Selection of therapeutic gene and vector design

During gene electrotransfer, plasmid vectors encode the information we wish to transfer into cells. Plasmids are circular DNA molecules, which do not integrate into the cell’s genome. The active component of a plasmid vector are the instructions for the production of therapeutic substances. Instructions can contain information to replace or repair defective genes or to elicit the death of tumor cells. The latter can be achieved by direct targeting of tumor cells with genes encoding cytotoxic molecules (gene chemotherapy), or indirectly by targeting tumor angiogenesis (angiogenic gene therapy) or by triggering an anti-tumor immune response (immunogene therapy). The latter approaches include the transfer of genes encoding various tumor antigens (DNA vaccination), or genes encoding immunostimulatory molecules such as interleukin 12, which we will use in our clinical study.

Aside from the gene encoding the therapeutic substance, plasmids also contain a bacterial backbone necessary for the production of plasmids in bacteria. The bacterial backbone carries the information for a selection marker, usually an antibiotic resistance gene, which could be controversial for clinical use. Namely, there is a risk for horizontal transfer of antibiotic resistance genes to microorganisms and risk for an allergic reaction to residues of the antibiotic used during the production of plasmids. European Medicines Agency (EMA) thus recommends avoidance of antibiotic resistance genes. To comply with these recommendations various alternative approaches for plasmid production without antibiotics have been developed. One such approach is the operator-repressor titration (ORT) developed by the Cobra Bio company, which we use to prepare our plasmids.

Cancer and gene therapy

Cancer is a group of systemic diseases with a common feature of immune system dysfunction since tumor cells obviously manage to avoid recognition and removal by the immune system. The biggest challenge in treating cancer are metastases, which are responsible for more than 90 % of all cancer-related deaths. Using the patient’s own immune system to fight cancer is currently the most promising and successful field in oncology. One of the more robust approaches is the use of local ablative treatments that can elicit an immune response against tumor cells located in the therapeutic area (in situ vaccination). In our clinical study we will use electrochemotherapy, which is also considered a local ablative treatment. To achieve a systemic and durable response such treatments need to be combined with treatments that stimulate the immune system or prevent its’ suppression.

A new therapeutic approach in treating cancer is gene therapy in which genetic material (usually DNA), which carries information for the production of therapeutic substances, is inserted inside the patient’s cells. Based on vectors carrying the information gene therapy is divided into viral and non-viral approaches. The most successful non-viral gene therapy approach is gene electrotransfer, which enables the genetic material to be delivered directly into tissues such as skin, muscles, and also tumors. The transfer is enabled by a physical method of electroporation that makes the cell membranes more permeable. Gene electrotransfer is already under clinical evaluation.

Plasmid DNA manufacturing

Plasmid DNA is almost exclusively produced in Escherichia coli cells. Fermentation in bioreactor is followed by cells harvest and lysis to release plasmid DNA. Pure plasmid DNA suitable for human use is obtained in purification process where the main challenge is separation of different plasmid DNA isoforms. Conventionally, the plasmid DNA manufacturing process is done in batch mode, where each production step starts and ends independently from the previous and following step. Batch processing has been used in pharmaceutical and biopharmaceutical industry for decades as it served well for both the industry and the regulatory bodies. Such manufacturing needs large facilities and it still has limited flexibility regarding the production of different products and capacities. The biopharmaceutical industry is in the transition from batch processing to fully continuous and integrated processing, mainly to increase productivity, maximize flexibility, simplify scale-up and process transfers, increase robustness, and minimize cost of goods while still maintaining operational excellence. Continuing integrated processing with integrated PAT, data analysis and process control that can revolutionize plasmid DNA manufacturing is one of the SmartGene.si goals.

Biological drugs, including plasmid DNA for gene therapy, are commonly produced in relatively smaller batches compared to traditional small molecule drugs. In parallel, health authorities  are making  initiatives towards personalized treatments. Due to these fact, important part of future drug manufacturing is in small dedicated efficient manufacturing facilities – we call them SMART GMP facilities. We will develop a SMART GMP facility having turn-key GMP facility (Clean Room) with supporting Quality documentation required for certification so that such facility will be able to be build, placed and up and running within couple of months instead of 1-2 years. Subsequently setting up such facility will decrease starting cost for more than 50%, which will mean huge boost for drugs waiting for investments to start clinical trials and consequently in next year’s result in bigger number of affordable drugs on the market.

Vector delivery

Plasmid DNA vectors, containing therapeutic genes are delivered to patients with different delivery systems. One of the methods is gene electrotransfer (method is called electroporation), a powerful method of DNA delivery, with high promises on the field of DNA vaccination and in cancer treatment therapy. Devices enabling electroporation called electroporators use electrical fields to increase DNA drug delivery efficiency. An exposure of a cell to an adequate amplitude and duration of electric pulses lead to a temporary increase of cell membrane permeability. We will develop and test a new electroporation system (generator, electrodes) and pulse delivery protocol that would maximize gene delivery and reduce pain.