Strategy for the consistent preparation of sufficient non-viral large chromosomal vectors for biopharmaceutical applications

Modern medicines and therapies are becoming increasingly complex and specific for a particular disease or group of patients. One very specific type of therapy is Gene Therapy. Gene therapy is the use of genes as medicines. These genes can be delivered to patients either by the use of genetically modified viruses to carry the genes or by using non-viral methods which employ circular DNA molecules called plasmids isolated from bacteria. Both techniques are still in their infancy but are already promising huge medical advances in vaccination, cancer therapy and the correction of genetic disorders. However, viruses suffer from several drawbacks including safety considerations and limited to carry large genetic information. On the other hand large plasmid DNA molecules called BACs ( Bacterial Artificial Chromosomes) can be modified to incorporate a wide range of important control regions which allow expression of the gene in the correct tissue and at the correct time. This larger size poses a scientific and industrial challenge because the size limits the amount that can be made in a single bacterium, in addition, the size also calls for special processing considerations because BACs are fragile. The challenge is to be able to prepare the BAC molecules in the amounts needed to treat the numbers of people who could benefit from these medicines. We need to investigate then develop methods of making the DNA molecules at large scales in the biomanufacturing industry. The quality of the DNA molecules is also paramount. They need to be in the form that is most appropriate for delivering to humans (and animals) and free of contaminating material. The research we propose will enable industry to make DNA molecules that can be made at the large scales. The proposed research outcomes will allow others involved in gene therapy to prepare large DNA molecules for the treatment of cancers, for vaccines and to correct genetic disorders The science proposed will also allow other researchers in related disciplines to benefit from being able to make and manipulate large DNA constructs .We will also explore ways of making the DNA in the correct three dimensional form needed for efficient uptake into cells so that the DNA is an effective medicine.

We will construct novel BAC vectors. One will have two origins of replication. The other will contain a single switchable origin. The first type of BAC vector will have the F origin giving stable copies of 1-2 per cell. We will place into this,a second origin that will be based on a silent multicopy origin from the pMB1 family of plasmids. The mechanics of this origin are known well enough to manipulate it to be silent during the first stages of growth and then being induced to express the RNAII primer of replication and thus the initiation of DNA replication at this multicopy origin. The second type of construct will consist of a single origin of replication in the plasmid. This will be the wild type pMB1 origin with the Rop protein that keeps the copy number to 10-15 per cell. The addition of the cer DNA sequence that prevents dimer formation will achieve a highly stable low copy plasmid. This replicon will be induced to a high copy number by use of antisense inhibition of RNAI (the normal antisense controller of the RNAII replication primer) In addition we will reduce the expression of the antibiotic resistance gene in these constructs. Such genes are usually expressed to a much higher level that is needed and the over expressed protein places a burden on the growth of the cell. Sequences that will increase the binding of DNA gyrase and thus increase the level of supercoiling will be included in the plasmid constructs. The constructs will be tested in batch and fed-batch bioreactors to assess the maximal levels of high quality large BAC DNA that can be prepared. Growth characteristics of the engineered strain and the optimal harvesting time to maximise specific ( e per unit biomass) supercoiled yield will be investigated. The aim is to have generic plasmid systems that can be used in most E. coli strains for others to use in making large plasmids (BACs) in bioreactors.