Latest Research
SCIENTISTS Bobby Gaspar and Adrian Thrasher together with Dr Marlene Carmo are making great strides in the Development of Gene Therapy for Familial HLH which is being funded by the H R Trust at the Institute of Child Health, London.
The team are looking at replacing Perforin, the commonest gene to cause inherited HLH. They have discovered that in order to cure HLH, patients who have an abnormal gene need to be given a normal perform gene. To achieve this, viruses called lentiviruses are used to carry the normal gene into the patients’ cells.
Cells called stem cells are taken from the bone marrow and infected with the viruses. When they are put back into the patient, the stem cells make white blood cells and these should make normal perforin and prevent the patient getting HLH, when they have a virus infection. It is also important that the perforin gene that is given to the cells is properly controlled, so that perforin is only produced in fully developed white blood cells, not in the stem cells. To do this, the researchers are working to insert into their lentiviruses, control elements, that will prevent them producing perforin in the stem cells.
The researchers have made several different lentiviruses to find out which one works best to produce perforin in white blood cells but not stem cells. They tested these first by infecting cell lines lacking perforin and measuring how much perforin was produced. The best virus was then used to infect stem cells from mice that have an abnormal perforin gene, just like HLH patients, and the stem cells were then put back into the mice.
Two months later white blood cells from these mice were shown to produce normal perforin, although in quite low amounts. However, the white blood cells were very good at killing virus-infected cells in tissue culture, suggesting that they may have enough perforin to prevent HLH.
The researchers are now carrying out an exciting experiment with collaborators at Cincinnati Children’s Hospital. Perforin deficient mice were given stem cells infected with the lentivirus carrying the normal perforin gene. The mice are then challenged with a virus called lymphocytic choriomeningitis virus. This causes HLH in perforin deficient mice and the object of the experiment is to see if mice given the stem cells carrying the normal gene are protected from HLH. If the experiment works and the mice are protected, it will be an important step forward in the development of gene therapy for perforin deficient HLH patients.
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THE Histiocytosis Research Trust's latest research project for 2011 will focus on LCH and improving diagnostic and prognostic tests as well as therapeutic targets.
The recipients of The Jon Pritchard Research Award are Professor Frederic Geissmann of King’s College, London and Professor Matthew Collin, of the Institute of Cellular Medicine at Newcastle University.
The duo’s project, entitled Cell Autonomous Mechanisms in the Pathogenesis of Langerhan’s Cell Histiocytosis, was selected from 12 applications received after the Trust invited calls for proposals last May.
Professor Collin explained: “We are delighted to receive this award. It will allow LCH research in the UK to take a unified stance against the disease with clinicians and scientists all working together. With LCH IV and the help of all the patients, we hope to be able to look at the cells and tissues of LCH in new ways, to explore exciting new ground related to the recent discovery of the V600E BRAF mutation in the majority of LCH cases.
“Our project aims to directly test the role of abnormal survival and proliferation in the development of LCH by a number of approaches. In particular, we will generate models to study the role of mutation of BRAF and we will investigate the existence of mutations in BRAF and in related cellular pathways in human patients. These studies are likely to lead to new diagnostic and prognostic tests and therapeutic targets for LCH.”
To date the causes of LCH and the mechanisms responsible for the disease are not well understood making the establishment of diagnostic and prognostic tests and the design of rational treatment extremely difficult.
However, after many years of searching for candidate genetic lesions in LCH, a recent breakthrough study has identified a mutation of the gene BRAF in over half of LCH samples. BRAF is a protein involved in cell activation, survival and proliferation and is mutated in numerous human cancers.
“This suggests that in the majority of patients, LCH is connected to a genetic defect within the cells themselves, although a complex of other factors may still play a role in LCH pathology particularly in patients who do not have a BRAF mutation,” added Professor Collin.
The three-year project will cost just over £327,000 and will be funded by The H R Trust and The Artemis Association of Greece.
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THE Trust is continuing to fund a project on the Development of Gene Therapy for Familial Haemophagocytic Lymphohistiocytosis (HLH) due to perforin deficiency. Scientists Bobby Gaspar and Adrian Thrasher together with Dr Marlene Carmo are working on replacing Perforin, the commonest gene to cause inherited HLH.
Dr Carmo started the project by investigating how to get a normal Perforin gene into white blood cells and whether Perforin protein is produced in the cells in sufficient quantities to enable them to carry out their killing function. She has used viruses to carry the Perforin gene into the white blood cells and has tested several different viruses to see which is best. With two of the viruses the amount of Perforin produced is the same as in normal white blood cells and the white blood cells are able to function normally.
The team now plan to see if Perforin-deficient stem cells can be given a normal gene with the lentiviral vectors and whether the stem cells can produce white blood cells with normal Perforin function. They will also test whether the treatment is safe.