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

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.

Integration deficient lentiviral vectors (IDLVs) deliver genes to a wide range of cell types, including non-dividing cells. They have the additional benefit of a reduced risk of insertional mutagenesis compared to integrating viruses. Following reverse transcription and nuclear delivery of the lentiviral payload, IDLVs form stable episomal circles, which provide long-term expression of transgenes in post-mitotic cells. As IDLVs lack an origin of replication, the episomes are diluted out of rapidly dividing cell populations. Consequently, IDLV are effective and safe tools for either long-term or transient expression, depending on the target tissue. It was thought that IDLV would not be affected by position effects that can reduce expression from integrating vectors, but variability and lower expression have also been observed from episomal vectors. The reason for this is unclear; while IDLV work efficiently in brain and retina, expression is generally lower in liver and haematopoietic stem cells, when compared to integrating lentiviruses. The aim of this project is to determine what factors may influence differential levels of expression observed in integrating and non-integrating vectors. Candidate causes include obstruction of viral entry and reverse transcription and nuclear import. Cell-type specific factors such as epigenetic effects influence integrating vectors, so these are also likely to affect gene expression from IDLV. Vector efficacy may also depend on both contents and context of what is encoded on the backbone. Using different target cells in vitro and in vivo we aim to elucidate the influence of factors on transgene expression using several vectors that are identical except for their promoter configuration and their ability to integrate DNA into the host cell genome. Information gained will lead to the development of a safer and more efficient lentiviral vector and applicable to generic vector design.

Investigating the controlling factors on episomal expression patterns will produce results that will have an impact on a community investigating many areas of biological science. The project aims to answer questions that could ultimately improve a versatile tool that could be used to express genes (or deliver nucleic acids) for numerous applications, including developmental and stem cell biology, homologous recombination, neuroscience and basic biology, epigenetics and control of gene expression. Medical sciences could also benefit if Integration deficient lentiviral vectors (IDLV) are applied to treatment of inherited diseases or for areas where short term expression is required, such as imaging and vaccination. All of these areas are of direct importance to healthcare and the general public. Because medical research funding is frequently obtained from charities, any advances in technology that could be utilized for biomedical applications could be used by those charities to improve public education and fundraising. Depending on the results obtained, further understanding epigenetic influences on delivered genes could have an impact on commercial drug discovery or production where negating interference from surrounding genes or silencing effects is of importance. Understanding epigenetic effects on extrachromosomal vectors would move the field of DNA delivery forward significantly, enabling generic improvements to be made to vectors of all types. This project has the potential to improve expression from delivered transgenes and has scientific impact for academic, commercial and medical applications. Consequently it would foster international collaboration as well as improve products or processes that are economically important to the UK, such as drug production. Following this 3 year project, it is likely that information would be rapidly disseminated through publication, presentation and collaborations and could be integrated into many different research areas with immediate effect. Where optimisation and application of knowledge gained to a novel subject or process is required, the impact would be visible within 5 years. Clinical applications are likely to take 10 years to become implemented. Disseminating data from this project to reach the wide audience who would benefit from it requires several different approaches. To improve the impact of the research, data would be presented at various national and international conferences and in open access journals that target a general readership, rather than a specific field. Our group collaborates extensively with other academic institutions both locally and worldwide and would actively encourage application of any relevant results to other groups' specialist area. Collaboration is not limited to academia and results would be disseminated to pharmaceutical, healthcare and specialised companies though direct and indirect contacts within the commercial sector. We are also involved in organising public outreach and education days as part of national conferences, where the impact of the research and its relevance to biomedical science and how it could enhance quality of life would be promoted to the general public, charities and the media.