Investigation into factors controlling the efficacy of integration deficient lentiviral vectors for gene delivery

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Vectors or gene delivery vehicles, have been developed to transfer DNA and genes into cells. Vectors are important tools which have been used to investigate basic biology, including how genes function, how cells work and what controls the process of animal development. Many diseases are caused by faulty genes and so recently vectors have also been developed for use in gene therapy; where a functional copy of a gene is used to fix the defect. The most effective vectors have been developed from viruses which have evolved over millions of years to deliver genetic material into cells. To make viruses into safe tools, or medicines, the pathogenic properties of the virus have been removed so the virus can be produced simply in a laboratory for carrying specific genes of interest. The Human Immunodeficiency Virus (HIV) is a lentivirus that has been made safe and is a commonly used vector for delivering genes to cells. This vector is very effective and has been used in clinical gene therapy trials. When HIV normally infects a target cell, it delivers its genetic payload directly into the host cell chromosomes and integrates it seamlessly, so that the viral DNA becomes part of the cell's genome or 'book of life'. This is very important for long-term experiments or treatments, because the DNA is delivered permanently. However, despite the fact that vectors are unable to cause infection, there are problems and side effects associated with integrating genes into the host cell's chromosomes. Not all of a cell's DNA is active; there are large regions of the genome that do not produce any product from the information that they contain. If the virus, or vector, inserts its payload into one of these regions, the chances of it functioning are reduced. Additionally, cells have also changed and evolved over millions of years to defend against DNA that is being delivered into them by viruses (or vectors derived from them). Mechanisms are present that can shut off production (or expression) from delivered genes and silence them. Lastly, a side effect of inserting DNA into a cell is that it can interfere with cell's own DNA and damage or alter existing genes. This is unlikely to cause any problems but if, when treating a person with an inherited disease, it affects a gene involved in controlling how a cell grows or replicates, then this can lead to cancer. Although this is a very small risk, it has unfortunately been observed in gene therapy clinical trials. An alternative is to use an HIV vector that has been altered to prevent it from inserting the DNA it is carrying into the target cell chromosome. These vectors are called integration deficient lentiviral vectors or IDLVs. These are no longer capable of integrating into the cell's genome, but this vector retains all other benefits of the integrating version. This has several consequences: Firstly, it should be safer. Because the delivered DNA does not integrate, it cannot disrupt any of the cell's own genes and this reduces the risk of causing cancer in patients. Interestingly, despite avoiding the problems associated with integrating into silent areas of the genome, IDLV do not work equally well in all cell types when compared to integrating versions of the vector, for reasons that are not clear. While they have been shown to effectively express genes in brain, eye and muscle tissue, they perform worse in liver and stem cells when compared to integrating vectors. This block could be due to many factors but because IDLVs represent a safer and viable alternative to those that integrate, it is important to investigate the reasons to find solutions or to ensure that expression of delivered genes is not eventually affected in tissues where they initially seem to work well. The aim of this project therefore is to determine factors that influence how integration defective lentiviral vectors function in different situations and use that information to produce an improved vector.