METHOD OF DELIVERING MRNA IN VIVO

Abstract

The present invention concerns an in vivo method for introducing a naked mRNA molecule 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 0.000001-0.001 ?g and the cells(s) are contacted with the mRNA molecule and photosensitising agent for 30 seconds to 10 minutes before irradiation of the cell(s). The method may be used to express a polypeptide in the subject. The method 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 naked, 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 0.000001-0.001 ?g and said contacting step is performed for 30 seconds to 10 minutes.

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 wherein said photosensitising agent is used in an amount of 0.0001-0.0003 ?g.

4. The method as claimed in claim 1 wherein said photosensitising agent is used at a concentration of 0.0005 to 1 ?g/ml, preferably 0.005 to 0.5 ?g/ml.

5. The method as claimed in claim 1 wherein the mRNA molecule is from 50 to 10,000 nucleotides long.

6. The method as claimed in claim 1 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 claim 1 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 claim 1 wherein said cell(s) is a mammalian cell(s) or a fish cell(s).

9. The method as claimed in claim 1 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 claim 1 wherein said contacting step is performed for 30 to 60 seconds.

11. The method as claimed in claim 1 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 claim 1 wherein the cell(s) is irradiated with a light dose of from 0.01 to 50 J/cm.sup.2, preferably 0.3 to 3 J/cm.sup.2.

13. The method as claimed in claim 1 wherein said cell(s) is contacted with said mRNA and photosensitising agent simultaneously, separately or sequentially, preferably simultaneously.

14. The method as claimed in claim 1 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 claim 1 wherein said mRNA and/or said photosensitising agent is administered locally, preferably intradermally, intramuscularly or intratumourally, preferably by injection.

16. An in vivo method of expressing a polypeptide in a cell(s) in a subject, comprising introducing an mRNA molecule into a cell(s) of the subject by the method as defined in claim 1, wherein said mRNA molecule encodes said polypeptide.

17. A pharmaceutical composition comprising an mRNA molecule and a photosensitising agent, wherein said mRNA is naked 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 0.000001 to less than 0.0001 ?g, wherein preferably said photosensitising agent is: i) TPCS.sub.2a or a pharmaceutically acceptable salt thereof; or ii) used at a concentration of 0.0005 to 1 ?g/ml, preferably 0.005 to 0.5 ?g/ml; and/or said mRNA: i) is from 50 to 10,000 nucleotides long; ii) is used in an amount of 0.1 to 100 preferably at a concentration of 5 to 5000 ?g/ml; or iii) expresses a polypeptide which is a therapeutic molecule, preferably an antibody, a vaccine polypeptide or a cytotoxic molecule.

18-20. (canceled)

21. 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 method as defined in claim 1.

22-25. (canceled)

26. The method of treating or preventing a disease, disorder or infection as claimed in claim 21, wherein: i) 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; ii) 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; iii) said disease is cancer or said infection is a viral or bacterial infection; and/or iv) said mRNA and photosensitising agent is administered intradermally, intramuscularly or intratumourally.

27. The method of treating or preventing a disease, disorder or infection as claimed in claim 21 wherein: i) said photosensitising agent is TPCS.sub.2a or a pharmaceutically acceptable salt thereof; ii) said photosensitising agent is used in an amount of 0.0001-0.0003 ?g; iii) said contacting step is performed for 30 to 60 seconds; iv) 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; and/or v) the cell(s) is irradiated with a light dose of from 0.01 to 50 J/cm.sup.2, preferably 0.3 to 3 J/cm.sup.2.

Description

[0106] The invention will now be described in more detail in the following non-limiting Examples with reference to the following drawings in which:

[0107] FIG. 1 shows (A) bio-luminescence imaging for the two injection sites in the thigh muscle of each mouse injected with a mixture of 2 ?g luciferase mRNA (TriLink L-6107, 5 meC, ?)) alone or with 0.003 ?g TPCS.sub.2a (see Example 1, Table 1 for protocol) and illuminated with red light 5 minutes after administration, and (B) shows the luciferase activity in muscle tissue from those sites for each animal.

[0108] FIG. 2 shows the mean luciferase activity in the skin from the intradermal injection site of mice after injection of 2 ?g luciferase mRNA alone or with 0.003 ?g TPCS.sub.2a and illuminated with blue light at various times after administration, as indicated.

[0109] FIGS. 3 and 4 show the mean luciferase activity in the skin from the intradermal injection site of mice after injection of 2 ?g luciferase mRNA alone or with various concentrations of TPCS.sub.2a (as indicated) and illuminated with blue light 30 seconds after administration.

[0110] FIG. 5 shows (A) bio-luminescence imaging for the two injection sites in the skin of each mouse after injection of 2 ?g luciferase mRNA alone or with 0.003 ?g TPCS.sub.2a and illuminated with blue light 30 seconds after administration and (B) the mean results from A.

[0111] FIG. 6 shows (A) mean bio-luminescence at the injection site in the muscle for each group injected with 2 ?g luciferase mRNA alone or with 0.0003 or 0.0001 ?g TPCS.sub.2a and illuminated with blue light 30 seconds after administration, and (B) the mean luciferase activity in muscle tissue from those sites for each group.

[0112] FIG. 7 shows the mean luciferase activity in muscle tissue from the injection site of mice after injection of 2 ?g luciferase mRNA alone or with various concentrations of TPCS.sub.2a (as indicated) and illuminated with blue light 30 seconds after administration.

[0113] FIG. 8 shows the mean luciferase activity in muscle tissue from the injection site of mice after injection of 2 ?g luciferase mRNA alone or with various concentrations of TPCS.sub.2a (as indicated) and illuminated with blue light 10 minutes after administration.

[0114] FIG. 9 shows the mean luciferase activity in muscle tissue from the injection site of mice after injection of 2 ?g luciferase mRNA alone or with 0.0001 ?g TPCS.sub.2a and illuminated with red light (at the doses indicated) 5 (FIG. 9A) or 10 minutes (FIG. 9B) after administration.

[0115] FIG. 10 shows the mean results of bio-luminescence imaging for the two intramuscular injection sites for the animals in each group for experiments 1 and 2 (FIGS. 10A and B, respectively), after injection of 2 ?g luciferase mRNA alone or various concentrations of TPCS.sub.2a (as indicated) and illuminated with red light 5 minutes after administration.

EXAMPLES

Example 1: Intramuscular mRNA Delivery to BL/6 Mice In Vivo with Red Light Illumination 5 Minutes after mRNA/Photosensitiser Injection

[0116] Experiments were performed to study in vivo naked mRNA intramuscular delivery to mice using shorter contact times and lower photosensitiser doses than previously used.

Materials and Methods

Mice

[0117] Female mice of the strain C57BL/6 (Charles River) were 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. Age and weight at start of dosing: 5-6 weeks, 18-20 g. The injection area was shaved before illumination.

mRNA

[0118] Firefly luciferase mRNA (L-7202with modified base 5-methoxyuridine (5 moU), and with the TriLink CleanCap? modification as the capping structure, purchased from TriLink Biotechnologies, San Diego, USA) was used.

Photosensitiser

[0119] TPCS.sub.2a (Amphinex?, PCI Biotech AS, Norway) was used mixed in a volume of 20 ?l PBS (e.g. using a 0.000015 ?g/?l solution for a 0.0003 ?g dose).

Methods

[0120] mRNA (in aqueous solution, 20 ?l of 0.1 ?g/?l from stock solution of 1 ?g/?l) in the amounts indicated in the table below were injected into the thigh muscle of each animal, with or without TPCS.sub.2a in the amounts indicated.

TABLE-US-00001 TABLE 1 Injection protocols Animal Injection Injection no. site A (left) site B (right) 1 2 ?g mRNA alone 2 ?g mRNA + 0.0003 ?g TPCS.sub.2a 2 2 ?g mRNA alone 2 ?g mRNA + 0.0003 ?g TPCS.sub.2a 3 2 ?g mRNA alone 2 ?g mRNA + 0.0003 ?g TPCS.sub.2a 4 2 ?g mRNA + 0.0003 ?g 2 ?g mRNA alone TPCS.sub.2a 5 2 ?g mRNA + 0.0003 ?g 2 ?g mRNA alone TPCS.sub.2a 6 2 ?g mRNA + 0.0003 ?g 2 ?g mRNA alone TPCS.sub.2a 7 2 ?g mRNA alone, 2 ?g mRNA + 0.0003 ?g no illumination TPCS.sub.2a, no illum. 8 2 ?g mRNA alone, 2 ?g mRNA + 0.0003 ?g no illumination TPCS.sub.2a, no illum. 9 2 ?g mRNA alone, 2 ?g mRNA + 0.0003 ?g no illumination TPCS.sub.2a, no illum. 10 2 ?g mRNA + 0.0003 ?g 2 ?g mRNA alone, TPCS.sub.2a, no illum. no illumination 11 2 ?g mRNA + 0.0003 ?g 2 ?g mRNA alone, TPCS.sub.2a, no illum. no illumination 12 2 ?g mRNA + 0.0003 ?g 2 ?g mRNA alone, TPCS.sub.2a, no illum. no illumination

[0121] In animals 1-6 both thigh muscles in each animal (injection site right and injection site left) were illuminated with red light for 6 minutes (using a laser emitting light with a wavelength peak of 652 nm, 1 J/cm.sup.2) 5 minutes after mRNA/TPCS.sub.2a injection. Animals 7-12 were not illuminated. 20 hours after the injections the animals were analysed by IVIS and assayed for luciferase activity (Luciferase Assay System, Promega, Cat #E1500) in muscle homogenates.

[0122] Muscle homogenates were prepared from frozen tissue samples using a Precelly homogenizer and lysis buffer with phosphatase and protease inhibitors (MSD, Cat. No. R60TX-2, R70AA-1). Protein analysis was performed using the DC Protein Assay (Biorad). Luciferase is expressed as relative units (RLU=relative luminescence units) per mass of protein in the samples.

IVIS Protocol

[0123] 1. 20-24 hours after light (or TPCS.sub.2a injection), mice were given an intraperitoneal injection of 3 mg D-Luciferin (200 ?l of 15 mg/ml stock). [0124] 2. After approximately 10 minutes the mice were anaesthetized by a subcutaneous injection of Zoletil (15 mg/kg xylasin, 7.5 mg/kg butorphanol, 9 mg/kg zolazepam, and 9 mg tiletamine) [0125] 3. 20 minutes after D-luciferin (Caliper Life Sciences) injection, the mice were placed in the IVIS instrument (IVIS Spectrum, model 124375R from PerkinElmer, Andor camera IS0825R4582; iKon Living Image version: 4.5.2.18424. Binning factor: 8; Excitation filter: Block; Emission filter: Open; f Number: 1) and pictures were taken with automatic exposure of Luminescence. [0126] 4. Bio-luminescence is expressed as Total Flux (photon/second)?10.sup.5.

Results

[0127] FIG. 1A shows bio-luminescence imaging for the two injection sites for each of animals 1 to 6 (see Table 1). The sites subjected to PCI exhibited significantly stronger luminescence than the control sites receiving mRNA only. This was also reflected in the luciferase assay (FIG. 1B). The mean fold of increase for the PCI treated sites was 4.9 times higher than for the mRNA only sites. Illumination without the photosensitiser did not show the same improvement (data not shown).

Example 2: Intradermal mRNA Delivery to BL/6 Mouse Skin In Vivo with Blue Light Illumination Various Times after mRNA/Photosensitiser Injection

[0128] Experiments were performed to study in vivo naked mRNA delivery to skin in mice using different contact times.

Materials and Methods

[0129] The materials and methods were as used in Example 1, except that the administration was intradermal and blue light was used (wavelength between 400 and 540 nm with a peak at around 435 nm from the LumiSource illumination device produced by PCI Biotech AS, with a 13 mW/cm.sup.2 fluence ratea 6 minute illumination delivers 4.7 J/cm.sup.2). Luciferase activity was assessed as set out in Example 1 except that skin homogenates were used.

[0130] The administration and illumination protocols were as set out in the table below, Table 2.

TABLE-US-00002 TABLE 2 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Illum. Group injection injection time point No. of no. Name site) site) (min) animals 1 mRNA alone 2 3 2 PCI 0.003, 0.003 2 10 3 10 min 3 PCI 0.003, 0.003 2 30 3 30 min 4 PCI 0.003, 0.003 2 60 3 60 min

Results

[0131] The results of the luciferase assay are shown in FIG. 2. It is evident that a 10 minute contact time enhanced mRNA delivery by about 20 times, whereas previously used longer contact times were much less effective.

Example 3: Intradermal mRNA Delivery to BL/6 Mouse Skin In Vivo with Blue Light Illumination 30 Seconds after mRNA/Photosensitiser Injection with Various Concentrations of Photosensitiser

[0132] Experiments were performed to study in vivo naked mRNA delivery to skin in mice using different photosensitiser doses with a short 30 second contact time.

Materials and Methods

[0133] The materials and methods were as used in Example 2. Luciferase activity was assessed as set out in Example 1 except that skin homogenates were used.

[0134] The administration and illumination protocols were as set out in the table below, Table 3.

TABLE-US-00003 TABLE 3 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Group injection injection Illum. No. of no. Name site) site) time point animals 1 mRNA alone 2 3 2 PCI 0.05, 0.05 2 30 seconds 3 30 seconds 3 PCI 0.01, 0.01 2 30 seconds 3 30 seconds 4 PCI 0.003, 0.003 2 30 seconds 3 30 seconds

Results

[0135] The results of the luciferase assay are shown in FIG. 3. The lower dose of photosensitiser (0.003 ?g) was found to be more effective than higher doses even with the short contact time before illumination.

Example 4: Intradermal mRNA Delivery to BL/6 Mouse Skin In Vivo with Blue Light Illumination 30 Seconds after mRNA/Photosensitiser Injection with Various Concentrations of Photosensitiser

[0136] Experiments were performed to study in vivo naked mRNA delivery to skin in mice using different photosensitiser doses with a short 30 second contact time.

Materials and Methods

[0137] The materials and methods were as used in Example 3 but with even lower doses. Luciferase activity was assessed as set out in Example 1 except that skin homogenates were used.

[0138] The administration and illumination protocols were as set out in the table below, Table 4.

TABLE-US-00004 TABLE 4 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Group injection injection Illum. No. of no. Name site) site) time point animals 1 mRNA alone 2 3 2 PCI 0.01, 0.01 2 30 seconds 3 30 seconds 3 PCI 0.001, 0.001 2 30 seconds 3 30 seconds 4 PCI 0.0003, 0.0003 2 30 seconds 3 30 seconds

Results

[0139] The results of the luciferase assay are shown in FIG. 4. The lower dose of photosensitiser (0.0003 ?g) was found to be effective even with the short contact time before illumination.

Example 5: Intradermal mRNA Delivery to BL/6 Mouse Skin In Vivo with Blue Light Illumination 30 Seconds after mRNA/Photosensitiser Injection

[0140] Experiments were performed to study in vivo naked mRNA delivery to skin in mice using a low photosensitiser dose with a short 30 second contact time.

Materials and Methods

[0141] The materials and methods were as used in Example 2. Animals were injected at two sites (left or right). IVIS was examined as set out in Example 1.

[0142] The administration protocols were as set out in the table below, Table 5. The animals were illuminated with blue light 30 seconds after administration.

TABLE-US-00005 TABLE 5 Administration protocols Animal Injection Injection no. site A site B 1 2 ?g mRNA alone 2 ?g mRNA + 0.003 ?g TPCS.sub.2a 2 2 ?g mRNA alone 2 ?g mRNA + 0.003 ?g TPCS.sub.2a 3 2 ?g mRNA alone 2 ?g mRNA + 0.003 ?g TPCS.sub.2a 4 2 ?g mRNA + 0.003 ?g 2 ?g mRNA alone TPCS.sub.2a 5 2 ?g mRNA + 0.003 ?g 2 ?g mRNA alone TPCS.sub.2a 6 2 ?g mRNA + 0.003 ?g 2 ?g mRNA alone TPCS.sub.2a

Results

[0143] FIG. 5A shows bio-luminescence imaging for the two injection sites for each of animals 1 to 6 (see Table 5). The mean results are shown in FIG. 5B. It can be seen that the sites subjected to PCI exhibited significantly stronger luminescence than the control sites receiving mRNA only (an increase of around 7.2 fold).

Example 6: Intramuscular mRNA Delivery to BL/6 Mice In Vivo with Blue Light Illumination 30 Seconds after mRNA/Photosensitiser Injection with Different Photosensitiser Concentrations

[0144] Experiments were performed to study in vivo naked mRNA delivery to muscles in mice using different photosensitiser concentrations.

Materials and Methods

[0145] The materials and methods were as used in Example 1, except that blue light was used (as in Example 2). IVIS was examined as set out in Example 1. Luciferase activity was assessed as set out in Example 1.

[0146] The administration and illumination protocols were as set out in the table below, Table 6.

TABLE-US-00006 TABLE 6 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Group injection injection Illum. No. of no. Name site) site) time point animals 1 mRNA alone 2 4 2 PCI 0.0003, 0.0003 2 30 s 4 blue 30 sec 3 PCI 0.0001, 0.0001 2 30 s 4 blue 30 sec

Results

[0147] FIG. 6A shows mean bio-luminescence at the injection sites for each group (see Table 6). FIG. 6B shows the results of the mean luciferase results for each group. It can be seen that the sites subjected to PCI exhibited significantly stronger luminescence and luciferase activity than the control sites receiving mRNA only, and that a low photosensitiser dose of 0.0001 ?g was highly effective.

Example 7: Intramuscular mRNA Delivery to BL/6 Mice In Vivo with Blue Light Illumination 30 Seconds after mRNA/Photosensitiser Injection with Different Photosensitiser Concentrations

[0148] Experiments were performed to study in vivo naked mRNA delivery to muscles in mice using different photosensitiser concentrations.

Materials and Methods

[0149] The materials and methods were as used in Example 1, except that blue light was used (as in Example 2). Luciferase activity was assessed as set out in Example 1. The administration and illumination protocols were as set out in the table below, Table 7.

TABLE-US-00007 TABLE 7 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Group injection injection Illum. No. of no. Name site) site) time point animals 1 mRNA alone 2 3 2 PCI 0.1, 0.1 2 30 s 3 blue 30 sec 3 PCI 0.003, 0.003 2 30 s 3 blue 30 sec 4 PCI 0.0003, 0.0003 2 30 s 3 blue 30 sec

Results

[0150] The results of the luciferase assay are shown in FIG. 7. The lower dose of photosensitiser (0.0003 ?g) was found to be effective even with the short contact time before illumination.

Example 8: Intramuscular mRNA Delivery to BL/6 Mice In Vivo with Blue Light Illumination 10 Minutes after mRNA/Photosensitiser Injection with Different Photosensitiser Concentrations

[0151] Experiments were performed to study in vivo naked mRNA delivery to muscles in mice using different photosensitiser concentrations.

Materials and Methods

[0152] The materials and methods were as used in Example 1, except that blue light was used (as in Example 2). Luciferase activity was assessed as set out in Example 1.

[0153] The administration and illumination protocols were as set out in the table below, Table 8.

TABLE-US-00008 TABLE 8 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Illum. Group injection injection time point No. of no. Name site) site) (min) animals 1 mRNA alone 2 3 2 PCI 0.1, 0.1 2 10 3 10 min 3 PCI 0.003, 0.003 2 10 3 10 min 4 PCI 0.0003, 0.0003 2 10 3 10 min

Results

[0154] The results of the luciferase assay are shown in FIG. 8. The lower dose of photosensitiser (0.0003 ?g) was found to be more effective than the higher doses.

Example 9: Intramuscular mRNA Delivery to BL/6 Mice In Vivo with Red Light Illumination 5 or 10 Minutes after mRNA/Photosensitiser Injection with Different Light Doses

[0155] Experiments were performed to study in vivo naked mRNA delivery to muscles in mice using different light doses concentrations and intervals before illumination.

Materials and Methods

[0156] The materials and methods were as used in Example 1 except that different light doses were used as described below. Luciferase activity was assessed as set out in Example 1.

[0157] The administration and illumination protocols were as set out in the table below, Table 9. Two sets of experiments were conducted using either a 5 or 10 minute interval after administration and before illumination.

TABLE-US-00009 TABLE 9 Administration and illumination protocols TPCS.sub.2a mRNA (?g per (?g per Group injection injection Illum. No. of no. Name site) site) dose animals 1 mRNA alone 2 3 2 PCI 0.0001, IM 0.0001 2 0.3 J/cm.sup.2 3 red 10 min, 0.3 J 3 PCI 0.0001, IM 0.0001 2 1 J/cm.sup.2 3 red 10 min, 1 J 4 PCI 0.0001, IM 0.0001 2 3 J/cm.sup.2 3 red 10 min, 3 J

Results

[0158] The results of the luciferase assay are shown in FIG. 9. With a 5 minute interval and a TPCS.sub.2a dose of 0.0001 ?g an 8-9 fold increase by PCI was observed over a red light dose range of 0.3 to 3 J/cm.sup.2 (FIG. 9A). A 10 minute interval was also effective but not at the same levels as the 5 minute interval and seemed to increase with light dose from 0.3 to 3 J/cm.sup.2 (FIG. 9B).

Example 10: Intramuscular mRNA Delivery to BL/6 Mice In Vivo with Red Light Illumination 5 Minutes after mRNA/Photosensitiser Injection Using Very Low Photosensitiser Concentrations

[0159] Experiments were performed to study in vivo naked mRNA intramuscular delivery to mice using very low photosensitiser doses after a 5 minute contact time.

Materials and Methods

[0160] The materials and methods were as used in Example 1. Animals were injected at two sites (left and right thigh). IVIS was examined as set out in Example 1.

[0161] The administration protocols were as set out in the table below, Table 10. The animals were illuminated with red light 5 minutes after administration (1 J/cm.sup.2).

TABLE-US-00010 TABLE 10 Administration and illumination protocols Experiment 1: TPCS.sub.2a mRNA (?g per (?g per Illum. Group injection injection time No. of no. Name site) site) point animals 1 mRNA alone 2 3 2 PCI 0.0003, IM 0.0003 (0.3 ng) 2 5 min 3 red 5 min 3 PCI 0.0001, IM 0.0001 (0.1 ng) 2 5 min 3 red 5 min 4 PCI 0.00001, IM 0.00001 (0.01 ng) 2 5 min 3 red 5 min Experiment 2: TPCS.sub.2a mRNA (?g per (?g per Illum. Group injection injection time No. of no. Name site) site) point animals 1 mRNA alone 2 3 2 PCI 0.0001, IM 0.0001 (0.1 ng) 2 5 min 3 red 5 min 3 PCI 0.00003, IM 0.00003 (0.03 ng) 2 5 min 3 red 5 min 4 PCI 0.000003, IM 0.000003 (0.003 ng) 2 5 min 3 red 5 min

Results

[0162] FIG. 10 shows the mean results of bio-luminescence imaging for the two injection sites for the animals in each group for experiments 1 and 2 (FIGS. 10A and B, respectively). With a 5 minute interval a 5- and 7-fold increase was observed at TPCS.sub.2a doses of 0.0001 and 0.0003 ?g, respectively. An effect was observed even when using TPCS.sub.2a at a very low dose of 0.000003 ?g (FIG. 10B).

CONCLUSIONS FROM EXAMPLES

[0163] Good correspondence between the IVIS and luciferase assay results was observed. The fold increase (PCI/mRNA alone) with illumination only 30 seconds after mRNA/photosensitiser injection is higher than has been achieved in earlier experiments in which illumination was conducted 60 minutes after injection (3-5 times improvement). The improvement relative to using no photosensitiser is in the order of 30 fold. Shorter contact times were also effective at very low photosensitiser dose allowing for reduced side effects in subjects.