Inhibitors Of Bacterial Protein Synthesis - A New Class Of Antibiotics
Funder
National Health and Medical Research Council
Funding Amount
$120,000.00
Summary
Pioneering work by CSIRO scientists has identified specific peptide motifs in the DNA replication machinery of bacteria that are critical for the correct functioning of the organism. In collaboration with CI Alewood potent (Kd ~ nM) lead compounds that inhibit bacterial DNA replication have been designed and synthesised. Through the application of a number of novel bioinformatics approaches to the analysis of the complete genome sequences of bacteria, the key sites of interaction of a number of ....Pioneering work by CSIRO scientists has identified specific peptide motifs in the DNA replication machinery of bacteria that are critical for the correct functioning of the organism. In collaboration with CI Alewood potent (Kd ~ nM) lead compounds that inhibit bacterial DNA replication have been designed and synthesised. Through the application of a number of novel bioinformatics approaches to the analysis of the complete genome sequences of bacteria, the key sites of interaction of a number of protein families (DNA synthesis and repair enzymes) with the beta subunit of bacterial DNA Polymerase III have been identified. The nature of the sites, and preliminary experimental data, suggests that the approach will be generally applicable to all species of bacteria. In addition a wide range of novel assays for the identification of inhibitors of the interaction of proteins with the beta subunit have been developed. In this proposal we wish to demonstrate that our in vitro nanomolar inhibitors of the beta subunit can inhibit bacterial cell growth. The development program proposes to develop methods and strategies to gain bacterial cell entry of inhibitors of the interaction of proteins with the beta subunit of bacterial DNA Polymerase III. Proof of concept will be demonstrated by inhibition of bacterial cell growth. Stable compounds with good binding characteristics and able to be taken up by cells will be developed based on structure-function assay results, structural studies and modelling of inhibitors bound to the target. Antimicrobial activity of compounds will be demonstrated in standard FDA approved NCLLS (National Centre of Clinical Laboratory Standards USA) tests. Spectrum of activity will be demonstrated by testing compounds against bacterial species representative of the range of pathogenic organisms in standard FDA assays.Read moreRead less
Development Of Therapeutically Useful Human Artificial Chromosomes For Gene Delivery And Optimal Gene Expression
Funder
National Health and Medical Research Council
Funding Amount
$496,986.00
Summary
Gene therapy is an exciting new form of treatment for genetic disorders aimed at providing long-term correction of the problems at source - namely the affected gene. The biggest technical hurdle facing gene therapy is to be able to deliver the therapeutic genes efficiently and safely into patient cells. Many gene therapy protocols are currently being trialled clinically. These protocols, based mostly on the use of attenuated viruses to deliver the genes, carry potential risks to the patients in ....Gene therapy is an exciting new form of treatment for genetic disorders aimed at providing long-term correction of the problems at source - namely the affected gene. The biggest technical hurdle facing gene therapy is to be able to deliver the therapeutic genes efficiently and safely into patient cells. Many gene therapy protocols are currently being trialled clinically. These protocols, based mostly on the use of attenuated viruses to deliver the genes, carry potential risks to the patients in terms of infection, immune response, and germline modification. We have developed the first stage of a new technology for gene delivery that does not require the use of viruses. This technology is based on the generation of human artificial chromosomes, which are smaller versions of the naturally occurring chromosomes that carry all the genes inside our cells. Safety in these artificial chromosomes comes from the use of entirely human materials for their engineering. These artificial chromosomes also have other advantages over the viral approaches, including allowing large genes to be carried, and providing a permanent cure in a single treatment. We have already successfully constructed, published, and patented a number of first-generation human artificial chromosomes. The current project aims to complete the next proof-of-concept milestone towards the further development of this technology. Specifically, we propose to demonstrate the ability of the artificial chromosomes to carry genes and provide sustainable expression of these genes in cells and in animal models. Success in this study will allow the technology to proceed rapidly into commercialisation and clinical trial as a new improved tool for gene delivery and gene therapy.Read moreRead less
Mechanically-restricted Percutaneous Gene Therapeutic Solutions For Heart Failure.
Funder
National Health and Medical Research Council
Funding Amount
$187,000.00
Summary
We have developed a novel system for the localized delivery of specialised genes to the heart in order to improve contractility and function of a failing heart. Many genes, for reasons of toxicity, clearance, or uptake, require direct delivery to the target region without spillover to the systemic circulation. Our system addresses these issues by isolating the local circulation of the target organ and directly delivering the agent with minimal systemic loss and improved delivery and uptake effic ....We have developed a novel system for the localized delivery of specialised genes to the heart in order to improve contractility and function of a failing heart. Many genes, for reasons of toxicity, clearance, or uptake, require direct delivery to the target region without spillover to the systemic circulation. Our system addresses these issues by isolating the local circulation of the target organ and directly delivering the agent with minimal systemic loss and improved delivery and uptake efficiency, while minimizing potentially dangerous and toxic systemic effects.Read moreRead less
Kunjin Replicons For Gene Therapy And Protein Manufacture
Funder
National Health and Medical Research Council
Funding Amount
$310,000.00
Summary
This grant seeks to provide proof of concept (PoC) for the use of the Kunjin replicon technology for gene therapy and protein production. (A) Protein production. Two Kunjin replicon constructs expressing green fluorescent protein (GFP) and secreted alkaline phosphatase (SEAP) are to be constructed and protein production monitored using FACS and SEAP bioactivity reporter kit (Roche), respectively. Protein production and biological activity of the proteins will be monitored in transient transfecti ....This grant seeks to provide proof of concept (PoC) for the use of the Kunjin replicon technology for gene therapy and protein production. (A) Protein production. Two Kunjin replicon constructs expressing green fluorescent protein (GFP) and secreted alkaline phosphatase (SEAP) are to be constructed and protein production monitored using FACS and SEAP bioactivity reporter kit (Roche), respectively. Protein production and biological activity of the proteins will be monitored in transient transfections and over an extended time period. Several cell lines, culture conditions and Kunjin replicon vector modifications will be tested. Arrangements have also been made to send the constructs to Roche, GSK, Eli Lilly, and Exelixis for side by side comparisons of this system with existing proprietary protein production echnologies. (B) Gene therapy. Two PoC gene therapy systems are proposed to be used for evaluation of Kunjin replicon vectors. (i) Tumours expressing granulocyte macrophage colony stimulating factor (GMCSF) by transfection cause the generation of anti-tumour CD8 T cells and subsequent tumour rejection. Current approaches include adoptive transfer of adeno-GM-CSF transfected tumour cells, a costly and laborious process resulting in only transient expression (Can. Imm. Immunother 2001 50:373). We intend to inject Kunjin replicon virus like particles into growing s.c. B16 melanomas and expect to see a high infection rate, a sustained high-level expression of GMCSF, and rejection of the tumour. In contrast to Kunjin, nearly all humans have antibody responses to adenovirus, and very high titres of adenovirus are required to obtain high infection and GM-CSF expression. Both factors limit adenovirus use in vivo. (ii) Transplant rejection can be inhibited by expression in the graft of CTLA4-Fc a reagent that blocks T cell co-stimulation enhancing allo-graft acceptance (Transplantation 2000 69:1806). High-level expression for over 100 days is expected to correlate with optimal graft acceptance. Our ability to use Kunjin to express beta galactosidase for several months in vivo without inflammation illustrates the potential for this approach (CIB ref 15). Initially we intend to use P815 cells injected i.p. into C57BL-6, where they are usually rejected within a few days. In contrast, P815 cells with Kunjin replicon-mediated CTLA4-Fc expression should survive for an extended period. Graft survival is easily monitored using FACS and anti-H-2d antibodies.Read moreRead less
Development And Evaluation Of Novel Fetal Haemoglobin Inducers For The Therapy Of Beta-thalassaemia
Funder
National Health and Medical Research Council
Funding Amount
$288,899.00
Summary
The most important haemoglobinopathies from the clinical point of view are the beta-thalassaemias, sickle cell disease (SCD), HbE disease and the interactions between them. These beta-haemoglobinopathies are the result of mutations in the beta-globin gene, causing beta-globin chain synthesis that is abnormal, low or absent leading to life-threatening severe anaemia, and blood transfusion-dependency for life. An alternative approach to the therapy of beta-thalassemia is to reactivate fetal haemog ....The most important haemoglobinopathies from the clinical point of view are the beta-thalassaemias, sickle cell disease (SCD), HbE disease and the interactions between them. These beta-haemoglobinopathies are the result of mutations in the beta-globin gene, causing beta-globin chain synthesis that is abnormal, low or absent leading to life-threatening severe anaemia, and blood transfusion-dependency for life. An alternative approach to the therapy of beta-thalassemia is to reactivate fetal haemoglobin (HbF) synthesis. Some chemical agents have been identified to induce HbF and significantly reduce the need for blood transfusion in some thalassaemia patients, while in SCD patients it can ameliorate the clinical symptoms. Despite a number of clinical trials investigating the potential of HbF-inducing agents, many of these drugs have low efficacy, specificity, and cytotoxicity. There is therefore an urgent need to identify novel pharmacological agents with greater efficacy and reduced toxicity. Without a clear understanding of the underlying mechanism(s) involved in the induction of HbF, it is virtually impossible to focus on any molecular target. A promising approach is the use of chemical libraries in a high-throughput (HTP) screening to identify positive regulators of gene products. Our research group created an assay that has allowed us for the first time to perform a side-by-side comparison of several previously described fetal hemoglobin inducers including 2000 existing pharmaceuticals used by patients unrelated to thalassaemia. The screen identified a distinct group of compounds that induced the gamma-globin promoter in primary and secondary screens. The identification of novel inducers of HbF warrants further investigation as alternative therapies for beta-thalassemia. This project will evaluate novel inducers of HbF in our thalassaemia mouse model and provide early 'proof-of-concept' and enable the initiation of preclinical and clinical studies.Read moreRead less
Prevention Of Neuron Death By Targeted Gene Delivery
Funder
National Health and Medical Research Council
Funding Amount
$195,691.00
Summary
Neurotrophic factors are potent proteins that have the ability to keep nerves alive. They have therefore been used in clinical trials to treat motor neuron disease, but without success. A major reason for this appears to be the way in which the neurotrophic factors are delivered. Direct injections into the blood stream are a convenient way of getting these large proteins into the bloodstream, but this is not their normal mode of action. These proteins are normally provided by cells adjacent to t ....Neurotrophic factors are potent proteins that have the ability to keep nerves alive. They have therefore been used in clinical trials to treat motor neuron disease, but without success. A major reason for this appears to be the way in which the neurotrophic factors are delivered. Direct injections into the blood stream are a convenient way of getting these large proteins into the bloodstream, but this is not their normal mode of action. These proteins are normally provided by cells adjacent to the nerves. We have designed a system that more closely resembles this physiological mode of action which involves the delivery of neurotrophic factor genes, via the bloodstream, to the affected nerves. Once inside the nerves the factors are produced on site and, following their secretion, act locally and directly on the injured nerves.Read moreRead less