Final Report Summary - DNA-TRAP (DNA-TRAP – Delivery of Nucleic Acid-Based Therapeutics for the TReatment of Antibiotic-Resistant Pathogens)
Antimicrobial agents have dramatically reduced the number of deaths from infection over the last seventy years. However, overuse and misuse of these agents has resulted in the emergence of many types of resistant bacteria, which now threaten human health and life. The project entitled Delivery of Nucleic Acid based therapeutics for the TReatment of antibiotic-resistant Pathogens (DNA-TRAP) is an interdisciplinary, cross-sector project aimed at the development of a new class of antibacterial therapies. The project consortium included two academic partners (University of East Anglia (UK) and University of Florence (IT) as well as four commercial partners, Procarta (UK), Nanovector srl (IT), Kuecept Ltd (UK) and Tecrea Ltd (UK). The project duration was four years and involved the transfer of knowledge between academic and industrial researchers. The scientific team includes microbiologists, physical chemists, nanoparticle production scientists and pharmaceutical formulation experts.
The Nucleic Acid based component of the DNA-TRAP project was a novel experimental therapeutic approach entitled Transcription Factor Decoys (TFDs). These are short pieces of DNA that, upon delivery inside a bacterial cell, interfere with essential biological processes and cause bacterial death as a result. Nanoparticles (NPs) are a revolutionary technology platform that has offered major improvements and advances in the detection and treatment of a number of diseases. NPs are generally considered to measure between 1-100 billionths of a metre in diameter i.e. about five hundred to one hundred thousand times smaller than the width of a single human hair.
The major challenge to the development of TFD-based therapeutics concerns the delivery of the TFD inside the bacterial cell & its formulation to improve efficacy at the sites of infection. Within the DNA TRAP project, a completely novel nanoparticle formulation based upon a new detergent-like molecule called 12-bis-THA (12-bis-tetrahydroacridine). This molecule is related to an existing and widely used antibacterial molecule called dequalinium. When dissolved in water 12-bis-THA molecules associate with each other to form loosely associated clusters. When DNA such as TFD is added to 12-bis-THA in water they form tightly packed NPs that are positively charged and protect DNA from degradation. The positively charge is extremely important in allowing the particles to bind to bacterial cells and the DNA protection is necessary to allow it to be delivered to the infection site. However, a number of key parameters of TFD NPs must be understood so that they can be synthesized with precision and formulated appropriately for therapeutic delivery.
The main of the project was to better understand how TFD-based therapeutics could be reproducibly prepared and delivered to bacterial cells for antibiotic therapy. Among other principle scientific objectives were:
• To design and test TFDs to inhibit two different aspects of infections caused by two challenging bacteria, C. difficile and P. aeruginosa
• To understand the characteristics of TFD NP formulations, specifically understanding the interaction of the NP with the TFD and optimising the amount of TFD loading
• To understand the mechanism of TFD NP entry within the bacterial cell and to identify the parameters that dictate efficient DNA transfer
Through careful project management, face-to-face meetings, video and teleconferences each of the project milestones were reached. More importantly, a total of 105 months of cross-sector secondment has up-skilled a large number of early-career research and facilitated knowledge exchange between the industrial and academic research environments. In addition to the planned research and knowledge transfer project objectives the consortium has, by the project end, delivered four peer-reviewed international journal article with at least four more manuscripts in preparation. Similarly, a significant proportion of the project data was disseminated to the expert research community at international/national scientific meetings as well as to the general public through dissemination and outreach events. These includes research talks at schools, science café events and research showcase events in Italy and the UK.
Summary of project findings:
Using a range of biological and chemical techniques we have identified that 12-bis-THA binds to a lipid (fat) molecule in the bacterial cell and that this interaction allows the TFD NPs to deliver the DNA into the cell (Figure 1, below).
Figure 1: Proposed model for TFD NP delivery of DNA into a bacterial cell. (a) the TFD-NP (circular NP coated with DNA) binds to the bacterial membrane lipid, cardiolipin and disrupts the membrane barrier allowing NP entry; (c) once inside the cell the TFD NP breaks up to liberate free TFD that can act against its target; alternatively, some free TFD from disrupted TFD NPs can pass directly through the disrupted membrane (b).
TFD NPs based on the 12-bis-THA detergent were found to efficiently deliver DNA into a number of different bacterial cell types. However, the detergent molecule and TFD NPs was found to be unstable in some biological fluids (e.g. blood components). To stabilise the molecule and to move towards a formulation more appropriate for delivery to patients the consortium explored liposomes as a drug carrier. Liposomes are 100 nm diameter balls of lipids that have been approved as medicines for disease such as cancer. Liposomes are easy to manufacture, easy to load with drug and generally show excellent stability in biological fluids such as blood serum. In a separate publication the consortium reported the manufacture and characterisation of liposomes loaded with 12-bis-THA and TFD which displayed better stability in biological fluids. Importantly, fluorescent microscope experiments confirmed that these liposomes retain the ability to deliver TFD to bacterial cells and so present an attractive option for development of TFD NPs. To widen the scope and impact of the project the consortium applied their expertise in liposome formulations to the development of new formulations of the existing antibiotic, levofloxacin. These formulations were also tested for safety using a range of human cell culture models and a novel toxicity technique that uses tadpoles of the frog, Xenopus laevis. The selection process for formulations included aspects of stability, activity and safety
Impact of the project findings: The DNA-TRAP project has significantly enhanced the understanding of how the TFD approach could be developed into a future anti-infective therapeutic. This will benefit a number of research groups and biotech companies globally are investigating NP based antibiotic delivery approaches. This knowledge and understanding will expedite the development of new antimicrobial therapies which are so desperately needed to counter the rapid emergence of antibiotic-resistant bacterial infections which are causing increased illness and deaths around the globe.
For more information:
Visit http://dnatrap-iapp.eu/ or contact project co-ordinator, Dr Chris Morris christopher.j.morris@uea.ac.uk
The Nucleic Acid based component of the DNA-TRAP project was a novel experimental therapeutic approach entitled Transcription Factor Decoys (TFDs). These are short pieces of DNA that, upon delivery inside a bacterial cell, interfere with essential biological processes and cause bacterial death as a result. Nanoparticles (NPs) are a revolutionary technology platform that has offered major improvements and advances in the detection and treatment of a number of diseases. NPs are generally considered to measure between 1-100 billionths of a metre in diameter i.e. about five hundred to one hundred thousand times smaller than the width of a single human hair.
The major challenge to the development of TFD-based therapeutics concerns the delivery of the TFD inside the bacterial cell & its formulation to improve efficacy at the sites of infection. Within the DNA TRAP project, a completely novel nanoparticle formulation based upon a new detergent-like molecule called 12-bis-THA (12-bis-tetrahydroacridine). This molecule is related to an existing and widely used antibacterial molecule called dequalinium. When dissolved in water 12-bis-THA molecules associate with each other to form loosely associated clusters. When DNA such as TFD is added to 12-bis-THA in water they form tightly packed NPs that are positively charged and protect DNA from degradation. The positively charge is extremely important in allowing the particles to bind to bacterial cells and the DNA protection is necessary to allow it to be delivered to the infection site. However, a number of key parameters of TFD NPs must be understood so that they can be synthesized with precision and formulated appropriately for therapeutic delivery.
The main of the project was to better understand how TFD-based therapeutics could be reproducibly prepared and delivered to bacterial cells for antibiotic therapy. Among other principle scientific objectives were:
• To design and test TFDs to inhibit two different aspects of infections caused by two challenging bacteria, C. difficile and P. aeruginosa
• To understand the characteristics of TFD NP formulations, specifically understanding the interaction of the NP with the TFD and optimising the amount of TFD loading
• To understand the mechanism of TFD NP entry within the bacterial cell and to identify the parameters that dictate efficient DNA transfer
Through careful project management, face-to-face meetings, video and teleconferences each of the project milestones were reached. More importantly, a total of 105 months of cross-sector secondment has up-skilled a large number of early-career research and facilitated knowledge exchange between the industrial and academic research environments. In addition to the planned research and knowledge transfer project objectives the consortium has, by the project end, delivered four peer-reviewed international journal article with at least four more manuscripts in preparation. Similarly, a significant proportion of the project data was disseminated to the expert research community at international/national scientific meetings as well as to the general public through dissemination and outreach events. These includes research talks at schools, science café events and research showcase events in Italy and the UK.
Summary of project findings:
Using a range of biological and chemical techniques we have identified that 12-bis-THA binds to a lipid (fat) molecule in the bacterial cell and that this interaction allows the TFD NPs to deliver the DNA into the cell (Figure 1, below).
Figure 1: Proposed model for TFD NP delivery of DNA into a bacterial cell. (a) the TFD-NP (circular NP coated with DNA) binds to the bacterial membrane lipid, cardiolipin and disrupts the membrane barrier allowing NP entry; (c) once inside the cell the TFD NP breaks up to liberate free TFD that can act against its target; alternatively, some free TFD from disrupted TFD NPs can pass directly through the disrupted membrane (b).
TFD NPs based on the 12-bis-THA detergent were found to efficiently deliver DNA into a number of different bacterial cell types. However, the detergent molecule and TFD NPs was found to be unstable in some biological fluids (e.g. blood components). To stabilise the molecule and to move towards a formulation more appropriate for delivery to patients the consortium explored liposomes as a drug carrier. Liposomes are 100 nm diameter balls of lipids that have been approved as medicines for disease such as cancer. Liposomes are easy to manufacture, easy to load with drug and generally show excellent stability in biological fluids such as blood serum. In a separate publication the consortium reported the manufacture and characterisation of liposomes loaded with 12-bis-THA and TFD which displayed better stability in biological fluids. Importantly, fluorescent microscope experiments confirmed that these liposomes retain the ability to deliver TFD to bacterial cells and so present an attractive option for development of TFD NPs. To widen the scope and impact of the project the consortium applied their expertise in liposome formulations to the development of new formulations of the existing antibiotic, levofloxacin. These formulations were also tested for safety using a range of human cell culture models and a novel toxicity technique that uses tadpoles of the frog, Xenopus laevis. The selection process for formulations included aspects of stability, activity and safety
Impact of the project findings: The DNA-TRAP project has significantly enhanced the understanding of how the TFD approach could be developed into a future anti-infective therapeutic. This will benefit a number of research groups and biotech companies globally are investigating NP based antibiotic delivery approaches. This knowledge and understanding will expedite the development of new antimicrobial therapies which are so desperately needed to counter the rapid emergence of antibiotic-resistant bacterial infections which are causing increased illness and deaths around the globe.
For more information:
Visit http://dnatrap-iapp.eu/ or contact project co-ordinator, Dr Chris Morris christopher.j.morris@uea.ac.uk