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Development of Microsystems for In-Vivo Human Gene Therapy

Deliverables

The Cork Cancer Research Centre has invented (jointly with the NMRC and Gaeltec) a device "EndoVac", suitable for electroporation of intraluminal tissue and designed for simple integration onto a endoscope/laproscope head. The device consists of a hollow chamber within which a pair of parallel planar macroeletrodes are contained. These electrodes can effectively electroporate several cm cubed of tissue with one pulse from the connected cythorlab unit. Potentially the silicon needle electrodes produced by the NMRC may expand the application of the device towards transdermal delivery of genes and drugs. Research carried out at the CCRC initially identified parallel planar electrodes as optimum for effective electroporation, and extensive work was carried out on the reduction to practice of the unit. The electroporation parameters were examined using a flow cytometry unit to examine molecular cell entry (altered pulse frequency, number, voltage and length) and also genetic based reporter genes (luciferase) identified the optimum pulse types both in-vitro and in-vivo. The market for this unit includes both the cancer therapeutics area but the application can also be expanded to include the treatment of diseased organs accessible endoscopically. Once all IPR issues have been resolved an extensive dissemination of the information gained will be carried out via peer reviewed journals and through presentations both in the EU and the USA.
NMRC Ireland (to be known as the Tyndall National Institute from January 2005), and Cork Cancer Research Centre has invented jointly with Gaeltec a device called "EndoVac", suitable for electroporation of intraluminal tissue and designed for simple integration onto a endoscope/laproscope head. The device consists of a hollow chamber within which a pair of parallel planar eletrodes are contained. These electrodes can effectively electroporate several cm cubed of tissue with one pulse from the connected pulse generating instrumentation. Potentially the silicon needle electrodes produced by the NMRC may expand the application of the device towards transdermal delivery of genes and drugs. Research carried out at the CCRC initially identified parallel planar electrodes as optimum for effective electroporation, and extensive work was carried out on the reduction to practice of the unit. The market for this unit includes both the cancer therapeutics area but the application can also be expanded to include the treatment of diseased organs accessible endoscopically or laparoscopically.
Analytical and numerical simulations of electric field and current distribution in the tissues during electroporation have been performed. The model was has been used to optimise the design of electroporation electrode. We have shown that tissue electroporation requires relatively large electrodes. In the case on co-planar interdigitated electrodes, the depth of electroporation is comparable to the spacing between electrodes. The field concentration at the edge of electrodes is the cause of superstimulation and subsequent possible tissue damage. Needle tips can help to penetrate the tissue. An electrode-electrolyte model has been developed and measurements have shown its predictive value. This model has then been further extended to take into account a tissue, which is modelled as a network of finite RC-elements. To verify the models an "artificial tissue" based on the hydrogel pHEMA has been developed. Our tissue model has been applied to determine the best electroporation pulse signal. We anticipate improvement of electroporation efficiency and reduction of cell damage when replacing usual pulsed signal with a modulated signal at a frequency that is related to relaxation effects in tissues.
CythorLab is an intelligent electroporation system combining refined electroporation with impedance measurement to create the next generation gene- and drug delivery systems for use in vivo and in vitro. ADITUS is the inventor of the CythorLab intelligent electroporation system. CythorLab combines an advanced pulse generator with an impedance measurement system all controlled from the attached personal computer. When connected to the electroporation treatment head (created by other members of this consortium) it constitutes the complete delivery system for the ENDOPORATOR project. Today's gene- and drug delivery methods, including viral vectors and traditional electroporation, all have shortcomings like high costs and unwanted side effects. The yield in traditional electroporation has been very low and has only been adapted for use in vitro. Only few attempts have been made to use the technology in vivo due to the difficulty in producing optimal results on a consistent basis. There is a need for a delivery method that is Effective, Simple and Cost Effective. This will be solved by the CythorLab system. CythorLab monitors the changes in the cell impedance thereby giving the user an indirect measurement of the size of the pores in the cell membranes. This information will show the user when to stop the electroporation process - just at the right time - not too early and not too late. With this new type of control mechanism, the electroporation method generates optimal results every time and can now safely be used for transfection both in vitro as well as in vivo. The software, specifically designed for CythorLab, has powerful features for setup and storage. It is possible to plan and setup experiments, to have accounts with Login and Access control that protects setup and results and to visualize the effect of the electroporation prior to the experiment. The CythorLab system has received patent approval from Sweden and from Australia. Patent is pending in all other major regions and close to approval both in Europe and in the US. More patents will follow in the next 2 years. A trademark for CYTHOR and ADITUS has been approved in Europe and is close to approval in the US.
NMRC Ireland (now known as Tyndall National Institute) has developed processes for optimising the fabrication of silicon microneedle arrays using both etch and dry etch technologies in the ENDOPORATOR project, these have been used as electroporation electrodes, but as the needles may also be hollow then they can be used for delivery of drugs or other therapeutic agents to tissue. NMRC has fabricated silicon needles up to and over 300 microns in height, with complete control over the needle height, shape, surface smoothness, needle quality, and reproducibility across a wafer or batch of wafers.
Gaeltec has invented (jointly with the partners at the University of Cork) the ENDOVAC Electroporation Treatment Head. This forms part of the subject of an as yet incomplete UK Patent Application filed by Gaeltec in respect of the collaborative work within the consortium. Gaeltec has reduced this concept to practice with stainless steel electrodes, micro-engineered needles on a silicon substrate, some such silicon substrates also incorporating an integral temperature sensor. A disposable treatment head is a probable future development that is anticipated in the patent. The hardware contributed by Gaeltec includes endoscopic attachments and laparoscopes with fixed treatment heads (that may also be disposable). The thinking has also been extended to include an alternative aspect ratio where the microelectrodes are applied by vacuum into a tumour located on the skin.Gaeltec has also produced delivery sub-systems suitable for use with both macroscopic needles and the ENDOVAC treatment head. Delivery sub-systems for use with both endoscopes and laparoscopes have also been created.