METHOD

Abstract

The present invention concerns an in vivo method for introducing an mRNA molecule (which is not associated with a carrier) into the cytosol of a cell(s) in a subject, by the use of photochemical internalization, wherein the photosensitising agent is a sulphonated meso-tetraphenyl chlorin, sulfonated tetraphenylporphine or a di- or tetrasulfonated aluminium phthalocyanine used in an amount of 0.0001 to 1 g. The method may be used to express a polypeptide in the subject. The invention is also directed to pharmaceutical compositions containing the photosensitising agents and the mRNA and uses of the molecules in therapy, e.g. to treat or prevent cancer or an infection.

Claims

1. An in vivo method for introducing an mRNA molecule into the cytosol of a cell(s) in a subject, said method comprising i) contacting said cell(s) with an mRNA molecule and a photosensitising agent, wherein said mRNA is not associated with a carrier, and ii) irradiating the cell(s) with light of a wavelength effective to activate the photosensitising agent, wherein said photosensitising agent is a sulphonated meso-tetraphenyl chlorin, sulfonated tetraphenylporphine or a di- or tetrasulfonated aluminium phthalocyanine used in an amount of 0.0001 to 1 g.

2. The method as claimed in claim 1 wherein said photosensitising agent is TPCS.sub.2a or a pharmaceutically acceptable salt thereof.

3. The method as claimed in claim 1 or 2 wherein said photosensitising agent is used in an amount of 0.001 to 0.1 g.

4. The method as claimed in any one of claims 1 to 3 wherein said photosensitising agent is used at a concentration of 0.005 to 200 g/ml, preferably 0.05 to 20 g/ml.

5. The method as claimed in any one of claims 1 to 4 wherein the mRNA molecule is from 50 to 10,000 nucleotides long.

6. The method as claimed in any one of claims 1 to 5 wherein the mRNA is used in an amount of 0.1 to 100 g, preferably at a concentration of 5 to 5000 g/ml.

7. The method as claimed in any one of claims 1 to 6 wherein said mRNA is expressed in said cell(s), wherein preferably the polypeptide expressed by said mRNA is a therapeutic molecule, preferably an antibody, a vaccine polypeptide or a cytotoxic molecule.

8. The method as claimed in any one of claims 1 to 7 wherein said cell(s) is a mammalian cell(s) or a fish cell(s).

9. The method as claimed in any one of claims 1 to 8 wherein the light has a wavelength of 400-475 nm, preferably 400-435 nm, or a wavelength of 620-750 nm, preferably 640-660 nm.

10. The method as claimed in any one of claims 1 to 9 wherein said contacting step is performed for 30 minutes to 4 hours, preferably 45 minutes to 90 minutes, for example 60 minutes.

11. The method as claimed in any one of claims 1 to 10 wherein the cell(s) is irradiated for between 15 seconds and 60 minutes, preferably for 0.5 to 12 minutes, preferably for 4 to 6 minutes.

12. The method as claimed in any one of claims 1 to 11 wherein the cell(s) is irradiated with a light dose of from 0.01 to 50 J/cm.sup.2.

13. The method as claimed in any one of claims 1 to 12 wherein said cell(s) is contacted with said mRNA and photosensitising agent simultaneously, separately or sequentially.

14. The method as claimed in any one of claims 1 to 13 wherein said subject is a mammal or a fish, preferably the mammal is a monkey, cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit or guinea pig, most preferably the subject is a human.

15. The method as claimed in any one of claims 1 to 14 wherein said mRNA and/or said photosensitising agent is administered locally, preferably intradermally or intratumourally.

16. An in vivo method of expressing a polypeptide in a cell(s) in a subject by introducing an mRNA molecule into a cell(s) by a method as defined in any one of claims 1 to 15, wherein said mRNA molecule encodes said polypeptide.

17. A pharmaceutical composition comprising an mRNA molecule and a photosensitising agent, wherein said mRNA is not associated with a carrier and said photosensitising agent is a sulphonated meso-tetraphenyl chlorin, sulfonated tetraphenylporphine or a di- or tetrasulfonated aluminium phthalocyanine and is provided in the amount of 0.0001 to 1 g, wherein preferably said photosensitising agent is as defined in any one of claims 2 to 4 and/or said mRNA is as defined in any one of claims 5 to 7.

18. The composition of claim 17 for use in therapy.

19. An mRNA molecule and a photosensitising agent for use in treating or preventing a disease, disorder or infection in a subject by expressing a polypeptide encoded by said mRNA molecule, wherein said mRNA is not associated with a carrier and said photosensitising agent is a sulphonated meso-tetraphenyl chlorin, sulfonated tetraphenylporphine or a di- or tetrasulfonated aluminium phthalocyanine used in an amount of 0.0001 to 1 g, wherein preferably said photosensitising agent is as defined in any one of claims 2 to 4 and/or said mRNA is as defined in any one of claims 5 to 7.

20. The mRNA molecule and photosensitising agent for the use of claim 19, wherein one or more cells in said subject are to be contacted with said mRNA molecule and photosensitising agent and irradiated with light of a wavelength effective to activate the photosensitising agent, wherein preferably said cell(s), light, contact, irradiation, subject and/or administration is as defined in any one of claims 8 to 15.

21. The use of an mRNA molecule and a photosensitising agent in the preparation of a medicament for treating or preventing a disease, disorder or infection in a subject by expressing a polypeptide encoded by said mRNA molecule, wherein said mRNA is not associated with a carrier and said photosensitising agent is a sulphonated meso-tetraphenyl chlorin, sulfonated tetraphenylporphine or a di- or tetrasulfonated aluminium phthalocyanine used in an amount of 0.0001 to 1 g, wherein preferably said photosensitising agent is as defined in any one of claims 2 to 4 and/or said mRNA is as defined in any one of claims 5 to 7.

22. The use as claimed in claim 21, wherein one or more cells in said subject are to be contacted with said mRNA molecule and photosensitising agent and irradiated with light of a wavelength effective to activate the photosensitising agent, wherein preferably said cell(s), light, contact, irradiation, subject and/or administration is as defined in any one of claims 8 to 15.

23. A method of treating or preventing a disease, disorder or infection in a subject comprising introducing an mRNA molecule into one or more cells in vivo in said subject according to the methods as defined in any one of claims 1 to 15.

24. The i) mRNA molecule and a photosensitising agent for use, ii) use of an mRNA molecule and a photosensitising agent, or iii) method of treating or preventing a disease, disorder or infection as claimed in any one of claims 19 to 23, wherein said disease, disorder or infection is one which would benefit from expression of one or more polypeptides, preferably for protein therapy, immunotherapy or gene therapy.

25. The i) mRNA molecule and a photosensitising agent for use, ii) use of an mRNA molecule and a photosensitising agent, or iii) method of treating or preventing a disease, disorder or infection as claimed in claim 24 wherein an immune response is generated to said expressed polypeptide, preferably said treatment or prevention occurs via vaccination, wherein preferably said vaccination is prophylactic or therapeutic.

26. The i) mRNA molecule and a photosensitising agent for use, ii) use of an mRNA molecule and a photosensitising agent, or iii) method of treating or preventing a disease, disorder or infection as claimed in claim 24 or 25 wherein said disease is cancer or said infection is a viral or bacterial infection.

27. The i) mRNA molecule and a photosensitising agent for use, ii) use of an mRNA molecule and a photosensitising agent, or iii) method of treating or preventing a disease, disorder or infection as claimed in any one of claims 24 to 26 wherein said mRNA and photosensitising agent is administered or is to be administered intradermally or intratumourally.

Description

[0107] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

[0108] FIG. 1 shows the effect of PCI on intradermal mRNA delivery. The results of bio-luminescence imaging in animal 6 of Example 1A are shown. The animal was injected with a mixture of 2 g luciferase mRNA (TriLink L-6107, 5meC, ) and 0.003 g TPCS.sub.2a in sites A and D (top left and bottom right circles) and 2 g mRNA only in sites B and C (top right and bottom left circles). 1 h after injection all injection sites were illuminated for 6 min with the LumiSource illumination device. 4 hours after illumination the luciferase expression was analysed by bio-luminescence imaging using the IVIS instrument. a. Image of the mouse showing the injection sites and the regions-of-interest. b. Quantitation of the bio-luminescence in the different regions of interest; horizontal black lines denote mean values.

[0109] FIG. 2 shows a comparison of luciferase expression in all treated sites for Example 1A. The animals were injected with 2 g luciferase mRNA (TriLink L-6107, 5meC, Y) alone or in combination with 0.003 g TPCS.sub.2a according to Table 1. Animals were imaged for luciferase bio-luminescence in the IVIS instrument and bio-luminescence in the different ROIs was quantified as described under Methods. Mean values+/standard error of the mean (n=12) are shown.

[0110] FIG. 3 shows the effects of different photosensitiser doses. 3 g mRNA and different amounts of TPCS.sub.2a (indicated in the figure; PCI10 means PCI with a TPCS.sub.2a dose of 10 g, etcetera) were mixed and injected into the skin of mice as described under Methods. 60 min after the injections the mice were illuminated for 6 (in general) or 3 min (indicated in the figure) with blue light. The animals were injected with luciferin and imaged in the IVIS instrument. For each animal the luminescence at the sites receiving mRNA+TPCS.sub.2a was compared to the luminescence at the sites receiving mRNA only, and the fold increase (FI) in luminescence at the TPCS.sub.2a injected sites in each animal was calculated.

[0111] FIG. 4 shows the effects of different light doses. 2 g mRNA and 0.003 g TPCS.sub.2a mixed and injected into the skin of mice. 60 min after the injections the mice were illuminated for 0.5 or 4 min. The animals were injected with luciferin and imaged in the IVIS instrument. The luminescence in defined ROIs around the injection sites was quantified in the IVIS instrument. For each animal the luminescence at the sites receiving mRNA+TPCS.sub.2a was compared to the luminescence at the sites receiving mRNA only, and the fold increase (FI) in luminescence at the TPCS.sub.2a injected sites was calculated.

[0112] FIG. 5 shows the efficacy of the PCI method of intradermal mRNA delivery relative to the use of lipofectamine. The animals were treated according to Table 3. Animals were imaged for luciferase bio-luminescence in the IVIS instrument and bio-luminescence in the different ROIs was quantified as described. Mean values+/standard error of the mean are shown. n=4 for PCI 0.003 and 6 for Lipo only mRNA.

[0113] FIG. 6 shows the effect of PCI on mRNA delivery in tumours. Animals with TC-1 tumours were treated as described in Example 2 and Table 4. 20 hours after illumination the tumours were excised and homogenized, and the luciferase activity (RLU) and protein concentration were measured in the tumour homogenates. Results are shown as median RLU/mg protein for each group (n=3).

[0114] FIG. 7 shows the effect of PCI on mRNA delivery in tumours. Animals with MC-38 tumours were treated as described in Example 3 and Table 5. 20 hours after illumination the tumours were excised and homogenized, and the luciferase activity and protein concentration were measured in the tumour homogenates. A. Results of tumour delivery of luciferase mRNA in single tumours. B. Results of tumour delivery of luciferase mRNA as a meanSEM for each experimental group.

[0115] FIG. 8 shows the sites at which animals were injected by mRNA and TPCS.sub.2a, as described under Methods and treated as described in Table 1.

EXAMPLES

Example 1: Intradermal mRNA Delivery In Vivo

[0116] Experiments were performed to study in vivo naked mRNA delivery to skin in mice.

[0117] Materials and Methods

[0118] Firefly luciferase mRNA (1921 nucleotides in length; L-6107with modified bases 5-methylcytidine (5meC) and pseudo-uridine (); L-6307unmodified mRNA; L-7202with modified base 5-methoxyuridine (5moU), and with the TriLink CleanCap modification as the capping structure, purchased from TriLink Biotechnologies, San Diego, USA) and TPCS.sub.2a (Amphinex, PCI Biotech AS, Norway) were mixed in a volume of 20 l PBS and the mixture was injected into the skin of mice at time 0. Different doses were used in different experiments, and in control samples no TPCS.sub.2a was used. Injections were performed in 4 sites (A-D) per mouse (see FIG. 1 and diagram and table below), and the injection sites were illuminated in general 60 min after mRNA/TPCS.sub.2a injection. With this set-up the two sites in each animal not receiving TPCS.sub.2a act as controls internal to each animal. This is useful for being able to correct for possible differences between animals e.g. in the injection and distribution of luciferin prior to the bio-luminescence imaging.

[0119] Injection sites were illuminated with blue light (wavelength between 400 and 540 nm with a peak at around 435 nm) from the LumiSource illumination device. 4-6 or 20-24 hours after illumination luciferase activity at the injection sites was analysed by bio-luminescence imaging in the IVIS instrument (IVIS Spectrum, model 124375R from PerkinElmer). Pictures were taken 20 min after luciferin injection, and bio-luminescence in a defined region-of-interest (ROI) around each injection site was quantified. The procedure for IVIS imaging was as follows: i) The mice were given an intraperitoneal injection of 3 mg D-Luciferin (200 l of 15 mg/ml stock).

[0120] ii) After approximately 10 min, the mice were anaesthetized by a subcutaneous injection of Zoletil (10-15 mg/kg xylasin, 5-10 mg/kg butorphanol and 15-20 mg/kg Zoletil (containing zolazepam and tiletamine)).

[0121] iii) 20 min after D-luciferin injection, mice were placed in the IVIS instrument and pictures were taken with automatic exposure of Luminescence (Andor camera IS0825R4582; iKon Living Image version: 4.5.2.18424. Binning factor: 8; Excitation filter: Block; Emission filter: Open; f Number: 1)

[0122] iv) The bio-luminescence in ROIs covering each injection site was measured to quantify luciferase expression. The detailed set-up of a typical experiment (Example 1) can be found in Table 1.

Example 1A: PCI Enhances Delivery of mRNA

[0123] Animals were injected by mRNA and TPCS.sub.2a as described under Methods and treated as described in Table 1 with the sites shown in FIG. 8.

TABLE-US-00001 TABLE 1 Set-up of the experiment in Example 1A. The different injection sites in each animal are shown in the diagram above. Amount of Illumination Animal mRNA TPCS.sub.2a (g) (min blue no./site Group name (g) per injection. light) 1-A Control, only mRNA 2 6 1-B PCI 0.003 B 2 0.003 6 1-C PCI 0.003 C 2 0.003 6 1-D Control, only mRNA 2 6 2-A PCI 0.003 A 2 0.003 6 2-B Control, only mRNA 2 6 2-C Control, only mRNA 2 6 2-D PCI 0.003 D 2 0.003 6 3-A Control, only mRNA 2 6 3-B PCI 0.003 B 2 0.003 6 3-C PCI 0.003 C 2 0.003 6 3-D Control, only mRNA 2 6 4-A PCI 0.003 A 2 0.003 6 4-B Control, only mRNA 2 6 4-C Control, only mRNA 2 6 4-D PCI 0.003 D 2 0.003 6 5-A Control, only mRNA 2 6 5-B PCI 0.003 B 2 0.003 6 5-C PCI 0.003 C 2 0.003 6 5-D Control, only mRNA 2 6 6-A PCI 0.003 A 2 0.003 6 6-B Control, only mRNA 2 6 6-C Control, only mRNA 2 6 6-D PCI 0.003 D 2 0.003 6

[0124] Results

[0125] FIG. 1a shows bio-luminescence imaging for the four injection sites in a typical animal (animal 6 from Table 1). It can be seen that the sites subjected to PCI (sites A and D) exhibited significantly stronger luminescence than the control sites receiving mRNA only (B and C). This is also reflected in the quantitation of the luminescence in the ROIs (panel b) showing that in this animal the mean bio-luminescence for the PCI treated sites was more than 5 times higher than for the mRNA only sites.

[0126] FIG. 2 shows the mean values of the mRNA only and PCI-treated sites (12 sites of each) for all the animals in Example 1A. It can be seen that on the average the employment of PCI enhanced mRNA delivery about 3 times.

Example 1B: mRNA Delivery at Different Photosensitiser Doses

[0127] In Example 1B experiments were performed to further explore the TPCS.sub.2a dose response relationships for intradermal mRNA delivery in detail.

[0128] 3 g mRNA and different amounts of TPCS.sub.2a (ranging from 0.0003 g to 10 g) were mixed and injected into the skin of mice as described under Methods. 60 min after the injections the mice were illuminated for 6 (in general) or 3 min (in one case) with blue light from the LumiSource illumination device. The animals were injected with luciferin and imaged in the IVIS instrument as described under Methods. The luminescence in defined regions of interest round the injection sites was assessed in the IVIS instrument. For each animal the luminescence at the sites receiving mRNA+TPCS.sub.2a was compared to the luminescence at the sites receiving mRNA only, and the fold increase (FI) in luminescence at the TPCS.sub.2a injected sites in each animal was calculated by the formula

FI=luminescence.sub.mRNA+TPCS2a/luminescence.sub.mRNA only.

[0129] Results

[0130] As can be seen from FIG. 3 with 6 min blue light illumination, PCI at TPCS.sub.2a doses above 0.3 g had a strongly detrimental effect on mRNA delivery with a strong decrease in luciferase expression being observed (PCI10, PCI3 and PCI1). TPCS.sub.2a doses of 0.3 to 0.03 g did not alter luciferase expression significantly as compared to what was obtained with mRNA alone, while at doses of 0.01 g and below a significant enhancement of mRNA delivery was observed, also in accordance with what was found with the 0.003 g dose in Example 1A. With the 0.003 g dose also two different light doses were tested and it is apparent that a comparable enhancement of mRNA delivery was obtained with both 6 and 3 min illumination. Furthermore it can be seen that PCI also improved mRNA delivery at the even lower TPCS.sub.2a dose of 0.0003 g.

Example 1C: mRNA Delivery at Different Light Doses

[0131] An experiment was performed to study the light dose relationship for mRNA delivery with PCI with 0.003 g TPCS.sub.2a. The Luciferase mRNA used was L-6307 FLuc unmodified mRNA from TriLink, 2 g per injection. mRNA and TPCS.sub.2a were mixed and injected intradermally at time 0 at four sites per mouse (as described under Methods and in Table 2). The injection sites were illuminated 60 min after mRNA/TPCS.sub.2a injection, with blue light from the LumiSource illumination device. Illumination times of 0.5 of 4 min were used, and the efficacy of mRNA delivery was assessed by in vivo fluorescence imaging as described under Methods. The luminescence in defined ROIs around the injection sites was quantified in the IVIS instrument. For each animal the luminescence at the sites receiving mRNA+TPCS.sub.2a was compared to the luminescence at the sites receiving mRNA only, and the fold increase (FI) in luminescence at the TPCS.sub.2a injected sites was calculated by the formula FI=luminescence.sub.mRNA+TPCS2a/luminescence.sub.mRNA only.

TABLE-US-00002 TABLE 2 Set-up of the experiment in Example 1C. Amount of Illumination Animal mRNA TPCS.sub.2a (g) (min blue no./site Group name (g) per injection. light) 1-A Control, only mRNA 2 0.5 1-B PCI 0.5/0.003 B 2 0.003 0.5 1-C PCI 0.5/0.003 C 2 0.003 0.5 1-D Control, only mRNA 2 0.5 2-A PCI 0.5/0.003 A 2 0.003 0.5 2-B Control, only mRNA 2 0.5 2-C Control, only mRNA 2 0.5 2-D PCI 0.5/0.003 D 2 0.003 0.5 3-A Control, only mRNA 2 4 3-B PCI 4/0.003 B 2 0.003 4 3-C PCI 4/0.003 C 2 0.003 4 3-D Control, only mRNA 2 4 4-A PCI 4/0.003 A 2 0.003 4 4-B Control, only mRNA 2 4 4-C Control, only mRNA 2 4 4-D PCI 4/0.003 D 2 0.003 4

[0132] Results

[0133] It can be seen from FIG. 4 that at a TPCS.sub.2a dose of 0.003 g PCI enhanced mRNA delivery also with illumination times of 0.5 and 4 min. Taken together with the results with 6 min illumination (Examples 1A and 1B) this shows that with this photosensitiser dose PCI can induce mRNA delivery over a quite large light dose span, i.e. at least from 0.4 to 5 J/cm.sup.2 blue light, as calculated from the known light output from LumiSource of about 13 mW/cm.sup.2.

Example 1D: Comparison of PCI with mRNA Delivery with Lipofectamine

[0134] Lipofectamine is a very efficient transfection agent commonly used for delivery of mRNA and other nucleic acids; and it has also been used for mRNA delivery in vivo (Zangi et al., 2013, supra). However several type of toxic reactions have been observed with in vivo use of lipofectamine, limiting the possibilities for clinical use of this transfection agent. In this example the in vivo mRNA delivery with of PCI with naked mRNA was compared to what was achieved with lipofectamine. The experiment was performed as follows: [0135] Modified Luciferase mRNA from TriLink (L-7202 CleanCap FLuc mRNA, 5moU modified) was used, 2 g per injection. [0136] mRNA was mixed with lipofectamine (Thermo Fisher Scientific, Waltham, Mass., USA) or TPCS.sub.2a according to Table 3, and the mixtures were injected (20 l injection volume) into the skin at time 0 in four sites per mouse. Some of the animals were illuminated for 6 min with blue light (LumiSource device) 60 min after lipofectamine/mRNA/TPCS.sub.2a injection (see Table 3). [0137] mRNA translation was detected by IVIS imaging of luciferase bio-luminescence 4 hours after the illumination time point, and bioluminescence in the regions-of-interest was quantified (see Methods).

TABLE-US-00003 TABLE 3 Set-up of the experiment in Example 1D. Lipofect- Amount of Illumination Animal mRNA amine TPCS.sub.2a (g) (min blue no./site Group name (g) (l) per injection. light) 1-A Control, only 2 6 mRNA 1-B PCI 0.003 2 0.003 6 1-C PCI 0.003 2 0.003 6 1-D Control, only 2 6 mRNA 2-A PCI 0.003 2 0.003 6 2-B Control, only 2 6 mRNA 2-C Control, only 2 6 mRNA 2-D PCI 0.003 2 0.003 6 3-A Control, only 2 mRNA 3-B Lipo only 2 6 mRNA 3-C Lipo only 2 6 mRNA 3-D Control, only 2 mRNA 4-A Lipo only 2 6 mRNA 4-B Control, only 2 mRNA 4-C Control, only 2 mRNA 4-D Lipo only 2 6 mRNA 5-A Control, only 2 mRNA 5-B Lipo only 2 6 mRNA 5-C Lipo only 2 6 mRNA 5-D Control, only 2 mRNA

[0138] Results

[0139] It can be seen from FIG. 5 that for intradermal mRNA delivery PCI with 0.003 g TPCS.sub.2a improved mRNA translation almost 4 times over what was achieved with lipofectamine.

Example 2: Intratumoural mRNA Delivery In Vivo with Red Light Illumination

[0140] An experiment was performed to study in vivo naked mRNA delivery to TC-1 tumours (model for HPV-induced tumours) in mice.

[0141] Materials and Methods

[0142] In these experiments the TC-1 tumour model for HPV-induced cancer was employed. mRNA delivery was assessed by performing a luciferase enzymatic assay on homogenates from tumours harvested the day after illumination. Red light laser light illumination with a wavelength of 652 nm was used. The experiment was performed as follows: [0143] Luciferase mRNA (L-7202 CleanCap FLuc mRNA (5moU)) from TriLink, 3 g per injection) was mixed with TPCS.sub.2a and injected into the tumours in an injection volume of 20 l. [0144] 60 min after mRNA/TPCS.sub.2a injection the tumours were illuminated with different doses of red laser light (at 652 nm) for 12.5-500 seconds. [0145] The day after illumination, all tumours were removed and frozen for later enzymatic assay for luciferase (Luciferase Assay System, Promega, Cat #E1500). [0146] The protein content in the samples was measured with the RC DC Protein Assay kit (BioRad) assay, and the relative amounts of active luciferase in each sample was calculated as Relative Luminescence Units (RLU: Arbitrary units) per mg of protein.

[0147] The detailed set-up of the experiment is shown in Table 4.

TABLE-US-00004 TABLE 4 Set-up of the experiment in Example 2. Animal g Light dose no. Group name TPCS.sub.2a (g) mRNA (J/cm.sup.2) 1 Untreated No illum. 2 Untreated No illum. 3 mRNA alone 3 No illum. 4 mRNA alone 3 No illum. 5 mRNA alone 3 No illum. 6 mRNA + 0.1 TPC 0.1 3 No illum. 7 mRNA + 0.1 TPC 0.1 3 No illum. 8 mRNA + 0.1 TPC 0.1 3 No illum. 9 PCI 0.1 J 0.1 3 0.1 10 PCI 0.1 J 0.1 3 0.1 11 PCI 0.1 J 0.1 3 0.1 12 PCI 0.5 J 0.1 3 0.5 13 PCI 0.5 J 0.1 3 0.5 14 PCI 0.5 0.1 3 0.5 15 PCI 2 J 0.1 3 2 16 PCI 2 J 0.1 3 2 17 PCI 2 J 0.1 3 2 18 PCI 4 J 0.1 3 4 19 PCI 4 J 0.1 3 4 20 PCI 4 J 0.1 3 4

[0148] Results

[0149] From FIG. 6 it can be seen from that with a TPCS.sub.2a dose of 0.1 g, PCI induced a strong enhancement of mRNA delivery and translation. This effect was observed at all the illumination doses tested (from 0.1 to 4 J/cm.sup.2), but seemingly with an optimum at around 2 J/cm.sup.2. In comparison adding 0.1 g TPCS.sub.2a without illuminating the tumours (the mRNA+0.1 g TPCS.sub.2a group) did not seem to enhance mRNA delivery significantly over what was achieved with mRNA alone.

Example 3: Intratumoural mRNA delivery in vivo with blue light illumination

[0150] An experiment was performed to study in vivo naked mRNA delivery to colon adenocarcinoma tumours (MC-38 tumours) in mice.

[0151] Materials and Methods

[0152] The experiment was performed with Luciferase-encoding modified mRNA, and the effect was analysed by an enzymatic luciferase assay on tumour extracts, with tumours harvested 20 hours after illumination. Each animal had two tumours (A and B) that were injected and analysed separately.

[0153] Materials

[0154] TPCS.sub.2a photosensitizer (PCI Biotech AS).

[0155] TriLink modified luciferase mRNA (T14-GU03A), 3 g was used per injection.

[0156] Female mice of the strain C57BL/6 (Charles River) were be used. Animal identification and conditions of housing, acclimatisation, environment, diet and water was in accordance with the current Standard Operating Procedures at the animal facilities at Oslo University HospitalThe Radium Hospital.

[0157] Age and weight at start of dosing: 5-6 weeks, 18-20 g

[0158] MC-38 tumour cells (colon adenocarcinoma) were obtained from Kerafast (Boston, USA).

[0159] Experimental Procedure

[0160] The animals were inoculated with 500 000 MC-38 cells per tumour, 2 tumours per animal. The size of the tumours on mRNA injection was 60-150 mm.sup.3. Animals were randomized by tumour volume, animals with tumour ulcers were excluded. mRNA was mixed with TPCS.sub.2a and the mixture (25 l) was injected into the tumours at time 0. The pure (naked) mRNA was injected at the same time point.

[0161] The injections were performed with gas anesthesia (Sevoflurane). The aim of the injection was to deposit a solution in the centre of the outer third of the tumour, avoiding the central core of the tumour. Insulin syringes were used to deliver the 25 l of treatment.

[0162] The tumours in the PCI groups were illuminated (blue light from LumiSource) 60 min after mRNA/TPCS.sub.2a injection according to Table 5. The day after mRNA administration (i.e. around 20 hours after illumination) animals were killed and all tumours were removed and frozen for later homogenization and enzymatic assay for luciferase (Luciferase Assay System, Promega, Cat #E1500). After homogenization the amount of protein in the tumour extract was determined using the RC-DC Protein Assay (Bio-Rad Hercules, Calif., USA) and the luciferase enzymatic activity per mg tumour was calculated.

[0163] The experimental groups were as set out in Table 5 below.

TABLE-US-00005 TABLE 5 Set-up of the experiment in Example 3. Animal no. naked Illumina- (tumour TPCS.sub.2a mRNA tion time (A or B)) Group name (g) (g) (min) 1A mRNA only 3 1B mRNA only 3 2A mRNA only 3 2B mRNA only 3 3A mRNA only 3 3B mRNA only 3 4A mRNA PCI 0.003/6 min 0.003 3 6 4B mRNA PCI 0.003/6 min 0.003 3 6 5A mRNA PCI 0.003/6 min 0.003 3 6 5B mRNA PCI 0.003/6 min 0.003 3 6 6A mRNA PCI 0.003/6 min 0.003 3 6 6B mRNA PCI 0.003/6 min 0.003 3 6 7A mRNA PCI 0.003/6 min 0.003 3 6 7B mRNA PCI 0.003/6 min 0.003 3 6 8A mRNA PCI 0.001/6 min 0.001 3 6 8B mRNA PCI 0.001/6 min 0.001 3 6 9A mRNA PCI 0.001/6 min 0.001 3 6 9B mRNA PCI 0.001/6 min 0.001 3 6 10A mRNA PCI 0.001/6 min 0.001 3 6 10B mRNA PCI 0.001/6 min 0.001 3 6 11A mRNA PCI 0.01/6 min 0.01 3 6 11B mRNA PCI 0.01/6 min 0.01 3 6 12A mRNA PCI 0.01/6 min 0.01 3 6 12B mRNA PCI 0.01/6 min 0.01 3 6 13A mRNA PCI 0.01/6 min 0.01 3 6 13B mRNA PCI 0.01/6 min 0.01 3 6 14A mRNA PCI 0.01/6 min 0.01 3 6 15A mRNA PCI 0.01/6 min 0.01 3 6

[0164] Results

[0165] As can be seen from FIG. 7A the employment of the PCI technology substantially enhanced luciferase mRNA delivery in MC38 tumours, manifested by a strongly increased luciferase enzymatic activity in the tumour homogenates. FIG. 7B shows the mean values for the different experimental groups, indicating PCI-induced improvements in mRNA delivery from about 3 (0.003/6 min group) to about 10 times (0.01/6 min group), as compared to the delivery of mRNA without using PCI.