Evaluating the gene delivery potential of E1L4-deficient adenovirus vectors

The aim of this project is to evaluate the performance of a new version of the adenovirus gene delivery system that has been widely considered as a way of achieving gene therapy in people. Adenoviruses, which naturally infect people, can be made incapable of growing and causing disease by removing parts of their genome DNA, and then foreign gene sequences can be added in their place to create a gene delivery vector. When the vector infects a cell, the foreign gene is efficiently taken into the cell but, ideally, no further events of the infectious cycle occur. Instead, the foreign gene becomes operative in the cell and has a beneficial effect. However, many earlier versions of adenovirus vectors suffered from two related problems: the gene delivery effect did not last very long and powerful immune responses were generated to viral proteins made from the so-called 'late' genes that had not been removed from the genomes of these vectors. Adenovirus late genes provide proteins that are essential for the vector to be grown in the laboratory. If you take the late genes out of the vector, you cannot grow the vector particles that you need to use in therapy unless you provide the proteins in a different way. The two options are to mix the vector with a second virus that still has these late genes, so it can help the vector to grow, or to make special cells that contain the missing genes and to use these to grow the vector. In the first case you end up with a mixture from which you have to separate out the vector particles that you want, which is difficult on a large scale, while in the second case, persuading cells to make all these proteins in the large amounts needed has so far been impossible. We have been working on a new way to prevent the production of these late viral proteins when a vector is used, that works without actually removing all the genes. This means that the genes can still be used to provide the necessary proteins when growing the vector in the laboratory. The trick is to remove the genes for just two late proteins, which we have discovered turn on the production of all the others. When these two proteins are provided the vector grows well but otherwise, none of the late genes it carries will work. We have made cells that will make these two key proteins 'on demand' / the cells cannot be asked to make them all the time because they harm the cells / and have used them to grow a virus that has the two genes removed. Now we want to find out how this deleted virus behaves when used for gene delivery. First of all, we need to study its basic properties, such as how easy it is to grow and how stable its particles are. Then, we are planning to use it to study the delivery of a test gene into cells and tissues, to find out how long the gene stays around and keeps working.

Gene delivery vectors based on human adenovirus have considerable promise in a wide range of applications but technical difficulties have so far limited their utility. We recently showed that two late proteins from the adenovirus L4 region are required to turn on full late gene expression, and have used this finding to develop a novel adenovirus vector that cannot encode these proteins, together with a cell line that supports its growth in the laboratory. This proposal will test the properties of that vector in vitro and in vivo. First, the ease of high titre vector particle stock preparation and vector particle stability will be determined, since these practical issues will impact on the utility of the vector system. These will employ widely accepted methods that are well-established in my laboratory, linked to our newly derived inducible L4 protein-expressing 293 cell derivative. Second, the extent and longevity of transgene delivery by this vector to mouse liver will be compared with a more conventional, E1-deficient, vector that retains L4 genes. Beta-galactosidase transgene product present over a time course will be visualised in sections of fixed, embedded tissue and the amounts of transgene retained will be determined by Southern blotting. Third, since it has been demonstrated that immune responses to residual late gene expression products are a significant factor in the short duration of transgene expression achieved by E1-deficient vectors, the nature and extent of these responses elicited by the new vector will be compared to those generated by an E1-deficient vector. Serum IgG will be monitored by ELISA and the frequency of different classes of specific immune cells in splenocytes determined by cytokine-specific ELISPOT assays. Finally, the effect of these responses to the two vectors on the subsequent transduction of a second transgene into mouse liver will be compared, to see if the anticipated reductions in immune responses have a practical impact.