RNAs for wound healing

Abstract

The present invention relates to an RNA encoding a therapeutic protein, in particular a collagenase, growth factor, cytokine, receptor, chaperone or signal transduction inhibitor. In particular, the present invention relates to RNA suitable for treatment of wounds, specifically for promoting wound healing. The present invention concerns such RNA as well as pharmaceutical compositions and kits and combinations comprising the RNA. Furthermore, the present invention relates to the RNA, pharmaceutical compositions, kits as disclosed herein for use in the treatment of wounds, specifically for promoting wound healing.

Claims

1. A method of treating a wound in a subject in need thereof comprising administering to the subject an effective amount of RNA comprising at least one coding sequence, wherein the coding sequence encodes and wherein the at least one coding sequence comprises a sequence at least 95% identical to the RNA sequence of SEQ ID NO: 259; 356; 453; 647; 744; 841; 938; 1035; 1132; 1229; or 1326.

2. The method of claim 1, wherein the RNA is mono-, bi-, or multicistronic.

3. The method of claim 1, wherein the RNA is an mRNA, a viral RNA or a replicon RNA.

4. The method of claim 1, wherein the RNA is a modified RNA.

5. The method of claim 1, wherein the RNA comprises a 5′-cap structure and/or at least one 3′-untranslated region element (3′-UTR element).

6. The method of claim 1, wherein the RNA comprises at least one histone stem-loop.

7. The method of claim 1, wherein the RNA comprises a poly(A) sequence comprising 10 to 200 adenosine nucleotides.

8. The method of claim 7, wherein the RNA comprises, in 5′ to 3′ direction, the following elements: a) a 5′-cap structure, b) optionally a 5′-UTR element, c) the at least one coding sequence, d) optionally a 3′-UTR element, e) said poly(A) sequence, and f) optionally a poly(C) tail.

9. The method of claim 8, wherein the RNA comprises a 3′-UTR element and wherein the 3 ′-UTR element comprises a nucleic acid sequence derived from a 3 ′-UTR of a gene.

10. The method of claim 9, wherein the 3 ′-UTR element comprises a nucleic acid sequence derived from a 3 ′-UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene.

11. The method of claim 8, wherein the RNA comprises a 5′-UTR element.

12. The method of claim 11, wherein the 5′-UTR element comprises a nucleic acid sequence, which is derived from the 5′-UTR of a TOP gene.

13. The method of claim 8, wherein the 5′ Cap structure is m7GpppN.

14. The method of claim 8, wherein the at least one coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 259.

15. The method of claim 14, wherein the at least one coding sequence comprises a sequence of SEQ ID NO: 259.

16. The method of claim 8, wherein the at least one coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 356.

17. The method of claim 16, wherein the at least one coding sequence comprises a sequence of SEQ ID NO: 356.

18. The method of claim 1, wherein the at least one coding sequence comprises a sequence at least 95% identical to SEQ ID NO: 259.

19. The method of claim 18, wherein the at least one coding sequence comprises a sequence of SEQ ID NO: 259.

20. The method of claim 1, wherein the RNA comprises a poly(C) sequence comprising 10 to 200 cytosine nucleotides.

Description

FIGURES

(1) FIG. 1A: Schematic drawing of the scratch assay experimental set-up in vitro. The 6-well plate was prepared for the scratch assay by drawing five horizontal lines on the bottom of the plate. Wounding of the confluent cell monolayer was achieved by scratching a 100 μl pipette tip perpendicular to the horizontal lines (vertical dotted line). Image recording was performed at indicated positions highlighted by circles.

(2) FIG. 1B: The kinetics of wound closure in vitro was followed over time. Scratch widths were measured at start of the experiment (0 hours), 16 and 24 hours after scratching. All subsequent measurements were normalized to the scratch width at start of the experiment (set to 100%).

(3) FIG. 1C: Endpoint analysis at the time point 24 hours after scratching. Wound closure in vitro was enhanced after transfection of CHO cells with MmHGF mRNA compared to control cells (***p<0.001 Student's t-test).

(4) FIG. 2: Normalized remaining wound area. Wound areas induced on the back of hairless guinea pigs by full thickness punch biopsies were measured on days 0, 2, and 7 in experimental groups treated with irrelevant mRNA, Hepatocyte Growth Factor (HGF) mRNA and Vascular Endothelial Growth Factor A (VEGF-A) mRNA. Wounds at start of experiment on day 0 were set to 100% to allow for comparison. On each treatment day, wound areas were measured and normalized to the wound area in animals treated with irrelevant mRNA. Enhanced wound closure in HGF and VEGF-A mRNA treated animals was observed compared to animals treated with irrelevant mRNA. Shown are mean+/−s.e.m. (standard error of the mean) (n=4 wounds for irrelevant mRNA, n=8 wounds for HGF mRNA treated wounds, n=8 wounds for VEGF-A mRNA treated wounds).

(5) FIG. 3: Normalized remaining wound area. Wound areas induced on the back of hairless guinea pigs by full thickness punch biopsies were measured on days 0, 2, 4, and 7 in experimental groups treated with Hepatocyte Growth Factor (HGF) mRNA. Treated wounds were compared to untreated wounds of the same animal. Wounds at start of experiment on day 0 were set to 100% to allow for comparison. On each treatment day, wound areas were measured and normalized to the wound area of untreated wounds. Enhanced wound closure in HGF mRNA treated animals was observed compared to untreated wounds on each single treatment day. Shown are mean+/−s.e.m. (standard error of the mean) (n=2 untreated wounds, n=8 HGF mRNA treated wounds) (**p<0.01 Student's t-test).

(6) FIG. 4: Normalized remaining wound area. Wound areas induced on the back of hairless guinea pigs by full thickness punch biopsies were measured on days 0, 2, 4, and 7 in experimental groups treated with Vascular Endothelial Growth Factor A (VEGF-A) mRNA. Treated wounds were compared to untreated wounds of the same animal. Wounds at start of experiment on day 0 were set to 100% to allow for comparison. On each treatment day, wound areas were measured and normalized to the wound area of untreated wounds. Enhanced wound closure in VEGF-A mRNA treated animals was observed compared to untreated wounds on each single treatment day. Shown are mean+/−s.e.m. (standard error of the mean) (n=2 untreated wounds, n=8 VEGF-A mRNA treated wounds) (*p<0.05 and ***p<0.001 Student's t-test).

(7) FIG. 5: Wound closure area normalized to start of wounding set to 0%. Wound areas induced on the back of diabetic mice by full thickness punch biopsies were measured on days 0, 3, 6, 9, 12, 15, 18 and on the day of termination day 21 in experimental groups treated with MMP1 mRNA and a mixture of ColG/H mRNAs, respectively. Treated wounds were compared to RiLa (Ringer Lactate) treated wounds. Wounds at start of experiment on day 0 were set to 0% to allow for comparison. On each treatment day, wound areas were measured. Enhanced wound closure in MMP1 mRNA and ColG/H mRNA treated animals was observed compared to RiLa treated wounds on each single treatment day. Shown are means for n=20 wounds for RiLa, MMP1 and ColG/H mRNA treatment.

(8) FIG. 6: Normalized remaining wound area. Wound areas induced on the back of hairless guinea pigs by full thickness punch biopsies were measured on days 0, 5, and 6 in experimental groups treated with irrelevant mRNA, Matrix Metalloproteinase 1 (MMP1) mRNA, a 1:1 mixture of Collagenase G and H (ColG/H) mRNAs, Hepatocyte Growth Factor (HGF) mRNA, Fibroblast Growth Factor 21 (FGF21) mRNA, and Vascular Endothelial Growth Factor A (VEGF-A) mRNA. Wounds at start of experiment on day 0 were set to 100% to allow for comparison. On each treatment day, wound areas were measured and normalized to the wound area in animals treated with irrelevant mRNA. Enhanced wound closure in all mRNA treated animals was observed compared to animals treated with irrelevant mRNA. Shown are mean+/−s.e.m. (standard error of the mean) (n=4 wounds for each experimental condition).

(9) FIG. 7A: Wound areas induced on the back of hairless guinea pigs by full thickness punch biopsies were scored on day 5 in experimental groups treated with irrelevant mRNA, Matrix Metalloproteinase 1 (MMP1) mRNA, a 1:1 mixture of Collagenase G and H (ColG/H) mRNAs, Hepatocyte Growth Factor (HGF) mRNA, and Fibroblast Growth Factor 21 (FGF21) mRNA. Wounds were analysed qualitatively by a scoring system: score 1=low wound closure; score 2=medium wound closure; score 3=full wound closure. Each wound was scored and % of wounds of each score was calculated (see FIG. 7B).

(10) FIG. 7B: Enhanced wound closure in all mRNA treated animals was observed compared to animals treated with irrelevant mRNA.

(11) FIG. 8: Normalized remaining wound area. Wound areas induced on the back of hairless guinea pigs by full thickness punch biopsies were measured on days 0 and 5 in experimental groups treated with irrelevant mRNA, Matrix Metalloproteinase 1 (MMP1) mRNA, a 1:1 mixture of Collagenase G and H (ColG/H) mRNAs, Hepatocyte Growth Factor (HGF) mRNA, Fibroblast Growth Factor 21 (FGF21) mRNA, and Vascular Endothelial Growth Factor A (VEGF-A) mRNA. To mimic standard of care of human patients, wounds were kept wet during the life phase of the experiment by applying a self-adhesive, transparent bio-film on the wound area from day 1 onwards (i.e. Tegaderm™ I.V.; Tegaderm™ I.V. is a 3M product, referring to a polyurethane film sheet with an adhesive layer, the sheet being porous to air but providing a barrier against bacterial infection). Wounds at start of experiment on day 0 were set to 100% to allow for comparison. On each treatment day, wound areas were measured and normalized to the wound area in animals treated with irrelevant mRNA. Enhanced wound closure in all mRNA treated animals was observed compared to animals treated with irrelevant mRNA. Shown are mean+/−s.e.m. (standard error of the mean) (n=8 wounds for each experimental condition).

EXAMPLES

(12) The Examples shown in the following are merely illustrative and shall describe the present invention in a further way. These Examples shall not be construed to limit the present invention thereto.

Example 1—Scratch Assay—In Vitro Wound Closure

(13) Experimental Setup

(14) The 6-well plate was prepared for the scratch assay by drawing five horizontal lines on the bottom of the plate (FIG. 1). 1.000.000 CHO cells (Chinese Hamster Ovary cells) were seeded into the 6-well plate in full culture medium (Ham's F-12+10% Fetal Calf Serum, 1% Penicillin/Streptomycin, 1% L-Glutamine) to obtain a confluent cell monolayer. 24 hours after seeding, cells were transfected with 2 μg mouse Hepatocyte Growth Factor (MmHGF) using Lipofectamine 2000. Transfection complexes remained on the cells for three hours. The transfection medium was changed to full culture medium thereafter.

(15) 8 hours after transfection, the confluent monolayer was wounded by scratching a 100 μl pipette tip perpendicular to the lines drawn on the bottom of the 6-well plate as depicted in FIG. 1A (vertical dotted lines). The serum-containing culture medium and loosened cells were removed by washing twice with PBS (Phosphate Buffered Saline). Serum-free culture medium was applied to all wells and images of the scratch width were recorded and set to 100% to allow for quantitation of the kinetics of wound closure in vitro (position of image recording highlighted by circle as depicted in FIG. 1A). The width at start of the experiment (0 h) was used for normalization of all other data points. The remaining widths of wounds in vitro were recorded during a time course of 24 hours to record kinetics of wound closure in vitro.

(16) Results:

(17) Wound closure in vitro is enhanced after transfection of CHO cells with MmHGF mRNA compared to control cells at all analysed time points (16 hours and 24 hours after transfection; FIGS. 1B and 1C).

Example 2—In Vivo Wound Healing Experiments with Guinea Pigs

(18) Experimental Setup

(19) Five punch biopsies (diameter 6 mm per biopsy) were induced on the back of each guinea pig (2 animals per group, animal model: female hairless guinea pigs from Charles River, strain code 161, animal weight ˜400-450 g). On days 0, 2, 4, 7, and 9 guinea pigs were injected intradermally into the wound edges of four wounds with 4×10 μg mRNA encoding mRNAs described in Example—Table 1; one wound per animal was left untreated for comparison. Injection sites per wound were rotated by 45° on alternating injection days. On days 0, 2, 4, 7, 9 and on the day of termination day 10, wounds were photographed to allow for quantitation of wound closure. Wound area on day 0 at induction of wounds was set to 100%. On day of termination, skin biopsies were extracted, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned for histological analysis. Parameters analysed on histological sections stained with Hematoxylin and Eosin include number of blood vessels/capillaries, extent of epithelization, fibrosis, and inflammation. Adjacent sections are stained with Masson Trichrome to visualize collagen content.

(20) TABLE-US-00011 Example-Table 1: Study design. # of Treatment (i.d.) SEQ guinea days 0, 2, 4, 7, 9. ID NO Group pigs Harvest Day 10 Formulation Design (RNA) 1 2 ntGFP RiLa Design2 7425 2 2 HGF-iso1(GC) RiLa Design5 5485 3 2 VEGF-A RiLa Design5 5487

(21) The efficacy of mRNA treatment is assessed by its ability to enhance wound closure. For evaluation of therapeutic efficacy, 40 μg of RNA (diluted in Ringer Lactate solution) are injected into the animals (n=5) intradermally (i.d.) on days 0, 2, 4, 7, 9 (4×10 μg at four injection sites into the wound edges):

(22) Animals in group 1 (n=2) serve as controls (i.e. administration of 40 μg irrelevant, non-therapeutic mRNA formulated in Ringer Lactate).

(23) Animals in groups 2, 4, 5 and 6 (n=2): administration of 40 μg respective therapeutic mRNA formulated in Ringer Lactate.

(24) Animals in group 3 (n=2): administration of a 1:1 mixture of ColG and ColH (injection of 20 μg each to result in a total amount of 40 μg) formulated in Ringer Lactate.

(25) Results:

(26) Enhanced wound closure in vivo was observed (FIGS. 2-4).

Example 3—In Vivo Wound Healing Experiments with Diabetic Mice

(27) Experimental Setup

(28) Two punch biopsies (diameter 1 cm per biopsy) were induced on the back of each diabetic mouse (db/db mouse strain) (10 animals per group). A silicon ring was adjusted around the wound to prevent wound closure by muscle contraction. On days 0, 3, 6, 9, 12, 15, and 18 mice were injected intradermally into the wound edges of four wounds with 4×10 μg mRNA encoding mRNAs described in Example—Table 2.

(29) TABLE-US-00012 Example-Table 2: Study design. # of Treatment (i.d.) days SEQ db/db 0, 3, 6, 9, 12, 15, 18 ID NO Group mice Harvest Day 21 Formulation Design (RNA) 1 10 Buffer only RiLa — — 2 10 MMP1 RiLa Design5 6323 3 10 ColG/H RiLa Design5 6325 + 6329

(30) The efficacy of mRNA treatment is assessed by its ability to enhance wound closure. For evaluation of therapeutic efficacy, 40 μg of RNA (diluted in Ringer Lactate solution) are injected into the animals (n=10) intradermally (i.d.) on days 0, 3, 6, 9, 12, 15, 18 (4×10 μg at four injection sites into the wound edges):

(31) Animals in group 1 (n=10) serve as controls (i.e. administration of Ringer Lactate).

(32) Animals in groups 2 (n=10): administration of 40 μg therapeutic mRNA formulated in Ringer Lactate.

(33) Animals in group 3: administration of a 1:1 mixture of ColG and ColH mRNAs (injection of 20 μg each to result in a total amount of 40 μg) formulated in Ringer Lactate.

(34) Results:

(35) On days 0, 3, 6, 9, 12, 15, 18 and on the day of termination day 21, wounds were photographed to allow for quantitation of wound closure. Wound area on day 0 at induction of wounds was set to 0% and subsequent wound closure was measured on digital images. Enhanced wound closure in vivo was observed in MMP1 and ColG/H mRNA treated wounds compared to buffer-treated wounds (Ringer Lactate (RiLa) only (FIG. 5).

Example 4—In Vivo Wound Healing Experiments with Guinea Pigs

(36) Experimental Setup

(37) Four punch biopsies (diameter 6 mm per biopsy) were induced on the back of each guinea pig (two animals per group, animal model: female hairless guinea pigs from Charles River, strain code 161, animal weight ˜300-350 g). On days 0, 2, and 5 guinea pigs were injected intradermally into the wound edges of four wounds with 4×10 μg mRNA encoding mRNAs described in Example—Table 3. Injection sites per wound were rotated by 45° on alternating injection days.

(38) TABLE-US-00013 Example-Table 3: Study design. Treatment (i.d.) SEQ # of days 0, 2, 5 ID NO Group wounds Harvest Day 6 Formulation (RNA) 1 4 ntGFP RiLa 7425 2 4 MMP1 RiLa 6323 3 4 ColG/H RiLa 6325 + 6329 4 4 HGF RiLa 5485 5 4 FGF21 RiLa 5479 6 4 VEGF-A RiLa 5487

(39) The efficacy of mRNA treatment is assessed by its ability to enhance wound closure. For evaluation of therapeutic efficacy, 40 μg of RNA (diluted in Ringer Lactate solution) are injected into the animals (n=4 wounds) intradermally (i.d.) on days 0, 2, 5 (4×10 μg at four injection sites into the wound edges):

(40) Animals in group 1 serve as controls (i.e. administration of 40 μg irrelevant, non-therapeutic mRNA formulated in Ringer Lactate).

(41) Animals in groups 2, 4, 5, 6: administration of 40 μg respective therapeutic mRNA formulated in Ringer Lactate.

(42) Animals in group 3: administration of a 1:1 mixture of ColG and ColH (injection of 20 μg each to result in a total amount of 40 μg) formulated in Ringer Lactate.

(43) Results:

(44) On days 0, 2, and 5 and on the day of termination day 6, wounds were photographed to allow for comparison of wound closure. Enhanced wound closure in vivo was observed in several groups (FIG. 6).

Example 5—In Vivo Wound Healing Experiments with Guinea Pigs

(45) Experimental Setup

(46) Four punch biopsies (diameter 6 mm per biopsy) were induced on the back of each guinea pig (two animals per group, eight wounds per group; animal model: female hairless guinea pigs from Charles River, strain code 161). On days 0, 2, and 5 guinea pigs were injected intradermally into the wound edges of four wounds with 4×10 μg mRNA encoding mRNAs described in Example—Table 4. Injection sites per wound were rotated by 45° on alternating injection days. On days 0, 2, and 5 and on the day of termination day 6, wounds were photographed to allow for comparison of wound closure. To mimic standard of care of human patients, wounds were kept wet during the life phase of the experiment by applying a self-adhesive, transparent bio-film on the wound area from day 1 onwards (i.e. Tegadermm I.V. as described above).

(47) TABLE-US-00014 Example-Table 4: Study design. # of Treatment (i.d.) SEQ guinea days 0, 2, 5 ID NO Group pigs Harvest Day 6 Formulation (RNA) 1 2 ntGFP RiLa 7425 2 2 MMP1 RiLa 6323 3 2 ColG/H RiLa 6325 + 6329 4 2 HGF RiLa 5485 5 2 FGF21 RiLa 5479

(48) The efficacy of mRNA treatment is assessed by its ability to enhance wound closure. For evaluation of therapeutic efficacy, 40 μg of RNA (diluted in Ringer Lactate solution) are injected into the animals (n=2) intradermally (i.d.) on days 0, 2, 5 (4×10 μg at four injection sites into the wound edges):

(49) Animals in group 1 (n=2) serve as controls (i.e. administration of 40 μg irrelevant, non-therapeutic mRNA formulated in Ringer Lactate).

(50) Animals in groups 2, 4, 5 (n=2): administration of 40 μg respective therapeutic mRNA formulated in Ringer Lactate.

(51) Animals in group 3: administration of a 1:1 mixture of ColG and ColH (injection of 20 μg each to result in a total amount of 40 μg) formulated in Ringer Lactate.

(52) Results:

(53) Wounds were analysed qualitatively by a scoring system: score 1=low wound closure; score 2=medium wound closure; score 3=full wound closure. Each wound was scored and % of wounds of each score was calculated. Enhanced wound closure in vivo was observed (FIGS. 7A and 7B).

Example 6—In Vivo Wound Healing Experiments with Guinea Pigs

(54) Experimental Setup:

(55) Four punch biopsies (diameter 6 mm per biopsy) were induced on the back of each guinea pig (two animals per group, eight wounds per group; animal model: female hairless guinea pigs from Charles River, strain code 161). On days 0, 2, and 5 guinea pigs were injected intradermally into the wound edges of four wounds with 4×10 μg mRNA encoding mRNAs described in Example—Table 5. Injection sites per wound were rotated by 45° on alternating injection days. On days 0, 2, and 5 and on the day of termination day 6, wounds were photographed to allow for comparison of wound closure. To mimic standard of care of human patients, wounds were kept wet during the life phase of the experiment by applying a self-adhesive, transparent bio-film on the wound area from day 1 onwards (i.e. Tegaderm™ I.V. as described above).

(56) TABLE-US-00015 Example-Table 5: Study design. # of Treatment (i.d.) SEQ guinea days 0, 2, 5 ID NO Group pigs Harvest Day 6 Formulation (RNA) 1 2 R5132 = ntGFP RiLa 7425 2 2 R6148 = MMP1 RiLa 6323 3 2 R6149 + R6150 = ColG/H RiLa 6325 + 6329 4 2 R6151 = HGF RiLa 5485 5 2 R6155 = FGF21 RiLa 5479 6 2 R6152 = VEGF-A RiLa 5487

(57) The efficacy of mRNA treatment was assessed by its ability to enhance wound closure. For evaluation of therapeutic efficacy, 40 μg of RNA (diluted in Ringer Lactate solution) were injected into the animals (n=2) intradermally (i.d.) on days 0, 2, 5 (4×10 μg at four injection sites into the wound edges):

(58) Animals in group 1 (n=2) served as controls (i.e. administration of 40 μg irrelevant, non-therapeutic mRNA formulated in Ringer Lactate).

(59) Animals in groups 2, 4, 5, and 6 (n=2): administration of 40 μg respective therapeutic mRNA formulated in Ringer Lactate.

(60) Animals in group 3: administration of a 1:1 mixture of ColG and ColH (injection of 20 μg each to result in a total amount of 40 μg) formulated in Ringer Lactate.

(61) Results:

(62) Wound areas were normalized to day 0 and on each subsequent day of analysis to the wound areas treated with the irrelevant, non-therapeutic mRNA (ntGFP). Enhanced wound closure in vivo was observed in all experimental groups treated with therapeutic mRNAs compared to wounds treated with irrelevant, non-therapeutic mRNA (FIG. 8).

Example 7—In Vivo Wound Healing Experiments with Mice [Prophetic]

(63) Experimental Setup:

(64) Two punch biopsies are taken on the back of each mouse (10 mice per group (db/db mice)). On day 0, 3, 6, 9, and 12 the mice are injected intradermally around each wound with 4×10 μg mRNA encoding the collagenase (e.g. ColH, ColG etc.). On day 0, 3, 6, 9, 12 and 15 the wound is photographed for wound closure measurement.

(65) Results:

(66) An enhanced wound closure is observed in several groups.