Patent Application
20120264805
A field of the invention is liver treatment. The present invention relates to a medicament for the treatment or prevention of liver failure, especially of acute liver failure, including subfulminant and fulminant liver failure. Generally, liver failure can be induced by viral agents, e.g. an infection by hepatitis B virus and/or by drug abuse, including abuse of alcohol and acetaminophen (paracetamol).
This application incorporates by reference the sequence listing in the electronic sequence listing filed herewith and as an appendix to this application.
Lee in the New England Journal of Medicine, 1862-1872 (1993) describes causes for acute liver failure. For the treatment of acute liver failure, a treatment directed against the causative agent is currently used, e.g. an antiviral therapy in the case of viral infection, or the administration of a chemical antidote in the case of drug abuse, e.g. acetylcystein in the case of paracetamol intoxication. In severe cases, liver transplantation if a suitable donor organ is available, is considered the therapy of choice.
Generally, it is known that microRNAs (miRNAs) are non-coding small RNA molecules, e.g. 21 or 23 nucleotides in length, which naturally regulate gene expression by interfering with mRNA molecules.
The invention provides a medicament for the treatment of a functionally impaired liver, for delaying a deterioration of liver function and/or for prevention of liver failure, containing in a pharmaceutically acceptable formulation an siRNA or microRNA having a nucleic acid sequence which hybridizes to the mRNA encoding. PUMA. In a preferred embodiment, the siRNA or microRNA is reverse complementary to 3′ UTR of the mRNA encoding PUMA, which 3′UTR has the sequence of SEQ ID NO: 1. In a preferred embodiment, the siRNA or microRNA comprises a sequence selected from the group consisting of nucleotide sequences having at least 85% sequence similarity to one of SEQ ID NO 2 to SEQ ID NO 84.
The invention provides a purified nucleic acid construct containing a sequence encoding an siRNA having a nucleic acid sequence which hybridizes to the mRNA encoding PUMA as a medicament for the treatment of a functionally impaired liver, for delaying a deterioration of liver function and/or for prevention of liver failure.
The invention provides a method for treating, delaying and/or preventing liver failure in humans or animals, the method comprising the step of providing a nucleic acid having which hybridizes to the mRNA encoding PUMA in a pharmaceutical formulation suitable for administration to the human or animal
In preferred embodiments, the siRNA or microRNA is reverse complementary to 3′ UTR of the mRNA encoding PUMA, which 3′UTR has the sequence of SEQ ID NO: 1. In preferred embodiments, the siRNA or microRNA comprises a sequence selected from the group consisting of nucleotide sequences having at least 85% sequence similarity to one of SEQ ID NO 2 to SEQ ID NO 84.
Generally, in the figures * indicates P<0.05, and ** indicates P<0.005.
The invention provides small inhibitory RNA (siRNA) molecules, which e.g. are purified and/or isolated, as an active in a medicament, which siRNA molecules are inhibitory RNA molecules that through RNA interference (RNAi) reduce or prevent expression of the p53 upregulated modulator of apoptosis (PUMA). The siRNA molecules can be administered as a medicament to a patient suffering from an impaired liver function or from liver damage for the treatment of a functionally impaired liver, for delaying a deterioration of liver function and/or for prevention of liver failure, especially in patients who suffer from a critical impairment of damage to the liver, e.g. for delaying the complete failure of liver function.
The siRNA molecules (siRNAs) can comprise or consist of single-stranded or double-stranded oligonucleotides of RNA and/or DNA hybridizing to the mRNA encoding PUMA, including siRNA molecules hybridizing under physiological conditions to the mRNA encoding PUMA, which siRNA molecules are at least 85%, preferably at least 90%, more preferably at least 95% reverse complementary of a section of at least 19 to at least 23 nucleotides length, preferably hybridizing to the 3′UTR (3′-untranslated region) of the mRNA (SEQ ID NO: 1) encoding PUMA, and can be in the form of short-hairpinRNA (shRNA) or comprised in a microRNA or in a microRNA precursor. In one embodiment, the siRNA molecule is the active in the medicament, e.g. formulated as liposomes, and in an alternative embodiment, the siRNA is encoded in an expression cassette of a nucleic acid construct which is formulated as a medicament, e.g. as a viral particle or as a virus-like particle. The siRNA, or combination of two or more of these, is preferably contained in a microRNA molecule or encoded in an expression cassette of a nucleic acid construct as a microRNA. Exemplary microRNAs are given as SEQ ID NO 2 to SEQ ID NO 84, of which the group comprising or consisting of miR-221 (SEQ ID NO 2), miR-222 (SEQ ID NO 3), miR-709 (SEQ ID NO 4), miR-1827 (SEQ ID NO 5), miR-148 (SEQ ID NO 6), miR-152 (SEQ ID NO 7), miR-144 (SEQ ID NO 8), miR-145 (SEQ ID NO 9), miR-139-5p (SEQ ID NO 10), miR-27a (SEQ ID NO 11), miR-27b (SEQ ID NO 78), miR-34a (SEQ ID NO 12), miR-34b-5p (SEQ ID NO 13), miR-34c (SEQ ID NO 14), miR-34c-5p (SEQ ID NO 15), miR-449 (SEQ ID NO 84), miR-449a (SEQ ID NO 16) miR-449b (SEQ ID NO 17), miR-449c (SEQ ID NO 79), miR-699 (SEQ ID NO 18), miR-24 (SEQ ID NO 19), miR-143 (SEQ ID NO 20), miR-101 (SEQ ID NO 21), miR-150 (SEQ ID NO 22), miR-128 (SEQ ID NO 23), miR-125 (SEQ ID NO 24), miR-351 (SEQ ID NO 25), miR-140 (SEQ ID NO 26), miR-140-5p (SEQ ID NO 27), miR-876-3p (SEQ ID NO 28), miR-205 (SEQ ID NO 29), miR-29a (SEQ ID NO 30), miR-29b (SEQ ID NO 80), miR-29c (SEQ ID NO 81), is preferred, of which miR-221 (SEQ ID NO 2), miR-222 (SEQ ID NO 3), miR-709 (SEQ ID NO 4), miR-1827 (SEQ ID NO 5) are more preferred, and of which miR-221, optionally in combination with miR-709, is most preferred as an active ingredient in a medicament for the treatment or prevention of liver failure. Using mice as an experimental model representing human patients, it could be shown that the presence of miR-221, transient or permanent, e.g. by transient or permanent over-expression of miR-221, optionally in combination with the presence of miR-709, e.g. by transient or permanent expression, protects hepatocytes in vivo from apoptosis and delays liver failure, especially fulminant liver failure in a patient. For the purposes of the invention, the prevention of liver failure includes the stabilization of a functionally impaired or injured liver, especially of an intoxicated liver, e.g. for administration of the medicament to patients suffering from impaired liver function.
In further experiments it was shown that the inhibition of the mRNA encoding PUMA, which is effectively brought about by the presence of at least one siRNA having a sequence that is reverse complementary to the mRNA encoding PUMA, especially to the 3′UTR of the mRNA encoding PUMA, leads to the reduction or prevention of apoptosis in the presence of an apoptosis inducing agent. Therefore, miR-221 and/or miR-709 are exemplary representatives of those microRNAs which reduce or inhibit the presence or activity of PUMA.
Accordingly, the invention also relates to a nucleic acid construct containing a sequence that encodes at least one of the microRNA molecules hybridizing to the mRNA encoding PUMA, especially to the 3′UTR of the mRNA encoding PUMA, most preferred miR-221 and/or miR-709, as a medicament for the treatment or prevention of liver failure. Such a nucleic acid construct preferably contains a nucleic acid sequence encoding miR-221 functionally linked to an upstream promoter sequence, and preferably to a downstream terminator sequence, e.g. the sequence encoding miR-221 being arranged between an upstream promoter element and a downstream terminator for forming an expression cassette suitable for producing miR-221 by transcription, e.g. within the liver cells of a recipient of the nucleic acid construct.
For permanent expression of the microRNA that reduces or inhibits the presence of PUMA in hepatocytes, the nucleic acid construct containing an expression cassette for this at least one microRNA can have sequences for integration into the genome of hepatocytes, which sequences for integration can e.g. be selected from the group comprising terminal ITR elements, e.g. viral ITR elements, or recombinase recognition elements, especially when the expression cassette is co-transfected or co-transduced with an expression cassette encoding and producing the respective specific recombinase.
Primary mouse hepatocytes originating from Balb/C mice, which were purchased from Charles River Laboratories, Germany, were transfected with liposomes containing one microRNA selected from a number of microRNAs which are found both in humans and in mice. In detail, nucleic acid constructs containing an expression cassette containing a coding sequence for the microRNA as given below were formulated as liposomes using liposome complexing reagents. The liposomes containing the nucleic acid constructs having expression cassettes for the microRNAs were transfected to separate cultivated primary mouse hepatocytes at transfection rates of up to 80%. Expression of the microRNAs from the transfected expression cassettes was confirmed by detection of the over-expression of the microRNAs by quantitative reverse transcription PCR (RT-PCR). As a control, the coding sequence for Cre recombinase was included for transcription as a non-specific control RNA. Apoptosis was induced by FAS using the Jo2 antibody at 0.5 μg/mL culture.
The effect of the expression of the microRNA which was encoded by the transfected nucleic acid construct was assessed by analyzing the hepatocytes. In detail, at 6 hours after seeding, hepatocytes were transfected with the nucleic acid constructs encoding the microRNA, and 12 hours after transfection, apoptosis was induced by supplementing Jo2 antibody to the culture. Apoptosis was detected by exclusion of trypane blue. Hepatocytes were counted at 12 hours after addition of Jo2 antibody. The results show that expression of miR-221 in hepatocytes results in an increased cell viability.
On the example of miR-221, it was shown that inhibition of the mRNA encoding PUMA significantly increased cell viability following induction of apoptosis.
Generally, primarily mouse hepatocytes were isolated by a two-step collagenase (Roche Diagnostics) perfusion followed by a Percoll (obtainable from Sigma) density gradient centrifugation as described by Huh et al, PNAS 4477-4482 (2004). Purified mouse hepatocytes were cultured in Primaria dishes (BD Labware) in hepatocyte basal medium supplemented with hepatocyte single quotes (Lonza). For transfection Targefect hepatocyte reagent and Targefect F2 reagent (both from Targeting Systems) were used according to the manufacturer's instructions.
Analysis of cell viability by WST (obtainable from Roche Diagnostics) analysis shows that miR-221 protects hepatocytes from apoptosis induced by Jo2, whereas an inhibitor of miR-221 enhances the apoptotic effect of Jo2. Results are shown in
The protective effect of over-expression of the microRNA in hepatocytes from an expression cassette encoding the microRNA that is directed against the mRNA encoding PUMA is also shown on the example of miR-221 analysis of the caspase-3/7 activity. Results are shown in
For further analysis, hepatocytes with a general knockdown of microRNA expression were used. In short, hepatocytes with a knockdown of microRNA production in general were generated by knockdown of DGCR8 in mouse heptoma cells (hepa 1-6, obtained from ATCC), which were cultured in DMEM medium (PAA Laboratories), supplemented with 10% fetal calf serum (PAA), and glutamin (PAA), and penicillin/streptomycin (PAA). The DGCR8 knockdown cells were obtained by transduction with a retroviral vector expressing an shRNA specifically directed against DGCR8 as described by Kumaretal, Nature Genetics 673-677 (2007) and selected in medium supplemented to 1 μg/mL puromycin. Loss of DGCR8 expression was confirmed by Western blotting. Cells were then co-transfected with a nucleic acid construct containing an expression cassette for miR-221.
Following induction of apoptosis by addition of Jo2, cells were analyzed by FACS using FL4 for annexin V immune staining. The result is shown in
The result of the analysis of caspase-3/7 activity in DGCR8-knockdown cells (shDGCR8) is shown in
The therapeutic effect of a microRNA which is directed against the mRNA encoding PUMA was shown on mice as experimental animals on FAS-induced fulminant liver failure. For over-expression of the microRNA in vivo, a viral vector containing an expression cassette for the microRNA was used, namely the adeno-associated vector, serotype 8 (AAV8), which has previously been shown by Nakai et al, Journal of Virology 214-224 (2005) to transduce up to 100% of hepatocytes in mice when injected intravenously. Further, Kota et al in Cell 1005-1017 (2009) have shown that the vector AAV8 can successfully be employed for delivery of microRNA to a hepatocellular carcinoma. In short, the viral vector termed AAV8-ttr-miR-221 contained an expression cassette for miR-221, wherein the coding sequence for miR-221 was arranged under the control of the hepatocyte specific transthyritin (Ttr) promoter. The expression cassette was flanked on both sides by inverted terminal repeat elements (ITR) as shown in
For transduction, 1×1011 viral particles were injected into the tail vein of Balb/C mice. As a control, ROSA26 reporter mice as described by Soriano in Nature Genetics 70-71 (1999) were used for controlling transduction efficiency by injecting 1×1011 viral particles of the control AAV8-Ttr-Cre vector into the tail vein of the ROSA26 Cre reporter mice, which contain a foxed stop codon upstream of the β-galactosidase reporter gene. Efficient hepatocyte transduction could be confirmed by staining for X-Gal of liver sections of ROSA26 mice, as shown in
Detection of expression of miR-221 in mice transduced by the viral particles showed an approximately eight-fold higher expression of miR-211 compared to the mice transduced with the control viral particles AAV8-Ttr-Cre four days after injection of the viral particles.
For detection of miR-221, total RNA was isolated from purified liver tissue using the miRNeasy kit (Qiagen). Following an on-column DNase treatment, total RNA quality was determined, preferably using the Nanodrop assay (NanoDrop Technologies), and Bioanalyzer (Agilent). For each array, 250 ng RNA was suspended in a specific miRNA hybridization buffer, 25 μL per array), available from FeBIT GmbH, Heidelberg, Germany. Hybridization was performed for 16 hours at 42° C. using the GeniomRT-Analyzer. For quantitative RT-PCR, a real-time RT-PCR reaction was used, with 1 μg RNA for gene expression analysis, and 10 ng total RNA for miRNA expression analysis, respectively, for first strand cDNA synthesis. Preferably, the TaqMan mRNA RT kit was used for cDNA synthesis of miRNAs, and the TaqMan Universal real-time PCR kit was used for quantitative RT-PCR of miRNA, available from Applied Biosystems (USA). For gene expression analysis, e.g. for assaying the mRNA of PUMA, the SYBR Green PCR master mix (Applied Biosystems, USA) was used for real-time PCR. Normalization of gene expression was to expression of GAPDH. Preferably, data were analyzed according to the delta-delta Ct method. For expression analysis of PUMA, forward-primer SEQ ID NO 87 and reverse-primer SEQ ID NO 88 were used, for GAPDH expression analysis, forward-primer of SEQ ID NO 89, and reverse-primer of SEQ ID NO 90 were used.
Consistent over-expression of miR-221 could also be detected in liver tissue up to 2 weeks after in vivo injection of the viral particles containing the nucleic acid construct including the expression cassette for miR-221.
Four days after transduction of mice by injection of the viral particles containing AAV8-Ttr-miR-221 vector for expression of miR-221, and of control-transduced mice, fulminant liver failure was induced by administration of Jo2 antibody intraperitoneally (0.4 μg/g body weight). In the experimental animals, delayed death due to fulminant liver failure was seen in those animals having received the nucleic acid construct for expression of miR-221 compared to control mice; the survival is graphically shown in
For mice of the control group and of the group over-expressing miR-221 were sacrificed at 9 hours after injection of Jo2 antibody. Pathologic analysis of the livers confirmed that the observed survival effect could be assigned to the presence of miR-221, as these liver tissues showed inhibition of apoptosis, i.e. reduced pathological signs of liver injury. Consistent with liver morphology in the mice over-expressing mirR-221, decreased levels of serum transaminases (ALT and AST) and a reduced apoptosis of hepatocytes by detecting lower caspase-3/7 activity were determined as shown in
The terminal deoxynucleotidyl transferase-mediated dUTZ nick-end labeling (TUNEL) analysis (available from Millipore, USA) showed a reduced number of TUNEL positive nuclei in the miR-221 expressing mice compared to control mice, as shown in the micrographs of
The mechanism of protection of hepatocytes from apoptosis by reducing expression of PUMA was confirmed by analysis of expression of PUMA following induction of apoptosis, i.e. after injection of Jo2. Analysis by Western blotting using an anti-PUMA antibody (1:1000, Abeam), and an anti-tubulin antibody (1:1000, Sigma) show a dramatic reduction of PUMA protein expression at 12 hours after contact with Jo2 antibody, compared to 0 hour control hepatocyte lysates. Transiently elevated levels of PUMA expression at earlier time points did not contradict the reduction of expression of PUMA protein in presence of miR-221 in hepatocytes upon induction of apoptosis. The Western blots are shown in
A further analysis of mRNA encoding PUMA by quantitative RT-PCR after induction of apoptosis showed that mRNA levels for PUMA did not decrease, but mRNA encoding PUMA at least in isolated primary hepatocytes increased by a factor of about 1.8 at 12 hours after induction of apoptosis.
For confirmation, an in vitro assay was used, in which primary hepatocytes were transfected by a nucleic acid construct on the basis of the miR-glo vector, which contains the 3′ UTR of PUMA downstream of the luciferase gene. This luciferase reporter assay using the miR-glo vector demonstrates the direct binding of mature miRNA with the 3′ UTR of the mRNA encoding PUMA. The hepatocytes were transfected to over-express miR-221 and, as a control, expression of miR-221 was inhibited in hepatocytes. The luciferase reporter assay showed that hepatocytes which were transfected with a nucleic acid construct expressing miR-221 (wt 3′UTR, miR-221 mimic) produced lower luciferase activities, whereas hepatocytes which expressed a non-specific miRNA (wt 3′UTR, control miRNA mimic) or inhibitor of miR-221 (wt 3′UTR, miRNA inhibitor) or hepatocytes expressing miR-221 with a mutated 3′UTR (mut 3′UTR, miR-221 mimic) showed a high luciferase activity. This result confirms that miR-221 is specific for reducing expression of Puma through binding to the 3′ UTR of PUMA encoding mRNA. A graphic representation of relative luciferase activities is shown in
Further, analysis of PUMA protein levels at 48 hours after transfection were decreased in hepatocytes over-expressing miR-221, and increased in hepatocytes in which miR-221 was inhibited, as shown in
In a further in vitro assay, PUMA expression was knocked-down in primary hepatocytes by miR-221, which is specific for the mRNA encoding PUMA. Analysis of WSC and caspase-3/7 activities showed that loss of PUMA protein protected hepatocytes from apoptosis as induced by Jo2. This result, shown in
The mice transduced by the viral vector containing the nucleic acid construct AAV8-Ttr-miR-221 were analyzed by Western blotting for PUMA, FAS, p10, p27 and tubulin (loading control) in liver tissue. The result is shown in
Further in vitro analysis of hepatocytes in the WST assay and caspase-3/7 activity assay using primary hepatocytes which were knockdown for p10 and BMF using specific siRNA showed that cells transfected with p10 siRNA but not with BMF siRNA were protected against FAS induced apoptosis. Further, transfection with miR-221 in hepatocytes treated with PUMA siRNA or p10 siRNA increased the protection against FAS compared to hepatocytes treated with PUMA siRNA or p10 siRNA alone, as shown in
While specific embodiments of the present invention have been shown and described, it should be understood that other modifications, substitutions and alternatives are apparent to one of ordinary skill in the art. Such modifications, substitutions and alternatives can be made without departing from the spirit and scope of the invention, which should be determined from the appended claims.
Various features of the invention are set forth in the appended claims.
