Patent Application
20210238257
The field of the disclosure relates to the process of transdifferentiation useful in the treatment of disease. More specifically, the disclosure relates to the treatment of cholestatic disease, for example in humans.
In regenerating organs of adult mammals, differentiated cells can replenish other types of differentiated cells by transdifferentiation, as in the pancreatic islet10, gastric gland11, lung alveolus12 and intestinal crypt13. Whether mammalian transdifferentiation can build these or other structures de novo is unknown. In the liver, hepatocytes can undergo biliary differentiation to form reactive ductules both in humans14-17 and animals with cholestatic liver injury3-6,18-21 Hepatocyte-derived biliary cells, however, exhibit incomplete biliary and residual hepatocyte differentiation, i.e., the cells are not mature cholangiocytes, and revert back to their original identity after the injury is reversed3-6. Moreover, hepatocyte-derived ductules do not contribute to bile drainage5. These findings are consistent with metaplasia, but not transdifferentiation, and call into question the functional significance of hepatocyte plasticity.
Transforming Growth Factor β Type I Receptor (TGFBR1) is a transmembrane serine/threonine kinase that forms a heteromeric complex with TGFβ type II serine/threonine kinase receptor (TGFBR2), the non-promiscuous receptor for the TGFβ cytokines TGFB1, TGFB2 and TGFB3. This non-promiscuous receptor transduces the TGFB1, TGFB2 and TGFB3 signal from the cell surface to the cytoplasm, thereby regulating a plethora of physiological and pathological processes, including cell cycle arrest in epithelial and hematopoietic cells, control of mesenchymal cell proliferation and differentiation, wound healing, extracellular matrix production, immunosuppression and carcinogenesis. The formation of the receptor complex composed of two TGFBR1 and two TGFBR2 molecules symmetrically bound to the cytokine dimer binding partner results in the phosphorylation and the activation of TGFBR1 by the constitutively active TGFBR2. Activated TGFBR1 phosphorylates SMAD2, which dissociates from the receptor and interacts with SMAD4. The SMAD2-SMAD4 complex is subsequently translocated to the nucleus, where it modulates the transcription of TGFβ-regulated genes. This constitutes the canonical SMAD-dependent TGFβ signaling cascade. The receptor is involved in non-canonical, SMAD-independent TGFβ signaling pathways. For instance, TGFBR1 induces TRAF6 autoubiquitination, which in turn results in MAP3K7 ubiquitination and activation to trigger apoptosis. Also, TGFBR1 regulates the epithelial to mesenchymal transition through a SMAD-independent signaling pathway via PARD6A phosphorylation and activation.
The JAG1 gene encodes the Jagged-1 protein (JAG1), which is a ligand that binds to the receptors NOTCH1, 2, 3 or 4 in the mammalian Notch signaling pathway. Other ligands in this pathway are JAG2 and Delta-like (DLL) 1, 3 and 4. The Notch signaling pathway is a highly conserved pathway that functions to establish and regulate cell fate decisions in many organ systems. Once the JAG1-NOTCH (ligand-receptor) interactions take place, a cascade of proteolytic cleavages is triggered, resulting in transcription activation of downstream target genes. The JAG1 gene is expressed in multiple organ systems in the body and mutations in JAG1 cause the autosomal dominant disorder Alagille syndrome (ALGS).
Despite progress in understanding the processes of tissue and organ regeneration, and despite advances in our knowledge of proteins such as TGFBR1, TGFBR2 and JAG1 that are involved in cell cycle control and cell fate decisions, a need continues to exist in the art for materials, methods and techniques useful in treating conditions or injuries associated with diseased, malformed or traumatized tissues and organs.
The disclosure provides materials and methods for treating conditions such as diseases of, or injuries to, mammalian tissues and organs. The disclosed methods rely on transdifferentiation to provide a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. Transdifferentiation is a form of lineage reprogramming, i.e., a process in which one mature somatic cell transforms into another mature somatic cell. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, i.e., in the presence of a fully developed scaffold and niche1. Disclosed herein is data establishing that transdifferentiation of hepatocytes in the liver can build a structure that failed to form in development—the biliary system in mice that mimic the hepatic phenotype of human Alagille syndrome (ALGS)2. In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, i.e., incomplete and transient biliary differentiation as an adaptation to cell injury, based on studies in mice with a fully developed biliary system3-6. In contrast to bile duct development7-9, demonstrated herein is the independence of de novo bile duct formation by hepatocyte transdifferentiation from NOTCH signaling. TGFβ signaling is identified as the driver and evidence is provided herein in support of this mechanism being active in patients with ALGS. Also shown herein is the ability to target TGFβ signaling in a manner that enhances the formation of the biliary system from hepatocytes, and the data disclosed herein also establish that the transdifferentiation-inducing signals and remodeling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. The results disclosed herein define the regenerative potential of mammalian transdifferentiation and reveal opportunities for therapy of ALGS and other cholestatic liver diseases. In some embodiments, a method of treatment of cholestatic disease comprises administration of an effective amount of wild-type hepatocytes, which is supported by data disclosed herein showing that transplanted wild-type hepatocytes form bile ducts in mice lacking bile ducts.
The disclosure contemplates the treatment of any of the following diseases associated with lack of bile ducts. Inherited diseases characterized by an absence of bile ducts include, but are not limited to, biliary atresia, Alagille syndrome, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18 and trisomy 21. Acquired disease exhibiting a lack of bile ducts include, but are not limited to, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, liver transplantation (ischemia, acute or chronic rejection), bone marrow transplantation (chronic graft versus host disease), cancer (Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome), infections (CMV, reovirus type 3, rubella, hepatitis C and B, EBV; Escherichia coli, Cryptosporidium parvum, Treponema pallidum), toxins and drugs, sarcoidosis, and idiopathic adulthood ductopenia.
In one aspect, the invention provides a method of forming a bile duct or ducts comprising introducing at least one expressible coding region in the TGFβ signaling pathway, such as Transforming Growth Factor β Type I Receptor (TGFBR1), Transforming Growth Factor β Type II Receptor (TGFBR2), Mothers Against Decapentaplegic homolog 3 (SMAD3), SMAD1, SMAD2, SMAD5 or SMAD8/9, or in the NOTCH signaling pathway, such as JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or any one or more of the respective NOTCH intracellular domains, into hepatocytes of a patient with a cholestatic disease or injury under conditions where the expression level of the coding region is greater than the wild-type level of expression, thereby inducing transdifferentiation of hepatocytes into mature cholangiocytes that form at least one bile duct. Without wishing to be bound by theory, the method provides a pathway that can compensate for defective signaling, such as defective Notch signaling, by overexpressing TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5 and SMAD8/9, i.e., the R-SMADs. Alternatively, the method compensates for defective signaling, e.g., Notch signaling, by providing an expressible coding region capable of restoring proper signaling, such as introducing wild-type JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains to restore Notch signaling. In some embodiments, the coding region encodes TGFBR1. In some embodiments, the coding region encodes TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains. In some embodiments, the patient has a cholestatic liver injury. In some embodiments, the bile duct contributes to bile drainage. In some embodiments, the bile duct is formed in the absence of Notch signaling. In some embodiments, the TGFBR1 coding region is a constitutive allele, or the TGFBR1 is under the expression control of a constitutive expression control element, e.g., a constitutive promoter, such as the Elongation Factor 1α (EF1α) promoter. In some embodiments, the TGFBR1 coding region is borne by a vector, such as a plasmid, phagemid, cosmid, human artificial chromosome, bacterial artificial chromosome or viral vector. In some embodiments, the viral vector is an Adeno-associated Virus (AAV), such as an Adeno-associated Virus serotype 8 (AAV8).
In some embodiments, the injury, such as a tissue or organ injury, results from a disease, such as human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus (EBV) infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the disease is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, or trisomy 21. In some embodiments, the disease is human Alagille syndrome (ALGS) wherein the expressible coding region introduced into hepatocytes of the patient encodes JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or any one or more of the respective NOTCH intracellular domains. In some embodiments, the disease is primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, infections (CMV infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the disease is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, or trisomy 21. In some embodiments, the disease is primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, infections (CMV infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the disease is human Alagille syndrome (ALGS), primary biliary cholangitis, or primary sclerosing cholangitis. In some embodiments, the patient has a cholestatic livery injury and practice of a method according to the disclosure results in formation of a bile duct that facilitates redress of the injury.
In some embodiments of the method of treating a cholestatic disease or injury in a patient, the expressible coding region of Transforming Growth Factor β Type I Receptor (TGFBR1), Transforming Growth Factor β Type II Receptor (TGFBR2), SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains is introduced into the hepatocytes ex vivo. In some embodiments, the coding region encodes TGFBR1. In some embodiments, the coding region encodes TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains. In some embodiments, the expressible coding region of Transforming Growth Factor β Type I Receptor (TGFBR1), Transforming Growth Factor β Type II Receptor (TGFBR2), SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains is introduced into the hepatocytes in vivo. In some embodiments, the coding region encodes TGFBR1. In various embodiments, the nucleic acid comprises a coding region for TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5 or SMAD8/9, or, to induce Notch signaling, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains.
Another aspect of the disclosure is directed to a method of treating a liver disease or injury in a patient by administering a therapeutically effective amount of a compound that induces, or results in, increased activity of at least one protein effector in hepatocytes of the patient, wherein the protein effector is (a) exogenously administered TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or any one or more of the respective NOTCH intracellular domains; (b) an mRNA encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or any one or more of the respective NOTCH intracellular domains; or (c) endogenously expressed TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or any one or more of the respective NOTCH intracellular domains, wherein the endogenous expression is induced by a small activating RNA. It is understood that when a protein effector itself is administered, the compound inducing, or resulting in, increased protein effector activity is the protein effector itself. In some embodiments, the hepatocyte is a wild-type hepatocyte. In some embodiments, the cholestatic disease or injury is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, Cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the cholestatic disease or injury is human Alagille syndrome. In some embodiments, the hepatocyte is a syngeneic hepatocyte. In some embodiments, the hepatocyte is an autologous hepatocyte.
A related aspect of the disclosure is drawn to a method of inducing an increase in activity of an endogenous effector protein using genome editing technology in the form of activating CRISPR (CRISPRa). Accordingly, the disclosure provides a method of inducing increased expression of an endogenous TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, or NOTCH4 expression control element, or gene, comprising administering a vector comprising a coding region for a guide RNA targeting the expression control region of TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, or NOTCH4 and a coding region for a fusion of deactivated Cas9 (dCas9) and at least one transcriptional activator. In some embodiments, the endogenous coding region, expression control element or gene is a JAG1 or NOTCH2 coding region, expression control element or gene. In some embodiments, the transcriptional activator is VP64, p65, or Rta. In some embodiments, the cholestatic disease or injury is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, Cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the cholestatic disease or injury is human Alagille syndrome.
Yet another aspect of the disclosure provides a method for correcting a mutated endogenous allele of TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, or NOTCH4 expression control element, or gene, comprising administering a vector comprising a coding region for a zinc finger nuclease (ZNF), a coding region for a transcription activator-like effector nuclease (TALEN), or coding regions for clustered regularly interspersed short palindromic repeats (CRISPR)-mediated gene repair, i.e., a guide RNA and Cas9, to a hepatocyte of a patient with a cholestatic disease or injury, thereby correcting the coding region of the mutated endogenous allele to treat the cholestatic disease or injury. In some embodiments, the mutated allele is a mutated JAG1 or a mutated NOTCH2 allele. In some embodiments, the cholestatic disease or injury is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, Cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the cholestatic disease or injury is human Alagille syndrome.
Yet another aspect of the disclosure is drawn to a method of forming at least one bile duct comprising transplanting an effective amount of a hepatocyte not engineered to overexpress a gene product to a patient with a cholestatic disease or injury, thereby inducing formation of at least one bile duct. Without wishing to be bound by theory, this approach provides cells competent for a signaling deficiency underlying a cholestatic liver disease, such as the defective Notch signaling underlying Alagille syndrome. In some embodiments, the hepatocytes are wild-type hepatocytes. In some embodiments, the cholestatic disease or injury is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, Cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the cholestatic disease or injury is human Alagille syndrome, primary biliary cholangitis, or primary sclerosing cholangitis. In some embodiments, the hepatocyte is a syngeneic hepatocyte. In some embodiments, the hepatocyte is an autologous hepatocyte. In some embodiments, the patient has a mutant JAG1 or NOTCH2 allele, wherein the mutant allele is corrected by introducing a vector comprising a zinc finger nuclease (ZFN), a coding region for a transcription activator-like effector nuclease (TALEN), or coding regions for clustered regularly interspersed short palindromic repeats (CRISPR)-mediated gene repair, i.e., a guide RNA and Cas9 into a hepatocyte of a patient with a cholestatic disease or injury, wherein the vector is introduced into the hepatocyte in vitro, thereby correcting the coding region of the mutated endogenous allele to treat the cholestatic disease or injury. In some embodiments, the patient has Alagille syndrome. In some embodiments, the expression of an endogenous normal allele of JAG1 or NOTCH2 is induced by introducing in vitro a vector comprising a coding region for a guide RNA targeting the expression control region of the JAG1 or NOTCH2 allele, and a coding region for a fusion of deactivated Cas9 (dCas9) and at least one transcriptional activator into a hepatocyte of a patient with a cholestatic disease or injury.
Yet another aspect of the disclosure is a method of treating a cholestatic disease or injury comprising administering an effective amount of a hepatocyte not engineered to overexpress a gene product to a patient. In some embodiments, the hepatocyte is a wild-type hepatocyte. In some embodiments, the cholestatic disease or injury is human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, Cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr Virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. In some embodiments, the cholestatic disease or injury is human Alagille syndrome, primary biliary cholangitis, or primary sclerosing cholangitis. In some embodiments, the hepatocyte is a syngeneic hepatocyte. In some embodiments, the hepatocyte is an autologous hepatocyte. In some embodiments, the patient has a mutant JAG1 or NOTCH2 allele, wherein the mutant allele is corrected by introducing a vector comprising a zinc finger nuclease (ZFN), a coding region for a transcription activator-like effector nuclease (TALEN), or coding regions for clustered regularly interspersed short palindromic repeats (CRISPR)-mediated gene repair, i.e., a guide RNA and Cas9 into a hepatocyte of a patient with a cholestatic disease or injury, wherein the vector is introduced into the hepatocyte in vitro, thereby correcting the coding region of the mutated endogenous allele to treat the cholestatic disease or injury. In some embodiments, the patient has Alagille syndrome. In some embodiments, the expression of an endogenous normal allele of JAG1 or NOTCH2 is induced by introducing in vitro a vector comprising a coding region for a guide RNA targeting the expression control region of the JAG1 or NOTCH2 allele, and a coding region for a fusion of deactivated Cas9 (dCas9) and at least one transcriptional activator into a hepatocyte of a patient with a cholestatic disease or injury.
Other features and advantages of the present disclosure will become apparent from the following detailed description, including the drawing. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments, are provided for illustration only, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from the detailed description.
The disclosure provides materials and methods for treating cholestatic disease, which is a group of diseases characterized by impaired flow of bile. Cholestasis is characterized by a decrease in bile flow due to impaired secretion by hepatocytes or to obstruction of bile flow through intra- or extrahepatic bile ducts. Bile is secreted by the liver and facilitates the digestion of fats, and impairments in bile flow can lead to tissue and organ necrosis. Unless treated, cholestatic disease can lead to chronic liver injury and end-stage liver disease.
Provided by the disclosure are methods of using Transforming Growth Factor β Receptor type I (TGFBR1), Transforming Growth Factor β Type II Receptor (TGFBR2), SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains to form at least one bile duct or to treat a cholestatic disease or injury. TGFBR1, an exemplary protein according to the disclosure, forms a heteromeric complex with type II TGFβ Receptor (TGFBR2) when bound to TGFβ, transducing the TGFβ signal from the cell surface to the cytoplasm. TFGβRI is a single-pass type I membrane protein, and the TGFβ receptor protein is a serine/threonine protein kinase. The protein is 503 amino acids (55960 Da) and interacts with CD109. The unphosphorylated protein interacts with peptidyl-prolyl cis-trans isomerase FKBP1A and is stabilized in the inactive conformation. Phosphorylation of the GS region of TFGβRI abolishes FKBP1A binding. When phosphorylated on several residues in the GS region, TFGβRI interacts with SMAD2 (Mothers Against Decapentaplegic homolog 2).
The disclosure also provides a nucleic acid, such as a double-stranded DNA, encoding a biologically active Transforming Growth Factor β Type I Receptor or a fragment, derivative or analog thereof, for delivery to at least one hepatocyte. The accession number for the TGFBR1 gene is NM_004612, and the sequence therein is incorporated herein by reference. The complete polynucleotide sequence of TGFBR1 with the sequence of its chromosomal context on chromosome 9 is provided at GenBank Accession Number NC_000009, incorporated herein by reference. Apparent from a consideration of the disclosure is that nucleic acids encoding a biologically active Transforming Growth Factor β Type II Receptor (Accession Number NM_001024847 for the TGFβR2 polynucleotide encoding variant 1 protein and NM_003242 for the TGFβR2 polynucleotide encoding variant 2 protein), a TGFβ effector coding region, such as a coding region for SMAD3 (Accession Number KR712265 providing the polynucleotide and encoded amino acid sequences), SMAD1 (Accession Number U59423 providing the polynucleotide and encoded amino acid sequences), SMAD2 (Accession Number KJ891567 providing the polynucleotide and encoded amino acid sequences), SMAD5 (Accession Number AH005750 providing the polynucleotide and encoded amino acid sequences) or SMAD8/9 (Accession Numbers NM_001127217, NM_005905, XM_006719827, XM_005266401, XM_005266403, and XM_005266404 providing the polynucleotides of the several variants and the amino acid sequences encoded by each variant), or the NOTCH receptor ligand JAG1 (Accession Number NM_000214 providing the polynucleotide and encoded amino acid sequences), JAG2 (Acc. Nos. NM_002226.5, NM_145159.2), DLL1 (Acc. No. NM_005618.4), DLL3 (Acc. Nos. NM_016941.3, NM_203486.2), DLL4 (Acc. No. NM_019074.4), NOTCH1 (Acc. No. NM_017617.5), NOTCH2 (Acc. Nos. NM_024408.4, NM_001200001.1), NOTCH3 (Acc. No. NM_000435.3), NOTCH4 (Acc. No. NM_004557.4) or the respective NOTCH intracellular domains, as well as coding regions for fragments, derivatives or analogs of any of these biologically active proteins, are expected to be beneficially useful in the methods according to the disclosure.
In addition to providing nucleic acids encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, and fragments, derivatives, and analogs thereof, the disclosure provides nucleic acids comprising at least one expression control element for delivery to hepatocytes. This aspect of the disclosure provides one or more expression control elements to hepatocytes to alter, e.g., increase, the expression of endogenous TGFBRI, TGFBRII, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, or fragments, derivatives, or analogs thereof. In this manner, the expression level and activity of TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains is increased in the methods according to the disclosure. Expression control elements contemplated for inclusion in this aspect of the disclosure are heterologous promoters, such as strong promoters, constitutive promoters, inducible promoters, repressible promoters, and any combination thereof known in the art. Other contemplated expression control elements include enhancers, strong ribosome binding sites (e.g., sites conforming more closely to the Kozak consensus sequence for expression in eukaryotic cells), an altered intron sequence or length, stronger intron donor and acceptor splice sites, a polyadenylation sequence, or a transcriptional termination element. Preferred expression control elements for delivery to hepatocytes according to the disclosure are heterologous promoters and enhancers. Also preferred are expression constructs in which at least one expression control element is constitutively active and mutant coding regions expressing a constitutively active protein, such as the D204T mutant TFGβR1.
The polynucleotide (e.g., gene, cDNA, RNA, or fragment, analog or derivative thereof) encoding a Transforming Growth Factor β Type I Receptor (TGFBR1) polypeptide, TGFBR2 polypeptide, SMAD3 polypeptide, SMAD1 polypeptide, SMAD2 polypeptide, SMAD5 polypeptide, SMAD8/9 polypeptide, JAG1 polypeptide, JAG2 polypeptide, DLL1 polypeptide, DLL3 polypeptide, DLL4 polypeptide, NOTCH1 polypeptide, NOTCH2 polypeptide, NOTCH3 polypeptide, NOTCH4 polypeptide or polypeptides of the respective NOTCH intracellular domains can be inserted into an appropriate expression or amplification vector using standard ligation techniques. As used herein, a “vector” is any molecule or moiety that transports, transduces or otherwise acts as a carrier of a polynucleotide according to the disclosure. The vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
Typically, expression vectors used in any of the host cells will contain sequences for plasmid maintenance and cloning and expression of inserted nucleotide sequences. Such sequences, referred to collectively as “flanking sequences”, may include a promoter and other regulatory elements such as an enhancer(s), an origin of replication element, a transcriptional termination element, a complete intron sequence containing a donor and acceptor splice site, a signal peptide sequence, a ribosome binding site element, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed into a vector, and a selectable marker element. A person skilled in the art will recognize that a target cell may require a specific promoter including, but not limited to, a promoter that is species-specific, inducible, tissue-specific, or cell-cycle-specific. See Parr et al., Nat. Med. 3:1145-9 (1997), the entirety of the contents of which are herein incorporated by reference. Optionally, the vector may contain a “tag” sequence, i.e., an oligonucleotide molecule located at the 5′ or 3′ end of a polypeptide coding sequence of TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains. The oligonucleotide molecule may encode polyHis (such as hexaHis), or another “tag” such as FLAG, HA (hemagglutinin influenza virus) or myc, for which commercially available antibodies exist. This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as means for affinity purification of the polypeptide from the host cell. Optionally, the tag can subsequently be removed from a purified polypeptide of TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains by various means such as using certain peptidases.
The flanking sequence may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), synthetic, or it may be the native nucleic acid flanking sequences of TGFβRI, TGFβRII, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains. The flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art. A selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, kanamycin, or G418, (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from the media used for culturing the host cells. The ribosome binding element, commonly called the Shine-Dalgarno sequence (prokaryotes) or the Kozak sequence (eukaryotes), is usually necessary for translational initiation of mRNA. The element is typically located 3′ to the promoter and 5′ to the coding sequence of the polypeptide TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains to be synthesized. Methods used for obtaining each of the elements are well known to the skilled artisan.
Many vectors, e.g., plasmids, cosmids, minicircles, phage, viruses, useful for transferring nucleic acids into target cells are available. The vectors comprising the nucleic acid(s) may be maintained episomally, e.g., as plasmids, minicircle DNAs, viruses such as cytomegalovirus, adenovirus, and adeno-associated virus, or they may be integrated into the target cell genome through homologous recombination or random integration, e.g., retrovirus-derived vectors such as lentivirus, MMLV, HIV-1, and ALV. Vectors may be provided directly to the subject cells by contacting the cells with a vector comprising the polynucleotide of the disclosure such that the vector is taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. DNA can be introduced as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV). For viral vector delivery, the cells may be contacted with viral particles comprising the polynucleotide of the disclosure.
A preferred vector according to the disclosure is an adeno-associated virus (AAV), which is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length, including two 145-nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). Cloning of the AAVrh.74 serotype is described in Rodino-Klapac et al., Journal of Translational Medicine 5, 45 (2007). Isolation of the AAV-B 1 serotype is described in Choudhury et al., Mol. Therap. 24(7): 1247-57, 2016. AAV vectors may also comprise self-complementary AAV vectors scAAVs). scAAV vectors contain both DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAV s allow for rapid expression in the cell. The sequences of these AAV serotypes are incorporated herein by reference.
AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.
In various embodiments, the polynucleotide encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains is contained in an adeno-associated virus (AAV) vector. The AAV may be a recombinant AAV virus and may comprise a capsid serotype such as, but not limited to, of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV2/5, AAV5/8, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8. As a non-limiting example, the capsid or capsids of the recombinant AAV virus is AAV2, AAV5 AAV8, AAV2/5 or AAV5/8.
As noted above, other suitable vectors include retroviruses, for example, lentiviruses, for use in the methods of the disclosure. Commonly used retroviral vectors are defective, i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising genes of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing the retroviral vectors comprising the polynucleotide according to the disclosure into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art.
In some embodiments, targeted integration is promoted by the presence of sequences on the polynucleotide of the disclosure that are homologous to sequences flanking the integration site. The “polynucleotide” as recited in the preceding sentence means the polynucleotide encoding a TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, or a fragment, a derivative, or an analog thereof, or an expression control element as disclosed herein. For example, targeted integration using the polynucleotides described herein may be achieved following conventional transfection techniques, e.g., techniques used to create gene knockouts or knock-ins by homologous recombination. In other embodiments, targeted integration is promoted both by the presence of sequences on the polynucleotide of the disclosure that are homologous to sequences flanking the integration site, and by contacting the cells with the polynucleotide described herein in the presence of a site-specific recombinase, such as a recombinase from the Integrase or Resolvase families.
The disclosure provides methods for administering or delivering the nucleic acids encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, or fragments, derivatives or analogs thereof, to individuals, e.g., humans that have a cholestatic disease. A variety of cholestatic diseases are amenable to treatment according to the methods of the disclosure. In general, any cholestatic disease is treatable by the methods disclosed herein. In particular, the disclosure contemplates treatment of human Alagille syndrome (ALGS), biliary atresia, cystic fibrosis, alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis, arthrogryposis-renal dysfunction-cholestasis syndrome, trihydroxycoprostanic acidemia, trisomy 17, trisomy 18, trisomy 21, primary biliary cholangitis, primary sclerosing cholangitis, autoimmune hepatitis, acute rejection of liver transplant, chronic rejection of liver transplant, liver transplant ischemia, bone marrow transplant-induced chronic graft-versus-host disease, Hodgkin lymphoma, Langerhans cell histiocytosis, macrophage activation syndrome, cytomegalovirus (CMV) infection, reovirus type 3 infection, rubella infection, hepatitis C infection, hepatitis B infection, Epstein-Barr virus infection; microbe infection, sarcoidosis, or idiopathic adulthood ductopenia. Any known method of administering or delivering the nucleic acids encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, or fragments, derivatives or analogs thereof is contemplated. In some embodiments, the nucleic acids, e.g., an AAV vector comprising a polynucleotide encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains is administered by injection into a peripheral vein. In some embodiments, the nucleic acid is targeted to one or more particular tissues or organs, such as targeting the liver by injection of the vector comprising the nucleic acid encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, or fragments, derivatives or analogs thereof, into the portal vein, hepatic artery or spleen. In addition to providing the encoding nucleic acid as a DNA polynucleotide in a suitable vector, the disclosure contemplates delivery of an mRNA encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, or fragments, derivatives or analogs thereof, to treat cholestatic disease.
As an alternative to using viral vector to deliver the nucleic acids encoding TGFBR1, TGFBR2, SMAD3, SMAD1, SMAD2, SMAD5, SMAD8/9, JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains, synthetic mRNAs, small activating RNAs, or recombinant proteins can be used to provide these therapeutic factors in vivo. In addition, small molecules can be used to activate the TGFβ or NOTCH signaling pathways in vivo. Intravenously injected in vitro-synthesized mRNAs encapsulated in lipid nanoparticles are readily taken up by hepatocytes through receptor-mediated endocytosis, leading to rapid delivery of the mRNA to the cytoplasm and production of the therapeutic protein. Small activating RNAs are short double-stranded oligonucleotides that can be targeted to a gene's promoter to induce transcriptional elongation. Formulation in liposomal nanoparticles facilitates efficient uptake and target gene activation in hepatocytes after intravenous injection. Recombinant proteins, like Fc chimeric fusion proteins composed of an immunoglobulin Fc domain that is directly linked to another peptide, are effective in the liver after intravenous injection; they are particularly useful to provide ligands such as JAG1, JAG2, DLL1, DLL3 or DLL4. Small molecules activating TGFβ or NOTCH signaling exist and can be targeted to exert their function specifically in the liver after intravenous injection. Because of low immunogenicity, mRNAs, small activating RNAs, recombinant proteins, and small molecules can be repeatedly applied, thereby overcoming their transient effect.
As an alternative to delivering the nucleic acids encoding JAG1 or NOTCH2, the mutated JAG1 or NOTCH2 allele can be corrected in situ using genome editing technology, such as, but not limited to, zinc finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR). It is known in the art that ZNFs and the Transcription Activator-Like (TAL) component of TALENs are amenable to genetic engineering to tailor the binding site of the ZNF or TALEN to a DNA site of interest, and it is further known that these components can be fused to non-specific nucleases such as the FokI nuclease cleavage domain to yield nucleases targeting a DNA site of interest for editing. Analogously, CRISPR has been developed to the point where the editing of a given region of DNA, such as a gene, coding region or expression control element, is known in the art. Each of these genome editing techniques is known in the art to be useful in correcting mutations in DNA, including mutations in coding regions, expression control elements or, more generally in genes or intergenic DNA. Further, each of these techniques is amenable to administration to hepatocytes in vivo in patients with cholestatic injury or disease and to introduction into hepatocytes from such patients in vitro, suitable for in vitro amplification of the hepatocytes and/or re-introduction of such cells into the patients.
In addition, the expression level of a normal or non-mutated allele such as the non-mutated JAG1 or NOTCH2 allele can be induced using activating CRISPR (CRISPRa). To induce the expression of a normal, e.g., wild-type, allele of JAG1 or NOTCH2, in a hepatocyte of a patient with a cholestatic disease or injury, a vector is administered to the hepatocyte(s) that comprises a deactivated Cas9 (dCas9), i.e., a Cas9 coding region in which the nuclease domains have been inactivated by mutation. The dCas9 is fused to one or more transcriptional activators, e.g., VP64, p65 and Rta. The vector further comprises a guide RNA that targets upstream of the transcriptional start site (TSS) of the target gene, such as the normal, or wild-type, allele of JAG1 or NOTCH2 in a patient exhibiting the haploinsufficiency characteristic of Alagille syndrome. The administration of such a vector results in induced expression of the normal allele such as the normal JAG1 or NOTCH2 allele in hepatocytes of patients with a cholestatic disease or injury. As with the gene correction methodologies noted above, induction of endogenous gene expression, e.g., JAG1 or NOTCH2 expression, using CRISPRa is amenable to in vivo administration of the vector or introduction of the vector into hepatocytes from such patient in vitro, followed by amplification and/or re-introduction of such cells into the patients. These gene-editing tools can be efficiently delivered to hepatocytes in vivo using viral vectors, particularly AAV vectors.
Because Alagille syndrome is caused by haploinsufficiency of JAG1, i.e., patients have a mutated and a normal allele, CRISPR-mediated activation (CRISPRa) can be used to increase expression of the normal allele. The components of CRISPRa can be efficiently delivered to hepatocytes in vivo using viral vectors, particularly AAV vectors.
The following examples illustrate the subject matter of the disclosure and are not intended to limit the scope of the disclosure. Example 1 provides the methods used in the experiments disclosed herein, Example 2 describes the mouse model, Example 3 discloses the tracing of peripheral bile duct formation, Example 4 shows that the peripheral bile ducts that were formed were contiguous with the extrahepatic biliary system, Example 5 discloses the examination of cell identity in the transdifferentiation of hepatocytes to cholangiocytes, Example 6 relates the determination of the relative numbers of transdifferentiating hepatocytes, Example 7 shows the results of assessing proliferation of transdifferentiated hepatocytes in a mouse model of cholestatic liver injury induced by DDC, and Example 8 discloses the assessment of the role played by TGFβ signaling in transdifferentiated hepatocyte formation of bile ducts.
Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice (mixed background) were previously reported2,26. R26R-RFP+/+39 (C57BL/6) and R26NZG+/+40 (FVB) mice were used. Flp-reporter mice were generated by crossing R26NZG+/+ mice with EIIa-Cre+/+41 mice (C57BL/6) to remove the Cre-reporter element and then crossing out the EIIa-Cre. These R26ZG+/+ mice were crossed with Alb-Cre+/−; Rbpjf/f;Hnf6f/f mice to generate Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26ZG+/+ mice. R26R-Confetti+/+42 mice (C57BL/6) were crossed with Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice to generate Alb-Cre+/−; Rbpjf/f;Hnf6f/f;R26R-Confetti+/− mice. Tgfbr2f/f43 mice (C57BL/6) were crossed with Alb-Cre+/−; Rbpjf/f;Hnf6f/f mice to generate Alb-Cre+/−;Rbpjf/f;Hnf6f/f;Tgfbr2f/f mice. Because Tgfbr2 (68.39 cM) and Hnf6 (41.93 cM) are both on chromosome 9, recombinants were generated at 0.1356 (35/258 mice) observed frequency (0.26 expected frequency). Different founder recombinants were intercrossed to generate Alb-Cre+/−;Rbpjf/f;Hnf6f/f;Tgfbr2f/f mice. R26R-ZsGreen+/+44 mice (C57BL/6) were crossed with Alb-Cre+/− mice to generate Alb-Cre+/−;R26R-ZsGreen+/+ mice. Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice were crossed with Rag2−/−45;Il2rg−/−46 mice (mixed background) to generate Alb-Cre+/−;Rbpjf/f;Hnf6f/f;Rag2−/−;Il2rg−/− mice. Jag1+/−55 (C57BL/6) were bred to R26R-RFP+/+ mice to generate Jag1+/−;R26R-RFP+/− mice. Male and female mice of the indicated age and genotype were chosen randomly for inclusion in experiments. The mouse used as a positive control for biliary gene expression was a C57BL/6 wild-type mouse fed choline-deficient diet (MP Biomedicals) and given 0.15% (w/v) ethionine (Sigma-Aldrich) in the drinking water (CDE diet) for 3 weeks. All mice were kept under barrier conditions. All procedures were approved by the Institutional Animal Care and Use Committee at UCSF or CCHMC.
The AAV-Ttr-Flp plasmid was generated by removing Cre from AAV-Ttr-Cre47 and replacing it with Flpo48 from pPGKFlpobpA (Addgene 13793); viruses were produced by Vector Biolabs and used at a high dose of 1-3×1012 vgs (viral genomes) or low dose of 4×1011 vgs. The AAV-Ef1α-caTgfbr1 plasmid was built by VectorBuilder to contain the activating T204D49 mutation; virus was produced by Vector Biolabs and used at a dose of 1×1011 vgs. Titers were determined using qPCR. Viruses were delivered by tail vein injection in volumes ≤100 μL to prevent hydrodynamic effects. AAV-CMV-Jag1 plasmid was built by VectorBuilder; virus was produced by Vector Biolabs and used at a dose of 1×1011 vgs.
Livers from Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26ZG+/+, R26R-Confetti+/− and transplanted mice were perfused with ice-cold PBS followed by 4% paraformaldehyde (PFA). Samples were cut into slices and fixed overnight in 4% PFA at 4° C. Livers from R26R-RFP+/+, Alb-Cre+/−; Rbpjf/f;Hnf6f/f, Rbpjf/f;Hnf6f/f and Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26ZG+/+ mice were fixed overnight in 4% PFA at 4° C. For thin sectioning, samples were moved to 30% sucrose overnight at 4° C. to cryopreserve and then embedded in OCT (Tissue-Tek) or processed for paraffin embedding and sectioning. A Leica CM3050 S cryostat was used to cut 6 μm cryosections for staining and imaging. For 3D and sparse-labeling clonal analysis, liver samples were embedded in 4% agarose and sectioned on a Leica VT100 S vibratome. For clonal analysis in R26R-Confetti+/− mice, liver samples were cut into 1 mm slivers, immunostained, washed in PBS, equilibrated in 30% sucrose and embedded in OCT. The 1 mm slivers were then cryosectioned at 100 μm, stained with Hoechst and mounted in PBS for confocal imaging. For analysis of Flp-based hepatocyte fate tracing in R26ZG+/+ mice, liver samples were fixed in neutral-buffered formalin containing zinc (Z-Fix, Anatech), embedded in paraffin and sectioned to 5 μm.
Explant samples from the regenerative nodule and nonregenerated liver tissue of a 3-year-old male patient with ALGS were previously described; regenerative nodule and nonregenerated liver tissue contained the same heterozygous JAG1 exon 1-26 deletion36. Samples were obtained with patient consent and approval from the Commission Cantonale d'Ethique de la Recherch CCER. Biopsy samples from the regenerative nodule and nonregenerated liver tissue of a 15-year-old male patient with ALGS caused by a heterozygous c.499T>A (p.W167R) JAG1 mutation and resection samples from a histologically normal region of the liver of a 35-year-old male undergoing surgery for metastasis of rectal adenocarcinoma were obtained with patient consent and approval from the UCSF Institutional Review Board.
Cryosections were blocked in 10% normal serum and permeabilized in 0.3% Triton-X before staining with primary and secondary antibodies listed in Tables 2 and 3, respectively. Paraffin-embedded samples were deparaffinized and underwent antigen retrieval in sodium citrate buffer (Bio-Genex or Vector Labs) or Tris EDTA buffer (10 mM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) in a steamer or pressure cooker for 15 minutes before blocking and permeabilization. Thin sections were mounted in FluorSave (MilliporeSigma) or 50% glycerol. Vibratome sections were stained free-floating in 12-well dishes and cleared in Focus Clear (Cedarlane Laboratories) before being mounted in Mount Clear (Cedarlane Laboratories) for confocal imaging. All samples stained with anti-wsCK antibody underwent antigen retrieval in 100 mM Tris buffer (pH 9.5) at 55° C. for 2 hours before blocking. Staining with anti-pSMAD3 antibody used a biotinylated secondary antibody followed by avidin/biotin-based peroxidase and tyramide amplification. Staining with anti-SSTR2 antibody used an HRP secondary antibody followed by tyramide-based amplification. M.O.M. Kit (Vector Laboratories) was used for antibodies raised in mice. DAPI or Hoechst was used to stain nuclear DNA. Chromogenic detection used an HRP secondary antibody followed by avidin/biotin-based peroxidase amplification and DAB substrate exposure. Sirius-red staining was performed on deparaffinized and rehydrated samples using a 0.1% Picrosirius-red solution (Dudley Corporation and Newcomer Supply) and 0.5% acetic acid water washes or was done at Peninsula Histopathology Laboratory.
indicates data missing or illegible when filed
Thin sections were imaged on an Olympus BX51 upright microscope and cultured cells were imaged on an Olympus IX71 inverted microscope using Openlab software (PerkinElmer). Chromogenic stains were imaged on a Leica DM 1000 LED. Fiji50 and/or Photoshop (Adobe) were used to process (brightness, contrast and gamma) and merge channels. Thick sections for 3D analysis of connectivity and clonal analysis were imaged on a Leica upright AOBS confocal microscope and processed and analyzed using Imaris (Bitplane) or Volocity (PerkinElmer) software. For R26R-Confetti+/− mouse and pSMAD3 analysis, images were acquired using a Nikon A1R GaAsP inverted SP confocal microscope and NIS elements software and processed and analyzed using Imaris software. Sirius-red-stained sections were imaged using a Cytation 5 cell imaging multi-mode reader (BioTek).
A catheter was inserted in a retrograde fashion into the common bile duct of postmortem mice and waterproof ink (Higgins) was slowly injected. Left liver lobes were dehydrated in 1:1 methanol:water followed by 100% methanol. Ink was visualized by tissue clearing in 1:2 benzyl alcohol:benzyl benzoate (BABB) solution and imaged on a Leica M205A or Nikon SMZ800 stereoscope.
Nonparenchymal liver cells were isolated from >P115 Alb-Cre+/−;Rbpjf/f;Hnf6f/f and Rbpjf/f;Hnf6f/f mice as previously described51. Cells were resuspended at 1×107 cells/mL in DMEM/2% FBS and blocked with Mouse Fc Block (BD Biosciences) for 30 minutes. Cells were incubated with fluorochrome-conjugated antibodies (Table 2) and DBA-FITC (Vector Laboratories) for 30 minutes, washed with cold DPBS 3 times and resuspended in DMEM/2% FBS. Sytox Red (Thermo Fisher Scientific) was added to label dead cells prior to sorting. Unstained and single-stained cells were used for compensation. Specificity of DBA binding was verified with a GalNAc (Sigma)-blocked control as previously described52. Cells were analyzed and sorted on a FACSAria III using FACSDiva software (BD Biosciences). From the CD11b− CD31−CD45− population, EPCAM+DBA− cells were collected as peripheral cholangiocytes and EPCAM+DBA+ as hilar cholangiocytes. FlowJo (FlowJo, LLC) was used to analyze data and generate charts. Cells were either sorted into DMEM/2% FBS, pelleted and snap frozen, or sorted directly into extraction buffer for RNA purification.
Hepatocytes were isolated from >P115 Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice by 2-step collagenase perfusion followed by purification through a Percoll gradient. Cells were resuspended at 1×106 cells/100 μL in Hanks Buffer with 10% FBS and incubated with OC2-2F8 antibody for 1 hour on ice. Cells were washed with cold DPBS 2 times and resuspended in Hanks/10% FBS. Fluorochrome-conjugated secondary antibody (Table 3) was added and cells were incubated for 30 minutes on ice followed by 2 washes with cold DPBS. Cells were blocked with 5% normal rat serum (Jackson Immuno) in Hanks Buffer for 10 minutes on ice. Cells were then incubated with fluorochrome-conjugated antibodies (Table 2) for 30 minutes on ice. Cells were then washed in cold DPBS 3 times and resuspended in Williams E medium/2% FBS. Sytox Red (Thermo Fisher Scientific) was added to label dead cells prior to sorting. Unstained and single-stained cells were used for compensation. Cells were analyzed and sorted on a FACSAria III using FACSDiva software (BD Biosciences). From the CD11b−CD31−CD45−EPCAM− population, OC2-2F8+ cells were collected as hepatocytes (
Hepatocytes were isolated from donor mice by 2-step collagenase perfusion followed by purification through a Percoll gradient. 1×106 viable cells were resuspended in 80-100 μl of Williams E Medium with glutamine. Transplantation was performed by transdermal intrasplenic injection of the cell suspension under isoflurane anesthesia.
Mice received PicoLab Mouse Diet 20, 5058 (LabDiet) with 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; Sigma-Aldrich) for the indicated durations.
qPCR and Gene Expression Analysis
Genomic DNA was isolated from cells using QIAamp DNA Micro Kit or DNeasy Blood & Tissue Kit (Qiagen). RNA was extracted using Trizol Reagent (Thermo Fisher Scientific) and purified by isopropanol precipitation (cells in collagen gel) or purified using RNAeasy Mini Kit (Qiagen) (liver tissue). Reverse transcription was performed using qScript cDNA Supermix (Quanta Biosciences). qPCR was performed using SYBR green reagent (Thermo Fisher Scientific) in a ViiA 7 system (Thermo Fisher Scientific). Reactions were performed in triplicate, and expression was normalized to an Rbpj (genotyping) or Gapdh (Glyceraldehyde-3-phosphate Dehydrogenase; gene expression) reference and quantified using the ΔΔCt method. Primers are listed in Table 4.
RNA was purified from FACS-isolated cells using PicoPure Kit (Thermo Fisher Scientific). RNA quality was assessed using RNA 6000 Pico Kit on a 2100 Bioanalyzer (Agilent). Samples with RIN≥7.7 and at least 15 ng of RNA were used to construct sequencing libraries using Clontech Low Input Library Prep Kit v2. Libraries were sequenced on a HiSeq 3000, 10 samples per lane, with single-end 50 bp reads. Raw reads were aligned to the mm10 mouse genome with annotations provided by UCSC using CobWEB, a proprietary Burrows-Wheeler Transform method. Reads per kb per million (RPKM) were calculated from aligned reads using the expectation-maximization algorithm. RPKM was thresholded at 1, log 2 transformed, normalized using the DESeq algorithm and baselined to the median of all samples. Analyses were performed on transcripts with RPKM>5 in all samples of at least 1 experimental condition (n=17,793 transcripts). These reasonably expressed transcripts were used in principle-component analysis. All transcripts with fold change >3 in at least 1 of the 3 possible pairwise comparisons (N=6,464) were selected, and a 1-way ANOVA was performed to identify significantly differential genes with FDR-corrected P<0.05 (n=4,997). Venn diagrams were used to identify unique and shared gene signatures. Gene sets were submitted to ToppGene.cchmc.org for identification of pathway and biological process enrichments.
Nuclear and cytoplasmic extracts were generated from whole liver using previously described buffers with protease and phosphatase inhibitors53. Samples were run on SDS-PAGE 4-20% Tris-Glycine gradient gels, electrophoretically transferred to nitrocellulose membrane and probed with antibodies. Signals were detected by ECL Western blotting substrate (GE Healthcare). The membrane was stripped and reprobed with anti-actin antibody to verify and normalize protein loading using densitometry. Quantification was performed using ImageJ software.
Blood was collected from postmortem Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice and controls of ages P20 to ≥P150 and tested for serum total bilirubin (TecoDiagnostics) and ALP, ALT and AST (ADVIA XPT clinical chemistry system, Siemens). For serial bilirubin measurements, blood was collected by retro-orbital venipuncture and tested for total bilirubin every 2 weeks from P60 to P189 from 4 different litters. Blood was collected from Alb-Cre+/−; Rbpjf/f;Hnf6f/f;Tgfbr2f/f mice and controls by retro-orbital venipuncture and tested for serum total bilirubin (TecoDiagnostics). Blood was collected from Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice intravenously injected with AAV8-Ef1α-caTgfbr1 and controls by retro-orbital venipuncture and serum total bilirubin was measured as previously reported54 using a Synergy 2 microplate reader (BioTek). Serum absorbance at 540 nm was subtracted from serum absorbance at 450 nm. A linear trendline equation of serum absorbance versus serum total bilirubin was determined by independently measuring absorbance and total bilirubin (TecoDiagnostics) of known cholestatic and noncholestatic serum samples. This equation was used to convert absorbance readings to serum total bilirubin levels.
In vitro 3D culture of hepatocytes to induce biliary conversion was carried out as previously reported21 with the following modifications. Hepatocytes were isolated from 8-10-week-old Rbpjf/f;Hnf6f/f mice injected with 1×1012 vgs of AAV8-Ttr-Cre 2 weeks prior to delete RBPJ and HNF6. Hepatocytes were isolated by 2-step collagenase perfusion followed by purification through a Percoll gradient, all in the absence of serum. Cells were grown on Primaria plates for 6 days to form spheroids. Spheroids were cultured in collagen gels (Cultrex Rat Collagen I, Lower Viscosity, Trevigen) in the presence of 10 μM SB-431542 (Selleckchem) or vehicle (DMSO). The medium was changed every other day.
For sparse-labeling clonal analysis, 8 liver regions from each of 3 Alb-Cre+/−; Rbpjf/f;Hnf6f/f;R26ZG+/+ mice were analyzed. GFP-positive cells in all clones within 100 μm-thick sections were manually counted using Imaris software for visualization. GFP-positive cells in direct contact were considered clones. Clones that extended to and potentially beyond the x, y or z boundaries were excluded. For clonal analysis in R26R-Confetti+/− mice, 30-40 μm z-stack images of wsCK-positive DBA-negative pBDs (peripheral bile ducts) and wsCK-positive DBA-positive hBDs were visualized in 3D with Imaris software. The module Surfaces within Imaris was used to render a 3D surface created on an intensity value on a per channel basis. The rendered surface-per-channel was masked to the Hoechst nuclear stain. 3D pBD masks were used to manually count cells per clone. Multiple portal regions were analyzed for clones at P90 (14 and 17 per Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26R-Confetti+/− and 8 and 9 per Alb-Cre+/−;R26R-Confetti+/− mouse) and P150 (7-10 per Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26R-Confetti+/− and 8-10 per Alb-Cre+/−;R26R-Confetti+/− mouse). To determine labeling efficiencies in Alb-Cre+/−; Rbpjf/f;Hnf6f/f;R26ZG+/+ mice, for each mouse, 20 random fields were analyzed for wsCK-positive DBA-negative peripheral cholangiocytes (about 350 cells) and 5 random fields were analyzed for hepatocytes (about 1,500 cells).
For proliferation analysis, OPN-positive cells in 4 random portal fields from each of 4 mice were analyzed for KI67 and CK19 staining. For hepatocyte-fate-tracing analysis in DDC diet-fed mice, 10 random portal fields from 4 Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26ZG+/+ and 3 Rbpjf/f;Hnf6f/f;R26ZG+/+ mice were analyzed. For quantification in transplantation experiments, all portal areas with donor-derived cells in a section from at least 3 lobes per mouse were examined and scored for the presence or absence of donor hepatocyte-derived EPCAM-positive cholangiocytes. For human samples, 63 bile ducts between the 2 patient samples were scored for the presence of nuclear pSMAD3. For Sirius-red-staining quantification, liver samples were stained in batches and sections of whole lobes were imaged at equal exposure. Using Fiji software, a threshold was set for Sirius-red-positive area within each lobe and the percent of the total area that was Sirius red positive was measured and is reported. Charts were generated in Prism 6 or 7 (GraphPad). Researchers were not blinded when analyzing results. P<0.05 was considered statistically significant. Experiments were replicated independently once (
of right lobe and
liver, respectively)
p.W167R)
normal tissue
indicates data missing or illegible when filed
The Gene Expression Omnibus accession number for the RNA-seq data is GSE108315.
All published studies of hepatocyte plasticity used animals with a fully developed biliary system in which residual cholangiocytes were available to regenerate injured bile ducts, likely leading to insufficient pressure for transdifferentiation. To determine the full extent of hepatocyte plasticity, we used mice lacking the intrahepatic biliary system.
Specifically, we used mice that mimic the hepatic phenotype of ALGSa human disease caused by impaired NOTCH signaling22-24—generated by deletion of floxed alleles of the NOTCH signaling effector RBPJ and, to prevent compensation25, the transcription factor HNF6 in embryonic liver progenitors using Cre expressed from an albumin promoter (Alb)2. These Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice are severely cholestatic because they lack peripheral bile ducts (pBDs), the branches of the intrahepatic biliary tree, at birth. Greater than 90% of the mice survive, however, and form pBDs and a functional biliary tree by postnatal day (P) 12026 (
To trace hepatocytes during pBD formation, we intravenously injected Alb-Cre+/−; Rbpjf/f;Hnf6f/f;R26ZG+/+ mice with AAV8-Ttr-Flp at P17, i.e., before cells expressing biliary markers emerge in the liver periphery2,26. At P120, the newly formed pBDs—identified by wsCK IF—were GFP positive, indicating hepatocyte origin (
To investigate the function of the hepatocyte-derived pBDs (HpBDs), we determined if they are contiguous with the extrahepatic biliary system. At P30, retrograde ink injection into the common bile duct filled only hBDs, reflecting a severely truncated biliary tree lacking pBDs; however, ink injection at ≥P120 revealed a biliary tree of normal dimensions, demonstrating that HpBDs are connected to and patent with hBDs (
We also investigated the authenticity and maturity of the cholangiocytes forming the HpBDs. The cells displayed acetylated tubulin (acTUB)-marked primary cilia (
The contribution of proliferation to HpBD formation, i.e., whether a few hepatocytes proliferate extensively after transdifferentiation, or whether many hepatocytes transdifferentiate, was also investigated. For this, we measured clonal expansion in HpBDs by sparsely labeling hepatocytes in Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26ZG+/+ mice with low-dose AAV8-Ttr-Flp at P18 and quantifying the cells in GFP-positive clones at P120 (
Unlike in HpBDs, we found significant proliferation in hepatocyte-derived reactive ductules, which are detectable in Alb-Cre+/−;Rbpjf/f;Hnf6f/f;R26ZG+/+ mice during cholestasis (
Proliferation in established HpBDs by inducing cholestatic liver injury with DDC diet in >P120 Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice was also assessed experimentally. Reactive ductules formed 2 weeks later than in Cre-negative littermates and consisted mainly of OPN-positive EPCAM-negative cells, indicative of hepatocyte metaplasia (
As illustrated by lack of pBDs in Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice (
We functionally validated these findings by showing that the TGFβ inhibitor SB-431542 blocks biliary differentiation and morphogenesis of hepatocytes lacking RBPJ and HNF6 in vitro (
The findings disclosed in the preceding Examples led us to investigate whether activating TGFβ signaling in hepatocytes enhances HpBD formation. We intravenously injected P19-24 Alb-Cre+/−;Rbpjf/f;Hnf6f/f mice with an AAV8 vector expressing constitutively active (ca) TGFBR1 from the elongation factor 1α (Ef1α) promoter. At P80-100, 9/11 treated mice had a mature hierarchical biliary network, whereas 9/9 untreated mice still had an immature homogeneous biliary network35 (
To exclude that transdifferentiation is limited to immature hepatocytes, we investigated whether adult hepatocytes can form HpBDs. To bypass potential adaptive processes in development, we deleted RBPJ and HNF6 and activated GFP in hepatocytes of P75 Rbpjf/f;Hnf6f/f;R26ZG+/+ mice by co-injecting AAV8-Ttr-Cre and AAV8-Ttr-Flp and transplanted the cells 1 week later into P31 Alb-Cre+/−;Rbpjf/f;Hnf6f/f;Rag2−/−;Il2rg−/− mice (
To determine whether our findings are relevant for human ALGS, we obtained liver samples from 2 patients who developed regenerative nodules containing pBDs36 (Extended Data Table 1). The regenerative nodules contained CK7-positive pBDs, whereas nonregenerated liver tissue from the same patients showed abundant CK7-positive cells with hepatocyte morphology, indicative of metaplasia14-17 (
In conclusion, by showing that hepatocytes can convert into mature cholangiocytes and form a functional and stable biliary system, our findings establish that hepatocyte plasticity extends beyond metaplasia to transdifferentiation and provide the first example of mammalian transdifferentiation building an organ structure de novo. Although hilar cholangiocytes are present in our mice, they fail to expand, resulting in severe cholestatic liver injury and strong pressure for hepatocytes to transdifferentiate into peripheral cholangiocytes. Analogously, cholangiocytes transdifferentiate into hepatocytes only if hepatocyte proliferation is completely suppressed37. The failure of hilar cholangiocytes to proliferate is likely caused by high TGFβ signaling in the cholestatic liver38. Accordingly, we identified TGFβ signaling as the driver of hepatocyte transdifferentiation and HpBD formation in our mice and potentially also in patients with ALGS. Unlike for bile duct development7-9, NOTCH signaling is not needed. Using clinically established AAV vectors and hepatocyte transplantation we show that our findings are potentially translatable into therapies for ALGS and other diseases associated with lack of bile ducts.
We also established that expressing JAG1 in hepatocytes induces de novo bile duct formation. For this, we used Jag1+/− mice on the C57BL/6 background that accurately model the genetics of human ALGS. We bred these mice to R26R-RFP+/+ mice—also on the C57BL/6 background—to be able to genetically label hepatocytes by Cre recombinase expression. We found that intravenously injecting these Jag1+/−;R26R-RFP+/− mice with AAV8-CMV-Jag1 caused hepatocytes—labeled by additional intravenous injection of AAV8-Ttr-Cre—to convert into cholangiocytes and assemble into bile ducts with a much higher frequency than occurred spontaneously in Jag1+/−;R26R-RFP+/− mice intravenously injected only with AAV8-Ttr-Cre. The hepatocyte-derived bile ducts in Jag1+/−;R26R-RFP+/− mice intravenously injected with AAV8-CMV-Jag1 and AAV8-Ttr-Cre were connected to the pre-existing bile ducts as evidenced by retrograde ink injection into the common bile duct. Moreover, ink injection showed that the biliary system was much denser and extended further into the periphery of the liver lobules than in Jag1+/−;R26R-RFP+/− mice intravenously injected only with AAV8-Ttr-Cre. The function of the hepatocyte-derived bile ducts was further illustrated by lower serum levels of cholestasis and liver injury markers in Jag1+/−;R26R-RFP+/− mice intravenously injected with AAV8-CMV-Jag1 and AAV8-Ttr-Cre than in Jag1+/−;R26R-RFP+/− mice intravenously injected only with AAV8-Ttr-Cre. These results showed that JAG1 does not have to be provided by mesenchymal cells for bile duct formation to occur, as had been thought to be necessary for bile duct development. In contrast, our results establish that hepatocytes can serve as JAG1 ligand providers to adjacent hepatocytes, and that the resulting activation of Notch signaling leads to bile duct formation from these cells. Collectively, these results establish that expressing JAG1 in hepatocytes can be used to overcome bile duct paucity in mice modeling ALGS, thereby providing a curative strategy for patients suffering from this disease.
In addition to JAG1, we contemplate expression of any one or more of the other Notch receptor ligands JAG2, DLL1, DLL3 and DLL4 to induce Notch signaling in hepatocytes in vivo because they compensate for each other. Moreover, we expect that any of Notch receptors NOTCH1, NOTCH2, NOTCH3, NOTCH4 could be expressed to induce Notch signaling in hepatocytes in vivo, provided surrounding cells express JAG2, DLL1, DLL3 or DLL4. Alternatively, the respective NOTCH intracellular domains, which do not require ligand-mediated activation, could be expressed to induce Notch signaling in hepatocytes in vivo.
As an alternative to AAV vectors and other viral vectors, it is contemplated that nonviral methods, including mRNAs, small activating RNAs or recombinant proteins, could be used to express JAG1, JAG2, DLL1, DLL3, DLL4, NOTCH1, NOTCH2, NOTCH3, NOTCH4 or the respective NOTCH intracellular domains in hepatocytes in vivo. In addition, small molecules could be used to induce Notch signaling or TGFβ signaling in hepatocytes in vivo. These nonviral methods proved to be safe and effective in clinical trials for therapy of genetically encoded liver diseases or hepatocellular carcinoma.
In ALGS patients, CRISPR or CRISPR-mediated activation (CRISPRa) could be used to correct the mutated JAG1 allele or induce the expression of the normal JAG1 allele, respectively. The components of CRISPR or CRISPRa could be delivered to hepatocytes in vivo using viral vectors, particularly AAV vectors, which proved to be safe and effective in clinical trials for therapy of genetically encoded liver diseases.
Each of the references cited herein is hereby incorporated by reference in its entirety, or in relevant part, as would be apparent from the context of the citation.
The disclosed subject matter has been described with reference to various specific embodiments and techniques. It should be understood, however, that many variations and modifications may be made while remaining within the spirit and scope of the disclosed subject matter.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US19/29501 | 4/26/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62663675 | Apr 2018 | US |
