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Pna applications for inhibition of streptococci biofilms

The project is focused on the use and medical application of antisense oligonucleotides, which selectively target expression of the genes encoding enzymes for synthesis of polysaccharides within streptococci in order to inhibit the formation of biofilm.

Bacteria of the genus Streptococcus are responsible for the most part of human skin, respiratory tract, oral and cardiovascular infections. One of the main reasons related to these infections is that streptococci synthesize sticky polysaccharide polymers, which cover tissue surfaces and compose structural matrix for bacterial attachment, biofilm formation and development of the infection. The bacterial biofilm is very difficult to eradicate because the antimicrobials cannot effectively penetrate through the polysaccharide matrix and reach the embedded bacteria. In the medical practice, it causes chronic and resistant to antimicrobial treatment diseases. This project is focused on the use and medical application of antisense oligonucleotides, which selectively target expression of the genes encoding enzymes for synthesis of polysaccharides within streptococci in order to inhibit the formation of biofilm. Preliminary data obtained at UAB Bioseka indicate that antisense oligonucleotides suppress biofilm formation in Streptococcus mutans – one of the main causative agents of oral and cardiovascular infections. The existing antisense technology of UAB Bioseka has an oligonucleotide, comprising of 19 nucleotide sequence, with phosphorothioate chemical backbone which simultaneously targets the expression of two genes coding for the synthesis of polysaccharide matrix of S. mutans, that is, glucosyltransferase (gtf) B and gtfC encoding the production of water-insoluble and partly water-soluble glucans, respectively. Current technology shows significant attenuation in adhesion of the streptococci to solid surface (i.e., glass) during 24 h of the treatment as compared with the unexposed control bacteria as well as considerable decrease in aggregation of the streptococci. We have enough data to demonstrate the inhibition of biofilm formation in S. mutans culture through decreased synthesis of polysaccharide polymers in bacteria. Bioinformatical data shows promise for the same sequence oligonucleotide to be applied for control of the biofilm formation in other bacteria of the genus Streptococcus, e.g., S. sobrinus – where the target is gtf I coding for the synthesis of water-insoluble glucan. On the basis of the existing in-house technology, UAB Bioseka in cooperation with GeneArrest will test effectiveness of the newest antisense oligonucleotides of the 3rd generation – peptide nucleic acids (PNAs) for suppression of the biofilm formation primarily in S. mutans, S. sobrinus, and secondly in other streptococci such as S. pyogenes, S. pneumoniae. For the latter streptococci, the antisense oligonucleotides will be optimized to target the expression of different genes encoding production of polysaccharides, that is, the gene coding for hyaluronic acid synthetase in S. pyogenes, and the gene coding for autophosphorylating tyrosine kinase – CpsD in S. pneumoniae. The oligonucleotide of the existing antisense technology will be converted to PNA, truncated and further optimized to exhibit at least the same or even biofilm synthesis inhibition effect. Additional possible antisense sequences targeting biofilm formation will be sought and PNA based antisense oligonucleotides will be optimized for the streptococci using proprietary bioinformatical tools. Synthesis of selected PNA based oligonucleotides will be carried out employing the patented technology of GeneArrest. The oligonucleotide candidates will be tested in vitro using TM (melting temperature) measurement by RT-PCR and later will be tested by streptococci bacterial systems, primarily focusing on S. mutans and S. sobrinus individually as well as in the polybacterial environments. For the evaluation of the PNA effects within bacterial system, the following methods will be used: 1) quantitative assessment of the bacterial biofilms, through colorimetric and profilometric techniques; 2) bacterial aggregation, applying spectrophotometry and microscopy; 3) quantification of the bacterial polysaccharide polymers using colorimetric technique; 4) expression of the mRNA and enzymes responsible for generation of polysaccharide polymers will be evaluated employing reverse transcription polymerase chain reaction (RT-PCR) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); 5) stability and degradation of the PNA oligonucleotides within the bacterial system will be assessed using high performance liquid chromatography (HPLC); 6) permeability into bacteria will be tested for optimization of the formulation and interaction of PNA with ingredients widely used in healthcare products (e.g., sodium saccharin) will be investigated employing methods as described herein. The goal is to develop the novel, patentable and commercially viable antisense technology for new antimicrobial drugs to control medically important bacterial biofilms, including potential for control of bacteria resistant to available antimicrobials, and gather pre-clinical data demonstrating efficacy.
Acronym: 
PAISBI
Project ID: 
8 764
Start date: 
01-07-2013
Project Duration: 
24months
Project costs: 
400 000.00€
Technological Area: 
Virus, Virology, Vaccines/ Antiobiotics / Bacteriology
Market Area: 
Monoclonal Antibodies and Hybridomas

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