Patent Application
20250188169
The instant application contains a Sequence Listing that has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on Mar. 9, 2023 is named 701039-191350WOPT_SL.xml and is 455,674 bytes in size.
The technology described herein relates to methods of treating clonal hematopoiesis disorders such as clonal hematopoiesis, myelodysplastic syndromes, and leukemia.
Clonal hematopoiesis (CH) is a state of clonal dominance of a mutant hematopoietic stem and progenitor cell (HSPC) promoted by unknown mechanisms. Individuals with CH have increased levels of inflammatory cytokines in blood. Mutations enabling competitive outgrowth may be due to aging, selective resistance to chemotherapy, or founder effects in HSPC transplantation. There is a need for methods and compositions for treating clonal hematopoietic blood disorders.
As described herein, the inventors have identified a gene that is important in the pathogenesis of CH. CH is established by introduction of mutations in certain genes, including ASML1, DNMT3A, IDH1, IDH2, and others. This state is considered a preleukemia with a high risk of progressing to myelodysplastic syndromes or leukemia over time. The inventors have discovered inter alia that the mutant blood stem cells in clonal hematopoiesis overexpress the gene NR4A1 which promotes the increased fitness and survival of the mutant stem cells compared to wildtype stem cells. When the inventors mutated both copies of NR4A1 in stem cells that were mutant for ASXL1, the competitive advantage was abrogated. Accordingly, in one aspect, provided herein is method for treating a clonal hematopoietic disease or disorder in a subject in need of treatment. Generally, the method comprises inhibiting a member of the nuclear receptor 4A (NR4A) family. For example, by administering an effective amount of an inhibitor of a member of the NR4A family to the subject.
In some embodiments of any one of the aspects described herein, the clonal hematopoietic disease or disorder is clonal hematopoiesis, myelodysplastic syndromes (MDS) or leukemia.
In some embodiments, the clonal hematopoietic disease or disorder is leukemia. For example, clonal hematopoietic disease or disorder is acute myelogenous leukemia (AML) or chronic myeloid leukemia (CML).
In some embodiment, the clonal hematopoietic disease or disorder is myelodysplastic syndromes. Exemplary myelodysplastic syndromes include, but are not limited to, MDS with multilineage dysplasia (MDS-MLD), MDS with single lineage dysplasia (MDS-SLD), MDA with ring sideroblasts (MDS-RS), MDS with excess blasts (MDS-EB), MDS with isolated del(5q), and/or MDS unclassifiable (MDS-U).
In another aspect provided herein is a method for inhibiting clonal expansion of hematopoietic stem and progenitor cells. The method comprises administering an effective amount of an inhibitor of a member of the NR4A family to a HSPC. It is noted that administering to the cell can be in vitro or in vivo. For example, when the administering to the cell is in vivo, the compound can be administered to a subject. The subject can be one having a clonal hematopoietic disease or disorder or in need of treatment for clonal hematopoietic disease or disorder.
In some embodiments of any one of the aspects described herein, the NR4A family member is selected from the group consisting of NR4A1, NR4A2 and NR4A3. In some preferred embodiments, the NR4A family member is NR4A1.
In some embodiments of any one of the aspects described herein, the inhibitor binds with the NR4A family member or with a nucleic acid encoding the NR4A family member.
Exemplary inhibitors that can be used include, but are not limited to, nucleic acids, antibodies, and small molecules. Accordingly, in some embodiments of any one of the aspects described herein, the inhibitor is a nucleic acid. For example, the inhibitor is a nucleic acid selected from the group consisting of siRNAs, antisense oligonucleotides, aptamers, ribozymes, and triplex forming oligonucleotides. Generally, the nucleic acid inhibitor comprises a nucleotide sequence substantially complementary to at least a portion of a nucleic acid encoding the NR4A family member. For example, the inhibitor comprises a nucleotide sequence substantially complementary to at least 15 contiguous nucleotides of a nucleic acid encoding the NR4A family member.
In some embodiments of any one of the aspects described herein, inhibitor is an antibody or an antigen binding fragment thereof. For example, the inhibitor is an antibody or an antigen binding fragment thereof that binds the NR4A family member. Generally, the antibody or the antigen binding fragment thereof binds an epitope on the NR4A family member such that the binding inhibits a function and/or activity of the NR4A family member. It is noted that the antibody can be a polyclonal or monoclonal antibody. Accordingly, in some embodiments of any one of the aspects described herein, the inhibitor is a monoclonal antibody. In some other embodiments of any one of the aspects described herein, the inhibitor is a polyclonal antibody. The antibody can be a chimeric antibody. In some embodiments of any one of the aspects described herein, the antibody is a human antibody or a humanized antibody.
In some embodiments of any one of the aspects described herein, the inhibitor is a monoclonal human antibody or monoclonal humanized antibody.
Small molecule inhibitors of NR4A family members are also known in the art. Accordingly, in some embodiments of any one of the aspects described herein, the inhibitor is a small molecule. Generally, the small molecule inhibitor binds with the NR4A family member and inhibits a function and/or activity of the NR4A family member. Exemplary small molecule inhibitors include, but are not limited to 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (DIM-C-pPhOh-3-Cl-5-OCH3), 1,1-bis(3′-indolyl)-1-(3,5-dibromo-4-hydroxyphenyl)methane (DIM-C-pPhOH-3,5-Br2), camptothecin (CPT), a cyclooxygenase (COX)-2 inhibitor (celecoxib analogue SC-236), Erlotinib, Afatinib, Bosutinib, Dasatinib, and KU171309.
The subject undergoing treatment can also be given additional therapies. For example, the described herein further comprises co-administering one or more additional therapeutic agents to the subject. It is noted that the additional therapeutic agent can be administered prior to, simultaneously with or after administering the inhibitor of the NR4A family member. Exemplary additional therapeutic agents that can be co-administered with the inhibitor of the NR4A family member include, but are not limited to immunomodulatory agents and anti-inflammatory agents. Accordingly, in some embodiments of any one of the aspects described herein, the inhibitor of the NR4A family member include, but method further comprises co-administering an immunomodulatory agent to the subject. In some embodiments of any one of the aspects described herein, the method further comprises co-administering an anti-inflammatory agent to the subject.
The inventors have discovered inter alia that when the expression of NR4A1 is inhibited in HSPC cells that are mutant for certain genes the increased fitness and survival of the mutant stem cells compared to wildtype stem cells is abrogated. Accordingly, in some embodiments of any one of the aspects described herein, the subject has at least one mutation in a nucleic acid encoding ASXL transcriptional regulator 1 (ASXL1), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), isocitrate dehydrogenase (NADP(+)) 1(IDH1) or isocitrate dehydrogenase (NADP(+)) 2 (IDH 2). The method described herein can include a step of identifying and/or selecting a subject with at least one mutation in a nucleic acid encoding ASXL1, DNMT3A, IDH1 or IDH 2 prior to onset of the treatment regime. For example, the method can further comprise a step of assaying a sample from the subject for at least one mutation in a nucleic acid encoding ASXL1, DNMT3A, IDH1 or IDH 2 prior to onset of the treatment regime.
In some embodiments, the method further comprises a step of assaying a HSPC from the subject for at least one mutation in a nucleic acid encoding ASXL1, DNMT3A, IDH1 or IDH2 prior to onset of the treatment regime.
The various aspects described herein are based in part on the inventors' discovery that mutant blood stem cells in clonal hematopoiesis overexpress the gene NR4A1 which promotes the increased fitness and survival of the mutant stem cells compared to wildtype stem cells. Inhibiting the NR4A1 or expression of a nucleic acid encoding the NR4A1 nullifies this advantage. Accordingly, in one aspect, provided herein is method for treating a clonal hematopoietic disease or disorder in a subject in need of treatment by inhibiting NR4A1 directly or by inhibiting the expression of a nucleic acid encoding NR4A1 in a HSPC.
The nuclear receptor subfamily 4A (NR4A) is a family of orphan nuclear receptors which act as transcription factors in neuron development and maintenance. The NR4A family is composed of three members—NR4A1 (Nur77/TR3/NGFI-B), NR4A2 (Nurr1/TINUR/NOT) and NR4A3 (MINOR/CSMF). While currently defined as ligandless, these transcription factors have been shown to regulate varied processes across a host of tissues. Of particular interest, the NR4A family impinge, in a tissue dependent fashion, on cellular proliferation, apoptosis and fuel utilization. The regulation of these processes occurs through both nuclear and non-genomic pathways.
The three members have a high degree of sequence homology, with each containing a ligand-independent activation function-1 domain, transactivation domain necessary for transcriptional activity and cofactor binding, a DNA binding domain and a ligand binding domain containing a ligand-dependent AF-2 transactivation domain. While currently defined as orphaned receptors with no known endogenous ligand, there have been suggestions that the NR4A family's transcription factor function may be regulated through binding unsaturated fatty acids in the ligand binding domain.
The NR4A family binds directly as monomers or homodimers to promoters of target genes that contain the NBRE (NGFIB Response Element-AAAGGTCA) motif. The NR4As can also form heterodimers and bind to the NuRE (Nur Response element-AAT(G/A)(C/T)CA). Family members NR4A1 and NR4A2 have been shown to forms dimers with retinoid X receptors and bind to the DR5 elements. While there is a high degree of similarity between the three family members, the different members have differing affinity for co-factors and response elements, thus giving specificity to each.
The NR4A family is widely expressed across various tissues. NR4A family members have been shown to be critical in the hematopoietic system, adipose tissue, liver, muscle and β-cells, among other tissues. In these tissues, the function of Nr4a family members fall into one of two categories. The majority of NR4A activity is due to direct activation or repression of transcriptional target expression. However, a growing body of information is demonstrating a direct non-genomic role of NR4A family members through interaction with binding partners.
The NR4A family members are known to regulate cellular proliferation in a tissue dependent manner. Key genes shown to be regulated by NR4A family members include cyclins, cyclin dependent kinases and other ancillary cell cycle genes. Due to this, NR4A1, NR4A2 and NR4A3 are potential therapeutic targets in many cancers, however their specific roles vary between tissues and among tumors from the same organ.
The NR4A family members referred to in this aspect, and all aspects and embodiments described herein in this application, comprises the nucleotide sequences of NR4A1, NR4A2, and NR4A3 with NCBI nucleotide sequence IDs:
NM_002135.5 (SEQ ID NO: 2, NR4A1 isoform 1 mRNA), NM_173157.3 (SEQ ID NO: 3, NR4A1 isoform 1b mRNA), NM_001202233.2 (SEQ ID NO: 4, NR4A1 isoform 2 mRNA), NM_001202234.2 (SEQ ID NO: 5, NR4A1 isoform 3 mRNA), NM_006186.4 (SEQ ID NO: 7, NR4A2 isoform a mRNA), NM_173173.3 (SEQ ID NO: 8, NR4A2 isoform d mRNA), NC_000009.12 (SEQ ID NO: 9, NR4A3 genomic sequence), NM_006981.4 (SEQ ID NO: 10, NR4A3 isoform a mRNA), NM_173200.3 (SEQ ID NO: 11, NR4A3 isoform b mRNA) and NM_173199.4 (SEQ ID NO: 12, NR4A3 isoform c mRNA) and the amino acid sequences of NR4A1, NR4A2, and NR4A3 with NCBI amino acid sequence IDs: NP_001189162.1 (SEQ ID NO: 13, NR4A1 isoform a), NP_775292.1 (SEQ ID NO: 14, NR4A1 isoform b), NP_002126.2 (SEQ ID NO: 15, NR4A1 isoform 1), NP_775180.1 (SEQ ID NO: 16, NR4A1 isoform 1b), NP_001189163.1 (SEQ ID NO: 17, NR4A1 isoform 3), NP_006177.1 (SEQ ID NO: 18, NR4A2 isoform a), NP_775265.1 (SEQ ID NO: 19, NR4A2 isoform d), NP_008912.2 (SEQ ID NO: 20, NR4A3 isoform a), NP_775292.1 (SEQ ID NO: 21, NR4A3 isoform b), and NP_775291.1 (SEQ ID NO: 22, NR4A3 isoform c).
In some preferred embodiments of any one of the aspects described herein, the NR4A family member is nuclear receptor subfamily 4 group A member 1 (NR4A1), which is encoded on human chromosome 12. NR4A1, also referred to as NUR77 in the art, is a nuclear transcription factor induced by phytohemagglutinin in human lymphocytes and by serum stimulation of arrested fibroblasts. The encoded protein is known to respond to diverse cellular stresses to regulate apoptosis, inflammation, and cell cycle mediation. Mutations in this gene are associated with a variety of human diseases such as pseudohypoaldosteronism and adrenal cortical carcinoma. In some embodiments of any one of the aspects described herein, the inhibitor of a NR4A family member inhibits NR4A1.
In some embodiments of any one of the aspects described herein, the NR4A family member is nuclear receptor subfamily 4 group A member 2 (NR4A2), which is encoded on human chromosome 2. This gene encodes a member of the steroid-thyroid hormone-retinoid receptor superfamily. The encoded protein may act as a transcription factor. Mutations in this gene have been associated with disorders related to dopaminergic dysfunction, including Parkinson disease, schizophrenia, and manic depression. Misregulation of this gene may be associated with rheumatoid arthritis. Alternatively, spliced transcript variants have been described, but their biological validity has not been determined. In some embodiments of any one of the aspects described herein, the inhibitor of a NR4A family member inhibits NR4A2.
In some embodiments of any one of the aspects described herein, the NR4A family member is nuclear receptor subfamily 4 group A member 3 (NR4A3), which is encoded on human chromosome 9. This gene encodes a member of the steroid-thyroid hormone-retinoid receptor superfamily. The encoded protein may act as a transcriptional activator. The protein can efficiently bind the NGFI-B Response Element (NBRE). Three different versions of extraskeletal myxoid chondrosarcomas (EMCs) are the result of reciprocal translocations between this gene and other genes. The translocation breakpoints are associated with NR4A3 (on chromosome 9) and either Ewing Sarcome Breakpoint Region 1 (on chromosome 22), RNA Polymerase II, TATA Box-Binding Protein-Associated Factor, 68-KD (on chromosome 17), or Transcription factor 12 (on chromosome 15). In some embodiments of any one of the aspects described herein, the inhibitor of a NR4A family member inhibits NR4A3.
Embodiments of the various aspects described herein include administering an inhibitor of a NR4A family member to a subject. The inhibitor can directly bind with the NR4A family member or with a nucleic acid encoding the NR4A family member. Exemplary inhibitors that can be used include, but are not limited to, nucleic acids, antibodies, and small molecules.
In some embodiments of any one of the aspects described herein, the inhibitor of the NR4A family member is an inhibitory nucleic acid. As used herein, the term “inhibitory nucleic acid” or “nucleic acid inhibitor” refers to a nucleic acid molecule which can inhibit the expression of a nucleic acid, e.g., mRNA encoding the NR4A family member. Exemplary, inhibitory nucleic acids include, but are not limited to, double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), antisense oligonucleotides, aptamers, and the like. In some embodiments of any of the aspects, the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA). Inhibitory nucleic acids can also include guide sequence molecules (e.g., a guide RNA) that function, e.g., in combination with an enzyme, to induce insertions, deletions, indels, and/or mutations of a target, thereby inhibiting the expression of the target.
Generally, a nucleic acid inhibitor comprises a nucleotide sequence that is substantially complementary to at least a portion of a nucleic acid encoding a NR4A family member. For example, the nucleic acid inhibitor comprises a nucleotide sequence that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 1-12. In some embodiments, the nucleic acid inhibitor comprises a nucleotide sequence that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 2-5, 7-8 and 10-12.
In some embodiments of any of the aspects, the nucleic acid inhibitor comprises a sequence at least 15 nucleotides in length that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 1-12. For example, the nucleic acid inhibitor comprises a sequence 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 2-5, 7-8 and 10-12.
In some embodiments, the nucleic acid inhibitor inhibits NR4A1. For example, the nucleic acid inhibitor comprises a sequence 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 2-5.
In some embodiments, the nucleic acid inhibitor inhibits NR4A2. For example, the nucleic acid inhibitor comprises a sequence 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 7 and 8.
In some embodiments, the nucleic acid inhibitor inhibits NR4A3. For example, the nucleic acid inhibitor comprises a sequence 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length that is substantially complementary to at least a portion of a nucleotide sequence selected from SEQ ID NOs: 10-12.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
Accordingly, in some embodiments of any one of the aspects described herein, the nucleic acid inhibitor is a dsRNA, where one strand (e.g., antisense strand) of the dsRNA comprises a nucleotide sequence substantially complementary to a portion, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 1-12. For example, the nucleic acid inhibitor is a dsRNA, where the antisense strand of the dsRNA comprises a nucleotide sequence substantially complementary to a portion, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 2-5. In another non-limiting example, the nucleic acid inhibitor is a dsRNA, where the antisense strand of the dsRNA comprises a nucleotide sequence substantially complementary to a portion, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 7-8. In yet another non-limiting example, the nucleic acid inhibitor is a dsRNA, where the antisense strand of the dsRNA comprises a nucleotide sequence substantially complementary to a portion, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides of a nucleotide sequence selected from SEQ ID NOs: 10-12.
Based on the knowledge in the art, one skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of any one of SEQ ID NOs: 2-6, 7-8 or 10-12. Double-stranded RNAs useful for targeting expression of a NR4A family member enzyme can be readily designed and tested. Chalk et al. (Nucl. Acids Res. 33: D131-D134 (2005)) describe a database of siRNA sequences and a predictor of siRNA sequences. Linked to the sequences in the database is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. The database and associated predictive tools enable the user to evaluate an siRNA's potential for inhibition and non-specific effects. The database is available at on the world wide web at siRNA.cgb.ki.se. Synthetic siRNA molecules, including shRNA molecules, can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA molecule can be chemically synthesized or recombinantly produced using methods known in the art, such as using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer (see, e.g., Elbashir, S. M. et al., Nature 411:494-498 (2001); Elbashir, S. M., et al., Genes & Development 15:188-200 (2001); Harborth, J. et al., J. Cell Science 114:4557-4565 (2001); Masters, J. R. et al., Proc. Natl. Acad. Sci., USA 98:8012-8017 (2001); and Tuschl, T. et al., Genes & Development 13:3191-3197 (1999)).
Alternatively, several commercial RNA synthesis suppliers are available including, but not limited to, Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). As such, siRNA molecules are not overly difficult to synthesize and are readily provided in a quality suitable for RNAi. In addition, dsRNAs can be expressed as stem loop structures encoded by plasmid vectors, retroviruses and lentiviruses (Paddison, P. J. et al., Genes Dev. 16:948-958 (2002); McManus, M. T. et al., RNA 8:842-850 (2002); Paul, C. P. et al., Nat. Biotechnol. 20:505-508 (2002); Miyagishi, M. et al., Nat. Biotechnol. 20:497-500 (2002); Sui, G. et al., Proc. Natl. Acad. Sci., USA 99:5515-5520 (2002); Brummelkamp, T. et al., Cancer Cell 2:243 (2002); Lee, N. S., et al., Nat. Biotechnol. 20:500-505 (2002); Yu, J. Y., et al., Proc. Natl. Acad. Sci., USA 99:6047-6052 (2002); Zeng, Y., et al., Mol. Cell 9:1327-1333 (2002); Rubinson, D. A., et al., Nat. Genet. 33:401-406 (2003); Stewart, S. A., et al., RNA 9:493-501 (2003)).
In some embodiments of the various aspects described herein, the inhibitory nucleic acid is a guide nucleic acid (gNA). As used herein, the terms “guide nucleic acid,” “guide sequence,” “crRNA,” “guide RNA,” “single guide RNA,” “gRNA” or “CRISPR guide sequence” refer to a nucleic acid comprising a sequence that determines the specificity of an enzyme, e.g., the Cas DNA binding protein of a CRISPR/Cas system, to a polynucleotide target. The gNA can comprise a polynucleotide sequence with at least partial complementarity with a target nucleic acid sequence, sufficient to hybridize with the target nucleic acid sequence and to direct sequence-specific binding of an enzyme, e.g, a nuclease, to the target nucleic acid sequence.
In some embodiments, the enzyme directed by the gNA is a gene-editing protein, e.g., any nuclease that induces a nick or double-strand break into a desired recognition site. Such enzymes can be native or engineered. These breaks can then be repaired by the cell in one of two ways: non-homologous end joining and homology-directed repair (homologous recombination). In non-homologous end joining (NHEJ), the double-strand breaks are repaired by direct ligation of the break ends to one another. As such, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion. In homology-directed repair, a donor polynucleotide with homology to the cleaved target DNA sequence can be used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the target DNA. Therefore, new nucleic acid material may be inserted/copied into the site. The modifications of the target DNA due to NHEJ and/or homology-directed repair can be used for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.
In one embodiment, the gene-editing protein is a CRISPR-associated nuclease. The native prokaryotic CRISPR-associated nuclease system comprises an array of short repeats with intervening variable sequences of constant length (i.e., clusters of regularly interspaced short palindromic repeats), and CRISPR-associated (“Cas”) nuclease proteins. The RNA of the transcribed CRISPR array is processed by a subset of the Cas proteins into small guide RNAs, which generally have two components as discussed below. There are at least three different systems: Type I, Type II and Type III. The enzymes involved in the processing of the RNA into mature crRNA are different in the 3 systems. In the native prokaryotic system, the guide RNA (“gRNA”) comprises two short, non-coding RNA species referred to as CRISPR RNA (“crRNA”) and trans-acting RNA (“tracrRNA”). In an exemplary system, the gRNA forms a complex with a nuclease, for example, a Cas nuclease. The gRNA: nuclease complex binds a target polynucleotide sequence having a protospacer adjacent motif (“PAM”) and a protospacer, which is a sequence complementary to a portion of the gRNA. The recognition and binding of the target polynucleotide by the gRNA: nuclease complex induces cleavage of the target.
Any CRISPR-associated nuclease can be used in the system and methods of the invention. CRISPR nuclease systems are known to those of skill in the art, e.g. Cas9, Cas12, Cas12a, or the like, see Patents/applications 8,993,233, US 2015/0291965, US 2016/0175462, US 2015/0020223, US 2014/0179770, 8,697,359; 8,771,945; 8,795,965; WO 2015/191693; U.S. Pat. No. 8,889,418; WO 2015/089351; WO 2015/089486; WO 2016/028682; WO 2016/049258; WO 2016/094867; WO 2016/094872; WO 2016/094874; WO 2016/112242; US 2016/0153004; US 2015/0056705; US 2016/0090607; US 2016/0029604; 8,865,406; 8,871,445; each of which are incorporated by reference in their entirety. The nuclease can also be a phage Cas nuclease, e.g., CasΦ (e.g., Pausch et al. Science 369:333-7 (2020); which is incorporated by reference herein in its entirety).
The full-length guide nucleic acid strand can be any length. For example, the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments of the various aspects described herein, a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. For example, the guide nucleic acid sequence is 10-30 nucleotides long.
In addition to a sequence that is complementary to a target nucleic acid, in some embodiments, the gNA also comprises a scaffold sequence. Expression of a gNA encoding both a sequence complementary to a target nucleic acid and scaffold sequence has the dual function of both binding (hybridizing) to the target nucleic acid and recruiting the endonuclease to the target nucleic acid, which may result in site-specific CRISPR activity. In some embodiments, such a chimeric gNA may be referred to as a single guide RNA (sgRNA).
In some embodiments of the various aspects described herein, the guide nucleic acid is designed using a guide design tool (e.g., Benchling™; Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™; E-CRISP™; Geneious™; GenHub™; GUIDES™ (e.g., for library design); Horizon Discovery™; IDT™; Off_Spotter™; and Synthego™; which are available on the world wide web).
In some embodiments of any of the aspects, the nucleic acid inhibitor comprises a nucleic modification. For example, the nucleic acid inhibitor is chemically modified to enhance stability or other beneficial characteristics. The nucleic acid inhibitors described herein may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages.
Modified nucleic acid backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Modified nucleic acid backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular —CH2-NH—CH2-, —CH2-N(CH3)-O—CH2-[known as a methylene (methylimino) or MMI backbone], —CH2-O—N(CH3)-CH2-, —CH2-N(CH3)-N(CH3)-CH2- and —N(CH3)-CH2-CH2-[wherein the native phosphodiester backbone is represented as —O—P—O—CH2-].
In some embodiments of any one of the aspects, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units in the nucleic acid inhibitor are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such modification that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
The nucleic acid inhibitors can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
Modified nucleic acid inhibitors can also contain one or more substituted sugar moieties. For example, the nucleic acid inhibitor described herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1-C10alkyl, C2-C10alkenyl or C2-C10alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)·nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, dsRNAs include one of the following at the 2′ position: C1-C10alkyl, substituted C1-C10alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl. In some embodiments of any of the aspects, the modification includes a 2′ methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2-O—CH2-N(CH2)2.
The inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. For example, the nucleic acid inhibitor can comprise a modified or non-natural nucleobase. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
In some embodiments of any one of the aspects described herein, the inhibitory nucleic acid comprises one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the nucleic acid inhibitor. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et a1., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
The preparation modified nucleic acids are well known in the art and available to one of skill in the art.
Antibodies that specifically bind NR4A family members can be used for inhibition in vivo, in vitro, or ex vivo. The NR4A family inhibitory activity of a given antibody, or, for that matter, any NR4A family inhibitor, can be assessed using methods known in the art or described herein. Specific binding is typically defined as binding that does not recognize other antigens, such as a protein, nucleotide, chemical residue, etc., at a detectable level in an assay used. Accordingly, in some embodiments of any one of the aspects described herein, the inhibitor of the NR4A family member is an antibody or an antigen binding fragment thereof. Generally, the antibody or the antigen binding fragment thereof binds an epitope on the NR4A family member such that the binding inhibits a function and/or activity of the NR4A family member.
Antibodies that can be used according to the methods described herein, include complete immunoglobulins, antigen binding fragments of immunoglobulins, as well as antigen binding proteins that comprise antigen binding domains of immunoglobulins. Antigen binding fragments of immunoglobulins include, for example, Fab, Fab′, F(ab′)2, scFv and dAbs. Modified antibody formats have been developed which retain binding specificity, but have other characteristics that may be desirable, including for example, bispecificity, multivalence (more than two binding sites), and compact size (e.g., binding domains alone). Single chain antibodies lack some or all of the constant domains of the whole antibodies from which they are derived. Therefore, they can overcome some of the problems associated with the use of whole antibodies. For example, single-chain antibodies tend to be free of certain undesired interactions between heavy-chain constant regions and other biological molecules. Additionally, single-chain antibodies are considerably smaller than whole antibodies and can have greater permeability than whole antibodies, allowing single-chain antibodies to localize and bind to target antigen-binding sites more efficiently. Furthermore, the relatively small size of single-chain antibodies makes them less likely to provoke an unwanted immune response in a recipient than whole antibodies.
Multiple single chain antibodies, each single chain having one VH and one VL domain covalently linked by a first peptide linker, can be covalently linked by at least one or more peptide linker to form multivalent single chain antibodies, which can be monospecific or multispecific. Each chain of a multivalent single chain antibody includes a variable light chain fragment and a variable heavy chain fragment, and is linked by a peptide linker to at least one other chain. The peptide linker is composed of at least fifteen amino acid residues. The maximum number of linker amino acid residues is approximately one hundred.
Two single chain antibodies can be combined to form a diabody, also known as a bivalent dimer. Diabodies have two chains and two binding sites, and can be monospecific or bispecific. Each chain of the diabody includes a VH domain connected to a VL domain. The domains are connected with linkers that are short enough to prevent pairing between domains on the same chain, thus driving the pairing between complementary domains on different chains to recreate the two antigen-binding sites.
Three single chain antibodies can be combined to form triabodies, also known as trivalent trimers. Triabodies are constructed with the amino acid terminus of a VL or VH domain directly fused to the carboxyl terminus of a VL or VH domain, i.e., without any linker sequence. The triabody has three Fv heads with the polypeptides arranged in a cyclic, head-to-tail fashion. A possible conformation of the triabody is planar with the three binding sites located in a plane at an angle of 120 degrees from one another. Triabodies can be monospecific, bispecific or trispecific.
Thus, antibodies useful in the methods described herein include, but are not limited to, naturally occurring antibodies, bivalent fragments such as (Fab′)2, monovalent fragments such as Fab, single chain antibodies, single chain Fv (scFv), single domain antibodies, multivalent single chain antibodies, diabodies, triabodies, and the like that bind specifically with an antigen.
Antibodies can also be raised against a nucleotide, polypeptide or portion of a polypeptide by methods known to those skilled in the art. Antibodies are readily raised in animals such as rabbits or mice by immunization with the gene product, or a fragment thereof. Immunized mice are particularly useful for providing sources of B cells for the manufacture of hybridomas, which in turn are cultured to produce large quantities of monoclonal antibodies. Antibody manufacture methods are described in detail, for example, in Harlow et al., 1988. While both polyclonal and monoclonal antibodies can be used in the methods described herein, it is preferred that a monoclonal antibody is used where conditions require increased specificity for a particular protein.
The term “intrabodies” as used herein, refers to a method wherein to target intracellular endogenous proteins as described in U.S. Pat. No. 6,004,940. Briefly, the method comprises the intracellular expression of an antibody capable of binding to the target. A DNA sequence is delivered to a cell, the DNA sequence contains a sufficient number of nucleotides coding for the portion of an antibody capable of binding to the target operably linked to a promoter that will permit expression of the antibody in the cell(s) of interest. The antibody is then expressed intracellularly and binds to the target, thereby disrupting the target from its normal actions.
“Antigen-binding fragments” include, inter alia, Fab, Fab′, F(ab′)2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The terms Fab, Fc, pFc′, F(ab′) 2 and Fv are employed with standard immunological meanings [Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford)].
Antibody inhibitors of NR4A family members can include polyclonal and monoclonal antibodies and antigen-binding derivatives or fragments thereof.
In some embodiments of any one of the aspects described herein, the inhibitor is an anti-NR4A1 antibody or an antigen binding fragment thereof. Generally, the antibody or the antigen binding fragment thereof binds an epitope on the NR4A1 such that the binding inhibits a function and/or activity of the NR4A1. In some embodiments of any one of the aspects described herein, the antibody binds to polypeptide of comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17. Exemplary anti-NR4A1 antibodies are commercially available and include, but are not limited to anti-NR41A, 25851-1-AP, ProteinTech; anti-NR41A, 12235-1-AP, ProteinTech; anti-NR41A, OriGene, Cat #: TA314017; anti-NR41A, Bioss, Cat #BS-3313R; anti-NR41A, Abcam, ab41917; anti-NR41A, Thermo-Fisher, Cat #53-5965-82; anti-NR41A, Invitrogen, #PA5-27274; anti-NR41A, Novus Biologicals, Cat #NB100-56745; anti-NR41A, Boster Bio,Cat #PA1676; anti-NR41A, Aviva Systems Biology, Cat #ARP31941_T100; anti-NR41A, Atlas Antibodies, Cat #HPA070142; anti-NR41A, Santa Cruz Biotechnologies, sc-365113; anti-NR41A, Creative Biolabs, NHP-AB411; anti-NR41A, EMD Millipore, Cat #ABE1454; anti-NR41A, ProSci, Cat. #27-450; and anti-NR41A, R&D Systems, Cat #PP-H1648-00.
In some embodiments of any one of the aspects described herein, the inhibitor is an anti-NR4A2 antibody or an antigen binding fragment thereof. Generally, the antibody or the antigen binding fragment thereof binds an epitope on the NR4A2 such that the binding inhibits a function and/or activity of the NR4A2. In some embodiments of any one of the aspects described herein, the antibody binds to polypeptide of comprising the amino acid sequence of SEQ ID NOs: 18-19. Exemplary anti-NR4A2 antibodies are commercially available and include, but are not limited to, anti-Nurr1, ab41917, Abcam; NR4A2 monoclonal antibody, 66878-1-Ig, ProteinTech; Nurr1 Polyclonal Antibody, CAT #: TA349421, OriGene; Nurr1 Monoclonal Antibody, Catalog #MA1-195, Invitrogen; Nurr1 Monoclonal Antibody, Catalog #H00004929-M08, Catalog #H00004929-M10, Catalog #H00004929-M07, Abnova; Anti-NR4A2 (Nurr1) antibody, Cat #682002, BioLegend; Nurr1/NGFI-B beta/NR4A2 Antibody, Cat #PP-N1404-00, Novus Biologicals; and anti-Nurr1 antibody, sc-376984, Santa Cruz Biotechnologies.
In some embodiments of any one of the aspects described herein, the inhibitor is an anti-NR4A3 antibody or an antigen binding fragment thereof. Generally, the antibody or the antigen binding fragment thereof binds an epitope on the NR4A3 such that the binding inhibits a function and/or activity of the NR4A3. In some embodiments of any one of the aspects described herein, the antibody binds to polypeptide of comprising the amino acid sequence of SEQ ID NOs: 20-22. Exemplary anti-NR4A3 antibodies are commercially available and include include, but are not limited to Recombinant Anti-NOR1/TEC antibody, ab259939, Abcam; NOR1 (NR4A3) Mouse Monoclonal Antibody, CAT #: TA804893, OriGene; NOR-1 Monoclonal Antibody, Catalog #MA5-26704, Invitrogen; NR4A3 Monoclonal Antibody, Catalog #H00008013-M02, Abnova; NR4A3/NOR1 Antibody, Cat #NBP2-46246, Novus Biologicals; Anti-NOR-1 Antibody, sc-393902, Santa Cruz Biotechnologies; and anti-NOR1 (NR4A3) Mouse Monoclonal Antibody, Cat #M02578, Boster Bio.
Methods of obtaining antibodies against an antigen are well known in the art and available to one of skill in the art.
In some embodiments of any one of the aspects described herein, the inhibitor of the NR4A family member is a small molecule. Generally, the small molecule inhibitor binds with the NR4A family member and inhibits a function and/or activity of the NR4A family member. As used herein, the term “small molecules” refers to natural or synthetic molecules including, but not limited to, amino acids, peptides, peptidomimetics, polynucleotides, aptamers, nucleotide analogs, organic or inorganic compounds (i.e., including heterorganic and organometallic compounds), saccharides (e.g., mono, di, tri and polysaccharides), steroids, hormones, pharmaceutically derived drugs (e.g., synthetic or naturally occurring), lipids, derivatives of these (e.g., esters and salts of these), fragments of these, and conjugates of these. In some implementations the small molecules have a molecular weight less than about 5,000 Da, organic or inorganic compounds having a molecular weight less than about 2,500 Da, organic or inorganic compounds having a molecular weight less than about 1,000 Da, organic or inorganic compounds having a molecular weight less than about 500 Da. In some implementations the small molecule has a molecular weight of less than about 1000 Da.
In some embodiments of any one of the aspects described herein, the small molecule inhibitor is a 1,1-bis(3′-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compounds are described in U.S. Pat. No. 7,232,843, content of which is incorporated herein by reference in its entirety. In some embodiments of any one of the aspects described herein, the inhibitor is selected from the group consisting of 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (DIM-C-pPhOh-3-Cl-5-OCH3), 1,1-bis(3′-indolyl)-1-(3,5-dibromo-4-hydroxyphenyl)methane (DIM-C-pPhOH-3,5-Br2), camptothecin (CPT), a cyclooxygenase (COX)-2 inhibitor (celecoxib analogue SC-236), Erlotinib, Afatinib, Bosutinib, Dasatinib, and KU171309.
In some embodiments of any one of the aspects, the inhibitor is selected from the group consisting of DIM-C-pPhOH, DIM-C-pPhOh-3-Cl-5-OCH3, DIM-C-pPhOH-3,5-Br2, camptothecin and celecoxib analogue SC-236. In some other embodiments of any one of the aspects described herein, the inhibitor is selected from the group consisting of Erlotinib, Afatinib, Bosutinib, Dasatinib, and KU171309.
In some embodiments of any one of the aspects described herein, the small molecule inhibitor inhabits NR4A1. For example, the inhibitor inhibits NR4A1 and is selected from the group consisting of DIM-C-pPhOH, DIM-C-pPhOh-3-Cl-5-OCH3, DIM-C-pPhOH-3,5-Br2, camptothecin and celecoxib analogue SC-236.
In some embodiments of any one of the aspects described herein, the small molecule inhibitor inhibits NR4A2. For example, the inhibitor inhibits NR4A2 and is selected from the group consisting of Erlotinib, Afatinib, Bosutinib, Dasatinib, KU171309, C-DIM12 and DIM-C-pPhCl.
In some embodiments of any one of the aspects described herein, the smile molecule inhibitor inhibits NR4A3.
Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) divide to produce blood cells by a continuous regeneration process. As the cells divide, they are prone to accumulating mutations, including deletions, insertions, and substitutions, that generally do not affect function. However, some mutations confer advantages in self-renewal, proliferation or both, resulting in clonal expansion of the cells comprising the mutations in question. The frequency of such somatic mutation events increases with age. The studies described herein demonstrate that preferential and progressive expansion of a subset of hematopoietic cells bearing somatic mutations in HSC genes, such as ASXL transcriptional regulator 1 (ASXL1), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), isocitrate dehydrogenase (NADP(+)) 1(IDH1) and/or isocitrate dehydrogenase (NADP(+)) 2 (IDH 2) leads to increased production of inflammatory cytokines, such as IL-10 and/or TNF-P.
Accordingly, in some embodiments of any one of the aspects described herein, the subject has at least one mutation in a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 or a nucleic acid encoding IDH 2. In some embodiments, the subject has at least one mutation in two of a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 and a nucleic acid encoding IDH 2. In some embodiments, the subject has at least one mutation in three of a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 and a nucleic acid encoding IDH 2. In some embodiments, the subject has at least one mutation in all four of a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 and a nucleic acid encoding IDH 2. It is noted that the mutation in ASXL1, DMNT3A, IDH1 or IDH2 is in a subject's HSPC.
Exemplary types of mutations include, but are not limited to, substitutions, insertions and deletions. It is noted that the insertion and/or deletion mutations can be a frameshift mutation or an in-frame mutation. A frameshift mutation (also called a framing error or a reading frame shift) is a mutation caused by indels (insertion or deletions) of a number of nucleotides in a nucleic acid sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame (the grouping of the codons), resulting in a completely different translation from the original. By contrast, any insertion or deletion that is evenly divisible by three is termed an in-frame mutation.
Methods for determining mutations in a nucleic acid are well known in the art and include, but are not limited to, targeted mutational analysis, full sequence analysis, and deletion/duplication analysis. The mutation can be detected either directly, e.g., by genomic analysis or genetic probing, or indirectly, e.g., by measuring relative levels of gene products to detect abnormal expression levels. Enzyme expression levels can be determined in multiple manners, and quantitation is relative, based on a specific standard for each assay.
Methods for detecting the presence of a mutation in a gene of interest are known in the art. Suitable methods for determining whether or not a particular mutation in a gene exists include, e.g., Southern blot (see, e.g., Sambrook et al. {supra)), real-time PCR analysis (see, e.g., Oliver et a1. (2000) JMol Diagnostics 2(4}:202-208), nucleic acid array analysis, allele-specific PCR (e.g., quantitative allele-specific PCR), pyrosequencing, DNA sequencing (e.g., Sanger chemistry sequencing), or through the use of molecular beacons (e.g., Tyagi et al. (1998) Nat Biotechnol 16:49-53; Abravaya et al. (2003) Clin Chem Lab Med4\:468-474; and Mullah et al. (1999) Nucleosides & Nucleotides 18: 1311-1312, the disclosures of each of which are incorporated herein by reference in their entirety). Suitable conditions and methods for detecting nucleic acid mutations using nucleic acid arrays are described in, e.g., Lamy et al. (2006) Nucleic Acids Research 34(14): elOO; European Patent Application Publication No. 1234058; U.S. Patent Application Publication Nos. 20060008823 and 20030059813; and U.S. Pat. No. 6,410,231; the disclosures of each of which are incorporated herein by reference in their entirety.
Inventors have discovered inter alia that indel (insertion/deletion) mutations in the mutant HSPCs enhance the competitive advantage of the mutant clones to establish clonal dominance. Accordingly, in some embodiments of any one of the aspects described herein, the subject to be treated has an indel mutation in a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 or a nucleic acid encoding IDH 2. For example, the subject to be treated has an in-frame indel mutation in at least one mutation in a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 or a nucleic acid encoding IDH 2. In another non-limiting example, the subject to be treated has a frameshift indel mutation in at least one mutation in a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 or a nucleic acid encoding IDH 2.
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid encoding ASXL1. For example, the subject has a frameshift mutation in a nucleic acid encoding ASXL1. In another non-limiting example, the subject has a frameshift mutation in a nucleic acid encoding ASXL1. The AXSL1, which is encoded on chromosome 20, is similar to the Drosophila additional sex combs gene, which encodes a chromatin-binding protein required for normal determination of segment identity in the developing embryo. The protein is a member of the Polycomb group of proteins, which are necessary for the maintenance of stable repression of homeotic and other loci. The protein is thought to disrupt chromatin in localized areas, enhancing transcription of certain genes while repressing the transcription of other genes. The protein encoded by this gene functions as a ligand-dependent co-activator for retinoic acid receptor in cooperation with nuclear receptor coactivator 1. Mutations in this gene are associated with myelodysplastic syndromes and chronic myelomonocytic leukemia. Alternative splicing results in multiple transcript variants. The ASXL1 referred to in this aspect, and all aspects and embodiments described herein in this application, comprises the nucleotide sequences of NCBI nucleotide sequence IDs: NC_000020.11 (SEQ ID NO: 23, AXSL1 genomic sequence), NM_015338.6 (SEQ ID NO: 24, AXSL1 isoform 1 mRNA), NM_001164603.1 (SEQ ID NO: 25, AXSL1 isoform 2 mRNA) and NM_001363734.1 (SEQ ID NO: 26, AXSL1 isoform 3 mRNA).
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26. For example, the subject has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26. In another example, the subject has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26.
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid encoding DNMT3A. For example, the subject has a frameshift mutation in a nucleic acid encoding DNMT3A. In another non-limiting example, the subject has a frameshift mutation in a nucleic acid encoding DNMT3A. The DNMT3A, which is encoded on human chromosome 2. CpG methylation is an epigenetic modification that is important for embryonic development, imprinting, and X-chromosome inactivation. Studies in mice have demonstrated that DNA methylation is required for mammalian development. This gene encodes a DNA methyltransferase that is thought to function in de novo methylation, rather than maintenance methylation. The protein localizes to the cytoplasm and nucleus and its expression is developmentally regulated. The DMNT3A referred to in this aspect, and all aspects and embodiments described herein in this application, comprises the nucleotide sequences of NCBI nucleotide sequence IDs: NC_000002.12 (SEQ ID NO: 27, DNMT3A genomic sequence), NM_001320892.2 (SEQ ID NO: 28, DNMT3A isoform c mRNA), NM_001320893.1 (SEQ ID NO: 29, DNMT3A isoform d mRNA), NM_001375819.1 (SEQ ID NO: 30, DNMT3A isoform e mRNA), NM_022552.5 (SEQ ID NO: 31, DNMT3A isoform a mRNA), NM_153759.3 (SEQ ID NO: 32, DNMT3A isoform b mRNA), NM_175629.2 (SEQ ID NO: 33, DNMT3A isoform a mRNA), and NM_175630.1 (SEQ ID NO: 34, DNMT3A isoform c mRNA).
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34. For example, the subject has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34. In another example, the subject has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34.
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid encoding IDH1. For example, the subject has a frameshift mutation in a nucleic acid encoding IDH1. In another non-limiting example, the subject has a frameshift mutation in a nucleic acid encoding IDH1. The IDH1, which is encoded on human chromosome 2. Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate. These enzymes belong to two distinct subclasses, one of which utilizes NAD(+) as the electron acceptor and the other NADP(+). Five isocitrate dehydrogenases have been reported: three NAD(+)-dependent isocitrate dehydrogenases, which localize to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic. Each NADP(+)-dependent isozyme is a homodimer. The protein encoded by this gene is the NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. It contains the PTS-1 peroxisomal targeting signal sequence. The presence of this enzyme in peroxisomes suggests roles in the regeneration of NADPH for intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl-CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. The cytoplasmic enzyme serves a significant role in cytoplasmic NADPH production. Alternatively, spliced transcript variants encoding the same protein have been found for this gene. The IDH1 referred to in this aspect, and all aspects and embodiments described herein in this application, comprises the nucleotide sequences of NCBI nucleotide sequence IDs: NC_000002.12 (SEQ ID NO: 35, IDH1 genomic sequence), NM_001282386.1 (SEQ ID NO: 36, IDH1 isoform 2 mRNA), NM_001282387.1 (SEQ ID NO: 37, IDH1 isoform 2 mRNA) and NM_005896.4 (SEQ ID NO: 38, IDH1 isoform 1 mRNA).
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-38. For example, the subject has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-38. In another example, the subject has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-38.
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid encoding IDH2. For example, the subject has a frameshift mutation in a nucleic acid encoding IDH2. In another non-limiting example, the subject has a frameshift mutation in a nucleic acid encoding IDH2. The IDH2, encoded on human chromosome 15. Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate. These enzymes belong to two distinct subclasses, one of which utilizes NAD(+) as the electron acceptor and the other NADP(+). Five isocitrate dehydrogenases have been reported: three NAD(+)-dependent isocitrate dehydrogenases, which localize to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic. Each NADP(+)-dependent isozyme is a homodimer. The protein encoded by this gene is the NADP(+)-dependent isocitrate dehydrogenase found in the mitochondria. It plays a role in intermediary metabolism and energy production. This protein may tightly associate or interact with the pyruvate dehydrogenase complex. Alternative splicing results in multiple transcript variants.
The IDH2 referred to in this aspect, and all aspects and embodiments described herein in this application, comprises the nucleotide sequences of NCBI nucleotide sequence IDs: NC_000015.10 (SEQ ID NO: 39, IDH2 genomic sequence), NM_002168.4 (SEQ ID NO: 40, IDH2 isoform 1 mRNA), NM_001289910.1 (SEQ ID NO: 41, IDH2 isoform 2 mRNA) and NM_001290114.2 (SEQ ID NO: 42, IDH2 isoform 3 mRNA).
In some embodiments of any one of the aspects described herein, the subject to be treated comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-42. For example, the subject has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-42. In another example, the subject has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-42.
A subject having a clonal hematopoiesis disease or disorder can suffer from conditions such as fatigue, shortness of breath, bruising or bleeding, infections due to low white blood cell count, pale skin, and anemia. Generally, the clonal hematopoiesis diseases and disorders are characterized by clonal expansion of mutant HSPCs, resulting in the depletion of wild-type HSPCs. Accordingly, some embodiments of any one of the aspects described herein, the subject to be treated is determined to have an increased level of mutant HSPCs and/or proliferation of mutant HSPCs cells relative to a reference level. For example, the method comprises a step of determining or obtaining results of an assay indicating a level of mutant HSPCs in a sample from the subject prior to onset of treatment.
In some embodiments of any one of the aspects described herein, the subject to be treated is determined to have an elevated level of at least one NR4A family member relative to a reference level. For example, the method comprises a step of determining or obtaining results of an assay indicating an expression level of at least member of a NR4A family member in a sample from the subject prior to onset of treatment.
It is noted that the reference level can be the level in a sample of similar sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject for which the level is to be determined. In some embodiments of any of the aspects, the reference level can be the level in a sample obtained from a reference subject of similar age as the subject for which the level is to be determined. In some embodiments of any of the aspects, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.
In some embodiments of any of the aspects, the reference can be a level in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms of a clonal hematopoiesis disease or disorder. In some embodiments of any of the aspects, the reference level can be a level in a population of subjects who do not contain mutations that confer competitive advantage to mutant HSPC, e.g., mutation in a nucleic acid encoding AXSL1, DMNT3A, IDH1 or IDH2. In some embodiments of any of the aspects, the reference level can also be a level in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same. In some embodiments of any of the aspects, the reference level can be the level in a sample obtained from the same subject at an earlier point in time, e.g., the methods described herein can be used to determine if a subject's sensitivity or response to a given therapy is changing over time.
A level which is more than a reference level can be a level which is greater by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or more than the reference level. In some embodiments of any of the aspects, a level which is more than a reference level can be a level which is statistically significantly greater than the reference level.
The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. The additional therapy can be administered prior to, simultaneously with, or after administering the NR4A inhibitor.
The phrase “combination therapy” as used herein means administration a NR4A inhibitor and one or more additional therapies as part of a specific treatment regimen intended to provide a beneficial effect from the co-action of these. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of therapeutic agents. Administration of these therapeutic agents in combination typically is carried out over a defined time period. The time period may be in minutes, hours, days or weeks depending upon the combination selected.
Combination therapy includes administration of these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent is administered at a different time, as well as administration of these therapeutic agents, or at least two of the therapeutic agents, in a substantially simultaneous manner. Substantially simultaneous administration can be done, for example, by administering to the subject a single pill having a fixed ratio of each therapeutic agent or in multiple, single pills for each of the therapeutic agents. Sequential or substantially simultaneous administration of each therapeutic agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination selected may be administered by intravenous injection while the other therapeutic agents of the combination may be administered orally. Alternatively, for example, all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection. The sequence in which the therapeutic agents are administered may or may not be important.
Combination therapy also can mean the administration of one or more inhibitors NR4A family in further combination with other compounds and non-drug therapies, such as, but not limited to, surgery or radiation treatment. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved.
If a subject is to be treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and/or treatment known to be beneficial for subjects suffering from pain or inflammation. For example, the method can further comprise co-administering an anti-inflammatory agent to the subject. As used herein the term “anti-inflammatory agent” refers to a compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) which can be used to treat inflammation or an inflammation related disease or disorder. Exemplary anti-inflammatory agents include, but are not limited to, the known steroidal anti-inflammatory and non-steroidal anti-inflammatory drugs (NSAIDs). Exemplary steroidal anti-inflammatory agents include but are not limited to 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetansone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furcate, paramethosone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide, and analogues and derivatives thereof. Exemplary nonsteroidal anti-inflammatory agents include but are not limited to COX inhibitors (COX-1 or COX nonspecific inhibitors) and selective COX-2 inhibitors. Exemplary COX inhibitors include but are not limited to salicylic acid derivatives such as aspirin, sodium salicylate, choline magnesium trisalicylate, salicylate, diflunisal, sulfasalazine and olsalazine; para-aminophenol derivatives such as acetaminophen; indole and indene acetic acids such as indomethacin and sulindac; heteroaryl acetic acids such as tolmetin, dicofenac and ketorolac; arylpropionic acids such as ibuprofen, naproxen, flurbiprofen, ketoprofen, fenoprofen and oxaprozin; anthranilic acids (fenamates) such as mefenamic acid and meloxicam; enolic acids such as the oxicams (piroxicam, meloxicam); alkanones such as nabumetone; and analogues and derivatives thereof. Exemplary COX-2 inhibitors include but are not limited to diaryl substituted furanones such as refecoxib; diaryl-substituted pyrazoles such as celecoxib; indole acetic acids such as etodolac and sulfonanilides such as nimesulide; and analogues and derivatives thereof.
In some embodiments, of the various aspects disclosed herein, the method can further comprise co-administering an immunomodulatory agent to the subject. Exemplary immunomodulatory agents include, but are not limited to, proteinaceous agents such as cytokines, peptide mimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab or F(ab)2 fragments or epitope binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules and triple helices), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), T cell receptor modulators, cytokine receptor modulators, and modulators mast cell modulators.
Examples of T cell receptor modulators include, but are not limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1.R™ (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies (e.g., siplizumab (MedImmune, Inc., International Publication Nos. WO 02/098370 and WO 02/069904)), anti-CD11a antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))), CTLA4-immunoglobulin, and LFA-3TIP (Biogen, International Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432).
Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNF-alpha receptor or a fragment thereof, the extracellular domain of an IL-1.beta. receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof), cytokines or fragments thereof (e.g., interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-23, TNF-alpha, TNF-beta, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and GM-CSF), anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax™ (Protein Design Labs)), anti-IL-3 receptor antibodies, anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, anti-IL-12 receptor antibodies, anti-IL-13 receptor antibodies, anti-IL-15 receptor antibodies, and anti-IL-23 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-.alpha. antibodies, anti-IL-1.beta. antibodies, anti-IL-3 antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-IL-8 (Abgenix)), anti-IL-12 antibodies, anti-IL-13 antibodies, anti-IL-15 antibodies, and anti-IL-23 antibodies).
In some embodiments, a cytokine receptor modulator is IL-3, IL-4, IL-10, or a fragment thereof. In another embodiment, a cytokine receptor modulator is an anti-IL-1-beta antibody, anti-IL-6 antibody, anti-IL-12 receptor antibody, or anti-TNF-alpha antibody. In one embodiment, a TNF-alpha antagonist used in the compositions and methods of the invention is a soluble TNF-alpha receptor. In some embodiments, a TNF-alpha antagonist used in the compositions and methods of the invention is etanercept (ENBREL™; Immunex) or a fragment, derivative or analog thereof. In another embodiment, a TNF-alpha antagonist used in the compositions and methods of the invention is an antibody that immunospecifically binds to TNF-.alpha.. In some embodiments, a TNF-alpha antagonist used in the compositions and methods of the invention is infliximab (REMICADE™; Centacor) a derivative, analog or antigen-binding fragment thereof. In another embodiment, a cytokine receptor modulator is the extracellular domain of a TNF-alpha receptor or a fragment thereof. In certain embodiments, a cytokine receptor modulator is not a TNF-alpha antagonist.
In one embodiment, a cytokine receptor modulator is a mast cell modulator. In an alternative embodiment, a cytokine receptor modulator is not a mast cell modulator. Examples of mast cell modulators include, but are not limited to stem cell factor (c-kit receptor ligand) inhibitors (e.g., mAb 7H6, mAb 8H7a, pAb 1337, FK506, CsA, dexamthasone, and fluconcinonide), c-kit receptor inhibitors (e.g., STI 571 (formerly known as CGP 57148B)), mast cell protease inhibitors (e.g., GW-45, GW-58, wortmannin, LY 294002, calphostin C, cytochalasin D, genistein, KT5926, staurosproine, and lactoferrin), relaxin (“RLX”), IgE antagonists (e.g., antibodies rhuMAb-E25 omalizumab, HMK-12 and 6HD5, and mAB Hu-901), IL-3 antagonists, IL-4 antagonists, IL-10 antagonists, and TGF-beta.
In some embodiments, of the various aspects disclosed herein, the method can further comprise co-administering an additional anti-cancer therapy to the subject. For example, administering a standard of care chemotherapeutic to the subject. Non-limiting examples of a standard of care chemotherapeutics or other anti-cancer therapy can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin gammalI and calicheamicin omegaIl (see, e.g. Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g. TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb®); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g. erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additional anti-cancer treatment can further include the use of radiation or radiation therapy. Further, the additional anti-cancer treatment can also include the use of surgical treatments.
In another aspect provided herein is a method for inhibiting clonal expansion of hematopoietic stem and progenitor cells. The method comprises administering an effective amount of an inhibitor of a member of the NR4A family to a HSPC. In some embodiments of any one of the aspects described herein, the method comprises administering an effective amount of an inhibitor of a member of the NR4A family to a HSPC, wherein the NR4A family member is selected from the group consisting of NR4A1, NR4A2 and NR4A3. In some preferred embodiments, the NR4A family member is NR4A1.
It is noted that administering to the HSPC can be in vitro or in-vivo. Methods for administering a compound to a cell are well known and available to one of skill in the art. As used herein, administering the compound to the cell means contacting the cell with the compound so that the compound is taken up by the cell. Generally, the cell can be contacted with the compound in a cell culture e.g., in vitro or ex vivo, or the compound can be administrated to a subject, e.g., in vivo. The term “contacting” or “contact” as used herein in connection with contacting a cell includes subjecting the cells to an appropriate culture media, which comprises NR4A inhibitor. Where the cell is in vivo, “contacting” or “contact” includes administering the Inhibitor, e.g., in a pharmaceutical composition to a subject via an appropriate administration route such that the compound contacts the cell in vivo.
For example, when the cell is in vitro, said administering to the cell can include subjecting the cell to an appropriate culture media which comprises the indicated compound. Where the cell is in vivo, said administering to the cell includes administering the compound to a subject via an appropriate administration route such that the compound is administered to the cell in vivo.
In some embodiments of any one of the aspects, the HSPC to which the NR4A inhibitor is administered has an elevated level of at least one NR4A family member relative to a reference level. For example, the HSPC has an elevated level of NR4A1 relative to a reference level. Thus, in some embodiments, the method further comprises measuring or determining a level of at least one NR4A family member relative to a reference level prior to administering the NR4A inhibitor to the cell.
In some embodiments of any one of the aspects, the HSPC to which the NR4A inhibitor is administered has at least one mutation in a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 or a nucleic acid encoding IDH 2. In some embodiments, the HSPC has at least one mutation in two of a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 and a nucleic acid encoding IDH 2. In some embodiments, the HSPC has at least one mutation in three of a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 and a nucleic acid encoding IDH 2. In some embodiments, the HSPC has at least one mutation in all four of a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 and a nucleic acid encoding IDH 2. In some embodiments, the method further comprises detecting or assaying, prior to administering the NR4A inhibitor to the cell, for a mutation in a nucleic acid encoding ASXL1, a nucleic acid encoding DNMT3A, a nucleic acid encoding IDH1 or a nucleic acid encoding IDH 2.
In some embodiments of any one of the aspects described herein, the HSPC has at least one mutation in a nucleic acid encoding ASXL 1. For example, the HSPC has a frameshift mutation in a nucleic acid encoding ASXL1. In another non-limiting example, the HSPC has a frameshift mutation in a nucleic acid encoding ASXL1. In some embodiments of any one of the aspects described herein, the HSPC has at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26. For example, the HSPC has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26. In another example, the HSPC has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 23-26.
In some embodiments of any one of the aspects described herein, the HSPC to which the NR4A inhibitor is administered has at least one mutation in a nucleic acid encoding DNMT3A. For example, the HSPC has a frameshift mutation in a nucleic acid encoding DNMT3A. In another non-limiting example, the HSPC has a frameshift mutation in a nucleic acid encoding DNMT3A. In some embodiments of any one of the aspects described herein, the HSPC to which the NR4A inhibitor is administered comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34. For example, the HSPC has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34. In another example, the HSPC has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 27-34.
In some embodiments of any one of the aspects described herein, the HSPC to which the NR4A inhibitor is administered comprises at least one mutation in a nucleic acid encoding IDH1. For example, the HSPC has a frameshift mutation in a nucleic acid encoding IDH1. In another non-limiting example, the HSPC has a frameshift mutation in a nucleic acid encoding IDH1. In some embodiments of any one of the aspects described herein, the HSPC to which the NR4A inhibitor is administered comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-38. For example, the HSPC has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-38. In another example, the HSPC has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-38.
In some embodiments of any one of the aspects described herein, the HSPC to which the NR4A inhibitor is administered comprises at least one mutation in a nucleic acid encoding IDH2. For example, the HSPC has a frameshift mutation in a nucleic acid encoding IDH2. In another non-limiting example, the HSPC has a frameshift mutation in a nucleic acid encoding IDH2. In some embodiments of any one of the aspects described herein, the HSPC to which the NR4A inhibitor is administered comprises at least one mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-42. For example, the HSPC has a frameshift mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-42. In another example, the HSPC has an in-frame mutation in a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39-42.
In some embodiments of any of the aspects, the methods described herein are directed to determination of the expression level of a clonal population of HSPCs in a biological sample of a subject.
In some embodiments of any of the aspects, the methods described herein are directed to determination of the expression level of a gene product (e.g. protein and/or gene transcript such as mRNA) in a biological sample from a subject.
In some embodiments of any of the aspects, measurement of the level of a target and/or detection of the level or presence of a target, e.g. of an expression product (nucleic acid or polypeptide of one of the genes described herein) or a mutation can comprise a transformation. As used herein, the term “transforming” or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but is not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological/chemical transformation can involve the action of at least one enzyme and/or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzymes, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments of any of the aspects, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
Transformation, measurement, and/or detection of a target molecule, e.g. a mRNA or polypeptide can comprise contacting a sample obtained from a subject with a reagent (e.g. a detection reagent) which is specific for the target, e.g., a target-specific reagent. In some embodiments of any of the aspects, the target-specific reagent is detectably labeled. In some embodiments of any of the aspects, the target-specific reagent is capable of generating a detectable signal. In some embodiments of any of the aspects, the target-specific reagent generates a detectable signal when the target molecule is present.
Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques.
In some embodiments of any of the aspects, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
In some embodiments of any of the aspects, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.
Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiments of any of the aspects, the immunoassay can be a quantitative or a semi-quantitative immunoassay.
An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody.
Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (e.g., any of the targets as described herein) can also be performed. A known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., a blood sample) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away. Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample. If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce significant color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate.
There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.
In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample. There are currently many LFIA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of “dipping” the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments of any of the aspects, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.
The use of “dip sticks” or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of “dip stick” technology to detect soluble antigens via immunochemical assays include, but are not limited to U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a “dip stick” which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the “dip stick,” prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this “dip stick” technology for the detection of polypeptides using antibody reagents as described herein.
Other techniques can be used to detect the level of a polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. A dot blot immobilizes a protein sample on a defined region of a support, which is thenprobed with antibody and labelled secondary antibody as in Western blotting. The intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.
In some embodiments of any of the aspects, the level of a target can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy and/or immunoelectrophoresis assay.
In certain embodiments, the expression levels as described herein can be determined by determining the level of messenger RNA (mRNA) expression of the genes described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a blood sample. Techniques for the detection of mRNA expression is known by persons skilled in the art, and can include but not limited to, PCR procedures, RT-PCR, quantitative RT-PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing; hybridization methods, etc.
In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.
In some embodiments of any of the aspects, the level of an mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology. Methods of sequencing a nucleic acid sequence are well known in the art. Briefly, a sample obtained from a subject can be contacted with one or more primers which specifically hybridize to a single-strand nucleic acid sequence flanking the target gene sequence and a complementary strand is synthesized. In some next-generation technologies, an adaptor (double or single-stranded) is ligated to nucleic acid molecules in the sample and synthesis proceeds from the adaptor or adaptor compatible primers. In some third-generation technologies, the sequence can be determined, e.g. by determining the location and pattern of the hybridization of probes, or measuring one or more characteristics of a single molecule as it passes through a sensor (e.g. the modulation of an electrical field as a nucleic acid molecule passes through a nanopore). Exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, and the like. Methods and protocols for performing these sequencing methods are known in the art, see, e.g. “Next Generation Genome Sequencing” Ed. Michal Janitz, Wiley-VCH; “High-Throughput Next Generation Sequencing” Eds. Kwon and Ricke, Humanna Press, 2011; and Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); which are incorporated by reference herein in their entireties.
Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
In some embodiments of any of the aspects, one or more of the reagents (e.g. an antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.
In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample from a subject. In some embodiments of any of the aspects, the present invention encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject. In some embodiments of any of the aspects, the test sample can be peripheral blood sample and the kidney marrow.
The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).
In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.
In some embodiments of any of the aspects, the method described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject. In some embodiments of any of the aspects, the subject can be a subject in need of treatment for (e.g. having or diagnosed as having clonal hematopoiesis, myelodysplasia, and leukemia) or a subject at risk of or at increased risk of developing clonal hematopoiesis, myelodysplasia, and leukemia as described elsewhere herein.
In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having clonal hematopoiesis, myelodysplasia, and leukemia. In some embodiments of any of the aspects, the subject is a current or former blood disease patient. In some embodiments of any of the aspects, the subject has been exposed to chemotherapy, radiation, viral infection, or certain chemicals. In some embodiments of any of the aspects, the subject has or is diagnosed as having clonal hematopoiesis, myelodysplasia, and leukemia.
In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a clonal hematopoietic disease or disorder, such as clonal hematopoiesis, myelodysplasia, or leukemia. Subjects having a clonal hematopoietic disease or disorder, such as clonal hematopoiesis, myelodysplasia, or leukemia can be identified by a physician using current methods of diagnosing such diseases and disorders. Symptoms and/or complications of clonal hematopoiesis, myelodysplasia, and leukemia which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, anemia, bruising and bleeding, infections due to low levels of white blood cells, fevers, drenching night sweats, unintentional weight loss, and fatigue. Tests that may aid in a diagnosis of, e.g. clonal hematopoiesis, myelodysplasia, and leukemia include, but are not limited to, blood tests, bone marrow aspiration and biopsy, and lumbar puncture. A family history of leukemia, mutation in the GATA2 gene, TERC gene, or TERT gene for myelodysplasia, or exposure to risk factors for clonal hematopoiesis, myelodysplasia, and leukemia (e.g. exposure to chemotherapy, radiation, viral infection, or certain chemicals (e.g., benzene)) can also aid in determining if a subject is likely to have clonal hematopoiesis, myelodysplasia, and leukemia or in making a diagnosis of clonal hematopoiesis, myelodysplasia, and leukemia.
It is noted that the terms “administered” and “subjected” are used interchangeably in the context of treatment of a disease or disorder. In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shall be restricted to prescribing a controlled substance that a human subject will be administer to the subject by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.
As used herein, the term “administer” refers to the placement of a composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced. A compound or composition described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.
Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, administration will generally be local rather than systemic.
For administration to a subject, the NR4A inhibitor can be provided in pharmaceutically acceptable compositions. These pharmaceutically acceptable compositions comprise a NR4A inhibitor, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions described herein can be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), gavages, lozenges, dragees, capsules, pills, tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) transmucosally; or (9) nasally. Additionally, compounds can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960, content of all of which is herein incorporated by reference.
As used here, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
Examples of solid carriers include starch, sugar, bentonite, silica, and other commonly used carriers. Further non-limiting examples of carriers and diluents which can be used in the formulations comprising a NR4A inhibitor include saline, syrup, dextrose, and water.
Pharmaceutically-acceptable antioxidants include, but are not limited to, (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lectithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acids, and the like.
The NR4A inhibitor can be formulated in a gelatin capsule, in tablet form, dragee, syrup, suspension, topical cream, suppository, injectable solution, or kits for the preparation of syrups, suspension, topical cream, suppository or injectable solution just prior to use. Also, compounds can be included in composites, which facilitate its slow release into the blood stream, e.g., silicon disc, polymer beads.
The formulations can conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques, excipients and formulations generally are found in, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1985, 17th edition, Nema et al., PDA J. Pharm. Sci. Tech. 1997 51:166-171. Methods to make invention formulations include the step of bringing into association or contacting an ActRIIB compound with one or more excipients or carriers. In general, the formulations are prepared by uniformly and intimately bringing into association one or more compounds with liquid excipients or finely divided solid excipients or both, and then, if appropriate, shaping the product.
The preparative procedure may include the sterilization of the pharmaceutical preparations. The compounds may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, salts for influencing osmotic pressure, etc., which do not react deleteriously with the compounds.
Examples of injectable forms include solutions, suspensions and emulsions. Injectable forms also include sterile powders for extemporaneous preparation of injectable solutions, suspensions or emulsions. The compounds of the present invention can be injected in association with a pharmaceutical carrier such as normal saline, physiological saline, bacteriostatic water, Cremophor™ EL (BASF, Parsippany, N.J.), phosphate buffered saline (PBS), Ringer's solution, dextrose solution, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof, and other aqueous carriers known in the art. Appropriate non-aqueous carriers may also be used and examples include fixed oils and ethyl oleate. In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. A suitable carrier is 5% dextrose in saline. Frequently, it is desirable to include additives in the carrier such as buffers and preservatives or other substances to enhance isotonicity and chemical stability.
In some embodiments, the NR4A inhibitor can be administrated encapsulated within liposomes. The manufacture of such liposomes and insertion of molecules into such liposomes being well known in the art, for example, as described in U.S. Pat. No. 4,522,811. Liposomal suspensions (including liposomes targeted to particular cells, e.g., a pituitary cell) can also be used as pharmaceutically acceptable carriers.
Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the composition can be administered in a sustained release formulation.
Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185; content of each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.
In some embodiments, the NR4A inhibitor are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
In the case of oral ingestion, excipients useful for solid preparations for oral administration are those generally used in the art, and the useful examples are excipients such as lactose, sucrose, sodium chloride, starches, calcium carbonate, kaolin, crystalline cellulose, methyl cellulose, glycerin, sodium alginate, gum arabic and the like, binders such as polyvinyl alcohol, polyvinyl ether, polyvinyl pyrrolidone, ethyl cellulose, gum arabic, shellac, sucrose, water, ethanol, propanol, carboxymethyl cellulose, potassium phosphate and the like, lubricants such as magnesium stearate, talc and the like, and further include additives such as usual known coloring agents, disintegrators such as alginic acid and Primogel™, and the like. The compounds can be orally administered, for example, with an inert diluent, or with an assimilable edible carrier, or they may be enclosed in hard or soft shell capsules, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet. For oral therapeutic administration, these compounds may be incorporated with excipients and used in the form of tablets, capsules, elixirs, suspensions, syrups, and the like. Such compositions and preparations should contain at least 0.1% of compound. The percentage of the agent in these compositions may, of course, be varied and may conveniently be between about 2% to about 60% of the weight of the unit. The amount of compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions according to the present invention are prepared so that an oral dosage unit contains between about 100 and 2000 mg of compound. Examples of bases useful for formulation of suppositories are oleaginous bases such as cacao butter, polyethylene glycol, lanolin, fatty acid triglycerides, witepsol (trademark, Dynamite Nobel Co. Ltd.) and the like. Liquid preparations may be in the form of aqueous or oleaginous suspension, solution, syrup, elixir and the like, which can be prepared by a conventional way using additives. The compositions can be given as a bolus dose, to maximize the circulating levels for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
The NR4A inhibitor can also be administrated directly to the airways in the form of an aerosol. For administration by inhalation, the compounds in solution or suspension can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or hydrocarbon propellant like propane, butane or isobutene. The compounds can also be administrated in a no-pressurized form such as in an atomizer or nebulizer.
In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound described herein and a suitable powder base such as lactose or starch.
Representative intranasal formulations are described in, for example, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452. Formulations that include a compound of Formulae (I), (II), (III) or (IV) are prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients. These ingredients are known to those skilled in the preparation of nasal dosage forms and some of these can be found in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st edition, 2005. The choice of suitable carriers is dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present. Preferably, the nasal dosage form should be isotonic with nasal secretions
The NR4A inhibitor can also be administered parenterally. Solutions or suspensions of these compounds can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solution, and glycols such as, propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, “dosage unit” refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
Administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
For oral or enteral formulations as disclosed herein for use with the present invention, tablets can be formulated in accordance with conventional procedures employing solid carriers well-known in the art. Capsules employed for oral formulations to be used with the methods of the present invention can be made from any pharmaceutically acceptable material, such as gelatin or cellulose derivatives. Sustained release oral delivery systems and/or enteric coatings for orally administered dosage forms are also contemplated, such as those described in U.S. Pat. No. 4,704,295, “Enteric Film-Coating Compositions,” issued Nov. 3, 1987; U.S. Pat. No. 4,556,552, “Enteric Film-Coating Compositions,” issued Dec. 3, 1985; U.S. Pat. No. 4,309,404, “Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406, “Sustained Release Pharmaceutical Compositions,” issued Jan. 5, 1982. As regards formulations for administering a compound of Formula I as disclosed herein, one particularly useful embodiment.
For intravenous injections or drips or infusions, NR4A inhibitor can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.
Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspension, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
In some embodiments of any of the aspects, the NR4A inhibitor is administered as a monotherapy, e.g., another treatment for the clonal hematopoiesis, myelodysplasia, and leukemia is not administered to the subject.
Nucleic acid inhibitor can be formulated into liposomes. Exemplary liposomes can comprise, e.g., DSPC, DPPC, DSPG, Cholesterol, hydrogenated soy phosphatidylcholine, soy phosphatidyl choline, methoxypolyethylene glycol (mPEG-DSPE) phosphatidyl choline (PC), phosphatidyl glycerol (PG), distearoylphosphatidylcholine, and combinations thereof.
The phrase “therapeutically-effective amount” as used herein means that amount of a compound, material, or composition which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. According, a “therapeutically effective amount” refers to an amount effective, at dosage and periods of time necessary, to achieve a desired therapeutic result. A therapeutic result can be, e.g., lessening of symptoms, prolonged survival, improved mobility, and the like. A therapeutic result need not be a “cure.”
Determination of a therapeutically effective amount is well within the capability of those skilled in the art. Generally, a therapeutically effective amount can vary with the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents.
Depending on the route of administration, effective doses can be calculated according to the body weight, body surface area, or organ size of the subject to be treated. Optimization of the appropriate dosages can readily be made by one skilled in the art in light of pharmacokinetic data observed in human clinical trials. Alternatively, or additionally, the dosage to be administered can be determined from studies using animal models for the particular type of condition to be treated, and/or from animal or human data obtained from agents which are known to exhibit similar pharmacological activities. The final dosage regimen will be determined by the attending surgeon or physician, considering various factors which modify the action of active agent, e.g., the agent's specific activity, the agent's specific half-life in vivo, the severity of the condition and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any present infection, time of administration, the use (or not) of other concomitant therapies, and other clinical factors.
Determination of an effective amount is well within the capability of those skilled in the art. Generally, the actual effective amount can vary with the specific compound, the use or application technique, the desired effect, the duration of the effect and side effects, the subject's history, age, condition, sex, as well as the severity and type of the medical condition in the subject, and administration of other pharmaceutically active agents. Accordingly, an effective dose of compound described herein is an amount sufficient to produce at least some desired therapeutic effect in a subject.
The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of use or administration utilized.
The effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the therapeutic which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The effective plasma concentration for a compound as disclosed herein can be about 0.01 μM to about 10 μM, about 0.2 μM to about 5 μM, or about 0.8 to about 3 μM in a subject, such as a rat, dog, or human.
Generally, the compositions are administered so that a compound described herein is used or given at a dose from 1 μg/kg to 1000 mg/kg; 1 μg/kg to 500 mg/kg; 1 μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100 mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10 mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10 mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood that ranges given here include all intermediate ranges, for example, the range 1 mg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3 mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1 mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10 mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6 mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to 10 mg/kg, and the like. Further contemplated is a dose (either as a bolus or continuous infusion) of about 0.1 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, or 0.5 mg/kg to about 3 mg/kg. It is to be further understood that the ranges intermediate to those given above are also within the scope of this disclosure, for example, in the range 1 mg/kg to 10 mg/kg, for example use or dose ranges such as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg, and the like.
The NR4A inhibitor can be administered at once, or can be divided into a number of smaller doses to be administered at intervals of time. Thus, in some embodiments, the NR4A inhibitor is administered once a day. In some other embodiments, NR4A inhibitor is administered multiple times, e.g., two, three, four, five or more times a day.
It is understood that the precise dosage and duration of treatment will be a function of route of administration, the carrier and other variables that can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens can need to be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. Hence, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.
The NR4A inhibitor can be administered as a single bolus or multiple boluses, as a continuous infusion, or a combination thereof. For example, the NR4A inhibitor can be administered as a single bolus initially, and then administered as a continuous infusion following the bolus. The rate of the infusion can be any rate sufficient to maintain effective concentration, for example, to maintain effective plasma concentration. Some contemplated infusion rates include from 1 μg/kg/min to 100 mg/kg/min, or from 1 μg/kg/hr to 1000 mg/kg/hr. Rates of infusion can include 0.2 to 1.5 mg/kg/min, or more specifically 0.25 to 1 mg/kg/min, or even more specifically 0.25 to 0.5 mg/kg/min. It will be appreciated that the rate of infusion can be determined based upon the dose necessary to maintain effective plasma concentration and the rate of elimination of the NR4A inhibitor, such that the compound is administered via infusion at a rate sufficient to safely maintain a sufficient effective plasma concentration of NR4A inhibitor in the bloodstream.
In all embodiments where a sample is obtained or has been obtained or provided, the sample can be sample taken, obtained, or provided via minimally invasive methods and/or involves only a minor intervention. In some embodiments of any of the aspects, a sample is taken, obtained, or provided by one or more of a blood draw or prick, an epidermal or mucus membrane swab, buccal sampling, saliva sample, a epidermal skin sampling technique, and/or collection of a secreted or expelled bodily fluid (e.g., mucus, urine, sweat, etc), fecal sampling, semen/seminal fluid sampling, or clippings (e.g., of hair or nails). In some embodiments of any of the aspects, the sample comprises, consists of, or consists essentially of blood (or any fraction or component thereof), serum, urine, mucus, epithelial cells, saliva, buccal cells, a secreted or expelled bodily fluid, and/or hair or nail clippings.
For convenience, certain terms employed herein, in the specification, examples and appended claims are collected herein. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skill in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein.
Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used to described the present invention, in connection with percentages means ±1%, +1.5%, +2%, +2.5%, ±3%, +3.5%, ±4%, ±4.5%, or +5%.
The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.
As used herein the terms “comprising” or “comprises” means “including” or “includes” and are used in reference to compositions, methods, systems, and respective component(s) thereof, that are useful to the invention, yet open to the inclusion of unspecified elements, whether useful or not.
As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
The term “consisting of” refers to compositions, methods, systems, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”
The terms “significantly different than,”, “statistically significant,” and similar phrases refer to comparisons between data or other measurements, wherein the differences between two compared data or other measurements are evidently or reasonably different to the trained observer, or statistically significant (if the phrase includes the term “statistically” or if there is some indication of statistical test, such as a p-value, or if the data, when analyzed, produce a statistical difference by standard statistical tests known in the art).
The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced”, “reduction” or “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
The term “a reference level” as used herein refer to a negative control. For example, in the context of treatment, a reference level is the level if a subject is not treated. In some embodiments, a reference level in the context of diagnosis is the level present in a normal healthy subject. The term “normal healthy subject” refers to a subject who has no symptoms of any diseases or disorders, or who is not identified with any diseases or disorders, or who is not on any medication treatment, or a subject who is identified as healthy by physicians based on medical examinations. In some embodiments, a reference level used herein refers to the level measured prior to onset of treatment.
As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” are used herein to characterize a method or process that is aimed at (1) delaying or preventing the onset of a disease or condition; (2) slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the disease or condition; (3) bringing about ameliorations of the symptoms of the disease or condition; or (4) curing the disease or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased morbidity or mortality. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
A treatment can be administered prior to the onset of the disease, for a prophylactic or preventive action. Alternatively, or additionally, the treatment can be administered after initiation of the disease or condition, for a therapeutic action. Accordingly, in some embodiments of any of the aspects, described herein is a prophylactic method of treatment. As used herein “prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom. Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. Accordingly, the methods described herein can be prophylactic relative to clonal hematopoiesis, myelodysplasia, and leukemia. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
In some embodiments, treatment is therapeutic and does not include prophylactic treatment.
As used herein, “alleviating” a symptom of a clonal hematopoiesis, myelodysplasia, and leukemia is ameliorating any condition or symptom associated with the clonal hematopoiesis, myelodysplasia, and leukemia. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
As used herein, “inhibitor” refers to a molecule or compound which can decrease the expression and/or activity of a target, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor of one or more targets, e.g. its ability to decrease the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and/or the activity of the target. In some embodiments of any of the aspects, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule. An inhibitor of a target described herein can inhibit the activity, expression, or accumulation of the target polypeptide. Inhibitors can include inhibitors that act directly on the target itself (e.g., that bind to the protein or transcript, e.g., direct inhibitors).
As used herein, the term “subject” refers to any living organism which can be administered compound and/or pharmaceutical compositions of the present invention. The term includes, but is not limited to, humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses, domestic subjects such as dogs and cats, laboratory animals including rodents such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or sex. Thus, adult, child and newborn subjects, whether male or female, are intended to be covered. The term “subject” is also intended to include living organisms susceptible to conditions or disease states as generally disclosed, but not limited to, throughout this specification. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species. The term “subject” and “individual” are used interchangeably herein, and refer to an animal, for example a human or non-human mammals/animals, to whom treatment, including prophylactic treatment, with the compounds and compositions according to the present invention, is provided. The term “non-human animals” and “non-human mammals” are used interchangeably herein and include all vertebrates, e.g., mammals, such as non-human primates, (particularly higher primates), sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and non-mammals such as chickens, amphibians, reptiles etc.
In some embodiments, the subject is a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein.
Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a fibrotic disease or disorder.
It is noted that a human subject can be of any age, gender, race or ethnic group, e.g., Caucasian (white), Asian, African, black, African American, African European, Hispanic, Middle eastern, etc.
In addition, the methods described herein can be used to treat domesticated animals and/or pets. A subject can be male or female. A subject can be one who has been previously diagnosed with or identified as suffering from or having a disease or disorder needing treatment, but need not have already undergone treatment. For example, the subject can be one who has been previously diagnosed with or identified as suffering from or having a microbial infection, e.g., a fungal infection.
In some embodiments of any one of the aspects, the subject is human.
A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. clonal hematopoiesis, myelodysplasia, and leukemia) or one or more complications related to such a condition, and optionally, have already undergone treatment for clonal hematopoiesis, myelodysplasia, and leukemia or the one or more complications related to clonal hematopoiesis, myelodysplasia, and leukemia. Alternatively, a subject can also be one who has not been previously diagnosed as having clonal hematopoiesis, myelodysplasia, and leukemia or one or more complications related to clonal hematopoiesis, myelodysplasia, and leukemia. For example, a subject can be one who exhibits one or more risk factors for clonal hematopoiesis, myelodysplasia, and leukemia or one or more complications related to clonal hematopoiesis, myelodysplasia, and leukemia or a subject who does not exhibit risk factors.
A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
The term “disorder” or “disease” used interchangeably herein, refers to any alteration in the state of the body or of some of its organs, interrupting or disturbing the performance of the functions and/or causing symptoms such as discomfort, dysfunction, distress, or even death to the person afflicted or those in contact with the person. A disease or disorder can also relate to distemper, ailing, ailment, malady, disorder, sickness, illness, complaint, indisposition, affection.
A “somatic mutation,” as used herein, refers to a change in the genetic structure of a subject that is not inherited from a parent, and also not passed to offspring. Hence, a somatic mutation is a genetic change that occurs in any cell after the first cell division, wherein the mutation is replicated in all cells that descend from the mutated cell. The somatic cells that descend from the original mutated cell comprise a clonal variant within the body of the subject. Where these mutations are present in cells of somatic origin and not present in the germline, they are often called a somatic cell mutation or an acquired mutation. Somatic mutations will be present in only a subset of the cells contributing DNA to an analysis, since the mutant sequence will be present in fewer than 50% of the sequence reads arising from that genomic site. In other words, somatic mutations are identified as when a specific sequence is measured as occurring at a fraction of total sequences that deviates significantly from the frequency expected for the far-larger number of inherited variants—namely around 0%, around 50% or around 100%.
Somatic mutations can occur in a sub-population of cells for example, such as a sub-population of hematopoietic cells. Somatic mutations in HSPC genes, such as ASM-1, DNMT3A, IDH1, IDH2, relevant to the composition and methods described herein include any nucleic acid or consequent amino acid somatic mutations in HSPC genes, such as ASM-1, DNMT3A, IDH1, IDH2, found in a subset of hematopoietic cells. Such somatic mutations in HSPC genes, such as ASXL1, DNMT3A, IDH1, IDH2, can be disruptive, in that they have an observed or predicted effect on protein function, or non-disruptive. As used herein, a “non-disruptive mutation” is typically a missense mutation, in which a codon is altered such that it codes for a different amino acid, but the encoded protein, i.e., ASXL1, DNMT3A, IDH1, IDH2, is still expressed. Somatic mutations in HSPC genes, such as ASXL1, DNMT3A, IDH1, IDH2, include, for example, frameshift mutations, nonsense mutations, missense mutations or splice-site variant mutations, as those terms are known to those of ordinary skill in the art.
As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., NR4A1, NR4A2, or NR4A3 genomic DNA or cDNA. Suitable RNA can include, e.g., NR4A1, NR4A2, or NR4A3 mRNA.
The term “expression” refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
“Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5′ untranslated (5′UTR) or “leader” sequences and 3′ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
“Marker” in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having having clonal hematopoiesis, myelodysplasia, and leukemia, as compared to a comparable sample taken from control subjects (e.g., a healthy subject). The term “biomarker” is used interchangeably with the term “marker.”
In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term “detecting” or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., provided herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. The invention is further illustrated by the following example, which should not be construed as further limiting.
The technology may be as described in any one of the following numbered paragraphs:
Paragraph 1: A method for treating a clonal hematopoietic disease or disorder, the method comprising administering an inhibitor of a member of the nuclear receptor 4A (NR4A) family.
Paragraph 2: The method of paragraph 1, wherein the inhibitor binds with the NR4A family member or with a nucleic acid encoding the NR4A family member.
Paragraph 3: The method of paragraph 1 or 2, wherein the NR4A family member is selected from the group consisting of NR4A1, NR4A2 and NR4A3.
Paragraph 4: The method of any one of paragraphs 1-3, wherein the NR4A family member is NR4A1.
Paragraph 5: The method of any one of paragraphs 1-4, wherein the inhibitor is a nucleic acid.
Paragraph 6: The method of paragraph 5, wherein the nucleic acid is selected from the group consisting of siRNAs, antisense oligonucleotides, aptamers, ribozymes, and triplex forming oligonucleotides.
Paragraph 7: The method of paragraphs 5 or 6, wherein the inhibitor comprises a nucleotide sequence substantially complementary to at least a portion of a nucleic acid encoding the NR4A family member.
Paragraph 8: The method of any one of paragraphs 5-7, wherein the inhibitor comprises a nucleotide sequence substantially complementary to at least 15 contiguous nucleotides of SEQ ID NO: 1 (NR4A1 sequence), SEQ ID NO: 6 (NR4A2 sequence) or SEQ ID NO: 9 (NR4A3 sequence).
Paragraph 9: The method of any one of paragraphs 1-4, wherein the inhibitor is an antibody or antigen binding fragment thereof that binds the NR4A family member.
Paragraph 10: The method of paragraph 9, wherein the antibody is an anti-NR4A1 antibody, an anti-NR4A2 antibody, an anti-NR4A3 antibody, or antigen binding fragment thereof
Paragraph 11: The method of paragraph 9 or 10, wherein the antibody is a monoclonal antibody.
Paragraph 12: The method of any one of paragraphs 9-11, wherein the antibody is a humanized antibody.
Paragraph 13: The method of any one of paragraphs 1-4, wherein the inhibitor is a small molecule.
Paragraph 14: The method of paragraph 13, wherein the inhibitor is a 1,1-bis(3′-indolyl)-1-(p-substituted phenyl)methane (C-DIM) compound.
Paragraph 15: The method of paragraph 13 or 14, wherein the inhibitor is 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (DIM-C-pPhOh-3-Cl-5-OCH3), 1,1-bis(3′-indolyl)-1-(3,5-dibromo-4-hydroxyphenyl)methane (DIM-C-pPhOH-3,5-Br2), camptothecin (CPT), or a cyclooxygenase (COX)-2 inhibitor (celecoxib analogue SC-236).
Paragraph 16: The method of any one of paragraphs 1-15, wherein the clonal hematopoietic disease or disorder is clonal hematopoiesis, myelodysplastic syndromes (MDS) or leukemia.
Paragraph 17: The method of any one of paragraphs 1-16, wherein the clonal hematopoietic disease or disorder is leukemia.
Paragraph 18: The method of paragraph 17, wherein the leukemia is acute myelogenous leukemia (AML) or chronic myeloid leukemia (CVL).
Paragraph 19: The method of any one of paragraphs 1-16, the clonal hematopoietic disease or disorder is MDS.
Paragraph 20: The method of paragraph 19, wherein the MDS is MDS with multilineage dysplasia (MDS-MLD), MDS with single lineage dysplasia (MDS-SLD), MDA with ring sideroblasts (MDS-RS), MDS with excess blasts (MDS-EB), MDS with isolated del(5q), and/or MDS unclassifiable (MDS-U).
Paragraph 21: The method of any one of paragraphs 1-20, further comprising co-administering an immunomodulatory agent to the subject.
Paragraph 22: The method of any one of paragraphs 1-20, further comprising co-administering an anti-inflammatory agent to the subject.
Paragraph 23: The method of any one of paragraphs 1-20, wherein the subject comprises at least one mutation in a nucleic acid encoding ASXL transcriptional regulator 1 (ASXL1), DNA (cytosine-5)-methyltransferase 3A (DNMT3A), isocitrate dehydrogenase (NADP(+)) 1(IDH1) or isocitrate dehydrogenase (NADP(+)) 2 (IDH 2).
Paragraph 24: The method of paragraph 16, further comprising, prior to onset of the treatment regime, identifying the subject with at least one mutation in a nucleic acid encoding ASXL1, DNMT3A, IDH1 or IDH 2.
Paragraph 25: The method of any one of paragraphs 1-24, further comprising a step of assaying a sample from the subject for at least one mutation in a nucleic acid encoding ASXL1, DNMT3A, IDH1 or IDH 2 prior to onset of the treatment regime.
Paragraph 26: The method of any one of paragraphs 1-25, wherein the subject is a mammal.
Paragraph 27: The method of any one of paragraphs 1-26, wherein the subject is human.
Clonal hematopoiesis. Zebrafish were generated with mosaic mutations in asxl1 using the TWISTR method (Avagyan et a1 Science 2021, PMID 34735227). Briefly, they were injected with guide-RNAs targeting asxl1 gene with Cas9 mRNA into 1-cell embryos of zebrafish, and grown to adulthood. At 3 months retroortibital bleeding was performed on the adult zebrafish and the size of the mutant clones in the peripheral blood was measured by sequencing the genomic DNA at the targeting locus of asxl1. Zebrafish with at least 1 mutant allele of 5% of greater allele size were chosen for the cohorts, randomly assigned to be treated with either DMSO or NR4A1 inhibitor (DIM-C). Zebrafish with mosaic mutations in asxl1 were treated with either DMSO or an NR4A1 inhibitor (DIM-C) and analyzed over a three-month period. Peripheral blood was collected every month to determine the size of the mutant clones over time. The results are shown in
It was previously shown by early genetic targeting nr4a1 during development prevented clonal growth of mutant cells. Next, the hypothesis that pharmacologic inhibition of nr4a1 would halt or reduce mutant clonal growth over time was tested (
All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
This application is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/US2023/065185 filed Mar. 31, 2023, which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63/326,596, filed Apr. 1, 2022, the contents of both of which are incorporated herein by reference in their entireties.
This invention was made with government support under grant numbers HL144780, HL131477 and HL126338, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/065185 | 3/31/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63326596 | Apr 2022 | US |
