANSFERASE (GTFB AND GTFC) GENES, COMPLETE CDS;

ENAID88651 ID88651.1_7/l-5684 - STREPTOCOCCUS MUTANS GENE FOR

GLUCOSYLTRANSFERASE-I, COMPLETE CDS, CLONE:PSK6;

ENAID63570ID63570.1_13/l-6838 - STREPTOCOCCUS SOBRINUS GENE FOR

GLUCOSYLTRANSFERASE GTF-I, COMPLETE CDS. Fig. 4 shows optical profile of glass slides with the mixed S. mutans and S. sobrinus culture biofilm after 24 h of incubation under different treatments in Todd Hewitt broth containing 10% serum and 1% sucrose. Fig. 4a - biofilm without treatment with ASOs; fig.4b - biofilm under treatment with ASOl + TF (TurboFect ); fig.4c - biofilm under treatment with AS02 + TF

(TurboFect ). Magnification, x50.

Fig. 5 shows quantities of the mixed S. mutans and S. sobrinus culture biofilm formed on the glass slide surface after 24 h of incubation under different treatments in Todd Hewitt (TH) broth containing 10% serum and 1 % sucrose. fig.5a - biofilm surface's roughness parameter Rq; fig.5b - biofilm thickness. Control - glass slide surface after 24 h of incubation in TH broth containing 10% serum and 1% sucrose, but without bacteria; untreated bacteria (black bar in Fig.5a,b) - that are bacteria after 24 h of incubation in TH broth containing 10% serum, but without sucrose and treatments with ASOs; untreated bacteria (white bar in A and B) - that are bacteria after 24 h of incubation in TH broth containing 10% serum and 1% sucrose, but without treatments with ASOs. Data (n = 6 for biofilm surface's roughness parameter Rq; n = 5 for biofilm thickness) are means ± standard error of the mean. *p < 0.05 compared to the untreated bacteria (white bar), **p < 0.05 compared to AS02 + TF (TurboFect ).

Definitions

As used herein the term "antisense effect" means the oligonucleotide's effect which is produced after its binding to the complementary sequence within mRNA, resulting in specific inhibition of the target gene and protein expression.

As used herein antisense oligonucleotides (ASOs) mean agents that are unmodified or chemically modified single-stranded nucleic acid molecules (usually 15-30 nt in length), which can selectively hybridize to their target complementary sequence within mRNA through Watson- Crick base pairing. Formation of an ASO -mRNA heteroduplexes induces the effects as follows: 1) activates RNase H endonuclease or as in bacteria endoribonucleases - RNase III and RNase E - leading to degradation of the bound mRNA, and leaving the ASO intact; 2) causes translational arrest by steric hindrance of ribosomal activity; 3) inhibits mRNA splicing; 4) destabilizes pre- mRNA. Indeed, what effect will occur depends on the ASO chemical composition and location of hybridization, but the subsequent result is specific down-regulation of the target gene and protein expression.

"Nucleotide sequence ", "nucleic acid", "nucleic acid chain " and "nucleic acid sequence " means anything that binds or hybridizes using base pairing including oligomers or polymers having a backbone formed from naturally occurring nucleotides such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), and/or nucleic acid analogs comprising nonstandard

nucleobases and/or nonstandard backbones, e.g., a peptide nucleic acid (PNA) or locked nucleic acid (LNA), or any derivatized or modified form of a nucleic acid, including modifications to increase stability, binding effectiveness and resistance to nucleases. As used herein, the term "peptide nucleic acid" or "PNA" means a synthetic oligomer or polymer having a polyamide backbone with pendant nucleobases (naturally occurring and modified), including, but not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acids in, e.g., U.S. Pat. nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,

5,986,053, 6,107,470 6,201, 103, 6,228,982 and 6,357,163, WO96/04000, all of which are herein incorporated by reference or any of the references cited therein. The pendant nucleobase, such as, e.g., a purine or pyrimidine base on PNA may be connected to the backbone via a linker such as, e.g., one of the linkers taught in PCT/US02/30573 or any of the references cited therein. In one embodiment, the PNA has an N-(2-aminoethyl)-glycine) backbone. PNAs may be synthesized (and optionally labeled) as taught in PCT/US02/30573 or any of the references cited therein. PNAs hybridize tightly, and with high sequence specificity, with DNA and RNA, because the PNA backbone is uncharged. Thus, short PNA probes may exhibit comparable specificity to longer DNA or RNA probes. PNA probes may also show greater specificity in binding to complementary DNA or RNA.

As used herein, the term "locked nucleic acid" or "LNA" means an oligomer or polymer comprising at least one or more LNA subunits. As used herein, the term "LNA subunit" means a ribonucleotide containing a methylene bridge that connects the 2'-oxygen of the ribose with the 4'-carbon. See generally, Kurreck in Antisense technologies. Improvement through novel chemical modifications (Eur J Biochem 270 (2003), pg 1628-1644). Examples of nucleic acids and nucleic acid analogs for the embodiments herein to be used as ASO in isolated forms also include oligomers and polymers of nucleotide monomers, including double and single stranded deoxyribonucleotides (DNA), ribonucleotides (RNA) including naturally occurring antisense RNA molecules such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) found in eukaryotic cells as well as small antisense RNAs found in prokaryotic cells (bacteria), a-anomeric forms thereof, natural and synthetic analogs thereof, and the like. The nucleic acid chain may be composed entirely of deoxyribonucleotides, ribonucleotides, peptide nucleic acids (PNA), locked nucleic acids (LNA), natural or synthetic analogs thereof such as phosphorodiamidate morpholino and thiophosphoroamidate

oligonucleotides, or mixtures thereof. DNA, RNA, or other natural or synthetic nucleic acids as defined herein can be used in the methods and compositions of the invention.

By "carrier" or " adjuvant" is herein intended to mean a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Further, adjuvants and carriers in compositions herein are suitable for delivery to tissues of cardiovascular system and blood, which component is serum, without causing any undesirable biological effects or interacting in a deleterious manner. Examples of suitable carriers and adjuvants are given herein and include nuclease-free water, or any reagent which forms compact, stable, positively charged complex with oligonucleotide in order to protect from degradation and facilitate better penetration of oligonucleotide to the bacterial cells, such as TurboFect transfection reagent commercially available (Thermo Fisher Scientific, Fermentas).

As used herein the term "nuclease-free water" means sterile deionized water which is absent from any type of nucleases capable to degrade oligonucleotide.

As used herein the term "biofilm " means biofilm consisting of the polysaccharide matrix composed mostly of water-insoluble and partly water-soluble glucans as well as bacteria capable to synthesize polysaccharide polymers using glucose molecules from the hydrolyzed sucrose, thereby composing exopolysaccharide matrix.

Detailed description of the Invention

As said and used herein, bacterial cultures are both in vivo and in vitro cultures, if not specified separately what particular type of culture is intended. Said cultures include cultures in suspension and cultures on solid phases, such as glass (example of an in vitro solid phase) or blood vessel wall (example of an in vivo solid phase) or any other solid phase surface in the cardiovascular system, or implant or other surface contacting with human tissues and blood and/or blood serum.

As revealed above, the present invention discloses a novel technique for decreasing, preventing or inhibiting biofilm formation, such as biofilm formation from oral streptococci, particularly S. mutans and S. sobrinus biofilm formation, all on solid phase surfaces, e.g., glass and blood vessel walls.

This technique employs administration of the effective dose of antisense oligonucleotides (ASOs) to the bacterial culture in order to target and suppress simultaneously the expression of glucosyltransferase mRNAs in several species of streptococci, particularly in S. mutans and S. sobrinus, such as, e.g., gtfB and gtfC glucosyltransferase mRNAs in S. mutans and gtfl mRNA in

S. sobrinus, leading to inhibition of both water-insoluble and partly water-soluble glucan polymers' production and bacterial cell adherence in S. mutans and S. sobrinus cultures thus leading to a decrease, inhibition or prevention of biofilm formation. Said cultures may be in vitro cultures or in vivo cultures, e.g., in the cardiovascular system or on other solid phase surfaces contacting with human tissues and blood and/or blood serum.

In this invention, list of the patented new ASO sequences is provided in Sequence list.

Said the new ASOs may be chemically synthesized phosphorothioate-modified

oligodeoxyribonucleotides of the sequence corresponding to the nucleotide sequences of SEQ ID NO: 1, 12, 18 for exhibiting antisense effect, as in the Examples, or any of the embodiments of an ASO as described herein, i.e. SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23, or fragments or modifications of the ASOs according to the conditions indicated in specification of this invention.

The new ASO sequences provided in this invention may be synthesized and modified in different ways. Fully phosphorothioate-modified oligodeoxyribonucleotides as shown in Example 2 may be used as ASO in different embodiments. Such phosphorothioate-modified

oligodeoxyribonucleotides differ from unmodified oligodeoxyribonucleotides in that one of the non-bridging oxygen atoms in the phosphodiester linkage is replaced by a sulfur atom in order to increase resistance to endo- and exonucleases. The phosphorothioate-modified

oligodeoxyribonucleotides may be chemically synthesized f.ex. at the Metabion International AG (Germany) using an automated DNA synthesizer and phosphoramidite method under the standard protocols known in the art, e.g., in G. Zon, Oligonucleoside Phosphorothioates, Ed., S. Agrawal, Protocols for Oligonucleotides and Analogs: Synthesis and Properties (Methods Mol Biol 20 (1993), pg 165-189, Humana Press Inc., Totowa, NJ); Guzaev, Reactivity of 3H-1 ,2,4- diathiazole-3-thiones and 3H-l,2-dithiole-3-thiones as sulfurizing agents for oligonucleotide synthesis (Tetrahedron Lett 52 (2011), pg 434-437); Sanghvi, A status update of modified oligonucleotides for chemotherapeutics applications (Curr Protoc Nucleic Acid Chem Suppl. 46 (2011), Unit 4.1.1-4.1.22).

Measuring glucans, composing exopolysaccharide matrix, may be done as described in Example 2 where profilometry results clearly show that bacterial adherence to glass surfaces is significantly reduced by the ASO, which nucleotide sequence corresponds to SEQ ID NO: 1. The decrease in biofilm formation indirectly indicates a reduction of glucan polymers' synthesis since glucans are essential for biofilm formation and no glucans leads to no biofilm formation.

Another aspect of the novelty of this invention provides means to use only one type of nucleotide sequence (one nucleotide sequence) of the ASOs in an effective amount for inhibiting the synthesis of both water-insoluble and partly water-soluble glucans in S. mutans encoded by gtfB and gtfC genes, and in S. sobrinus encoded by gtfl gene.

This novel technique allows administration of ASO alone to the bacterial culture at effective concentration in order to cover two targets - gtfB and gtfC mRNAs in S. mutans, and at the same time S. sobrinus gtfl mRNA.

These target sequences in S. mutans gtfB, gtfC and S. sobrinus gtfl genes and their corresponding mRNAs are well conserved, that is, coding nucleotide sequences are invariant in the genes of these bacteria. Therefore, use of the ASOs decreases the ability of bacteria to use alternate routes for synthesis of water-insoluble and partly water-soluble glucans that to build up the polymeric matrix of biofilm. The conserved consensus targets are shown and based in fig 2 and fig 3. The shorter sequences (SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23) have the same arrangement of nucleotides (nt) as in the ASOs with SEQ ID NO: 1, 12, 18, and therefore they may be independently used to the determined target regions within gtfB and gtfC of S. mutans as well as gtfl of S. sobrinus in terms of the genes and mRNAs. On the basis of thermodynamical features (the same as ASOs with SEQ ID NO: 1, 12, 18), these shorter fragments can also form heteroduplex with the determined region.

Importantly, the ASO with SEQ ID NO: 1 binds to unpaired nucleotides of the bulge loop in the gtfB mRNA of S. mutans as it is delineated in fig 1. Similarly, the ASO of longer sequence (SEQ ID NO: 13) can form heteroduplex with the determined region, and it is complementary and cover the bulge loop with unpaired nucleotides in the gtfB mRNA of S. mutans as it is delineated in fig 1.

Numerous investigations in vitro and in vivo conditions presented in the literature demonstrate that the ASOs as short as 8 nt or as long as 30 nt of the same nucleotide arrangement exhibit the antisense effect, and even ASOs with one mismatched nucleotide also provide a substantial antisense effect (see references: Wagner et al in Potent and selective inhibition of gene expression by an antisense heptanucleotide (Nat Biotechnol 14 (1996), pg 840-844); Fakler et al in Short antisense oligonucleotide-mediated inhibition is strongly dependent on oligo length and concentration but almost independent of location of the target sequence (J Biol Chem 269

(1994), pg 16187-16194); Woolf et al in Specificity of antisense oligonucleotides in vivo (Proc Natl Acad Sci USA 89 (1992), pg 7305-7309)).

Carriers of ASO

The ASOs herein may be delivered to S. mutans and S. sobrinus bacteria more effectively using carriers. Said carriers may be transfection reagents or cationic polymers which are known in the art. The combination of all the embodiments of ASO with carrier or transfection reagent composed of cationic polymer such as TurboFect (Thermo Fisher Scientific, Fermentas) may be used in order to enhance the ASO uptake by bacteria and to effectively improve the inhibition of biofilm formation in S. mutans and S. sobrinus cultures. Other carriers to be used for all the embodiments of ASO as described herein for delivery to eukaryotic and prokaryotic cells represent various chemical substances that can form complexes with the "naked" oligonucleotides in order to protect them from degradation and to improve their penetration into the cells. These substances include, but not limited to, calcium salts (e.g., calcium phosphate, calcium chloride), cationic lipids (e.g., N-(2,3-dioleyloxypropyl) N, N, N- trimethylammonium chloride, dioleoylphosphatidylethanolamine, cholesterol), cationic polymers (e.g., diethylaminoethyl dextran, polyethyleneimine, chitosan, cyclodextrin, polyamidoamine dendrimer, polypropylenimine dendrimer), cell penetrating peptides (peptides usually less than 30 amino acids; e.g., penetratin) and different types of nanoparticles as described by D. Liu et al in Chemical Methods for DNA Delivery, Ed., W. C. Heiser, Volume 1 of Gene Delivery to Mammalian Cells (Methods Mol Biol 245 (2004), pg 3-23, Humana Press Inc., Totowa, NJ); Zhu and Mahato in Lipid and polymeric carrier-mediated nucleic acid delivery (Expert Opin Drug Deliv 7 (2010), pg 1209-1226); Bai et al in Antisense antibiotics: a brief review of novel target discovery and delivery (Curr Drug Discov Technol 7 (2010), pg 76-85); Aalinkeel et al in Quantum rods as nanocarriers of gene therapy (Drug Deliv 19 (2012), pg 220-231). All of these transfection reagents, with the exception of calcium phosphate which forms calcium-phosphate - DNA precipitates, function in a similar fashion - they form complexes with oligonucleotides via electrostatic interaction between negatively charged oligonucleotide molecules and positively charged reagent molecules. Since such complexes maintain positive charge, therefore they can bind to the negatively charged eukaryotic cell plasma membrane or bacterial cell wall, and can be taken up by the cells.

The most widely used transfection reagents for the delivery of ASOs to bacterial cells are cationic polymers and cell penetrating peptides, which is usable for all the embodiments of the ASO herein as well. It is important to note that cell penetrating peptides are capable to enter into the cells not just through the electrostatic interaction but also by forming transient pores in the cellular plasma membrane. Guo et al in Treatment of Streptococcus mutans with antisense oligodeoxyribonucleotides to gtfB mRNA inhibits GtfB expression and function (FEMS Microbial Lett 264 (2006), pg 8-14) describes the efficient delivery of phosphorothioate-modified ASOs to

S. mutans bacteria using a cationic polymer - So-Fast (Taiyangma). In the underlying experiments for this invention, it was used the known and commercially available transfection reagent - TurboFect (Thermo Fisher Scientific, Fermentas). By combining with this cationic polymer, the ASO of SEQ ID NO: 1 was delivered to S. mutans and S. sobrinus bacteria, thereby demonstrating experimentally its efficacy.

The Use

The present invention in all its embodiments provides means and method for inhibition of S. mutans and S. sobrinus biofilm formation on solid phase surfaces. In this invention, the indicated ASOs and method enable administration of ASOs with SEQ ID NO: 1, 12, 18 or fragments thereof according to SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23, to the bacterial culture using an effective concentration in order to decrease S. mutans and S. sobrinus adhesion and biofilm formation as clearly demonstrated with the mixed S. mutans and S. sobrinus culture in fig 4 and fig 5.

The invention allows to use the ASOs of SEQ ID NO: 1, 12, 18, or ASO fragments, or ASO modifications as described herein for antisense effect as well as for prevention and control of atherosclerosis and cardiovascular infections.

As it is clearly seen in Example 2 and fig 4, fig 5 an effective concentration of the ASO decreases S. mutans and S. sobrinus adhesion and biofilm formation. In Example 2 and fig 4, fig 5, it is specifically shown the effect of effective ASO concentration in the medium with blood serum, thereby demonstrating the potential effect of ASO to prevent the formation of atheromatous plaques in blood and/or blood serum as well as blood vessels of cardiovascular system.

In all aspects pointed out herein, the present invention provides a novel method to control and prevent the development of bacterial biofilms in the tissues of cardiovascular system as well as blood and/or blood serum using ASOs of the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs, thereby achieving antisense effect to the glucosyltransferases of S. mutans and S. sobrinus. This novel method renders a tool for the inhibition of biofilm formation on solid phase surfaces, including blood vessel walls, implants, also on other artificial surfaces contacting with human tissues and blood and/or blood serum. The invented new ASOs of the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs can be used as parts of biopharmaceutical compositions (solutions, suspensions, etc.), or can be included in the formulations of biopharmaceutical compositions, providing local as well as systemic effects for living organism - human, or non-living surfaces contacting with tissues as well as blood and/or blood serum of the living organism.

The disclosed in this invention new ASOs of the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs are capable to bind simultaneously with the complementary sequences in S. mutans genome encoding gtffi and gtfC mRNAs as well as in S. sobrinus genome encoding gtfl mRNA, thereby inhibiting glucan production, reducing biofilm formation and in this way preventing the development of atheromatous plaques and

cardiovascular infections.

Moreover, in this invention, the disclosed new ASOs of the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs prevent the bacterial adhesion and attachment to solid phase surfaces. This aspect provides possibility to apply the presented herein ASOs for the treatment of atherosclerosis and cardiovascular infections.

Purpose of the present invention is to provide method and means for the inhibition of biofilm formation, particularly in the cardiovascular system containing blood and/or blood serum. The purpose is achieved using the ASOs described herein that suppress production of glucans in S. mutans and S. sobrinus, targeting simultaneously to the mechanisms of glucan synthesis in both bacterial species with one type molecule of the ASO.

The statement "one type molecule of antisense oligonucleotide " is used to emphasize that the indicated antisense effect in the above paragraph is achieved with one or two molecules, which oligonucleotide sequence is disclosed for the first time in this invention. The ASOs are provided in this invention as one or several sequences of molecules given below herein, i.e. ASOs which sequences are the SEQ ID NO: 1, 12, 18, also their fragments and modifications (e.g., which sequences are one of the SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23), in the amount that is effective and sufficient according to the medium and specific composition.

Effect of the new ASOs developed in present invention that have sequences of the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs (e.g., which sequences are one of the SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23) was simulated in silico for S. mutans and S. sobrinus bacteria, the effect of ASO with SEQ ID: 1 in vitro corresponded to the projected effect in silico (see Example 2 and fig 4, fig 5). Said ASO sequences inhibit the ability of S. mutans and S. sobrinus bacteria to synthesize glucans for biofilm formation. The bacteria not present in biofilm are easier recognized and eliminated in the natural way by human immune system.

Accordingly, this invention provides method and means for inhibiting and reducing bacterial biofilm formation, it also prevents the development of biofilm on solid phase surfaces in vitro and in vivo. If in vivo the solid phase surface is a part of human tissues, for instance, wall of blood vessel, this method comprises of the administration of effective ASO dose to human blood, which component is serum, as described herein, i.e. ASOs which sequences are the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs (e.g., which sequences are one of the SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23), as the structural parts of biopharmaceutical composition are used for prevention and treatment of the cardiovascular pathologies associated with atherosclerosis and/or infections caused by S. mutans and S. sobrinus due to formation of the bacterial biofilms.

Technology The antisense technology offers several advantages in comparison with other antibacterial technologies, and among them the main advantages are as follows: 1) specific and simultaneous suppression of the expressed gtf mRNAs in S. mutans and S. sobrinus bacteria;

2) usage of the ASO sequences disclosed in this invention for analogous targets in two streptococci species (S. mutans and S. sobrinus);

3) external use in order to inhibit the formation of S. mutans and S. sobrinus biofilms on the artificial non-living surfaces such as catheters, prostheses or implants, and that to reduce maximally the risks of infection or rejection as well as to decrease systemic side effects.

The present invention discloses the method of ASO use and ASOs, which sequences are the SEQ ID NO: 1, 12, 18, or any of the indicated herein fragments or modifications of the ASOs (e.g., which sequences are one of the SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23), to suppress biofilm formation, and thereby the development of diseases and pathologies caused by the biofilm formation, e.g., atherosclerosis, bacterial endocarditis and inflammatory reactions to implants or prostheses, their rejection reactions, etc.

Glucans, comprising the exopolysaccharides of S. mutans and S. sobrinus biofilms, are composed of repeating glucose units, and they are synthesized by the enzymatic action of glucosyltransferases. Glucans can be water-insoluble, water-soluble and partly water-soluble. The water-insoluble and partly water-soluble glucans serve as a polysaccharide matrix for the biofilm with several functions: 1) enhance bacterial adherence and further accumulation on solid phase surfaces; 2) provide structural integrity and bulk to the biofilm; 3) increase adhesive properties of the biofilm matrix.

The separate purpose and novelty of the present invention is to inhibit simultaneously and parallelly the expression of glucosyltransferase genes and mRNAs in S. mutans and S. sobrinus bacteria.

Inhibitory effects on the expression of glucosyltransferase genes and mRNAs as well as on the synthesis of water-insoluble and partly water-soluble glucans, and consequently on the biofilm formation, is achieved using ASOs of these sequences: SEQ ID NO: 1 (5'-GCAGACCATTGCTTAATCT-3'), which effect is the inhibition of water- insoluble and partly water-soluble glucans' synthesis, and consequently the suppression of biofilm formation. SEQ ID NO: 12 (5'-TTGGCAGACCATTGCTTAATCTTAAC-3') and/or

SEQ ID NO: 18 (5'-TTAGAGAAACCTTCAAACATGACGCG-3), which effect is analogous to SEQ ID NO: 1.

Also, the analogous effect as indicated above is achieved using fragments of the ASO sequences and modified sequences (e.g., which sequence is one of the SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 21, 22, 23).

The ASO can be of specific sequence, as the indicated SEQ ID NO: 1, 12, 18, or parts or fragments of these sequences. The parts or fragments thereof are oligonucleotides shortened in length by at least one nucleotide either from 5'-end or 3'-end. Thus, it can be the ASOs, which sequences are part and fragment of the SEQ ID NO: 1, 12, 18, selected from this list:

5'- -C AGACCATTGCTTAATCT-3 ' (SEQ ID NO: 3)

5'- -AGACCATTGCTTAATCT-3' (SEQ ID NO: 4)

5'- - G ACC ATTGCTT A ATCT-3 ' (SEQ ID NO: 5)

5'- -GCAGACCATTGCTTAATC-3' (SEQ ID NO: 6)

5'- -GCAGACCATTGCTTAAT-3' (SEQ ID NO: 7)

5'- -GCAGACCATTGCTTAA-3' (SEQ ID NO: 8)

5'- -CAGACCATTGCTTAATC-3' (SEQ ID NO: 9)

5'- -AGACCATTGCTTAATC-3' (SEQ ID NO: : 10)

5'- -C AGACCATTGCTTAAT-3 ' (SEQ ID NO: 11)

5'- -GAAACCTTCAAACATGACGC-3 ' (SEQ ID NO: 19)

5'- -AAACCTTCAAACATGACGC-3 ' (SEQ ID NO: 20)

5'- -GAAACCTTCAAACATGACGCG-3 ' (SEQ ID NO: 21)

5'- -GAAACCTTCAAACATGACG-3 ' (SEQ ID NO: 22)

5'- -AGAAACCTTCAAACATGACGC-3 ' (SEQ ID NO: 23) The ASO with SEQ ID NO: 1 is derived from SEQ ID NO: 12, which is 100% complementary to the region of 26 nt in S. mutans gtfB and gtfC genes, and also with one mismatched nucleotide in S. sobrinus gtfl gene (see Example 1). The ASO with SEQ ID NO: 1 is the optimized molecule of 19 nucleotides on the basis of thermodynamic peculiarities needed to inhibit S. mutans gtfB, gtfC and S. sobrinus gtfl mRNAs.

The ASO with SEQ ID NO: 1 is derived from the SEQ ID NO: 12 and it is composed of 19 nt: 5'-TTG-GCAGACCATTGCTTAATCT-TAAC-3' (underlined and indicated in Example 1).

The ASO with SEQ ID NO: 2 is derived from the SEQ ID NO: 1 and it is used as a negative control in the provided experiments (see Example 2).

The ASOs with SEQ ID NO: 3-11 are derived from SEQ ID NO: 1.

The ASO with SEQ ID NO: 12 is isolated synthetic ASO composed of 26 nt:

5'-TTGGCAGACCATTGCTTAATCTTAAC-3', which is used to achieve the antisense effect for the natural mRNA sequence with SEQ ID NO: 13 as well as for the natural DNA sequence with SEQ ID NO: 15.

The ASO with SEQ ID NO: 18 is isolated synthetic ASO composed of 26 nt:

5 '-TTAGAGAAACCTTCAAACATGACGCG-3 ', which is used to achieve the antisense effect for the natural DNA sequence with SEQ ID NO: 17. The ASOs with SEQ ID NO: 19-23 are derived from SEQ ID NO: 18.

Consequently, any fragments of the ASOs, even shorter than 26 nt, with the sequences corresponding to SEQ ID NO: 1 or SEQ ID NO: 12, or SEQ ID NO: 18, which also cover and fit in the delineated herein coding regions of the glucosyltransferases, can provide antisense effect and fall in the delineated by this invention ASO forms (and brought for the patenting) that effect corresponds to the named method and means in the present invention. Said isolated ASO herein may be isolated from a natural or native source as in a purified restriction digest, produced synthetically e.g., chemically synthesized or produced by

recombinant or genetic engineering or polymerization and amplification techniques such as e.g., polymerase chain reaction (PCR) and PCR amplification or in any other artificial way. The ASO may also be prepared by a process known in the art or any other method described herein in accordance with the present invention.

The ASOs of the present invention as described herein may be any of molecules with

corresponding nucleotide sequences that bind or hybridize using base pairing including oligomers or polymers having a backbone formed from naturally occurring nucleotides and/or nucleic acid analogs comprising nonstandard nucleobases and/or nonstandard backbones e.g., a peptide nucleic acid (PNA) or locked nucleic acid (LNA), or any derivatized form of a nucleic acid, as exemplified herein. The ASOs may also contain partial, such as 10, 20, 30, 40, 50, 60, 70, 80 or even 90% naturally occurring nucleotides and the rest, thus e.g., 90, 80, 70, 60, 50, 40, 30, 20, or even 10%, being nucleic acid analogues comprising non-standard nucleobases and/or nonstandard backbones as exemplified herein by PNA, LNA or any derivatized form of nucleic acid. The ASOs may further be chemically modified. Such modifications may be

phosphorothioate-modified oligodeoxyribonucleotides as exemplified herein. All of the sequences given herein may be chemically synthesized phosphorothioate-modified

oligodeoxyribonucleotides.

Other forms of the ASO may be made of nucleotides wherein at least one of the nucleotides has a base modified to enhance binding properties and/or to decrease the action of endo- and exonucleases including 5' to 3' and 3' to 5' DNA pol 1 exonuclease, nucleases S I and PI, RNases, serum nucleases and snake venom phosphodiesterase.

Examples Example 1 - Determination of the Antisense Oligonucleotide Sequences for

Glucosyltransferases Bioinformatical Methods and Results

In order to select the new ASO sequences disclosed in the present invention, at first the conserved homologous regions among the gtf genes of oral streptococci were identified. For this purpose, it was primarily conducted a comparative analysis of amino acid sequences between S. mutans GtfB protein and other Gtf proteins present in oral streptococci. S. mutans (strain UA159, Bratthall serotype c, ATCC no. 700610) genome is completely sequenced, and the sequences are deposited in GenBank database at the National Center for Biotechnology Information (NCBI). Therefore, using S. mutans (strain UA159) GtfB protein as a query sequence, the BLAST (Basic Local Alignment Search Tool) search of homologs among all known proteins have been performed (BLAST service is available at http://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed in March, 2013). In consequence, it was revealed that S. mutans (strain UA159) GtfB protein features high identity to the GtfC protein from S. mutans (strain UA159) as well as other Gtf (i.e. Gtfl) proteins from S. criceti, S. dentirousetti, S. dentisuis, S. downei and S. sobrinus responsible for the generation of water-insoluble glucans. Eventually, sequences of these proteins were collected and multiple sequence alignment produced employing MAFFT online server at the Max-Planck Institute for Development Biology (http://toolkit.tuebingen.mpg.de/mafft; accessed in March, 2013). Applying such approach, it was determined a region of conserved amino acid sequence within the GtfB protein of S. mutans (strain UA159) that possesses 100% homology to the appropriate fragments in S. mutans (strain UA159) GtfC, S. criceti Gtfl, S. dentirousetti Gtfl, S. dentisuis Gtfl and S. orisuis Gtf proteins. The respective coding segment for this region in S. mutans (strain UA159) gtfB gene contains the following sequence of 26 nt: 5'- GTTAAGATTAAGCAATGGTCTGCCAA-3 ' (SEQ ID NO: 15). As in the case of comparative amino acid sequence analysis, this segment of S. mutans (strain UA159) gtfB gene has also 100% homologous fragments in S. mutans (strain UA159) gtfC, S. criceti gtfl, S. dentirousetti gtfl, S. dentisuis gtfl and S. orisuis gtf genes. Additionally, it was also found a highly similar sequence with only two mismatched nucleotides in S. downei gtf precursor and S. sobrinus gtfl genes. On the basis of best priming analysis (Primer3 service was used at http://primer3.ut.ee in order to analyze thermodynamics and other peculiarities of the heteroduplex forming; accessed in March, 2013), a complementary sequence of 19 nt was picked out for the above indicated coding region:

5 '-AGATTAAGCAATGGTCTGC-3 ' (sequence of the coding region) [SEQ ID NO: 16] 3 ' -TCTAATTCGTTACCAGACG-5 ' (complementary sequence) [SEQ ID NO : 1 ]

In the experimental work performed on basis of these bioinformatical results, it was used antisense oligonucleotide of the following sequence:

5'-GCAGACCATTGCTTAATCT-3 ' [SEQ ID NO: 1]

The exact locations of this segment in the gtf genes of S. mutans (strain UA159) and S. sobrinus (strain SLl) along with the data linked to the GenBank of NCBI are presented below:

1. In S. mutans (strain UA159) gtfB (herein indicated as gtfl) gene:

GenBank accession no. gb | AEO 14133.1 |

Streptococcus mutans UA159, complete genome

Length=2030921

Features in this part of subject sequence:

glucosyltransferase-I

Query 1 GCAGACCATTGCTTAATCT 19 [SEQ ID NO: 1]

I I I I I I I I I I I I I I I I I I I

Sbjct 954167 GCAGACCATTGCTTAATCT 954149 [SEQ ID NO: 1]

2. In S. mutans (strain UA159) gtfC (herein indicated as gtfSI) gene:

GenBank accession no. gb | AEO 14133.1 |

Streptococcus mutans UA159, complete genome

Length=2030921

Features in this part of subject sequence:

glucosyltransferase-SI

Query 1 GCAGACCATTGCTTAATCT 19 [SEQ ID NO: 1]

I I I I I I I I I I I I I I I I I I I

Sbjct 958871 GCAGACCATTGCTTAATCT 958853 [SEQ ID NO: 1]

3. In S. sobrinus (strain SLl) gtfl gene:

GenBank accession no. dbj | D63570.1 |

Streptococcus sobrinus gene for glucosyltrans feras e GTF-I, complete cds

Length=6838

Query 1 GCAGACCATTGCTTAATCT 19 [SEQ ID NO: 1]

I I I I I I I I I I I I I I I I I I

Sbjct 4079 GCAGACCATTGTTTAATCT 4061 [SEQ ID NO: 14]

Mismatch in SEQ ID NO: 1 is underlined. Based on the above findings, there was projected inhibitory effect of the selected ASO for glucosyltransferase mRNA expression in S. sobrinus because of just one mismatched nucleotide which is present approximately in the middle of sequence.

Modeling of S. mutans gtfB mRNA secondary structure was carried out using the Mfold program at the Rensselaer bioinformatics web server (http://www.bioinfo.rpi.edu/ applications/mfold; accessed in March, 2013) in order to assess binding site for the selected ASO. Consequently, one hundred models of the mRNA secondary structure were generated and the most reliable structural motif around the ASO deduced (Fig 1). In order to determine the conserved homologous regions among the gtf genes of oral streptococci - S. mutans and S. sobrinus, which are associated with human cardiovascular pathologies, and to choose the complementary ASO sequences to them, there were selected sequences of the water- insoluble glucan synthesis encoding gtf genes of the various clinical strains and clones, belonging to these bacterial species, from the European Nucleotide Archive [ENA] (accessed in September, 2013). Afterwards, using S. mutans (strain UA159) gtfB gene as a reference sequence, the multiple sequence alignment was produced employing MAFFT online server at the Max-Planck Institute for Development Biology (http://toolkit.tuebingen.mpg.de/mafft; accessed in September, 2013). Applying such approach, it was identified the conserved homologous region within S. mutans and S. sobrinus glucosyltransferase genes consisting of 26 nt: 5'- CGCGTCATGTTTGAAGGTTTCTCTAA-3 ' (SEQ ID NO : 17). The complementary to this region antisense sequence (i.e. antisense oligonucleotide) is composed of these 26 nt: 5'- TTAGAGAAACCTTCAAACATGACGCG-3 ' (SEQ ID NO: 18). The exact locations of this segment in the gtf genes of S. mutans (strain UA159) and S. sobrinus (strain SL1) along with the data linked to the GenBank of NCBI are presented below:

1. In S. mutans (strain UA159) gtfB (herein indicated as gtfl) gene:

GenBank accession no. gb | AEO 14133.2 |

Streptococcus mutans UA159, complete genome

Length=2032925

Features in this part of subject sequence:

glucosyltransferase-I Query 1 TTAGAGAAACCTTCAAACATGACGCG 26 [SEQ ID NO: 18]

I I I I I I I I I I I I I I I I I I I I I I I I I I

Sbjct 953618 TTAGAGAAACCTTCAAACATGACGCG 953593 [SEQ ID NO: 18]

2. In S. mutans (strain UA159) gtfC (herein indicated as gtfSI) gene:

GenBank accession no. gb | AEO 14133.2 |

Streptococcus mutans UA159, complete genome

Length=2032925

Feature in this part of subject sequence:

glucosyltrans ferase- SI

Query 1 TTAGAGAAACCTTCAAACATGACGCG 26 [SEQ ID NO: 18]

I I I I I I I I I I I I I I I I I I I I I I I I I I

Sbjct 958322 TTAGAGAAACCTTCAAACATGACGCG 958297 [SEQ ID NO: 18]

3. In S. sobrinus (strain SL1) gtfl gene:

GenBank accession no. dbj | D63570.1 | Streptococcus sobrinus gene for glucosyltrans feras e GTF-I, complete cds

Length=6838

Query 1 TTAGAGAAACCTTCAAACATGACGCG 26 [SEQ ID NO: 18]

I I I I I I I I I I I I I I I I I I I I I I I I I I

Sbjct 3530 TTAGAGAAACCTTCAAACATGACGCG 3505 [SEQ ID NO: 18]

Employing the "AntiSense Design" programme available at the Integrated DNA Technologies website (http://eu.idtdna.com/Scitools/Applications/AntiSense/ Antisense.aspx), there were optimized the complementary antisense sequences (i.e. antisense oligonucleotides) of 19-21 nucleotides in length to the region (SEQ ID NO: 17) said herein above that are derivates of the SEQ ID NO: 18:

5 '-G AAACCTTCA AAC ATGACGC-3 ' (SEQ ID NO: 19)

5 '- A AACCTTCA AACATGACGC-3 ' (SEQ ID NO: 20)

5 '-G AAACCTTCA AAC ATGACGCG-3 ' (SEQ ID NO: 21)

5 '-G AAACCTTCA AAC ATGACG-3 ' (SEQ ID NO: 22) 5 '- AGAAACCTTCA AACATGACGC-3 ' (SEQ ID NO: 23)

Example 2 - Treatment Effects of Antisense Oligonucleotide on Biofilm Formation in the Mixed S. mutans and S. sobrinus Cultures with Blood Serum

Laboratory Methods and Results

The mixed S. mutans and S. sobrinus cultures were used in order to evaluate the test ASOl molecule (SEQ ID NO: 1) effect on the bacterial biofilm formation in vitro medium containing the main blood component - a serum. S. mutans strain UA159 (Bratthall serotype c) and S.

sobrinus strain SL1, which are available through the American Type Culture Collection (ATCC no. 700610 and ATCC no. 33478, respectively), were cultured in Todd Hewitt (TH) broth (Difco) with 10% heat-inactivated horse serum (Gibco) under anaerobic conditions (95% N₂ and 5% C0₂) at 37 °C for 18 h. The culture purity was checked on Mitis salivarius agar (Difco) and Columbia blood agar (E&O Laboratories). Afterwards, the optical density (OD) of bacterial culture was adjusted to 0.2 at 630 nm using microplate reader spectrophotometer (Dynex MRX).

Prior to inoculation of the bacteria, 24-well flat-bottomed polystyrene cell culture plates

(Sarstedt) were filled with the TH broth containing 10% heat-inactivated horse serum. Then, two phosphorothioate-modified oligodeoxyribonucleotides of the different sequences were added to the plate wells at the final concentration of 10 μΜ in combination with the transfection reagent -

TurboFect (TF):

1. The ASOl, comprising of the sequence: 5 '-GCAGACCATTGCTTAATCT-3 ' (SEQ ID NO:

1) , which served as a test molecule.

2. The AS02, comprising of the sequence: 5 '- ACTCGTATGCTAC AGCTAT-3 ' (SEQ ID NO:

2) , which served as a negative control molecule and differed from the test molecule by scrambling the nucleotide sequence.

Originally, these ASOs were synthesized at the Metabion International AG (Germany) as full phosphorothioate oligodeoxyribonucleotides using 1 μπιοΐ synthesis scale and HPLC

purification. Before experiments, the lyophilized ASOs were dissolved in sterile nuclease-free distilled water (Thermo Fisher Scientific, Fermentas) in order to get stock solutions with the final concentration of 100 μΜ. Where needed, the ASOs were combined with TurboFect^™ reagent and prepared according to the manufacturer's protocol by using 2 μΐ of the reagent for 1 μg of the oligonucleotide DNA.

After addition of the ASOs, S. mutans and S. sobrinus cultures were mixed in equal parts and inoculated to the plate wells at the final dilution of 1: 100. Immediately, the sterile glass slides of 1 mm thickness cut from standard microscope slides (76 x 26 mm; Thermo Fisher Scientific) were vertically inserted into wells, and the plates were incubated anaerobically at 37 °C for additional 4 h. Afterwards, a sterile solution of sucrose was added to the appropriate wells at the final concentration of 1%, and the plates were incubated anaerobically at 37 °C in 95% N₂ and 5% C0₂ for another 20 h. In this experiment, the final volume of TH broth per well was 1 ml, the wells without bacterial cells were used as blank controls, and the untreated S. mutans and S. sobrinus bacteria served as experimental controls.

Following 24 h of the total incubation time, the glass slides were removed from wells, dried and further used for analysis of the mixed S. mutans and S. sobrinus biofilm by Sensofar PLu 2300 optical confocal profilometer. For this purpose, there were performed 6 measurements for evaluation of the biofilm roughness and 5 measurements for assessment of the biofilm thickness (every measurement area of 180 x 240 μιτι) per slide halfway from bottom to top of the visible biofilm employing 50X confocal objective. Data of the scanned and measured samples were further processed with Gwyddion programme (version 2.27, available at http://gwyddion.net) in order to quantify the biofilm surface's roughness parameters and its thickness reflecting a maturity of the formed biofilm. In addition, a Median filter (size of 10 pixels or 3 μιτι) was selected to remove errors of form and waviness of the surface. For the quantitative evaluation of the biofilm surface's roughness, it was calculated one of the most critical surface's parameters - Rq (an average of the measured height deviations). Statistical significance of the measured biofilm parameters was evaluated using the One -Way ANOVA with LSD Post Hoc test of SPSS programme (version 20.0). A p value less than 0.05 was considered statistically significant. Analysis of the glass slides' surfaces with the mixed S. mutans and S. sobrinus culture biofilms applying technique of the optical profilometry revealed that the presence of 1% sucrose and 10% blood serum in TH broth considerably increase biofilm surface's roughness parameter - Rq as well as biofilm thickness in comparison to the untreated bacteria growing with 10% serum, but without sucrose (fig 4a; fig 5a,b). In this respect, it shows that the presence of 1% sucrose and 10% serum stimulated attachment (adhesion) of the bacteria to glass surface and biofilm formation. However, the test ASOl molecule (SEQ ID NO: 1) in combination with TurboFect reagent decreased biofilm surface's roughness (Rq) of the mixed S. mutans and S. sobrinus cultures by 25% compared to the bacteria exposed to the AS02 (SEQ ID NO: 2) in combination with TurboFect reagent (p < 0.05) within TH broth containing 1% sucrose and 10% blood serum (fig 4b,c ; fig 5a). Moreover, the test ASOl molecule (SEQ ID NO: 1) in combination with TurboFect reagent reduced biofilm thickness of the mixed S. mutans and S. sobrinus cultures by approximately 44% compared to the untreated bacteria and bacteria exposed to the

AS02 (SEQ ID NO: 2) in combination with TurboFect reagent (p < 0.05) within TH broth containing 1% sucrose and 10% blood serum (fig 4a, b, c and fig 5b). On the basis of these results, it can be concluded that the test ASOl molecule (SEQ ID NO: 1) effectively decreases S. mutans and S. sobrinus adherence and biofilm formation on solid phase surface (glass slide) in the medium containing the main blood component - serum in vitro conditions.

Claims

1. The antisense oligonucleotides (ASOs) for prevention of atherosclerosis and cardiovascular infections, corresponding to these features:

a) ASOs composed of the nucleotide sequences according to SEQ ID NO: 1, 12, 18; b) ASOs composed of the sequence SEQ ID NO: 1, 12, 18 fragments; or

c) ASOs having at least 85% sequence identities to sequences SEQ ID NO: 1, 12, 18.

2. The ASOs, according to claim 1, characterized in that the nucleotide sequences are distinct from SEQ ID indicated SEQ ID NO: 1, 12, 18, by one, two, three or four nucleotides.

3. The ASOs, according to claim 1, characterized in that the nucleotide sequences are comprised of fragments or parts selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 19, 20, 22, 23.

4. The ASOs, according to any of claims 1-3, characterized in that the ASOs are conjugated with cell-penetrating peptide.

5. The ASOs, according to any of claims 1-4, for use in prevention, inhibition and reduction of atheromatous plaques' formation in human cardiovascular tissues and/or in human blood, which component is serum.

6. A biopharmaceutical composition, which constituent part is the ASOs according to any of claims 1-5, for biopharmaceutical and medical use.

7. A biopharmaceutical composition comprised of the ASOs, according to any of claims 1-6, characterized in that it consists of other parts, among them the adjuvants and carriers used in biopharmacy.

8. A biopharmaceutical composition, according to claim 7, c h ar a c t e ri z e d in that the used carrier is incorporated cationic polymer.

9. A biopharmaceutical composition, which constituent part is the ASOs according to any of claims 1-8, for prevention and treatment of atherosclerosis.

10. A biopharmaceutical composition, which constituent part is the ASOs according to any of claims 1-8, for prevention and treatment of bacterial endocarditis.

11. A biopharmaceutical composition, which constituent part is the ASOs according to any of claims 1-8, also adjuvants and/or carriers used in biopharmacy, for treatment of the surfaces that are in contact with human tissues and blood and/or blood serum, e.g., tubes, catheters, dialysis membranes, probes, prosthetic heart valves, prostheses, implants, etc., in order to achieve the antibiofilm effect against S. mutans and S. sobrinus present on these surfaces.

12. A method for control of S. mutans and S. sobrinus cultures, comprising the ASOs according to any of claims 1-3, ch ar ac t eri z e d in that the specifically and simultaneously inhibits the expression of S. mutans gtfB, gtfC and S. sobrinus gtfi mRNAs, as well as at the same time suppresses the ability of these bacteria to synthesize water-insoluble and partly water-soluble glucan polymers and reduces the capability of these bacteria to produce exopolysaccharides composing the biofilm matrix.