RNA for treatment or prophylaxis of a liver disease

Abstract

The present invention relates to an RNA suitable for treatment or prophylaxis of liver diseases. In particular, the present invention provides an RNA encoding at least one peptide or protein selected from the group consisting of an extracellular matrix protease, CCAAT/enhancer-binding protein alpha (CEBPA), TNF-related apoptosis-inducing ligand (TRAIL), Hepatocyte Growth Factor (HGF), hepatocyte nuclear factor 4 alpha (HNF4A), fibroblast growth factor 21 (FGF21), opioid growth factor receptor-like 1 (OGFRL1), Relaxin 1 (RLN1), Relaxin 2 (RLN2) and Relaxin 3 (RLN3), or a fragment or a variant of any of these peptides or proteins. The present invention concerns said RNA as well as compositions and kits comprising the RNA. Furthermore, the present invention relates to the RNA, compositions or kits as disclosed herein for use as a medicament, in particular for treatment or prophylaxis of a liver disease. The present invention also provides the use of the RNA, compositions or kits as disclosed herein for increasing the expression of said encoded protein, in particular in gene therapy.

Claims

1. A method for treatment of liver fibrosis, the method comprising administering to a patient in need thereof an effective amount of RNA comprising at least one coding sequence, wherein said RNA is formulated a lipid nanoparticle and administered by injection, wherein the coding sequence encodes hepatocyte nuclear factor 4 alpha (HNF4A) having a polypeptide sequence at least 95% identical to SEQ ID NO: 14, and wherein the coding sequence comprises a nucleic acid sequence at least 90% identical to the RNA sequence of SEQ ID NO: 169.

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

3. The method of claim 1, wherein the RNA does not comprise a chemically modified sugar, a chemically modified backbone or a chemically modified nucleobase.

4. The method of claim 1, wherein the RNA does not comprise a chemically modified nucleoside or nucleotide.

5. The method of claim 1, wherein the RNA is not chemically modified.

6. The method of claim 1, wherein the at least one coding sequence of the RNA comprises a nucleic acid sequence encoding a polypeptide of SEQ ID NO: 14.

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

8. The method of claim 1, wherein the RNA is an mRNA.

9. The method of claim 8, wherein the RNA is mRNA comprising from 5′ to 3′: a 5′ Cap; a 5′ untranslated regions (UTR); the at least one coding sequence; a 3′ UTR and a Poly-A region.

10. The method of claim 8, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic lipid.

11. The method of claim 10, wherein lipid nanoparticle comprises: a cationic lipid, an aggregation reducing agent, a non-cationic lipid and a sterol.

12. The method of claim 11, wherein the aggregation reducing agent comprises a PEG-modified lipid.

13. The method of claim 1, wherein the administering is by intravenous injection.

14. The method of claim 1, wherein the method reduces the level of alanine aminotransferase (ALT) in the patient's serum.

15. The method of claim 1, wherein the RNA comprises at least one chemically modified nucleotide.

16. The method of claim 15, wherein the at least one chemically modified nucleotide comprises a chemically modified uridine.

17. The method of claim 15, wherein the at least one chemically modified nucleotide comprises pseudouridine or 1-methyl-pseudouridine.

Description

FIGURES

(1) FIG. 1: Macroscopic classification of liver fibrosis in the CCl.sub.4 mouse model: Representative macroscopic pictures of livers from control and CCl.sub.4 treated mice. The grade of the fibrosis in the liver were classified into grade 0 (no fibrosis) to grade 3 (advanced fibrosis). Grade 0: the external surface of the liver was smooth and glistening. Grade 1: a part of the external surface of the liver (<50%) did show a structural changes such as cell dysplasia and increasing collaged deposition (scaring) of the liver. Grade 2: the majority of the external surface of the liver (>50%) did show a structural changes such as cell dysplasia and increasing collaged deposition (scaring) of the liver. Grade 3: strong macroscopic changes of the external liver surface indicated by the dysplastic nodules and strong extracellular matrix (ECM) and collagen deposition (scaring) of the liver.

(2) FIG. 2: Evaluation of the fibrosis stage: The liver of all animals were classified into the different grades of fibrosis according to the macroscopic classification criteria in FIG. 1 by macroscopic evaluation of the liver condition. Classification of the fibrosis: no fibrosis (Grade 0); beginning to advanced fibrosis (Grade 1 to Grade 3). mRNA treated groups (group 3 to 6) did show a reduced appearance of the Grade 3 fibrosis and a increasing appearance of the Grade 2 and 1 fibrosis compared to the mRNA untreated control group 2. The dotted line separates control groups 1-2 from treated animals groups 3-6.

(3) FIGS. 3(A) and 3(B): Reduction of the liver weight and the liver body weight ratio in mRNA treated animals:

(4) FIG. 3(A) Liver weight was determined at 29 days after start of the CCl.sub.4 administration (14 days after start of the mRNA treatment). mRNA treated groups (group 3 to 6) did show a lower liver weight compared to the mRNA untreated control group 2. Data (mean±SEM).

(5) FIG. 3(B) Liver weight to body weight ratio was determined at 29 days after start of the CCl.sub.4 administration (14 days after start of the mRNA treatment). The liver weight was divided by body weight and multiplied with 100 to determine the indicated ratio. mRNA treated groups (group 3 to 6) did show a lower liver weight compared to the mRNA untreated control group 2. Data (mean±SEM).

(6) FIG. 4: Reduction of the chronic liver injury marker aminotransferase (ALT) in mRNA treated animals: Serum ALT was assessed in serum 29 days after start of the CCl.sub.4 administration (14 days after start of the mRNA treatment). ALT levels were automatically determined by enzymatic analysis. mRNA treated groups (group 3 to 6) did show lower ALT levels compared to the mRNA untreated control group 2. Data (mean±SEM)

EXAMPLES

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

Example 1: Evaluation of the Therapeutic Efficacy of Treatment with mRNA on Carbon Tetrachloride Induced Liver Fibrosis Model in Male BALB/c Mice

(8) BALB/c mice were divided into 6 groups as apparent from the study design shown in Table 3. Induction of liver failure was achieved by CCl.sub.4 administration and subsequent treatment with test compounds shows therapeutic efficacy. The administration of CCl.sub.4:mineral oil was via intraperitoneal (i.p.) route twice a week for a month. As control, saline:mineral oil was administered to 5 animals (i.p.).

(9) Bleeds via sub-mandibular route or via tail nick were scheduled on days minus 1, day 15, day 22, and a final bleed upon harvest day 29. Approximately 100 μl of whole blood was collected in serum separator tubes and serum was prepared and stored at −20° C.

(10) For evaluation of liver function, alanine aminotransferase (ALT) was measured.

(11) TABLE-US-00005 TABLE 3 Study design. Treatment (i.v..sup.B) days 14, 17, 19, CCl.sub.4 treatment.sup.A (i.p.) # of 21, 24 and 26 SEQ ID Group 2x/week mice Harvest Day 29 Formulation RNA design NO (RNA) 1 0.9% saline:mineral oil 5 saline — — 2 CCU:mineral oil 12 saline — — 3 CCl.sub.4:mineral oil 12 human collagenase LNP Cap1; PolyA; Hsd17B4 2134 MMP1 construct 2 (V2) . . . PSBM3 . . . A64-N5- (MMP1-del 20-99) C30-histoneSL-N5(EcoR1) 4 CCl.sub.4:mineral oil 12 MmHGF-iso1 LNP Cap1; PolyA; Hsd17B4 1955 (V2) . . . PSBM3 . . . A64-N5- C30-histoneSL-N5(EcoR1) 5 CCl.sub.4:mineral oil 12 MmCEBPA_Isoform 1m LNP Cap1; PolyA; Hsd17B4 2173 (V2) . . . PSBM3 . . . A64-N5- C30-histoneSL-N5(EcoR1) 6 CCl.sub.4:mineral oil 12 sTRAIL construct 2 LNP Cap1; PolyA; Hsd17B4 2120 (HsALB(1- (V2) . . . PSBM3 . . . A64-N5- 18)(GC)_ILZ(GC)_HsTNFSF10(95- C30-histoneSL-N5(EcoR1) 281) (GC)) 7 CCl.sub.4:mineral oil 12 clostridial type II LNP Cap1; PolyA; Hsd17B4 2136 + collagenase (V2) . . . PSBM3 . . . A64-N5- 2140 (ColG + H mixture 5) C30-histoneSL-N5(EcoR1) 8 CCl.sub.4:mineral oil 12 FGF21 LNP Cap1; PolyA; Hsd17B4 1946 (V2) . . . PSBM3 . . . A64-N5- C30-histoneSL-N5(EcoR1) .sup.ACCL.sub.4 or saline:mineral oil (control) was administered (i.p.) twice a week for 4 weeks; .sup.BCureVac compounds or saline were administered by i.v. every 2 or 3 days beginning from day 14 post CCl.sub.4 administration, see schedule.

(12) The efficacy of mRNA treatment was assessed by its ability to limit the progression of disease or liver failure. For evaluation of therapeutic efficacy, 100 μl (40 μg) of LNP-formulated RNA (RNA-LNP) were injected into the animals (n=12) via intravenous route (i.v.) on days 14, 17, 19, 21, 24 and 26 post CCl.sub.4 administration initiation:

(13) Animals in group 1 (n=5) did serve as sham control (i.e. administration of (a) saline (i.p.) and (b) 1 ml/kg 1:1 ratio of saline:mineral oil (i.p.));

(14) Animals in group 2 (n=12): administration of (a) saline (i.p.) (b) 1 ml/kg 1:1 ratio of CCl.sub.4:mineral oil (i.p.);

(15) Animals in groups 3-6 (each time: n=12): administration of (a) respective mRNA and formulation as indicated (IV) (b) 1 ml/kg 1:1 ratio of CCl.sub.4:mineral oil (i.p.).

(16) Final harvest was performed on day 29, i.e. 96 hours following the last CCl.sub.4 administration, i.e. all surviving animals were weighed, the activity level was observed, blood was extracted by cardiac bleed for serum preparation, and the animals were euthanized followed by gross necropsy. Subsequently, the liver of each animal was harvested and weighed. Median lobe of the liver was fixed in 10% neutral buffered formalin for histology and remaining lobes were distributed into two tubes and snap frozen.

(17) Representative macroscopic pictures of livers from control and CCl.sub.4 treated mice were taken and classified into the different grades of fibrosis according to the macroscopic classification criteria in FIG. 1 (macroscopic evaluation of the liver condition). The grade of the fibrosis in the liver were classified into grade 0 (no fibrosis) to grade 3 (advanced fibrosis). Grade 0: the external surface of the liver was smooth and glistening. Grade 1: a part of the external surface of the liver (<50%) did show a structural changes such as cell dysplasia and increasing collaged deposition (scaring) of the liver. Grade 2: the majority of the external surface of the liver (>50%) did show a structural changes such as cell dysplasia and increasing collaged deposition (scaring) of the liver. Grade 3: strong macroscopic changes of the external liver surface indicated by the dysplastic nodules and strong extracellular matrix (ECM) and collagen deposition (scaring) of the liver.

(18) Further, liver weight as indicator for liver fibrosis progression was determined at 29 days after start of the CCl.sub.4 administration (14 days after start of the mRNA treatment; FIG. 3(A)) and liver weight to body weight ratio was determined (FIG. 3(B)). Further, according to FIG. 4, serum ALT was assessed.

(19) Results:

(20) All mRNA treated animals (Gr3 to Gr6) showed a lower liver weight and a lower liver/body weight ratio as compared to the control group (Gr2), FIGS. 3A and 3B, i.e. indicated an improvement or a reduced progression of the fibrosis. In addition, a lower amount of liver with a high grade of fibrosis could be observed in the mRNA treated animals (Gr3 to Gr6) as apparent from FIG. 1 and FIG. 2. Further, as apparent from FIG. 4, the fibrosis marker Alanine aminotransferase (ALT) was reduced in all mRNA treated groups compared to the control group, i.e. indicated an improvement or a reduced progression of liver fibrosis in the animals.

(21) For further evaluation of liver function, a further enzyme panel is measured, i.e. aspartate transaminase (AST), alkaline phosphatase (ALP) and also total bilirubin (TBIL). Measurement of total bilirubin includes both unconjugated and conjugated bilirubin, as unconjugated bilirubin is a breakdown product of heme and problems with the liver are reflected as deficiencies in bilirubin metabolism (e.g., reduced hepatocyte uptake, impaired conjugation of bilirubin, and reduced hepatocyte secretion of bilirubin; examples would be cirrhosis and viral hepatitis).

Example 2: Histopathological Analysis [Prophetic]

(22) A histopathological analysis for animals of Example 1 is performed, i.e. Sectioning and Picrosirius (PSR) staining (i.e. histological visualization of collagen I and III fibers). Furthermore, digital images of the liver of each animal are taken and additionally, scoring from standard hematoxylin-eosin (H&E) and PSR stained slides is performed. The histopathological analysis shows reduced progression of liver fibrosis.

Example 3: Evaluation of the Therapeutic Efficacy of Treatment with mRNA on Carbon Tetrachloride Induced Liver Fibrosis Model in Male BALB/c Mice [Prophetic]

(23) BALB/c mice are divided into 17 groups as apparent from the study design shown in Table 4. Induction of liver failure is achieved by CCl.sub.4 administration and subsequent treatment with test compounds shows therapeutic efficacy. The administration of CCl.sub.4:mineral oil is via intraperitoneal (i.p.) route twice a week for a month. As control, saline:mineral oil is administered to 5 animals (i.p.).

(24) Bleeds via sub-mandibular route or via tail nick are scheduled on days minus 1, day 14, day 25, and a final bleed upon harvest day 32. Approximately 100 μl of whole blood are collected in serum separator tubes and serum prepared and stored at −20° C.

(25) For evaluation of liver function, an enzyme panel is measured, i.e. alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and also total bilirubin (TBIL). Further, alkaline phosphatase (ALP), gamma-glutamyl transfertase (GGT), total bilirubin (TBIL), ammonia, globulin and albumin/globulin ratio is measured. Measurement of total bilirubin includes both unconjugated and conjugated bilirubin, as unconjugated bilirubin is a breakdown product of heme and problems with the liver are reflected as deficiencies in bilirubin metabolism (e.g., reduced hepatocyte uptake, impaired conjugation of bilirubin, and reduced hepatocyte secretion of bilirubin; examples would be cirrhosis and viral hepatitis). Also, albumin levels are measured, as albumin levels are decreased in chronic liver disease, such as cirrhosis. Further, total protein (the remaining from globulins) is measured.

(26) TABLE-US-00006 TABLE 4 Study design CCl.sub.4 treatment.sup.A Treatment (i.v..sup.B) days 17, 20, 24, 27 SEQ (i.p.) # of and 30, ID NO Group 2×/week mice Harvest Day 32 Formulation (RNA)  1 0.9% saline:  5 saline — — mineral oil  2 CCl.sub.4: mineral oil 12 saline — —  3 CCl.sub.4: mineral oil 12 TRAIL (HsTNFSF10) LNP 1131  4 CCl.sub.4: mineral oil 12 sTRAIL construct 1 (HsALB(1- LNP 1133 18)_HsTNFSF10(95-281) (GC))  5 CCl.sub.4: mineral oil 12 sTRAIL construct 2 (HsALB(1- LNP 1134 18)(GC)_ILZ(GC)_HsTNFSF10(95- 281)(GC))  6 CCl.sub.4: mineral oil 12 human collagenase MMP1 construct 1 LNP  807  7 CCl.sub.4: mineral oil 12 human collagenase MMP1 construct 2 LNP  808  8 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG) LNP  809 construct 1  9 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG) LNP  810 construct 2 10 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG) LNP  811 construct 3 11 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColH) LNP  813 construct 1 12 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColH) LNP  814 construct 2 13 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColH) LNP  815 construct 3 14 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  809 + H mixture 1) 813 15 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  810 + H mixture 2) 813 16 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  811 + H mixture 3) 813 17 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  809 + H mixture 4) 814 18 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  810 + H mixture 5) 814 19 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  811 + H mixture 6) 814 20 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  809 + H mixture 7) 815 21 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  810 + H mixture 8) 815 22 CCl.sub.4: mineral oil 12 clostridial type II collagenase (ColG + LNP  811 + H mixture 9) 815 23 CCl.sub.4: mineral oil 12 C/EBPa construct 1 LNP  36 24 CCl.sub.4: mineral oil 12 C/EBPa construct 2 LNP  37 25 CCl.sub.4: mineral oil 12 HNF4a LNP 1104 26 CCl.sub.4: mineral oil 12 FGF21 LNP 1106 27 CCl.sub.4: mineral oil 12 opioid growth factor receptor-like 1 LNP 1108 (OGFRL1) 28 CCl.sub.4: mineral oil 12 Relaxin 1 (RLN1) LNP 1110 29 CCl.sub.4: mineral oil 12 Relaxin 2 (RLN2) LNP 1111 30 CCl.sub.4: mineral oil 12 Relaxin 3 (RLN3) LNP 1113 31 CCl.sub.4: mineral oil 12 Hepatocyte Growth Factor (HGF) LNP 1955 32 CCl.sub.4: mineral oil 12 Control LNP LNP — .sup.ACCL.sub.4 or saline: mineral oil (control) is administered (i.p.) twice a week for 4 weeks; .sup.Bcompounds or saline are administered (i.v.) every 3 or 4 days beginning from day 17 post CCl.sub.4 administration

(27) The efficacy of mRNA treatment is assessed by its ability to limit the progression of disease or liver failure. For evaluation of therapeutic efficacy, 100 μl of LNP-formulated RNA (RNA-LNP) are injected into the animals (n=12) via intravenous route (i.v.) on days 17, 20, 24, 27 and 30 post CCl.sub.4 administration initiation:

(28) Animals in group 1 (n=5) serve as sham control (i.e. administration of (a) saline (i.p.) and (b) 1 ml/kg 1:1 ratio of saline:mineral oil (i.p.));

(29) Animals in group 2 (n=12): administration of (a) saline (i.p.) (b) 1 ml/kg 1:1 ratio of CCl.sub.4:mineral oil (i.p.);

(30) Animals in groups 3-31 (each time: n=12): administration of (a) respective mRNA and formulation as indicated (IV) (b) 1 ml/kg 1:1 ratio of CCl.sub.4:mineral oil (i.p.);

(31) Animals in group 32 (n=12): administration of (a) Control LNP (IV) (b) 1 ml/kg 1:1 ratio of CCl.sub.4:mineral oil (i.p.).

(32) In a separate experiment, the study design of Table 4 is assessed with CVCM-formulated mRNA instead of LNP-formulated mRNA.

(33) Final harvest is on day 32, i.e. 96 hours following the last CCl.sub.4 administration, i.e. all surviving animals are weighed, the activity level is observed, blood is extracted by cardiac bleed for serum preparation, and the animals are euthanized followed by gross necropsy. Subsequently, the liver is harvested and weighed. Median lobe of the liver is fixed in 10% neutral buffered formalin for histology and remaining lobes are distributed into two tubes and snap frozen.

(34) Collagen deposition is evaluated by hydroxyproline measurement. Also, a histopathological analysis is performed, i.e. Sectioning and Picrosirius (PSR) staining (i.e. histological visualization of collagen I and III fibers) including an αSMA staining (marker for activated hepatic stellate cells). Furthermore, digital images of the liver of each animal are taken and additionally, scoring from standard hematoxylin-eosin (H&E) and PSR stained slides is performed.

(35) Results:

(36) Reduction of collagen in the liver is observed in several groups. Also, a hydroxyproline reduction is observed in several groups. Further, decreased extracellular matrix deposition, including collagen and hydroxyproline content is observed for some groups. Some groups show a specific increase of apoptosis in HSCs without affecting hepatocytes. Some groups show increased survival when the animal model reached a state of liver cirrhosis.

Example 4: Evaluation of the Effect of Treatment with mRNA in a Rat Model of Carbon Tetrachloride Induced Liver Fibrosis [Prophetic]

(37) CCl.sub.4:Olive oil (1:1 ratio) is administered (i.p.) to male rats (species Rattus norvegicus, strain: Sprague Dawley) once in 3 days at a dose of 2 ml/kg for a period of 2 weeks (5 injections), followed by 1 ml/kg (i.p.), once in 3 days for 6 weeks. Rats are divided into 17 groups as apparent from the study design shown in Table 5. The study is terminated in week 10 (i.e., 8 weeks of CCl.sub.4 treatment plus 2 weeks of treatment with mRNA).

(38) Blood is collected on d2, d7, d10, d14 post treatment (post 8 weeks). On study termination, blood samples are collected and plasma samples are submitted for liver function tests and measurement of prothrombin time.

(39) The efficacy of mRNA treatment is assessed by its ability to limit the progression of disease or liver failure.

(40) TABLE-US-00007 TABLE 5 Study design Bleeding Liver # of Dosing (i.v.)/ Treatment Function No Group Fibrosis Induction rats Treatment Formulation schedule Test (LFT) SEQ ID Total of 8 weeks Start - week 8 End - week 8 + 2 (10) NO (RNA) 1 Sham Olive oil 2 + 6 weeks 5 no no no d2, d7, — Control d10, d14 2 Path CCl.sub.4 in 8 no no no d2, d7, — Control Olive oil 2 + 6 weeks d10, d14 3 Test 1 CCl.sub.4 in 8 TRAIL (HsTNFSF10) 40 μg d1; d4; d7; d2, d7, 1131 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 4 Test 2 CCl.sub.4 in 8 sTRAIL construct 1 40 μg d1; d4; d7; d2, d7, 1133 Olive oil 2 + 6 weeks (HsALB(1-18)_HsTNFSF10(95- (40 μl)/LNP d10; d13 - End d10, d14 281) (GC)) point d14 5 Test 3 CCl.sub.4 in 8 sTRAIL construct 2 40 μg d1; d4; d7; d2, d7, 1134 Olive oil 2 + 6 weeks (HsALB(1- (40 μl)/LNP d10; d13 - End d10, d14 18)(GC)_ILZ(GC)_HsTNFSF10(95- point d14 281) (GC)) 6 Test 4 CCl.sub.4 in 8 human collagenase MMP1 40 μg d1; d4; d7; d2, d7, 807 Olive oil 2 + 6 weeks construct 1 (40 μl)/LNP d10; d13 - End d10, d14 point d14 7 Test 5 CCl.sub.4 in 8 human collagenase MMP1 40 μg d1; d4; d7; d2, d7, 808 Olive oil 2 + 6 weeks construct 2 (40 μl)/LNP d10; d13 - End d10, d14 point d14 8 Test 6 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 809 Olive oil 2 + 6 weeks (ColG) construct 1 (40 μl)/LNP d10; d13 - End d10, d14 point d14 9 Test 7 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 810 Olive oil 2 + 6 weeks (ColG) construct 2 (40 μl)/LNP d10; d13 - End d10, d14 point d14 10 Test 8 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 811 Olive oil 2 + 6 weeks (ColG) construct 3 (40 μl)/LNP d10; d13 - End d10, d14 point d14 11 Test 9 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 813 Olive oil 2 + 6 weeks (ColH) construct 1 (40 μl)/LNP d10; d13 - End d10, d14 point d14 12 Test 10 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 814 Olive oil 2 + 6 weeks (ColH) construct 2 (40 μl)/LNP d10; d13 - End d10, d14 point d14 13 Test 11 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 815 Olive oil 2 + 6 weeks (ColH) construct 3 (40 μl)/LNP d10; d13 - End d10, d14 point d14 14 Test 12 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 809 + 813 Olive oil 2 + 6 weeks (ColG + H mixture 1) (40 μl)/LNP d10; d13 - End d10, d14 point d14 15 Test 13 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 810 + 813 Olive oil 2 + 6 weeks (ColG + H mixture 2) (40 μl)/LNP d10; d13 - End d10, d14 point d14 16 Test 14 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 811 + 813 Olive oil 2 + 6 weeks (ColG + H mixture 3) (40 μl)/LNP d10; d13 - End d10, d14 point d14 17 Test 15 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 809 + 814 Olive oil 2 + 6 weeks (ColG + H mixture 4) (40 μl)/LNP d10; d13 - End d10, d14 point d14 18 Test 16 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 810 + 814 Olive oil 2 + 6 weeks (ColG + H mixture 5) (40 μl)/LNP d10; d13 - End d10, d14 point d14 19 Test 17 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 811 + 814 Olive oil 2 + 6 weeks (ColG + H mixture 6) (40 μl)/LNP d10; d13 - End d10, d14 point d14 20 Test 18 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 809 + 815 Olive oil 2 + 6 weeks (ColG + H mixture 7) (40 μl)/LNP d10; d13 - End d10, d14 point d14 21 Test 19 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 810 + 815 Olive oil 2 + 6 weeks (ColG + H mixture 8) (40 μl)/LNP d10; d13 - End d10, d14 point d14 22 Test 20 CCl.sub.4 in 8 clostridial type II collagenase 40 μg d1; d4; d7; d2, d7, 811 + 815 Olive oil 2 + 6 weeks (ColG + H mixture 9) (40 μl)/LNP d10; d13 - End d10, d14 point d14 23 Test 21 CCl.sub.4 in 8 C/EBPa construct 1 40 μg d1; d4; d7; d2, d7, 36 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 24 Test 22 CCl.sub.4 in 8 C/EBPa construct 2 40 μg d1; d4; d7; d2, d7, 37 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 25 Test 23 CCl.sub.4 in 8 HNF4a 40 μg d1; d4; d7; d2, d7, 1104 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 26 Test 24 CCl.sub.4 in 8 FGF21 40 μg d1; d4; d7; d2, d7, 1106 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 27 Test 25 CCl.sub.4 in 8 opioidgrowth factor receptor- 40 μg d1; d4; d7; d2, d7, 1108 Olive oil 2 + 6 weeks like 1 (OGFRL1) (40 μl)/LNP d10; d13 - End d10, d14 point d14 28 Test 26 CCl.sub.4 in 8 Relaxin 1 (RLN1) 40 μg d1; d4; d7; d2, d7, 1110 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 29 Test 27 CCl.sub.4 in 8 Relaxin 2 (RLN2) 40 μg d1; d4; d7; d2, d7, 1111 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 30 Test 28 CCl.sub.4 in 8 Relaxin 3 (RLN3) 40 μg d1; d4; d7; d2, d7, 1113 Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14 31 Test 29 CCl.sub.4 in 8 Hepatocyte Growth Factor 40 μg d1; d4; d7; d2, d7, 1955 Olive oil 2 + 6 weeks (HGF) (40 μl)/LNP d10; d13 - End d10, d14 point d14 32 Test 30 CCl.sub.4 in 8 LNP Control 40 μg d1; d4; d7; d2, d7, — Olive oil 2 + 6 weeks (40 μl)/LNP d10; d13 - End d10, d14 point d14

(41) In a separate experiment, the study design of Table 5 is assessed with CVCM-formulated mRNA instead of LNP-formulated mRNA.

(42) A liver function test is performed, i.e. alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transfertase (GGT), total bilirubin (TBIL), total protein (TP), ammonia, albumin, globulin and albumin/globulin ratio are measured.

(43) A portion of ˜100 mg live tissue is flash frozen in liquid nitrogen and stored at −80C for hydroxyproline estimation for evaluation of collagen deposition. For the hydroxyproline assay in liver tissue, standard commercially available kits are applied, expressing the results as μg per whole liver or per g of liver.

(44) Remaining liver tissue is fixed in 10% Neutral Buffered Formalin and sections are prepared after routine histopathological processing. The paraffin sections are stained with Masson's Trichrome staining and assessed by a pathologist for severity of liver fibrosis. Also, the Liver Function Tests as described in Example 1 are performed.

(45) Results:

(46) Analogous to Example 1, a collagen reduction in liver is observed in several groups. Also, a hydroxyproline reduction is observed in several groups. Further, decreased extracellular matrix deposition, including collagen and hydroxyproline content is observed for some groups.

(47) Some groups show a specific increase of apoptosis in aHSCs without affecting hepatocytes. Some groups show increased survival when the animal model reached a state of liver cirrhosis.