Engineered Histones As DNA Carriers With Application In Therapeutic Gene Delivery
Funder
National Health and Medical Research Council
Funding Amount
$417,750.00
Summary
We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy ....We intend to apply our knowledge of protein transport to the nucleus to enhance the delivery of DNA to target cells. This relates to the use of gene therapy to treat genetic defects such as inborn errors of metabolism, where a disease-causing lack-of-function mutation can be overcome by engineering cells within the organism which express, in the necessary quantities and in response to the appropriate regulatory signals, the particular component which is lacking. A limiting factor in gene therapy approaches is the low efficiency of nuclear uptake of introduced DNA, where it has been estimated that < 1% of the DNA taken up is actually expressed. Our proposal seeks to develop approaches to enhance non-viral-mediated gene delivery, in particular by optimising this critical, limiting step of the delivery of exogenous DNA to the nucleus. We intend to apply knowledge from studies of nuclear targeting and chromatin assembly to improve gene transfer technologies. We will build on our work showing that specific signals for nuclear import - nuclear targeting signals (NTSs) - can be used to enhance nuclear gene delivery and expression. Since DNA in the normal cellular context is in the form of chromatin - a specific complex with proteins such as histones - we intend to use reconstituted chromatin as the transfecting DNA, whereby histones engineered to include NTSs and other modular sequence elements will be used. Chromatin should not only enable NTSs and other sequence modules to be linked to the DNA but also protect against nuclease-mediated degradation prior to nuclear entry, condense the DNA to enable more efficient cellular-nuclear entry, and ensure expression of the transfected reporter gene by presenting it to the cell in a physiological context. Our approaches should contribute to bringing gene therapy closer to reality in the clinic.Read moreRead less
I am a molecular and cell biologist with a major research focus on haemopoiesis and leukaemia development. This work principally involves the analysis of mutant mouse models.
NUCLEAR AND TRANSGOLGI TARGETING AND MEMBRANE INDUCTION BY DENGUE NS5 RNA-DEPENDENT RNA POLYMERASE INTERDOMAIN REGION
Funder
National Health and Medical Research Council
Funding Amount
$450,750.00
Summary
Dengue virus is the causative agent of a mosquito-borne disease, Dengue fever, relevant to northern Queensland, where antibodies from a previous infection can complex with virus of a different serotype in a subsequent infection, and cause a severe, potentially fatal form of the disease (Dengue haemorrhagic fever-Dengue shock syndrome). The present proposal seeks to further understanding of the role of the dengue RNA-dependent RNA polymerase NS5, which is essential for viral RNA replication, with ....Dengue virus is the causative agent of a mosquito-borne disease, Dengue fever, relevant to northern Queensland, where antibodies from a previous infection can complex with virus of a different serotype in a subsequent infection, and cause a severe, potentially fatal form of the disease (Dengue haemorrhagic fever-Dengue shock syndrome). The present proposal seeks to further understanding of the role of the dengue RNA-dependent RNA polymerase NS5, which is essential for viral RNA replication, within the viral infectious cycle. We intend to examine the subcellular targeting properties of a short central region (the interdomain) of NS5, which appears to play multiple roles in targeting to both the perinuclear Golgi-membranes and to the nucleus, as well as in inducing intracellular membranes derived from the Golgi which are the site of viral replication. We will determine how NS5 localisation-membrane induction may differ in insect and primate cells, and attempt to isolate binding partners of NS5 from the nucleus and Golgi compartment of insect and primate cells using various different approaches. Our studies should assist in understanding NS5's critical role in the Dengue infectious cycle, and contribute towards devising new anti-viral strategies such as vaccination and-or therapies targeted at the NS5 interdomain.Read moreRead less
A fundamental study of the role of signal transduction pathways in the regulation of Chlamydia's complex developmental cycle. Chlamydia are unique organisms in the microbial world. They are among the smallest bacteria and yet have a complex two-stage developmental cycle. In addition they are major causes of disease in animals and humans with no vaccines available. We have used the recent flood of full genome sequence data to identify over 30 new cell signalling proteins. By understanding how the ....A fundamental study of the role of signal transduction pathways in the regulation of Chlamydia's complex developmental cycle. Chlamydia are unique organisms in the microbial world. They are among the smallest bacteria and yet have a complex two-stage developmental cycle. In addition they are major causes of disease in animals and humans with no vaccines available. We have used the recent flood of full genome sequence data to identify over 30 new cell signalling proteins. By understanding how these cell signaling proteins are organized into pathways and how this microorganism controls its complex growth and developmental cycle, we will be able to develop novel methods of control. We are at the fore front of international research and therefore uniquely placed to conduct this project.Read moreRead less
A study of the nongenomic action of Vitamin D: proposed role of the nuclear VDR and downstream signalling molecules. Vitamin D (1,25D) activates genes in the nucleus through the vitamin D receptor (VDR). 1,25D can also elicit rapid responses at the plasma membrane. This action is critical to the activation of nuclear genes. We hypothesise that a proportion of the nuclear VDR is located at the plasma membrane where it stimulates downstream signalling molecules eg Ras, ERK1/2 and ERK5. We plan to ....A study of the nongenomic action of Vitamin D: proposed role of the nuclear VDR and downstream signalling molecules. Vitamin D (1,25D) activates genes in the nucleus through the vitamin D receptor (VDR). 1,25D can also elicit rapid responses at the plasma membrane. This action is critical to the activation of nuclear genes. We hypothesise that a proportion of the nuclear VDR is located at the plasma membrane where it stimulates downstream signalling molecules eg Ras, ERK1/2 and ERK5. We plan to explore this hypothesis and to identify the signalling molecules. We will also investigate our novel finding that a specific Ras isoform is involved in ERK5 activation. The work will provide new information on signalling pathways.Read moreRead less
Structural studies on the mitochondrial protein import machinery. Proteins transported across biological membranes are generally synthesized as precursors with signal sequences. These signal sequences are decoded by one of a number of membrane-specific protein transport machinery, but how this decoding occurs is largely unknown. This proposal aims to understand the structural basis of protein import into the mitochondrion, a poorly understood biological process. This study will enhance signif ....Structural studies on the mitochondrial protein import machinery. Proteins transported across biological membranes are generally synthesized as precursors with signal sequences. These signal sequences are decoded by one of a number of membrane-specific protein transport machinery, but how this decoding occurs is largely unknown. This proposal aims to understand the structural basis of protein import into the mitochondrion, a poorly understood biological process. This study will enhance significantly our understanding of mitochondrial biology, and will also have ramifications for other areas of protein transport.Read moreRead less
Guanylate cyclases - an expanding family critical in plant growth and development. The enzyme guanylate cyclase (GC) forms an important signalling molecule. We have identified unique GC molecules from higher plants. We shall use strategic basic research to determine the biological importance of these novel molecules in plant growth and development. We have formed an international team to achieve these goals and also to develop strong scientific links between Australia and other countries such as ....Guanylate cyclases - an expanding family critical in plant growth and development. The enzyme guanylate cyclase (GC) forms an important signalling molecule. We have identified unique GC molecules from higher plants. We shall use strategic basic research to determine the biological importance of these novel molecules in plant growth and development. We have formed an international team to achieve these goals and also to develop strong scientific links between Australia and other countries such as South Africa. The outcomes will provide new insight into the biological function of the novel GCs. Consequently, the new knowledge is critical to the development of novel biotechnological approaches to benefit sustainable agriculture in Australia.Read moreRead less
Regulation of the actin cytoskeleton by LIM kinase 2. Because the regulation of actin cytoskeleton is essential for many cellular processes including cell motility and the normal function of neurons, it is of great importance to understand its regulation. Elucidation of the molecular and biological mechanisms underlying the actin cytoskeleton including cell motility may enable the identification of novel therapeutic targets for the treatment of diseases such as cancer metastasis, Alzheimer disea ....Regulation of the actin cytoskeleton by LIM kinase 2. Because the regulation of actin cytoskeleton is essential for many cellular processes including cell motility and the normal function of neurons, it is of great importance to understand its regulation. Elucidation of the molecular and biological mechanisms underlying the actin cytoskeleton including cell motility may enable the identification of novel therapeutic targets for the treatment of diseases such as cancer metastasis, Alzheimer disease (AD) and/or Multiple Sclerosis (MS) in which the regulation of the actin cytoskeleton is affected.
Read moreRead less
Analysis of the Role of Snapin in the Regulation of SNARE Complex Assembly. The aims of the proposed studies are to investigate the role played by a protein, snapin in the trafficking of membranes and cargo proteins between different compartments inside mammalian cells. Membrane trafficking is a fundamental cellular process that requires a family of related molecules termed SNARES. We have recently discovered that snapin interacts with certain members of the SNARE family, implying a critical rol ....Analysis of the Role of Snapin in the Regulation of SNARE Complex Assembly. The aims of the proposed studies are to investigate the role played by a protein, snapin in the trafficking of membranes and cargo proteins between different compartments inside mammalian cells. Membrane trafficking is a fundamental cellular process that requires a family of related molecules termed SNARES. We have recently discovered that snapin interacts with certain members of the SNARE family, implying a critical role in membrane trafficking. The proposed studies will provide important new insights into the molecular mechanisms underlying the function of both snapin and SNAREs, and membrane trafficking in general.Read moreRead less