Dystrophin Gene Repair In Mdx Mouse Myoblasts And Bone Marrow Cells As A Basis For Autologous Transplant In Human DMD
Funder
National Health and Medical Research Council
Funding Amount
$422,036.00
Summary
The muscular dystrophies are inherited diseases that lead to muscle wastage and severe disabilities. The most severe forms result in the early death of newborns, but a large number are diagnosed in children showing early mild symptoms and progress steadily to severe disabling forms in the juvenile and young adult. Perhaps the most devastating of these dystrophies is Duchenne Muscular Dystrophy (DMD). This condition affects 1 in 3,300 boys, who show symptoms at around 5 years of age until wheelch ....The muscular dystrophies are inherited diseases that lead to muscle wastage and severe disabilities. The most severe forms result in the early death of newborns, but a large number are diagnosed in children showing early mild symptoms and progress steadily to severe disabling forms in the juvenile and young adult. Perhaps the most devastating of these dystrophies is Duchenne Muscular Dystrophy (DMD). This condition affects 1 in 3,300 boys, who show symptoms at around 5 years of age until wheelchair confinement by early teens. DMD boys undergo major clinical and surgical treatments which at present only provide small but significant improvements to their lives. The median age at death for Duchenne boys is 22 years. The cause of DMD has been known for almost 2 decades and is a defect in just a single component of muscle, Dystrophin which is produced by muscle cells. In general, boys with DMD possess Dystrophin which is missing an important part that prevents the breakdown of muscles during activity. As a consequence, all the muscles in DMD boys slowly break down over their lifetime until they die because the muscle which helps in drawing breath (Diaphragm) is no longer capable of helping them to breathe. The muscle component Dystrophin is produced by a gene (the dys gene) and the defect of Dystrophin is caused by a defect in the dys gene. If the dys gene defect was able to be corrected in boys with DMD, their Dystrophin may also be corrected and the breakdown of their muscle prevented. We have been able to correct the dys gene in muscle cells from a mouse with DMD. We wish to improve this technology and allow muscle to be repopulated with genetically corrected cells to form a basis for treatment of human DMD. In this way we hope to significantly improve and lengthen these boys' lives and even lead to a cure for DMD and other genetic muscle diseases.Read moreRead less
A FACS-based Screen For Retroviral Silencing: A Tool For Detecting Trans-sensing In Mammalian Cells
Funder
National Health and Medical Research Council
Funding Amount
$205,010.00
Summary
Retroviruses are RNA viruses that infect cells and then become integrated into the genome of the infected cell. This property has been exploited in attempts to cure genetic diseases by replacing the defective gene: the replacement can be incorporated into the retrovirus, transported into a cell, and become part of the cell?s genetic material. However, retroviruses are frequently suppressed by the host cell some time after integration, so that their genetic information becomes silent. The factors ....Retroviruses are RNA viruses that infect cells and then become integrated into the genome of the infected cell. This property has been exploited in attempts to cure genetic diseases by replacing the defective gene: the replacement can be incorporated into the retrovirus, transported into a cell, and become part of the cell?s genetic material. However, retroviruses are frequently suppressed by the host cell some time after integration, so that their genetic information becomes silent. The factors that cause silencing of retroviruses are not well understood, but it is clear that this problem is a major impediment to retrovirus-based (and perhaps all) gene therapies. In a wide variety of organisms, including plants, flies, and yeast, it has been found that multiple copies of a gene can silence each other, a phenomenon termed cosuppression. Some reports suggest that this might happen in mammals as well. We have initiated a study of retroviral silencing, using a marker protein that produces green fluorescence as a model for the replaced gene. We find that the gene is usually silenced after integration, immediately or over time, but can be reactivated by drugs that demethylate DNA or alter chromosomal structure. We now propose to extend this work by doing a systematic analysis of the frequency of retroviral silencing in human T cells, and then to develop a system to detect and analyze cosuppression by retroviruses. This work will rely on the ability of fluorescence-activated cell sorting (FACS) to detect rare events in a large population of cells, and recover those rare cells. We will also test methods of destabilizing the silent state of a retrovirus. These experiments are likely to yield information that will benefit a broad array of gene replacement efforts. A demonstration of cosuppression would be particularly interesting because of the possibility that endogenous elements in the mammalian genome are regulated by this mechanism.Read moreRead less
Cell-targeted Gene Delivery Into Human Haematopoietic Stem Cells For The Treatment Of Thalassaemia
Funder
National Health and Medical Research Council
Funding Amount
$171,208.00
Summary
Thalassaemia is the most common inherited single gene disorder affecting haemoglobin synthesis in red blood cells. It mainly affects people of Mediterranean, Middle Eastern, African, South East Asian, Chinese, and Indian origin. However, large numbers of thalassaemia patients are found nowadays in Australia and other developed countries, due to large population movements in the twentieth century. Approximately 300,000 severely affected children are born each year with thalassaemia and various ot ....Thalassaemia is the most common inherited single gene disorder affecting haemoglobin synthesis in red blood cells. It mainly affects people of Mediterranean, Middle Eastern, African, South East Asian, Chinese, and Indian origin. However, large numbers of thalassaemia patients are found nowadays in Australia and other developed countries, due to large population movements in the twentieth century. Approximately 300,000 severely affected children are born each year with thalassaemia and various other abnormalities of haemoglobin synthesis. If untreated, most thalassaemia patients will die within the first few years of life. The vast majority of thalassaemia patients depend on regular blood transfusions every two to three weeks, and on nightly infusions of an iron chelator (a drug for removing excess iron from the blood). These procedures place considerable burden on thalassaemia patients, their families and society, and expose them to blood transmitted infections. The only curative treatment for thalassaemia is bone marrow transplantation from a matching donor. However, the vast majority of patients do not have matching donors and thus the only prospect for them to receive such therapy is to replace in their bone marrow cells a copy of the normal set of genes for the synthesis of haemoglobin. The studies in this proposal are therefore designed to test gene therapy protocols on bone marrow stem cells derived from thalassaemia patients. A normal set of globin genes will be delivered to the bone marrow stem cells via non-viral delivery systems and examined for function in an immunodeficient mouse strain that can accept human bone marrow. This research may enable bone marrow transplantation to be applied for the therapy of most patients with thalassaemia, while it may also have a major impact on therapeutic approaches for other haematological anomalies.Read moreRead less
Induction Of Antigen-specific Humoral Tolerance By RAAV-mediated Delivery Of CTLA4-Ig-antigen Fusion Molecules
Funder
National Health and Medical Research Council
Funding Amount
$524,456.00
Summary
There are many medical situations where immune suppression is required. Available methods lack specificity and risk infection, drug-related side-effects and cancer. We have discovered a novel way of suppressing immunity such that only unwanted responses are eliminated. This involves virus-mediated delivery of antigen fused to CTLA4-Ig. We plan to test this strategy in the context of gene therapy, to work out how it works and to optimise the approach. Success will have broad health implications.
Gene therapy is a novel form of medical treatment in which healthy genes are used to replace defective genes in cells. It is sobering to realise that in the next few years the whole human DNA sequence will be known. Consequently the already large list of genes which are known to cause disease will be greatly expanded through the application of molecular genetics. Surprisingly, however, treatments based on the correction of disease genes in the cells of a patient are not keeping up with expectati ....Gene therapy is a novel form of medical treatment in which healthy genes are used to replace defective genes in cells. It is sobering to realise that in the next few years the whole human DNA sequence will be known. Consequently the already large list of genes which are known to cause disease will be greatly expanded through the application of molecular genetics. Surprisingly, however, treatments based on the correction of disease genes in the cells of a patient are not keeping up with expectations. In attempting to achieve clinically relevant results, viruses (masters of forcing infected cells to do their bidding) have been harnessed to deliver healthy genes into diseased cells. The problem has been that the modified, safe viruses used clinically have not been efficient at achieving sustained production of healthy genes in sufficient numbers of cells. In the studies described , we will attack this problem using a number of different, but complementary approaches. Our main focus will be to facilitate efficient virus entry of appropriate target cells. We have recently been successful in cloning the receptors for two important viruses which can enter human cells. Identification of these receptors gives us clues to methods of improving virus entry. Now that we know the identity of these receptors, we can create tools to define the type of cells that these viruses can readily target.Read moreRead less
Gene Therapy For The Treatment Of Retinal Dystrophy In The RPE65 Knockout Mouse Using RAAV Virus Mediated Gene Therapy.
Funder
National Health and Medical Research Council
Funding Amount
$211,527.00
Summary
RPE65 is a gene that is found exclusively within the retina. At the moment the exact role of RPE65 is not known, however recent research has shown that mutations in the RPE65 gene have been found in a number of inherited retinal dystrophies (these dystrophies include Leber congenital amaurosis and autosomal recessive retinitis pigmentosa). It therefore appears that a functional, non-mutated RPE65 gene is essential for normal vision. A mouse model of RPE65-related retinal dystrophies has been rec ....RPE65 is a gene that is found exclusively within the retina. At the moment the exact role of RPE65 is not known, however recent research has shown that mutations in the RPE65 gene have been found in a number of inherited retinal dystrophies (these dystrophies include Leber congenital amaurosis and autosomal recessive retinitis pigmentosa). It therefore appears that a functional, non-mutated RPE65 gene is essential for normal vision. A mouse model of RPE65-related retinal dystrophies has been recently developed, by producing a RPE65 knockout mouse breed in which the mouse's RPE65 gene has been mutated into an inactive form. Research on these mice have shown that they develop retinal dystrophies very similar to those seen in patients with mutated RPE65 genes. We propose to use these RPE65 knockout mice to test potential methods for treating the RPE65-related retinal dystrophies in patients. In particular, we will study the potential of using gene therapy to treat these diseases. The project will involve delivering a new, functional RPE65 gene to the retinas of the RPE65 knockout mice. The new, functional RPE65 gene will then replace the inactive, mutated RPE65 gene within the mouse retinas, an action that we predict will be able to stop these mice developing retinal dystrophy. Performing such a study will allow us to improve our understanding of the RPE65-related retinal dystrophies, and provide an indication of whether they can be treated with gene therapy.Read moreRead less
Infectious Large Capacity Vectors For Gene Therapy
Funder
National Health and Medical Research Council
Funding Amount
$374,625.00
Summary
The next 25 to 50 years will witness the culmination of a demographic shift in the age of the population which will be associated with an increasing importance of both inherited predispositions to late-onset chronic, complex diseases and natural degenerative processes. Medicine has historically sought to manage and cure the symptoms of disease. The focus for therapy has begun to switch from alleviating the ailments to establishing and resolving their causes. On the back of the Human Genome Proje ....The next 25 to 50 years will witness the culmination of a demographic shift in the age of the population which will be associated with an increasing importance of both inherited predispositions to late-onset chronic, complex diseases and natural degenerative processes. Medicine has historically sought to manage and cure the symptoms of disease. The focus for therapy has begun to switch from alleviating the ailments to establishing and resolving their causes. On the back of the Human Genome Project, genetics research will identify genes that are central to these processes, leading to gene-based medicine. Some of this new treatment will be drug-based but an alternative is the correction of the defective genes themselves gene therapy to either replace inherited faulty genes or to provide novel or modified genes that may help the repair and maintenance of tissue, or combat abnormal processes such as cancer. Gene therapy is a field still in its infancy with just a few qualified successes reported in the past few years. Persistent expression of a transgene at therapeutic levels is required for successful gene therapy. Most of the currently used vector and virus systems have a small capacity and usually employ a reduced (cDNA) copy of the transgene lacking natural control mechanisms. These are prone to vector loss and promiscuous expression or loss of expression. The delivery of genomic DNA up to 20 times this size would enable genes to be transferred in entirety, including their natural regulatory elements. This project aims to develop a vector system based on Herpesviruses that tackles some of the problems with the current generation of gene therapy vectors. This system is particularly aimed at providing long-term gene expresssion at physiological levels and safe, efficient delivery systems through the use of genomic DNA.Read moreRead less
The Role Of Bone Morphogenetic Proteins In The Pathogenesis Of Pulmonary Hypertension
Funder
National Health and Medical Research Council
Funding Amount
$236,540.00
Summary
Many people develop problems with the blood vessels in the lungs, which then leads to a narrowing of these vessels and consequently a back-pressure strain on the heart. These disorders can arise from inherited diseases of the blood vessels themselves, or from accquired lung disease (for example due to smoking or chronic infections). At present there are few treatments which have any benefits for these patients and many must undergo lung or heart-lung transplantation. This project is desigened bo ....Many people develop problems with the blood vessels in the lungs, which then leads to a narrowing of these vessels and consequently a back-pressure strain on the heart. These disorders can arise from inherited diseases of the blood vessels themselves, or from accquired lung disease (for example due to smoking or chronic infections). At present there are few treatments which have any benefits for these patients and many must undergo lung or heart-lung transplantation. This project is desigened both to find out new information about the disease process that affects the lung blood vessels and to offer a strategy for new treatments. The project will use a crippled form of the cold virus to deliver genes to the lining of the lung blood vessels, then see what impact that has on the pressure within the vessels and the ways in which they respond to certain stresses. These studies will be carried out using laboratory animals. If successful, it may be possible to eventually design such viruses to deliver genes which have a helpful therapeutic impact on the disease in patients.Read moreRead less
A Gene Therapy Approach To Acute Myeloid Leukaemia: Reversion Of The Disease Phenotype
Funder
National Health and Medical Research Council
Funding Amount
$744,911.00
Summary
Acute myeloid leukaemia is a cancer of white blood cells in which there is uncontrolled cell growth filling up the blood with cells that do not function properly. The cells also invade various tissues causing further damage. The patient becomes increasingly compromised due to the lack of appropriate normal cell types. The present standard therapy involves radiation or chemicals which are toxic to the leukaemic cells. However, these treatments are also toxic to normal cells so very high doses tha ....Acute myeloid leukaemia is a cancer of white blood cells in which there is uncontrolled cell growth filling up the blood with cells that do not function properly. The cells also invade various tissues causing further damage. The patient becomes increasingly compromised due to the lack of appropriate normal cell types. The present standard therapy involves radiation or chemicals which are toxic to the leukaemic cells. However, these treatments are also toxic to normal cells so very high doses that might be more effective to kill the leukaemic cells can not be given. At the doses which can be administered, the leukaemia often (in greater than 50% of patients) becomes resistant. The present project seeks to use a novel treatment strategy in which genes are used to modify the genetic abnormalities present in these leukaemic cells thereby stopping their growth. This growth suppression will be specific to the leukaemic cells as it targets their abnormal genetics leaving normal cells alone. The gene therapeutics have already been identified in part and the aims of this grant are: 1. To show that the gene therapeutic approach is justified to selectively stop the leukaemic cells from growing. 2. To confirm novel means to identify the inhibitory genes. 3. To determine the best inhibitory gene(s). 4. To show how these inhibitory genes can be delivered to the patients' cells and to model their effect in animal models. This project would represent a paradigm shift in the treatment of leukaemia and also has implications for the treatment of other cancer types.Read moreRead less
Conditionally Replicative Adenoviruses For Mesothelioma Therapy
Funder
National Health and Medical Research Council
Funding Amount
$260,600.00
Summary
Australia has one of the highest incidences of mesothelioma in the world. The clinical outcome for patients with this disease is extremely poor, with median survival of only 6-9 months. The latest developments in chemotherapy, radiotherapy and radical surgery have done little to improve the overall survival rate. New approaches to therapy are thus required. Oncolytic therapy using conditionally replicative adenoviruses (CRAds) is a novel and promising approach to cancer treatment. This strategy ....Australia has one of the highest incidences of mesothelioma in the world. The clinical outcome for patients with this disease is extremely poor, with median survival of only 6-9 months. The latest developments in chemotherapy, radiotherapy and radical surgery have done little to improve the overall survival rate. New approaches to therapy are thus required. Oncolytic therapy using conditionally replicative adenoviruses (CRAds) is a novel and promising approach to cancer treatment. This strategy relies on selective viral replication in (and therefore death of) tumour cells but not normal cells. In principle, mesothelioma is an attractive target for this therapeutic approach owing to its propensity to remain localised to the pleural space until late in the disease. However, for any CRAd strategy to succeed, viral replication must be limited to the tumour cells so as not to cause unnecessary toxicity to normal tissues. This level of specificity can potentially be achieved by using cell-specific promoters to control the expression of viral genes essential for replication. To date however, there have been no reports evaluating candidate mesothelioma-specific promoters in adenoviral vectors. Furthermore, other issues such as tumour a lack of viral receptors or tumour-associated fibrosis could limit viral spread through a mesothelioma mass and reduce the efficacy of the approach. In this proposal we will contruct and test CRAds which are controlled by promoters which we believe will be highly active in mesothelioma, but very poorly active in other tissues. We will test the ability of these new agents to kill mesothelioma cells in tissue culture, in pieces of mesothelioma tumours removed from patients, and in animal models. If successful, this approach could offer new hope for mesothelioma patients.Read moreRead less