METHODS AND COMPOSITIONS FOR TREATING UREA CYCLE DISORDERS, IN PARTICULAR OTC DEFICIENCY

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 9, 2018, is named 20931011USPROSL.txt, and is 758,847 bytes in size.

Field of the Invention

The present invention provides compositions and methods for the treatment of urea cycle disorders in humans.

Background of the Invention

Urea cycle disorders are a group of genetic disorders caused by defects in the metabolism of waste nitrogen via the urea cycle. The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. The urea cycle mainly occurs in the mitochondria of liver cells. The urea produced by the liver enters the bloodstream where it travels to the kidneys and is ultimately excreted in urine. Genetic defects in any of the enzymes or transporters in the urea cycle can cause hyperammonemia (elevated blood ammonia), or the buildup of a cycle intermediate. Ammonia then reaches the brain through the blood, where it can cause cerebral edema, seizures, coma, long term disabilities in survivors, and/or death.

The onset and severity of urea cycle disorders is highly variable. It is influenced by the position of the defective protein in the cycle and the severity of the defect. Mutations that lead to severe deficiency or total absence of activity of any of the enzymes can result in the accumulation of ammonia and other precursor metabolites during the first few days of life. Because the urea cycle is the principal clearance system for ammonia, complete disruption of this pathway results in the rapid accumulation of ammonia and development of related symptoms. Mild to moderate mutations represent a broad spectrum of enzyme function, providing some ability to detoxify ammonia, and result in mild to moderate urea cycle disorders.

According to National Urea Cycle Disorders Foundation, the incidence of urea cycle disorders is estimated to be 1 in 8,500 live birth in the United States. The estimated incidence of individual urea cycle disorder varies from less than 1:2,000,000 to about 1:56,500 (See NA Mew et al., Urea Cycle Disorders Overview, 2015). They occur in both children and adults. These disorders are most often diagnosed in infancy, but some children do not develop symptoms until early childhood. Newborns with severe urea cycle disorders become catastrophically ill within 36-48 hours of life. In children with mild or moderate urea cycle disorders, symptoms may be seen as early as one year of age. Early symptoms include disliking meat or other high-protein foods, inconsolable crying, failure to thrive, mental confusion, and hyperactive behavior. Symptoms can progress to frequent episodes of vomiting, lethargy, delirium, and coma. Some individuals with mild urea cycle defects are diagnosed in adulthood. Ammonia accumulation may be triggered by illness or stress (e.g., viral infection, surgery, prolonged fasting, excessive exercising, and excessive dieting), resulting in multiple mild elevations of plasma ammonia concentration. Without proper diagnosis and treatment, these individuals are at risk for permanent brain damage, coma, and death.

Treatment for urea cycle disorders is a lifelong process. Symptoms are usually managed by using a combination of strategies including diet restriction, amino acid supplements, medications, dialysis, and/or hemofiltration. Dietary management is key to restricting the level of ammonia produced in the body. A careful balance of dietary protein, carbohydrates and fats is necessary to lower protein intake, while providing adequate calories for energy needs, as well as adequate essential amino acids for cell growth and development. Depending on the type of urea cycle disorder, amino acid supplements such as arginine or citrulline may be added to the diet. Sodium phenylbutyrate (BUPHENYL®), glycerol phenylbutyrate (RAVICTI®) and sodium benzoate are FDA approved drugs for the treatment of urea cycle disorders. They function as nitrogen binding agents to allow the kidneys to excrete excess nitrogen in place of urea. Dialysis and/or hemofiltration are used to quickly reduce plasma ammonia concentration to normal physiological level. When other treatment and management options fail, or for neonatal onset CPS1 and OTC deficiency, liver transplant is an option. Although the transplant alternative has been proven to be effective, the cost of the surgery, shortage of donors, and possible side effects of immunosuppressants can be difficult to overcome.

Therefore, there is a high unmet need for developing effective therapeutics for the treatment of urea cycle disorders.

SUMMARY OF THE INVENTION

The invention provides, among other things, methods of treating a subject with a urea cycle disorder. The methods include administering to the subject an effective amount of a compound capable of modulating the expression of one or more genes selected from Carbamoyl Phosphate Synthetase 1 (CPS1), Ornithine Transcarbamoylase (OTC), Argininosuccinate Synthetase 1 (ASS1), Argininosuccinate Lyase (ASL), N-Acetylglutamate Synthetase (NAGS), Arginase 1 (ARG1), Solute Carrier Family 25 Member 15 (SLC25A15), and Solute Carrier Family 25 Member 13 (SLC25A13). Such compound may be a small molecule, a polypeptide, an antibody, a hybridizing oligonucleotide, or a genome editing agent. In some embodiments, such compound may include at least one selected from Tables 2-10, or a derivative or an analog thereof.

In some embodiments, the invention provides methods for increasing OTC expression in a cell harboring an OTC mutation associated with a partial reduction of OTC function by contacting the cell with an effective amount of a compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, MAPK, EGFR, FGFR, BRAF, RAF1, KDR, FLT1, TBK1, IKBKE, PRKAA1, PRKAA2, PRKAB1, BMPR1A and BMPR1B. In some embodiments the cell is a hepatocyte. In some embodiments the target is JAK1, JAK2 or JAK3 and the compound selected from the group consisting of Momelotinib and Baricitinib. In some embodiments the target is HSP90 and the compound is selected from the group consisting of 17-AAG, BIIB021, HSP-990, and Retaspimycin HCl. In some embodiments, the target is MAPK and the compound is selected from the group consisting of BIRB796, Pamapimod and PH-797804. In some embodiments, the target is EGFR and the compound is Mubritinib (TAK 165). In some embodiments, the target is FGFR and the compound is XL228. In some embodiments, the target is BRAF or RAF1 and the compound is selected from the group consisting of Lifirafenib (BGB-283) and BMS-214662. In some embodiments, the target is KDR or FLT1 and the compound is Foretinib/XL880 (GSK1363089). In some embodiments, the target is TBK1 or IKBKE and the compound is BX795. In some embodiments the target is PRKAA1, PRKAA2, or PRKAB1 and the compound is Dorsomorphin. In some embodiments, the OTC mutation is a mutation selected from the list of mutations in Table 26 that are associated with non-zero residual OTC function.

In some embodiments, the invention provides methods for increasing OTC expression in a human subject harboring an OTC mutation associated with a partial reduction of OTC function by administering to the subject an effective amount of a compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, MAPK, EGFR, FGFR, BRAF, RAF1, KDR, FLT1, TBK1, IKBKE, PRKAA1, PRKAA2, PRKAB1, BMPR1A and BMPR1B. In some embodiments the target is JAK1, JAK2 or JAK3 and the compound selected from the group consisting of Momelotinib and Baricitinib. In some embodiments the target is HSP90 and the compound is selected from the group consisting of 17-AAG, BIIB021, HSP-990, and Retaspimycin HCl. In some embodiments, the target is MAPK and the compound is selected from the group consisting of BIRB796, Pamapimod and PH-797804. In some embodiments, the target is EGFR and the compound is Mubritinib (TAK 165). In some embodiments, the target is FGFR and the compound is XL228. In some embodiments, the target is BRAF or RAF1 and the compound is selected from the group consisting of Lifirafenib (BGB-283) and BMS-214662. In some embodiments, the target is KDR or FLT1 and the compound is Foretinib/XL880 (GSK1363089). In some embodiments, the target is TBK1 or IKBKE and the compound is BX795. In some embodiments the target is PRKAA1, PRKAA2, or PRKAB1 and the compound is Dorsomorphin. In some embodiments, the OTC mutation is a mutation selected from the list of mutations in Table 20 that are associated with non-zero residual OTC function.

In some embodiments, the compound may be capable of modulating the expression of CPS1 and is at least one selected from Table 2, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of OTC and is at least one selected from Table 3, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of ASS1 and is at least one selected from Table 4, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of ASL and is at least one selected from Table 5, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of NAGS and is at least one selected from Table 6, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of ARG1 and is at least one selected from Table 7, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of SLC25A15 and is at least one selected from Table 8, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of SLC25A13 and wherein the compound is at least one selected from Table 9, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of three or more genes selected from the group consisting of CPS1, OTC, ASS1, ASL, NAGS, ARG1, SLC25A15, and SLC25A13, and is at least one selected from Table 10, or a derivative or an analog thereof.

In some embodiments, the compound may increase the expression of the target genes. In some embodiments, the expression of the target genes may be increased by at least about 40%. In some embodiments, the expression of the target genes may be increased in the liver of the subject. In some embodiments, the subject may have at least one mutation within or near the target genes.

In some embodiments, the urea cycle disorder may be Carbamoyl Phosphate Synthetase 1 (CPS1) deficiency. In some embodiments, the urea cycle disorder may be Ornithine Transcarbamylase (OTC) deficiency. In some embodiments, the urea cycle disorder may be Argininosuccinate Synthetase (ASS1) deficiency. In some embodiments, the urea cycle disorder may be Argininosuccinate Lyase (ASL) deficiency. In some embodiments, the urea cycle disorder may be Arginase-1 (ARG1) deficiency. In some embodiments, the urea cycle disorder may be N-Acetylglutamate Synthetase (NAGS) deficiency. In some embodiments, the urea cycle disorder may be Ornithine translocase (ORNT1) deficiency. In some embodiments, the urea cycle disorder may be Citrin deficiency.

Also provided herein is a method of modulating the expression of one or more urea cycle-related genes in a cell. The method includes introducing into the cell an effective amount of a compound capable of altering one or more signaling molecules associated with the regulatory sequence regions (RSRs) or portion thereof of the urea cycle-related genes. The urea cycle-related genes may be one or more selected from CPS1, OTC, ASS1, ASL, NAGS, ARG1, SLC25A13, and SLC25A15. The compound may be a small molecule, a polypeptide, an antibody, a hybridizing oligonucleotide, or a genome editing agent. In some embodiments, such compound may include at least one selected from Tables 2-10, or a derivative or an analog thereof.

In some embodiments, the compound may be capable of modulating the expression of CPS1 and is at least one selected from Table 2, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of OTC and is at least one selected from Table 3, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of ASS1 and is at least one selected from Table 4, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of ASL and is at least one selected from Table 5, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of NAGS and is at least one selected from Table 6, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of ARG1 and is at least one selected from Table 7, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of SLC25A15 and is at least one selected from Table 8, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of SLC25A13 and wherein the compound is at least one selected from Table 9, or a derivative or an analog thereof. In some embodiments, the compound may be capable of modulating the expression of three or more genes selected from the group consisting of CPS1, OTC, ASS1, ASL, NAGS, ARG1, SLC25A13, and SLC25A15, and is at least one selected from Table 10, or a derivative or an analog thereof.

In some embodiments, the compound increases the expression of the urea cycle-related genes. In some embodiments, the expression of the urea cycle-related genes is increased by at least about 40%. In some embodiments, the cell has at least one mutation within or near the urea cycle-related genes. In some embodiments, the cell is a hepatocyte.

Also provided herein is a method of modulating the expression of one or more urea cycle-related genes in a cell, comprising introducing into the cell an effective amount of a compound that may be capable of modulating the Platelet-derived Growth Factor Receptor (PDGFR)-mediated signaling pathway. The urea cycle-related genes may be selected from CPS1, OTC, ASS1, ASL, NAGS, ARG1, SLC25A13, and SLC25A15. The compound may be a small molecule, a polypeptide, an antibody, a hybridizing oligonucleotide, or a genome editing agent.

In some embodiments, the compound may be a PDGFR inhibitor. In some embodiments, the compound comprises CP-673451, or a derivative or an analog thereof. In some embodiments, the compound comprises Amuvatinib, or a derivative or an analog thereof. In some embodiments, the compound comprises Crenolanib, or a derivative or an analog thereof. In some embodiments, the compound may be a PDGFR activator. In some embodiments, the compound comprises PDGF, or a derivative or an analog thereof.

Further provided herein is a method of modulating the expression of one or more urea cycle-related genes in a cell. The method includes introducing into the cell an effective amount of a compound that may be capable of modulating the Transforming Growth Factor-beta (TGF-B) signaling pathway. The urea cycle-related genes may be selected from CPS1, OTC, ASS1, ASL, NAGS, ARG1, SLC25A13, and SLC25A15. The compound may be a small molecule, a polypeptide, an antibody, a hybridizing oligonucleotide, or a genome editing agent.

In some embodiments, the compound activates the TGF-B signaling pathway. In some embodiments, the compound comprises GDF2 (BMP9), or a derivative or an analog thereof. In some embodiments, the compound comprises BMP2, or a derivative or an analog thereof. In some embodiments, the compound comprises Activin, or a derivative or an analog thereof. In some embodiments, the compound comprises Nodal, or a derivative or an analog thereof. In some embodiments, the compound comprises Anti mullerian hormone, or a derivative or an analog thereof.

The present invention also provides a method of modulating the expression of a CPS1 gene in a cell. The method includes introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the CPS1 gene. In some embodiments, the upstream neighborhood genes include LANCL1-AS1 and LANCL1. In some embodiments, the downstream neighborhood genes include LOC107985978. In some embodiments, the cell has at least one mutation within or near the CPS1 gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an OTC gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the OTC gene. In some embodiments, the upstream neighborhood genes include RPGR. In some embodiments, the downstream neighborhood genes include LOC392442. In some embodiments, the cell has at least one mutation within or near the OTC gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an ASS1 gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the ASS1 gene. In some embodiments, the upstream neighborhood genes include HMCN2 and LOC107987134. In some embodiments, the downstream neighborhood genes include FUBP3 and LOC100272217. In some embodiments, the cell has at least one mutation within or near the ASS1 gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an ASL gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the ASL gene. In some embodiments, the upstream neighborhood genes include LOC644667. In some embodiments, the downstream neighborhood gene include CRCP. In some embodiments, the cell has at least one mutation within or near the ASL gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an NAGS gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the NAGS gene. In some embodiments, the upstream neighborhood genes include PPY. In some embodiments, the downstream neighborhood genes include TMEM101. In some embodiments, the cell has at least one mutation within or near the NAGS gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an ARG1 gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the ARG1 gene. In some embodiments, the upstream neighborhood genes include RPL21P67. In some embodiments, the downstream neighborhood genes include ENPP3. In some embodiments, the cell has at least one mutation within or near the ARG1 gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an SLC25A15 gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the SLC25A15 gene. In some embodiments, the upstream neighborhood genes include MRPS31. In some embodiments, the downstream neighborhood genes include MIR621. In some embodiments, the cell has at least one mutation within or near the SLC25A15 gene. In some embodiments, the cell is a hepatocyte.

The present invention also provides a method of modulating the expression of an SLC25A13 gene in a cell, comprising introducing to the cell one or more compounds that alter one or more of the upstream or downstream neighborhood genes or its RSRs of the insulated neighborhood comprising the SLC25A13 gene. In some embodiments, the upstream neighborhood genes include DYNC1I1. In some embodiments, the downstream neighborhood genes include RNU6-532P. In some embodiments, the cell has at least one mutation within or near the SLC25A13 gene. In some embodiments, the cell is a hepatocyte.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the packaging of chromosomes in a nucleus, the localized topological domains into which chromosomes are organized, insulated neighborhoods in TADs and finally an example of an arrangement of a signaling center(s) around a particular disease gene.

FIG. 2A and FIG. 2B illustrate a linear and 3D arrangement of the CTCF boundaries of an insulated neighborhood.

FIG. 3A and FIG. 3B illustrate tandem insulated neighborhoods and gene loops formed in such insulated neighborhoods.

FIG. 4 illustrates the concept of an insulated neighborhood contained within a larger insulated neighborhood and the signaling which may occur in each.

FIG. 5 illustrates the components of a signaling center; including transcriptional factors, signaling proteins, and/or chromatin regulators.

DETAILED DESCRIPTION OF THE INVENTION
I. INTRODUCTION

The present invention provides compositions and methods for the treatment of urea cycle disorders in mammalian subjects, particularly in human subjects. In particular, the invention provides compounds and related use for the modulation of at least one gene encoding a protein (e.g., an enzyme or a transporter) involved in the urea cycle.

Binding Sites for Signaling Molecules

A series of consensus binding sites, or binding motifs for binding sites, for signaling molecules has been identified by the present inventors. These consensus sequences reflect binding sites along a chromosome, gene, or polynucleotide for signaling molecules or for complexes which include one or more signaling molecules. These sites are provided by Table 11 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and is reproduced below as Table 13 of the instant specification.

In some embodiments, binding sites are associated with more than one signaling molecule or complex of molecules. Further, non-limiting examples of such motifs or sites are also provided in Table 12 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and is reproduced below as Table 14 of the instant specification.

It has further been determined that certain patterns are found in the binding motifs. A list of such patterns for complexes is provided by Tables 13 and 14 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and for single molecules is provided in Tables 15 and 16 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety. Each of these are reproduced below, respectively, as Tables 15-18 of the instant specification.

In the Motif Tables 13-16 of U.S. 62/501,795 which is hereby incorporated by reference in its entirety, certain designators are used according to the IUPAC nucleotide code. This code is shown in Table 17 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and is reproduced below as Table 19 of the instant specification.

Table 18 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, provides a list of signaling molecules including those which act as transcription factors (TF) and/or chromatin remodeling factors (CR) that function in various cellular signaling pathways. The methods described herein may be used to inhibit or activate the expression of one or more signaling molecules associated with the regulatory sequence region of the primary neighborhood gene encoded within an insulated neighborhood. The methods may thus alter the signaling signature of one or more primary neighborhood genes which are differentially expressed upon treatment with the therapeutic agent compared to an untreated control.

Various embodiments of the transcripts encoding the signaling proteins of Table 18 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, contain internal stop codons. These internal stop codons result in translation of multiple polypeptides. In one embodiment, a polypeptide that is a fragment of the signaling proteins taught in Table 18 of U.S. 62/501,795 may have signaling properties. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 9 and SEQ ID NO: 10 from SEQ ID NO: 11. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 12 and SEQ ID NO: 13 from SEQ ID NO: 14. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 15 and SEQ ID NO: 16 from SEQ ID NO: 17. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 18-19 from SEQ ID NO: 20. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 21 and SEQ ID NO: 22 from SEQ ID NO: 23. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 24 and SEQ ID NO: 25 from SEQ ID NO: 26. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 27 and SEQ ID NO: 28 from SEQ ID NO: 29. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 30 and SEQ ID NO: 31 from SEQ ID NO: 32. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 33-37 from SEQ ID NO: 38.

In some embodiments, at least one compound selected from Tables 19-21, of U.S. 62/501,795, which are hereby incorporated by reference in their entirety, and Tables 22-26 and 28 of U.S. 62/501,795, which are hereby incorporated by reference in their entirety, may be used to modulate RNAs derived from regulatory sequence regions to alter or elucidate the gene signaling networks of the present invention.

II. UREA CYCLE DISORDERS AND RELATED GENES

Compositions and methods described herein may be used to treat one or more urea cycle disorders. As used herein, the term “urea cycle disorder” refers to any disorder that is caused by a defect or malfunction in the urea cycle. The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. It is composed of 5 key enzymes including carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamoylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1), but also requires other enzymes, such as N-acetylglutamate synthetase (NAGS), and mitochondrial amino acid transporters, such as ornithine translocase (ORNT1) and citrin.

As used herein, a “urea cycle-related gene” refers to a gene whose gene product (e.g., RNA or protein) is involved in the urea cycle. Urea cycle-related genes include, but are not limited to, CPS1 (encoding CPS1), OTC (encoding OTC), ASS1 (encoding ASS1), NAGS (encoding NAGS), ARG1 (encoding ARG1), SLC25A15 (encoding ORNT1), and SLC25A13 (encoding citrin). Mutations in the urea cycle-related genes or their regulatory regions may lead to production of dysfunctional proteins and disruption of the urea cycle. In some cases, patients with a urea cycle disorder may carry a single functional allele and a mutated allele of a urea cycle-related gene. This results in the production of insufficient amount of the functional protein. The phenomenon is known as “haploinsufficiency.”

The urea cycle mainly occurs in the mitochondria of liver cells. The urea produced by the liver enters the bloodstream where it travels to the kidneys and is ultimately excreted in urine. Genetic defects in any of the enzymes or transporters in the urea cycle can cause hyperammonemia (elevated blood ammonia), or the buildup of a cycle intermediate. Ammonia then reaches the brain through the blood, where it can cause cerebral edema, seizures, coma, long term disabilities in survivors, and/or death.

The onset and severity of urea cycle disorders is highly variable. It is influenced by the position of the defective protein in the cycle and the severity of the defect. Mutations that lead to severe deficiency or total absence of activity of any of the first four enzymes in the pathway (CPS1, OTC, ASS1, and ASL) or the cofactor producer (NAGS) can result in the accumulation of ammonia and other precursor metabolites during the first few days of life. Because the urea cycle is the principal clearance system for ammonia, complete disruption of this pathway results in the rapid accumulation of ammonia and development of related symptoms. Mild to moderate mutations represent a broad spectrum of enzyme function, providing some ability to detoxify ammonia, and result in mild to moderate urea cycle disorders.

According to National Urea Cycle Disorders Foundation, the incidence of urea cycle disorders is estimated to be 1 in 8,500 live birth in the United States. The estimated incidence of individual urea cycle disorder varies from less than 1:2,000,000 to about 1:56,500 (See NA Mew et al., Urea Cycle Disorders Overview, 2015, which is incorporated by reference in its entirety). They occur in both children and adults. These disorders are most often diagnosed in infancy, but some children do not develop symptoms until early childhood. Newborns with severe urea cycle disorders become catastrophically ill within 36-48 hours of life. In children with mild or moderate urea cycle disorders, symptoms may be seen as early as one year of age. Early symptoms include disliking meat or other high-protein foods, inconsolable crying, failure to thrive, mental confusion or hyperactive behavior. Symptoms can progress to frequent episodes of vomiting, lethargy, delirium, and coma. Some individuals with mild urea cycle defects are diagnosed in adulthood. Ammonia accumulation may be triggered by illness or stress (e.g., viral infection, surgery, prolonged fasting, excessive exercising, and excessive dieting), resulting in multiple mild elevations of plasma ammonia concentration. Without proper diagnosis and treatment, these individuals are at risk for permanent brain damage, coma, and death.

Specific types of urea cycle disorder include, but are not limited to, Phosphate Synthetase 1 (CPS1) deficiency, Ornithine Transcarbamylase (OTC) deficiency, Argininosuccinate Synthetase (ASS1) deficiency, Argininosuccinate Lyase (ASL) deficiency, Arginase-1 (ARG1) deficiency, N-Acetylglutamate Synthetase (NAGS) deficiency, Ornithine translocase (ORNT1) deficiency, and Citrin deficiency. Any one or more of these disorders may be treated or targeted by the compositions and methods described herein.

Carbamoyl Phosphate Synthetase 1 (CPS1) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Carbamoyl Phosphate Synthetase 1 (CPS1) deficiency. CPS1 deficiency (MIM #237300) is an autosomal recessive disorder caused by mutations in the CPS1 gene. CPS1 catalyzes the synthesis of carbamoyl phosphate from ammonia and bicarbonate. CPS1 deficiency is the most severe type of the urea cycle disorders. Approximately 10 mutations that cause CPS1 deficiency have been identified in the CPS1 gene. Individuals with complete CPS1 deficiency rapidly develop hyperammonemia in the newborn period. Children who are successfully rescued from crisis are chronically at risk for repeated episodes of hyperammonemia.

In some embodiments, methods of the present invention involve modulating the expression of the CPS1 gene. CPS1 may also be referred to as Carbamoyl-Phosphate Synthase 1, Mitochondrial; Carbamoyl-Phosphate Synthase (Ammonia); EC 6.3.4.16; Carbamoyl-Phosphate Synthase [Ammonia], Mitochondrial; Carbamoyl-Phosphate Synthetase I; Carbamoylphosphate Synthetase I; CPSase I; CPSASE1; and PHN. The CPS1 gene has a cytogenetic location of 2q34 and the genomic coordinate are on Chromosome 2 on the forward strand at position 210,477,682-210,679,107. LANCL1-AS1 (ENSG00000234281) and LANCL1 (ENSG00000115365) are the genes upstream of CPS1 and LOC107985978 is the gene downstream of CPS1. CPS1-IT1 (ENSG00000280837) is a gene located within CPS1 on the forward strand. The CPS1 gene has a NCBI gene ID of 1373, Uniprot ID of P31327 and Ensembl Gene ID of ENSG00000021826. The genomic sequence of CPS1 is shown as in SEQ ID NO: 1.

Ornithine Transcarbamylase (OTC) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Ornithine Transcarbamylase (OTC) deficiency. OTC deficiency (MIM #311250) is an X-linked genetic disorder caused by mutations in the OTC gene. OTC catalyzes the reaction between carbamoyl phosphate and ornithine to form citrulline and phosphate. More than 500 OTC gene mutations have been identified in people with OTC deficiency. The severe, early-onset form of the disorder, caused by complete absence of OTC activity, usually affects males. This form is as severe as CPS1 deficiency. The later-onset form of the disorder occurs in both males and females. These individuals develop hyperammonemia during their lifetime and many require chronic medical management for hyperammonemia.

In some embodiments, methods of the present invention involve modulating the expression of the OTC gene. OTC may also be referred to as Ornithine Carbamoyltransferase; Ornithine Transcarbamylase; EC 2.1.3.3; OTCase, Ornithine Carbamoyltransferase, Mitochondrial; EC 2.1.3; and OCTD. The OTC gene has a cytogenetic location of Xp11.4 and the genomic coordinate are on Chromosome X on the forward strand at position 38,352,545-38,421,450. RPGR (ENSG00000 156313) is the gene upstream of OTC and LOC392442 is the gene downstream of OTC. TDGF1P1 (ENSG00000227988) is the gene located within OTC on the reverse strand. The OTC gene has a NCBI gene ID of 5009, Uniprot ID of P00480 and Ensembl Gene ID of ENSG00000036473. The genomic sequence of OTC is shown as in SEQ ID NO: 2.

Argininosuccinate Synthetase (ASS1) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Argininosuccinate Synthetase (ASS1) deficiency. ASS1 deficiency (MIM #215700), also known as Citrullinemia type I, is an autosomal recessive disorder caused by mutations in the ASS1 gene. ASS1 catalyzes the synthesis of argininosuccinate from citrulline and aspartate. About 118 mutations that cause ASS1 deficiency have been identified in the ASS1 gene. The early onset form of this disorder can also be quite severe. The symptoms associated with hyperammonemia are life-threatening in many cases. Affected individuals are able to incorporate some waste nitrogen into urea cycle intermediates, which makes treatment slightly easier than in the other urea cycle disorders.

In some embodiments, methods of the present invention involve modulating the expression of the ASS1 gene. ASS1 may also be referred to as EC 6.3.4.5, Argininosuccinate Synthase, ASS, Argininosuccinic Acid Synthetase 1, Argininosuccinate Synthetase 1, Argininosuccinate Synthetase, Citrulline-Aspartate Ligase, and CTLN1. The ASS1 gene has a cytogenetic location of 9q34.11 and the genomic coordinate are on Chromosome 9 on the forward strand at position 130,444,929-130,501,274. HMCN2 (ENSG00000148357) and LOC107987134 are the genes upstream of ASS1, and FUBP3 (ENSG00000107164) and LOC100272217 are the genes downstream of ASS1. LOC105376294 is the gene that overlaps with the 3′ region of ASS1 on the reverse strand. The ASS1 gene has a NCBI gene ID of 445, Uniprot ID of P00966 and Ensembl Gene ID of ENSG00000130707. The genomic sequence of ASS1 is shown as in SEQ ID NO: 3.

Argininosuccinate Lyase (ASL) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Argininosuccinate Lyase (ASL) deficiency. ASL deficiency (MIM #207900) is an autosomal recessive disorder caused by mutations in the ASL gene. ASL cleaves argininosuccinic acid to produce arginine and fumarate in the fourth step of the urea cycle. More than 30 different mutations in the ASL gene have been identified worldwide. This disorder has a severe neonatal onset form and a late onset form. The severe neonatal onset form is indistinguishable from that of other urea cycle disorders. The late onset form ranges from episodic hyperammonemia triggered by acute infection or stress to cognitive impairment, behavioral abnormalities, and/or learning disabilities in the absence of any documented episodes of hyperammonemia.

In some embodiments, methods of the present invention involve modulating the expression of the ASL gene. ASL may also be referred to as Arginosuccinase, EC 4.3.2.1, ASAL, and Argininosuccinase. The ASL gene has a cytogenetic location of 7q11.21 and the genomic coordinate are on Chromosome 7 on the forward strand at position 66,075,798-66,093,558. LOC644667 is the gene upstream of ASL and CRCP (ENSG00000241258) is the genes downstream of ASL. The ASL gene has a NCBI gene ID of 435, Uniprot ID of P04424 and Ensembl Gene ID of ENSG00000126522. The genomic sequence of ASL is shown as in SEQ ID NO: 4.

N-Acetylglutamate Synthetase (NAGS) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat N-Acetylglutamate Synthetase (NAGS) deficiency. NAGS deficiency (MIM #237310) is an autosomal recessive disorder caused by mutations in the NAGS gene. NAGS catalyzes the production of N-Acetylglutamate (NAG) from glutamate and acetyl-CoA. NAG is a cofactor of CPS1. Approximately 12 mutations in the NAGS gene have been identified in people with NAGS deficiency. Symptoms of NAGS deficiency mimic those of CPS1 deficiency, as CPS1 is rendered inactive in the absence of NAG.

In some embodiments, methods of the present invention involve modulating the expression of the NAGS gene. NAGS may also be referred to as Amino-Acid Acetyltransferase, N-Acetylglutamate Synthase, Mitochondrial, EC 2.3.1.1, AGAS, and ARGA. The NAGS gene has a cytogenetic location of 17q21.31 and the genomic coordinate are on Chromosome 17 on the forward strand at position 44,004,546-44,009,063. PPY (ENSG00000108849) is the gene upstream of NAGS and TMEM101 (ENSG00000091947) is the genes downstream of NAGS. PYY (ENSG00000131096) is a gene that overlaps with NAGS on the reserve strand. The NAGS gene has a NCBI gene ID of 162417, Uniprot ID of Q8N159 and Ensembl Gene ID of ENSG00000161653. The genomic sequence of NAGS is shown as in SEQ ID NO: 5.

Arginase-1 (ARG1) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Arginase-1 (ARG1) deficiency. ARG1 deficiency (MIM #207800) is an autosomal recessive disorder caused by mutations in the ARG1 gene. ARG1 catalyzes the hydrolysis of arginine to ornithine and urea, which is the final step in the urea cycle. More than 40 mutations have been found in the ARG1 gene that cause partial or complete loss of enzyme function. Defects in ARG1 cause hyperargininemia, a more subtle disorder involving neurologic symptoms. Arginase deficiency usually becomes evident by about the age of 3. It most often appears as stiffness, especially in the legs, caused by abnormal tensing of the muscles (spasticity). Other symptoms may include slower than normal growth, developmental delay and eventual loss of developmental milestones, intellectual disability, seizures, tremor, and difficulty with balance and coordination (ataxia). Occasionally, high protein meals or stress caused by illness or periods without food (fasting) may cause ammonia to accumulate more quickly in the blood. This rapid increase in ammonia may lead to episodes of irritability, refusal to eat, and vomiting. In some affected individuals, signs and symptoms of arginase deficiency may be less severe, and may not appear until later in life. Hyperammonemia is rare or usually not severe in Arginase deficiency. Arginase deficiency is a very rare disorder; it has been estimated to occur once in every 300,000 to 1,000,000 individuals.

In some embodiments, methods of the present invention involve modulating the expression of the ARG1 gene. ARG1 may also be referred to as Liver-Type Arginase; Type I Arginase; Arginase, liver; and EC 3.5.3.1. The ARG1 gene has a cytogenetic location of 6q23.2 and the genomic coordinate are on Chromosome 6 on the forward strand at position 131,573,144-131,584,332. RPL21P67 (ENSG00000219776) is the gene upstream of ARG1 and ENPP3 (ENSG00000154269) is the genes downstream of ARG1. MED23 (ENSG00000112282) is a gene that overlaps with ARG1 on the reserve strand. The ARG1 gene has a NCBI gene ID of 383, Uniprot ID of P05089 and Ensembl Gene ID of ENSG00000118520. The genomic sequence of ARG1 is shown as in SEQ ID NO: 6.

Ornithine Translocase (ORNT1) Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Ornithine translocase (ORNT1) deficiency. ORNT1 deficiency (MIM #238970), also known as the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, is an autosomal recessive disorder caused by mutations in the SLC25A15 gene. ORNT1 is a transporter protein that transports ornithine across the inner mitochondrial membrane to the mitochondrial matrix, where it participates in the urea cycle. Failure to transport ornithine results in an interruption of the urea cycle and the accumulation of ammonia. Approximately 17 mutations in the SLC25A15 gene have been identified in individuals with ORNT1 deficiency, including F188delta, E180K, T32R, Q89X, G27R, G190D, R275Q, and a 13q14 microdeletion, etc (Camacho et al., Nat Genet. 1999 Jun;22(2):151-8; Camacho et al., Pediatric Research (2006) 60, 423-429; Salvi et al., Human Mutation, Mutation in Brief #457 (2001), which are hereby incorporated by reference in their entirety). Affected newborns typically present with lethargy, muscular hypotonia, and seizures. If untreated, death occurs within the first few days. The majority of survivors have pyramidal tract signs, with spastic paraparesis (Lemay et al., J Pediatr. 1992 Nov;121(5 Pt 1):725-30, Salvi et al., Neurology. 2001;57(5):911, which are hereby incorporated by reference in their entirety). Most have myoclonic seizures, ataxia, and mental retardation. A milder form of the disorder has been reported in adults, who become symptomatic following protein-rich meals.

In some embodiments, methods of the present invention involve modulating the expression of the SLC25A15 gene. SLC25A15 may also be referred to as Solute Carrier Family 25 Member 15, Solute Carrier Family 25 (Mitochondrial Carrier; Ornithine Transporter) Member 15, Ornithine Transporter 1, ORNT1, Mitochondrial Ornithine Transporter 1, D13S327, ORC1, and HHH. SLC25A15 has a cytogenetic location of 13q14.11 and the genomic coordinate are on Chromosome 13 on the forward strand at position 40,789,412-40,810,111. MRPS31 (ENSG00000102738) is the gene upstream of SLC25A15 and MIR621 (ENSG00000207652) is the genes downstream of SLC25A15. TPTE2P5 (ENSG00000168852) is a gene that overlaps with SLC25A15 on the reserve strand. SLC25A15 has a NCBI gene ID of 10166, Uniprot ID of Q9Y619 and Ensembl Gene ID of ENSG00000102743. The genomic sequence of SLC25A15 is shown as in SEQ ID NO: 7.

Citrin Deficiency

In some embodiments, methods and compositions of the present invention may be used to treat Citrin deficiency. Citrin deficiency (neonatal-onset MIM #605814 and adult-onset #603471), also known as Citrullinemia type II, is an autosomal recessive disorder caused by mutations in the SLC25A13 gene. Citrin is a transporter protein responsible for the transport of aspartate into the urea cycle. The loss of citrin blocks the aspartate transport and decrease the ability of ASS to produce argininosuccinate. More than 20 mutations in the SLC25A13 gene have been identified in people with adult-onset type II citrullinemia. It can manifest in newborns as neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), in older children as failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD), and in adults as recurrent hyperammonemia with neuropsychiatric symptoms in citrullinemia type II (CTLN2). Citrin deficiency as a cause of neonatal intrahepatic cholestasis occurs almost exclusively in Asian infants (Yeh et al., J Pediatr 2006; 148:642; Zhang et al., Tohoku J Exp Med. 2014 Aug;233(4):275-81; Tzun and Marques., EC Paediatrics 2.5 (2016); Lin et al., Sci Rep. 2016 Jul 11;6:29732; which are hereby incorporated by reference in their entirety). Adult-onset form of this disease is characterized by fatty liver, hyperammonemia, and neurological symptoms (Yasuda et a., Hum Genet 2000; 107:537; Lee et al., J Pediatr Gastroenterol Nutr 2010; 50:682, which are hereby incorporated by reference in their entirety).

In some embodiments, methods of the present invention involve modulating the expression of the SLC25A13 gene. SLC25A13 may also be referred to as Solute Carrier Family 25 Member 13, Mitochondrial Aspartate Glutamate Carrier 2, Solute Carrier Family 25 (Aspartate/Glutamate Carrier) Member 13, ARALAR2, CITRIN, Calcium-Binding Mitochondrial Carrier Protein Aralar2, and CTLN2. SLC25A13 has a cytogenetic location of 7q21.3 and the genomic coordinate are on Chromosome 7 on the reverse strand at position 96,120,220-96,322,147. DYNC1I1 (ENSG00000158560) is the gene upstream of SLC25A13 and RNU6-532P (ENSG00000207045) is the gene downstream of SLC25A13. CYCSP18, MIR591 (ENSG00000208025), and RPL21P74 are genes located within SLC25A13. SLC25A13 has a NCBI gene ID of 10165, Uniprot ID of Q9UJSO and Ensembl Gene ID of ENSG00000004864. The genomic sequence of SLC25A13 is shown as in SEQ ID NO: 8.

III. COMPOSITIONS AND METHODS OF THE INVENTION

The present invention provides compositions and methods for modulating the expression of one or more urea cycle-related genes to treat a urea cycle disorder. Any one or more of the compositions and methods described herein may be used to treat a urea cycle disorder in a subject.

The terms “subject” and “patient” are used interchangeably herein and refer to an animal to whom treatment with the compositions according to the present invention is provided. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human being.

In some embodiments, subjects may have been diagnosed with or have symptoms for a urea cycle disorder, e.g., CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency. In other embodiments, subjects may be susceptible to or at risk for a urea cycle disorder, e.g., CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency.

In some embodiments, subjects may carry mutations within or near a urea cycle-related gene. In some embodiments, subjects may carry one or more mutations within or near the CPS1 gene. In some embodiments, subjects may carry one or more mutations within or near the OTC gene. In some embodiments, subjects may carry one or more mutations within or near the ASS1 gene. In some embodiments, subjects may carry one or more mutations within or near the ASL gene. In some embodiments, subjects may carry one or more mutations within or near the NAGS gene. In some embodiments, subjects may carry one or more mutations within or near the ARG1 gene. In some embodiments, subjects may carry one or more mutations within or near the SLC25A15 gene. In some embodiments, subjects may carry one or more mutations within or near the SLC25A13 gene. In some embodiment, subjects may carry one functional allele and one mutated allele of a urea cycle-related gene. In some embodiment, subjects may carry two mutated alleles of a urea cycle-related gene.

In some embodiments, subjects may have dysregulated expression of at least one urea cycle-related gene. In some embodiments, subjects may have a deficiency of at least one urea cycle-related protein. In some embodiments, subjects may have at least one urea cycle-related protein that is partially functional.

In some embodiments, compositions and methods of the present invention may be used to increase the expression of a urea cycle-related gene in a cell or a subject. Changes in gene expression may be assessed at the RNA level or protein level by various techniques known in the art and described herein, such as RNA-seq, qRT-PCR, Western Blot, or enzyme-linked immunosorbent assay (ELISA). Changes in gene expression may be determined by dividing the level of target gene expression in the treated cell or subject by the level of expression in an untreated or control cell or subject. In some embodiments, compositions and methods of the present invention cause an increase in the expression of a urea cycle-related gene by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, from about 25% to about 50%, from about 40% to about 60%, from about 50% to about 70%, from about 60% to about 80%, from about 80% to about 100%, from about 100% to about 125%, from about 100 to about 150%, from about 150% to about 200%, from about 200% to about 300%, from about 300% to about 400%, from about 400% to about 500%, or more than 500%. In some embodiments, compositions and methods of the present invention cause a fold change in the expression of a urea cycle-related gene by about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 12 fold, about 15 fold, about 18 fold, about 20 fold, about 25 fold, or more than 30 fold.

In some embodiments, the increase in the expression of a urea cycle-related gene induced by compositions and methods of the present invention may be sufficient to prevent or alleviate one or more signs or symptoms of a urea cycle disorder.

Small Molecules

In some embodiments, compounds used to modulate the expression of a urea cycle-related gene may include small molecules. As used herein, the term “small molecule” refers a low molecular weight drug, i.e. <5000 Daltons organic compound that may help regulate a biological process. In some embodiments, small molecule compounds described herein are applied to a genomic system to interfere with components (e.g., transcription factor, signaling proteins) of the gene signaling networks associated with one or more urea cycle-related genes, thereby modulating the expression of these genes. In some embodiments, small molecule compounds described herein are applied to a genomic system to alter the boundaries of an insulated neighborhood and/or disrupt signaling centers associated with one or more urea cycle-related genes, thereby modulating the expression of these genes.

A small molecule screen may be performed to identify small molecules that act through signaling centers of an insulated neighborhood to alter gene signaling networks which may modulate expression of a select group of urea cycle-related genes. For example, known signaling agonists/antagonists may be administered. Credible hits are identified and validated by the small molecules that are known to work through a signaling center and modulate expression of the target gene.

In some embodiments, small molecule compounds capable of modulating expression of one or more urea cycle-related genes include, but are not limited to, 17-AAG (Tanespimycin), Afatinib, Amlodipine Besylate, Amuvatinib, AZD2858, BAY 87-2243, BIRB 796, bms-986094 (inx-189), Bosutinib, Calcitriol, CD 2665, Ceritinib, CI-4AS-1, CO-1686 (Rociletinib), CP-673451, Crenolanib, Crizotinib, Darapladib, Dasatinib, Deoxycorticosterone, Echinomycin, Enzastaurin, Epinephrine, Erlotinib, EVP-6124 (hydrochloride) (encenicline), EW-7197, FRAX597, GDC-0879, GO6983, GSK2334470, GZD824 Dimesylate, INNO-206 (aldoxorubicin), LDN193189, LDN-212854, Merestinib, MK-0752, Momelotinib, Oligomycin A, OSU-03012, Pacritinib (SB1518), PHA-665752, Phenformin, Phorbol 12,13-dibutyrate, Pifithrin-μ, PND-1186, prednisone, R788 (fostamatinib disodium hexahydrate), Rifampicin, Semaxinib, SIS3, SKL2001, SMI-4a, T0901317, TFP, Thalidomide, Tivozanib, TP-434 (Eravacycline), WYE-125132 (WYE-132), and Zibotentan, or derivatives or analogs thereof. Any one of these compounds or a combination thereof may be administered to a subject to treat a urea cycle disorder, such as CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include 17-AAG (Tanespimycin), or a derivative or an analog thereof. 17-AAG (Tanespimycin), also known as NSC 330507 or CP 127374, is a potent HSP90 inhibitor with half-maximal inhibitory concentration (IC50) of 5 nM, a 100-fold higher binding affinity for HSP90 derived from tumor cells than HSP90 from normal cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Afatinib, or a derivative or an analog thereof. Afatinib, also known as BIBW2992, irreversibly inhibits epidermal growth factor receptor (EGFR)/HER2 including EGFR (wildtype), EGFR (L858R), EGFR (L858R/T790M) and HER2 with IC₅₀of 0.5 nM, 0.4 nM, 10 nM and 14 nM, respectively. It is 100-fold more active against Gefitinib-resistant L858R-T790M EGFR mutant.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Amlodipine Besylate, or a derivative or an analog thereof. Amlodipine, also known as Norvasc, is a long-acting calcium channel blocker with an IC₅₀of 1.9 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Amuvatinib, or a derivative or an analog thereof. Amuvatinib, also known as MP-470, is a potent and multi-targeted inhibitor of c-Kit, PDGFRα and FLT3 with IC₅₀of 10 nM, 40 nM and 81 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include AZD2858, or a derivative or an analog thereof. AZD2858 is a selective GSK-3 inhibitor with an IC₅₀of 68 nM. It activates Wnt signaling and increases bone mass in rats.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BAY 87-2243, or a derivative or an analog thereof. BAY 87-2243 is a potent and selective hypoxia-inducible factor-1 (HIF-1) inhibitor.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BIRB 796, or a derivative or an analog thereof. BIRB 796, also known as Doramapimod, is a highly selective p38a MAPK inhibitor with dissociation constant (Kd) of 0.1 nM, 330-fold greater selectivity versus JNK2. It shows weak inhibition for c-RAF, Fyn and Lck and insignificant inhibition of ERK-1, SYK, IKK2, ZAP-70, EGFR, HER2, PKA, PKC, and PKCα/β/γ.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include bms-986094 (inx-189), or a derivative or an analog thereof. Bms-986094, also known as INX-08189, INX-189, or IDX-189, is a prodrug of a guanosine nucleotide analogue (2′-C-methylguanosine). Bms-986094 is an RNA-directed RNA polymerase (NS5B) inhibitor originally developed by Inhibitex (acquired by Bristol-Myers Squibb in 2012). It was in phase II clinical trials for the treatment of hepatitis C virus infection. However, the study was discontinued due to unexpected cardiac and renal adverse events.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Bosutinib, or a derivative or an analog thereof. Bosutinib, also known as SKI-606, is a novel, dual Src/Abl inhibitor with IC₅₀of 1.2 nM and 1 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Calcitriol, or a derivative or an analog thereof. Calcitriol, also known as 1,25-Dihydroxyvitamin D3 or Rocaltrol, is the hormonally active form of vitamin D, Calcitriol is the active metabolite of vitamin D3 that activates the vitamin D receptor.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CD 2665, or a derivative or an analog thereof. CD 2665 is a selective RARβγ antagonist with Kd values of 110 nM, 306 nM, and >1000 nM for RARγ, RARβ, and RARα, respectively. It blocks retinoic acid-induced apoptosis ex vivo.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Ceritinib, or a derivative or an analog thereof. Ceritinib, also known as LDK378, is potent inhibitor against ALK with IC₅₀of 0.2 nM, exhibiting 40- and 35-fold selectivity against IGF-1R and InsR, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CI-4AS-1, or a derivative or an analog thereof. CI-4AS-1 is a potent steroidal androgen receptor agonist (IC₅₀=12 nM). It mimics the action of 5α-dihydrotestosterone (DHT). It transactivates the mouse mammary tumor virus (MMTV) promoter; represses MMP1 promoter activity. It also inhibits 5a-reductase type I and II with IC₅₀values of 6 nM and 10 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CO-1686 (Rociletinib), or a derivative or an analog thereof. CO-1686, also known as Rociletinib, is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR including T790M (IC₅₀=21 nM).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CP-673451, or a derivative or an analog thereof. CP 673451 is a selective inhibitor of PDGFRα/β with IC₅₀of 10 nM/1 nM, exhibiting >450-fold selectivity over other angiogenic receptors. CP 673451 also has antiangiogenic and antitumor activity.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Crenolanib, or a derivative or an analog thereof. Crenolanib, also known as CP-868596, is a potent and selective inhibitor of PDGFRα/β with Kd of 2.1 nM/3.2 nM. It also potently inhibits FLT3 and is sensitive to D842V mutation not V561D mutation. It is >100-fold more selective for PDGFR than c-Kit, VEGFR-2, TIE-2, FGFR-2, EGFR, erbB2, and Src.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Crizotinib, or a derivative or an analog thereof. Crizotinib, also known as PF-2341066, is a potent inhibitor of c-Met and ALK with IC₅₀of 11 nM and 24 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Darapladib, or a derivative or an analog thereof. Darapladib is a selective and orally active inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2) with IC₅₀of 270 pM. Lp-PLA2 may link lipid metabolism with inflammation, leading to the increased stability of atherosclerotic plaques present in the major arteries. Darapladib is being studied as a possible add-on treatment for atherosclerosis.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Dasatinib, or a derivative or an analog thereof. Dasatinib is a novel, potent and multi-targeted inhibitor that targets Abl, Src, and c-Kit, with IC₅₀of <1 nM, 0.8 nM, and 79 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Deoxycorticosterone, or a derivative or an analog thereof. Deoxycorticosterone acetate is a steroid hormone used for intramuscular injection for replacement therapy of the adrenocortical steroid. 11β-hydroxylation of deoxycorticosterone leads to corticosterone.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Echinomycin, or a derivative or an analog thereof. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion. Echinomycin is a cell-permeable inhibitor of HIF-1-mediated gene transcription. It acts by intercalating into DNA in a sequence-specific manner, blocking the binding of either HIF-1α or HIF-1β to the hypoxia-responsive element. Echinomycin reversibly inhibits hypoxia-induced HIF-1 transcription activity in U215 cells with a half maximal effective concentration (EC₅₀) value of 1.2 nM. It inhibits hypoxia-induced expression of vascular endothelial growth factor, blocking angiogenesis and altering excitatory synaptic transmission in hippocampal neurons. Echinomycin also impairs expression of survivin, enhancing the sensitivity of multiple myeloma cells to melphalan.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Enzastaurin, or a derivative or an analog thereof. Enzastaurin, also known as LY317615, is a potent PKCβ selective inhibitor with IC₅₀of 6 nM, exhibiting 6- to 20-fold selectivity against PKCα, PKCγ and PKCε.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Epinephrine, or a derivative or an analog thereof. Epinephrine HCl is a hormone and a neurotransmitter.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Erlotinib, or a derivative or an analog thereof. Erlotinib is an EGFR inhibitor with IC₅₀of 2 nM, >1000-fold more sensitive for EGFR than human c-Src or v-Abl.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include EVP-6124 (hydrochloride) (encenicline), or a derivative or an analog thereof. EVP-6124 hydrochloride, also known as encenicline, is a novel partial agonist of a7 neuronal nicotinic acetylcholine receptors (nAChRs). EVP-6124 shows selectivity for a7 nAChRs and does not activate or inhibit heteromeric a402 nAChRs.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include EW-7197. EW-7197 is a highly potent, selective, and orally bioavailable TGF-β receptor ALK4/ALK5 inhibitor with IC₅₀of 13 nM and 11 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include FRAX597, or a derivative or an analog thereof. FRAX597 is a potent, ATP-competitive inhibitor of group I PAKs with IC₅₀of 8 nM, 13 nM, and 19 nM for PAK1, PAK2, and PAK3, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GDC-0879, or a derivative or an analog thereof. GDC-0879 is a novel, potent, and selective B-Raf inhibitor with IC₅₀of 0.13 nM with activity against c-Raf as well.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GO6983, or a derivative or an analog thereof. GO6983 is a pan-PKC inhibitor against for PKCα, PKCβ, PKCγ and PKC with IC₅₀of 7 nM, 7 nM, 6 nM and 10 nM, respectively. It is less potent to PKCζ and inactive to PKCμ.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GSK2334470, or a derivative or an analog thereof. GSK2334470 is a novel PDK1 inhibitor with IC₅₀of about 10 nM and with no activity at other close related AGC-kinases.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GZD824 Dimesylate, or a derivative or an analog thereof. GZD824 is a novel orally bioavailable Bcr-Abl inhibitor for Bcr-Abl (wildtype) and Bcr-Abl (T315I) with IC₅₀of 0.34 nM and 0.68 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include INNO-206 (aldoxorubicin), or a derivative or an analog thereof INNO-206, also known as Aldoxorubicin, is the 6-maleimidocaproyl hydrazone derivative prodrug of the anthracycline antibiotic doxorubicin (DOXO-EMCH) with antineoplastic activity.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include LDN193189, or a derivative or an analog thereof. LDN193189 is a selective BMP signaling inhibitor that inhibits the transcriptional activity of the BMP type I receptors ALK2 and ALK3 with IC₅₀of 5 nM and 30 nM, respectively, exhibiting 200-fold selectivity for BMP versus TGF-B.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include LDN-212854, or a derivative or an analog thereof. LDN-212854 is a potent and selective BMP receptor inhibitor with IC₅₀of 1.3 nM for ALK2, exhibiting about 2-, 66-, 1641-, and 7135-fold selectivity over ALK1, ALK3, ALK4, and ALK5, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Merestinib, or a derivative or an analog thereof. Merestinib, also known as LY2801653, is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a Kd of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min⁻¹and half life (t_t/2) of 525 min.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include MK-0752, or a derivative or an analog thereof. MK-0752 is a potent, reversible inhibitor of y-secretase, reducing the cleavage of amyloid precursor protein (APP) to Aβ40 in human neuroblastoma SH-SYSY cells with an IC₅₀value of 5 nM. It is orally bioavailable and crosses the blood-brain barrier, as orally administered MK-0752 dose-dependently reduces the generation of new amyloid β protein in the brain of rhesus monkeys. Through its effects on the NOTCH pathway, MK-0752 reduces the number of breast cancer stem cells in tumor grafts, enhancing the efficacy of the chemotherapy drug docetaxel in mice with breast cancer tumors.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Momelotinib, or a derivative or an analog thereof. Momelotinib, also known as CYT387, is an ATP-competitive inhibitor of JAK1/JAK2 with IC₅₀of 11 nM/18 nM and approximately 10-fold selectivity versus JAK3.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Oligomycin A, or a derivative or an analog thereof. Oligomycin A is an inhibitor of ATP synthase, inhibits oxidative phosphorylation and all the ATP-dependent processes occurring on the coupling membrane of mitochondria.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include OSU-03012, or a derivative or an analog thereof. OSU-03012 is a potent inhibitor of recombinant PDK-1 with IC₅₀of 5 μM and 2-fold increase in potency over OSU-02067.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Pacritinib (SB1518), or a derivative or an analog thereof. Pacritinib, also known as SB1518, is a potent and selective inhibitor JAK2 and FLT3 with IC_50Sof 23 and 22 nM in cell-free assays, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PHA-665752, or a derivative or an analog thereof. PHA-665752 is a potent, selective and ATP-competitive c-Met inhibitor with IC₅₀of 9 nM, >50-fold selectivity for c-Met than RTKs or STKs.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Phenformin, or a derivative or an analog thereof. Phenformin hydrochloride is a hydrochloride salt of phenformin that is an anti-diabetic drug from the biguanide class.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Phorbol 12,13-dibutyrate, or a derivative or an analog thereof. Phorbol 12,13-dibutyrate is a protein kinase C activator. It induces contraction of vascular smooth muscle and inhibits MLC phosphatase (MLCP) in vascular smooth muscle. The activity does not alter intracellular Ca' concentration. It also inhibits the activity of Na+, K+ ATPase in opossum kidney cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Pifithrin-μ, or a derivative or an analog thereof. Pifithrin-μ specifically inhibits p53 activity by reducing its affinity to Bcl-xL and Bcl-2, and it also inhibits HSP70 function and autophagy.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PND-1186, or a derivative or an analog thereof. PND-1186, VS-4718, is a reversible and selective focal adhesion kinase (FAK) inhibitor with IC₅₀of 1.5 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include prednisone, or a derivative or an analog thereof. Prednisone is a synthetic glucocorticoid with anti-inflammatory and immunosuppressive activity.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include R788 (fostamatinib disodium hexahydrate), or a derivative or an analog thereof. R788 sodium salt hydrate (fostamatinib), a prodrug of the active metabolite R406, is a potent Syk inhibitor with IC₅₀of 41 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Rifampicin, or a derivative or an analog thereof. Rifampicin is a member of the rifamycin class of antibiotics, as it inhibits bacterial DNA-dependent RNA synthesis (Ki=˜1 nM).While this compound does not directly affect RNA synthesis in humans, its use as an antibiotic is limited by its potency toward activation of the pregnane X receptor (PXR, EC₅₀=˜2 μM), which results in the up-regulation of enzymes that alter drug metabolism. Access of rifampicin to the nuclear receptor PXR requires its import into the cell via organic anion transporters (OATs) in the OAT polypeptide (OATP) family. By acting as a transporter substrate, rifampicin inhibits OATPs with Ki/IC₅₀values ranging from 0.58-18 μM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Semaxanib, or a derivative or an analog thereof. Semaxanib is a quinolone derivative with potential antineoplastic activity. Semaxanib reversibly inhibits ATP binding to the tyrosine kinase domain of vascular endothelial growth factor receptor 2 (VEGFR2).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include SIS3, or a derivative or an analog thereof. SIS3 is a specific inhibitor of Smad3. It inhibits TGF-B and activin signaling by suppressing Smad3 phosphorylation without affecting the MAPK/p38, ERK, or PI3-kinase signaling pathways.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include SKL2001, or a derivative or an analog thereof. SKL2001 is a novel agonist of the Wnt/β-catenin pathway that disrupts the Axin/β-catenin interaction.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include SMI-4a, or a derivative or an analog thereof. SMI-4a is a potent inhibitor of Pim1 with IC₅₀of 17 nM, with modest potency to Pim-2. It does not significantly inhibit other serine/threonine- or tyrosine-kinases.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include T0901317, or a derivative or an analog thereof. T0901317 is a potent, non-selective LXR agonist with EC₅₀of 50 nM. It increases ABCA1 expression associated with cholesterol efflux regulation and HDL metabolism. It also increases muscle expression of PPAR-δ and shows antiobesogenic effects in vivo.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include TFP, or a derivative or an analog thereof. Trifluoperazine (TFP) has central antiadrenergic, antidopaminergic, and minimal anticholinergic effects. It is thought to function by blockading dopamine D1 and D2 receptors in the mesocortical and mesolimbic pathways, relieving or minimizing such symptoms of schizophrenia as hallucinations, delusions, and disorganized thought and speech.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Thalidomide, or a derivative or an analog thereof. Thalidomide was introduced as a sedative drug, immunomodulatory agent and also is investigated for treating symptoms of many cancers. Thalidomide inhibits an E3 ubiquitin ligase, which is a CRBN-DDB1-Cul4A complex.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Tivozanib, or a derivative or an analog thereof. Tivozanib, also known as AV-951, is a potent and selective VEGFR inhibitor for VEGFR1/2/3 with IC₅₀of 30 nM/6.5 nM/15 nM. It also inhibits PDGFR and c-Kit but exhibits low activity against FGFR-1, Flt3, c-Met, EGFR and IGF-1R.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include TP-434 (Eravacycline), or a derivative or an analog thereof. TP-434, also known as Eravacycline, is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms including tetracycline-specific efflux and ribosomal-protection.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include WYE-125132 (WYE-132), or a derivative or an analog thereof. WYE-125132, also known as WYE-132, is a highly potent, ATP-competitive mTOR inhibitor with IC₅₀of 0.19 nM. It is highly selective for mTOR versus PI3Ks or PI3K-related kinases hSMG1 and ATR.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Zibotentan, or a derivative or an analog thereof. Zibotentan, also known as ZD4054, is an orally administered, potent and specific endothelin A receptor (ETA)-receptor antagonist with IC₅₀of 21 nM.

Polypeptides

In some embodiments, compounds for altering expression of a urea cycle-related gene comprise a polypeptide. As used herein, the term “polypeptide” refers to a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid.

In some embodiments, polypeptide compounds capable of modulating expression of one or more urea cycle-related genes include, but are not limited to, Activin, Anti mullerian hormone, BMP2, EGF, FGF, GDF10 (BMP3b), GDF2 (BMP9), HGF/SF, IGF-1, Nodal, PDGF, TNF-α, and Wnt3a, or derivatives or analogs thereof. Any one of these compounds or a combination thereof may be administered to a subject to treat a urea cycle disorder, such as CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Activin, or a derivative or an analog thereof. Activins are homodimers or heterodimers of the different β subunit isoforms, part of the transforming growth factor-beta (TGF-B) family. Mature Activin A has two 116 amino acids residues βA subunits (βA-βA). Activin displays an extensive variety of biological activities, including mesoderm induction, neural cell differentiation, bone remodeling, hematopoiesis, and reproductive physiology. Activins takes part in the production and regulation of hormones such as FSH, LH, GnRH and ACTH. Cells that are identified to express Activin A include fibroblasts, endothelial cells, hepatocytes, vascular smooth muscle cells, macrophages, keratinocytes, osteoclasts, bone marrow monocytes, prostatic epithelium, neurons, chondrocytes, osteoblasts, Leydig cells, Sertoli cells, and ovarian granulosa cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include anti Mullerian hormone, or a derivative or an analog thereof. Anti Mullerian hormone is a member of the TGF-B gene family which mediates male sexual differentiation. Anti Mullerian hormone causes the regression of Mullerian ducts which would otherwise differentiate into the uterus and fallopian tubes. Some mutations in the anti-Mullerian hormone result in persistent Mullerian duct syndrome.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BMP2, or a derivative or an analog thereof. Bone morphogenetic protein 2 (BMP2) belongs to the TGF-B superfamily. The BMP family members are regulators of cell growth and differentiation in both embryonic and adult tissues. BMP2 is a candidate gene for the autosomal dominant disease of fibrodysplasia (myositis) ossificans progressiva.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include EGF, or a derivative or an analog thereof. Epidermal Growth Factor (EGF) is a polypeptide growth factor which stimulates the proliferation of a wide range of epidermal and epithelial cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include FGF, or a derivative or an analog thereof. Fibroblast Growth Factor-acidic (FGF-acidic), also known as FGF-1 and endothelial cell growth factor, is a member of the FGF family which currently contain 23 members. FGF acidic and basic, unlike the other members of the family, lack signal peptides and are apparently secreted by mechanisms other than the classical protein secretion pathway. FGF acidic has been detected in large amounts in the brain. Other cells known to express FGF acidic include hepatocytes, vascular smooth muscle cells, CNS neurons, skeletal muscle cells, fibroblasts, keratinocytes, endothelial cells, intestinal columnar epithelium cells and pituitary basophils and acidophils.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GDF10 (BMP3b), or a derivative or an analog thereof. GDF10, also known as BMP3b, is a member of the BMP family and the TGF-B superfamily. GDF10 is expressed in femur, brain, lung, skeletal, muscle, pancreas and testis, and has a role in head formation and possibly multiple roles in skeletal morphogenesis. In humans, GDF10 mRNA is found in the cochlea and lung of fetuses, and in testis, retina, pineal gland, and other neural tissues of adults. These proteins are characterized by a polybasic proteolytic processing site which is cleaved to produce a mature protein containing 7 conserved cysteine residues.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GDF2 (BMP9), or a derivative or an analog thereof. GDF2, also known as BMP9, is a member of BMP family and the TGF-B superfamily. BMP9 has a role in the maturation of basal forebrain cholinergic neurons (BFCN) as well as the induction and maintenance of the ability of these cells to respond to acetylcholine. BFCN are important for the processes of learning, memory and attention. BMP9 is a potent inducer of hepcidin (a cationic peptide that has an antimicrobial properties) in hepatocytes and can regulate iron metabolism. The physiological receptor of BMP9 is thought to be activin receptor-like kinase 1, ALK1 (also known as ACVRL1), an endothelial-specific type I receptor of the TGF-B receptor family. BMP9 is one of the most potent BMPs to induce orthotopic bone formation in vivo. BMP3, a blocker of most BMPs appears not to affect BMP9.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include HGF/SF, or a derivative or an analog thereof. Hepatocyte Growth Factor (HGF), also known as hepatopoietin-A and scatter factor (SF), is a pleiotropic mitogen belonging to the peptidase 51 family (plasminogen subfamily). It is produced by mesenchymal cells and acts on epithelial cells, endothelial cells and hematopoietic progenitor cells. HGF binds to the proto-oncogenic c-Met receptor to activate a tyrosine kinase signaling cascade. It regulates cell growth, motility and morphogenesis, and it also plays a pivotal role in angiogenesis, tumorigenesis and tissue regeneration.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include IGF-1, or a derivative or an analog thereof. Insulin-like growth factor I (IGF-I) also known as Somatamedin C is a hormone similar in molecular structure to insulin. Human IGF-I has two isoforms (IGF-IA and IGF-IB) which is differentially expressed by various tissues. Mature human IGF-I respectively shares 94% and 96% aa sequence identity with mouse and rat IGF-I. Both IGF-I and IGF-II (another ligand of IGF) can signal through the IGF-I receptor (IGFIR), but IGF-II can alone bind the IGF-II receptor (IGFIIR/Mannose-6-phosphate receptor). IGF-I plays an important role in childhood growth and continues to have anabolic effects in adults.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Nodal, or a derivative or an analog thereof. Nodal is a 13 kDa member of the TGF-B superfamily of molecules. In human, it is synthesized as a 347 amino acid preproprecursor that contains a 26 amino acid signal sequence, a 211 amino acid prodomain, and a 110 amino acid mature region. Consistent with its TGF-B superfamily membership, it exists as a disulfide-linked homodimer and would be expected to demonstrate a cysteine-knot motif. Mature human Nodal is 99%, 98%, 96% and 98% amino acid identical to mature canine, rat, bovine and mouse Nodal, respectively. Nodal signals through two receptor complexes, both of which contain members of the TGF-beta family of Ser/Thr kinase receptors.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PDGF, or a derivative or an analog thereof. the Platelet-derived growth factor (PDGF) is a disulfide-linked dimer consisting of two peptides-chain A and chain B. PDGF has three subforms: PDGF-AA, PDGF-BB, PDGF-AB. It is involved in a number of biological processes, including hyperplasia, embryonic neuron development, chemotaxis, and respiratory tubule epithelial cell development. The function of PDGF is mediated by two receptors (PDGFRα and PDGFRβ).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include TNF-α, or a derivative or an analog thereof. TNF-α, the prototypical member of the TNF protein superfamily, is a homotrimeric type-II membrane protein. Membrane bound TNF-α is cleaved by the metalloprotease TACE/ADAM17 to generate a soluble homotrimer. Both membrane and soluble forms of TNF-α are biologically active. TNF-α α is produced by a variety of immune cells including T cells, B cells, NK cells and macrophages. Cellular response to TNF-α is mediated through interaction with receptors TNF-R1 and TNF-R2 and results in activation of pathways that favor both cell survival and apoptosis depending on the cell type and biological context. Activation of kinase pathways (including JNK, ERK (p44/42), p38 MAPK and NF-kB) promotes the survival of cells, while TNF-α mediated activation of caspase-8 leads to programmed cell death. TNF-α plays a key regulatory role in inflammation and host defense against bacterial infection, notably Mycobacterium tuberculosis. The role of TNF-α in autoimmunity is underscored by blocking TNF-α action to treat rheumatoid arthritis and Crohn's disease.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Wnt3a, or a derivative or an analog thereof. The WNT gene family consists of structurally related genes which encode secreted signaling proteins. These proteins have been implicated in oncogenesis and in several developmental processes, including regulation of cell fate and patterning during embryogenesis. This gene is a member of the WNT gene family. It encodes a protein which shows 96% amino acid identity to mouse Wnt3a protein, and 84% to human WNT3 protein, another WNT gene product. This gene is clustered with WNT14 gene, another family member, in chromosome 1q42 region.

Antibodies

In some embodiments, compounds for altering expression of one or more urea cycle-related genes comprise an antibody. In one embodiment, antibodies of the present invention comprising antibodies, antibody fragments, their variants or derivatives described herein are specifically immunoreactive with at least one component of the gene signaling networks associated with the urea cycle-related gene.

As used herein, the term “antibody” is used in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies formed from at least two intact antibodies), and antibody fragments such as diabodies so long as they exhibit a desired biological activity. Antibodies are primarily amino-acid based molecules but may also comprise one or more modifications such as with sugar moieties.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising an antigen binding region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site. Also produced is a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen. Antibodies of the present invention may comprise one or more of these fragments. For the purposes herein, an “antibody” may comprise a heavy and light variable domain as well as an Fc region.

“Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V_H) followed by a number of constant domains. Each light chain has a variable domain at one end (V_L) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

As used herein, the term “variable domain” refers to specific antibody domains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. As used herein, the term “Fv” refers to antibody fragments which contain a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.

Antibody “light chains” from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “scFv” as used herein, refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain. In some embodiments, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain V_Hconnected to a light chain variable domain V_Lin the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), the contents of each of which are incorporated herein by reference in their entirety.

Antibodies of the present invention may be polyclonal or monoclonal or recombinant, produced by methods known in the art or as described in this application. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.

The term “hypervariable region” when used herein in reference to antibodies refers to regions within the antigen binding domain of an antibody comprising the amino acid residues that are responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining region (CDR). As used herein, the “CDR” refers to the region of an antibody that comprises a structure that is complimentary to its target antigen or epitope.

In some embodiments, the compositions of the present invention may be antibody mimetics. The term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. As such, antibody mimics include nanobodies and the like.

In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, DARPins, Fynomers and Kunitz and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide region.

As used herein, the term “antibody variant” refers to a biomolecule resembling an antibody in structure and/or function comprising some differences in their amino acid sequence, composition or structure as compared to a native antibody.

The preparation of antibodies, whether monoclonal or polyclonal, is known in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999.

Antibodies of the present invention may be characterized by their target molecule(s), by the antigens used to generate them, by their function (whether as agonists or antagonists) and/or by the cell niche in which they function.

Measures of antibody function may be made relative to a standard under normal physiologic conditions, in vitro or in vivo. Measurements may also be made relative to the presence or absence of the antibodies. Such methods of measuring include standard measurement in tissue or fluids such as serum or blood such as Western blot, enzyme-linked immunosorbent assay (ELISA), activity assays, reporter assays, luciferase assays, polymerase chain reaction (PCR) arrays, gene arrays, Real Time reverse transcriptase (RT) PCR and the like.

Antibodies of the present invention exert their effects via binding (reversibly or irreversibly) to one or more target sites. While not wishing to be bound by theory, target sites which represent a binding site for an antibody, are most often formed by proteins or protein domains or regions. However, target sites may also include biomolecules such as sugars, lipids, nucleic acid molecules or any other form of binding epitope.

Alternatively, or additionally, antibodies of the present invention may function as ligand mimetics or nontraditional payload carriers, acting to deliver or ferry bound or conjugated drug payloads to specific target sites.

Changes elicited by antibodies of the present invention may result in a neomorphic change in the cell. As used herein, “a neomorphic change” is a change or alteration that is new or different. Such changes include extracellular, intracellular and cross cellular signaling.

In some embodiments, compounds or agents of the invention act to alter or control proteolytic events. Such events may be intracellular or extracellular.

Antibodies of the present invention, as well as antigens used to generate them, are primarily amino acid-based molecules. These molecules may be “peptides,” “polypeptides,” or “proteins.”

As used herein, the term “peptide” refers to an amino-acid based molecule having from 2 to 50 or more amino acids. Special designators apply to the smaller peptides with “dipeptide” referring to a two amino acid molecule and “tripeptide” referring to a three amino acid molecule. Amino acid based molecules having more than 50 contiguous amino acids are considered polypeptides or proteins.

The terms “amino acid” and “amino acids” refer to all naturally occurring L-alpha-amino acids as well as non-naturally occurring amino acids. Amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagines (Asn:N), where the amino acid is listed first followed parenthetically by the three and one letter codes, respectively. Hybridizing oligonucleotides

In some embodiments, oligonucleotides, including those which function via a hybridization mechanism, whether single of double stranded such as antisense molecules, RNAi constructs (including siRNA, saRNA, microRNA, etc.), aptamers and ribozymes may be used to alter or as perturbation stimuli of the gene signaling networks associated with a urea cycle-related gene.

As such oligonucleotides may also serve as therapeutics, their therapeutic liabilities and treatment outcomes may be ameliorated or predicted, respectively by interrogating the gene signaling networks of the invention.

Genome Editing Approaches

In certain embodiments, expression of a urea cycle-related gene may be modulated by altering the chromosomal regions defining the insulated neighborhood(s) and/or genome signaling center(s) associated with the urea cycle-related gene. For example, protein production may be increased by targeting a component of the gene signaling network that functions to repress the expression of the urea cycle-related gene.

Methods of altering the gene expression attendant to an insulated neighborhood include altering the signaling center (e.g. using CRISPR/Cas to change the signaling center binding site or repair/replace if mutated). These alterations may result in a variety of results including: activation of cell death pathways prematurely/inappropriately (key to many immune disorders), production of too little/much gene product (also known as the rheostat hypothesis), production of too little/much extracellular secretion of enzymes, prevention of lineage differentiation, switch of lineage pathways, promotion of stemness, initiation or interference with auto regulatory feedback loops, initiation of errors in cell metabolism, inappropriate imprinting/gene silencing, and formation of flawed chromatin states. Additionally, genome editing approaches including those well-known in the art may be used to create new signaling centers by altering the cohesin necklace or moving genes and enhancers.

In certain embodiments, genome editing approaches describe herein may include methods of using site-specific nucleases to introduce single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ). HDR is essentially an error-free mechanism that repairs double-strand DNA breaks in the presence of a homologous DNA sequence. The most common form of HDR is homologous recombination. It utilizes a homologous sequence as a template for inserting or replacing a specific DNA sequence at the break point. The template for the homologous DNA sequence can be an endogenous sequence (e.g., a sister chromatid), or an exogenous or supplied sequence (e.g., plasmid or an oligonucleotide). As such, HDR may be utilized to introduce precise alterations such as replacement or insertion at desired regions. In contrast, NHEJ is an error-prone repair mechanism that directly joins the DNA ends resulting from a double-strand break with the possibility of losing, adding or mutating a few nucleotides at the cleavage site. The resulting small deletions or insertions (termed “Indels”) or mutations may disrupt or enhance gene expression. Additionally, if there are two breaks on the same DNA, NHEJ can lead to the deletion or inversion of the intervening segment. Therefore, NHEJ may be utilized to introduce insertions, deletions or mutations at the cleavage site.

CRISPR/Cas Systems

In certain embodiments, a CRISPR/Cas system may be used to delete CTCF anchor sites to modulate gene expression within the insulated neighborhood associated with that anchor site. See, Hnisz et al., Cell 167, Nov. 17, 2016, which is hereby incorporated by reference in its entirety. Disruption of the boundaries of insulated neighborhood prevents the interactions necessary for proper function of the associated signaling centers. Changes in the expression genes that are immediately adjacent to the deleted neighborhood boundary have also been observed due to such disruptions.

In certain embodiments, a CRISPR/Cas system may be used to modify existing CTCF anchor sites. For example, existing CTCF anchor sites may be mutated or inverted by inducing NHEJ with a CRISPR/Cas nuclease and one or more guide RNAs, or masked by targeted binding with a catalytically inactive CRISPR/Cas enzyme and one or more guide RNAs. Alteration of existing CTCF anchor sites may disrupt the formation of existing insulated neighborhoods and alter the expression of genes located within these insulated neighborhoods.

In certain embodiments, a CRISPR/Cas system may be used to introduce new CTCF anchor sites. CTCF anchor sites may be introduced by inducing HDR at a selected site with a CRISPR/Cas nuclease, one or more guide RNAs and a donor template containing the sequence of a CTCF anchor site. Introduction of new CTCF anchor sites may create new insulated neighborhoods and/or alter existing insulated neighborhoods, which may affect expression of genes that are located adjacent to these insulated neighborhoods.

In certain embodiments, a CRISPR/Cas system may be used to alter signaling centers by changing signaling center binding sites. For example, if a signaling center binding site contains a mutation that affects the assembly of the signaling center with associated transcription factors, the mutated site may be repaired by inducing a double strand DNA break at or near the mutation using a CRISPR/Cas nuclease and one or more guide RNAs in the presence of a supplied corrected donor template.

In certain embodiments, a CRISPR/Cas system may be used to modulate expression of neighborhood genes by binding to a region within an insulated neighborhood (e.g., enhancer) and block transcription. Such binding may prevent recruitment of transcription factors to signaling centers and initiation of transcription. The CRISPR/Cas system may be a catalytically inactive CRISPR/Cas system that do not cleave DNA.

In certain embodiments, a CRISPR/Cas system may be used to knockdown expression of neighborhood genes via introduction of short deletions in coding regions of these genes. When repaired, such deletions would result in frame shifts and/or introduce premature stop codons in mRNA produced by the genes followed by the mRNA degradation via nonsense-mediated decay. This may be useful for modulation of expression of activating and repressive components of signaling pathways that would result in decreased or increased expression of genes under control of these pathways including disease genes such as CPS1, OTC, ASS, ASL, NAGS, and ARG1.

In other embodiments, a CRISPR/Cas system may also be used to alter cohesion necklace or moving genes and enhancers.

CRISPR/Cas Enzymes

CRISPR/Cas systems are bacterial adaptive immune systems that utilize RNA-guided endonucleases to target specific sequences and degrade target nucleic acids. They have been adapted for use in various applications in the field of genome editing and/or transcription modulation. Any of the enzymes or orthologs known in the art or disclosed herein may be utilized in the methods herein for genome editing.

In certain embodiments, the CRISPR/Cas system may be a Type II CRISPR/Cas9 system. Cas9 is an endonuclease that functions together with a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA) to cleave double stranded DNAs. The two RNAs can be engineered to form a single-molecule guide RNA by connecting the 3′ end of the crRNA to the 5′ end of tracrRNA with a linker loop. Jinek et al., Science, 337(6096):816-821 (2012) showed that the CRISPR/Cas9 system is useful for RNA-programmable genome editing, and international patent application WO2013/176772 provides numerous examples and applications of the CRISPR/Cas endonuclease system for site-specific gene editing, which are incorporated herein by reference in their entirety. Exemplary CRISPR/Cas9 systems include those derived from Streptococcus pyogenes, Streptococcus thermophilus, Neisseria meningitidis, Treponema denticola, Streptococcus aureas, and Francisella tularensis.

In certain embodiments, the CRISPR/Cas system may be a Type V CRISPR/Cpfl system. Cpfl is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA. Cpfl produces staggered DNA double-stranded break with a 4 or 5 nucleotide 5′ overhang. Zetsche et al. Cell. 2015 Oct 22;163(3):759-71 provides examples of Cpfl endonuclease that can be used in genome editing applications, which is incorporated herein by reference in its entirety. Exemplary CRISPR/Cpfl systems include those derived from Francisella tularensis, Acidaminococcus sp., and Lachnospiraceae bacterium.

In certain embodiments, nickase variants of the CRISPR/Cas endonucleases that have one or the other nuclease domain inactivated may be used to increase the specificity of CRISPR-mediated genome editing. Nickases have been shown to promote HDR versus NHEJ. HDR can be directed from individual Cas nickases or using pairs of nickases that flank the target area.

In certain embodiments, catalytically inactive CRISPR/Cas systems may be used to bind to target regions (e.g., CTCF anchor sites or enhancers) and interfere with their function. Cas nucleases such as Cas9 and Cpfl encompass two nuclease domains. Mutating critical residues at the catalytic sites creates variants that only bind to target sites but do not result in cleavage. Binding to chromosomal regions (e.g., CTCF anchor sites or enhancers) may disrupt proper formation of insulated neighborhoods or signaling centers and therefore lead to altered expression of genes located adjacent to the target region.

In certain embodiments, a CRISPR/Cas system may include additional functional domain(s) fused to the CRISPR/Cas enzyme. The functional domains may be involved in processes including but not limited to transcription activation, transcription repression, DNA methylation, histone modification, and/or chromatin remodeling. Such functional domains include but are not limited to a transcriptional activation domain (e.g., VP64 or KRAB, SID or SID4X), a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain or a chemically inducible/controllable domain.

In certain embodiments, a CRISPR/Cas enzyme may be administered to a cell or a patient as one or a combination of the following: one or more polypeptides, one or more mRNAs encoding the polypeptide, or one or more DNAs encoding the polypeptide.

Guide Nucleic Acid

In certain embodiments, guide nucleic acids may be used to direct the activities of an associated CRISPR/Cas enzymes to a specific target sequence within a target nucleic acid. Guide nucleic acids provide target specificity to the guide nucleic acid and CRISPR/Cas complexes by virtue of their association with the CRISPR/Cas enzymes, and the guide nucleic acids thus can direct the activity of the CRISPR/Cas enzymes.

In one aspect, guide nucleic acids may be RNA molecules. In one aspect, guide RNAs may be single-molecule guide RNAs. In one aspect, guide RNAs may be chemically modified.

In certain embodiments, more than one guide RNAs may be provided to mediate multiple CRISPR/Cas-mediated activities at different sites within the genome.

In certain embodiments, guide RNAs may be administered to a cell or a patient as one or more RNA molecules or one or more DNAs encoding the RNA sequences. Ribonucleoprotein complexes (RNPs)

In one embodiment, the CRISPR/Cas enzyme and guide nucleic acid may each be administered separately to a cell or a patient.

In another embodiment, the CRISPR/Cas enzyme may be pre-complexed with one or more guide nucleic acids. The pre-complexed material may then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

Zinc Finger Nucleases

In certain embodiments, genome editing approaches of the present invention involve the use of Zinc finger nucleases (ZFNs). Zinc finger nucleases (ZFNs) are modular proteins comprised of an engineered zinc finger DNA binding domain linked to a DNA-cleavage domain. A typical DNA-cleavage domain is the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, a pair of ZFNs must are required to be engineered to bind to cognate target “half-site” sequences on opposite DNA strands and with precise spacing between them to allow the two enable the catalytically active FokI domains to dimerize. Upon dimerization of the FokI domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half-sites as the initiating step in genome editing.

Transcription Activator-Like Effector Nucleases (TALENs)

In certain embodiments, genome editing approaches of the present invention involve the use of Transcription Activator-Like Effector Nucleases (TALENs). TALENs represent another format of modular nucleases which, similarly to ZFNs, are generated by fusing an engineered DNA binding domain to a nuclease domain, and operate in tandem to achieve targeted DNA cleavage. While the DNA binding domain in ZFN consists of Zinc finger motifs, the TALEN DNA binding domain is derived from transcription activator-like effector (TALE) proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single basepair in the target DNA sequence that is typically up to 20 bp in length, giving a total target sequence length of up to 40 bp. Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. This constitutes a much simpler recognition code than for zinc fingers, and thus represents an advantage over the latter for nuclease design. Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the FokI domain to reduce off-target activity.

Modulation of Urea Cycle-Related Genes

Compositions and methods described herein may be effective in modulating the expression of one or more urea cycle-related genes. Such compositions and methods may be used to treat a urea cycle disorder.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of CPS1. Compounds that may be used to modulate the CPS1 expression include, but are not limited to, Dasatinib, R788 (fostamatinib disodium hexahydrate), Bosutinib, Epinephrine, FRAX597, Merestinib, Deoxycorticosterone, 17-AAG (Tanespimycin), GDF2 (BMP9), GZD824 Dimesylate, PND-1186, Wnt3a, Nodal, Anti mullerian hormone, TNF-α, Activin, IGF-1, prednisone, PDGF, HGF/SF, EGF, BAY 87-2243, CP-673451, FGF, GDF10 (BMP3b), LDN193189, Amuvatinib, Momelotinib, Echinomycin, Pacritinib (SB1518), BMP2, Crizotinib, LDN-212854, Thalidomide, CO-1686 (Rociletinib), Zibotentan, and derivatives or analogs thereof. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate CPS1 expression. In some embodiments, R788 (fostamatinib disodium hexahydrate) perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate CPS1 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate CPS1 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate CPS1 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate CPS1 expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate CPS1 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate CPS1 expression. In some embodiments, 17-AAG (Tanespimycin) perturbs the Cell Cycle/DNA Damage Metabolic Enzyme/Protease signaling pathway to modulate CPS1 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, GZD824 Dimesylate perturbs the ABL signaling pathway to modulate CPS1 expression. In some embodiments, PND-1186 perturbs the FAK signaling pathway to modulate CPS1 expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate CPS1 expression. In some embodiments, Nodal perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Anti mullerian hormone perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, TNF-α perturbs the NF-kB, MAPK, or Apoptosis pathway to modulate CPS1 expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, IGF-1 perturbs the IGF-1R/InsR signaling pathway to modulate CPS1 expression. In some embodiments, prednisone perturbs the GR signaling pathway to modulate CPS1 expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate CPS1 expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate CPS1 expression. In some embodiments, EGF perturbs the EGFR signaling pathway to modulate CPS1 expression. In some embodiments, BAY 87-2243 perturbs the Hypoxia activated signaling pathway to modulate CPS1 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate CPS1 expression. In some embodiments, FGF perturbs the FGFR signaling pathway to modulate CPS1 expression. In some embodiments, GDF10 (BMP3b) perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, LDN193189 perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate CPS1 expression. In some embodiments, Momelotinib perturbs the JAK/STAT signaling pathway to modulate CPS1 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate CPS1 expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate CPS1 expression. In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Crizotinib perturbs the c-MET signaling pathway to modulate CPS1 expression. In some embodiments, LDN-212854 perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate CPS1 expression. In some embodiments, CO-1686 (Rociletinib) perturbs the JAK/STAT and/or Tyrosine Kinase/RTK signaling pathway to modulate CPS1 expression. In some embodiments, Zibotentan perturbs the GPCR/G protein signaling pathway to modulate CPS1 expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the CPS1 gene. The CPS1 gene has a cytogenetic location of 2q34 and the genomic coordinate are on Chromosome 2 on the forward strand at position 210,477,682-210,679,107. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of CPS1. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, p300, and SMC1. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, and YY1. The signaling proteins may include but are not limited to TCF7L2, ESRA, FOS, NR3C1, JUN, NR5A2, RBPJK, RXR, STAT3, NR1I1, NF-kB, SMAD2/3, SMAD4, STAT1, TEAD1, and TP53.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of OTC. Compounds that may be used to modulate the OTC expression include, but are not limited to, CP-673451, Pacritinib (SB1518), Echinomycin, Crenolanib, Thalidomide, Amuvatinib, Dasatinib, Momelotinib, Activin, Wnt3a, INNO-206 (aldoxorubicin), TNF-α, Anti mullerian hormone, Pifithrin-μ, PDGF, IFG-1, FRAX597, Nodal, EGF, FGF, HGF/SF, BIRB 796, and derivatives or analogs thereof. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate OTC expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate OTC expression. In some embodiments, Crenolanib perturbs the PDGFR. signaling pathway to modulate OTC expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate OTC expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate OTC expression. In some embodiments, Momelotinib perturbs the JAK/STAT signaling pathway to modulate OTC expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate OTC expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate OTC expression. In some embodiments, INNO-206 (aldoxorubicin) perturbs the Cell Cycle/DNA Damage pathway to modulate OTC expression. In some embodiments, TNF-α perturbs the NF-kB, MAPK, or Apoptosis signaling pathway to modulate OTC expression. In some embodiments, Anti mullerian hormone perturbs the TGF-B signaling pathway to modulate OTC expression. In some embodiments, Pifithrin-μ perturbs the p53 signaling pathway to modulate OTC expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, IGF-1 perturbs the IGF-1R/InsR signaling pathway to modulate OTC expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate OTC expression. In some embodiments, Nodal perturbs the TGF-B signaling pathway to modulate OTC expression. In sonic embodiments, EGF perturbs the EGFR signaling pathway to modulate OTC expression. In some embodiments, FGF perturbs the FGFR signaling pathway to modulate OTC expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate OTC expression. In some embodiments, BIRB 796 perturbs the MAPK signaling pathway to modulate OTC expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the OTC gene. The OTC gene has a cytogenetic location of Xp11.4 and the genomic coordinate are on Chromosome X on the forward strand at position 38,352,545-38,421,450. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of OTC. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac and BRD4. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, YY1, and HNF1A. The signaling proteins may include but are not limited to TCF7L2, HIF1a, ESRA, NR3C1, JUN, RXR, STAT3, NF-kB, SMAD2/3, SMAD4, and TEAD1.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of ASS1. Compounds that may be used to modulate the ASS1 expression include, but are not limited to, Dasatinib, CP-673451, Echinomycin, GDF2 (BMP9), Pacritinib (SB1518), Epinephrine, FRAX597, Bosutinib, TP-434 (Eravacycline), BMP2, SMI-4a, Amuvatinib, Crenolanib, Deoxycorticosterone, INNO-206 (aldoxorubicin), TNF-α, T0901317, and derivatives or analogs thereof. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate ASS1 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate ASS1 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate ASS1 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate ASS1 expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate ASS1 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate ASS1 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate ASS1 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate ASS1 expression.

In some embodiments, TP-434 (Eravacycline) perturbs the Tetracycline-specific efflux signaling pathway to modulate ASS1 expression. In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate ASS1 expression. In some embodiments, SMI-4a perturbs the PIM signaling pathway to modulate ASS1 expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate ASS1 expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate ASS1 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate ASS1 expression. In some embodiments, INNO-206 (aldoxorubicin) perturbs the Cell Cycle/DNA Damage pathway to modulate ASS1 expression. In some embodiments, TNF-a perturbs the NF-kB, MAPK, or apoptosis pathway to modulate ASS1 expression. In some embodiments, T0901317 perturbs the LXR signaling pathway to modulate ASS1 expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the ASS1 gene. The ASS1 gene has a cytogenetic location of 9q34.11 and the genomic coordinate are on Chromosome 9 on the forward strand at position 130,444,929-130,501,274. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ASS1. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, p300, and SMC1. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, MYC, and YY1. The signaling proteins may include but are not limited to CREB1, NR1H4, HIF1a, ESRA, JUN, RXR, STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3, SMAD4, and TEAD1.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of ASL. Compounds that may be used to modulate the ASL expression include, but are not limited to, CP-673451, Echinomycin, Pacritinib (SB1518), Dasatinib, Oligomycin A, Merestinib, Amuvatinib, Crenolanib, Epinephrine, BAY 87-2243, Thalidomide, and derivatives or analogs thereof. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate ASL expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate ASL expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate ASL expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate ASL expression. In some embodiments, Oligomycin A perturbs the ATP channel signaling pathway to modulate ASL expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate ASL expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate ASL expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate ASL expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate ASL expression. In some embodiments, BAY 87-2243 perturbs the Hypoxia activated signaling pathway to modulate ASL expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate ASL expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the ASL gene. The ASL gene has a cytogenetic location of 7q11.21 and the genomic coordinate are on Chromosome 7 on the forward strand at position 66,075,798-66,093,558. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ASL. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, and p300. The transcription factors may include but are not limited to HNF3, HNF4A, ONECUT1, HNF1A, and MYC. The signaling proteins may include but are not limited to TCF7L2, CREB1, NR1H4, HIF1a, ESRA, FOS, JUN, RBPJK, RXR, STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3, SMAD4, STAT1, TEAD1, and TP53.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of NAGS. Compounds that may be used to modulate the NAGS expression include, but are not limited to, AZD2858, Enzastaurin, Bosutinib, Semaxanib, INNO-206 (aldoxorubicin), TP-434 (Eravacycline), Phenformin, Crizotinib, SMI-4a, Dasatinib, Calcitriol, Pifithrin-μ, PHA-665752, Darapladib, Thalidomide. CO-1686 (Rociletinib), OSU-03012, prednisone, t1SK2334470, Afatinib, Tivozanib, SKL2001, GDC-0879, EVP-6124 (hydrochloride) (encenicline), Amlodipine Besylate, T0901317, GO6983, Activin, WYE-125132 (WYE-132), SIS3, GDF2 (BMP9), Phorbol 12,13-dibutyrate, CD 2665, Erlotinib, Ceritinib, BMP2, TFP, HGF/SF, CI-4AS-1, and derivatives or analogs thereof. In some embodiments, AZD2858 perturbs the GSK-3 signaling pathway to modulate NAGS expression. In some embodiments, Enzastaurin perturbs the Epigenetics or TGF'-beta/Small signaling pathway to modulate NAGS expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate NAGS expression. In some embodiments, Semaxanib perturbs the VEGFR signaling pathway to modulate NAGS expression. In some embodiments, INNO-206 (aldoxorubicin) perturbs the Cell Cycle/DNA Damage pathway to modulate ^-NAGS expression. In some embodiments, TP-434 (Eravacycline) perturbs the Tetracycline-specific efflux signaling pathway to modulate NAGS expression. In some embodiments, Phenformin perturbs the AMPK signaling pathway to modulate NAGS expression. In some embodiments, Crizotinib perturbs the c-MET signaling pathway to modulate NAGS expression. In some embodiments, SMI-4a perturbs the PIM signaling pathway to modulate NAGS expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate NAGS expression. In some embodiments, Calcitriol perturbs the Vitamin D Receptor signaling pathway to modulate NAGS expression. In some embodiments, Pifithrin-μ perturbs the p53 signaling pathway to modulate NAGS expression. In some embodiments, PHA-665752 perturbs the c-MFT signaling pathway to modulate NAGS expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate NAGS expression. In some embodiments, CO-1686 (Rociletinib) perturbs the JAK/STAT or Tyrosine Kinase/RTK signaling pathway to modulate NAGS expression. In some embodiments, OSU-03012 perturbs the PDK-i signaling pathway to modulate NAGS expression. In sonic embodiments, prednisone perturbs the GR signaling pathway to modulate NAGS expression. In some embodiments, GSK2334470 perturbs the PDK-1 signaling pathway to modulate NAGS expression. In some embodiments, Afatinib perturbs the EGFR signaling pathway to modulate NAGS expression. In some embodiments, Tivozanib perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate NAGS expression. In some embodiments, SKL2001 perturbs the WNT signaling pathway to modulate NAGS expression. In some embodiments, GDC-0879 perturbs the MAPK signaling pathway to modulate NAGS expression. In some embodiments, EVP-6124 (hydrochloride) (encenicline) perturbs the Membrane Transporter/Ion Channel signaling pathway to modulate NAGS expression. In some embodiments, Amlodipine Besylate perturbs the Calcium channel signaling pathway to modulate NAGS expression. In some embodiments, T0901317 perturbs the LXR signaling pathway to modulate NAGS expression. In some embodiments, GO6983 perturbs the PKC signaling pathway to modulate NAGS expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, WYE-125132 (WYE-132) perturbs the mTOR signaling pathway to modulate NAGS expression. In some embodiments, SIS3 perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, Phorbol 12,13-dibutyrate perturbs the PKC signaling pathway to modulate NAGS expression. In some embodiments, CD 2665 perturbs the RAR signaling pathway to modulate NAGS expression. In some embodiments, Erlotinib perturbs the EGFR signaling pathway to modulate NAGS expression. In some embodiments, Ceritinib perturbs the ALK signaling pathway to modulate NAGS expression, .In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, TFP perturbs the Calmodulin signaling pathway to modulate NAGS expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate NAGS expression. In some embodiments, CI-4AS-1 perturbs the Androgen receptor signaling pathway to modulate NAGS expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the NAGS gene. The NAGS gene has a cytogenetic location of 17q21.31 and the genomic coordinate are on Chromosome 17 on the forward strand at position 44,004,546-44,009,063. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of NAGS. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, and p300. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, YY1, and HNF1A. The signaling proteins may include but are not limited to TCF7L2, HIF1a, AHR, ESRA, JUN, RXR, STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3, SMAD4, TEAD1, and TP53.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of ARG1. Compounds that may be used to modulate the ARG1 expression include, but are not limited to, 8788 (fostamatinib disodium hexahydrate), Dasatinib, CP-673451, Merestinib, Echinomycin, Amuvatinib, Epinephrine, Bosutinib, Wnt3a, Anti mullerian Hormone, Nodal, Activin, IGF-1, 17-AAG (Tanespimycin), TNF-a, Pifithrin-μ, PDGF, Pacritinib (SB1518), GDF2 (BMP9). Crenolanib. prednisone, HGF/SF, Momelotinib, EGF, Deoxycorticosterone, FGF, Thalidomide, Phenformin, Tivozanib, BAY 87-2243, GZD824 Dimesylate, GDF10 (BMP3b), PND-1186, FRAX597, BMP2, Oligomycin A. Rifampicin, MK-0752, and derivatives or analogs thereof. In some embodiments, R788 (fostamatinib disodium hexahydrate) perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate ARG1 expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate ARG1 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate ARG1 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate ARG1 expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate ARG1 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate ARG1 expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate ARG1 expression. In some embodiments, Anti mullerian Hormone perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Nodal perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, perturbs the IGF-1R/InsR signaling pathway to modulate ARG1 expression. In some embodiments, 17-AAG (Tanespimycin) perturbs the Cell Cycle/DNA. Damage Metabolic Enzyme/Protease signaling pathway to modulate ARG1 expression. In some embodiments, TNF-a perturbs the NF-kB, MAPK, or Apoptosis pathway to modulate ARG1 expression. In some embodiments, Pifithrin-μ perturbs the p53 signaling pathway to modulate ARG1 expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate ARG1 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, prednisone perturbs the GR signaling pathway to modulate ARG1 expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate ARG1 expression. In some embodiments, Momelotinib perturbs the JAK/STAT signaling pathway to modulate ARG1 expression. In some embodiments, EGF perturbs the EGFR signaling pathway to modulate ARG1 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate ARG1 expression. In some embodiments, FGF perturbs the FGFR signaling pathway to modulate ARG1 expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate ARG1 expression. In some embodiments, Phenformin perturbs the AMPK signaling pathway to modulate ARG1 expression. In some embodiments, Tivozanib perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate ARG1 expression. In some embodiments, BAY 87-2243 perturbs the Hypoxia activated signaling pathway to modulate ARG1 expression. In some embodiments, GZD824 Dimesylate perturbs the ABL signaling pathway to modulate ARG1 expression. In some embodiments, GDF10 (BMP3b) perturbs the TGF-B signaling pathway to modulate ARG1 expression. In sonic embodiments, PND-1186 perturbs the FAK signaling pathway to modulate ARG1 expression. In some embodiments, FRAXS97 perturbs the PAK signaling pathway to modulate ARG1 expression. In sonic embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Oligomycin A perturbs the ATP channel signaling pathway to modulate ARG1 expression. In some embodiments, Rifampicin perturbs the PXR signaling pathway to modulate ARG1 expression. In some embodiments, MK-0752 perturbs the NOTCH signaling pathway to modulate ARG1 expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the ARG1 gene. The ARG1 gene has a cytogenetic location of 6q23.2 and the genomic coordinate are on Chromosome 6 on the forward strand at position 131,573,144-131,584,332. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ARG1. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, and p300. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, YY1, HNF1A, and MYC. The signaling proteins may include but are not limited to HIF1a, ESRA, NR3C1, JUN, RXR, STAT3, NR1I1, SMAD2/3, STAT1, and TEAD1.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of SLC25A15, Compounds that may be used to modulate the SLC25A15 expression include, but are not limited to, Dasatinib, FRAX597, Merestinib, R788 (fostamatinib disodium hexahydrate), Bosutinib, bms-986094 (inx-189), Epinephrine, GDF2 (BMP9), Echinomycin, Corticosterone, IGF1, CP-673451, GZD824 Dimesylate, EW-7197, PDGF, Wnt3a, and derivatives or analogs thereof. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate SLC25A15 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate SLC25A15 expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate SLC25A15 expression. In some embodiments, R788 (fostamatinib disodium hexahydrate) perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate SLC25A15 expression. In some embodiments, Epinephrine perturbs the Src signaling pathway to modulate SLC25A15 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate SLC25A15 expression. In some embodiments, GI)F2 (BMP9) perturbs the TGF-B signaling pathway to modulate SLC25A15 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate SLC25A15 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate SLC25A15 expression. In some embodiments, IGF1 perturbs the IGF-1R/InsR signaling pathway to modulate SLC25A15 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate SLC25A15 expression. In some embodiments, GZD824 Dimesylate perturbs the ABL signaling pathway to modulate SLC25A15 expression. In some embodiments, EW-7197 perturbs the TGF-B signaling pathway to modulate SLC25A15 expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate SLC25A15 expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate SLC25A15 expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the SLC25A15 gene. SLC25A15 has a cytogenetic location of 13q14.11 and the genomic coordinate are on Chromosome 13 on the forward strand at position 40,789,412-40,810,111. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of SLC25A15. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, and BRD4. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, and YY1. The signaling proteins may include but are not limited to ESRA, Jun, RXR, NR1I1, NF-kB, NR3C1, SMAD2/3, and TP53.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of SLC25A13. Compounds that may be used to modulate the SLC25A13 expression include, but are not limited to, TFP, 17-AAG (Tanespimycin), and derivatives or analogs thereof. In some embodiments, TFP perturbs the Calmodulin signaling pathway to modulate SLC25A13 expression. In some embodiments, 17-AAG (Tanespimycin) perturbs the Cell Cycle/DNA Damage Metabolic Enzyme/Protease signaling pathway to modulate SLC25A13 expression.

In some embodiments, methods of the present invention involve altering the composition and/or the structure of the insulated neighborhood containing the SLC25A13 gene. SLC25A13 has a cytogenetic location of 7q21.3 and the genomic coordinate are on Chromosome 7 on the reverse strand at position 96,120,220-96,322,147. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of SLC25A13. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, p300, and SMC1. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ATF5, ONECUT2, YY1, HNF1A, and MYC. The signaling proteins may include but are not limited to TCF7L2, HIF1a, ESRA, NR3C1, JUN, RXR, STAT3, NR1I1, NF-kB, SMAD2/3, STAT1, TEAD1, and TP53.

In some embodiments, compositions and methods of the present invention may be used to treat a urea cycle disorder by modulating the expression of multiple urea cycle-related genes. As a non-limiting example, methods of the present invention may be used to modulate the expression of any one of the following groups of genes: NAGS, CPS1, ASS1, ASL, OTC, ARG1, and SLC25A15; CPS1, ASS1, ASL, OTC, ARG1, and SLC25A15; ASS1, CPS1, NAGS, ARG1, and SLC25A15; CPS1, ASS1, ASL, ARG1, and SLC25A15; CPS1, ASS1, ASL, OTC, and ARG1; CPS1, ASS1, OTC, ARG1, and SLC25A15; NAGS, CPS1, ALS, OTC, and ARG1; ASS1, ASL, OTC, and ARG1; ASS1, CPS1, NAGS, and ARG1; CPS1, ASS1, ARG1, and SLC25A15; CPS1, ASS1, OTC, and ARG1; CPS1, OTC, ARG1, and SLC25A15; NAGS, CPS1, OTC, and ARG1; CPS1, ARG1, and SLC25A13; CPS1, ARG1, and SLC25A15; CPS1, ASL, and ARG1; CPS1, NAGS, and ARG1; CPS1, OTC, and ARG1; NAGS, ASS1, and OTC; OTC, NAGS, and ARG1. Compounds that may be used to modulate multiple urea cycle-related genes include, but are not limited to, Dasatinib, Echinomycin, CP-673451, GDF2 (BMP9), Bosutinib, Epinephrine, Pacritinib (SB1518), Amuvatinib, FRAX597, Thalidomide, Crenolanib, BMP2, Deoxycorticosterone, TNF-α, Wnt3a, PDGF, IGF-1, Activin, HGF/SF, 17-AAG (Tanespimycin), R788 (fostamatinib disodium hexahydrate), GZD824 Dimesylate, BAY 87-2243, prednisone, Nodal, Momelotinib, FGF, EGF, Anti mullerian hormone, INNO-206 (aldoxorubicin), and Pifithrin-μ.

In some embodiments, targeting multiple urea cycle-related genes may be accomplished by utilizing a combination of compounds that each specifically modulates a urea cycle-related gene. In some embodiments, targeting multiple urea cycle-related genes may be accomplished by utilizing a single compound that is capable of modulating multiple urea cycle-related genes.

In some embodiment, compounds of the present invention may be used in combination with other drugs, such as Sodium phenylbutyrate (BUPHENYL®), glycerol phenylbutyrate (RAVICTI®), and sodium benzoate, to treat a urea cycle disorder.

IV. FORMULATIONS AND DELIVERY
Pharmaceutical Compositions

According to the present invention the compositions may be prepared as pharmaceutical compositions. It will be understood that such compositions necessarily comprise one or more active ingredients and, most often, a pharmaceutically acceptable excipient.

Relative amounts of the active ingredient, a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

In some embodiments, the pharmaceutical compositions described herein may comprise at least one payload. As a non-limiting example, the pharmaceutical compositions may contain 1, 2, 3, 4 or 5 payloads.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, rats, birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

In some embodiments, compositions are administered to humans, human patients or subjects.

Formulations

Formulations of the present invention can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.

Formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

Excipients and Diluents

In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.

Inactive Ingredients

In some embodiments, the pharmaceutical compositions formulations may comprise at least one inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations. In some embodiments, all, none or some of the inactive ingredients which may be used in the formulations of the present invention may be approved by the US Food and Drug Administration (FDA).

In one embodiment, the pharmaceutical compositions comprise at least one inactive ingredient such as, but not limited to, 1,2,6-Hexanetriol; 1,2-Dimyristoyl-Sn-Glycero-3-(Phospho-S-(1-Glycerol)); 1,2-Dimyristoyl-Sn-Glycero-3-Phosphocholine; 1,2-Dioleoyl-Sn-Glycero-3-Phosphocholine; 1,2-Dipalmitoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); 1,2-Distearoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); 1,2-Distearoyl-Sn-Glycero-3-Phosphocholine; 1-O-Tolylbiguanide; 2-Ethyl-1,6-Hexanediol; Acetic Acid; Acetic Acid, Glacial; Acetic Anhydride; Acetone; Acetone Sodium Bisulfite; Acetylated Lanolin Alcohols; Acetylated Monoglycerides; Acetylcysteine; Acetyltryptophan, DL-; Acrylates Copolymer; Acrylic Acid-Isooctyl Acrylate Copolymer; Acrylic Adhesive 788; Activated Charcoal; Adcote 72A103; Adhesive Tape; Adipic Acid; Aerotex Resin 3730; Alanine; Albumin Aggregated; Albumin Colloidal; Albumin Human; Alcohol; Alcohol, Dehydrated; Alcohol, Denatured; Alcohol, Diluted; Alfadex; Alginic Acid; Alkyl Ammonium Sulfonic Acid Betaine; Alkyl Aryl Sodium Sulfonate; Allantoin; Allyl .Alpha.-Ionone; Almond Oil; Alpha-Terpineol; Alpha-Tocopherol; Alpha-Tocopherol Acetate, Dl-; Alpha-Tocopherol, Dl-; Aluminum Acetate; Aluminum Chlorhydroxy Allantoinate; Aluminum Hydroxide; Aluminum Hydroxide-Sucrose, Hydrated; Aluminum Hydroxide Gel; Aluminum Hydroxide Gel F 500; Aluminum Hydroxide Gel F 5000; Aluminum Monostearate; Aluminum Oxide; Aluminum Polyester; Aluminum Silicate; Aluminum Starch Octenylsuccinate; Aluminum Stearate; Aluminum Subacetate; Aluminum Sulfate Anhydrous; Amerchol C; Amerchol-Cab; Aminomethylpropanol; Ammonia; Ammonia Solution; Ammonia Solution, Strong; Ammonium Acetate; Ammonium Hydroxide; Ammonium Lauryl Sulfate; Ammonium Nonoxynol-4 Sulfate; Ammonium Salt Of C-12-C-15 Linear Primary Alcohol Ethoxylate; Ammonium Sulfate; Ammonyx; Amphoteric-2; Amphoteric-9; Anethole; Anhydrous Citric Acid; Anhydrous Dextrose; Anhydrous Lactose; Anhydrous Trisodium Citrate; Aniseed Oil; Anoxid Sbn; Antifoam; Antipyrine; Apaflurane; Apricot Kernel Oil Peg-6 Esters; Aquaphor; Arginine; Arlacel; Ascorbic Acid; Ascorbyl Palmitate; Aspartic Acid; Balsam Peru; Barium Sulfate; Beeswax; Beeswax, Synthetic; Beheneth-10; Bentonite; Benzalkonium Chloride; Benzenesulfonic Acid; Benzethonium Chloride; Benzododecinium Bromide; Benzoic Acid; Benzyl Alcohol; Benzyl Benzoate; Benzyl Chloride; Betadex; Bibapcitide; Bismuth Subgallate; Boric Acid; Brocrinat; Butane; Butyl Alcohol; Butyl Ester Of Vinyl Methyl Ether/Maleic Anhydride Copolymer (125000 Mw); Butyl Stearate; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Butylene Glycol; Butylparaben; Butyric Acid; C20-40 Pareth-24; Caffeine; Calcium; Calcium Carbonate; Calcium Chloride; Calcium Gluceptate; Calcium Hydroxide; Calcium Lactate; Calcobutrol; Caldiamide Sodium; Caloxetate Trisodium; Calteridol Calcium; Canada Balsam; Caprylic/Capric Triglyceride; Caprylic/Capric/Stearic Triglyceride; Captan; Captisol; Caramel; Carbomer 1342; Carbomer 1382; Carbomer 934; Carbomer 934p; Carbomer 940; Carbomer 941; Carbomer 980; Carbomer 981; Carbomer Homopolymer Type B (Allyl Pentaerythritol Crosslinked); Carbomer Homopolymer Type C (Allyl Pentaerythritol Crosslinked); Carbon Dioxide; Carboxy Vinyl Copolymer; Carboxymethylcellulose; Carboxymethylcellulose Sodium; Carboxypolymethylene; Carrageenan; Carrageenan Salt; Castor Oil; Cedar Leaf Oil; Cellulose; Cellulose, Microcrystalline; Cerasynt-Se; Ceresin; Ceteareth-12; Ceteareth-15; Ceteareth-30; Cetearyl Alcohol/Ceteareth-20; Cetearyl Ethylhexanoate; Ceteth-10; Ceteth-2; Ceteth-20; Ceteth-23; Cetostearyl Alcohol; Cetrimonium Chloride; Cetyl Alcohol; Cetyl Esters Wax; Cetyl Palmitate; Cetylpyridinium Chloride; Chlorobutanol; Chlorobutanol Hemihydrate; Chlorobutanol, Anhydrous; Chlorocresol; Chloroxylenol; Cholesterol; Choleth; Choleth-24; Citrate; Citric Acid; Citric Acid Monohydrate; Citric Acid, Hydrous; Cocamide Ether Sulfate; Cocamine Oxide; Coco Betaine; Coco Diethanolamide; Coco Monoethanolamide; Cocoa Butter; Coco-Glycerides; Coconut Oil; Coconut Oil, Hydrogenated; Coconut Oil/Palm Kernel Oil Glycerides, Hydrogenated; Cocoyl Caprylocaprate; Cola Nitida Seed Extract; Collagen; Coloring Suspension; Corn Oil; Cottonseed Oil; Cream Base; Creatine; Creatinine; Cresol; Croscarmellose Sodium; Crospovidone; Cupric Sulfate; Cupric Sulfate Anhydrous; Cyclomethicone; Cyclomethicone/Dimethicone Copolyol; Cysteine; Cysteine Hydrochloride; Cysteine Hydrochloride Anhydrous; Cysteine, Dl-; D&C Red No. 28; D&C Red No. 33; D&C Red No. 36; D&C Red No. 39; D&C Yellow No. 10; Dalfampridine; Daubert 1-5 Pestr (Matte) 164z; Decyl Methyl Sulfoxide; Dehydag Wax Sx; Dehydroacetic Acid; Dehymuls E; Denatonium Benzoate; Deoxycholic Acid; Dextran; Dextran 40; Dextrin; Dextrose; Dextrose Monohydrate; Dextrose Solution; Diatrizoic Acid; Diazolidinyl Urea; Dichlorobenzyl Alcohol; Dichlorodifluoromethane; Dichlorotetrafluoroethane; Diethanolamine; Diethyl Pyrocarbonate; Diethyl Sebacate; Diethylene Glycol Monoethyl Ether; Diethylhexyl Phthalate; Dihydroxyaluminum Aminoacetate; Diisopropanolamine; Diisopropyl Adipate; Diisopropyl Dilinoleate; Dimethicone 350; Dimethicone Copolyol; Dimethicone Mdx4-4210; Dimethicone Medical Fluid 360; Dimethyl Isosorbide; Dimethyl Sulfoxide; Dimethylaminoethyl Methacrylate-Butyl Methacrylate-Methyl Methacrylate Copolymer; Dimethyldioctadecylammonium Bentonite; Dimethylsiloxane/Methylvinylsiloxane Copolymer; Dinoseb Ammonium Salt; Dipalmitoylphosphatidylglycerol, Dl-; Dipropylene Glycol; Disodium Cocoamphodiacetate; Disodium Laureth Sulfosuccinate; Disodium Lauryl Sulfosuccinate; Disodium Sulfosalicylate; Disofenin; Divinylbenzene Styrene Copolymer; Dmdm Hydantoin; Docosanol; Docusate Sodium; Duro-Tak 280-2516; Duro-Tak 387-2516; Duro-Tak 80-1196; Duro-Tak 87-2070; Duro-Tak 87-2194; Duro-Tak 87-2287; Duro-Tak 87-2296; Duro-Tak 87-2888; Duro-Tak 87-2979; Edetate Calcium Disodium; Edetate Disodium; Edetate Disodium Anhydrous; Edetate Sodium; Edetic Acid; Egg Phospholipids; Entsufon; Entsufon Sodium; Epilactose; Epitetracycline Hydrochloride; Essence Bouquet 9200; Ethanolamine Hydrochloride; Ethyl Acetate; Ethyl Oleate; Ethylcelluloses; Ethylene Glycol; Ethylene Vinyl Acetate Copolymer; Ethylenediamine; Ethylenediamine Dihydrochloride; Ethylene-Propylene Copolymer; Ethylene-Vinyl Acetate Copolymer (28% Vinyl Acetate); Ethylene-Vinyl Acetate Copolymer (9% Vinylacetate); Ethylhexyl Hydroxystearate; Ethylparaben; Eucalyptol; Exametazime; Fat, Edible; Fat, Hard; Fatty Acid Esters; Fatty Acid Pentaerythriol Ester; Fatty Acids; Fatty Alcohol Citrate; Fatty Alcohols; Fd&C Blue No. 1; Fd&C Green No. 3; Fd&C Red No. 4; Fd&C Red No. 40; Fd&C Yellow No. 10 (Delisted); Fd&C Yellow No. 5; Fd&C Yellow No. 6; Ferric Chloride; Ferric Oxide; Flavor 89-186; Flavor 89-259; Flavor Df-119; Flavor Df-1530; Flavor Enhancer; Flavor FIG. 827118; Flavor Raspberry Pfc-8407; Flavor Rhodia Pharmaceutical No. Rf 451; Fluorochlorohydrocarbons; Formaldehyde; Formaldehyde Solution; Fractionated Coconut Oil; Fragrance 3949-5; Fragrance 520a; Fragrance 6.007; Fragrance 91-122; Fragrance 9128-Y; Fragrance 93498 g; Fragrance Balsam Pine No. 5124; Fragrance Bouquet 10328; Fragrance Chemoderm 6401-B; Fragrance Chemoderm 6411; Fragrance Cream No. 73457; Fragrance Cs-28197; Fragrance Felton 066m; Fragrance Firmenich 47373; Fragrance Givaudan Ess 9090/1c; Fragrance H-6540; Fragrance Herbal 10396; Fragrance Nj-1085; Fragrance P O Fl-147; Fragrance Pa 52805; Fragrance Pera Derm D; Fragrance Rbd-9819; Fragrance Shaw Mudge U-7776; Fragrance Tf 044078; Fragrance Ungerer Honeysuckle K 2771; Fragrance Ungerer N5195; Fructose; Gadolinium Oxide; Galactose; Gamma Cyclodextrin; Gelatin; Gelatin, Crosslinked; Gelfoam Sponge; Gellan Gum (Low Acyl); Gelva 737; Gentisic Acid; Gentisic Acid Ethanolamide; Gluceptate Sodium; Gluceptate Sodium Dihydrate; Gluconolactone; Glucuronic Acid; Glutamic Acid, Dl-; Glutathione; Glycerin; Glycerol Ester Of Hydrogenated Rosin; Glyceryl Citrate; Glyceryl Isostearate; Glyceryl Laurate; Glyceryl Monostearate; Glyceryl Oleate; Glyceryl Oleate/Propylene Glycol; Glyceryl Palmitate; Glyceryl Ricinoleate; Glyceryl Stearate; Glyceryl Stearate-Laureth-23; Glyceryl Stearate/Peg Stearate; Glyceryl Stearate/Peg-100 Stearate; Glyceryl Stearate/Peg-40 Stearate; Glyceryl Stearate-Stearamidoethyl Diethylamine; Glyceryl Trioleate; Glycine; Glycine Hydrochloride; Glycol Distearate; Glycol Stearate; Guanidine Hydrochloride; Guar Gum; Hair Conditioner (18n195-1 m); Heptane; Hetastarch; Hexylene Glycol; High Density Polyethylene; Histidine; Human Albumin Microspheres; Hyaluronate Sodium; Hydrocarbon; Hydrocarbon Gel, Plasticized; Hydrochloric Acid; Hydrochloric Acid, Diluted; Hydrocortisone; Hydrogel Polymer; Hydrogen Peroxide; Hydrogenated Castor Oil; Hydrogenated Palm Oil; Hydrogenated Palm/Palm Kernel Oil Peg-6 Esters; Hydrogenated Polybutene 635-690; Hydroxide Ion; Hydroxyethyl Cellulose; Hydroxyethylpiperazine Ethane Sulfonic Acid; Hydroxymethyl Cellulose; Hydroxyoctacosanyl Hydroxystearate; Hydroxypropyl Cellulose; Hydroxypropyl Methylcellulose 2906; Hydroxypropyl-Beta-cyclodextrin; Hypromellose 2208 (15000 Mpa.S); Hypromellose 2910 (15000 Mpa.S); Hypromelloses; Imidurea; Iodine; Iodoxamic Acid; Iofetamine Hydrochloride; Irish Moss Extract; Isobutane; Isoceteth-20; Isoleucine; Isooctyl Acrylate; Isopropyl Alcohol; Isopropyl Isostearate; Isopropyl Myristate; Isopropyl Myristate-Myristyl Alcohol; Isopropyl Palmitate; Isopropyl Stearate; Isostearic Acid; Isostearyl Alcohol; Isotonic Sodium Chloride Solution; Jelene; Kaolin; Kathon Cg; Kathon Cg II; Lactate; Lactic Acid; Lactic Acid, Dl-; Lactic Acid, L-; Lactobionic Acid; Lactose; Lactose Monohydrate; Lactose, Hydrous; Laneth; Lanolin; Lanolin Alcohol-Mineral Oil; Lanolin Alcohols; Lanolin Anhydrous; Lanolin Cholesterols; Lanolin Nonionic Derivatives; Lanolin, Ethoxylated; Lanolin, Hydrogenated; Lauralkonium Chloride; Lauramine Oxide; Laurdimonium Hydrolyzed Animal Collagen; Laureth Sulfate; Laureth-2; Laureth-23; Laureth-4; Lauric Diethanolamide; Lauric Myristic Diethanolamide; Lauroyl Sarcosine; Lauryl Lactate; Lauryl Sulfate; Lavandula Angustifolia Flowering Top; Lecithin; Lecithin Unbleached; Lecithin, Egg; Lecithin, Hydrogenated; Lecithin, Hydrogenated Soy; Lecithin, Soybean; Lemon Oil; Leucine; Levulinic Acid; Lidofenin; Light Mineral Oil; Light Mineral Oil (85 Ssu); Limonene, (+/−)-; Lipocol Sc-15; Lysine; Lysine Acetate; Lysine Monohydrate; Magnesium Aluminum Silicate; Magnesium Aluminum Silicate Hydrate; Magnesium Chloride; Magnesium Nitrate; Magnesium Stearate; Maleic Acid; Mannitol; Maprofix; Mebrofenin; Medical Adhesive Modified S-15; Medical Antiform A-F Emulsion; Medronate Disodium; Medronic Acid; Meglumine; Menthol; Metacresol; Metaphosphoric Acid; Methanesulfonic Acid; Methionine; Methyl Alcohol; Methyl Gluceth-10; Methyl Gluceth-20; Methyl Gluceth-20 Sesquistearate; Methyl Glucose Sesquistearate; Methyl Laurate; Methyl Pyrrolidone; Methyl Salicylate; Methyl Stearate; Methylboronic Acid; Methylcellulose (4000 Mpa.S); Methylcelluloses; Methylchloroisothiazolinone; Methylene Blue; Methylisothiazolinone; Methylparaben; Microcrystalline Wax; Mineral Oil; Mono And Diglyceride; Monostearyl Citrate; Monothioglycerol; Multisterol Extract; Myristyl Alcohol; Myristyl Lactate; Myristyl-.Gamma.-Picolinium Chloride; N-(Carbamoyl-Methoxy Peg-40)-1,2-Distearoyl-Cephalin Sodium; N,N-Dimethylacetamide; Niacinamide; Nioxime; Nitric Acid; Nitrogen; Nonoxynol Iodine; Nonoxynol-15; Nonoxynol-9; Norflurane; Oatmeal; Octadecene-1/Maleic Acid Copolymer; Octanoic Acid; Octisalate; Octoxynol-1; Octoxynol-40; Octoxynol-9; Octyldodecanol; Octylphenol Polymethylene; Oleic Acid; Oleth-10/Oleth-5; Oleth-2; Oleth-20; Oleyl Alcohol; Oleyl Oleate; Olive Oil; Oxidronate Disodium; Oxyquinoline; Palm Kernel Oil; Palmitamine Oxide; Parabens; Paraffin; Paraffin, White Soft; Parfum Creme 45/3; Peanut Oil; Peanut Oil, Refined; Pectin; Peg 6-32 Stearate/Glycol Stearate; Peg Vegetable Oil; Peg-100 Stearate; Peg-12 Glyceryl Laurate; Peg-120 Glyceryl Stearate; Peg-120 Methyl Glucose Dioleate; Peg-15 Cocamine; Peg-150 Distearate; Peg-2 Stearate; Peg-20 Sorbitan Isostearate; Peg-22 Methyl Ether/Dodecyl Glycol Copolymer; Peg-25 Propylene Glycol Stearate; Peg-4 Dilaurate; Peg-4 Laurate; Peg-40 Castor Oil; Peg-40 Sorbitan Diisostearate; Peg-45/Dodecyl Glycol Copolymer; Peg-5 Oleate; Peg-50 Stearate; Peg-54 Hydrogenated Castor Oil; Peg-6 Isostearate; Peg-60 Castor Oil; Peg-60 Hydrogenated Castor Oil; Peg-7 Methyl Ether; Peg-75 Lanolin; Peg-8 Laurate; Peg-8 Stearate; Pegoxol 7 Stearate; Pentadecalactone; Pentaerythritol Cocoate; Pentasodium Pentetate; Pentetate Calcium Trisodium; Pentetic Acid; Peppermint Oil; Perflutren; Perfume 25677; Perfume Bouquet; Perfume E-1991; Perfume Gd 5604; Perfume Tana 90/42 Scba; Perfume W-1952-1; Petrolatum; Petrolatum, White; Petroleum Distillates; Phenol; Phenol, Liquefied; Phenonip; Phenoxyethanol; Phenylalanine; Phenylethyl Alcohol; Phenylmercuric Acetate; Phenylmercuric Nitrate; Phosphatidyl Glycerol, Egg; Phospholipid; Phospholipid, Egg; Phospholipon 90 g; Phosphoric Acid; Pine Needle Oil (Pinus Sylvestris); Piperazine Hexahydrate; Plastibase-50w; Polacrilin; Polidronium Chloride; Poloxamer 124; Poloxamer 181; Poloxamer 182; Poloxamer 188; Poloxamer 237; Poloxamer 407; Poly(Bis(P-Carboxyphenoxy)Propane Anhydride): Sebacic Acid; Poly(Dimethylsiloxane/Methylvinylsiloxane/Methylhydrogensiloxane) Dimethylvinyl Or Dimethylhydroxy Or Trimethyl Endblocked; Poly(Dl-Lactic-Co-Glycolic Acid), (50:50; Poly(Dl-Lactic-Co-Glycolic Acid), Ethyl Ester Terminated, (50:50; Polyacrylic Acid (250000 Mw); Polybutene (1400 Mw); Polycarbophil; Polyester; Polyester Polyamine Copolymer; Polyester Rayon; Polyethylene Glycol 1000; Polyethylene Glycol 1450; Polyethylene Glycol 1500; Polyethylene Glycol 1540; Polyethylene Glycol 200; Polyethylene Glycol 300; Polyethylene Glycol 300-1600; Polyethylene Glycol 3350; Polyethylene Glycol 400; Polyethylene Glycol 4000; Polyethylene Glycol 540; Polyethylene Glycol 600; Polyethylene Glycol 6000; Polyethylene Glycol 8000; Polyethylene Glycol 900; Polyethylene High Density Containing Ferric Oxide Black (<1%); Polyethylene Low Density Containing Barium Sulfate (20-24%); Polyethylene T; Polyethylene Terephthalates; Polyglactin; Polyglyceryl-3 Oleate; Polyglyceryl-4 Oleate; Polyhydroxyethyl Methacrylate; Polyisobutylene; Polyisobutylene (1100000 Mw); Polyisobutylene (35000 Mw); Polyisobutylene 178-236; Polyisobutylene 241-294; Polyisobutylene 35-39; Polyisobutylene Low Molecular Weight; Polyisobutylene Medium Molecular Weight; Polyisobutylene/Polybutene Adhesive; Polylactide; Polyols; Polyoxyethylene-Polyoxypropylene 1800; Polyoxyethylene Alcohols; Polyoxyethylene Fatty Acid Esters; Polyoxyethylene Propylene; Polyoxyl 20 Cetostearyl Ether; Polyoxyl 35 Castor Oil; Polyoxyl 40 Hydrogenated Castor Oil; Polyoxyl 40 Stearate; Polyoxyl 400 Stearate; Polyoxyl 6 And Polyoxyl 32 Palmitostearate; Polyoxyl Distearate; Polyoxyl Glyceryl Stearate; Polyoxyl Lanolin; Polyoxyl Palmitate; Polyoxyl Stearate; Polypropylene; Polypropylene Glycol; Polyquaternium-10; Polyquaternium-7 (70/30 Acrylamide/Dadmac; Polysiloxane; Polysorbate 20; Polysorbate 40; Polysorbate 60; Polysorbate 65; Polysorbate 80; Polyurethane; Polyvinyl Acetate; Polyvinyl Alcohol; Polyvinyl Chloride; Polyvinyl Chloride-Polyvinyl Acetate Copolymer; Polyvinylpyridine; Poppy Seed Oil; Potash; Potassium Acetate; Potassium Alum; Potassium Bicarbonate; Potassium Bisulfite; Potassium Chloride; Potassium Citrate; Potassium Hydroxide; Potassium Metabisulfite; Potassium Phosphate, Dibasic; Potassium Phosphate, Monobasic; Potassium Soap; Potassium Sorbate; Povidone Acrylate Copolymer; Povidone Hydrogel; Povidone K17; Povidone K25; Povidone K29/32; Povidone K30; Povidone K90; Povidone K90f; Povidone/Eicosene Copolymer; Povidones; Ppg-12/Smdi Copolymer; Ppg-15 Stearyl Ether; Ppg-20 Methyl Glucose Ether Distearate; Ppg-26 Oleate; Product Wat; Proline; Promulgen D; Promulgen G; Propane; Propellant A-46; Propyl Gallate; Propylene Carbonate; Propylene Glycol; Propylene Glycol Diacetate; Propylene Glycol Dicaprylate; Propylene Glycol Monolaurate; Propylene Glycol Monopalmitostearate; Propylene Glycol Palmitostearate; Propylene Glycol Ricinoleate; Propylene Glycol/Diazolidinyl Urea/Methylparaben/Propylparben; Propylparaben; Protamine Sulfate; Protein Hydrolysate; Pvm/Ma Copolymer; Quaternium-15; Quaternium-15 Cis-Form; Quaternium-52; Ra-2397; Ra-3011; Saccharin; Saccharin Sodium; Saccharin Sodium Anhydrous; Safflower Oil; Sd Alcohol 3a; Sd Alcohol 40; Sd Alcohol 40-2; Sd Alcohol 40b; Sepineo P 600; Serine; Sesame Oil; Shea Butter; Silastic Brand Medical Grade Tubing; Silastic Medical Adhesive, Silicone Type A; Silica, Dental; Silicon; Silicon Dioxide; Silicon Dioxide, Colloidal; Silicone; Silicone Adhesive 4102; Silicone Adhesive 4502; Silicone Adhesive Bio-Psa Q7-4201; Silicone Adhesive Bio-Psa Q7-4301; Silicone Emulsion; Silicone/Polyester Film Strip; Simethicone; Simethicone Emulsion; Sipon Ls 20np; Soda Ash; Sodium Acetate; Sodium Acetate Anhydrous; Sodium Alkyl Sulfate; Sodium Ascorbate; Sodium Benzoate; Sodium Bicarbonate; Sodium Bisulfate; Sodium Bisulfite; Sodium Borate; Sodium Borate Decahydrate; Sodium Carbonate; Sodium Carbonate Decahydrate; Sodium Carbonate Monohydrate; Sodium Cetostearyl Sulfate; Sodium Chlorate; Sodium Chloride; Sodium Chloride Injection; Sodium Chloride Injection, Bacteriostatic; Sodium Cholesteryl Sulfate; Sodium Citrate; Sodium Cocoyl Sarcosinate; Sodium Desoxycholate; Sodium Dithionite; Sodium Dodecylbenzenesulfonate; Sodium Formaldehyde Sulfoxylate; Sodium Gluconate; Sodium Hydroxide; Sodium Hypochlorite; Sodium Iodide; Sodium Lactate; Sodium Lactate, L-; Sodium Laureth-2 Sulfate; Sodium Laureth-3 Sulfate; Sodium Laureth-5 Sulfate; Sodium Lauroyl Sarcosinate; Sodium Lauryl Sulfate; Sodium Lauryl Sulfoacetate; Sodium Metabisulfite; Sodium Nitrate; Sodium Phosphate; Sodium Phosphate Dihydrate; Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate, Dibasic, Dihydrate; Sodium Phosphate, Dibasic, Dodecahydrate; Sodium Phosphate, Dibasic, Heptahydrate; Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous; Sodium Phosphate, Monobasic, Dihydrate; Sodium Phosphate, Monobasic, Monohydrate; Sodium Polyacrylate (2500000 Mw); Sodium Pyrophosphate; Sodium Pyrrolidone Carboxylate; Sodium Starch Glycolate; Sodium Succinate Hexahydrate; Sodium Sulfate; Sodium Sulfate Anhydrous; Sodium Sulfate Decahydrate; Sodium Sulfite; Sodium Sulfosuccinated Undecyclenic Monoalkylolamide; Sodium Tartrate; Sodium Thioglycolate; Sodium Thiomalate; Sodium Thiosulfate; Sodium Thiosulfate Anhydrous; Sodium Trimetaphosphate; Sodium Xylenesulfonate; Somay 44; Sorbic Acid; Sorbitan; Sorbitan Isostearate; Sorbitan Monolaurate; Sorbitan Monooleate; Sorbitan Monopalmitate; Sorbitan Monostearate; Sorbitan Sesquioleate; Sorbitan Trioleate; Sorbitan Tristearate; Sorbitol; Sorbitol Solution; Soybean Flour; Soybean Oil; Spearmint Oil; Spermaceti; Squalane; Stabilized Oxychloro Complex; Stannous 2-Ethylhexanoate; Stannous Chloride; Stannous Chloride Anhydrous; Stannous Fluoride; Stannous Tartrate; Starch; Starch 1500, Pregelatinized; Starch, Corn; Stearalkonium Chloride; Stearalkonium Hectorite/Propylene Carbonate; Stearamidoethyl Diethylamine; Steareth-10; Steareth-100; Steareth-2; Steareth-20; Steareth-21; Steareth-40; Stearic Acid; Stearic Diethanolamide; Stearoxytrimethylsilane; Steartrimonium Hydrolyzed Animal Collagen; Stearyl Alcohol; Sterile Water For Inhalation; Styrene/Isoprene/Styrene Block Copolymer; Succimer; Succinic Acid; Sucralose; Sucrose; Sucrose Distearate; Sucrose Polyesters; Sulfacetamide Sodium; Sulfobutylether .Beta.-Cyclodextrin; Sulfur Dioxide; Sulfuric Acid; Sulfurous Acid; Surfactol Qs; Tagatose, D-; Talc; Tall Oil; Tallow Glycerides; Tartaric Acid; Tartaric Acid, Dl-; Tenox; Tenox-2; Tert-Butyl Alcohol; Tert-Butyl Hydroperoxide; Tert-Butylhydroquinone; Tetrakis(2-Methoxyisobutylisocyanide)Copper(I) Tetrafluoroborate; Tetrapropyl Orthosilicate; Tetrofosmin; Theophylline; Thimerosal; Threonine; Thymol; Tin; Titanium Dioxide; Tocopherol; Tocophersolan; Total parenteral nutrition, lipid emulsion; Triacetin; Tricaprylin; Trichloromonofluoromethane; Trideceth-10; Triethanolamine Lauryl Sulfate; Trifluoroacetic Acid; Triglycerides, Medium Chain; Trihydroxystearin; Trilaneth-4 Phosphate; Trilaureth-4 Phosphate; Trisodium Citrate Dihydrate; Trisodium Hedta; Triton 720; Triton X-200; Trolamine; Tromantadine; Tromethamine (TRIS); Tryptophan; Tyloxapol; Tyrosine; Undecylenic Acid; Union 76 Amsco-Res 6038; Urea; Valine; Vegetable Oil; Vegetable Oil Glyceride, Hydrogenated; Vegetable Oil, Hydrogenated; Versetamide; Viscarin; Viscose/Cotton; Vitamin E; Wax, Emulsifying; Wecobee Fs; White Ceresin Wax; White Wax; Xanthan Gum; Zinc; Zinc Acetate; Zinc Carbonate; Zinc Chloride; and Zinc Oxide.

Pharmaceutical composition formulations disclosed herein may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mn2+, Mg+ and combinations thereof. As a non-limiting example, formulations may include polymers and complexes with a metal cation (See e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporated by reference in its entirety).

Formulations of the invention may also include one or more pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

Solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”

V. ADMINISTRATION AND DOSING
Administration

The terms “administering” and “introducing” are used interchangeably herein and refer to the delivery of the pharmaceutical composition into a cell or a subject. In the case of delivery to a subject, the pharmaceutical composition is delivered by a method or route that results in at least partial localization of the introduced cells at a desired site, such as hepatocytes, such that a desired effect(s) is produced.

In one aspect of the method, the pharmaceutical composition may be administered via a route such as, but not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis and spinal.

Modes of administration include injection, infusion, instillation, and/or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some examples, the route is intravenous. For the delivery of cells, administration by injection or infusion can be made.

In some embodiments, compounds of the present invention can be administered to cells systemically. The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” refer to the administration other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes. In other embodiments, compounds of the present invention can be administered to cells ex vivo, i.e., the compounds can be administered to cells that have been removed from an organ or tissue and held outside the subject's body e.g., in primary culture.

Dosing

The term “effective amount” refers to the amount of the active ingredient needed to prevent or alleviate at least one or more signs or symptoms of a specific disease and/or condition, and relates to a sufficient amount of a composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of active ingredient or a composition comprising the active ingredient that is sufficient to promote a particular effect when administered to a typical subject. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation.

The pharmaceutical, diagnostic, or prophylactic compositions of the present invention may be administered to a subject using any amount and any route of administration effective for preventing, treating, managing, or diagnosing diseases, disorders and/or conditions. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. Compositions in accordance with the invention are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate diagnostic dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, and route of administration; the duration of the treatment; drugs used in combination or coincidental with the active ingredient; and like factors well known in the medical arts.

In certain embodiments, pharmaceutical compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 0.05 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, or prophylactic, effect.

The desired dosage of the composition present invention may be delivered only once, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of “single unit dose” or total daily dose into two or more doses, e.g., two or more administrations of the “single unit dose”. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.

VI. DEFINITIONS

The term “analog”, as used herein, refers to a compound that is structurally related to the reference compound and shares a common functional activity with the reference compound.

The term “biologic”, as used herein, refers to a medical product made from a variety of natural sources such as micro-organism, plant, animal, or human cells.

The term “boundary”, as used herein, refers to a point, limit, or range indicating where a feature, element, or property ends or begins.

The term “compound”, as used herein, refers to a single agent or a pharmaceutically acceptable salt thereof, or a bioactive agent or drug.

The term “derivative”, as used herein, refers to a compound that differs in structure from the reference compound, but retains the essential properties of the reference molecule.

The term “downstream neighborhood gene”, as used herein, refers to a gene downstream of primary neighborhood gene that may be located within the same insulated neighborhood as the primary neighborhood gene.

The term “drug”, as used herein, refers to a substance other than food intended for use in the diagnosis, cure, alleviation, treatment, or prevention of disease and intended to affect the structure or any function of the body.

The term “enhancer”, as used herein, refers to regulatory DNA sequences that, when bound by transcription factors, enhance the transcription of an associated gene.

The term “gene”, as used herein, refers to a unit or segment of the genomic architecture of an organism, e.g., a chromosome. Genes may be coding or non-coding. Genes may be encoded as contiguous or non-contiguous polynucleotides. Genes may be DNA or RNA.

The term “genomic signaling center”, i.e., a “signaling center”, as used herein, refers to regions within insulated neighborhoods that include regions capable of binding context-specific combinatorial assemblies of signaling molecules/signaling proteins that participate in the regulation of the genes within that insulated neighborhood or among more than one insulated neighborhood.

The term “genomic system architecture”, as used herein, refers to the organization of an individual's genome and includes chromosomes, topologically associating domains (TADs), and insulated neighborhoods.

The term “herbal preparation”, as used herein, refers to herbal medicines that contain parts of plants, or other plant materials, or combinations as active ingredients.

The term “insulated neighborhood” (IN), as used herein, refers to chromosome structure formed by the looping of two interacting sites in the chromosome sequence that may comprise CCCTC-Binding factor (CTCF) co-occupied by cohesin and affect the expression of genes in the insulated neighborhood as well as those genes in the vicinity of the insulated neighborhoods.

The term “insulator”, as used herein, refers to regulatory elements that block the ability of an enhancer to activate a gene when located between them and contribute to specific enhancer-gene interactions.

The term “master transcription factor”, as used herein, refers to signaling molecules which alter, whether to increase or decrease, the transcription of a target gene, e.g., a neighborhood gene and establish cell-type specific enhancers. Master transcription factors recruit additional signaling proteins, such as other transcription factors to enhancers to form signaling centers.

The term “minimal insulated neighborhood”, as used herein, refers to an insulated neighborhood having at least one neighborhood gene and associated regulatory sequence region or regions (RSRs) which facilitate the expression or repression of the neighborhood gene such as a promoter and/or enhancer and/or repressor regions, and the like.

The term “modulate”, as used herein, refers to an alteration (e.g., increase or decrease) in the expression of the target gene and/or activity of the gene product.

The term “neighborhood gene”, as used herein, refers to a gene localized within an insulated neighborhood.

The term “penetrance”, as used herein, refers to the proportion of individuals carrying a particular variant of a gene (e.g., mutation, allele or generally a genotype, whether wild type or not) that also exhibits an associated trait (phenotype) of that variant gene and in some situations is measured as the proportion of individuals with the mutation who exhibit clinical symptoms thus existing on a continuum.

The term “polypeptide”, as used herein, refers to a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long.

The term “primary neighborhood gene” as used herein, refers to a gene which is most commonly found within a specific insulated neighborhood along a chromosome.

The term “primary downstream boundary”, as used herein, refers to the insulated neighborhood boundary located downstream of a primary neighborhood gene.

The term “primary upstream boundary”, as used herein, refers to the insulated neighborhood boundary located upstream of a primary neighborhood gene.

The term “promoter” as used herein, refers to a DNA sequence that defines where transcription of a gene by RNA polymerase begins and defines the direction of transcription indicating which DNA strand will be transcribed.

The term “regulatory sequence regions”, as used herein, include but are not limited to regions, sections or zones along a chromosome whereby interactions with signaling molecules occur in order to alter expression of a neighborhood gene.

The term “repressor”, as used herein, refers to any protein that binds to DNA and therefore regulates the expression of genes by decreasing the rate of transcription.

The term “secondary downstream boundary”, as used herein, refers to the downstream boundary of a secondary loop within a primary insulated neighborhood.

The term “secondary upstream boundary”, as used herein, refers to the upstream boundary of a secondary loop within a primary insulated neighborhood.

The term “signaling center”, as used herein, refers to a defined region of a living organism that interacts with a defined set of biomolecules, such as signaling proteins or signaling molecules (e.g., transcription factors) to regulate gene expression in a context-specific manner.

The term “signaling molecule”, as used herein, refers to any entity, whether protein, nucleic acid (DNA or RNA), organic small molecule, lipid, sugar or other biomolecule, which interacts directly, or indirectly, with a regulatory sequence region on a chromosome.

The term “signaling transcription factor”, as used herein, refers to signaling molecules which alter, whether to increase or decrease, the transcription of a target gene, e.g., a neighborhood gene and also act as cell-cell signaling molecules.

The term “small molecule”, as used herein, refers to a low molecular weight drug, i.e. <5000 Daltons organic compound that may help regulate a biological process.

The terms “subject” and “patient” are used interchangeably herein and refer to an animal to whom treatment with the compositions according to the present invention is provided.

The term “super-enhancers”, as used herein, refers to are large clusters of transcriptional enhancers that drive expression of genes that define cell identity.

The term “therapeutic agent”, as used herein, refers to a substance that has the ability to cure a disease or ameliorate the symptoms of the disease.

The term “therapeutic or treatment outcome”, as used herein, refers to any result or effect (whether positive, negative or null) which arises as a consequence of the perturbation of a GSC or GSN. Examples of therapeutic outcomes include, but are not limited to, improvement or amelioration of the unwanted or negative conditions associated with a disease or disorder, lessening of side effects or symptoms, cure of a disease or disorder, or any improvement associated with the perturbation of a GSC or GSN.

The term “topologically associating domains” (TADs), as used herein, refers to structures that represent a modular organization of the chromatin and have boundaries that are shared by the different cell types of an organism.

The term “transcription factors”, as used herein, refers to signaling molecules which alter, whether to increase or decrease, the transcription of a target gene, e.g., a neighborhood gene.

The term “therapeutic or treatment liability”, as used herein, refers to a feature or characteristic associated with a treatment or treatment regime which is unwanted, harmful or which mitigates the therapies positive outcomes. Examples of treatment liabilities include for example toxicity, poor half-life, poor bioavailability, lack of or loss of efficacy or pharmacokinetic or pharmacodynamic risks.

The term “upstream neighborhood gene”, as used herein, refers to a gene upstream of a primary neighborhood gene that may be located within the same insulated neighborhood as the primary neighborhood gene.

The term “urea cycle disorder”, as used herein, refers to any disorder that is caused by a defect or malfunction in the urea cycle.

The term “urea cycle-related gene”, as used herein, refers to a gene whose gene product (e.g., RNA or protein) is involved in the urea cycle.

Described herein are compositions and methods for perturbation of genomic signaling centers (GSCs) or entire gene signaling networks (GSNs) for the treatment of urea cycle disorders (e.g., OTC deficiency). The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.

The present invention is further illustrated by the following non-limiting examples.

VII. EXAMPLES
Example 1
Experimental procedures

A. Hepatocyte Cell Culture

Cryopreserved hepatocytes were cultured in plating media for 16 hours, transferred to maintenance media for 4 hours. Cultured on serum-free media for 2 hours, then a compound was added. The hepatocytes were maintained on the serum-free media for 16 hours prior to gene expression analysis. Primary Human Hepatocytes were stored in the vapor phase of a liquid nitrogen freezer (about −130° C.).

To seed the primary human hepatocytes, vials of cells were retrieved from the LN₂freezer, thawed in a 37° C. water bath, and swirled gently until only a sliver of ice remained. Using a 10 ml serological pipet, cells were gently pipetted out of the vial and gently pipetted down the side of 50 mL conical tube containing 20 mL cold thaw medium. The vial was rinsed with about 1 mL of thaw medium, and the rinse was added to the conical tube. Up to 2 vials may be added to one tube of 20 mL thaw medium.

The conical tube(s) were gently inverted 2-3 times and centrifuged at 100 g for 10 minutes at 4° C. with reduced braking (e.g. 4 out of 9). The thaw medium slowly was slowly aspirated to avoid the pellet. 4 mL cold plating medium was added slowly down the side (8 mL if combined 2 vials to 1 tube), and the vial was inverted gently several times to resuspend cells.

Cells were kept on ice until 100 μl of well-mixed cells were added to 400 μl diluted Trypan blue and mixed by gentle inversion. They were counted using a hemocytometer (or Cellometer), and viability and viable cells/mL were noted. Cells were diluted to a desired concentration and seeded on collagen I-coated plates. Cells were pipetted slowly and gently onto plate, only 1-2 wells at a time. The remaining cells were mixed in the tubes frequently by gentle inversion. Cells were seeded at about 8.5×10⁶cells per plate in 6 mL cold plating medium (10 cm). Alternatively, 1.5×10⁶cells per well for a 6-well plate (1 mL medium/well); 7×10⁵cells per well for 12-well plate (0.5 mL/well); or 3.75×10⁵cells per well for a 24-well plate (0.5 mL/well)

After all cells and medium were added to the plate, the plate was transferred to an incubator (37° C., 5% CO₂, about 90% humidity) and rocked forwards and backwards, then side to side several times each to distribute cells evenly across the plate or wells. The plate(s) were rocked again every 15 minutes for the first hour post-plating. About 4 hours post-plating (or first thing the morning if cells were plated in the evening), cells were washed once with PBS and complete maintenance medium was added. The primary human hepatocytes were maintained in the maintenance medium and transferred to fresh medium daily.

B. Starvation and Compound Treatment of Hepatocytes

Two to three hours before treatment, cells cultured as described above were washed with PBS and the medium was changed to either: fresh maintenance medium (complete) or modified maintenance medium 4b.

Compound stocks were prepared at 1000× final concentration and added in a 2-step dilution to the medium to reduce risk of a compound precipitating out of solution when added to the cells, and to ensure reasonable pipetting volumes. One at a time, each compound was first diluted 10-fold in warm (about 37° C.) modified maintenance medium (initial dilution=ID), mixed by vortexing, and the ID was diluted 100-fold into the cell culture (e.g. 5.1 μl into 1 well of a 24-well plate containing 0.5 mL medium). The plate was mixed by carefully swirling and after all wells were treated and returned to the incubator overnight. If desired, separate plates/wells were treated with vehicle-only controls and/or positive controls. If using multi-well plates, controls were included on each plate. After about 18 hours, cells were harvested for further analysis, e.g., ChIP-seq, RNA-seq, ATAC-seq, etc.

C. Media Composition

The thaw medium contained 6 mL isotonic percoll and 14 mL high glucose DMEM (Invitrogen #11965 or similar). The plating medium contained 100 mL Williams E medium (Invitrogen #A1217601, without phenol red) and the supplement pack #CM3000 from ThermoFisher Plating medium containing 5 mL FBS, 10 μl dexamethasone, and 3.6 mL plating/maintenance cocktail. Stock trypan blue (0.4%, Invitrogen #15250) was diluted 1:5 in PBS.

The ThermoFisher complete maintenance medium contained supplement pack #CM4000 (1 μl dexamethasone and 4 mL maintenance cocktail) and 100 mL Williams E (Invitrogen #A1217601, without phenol red).

The modified maintenance media had no stimulating factors (dexamethasone, insulin, etc.), and contained100 mL Williams E (Invitrogen #A1217601, without phenol red), 1 mL L-Glutamine (Sigma #G7513) to 2 mM, 1.5 mL HEPES (VWR #J848) to 15 mM, and 0.5 mL penicillin/streptomycin (Invitrogen #15140) to a final concentration of 50 U/mL each.

D. DNA Purification

DNA purification was conducted as described in Ji et al., PNAS 112(12):3841-3846 (2015) Supporting Information, which is hereby incorporated by reference in its entirety. One milliliter of 2.5 M glycine was added to each plate of fixed cells and incubated for 5 minutes to quench the formaldehyde. The cells were washed twice with PBS. The cells were pelleted at 1,300 g for 5 minutes at 4° C. Then, 4×10⁷cells were collected in each tube. The cells were lysed gently with 1 mL of ice-cold Nonidet P-40 lysis buffer containing protease inhibitor on ice for 5 minutes (buffer recipes are provided below). The cell lysate was layered on top of 2.5 volumes of sucrose cushion made up of 24% (wt/vol) sucrose in Nonidet P-40 lysis buffer. This sample was centrifuged at 18,000 g for 10 minutes at 4° C. to isolate the nuclei pellet (the supernatant represented the cytoplasmic fraction). The nuclei pellet was washed once with PBS/1 mM EDTA. The nuclei pellet was resuspended gently with 0.5 mL glycerol buffer followed by incubation for 2 minutes on ice with an equal volume of nuclei lysis buffer. The sample was centrifuged at 16,000 g for 2 minutes at 4° C. to isolate the chromatin pellet (the supernatant represented the nuclear soluble fraction). The chromatin pellet was washed twice with PBS/1 mM EDTA. The chromatin pellet was stored at −80° C.

The Nonidet P-40 lysis buffer contained 10 mM Tris.HCl (pH 7.5), 150 mM NaCl, and 0.05% Nonidet P-40. The glycerol buffer contained 20 mM Tris.HCl (pH 7.9), 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, and 50% (vol/vol) glycerol. The nuclei lysis buffer contained 10 mM Hepes (pH 7.6), 1 mM DTT, 7.5 mM MgCl₂, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, and 1% Nonidet P-40.

E. Chromatin Immunoprecipitation Sequencing (ChIP-seq)

ChIP-seq was performed using the following protocol for primary hepatocytes and HepG2 cells to determine the composition and confirm the location of signaling centers.

i. Cell Cross-Linking

2×10⁷cells were used for each run of ChIP-seq. Two ml of fresh 11% formaldehyde (FA) solution was added to 20 ml media on 15 cm plates to reach a 1.1% final concentration. Plates were swirled briefly and incubated at room temperature (RT) for 15 minutes. At the end of incubation, the FA was quenched by adding 1 ml of 2.5M Glycine to plates and incubating for 5 minutes at RT. The media was discarded to a 1 L beaker, and cells were washed twice with 20 ml ice-cold PBS. PBS (10 ml) was added to plates, and cells were scraped off the plate. The cells were transferred to 15 ml conical tubes, and the tubes were placed on ice. Plates were washed with an additional 4 ml of PBS and combined with cells in 15 ml tubes. Tubes were centrifuged for 5 minutes at 1,500 rpm at 4° C. in a tabletop centrifuge. PBS was aspirated, and the cells were flash frozen in liquid nitrogen. Pellets were stored at −80° C. until ready to use.

ii. Pre-Block Magnetic Beads

Thirty μl Protein G beads (per reaction) were added to a 1.5 ml Protein LoBind

Eppendorf tube. The beads were collected by magnet separation at RT for 30 seconds. Beads were washed 3 times with 1 ml of blocking solution by incubating beads on a rotator at 4° C. for 10 minutes and collecting the beads with the magnet. Five μg of an antibody was added to the 250 μl of beads in block solution. The mix was transferred to a clean tube, and rotated overnight at 4° C. On the next day, buffer containing antibodies was removed, and beads were washed 3 times with 1.1 ml blocking solution by incubating beads on a rotator at 4° C. for 10 minutes and collecting the beads with the magnet. Beads were resuspended in 50 μl of block solution and kept on ice until ready to use.

iii. Cell Lysis, Genomic Fragmentation, and Chromatin Immunoprecipitation

COMPLETE® protease inhibitor cocktail was added to lysis buffer 1 (LB1) before use. One tablet was dissolved in 1 ml of H₂O for a 50× solution. The cocktail was stored in aliquots at −20° C. Cells were resuspended in each tube in 8 ml of LB1 and incubated on a rotator at 4° C. for 10 minutes. Nuclei were spun down at 1,350 g for 5 minutes at 4° C. LB1 was aspirated, and cells were resuspended in each tube in 8 ml of LB2 and incubated on a rotator at 4° C. for 10 minutes.

A COVARIS® E220EVOLUTION™ ultrasonicator was programmed per the manufacturer's recommendations for high cell numbers. HepG2 cells were sonicated for 12 minutes, and primary hepatocyte samples were sonicated for 10 minutes. Lysates were transferred to clean 1.5 ml Eppendorf tubes, and the tubes were centrifuged at 20,000 g for 10 minutes at 4° C. to pellet debris. The supernatant was transferred to a 2 ml Protein LoBind Eppendorf tube containing pre-blocked Protein G beads with pre-bound antibodies. Fifty μl of the supernatant was saved as input. Input material was kept at −80° C. until ready to use. Tubes were rotated with beads overnight at 4° C.

iv. Wash, Elution, and Cross-Link Reversal

All washing steps were performed by rotating tubes for 5 minutes at 4° C. The beads were transferred to clean Protein LoBind Eppendorf tubes with every washing step. Beads were collected in 1.5 ml Eppendorf tube using a magnet. Beads were washed twice with 1.1 ml of sonication buffer. The magnetic stand was used to collect magnetic beads. Beads were washed twice with 1.1 ml of wash buffer 2, and the magnetic stand was used again to collect magnetic beads. Beads were washed twice with 1.1 ml of wash buffer 3. All residual Wash buffer 3 was removed, and beads were washed once with 1.1 ml TE+0.2% Triton X-100 buffer. Residual TE+0.2% Triton X-100 buffer was removed, and beads were washed twice with TE buffer for 30 seconds each time. Residual TE buffer was removed, and beads were resuspended in 300 μl of ChIP elution buffer. Two hundred fifty μl of ChIP elution buffer was added to 50 μl of input, and the tubes were rotated with beads 1 hour at 65° C. Input sample was incubated overnight at 65° C. oven without rotation. Tubes with beads were placed on a magnet, and the eluate was transferred to a fresh DNA LoBind Eppendorf tube. The eluate was incubated overnight at 65° C. oven without rotation

v. Chromatin Extraction and Precipitation

Input and immunoprecipitant (IP) samples were transferred to fresh tubes, and 300 μl of TE buffer was added to IP and Input samples to dilute SDS. RNase A (20 mg/ml) was added to the tubes, and the tubes were incubated at 37° C. for 30 minutes. Following incubation, 3 μl of 1M CaCl₂and 7 μl of 20 mg/ml Proteinase K were added, and incubated 1.5 hours at 55° C. MaXtract High Density 2 ml gel tubes (Qiagen) were prepared by centrifugation at full speed for 30 seconds at RT. Six hundred μl of phenol/chloroform/isoamyl alcohol was added to each proteinase K reaction and transferred in about 1.2 ml mixtures to the MaXtract tubes. Tubes were spun at 16,000 g for 5 minutes at RT. The aqueous phase was transferred to two clean DNA LoBind tubes (300 μl in each tube), and 1.5 μl glycogen, 30 μl of 3M sodium acetate, and 900 μl ethanol were added. The mixture was precipitated overnight at −20° C. or for 1 hour at −80° C., and spun down at maximum speed for 20 minutes at 4° C. The ethanol was removed, and pellets were washed with 1 ml of 75% ethanol by spinning tubes down at maximum speed for 5 minutes at 4° C. Remnants of ethanol were removed, and pellets were dried for 5 min at RT. Twenty-five μl of H₂O was added to each immunoprecipitant (IP) and input pellet, left standing for 5 minutes, and vortexed briefly. DNA from both tubes was combined to obtain 50 μl of IP and 50 μl of input DNA for each sample. One μl of this DNA was used to measure the amount of pulled down DNA using Qubit dsDNA HS assay (ThermoFisher, #Q32854). The total amount of immunoprecipitated material ranged from several ng (for TFs) to several hundred ng (for chromatin modifications). Six μl of DNA was analyzed using qRT-PCR to determine enrichment. The DNA was diluted if necessary. If enrichment was satisfactory, the rest was used for library preparation for DNA sequencing.

vi. Library Preparation for DNA Sequencing

Libraries were prepared using NEBNext Ultra II DNA library prep kit for Illumina (NEB, #E7645) using NEBNext Multiplex Oligos for Illumina (NEB, #6609S) according to manufacturer's instructions with the following modifications. The remaining ChIP sample (about 43 μl) and 1 μg of input samples for library preparations were brought up the volume of 50 μl before the End Repair portion of the protocol. End Repair reactions were run in a PCR machine with a heated lid in a 96-well semi-skirted PCR plate (ThermoFisher, #AB1400) sealed with adhesive plate seals (ThermoFisher, #AB0558) leaving at least one empty well in-between different samples. Undiluted adapters were used for input samples, 1:10 diluted adapters for 5-100 ng of ChIP material, and 1:25 diluted adapters for less than 5 ng of ChIP material. Ligation reactions were run in a PCR machine with the heated lid off. Adapter ligated DNA was transferred to clean DNA LoBind Eppendorf tubes, and the volume was brought to 96.5 μl using H₂O.

200-600 bp ChIP fragments were selected using SPRIselect magnetic beads (Beckman Coulter, #B23317). Thirty μl of the beads were added to 96.5 μl of ChIP sample to bind fragments that are longer than 600 bp. The shorter fragments were transferred to a fresh DNA LoBind Eppendorf tube. Fifteen μl of beads were added to bind the DNA longer than 200 bp, and beads were washed with DNA twice using freshly prepared 75% ethanol. DNA was eluted using 17 μl of 0.1× TE buffer. About 15 μl was collected.

Three μl of size-selected Input sample and all (15 μl) of the ChIP sample was used for PCR. The amount of size-selected DNA was measured using a Qubit dsDNA HS assay. PCR was run for 7 cycles of for Input and ChIP samples with about 5-10 ng of size-selected DNA, and 12 cycles with less than 5 ng of size-selected DNA. One-half of the PCR product (25 μl) was purified with 22.5 μl of AMPure XP beads (Beckman Coulter, #A63880) according to the manufacturer's instructions. PCR product was eluted with 17 μl of 0.1× TE buffer, and the amount of PCT product was measured using Qubit dsDNA HS assay. An additional 4 cycles of PCR were run for the second half of samples with less than 5 ng of PCR product, DNA was purified using 22.5 μl of AMPure XP beads. The concentration was measured to determine whether there was an increased yield. Both halves were combined, and the volume was brought up to 50 μl using H₂O.

A second round of purifications of DNA was run using 45 μl of AMPure XP beads in 17 μl of 0.1× TE, and the final yield was measured using Qubit dsDNA HS assay. This protocol produces from 20 ng to 1 mg of PCR product. The quality of the libraries was verified by diluting 1 μl of each sample with H₂O if necessary using the High Sensitivity BioAnalyzer DNA kit (Agilent, #5067-4626) based on manufacturer's recommendations.

vii. Reagents

11% Formaldehyde Solution (50 mL) contained 14.9 ml of 37% formaldehyde (final conc. 11%), 1 ml of 5M NaCl (final conc. 0.1 M), 100 μl of 0.5M EDTA (pH 8) (final conc. 1 mM), 50 μl of 0.5M EGTA (pH 8) (final conc. 0.5 mM), and 2.5 ml 1M Hepes (pH 7.5) (final conc. 50 mM).

Block Solution contained 0.5% BSA (w/v) in PBS and 500 mg BSA in 100 ml PBS. Block solution may be prepared up to about 4 days prior to use.

Lysis buffer 2 (LB2) (1000 ml) contained 10 ml of 1 M Tris-HCL, pH 8.0; 40 ml of 5 M NaCl; 2 ml of 0.5M EDTA, pH 8.0; and 2 ml of 0.5M EGTA, pH 8.0. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

Proteinase inhibitors were included in the LB1, LB2, and Sonication buffer.

Wash Buffer 2 (500 ml) contained 25 ml of 1M Hepes-KOH, pH 7.5; 35 ml of 5M NaCl; 1 ml of 0.5M EDTA, pH 8.0; 50 ml of 10% Triton X-100; 10 ml of 5% Na-deoxycholate; and 5 ml of 10% SDS. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

Wash Buffer 3 (500 ml) contained 10 ml of 1M Tris-HCL, pH 8.0; 1 ml of 0.5M EDTA, pH 8.0; 125 ml of 1M LiCl solution; 25 ml of 10% NP-40; and 50 ml of 5% Na-deoxycholate. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

ChIP elution Buffer (500 ml) contained 25 ml of 1 M Tris-HCL, pH 8.0; 10 ml of 0.5M EDTA, pH 8.0; 50 ml of 10% SDS; and 415 ml of ddH₂O. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

F. Analysis of ChIP-seq Results

All pass filter reads from each sample were trimmed of sequencing adapters using trim_galore 0.4.4 with default options. Trimmed reads were mapped against the human genome (assembly GRCh38/GCA_000001405.15 “no alt” analysis set merged with hs38d1/GCA_000786075.2) using bwa version 0.7.15 (Li (2013) arXiv:1303.3997v1) with default parameters. Aligned read duplicates were assessed using picard 2.9.0 (http://broadinstitute.hithub.io/picard) and reads with a MAPQ<20 or those matching standard SAM flags 0×1804 were discarded. Standard QC were applied (read integrity, mapping statistics, library complexity, fragment bias) to remove unsatisfactory samples. Enriched ChIP-seq peaks were identified by comparing samples against whole cell extract controls using MACS2 version 2.1.0 (Zhang et al., Genome Biol. (2008) 9(9):R137), with significant peaks selected as those with an adjusted p-value<0.01. Peaks overlapping known repetitive “blacklist” regions (ENCODE Project Consortium, Nature (2012) 489(7414:57-74) were discarded. ChIP-seq signals were also normalized by read depth and visualized using the UCSC browser.

G. RNA-seq

This protocol is a modified version of the following protocols: MagMAX mirVana Total RNA Isolation Kit User Guide (Applied Biosystems #MAN0011131 Rev B.0), NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490), and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420) (New England Biosystems #E74901).

The MagMAX mirVana kit instructions (the section titled “Isolate RNA from cells” on pages 14-17) were used for isolation of total RNA from cells in culture. Two hundred μl of Lysis Binding Mix was used per well of the multiwell plate containing adherent cells (usually a 24-well plate).

For mRNA isolation and library prep, the NEBNext Poly(A) mRNA Magnetic Isolation Module and Directional Prep kit was used. RNA isolated from cells above was quantified, and prepared in 500 μg of each sample in 50 μl of nuclease-free water. This protocol may be run in microfuge tubes or in a 96-well plate.

The 80% ethanol was prepared fresh, and all elutions are done in 0.1× TE Buffer. For steps requiring Ampure XP beads, beads were at room temperature before use. Sample volumes were measured first and beads were pipetted. Section 1.9B (not 1.9A) was used for NEBNext Multiplex Oligos for Illumina (#E6609). Before starting the PCR enrichment, cDNA was quantified using the Qubit (DNA High Sensitivity Kit, ThermoFisher #Q32854). The PCR reaction was run for 12 cycles.

After purification of the PCR Reaction (Step 1.10), the libraries were quantified using the Qubit DNA High Sensitivity Kit. 1 μl of each sample were diluted to 1-2 ng/μl to run on the Bioanalyzer (DNA High Sensitivity Kit, Agilent # 5067-4626). If Bioanalyzer peaks were not clean (one narrow peak around 300 bp), the AMPure XP bead cleanup step was repeated using a 0.9× or 1.0× beads:sample ratio. Then, the samples were quantified again with the Qubit, and run again on the Bioanalyzer (1-2 ng/μl).

Nuclear RNA from INTACT-purified nuclei or whole neocortical nuclei was converted to cDNA and amplified with the Nugen Ovation RNA-seq System V2. Libraries were sequenced using the Illumina HiSeq 2500.

H. RNA-seq Data Analysis

All pass filter reads from each sample were mapped against the human genome (assembly GRCh38/GCA 000001405.15 “no alt” analysis set merged with hs38d1/GCA 000786075.2) using two pass mapping via STAR version 2.5.3a (alignment parameters alignIntronMin=20; alignIntronMax=1000000; outFilterMismatchNmax=999; outFilterMismatchNoverLmax=0.05; outFilterType=BySJout; outFilterMultimapNmax=20; alignSJoverhangMin=8; alignSJDBoverhangMin=1; alignMatesGapMax=1000000) (Dobin et al., Bioinformatics (2012) 29(1):15-21). Genomic alignments were converted to transcriptome alignments based on reference transcript annotations from The Human GENCODE Gene Set release 24 (Harrow et al., Genome Res. (2012) 22(9): 1760-1774). Using unique and multimapped transcriptomic alignments, gene-level abundance estimates were computed using RSEM version 1.3.0 (Li and Dewey, BMC Bioinformatics (2011) 12:323) in a strand-aware manner, and including confidence interval sampling calculations, to arrive at posterior mean estimates (PME) of abundances (counts and normalized FPKM—fragments per kilobase of exon per million mapped fragments) from the underlying Bayesian model. Standard QC were applied (read integrity, mapping statistics, library complexity, fragment bias) to remove unsatisfactory samples. Differential gene expression was computed using the negative binomial model implemented by DESeq2 version 1.16.1 (Love et al., Genome Biol. (2014) 15(12):550). Log2 fold change and significance values were computed using PME count data (with replicates explicitly modeled versus pan-experiment controls), median ratio normalized, using maximum likelihood estimation rather than maximum a posteriori, and disabling the use of Cook's distance cutoff when determining acceptable adjusted p-values. Significantly differential genes were assigned as those with an adjusted p-value<0.01, a log2 fold change of >=1 or <=−1, and at least one replicate with PME FPKM>=1. RNA-seq signals were also normalized by read depth and visualized using the UCSC browser.

I. ATAC-seq

Hepatocytes were seeded overnight, then the serum and other factors were removed. After 2-3 hours, the cells were treated with the compound and incubated overnight. The cells were harvested and the nuclei were prepared for the transposition reaction. 50,000 bead bound nuclei were transposed using Tn5 transposase (Illumina FC-121-1030) as described in Mo et al., 2015, Neuron 86, 1369-1384, which is hereby incorporated by reference in its entirety. After 9-12 cycles of PCR amplification, libraries were sequenced on an Illumina HiSeq 2000. PCR was performed using barcoded primers with extension at 72° C. for 5 minutes, PCR, then the final PCR product was sequenced.

All obtained reads from each sample were trimmed using trim_galore 0.4.1 requiring Phred score≥20 and read length≥30 for data analysis. The trimmed reads were mapped against the human genome (hg19 build) using Bowtie2 (version 2.2.9) with the parameters: -t -q -N 1 -L 25 -X 2000 no-mixed no-discordant. All unmapped reads, non-uniquely mapped reads and PCR duplicates were removed. All the ATAC-seq peaks were called using MACS2 with the parameters —nolambda —nomodel -q 0.01 —SPMR. The ATAC-seq signal was visualized in the UCSC genome browser. ATAC-seq peaks that were at least 2 kb away from annotated promoters (RefSeq, Ensemble and UCSC Known Gene databases combined) were selected as distal ATAC-seq peaks.

J. qRT-PCR

qRT-PCR was performed as described in North et al., PNAS, 107(40) 17315-17320 (2010), which is hereby incorporated by reference in its entirety. qRT-PCR was performed with cDNA using the iQ5 Multicolor rtPCR Detection system from BioRad with 60° C. annealing.

Analysis of the fold changes in expression as measured by qRT-PCR were performed using the technique below. The control was DMSO, and the treatment was the selected compound (CPD). The internal control was GAPDH or B-Actin, and the gene of interest is the target. First, the averages of the 4 conditions were calculated for normalization: DMSO:GAPDH, DMSO:Target, CPD: GAPDH, and CPD:Target. Next, the ΔCT of both control and treatment were calculated to normalize to internal control (GAPDH) using (DMSO:Target)−(DMSO:GAPDH)=ΔCT control and (CPD:Target)−(CPD: GAPDH)=ΔCT experimental. Then, the ΔΔCT was calculated by ΔCT experimental−ΔCT control. The Expression Fold Change was calculated by 2-(ΔΔCT) (2-fold expression change was shown by RNA-Seq results provided herein).

K. Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET)

ChIA-PET was performed as previously described in Chepelev et al. (2012) Cell Res. 22, 490-503; Fullwood et al. (2009) Nature 462, 58-64; Goh et al. (2012) J. Vis. Exp., http://dx.doi.org/10.3791/3770; Li et al. (2012) Cell 148, 84-98; and Dowen et al. (2014) Cell 159, 374-387, which are each hereby incorporated by reference in their entireties. Briefly, embryonic stem (ES) cells (up to 1×10⁸cells) were treated with 1% formaldehyde at room temperature for 20 minutes and then neutralized using 0.2M glycine. The crosslinked chromatin was fragmented by sonication to size lengths of 300-700 bp. The anti-SMC1 antibody (Bethyl, A300-055A) was used to enrich SMC1-bound chromatin fragments. A portion of ChIP DNA was eluted from antibody-coated beads for concentration quantification and for enrichment analysis using quantitative PCR. For ChIA-PET library construction ChIP DNA fragments were end-repaired using T4 DNA polymerase (NEB). ChIP DNA fragments were divided into two aliquots and either linker A or linker B was ligated to the fragment ends. The two linkers differ by two nucleotides which were used as a nucleotide barcode (Linker A with CG; Linker B with AT). After linker ligation, the two samples were combined and prepared for proximity ligation by diluting in a 20 ml volume to minimize ligations between different DNA-protein complexes. The proximity ligation reaction was performed with T4 DNA ligase (Fermentas) and incubated without rocking at 22° C. for 20 hours. During the proximity ligation DNA fragments with the same linker sequence were ligated within the same chromatin complex, which generated the ligation products with homodimeric linker composition. However, chimeric ligations between DNA fragments from different chromatin complexes could also occur, thus producing ligation products with heterodimeric linker composition. These heterodimeric linker products were used to assess the frequency of nonspecific ligations and were then removed.

i. Day 1

The cells were crosslinked as described for ChIP. Frozen cell pellets were stored in the −80° C. freezer until ready to use. This protocol requires at least 3×10⁸cells frozen in six 15 ml Falcon tubes (50 million cells per tube). Six 100 μl Protein G Dynabeads (for each ChIA-PET sample) was added to six 1.5 ml Eppendorf tubes on ice. Beads were washed three times with 1.5 ml Block solution, and incubated end over end at 4° C. for 10 minutes between each washing step to allow for efficient blocking. Protein G Dynabeads were resuspended in 250 μl of Block solution in each of six tubes and 10 μg of SMC1 antibody (Bethyl A300-055A) was added to each tube. The bead-antibody mixes were incubated at 4° C. end-over-end overnight.

ii. Day 2

Beads were washed three times with 1.5 ml Block solution to remove unbound IgG and incubated end-over-end at 4° C. for 10 minutes each time. Smc1-bound beads were resuspended in 100 μl of Block solution and stored at 4° C. Final lysis buffer 1 (8 ml per sample) was prepared by adding 50× Protease inhibitor cocktail solution to Lysis buffer 1 (LB1) (1:50). Eight ml of Final lysis buffer 1 was added to each frozen cell pellet (8 ml per sample×6). The cells were thoroughly resuspended and thawed on ice by pipetting up and down. The cell suspension was incubated again end-over-end for 10 minutes at 4° C. The suspension was centrifuged at 1,350×g for 5 minutes at 4° C. Concurrently, Final lysis buffer 2 (8 ml per sample) was prepared by adding 50× Protease inhibitor cocktail solution to lysis buffer 2 (LB2) (1:50)

After centrifugation, the supernatant was discarded, and the nuclei were thoroughly resuspended in 8 ml Final lysis buffer 2 by pipetting up and down. The cell suspension was incubated end-over-end for 10 minutes at 4° C. The suspension was centrifuged at 1,350×g for 5 minutes at 4° C. During incubation and centrifugation, the Final sonication buffer (15 ml per sample) was prepared by adding 50x Protease inhibitor cocktail solution to sonication buffer (1:50). The supernatant was discarded, and the nuclei were fully resuspended in 15 ml Final sonication buffer by pipetting up and down. The nuclear extract was extracted to fifteen 1 ml Covaris Evolution E220 sonication tubes on ice. An aliquot of 10 μl was used to check the size of unsonicated chromatin on a gel.

A Covaris sonicator was programmed according to manufacturer's instructions (12 minutes per 20 million cells=12×15=3 hours). The samples were sequentially sequenced as described above. The goal is to break chromatin DNA to 200-600 bp. If sonication fragments are too big, false positives become more frequent. The sonicated nuclear extract was dispensed into 1.5 ml Eppendorf tubes. 1.5 ml samples are centrifuged at full speed at 4° C. for 10 minutes. Supernatant (SNE) was pooled into a new pre-cooled 50 ml Falcon tube, and brought to a volume of 18 ml with sonication buffer. Two tubes of 50 μl were taken as input and to check the size of fragments. 250 μl of ChIP elution buffer was added and reverse crosslinking occurred at 65° C. overnight in the oven After reversal of crosslinking, the size of sonication fragments was determined on a gel.

Three ml of sonicated extract was added to 100 μl Protein G beads with SMC1 antibodies in each of six clean 15 ml Falcon tubes. The tubes containing SNE-bead mix were incubated end-over-end at 4° C. overnight (14 to 18 hours).

iii. Day 3

Half the volume (1.5 ml) of the SNE-bead mix was added to each of six pre-chilled tubes and SNE was removed using a magnet. The tubes were sequentially washed as follows: 1) 1.5 ml of Sonication buffer was added, the beads were resuspended and rotated for 5 minutes at 4° C. for binding, then the liquid was removed (step performed twice); 2) 1.5 ml of high-salt sonication buffer was added, and the beads were resuspended and rotated for 5 minutes at 4° C. for binding, then the liquid was removed (step performed twice); 3) 1.5 ml of high-salt sonication buffer was added, and the beads were resuspended and rotated for 5 minutes at 4° C. for binding, then the liquid was removed (step performed twice); 4) 1.5 ml of LiCl buffer was added, and the cells were resuspended and incubated end-over-end for 5 minutes for binding, then the liquid was removed (step performed twice); 5) 1.5 ml of 1× TE +0.2% Triton X-100 was used to wash the cells for 5 minutes for binding, then the liquid was removed; and 1.5 ml of ice-cold TE Buffer was used to wash the cells for 30 seconds for binding, then the liquid was removed (step performed twice). Beads from all six tubes were sequentially resuspended in beads in one 1,000 ul tube of 1× ice-cold TE buffer.

ChIP-DNA was quantified using the following protocol. Ten percent of beads (by volume), or 100 μl, were transferred into a new 1.5 ml tube, using a magnet. Beads were resuspended in 300 μl of ChIP elution buffer and the tube was rotated with beads for 1 hour at 65° C. The tube with beads was placed on a magnet and the eluate was transferred to a fresh DNA LoBind Eppendorf tube. The eluate was incubated overnight at 65° C. oven without rotating. Immuno-precipitated samples were transferred to fresh tubes, and 300 μl of TE buffer was added to the immuno-precipitants and Input samples to dilute. Five μl of RNase A (20 mg/ml) was added, and the tube was incubated at 37° C. for 30 minutes.

Following incubation, 3 μl of 1M CaCl₂and 7 μl of 20 mg/ml Proteinase K was added to the tube and incubated 1.5 hours at 55° C. MaXtract High Density 2 ml gel tubes (Qiagen) were prepared by centrifuging them at full speed for 30 seconds at RT. 600 μl of phenol/chloroform/isoamyl alcohol was added to each proteinase K reaction. About 1.2 ml of the mixtures was transferred to the MaXtract tubes. Tubes were spun at 16,000 g for 5 minutes at RT. The aqueous phase was transferred to two clean DNA LoBind tubes (300 μl in each tube), and 1 μl glycogen, 30 μl of 3M sodium acetate, and 900 μl ethanol was added. The mixture was allowed to precipitate overnight at −20° C. or for 1 hour at −80° C.

The mixture was spun down at maximum speed for 20 minutes at 4° C., ethanol was removed, and the pellets were washed with 1 ml of 75% ethanol by spinning tubes down at maximum speed for 5 minutes at 4° C. All remnants of ethanol were removed, and pellets were dried for 5 minutes at RT. H₂O was added to each tube. Each tube was allowed to stand for 5 minutes, and vortexed briefly. DNA from both tubes was combined to obtain 50 μl of IP and 100 μl of Input DNA.

The amount of DNA collected was quantitated by ChIP using Qubit (Invitrogen #Q32856). One μl intercalating dye was combined with each measure 1 μl of sample. Two standards that come with the kit were used. DNA from only 10% of the beads was measured. About 400 ng of chromatin in 900 μl of bead suspension was obtained with a good enrichment at enhancers and promoters as measured by qPCR.

iv. Day 3 or 4

End-blunting of ChIP-DNA was performed on the beads using the following protocol. The remaining chromatin/beads were split by pipetting, and 450 μl of bead suspension was aliquoted into 2 tubes. Beads were collected on a magnet. Supernatant was removed, and then the beads were resuspended in the following reaction mix: 70 μl 10× NEB buffer 2.1 (NEB, M0203L), 7 μl 10 mM dNTPs, 615.8 μl dH₂O, and 7.2 μl of 3 U/μl T4 DNA Polymerase (NEB, M0203L). The beads were incubated at 37° C. with rotation for 40 minutes. Beads were collected with a magnet, then the beads were washed 3 times with 1 ml ice-cold ChIA-PET Wash Buffer (30 seconds per each wash).

On-Bead A-tailing was performed by preparing Klenow (3′ to 5′ exo-) master mix as stated below: 70 μl 10× NEB buffer 2, 7 μl 10 mM dATP, 616 μl dH20, and 7 μl of 3 U/μl Klenow (3′ to 5′ exo-) (NEB, M0212L). The mixture was incubated at 37° C. with rotation for 50 minutes. Beads were collected with a magnet, then beads were washed 3 times with 1 ml of ice-cold ChIA-PET Wash Buffer (30 seconds per each wash).

Linkers were thawed gently on ice. Linkers were mixed well with water gently by pipetting, then with PEG buffer, then gently vortexed. Then, 1394 μl of master mix and 6 μl of ligase was added per tube and mixed by inversion. Parafilm was put on the tube, and the tube was incubated at 16° C. with rotation overnight (at least 16 hours). The biotinylated linker was ligated to ChIP-DNA on beads by setting up the following reaction mix and adding reagents in order: 1110 μl dH₂0, 4 μl 200 ng/μl biotinylated bridge linker, 280 μl 5× T4 DNA ligase buffer with PEG (Invitrogen), and 6 μl 30 U/μl T₄DNA ligase (Fermentas).

v. Day 5

Exonuclease lambda/Exonuclease I On-Bead digestion was performed using the following protocol. Beads were collected with a magnet and washed 3 times with 1 ml of ice-cold ChIA-PET Wash Buffer (30 seconds per each wash). The Wash buffer was removed from beads, then resuspended in the following reaction mix: 70 μl 10× lambda nuclease buffer (NEB, M0262L), 618 μl nuclease-free dH20, 6 μl 5 U/μl Lambda Exonuclease (NEB, M0262L), and 6 μl Exonuclease I (NEB, M0293L). The reaction was incubated at 37° C. with rotation for 1 hour. Beads were collected with a magnet, and beads were washed 3 times with 1 ml ice-cold ChIA-PET Wash Buffer (30 seconds per each wash).

Chromatin complexes were eluted off the beads by removing all residual buffer and resuspending the beads in 300 μl of ChIP elution buffer. The tube with beads was rotated 1 hour at 65° C. The tube was placed on a magnet and the eluate was transferred to a fresh DNA LoBind Eppendorf tube. The eluate was incubated overnight at 65° C. in an oven without rotating.

vi. Day 6

The eluted sample was transferred to a fresh tube and 300 μl of TE buffer was added to dilute the SDS. Three μl of RNase A (30 mg/ml) was added to the tube, and the mixture was incubated at 37° C. for 30 minutes. Following incubation, 3 μl of 1M CaCl₂and 7 μl of 20 mg/ml Proteinase K was added, and the tube was incubated again for 1.5 hours at 55° C. MaXtract High Density 2 ml gel tubes (Qiagen) were precipitated by centrifuging them at full speed for 30 seconds at RT. Six hundred μl of phenol/chloroform/isoamyl alcohol was added to each proteinase K reaction, and about 1.2 ml of the mixture was transferred to the MaXtract tubes. Tubes were spun at 16,000×g for 5 minutes at RT.

The aqueous phase was transferred to two clean DNA LoBind tubes (300 μl in each tube), and 1 μl glycogen, 30 μl of 3M sodium acetate, and 900 μl ethanol is added. The mixture was precipitated for 1 hour at −80° C. The tubes were spun down at maximum speed for 30 minutes at 4° C., and the ethanol was removed. The pellets were washed with 1 ml of 75% ethanol by spinning tubes down at maximum speed for 5 minutes at 4° C. Remnants of ethanol were removed, and the pellets were dried for 5 minutes at RT. Thirty μl of H₂O was added to the pellet and allowed to stand for 5 minutes. The pellet mixture was vortexed briefly, and spun down to collect the DNA.

Qubit and DNA High Sensitivity ChIP were performed to quantify and assess the quality of proximity ligated DNA products. About 120 ng of the product was obtained.

vii. Day 7

Components for Nextera tagmentation were then prepared. One hundred ng of DNA was divided into four 25 μl reactions containing 12.5 μl 2× Tagmentation buffer (Nextera), 1 μl nuclease-free dH₂0, 2.5 μl Tn5 enzyme (Nextera), and 9 μl DNA (25 ng). Fragments of each of the reactions were analyzed on a Bioanalyzer for quality control.

The reactions were incubated at 55° C. for 5 minutes, then at 10° C. for 10 minutes. Twenty-five μl of H₂O was added, and tagmented DNA was purified using Zymo columns. Three hundred fifty μl of Binding Buffer was added to the sample, and the mixture was loaded into a column and spun at 13,000 rpm for 30 seconds. The flow through was re-applied and the columns were spun again. The columns are washed twice with 200 μl of wash buffer and spun for 1 minute to dry the membrane. The column was transferred to a clean Eppendorf tube and 25 μl of Elution buffer was added. The tube was spun down for 1 minute. This step was repeated with another 25 μl of elution buffer. All tagmented DNA was combined into one tube.

ChIA-PETs was immobilized on Streptavidin beads using the following steps. 2X B&W Buffer (40 ml) was prepared as follows for coupling of nucleic acids: 400 μl 1M Tris-HCl pH 8.0 (10 mM final), 80 μl 1M EDTA (1 mM final), 16 ml 5M NaCl (2M final), and 23.52 ml dH₂O. 1× B&W Buffer (40 ml total) was prepared by adding 20 ml dH₂O to 20 ml of the 2× B&W Buffer.

MyOne Streptavidin Dynabeads M-280 were allowed to come to room temperature for 30 minutes, and 30 μl of beads were transferred to a new 1.5 ml tube. Beads were washed with 150 μl of 2× B&W Buffer twice. Beads were resuspended in 100 μl of iBlock buffer (Applied Biosystems), and mixed. The mixture was incubated at RT for 45 minutes on a rotator.

I-BLOCK Reagent was prepared to contain: 0.2% I-Block reagent (0.2 g), 1× PBS or 1× TBS (10 ml 10× PBS or 10× TBS), 0.05% Tween-20 (50 μl), and H₂O to 100 ml. 10× PBS and I-BLOCK reagent was added to H₂O, and the mixture was microwaved for 40 seconds (not allowed to boil), then stirred. Tween-20 was added after the solution is cooled. The solution remained opaque, but particles dissolved. The solution was cooled to RT for use.

During incubation of beads, 500 ng of sheared genomic DNA was added to 50 μl of H₂O and 50 μl of 2× B&W Buffer. When the beads finished incubating with the iBLOCK buffer, they were washed twice with 200 μl of 1× B&W buffer. The wash buffer was discarded, and 100 μl of the sheared genomic DNA was added. The mixture was incubated with rotation for 30 minutes at RT. The beads were washed twice with 200 μl of 1× B&W buffer. Tagmented DNA was added to the beads with an equal volume of 2× B&W buffer and incubated for 45 minutes at RT with rotation. The beads were washed 5 times with 500 μl of 2×SSC/0.5% SDS buffer (30 seconds each time) followed by 2 washes with 500 ml of 1× B&W Buffer and incubated each after wash for 5 minutes at RT with rotation. The beads were washed once with 100 μl elution buffer (EB) from a Qiagen Kit by resuspending beads gently and putting the tube on a magnet. The supernatant was removed from the beads, and they were resuspended in 30 μl of EB.

A paired end sequencing library was constructed on beads using the following protocol. Ten μ1 of beads are tested by PCR with 10 cycles of amplification. The 50 μl of the PCR mixture contains: 10 μl of bead DNA, 15 μl NPM mix (from Illumina Nextera kit), 5 μl of PPC PCR primer, 5 μl of Index Primer 1 (i7), 5 μl of Index Primer 2 (i5), and 10 μl of H₂O. PCR was performed using the following cycle conditions: denaturing the DNA at 72° C. for 3 minutes, then 10-12 cycles of 98° C. for 10 seconds, 63° C. for 30 seconds, and 72° C. for 50 seconds, and a final extension of 72° C. for 5 minutes. The number of cycles was adjusted to obtain about 300 ng of DNA total with four 25 μl reactions. The PCR product may be held at 4° C. for an indefinite amount of time.

The PCR product was cleaned-up using AMPure beads. Beads were allowed to come to RT for 30 minutes before using. Fifty μl of the PCR reaction was transferred to a new Low-Bind Tube and (1.8× volume) 90 μl of AMPure beads was added. The mixture was pipetted well and incubated at RT for 5 minutes. A magnet was used for 3 minutes to collect beads and remove the supernatant. Three hundred μ1 of freshly prepared 80% ethanol was added to the beads on the magnet, and the ethanol was carefully discarded. The wash was repeated, and then all ethanol was removed. The beads were dried on the magnet rack for 10 minutes. Ten μl EB was added to the beads, mixed well, and incubated for 5 minutes at RT. The eluate was collected, and 1 μl of eluate was used for Qubit and Bioanalyzer.

The library was cloned to verify complexity using the following protocol. One μl of the library was diluted at 1:10. A PCR reaction was performed as described below. Primers that anneal to Illumina adapters were chosen (Tm=52.2° C.). The PCR reaction mixture (total volume: 50 μl) contained the following: 10 μl of 5× GoTaq buffer, 1 μl of 10 mM dNTP, 5 μl of 10 μM primer mix, 0.25 μl of GoTaq polymerase, 1 μl of diluted template DNA, and 32.75 μl of H₂O. PCR was performed using the following cycle conditions: denaturing the DNA at 95° C. for 2 minutes and 20 cycles at the following conditions: 95° C. for 60 seconds, 50° C. for 60 seconds, and 72° C. for 30 seconds with a final extension at 72° C. for 5 minutes. The PCR product may be held at 4° C. for an indefinite amount of time.

The PCR product was ligated with the pGEM® T-Easy vector (Promega) protocol. Five μl of 2× T4 Quick ligase buffer, 1 μl of pGEM® T-Easy vector, 1 μl of T4 ligase, 1 μl of PCR product, and 2 μl of H₂O were combined to a total volume of 10 μl. The product was incubated for 1 hour at RT and 41 was used to transform Stellar competent cells. Two hundred μl of 500 μl of cells were plated in SOC media. The next day, 20 colonies were selected for Sanger sequencing using a T7 promoter primer. 60% clones had a full adapter, and 15% had a partial adapter.

viii. Reagents

Protein G Dynabeads for 10 samples were from Invitrogen Dynal, Cat# 10003D. Block solution (50 ml) contained 0.25 g BSA dissolved in 50 ml of ddH2O (0.5% BSA, w/v), and was stored at 4° C. for 2 days before use.

Lysis buffer 1 (LB1) (500 ml) contained 25 ml of 1M Hepes-KOH, pH 7.5; 14 ml of 5M NaCl; 1 ml of 0.5 M EDTA, pH 8.0; 50 ml of 100% Glycerol solution; 25 ml of 10% NP-40; and 12.5 ml of 10% Triton X-100. The pH was adjusted to 7.5. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use. Lysis buffer 2 (LB2) (1000 ml) contained 10 ml of 1M Tris-HCL, pH 8.0; 40 ml of 5 M NaCl; 2 ml of 0.5 M EDTA, pH 8.0; and 2 ml of 0.5 M EGTA, pH 8.0. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

Sonication buffer (500 ml) contained 25 ml of 1M Hepes-KOH, pH 7.5; 14 ml of 5M NaCl; 1 ml of 0.5 M EDTA, pH 8.0; 50 ml of 10% Triton X-100; 10 ml of 5% Na-deoxycholate; and 5 ml of 10% SDS. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use. High-salt sonication buffer (500 ml) contained 25 ml of 1M Hepes-KOH, pH 7.5; 35 ml of 5M NaCl; 1 ml of 0.5 M EDTA, pH 8.0; 50 ml of 10% Triton X-100; 10 ml of 5% Na-deoxycholate; and 5 ml of 10% SDS. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

LiCl wash buffer (500 ml) contained 10 ml of 1M Tris-HCL, pH 8.0; 1 ml of 0.5M EDTA, pH 8.0; 125 ml of 1M LiCl solution; 25 ml of 10% NP-40; and 50 ml of 5% Na-deoxycholate. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

Elution buffer (500 ml) used to quantify the amount of ChIP DNA contained 25 ml of 1M Tris-HCL, pH 8.0; 10 ml of 0.5M EDTA, pH 8.0; 50 ml of 10% SDS; and 415 ml of ddH₂O. The pH was adjusted to 8.0. The buffer was sterile-filtered, and stored at 4° C. The pH was re-checked immediately prior to use.

ChIA-PET Wash Buffer (50 ml) contains 500 μl of 1M Tris-HCl, pH 8.0 (final 10 mM); 100 μl of 0.5M EDTA, pH 8.0 (final 1 mM); 5 ml of 5M NaCl (final 500 mM); and 44.4 ml of dH₂0.

L. HiChIP

Alternatively to ChIA-PET, HiChIP was used to analyze chromatin interactions and conformation. HiChIP requires fewer cells than ChIA-PET.

i. Cell Crosslinking

Cells were cross-linked as described in the ChIP protocol above. Crosslinked cells were either stored as pellets at −80° C. or used for HiChIP immediately after flash-freezing the cells.

ii. Lysis and Restriction

Fifteen million cross-linked cells were resuspended in 500 μL of ice-cold Hi-C Lysis

Buffer and rotated at 4° C. for 30 minutes. For cell amounts greater than 15 million, the pellet was split in half for contact generation and then recombined for sonication. Cells were spun down at 2500 g for 5 minutes, and the supernatant was discarded. The pelleted nuclei were washed once with 500 μL of ice-cold Hi-C Lysis Buffer. The supernatant was removed, and the pellet was resuspended in 100 μL of 0.5% SDS. The resuspension was incubated at 62° C. for 10 minutes, and then 285 μL of H₂O and 50 μL of 10% Triton X-100 were added to quench the SDS. The resuspension was mixed well, and incubated at 37° C. for 15 minutes. Fifty μL of 10× NEB Buffer 2 and 375 U of Mbol restriction enzyme (NEB, R0147) was added to the mixture to digest chromatin for 2 hours at 37° C. with rotation. For lower starting material, less restriction enzyme was used: 15 μL was used for 10-15 million cells, 8 μL for 5 million cells, and 4 μL for 1 million cells. Heat (62° C. for 20 minutes) was used to inactivate Mbol.

iii. Biotin Incorporation and Proximity Ligation

To fill in the restriction fragment overhangs and mark the DNA ends with biotin, 52 μL of fill-in master mix was reacted by combining 37.5 μL of 0.4 mM biotin-dATP (Thermo 19524016); 1.5 μL of 10 mM dCTP, dGTP, and dTTP; and 10 μL of 5U/μL DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210). The mixture was incubated at 37° C. for 1 hour with rotation.

948 μL of ligation master mix was added. Ligation Master Mix contained 150 μL of 10× NEB T4 DNA ligase buffer with 10 mM ATP (NEB, B0202); 125 μL of 10% Triton X-100; 3 μL of 50 mg/mL BSA; 10 μL of 400 U/μL T4 DNA Ligase (NEB, M0202); and 660 μL of water. The mixture was incubated at room temperature for 4 hours with rotation. The nuclei were pelleted at 2500 g for 5 minutes, and the supernatant was removed.

iv. Sonication

For sonication, the pellet was brought up to 1000 μL in Nuclear Lysis Buffer. The sample was transferred to a Covaris millitube, and the DNA was sheared using a Covaris® E220Evolution™ with the manufacturer recommended parameters. Each tube (15 million cells) was sonicated for 4 minutes under the following conditions: Fill Level 5; Duty Cycle 5%; PIP 140; and Cycles/Burst 200.

v. Preclearing, Immunoprecipitation, IP Bead Capture, and Washes

The sample was clarified for 15 minutes at 16,100 g at 4° C. The sample was split into 2 tubes of about 400 μL each and 750 μL of ChIP Dilution Buffer was added. For the Smc1a antibody (Bethyl A300-055A), the sample was diluted 1:2 in ChIP Dilution Buffer to achieve an SDS concentration of 0.33%. 60 μL of Protein G beads were washed for every 10 million cells in ChIP Dilution Buffer. Amounts of beads (for preclearing and capture) and antibodies were adjusted linearly for different amounts of cell starting material. Protein G beads were resuspended in 50 μL of Dilution Buffer per tube (100 μL per HiChIP). The sample was rotated at 4° C. for 1 hour. The samples were put on a magnet, and the supernatant was transferred into new tubes. 7.5 μg of antibody was added for every 10 million cells, and the mixture was incubated at 4° C. overnight with rotation. Another 60 μL of Protein G beads for every 10 million cells in ChIP Dilution Buffer was added. Protein G beads were resuspended in 50 μL of Dilution Buffer (100 μL per HiChIP), added to the sample, and rotated at 4° C. for 2 hours. The beads were washed three times each with Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffer. Washing was performed at room temperature on a magnet by adding 500 μL of a wash buffer, swishing the beads back and forth twice by moving the sample relative to the magnet, and then removing the supernatant

vi. ChIP DNA Elution

ChIP sample beads were resuspended in 100 μL of fresh DNA Elution Buffer. The sample beads were incubated at RT for 10 minutes with rotation, followed by 3 minutes at 37° C. with shaking. ChIP samples were placed on a magnet, and the supernatant was removed to a fresh tube. Another 100 μL of DNA Elution Buffer was added to ChIP samples and incubations were repeated. ChIP sample supernatants were removed again and transferred to a new tube. There was about 200 μL of ChIP sample. Ten μL of Proteinase K (20 mg/ml) was added to each sample and incubated at 55° C. for 45 minutes with shaking. The temperature was increased to 67° C., and the samples were incubated for at least 1.5 hours with shaking. The DNA was Zymo-purified (Zymo Research, #D4014) and eluted into 10 μL of water. Post-ChIP DNA was quantified to estimate the amount of Tn5 needed to generate libraries at the correct size distribution. This assumed that contact libraries were generated properly, samples were not over sonicated, and that material was robustly captured on streptavidin beads. SMC1 HiChIP with 10 million cells had an expected yield of post-ChIP DNA from 15 ng-50 ng. For libraries with greater than 150 ng of post-ChIP DNA, materials were set aside and a maximum of 150 ng was taken into the biotin capture step

vii. Biotin Pull-Down and Preparation for Illumina Sequencing

To prepare for biotin pull-down, 5 μL of Streptavidin C-1 beads were washed with Tween Wash Buffer. The beads were resuspended in 10 μL of 2× Biotin Binding Buffer and added to the samples. The beads were incubated at RT for 15 minutes with rotation. The beads were separated on a magnet, and the supernatant was discarded. The beads were washed twice by adding 500 μL of Tween Wash Buffer and incubated at 55° C. for 2 minutes while shaking. The beads were washed in 100 μL of 1× (diluted from 2×) TD Buffer. The beads were resuspended in 25 μL of 2× TD Buffer, 2.5 μL of Tn5 for each 50 ng of post-ChIP DNA, and water to a volume of 50 μL.

The Tn5 had a maximum amount of 4 μL. For example, for 25 ng of DNA transpose, 1.25 μL of Tn5 was added, while for 125 ng of DNA transpose, 4 μL of Tn5 was used. Using the correct amount of Tn5 resulted in proper size distribution. An over-transposed sample had shorter fragments and exhibited lower alignment rates (when the junction was close to a fragment end). An undertransposed sample has fragments that are too large to cluster properly on an Illumina sequencer. The library was amplified in 5 cycles and had enough complexity to be sequenced deeply and achieve proper size distribution regardless of the level of transposition of the library.

The beads were incubated at 55° C. with interval shaking for 10 minutes. Samples were placed on a magnet, and the supernatant was removed. Fifty mM EDTA was added to samples and incubated at 50° C. for 30 minutes. The samples were then quickly placed on a magnet, and the supernatant was removed. The samples were washed twice with 50 mM EDTA at 50° C. for 3 minutes, then were removed quickly from the magnet. Samples were washed twice in Tween Wash Buffer for 2 minutes at 55° C., then were removed quickly from the magnet. The samples were washed with 10 mM Tris-HCl, pH8.0.

viii. PCR and Post-PCR Size Selection

The beads were resuspended in 50 μL of PCR master mix (use Nextera XT DNA library preparation kit from Illumina, #15028212 with dual-Index adapters # 15055289). PCR was performed using the following program. The cycle number was estimated using one of two methods: (1) A first run of 5 cycles (72° C. for 5 minutes, 98° C. for 1 minute, 98° C. for 15 seconds, 63° C. for 30 seconds, 72° C. for 1 minute) was performed on a regular PCR and then the product was removed from the beads. Then, 0.25× SYBR green was added, and the sample was run on a qPCR. Samples were pulled out at the beginning of exponential amplification; or (2) Reactions were run on a PCR and the cycle number was estimated based on the amount of material from the post-ChIP Qubit (greater than 50 ng was run in 5 cycles, while approximately 50 ng was run in 6 cycles, 25 ng was run in 7 cycles, 12.5 ng was run in 8 cycles, etc.).

Libraries were placed on a magnet and eluted into new tubes. The libraries were purified using a kit form Zymo Research and eluted into 10 μL of water. A two-sided size selection was performed with AMPure XP beads. After PCR, the libraries were placed on a magnet and eluted into new tubes. Then, 25 μL of AMPure XP beads were added, and the supernatant was kept to capture fragments less than 700 bp. The supernatant was transferred to a new tube, and 15 μL of fresh beads were added to capture fragments greater than 300 bp. A final elution was performed from the Ampure XP beads into 10 μL of water. The library quality was verified using a Bioanalyzer.

ix. Buffers

Hi-C Lysis Buffer (10 mL) contained 100 μL of 1M Tris-HCl pH 8.0; 20 μL of 5M NaCl; 200 μL of 10% NP-40; 200 μL of 50× protease inhibitors; and 9.68 mL of water. Nuclear Lysis Buffer (10 mL) contained 500 μL of 1M Tris-HCl pH 7.5; 200 μL of 0.5M EDTA; 1 mL of 10% SDS; 200 μL of 50× Protease Inhibitor; and 8.3 mL of water. ChIP Dilution Buffer (10 mL) contained 10 μL of 10% SDS; 1.1 mL of 10% Triton X-100; 24 μL of 500 mM EDTA; 167 μL of 1M Tris pH 7.5; 334 μL of 5M NaCl; and 8.365 mL of water. Low Salt Wash Buffer (10 mL) contained 100 μL of 10% SDS; 1 mL of 10% Triton X-100; 40 μL of 0.5M EDTA; 200 μL of 1M Tris-HCl pH 7.5; 300 μL of 5M NaCl; and 8.36 mL of water. High Salt Wash Buffer (10 mL) contained 100 μL of 10% SDS; 1 mL of 10% Triton X-100; 40 μL of 0.5M EDTA; 200 μL of 1M Tris-HCl pH 7.5; 1 mL of 5M NaCl; and 7.66 mL of water. LiCl Wash Buffer (10 mL) contained 100 μL of 1M Tris pH 7.5; 500 μL of 5M LiCl; 1 mL of 10% NP-40; 1 mL of 10% Na-deoxycholate; 20 μL of 0.5M EDTA; and 7.38 mL of water.

DNA Elution Buffer (SmL) contained 250 μL of fresh 1M NaHCO₃; 500 μL of 10% SDS; and 4.25 mL of water. Tween Wash Buffer (50 mL) contained 250 μL of 1M Tris-HCl pH 7.5; 50 μL of 0.5M EDTA; 10 mL of 5M NaCl; 250 μL of 10% Tween-20; and 39.45 mL of water. 2× Biotin Binding Buffer (10 mL) contained 100 μL 1M Tris-HCl pH 7.5; 20 μL of 0.5M; 4 mL of 5M NaCl; and 5.88 mL of water. 2× TD Buffer (1 mL) contains 20 μL of 1M Tris-HCl pH 7.5; 10 μL of 1M MgCl₂; 200 μL of 100% Dimethylformamide; and 770 μL of water.

M. Drug Dilutions for Administration to Hepatocytes

Prior to compound treatment of hepatocytes, 100 mM stock drugs in DMSO were diluted to 10 mM by mixing 0.1 mM of the stock drug in DMSO with 0.9 ml of DMSO to a final volume of 1.0 ml. Five μl of the diluted drug was added to each well, and 0.5 ml of media was added per well of drug. Each drug was analyzed in triplicate. Dilution to 1000× was performed by adding 5 μl of drug into 45 μl of media, and the 50 μl being added to 450 μl of media on cells.

Bioactive compounds were also administered to hepatocytes. To obtain 1000× stock of the bioactive compounds in 1 ml DMSO, 0.1 ml of 10,000× stock was combined with 0.9 ml DMSO.

Example 2
RNA-seq Study for Stimulated Hepatocytes

To identify small molecules that modulate urea cycle enzymes, primary human hepatocytes were prepared as a monoculture, and at least one small molecule compound was applied to the cells.

RNA-seq was performed to determine the effects of the compounds on the expression of urea cycle enzymes in hepatocytes. Fold change was calculated by dividing the level of expression in the cell system that had been perturbed by the level of expression in an unperturbed system. Changes in expression having a p-value≤0.05 were considered significant.

Compounds used to perturb the signaling centers of hepatocytes include at least one compound listed in Table 1. In the table, compounds are listed with their ID, target, pathway, and pharmaceutical action. Most compounds chosen as perturbation signals are known in the art to modulate at least one canonical cellular pathway. Some compounds were selected from compounds that failed in Phase III clinical evaluation due to lack of efficacy.

TABLE 1

Compounds used in RNA-seq

ID
Compound Name
Target
Pathway
Action

1
Simvastatin
HMG-CoA reductase
Metabolic
Inhibitor

2
Adapin (doxepin)
H₁histamine, α-adrenoreceptors
Histamine receptor signaling
Antagonist

4
Danazol
ER, AR, Progesteron receptor
Estrogen signaling
Agonist

5
Nefazodone
HTR2A
Calcium signaling
Antagonist

6
Rosiglitazone maleate
PPARg
PPAR signaling
Agonist

7
Sulpiride
D₂dopamine
cAMP signaling
Antagonist

8
Captopril
MMP2
Estrogen signaling
Inhibitor

9
atenolol
ADRB1
Adrenergic signaling
Antagonist

10
Ranitidine
H₂histamine receptor
Histamine receptor signaling
Antagonist

11
Metformin
AMPK
Insulin & AMPK signaling
Activator

12
imatinib
RTK, Bcr-Abl
PDGFR, ABL signaling
Inhibitor

13
Papaverine
phosphodiesterase
AMPK signaling
Inhibitor

14
Amiodarone
Adrenergic receptor β, CYP
Adrenergic signaling
Antagonist

15
Nitrofurantoin
pyruvate-flavodoxin
antibiotic
Activator

oxidoreductase

16
prednisone
GR
GR signaling
Agonist

17
Penicillamine(D-)
copper
copper chelation
Chelator

18
Disopyramide
SCN5A
Adrenergic signaling
Inhibitor

19
Rifampicin
PXR
PXR
Inhibitor

20
Benzbromarone
xanthine oxidase, CYP2C9
uric acid formation
Inhibitor

21
isoniazid
CYP2C19, CYP3A4
unknown
Inhibitor

22
Acetaminophen
COX1/2
COX
Inhibitor

(paracetamol)

23
Ritonavir
CYP3A4, Pol polyprotein
HIV Transcription
Inhibitor

24
SGI-1776
PIM
JAK/STAT signaling
Inhibitor

25
Valproate
HDAC9, glucuronyl transferase,
unknown
Inhibitor

epoxide hydrolase

26
Ibuprofen
COX, PTGS2
COX
Inhibitor

27
Propylthiouracil
thyroperoxidase
Thyroid hormone synthesis
Inhibitor

28
rapamycin
mTOR
mTOR signaling
Inhibitor

29
BIO
GSK-3
WNT, TGF beta signaling
Inhibitor

30
ATRA
RXRb, RXRg, RARg
RAR signaling
Agonist

31
Xav939
tankyrase
WNT & PARP pathway
Inhibitor

32
bms189453
RARB
Nuclear Receptor transcription
Agonist

33
dorsomorphin
ALK
TGF beta signaling
Inhibitor

34
BMP2
BMPR1A
TGF beta signaling
Agonist

35
BMS777607
Met
Ras signaling
Inhibitor

36
bms833923
SMO
Hedgehog signaling
Antagonist

37
dmPGE2
EPR, PGDH
EP receptor signaling
Agonist

38
MK-0752
y-secretase
NOTCH signaling
Inhibitor

39
N-Acetylpurinomycin
SnoN, SKI, SKIL
TGF beta signaling
Modulator

40
LY 364947
TGF-β RI, TGFR-I, TβR-I,
TGF beta signaling
Inhibitor

ALK-5

41
Enzastaurin
PKC
Epigenetics; TGF-beta/Smad
Inhibitor

42
DMXAA
Unclear
Tumor necrosis
Inhibitor

43
BSI-201
PARP
Cell Cycle/DNA Damage;
Inhibitor

Epigenetics

44
Darapladib
Phospholipase
Others
Inhibitor

45
Selumetinib
MEK
MAPK/ERK Pathway
Inhibitor

46
Peramivir (trihydrate)
Influenza Virus
Anti-infection
Inhibitor

47
Palifosfamide
DNA alkylator/crosslinker
Cell Cycle/DNA Damage

48
Evacetrapib
CETP
Others
Inhibitor

49
Cediranib
VEGFR
Protein Tyrosine Kinase/RTK
Inhibitor

50
R788 (fostamatinib,
Syk
Protein Tyrosine Kinase/RTK
Inhibitor

disodium hexahydrate)

51
Torcetrapib
CETP
Others
Inhibitor

52
Tivozanib
VEGFR
Protein Tyrosine Kinase/RTK
Inhibitor

53
17-AAG
HSP
Cell Cycle/DNA Damage
Inhibitor

(Tanespimycin)

Metabolic Enzyme/Protease

54
Zibotentan
Endothelin Receptor
GPCR/G protein
Antagonist

55
Semagacestat
γ-secretase
Neuronal Signaling Stem
Inhibitor

Cells/Wnt

56
Dalcetrapib
CETP
Others
Inhibitor

57
Latrepirdine
AMPK
Epigenetics; PI3K/Akt/mTOR
Activator

(dihydrochloride)

58
CMX001
CMV
Anti-infection
NA

(Brincidofovir)

59
Vicriviroc (maleate)
CCR
GPCR/G protein;
Antagonist

Immunology/Inflammation

60
Temsirolimus
mTOR
PI3K/Akt/mTOR
Inhibitor

61
Preladenant
Adenosine Receptor
GPCR/G protein
Antagonist

62
EVP-6124
nAChR
Membrane Transporter/Ion
Activator

(hydrochloride)

Channel

(encenicline)

63
Bitopertin
GlyT1
Membrane Transporter/Ion
Inhibitor

Channel

64
Latrepirdine
AMPK
Epigenetics; PI3K/Akt/mTOR
Inhibitor

65
Vanoxerine
Dopamine Reuptake Inhibitor
Neuronal Signaling
Inhibitor

(dihydrochloride)

66
CO-1686 (Rociletinib)
EGFR
JAK/STAT Signaling Protein
Inhibitor

Tyrosine Kinase/RTK

67
Laropiprant
Prostaglandin Receptor
GPCR/G protein
Antagonist

(tredaptive)

68
Bardoxolone
Keapl-Nrf2
NF-κB
Activator

69
VX-661 (tezacaptor)
CFTR
Membrane transporter/ion
Corrector

channel

70
INNO-206
Topoisomerase
Cell Cycle/DNA Damage
NA

(aldoxorubicin)

71
LY404039
mGluR
GPCR/G protein
Inhibitor

(pomaglumetad

methionil (mGlu2/3))

72
Perifosine
AKT
PI3K/AKT
Inhibitor

(KRX-0401)

73
Cabozantinib (XL184,
VEGFR2, MET, Ret, Kit, Flt-
MET
Inhibitor

BMS-907351)
1/3/4, Tie2, and AXL

74
Dacomitinib
EGFR, ErbB2, ErbB4
AKT/ERK, HER
Inhibitor

(PF299804, PF299)

75
Pacritinib (SB1518)
FLT3, JAK2, TYK2, JAK3,
JAK-STAT
Inhibitor

JAK1

76
TH-302
hypoxic regions
Unclear
NA

(Evofosfamide)

77
α-PHP
Unclear
Unclear
NA

78
LY 2140023
mGlu₂& mGlu₃
Gαi/o protein-dependent
Activator

(Pomaglumetad

methionil-LY404039)

79
TP-434 (Eravacycline)
Antibiotic resistance
Tetracycline-specific efflux
Inhibitor

mechanisms

80
TC-5214 (S-(+)-
Nicotinic acetylcholine receptors
Base excision repair and
Antagonist

MecaMylaMine

homologous recombination

Hydrochloride)

repair

81
Rolofylline
A1 adenosine receptor
Unclear
Antagonist

(KW-3902)

82
Amigal
a-galactosidase
Stress signaling
Inhibitor

(Deoxygalactonojirimycin

hydrochloride)

83
NOV-002 (oxidized L-
gamma-glutamyl-transpeptidase
Glutathione pathway
NA

Glutathione)
(GGT)

84
bms-986094 (inx-189)
NS5B
Unclear
Inhibitor

85
TC-5214 (R-
Nicotinic receptors
Base excision repair and
Antagonist

Mecamylamine

homologous recombination

hydrochloride)

repair

86
Ganaxolone
GBAA receptors
Unclear
Modulator

87
Irinotecan
DNA Topo I
Unclear
Inhibitor

Hydrochloride

Trihydrate

88
TFP
D2R, Calmodulin
Calmodulin
Inhibitor

89
Perphenazine
D2R, Calmodulin
Calmodulin
Inhibitor

90
A3-HCl
CKI, CKII, PKC, PKA
WNT, Hedgehog, PKC, PKA
Inhibitor

91
FICZ
Aryl hydrocarbon receptor
Aryl hydrocarbon receptor
Agonist

92
Pifithrin-a
p53
p53
Inhibitor

93
Deferoxamine
HIF
Hypoxia activated
Inhibitor

mesylate

94
Insulin
InsR
IGF-1R/InsR
Activator

95
Phorbol 12,13-
PKC
PKC
Activator

dibutyrate

96
RU 28318
MR
Mineralcorticoid receptor
Antagonist

97
Bryostatin1
PKC
PKC
Activator

98
DY 268
FXR
FXR
Antagonist

99
GW 7647
PPARα
PPAR
Agonist

100
CI-4AS-1
AR
Androgen receptor
Agonist

101
T0901317
LXR
LXR
Agonist

102
BMP2
BMPR1A
TGF-B
Activator

103
22S-
LXR
LXR
Inhibitor

Hydroxycholesterol

104
CALP1
Calmodulin
Calmodulin
Activator

105
CALP3
Calmodulin
Calmodulin
Activator

106
Forskolin
Adenylyl cyclase
cAMP related
Activator

107
Dexamethasone
GR
Glucocorticoid receptor
Activator

108
IFN-y
IFNGR1/IFNGR2
JAK/STAT
Activator

109
TGF-b
TGF-beta Receptor
TGF-B
Activator

110
TNF-α
TNF-R1/TNF-R2
NF-kB, MAPK, Apoptosis
Activator

111
PDGF
Pan-PDGFR
PDGFR
Activator

112
IGF-1
IGF-1R
IGF-1R/InsR
Activator

113
FGF
FGFR
FGFR
Activator

114
EGF
Pan-ErbB
EGFR
Activator

115
HGF/SF
c-Met
c-MET
Activator

116
TCS 359
FLT3
Protein Tyrosine Kinase/RTK
Inhibitor

117
Cobalt chloride
HIF1
Hypoxia activated
Inducer

118
CH223191
AhR
Aryl hydrocarbon receptor
Antagonist

119
Echinomycin
HIF
Hypoxia activated
Inhibitor

120
PAF C-16
MEK
MAPK
Activator

121
Bexarotene
RXR
RXR
Agonist

122
CD 2665
RAR
RAR
Antagonist

123
Pifithrin-μ
p53
p53
Inhibitor

124
EB1089
VDR
Vitamin D Receptor
Agonist

125
BMP4
TGF-beta
TGF-B
Activator

126
IWP-2
Wnt
WNT
Inhibitor

127
RITA (NSC 652287)
p53
p53
Inhibitor

128
Calcitriol
VDR
Vitamin D Receptor
Agonist

129
ACEA
CB1
Cannabinoid receptor
Agonist

130
Rimonabant
CB1
Cannabinoid receptor
Antagonist

131
Otenabant
CB1
Cannabinoid receptor
Antagonist

132
DLPC
LRH-1/NR5A2
LHR-1
Agonist

133
LRH-1 antagonist
LRH-1/NR5A3
LHR-1
Antagonist

134
Wnt3a
FRIZZLED
WNT
Activator

135
Activin
TGF-beta
TGF-B
Activator

136
Nodal
TGF-beta
TGF-B
Activator

137
Anti mullerian
TGF-beta
TGF-B
Activator

hormone

138
GDF2 (BMP9)
TGF-beta
TGF-B
Activator

139
GDF10 (BMP3b)
TGF-beta
TGF-B
Activator

140
Oxoglaucine
PI3K/Akt
PI3K/AKT
Activator

141
BMS 195614
RAR
RAR
Antagonist

142
LDN193189
ALK2/3
TGF-B
Inhibitor

143
Varenicline Tartrate
AchR
Acetylcholine receptor
Agonist

144
Histamine
Histamine receptor
Histamine receptor
Activator

145
Chloroquine
ATM/ATR
ATM/ATR
Activator

phosphate

146
LJI308
RSK1/2/3
S6K
Inhibitor

147
GSK621
AMPK
AMPK
Activator

148
STA-21
STAT3
JAK/STAT
Inhibitor

149
SMI-4a
Pim1
PIM
Inhibitor

150
AMG 337
c-Met
c-MET
Inhibitor

151
Wnt agonist 1
Wnt
WNT
Activator

152
PRI-724
Wnt
WNT
Inhibitor

153
ABT-263
Pan-Bcl-2
BCL2
Inhibitor

154
Axitinib
Pan-VEGFR
VEGFR
Inhibitor

155
Afatinib
Pan-ErbB
EGFR
Inhibitor

156
Bosutinib
Src
Src
Inhibitor

157
Dasatinib
Bcr-Abl
ABL
Inhibitor

158
Masitinib
c-Kit
c-KIT
Inhibitor

159
Crizotinib
c-Met
c-MET
Inhibitor

160
PHA-665752
c-Met
c-MET
Inhibitor

161
GSK1904529A
IGF-1R/InsR
IGF-1R/InsR
Inhibitor

162
GDC-0879
Raf
MAPK
Inhibitor

163
LY294002
Pan-PI3K
PI3K/AKT
Inhibitor

164
OSU-03012
PDK-1
PDK-1
Inhibitor

165
JNJ-38877605
c-Met
c-MET
Inhibitor

166
BMS-754807
IGF-1R/InsR
IGF-1R/InsR
Inhibitor

167
TGX-221
p110b
PI3K/AKT
Inhibitor

168
Regorafenib
Pan-VEGFR
VEGFR
Inhibitor

169
Thalidomide
AR
NF-kB
Antagonist

170
Amuvatinib
PDGFRA
PDGFR
Inhibitor

171
Etomidate
GABA
GABAergic receptor
Inhibitor

172
Glimepiride
Potassium channel
Potassium channel
Inhibitor

173
Omeprazole
Proton pump
Proton pump
Agonist

174
Tipifarnib
Ras
RAS
Inhibitor

175
SP600125
Jnk
MAPK
Inhibitor

176
Quizartinib
FLT3
FLT3
Inhibitor

177
CP-673451
Pan-PDGFR
PDGFR
Inhibitor

178
Pomalidomide
TNF-α
NF-kB
Inhibitor

179
KU-60019
ATM kinase
DNA Damage
Inhibitor

180
BIRB 796
p38
MAPK
Inhibitor

181
RO4929097
Gamma-secretase
NOTCH
Inhibitor

182
Hydrocortisone
GR
Glucocorticoid receptor
Agonist

183
AICAR
AMPK
AMPK
Activator

184
Amlodipine Besylate
Calcium channel
Calcium channel
Inhibitor

185
DPH
Bcr-Abl
ABL
Activator

186
Taladegib
Hedgehog/Smoothened
Hedgehog/Smoothened
Inhibitor

187
AZD1480
JAK2
JAK/STAT
Inhibitor

188
AST-1306
Pan-ErbB
EGFR
Inhibitor

189
AZD8931
Pan-ErbB
EGFR
Inhibitor

190
Momelotinib
Pan-Jak
JAK/STAT
Inhibitor

191
Cryptotanshinone
STAT3
JAK/STAT
Inhibitor

192
Bethanechol chloride
AchR
Acetylcholine receptor
Activator

193
Clozapine
5-HT
5-HT
Antagonist

194
Dopamine
Dopamine
Dopamine receptor
Agonist

195
Phenformin
AMPK
AMPK
Activator

196
Mifepristone
GR
Glucocorticoid receptor
Antagonist

197
GW3965
LXR
LXR
Agonist

198
WYE-125132
mTOR
mTOR
Inhibitor

(WYE-132)

199
Crenolanib
Pan-PDGFR
PDGFR
Inhibitor

200
PF-04691502
Pan-Akt
PI3K/AKT
Inhibitor

201
GW4064
FXR
FXR
Agonist

202
Sotrastaurin
PKC
PKC
Inhibitor

203
Ipatasertib
Pan-Akt
PI3K/AKT
Inhibitor

204
ARN-509
AR
Androgen receptor
Inhibitor

205
T0070907
PPARg
PPAR
Antagonist

206
GO6983
PKC
PKC
Inhibitor

207
Epinephrine
Adrenergic
Adrenergic receptor
Agonist

208
Eletriptan
5-HT
5-HT
Agonist

209
Trifluoperazine
Dopamine
Dopamine receptor
Inhibitor

210
Fexofenadine
Histamine
Histamine receptor
Inhibitor

211
Deoxycorticosterone
MR
Mineralcorticoid receptor
Agonist

212
Tamibarotene
RAR
RAR
Agonist

213
Leucine
mTOR
mTOR
Activator

214
Glycopyrrolate
AchR
Acetylcholine receptor
Antagonist

215
Tiagabine
GABA
GABAergic receptor
Inhibitor

216
Fluoxymesterone
AR
Androgen receptor
Agonist

217
Tamsulosin
Adrenergic
Adrenergic receptor
Antagonist

hydrochloride

218
Ceritinib
ALK
ALK
Inhibitor

219
GSK2334470
PDK-1
PDK-1
Inhibitor

220
AZD1208
Pan-PIM
PIM
Inhibitor

221
CGK733
ATM/ATR
DNA Damage
Inhibitor

222
LDN-212854
Pan-TGFB
TGF-B
Inhibitor

223
GZD824 Dimesylate
Bcr-Abl
ABL
Inhibitor

224
AZD2858
Pan-GSK-3
GSK-3
Inhibitor

225
FRAX597
PAK
PAK
Inhibitor

226
SC75741
NF-kB
NF-kB
Inhibitor

227
SH-4-54
Pan-STAT
JAK/STAT
Inhibitor

228
HS-173
p110a
PI3K/AKT
Inhibitor

229
K02288
Pan-TGFB
TGF-B
Inhibitor

230
EW-7197
Pan-TGFB
TGF-B
Inhibitor

231
Decernotinib
Pan-Jak
JAK/STAT
Inhibitor

232
MI-773
p53
p53
Inhibitor

233
PND-1186
FAK
FAK
Activator

234
Kartogenin
SMAD4/5
TGF-B
Activator

235
Picropodophyllin
IGF-1R
IGF-1R/InsR
Inhibitor

236
AZD6738
ATR
ATM/ATR
Inhibitor

237
Smoothened Agonist
Hedgehog/Smoothened
Hedgehog/Smoothened
Agonist

238
Erlotinib
EGFR/ErbB1
EGFR
Inhibitor

239
MHY1485
mTOR
mTOR
Activator

240
SC79
Pan-Akt
PI3K/AKT
Activator

241
meBIO
AhR
Aryl hydrocarbon receptor
Agonist

242
Huperzine
AchE
Acetylcholine receptor
Inhibitor

243
BGJ398
Pan-FGFR
FGFR
Inhibitor

244
Netarsudil
ROCK
ROCK
Inhibitor

245
Acetycholine
AchR
Acetylcholine receptor
Agonist

246
Purmorphamine
Hedgehog/Smoothened
Hedgehog/Smoothened
Agonist

247
LY2584702
p70 S6K
S6K
Inhibitor

248
Dorsomorphin
AMPK
AMPK
Inhibitor

249
Glasdegib
Hedgehog/Smoothened
Hedgehog/Smoothened
Inhibitor

(PF-04449913)

250
LDN193189
Pan-TGFB
TGF-B
Inhibitor

251
Oligomycin A
ATPase
ATP channel
Inhibitor

252
BAY 87-2243
HIF1
Hypoxia activated
Inhibitor

253
SIS3
SMAD3
TGF-B
Inhibitor

254
BDA-366
Bcl-2
BCL2
Antagonist

255
XMU-MP-1
MST1/2
Hippo
Inhibitor

256
Semaxanib
Pan-VEGFR
VEGFR
Inhibitor

257
BAM7
Bcl-2
BCL2
Activator

258
GDC-0994
Erk
MAPK
Inhibitor

259
SKL2001
Wnt
WNT
Agonist

260
Merestinib
c-Met
c-MET
Inhibitor

261
APS-2-79
MEK
MAPK
Antagonist

262
NSC228155
Pan-ErbB
EGFR
Activator

263
740 Y-P
Pan-PI3K
PI3K/AKT
Activator

264
b-Estradiol
ER
ER
Activator

265
Glucose
GLUTs
metabolic/glycolysis
Activator

266
Transferrin
Transferrin Receptor
Iron transport
Activator

267
AM 580
RAR
RAR
Activator

Example 3
Identification of Compounds that Modulate Expression of Urea Cycle Enzymes

Analysis of RNA-seq data revealed a number of compounds that caused significant changes in the expression of CPS1, OTC, ASS1, ASL, and/or NAGS. Significance was defined as an FPKM≥1, a log2 (fold change)≥0.5, and a q-value of≤0.05 for selected gene target. RNA-seq results for compounds that significantly modulated at least one target gene are shown in Tables 2-10. Table 2 provides the log2 fold change for compounds that were observed to significantly increase the expression of CPS1, which is associated with CPS deficiency.

TABLE 2

CPS1 expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

157
Dasatinib
6.44

50
R788 (fostamatinib
5.93

disodium hexahydrate)

156
Bosutinib
5.06

207
Epinephrine
4.85

225
FRAX597
4.61

260
Merestinib
3.96

211
Deoxycorticosterone
3.79

53
17-AAG (Tanespimycin)
3.73

138
GDF2 (BMP9)
3.46

223
GZD824 Dimesylate
3.39

233
PND-1186
2.85

134
Wnt3a
2.6

136
Nodal
2.44

137
Anti mullerian hormone
2.35

110
TNF-α
2.31

135
Activin
2.23

112
IGF-1
2.22

16
prednisone
2.17

111
PDGF
2.12

115
HGF/SF
2.02

114
EGF
1.93

252
BAY 87-2243
1.81

177
CP-673451
1.77

113
FGF
1.72

139
GDF10 (BMP3b)
1.66

250
LDN193189
1.56

170
Amuvatinib
1.46

190
Momelotinib
1.46

119
Echinomycin
1.45

75
Pacritinib (SB1518)
1.33

102
BMP2
1.31

159
Crizotinib
1.08

222
LDN-212854
1.08

169
Thalidomide
1.02

66
CO-1686 (Rociletinib)
0.89

54
Zibotentan
0.81

Table 3 provides the log2 fold change for compounds that were observed to significantly increase the expression of OTC, which is associated with OTC deficiency.

TABLE 3

OTC expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

177
CP-673451
2.63

75
Pacritinib (SB1518)
2.33

119
Echinomycin
2.08

199
Crenolanib
1.23

169
Thalidomide
1.21

170
Amuvatinib
1.15

157
Dasatinib
1.06

190
Momelotinib
0.95

135
Activin
0.94

134
Wnt3a
0.92

70
INNO-206
0.87

(aldoxorubicin)

110
TNF-α
0.87

137
Anti mullerian hormone
0.81

123
Pifithrin-μ
0.79

111
PDGF
0.77

112
IGF-1
0.76

225
FRAX597
0.69

136
Nodal
0.68

114
EGF
0.64

113
FGF
0.56

115
HGF/SF
0.56

180
BIRB 796
0.56

Table 3A provides additional data for compounds that were observed to increase the expression of OTC, which is associated with OTC deficiency. Compounds assayed at 10 uM final concentration, except for compounds HSP-990 and Retaspimycin Hydrochloride which were assayed at 1 uM final concentration.

TABLE 3A

OTC expression modulated by compounds

Max Value

(OTC/Geo

Mean of

HKs;

ID
Name/Company
MoA
DMSO = 1)

248
Dorsomorphin
AMPK and BMPR1
4.78

600
Foretinib
c-Met, VEGFR
4.09

(GSK1363089)

327
CCCP
TBK1/IRF3
4.06

190
Momelotinib
JAK
3.52

630
PF-00562271
FAK
3.3

626
Mubritinib (TAK 165)
HER2
3.23

316
XL228
BCR-Abl, IGF-1R
2.92

551
Sunitinib
PDGFR
2.62

180
BIRB 796
p38 MAPK & JNK2
2.61

326
BX795
TBK1
2.57

70
Aldoxorubicin
Doxorubicin prodrug
2.36

673
BMS-214662
Farnesyl
2.27

Transferase Inhibitor

654
Lifirafenib (BGB-283)
Raf, EGFR
2.26

685
Pamapimod
p38 MAPK
1.73

323
PF-04929113
HSP90 & HER2
1.73

(SNX-5422)

53
17-AAG
HSP90 & HER2
1.69

320
BIIB021
HSP90
1.66

277
Baricitinib
JAK 1 & 2
1.66

170
Amuvatinib
PDGFA
3.21

199
Crenolanib
Pan-PDGFR
2.79

497
Pazopanib
c-Kit, PDGFR, VEGFR
1.48

322
HSP-990
HSP90
1.5

(compound

tested at

1 uM)

925
Retaspimycin
HSP90 ATPase
1.48

Hydrochloride

(compound

tested at

1 uM)

187
AZD1480
JAK2
1.46

325
MRT67307
TBK1
1.68

68
Bardoxolone
NF-kB
2.76

177
CP-673451
Pan-PDGFR
3.97

313
Tofacitinib citrate
JAK1 & 2
1.6

311
LY2784544
JAK2
2.78

684
R1487 Hydrochloride
p38 MAPK
1.64

52
Tivozanib
VEGFR 1/2/3
1.57

268
Regorafenib
Pan-VEGFR
1.54

300
GLPG0634
Pan-JAK
1.54

632
PH-797804
p38 MAPK
1.53

55
Semagacestat
β-amyloid & Notch
1.5

71
Pomaglumetad
mGlu2/mGlu3 (agonist
1.49

253
SIS3
SMAD3 (TGFB)
1.46

587
Linifanib (ABT-869)
CSF-1R, PDGFR, VEGFR
2.87

609
Regorafenib
VEGFR, PDGFRβ
2.07

(BAY 73-4506)

631
TAK-901
Aurora Kinase
2.9

919
AS602801
JNK
2.75

(Bentamapimod)

603
YM155 (Sepantronium
Survivin
3.16

Bromide)

Example 4
Use of siRNA Agents to Up-Regulate OTC Expression

Primary human hepatocytes were reversed transfected with 6pmol siRNA using RNAiMAX Reagent (ThermoFisher Cat No13778030) in 24 well format, 1 ul per well (for a final cone of 10 nM). The following morning, the medium was removed and replaced with Modified Maintenance Medium (see above) for an additional 24 hrs. The entire treatment lasted 48hrs, at which point the medium was removed and replaced with RLT Buffer for RNA extraction (Qiagen RNeasy 96 QIAcube HT Kit Cat#74171). siRNAs were obtained from Dharmacon and are a pool of four siRNA duplex all designed to target distinct sites within the specific gene of interest (known as “SMARTpool”). The siRNA catalog numbers are listed in Table 3B, below. They were tested against the control non-targeting siRNA D-001206-13-05.

Isolated RNA was processed for cDNA synthesis and qPCR as described above. Taqman assay used for OTC measurement: Hs00166892_m1

TABLE 3B

siRNA Agents up-regulate OTC

Max fold

change in

siRNA
OTC
Catalog Number

JAK1
3.51
M-003145-02-0005

WWTR1
3.43
M-016083-00-0005

YAP1
2.93
M-046247-01-0005

CSF1R
2.80
D-003109-06

LYN
2.51
D-003153-03

SMAD3
2.49
M-020067-00-0005

NTRK1
2.25
D-003159-05

EPHB3
2.23
D-003123-09

EPHB4
2.21
D-003124-05

FGFR4
2.16
D-003134-05

INSR
2.13
D-003014-05

KDR
2.12
D-003148-05

FLT1
2.09
D-003136-05

FGFR2
2.07
D-003132-05

EPHB2
1.91
D-003122-07

PDGFRB
1.90
M-003163-03-0005

IRF5
1.90
M-011706-00-0005

FGFR1
1.87
D-003131-09

EPHB1
1.85
D-003121-05

FYN
1.81
D-003140-09

FLT4
1.71
D-003138-05

Yy1
1.71
M-011796-02-0005

IRF1
1.70
M-011704-01-0005

IGF-1
1.69
D-003012-06

SMAD1
1.65
M-012723-01-0005

DDR1
1.64
D-003111-08

HSP90AA1
1.52
M-005186-02-0005

SMAD2
1.44
M-003561-01-0005

Table 4 provides the log2 fold change for compounds that were observed to significantly increase the expression of ASS1, which is associated with ASS1 deficiency.

TABLE 4

ASS1 expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

157
Dasatinib
1.86

177
CP-673451
1.52

119
Echinomycin
1.44

138
GDF2 (BMP9)
1.34

75
Pacritinib (SB1518)
1.27

207
Epinephrine
1.08

225
FRAX597
1.06

156
Bosutinib
0.86

79
TP-434 (Eravacycline)
0.76

102
BMP2
0.75

149
SMI-4a
0.69

170
Amuvatinib
0.69

199
Crenolanib
0.64

211
Deoxycorticosterone
0.6

70
INNO-206
0.58

(aldoxorubicin)

110
TNF-α
0.58

101
T0901317
0.55

Table 5 provides the log2 fold change for compounds that were observed to significantly increase the expression of ASL, which is associated with ASL deficiency.

TABLE 5

ASL expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

177
CP-673451
1.81

119
Echinomycin
1.76

75
Pacritinib (SB1518)
1.45

157
Dasatinib
0.94

251
Oligomycin A
0.84

260
Merestinib
0.84

170
Amuvatinib
0.81

199
Crenolanib
0.76

207
Epinephrine
0.6

252
BAY 87-2243
0.58

169
Thalidomide
0.56

Table 6 provides the log2 fold change for compounds that were observed to significantly increase the expression of NAGS, which is associated with NAGS deficiency.

TABLE 6

NAGS expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

224
AZD2858
2.16

41
Enzastaurin
2.03

156
Bosutinib
2.02

256
Semaxanib
2

70
INNO-206
1.63

(aldoxorubicin)

79
TP-434 (Eravacycline)
1.5

195
Phenformin
1.38

159
Crizotinib
1.35

149
SMI-4a
1.25

157
Dasatinib
1.21

128
Calcitriol
1.2

123
Pifithrin-μ
1.18

160
PHA-665752
1.15

44
Darapladib
1.13

169
Thalidomide
1.1

66
CO-1686 (Rociletinib)
1.05

164
OSU-03012
1.03

16
prednisone
1

219
GSK2334470
1

155
Afatinib
0.98

52
Tivozanib
0.95

259
SKL2001
0.95

162
GDC-0879
0.85

62
EVP-6124
0.75

(hydrochloride)

(encenicline)

184
Amlodipine Besylate
0.73

101
T0901317
0.72

206
GO6983
0.7

135
Activin
0.69

198
WYE-125132 (WYE-132)
0.64

253
SIS3
0.64

138
GDF2 (BMP9)
0.63

95
Phorbol 12,13-dibutyrate
0.62

122
CD 2665
0.62

238
Erlotinib
0.62

218
Ceritinib
0.61

102
BMP2
0.6

88
TFP
0.58

115
HGF/SF
0.56

100
CI-4AS-1
0.55

Table 7 provides the log2 fold change for compounds that were observed to significantly increase the expression of ARG1, which is associated with Arginase deficiency.

TABLE 7

ARG1 expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

50
R788 (fostamatinib
3.74

disodium hexahydrate)

157
Dasatinib
3.29

177
CP-673451
3.21

260
Merestinib
2.7

119
Echinomycin
2.54

170
Amuvatinib
2.5

207
Epinephrine
2.28

156
Bosutinib
2.27

134
Wnt3a
2.07

137
Anti mullerian Hormone
2.06

136
Nodal
2.05

135
Activin
2.01

112
IGF-1
1.89

53
17-AAG (Tanespimycin)
1.87

110
TNF-a
1.87

123
Pifithrin-u
1.87

111
PDGF
1.85

75
Pacritinib (SB1518)
1.84

138
GDF2 (BMP9)
1.79

199
Crenolanib
1.7

16
prednisone
1.59

115
HGF/SF
1.51

190
Momelotinib
1.51

114
EGF
1.46

211
Deoxycorticosterone
1.37

113
FGF
1.24

169
Thalidomide
1.16

195
Phenformin
1.14

52
Tivozanib
1.1

252
BAY 87-2243
1.09

223
GZD824 Dimesylate
1.06

139
GDF10 (BMP3b)
0.97

233
PND-1186
0.93

225
FRAX597
0.82

102
BMP2
0.81

251
Oligomycin A
0.75

19
Rifampicin
0.72

38
MK-0752
0.71

Table 8 provides the log2 fold change for compounds that were observed to significantly increase the expression of SLC25A15, which is associated with ORNT1 deficiency.

TABLE 8

SLC25A15 expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

157
Dasatinib
2.76

225
FRAX597
2.51

260
Merestinib
2.01

50
R788 (fostamatinib
1.68

disodium hexahydrate)

156
Bosutinib
1.25

84
bms-986094 (inx-189)
1.18

207
Epinephrine
1.14

138
GDF2 (BMP9)
1.09

119
Echinomycin
0.96

211
Corticosterone
0.96

112
IGF-1
0.8

177
CP-673451
0.76

223
GZD824 Dimesylate
0.73

230
EW-7197
0.72

111
PDGF
0.71

134
Wnt3a
0.7

Table 9 provides the log2 fold change for compounds that were observed to significantly increase the expression of SLC25A13, which is associated with Citrin deficiency.

TABLE 9

SLC25A13 expression modulated by compounds

Fold change

(Log 2) vs

ID
Compound
untreated

88
TFP
0.57

53
17-AAG (Tanespimycin)
0.71

Table 10 provides the list of compounds that were observed to significantly increase expression of multiple urea cycle-related genes.

TABLE 10

Compounds that modulate multiple genes

ID
Compound
Pathway
Up-regulated Genes

157
Dasatinib
ABL
CPS1, ASS1, ASL, OTC, NAGS,

ARG1, SLC25A15

119
Echinomycin
Hypoxia
CPS1, ASS1, ASL, OTC, ARG1,

activated
SLC25A15

177
CP-673451
PDGFR
CPS1, ASS1, ASL, OTC, ARG1,

SLC25A15

138
GDF2 (BMP9)
TGF-B
ASS1, CPS1, NAGS, ARG1,

SLC25A15

156
Bosutinib
Src
ASS1, CPS1, NAGS, ARG1,

SLC25A15

207
Epinephrine
Adrenergic
CPS1, ASS1, ASL, ARG1,

receptor
SLC25A15

75
Pacritinib
JAK-STAT
CPS1, ASS1, ASL, OTC, ARG1

(SB1518)

170
Amuvatinib
PDGFR
CPS1, ASS1, ASL, OTC, ARG1

225
FRAX597
PAK
CPS1, ASS1, OTC, ARG1,

SLC25A15

169
Thalidomide
NF-kB
NAGS, CPS1, ALS, OTC, ARG1

199
Crenolanib
PDGFR
ASS1, ASL, OTC, ARG1

102
BMP2
TGF-B
ASS1, CPS1, NAGS, ARG1

211
Deoxy-
Mineral-
CPS1, ASS1, ARG1, SLC25A15

corticosterone
corticoid

receptor

110
TNF-α
NF-kB,
CPS1, ASS1, OTC, ARG1

MAPK,

Apoptosis

134
Wnt3a
WNT
CPS1, OTC, ARG1, SLC25A15

111
PDGF
PDGFR
CPS1, OTC, ARG1, SLC25A15

112
IGF-1
IGF-1R/InsR
CPS1, OTC, ARG1, SLC25A15

135
Activin
TGF-B
NAGS, CPS1, OTC, ARG1

115
HGF/SF
c-MET
NAGS, CPS1, OTC, ARG1

53
17-AAG
Cell Cycle/
CPS1, ARG1, SLC25A13

(Tanespimycin)
DNA

Damage;

Metabolic

Enzyme/

Protease

50
R788
Protein
CPS1, ARG1, SLC25A15

(fostamatinib
Tyrosine

disodium
Kinase/RTK

hexahydrate)

223
GZD824
ABL
CPS1, ARG1, SLC25A15

Dimesylate

252
BAY 87-2243
Hypoxia
CPS1, ASL, ARG1

activated

16
prednisone
GR
CPS1, NAGS, ARG1

136
Nodal
TGF-B
CPS1, OTC, ARG1

190
Momelotinib
JAK/STAT
CPS1, OTC, ARG1

113
FGF
FGFR
CPS1, OTC, ARG1

114
EGF
EGFR
CPS1, OTC, ARG1

137
Anti mullerian
TGF-B
CPS1, OTC, ARG1

hormone

70
INNO-206
Cell Cycle/
NAGS, ASS1, OTC

(aldoxorubicin)
DNA

Damage

123
Pifithrin-μ
p53
OTC, NAGS, ARG1

As shown above, Dasatinib was observed to significantly up-regulate the expression of seven urea cycle genes with log2 fold changes≥1. Echinomycin and CP-673451 were observed to significantly up-regulate the expression of six urea cycle genes. GDF2 (BMP9), Bosutinib, Epinephrine, Pacritinib (SB1518), Amuvatinib, FRAX597, and Thalidomide were observed to significantly up-regulate the expression of five urea cycle genes. Crenolanib, BMP2, Deoxycorticosterone, TNF-α, Wnt3a, PDGF, IGF-1, Activin, and HGF/SF were observed to significantly up-regulate the expression of four urea cycle genes. 17-AAG (Tanespimycin), R788 (fostamatinib disodium hexahydrate), GZD824 Dimesylate, BAY 87-2243, prednisone, Nodal, Momelotinib, FGF, EGF, Anti mullerian hormone, INNO-206 (aldoxorubicin), and Pifithrin-μ, were observed to significantly up-regulate the expression of three urea cycle genes.

Dasatinib is a novel, potent and multi-targeted inhibitor that targets Abl, Src and c-Kit. This suggest that inhibiting signaling molecules, particularly Abl, Src or c-Kit, in the Abl-, Src- or c-Kit-mediated signaling pathways may potentially up-regulate enzymes of the urea cycle.

Mubritinib (TAK-165) is a potent inhibitor of HER2/ErbB2 with IC₅₀of 6 nM in BT-474 cells.

XL228 is designed to inhibit the insulin-like growth factor type-1 receptor (IGF1R), Src and Abl tyrosine kinases—targets that play crucial roles in cancer cell proliferation, survival and metastasis.

Three identified compounds, Foretinib/XL880, Regorafenib, are known modulators of the Vascular endothelial growth factor receptor (VEGFR) family mediated signaling pathway. This suggest that modulating signaling molecules, particularly VEGFRs, in the VEGFR-mediated signaling pathway may potentially up-regulate enzymes of the urea cycle.

Six identified compounds, CP-673451, Amuvatinib, Crenolanib, Sunitinib, Amuvatinib, and PDGF, are known modulators of the Platelet-derived growth factor receptor (PDGFR)-mediated signaling pathway. This suggest that modulating signaling molecules, particularly PDGFRs, in the PDGFR-mediated signaling pathway may potentially up-regulate enzymes of the urea cycle.

Three identified compounds, Pazopanib, Linifanib, Regorafenib, are known modulators of the Platelet-derived growth factor receptor (PDGFR) and Vascular endothelial growth factor receptor (VEGFR) family mediated signaling pathway. This suggest that modulating signaling molecules, particularly PDGFRs, VEGFRs, in the PDGFR and VEGFR-mediated signaling pathway may potentially up-regulate enzymes of the urea cycle.

Five identified compounds, PF-04929113 (SNX-5422), 17-AAG, BIIB021, HSP-990, Retaspimycin Hydrochloride, are known modulators of the Heat shock protein 90 (HSP90) mediated signaling pathway. This suggest that modulating signaling molecules, particularly HSP90 may potentially up-regulate enzymes of the urea cycle.

Five identified compounds, GDF2 (BMP9), BMP2, Activin, Nodal and Anti mullerian hormone, Dorsomorphin are known modulators of the transforming growth factor-beta (TGF-B) signaling pathway. This suggest that modulating signaling molecules, particularly TGF-B and/or Bone morphogenetic protein receptor type-1A (BMPR1A), in the TGF-B signaling pathway may potentially up-regulate enzymes of the urea cycle.

Five identified compounds, Momelotinib, Baricitinib, GLPG0634, AZD1480 , LY2784544, TAK-901, Tofacitinib citrate , are known modulators of the JAK mediated signaling pathway. This suggest that modulating signaling molecules, particularly JAK/STAT may potentially up-regulate enzymes of the urea cycle.

Three identified compounds, CCCP, BX795, MRT67307 are known modulators of the TBK1/Ikke mediated signaling pathway. This suggest that modulating signaling molecules, particularly through TBK1 pathway may potentially upregulate urea cycle enzymes.

Seven identified compounds, AS602801 (Bentamapimod), PF-00562271, BIRB 796, Pamapimod, R1487 Hydrochloride, Bardoxolone, PH-797804, are known modulators of the MAPK mediated signaling pathway. This suggest that modulating signaling molecules, particularly MAPK may potentially up-regulate enzymes of the urea cycle.

Mubritinib (TAK-165) is a potent inhibitor of HER2/ErbB2 with IC50 of 6 nM in BT-474 cells.

Sunitinib inhibits cellular signaling by targeting multiple RTKs. These include all platelet-derived growth factor receptors (PDGF-R) and vascular endothelial growth factor receptors (VEGF-R). Sunitinib also inhibits KIT (CD117), the RTK that drives the majority of GISTs. In addition, sunitinib inhibits other RTKs including RET, CSF-1R, and flt3

Lifirafenib (BGB-283) potently inhibits RAF family kinases and EGFR activities in biochemical assays with IC50 values of 23, 29 and 495 nM for the recombinant BRAFV600E kinase domain, EGFR and EGFR T790M/L858R mutant.

Foretinib (GSK1363089) is an ATP-competitive inhibitor of HGFR and VEGFR, mostly for Met and KDR with IC₅₀of 0.4 nM and 0.9 nM in cell-free assays. Less potent against Ron, Flt-1/3/4, Kit, PDGFRα/β and Tie-2, and little activity to FGFR1 and EGFR.

BIIB021 is an orally available, fully synthetic small-molecule inhibitor of HSP90 with K_iand EC50 of 1.7 nM and 38 nM, respectively

NVP-HSP990 (HSP990) is a novel, potent and selective HSP90 inhibitor for HSP90α/β with IC₅₀of 0.6 nM/0.8 nM.

IPI-504 is a novel, water-soluble, potent inhibitor of heat-shock protein 90 (Hsp90

Baricitinib is a selective and reversible Janus kinase 1 (JAK1) and 2 (JAK2) inhibitor

Doramapimod (BIRB 796) is a pan-p38 MAPK inhibitor with IC50 of 38 nM, 65 nM, 200 nM and 520 nM for p38α/β/γ/δ in cell-free assays, and binds p38α with K_dof 0.1 nM in THP-1 cells, 330-fold greater selectivity versus JNK2.

Pamapimod is a potent inhibitor of p38α MAP kinase (IC₅₀=14 nM).¹It displays over 30-fold selectivity for p38α over p38β, has no activity against p38δ or p38γ, and has limited activity against a panel of 350 other kinases

Farnesyltransferase inhibitor BMS-214662 inhibits the enzyme farnesyltransferase and the post-translational farnesylation of number of proteins involved in signal transduction, which may result in the inhibition of Ras function and apoptosis in susceptible tumor cells.

The results also suggest that expression of urea cycle enzymes may be associated with other signaling pathways, such as the NF-kB signaling pathway, hypoxia activated signaling pathway, Src signaling pathway, c-MET signaling pathway, cell cycle/DNA damage pathway, Adrenergic receptor-mediated pathway, PAK signaling pathway, MAPK signaling pathway, farnesyl transferase, EGFR, FGFR and apoptosis. Modulating one of these pathways may also lead to up-regulation of the urea cycle enzymes.

Example 5
Determining Genomic Position and Composition of Signaling Centers

A multilayered approach is used herein to identify locations or the “footprint” of signaling centers. The linear proximity of genes and enhancers is not always instructive to determine the 3D conformation of the signaling centers.

ChIP-seq was used to determine the genomic position and composition of signaling centers. Antibodies specific to 67 targets, including transcription factors, signaling proteins, and chromatin modifications, were selected for validation in HepG2 cells using ChIP-seq. These validated antibodies were used in ChIP-seq for hepatocytes to create a two-dimensional (2D) map. These antibody targets are shown in Table 11.

TABLE 11

ChIP-seq targets for primary human hepatocytes

Transcription

Chromatin
factors
Signaling proteins

H3K4me3
HNF1A
RNA Pol II
STAT1-JAK/STAT
NR3C1 (glucocorticoid receptor) -

nuclear receptor signaling

H3K27ac
FOXA1
ONECUT2
STAT3-JAK/STAT
AR (androgen receptor) - nuclear

receptor signaling

H3K4me1
HNF4A
PROX1
TP53-p53, mTOR, AMPK
ESR1 (estrogen receptor) - nuclear

receptor signaling

H3K27me3
NROB2
YY1
TEAD 1/2-Hippo
NR1H3 (liver X receptor alpha) -

nuclear receptor signaling

p300
FOXA2
CTCF
NF-κB (p65)-NF-κB
NR1H4 (farnesoid X receptor) -

nuclear receptor signaling

BRD4
CUX2
ONECUT1
CREB1-MAPK
AHR (aryl hydrocarbon receptor) -

aryl hydrocarbon signaling

SMC1
HHEX
MYC
CREB2-MAPK
NR1I2 (pregnane X receptor) - nuclear

receptor signaling

ZGPAT

JUN-TLR, MAPK
HIF1a (hypoxia inducible factor) -

hypoxia activated signaling

NR113

FOS-TLR, MAPK
TCF7L2 (TCF4)-WNT

ATF5

ELK1-MAPK
CTNNB1-WNT

SMAD2/3-TGFβ,
RBPJ - NOTCH

SMAD4 - TGFβ,
SREBP1 - cholesterol biosynthesis

SMAD1/5/8 - TGFβ,
SREBP2 - cholesterol biosynthesis

ETV4 - ERK MAPK
RXR (RA pathway) - nuclear receptor

signaling

RARA - nuclear receptor
NR3C2 (Mineralocorticoid receptor) -

signaling
nuclear receptor signaling

NR1I1 (Vitamin D receptor) -
STAT5 - JAK/STAT

nuclear receptor signaling

NR5A2 (liver receptor homolog
PPARG - nuclear receptor signaling

1) - nuclear receptor signaling

YAP1 - Hippo signaling
PPARA - nuclear receptor signaling

TAZ - Hippo signaling
mTOR - mTOR signaling

MLXIPL - carbohydrate
GLI3 - Hedgehog signaling

response signaling

GLI1 - Hedgehog signaling
ATR - DNA damage response

signaling

WWTR1 - Hippo signaling

In the signaling proteins column, the associated canonical pathway is included after the “-”.

Table 12 shows the chromatin marks, and chromatin-associated proteins, transcription factors, and specific signaling proteins/or factors associated with the insulated neighborhood of each urea cycle-related gene in primary human hepatocytes.

TABLE 12

ChIP-seq results

Gene
Chromatin
Transcription factors
Specific signaling proteins/or factors

CPS1
H3K27ac, BRD4,
FOXA2, HNF4A, ONECUT1,
TCF7L2, ESRA, FOS, NR3C1, JUN,

p300, SMC1
ONECUT2, YY1
NR5A2, RBPJK, RXR, STAT3, NR1I1, NF-

kB, SMAD2/3, SMAD4, STAT1, TEAD1,

TP53

OTC
H3K27ac, BRD4
FOXA2, HNF4A, ONECUT1,
TCF7L2, HIF1a, ESRA, NR3C1, JUN,

ONECUT2, YY1, HNF1A
RXR, STAT3, NF-kB, SMAD2/3, SMAD4,

TEAD1

ASS1
H3K27ac, BRD4,
FOXA2, HNF4A, ONECUT1,
CREB1, NR1H4, HIF1a, ESRA, JUN, RXR,

p300, SMC1
MYC, YY1
STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3,

SMAD4, TEAD1

ASL
H3K27ac, BRD4,
HNF3, HNF4A, ONECUT1,
TCF7L2, CREB1, NR1H4, HIF1a, ESRA,

p300
HNF1A, MYC
FOS, JUN, RBPJK, RXR, STAT3, NR1I1,

NF-kB, NR3C1, SMAD2/3, SMAD4,

STAT1, TEAD1, TP53

NAGS
H3K27ac, BRD4,
FOXA2, HNF4A, ONECUT1,
TCF7L2, HIF1a, AHR, ESRA, JUN, RXR,

p300
ONECUT2, YY1, HNF1A
STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3,

SMAD4, TEAD1, TP53

ARG1
H3K27ac, BRD4,
FOXA2, HNF4A, ONECUT1,
HIF1a, ESRA, NR3C1, JUN, RXR, STAT3,

p300
ONECUT2, YY1, HNF1A,
NR1I1, SMAD2/3, STAT1, TEAD1

MYC

SLC25A15
H3K27ac, BRD4
FOXA2, HNF4A, ONECUT1,
ESRA, Jun, RXR, NR1I1, NF-kB, NR3C1,

ONECUT2, YY1
SMAD2/3, TP53

SLC25A13
H3K27ac, BRD4,
FOXA2, HNF4A, ONECUT1,
TCF7L2, HIF1a, ESRA, NR3C1, JUN,

p300, SMC1
ATF5, ONECUT2, YY1,
RXR, STAT3, NR1I1, NF-kB, SMAD2/3,

HNF1A, MYC
STAT1, TEAD1, TP53

Example 6
RNA-seq Result Validation

Compounds identified from initial RNA-seq analysis are subjected to validation with qRT-PCR. qRT-PCR is performed on samples of primary human hepatocytes from a second donor stimulated with identified compounds. Compounds are tested at different concentrations and with different cell lots. Fold change in gene expression observed via qRT-PCR is compared to that from RNA-seq analysis. Compounds that cause robust increase in the expression of at least one urea cycle enzyme are selected for further characterization.

Example 7
Disruption of Pathway of Interest

Canonical pathways that showed connection with changes in the expression of urea cycle enzymes are perturbed with additional compounds to confirm the involvement of the pathway in the regulation of urea cycle enzymes. In one embodiment, primary human hepatocytes are treated with additional compounds that target different components in the PDGFR-mediated signaling pathway. In another embodiment, primary human hepatocytes are treated with additional compounds that target different components in the TGF-B signaling pathway. Expression of selected urea cycle enzymes in stimulated hepatocytes is analyzed with RNA-seq as described in Example 1. Hepatic stellate cells are also treated with the same compounds and the effect of the perturbed pathway on gene expression is compared. Changes in binding patterns of the signaling proteins are examined using ChIP. Results are utilized to illustrate gene signaling networks controlling expression of selected urea cycle enzymes and identify additional compounds that modulate selected urea cycle enzymes in the desired direction.

Example 8
Compound Testing in Other Liver Cell Lines

In one embodiment, candidate compounds are evaluated in a hepatic stellate cells to confirm their efficacy. Changes in target gene expression in stellate cells are analyzed with qRT-PCR. Results are compared with that from the primary hepatocytes. Compounds that show consistent induction of at least one urea cycle enzyme are selected for further analysis.

Example 9
Compound Testing in Patient Cells

Candidate compounds are evaluated in patient derived induced pluripotent stem (iPS)—hepatoblast cells to confirm their efficacy. Selected patients have deficiency in at least one of the urea cycle enzymes. Changes in target gene expression in iPS-hepatoblast cells are analyzed with qRT-PCR. Results are used to confirm if the pathway is similarly functional in patient cells and if the compounds have the same impact.

Example 10
Compound Testing in a Mouse Model

Candidate compounds are evaluated in a mouse model of a urea cycle disorder (e.g., CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, Arginase deficiency, ORNT1 deficiency, or Citrin deficiency) for in vivo activity and safety.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

TABLE 13

Motifs for Binding Sites or Signaling Centers

SEQ ID
Gene

SEQ ID
Gene

Motif Sequence
NO
symbol
Motif Sequence
NO
symbol

AAAAGTCAA

NR2E1

NAACCGGTTN

TFCP2

AAAATGGCGCCATTTT

E2F2

NACACGTGTN

CLOCK

AAACCACAAA

RUNX1

NACCCGGAAGTAN

ELF3

AAAGTAAACA

FOXA1

NACCGAAACYN

IRF5

AAATGGCGCCATTT

E2F1

NACGCCCACGCANN

EGR1

AACAGCTGTT

MSC

NAGGTGTGAAAWN

EOMES

AACATCTGGA

ZBTB18

NAGGTGTGAAN

TBR1

AACCGGAAGT

ELF1

NAGGTGTGAAN

TBX20

AACCGGAAGT

ETV1

NAGGTGTGAWN

TBX2

AACCGGAAGT

GABPA

NATATGCTAATKN

POU5F1

AATGGCGCCAAA

E2F1

NATGACGTCAYN

JDP2

AATGGCGCCAAA

E2F4

NATGCGGAAGTR

ELF2

AATTGGCCAATTA

LBX2

NATGCGGGTAN

GCM1

ACAGGAAGTG

ERG

NATGCGGGTN

GCM2

ACAGGAAGTG

ETS1

NCCGCCATTNN

YY1

ACATCCTGGT

SPDEF

NCCGTTAACGGN

MYBL1

ACCCTTGAACCC

ZNF524

NCGAAACCGAAACY

IRF8

ACCGAACAAT

SOX17

NCGAAACCGAAACYN

IRF4

aCGCGTc

MBL2

NCGACCACCGAN

ZBTB7B

ACTACAATTCCC

GFY

NCTAATTANN

LHX2

ACTGAAACCA

IRF4

NCTCGTAAAAN

HOXA13

ACTTTCACTTTC

PRDM1

NCTCGTAAAAN

HOXB13

AGAAATGACTTCCCT

ZNF528

NCTCGTAAAAN

HOXC13

AGAGGAAGTG

MAZ

NCTCGTAAAAN

HOXD13

aGATAAG

SLC6A1

NGATGACGTCATCR

FOS

AGGCCTAG

ZNF711

NGCGACCACCNN

ZBTB7A

AGGCCTGG

ZFX

NGGCACGTGCCN

HES5

AGGTCTCTAACC

PRDM14

NGGCACGTGCCN

HES7

AGGTGTGA

MGAM

NGTCGTAAAAN

HOXC12

AGGTGTGA

TBX1

NGTCGTAAAAN

HOXD12

AGGTGTGA

TBX15

NKCACGTGMN

BHLHE40

AGGTGTGA

TBX4

NKCACGTGMN

BHLHE41

AGGTGTGA

TBX5

NNAAATGGCGCCAAAANN

E2F3

AGGTGTGAAAAAAGGTGT

TBX1

NNAAATGGCGCCATTTNN

E2F3

GA

AGGTGTGAAGGTGTGA

TBX20

NNAACCGTTNN

MYBL1

ATaacATG

SLC6A11

NNAACCGTTNN

MYBL2

ATAGTGCCACCTGGTGGC

CTCF

NNACGCCCACGCANN

EGR3

CA

ATCAATAACATTGAT

SOX15

NNACGCCCACGCANNN

EGR4

ATCAATTGCAGTGAT

SOX10

NNATGACGTCATNN

ATF7

ATCACGTGAT

MLX

NNCACCTGCN

TCF3

ATCACGTGAT

MLXIPL

NNCACCTGNN

FIGLA

ATGAATAACATTCAT

SOX15

NNCACCTGNN

MESP1

ATGAATTGCAGTCAT

SOX10

NNCACCTGNN

TCF3

ATGACGTCAT

JUN

NNCACCTGNN

TCF4

ATGACTCAT

BACH1

NNCACGTGNN

HEY1

ATGACTCATC

FOS

NNCACGTGNN

MAX

ATGCCCTGAGGC

TFAP2A

NNCACGTGNN

MNT

ATTGCGCAAC

CEBPA

NNCACGTGNN

TFE3

CAAGATGGCGGC

YY1

NNCATATGNN

BHLHA15

CACCGAACAAT

SOX6

NNCATATGNN

NEUROD2

CCAAT

NFYA

NNCCGCCATNW

YY2

CCAAT

NFYB

NNGCAACAGGTGGNN

SCRT1

CCAAT

NFYC

NNGCAACAGGTGN

SCRT2

CCATGCCGCCAT

YY2

NNGGAAGTGCTTCCNN

ETV7

CCCCGCGC

SLC13A4

NNGGATTANN

DMBX1

CGCAGCTGCG

NHLH1

NNGGATTANN

DPRX

CGCGAAAa

VANGL2

NNMCGCCCACGCANN

EGR2

CGGACACAAT

FOXKl

NNMCGCCCACGCANNN

EGR4

CGGTTGCCATGGCAAC

RFX1

NNNNNTGCTGAN

MAFK

CGGTTTCAAA

CHR

NNNNTGCTGAN

NRL

CGTGGGTGGTCC

GLI3

NNNTGGCGCCANNN

E2F1

CTAATTAG

PAX4

NNRRAAAGGAAACCGAAACT

IRF3

N

CTGCGCATGCGC

NFE2L1

NNTAAACGNN

BARHL2

CTTCGAG

XBP1

NNTAATCCGATTANN

OTX1

GAAAGTGAAAGT

IRF1

NNTAATCCNN

GSC

GAGGTCAAAGGTCA

NR2C2

NNTAATCCNN

GSC2

GATGACGTCATC

XBP1

NNTAATTANN

HOXD8

GATGACTCAGCA

NFE2

NNTAATTGNN

BARHL2

GATTGCATCA

APEH

NNTAATTRNN

HESX1

GCACTTAA

ISL2

NNTCCAGATGTKN

ZBTB18

GCAGCCAAGCGTGACC

PAX5

NNTGCCAAN

NFIX

GCTAATCC

CRX

NNTGCCAANN

NFIA

GCTTTTCCCACA

ZNF75A

NNTTTTGGCGCCAAAANN

E2F2

ggACCCt

NRG1

NNTTTTGGCGCCAAAANN

E2F3

GGCCATAAATCA

HOXA9

NNYAATTANN

ALX3

GGGGGTGTGTCC

KLF10

NNYAATTANN

DLX1

GGGGTCAAAGGTCA

RXRA

NNYAATTANN

EMX1

GGGGTCAAAGGTCA

RXRB

NNYAATTANN

EMX2

GGGGTCAAAGGTCA

RXRG

NNYAATTANN

EN1

GGTCACGTGA

USF1

NNYAATTANN

EN2

GGTCCCTAGGGA

EBF1

NNYAATTANN

ESX1

GTAAACA

FOXI1

NNYAATTANN

GBX1

GTAAACA

FOXO4

NNYAATTANN

GBX2

GTAAACA

FOXO6

NNYAATTANN

HOXA1

GTAAACAA

FOXO1

NNYAATTANN

LBX2

GTAAACATGTTTAC

FOXO1

NNYAATTANN

MIXL1

GTAAACATGTTTAC

FOXO3

NNYAATTANN

MNX1

GTAAACATGTTTAC

FOXO4

NNYAATTANN

PRKRA

GTAAACATGTTTAC

FOXO6

NNYMATTANN

GSX1

GTAATAAAA

HOXD12

NNYMATTANN

GSX2

GTACCTACCT

ZNF784

NNYMATTANN

HOXA2

GTCACGTG

RBPJ

NNYMATTANN

HOXB2

GTCACGTGAC

ARNTL

NNYMATTANN

HOXB3

GTCACGTGAC

BHLHE41

NNYMATTANN

HOXB5

GTCCCCAGGGGA

EBF1

NNYMATTANN

HOXD4

GTGCGCATGCGC

NKRF

NNYTAATTANN

VSX1

GTGGGCCCCA

ZNF692

NNYTTCCGGGAARNR

ETV7

TCACACCTAGGTGTGA

T

NRAAAGTGAAAGTGN

PRDM1

tCACGTG

RBPJ

NRCACCTGNN

ID4

TGACCTTTAAAGGTCA

NR4A2

NRCAGGTGN

SNAI2

TGATGACGTCATCA

BATF3

NRCATTCCWN

IEAD4

TGCCACGTGGCA

CREB3L1

NRGATAATCYN

DPRX

tGCTGGT

ACE2

NRGTCCAAAGTCCANY

HNF4A

TGGCAGTTGG

MYBL1

NRTTACGTAAYN

DBP

TTACGTAA

GMEB2

NRTTACGTAAYN

EPAS1

TTACTAA

ARRB1

NRYTTCCGGH

FLII

TTgccATggCAAC

RFX1

NSTAATTANN

HOXA3

tTgTTTac

FOXO1

NSTAATTANN

MEOX1

TTTAAAGGTCA

NR4A2

NSTAATTANN

MEOX2

TTTCCCCACAC

FOXO3

NTAATCCN

OTX1

TTTCCCCACACG

FOXO1

NTAATCCN

OTX2

TTTCCCCACACG

FOXO4

NTAATCCN

PITX1

TTTCCCGCCAAA

E2F8

NTAATTAN

LMX1A

TTTGGCGCCAAA

E2F1

NTCAAGGTCAN

ESRRA

TTTGGCGCCAAA

E2F4

NTGACAGN

MEIS3P1

TTTTCCATGGAAAA

NFATC1

NTGACAN

MEIS1

AAAAAGCGGAAGTN

SPI1

NTTAATCCN

PITX1

AAAAATGGCGCCAAAANN

E2F2

NWAACCACADNN

RUNX2

AAAGATCAAAGGRWW

LEF1

NYAATTAN

DLX2

AACCCGGAAGTN

ELF1

NYAATTAN

DLX3

AACCCGGAAGTR

ELF1

NYAATTAN

DLX4

AACCCGGAAGTR

ELF4

NYAATTAN

DLX5

AACCGTTAACGGNN

MYBL2

NYAATTAN

DLX6

ACCCGGAAGTN

ELF5

NYAATTAN

EN1

ACCGGAAGTN

ELK1

NYAATTAN

ISX

ACCGGAAGTN

ELK3

NYAATTAN

LHX9

ACCGGAAGTN

ERF

NYAATTAN

LMX1B

ACCGGAAGTN

ETV3

NYAATTAN

MSX1

ACCGGAAGTN

ETV4

NYAATTAN

MSX2

ACCGGAAGTN

FEV

NYAATTAN

PRRX1

ACCGGAAGTN

GABPA

NYAATTAN

PRRX2

ACCGGAARYN

ETS1

NYAATTAN

RAX2

ACCGGAARYN

ETV1

NYAATTAN

SHOX

ACCGGAARYN

FLI1

NYAATTAN

SHOX2

AGATAAN

GATA1

NYAATTAN

UNCX

AGATAANN

GATA3

NYMATAAAN

CDX1

AGATAANN

GATA4

NYMATAAAN

CDX2

AGATAANN

GATA5

NYMATTAN

HOXA7

AGATAASR

GATA3

NYMATTAN

NKX6-1

AGGTGTGANN

TBR1

NYMATTAN

NKX6-2

AGGTGTNR

TBX3

NYMATTAN

PDX1

CCAATAAAAN

HOXA13

NYNATTAN

BARX1

CCAATAAAAN

HOXB13

NYNATTAN

BSX

CCAATAAAAN

HOXC13

NYTAATCCN

PITX1

CCGAAACCGAAACY

IRF5

NYTAATCCN

PITX3

cTCGAGG.

XBP1

NYTAATTARN

LHX6

gCTTCCw

NR3C1P1

NYTAGTTAMN

HMBOX1

GGCGGGAAAH

E2F4

NYYTGTTTACHN

FOXP1

GGCGGGAARN

E2F6

RACATATGTY

NEUROG2

GHCACGTG

CLOCK

RAGGTCAAAAGGTCAAKN

RARA

GNCACGTG

ARNTL

RATAAAAR

CPEB1

GTAAACAW

FOXO3

RCGGACACAATR

FOXG1

TCAAGGTCAWN

ESRRB

RGGATTAR

GSC

tgCACCy

FAM64A

RGGcAm

EGLN2

tgCATTT.

CST6

RGGGCACTAACY

ZNF264

tGCTGg..

SWI5

RRGGTCAAAGTCCRNN

HNF4A

TGCTTTCTAGGAATTMM

BCL6B

RRGGTCAN

NR2F1

TGGGGAAGGGCM

ZNF467

RRGGTCATGACCYY

RXRA

TTGACAGS

MEIS2

RRGGTCATGACCYY

RXRG

TTTTCCCGCCAAAW

E2F7

RTCACGTGAY

SREBF2

aaa.GTAAACAa

FOXG1

RTCACGTGAY

TFEB

AACAATAMCATTGTT

SOX15

RTCACGTGAY

TFEC

AACAATANCATTGTT

SRY

RTCACGTGAY

USF1

AACAATKNYAGTGTT

SOX8

RTGCCMNNN

HIC2

AACAATNNCATTGTT

SOX18

SCACGTGS

MYCLP1

AACAATNNNAGTGTT

SOX10

SYMATTAN

HOXA6

AACAATNNNCATTGTT

SRY

VCAGCTGBNN

TCF12

AACAVBTGTT

MYF6

WATGCGCATW

POU5F1

AACCGGWNNNWCCGGTT

GRHL1

WRCGTAACCACGYN

GMEB2

AAGAACATWHTGTTC

PGR

YGCGCATGCGCN

NFE2L1

AAGGTCANNNNAAGGTCA

ESRRG

yGCGGCk.

SULT1A1

AANTAGGTCAGTAGGTCA

RORB

YRATTGCAATYR

LHX6

AARGGTCAAAAGGTCA

RARB

YTAATTAN

VAX1

ACAATANCATTG

SOX14

YTAATTAN

VAX2

ACCGGAWATCCGGT

ERG

YTAATTAN

VSX1

ACCGGAWATCCGGT

FLI1

YTAATTAN

VSX2

ACCGTTARRACCGTTA

MYBL2
...a...gTAAACAa

FOXG1

ACTACTAwwwwTAG

TMEM50A
.gCsGgg

SLC13A4

ACVAGGAAGT

ELF5

DATGASTCAT

BATF

AGATGKDGAGATAAG

GATA3

kGmCAGCGTGTC

TRIM3

AGGTCANNNNNTGACCT

NR4A2

NAACAATTKCAGTGTT

SOX4

AGGTGTGAANTTCACACC

TBX15

NATTTCCNGGAAAT

STAT1

T

AGGTGTGAAWTTCACACC

TBX1

NCCGCCATNNT

YY2

T

AGGTGTGAWWWTCACAC

TBX20

NCYAATAAAA

HOXD13

CT

AGTGTTAACAGARCACCT

ZSCAN16

NGYAATAAAA

HOXD11

ANACAGCTGC

TCF3

NNCATCCCATAATANTC

ZNF410

ANCAGCTG

KRT7

NNNGCATGTCCNGACATGCC

UVRAG

ARGGTCAAAAGGTCA

RARA

NNNGMCACGTCATC

XBP1

ATAAACAANWGTAAACA

FOXG1

NRAAGGTCAANNNNRGGTCA

RORA

ATCAATNNCATTGAT

SOX18

NTGCCACGTCANCA

CREB3L1

ATGAATKNYAGTC

SOX8

NYAATTAAAANNYAATTA

MSX1

ATGAATWYCATTCAT

SOX18

NYAATTAAAANNYAATTA

MSX2

ATGASTCAT

JDP2

RACATGTYNNRACATGTC

TP63

ATGCGGGYRCCCGCAT

GCM1

RAGGTCANTCAAGGTCA

ESRRA

ATGMATAATTAATG

POU4F1

RAGGTCANTCAAGGTCA

ESRRG

ATTTCTNAGAAA

STAT5A

RAGTCAANAAGTCA

NR2E1

AVCAGGAAGT

EHF

rCGGC...rCGGC

SULT1A1

CACGTGNNNNNCACGTG

MAX

RGTTCRNNNRGTTC

NR1I2

CGatAa..sC

APEX1

RMATWCCA

ILAD3

CGG...........cCg

LGALS4

rracGCsAaa

VANGL2

CGGas.rsw.y.s.CCGa

LGALS4

RRGGTCANNNRRGGTCA

RARG

CTTGGCACNGTGCCAA

NF1

RTAAACAATAAAYA

FOXJ2

CWGGCGGGAA

E2F1

RTAAACARTAAACA

FOXJ3

GACCCCCCGCGNNG

GLIS2

RTAAACAWMAACA

FOXJ2

GACCCCCCGCGNNG

GLIS3

RTAAAYA

FOXD2

GAGSCCGAGC

ZNF519

RTAAAYA

FOXD3

GARTGGTCATCGCCC

ZNF669

RTAAAYA

FOXL1

GCGACCACNCTG

GLI2

RTAAAYAAACA

FOXC2

ggGTGca.t

FAM64A

rTGTAcGGrT

FHL1

GGGTTTTGAAGGATGART

ZFP3

RTGTTAAAYGTAGATTAAG

ZNF232

AGGAGTT

GGNTCTCGCGAGAAC

ZBTB33

WRTAAAYAAACAA

FOXL1

GGTGTGANNTCACACC

TBX21

WTTCGAAYG

HSFY2

GGTGTGAWATCACACC

TBX21

YACGTAACNKACGTA

GMEB2

GNCCACGTGG

MYC

yGGCGCTAyca

SNTB1

GSCTGTCACTCA

PBX1

YTGGCANNNTGCCAA

NFIX

GTCACGCTCNCTGA

PAX5

YTGGCNNNNTGCCAA

NFIX

GTGGTCCCGGATYAT

SPDEF
..ACCCR.aCMy

RAP1A

GYAATAAAA

HOXC12
.tGrTAGCGCCr...

SNTB1

TAATTRNNYAATTA

GBX2

KCTAWAAATAGM

MEF2A

TAATTRSNYAATTA

EN1

MCCATATAWGGN

SRF

TAATYNAATTA

PROP1

NAAACGATNNAN

HOMEZ

TAATYRATTA

PAX3

NAACAATANCATTGTTN

SOX2

TAATYRATTA

PAX7
NAACAATKNYAGTGTTN

SOX8

TAATYTAATTA

PRRX1

NAACAATKNYAGTGTTN

SOX9

TAATYYAATTA

PHOX2A

NAACAATNNNATTGTTN

SOX2

TAATYYAATTA

PHOX2B

NAACCGCAAACCRCAN

RUNX2

TAAYYNAATTA

PROP1

NAACCGCAAACCRCAN

RUNX3

TATGCWAAT

POU2F3

NAACCRCAAN

RUNX3

TATGCWAAT

POU3F4

NAAGACGYCTTN

PROX1

TATGCWAAT

POU5F1B

NAANCGAAASYR

IRF2

TCAATANCATTGA

SRY

NAASATCAAAGN

TCF7L1

TCAATNNCATTGA

SOX14

NACAYATGNN

TCF15

TCAATNNNATTGA

SOX21

NACTTCCGSCGGAARMN

ELK1

TCACACCTNNNNAGGTGT

EOMES

NAGGTCAMSRTGACCTN

ESR1

GA

TGAATANCATTCA

SRY

NAMCCGGAAGTR

ELF2

TGACAGSTGTCA

MEIS3P1

NATCAATANCATTGATN

SOX2

TGACAGSTGTCA

PKNOX1

NATCAATKNYAGTGATN

SOX8

TGACAGSTGTCA

PKNOX2

NATCAATKNYAGTGATN

SOX9

TGACAGSTGTCA

TGIF1

NATCAATNNNATTGATN

SOX2

TGACAGSTGTCA

TGIF2

NATGAATKNYAGTCATN

SOX9

TGACAGSTGTCA

TGIF2LX

NATGASTCATN

NFE2

TGCACACNCTGAAAA

ZSCAN4

NCACTTNAN

NKX2-8

TGGAACAGMA

ZNF189

NCCACTTRAN

NKX2-3

TRNGTAAACA

FOXA1

NCCGGAWATCCGGN

ERG

TTaGTmAGc

YAP1

NCCGGAWRYRYWTCCGGN

ETS1

TTCCKNAGAA

STAT6

NCCGGNNNNNNCCGGN

TFCP2

TTCGAANNNTTCGAA

HSFY2

NCGAAANYGAAACY

IRF7

TTCTAGAANNTTC

HSF1

NCGACCACCNAN

ZBTB7C

TTCTAGAANNTTC

HSF2

NCTAWAAATAGM

MEF2D

TTCTAGAANNTTC

HSF4

NGACCCCCCACGANGN

GLIS1

TTGACAGSTGTCAA

MEIS2

NGAGGTCANNGAGTTCANNN

CYP27B1

TTTCAAGGCYCCC

PRDM4

NGCCNNNNGGCN

TFAP2A

TTTCCAYNRTGGAAA

NFATC1

NGCCNNNNGGCN

TFAP2B

TTTCCCCACACRAC

FOX06

NGCCNNNNGGCN

TFAP2C

TTTTATKRGG

HOXB13

NGCCNNNNNGGCN

TFAP2C

AAAAAGMGGAAGTN

SPIB

NGGGGAWTCCCCN

NFKB1

AAAAAGNGGAAGTN

SPIC

NGGGGAWTCCCCN

NFKB2

AACCGGAARTR

ETV2

NGTCGTWAAAN

HOXC11

ACCGGAARTN

ELK4

NGTCGTWAAANN

HOXA10

ACCGGAARTN

ERG

NHAACBGYYV

MYBL2

ACCGGAWATCCGGN

FLI1

NMCGCCCMCGCANN

EGR1

ACCGGAWGYN

ETV5

NMCRATTAR

VENTX

AGGTCANTGACCTN

NR1H4

NNAAAATCRATANN

ONECUT3

AMCCGGATGTN

SPDEF

NNAAAATCRATAWN

ONECUT1

AVYTATCGATAD

ZBED1

NNAAAATCRATAWN

ONECUT2

AWCGAAACCGAAACY

IRF9

NNATGAYGCAATN

ATF4

CCAAT.a.

NFYA

NNCAMTTAANN

HMX1

CCCATWAAAN

HOXC10

NNCAMTTAANN

HMX2

CCGGAASCGGAAGTN

ETV6

NNCAMTTAANN

HMX3

CCWGGAATGY

IhAD2

NNMCCATATAWGGKNN

SRF

CCWGGAATGY

1hAD4

NNNNTGCTGASTCAGCANNNN

MAFG

CGSGTAA.

VANGL1

NNNNTGCTGASTCAGCANNNN

MAFK

CTCGRACCCGTGCN

ZNF524

NNRTGACGTCAYCN

CREB3

CTTTCCCMYAACACKNN

ZNF282

NNTAATCCGMTTANN

OTX2

CYAATAAAAN

HOXD13

NNTCCCNNGGGANN

EBF1

GAAASYGAAASY

IRF2

NNTNATTANN

EVX1

GAATGACACRGCGN

FOXB1

NNTNATTANN

EVX2

GAGCCTGGTACTGWGCCT

ZNF322

NNYATGMATAATTAATN

POU1F1

GR

GGCVGTTR

MYB

NRGGTCANNGGTCAN

NR2F1

GNCACGTGYN

HEY2

NRMATWCCWN

TEAD1

GTAATWAAAN

HOXC10

NRNWAAYRTTKNYN

FOXD2

GTCACGCNNMATTAN

PAX3

NRNWAAYRTTKNYN

FOXD3

GTCGTWAAAN

HOXC10

NRTTAATNATTAACN

HNF1A

GTCGTWAAAN

HOXD11

NRTTAATNATTAACN

HNF1B

GTTAATNATTAAY

HNF1B

NRTTACRTAAYN

NFIL3

TAATCRATAN

CUX1

NRTTACRTAAYN

TEF

TAATCRATAN

CUX2

NSCCNNNGGSN

TFAP2A

TRTTTACTTW

FOXM1

NSCCNNNGGSN

TFAP2B

TSAAGGTCAN

ESRRG

NSCCNNNGGSN

TFAP2C

TTACGYAM

GMEB2

NSCCNNNNNGGSN

TFAP2A

TTGGCANNNTGCCAR

NFIA

NSCCNNNNNGGSN

TFAP2B

TTGGCANNNTGCCAR

NFIB

NSCCNNNNNGGSN

TFAP2C

A.yTrAAt

ARRB1

NSCTMATTAN

POU6F2

AACAATNNNAKTGTT

SOX21

NTAATTANNNTAATTAN

MEOX2

AACAATNNNNAKTGTT

SOX21

NTAATYNRATTAN

ALX3

AACAATNNNNAKTGTT

SOX7

NTAATYNRATTAN

ALX4

AAGGKGRCGCAGGCA

ZNF165

NTAATYNRATTAN

UBA2

ACCCTmAAGGTyrT

ZNF569

NTAATYTAATTAN

ISX

ACNGTTAAACNG

MYBL1

NTAATYTAATTAN

UNCX

AkGmCACGTA

TRIM3

NTAAYYNAATTAN

DRGX

AMCATATGKT

OLIG2

NTAAYYYAATTAN

ALX1

AMCATATGKT

OLIG3

NTATCGCGAYATR

ZBED1

ANCATATGNT

BHLHE23

NTATGCWAATN

POU2F2

ANCATATGNT

OLIG1

NTATYGATCH

ONECUT1

ANRTAAAYAAACA

FOXCl

NTGAATNNCATTCAN

SOX14

ARRGGTCANNNRRGGTCA

RARA

NTGAATNNNATTCAN

SOX2

AWCAGCTGWT

TFAP4

NTGAATWNCATTCAN

SOX2

AWNTAGGTCATGACCTAN

RORB

NTGACCTNNNNNAGGTCAN

THRB

WT

CCCGCNTNNNRCGAA

CENPB

NTGCCACGTCAYCN

CREB3

cCr.RTcrGG

ASCL1

NTGCTGANTCAGCRN

MAFK

CGTRNAAARTGA

MSC

NTGCTGASTCAGCAN

MAFF

CNNBRGCGCCCCCTGSTG

CTCFL

NTGMATAATTAATGAN

POU4F3

GC

GACCACMCACNNNG

GLI2

NTMATCATCATTAN

MEOX2

GCCMCGCCCMC

AZF1

NTTRCGCAAY

CEBPB

GCCMCGCCCMC

KLF16

NTTRCGCAAY

CEBPD

GCCMCGCCCMC

PSG1

NTTRCGCAAY

CEBPE

GNCACGTGNC

HES1

NTTRCGCAAY

CEBPG

GRGGTCAAAAGGTCANA

RARG

NWACAYRACAWN

IRX2

GRGGTCAAAAGKTCAC

RARG

NWACAYRACAWN

IRX5

GRGTTCANNRRGTTCA

CYP27B1

NWTATGCWAATN

POU2F1

GTCWGCTGTYYCTCT

ZNF317

NWTATGCWAATTW

POU3F3

GTtgyCATgG.aAc

RFX1

NYAATTARNNNYAATTAN

PDX1

GYCATCMATCAT

HOXA2

NYTMATTANN

NOTO

TAATTAGYRYTRATTA

LHX6

RACCGTTAAACNGYY

MYBL2

TAATTRCYAATTA

LHX9

RCATTCCNNRCATTCCN

TEAD3

TGAATNNNAKTCA

SOX21

RCTAWAAATAGM

BORCS8-

MEF2B

TGAATRTKCAGTCA

SOX10

RRGGTCANNNGGTCAN

NR2F1

TGGKCTARCCTCGA

ZKSCAN3

RTMATTAN

HOXA4

TTCya.....TTC

HSF1

SCTMATTANN

POU6F2

tttCC.rAt..gg

SRF

WAACCRCAN

RUNX2

TTTCGCNTGGCNNGTCA

ZBTB49

WGTAAAYAN

FOXB1

TWCCCAYAATGCATTG

ZNF143

WTATGCWAATNW

POU3F1

AGATSTNDNNDSAGATAA

GATA3

YAATTANNCTAATTR

LHX2

SN

ARGAGGMCAAAATGW

ZNF675

YMATTARYTAATKR

EMX1

ATTTCCCAGVAKSCY

ZNF143

YMATTARYTAATKR

EMX2

GGmraTA.CGs

APEX1

YTCACACCTNNAGGTGTGAR

TBX1

AACAATRTKCAGWGTT

SOX10

DGGGYGKGGC

KLF5

AACAATRTKCAGWGTT

SOX8

NGACCACMCACGWNG

GLI3

AACAATRTKCAGWGTT

SOX9

NGCCACGCCCMCNT

SP6

ADGGYAGYAGCATCT

PRDM9

NTGMATAATTAATKAG

POU4F2

AGGTGTKANGGTGTSA

TBX20

NWTATGCWAATKAG

POUlF1

AGGTGTKANNTMACACCT

MGAM

rGAA..TtctrGAA

HSF1

AGGTGTNANWWNTNACA

TBX4

RGGTCANNNARRGGTCA

RARA

CCT

AGGTGTNANWWNTNACA

TBX5

RGKGGGCGKGGC

SP6

CCT

ATCAATRTKCAGWGAT

SOX9

RRGGTCANNNARAGGTCA

RARG

ATCRATNNNNNATCRAT

CUX1

rTCAyt....Acg

MSC

ATCRATNNNNNNATCRAT

CUX1

rTGTayGGrtg

FHL1

ATCRATNNNNNNATCRAT

CUX2

SAAGGTCANNTSAAGGTCA

ESRRA

ATGAATRTKCAGWCAT

SOX9

sc.GC.gg

EGLN2

CCTCATGGTGYCYTWYTC

ZNF41

SMCAGTCWGAKGGAGGAGGC

ZSCAN22

CCTTGTG

GACCMCCYRMTGNG

ZIC1

WTATGCWAATKA

POU3F2

gcsGsg..sG

SLC13A4
.rTCAyt.y..ACG.

MSC

GGMTNAKCC

RHOXF1

MACCTTCYATGGCTCCCTAKT

ZNF16

GCCY

GGTGTNANWWNTNACAC

TBX2

NAACAATKNYAKTGTTN

SOX7

C

GGYGTGANNNNTCACRCC

MGAM

NANCATATGNTN

BHLHE22

GRTGMTRGAGCC

ZNF415

NATGAATKNYAKTCATN

SOX7

TAAWYGNNNNTAAWYG

BARHL2

NATGGAAANWWWWTTTYCM

NFATC1

N

TGAATRTKCAGWCA

SOX8

NCAGNAAGAMGTAWMM

SPDEF

TGMATWWWTNA

POU3F4

NGCGCCMYCTAGYGGTN

CTCF

tGyayGGrtg

RAP1A

NGNTCTAGAACCNGV

ZBTB12

tktCC..wTt.GGAAA

SRF

NGTCACGCWTSRNTGNNY

PAX2

TNACACCTNNNAGGTGTN

A

TBX21

NGTCACGCWTSRNTGNNY

PAX5

TRGGTYASTAGGTCA

NR1D2

NGTTNCCATGGNAACN

RFX3

TYTCACACCTNNNAGGTG

TBX1

NGTTNCCATGGNAACN

RFX4

TGARA

AAACYKGTTWDACMRGT

GRHL2

NGTTRCCATGGYAACN

RFX2

TTB

GACCCCCYGYTGNGN

ZIC3

NGTTRCCATGGYAACN

RFX5

GACCCCCYGYTGNGN

ZIC4

NGYAATWAAAN

HOXA10

TTTYACGCWTGANTGMNY

PAX6

NGYAATWAAAN

HOXC11

N

ACCYTmArGGT.rTG

ZNF569

NMCMCGCCCMCN

SP8

CNATTAAAWANCNATTA

BARX1

NNGNNACGCCCMYTTTNN

KLF13

TAGAYRAYTGMCANGAA

ZNF713

NRTMAATATTKAYN

FOXC1

TWGHWACAWTGTWDC

DMRT1

NRTMAATATTKAYN

FOXC2

GNCTGTASTRNTGBCTCHT

ZNF382

NTAATTRGYAATTAN

HESX1

T

....gTAAACAa

FOXO1

NTGACCTNATNAGGTCAN

THRA

KCCACGTGAC

NPAS2

NTGACCTNATNAGGTCAN

THRB

MCGCCCACGCA

EGR2

NTGTTTACRGTAAAYAN

FOXI1

NACCCGGAAGTA

EHF

NTRAKCCN

RHOXF1

NAGGTGTGAA

TBX21

NTRAYNTAATCAN

DUXA

NATGCGGGTA

GCM1

NWRGCCMCGCCCMCTNN

SP4

NCCACGTG

MYC

NWTRMATATKYAWN

POU2F1

NCCACTTAA

NKX3-1

NWTRMATATKYAWN

POU2F2

NCCACTTAA

NKX3-2

RCATTCCNNRCATWCCN

TEAD1

NCCCCCCCAC

ZNF740

RTGGAAAANTMCNN

NFAT5

NCGAAACCGAAACT

IRF8

SGTTGCYARGCAACS

RFX2

NNCACGTGCC

HEY2

SGTTGCYARGCAACS

RFX3

NNGCGGAAGTG

ETV7

SGTTGCYARGCAACS

RFX4

NTGGGTGTGGCC

KLF1

SGTTRCCATRGCAACS

RFX5

NTTTCCAGGAAA

STAT4

SSYTAATCGRWAANCGATTAR

VENTX

rACGCGt

MBL2

w.TTRamkAR

SPTLC2

rCACAAT

MAT1A

WAWRTAAAYAWW

FOXC2

RCCGGAAGTG

ETS2

WNWGTMAATATTRACWNW

FOXB1

RRGGTCAAAAGGTCA

NR2F6

WRWGTMAAYAN

FOXB1

RRGGTCAAAGGTCA

HNF4A

WRWRTMAAYAW

FOXC1

RRGGTCAAAGGTCA

NR2C2

WTAATKAGCTMATTAW

POU6F2

RRGGTCAAAGGTCA

NR2F6

ymtGTmTytAw

SPTLC2

RRGTCCAAAGGTCAA

HNF4A

NGTGTTCAVTSAAGCGKAAA

PAX6

RTAAACA

FOXP3

NWTGMATAAWTNA

POU3F2

RTAAACAA

FOXJ2

SGTCACGCWTGANTGMA

PAX1

RTAAACAA

FOXJ3

SGTCACGCWTGANTGMA

PAX9

RTAAACATAAACA

FOXJ3

VAGRACAKNCTGTBC

PGR

RTCATGTGAC

MITF

WAWNTRGGTYAGTAGGTCA

RORA

VDTTTCCCGCCA

E2F7

WTGMATAAWTNA

POU3F1

WACCCGGAAGTA

ELF3

WTGMATAAWTNA

POU3F3

WDNCTGGGCA

ZNF416

WTRMATATKYAW

POU2F3

wwwwsyGGGG

VPS4B

WTRMATATKYAW

POU3F3

YGTCTAGACA

SMAD3

WTRMATATKYAW

POU5F1B

yTGACT

ASCL1

YTKGATAHAGTATTCTWGGTN

ZNF136

GGCA

.gcTgAcTAA.

YAP1

NNCNGTTNNNACNGTTN

MYBL1

DNNNGGTCANNNH

NR2F1

NRGNACANNNTGTNCYN

NR3C2

HAATAAAGNN

PABPC1

NRGWACANNNTGTWCYN

NR3C1

HNHNAGGTGTGANHH

TBX20

NTGACCTYANNTRAGGTCAN

THRB

kGCTGr

SWI5

RAANCGAAAWTCGNTTY

IRF7

KSYGGAAGTN

ETV6

RRGWACANNNTGTWCYY

AKR1B1

NAAACCGGTTTN

GRHL1

WAACCRCAAWAACCRCAN

RUNX2

NAACAATRN

SOX9

WAACCRCAAWAACCRCAN

RUNX3

NAACCGCAAN

RUNX3

NNTCRCACNTANGTGYGANN

TBX19

NAACCGGTTN

GRHL 1

YwTTkcKkTyyckgykky

AZF1

TABLE 14

Motifs for Binding Sites or Signaling Centers

SEQ

ID

Motif Sequence
NO
Complex_name

ACCGGAAATGA

HOXB2:ETV1

CCATAAATCA

PBX4:HOXA10

TAAACAGGATTA

FOXJ2:PITX1

TGTCAATTA

METS1:DRGX

ACCGGAAACAGCTGNN

TFAP4:FLI1

ACCGGAAATGAN

HOXB2:ETV4

AGGTGTTAATKN

HOXB2:TBX3

AGGTGTTGACAN

METS1:EOMES

CTCGTAAAATGYN

TEAD4:HOXB13

GAAAACCGAAAN

FLI1:FOXI1

GGAATGCGGAAGTN

TEAD4:ELF1

GGAATGTTAATTR

TEAD4:DRGX

GTGCGGGCGGAAGTN

GCM1:ELF1

TCACACCGGAWRY

ETV2:EOMES

TGTTGCCGGANNN

FOXO1:ELK1

ACAAWRSNNNNNYMATTA

HOXB2:SOX15

ACAAWRSNNNNYMATTA

HOXB2:SOX15

ACAAWRSNNNYMATTA

HOXB2:SOX15

AGGTGTKAAGTCGTAAA

HOXD12:TBX21

CAGCTGNNNNNNNNNNNTGCGGG

GCM1:FIGLA

CAGCTGNNNNNNNNNTGCGGG

GCM1:FIGLA

CAGCTGNNNNNNNNTGCGGG

GCM1:FIGLA

CAGCTGNNNNNNNTGCGGG

GCM1:FIGLA

CCGGAANNNNNNCACGTG

ETV5:HES7

GGAATGNNNNNYTAATTA

TEAD4:ALX4

GGATTANNNNNNNNNTGCGGG

GCM1:PITX1

GGATTANNNNNNNTGCGGG

GCM1:PITX1

GGATTANNNNNNTGCGGG

GCM1:PITX1

GGTGTGNNNNNCACGTG

CLOCK:TBX3

TAATKNNNNNNNNAAGGTCA

HOXB2:ESRRB

TAATKRNGGATTA

HOXB2:PITX1

TAATKRNNNNGCAAC

HOXB2:RFX5

TAATKRNNNNGGATTA

HOXB2:PITX1

TAATKRNNNNGGATTA

PITX1:HOXA3

TAATKRNNNNNGCAAC

HOXB2:RFX5

TAATKRNNNNNNAAGGTCA

HOXB2:ESRRB

TAATKRNRNGGATTA

HOXB2:PITX1

TAATTANNNNNNCACGTG

CLOCK:EVX1

TGACANNNNNTCATTA

MEIS1:EVX1

TGACANSNTAATTG

MEIS1:DLX3

TMACACCGGAAG

ERF:TBX21

TNTCACACCGGAAAT

ERF:EOMES

ACCCGCANCCGGAAGN

GCM1:ELK3

ACCGGANNTACGCNNNNNYR

ETV2:PAX5

AGGTGNTAATKR

MGA:DLX3

AGGTGNTAATKW

MGA:EVX1

AGGTGNTAATTR

MGA:DLX2

ATTTGCATNACAATRN

POU2F1:SOX2

CACGTGNNNGCGGGY

GCM1:MAX

CACGTGNNNNNSRGGAARNN

ERF:CLOCK

CACGTGNNNRGATTAN

ARNTL:PITX1

CAGCTGNNNNNNCCCGCAY

GCM1:FIGLA

CYAATAAAATGYN

TEAD4:HOXB13

GTMAACAGGAWRN

ETV5:FOXI1

GTMAATAAGGGYRN

GCM1:FOXO1

TAATGNNNNNCGGAAGTN

HOXB2:ELK3

TAATKRCCGGAAGNN

HOXB2:ELK1

TAATKRNNNNGGAAGTN

HOXB2:ELF1

TAATKRNNNNNGGAAGTN

HOXB2:ELF1

TAATTRNNNNCGGAARYN

ETV2:DLX3

TGACAKNNNAACAATGN

MEIS1:SOX2

TGACANNNTAATKR

MEIS1:DLX2

TGACASTAATKR

MEIS1:DRGX

TGTTGANGCGGGN

GCM1:FOXI1

TGTTGNCGGAWRN

ETV5:FOXI1

TGTTKMCGGAWRN

ERF:FOXI1

TGTTKMCGGAWRNN

FLI1:FOXI1

TNRCACCGGAAGN

ELK1:EOMES

TNRCACCGGAAGNN

ELK1:TBX21

TNRCACCGGAWN

ETV5:EOMES

TTACGTNNNNNNNNNCCGGAANN

ETV2:TEF

ACCGGAARTNNNYAATTA

FLI1:DLX2

AGGTGNGAARGGATTA

PITX1:TBX21

AGGTGTGNNNNATCRAT

CUX1:TBX3

ANCCGGATNNNNNNMATTA

HOXB2:SPDEF

ARGTGNNNNNNATAAA

HOXB13:TBX21

ATCRATNNNNNNNNSYATTGTT

CUX1:SOX15

CACGTGNNNTAATKAT

CLOCK:EVX1

CASSTGNNNNNNNNNNNTGCGGG

GCM1:FIGLA

CASSTGNNNNNNNNNNTGCGGG

GCM1:FIGLA

CASSTGNNNNNNNNTGCGGG

GCM1:FIGLA

CASSTGNNNNNNNTGCGGG

GCM1:FIGLA

CYCATAAANNTGTCA

MEIS2:HOXA13

GGAATKNNCASSTG

TEAD4:FIGLA

GGWATGNNNRTAAA

TEAD4:HOXA13

GGWATGNNNRTAAA

TEAD4:HOXB13

GTTGCYNNNNNNNNNNNNCASSTG

RFX3:FIGLA

GWMAACANNSYMRTAAA

FOXO1:HOXB13

GWMAACAYMRTAAA

FOXO1:HOXB13

TAATCCNNNNCASSTG

PITX1:FIGLA

TAATKAGGTGNKA

HOXA3:EOMES

TAATKANNGGATTA

HOXB2:PITX1

TAATKRGGTGYKA

HOXB2:EOMES

TAATKRGGTGYKA

HOXB2:TBX21

TMACACCYAATA

CUX1:TBX21

TTTWATNRNMAACA

FOXJ2:HOXB13

AGGTGNTAATKWNNNNTN

MGA:EVX1

AGGTGTGNCSGTTR

MYBL1:TBX21

AGGTGTNNNGSGGGN

GCM2:TBX21

AGGTGTNRTRCGGGNN

GCM1:TBX21

ANGTGTGAATWCY

TEAD4:TBX21

ANGTGTSNNATAAAN

HOXB13:EOMES

ATRCGGGYNNNNNYWTTGTNN

GCM2:SOX15

CASSTGNNCCCGCAY

GCM1:FIGLA

CASSTGNNCCGGAWRYN

ETV2:TCF3

CASSTGNRNNGGAAGNN

ETV5:TCF3

CTCRTAAAWNNNNRMCGTTR

MYBL1:HOXA13

GGATTANNARGTGTKN

PITX1:EOMES

GGATTANNNNATCRATN

CUX1:PITX1

GGWATGNNNNNNCACGTGN

TEAD4:CLOCK

GGWATGNNNNNNNCACGTGN

TEAD4:CLOCK

GGWATGNRAGGTGNNR

TEAD4:EOMES

TAATKNNNNGNNNNNNCTTCCNN

HOXB2:ETV7

TAATKRSCGGAWGN

ETV5:DRGX

TGACAGNWAATCRATR

METS1:ONECUT2

TGTTKATRCGGGN

GCM2:FOXI1

TGTTKMCGGAWRTN

ETV2:FOXI1

TMACACMGGAARN

ETV2:TBX21

TTGCGYAANNSCGGAAGN

CEBPG:ELF1

ATCRATNNNNYCRTAAA

CUX1:HOXA13

ATRATYANNNNCACGTG

CLOCK:EVX1

GGWATGYGTMAACA

TEAD4:FOXI1

TMACACYYMRTWAA

HOXC10:EOMES

TMRCACCTCRTWAA

HOXD12:EOMES

ARGTGNNANNNMWTAAAN

HOXB13:TBX21

ARGTGTKANTTTATNN

HOXB13:TBX21

ARGTGTKRNNNNNRGWATGY

TEAD4:TBX21

CYCRTAAATWCCN

TEAD4:HOXA13

GTMAACANNNNNATCRATN

CUX1:FOXO1

TGMATATKCANNNNNTAATKR

POU2F1:DLX2

TGMATATKCANNNNTAATKR

POU2F1:DLX2

TNSCCNNNGGSNNNCACGTGN

TFAP2C:MAX

TNSCCNNNGGSNNNNCACGTGN

TFAP2C:MAX

TNSCCNNNGGSNNNNNNNNNNNNNCACGTGN

TFAP2C:MAX

TNSCCNNNGGSNNNNNNNNNNNNNNCACGTG

TFAP2C:MAX

N

TMRCACYTMATWAA

HOXC10:TBX21

ANGTGNNANNNNNNNNNNNCNNMGGAWNN

ELK1:TBX21

ARGTGTNNNAATATKYNNNCRCNN

POU2F1:EOMES

ATRSGGGNNNNTTRCGYAAN

GCM1:CEBPB

GGWATGYNNNNCRCGYGY

TEAD4:HES7

GGWATGYNNNNNNCRCGYGY

TEAD4:HES7

TRGYAACNNNNCASSTGNN

RFX3:FIGLA

AGGYGYGANNNNNNNNNNNNNNNTCRTWAA

HOXD12:EOMES

NNCGTAAAATTA

HOXB2:HOXB13

NRMATATACCAATAAA

POU2F1:HOXB13

NTCGTAAAATGC

TEAD4:HOXB13

NTCGTAAATCA

PBX4:HOXA10

RCCGGAAGTAATTA

ELK1:HOXA1

YNNRTAAATCAATCA

PBX4:HOXA10

NCCGGATATGCAN

POU2F1:ELK1

NCCGGATATGCAN

POU2F1:ETV1

NCCGGATATGCAN

POU2F1:ETV4

NGATGATGCAATNN

ATF4:CEBPD

NGCAGCTGCCGGAWRYN

ETV2:NHLH1

NGCCACGCAAYN

CEBPG:CREB3L1

NMATGACACGCGCCMNN

E2F3:FOXO6

NMCCGGAACCGTTR

MYBL1:ELF1

NNCAGCTGCCGGAWRYN

ERF:NHLH1

NNGAAAACCGAANM

FOXO1:ELF1

NNMATCACATAAAN

HOXB2:HOXB13

NNMCACCGCGCCCMN

E2F3:FOXI1

NNNRTAAATCACACNN

HOXB13:TBX3

NTGCCGGAAGTN

MEIS1:ELF1

NWAAACAGGAAGNN

FOXJ2:ELF1

RCCGGAAATRSY

TEAD4:FLI1

RCCGGATGTTKWN

ETV2:FOXO6

RCCGGATGTTKWY

FLI1:FOXI1

RGAATGCGGAAGTN

TEAD4:SPIB

RGAATGCGGAAGTR

TEAD4:ELF1

RGAATGCGGATN

TEAD4:SPDEF

RGGTGTTAATKN

HOXB2:EOMES

RMAGAAAACCGAANN

FOXJ2:ELF1

RNCGGATGTTKWN

ETV5:FOXO1

RNMTGATGCAATN

ATF4:TEF

RSCGGAAGTAATAAAN

HOXD12:ELK1

RSCGGAAGTAATAAAN

HOXD12:ELK3

RSCGGAAGTAATAAAN

HOXD12:ETV1

RSCGGAAGTAATAAAN

HOXD12:ETV4

RSCGGAAGTCGTAAAN

HOXD12:ELK3

RSCGGAAGTCGTAAAN

HOXD12:ETV4

RSCGGATGTKKN

ELK1:FOXI1

RSCGGATGTTGN

ETV5:FOXI1

RSCGGATGTTKW

ETV2:FOXI1

RSCGGATGTTKWN

ERF:FOXI1

RSCGGTAATKR

ELK1:HOXA3

RTGCGGGCGGAAGTN

GCM1:ELK1

RTGCGGGCGGAAGTN

GCM1:ELK3

RTGCGGGCGGAAGTR

GCM1:ETV4

RTGCGGGTAATAAAN

GCM1:HOXB13

RWMAACAGGAAGTN

FOXO1:ELF1

RWMAACAGGAARNN

FOXO1:ETV4

RWYAACAGGAAGYN

FOXO1:ELK1

SGCGCTAATTKN

E2F3:DRGX

WAACAACACMY

FOXJ3:TBX21

WMSCGGATGTKNW

FOXO1:SPDEF

NCACCTGNNNNNMATTA

HOXB2:TCF3

NCACGTGNNGGATTA

PITX1:HES7

NCAGGTGNNNNNMATTA

HOXB2:TCF3

NCCCGCANNMATAAA

GCM1:HOXB13

NCCCGCANNNMRTAAA

GCM1:HOXB13

NCCGGAAGNNNNNNYMATTA

ETV2:HOXA2

NCCGGAAGTMATTA

ETV2:HOXA2

NNATCATNGTAAA

HOXB2:HOXB13

NNATGAYGCAAT

CEBPG:ATF4

NNMAACAYNRTAAA

HOXB13:Fox

NTAATCCNNWMAACA

FOXJ2:PITX1

NYNATMAATCA

PBX4:HOXA1

RCATTCCNNNNNNCACGTG

TEAD4:MAX

RCATTCCNNNNNNNCACGTG

TEAD4:MAX

RCATTCCNNNYAATTA

TEAD4:DLX3

RCATTCCNNNYMATTA

TEAD4:DLX3

RCATTCNNNNNNCATTA

TEAD4:HOXA2

RCATTCNNNNNNCATTA

TEAD4:HOXA3

RCATTCNNNNNTAATCC

TEAD4:PITX1

RCATTCYNNNCAATTA

TEAD4:DLX2

RCATTCYNNNNCAATTA

TEAD4:DLX2

RCATTCYNNNNNCATTA

TEAD4:HOXA3

RCCGGAAATRCC

TEAD4:ETV4

RCCGGAANNNNNNYAATTA

ETV2:DLX2

RCCGGAANNNNNYAATTA

ETV2:DLX2

RGAATGCGGAWRT

TEAD4:SPDEF

RGAATGCNNRTAAA

TEAD4:HOXB13

RGAATGYGTGA

TEAD4:EOMES

RGAATGYNNACGTG

TEAD4:MAX

RGGTGTNNNNNNNNNYATTGT

SOX6:TBX21

RKRNGGGNNATAAA

GCM1:HOXA13

RNCGGAANNAAACA

ETV2:FOXI1

RRAATGCARTAAA

TEAD4:HOXB13

RSCGGAAATRCC

TEAD4:ERG

RSCGGAAGTMRTTA

HOXB2:ELK1

RSCGGAAGTMRTTA

HOXB2:ELK3

RSCGGAANNNNGGATTA

ETV2:GSC2

RSCGGAANNNNNGGATTA

ETV2:GSC2

RSCGGAANNNNNNNYWTTGT

ETV2:SOX15

RSCGGAANNNNNNRTCGAT

FLI1:ONECUT2

RSCGGAANNNNNNYAATTA

ETV2:DLX3

RSCGGAANNNNNNYWTTGT

ETV2:SOX15

RSCGGAANNNNNYAATTA

ERF:DLX3

RSCGGAANNNNNYAATTA

ETV2:DLX3

RSCGGAANNNNNYAATTA

ETV5:DLX2

RSCGGAANNNNNYMATTA

ERF:DLX2

RSCGGAANNNNYAATTA

ERF:DLX3

RSCGGAANYAATTA

ETV2:DRGX

RSCGGAANYNRTAAA

FLI1:HOXB13

RSCGGAARYAATTA

FLI1:DLX2

RSCGGAWNNNNNNNYMATTA

ERF:HOXA3

RSCGGAWRYATTA

ETV2:DRGX

SMGGAAGTMRTTA

HOXB2:ELF1

SYMRTAAANCTGTCA

MEIS1:HOXB13

SYNRTAAANNTGTCA

MEIS1:HOXA13

YAACGGNNNNNNNNNNCACGTG

MYBL1:MAX

YAACGGNNNNNNNNNNNCACGTG

MYBL1:MAX

YRATTANNNNNNNCACGTG

CLOCK:EVX1

NACAATRNNNNGAATGY

TEAD4:SOX15

NACAATRNNNNNGAATGY

TEAD4:SOX15

NACTTCCGGYNNNNGCAACSN

ETV2:RFX5

NAGGTGNTAATKR

HOXB2:TBX21

NCACGTGNNNNNCATATGN

CLOCK:BHLHA15

NCACGTGNNNNNSCGGAWRN

ETV5:CLOCK

NCAGCTGNNNNNCACGTGN

TFAP4:MAX

NCAGCTGNNNNNNNCACGTGN

TFAP4:MAX

NCCGGAAGTYRTAAAN

ETV2:HOXB13

NCCGGAANCATATGN

FLI1:BHLHA15

NCCGGAANNNNNNCAGCTGNN

ETV2:NHLH1

NCCGGAANYATAAAN

ETV2:HOXB13

NCCGGWNNNNNNNNNNNSCATTAN

ELK1:HOXA3

NGGTGTGNNGGCGCSN

E2F3:TBX21

NGGTGTGNNNGGCGCSN

E2F3:TBX21

NGGTGTNNNNNNNNNNNNNNNCCGGAWNNN

ETV2:EOMES

NMATGACACNGCGCCMNN

E2F3:FOXO6

NNACAATNNNNNNGAATGY

TEAD4:SOX6

NNARAAAACCGAAWMN

FOXJ3:ELF1

NNATGAYGCAAYN

ATF4:CEBPB

NNCACGTGACMGGAARNN

ERF:SREBF2

NNCACGTGNNNGCGGGYN

GCM1:MAX

NNCACGTGNNNNCCGGAANN

ERF:HES7

NNCACGTGNNNNCCGGAANN

ETV5:HES7

NNCACGTGNNNNNCCGGAANN

ERF:HES7

NNCACGTGNNNNNCCGGAWRY

ETV2:CLOCK

NNCACGTGNNNNNCGGAWRY

ETV2:HES7

NNCACGTGNNNNNNNNNNRTGCGGGYRN

GCM1:MAX

NNCAGCTGNNNNNNCACGTGNN

CLOCK:NHLH1

NNCAGCTGNNNNNNNNATCGATN

CUX1:NHLH1

NNCAGCTGNNNNNNNTAATTN

TFAP4:DLX3

NNCAGCTGNNNNTAATKR

TFAP4:DLX3

NNCAGGTGNNNNNMCGGAARYN

ETV2:FIGLA

NNCGGAANCAGGTGNN

ETV2:FIGLA

NNGATTANNNATGCAWNNN

POU2F1:GSC2

NNGTCACGCNNCATTAN

HOXB2:PAX5

NNGTCACGNNTCATTNN

HOXB2:PAX1

NNMGGAARNNRTAAAN

ERF:HOXB13

NNNACGANNNNNNTCGTNNN

HOXD12:EOMES

NNNCGGGNNNGGTGTNN

GCM2:TBX21

NNNGMATAACAAWRRN

POU2F1:SOX15

NNNNGCGCSNNNNNCACGTGNN

E2F3:HES7

NNNRTAAANCTGTN

HOXB13:WIEIS1

NNNTTCCGSNNNNGCAACNN

ETV2:RFX5

NNRNGGGCGGAARTN

GCM2:ELK1

NNSGCGCSNNNATCGAYN

E2F3:ONECUT2

NNSGCGCSNNNNATCGAYN

E2F3:ONECUT2

NNSMGGACGGAYNTCCKSNN

ELK1:ETV7

NNYMATTANNNNNNNGGAAGNN

HOXB2:ELF1

NRCAGCTGNNNNNCACGTGNN

CLOCK:NHLH1

NRCATTCNNNNNTAATTRN

TEAD4:DLX2

NRCATTCNNNNYAATTN

TEAD4:DLX2

NRCCCRNNCGGAAGNN

GCM1:ELF1

NRSCGGAAGNNGTAAAN

HOXD12:ELK1

NSCGGAARNCACGTGNN

ERF:SREBF2

NSCGGAARNNNNNMATTAN

HOXB2:ETV7

NSCGGAARNNNNNNMATTAN

HOXB2:ETV7

NSCGGAARNNNNNNTCACACNN

ETV7:TBX21

NSCGGAARNNNNNTCACACNN

ETV7:TBX21

NSCGGAWNTTACGTAAN

ELK1:TEF

NSMGGACGGAYNTCCKSN

ELK1:SPDEF

NSMGGACGGAYNTCCKSN

ERF:ETV7

NSMGGACGGAYNTCCKSN

ETV2:ETV7

NSMGGACGGAYNTCCKSN

FLI1:ETV7

NTAATKRSNMRTAAAN

HOXD12:HOXA3

NTAATNRSNYMRTAAAN

HOXD12:HOXA3

NTGACRNNNNNNCACGTGN

MEIS1:MAX

NTGTTGATRNGGGN

GCM1:FOXI1

NTGTTGNCGGAARNN

FOXO1:ELF1

NTTTAYNNCCGGAARNN

HOXD12:ELK3

NYMATTANNNNNACAATR

HOXB2:SOX15

NYMATTANNNNNCAGCTGNN

HOXB2:NHLH1

NYMATTANNNNNNACAATR

HOXB2:SOX15

NYMATTANNNNNNCAGCTGNN

HOXB2:NHLH1

NYMATTANNNNNNNCAGCTGNN

HOXB2:NHLH1

NYMATTANNNNNNNNGGAAGNN

HOXB2:ELF1

RAGGTSRNNNNNNNNNNNNNNNNCGGAAGYN

ELK1:TBX21

RCATTCCNNATCGAYN

TEAD4:ONECUT2

RCATTCCNNNNTAATKR

TEAD4:DLX3

RCATTCNNNNNNNNNNNNNNNNGCAACN

TEAD4:RFX5

RCATTCNNNNNNNNNNNNNNNNNGCAACN

TEAD4:RFX5

RCATTCNNNNNTAATKR

TEAD4:DLX3

RCATTCYNNNNCAAGGTCAN

TEAD4:ESRRB

RCATTCYNNNNTAATTR

TEAD4:DLX2

RCCGGAANNNNNATCGATN

ETV2:ONECUT2

RCCGGAANNNNNNATCGATN

ETV2:ONECUT2

RCCGGANNNNNNNNNNNACACCTN

ETV2:EOMES

RCCGGANNNNNNNNNNNACACCTN

ETV2:TBX21

RCCGGANNNNNNNNNNTAATCCN

ETV2:GSC2

RCCGGANNNNNNNNNTAATCCN

ETV2:GSC2

RCCGGANNNNNNNNTAATCCN

ETV2:GSC2

RCCGGAWGTKKN

FOXO1:ETV1

RCCGGAWGTKKN

FOXO1:ETV4

RCCGGAWGTKKW

FOXO1:ELK3

RGAATGCGGAARYNNNTCCN

TEAD4:ETV7

RGAATSCGGAAGYN

TEAD4:ELK1

RGGTGTNNNNNNNNGAATGYN

TEAD4:EOMES

RGGTGYTAATWR

ALX4:TBX21

RGTGTNNNAATATKNN

POU2F1:EOMES

RGWATGTTAATCCS

TEAD4:GSC2

RKCACGTGNNNMCATATGKN

ARNTL:BHLHA15

RKRNRGGCGGAARCGGAAGNN

GCM1:ELK1

RMATWCCGGAWRN

TEAD4:ELK1

RNCGGAANNNNNNNNNGCAACN

ETV2:RFX5

RNCGGANNTTGCGCAAN

FLI1:CEBPB

RNCGGANNTTGCGCAAN

FLI1:CEBPD

RRGTGTNNNNNNNNACAATRN

SOX6:TBX21

RSCGGAAATRCM

TEAD4:ETV1

RSCGGAAGNNGTAAAN

ELK1:HOXB13

RSCGGAAGTNGTAAAN

HOXD12:ETV1

RSCGGAANCACGTGN

ERF:MAX

RSCGGAANCACGTGN

FLI1:MAX

RSCGGAANNNNNNCACGTGNN

ETV2:HES7

RSCGGAANNNNNNNCACGTGNN

ETV2:HES7

RSCGGAANNNNNNNTAATKR

ETV2:DLX3

RSCGGAANNNNNNRTCGATN

ERF:ONECUT2

RSCGGAANNNNNYAATTAN

FLI1:DLX2

RSCGGAANNRYMAACAN

ETV2:FOXI1

RSCGGAANRWMAACAN

ELK1:FOXI1

RSCGGAARCAGGTGN

ETV5:FIGLA

RSCGGAARYNNTAAAN

HOXB13:ETV1

RSCGGANNCATATGK

ETV2:BHLHA15

RSCGGATGTTRTN

FOXO1:ELK1

RSCGGAWNNNNNNACAATRN

ETV2:SOX15

RSCGGAWNNNNNNNACAATRN

ETV2:SOX15

RSCGGWAATKR

ETV5:EVX1

RSCGGWAATKR

ETV5:HOXA2

RSGTGNNNAACGK

MYBL1:EOMES

RTAAACAYNRTAAAN

FOXJ2:HOXB13

RTAAACMGGAARYN

ERF:FOXI1

RTAAACMGGAARYN

FLI1:FOXI1

RTAAATANGGGNN

GCM1:FOXI1

RTAAATANGGGNN

GCM2:FOXI1

RTCACGYSNCCGGAWN

ETV2:SREBF2

RTGCGGGNAGGTGNNN

GCM2:EOMES

RTGCGGGNNATCGATR

GCM1:ONECUT2

RTGCGGGNNNNNNNCACGTGN

GCM1:MAX

RTGCGGGNNNTCGATR

GCM1:ONECUT2

RTMAACAGGAAGTN

FOXO1:ELK3

RTMAACAGGAAGTN

FOXO1:FLI1

RTMAACAGGAARNN

ERF:FOXO1

RTMAACAGGAWRN

ETV5:FOXO1

RTRCGGGTAATAAAN

GCM2:HOXA13

RTRNNGGCGGAAGTN

GCM1:ETV1

RTRYGGGCGGAARKN

GCM1:ERG

RWCACGTGNNCGGAANN

ELK1:SREBF2

RWMAACAGGNNNNNNTTCCNN

FOXO1:ETV7

SGCGCCNNNNNNNNNNNNCAGCTGNN

E2F1:NHLH1

SGCGCNNNNNCGGAAGN

E2F1:ELK1

SGCGCNNNNNNNNNNCGGAAGN

E2F1:ELK1

SGCGCNNNNNNNNNNNCGGAAGN

E2F1:ELK1

SGTCACGCNTCATTNN

HOXB2:PAX9

SYMATTANNNNNNRGCAACN

HOXB2:RFX5

WTGMATAACAATR

POU2F1:SOX15

YNATTANRGGTGTGAN

MGA:DLX3

NCACGTGNNNNNNCATWCC

TEAD4:CLOCK

NCACGTGNNNNNNNCATWCC

TEAD4:CLOCK

NCAGCTGNNNNNNNNTRCGGG

GCM1:NHLH1

NCAGCTGNNNNNNNTRCGGG

GCM1:NHLH1

NCCGGAANNNNNNNMATWCC

TEAD4:ELK3

NCRCGTGNNNGGATTA

PITX1:HES7

NGTGNNNNMATATKNNNACACC

POU2F1:TBX21

NNCRCGTGNNNNGGATTA

PITX1:HES7

NNMATTAGTCACGCWTSRNTG

HOXB2:PAX1

NNMATTAGTCACGCWTSRNTG

HOXB2:PAX5

NNMATTAGTCACGCWTSRNTG

HOXB2:PAX9

NNNTATGCAGYGTKA

POU2F1:EOMES

NNTCCCGCNNNCCNNNGGC

TFAP2C:E2F8

NSCCNNNRGGCANNNNYMATTA

TFAP2C:DLX3

NSCGGACGGAWATCCGSNT

ETV2:SPDEF

NSCGGANNNNNGGMTTA

ERF:PITX1

NSCGGANNNNNNGGMTTA

ERF:PITX1

NTRNGGGNNNCACGTG

GCM2:MAX

NTTTATNRNTMAACA

FOXJ2:HOXB13

RCATWCCNNNNNNNNYNNTAAA

TEAD4:HOXA13

RCATWCNNNNGGATTA

TEAD4:PITX1

RCATWCNNNNNGGATTA

TEAD4:PITX1

RCATWCNNNNNNAGGTCA

TEAD4:ESRRB

RCATWCYNNNNNTAATCC

TEAD4:PITX1

RCATWCYNNNNTAATCC

TEAD4:PITX1

RGGTGTKANNNNGGATTA

PITX1:EOMES

RMATATKCNNNNNNNNNNNNNNNNRWMAACA

POU2F1:FOXO6

RMATATKCNNNNNNNNNNNNNNNRWMAACA

POU2F1:FOXO6

RMATATKCNNNNNNNNNNNNNRWMAACA

POU2F1:FOXO6

RNCGGAWGTMATTA

ETV5:HOXA2

RSCGGAANNNNNNNNCATWCC

TEAD4:ERG

RSCGGAANTSRCGTGA

ELK1:SREBF2

RSCGGAASNGRTCGATA

ELK1:ONECUT2

RSCGGWAATKNNNNNNNNMATTA

ETV5:HOXA2

RSCGGWANNNNYMATTA

ELK1:EVX1

RSMGGAWGYAATTA

ETV5:DRGX

RTAAACWNATWAAA

FOXJ2:HOXB13

RTGNKGGCGGAWG

GCM1:SPDEF

RTRCGGGNNGATTA

GCM2:PITX1

RTRCGGGNNNNTTACGTAA

GCM2:TEF

RWMAACASYMRTWAAA

FOXO1:HOXB13

SCCNNNGGCNNYAATTA

TFAP2C:DRGX

SYAATTANWGGTGYGA

MGA:DLX2

WTWTGCATANNTAATTA

POU2F1:DLX2

YNRCACSTCGTWAA

HOXD12:TBX21

NAACGGNNATYGANN

MYBL1:ONECUT2

NATCGATNNNNNGCCTNNGGSNN

TFAP2C:

ONECUT2

NATCGATNNNNNNNGCCTNNGGSNN

TFAP2C:

ONECUT2

NATCGATNNNNNNNNGCCTNNGGSNN

TFAP2C:

ONECUT2

NATCRATNNNNNNNAACAATRS

CUX1:SOX15

NATCRATNNNNNNNNAACAATRS

CUX1:SOX15

NATTTRCNNNACAATRN

POU2F1:SOX2

NCACGTGNNYAACSGNN

MYBL1:MAX

NCAGGTGNGWATGYN

TEAD4:TCF3

NCAGSTGNNNNNNGTAATKR

TFAP4:DLX3

NCAGSTGNNNNNNNGTAATKR

TFAP4:DLX3

NCASSTGKNNNNNNNNCACGTGN

CLOCK:FIGLA

NCASSTGKNNNNNNNNNCACGTGN

CLOCK:FIGLA

NCCGGAAGYNNCNTAGCAACS

ETV2:RFX5

NCCGGAANNNNNNCACGYGNN

ERF:HES7

NCCGGAANTNRTAAAN

FLI1:HOXB13

NCCGGANNCASSTGY

FLI1:TCF3

NGATAASNNNRGWATGY

TEAD4:GATA3

NGCCTNNGGSNNCGGAAGYN

TFAP2C:ELK1

NGGTGTGNNGGCGCSNNNNCRMN

E2F3:TBX21

NNCACGTGNNNNRGMTTAN

ARNTL:PITX1

NNCAGCTGNNNNNNNNNATYGATN

CUX1:NHLH1

NNCGGAWGYMATTAN

FLI1:DRGX

NNGGAMGGATKTCCGSN

ETV5:ETV7

NNGTMAATANGGGYR

GCM1:FOXI1

NNGYGNNNNNNNNWAACAACACNN

FOXJ3:TBX21

NNGYGYSACATTCCN

TEAD4:EOMES

NNMGGAARTGCKGGN

GCM1:ELF1

NNMRTAAANTMACACNN

HOXB13:EOMES

NNNMGGAANNNNNTCCNNNRCAACN

RFX3:ETV7

NNNRTAAANNTNACACYN

HOXB13:TBX3

NNNRTAAAWATYGAYY

HOXB13:

ONECUT2

NNRGYAACNTCACGTGAY

RFX3:SREBF2

NRGTGTNANNNNNNNNNNNNNCCGGAANN

ERF:TBX21

NRGYAACNNNCATATGKN

RFX3:BHLHA15

NRTCRATANCGGAARYN

ETV2:ONECUT2

NRTMAACMGGAARYN

ETV2:FOXI1

NSCCNNNRGGCANNNNNNTAATKR

TFAP2C:DLX3

NSCCNNNRGGCANNNNNTAATKR

TFAP2C:DLX3

NTCACACMNNNATCRATN

CUX1:TBX21

NTGACAGNTAATCRATAN

MEIS2:ONECUT2

NTNNNGGCGGAAGNNNTTCCNNN

GCM1:ETV7

NTRNGGGCGGAAGNNNTTCCNNN

GCM1:ETV7

NTRYGGGNNNTGGCGGGARN

GCM2:E2F8

NYMRTAAANATYGATN

CUX1:HOXA13

RATTCCNNNNNNNNNNCASSTGN

TEAD4:FIGLA

RCATWCCNNNNNNNNNNNNRWGCGTGACN

TEAD4:PAX5

RCATWCCNNNNNNTTTAYNNN

TEAD4:HOXA13

RCCGGAANNNNNNRATCRATN

ETV2:ONECUT2

RCCGGAARCASSTGNN

TFAP4:ETV4

RCCGGANRNNCGGWATKN

TEhAD4:ELK1

RGGTGNTAAKCCN

MGA:PITX1

RGGTGNTAATNNNNNNNNNCASYNN

ALX4:TBX21

RGWATGYTAATKN

TEAD4:DRGX

RGWATGYTAATKR

TEAD4:DLX2

RNCGGAAGNNNNNCASSTGN

ETV5:TCF3

RNCGGAANYNRTWAAN

ELK1:HOXB13

RNGTGNNNNNNNNNNNNNRCRCCGGAWSN

ETV5:EOMES

RRGTGTKNNNNNNNNNNNNNNCMGGANNN

ERF:EOMES

RRTCRATNNNCGGAARYN

ETV2:ONECUT2

RSCGGAANCAGSTGNN

TFAP4:ETV1

RSCGGAANCASCTGNN

ERF:FIGLA

RSCGGAANCASSTGN

FLI1:FIGLA

RSCGGAANNNNNGGMTTAN

ETV2:PITX1

RSCGGAASNGRTCGATAN

ELK1:ONECUT2

RSCGGANNTTGCGYAAN

ETV2:CEBPD

RSCGGAWRCASSTGN

TFAP4:FLI1

RSCGGWAATNNNNATTAN

ETV5:EVX1

RSCGGWAATNNNNNATTAN

ETV5:EVX1

RSCGGWAATNNNYATTAN

ETV2:EVX1

RSMGGAANTTGCGYAAN

ERF:CEBPD

RSMGGAARTNNTAAAN

HOXB13:ELK1

RTAAACANNNNNATCRATN

CUX1:FOXI1

RTAAACANNNNNATCRATN

CUX1:FOXO6

RTMAACATRNGGGN

GCM1:FOXI1

RTMAATAMGGGYRN

GCM1:FOXO1

RTMRCGTGACGGAWGN

ETV2:SREBF2

RTRCGGGNNNNNNNYWTTGTNN

GCM2:SOX15

RTRCGGGNNNNNNTAATKR

GCM2:DLX3

RTRCGGGNNNNNTAATKR

GCM2:DLX3

RTRCGGGNNNNNTAATTR

GCM2:DLX2

RTRCGGGNNNNRNACAAWN

GCM2:SOX15

RTRCGGGNNNRNACAAWN

GCM2:SOX15

RTRCGGGSGATTAN

GCM2:PITX1

RTRNGGGTNNTAAAN

GCM1:HOXB13

RTRSGGGNAATTAN

GCM2:DRGX

RTRSGGGNNAATTAN

GCM2:DRGX

RTRSGGGNNNATTGTKY

GCM1:SOX2

RTRSGGGNNTAATKR

GCM1:HOXA2

RTRSKGGCGGANNNNNATCCNNN

GCM1:SPDEF

RTRSKGGCGGANNNNNNATCCNNN

GCM1:SPDEF

SYMRTAAANATYGATN

CUX1:HOXB13

SYMRTAAANNNNCASSTGN

HOXD12:FIGLA

WGTTKMCGGAWRTN

ELK1:FOXI1

YNATTAGTCACGCWTSRNTR

HOXA3:PAX5

NCASSTGNNNNNNNNRTRCGGG

GCM2:FIGLA

NGGTGTGNNNGGCGCSNNNCRC

E2F1:EOMES

NNCGGANGNNNTWAA

ETV5:HOXB13

NNCGGAWGTNRTWAA

ETV5:HOXA13

NTATKCAGYGTNA

POU2F1:EOMES

NTATKCAGYGTNA

POU2F1:TBX21

RATCRATNNNNNNNNNNRGYAAC

CUX1:RFX5

RATCRATNNNNNNNNNRGYAAC

CUX1:RFX5

RCATWCCNNNNNNNNNNNNNNRTMAACA

TEAD4:FOXI1

RCATWCCNNNNNNNNNNNNNRTMAACA

TEAD4:FOXI1

RCATWCCNNNNNNNNNNNNRTMAACA

TEAD4:FOXI1

RCATWCCNNNNNNNNNNNRTMAACA

TEAD4:FOXI1

RCATWCYNNNNTAATYRNATTA

TEAD4:ALX4

RSCGGAACYACGCWYSANTG

ELK1:PAX1

RSCGGAACYACGCWYSANTG

ELK1:PAX5

RSCGGAACYACGCWYSANTG

ELK1:PAX9

RSCGGAANNAGGYGYNA

ELK1:EOMES

RSCGGAANNRTMAAYA

ELK1:FOXI1

RSCGGAANRTMAAYA

ELK1:FOXI1

RTRCGGGNNCASSTG

GCM2:FIGLA

RWMAACAYCRTWAA

FOXO1:HOXA10

SCCNNNNGGCATNGTWAA

TFAP2C:HOXB13

NATYGATNNNNNNNNNGCCTNNGGSNN

TFAP2C:

ONECUT2

NCASSTGNNNNNNNCSGTTR

MYBL1:FIGLA

NCASSTGNNNNNNNNCSGTTR

MYBL1:FIGLA

NCASSTGNNNNNNNNNCSGTTR

MYBL1:FIGLA

NCASSTGNNNNNNYAACSGYN

MYBL1:FIGLA

NCASSTGNNNNNYAACSGYN

MYBL1:FIGLA

NCCATAYWNGGWNNNNATCRATN

CUX1:SRF

NGGTGTGNNNNNTMATTWGCRN

HOXB2:TBX21

NRGCAACNNNCRCGYGNN

RFX3:HES7

NRGYAACNNNNNCCTWWWNNGGN

RFX3:SRF

NSCGGANNTTRCGYAAN

ETV5:CEBPD

NTTRCGYAANNNNNNGGAATGY

TEAD4:CEBPB

RCATTCCNNCRCGYGYN

TEAD4:HES7

RCATTCCNNNNNCRCGYGYN

TEAD4:HES7

RGGTGNGANNNNNNNNTNNCACCGGAAGY

ELK1:EOMES

RGGTGTNNNGGCGSNNTNNCRSNN

E2F1:EOMES

RGTGTKRNNNNNNNNNNNCNCMGGAARN

ETV2:TBX21

RGWATSCGGATGNNNNTCCKNNN

TEAD4:SPDEF

RRCRCGYGYNNNNSGCGCSN

E2F1:HES7

RSCGGAANNRTMAAYAN

ELK1:FOXI1

RSCGGAWNTTRCGYAAN

ETV2:TEF

RTGNKGGCGGAWGNNNNNTCCGGNN

GCM1:SPDEF

RTGNKGGCGGAWGNNNNTCCGGNN

GCM1:SPDEF

RTRCGGGNNNNNNATCRATN

GCM2:ONECUT2

RTRNKGGTNNNGCACGYGNN

GCM2:HES7

SCCNNNGGSNNNGGAAGNNNTTCCNN

TFAP2C:ETV7

SRCRCGYGSYNNNNSGCGCSN

E2F1:HES7

WNGTGYKAMATWCY

TEAD4:EOMES

MTRSGGGNNNNNNTTRCGYAAN

GCM1:CEBPB

MTRSGGGNNNNNTTRCGYAAN

GCM1:CEBPB

NGGTGTNNNNNNNNNNNNNCACNTNNTWAN

HOXD12:TBX21

NGYGYTAAYNNNNNNTNACACNN

ALX4:EOMES

NNCRCGYGNNNNNNNSCCNNNGGS

TFAP2C:HES7

NNCRCGYGNNNNNNSCCNNNGGS

TFAP2C:HES7

NNGYGNNNNGGCGCSNNNNCRCNN

E2F3:EOMES

NNNTATGCNNNGYGANNNNNNNNNNNCRCN

POU2F1:EOMES

NNSYGNCNAACNNNNNNCACRCNN

MYBL1:EOMES

NTTRCGYAANNNNNNNNRGWATGY

TEAD4:CEBPD

NTTRCGYAANNNNNNNRGWATGY

TEAD4:CEBPD

NTTRCGYAANNNNNNRGWATGY

TEAD4:CEBPD

RGGTGWKNNNNNNNNNTNNCRTRNGGGN

GCM1:TBX21

RGWATGYNNTTRCGYAAN

TEAD4:CEBPD

NGYGNNAMATWCYNNTMRCRCN

TEAD4:EOMES

TABLE 15

Gene Complex Motif Patterns

Number of each pattern in

Pattern
Gene Complex Table

AB
11

ABA
27

ABAB
29

ABABA
25

ABABAB
23

ABABABA
5

ABABABAB
11

ABABABABA
1

ABABABABAB
6

ABABABABABA
1

TABLE 16

Gene Complex Motif Patterns

Number of each pattern in

Pattern
Gene Complex Table

BA
6

BAB
46

BABA
58

BABAB
155

BABABA
45

BABABAB
95

BABABABA
22

BABABABAB
28

BABABABABAB
14

BABABABABABABABAB
1

TABLE 17

Single Gene Motif Patterns

Number of each pattern in

Pattern
Single Gene Table

AB
42

ABA
97

ABAB
37

ABABA
40

ABABAB
4

ABABABA
28

ABABABAB
4

ABABABABA
4

ABABABABABA
1

TABLE 18

Single Gene Motif Patterns

Number of each pattern in

Pattern
Single Gene Table

BA
34

BAB
186

BABA
40

BABAB
120

BABABA
15

BABABAB
47

BABABABA
8

BABABABAB
8

BABABABABA
1

BABABABABAB
2

TABLE 19

IUPAC Nucoetide Code

IUPAC code
Base

A
Adenine

C
Cytosine

G
Guanine

T (or U)
Thymine (or Uracil)

R
A or G

Y
C or T

S
G or C

W
A or T

K
G or T

M
A or C

B
C or G or T

TABLE 20

Mutations in the OTC gene associated with OTC deficiency

% Enzyme

activity/|%]

Nucleotide
Amino acid or

N ammonia

No.
Exon
Codon¹
change²
other change
Function
incorporation³

1

c.−366A > G

Regulatory

2
Exon 1
1
c.1A > G
p.Met1Val
Missense

3

1
c.1A > T
p.Met1Leu
Missense

4

1
c.2T > C
p.Met1Thr
Missense

5

1
c.3G > A
p.Met1Ile
Missense

6

9
c.25T > G
p.Leu9*
Nonsense

7

10, 11
c.28_31del
p.Asn10_Asn11
Frameshift

AACA

8

10
c.29dupA
p.fsX
Frameshift

9

14
c.40delT
p.Phe14Leufs
Frameshift

10

18
c.53delA
p.His18Profs
Frameshift

11

23
c.67C > T
p.Arg23*
Nonsense

12

26
c.77G > A
p.Arg26Gln
Missense
0%

13

26
c.77G > C
p.Arg26Pro
Missense

14
Intron 1

c.77 + 1G > T

Splice site error

15

c.77 + 1G > A

Splice site error

16

c.77 + 2dupT

Splice site error

17

c.77 + 3_6del

Splice site error

AAGT

18

c.77 + 4A > C

Splice site error
0%

19

c.77 + 5G > A

Splice site error

20

c.78 − 3C > G

Splice site error
<5%

21

c.78 − 1G > C

Splice site error

22
Exon 2
32
c.94C > T
p.Gln32*
Nonsense

23

36
c.106C > T
p.Gln36*
Nonsense

24

39
c.115G > T
p.Gly39Cys
Missense

25

39
c.116G > A
p.Gly39Asp
Missense

26

40
c.118C > T
p.Arg40Cys
Missense

27

40
c.119G > A
p.Arg40His
Missense
6%

28

40
c.119G > T
p.Arg40Leu
Missense

29

41
c.122A > G
p.Asp41Gly
Missense

30

43
c.126_128del
p.Leu43del
In frame indel

TCT

31

43
c.127C > T
p.Leu43Phe
Missense

32

44
c.131C > T
p.Thr44Ile
Missense

33

45
c.133C > G
p.Leu45Val
Missense

34

45
c.134T > C
p.Leu45Pro
Missense

35

45
c.135dupA
p.fsX
Frameshift

36

45-47
c.135delA
p.Asn47Thrfs
Frameshift
0%

37

47
c.140_141insG
p.Asn47delinsLysLeufs
Frameshift

38

47
c.140A > T
p.Asn47Ile
Missense

39

47
c.140A > C
p.Asn47Thr
Missense

40

48
c.143T > C
p.Phe48Ser
Missense

41

49
c.145A > C
p.Thr49Pro
Missense

42

50
c.148G > A
p.Gly50Arg
Missense

43

50
c.148G > T
p.Gly50*
Nonsense

44

52
c.154G > A
p.Glu52Lys
Missense

45

52
c.154G > T
p.Glu52*
Nonsense

46

52
c.155A > G
p.Glu52Gly
Missense

47

52
c.156A > T
p.Glu52Asp
Missense
4%

48

53
c.158T > C
p.Ile53Thr
Missense

49

53
c.158T > G
p.Ile53Ser
Missense

50

55
c.163T > G
p.Tyr55Asp
Missense
28%

51

56
c.167T > C
p.Met56Thr
Missense
[54%]

52

57
c.170T > A
p.Leu57Gln
Missense

53

58
c.174G > A
p.Trp58*
Nonsense

54

59
c.176T > G
Leu59Arg
Missense

55

60
c.179C > T
p.Ser60Leu
Missense

56

62
c.184G > C
p.Asp62His
Missense

57

62
c.185A > G
p.Asp62Gly
Missense

58

63
c.188T > C
p.Leu63Pro
Missense

59

67
c.200T > G
p.Ile67Arg
Missense

60

69
c.205C > T
p.Gln69*
Nonsense

61
Intron 2

c.216 + 1G > A

Splice site error

62

c.216 + 1G > T

Splice site error
10%

63

c.217 − 1G > A

Splice site error

64
Exon 3
73
c.219T > G
p.Tyr73*
Nonsense

65

76
c.227T > C
p.Leu76Ser
Missense

66

77
c.231G > T
p.Leu77Phe
Missense
[35%]

67

78
c.232C > T
p.Gln78*
Nonsense

68

79
c.236G > A
p.Gly79Glu
Missense
0%

69

80
c.238A > G
p.Lys80Glu
Missense

70

80
c.240G > T
p.Lys80Asn
Missense

71

81-82
c.243_245del
p.Leu244del
In frame indel

CTT

72

82
c.245T > G
p.Leu82*
Nonsense

73

82
c.245T > A
p.Leu82*
Nonsense

74

83
c.248G > A
p.Gly83Asp
Missense

75

85
c.256dupT
p.fsX
Frameshift

76

88
c.264A > T
p.Lys88Asn
Missense
3%

77

90
c.268A > G
p.Ser90Gly
Missense
(<20%)

78

90
c.269G > A
p.Ser90Asn
Missense

79

90
c.270T > G
p.Ser90Arg
Missense

80

91
c.271delA
p.Thr91Leufs
Frameshift

81

92
c.274C > G
p.Arg92Gly
Missense

82

92
c.274C > T
p.Arg92*
Nonsense
0%

83

92
c.275G > T
p.Arg92Leu
Missense

84

92
c.275G > A
p.Arg92Gln
Missense
0%

85

92
c.275G > C
p.Arg92Pro
Missense

86

93
c.277A > G
p.Thr93Ala
Missense

87

94
c.281G > C
p.Arg94Thr
Missense

88

95
c.284T > C
p.Leu95Ser
Missense

89

98
c.292G > A
p.Glu98Lys
Missense
33%

90
Intron 3

c.298 + 1G > A

Splice site error

91

c.298 + 1G > T

Splice site error

92

c.298 + 1_5del

Splice site error

GTAAG

93

c.298 + 5G > C

Splice site error

94

c.299 − 8T > A

Splice site error

95

c.299 − 7A > G

Splice site error

96
Exon 4
100
c.299G > A
p.Gly100Asp
Missense

97

100
c.298G > C
Gly100Arg
Missense

98

102
c.304G > C
pAla102Pro
Missense

99

102
c.305C > A
p.Ala102Glu
Missense

100

105
c.314G > T
p.Gly105Val
Missense

101

105
c.314G > A
p.Gly105Glu
Missense

102

106
c.316G > A
p.Gly106Arg
Missense

103

106
c.317G > A
p.Gly106Glu
Missense
<20%

104

106
c.317G > T
p.Gly106Val
Missense

105

109
c.327T > C
p.Cys109Arg
Missense

106

109-110
c.327delT
p.Cys109Cysfsx
Frameshift

107

c.341 −
p.fsX
Frameshift

342delAA

108

111
c.332T > C
p.Leu111Pro
Missense

109

117
c.350A > G
p.His117Arg
Missense
18%

110

117
c.350A > T
p.His117Leu
Missense

111

120
c.358_359del
p.Val120Glufs
Frameshift

GT

112

122
c.364_365ins
p.Glu122delinsValLysfs
Frameshift

TT

113

125
c.374C > T
p.Thr125Met
Missense
<1%

114

125
c.375delG
p.Thr125Thrfs
Frameshift

115

126
c.377A > G
p.Asp126Gly
Missense

116

129
c.386G > C
p.Arg129Pro
Splice site error

117

129
c.386G > A
p.Arg129His
Splice site error
3.50%

118

129
c.386G > T
p.Arg129Leu
Splice site error

119

129
c.385C > T
p.Arg129Cys
Splice site error

120
Intron 4

c.386 + 1G > T

Splice site error

121

c.386 + 1G > A

Splice site error

122

c.386 + 1G > C

Splice site error

123

c.386 + 2T > C

Splice site error

124

c.386 + 5G > A

Splice site error

125

c.387 − 2A > T

Splice site error

126

c.387 − 2A > C

Splice site error

127

c.387 − 2A > G

Splice site error

128
Exon 5
130-131
c.390_391ins
p.Val130_Leu131insLeu
In frame indel

TTA

129

131
c.392T > C
p.Leu131Ser
Missense

130

132
c.394T > C
p.Ser132Pro
Missense

131

132
c.395C > T
p.Ser132Phe
Missense
[78.6%]

132

135
c.403delG
p.Ala135Glnfs
Frameshift

133

135
c.404C > A
p.Ala135Glu
Missense

134

136
c.407A > T
p.Asp136Val
Missense

135

137
c.409G > C
p.Ala137Pro
Missense

136

137
c.409G > A
p.Ala137Thr
Missense

137

139
c.416T > C
p.Leu139Ser
Missense

138

140
c.418G > C
p.Ala140Pro
Missense

139

140
c.419C > A
pAla140Asp
Missense

140

141
c.421C > G
p.Arg141Gly
Missense

141

141
c.421C > T
p.Arg141*
Nonsense

142

141
c.422G > A
p.Arg141Gln
Missense
0%

143

141
c.422G > C
p.Arg141Pro
Missense

144

142
c.425T > A
p.Val142Glu
Missense

145

143
c.429T > A
p.Tyr143*
Nonsense

146

144
c.430A > T
p.Lys144*
Nonsense

147

146
c.437C > G
p.Ser146*
Nonsense

148

148
c.443T > C
p.Leu148Ser
Missense

149

148
c.443T > G
p.Leu148Trp
Missense

150

148
c.444G > C
p.Leu148Phe
Missense

151

148
c.444G > T
p.Leu148Phe
Missense
17%

152

150-151
c.449delC
p.fsX
Frameshift

153

151
c.452T > G
p.Leu151Arg
Missense

154

152
c.455C > T
p.Ala152Val
Missense
3.70%

155

154
c.460G > T
p.Glu154*
Nonsense
0%

156

154
c.461_471del
p.Glu154Alafs*18
Frameshift

157

155
c.463G > T
p.Ala155Ser
Missense

158

155
c.463G > C
p.Ala155Pro
Missense

159

155
c.464C > A
p.Ala155Glu
Missense

160

158
c.472C > T
p.Pro158Ser
Missense

161

159
c.476T > C
p.Ile159Thr
Missense
1.50%

162

159
c.477T > G
p.Ile159Met
Missense

163

160
c.479T > G
p.Ile160Ser
Missense

164

160
c.479T > A
p.Ile160Asn
Missense

165

160
c.479T > C
p.Ile160Thr
Missense

166

161
c.481A > G
p.Asn161Asp
Missense

167

161
c.482A > G
p.Asn161Ser
Missense

168

161
c.483T > A
p.Asn161Lys
Missense
<10%

169

161
c.483T > G
p.Asn161Lys
Missense
<10%

170

162
c.484G > C
p.Gly162Arg
Missense

171

162
c.484G > A
p.Gly162Arg
Missense

172

162
c.485G > A
p.Gly162Glu
Missense

173

164
c.490T > C
p.Ser164Pro
Missense

174

164
c.491C > G
p.Ser164*
Nonsense

175

165
c.493G > T
p.Asp165Tyr
Missense

176

167
c.501C > A
p.Tyr167*
Nonsense
0%

177

167
c.501C > G
p.Tyr167*
Nonsense

178

168
c.503A > C
p.His168Pro
Missense

179

168
c.503A > G
p.His168Arg
Missense

180

168
c.504T > A
p.His168Gln
Missense
[69%]

181

169
c.505C > G
p.Pro169Ala
Missense

182

169
c.506C > T
p.Pro169Leu
Missense

183

169
c.506C > A
p.Pro169His
Missense

184

172
c.514A > T
p.Ile172Phe
Missense

185

172
c.516C > G
p.Ile172Met
Missense

186

172
c.515T > A
Ile172Asn
Missense

187

174
c.520G > C
p.Ala174Pro
Missense

188

175
c.524A > G
p.Asp175Gly
Missense

189

175
c.524A > T
p.Asp175Val
Missense

190

176
c.526T > C
p.Tyr176His
Missense

191

176
c.527A > G
p.Tyr176Cys
Missense
19%

192

176
c.527A > C
p.Tyr176Leu
Missense

193

178
c.533C > T
p.Thr178Met
Missense

194

177-178
c.530_533
p.fsX
Frameshift

dupTCAC

195

178-179
c.532_537del
p.Thr178_Leu179del
In frame indel

ACGCTC

196

179
c.535C > T
p.Leu179Phe
Missense

197

179
c.536T > C
p.Leu179Pro
Missense

198

180
c.538C > T
p.Gln180*
Nonsense

199

180
c.[539A > C (+)
p.Gln180Pro
Missense
<10%

540G > C]

200

180
c.540G > C
p.Gln180His
Splice site error
7.1%
[43%]

201
Intron 5

c.540 + 1G > C

Splice site error

202

c.540 + 2T > C

Splice site error
0%

203

c.540 + 2T > A

Splice site error

204

c.541 − 2A > G

Splice site error

205

c.540 + 265

Splice site error

G > A

206

c540 + 2T > G

Splice site error

207
Exon 6
181
c.542A > G
p.Glu181Gly
Missense

208

182
c.545A > T
p.His182Leu
Missense

209

183
c.547T > G
p.Tyr183Asp
Missense

210

183
c.548A > G
p.Tyr183Cys
Missense

211

186
c.557T > C
p.Leu186Pro
Missense

212

188
c.561delA
p.Gln188fs
Frameshift

213

188
c.562G > C
p.Gly188Arg
Missense
2%

214

188
c.562_563del
p.Gly188SfsX36
Frameshift

GG

215

188
c.563G > T
p.Gly188Val
Missense

216

188
c.563G > C
p.Gly188Ala
Missense

217

190
c.568delA
p.T190PfsX16
Frameshift

218

191
c.571C > T
p.Leu191Phe
Missense
5.70%

219

191
c.571delC
p.Leu191SerfsX15
Frameshift

220

191
c.572T > G
p.Leu191Arg
Missense

221

192
c.576C > G
p.Ser192Arg
Missense

222

193
c.577T > C
p.Trp193Arg
Missense

223

193
c.577T > G
p.Trp193Gly
Missense

224

193
c.578G > A
p.Trp193*
Nonsense

225

193
c.579G > C
p.Trp193Cys
Missense

226

194
c.581T > C
p.Ile194Thr
Missense

227

195
c.583G > A
p.Gly195Arg
Missense
0%

228

195
c.583delG
p.Asp196Metfs
Frameshift

229

196
c.586G > A
p.Asp196Asn
Missense

230

196
c.586G > T
p.Asp196Tyr
Missense

231

196
c.586G > C
p.Asp196His
Missense

232

196
c.587A > T
p.Asp196Val
Missense
7%

233

197
c.589G > A
p.Gly197Arg
Missense

234

197
c.590G > A
p.Gly197Glu
Missense

235

197
c.589G > T
p.Gly197Trp
Missense

236

198
c.593A > T
p.Asn198Ile
Missense

237

198
c.594C > A
p.Asn198Lys
Missense

238

199
c.595A > G
p.Asn199Asp
Missense

239

199
c.596A > G
p.Asn199Ser
Missense

240

199-200
c.(597_598)
p.fsX
Frameshift

delTA

241

201
c.602T > C
p.Leu201Pro
Missense

242

202
c.604C > T
p.His202Tyr
Missense
[49%]

243

202
c.605A > C
p.His202Pro
Missense

244

203
c.608C > G
p.Ser203Cys
Missense

245

205
c.613A > G
p.Met205Val
Missense

246

205
c.614T > C
p.Met205Thr
Missense

247

206
c.617T > G
p.Met206Arg
Missense

248

206
c.618G > C
p.Met206Ile
Missense

249

207
c.620G > A
p.Ser207Asn
Missense

250

207
c.621C > A
p.Ser207Arg
Missense

251

208
c.622G > A
p.Ala208Thr
Missense
4%

252

209
c.626C > T
p.Ala209Val
Missense
1%
[1.4%]

253

210
c.628A > C
p.Lys210Glu
Missense

254

210
c.630A > C
pLys210Asn
Missense

255

210
c.628A > C
p.Lys210Gln
Missense
0%

256

213
c.637T > A
p.Met213Lys
Missense

257

213
c.637T > C
p.Met213Thr
Missense

258

213
c.637T > G
p.Met213Arg
Missense

259

214
c.640C > T
p.His214Tyr
Missense

260

215
c.643C > T
p.Leu215Phe
Missense
17%

261

215-216
c.645_646insT
p.Gln216delinsSerGlyfs
Frameshift

262

216
c.646C > G
p.Gln216Glu
Missense

263

217
c.650C > A
p.Ala217Glu
Missense

264

218
c.653C > T
p.Ala218Val
Missense

265

220
c.658C > G
p.Pro220Ala
Missense
35%

266

220
c.659C > T
p.Pro220Leu
Missense

267

221
c.663G > C
p.Lys221Asn
Missense

268

221
c.663G > A
p.Lys221Lys
Splice site error
8%

269
Intron 6

c.663 + 1G > A

Splice site error

270

c.663 + 1G > T

Splice site error

271

c.663 + 1delG

Splice site error

272

c.663 + 2T > C

Splice site error

273

c.663 + 2dupT

Splice site error

274

c.664 − 1G > A

Splice site error

275

c.664 − 1delG
p.fsX
Frameshift

276
Exon 7
222
c.664 −
p.Gly222Thrfs*2
Frameshift

667delinsAC

277

224
c.670G > T
p.Glu224*
Nonsense

278

225
c.673C > A
p.Pro225Thr
Missense
[42%]

279

225
c.674C > G
p.Pro225Arg
Missense
0%

280

225
c.674C > T
p.Pro225Leu
Missense
0%
[0.45%]

281

233
c.698C > T
p.Ala233Val
Missense

282

234
c.700G > T
p.Glu234*
Nonsense

283

239
c.716A > T
p.Glu239Val
Missense

284

239
c.716A > G
p.Glu239Gly
Missense

285

239
c.717G > C
p.Glu239Asp
Missense

286

239
c.717G > A
p.Glu239Glu
Splice site error

287
Intron 7

c.717 + 1G > T

Splice site error

288

c.717 + 1G > A

Splice site error

289

c.717 + 2T > C

Splice site error

290

c.717 + 3A > G

Splice site error

291

c.717 + 7_22

Splice site error
0-1.5%

delTCTTTA

CATGTAAA

GC

292

c.718 − 2A > G

Splice site error

293
Exon 8

c.718 −

Splice site error

4_729delCT

AGAATGGT

ACCAAG

294

242
c.725C > T
p.Thr242Ile
Missense

295

244
c.731T > A
p.Leu244Gln
Missense
8%

296

247
c.740C > A
p.Thr247Lys
Missense
0%

297

244-247
c.731_739del

In frame indel

TGTTGCTG A

298

249
c.746A > G
p.Asp249Gly
Missense

299

250
c.749C > T
p.Pro250Leu
Missense

300

253
c.757G > A
p.Ala253Thr
Missense

301

253
c.757G > C
p.Ala253Pro
Missense

302

253
c.759delA
p.fsX
Frameshift

303

254
c.760A > T
p.Ala254*
Nonsense

304

255
c.764A > C
p.His255Pro
Missense

305

260
c.779T > C
p.Leu260Ser
Missense

306

262
c.784_792
p.thr262_Thr264dup
In frame indel

dup9
TDT

307

262
c.785C > A
p.Thr262Lys
Missense
26%

308

262
c.785C > T
p.Thr262Ile
Missense

309

263
c.787G > A
p.Asp263Asn
Missense

310

263
c.788A > G
p.Asp263Gly
Missense

311

264
c.790A > G
p.Thr264Ala
Missense
22%

312

264
c.791C > A
p.Thr264Asn
Missense

313

264
c.791C > T
p.Thr264Ile
Missense

314

265
c.793T > C
p.Trp265Arg
Missense

315

265
c.794G > T
p.Trp265Leu
Missense
56%

316

265
c.795G > A
p.Trp265*
Nonsense

317

265-268
c.796_805del
p.Ile265_Gly268delins
Frameshift

AspfsX19

318

267
c.799A > C
p.Ser267Arg
Missense

319

268
c.803T > C
p.Met268Thr
Missense
6.70%

320

268
c.802A > G
p.Met268Val
Missense

321

269
c.806G > A
p.Gly269Glu
Missense
2%

322

270
c.808C > T
p.Gln270*
Nonsense

323

270
c.809A > C
p.Gln270Pro
Missense

324

271-272
c.810_811del
p.fsX
Frameshift

AGinsC

325

272-273
c.817_819del
p.Glu273del
In frame indel
5%

GAG

326

273
c.818delA
p.Glu273Glyfs
Frameshift

327

277
c.829C > T
p.Arg277Trp
Missense
5%
[59%]

328

277
c.830G > A
p.Arg277Gln
Missense
7%

329

277
c.830G > T
p.Arg277Leu
Missense

330

279
c.835C > T
p.Gln279*
Nonsense

331

281
c.842T > C
p.Phe281Ser
Missense

332

284
c.853delC
p.Phe284fsX38
Frameshift

333

285
c.853C > T
p.Gln285*
Nonsense

334

287
c.860A > C
p.Thr287Pro
Missense

335

287
c.861insAC
p.fsX
Frameshift

336

286-289
c.867G > A
p.Val286_Lys289del
In frame indel

r.856_867del

GTTACAAT

GAG

337

289
c.867G > C
p.Lys289Asp
Missense

338

289
c.867G > T
p.Lys289Asn
Missense
0%

339
Intron 8

c.867 + 1G > A

Splice site error

340

c.867 + 1G > T

Splice site error

341

c.867 +

Splice site error

1126A > G

342

c.868 − 3T > C

Splice site error

343
Exon 9
292
c.875delA
p.Val293Leufs
Frameshift

344

294
c.882delT
p.Ala295Profs
Frameshift

345

297
c.889G > T
p.Aso297Tyr
Missense

346

297-298
c.889_892del
p.fsX
Frameshift
0%

GACT

347

298
c.892_893del
p.Trp298Aspfs
Frameshift

TG

348

298
c.892T > C
p.Trip298Arg
Missense

349

298
c.893G > C
p.Trp298Ser
Missense
0%

350

301
c.902T > C
p.Leu301Ser
Missense

351

301
c.903A > T
p.Leu301Phe
Missense
3%

352

302
c.904C > T
p.His302Tyr
Missense
0%

353

302
c.905A > G
p.His302Arg
Missense

354

302
c.905A > T
p.His302Leu
Missense

355

302
c.906C > G
p.His302Gln
Missense

356

302
c.906delC
p.Cys303Alafs
Frameshift

357

303
c.907T > C
p.Cys303Arg
Missense

358

303
c.907T > G
p.Cys303Gly
Missense

359

303
c.908G > A
p.Cys303Tyr
Missense

360

304
c.912G > T
p.Leu304Phe
Missense
6%
[74%]

361

305
c.914C > G
p.Pro305Arg
Missense

362

305
c.914C > A
p.Pro305His
Missense

363

306
c.916A > T
p.Arg306*
Nonsense

364

306
c.917G > C
p.Arg306Thr
Missense

365

309-310
c.(925 − 927)
p.Glu309del
In frame indel

delGAA

366

310
c.928G > T
p.Glu310*
Nonsense

367

310
c.929_931del
p.Glu310del
In frame indel

AAG

368

310
c.929A > G
p.Glu310Gly
Missense

369

311
c.931G > A
p.Val311Met
Missense

370

311
c.932T > A
p.Val311Glu
Missense

371

314
c.940_942del
p.Glu314del
In frame indel

GAA

372

315
c.943G > T
p.Val315Phe
Missense

373

315
c.944T > A
p.Val315Asp
Missense

374

315
c.944T > G
p.Val315Gly
Missense

375

316
c.947T > C
p.Phe316Ser
Missense

376

318
c.953C > T
p.Ser318Phe
Missense

377

320
c.958C > T
p.Arg320*
Nonsense
10-15%

378

320
c.959G > T
p.Arg320Leu
Missense
[3.9%]

379

321
c.962C > A
p.Ser321*
Nonsense

380

322
c.964C > G
p.Leu322Val
Missense

381

322
c.965T > C
p.Leu322Pro
Missense

382

323
c.967G > A
p.Val323Met
Missense

383

326
c.976G > A
p.Glu326Lys
Missense

384

328
c.982G > T
p.Glu328*
Nonsense

385

330
c.988A > T;
p.fsX
Frameshift

989_990delG

A

386

330
c.988A > G
p.Arg330Gly
Missense

387

331
c.991A > T
p.Lys331*
Nonsense

388

332
c.994T > A
p.Trp332Arg
Missense
0%

389

332
c.995G > A
p.Trp332*
Nonsense

390

332
c.996G > A
p.Trp332*
Nonsense

391

332
c.995G > C
p.Trp332Ser
Missense

392

335
c.1005G > A
p.Met335Ile
Missense

393
Intron 9

c.1005 + 1G > T

Splice site error

394

c.1005 + 2T > C

Splice site error

395

c.1006 − 3C > G

Splice site error
2.70%

396

c.1006 − 1G > A

Splice site error
[23%]

397

c.1005_1091

Splice site error

C > G

398
Exon 10
336
c.1006G > T
p.Ala336Ser
Missense

399

337
c.1009G > C
p.Val337Leu
Missense
<5%

400

339
c.1015G > C
p.Val339Leu
Missense

401

339
c.1016T > G
p.Val339Gly
Missense

402

340
c.1018T > C
p.Ser340Pro
Missense

403

341
c.1022T > C
p.Leu341Pro
Missense

404

343
c.1028C > G
p.Thr343Arg
Missense

405

343
c.1028C > A
p.Thr343Lys
Missense

406

345
c.1033T > C
p.Tyr345His
Missense

407

345
c.1033T > G
p.Tyr345Asp
Missense

408

345
c.1034A > G
p.Tyr345Cys
Missense

409

347
c.1039C > A
p.Pro347Thr
Missense

410

347
c.1039C > T
p.Pro347Ser
Missense

411

347
c.1040C > T
p.Pro347Leu
Missense

412

348
c.1042C > T
p.Gln348*
Nonsense

413

348
c.1043delA
p.Gln348Argfs*47
Frameshift

414

348
c.1046T > C
p.Leu349Pro
Missense

415

354
c.1061T > G
p.Phe354Cys
Missense
1.80%

416

355
c.1063T > C
p.*355Glu
Extending

417

355
c.1065A > T
p.*355Cysext*14
Extending

¹Nucleotide + 1 is the A of the translation initiation codon of the NM_000531.3.

²For deletions or insertions, the cDNA nucleotide number is given starting with the A of the translation initiation codon.

³%) residual activity in liver or intestine or determined by expression studies: [15N] residual nitrogen incorporation into urea. %) residual activity in liver or intestine or determined by expression studies; [15N] residual nitrogen incorporation into urea

TABLE 21

CAS Registry Numbers for Selected Compounds

Drugs
CAS Registry No

Baricitinib
1187594-09-7

Momelotinib
1056634-68-4

17-AAG
75747-14-7

BIIB021
848695-25-0

NVP-HSP990/HSP990
934343-74-5

Retaspimycin Hydrochloride
857402-63-2

Retaspimycin
857402-23-4

BIRB796/Doramapimod
285983-48-4

Pamapimod/Ro4402257/R1503
449811-01-2

PH-797804
586379-66-0

Mubritinib/TAK-165
366017-09-6

XL-228
898280-07-4

Lifirafenib/BGB-283
1446090-79-4

BMS-214662
195987-41-8

Foretinib
849217-64-7

BX795
702675-74-9

Dorsomorphin dihydrochloride
1219168-18-9

Dorsomorphin
866405-64-3

METHODS AND COMPOSITIONS FOR TREATING UREA CYCLE DISORDERS, IN PARTICULAR OTC DEFICIENCY

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

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Abstract

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Provisional Applications (1)