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Development of globin expression vectors for human gene therapy

Exploitable results

Non-viral therapy would in principle be preferable over viral therapy especially in underdeveloped countries simply because non-viral components are easier and cheaper to synthesize, store and transport. However they’re a number of problems with this approach, including efficient delivery of DNA to the nucleus of the target cell and stable integration and expression in the host cell nuclei. We have developed a novel non-viral delivery protein using parts of the nuclear transport machinery and a DNA binding moiety into a hybrid protein for the delivery of DNA to a cell nucleus. Non viral gene therapy: Several hurdles have to be overcome for the successful application of non-viral delivery methods. Firstly there is the aspect of delivery to specific cells using cell surface specific proteins. We have used integrin-binding proteins as part of the delivery vehicle and we have shown that specific targeting can be obtained, although the efficiency will still have to be improved. The next hurdle after escape from the endosome in the cytoplasm is the crossing of the nuclear membrane. It is clear from a number of studies that this works with reasonable efficiency in dividing cells, but that it is very inefficient in non-dividing cells. As a result the methods that use more toxic delivery compounds appear to work more efficiently. This is because these compounds kill more target cells leading to proliferation and division of the surrounding cells that as a result take up DNA. Unfortunately stem cells do not divide (nor should they be killed) and hence a more efficient way of delivery to the nucleus has to be developed. We have addressed this in two ways. First some viruses can do this efficiently without using cell division including the insect virus, baculovirus. We have therefore started an analysis of the capsid delivery proteins of this virus after showing that they can deliver DNA to the nucleus of non-dividing mammalian cells. We have now purified capsids and are presently characterizing the proteins at the delivery end of the viral core. We have also developed hybrid proteins that consist of a part that binds DNA, the outside of the cell for targeting and importantly a component that is normally part of the nuclear import machinery. These hybrid proteins still have to be optimised for efficient delivery, but we have shown that this is a promising route as we do obtain a very substantial improvement of delivery to the nuclei of non-dividing cells. A concept patent application has been prepared for submission.
Non-viral therapy would in principle be preferable over viral therapy especially in underdeveloped countries simply because non-viral components are easier and cheaper to synthesize, store and transport. However they are a number of problems with this approach, including efficient delivery of DNA to the nucleus of the target cell and stable integration and expression in the host cell nuclei. Most vectors are aimed at integration of the therapeutic DNA into the host DNA, however a stably maintained episomal DNA would be just as effective and would not need the development of an integration moiety in the delivery vehicle. We have developed an episomal vector that expresses high levels of beta globin for a prolonged period of time in the absence of drug selection. One important aspect of non-viral (or viral) gene delivery concerns the choice between the integration of the DNA in the host genome or keeping the DNA stably as an episome. We have tested several strategies and are testing additional strategies to obtain efficient stable integration in the host genome using recombinases, transposase or satellite DNA. The most promising of these is the use of minos transposase which does increase integration in cultured cells. It is also capable of existing DNA in mice (reintegration has confirmed yet) that provides an unexpected bonus, namely a novel way to develop forward genetics in mammals, which would be extremely important for functional genomic approaches in future (a patent has been filed). More directly applicable we have developed an episomal vector that is maintained over a long period of time and which provides high expression. Interestingly the stability of this Epstein Barr virus replicon based vector appears to depend on the presence of a functional beta-globin LCR. This unexpectedly provides an additional level of specificity, as such an episome would be lost from non-haematopoietic cells. A patent on this vector has been filed.