Project: Photochemical internalisation for delivery of short interfering RNA (siRNA) - towards safe and specific cancer therapy

Background._x000D_ The last years have seen a greatly increased understanding of the mechanisms behind cancer development. This has led to the identification of many new potential molecular targets for cancer therapy, typically being proteins that are mutated or aberrantly expressed in the cancer cells, and being responsible e.g. for the uncontrolled growth typical for cancer. To turn off the expression of such proteins can constitute an efficient and very specific cancer therapy. The recently discovered natural mechanism of RNA interference (RNAi) is a very attractive way to achieve this. One way to exploit this mechanism is to introduce so-called short interfering RNA (siRNA) molecules into the cancer cells. Since the effect of siRNAs is based on nucleic acid sequence homology, siRNAs can be designed to specifically target proteins that are altered in cancer cells, potentially constituting a 100 % specific cancer therapy, without the serious side effects commonly seen with current cancer therapies. The siRNA principle has evoked great interest in the biotech and pharmaceutical industry where large investments are being made to realize the enormous therapeutic and economic potential of this technology. However, this potential has yet to be realized. It is commonly acknowledged that the CO reason for this is the lack of efficient, specific and safe methods for delivering siRNAs to the target cells in the body. _x000D_ _x000D_ PCI Biotech AS has developed a proprietary technology (photochemical internalisation, PCI) for light-induced drug delivery, and in the present project PCI will be developed for delivering siRNA efficiently to solid tumours. The PCI technology has been documented in animal models for delivery of several different types of molecules, and is currently in a Phase I clinical trial with a cytotoxic anti-cancer drug. _x000D_ _x000D_ In the PCI technology illumination is used to release therapeutic molecules from so-called endocytic vesicles inside the cell (Fig. 1 in appendix). Many important therapeutic molecules (e.g. siRNA) are taken up into such vesicles and cannot exert a therapeutic effect unless they are released from the vesicles. With the PCI technology such release is obtained by using a photosensitising compound (AmphinexTM) that localises specifically in the membranes of endocytic vesicles and that after light activation will mediate destruction of these membranes (Fig.1 in appendix). In vitro results indicate that PCI is a very efficient technology for delivering siRNA, with enhancements in biological activity of 10 to 100 times being observed. This effect can be observed without inducing cytotoxicity, a feature that is very important for in vivo delivery of siRNA; resulting in increased safety. _x000D_ _x000D_ In vivo there are many delivery hurdles that are not present in vitro, implying that only a very small fraction of the administered siRNA will ever reach the target cells, and that “unnecessary” high doses must be used if proper delivery shall be achieved. This again leads to problems with toxicity and “off-target” effects. PCI is a technology that can efficiently remove a very important delivery hurdle and thus make it possible to exploit the limited number of siRNA molecules reaching their target cells in a much more effective way than what is possible with current “standard” delivery technologies. _x000D_ _x000D_ Goals of the project._x000D_ _x000D_ · The overall technological goal of the project is to develop and document Photochemical Internalisation as an efficient technology for specific delivery of siRNA to solid tumours. _x000D_ _x000D_ CO subgoals are: _x000D_ _x000D_ - Demonstrate siRNA therapeutic effects in animal models_x000D_ - Establish conditions for use of the technology in pancreatic cancer patients._x000D_ _x000D_ For the consortium companies a successful project can lead to the following commercial goals: _x000D_ · Deals for out-licensing the technology to pharmaceutical companies developing siRNA therapeutics._x000D_ · Income from sale of the substances and equipment involved in the technology to such companies_x000D_ · Development of therapeutic siRNAs that can be out-licensed or sold. _x000D_ _x000D_ Consortium._x000D_ The consortium consists of 3 SME’s from Norway and Sweden, all contributing essential competence to the project. PCI Biotech (Norway) has developed the PCI technology and the proprietary photosensitizing molecule necessary for performing this technology. Since PCI-mediated delivery is a function of the light dose, it is very important to be able give homogeneous illumination of all the cells in the target area. In the project this issue will be addressed by exploiting a proprietary light source developed by SpectraCure AB (Sweden). By using multiple laser fibres with computerized dose planning and monitoring this light source makes it possible to generate nearly homogeneous illumination throughout the region to be treated._x000D_

Acronym	PCsiRNA (Reference Number: 4831)
Duration	01/07/2009 - 30/06/2012
Project Topic	This project aims at developing a method for efficient light-directed delivery of short interfering RNA (siRNA) to tumours. siRNAs are gene-regulating compounds with huge therapeutic and commercial potential; the realization of which requires technologies for delivering siRNA into cells in the body
Project Results (after finalisation)	In the first phase of the project it was shown in in vitro studies that the PCI technology could enhance siRNA delivery with several different siRNA delivery vehicles by 10 to 100 times, and a delivery vehicle was chosen for use in animal studies. _x000D__x000D_SpectraCure AB developed a new laser device for tumour illumination in animal models. This device was constructed in such a way that the tumours could be illuminated from several positions simultaneously making it possible to get a much more homogenous illumination of tumours than what would be possible with a single point light source. _x000D__x000D_The SpectraCure light source was used by PCI Biotech in an animal model with tumours that express the gene for luciferase. In this model successful tumour delivery of siRNA against luciferase will lead decreased expression of the luciferase protein and can be observed as a decrease in luminescence from the tumour. Thus, the effect of the treatment was followed by in vivo luminescence imaging. The results of these experiments indicated that illumination with the SpectraCure light source gave a statistically significant increase in the biological activity of luciferase siRNA. However, unfortunately attempts to verify these results with other analysis methods were not successful._x000D__x000D_Further animal studies were done with siRNA against the epidermal growth factor (EGF) Receptor (EGFR), a validated cancer therapeutic target. For these experiments the same animal luciferase-expressing tumour model as described above was employed. This made it possible to follow the growth of the tumours in two ways; by analysing the amount of light emitted from the tumour, and by direct measurements of tumour size. The results indicated that EGFR siRNA combined with the PCI illumination treatment could delay the growth of tumours in mice. Unfortunately all necessary experiments could not be finished before the end of the project period. Thus it was, COly due to large variations between individual animals and the rather short duration of the effect, not possible to demonstrate statistical significance in these experiments. _x000D__x000D_In addition to the work with the animal illumination device it was also an important project goal for SpectraCure to lay the ground for a clinical study in pancreatic cancer patients. This work included the construction of an illumination device to be used in such a study, as well as the development of hardware and software for dosimetry, optical fibre placement and treatment monitoring during the illumination phase of the treatment. All the necessary work was completed and incorporated into an application for a clinical study (at University College London Hospital). The clinical trial was however not started before the conclusion of the Eurostars project; the trial was formally under way, but was still awaiting inclusion of the first patient._x000D__x000D_
Network	Eurostars
Call	Eurostars Cut-Off 2

Project partner

Number	Name	Role	Country
3	PCI Biotech AS	Coordinator	Norway
3	siRNAsense AS	Partner	Norway
3	SpectraCure AB	Partner	Sweden