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
Enhancing Disease Vector Biosecurity Through High Density Molecular Markers
Funder
National Health and Medical Research Council
Funding Amount
$763,845.00
Summary
The outbreak of several diseases spread by mosquitoes is increasing rapidly around the world, driven by increased people movement spreading both viruses and disease vectors, a lack of effective vaccines and changing climatic conditions. In this proposal I aim to develop cutting edge molecular tools for identifying pathways of exotic mosquito introductions into Australia and a program that uses bacteria living inside mosquitoes in novel ways for disease suppression.
The Genetic Basis Of Pathogen Blocking: Elucidating The Contributions Of The Wolbachia, Dengue Virus And Mosquito Genomes
Funder
National Health and Medical Research Council
Funding Amount
$736,339.00
Summary
A bacterium called Wolbachia has been shown to stop dengue virus from replicating inside mosquitoes and so is being field-tested as a biocontrol agent against dengue fever. A major threat to this strategy is the emergence of resistance either in the mosquito or virus. This proposal addresses two fundamental knowledge gaps – how does Wolbachia block virus growth and how might resistance evolve? This research is significant as it will inform the development of strategies to counter resistance.
Natural Variation And Genetic Basis Of Dengue Virus Transmission Rate In Australian Mosquitoes
Funder
National Health and Medical Research Council
Funding Amount
$680,088.00
Summary
Dengue fever outbreaks occur in Australia when infected travelers enter the country and are bitten by local mosquitoes. Here we examine the degree of genetic compatibility between mosquitoes and incoming dengue viruses that may affect disease risk for humans. We will identify the mosquito genes that determine the insect’s capacity to transmit dengue virus and develop a geographic map of transmissibility for a range of different dengue strains across Australian populations of the insect.
Evaluation And Comparison Of Lentiviral And AAV Vector Mediated Gene Therapy For The Mucopolysaccharidoses
Funder
National Health and Medical Research Council
Funding Amount
$521,320.00
Summary
The mucopolysaccharidoses are a group of inherited diseases that have profound consequences for affected individuals. They have pleiotropic effects and usually result in premature death. Although intravenous enzyme replacement therapy has been developed for a number of these disorders, this approach to therapy is invasive, very expensive, of limited efficacy, and is completely ineffective in treating brain pathology. The principal reason for this is the protected nature of the brain which preven ....The mucopolysaccharidoses are a group of inherited diseases that have profound consequences for affected individuals. They have pleiotropic effects and usually result in premature death. Although intravenous enzyme replacement therapy has been developed for a number of these disorders, this approach to therapy is invasive, very expensive, of limited efficacy, and is completely ineffective in treating brain pathology. The principal reason for this is the protected nature of the brain which prevents enzymes that are administered intravenously from entering. Therefore, alternative therapies must be considered in order to provide more effective therapy for the mucopolysaccharidoses, especially those that have significant brain pathology. Gene therapy is one such alternative therapy but this still faces the problem of introducing the therapeutic agent (in this case the gene encoding the requisite enzyme) into the brain. This project aims to provide a comparitive evaluation of two gene therapy vectors for their efficacy in treating all aspects of the pathology found in the mucopolysaccharidoses. Both vectors have the properties of being able to efficiently deliver genes to different cell types and result in the stable genetic modification of the target cell, making them ideal for long-term treatment. However, for effective gene therapy, significant and widely distributed gene delivery to the brain, as well as to other tissues, will be required. This project aims to compare the efficacy of these vectors in two different animal models of the mucopolysaccharidoses that exhibit a wide range of the clinical problems associated with these diseases, importantly including brain pathology.Read moreRead less
Novel Use Of Fungal Entomopathogens For Sustainable Control Of Mosquito-borne Viruses
Funder
National Health and Medical Research Council
Funding Amount
$605,993.00
Summary
Mosquito-born viruses such as Dengue, Ross River and Barmah Forest are increasing in regional significance. At a broader scale, an estimated 2.5 billion people live in areas at risk of epidemic Dengue transmission. Chemical insecticides are the mainstay of current mosquito control throughout many parts of the world. However, problems of insecticide resistance, environmental contamination and risks to human health, mean that chemical pesticides have not provided a universal solution, either as ou ....Mosquito-born viruses such as Dengue, Ross River and Barmah Forest are increasing in regional significance. At a broader scale, an estimated 2.5 billion people live in areas at risk of epidemic Dengue transmission. Chemical insecticides are the mainstay of current mosquito control throughout many parts of the world. However, problems of insecticide resistance, environmental contamination and risks to human health, mean that chemical pesticides have not provided a universal solution, either as outdoor sprays, residual house sprays or as insecticide treated nets. This creates a pressing need for practical alternatives. Building on approaches and technologies developed for control of locusts in Australia and Africa, we have recently discovered that the ability of mosquitoes to transmit malaria can be substantially reduced with insect fungal pathogens used as biological pesticides. We found that exposure to biopesticide-treated surfaces reduced the number of mosquitoes able to transmit malaria 80-fold. Other supporting data from semi-field trials confirm the feasibility of infecting mosquitoes under real field conditions. Together, these results represent a significant advance in the development of a cheap and sustainable biological alternative to chemical insecticides for disease control. We now wish to extend this research to explore the potential for use of fungal pathogens in control of mosquito-borne viruses. Preliminary studies already confirm that we can infect the key mosquito species responsible for transmitting Dengue. The aim of the current project is to conduct a more comprehensive evaluation of a wider range of fungal isolates to identify strains with the greatest potential to stop transmission of mosquito-borne viruses. The longer term goal is to translate this research into a practical product. Such a product would offer a cheap, environmentally friendly disease control measure, with reduced potential for resistance evolution.Read moreRead less
Substandard Bed Nets And Malaria: Causes, Impact And Solutions
Funder
National Health and Medical Research Council
Funding Amount
$827,057.00
Summary
Long-lasting insecticidal nets (LLIN) are a cornerstone of malaria control. LLIN undergo strict testing overseen by WHO and are subject to inspections prior to delivery to recipient countries. Despite this, we found that LLINs delivered to Papua New Guinea (PNG) between 2013 and 2019 were ineffective against malaria mosquitoes. Concurrently we observed a massive rise in malaria in PNG. This study is aimed at understanding the causes and impact of substandard LLINs on the global malaria burden.
In recent years it has become clear that certain white blood cells called CD8+ T lymphocytes or killer T cells are required to protect people against HIV. Unfortunately, current vaccines that produce or anti-HIV CD8 T cells only produce effective T cells for a short period. In this project we intend to test a novel vaccine vector called a Kunjin replicon, which promises to persistently produce or maintain effective T cells because the vaccine itself persists and continually immunises for extende ....In recent years it has become clear that certain white blood cells called CD8+ T lymphocytes or killer T cells are required to protect people against HIV. Unfortunately, current vaccines that produce or anti-HIV CD8 T cells only produce effective T cells for a short period. In this project we intend to test a novel vaccine vector called a Kunjin replicon, which promises to persistently produce or maintain effective T cells because the vaccine itself persists and continually immunises for extended periods. We intend to test the ability of this vaccine to persist and persistently produce effective CD8 T cells not only systemically in the blood system but also at mucosal surfaces, where HIV usually gains entry during sexual intercourse.Read moreRead less
Japanese Encephalitis Virus In Northern Australia And Papua New Guinea:its Ecology And Risk To Australia.
Funder
National Health and Medical Research Council
Funding Amount
$292,045.00
Summary
Japanese encephalitis (JE) virus is a mosquito-transmitted virus of Asia. Infection causes clinical disease in about 1 in 50 people infected, and of these, about 25% will die from a fatal encephalitis (inflammation of the brain), and a further 50% will have lifelong severe disabilities. There are over 50,000 cases annually in Asia, with about 12,000 fatalities. However, many more cases may go unrecognised. The virus normally circulates between mosquitoes and water birds and between mosquitoes an ....Japanese encephalitis (JE) virus is a mosquito-transmitted virus of Asia. Infection causes clinical disease in about 1 in 50 people infected, and of these, about 25% will die from a fatal encephalitis (inflammation of the brain), and a further 50% will have lifelong severe disabilities. There are over 50,000 cases annually in Asia, with about 12,000 fatalities. However, many more cases may go unrecognised. The virus normally circulates between mosquitoes and water birds and between mosquitoes and pigs. The World Health Organization has recognised JE as one of the most important mosquito-borne viruses because of its propensity to spread and to colonise new areas. The virus first appeared in the Torres Strait of northern Australia in 1995, causing three clinical cases of whom 2 died. This was unexpected as the nearest known focus of virus activity was in Bali, over 3000km away. The virus returned again in 1998, with a further case in the Torres Strait and the first case to occur on mainland Australia in Cape York. Both of these patients recovered. We have shown that the virus is established in Papua New Guinea (PNG), where it is spreading rapidly, and our results suggest that PNG was the source of the virus causing the outbreaks in 1995 and 1998. This project is aimed at finding out more about JE virus in PNG, particularly as it relates to spread into northern Australia. The project also seeks to investigate the potential mosquito and animal hosts in Australia that might be involved if the virus becomes established in our wildlife in Cape York. Australia is already known to have suitable mosquito vector species and suitable animal hosts in water birds and feral pigs, but the ecology is not yet understood. Thus the overall aim is to provide information on which a sound risk assessment can be based.Read moreRead less