ES2703193T3 - Hepatocyte grown for blood purification - Google Patents

Abstract

Cultured hepatocyte for blood purification, wherein the hepatocyte contains a compound that is an inhibitor of MKK4 kinase activity, MKK4 being encoded by the mRNA of SEQ ID NO: 1204, wherein the compound is not SP600125 , myricithin, genistein, or PD98059.

Description

DESCRIPTION

Hepatocyte grown for blood purification

The present invention relates to a hepatocyte cultured for blood purification, wherein the hepatocyte contains a compound that is an inhibitor of the kinase activity of MKK4, MKK4 which is encoded by the mRNA of SEQ ID NO: 1204, in that the compound is not SP600125, myricithin, geistein or PD98059.

Also disclosed herein is the use of a compound as a medicament for the treatment of a patient suffering from impaired hepatic function, the use of a compound as a medicament for the treatment of hepatic insufficiency, including acute liver failure. fulminant or chronic, and / or to increase the regeneration of liver tissue in a patient. Also disclosed herein is the use of the compound to increase the robustness and regeneration of hepatocytes cultured in vitro to improve cellular therapies, for example, for a process to grow hepatocytes in the presence of the compound, including the use of cultured hepatocytes as a transplantation, and for hepatocyte transplantation, respectively, in a patient suffering from liver failure. The present disclosure further discloses the use of the compound for the production of the medicament and the use of hepatocytes cultured in vitro in the presence of the compound for the production of a hepatocyte transplant.

In addition, the invention relates to a bioartificial liver comprising cultured hepatocytes containing the compound that has been described hereinabove and in the claims.

In addition, a process for producing hepatocytes comprising the compound used as a medicament, and the use of cultured hepatocytes contacted with the medicament for use as a medicament in the treatment of a liver functionally, is disclosed herein. impaired, for the treatment of liver failure, and / or to support liver regeneration. Hepatic impairment that can be treated in accordance with the present disclosure includes acute and / or fulminant hepatitis due to infection with hepatotropic viruses, alcohol abuse, obesity, genetic diseases such as Wilson's disease, hemochromatosis, deficiency in Alpha 1 antitrypsin and related conditions. The hepatic insufficiency that can be treated according to the invention also includes all forms of chronic liver failure with liver cirrhosis induced by, for example, the causes indicated above.

State of the art

To date, liver failure is treated by transplantation of a donor liver, however, there is a severe shortage of donor organs.

WO 98/39352, WO 99/14226 and US 7,569,575 B2 describe the use and synthesis of blocked nucleic acids (ANB).

Objects of the invention

It is an object of the invention to provide cultured hepatocytes, which can be maintained in culture for use in a bio-artificial liver, for example, for use in blood purification or for transplantation in patients with impaired hepatic function.

General description of the invention

The invention provides a cultured hepatocyte containing a compound that inhibits or inactivates kinase 4 of the mitogen-activated protein kinase (MAP2K4, also referred to as MKK4), as further defined in the claims. The nucleotide sequence of the mRNA encoding human MKK4 according to registration No. NM_003010 is provided as SEQ ID NO: 1204. Inhibition or inactivation of MKK4 activity can be by reduction of MKK4 expression, for example, by RNA interference induced by siRNA, especially shRNA or microRNA that hybridizes with the mRNA encoding MKK4, or by inhibition of MKK4 present in a hepatocyte, for example, by a specific kinase inhibitor compound.

The invention and the present disclosure are based on the discovery that the reduction or deletion of active MKK4 in hepatocytes, which may be both hepatocytes cultured in vitro and hepatocytes from a liver of an animal or human patient, results in increased regeneration of hepatocytes, for example, in prolonged culture periods and an increase in the regeneration of a damaged or damaged liver in vivo, for example, in experimental animals after the induction of an experimental hepatic insufficiency representing hepatic insufficiency in a human patient. It has been found that the reduction or deletion of active MKK4 in hepatocytes can result in an increased proliferative capacity due to an earlier entry into the cell cycle and an increased resistance against apoptosis. In summary, contacting hepatocytes in vivo with the compound that inhibits or inactivates MKK4 results in increased survival of mice in experimental models of hepatic insufficiency. In addition, contact hepatocytes cultured in vitro with the compound that inhibits or inactive MKK4 results in prolonged culture periods and production of cultured hepatocytes, which can be used, as disclosed herein, as a medicament, for example, as a transplant, or which can be used as part of a device for the continuous purification of blood taken from and returned to a patient.

Inactivation or deletion of MKK4 can be obtained by preventing the expression of functional MKK4 in liver cells, for example, by inactivating the endogenous gene encoding MKK4, for example, by insertional mutagenesis of the endogenous gene encoding MKK4, for example, by inserting a sequence of MKK4. nucleotides comprising at least one nucleotide, for the alteration of the endogenous gene encoding MKK4, preventing translation of the mRNA encoding MKK4, or by pharmacological means, for example, contacting hepatocytes in vivo or in vitro with a compound that inhibits MKK4 kinase function.

Preferably, the inactivation of MKK4 is obtained by reducing or preventing the expression of MKK4 by administration of an inhibitory RNA by RNA interference (iRNA), which is, for example, an oligonucleotide that hybridizes to the mRNA encoding MKK4, inhibitory RNA which may be, for example, a siRNA, an shRNA or any form of shRNA contained in a microRNA, for example, a siRNA based on microRNA, an antisense oligonucleotide, or a mixture thereof. Preferably, the oligonucleotide that hybridizes to the mRNA encoding MKK4 comprises or consists of 19, 21 or 22 nucleotides that are complementary, especially under physiological and cellular conditions, to the mRNA sequence encoding MKK4, and a second section, for example, an antisense chain, which is complementary in sequence to the first section. Of said double-stranded siRNA molecule, in a cell medium, the first section is released from the second section and binds to the mRNA encoding MKK4 to induce the degradation of this mRNA or to induce translation inhibition. The double-stranded RNA molecules (siRNA) that then release a section to target the mRNA can be administered directly to liver or liver cells, but can also be contained in shRNA or miRNA from which the double-stranded RNA is then released by enzymatic processing by the iRNA cellular machinery. The sequence of the oligonucleotide that hybridizes with the mRNA encoding MKK4 to induce its degradation or to prevent its translation may be 100% complementary in sequence as is usually the case with siRNA or shRNA, but may also contain mismatches as is often the case. case with endogenous miRNA, or it can be directed to MKK4 mRNA being only partially complementary in sequence. In the description, exemplary oligonucleotide sequences are provided which hybridize to the mRNA encoding MKK4, oligonucleotides which may be contained in an siRNA, for example, as a first section, preferably forming a double strand with a second reverse complementary section contained in the siRNA.

It has been found that inactivation of the MKK4 activity, preferably by reduction or inhibition of the expression of MKK4 by the presence of an oligonucleotide that hybridizes with the mRNA encoding MKK4, can be obtained by contacting hepatocytes in vivo or in vitro with the minus an oligonucleotide that hybridizes specifically with the mRNA encoding MKK4. Contacting in hepatocytes the mRNA encoding MKK4 can be obtained by administering to a human or animal patient the RNA that hybridizes to the mRNA encoding MKK4 using iRNA by siRNA by transient transfection of the siRNA in vivo, or alternatively using, for example, as a medicament, any means of stable administration of siRNA, for example, shRNA, especially siRNA based on microRNA or antisense oligonucleotides that hybridize to the mRNA encoding MKK4, eg, the use of a viral nucleic acid construct or based on transposon containing an expression cassette encoding the shRNA, for the transcription of the shRNA from the expression cassette. The siRNA, or the nucleic acid construct containing an expression cassette encoding the siRNA, is used as a medicine. The nucleic acid construct can be, for example, a viral vector or a nucleic acid construct that contains a transposon that additionally codes transposase for stable integrative transduction.

Generally, an oligonucleotide that hybridizes to the mRNA encoding MKK4 to reduce or prevent the expression of MKK4 in a hepatic cell is an oligonucleotide having a sequence that hybridizes to the mRNA encoding MKK4, especially that hybridizes to SEQ ID NO: 1204, under physiological conditions, for example, in the cell medium of a hepatic cell. The sequence can be completely complementary, i.e., be inverse complementary to a mRNA section of SEQ ID NO: 1204, or the sequence can have mismatches as often occurs in inhibition of microRNA-mediated translation, e.g. The oligonucleotide sequence has a nucleotide sequence of at least 80%, preferably at least 85%, more preferably at least 90% or 95% identity to an inverse complementary section of SEQ ID NO: 1204.

Preferred inhibitory oligonucleotides, for example, shRNA, comprise or consist of one or more of the oligonucleotides: SEQ ID NO: 1 to SEQ ID NO: 1203. For the design of these sequences, the DSIR tool was used to design siRNA targets and ARNhc (BMC Bioinformatics, November 30, 2006; 7 (1): 520) with a score threshold of 70, and therefore all of SEQ ID NO: 1 to 1203 have a score of at least 70. Sequences are preferred of inhibitory oligonucleotides, and groups of oligonucleotide inhibitor sequences that have higher scores. The sequences are provided in descending order of scoring value, eg, s Eq ID NO: 1 has the highest score (107.1) and SEQ ID NO: 1200, SEQ ID NO: 1201, SEQ ID NO: 1202, and SEQ ID NO: 1203 have the lowest score (70.0 each). From SEQ ID NO: 1 to SEQ ID NO: 11 have a score of at least 100, from SEQ ID NO: 12 to SEQ ID NO: 55 have a score of at least 95.1, eg, from 99.8 to 95.1, of SEQ ID NO: 56 to SEQ ID NO: 136 have a score of at least 90.0, for example, from 94.8 to 90.0, of SEQ ID NO: 137 to SEQ ID NO: 317 have a score of at least 85, for example, from 89.9 to 85.0, from SEQ ID NO: 318 to SEQ ID NO: 593 have a score of at least 80, for example, from 84.9 to 80.0, of SEQ ID NO: 594 to SEQ ID NO: 915 have a score of at least 75.0, for example, from 79.9 to 75.0, and of SEQ ID. NO: 916 to SEQ ID NO: 1203 have a score between 74.9 and 70.0. In addition, the shRNA or microRNAs may comprise one of these oligonucleotides that are complementary to the mRNA encoding MKK4, for example, comprising one of these oligonucleotides as a first section and a second complementary section in the siRNA as sections that hybridize to a microRNA. .

Oligonucleotides that hybridize with the mRNA encoding MKK4, which are disclosed herein for use as a medicament for the regeneration of liver tissue, or for the treatment of liver failure, liver failure, and / or liver cirrhosis, may be preferably in the form of RNA, DNA, or DNA and RNA hybrids, peptide-linked nucleic acids (APN), and nucleic acid derivatives containing a ribose moiety with substituents linking the 2'-carbon atom and the 4-carbon atom ', for example, by means of an oxymethylene group or an aminomethylene group, derivatives which are termed blocked nucleic acids (ANB), including additional phosphate-sugar main chain, single-stranded, preferably double-stranded, which by intracellular processing by the enzymatic machinery of iRNA release a single-stranded oligonucleotide for hybridization with the mRNA encoding MKK4.

In the alternative to consist of the use of a nucleic acid sequence that hybridizes to the mRNA encoding MKK4, for example, for direct use as a medicament disclosed herein, the inhibitory RNA may be contained as a sequence. encoding under the control of a promoter in an expression cassette. Depending on the promoter, which may be a constitutive or inducible promoter, after introduction into the hepatocyte the inhibitory RNA is produced by transcription.

For the introduction of nucleic acid constructs that reduce or delete the expression of active MKK4, for example, nucleic acid constructs that interrupt the endogenous gene encoding MKK4 of a hepatocyte or inhibitory RNA that hybridizes with the mRNA encoding MKK4, the Nucleic acid constructs are preferably provided in the form of one or more oligonucleotides in a pharmaceutically acceptable carrier formulation or in the form of a viral vector packaged in a viral particle or virus-like particle. A viral vector can be a retroviral, lentiviral vector, an adeno-associated viral vector, or adenoviral vector.

A formulation of the compounds or compositions for inhibiting or inactivating MKK4 in a pharmaceutically acceptable carrier can be, for example, in a lipid nanoparticle (LNP) formulation (eg, available from Alnylam Pharmaceuticals, USA), a formulation in liposomes, and / or in a formulation containing a combination with at least one transfection enhancing agent, for example, lipofectamine and / or as a complex with calcium.

In the alternative or in addition to an oligonucleotide having a sequence that hybridizes to the mRNA encoding MKK4, for example, an iRNA that hybridizes to the mRNA encoding MKK4, agents inactivating the activity can be used according to the invention. of MKK4, for example, agents that block the function of the MKK4 protein. Histological analysis of mouse livers with stable reduced expression of MKK4, experimentally generated by transfection with a nucleic acid construct containing an expression cassette for shRNA that hybridizes to the mRNA encoding MKK4 showed a normal histology. In addition, no increase in neoplasms was detected in the experimental animals, indicating that deletion of MKK4 does not increase the risk of cancer development.

Detailed description of the invention

The invention is now described in greater detail by the examples with reference to the figures, which show in Figure 1 schematic representations of nucleic acid constructs to produce inhibitory RNA, Figure 2 a schematic representation of a transposase-mediated intrahepatic transfer of a expression cassette encoding an inhibitory RNA (microRNA-based shRNA) of the invention,

Figure 3 the temporal evolution of the body weight of mice after intrahepatic transfer mediated by stable transposon of an expression cassette for inhibitor RNA and controls while an increase in body weight correlates with an increase in hepatic repopulation with the construct ,

Figure 4 GFP images of mouse livers explanted in the process of repopulation by hepatocytes stably expressing specific hcRNA to inactivate MKK4,

5 shows a Western blot specific for MKK4 from liver samples from mice stably transfected with an expression cassette encoding specific hcRNA to inactivate MKK4,

Figure 6 immunofluorescence analysis of the livers of mice stably transfected with an expression cassette encoding specific hcRNA to inactivate MKK4,

Figure 7 a Western blot for cyclin A and E from liver nuclear extracts of livers from mice transfected at the indicated time course after partial hepatectomy indicating earlier entry into the cell cycle of hepatocytes stably expressing mRNA specific for MKK4,

Figure 8 Ki67 staining of mouse livers expressing mRNA specific for MKK4 or control shRNA in the indicated time points after partial hepatectomy,

9 a graph of quantification of Ki67-positive hepatocytes depicted in FIG. 8, FIG. 10 TUNEL stain (upper panel) and with H & E (lower panel) in liver sections after induction of acute / fulminant hepatic failure in hepatocytes transfected with control hcRNA compared to hepatocytes that express an mRNA specific for MKK4 RNA, which are protected,

11 a graph of quantification of apoptotic hepatocytes according to the TUNEL stain as shown in FIG. 10,

Figure 12 shows a survival curve of mice expressing shRNA that inactivates MKK4 (shMKK4) compared to control mice (shCtr) after induction of hepatic insufficiency,

Figure 13 a quantification graph of the incorporation of EdU in murine hepatocytes cultured with inactivated MKK4 activity (FAHIG-shMKK4) and control hepatocytes (FAHIG-shCtr) with a box showing phase contrast micrographs of these hepatocytes,

Figure 14 phase contrast micrographs of hepatocytes cultured with inactivated MKK4 (shMKK4) on day 29 (d29) and control hepatocytes (ahCtr) on day 12 (d12), and day 3 of hepatocytes re-cultured on plates on day 15 (re-cultivated in plate),

FIG. 15 shows a survival curve of FAH - / - mice after transplantation of hepatocytes maintained for one week in culture expressing shRNA that specifically inactivates MKK4 or a control shRNA,

Figure 16 photographs, GFP image, a-FAH immunostaining and H & E staining of liver from mice aged for 1 year after transplantation of hepatocytes stably expressing shRNA that specifically inactivates MKK4, and in

17 shows a global view of the inhibitory effect of preferred small inhibitory compounds and of the genistein reference compound.

Using mice and liver tissue and murine hepatocytes as examples, especially representing human patients and human liver tissue and human hepatocytes, respectively, it was found that liver regeneration could be increased by inactivating MKK4 activity, both in vivo and in cultured hepatocytes. Mice that have livers with reduced MKK4 activity show increased regenerative capacity under conditions of liver failure, which also resulted in increased survival. Inactivation of MKK4 activity could be effectively achieved by inhibitory RNA present in hepatocytes, in vivo and in vitro, inhibitory RNA that could be generated by transcription of a transiently or stably transfected nucleic acid construct containing an expression cassette that encodes at least one RNA that under physiological conditions hybridizes with the MKK4 mRNA.

Alternatively, the inhibitory RNA could be introduced, for example, transfected into hepatocytes in vivo and in culture, for example, in the form of a siRNA, hsRNA or microRNA, preferably in a suitable formulation, for example, formulated as a preparation in liposomes. or a preparation of lipid nanoparticles. In the alternative to the use of inhibitor RNA, 3- (dimethylamino) -N- [3 - [(4-hydroxybenzoyl) -amino] -4-methylphenyl] benzamide (ZM 336372), 2-hydroxy-1-methyl-4- oxo-N-pyridin-4-ylquinoline-3-carboxamide (BAS00525963), 2- (1H-indazol-5-yliminomethyl) -6-nitrophenolate (BAS00697444), 5- [2- (3,5-dimethyl-1H- pyrazol-4-yl) ethyl] -7-oxo-N-phenyl-1H-pyrazolo [1,5-a] pyrimidine-3-carboxamide (SYN22174524), 5- [2- (3,5-dimethyl-1H- pyrazol-4-yl) ethyl] -3- (4-fluorophenyl) -1H-pyrazolo [1,5-a] pyrimidin-7-one (SYN22174787), 5- [2- (3,5-dimethyl-1H- pyrazol-4-yl) ethyl] -3- (4-methylphenyl) -1H-pyrazolo [1,5-a] pyrimidin-7-one (SYN22175977), 3- (4-chlorophenyl) -5- [2- ( 3,5-Dimethyl-1H-pyrazol-4-yl) ethyl] -2- (methoxymethyl) -1H-pyrazolo [1,5-a] pyrimidin-7-one (SYN22176267), - [2- (3,5 -dimethyl-1H-pyrazol-4-yl) ethyl] -2- (methoxymethyl) -3- (4-methylphenyl) -1H-pyrazolo [1,5-a] pyrimidin-7-one (SYN22176367), 5- [ 2- (3,5-Dimethyl-1H-pyrazol-4-yl) ethyl] -2- (3-methoxyphenyl) -3-methyl-pyrazolo [5,1-b] pyrimidin-7-ol (SYN22176842), - [2- (3,5-Dimethyl-1H-pyrazol-4-yl) ethyl] -2- (2-methoxyphen) il) -3-methyl-pyrazolo [5,1-b] pyrimidin-7-ol (SYN22176990), 3- (4-chlorophenyl-5- [2- (3,5-dimethyl-1H-pyrazol-4-yl) ) ethyl] -2-methyl-1H-pyrazolo [1,5-a] pyrimidin-7-one (SYN22177890), 5-amino-3 - [(Z) -1-cyano-2- [3 - [(4 -methoxy-6-piperidin-1-yl-1,3,5-triazin-2-yl) oxyphenyl] ethenyl] -1- (2-hydroxyethyl) pyrazole-4-carbonitrile (BAS00896568), 2- (1H-indazole -5-Uninomethyl) -6-methoxy-4-nitrophenolate (BAS00697462), 7-oxobenzo [e] perimidine-4-carboxylic acid (BAS00368055), the additional compounds contained in Table 1 provided herein, and combinations of they could be used as drugs, the presence of which would inactivate the MKK4 activity at least partially, also producing an increase in the proliferation of hepatocytes, protection against induced apoptosis, and restoration of hepatic function. These compounds having specific inhibitory activity against MKK4 are also collectively referred to as small inhibitor compounds. Accordingly, both the inhibitory RNAs having specificity for the RNA encoding MKK4 and the small inhibitory compounds having specificity for the MKK4 protein inhibit each MKK4 and, therefore, are used, as disclosed herein, as medicaments. in the treatment of hepatic insufficiency and / or for the protection of hepatocytes against apoptosis and / or for the regeneration of hepatocytes. The small inhibitor compounds can be formulated in a pharmaceutically acceptable formulation, comprising, for example, buffer substance and carrier substance, as well as formulation additives known to the pharmacist, for example, for iv, im, intrahepatic administration or administration oral.

During the in vitro functional assay of hepatocytes containing nucleic acid constructs with stable expression of FAH, GFP and shRNA, hepatocytes were isolated from the livers of mice and cultured. It was found that only hepatocytes that were transfected with an expression cassette encoding a targeting inhibitor RNA, i.e., hybridized specifically with the mRNA encoding MKK4, could be cultured for prolonged periods of time, for example, for more than 30 days. In addition, these hepatocytes could be trypsinized and replated in plates according to conventional methods. The transplantation of primary hepatocytes expressing the shRNA against MKK4 after 1 week in culture in mice deficient in FAH showed the ability of the hepatocytes in which MKK4 was inactivated to repopulate the liver of these mice and allow survival. On the contrary, this result could not be obtained by primary hepatocytes expressing the non-specific control hsRNA. This result also indicates that the primary hepatocytes expressing the shRNA against MKK4 do not undergo major dedifferentiation during the culture time.

Example 1: Inactivation of MKK4 by transcription of RNA inhibitor of an integrated expression cassette in liver tissue (reference)

The introduction of inhibitory RNA into hepatocytes, ie, in the liver of a patient, to inactivate MKK4 in vivo by expression of the inhibitory RNA of a nucleic acid construct encoding the inhibitory RNA in an expression cassette is shown in the example of mice (C57BL / 6) using an expression cassette encoding the inhibitory RNA for the production of the shRNA that hybridizes to the mRNA encoding MKK4. The promoter that controls the transcription of the inhibitory RNA was constitutive.

Briefly, homozygous negative mice were maintained for FAH (FAH - / -) with constant administration of NTCB to block 4-hydroxyphenylpyruvate dioxygenase that would otherwise produce the accumulation of toxic metabolites in the liver. As inhibitor RNA, SEQ ID NO: 1 was used, or, alternatively, SEQ ID NO: 2, both of which hybridize with the mRNA encoding MKK4. Each inhibitory RNA was introduced by contacting the liver cells in vivo with nucleic acid constructs with transposase-specific inverted repeat (IR) sections at both ends, containing an expression cassette for FAH to complement the FAH - / - genotype after expression, by hydrodynamic injection into the tail vein in combination with a second nucleic acid construct coding for transposase sleeping beauty 13 (SB13) under the control of the PGK promoter.

The nucleic acid constructs are shown in Figure 1. Figure 2 shows schematically the steps of genetic manipulation. A first control construct p / T-FAHIG contains the expression cassette that complements FAH and a green fluorescent protein (GFP) expression cassette comprising the coding sequence of GFP under the control of an IRES element, but does not encode inhibitory RNA . A sequence encoding an inhibitor RNA without targets as a control, which in addition to the GFP expression cassette in 3 'with respect to the coding sequence of GFP encodes a microRNA was contained in the p / T-FAHIG-shCtr construct. A sequence encoding an inhibitory RNA according to the invention was contained in the p / T-FAHIS-shMKK4 construct, which in addition to the GFP expression cassette 3 'to the GFP coding sequence encodes a microRNA (represented as a loop) comprising an hsRNA as an example of an inhibitory RNA. In the example, SEQ ID NO: 1 was used, alternatively SEQ ID NO: 2 as a preferred representative of RNA inhibitory sequences. After the introduction of the nucleic acid constructs, the mice were maintained in the absence of NTBC to select animals that had complemented hepatocytes. In cotransfected cells, transient expression of SB13 results in stable integration of the expression cassette into the genome.

Analysis of the mice after the introduction of the nucleic acid constructs confirmed the stable transcription of the nucleic acid construct inhibitory RNA. In detail, the analysis of the body weight of the mice of figure 3 shows that the animals that had received the control construct p / T-FAHIG (5), as well as the animals that had received the control construct p / T-FAHIG- shCtr (1), which expresses a non-specific RNA could not reconstitute liver function effectively, but died.

Animals from those groups that had received a nucleic acid construct containing an expression cassette for an inhibitory RNA that is specific for SEQ ID NO: 1, ie, p / T-FAHIG-shMKK4.A (2, 4) and p / T-FAHIG-shMKK4.B (3) were able to reconstitute liver function, as shown by the survival and restoration of body weights.

This result is further supported by Figure 4 which shows explanted livers on day 20 after administration of the nucleic acid construct, where the livers are in the process of repopulation by hepatocytes that were cotransfected in vivo with a nucleic acid construct containing an expression cassette for FAH and GFP and included an expression cassette for inhibitory RNA specific for the mRNA encoding MKK4 (p / T-FAHIG-shMKK4, both images on the left), or including an expression cassette encoding a Nonspecific RNA (p / T-FAHIG-shCtr, both images on the right). The explanted livers of Figure 4 show a faster increase in GFP fluorescence over time in vivo of animals cotransfected with the nucleic acid construct that includes the expression cassette encoding a specific inhibitory RNA for the mRNA encoding MKK4 compared to animals cotransfected with the nucleic acid construct that includes the expression cassette encoding an inhibitory RNA without a target.

The result of fluorescence is confirmed in this case in mouse livers completely repopulated by the immunospecific staining for MKK4 in the immunofluorescence shown in FIG. 5 and by immunofluorescence analysis for the expression of MKK4 in tissue samples from explanted mouse livers that they are shown in figure 6.

In Figure 5, shMKK4-224 indicates protein extracts from mouse livers repopulated with an expression cassette encoding an RNA inhibitor against MKK4 and shMKK4-3553 indicates protein extracts from mouse livers repopulated with an expression cassette encoding a Independent inhibitory RNA against MKK4; tubulin served as charge control and was detected by a specific antibody (a-tub), MKK4 was detected by an anti-MKK4 antibody (a-MKK4). In Figure 6, FAHIG-shCtr indicates a nucleic acid construct that contains the expression cassette to complement FAH and for GFP, which includes a non-specific inhibitory RNA (shCtr). shMKK4-A and shMKK4-B indicate nucleic acid constructs containing expression cassettes for shRNAs that specifically hybridize with MKK4 mRNA.

Both analyzes show that only the nucleic acid construct that includes an expression cassette encoding an inhibitory RNA for the mRNA encoding MKK4 produces a decrease in the expression of MKK4 in hepatocytes. Figure 7 shows Western blots for cyclin A and E from nuclear extracts from the mouse livers contacted with the nucleic acid construct expressing the shRNA that hybridizes to the MKK4 mRNA (shMKK4,) and expressing the non-specific shRNA (shCtr,), respectively, at 0, 24 h, 38 h, and 48 h after partial hepatectomy, detected with antibody a-cyclin A (a-Cic.A) and antibody a-cyclin B (a-Cic.B ). This analysis shows that the inactivation of MKK4, which is obtained, for example, by the expression of an inhibitory RNA that hybridizes with the mRNA encoding MKK4, leads to an earlier entry of the cell cycle after partial hepatectomy.

Figure 8 shows Ki67 staining of livers from experimental animals that had received the nucleic acid construct expressing the inhibitory RNA specific for MKK4 mRNA (shMKK4) and from animals that had received the non-specific shRNA-expressing construct. (shCtr), respectively at 0 h, 38 h and 48 h after partial hepatectomy. The analyzes show that the inactivation of MKK4, which in the example is obtained by the presence of the shRNA that is specific for the MKK4 mRNA and is expressed from the nucleic acid construct introduced into the hepatocytes, produces an increase in the proliferation of hepatocytes in alive.

Figure 9 shows a quantification of Ki67-positive cells from the analysis of Figure 8. The increase in proliferation of hepatocytes for hepatocytes containing the shRNA that inhibits the expression of MKK4 (p / t-FAHIG-shMKK4) is significant in comparison to the nonspecific control shRNA (p / t-FAHIG-shCtr).

Figure 10 shows TUNEL staining for the identification of apoptotic cells in liver tissue from mice transfected with an integrating nucleic acid construct that contains an expression cassette for nonspecific shRNA (shCtr) or an expression cassette for shRNA that specifically hybridizes with MKK4 mRNA (shMKK4.224 or shMKK4.355, each expresses a mouse-specific siRNA that hybridizes with MKK4 RNA). Apoptosis was induced in vivo 9 h before the analysis experimentally by injection of Jo2 antibody, which interacts with CD95 to induce fulminant hepatic failure. TUNEL staining reveals fewer apoptotic hepatocytes in liver tissue expressing MKK4-specific mRNA (shMKK4.224 or shMKK4.355) than in controls (shCtr). The upper row of images shows fluorescence micrographs of TUNEL analysis, the lower row shows light field micrographs of tissue samples stained with H & E.

The quantification of the TUNEL analysis after the induction of hepatic insufficiency is shown in Figure 11, which shows a significantly lower number of apoptotic hepatocytes in those hepatic tissues containing the shRNA (shMKK4.224 and shMKK4.3553) that hybridizes with the MKK4 mRNA when compared to the control with non-specific shRNA (shCtr).

Figure 12 shows the survival rate according to Kaplan Meier of mice transfected with the nucleic acid construct expressing in hsRNA that hybridizes to the mRNA of MKK4 (shMKK4.224 and shMKK4.3553) and control mice (shCtr) after the experimental induction of hepatic insufficiency. The result demonstrates that the inactivation of MKK4, inactivation that in the example is obtained by the expression of an inhibitory RNA (shRNA) from an expression cassette of a nucleic acid construct, effectively protects hepatocytes in vivo against apoptosis. Example 2: Inhibition of MKK4 in vivo by transcription of an RNA inhibitor of an expression cassette encoding shRNA (reference)

For the transient transfection of hepatocytes, a nucleic acid construct that contains or consists of an expression cassette encoding an inhibitory RNA that specifically hybridizes to the mRNA encoding MKK4, for example, containing SEQ ID NO: 1 or SEQ ID NO. : 2 (which are both specific for the human and mouse mRNA of MKK4) was transiently introduced into hepatocytes. For transient transfection in vivo, the nucleic acid construct was formulated in liposomes and administered to the experimental animals. The liposome formulation contained the lipids 3-N - [(qmethoxypoly (ethylene glycol) 2000) carbamoyl] -1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N, N-dimethyl-3 -aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a ratio of 2: 40: 10: 48 mole percent.

The effect of increasing the proliferation of hepatocytes, the protection against the induction of apoptosis could be shown as explained for the stable expression of the shRNA in Example 1, which indicates that the effect was limited to the period in which the shRNA was present in the hepatocytes using the analytical methods described in Example 1. This shows that the MKK4 activity can be effectively inhibited or inactivated by transient expression of a Rn inhibitor, e.g., shRNA or microRNA, of an expression cassette of a nucleic acid construct that does not integrate into the hepatocyte.

Example 3: Inhibition of MKK4 in vivo by inhibitor RNA transfected in hepatocytes (reference)

The suitability of the inhibitory RNA for use as a medicament could be shown by transfecting the inhibitory RNA that specifically hybridizes with MKK4 mRNA in liver tissue in vivo. The inhibitory RNA could be hcRNA or microRNA, preferably formulated as liposomes or lipid nanoparticles. Generally, the reduction or elimination of MKK4 could be obtained in at least a fraction of the liver tissue by the formulation of the inhibitory RNA using the analytical methods described in example 1. This shows that inhibitory RNA specific for MKK4 mRNA can be used as a medication, especially for the treatment of impaired liver function.

Example 4: Inhibition of MKK4 in vivo by SP600125, myricithin or genistein in hepatocytes (reference)

In the alternative to the inhibition of MKK4 activity in a liver tissue by the inhibitory RNA, SP600125 (reference) myricithin (reference) or genistein (reference) was used to inactivate MKK4 in the liver. Generally, SP600125, myricithin or genistein were administered to mice at a dose effective for in vivo inactivation of MKK4. Preferably, the dose was effective to inactivate at least 80%, more preferably at least 90% or 95% of the average MKK4 activity in vivo.

It could be found that the inactivation of MKK4 in the liver by administration of SP600125, myricithin or genistein as a drug produced a significant increase in liver regeneration, an increase in proliferation, and in protection against induced apoptosis using the analytical methods described in the example one.

Example 5: Inhibition of MKK4 in primary hepatocytes cultured in vitro by stable or transient transcription of RNA inhibitor of an expression cassette encoding shRNA

For in vitro transfection, cultured primary hepatocytes obtained from experimental animals were contacted with the nucleic acid construct as described in example 1 or 2. In general, the nucleic acid construct could be formulated as liposomes according to Example 2. In general, stable or transient expression of the inhibitory RNA could be obtained in cultured hepatocytes, and the reduction or elimination of MKK4 could be detected using the analytical methods described in example 1.

For experimental purposes, in the alternative to in vivo transfection of primary hepatocytes originating from an experimental animal, stably transfected hepatocytes expressing mRNA specific for MKK4 mRNA were isolated from experimental mice generated according to example 1. The analysis of cultured hepatocytes was by quantification of EdU incorporation by primary hepatocytes by flow cytometry. The result of transfected hepatocytes cultured after 3 days of culture is shown in Figure 13. The phase contrast micrographs of the box and the hepatocyte ratio containing mRNA specific for MKK4 mRNA, generated by expression of the transfected expression cassette. , show that hepatocytes cultured with inactivated MKK4 (FAHIG-shMKK4) show a dramatically improved EdU incorporation as a marker for proliferation on controls (FAHIG-shCtr) without inhibition of MKK4 activity in culture.

The plaque culture of freshly cultured hepatocytes in fresh culture medium shows the increased long-term survival of cultured hepatocytes in which MKK4 activity is essentially inhibited, for example, by the presence of inhibitory RNA (shMKK4) that hybridizes specifically with MKK4 mRNA, as shown in the micrographs of Figure 14. Hepatocytes with inactivated MKK4 (shMKK4) can be cultured efficiently at least until day 29 (d29), and can be cultured by trypsinizing and returning to cultivate in plates at a new medium on day 15; the micrographs on the right show day 3 of cells returned to cultivate in plates after 15 days of the initial culture. In contrast, cells transfected with a non-specific shRNA (shCtr) show a shorter long-term survival in culture and no growth after replanting in plaques after 15 days of initial culture.

These results show that the inactivation of MKK4 activity drastically increases the long-term survival and culture efficiency of plaques again of cultured hepatocytes.

In general, the Eagle medium generally known for hepatocyte cultures was used.

Example 6: Cultured hepatocytes with inactivated MKK4 activity for use as a medicine for liver regeneration

Hepatocytes were cultured from a mouse representing a patient having a compatible or identical blood group, preferably hepatocytes that were immunologically compatible with a subsequent recipient, eg, a patient, preferably autologous hepatocytes. The MKK4 activity was inhibited as described in the previous examples, preferably by transfection of hepatocytes cultured with a nucleic acid construct that contains an expression cassette for an inhibitory RNA that hybridizes with the mRNA encoding MKK4, by transfection with an inhibitory RNA, preferably repeatedly, or by contacting SP600125 (reference), myricithin (reference) or genistein (reference).

Cultured mouse hepatocytes that were stably transfected with a nucleic acid construct expressing complementary FAH and GFP (FAHIG) and an inhibitory RNA specific for the mRNA encoding MKK4 or a non-specific shRNA (Ctrl), respectively, were harvested by trypsinization. These hepatocytes were resuspended in a pharmaceutically acceptable vehicle and transplanted into the spleen or liver of FAH - / - mice, which were subsequently maintained without NTBC. The Kaplan Meier analysis of survival after intrasplenic transplantation of cultured hepatocytes is shown in Figure 15. Compared to mice that had received hepatocytes containing nonspecific shRNA (shRNA.Ctrl) that die on day 37-38 (line vertical), mice that had received hepatocytes containing MKK4 mRNA specific for MKK4 mRNA by expression of the expression cassette encoding the shRNA have a dramatically increased survival.

Experimental FAH - / - mice that had repopulated liver with hepatocytes with an expression cassette for GFP, including mRNA specific for the mRNA encoding MKK4 (mRNA. MKK4) were maintained for 12 months after repopulation. The analysis of explanted livers in clear field photography (Claro), with GFP image (GFP) (images on the left of figure 16) and anti-FAH immunofluorescence and H & E staining of liver sections (images on the right hand side of Figure 16) shows no tumor development with stable intrahepatic expression of GFP and of the shRNA that specifically inactivates MKK4. These data emphasize that the inhibition of MKK4 can be used to increase regeneration without triggering tumor growth.

Example 7: Cultured hepatocytes with inactivated MKK4 activity for use as a device for extracorporeal blood purification

Cultured hepatocytes were cultured as described above, preferably by culturing primary hepatocytes that were stably transfected with a nucleic acid construct expressing an mRNA specific for MKK4 mRNA, on a support substrate, eg, a polymer support. The cultured hepatocytes that adhere to the support substrate were placed in a container that was perfused with blood drawn from a patient, exemplified by a mouse or rat. The blood that comes out of the container could be returned immediately to the patient. In initial experiments, it could be shown that hepatocytes that are genetically engineered to stably express a shRNA that inactivates the mRNA encoding MKK4 are stable when grown on a support substrate, and that these cultured hepatocytes could be used as a purification device of blood. Example 8: Inactivation of MKK4 in in vitro analysis

The inhibitory effect of compounds against MKK4 was analyzed in an in vitro assay using purified MKK4 protein, for example, obtained from a cell line that was genetically engineered to overexpress MKK4 from an expression cassette containing the nucleotide sequence SEQ ID NO: 1204 as a coding sequence and affinity purification using, for example, an antibody directed against the MKK4 protein.

In the assay, MKK4 protein purified active incubated with its substrate JNK1a1 and GATP 32P - labeled (5 Jci approx 10 |. ^J M) without additional active compound, with the compound small inhibitor, or with genistein as positive control. For the assay, assay kinase buffer (20 mM HEPES pH 7.5, 10 mM MgCh, 1 mg / ml BSA, 1 mM Na3VO4, 1 mM DTT) was used. An inhibitory effect of the small inhibitor compound (final concentration 50 μM) was detected as a reduction of the phosphorylation activity of the MKK4 protein on its substrate JNK1a1 by measuring the amount of radioactive phosphate (32P) in JNK1a1. Phosphorylation of JNK1a1 was measured in the presence of 2 ml of scintillation cocktail per sample using a scintillation counter (Wallac, liquid scintillation counter). In this assay, genistein provided an inhibition of up to about 80% of the activity compared to the assay without additional active compound.

T l 1 m inhii r r i n r r r r rrrrrr rrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrrr

The compounds in Table 1 can be found at http: Wzinc.docking.org.

Figure 17 gives a global view of the inhibitory effects of these small inhibitory compounds on the MKK4 protein in relation to the inhibition by genistein (reference).

The in vitro assays according to Example 4 and the in vivo assays according to Example 6 could show that these small inhibitor compounds are suitable for the use disclosed herein as a medicament for the treatment of hepatic insufficiency and / or for the protection of hepatocytes against apoptosis and / or for the regeneration of hepatocytes.

Claims (3)

1. Hepatocyte cultured for blood purification, in which the hepatocyte contains a compound that is an inhibitor of the kinase activity of MKK4, MkK4 being encoded by the mRNA of SEQ ID NO: 1204, wherein the compound does not is SP600125, myricithin, genistein or PD98059. 2. Cultured hepatocyte according to claim 1, wherein the hepatocyte is disposed in a container having an inlet hole for the entry of blood from a patient and an exit orifice to recirculate the blood to the patient. 3. Cultured hepatocyte according to claim 1 or 2, wherein the compound is selected from the oligonucleotides of SEQ ID NO: 1 to SEQ ID NO: 1203 and the compounds of Table 1.