This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled “321501_2380_Sequence_Listing_ST25” created on Dec. 18, 2019. The content of the sequence listing is incorporated herein in its entirety.
Current therapies for musculoskeletal diseases, such as low back pain, are highly invasive and is a major contributor to the growing opioid crisis. Additionally, these therapies only treat the symptomatic pain of the patient while failing to target the underlying pathology of disease which leads to further disease progression and future pain. For example, lumbar fusion of the spine is a common surgical operation to fuse the spine in place of the intervertebral disc space between the vertebras. However, removal of the disc and fusion of the vertebrae often results in adjacent segment disease due to imbalanced biomechanics of the spine post-surgery. In addition, micro-discectomies which remove the diseased tissue from the site often lead to tissue collapse and additional surgical intervention with added pain. Therefore, new treatment methods of such diseases are needed to alleviate these issues.
Current studies in the field include engineered intervertebral discs, cell therapies, drug delivery, growth factors, viral reprogramming or gene editing. However, these all include their pitfalls and risks. Engineered constructs for replacement of musculoskeletal components are disadvantageous in their biocompatibility and most importantly mechanical integrity in the body environment to function effectively. Cell therapies are poor in terms of long-term cell viability due to the harsh avascular environment of tissues such as the intervertebral disc. Drug delivery systems are hard to sustain in the environment and has potential to leech onto nearby tissue with undesired effects similar to growth factors such as Bone morphogenic proteins (BMPs) and Tumor Growth Factor (TGFβ). Viral reprogramming and gene editing have large regulatory burdens as they often involve integration into the native host genome which has been shown in history to cause adverse immunogenic and mutagenic effects on the patients. The death of Jesse Gelsinger is one such example.
Disclosed herein are compositions and methods for reprogramming diseased musculoskeletal cells both in vitro and in vivo.
In some embodiments, the disclosed method involves non-virally delivering intracellularly into the diseased musculoskeletal cells a polynucleotide comprising one or more nucleic acid sequences encoding one or more transcription factors, such as HIF-1α, FOX, T, SOX, and Mohawk families of transcription factors, including the factors listed in Tables 1A, 1B, and 1C.
For example, in some embodiments, the method involves reprogramming a diseased nucleus pulposus (NP) cell into a healthy cell by non-virally delivering intracellularly into the NP cell one or more transcription factor proteins selected from the group comprising HIF-1α, HIF-2α, Hedgehog family (SHH, DHH, IHH), a T-box family of proteins (TBXT, TBR1, TBX1-6, TBX10, TBX15, TX618-22), and a Forkhead-box (FOX) family of proteins (FOXF1, FOXA1-3, FOXB1-2, FOXC1-2, FOXD1-6, FOXE1-3, FOXG1, FOXH1, FOXI1, FOXJ1, FOXK1, FOXL1-2, FOXM1, FOXN1-4, FOXO1, FOXO3-4, FOXO6, FOXP1-4, FOXQ1, FOXR1-2), or polynucleotides encoding the one or more transcription factor proteins; or exposing the NP cell to an extracellular vesicle produced from a cell containing or expressing the one or more transcription factor proteins, or polynucleotides encoding the one or more transcription factor proteins.
In some embodiments, the method involves reprogramming a diseased annulus fibrosis (AF) cell into a healthy cell by non-virally delivering intracellularly into the AF cell one or more transcription factor proteins selected from the group comprising a Iroquois Homeobox family of proteins (Mohawk, IRX1-6), Tenomodulin and Scleraxis, or polynucleotides encoding the one or more transcription factor proteins; or exposing the AF cell to an extracellular vesicle produced from a cell containing or expressing the one or more transcription factor proteins, or polynucleotides encoding the one or more transcription factor proteins.
In some embodiments, the method involves reprogramming a diseased cartilage endplate cell into a healthy cell by non-virally delivering intracellularly into the cartilage endplate cell one or more transcription factor proteins selected from the group comprising an NFAT Family proteins (NFATc1-4), ERG (C-1-1), PGC1α, Osterix, SOX family of proteins (SRY, SOX1-15, SOX17-18, SOX21, SOX30) and MEF2C, or polynucleotides encoding the one or more transcription factor proteins; or exposing the cartilage endplate cell to an extracellular vesicle produced from a cell containing or expressing the one or more transcription factor proteins, or polynucleotides encoding the one or more transcription factor protein.
Also disclosed herein is a method for treating a musculoskeletal disease in a subject that involves non-virally delivering intracellularly into disease musculoskeletal cells of the subject one or more transcription factor proteins selected from the group comprising HIF-1a, HIF-2a, a T-box family protein, and Forkhead-box (FOX) family protein, a Iroquois family proteins, Tenomodulin, Scleraxis, NFAT Family proteins, ERG, PGC1a, Osterix, Runx family of proteins, Hedgehog family of proteins, SOX family of proteins and MEF2C, or polynucleotides encoding the one or more transcription factor proteins; or exposing the disease musculoskeletal cells to an extracellular vesicle produced from a cell containing or expressing the one or more transcription factor proteins, or polynucleotides encoding the one or more transcription factor proteins.
In some embodiments, the musculoskeletal disease is osteoarthritis where chondrocytes, synoviocytes, fibrocartilage cells of the meniscus, osteoblasts, osteocytes and osteoclasts will be subject to non-viral reprogramming. In some embodiments, the musculoskeletal disease is intervertebral disc degeneration and chronic low back pain where notochordal cells, nucleus pulposus cells, annulus fibrosus cells, cartilage endplate cells, ligamentous cells, dorsal root ganglion cells and myocytes/myofibroblasts will be subject to non-viral reprogramming or injection of engineered vesicles. In some embodiments, the musculoskeletal disease is tendinopathy or rotator cuff tendonitis where tenocytes and myocytes/myofibroblasts will be subject to non-viral reprogramming or injection of engineered vesicles.
In some embodiments, the disclosed methods involve non-viral tissue nanotransfection (TNT) of notochordal cells, nucleus pulposus (NP), annulus fibrosis (AF), or cartilage endplate cells of a subject's intervertebral disc (IVD) or chondrocytes, synoviocytes, fibrocartilage cells of the meniscus, ligamentous cells, dorsal root ganglion cells, osteoblasts, osteoclasts, osteocytes, myocytes/myofibroblasts, haemapoetic and mesenchymal stem cells or tenocytes. This can be done via direct tissue nanotransfection of the NP, AF, and CEP tissue with previously stated transcription factors during patient surgery, or on cells isolated from patients. More precisely, the tissue nanotransfection device chip will be placed at the site of the IVD where degeneration is occurring and transcription factors targeting the specific tissue will be delivered in-situ. More precisely, cells from the patient IVD can be isolated and transfected ex-vivo with transcription factors and injected back into the patient.
In some embodiments, the disclosed methods involve delivery of extracellular vesicles (EVs) to the notochordal cells, nucleus pulposus (NP), annulus fibrosis (AF), or cartilage endplate cells of a subject's intervertebral disc (IVD) or chondrocytes, synoviocytes, fibrocartilage cells of the meniscus, ligamentous cells, dorsal root ganglion cells, osteoblasts, osteoclasts, osteocytes, myocytes/myofibroblasts, or tenocytes. EVs will be generated using the patient's cells which encapsulates the desired transcription factors specific for each tissue. EVs containing these factors are then injected back into the diseased/degenerate tissue and up taken by the patients cells within 4-6 hours of cell-vector contact.
Also disclosed herein are polynucleotides comprising one, two, or more nucleic acid sequences encoding transcription factors disclosed herein, such as Forkhead-box (FOX) family protein, Iroquois I family proteins, Scleraxis, NFAT Family proteins, ERG, PGC1a, Osterix, and MEF2C. In some embodiments, the transcription factors are mammalian proteins, such as human proteins.
Also disclosed a composition comprising a polynucleotide comprising one, two, or more nucleic acid sequences encoding transcription factors disclosed herein. Also disclosed are non-viral vectors containing the disclosed polynucleotides. In particular embodiments, the vector is a recombinant bacterial plasmid. For example, in some embodiments, the non-viral vector has a pCDNA3 backbone. In some embodiments, the vector comprises an internal ribosome entry site (IRES).
In some embodiments, after transfecting target cells with nucleic acid sequences encoding the disclosed transcription factors, the cells can then pack the transfected genes (e.g. cDNA) into EVs, which can then reprogram diseased musculoskeletal cells. Therefore, also disclosed is a method of reprogramming diseased musculoskeletal cells that involves exposing the cells with an extracellular vesicle produced from a cell containing or expressing the disclosed transcription factors.
In these embodiments, the polynucleotides and compositions may be delivered to diseased musculoskeletal cells, or donor cells, intracellularly via a gene gun, a microparticle or nanoparticle suitable for such delivery, transfection by electroporation, three-dimensional nanochannel electroporation, a tissue nanotransfection device, a liposome suitable for such delivery, or a deep-topical tissue nanoelectroinjection device. In some of these embodiments, the polynucleotides can be incorporated into a non-viral vector, such as a bacterial plasmid. In some embodiments, a viral vector can be used. For example, the polynucleotides can be incorporated into a viral vector, such as an adenoviral vector. However, in other embodiments, the polynucleotides are not delivered virally.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.
Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.
It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.
The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician or veterinarian.
The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
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 problems or complications commensurate with a reasonable benefit/risk ratio.
The term “carrier” means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
The term “inhibit” refers to a decrease in an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
The term “polypeptide” refers to amino acids joined to each other by peptide bonds or modified peptide bonds, e.g., peptide isosteres, etc. and may contain modified amino acids other than the 20 gene-encoded amino acids. The polypeptides can be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification can be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide can have many types of modifications. Modifications include, without limitation, acetylation, acylation, ADP-ribosylation, amidation, covalent cross-linking or cyclization, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of a phosphytidylinositol, disulfide bond formation, demethylation, formation of cysteine or pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristolyation, oxidation, pergylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, and transfer-RNA mediated addition of amino acids to protein such as arginylation. (See Proteins—Structure and Molecular Properties 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York (1993); Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12 (1983)).
As used herein, the term “amino acid sequence” refers to a list of abbreviations, letters, characters or words representing amino acid residues. The amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.
The phrase “nucleic acid” as used herein refers to a naturally occurring or synthetic oligonucleotide or polynucleotide, whether DNA or RNA or DNA-RNA hybrid, single-stranded or double-stranded, sense or antisense, which is capable of hybridization to a complementary nucleic acid by Watson-Crick base-pairing. Nucleic acids can also include nucleotide analogs (e.g., BrdU), and non-phosphodiester internucleoside linkages (e.g., peptide nucleic acid (PNA) or thiodiester linkages). In particular, nucleic acids can include, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or any combination thereof.
A “nucleotide” as used herein is a molecule that contains a base moiety, a sugar moiety, and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The term “oligonucleotide” is sometimes used to refer to a molecule that contains two or more nucleotides linked together. The base moiety of a nucleotide can be adenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3′-AMP (3′-adenosine monophosphate) or 5′-GMP (5′-guanosine monophosphate).
A nucleotide analog is a nucleotide that contains some type of modification to the base, sugar, and/or phosphate moieties. Modifications to nucleotides are well known in the art and would include, for example, 5-methylcytosine (5-me-C), 5 hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties.
Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
The term “vector” or “construct” refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked. The term “expression vector” includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element). “Plasmid” and “vector” are used interchangeably, as a plasmid is a commonly used form of vector. Moreover, the invention is intended to include other vectors which serve equivalent functions.
The term “operably linked to” refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences. For example, operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
For purposes herein, the % sequence identity of a given nucleotides or amino acids sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given sequence C that has or comprises a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:
100 times the fraction W/Z,
where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program's alignment of C and D, and where Z is the total number of nucleotides or amino acids in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
By “specifically hybridizes” is meant that a probe, primer, or oligonucleotide recognizes and physically interacts (that is, base-pairs) with a substantially complementary nucleic acid (for example, a c-met nucleic acid) under high stringency conditions, and does not substantially base pair with other nucleic acids.
The term “stringent hybridization conditions” as used herein mean that hybridization will generally occur if there is at least 95% and preferably at least 97% sequence identity between the probe and the target sequence. Examples of stringent hybridization conditions are overnight incubation in a solution comprising 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared carrier DNA such as salmon sperm DNA, followed by washing the hybridization support in 0.1×SSC at approximately 65° C. Other hybridization and wash conditions are well known and are exemplified in Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), particularly chapter 11.
Compositions
Disclosed are polynucleotides comprising nucleic acid sequences encoding transcription factors that can be used to reprogram diseased musculoskeletal cells according to the disclosed methods. Examples of these transcription factors are provided in Tables 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H.
The amino acid and nucleic acid sequences encoding Forkhead-box (FOX) family proteins, a Mohawk family proteins, Scleraxis, NFAT Family proteins, C-1-1, PGC1α, Osterix, and MEF2C are known in the art.
In some embodiments, Forkhead box F1 (FOXF1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:1).
In some embodiments, the nucleic acid sequence encoding FOXF1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:2 under stringent hybridization conditions.
In some embodiments, Forkhead Box A1 (FOXA1) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3.
In some embodiments, the nucleic acid sequence encoding FOXA1 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:4 under stringent hybridization conditions.
In some embodiments, Forkhead box A2 (FOXA2) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:5.
In some embodiments, the nucleic acid sequence encoding FOXA2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:6 under stringent hybridization conditions.
In some embodiments, Forkhead box A3 (FOXA3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7.
In some embodiments, the nucleic acid sequence encoding FOXA3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:8 under stringent hybridization conditions.
In some embodiments, Forkhead box B1 (FOXB1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:9.
In some embodiments, the nucleic acid sequence encoding FOXB1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:10 under stringent hybridization conditions.
In some embodiments, Forkhead box B2 (FOXB2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:11.
In some embodiments, the nucleic acid sequence encoding FOXB2 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:12 under stringent hybridization conditions.
In some embodiments, Forkhead box C1 (FOXC1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:13.
In some embodiments, the nucleic acid sequence encoding FOXC1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:14 under stringent hybridization conditions.
In some embodiments, Forkhead box C2 (FOXC2) comprises the amino acid sequence
NP 005242), or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:15.
In some embodiments, the nucleic acid sequence encoding FOXC2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:16 under stringent hybridization conditions.
In some embodiments, Forkhead box D1 (FOXD1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:17.
In some embodiments, the nucleic acid sequence encoding FOXD1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:18 under stringent hybridization conditions.
In some embodiments, Forkhead box D2 (FOXD2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:19.
In some embodiments, the nucleic acid sequence encoding FOXD2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:20 under stringent hybridization conditions.
In some embodiments, Forkhead box D3 (FOXD3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:21.
In some embodiments, the nucleic acid sequence encoding FOXD3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:22 under stringent hybridization conditions.
In some embodiments, Forkhead box D4 (FOXD4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:23.
In some embodiments, the nucleic acid sequence encoding FOXD4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:24 under stringent hybridization conditions.
In some embodiments, Forkhead box D4 like 1 (FOXD5) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:25.
In some embodiments, the nucleic acid sequence encoding FOXD5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:26 under stringent hybridization conditions.
In some embodiments, Forhead box D4 like 3 (FOXD6) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:27.
In some embodiments, the nucleic acid sequence encoding FOXD6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:28 under stringent hybridization conditions.
In some embodiments, Forkhead box E1 (FOXE1/FOXE2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:29.
In some embodiments, the nucleic acid sequence encoding FOXE1/FOXE2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:30 under stringent hybridization conditions.
In some embodiments, Forkhead box E3 (FOXE3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:31.
In some embodiments, the nucleic acid sequence encoding FOXE3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:32 under stringent hybridization conditions.
In some embodiments, Forrkhead box G1 (FOXG1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:33.
In some embodiments, the nucleic acid sequence encoding FOXG1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:34 under stringent hybridization conditions.
In some embodiments, Forhead box H1 (FOXH1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:35.
In some embodiments, the nucleic acid sequence encoding FOXH1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:36 under stringent hybridization conditions.
In some embodiments, Forkheadbox 11 (FOXI1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:37
In some embodiments, the nucleic acid sequence encoding FOXI1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:38 under stringent hybridization conditions.
In some embodiments, Forhead box J1 (FOXJ1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:39.
In some embodiments, the nucleic acid sequence encoding FOXJ1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:40 under stringent hybridization conditions.
In some embodiments, Forkhead box K1 (FOXK1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:41.
In some embodiments, the nucleic acid sequence encoding FOXK1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:42 under stringent hybridization conditions.
In some embodiments, Forkhead box L1 (FOXL1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.
In some embodiments, the nucleic acid sequence encoding FOXL1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:44 under stringent hybridization conditions.
In some embodiments, Forkhead box L2 (FOXL2) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:45.
In some embodiments, the nucleic acid sequence encoding FOXL2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:46 under stringent hybridization conditions.
In some embodiments, Forhead box M1 (FOXM1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:47.
In some embodiments, the nucleic acid sequence encoding FOXM1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:48 under stringent hybridization conditions.
In some embodiments, Forkhead box N1 (FOXN1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:49.
In some embodiments, the nucleic acid sequence encoding FOXN1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:50 under stringent hybridization conditions.
In some embodiments, Forhead box N2 (FOXN2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:51.
In some embodiments, the nucleic acid sequence encoding FOXN2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:52 under stringent hybridization conditions.
In some embodiments, Forkhead box N3 (FOXN3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:53.
In some embodiments, the nucleic acid sequence encoding FOXN3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:54 under stringent hybridization conditions.
In some embodiments, Forkhead box N4 (FOXN4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:55.
In some embodiments, the nucleic acid sequence encoding FOXN4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:56 under stringent hybridization conditions.
In some embodiments, Forkhead Box 01 (FOXO1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57.
In some embodiments, the nucleic acid sequence encoding FOXO1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:58 under stringent hybridization conditions.
In some embodiments, Forkhead Box 03 (FOXO3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:59.
In some embodiments, the nucleic acid sequence encoding FOXO3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:60 under stringent hybridization conditions.
In some embodiments, Forkhead Box 04 (FOXO4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:61.
In some embodiments, the nucleic acid sequence encoding FOXO4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:62 under stringent hybridization conditions.
In some embodiments, Forkhead Box 06 (FOXO6) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:63.
In some embodiments, the nucleic acid sequence encoding FOXO6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:64 under stringent hybridization conditions.
In some embodiments, Forkheadbox P1 (FOXP1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:65.
In some embodiments, the nucleic acid sequence encoding FOXP1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:66 under stringent hybridization conditions.
In some embodiments, Forkhead box P2 (FOXP2) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:67.
In some embodiments, the nucleic acid sequence encoding FOXP2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:68 under stringent hybridization conditions.
In some embodiments, Forkhead box P3 (FOXP3) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:69.
In some embodiments, the nucleic acid sequence encoding FOXP3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:70 under stringent hybridization conditions.
In some embodiments, Forkhead box 4 (FOXP4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:71.
In some embodiments, the nucleic acid sequence encoding FOXP4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:72 under stringent hybridization conditions.
In some embodiments, Forkhead box Q1 (FOXQ1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:73.
In some embodiments, the nucleic acid sequence encoding FOXQ1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:74 under stringent hybridization conditions.
In some embodiments, Forkhead Box R1 (FOXR1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:75.
In some embodiments, the nucleic acid sequence encoding FOXR1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:76 under stringent hybridization conditions.
In some embodiments, FOXR2 comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:77.
In some embodiments, the nucleic acid sequence encoding FOXR2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:78 under stringent hybridization conditions.
In some embodiments, Hypoxia inducible factor 1 subunit alpha (HIF-1a) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:79.
In some embodiments, the nucleic acid sequence encoding HIF-1a comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:80 under stringent hybridization conditions.
In some embodiments, endothelial PAS domain protein 1 (HIF-2a/EPAS1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:81.
In some embodiments, the nucleic acid sequence encoding HIF-2a comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:82 under stringent hybridization conditions.
In some embodiments, Sonic Headgehog (SHH) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:83.
In some embodiments, the nucleic acid sequence encoding SHH comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:84 under stringent hybridization conditions.
In some embodiments, desert hedgehog (DHH) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:85.
In some embodiments, the nucleic acid sequence encoding DHH comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:86 under stringent hybridization conditions.
In some embodiments, Indian Hedgehog (IHH) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:87.
In some embodiments, the nucleic acid sequence encoding IHH comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:88 under stringent hybridization conditions.
In some embodiments, Brachyury (TBXT) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:89.
In some embodiments, the nucleic acid sequence encoding TBXT comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:90 under stringent hybridization conditions.
In some embodiments, T-box brain 1 (TBR1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:91.
In some embodiments, the nucleic acid sequence encoding TBR1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:92 under stringent hybridization conditions.
In some embodiments, T-Box 1 (TBX1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:93.
In some embodiments, the nucleic acid sequence encoding TBX1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:94 under stringent hybridization conditions.
In some embodiments, T-box 2 (TBX2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:95.
In some embodiments, the nucleic acid sequence encoding TBX2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:96 under stringent hybridization conditions.
In some embodiments, T-box 3 (TBX3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:97.
In some embodiments, the nucleic acid sequence encoding TBX3 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:98 under stringent hybridization conditions.
In some embodiments, T-box 4 (TBX4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:99.
In some embodiments, the nucleic acid sequence encoding TBX4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:100 under stringent hybridization conditions.
In some embodiments, T-box 5 (TBX5) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:101.
In some embodiments, the nucleic acid sequence encoding TBX5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:102 under stringent hybridization conditions.
In some embodiments, T-box 6 (TBX6) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:103.
In some embodiments, the nucleic acid sequence encoding TBX6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:104 under stringent hybridization conditions.
In some embodiments, T-box 10 (TBX10) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:105.
In some embodiments, the nucleic acid sequence encoding TBX10 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:106 under stringent hybridization conditions.
In some embodiments, T-box 15 (TBX15) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:107.
In some embodiments, the nucleic acid sequence encoding TBX15 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:108 under stringent hybridization conditions.
In some embodiments, T-box 18 (TBX18) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:109.
In some embodiments, the nucleic acid sequence encoding TBX18 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:110 under stringent hybridization conditions.
In some embodiments, T-box 10 (TBX19) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:111.
In some embodiments, the nucleic acid sequence encoding TBX19 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:112 under stringent hybridization conditions.
In some embodiments, T-box 20 (TBX20) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:113.
In some embodiments, the nucleic acid sequence encoding TBX20 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:114 under stringent hybridization conditions.
In some embodiments, T-box 21 (TBX21) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:115.
In some embodiments, the nucleic acid sequence encoding TBX21 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:116 under stringent hybridization conditions.
In some embodiments, T-box 22 (TBX22) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:117.
In some embodiments, the nucleic acid sequence encoding TBX22 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:118 under stringent hybridization conditions.
In some embodiments, Paired box 1 (PAX1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:119.
In some embodiments, the nucleic acid sequence encoding PAX1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:120 under stringent hybridization conditions.
In some embodiments, Sex determining region Y (SRY/SOXA) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:121.
In some embodiments, the nucleic acid sequence encoding SRY/SOXA comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:122 under stringent hybridization conditions.
In some embodiments, SRY box 1 (SOX1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:123.
In some embodiments, the nucleic acid sequence encoding SOX1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:124 under stringent hybridization conditions.
In some embodiments, SRY-box 2 (SOX2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:125.
In some embodiments, the nucleic acid sequence encoding SOX2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:126 under stringent hybridization conditions.
In some embodiments, SRY-box 3 (SOX3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:127.
In some embodiments, the nucleic acid sequence encoding SOX3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:128 under stringent hybridization conditions.
In some embodiments, SRY box 14 (SOX14) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:129.
In some embodiments, the nucleic acid sequence encoding SOX14 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:130 under stringent hybridization conditions.
In some embodiments, SRY-box 21 (SOX21) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:131.
In some embodiments, the nucleic acid sequence encoding SOX21 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:132 under stringent hybridization conditions.
In some embodiments, SRY-box 4 (SOX4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:133.
In some embodiments, the nucleic acid sequence encoding SOX4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:134 under stringent hybridization conditions.
In some embodiments, SRY-box 11 (SOX11) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:135.
In some embodiments, the nucleic acid sequence encoding SOX11 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:136 under stringent hybridization conditions.
In some embodiments, SRY-box 12 (SOX12) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:137.
In some embodiments, the nucleic acid sequence encoding SOX12 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:138 under stringent hybridization conditions.
In some embodiments, SRY-box 5 (SOX5) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:139.
In some embodiments, the nucleic acid sequence encoding SOX5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:140 under stringent hybridization conditions.
In some embodiments, SRY-box 6 (SOX6) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:141.
In some embodiments, the nucleic acid sequence encoding SOX6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:142 under stringent hybridization conditions.
In some embodiments, SRY-box 13 (SOX13) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:143.
In some embodiments, the nucleic acid sequence encoding SOX13 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:144 under stringent hybridization conditions.
In some embodiments, SRY-box 8 (SOX8) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:145.
In some embodiments, the nucleic acid sequence encoding SOX8 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:146 under stringent hybridization conditions.
In some embodiments, SRY-box 9 (SOX9) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:147.
In some embodiments, the nucleic acid sequence encoding SOX9 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:148 under stringent hybridization conditions.
In some embodiments, SRY-box 10 (SOX10) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:149.
In some embodiments, the nucleic acid sequence encoding SOX10 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:150 under stringent hybridization conditions.
In some embodiments, SRY-box 7 (SOX7) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:151.
In some embodiments, the nucleic acid sequence encoding SOX7 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:152 under stringent hybridization conditions.
In some embodiments, SRY-box 17 (SOX17) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:153.
In some embodiments, the nucleic acid sequence encoding SOX17 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:154 under stringent hybridization conditions.
In some embodiments, SRY-box 18 (SOX18) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:155.
In some embodiments, the nucleic acid sequence encoding SOX18 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:156 under stringent hybridization conditions.
In some embodiments, SRY-box 15 (SOX15) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:157.
In some embodiments, the nucleic acid sequence encoding SOX15 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:158 under stringent hybridization conditions.
In some embodiments, SRY-box 30 (SOX30) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:159.
In some embodiments, the nucleic acid sequence encoding SOX30 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:160 under stringent hybridization conditions.
In some embodiments, Notochord Homeobox (NOTO) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:161.
In some embodiments, the nucleic acid sequence encoding NOTO comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:162 under stringent hybridization conditions.
In some embodiments, Tenomodulin (TNMD) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:163.
In some embodiments, the nucleic acid sequence encoding TNMD comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:164 under stringent hybridization conditions.
In some embodiments, Human nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:165.
In some embodiments, the nucleic acid sequence encoding NFATc1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:166 under stringent hybridization conditions.
In some embodiments, Human nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 2 (NFATc2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:167.
In some embodiments, the nucleic acid sequence encoding NFATc2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:168 under stringent hybridization conditions.
In some embodiments, Human nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3 (NFATc3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:169.
In some embodiments, the nucleic acid sequence encoding NFATc3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:170 under stringent hybridization conditions.
In some embodiments, Human nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4 (NFATc4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:171.
In some embodiments, the nucleic acid sequence encoding NFATc4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:172 under stringent hybridization conditions.
In some embodiments, Human v-ets erythroblastosis virus E26 oncogene homolog (ERG, C-1-1 is a variant) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:173.
In some embodiments, the nucleic acid sequence encoding ERG comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:174 under stringent hybridization conditions.
In some embodiments, PGC1α comprises the amino acid sequence: MAWDMCNQDSESVWSDIECAALVGEDQPLCPDLPELDLSELDVNDLDTDSFLGGLKW CSDQSEIISNQYNNEPSNIFEKIDEENEANLLAVLTETLDSLPVDEDGLPSFDALTDGDV
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:175.
In some embodiments, the nucleic acid sequence encoding PGC1a comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:176 under stringent hybridization conditions.
In some embodiments, Osterix (SP7) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:177.
In some embodiments, the nucleic acid sequence encoding Osterix comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:178 under stringent hybridization conditions.
In some embodiments, myocyte enhancer factor 2C (MEF2C) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:179.
In some embodiments, the nucleic acid sequence encoding MEF2C comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:180 under stringent hybridization conditions.
In some embodiments, Mohawk (MKX) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:181.
In some embodiments, the nucleic acid sequence encoding MKX comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:182 under stringent hybridization conditions.
In some embodiments, Iroquois homeobox 1 (IRX1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:183.
In some embodiments, the nucleic acid sequence encoding IRX1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:184 under stringent hybridization conditions.
In some embodiments, iroquois homeobox 2 (IRX2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:185.
In some embodiments, the nucleic acid sequence encoding IRX2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:186 under stringent hybridization conditions.
In some embodiments, iroquois homeobox 3 (IRX3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:187.
In some embodiments, the nucleic acid sequence encoding IRX3 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:188 under stringent hybridization conditions.
In some embodiments, iroquois homeobox 4 (IRX4) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:189.
In some embodiments, the nucleic acid sequence encoding IRX4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:190 under stringent hybridization conditions.
In some embodiments, iroquois homeobox 5 (IRX5) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:191.
In some embodiments, the nucleic acid sequence encoding IRX5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:192 under stringent hybridization conditions.
In some embodiments, iroquois homeobox 6 (IRX6) comprises the amino acid sequence
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:193.
In some embodiments, the nucleic acid sequence encoding IRX6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:194 under stringent hybridization conditions.
In some embodiments, Scleraxis (SCX) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:195.
In some embodiments, the nucleic acid sequence encoding SCX comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:196 under stringent hybridization conditions.
In some embodiments, runt-related transcription factor 1 (RUNX1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:197.
In some embodiments, the nucleic acid sequence encoding RUNX1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:198 under stringent hybridization conditions.
In some embodiments, runt-related transcription factor 2 (RUNX2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:199.
In some embodiments, the nucleic acid sequence encoding RUNX2 comprises the nucleic acid sequence
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:200 under stringent hybridization conditions.
In some embodiments, runt-related transcription factor 3 (RUNX3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:201.
In some embodiments, the nucleic acid sequence encoding RUNX3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:202 under stringent hybridization conditions.
In some embodiments, Paired box 9 (PAX9) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:203.
In some embodiments, the nucleic acid sequence encoding PAX9 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:204 under stringent hybridization conditions.
In some embodiments, Homeobox Protein Nkx-3.2 (Nkx-3.2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:205
In some embodiments, the nucleic acid sequence encoding Nkx-3.2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:206 under stringent hybridization conditions.
In some embodiments, AP-1 transcription factor (FOS, C-FOX, AP-1, p55) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:207)
In some embodiments, the nucleic acid sequence encoding FOS comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:208 under stringent hybridization conditions.
In some embodiments, FosB proto-oncogene (FosB, GOS3, G0S3, GOSB) comprises the amino acid sequence:
MFQAFPGDYDSGSRCSSSPSAESQYLSSVDSFGSPPTAAASQECAGLGEMPGSFVPT VTAITTSQDLQWLVQPTLISSMAQSQGQPLASQPPVVDPYDMPGTSYSTPGMSGYSS GGASGSGGPSTSGTTSGPGPARPARARPRRPREETETDQLEEEKAELESEIAELQKEK ERLEFVLVAHKPGCKIPYEEGPGPGPLAEVRDLPGSAPAKEDGFSWLLPPPPPPPLPF QTSQDAPPNLTASLFTHSEVQVLGDPFPVVNPSYTSSFVLTCPEVSAFAGAQRTSGSD QPSDPLNSPSLLAL. (SEQ ID NO:209; NP_001107643.1), or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:209)
In some embodiments, the nucleic acid sequence encoding FosB comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:210 under stringent hybridization conditions.
In some embodiments, FOS like 1, AP-1 transcription factor subunit (FRA, FRA1, FOSL1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:211)
In some embodiments, the nucleic acid sequence encoding FRA comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:212 under stringent hybridization conditions.
In some embodiments, FOS like 2, AP-1 transcription factor subunit (FRA2, FOSL2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:213) In some embodiments, the nucleic acid sequence encoding FRA2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:214 under stringent hybridization conditions.
In some embodiments Jun proto-oncogene, AP-1 transcription factor subunit (JUN, p39, cJUN) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:215)
In some embodiments, the nucleic acid sequence encoding JUN comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:216 under stringent hybridization conditions.
In some embodiments, JunB proto-oncogene, AP-1 transcription factor subunit (JUNB) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:217)
In some embodiments, the nucleic acid sequence encoding JUNB comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:218 under stringent hybridization conditions.
In some embodiments, JunD proto-oncogene, AP-1 transcription factor subunit (JunD) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:219)
In some embodiments, the nucleic acid sequence encoding JUND comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:220 under stringent hybridization conditions.
In some embodiments, ZFP36 ring finger protein (ZFP36, TTP) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:221)
In some embodiments, the nucleic acid sequence encoding ZFP36 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:222 under stringent hybridization conditions.
In some embodiments, EBF transcription factor 1 (EBF1, EBF, COE1, OL1)) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:223)
In some embodiments, the nucleic acid sequence encoding EBF1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:224 under stringent hybridization conditions.
In some embodiments, EBF transcription factor 3 (EBF3, COE3, OE2, HADDS) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:225)
In some embodiments, the nucleic acid sequence encoding EBF3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:226 under stringent hybridization conditions.
In some embodiments, MAF bZIP transcription factor (MAF, CCA4, AYGRP, c-MAF, CTRCT21) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:227)
In some embodiments, the nucleic acid sequence encoding MAF comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:228 under stringent hybridization conditions.
In some embodiments, nuclear protein 1 (NUPR1, P8, COM1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:229)
In some embodiments, the nucleic acid sequence encoding NUPR1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:230 under stringent hybridization conditions.
In some embodiments, twist family bHLH transcription factor 1 (TWIST1, CRS, CSP, SC3, ACS3, CRS1, BPES2/3, SWCOS, TWIST) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:231)
In some embodiments, the nucleic acid sequence encoding TWIST1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:232 under stringent hybridization conditions
In some embodiments, twist family bHLH transcription factor 2 (TWIST2, AMS, FFDD3, BBRSAY, DERMO1, SETLSS) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:233)
In some embodiments, the nucleic acid sequence encoding Fos comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:234 under stringent hybridization conditions
In some embodiments, MAGE family member D1 (MAGED1, NRAGE, DLXIN-1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:235)
In some embodiments, the nucleic acid sequence encoding MAGED1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:236 under stringent hybridization conditions
In some embodiments, SATB homeobox 2 (SATB2, GLSS) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:237)
In some embodiments, the nucleic acid sequence encoding SATB2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:238 under stringent hybridization conditions
In some embodiments, PDZ and LIM domain 7 (LMP3, PDLIM7, LMP1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:239)
In some embodiments, the nucleic acid sequence encoding LMP3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:240 under stringent hybridization conditions
In some embodiments, POU class 5 homeobox 1 (OCT3, OCT4, POU5F1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:241)
In some embodiments, the nucleic acid sequence encoding OCT3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:242 under stringent hybridization conditions
In some embodiments, Kruppel like factor 4 (KLF4, EZF, GKLF) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:243)
In some embodiments, the nucleic acid sequence encoding KLF4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:244 under stringent hybridization conditions
In some embodiments, MYC proto-oncogene, bHLH transcription factor (MYC, MRTL, MYCC, c-MC) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:245)
In some embodiments, the nucleic acid sequence encoding MYC comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:246 under stringent hybridization conditions
In some embodiments, distal-less homeobox 3 (DLX3, AI4, TDO) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:247)
In some embodiments, the nucleic acid sequence encoding DLX3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:248 under stringent hybridization conditions
In some embodiments, distal-less homeobox 5 (DLX5, SHFM1 D) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:249)
In some embodiments, the nucleic acid sequence encoding DLX5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:250 under stringent hybridization conditions
In some embodiments, distal-less homeobox 6 (DLX6) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:251)
In some embodiments, the nucleic acid sequence encoding DLX6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:252 under stringent hybridization conditions
In some embodiments, HOP homeobox2 (OB1, HOD, HOPX, LAGY, TOTO, CAMEO, NECC1, SMAP31) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:253)
In some embodiments, the nucleic acid sequence encoding OB1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:254 under stringent hybridization conditions
In some embodiments, CCAAT enhancer binding protein alpha (CEBPA, CEBP C/EBP) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:255)
In some embodiments, the nucleic acid sequence encoding CEBP comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:256 under stringent hybridization conditions
In some embodiments, activating transcription factor 4 (ATF4, CREB2, TXREB) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:257)
In some embodiments, the nucleic acid sequence encoding ATF4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:258 under stringent hybridization conditions
In some embodiments, SMAD family member 1 (SMAD1) comprises the amino acid sequence:
SVS. (SEQ ID NO:259; NP_001003688.1), or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:259)
In some embodiments, the nucleic acid sequence encoding SMAD1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:260 under stringent hybridization conditions
In some embodiments, menin 1 (MEN1, MEAT, SCG2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:261)
In some embodiments, the nucleic acid sequence encoding MEN1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:262 under stringent hybridization conditions
In some embodiments, msh homeobox 1 (MSX1, HOX7, HYD1, ECTD3, SThaG1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:263)
In some embodiments, the nucleic acid sequence encoding MSX comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:264 under stringent hybridization conditions
In some embodiments, msh homeobox 2 (MSX2, FPP, MSH, PFM, CRS2, HOX8, PFM1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:265)
In some embodiments, the nucleic acid sequence encoding MSX2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:266 under stringent hybridization conditions
In some embodiments, neurofibromin 1 (NF1, WSS, NFNS, VRNF) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:267)
In some embodiments, the nucleic acid sequence encoding NF1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:268 under stringent hybridization conditions
In some embodiments, early growth response 1 (EGR1, TIS8, AT225, KROX-24, NGFI-A, ZNF225) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:269)
In some embodiments, the nucleic acid sequence encoding Fos comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:270 under stringent hybridization conditions
In some embodiments, early growth response 2 (EGR2, CHN1, AT591, CMT1D, CMT4E, KROX20) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:271)
In some embodiments, the nucleic acid sequence encoding EGR2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:272 under stringent hybridization conditions
In some embodiments, Sp3 transcription factor (SP3, SPR2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:273)
In some embodiments, the nucleic acid sequence encoding SP3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:274 under stringent hybridization conditions
In some embodiments, SIX homeobox 1 (SIX1, BOS3, TIP39, DFNA23)) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:275)
In some embodiments, the nucleic acid sequence encoding SIX1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:276 under stringent hybridization conditions
In some embodiments, SIX homeobox 2 (SIX2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:277)
In some embodiments, the nucleic acid sequence encoding SIX2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:278 under stringent hybridization conditions
In some embodiments, EYA transcriptional coactivator and phosphatase 1 (EYA1, BOP, BOR, BOS1, OFC1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:279)
In some embodiments, the nucleic acid sequence encoding EYA1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:280 under stringent hybridization conditions
In some embodiments, EYA transcriptional coactivator and phosphatase 2 (EYA2, EAB1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:281)
In some embodiments, the nucleic acid sequence encoding EYA2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:282 under stringent hybridization conditions
In some embodiments, ETS variant transcription factor 4 (PEA3, ETV4, E1AF, PEAS3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:283)
In some embodiments, the nucleic acid sequence encoding PEA3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:284 under stringent hybridization conditions
In some embodiments, nuclear factor of activated T cells 5 (NFAT5, NFATZ, OREBP, NF-ATS, NFATL1, TONEBP) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:285)
In some embodiments, the nucleic acid sequence encoding NFAT5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:286 under stringent hybridization conditions
In some embodiments, BCL6 transcription repressor (BCL6, BCL5, LAZ3, BCL6A, ZNF51, ZBTB27) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:287)
In some embodiments, the nucleic acid sequence encoding BCL6 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:288 under stringent hybridization conditions
In some embodiments, myogenic differentiation 1 (MYOD1, PUM, MYF3, MYOD, bHLHc1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:289)
In some embodiments, the nucleic acid sequence encoding MYOD comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:290 under stringent hybridization conditions
In some embodiments, myogenic factor 5 (MYF5, EORVA, bHLHc2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:291)
In some embodiments, the nucleic acid sequence encoding MYF5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:292 under stringent hybridization conditions
In some embodiments, myogenin (MYOG, MYF5, bHLHc3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:293)
In some embodiments, the nucleic acid sequence encoding MYOG comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:294 under stringent hybridization conditions
In some embodiments, Spi-1 proto-oncogene (SPI1, OF, PU.1, SFPI1, SPI-A) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:295)
In some embodiments, the nucleic acid sequence encoding PU.1 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:296 under stringent hybridization conditions
In some embodiments, colony stimulating factor 1 receptor (CSF1R, FMS, CSFR, FIM2, HDLS, CD115, CSF-1R, BANDDOS, M-CSF-R) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:297)
In some embodiments, the nucleic acid sequence encoding CSFR comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:298 under stringent hybridization conditions
In some embodiments, serum response factor (SRF, MCMI) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:299)
In some embodiments, the nucleic acid sequence encoding SRF comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:300 under stringent hybridization conditions
In some embodiments, GLI family zinc finger 2 (GLI2, CJS, HPE9, PHS2, THP1, TH P2) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:301)
In some embodiments, the nucleic acid sequence encoding GLI2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:302 under stringent hybridization conditions
In some embodiments, Sp4 transcription factor (SP4, HF1B, SPR-1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:303)
In some embodiments, the nucleic acid sequence encoding SP4 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:304 under stringent hybridization conditions
In some embodiments, activating transcription factor 2 (ATF2, HB16, CREB2, TREB7, CRE-BP1) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:305)
In some embodiments, the nucleic acid sequence encoding ATF2 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:306 under stringent hybridization conditions
In some embodiments, activating transcription factor 3 (ATF3) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:307)
In some embodiments, the nucleic acid sequence encoding ATF3 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:308 under stringent hybridization conditions
In some embodiments, ETS variant transcription factor 5 (ETV5, ERM) comprises the amino acid sequence:
or an amino acid sequence that has at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:309)
In some embodiments, the nucleic acid sequence encoding ETV5 comprises the nucleic acid sequence:
or a nucleic acid sequence that hybridizes to a nucleic acid sequence consisting of SEQ ID NO:310 under stringent hybridization conditions. In order to express a polypeptide or functional nucleic acid, the nucleotide coding sequence may be inserted into appropriate expression vector. Therefore, also disclosed is a non-viral vector comprising a polynucleotide comprising one or more nucleic acid sequences encoding the disclosed transcription factors, wherein the one or more nucleic acid sequences are operably linked to an expression control sequence. In some embodiments, the nucleic acid sequences are operably linked to a single expression control sequence. In other embodiments, the nucleic acid sequences are operably linked to two or more separate expression control sequences. In some embodiments, the non-viral vector comprises a plasmid selected from the group pIRES-hrGFP-21, pAd-IRES-GFP, pCMV6-AC-GFP, and pCDNA3.0.
Methods to construct expression vectors containing genetic sequences and appropriate transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Press, Plainview, N.Y., 1989), and Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, New York, N.Y., 1989).
Expression vectors generally contain regulatory sequences necessary elements for the translation and/or transcription of the inserted coding sequence. For example, the coding sequence is preferably operably linked to a promoter and/or enhancer to help control the expression of the desired gene product.
The “control elements” or “regulatory sequences” are those non-translated regions of the vector—enhancers, promoters, 5′ and 3′ untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity.
A “promoter” is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site. A “promoter” contains core elements required for basic interaction of RNA polymerase and transcription factors and can contain upstream elements and response elements.
“Enhancer” generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5′ or 3′ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300 bp in length, and they function in cis. Enhancers function to increase transcription from nearby promoters. Enhancers, like promoters, also often contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression.
An “endogenous” enhancer/promoter is one which is naturally linked with a given gene in the genome. An “exogenous” or “heterologous” enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter.
Promoters used in biotechnology are of different types according to the intended type of control of gene expression. They can be generally divided into constitutive promoters, tissue-specific or development-stage-specific promoters, inducible promoters, and synthetic promoters.
Constitutive promoters direct expression in virtually all tissues and are largely, if not entirely, independent of environmental and developmental factors. As their expression is normally not conditioned by endogenous factors, constitutive promoters are usually active across species and even across kingdoms. Examples of constitutive promoters include CMV, EF1a, SV40, PGK1, Ubc, Human beta actin, and CAG.
Tissue-specific or development-stage-specific promoters direct the expression of a gene in specific tissue(s) or at certain stages of development. For plants, promoter elements that are expressed or affect the expression of genes in the vascular system, photosynthetic tissues, tubers, roots and other vegetative organs, or seeds and other reproductive organs can be found in heterologous systems (e.g. distantly related species or even other kingdoms) but the most specificity is generally achieved with homologous promoters (i.e. from the same species, genus or family). This is probably because the coordinate expression of transcription factors is necessary for regulation of the promoters activity.
The performance of inducible promoters is not conditioned to endogenous factors but to environmental conditions and external stimuli that can be artificially controlled. Within this group, there are promoters modulated by abiotic factors such as light, oxygen levels, heat, cold and wounding. Since some of these factors are difficult to control outside an experimental setting, promoters that respond to chemical compounds, not found naturally in the organism of interest, are of particular interest. Along those lines, promoters that respond to antibiotics, copper, alcohol, steroids, and herbicides, among other compounds, have been adapted and refined to allow the induction of gene activity at will and independently of other biotic or abiotic factors.
The two most commonly used inducible expression systems for research of eukaryote cell biology are named Tet-Off and Tet-On. The Tet-Off system makes use of the tetracycline transactivator (tTA) protein, which is created by fusing one protein, TetR (tetracycline repressor), found in Escherichia coli bacteria, with the activation domain of another protein, VP16, found in the Herpes Simplex Virus. The resulting tTA protein is able to bind to DNA at specific TetO operator sequences. In most Tet-Off systems, several repeats of such TetO sequences are placed upstream of a minimal promoter such as the CMV promoter. The entirety of several TetO sequences with a minimal promoter is called a tetracycline response element (TRE), because it responds to binding of the tetracycline transactivator protein tTA by increased expression of the gene or genes downstream of its promoter. In a Tet-Off system, expression of TRE-controlled genes can be repressed by tetracycline and its derivatives. They bind tTA and render it incapable of binding to TRE sequences, thereby preventing transactivation of TRE-controlled genes. A Tet-On system works similarly, but in the opposite fashion. While in a Tet-Off system, tTA is capable of binding the operator only if not bound to tetracycline or one of its derivatives, such as doxycycline, in a Tet-On system, the rtTA protein is capable of binding the operator only if bound by a tetracycline. Thus the introduction of doxycycline to the system initiates the transcription of the genetic product. The Tet-On system is sometimes preferred over Tet-Off for its faster responsiveness.
In some embodiments, the nucleic acid sequences encoding the disclosed transcription factors are operably linked to the same expression control sequence. Alternatively, internal ribosome entry sites (IRES) elements can be used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites. IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
Disclosed are non-viral vectors containing one or more polynucleotides disclosed herein operably linked to an expression control sequence. Examples of such non-viral vectors include the oligonucleotide alone or in combination with a suitable protein, polysaccharide or lipid formulation. Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.
Examples of suitable non-viral vectors include, but are not limited to pIRES-hrGFP-2a, pAd-IRES-GFP, and pCDNA3.0.
The compositions disclosed can be used therapeutically in combination with a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.
Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringers dextrose, dextrose and sodium chloride, lactated Ringers, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringers dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
The herein disclosed compositions, including pharmaceutical composition, may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. For example, the disclosed compositions can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, ophthalmically, vaginally, rectally, intranasally, topically or the like, including topical intranasal administration or administration by inhalant.
Also disclosed are methods of reprogramming diseased musculoskeletal cells that involve delivering intracellularly into the somatic cells a polynucleotide comprising one or more nucleic acid sequences encoding the disclosed transcription factors. In some embodiments, the nucleic acid sequences are present in non-viral vectors. In some embodiments, the nucleic acid sequences are operably linked to an expression control sequence. In other embodiments the nucleic acids are operably linked to two or more expression control sequences.
A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.
In some embodiments, after transfecting target cells with the disclosed polynucleotides, the cells can then pack the transfected genes (e.g. cDNA) into EVs, which can then induce endothelium in other somatic cells. Therefore, also disclosed is a method of reprogramming diseased musculoskeletal cells that involves exposing the cells with an extracellular vesicle produced from a cell containing or expressing the disclosed transcription factors.
Therefore, disclosed are methods of reprogramming diseased musculoskeletal cells that involve exposing the cells to extracellular vesicles (EVs) isolated from cells expressing or containing exogenous polynucleotides comprising one or more nucleic acid sequences encoding the disclosed transcription factors. For example, in some embodiments, the donor cells are transfected with the one or more disclosed polynucleotides and cultured in vitro. EVs secreted by the donor cells can then collected from the culture medium. These EVs can then be administered to the diseased musculoskeletal to reprogram them into healthy cells. In some embodiments, the donor cells can be any viable musculoskeletal cells or skin cells, including (but not limited to) NP, AF, CEPs, Articular Chondrocytes, tenocytes, and osteoblasts.
Exosomes and microvesicles are EVs that differ based on their process of biogenesis and biophysical properties, including size and surface protein markers. Exosomes are homogenous small particles ranging from 40 to 150 nm in size and they are normally derived from the endocytic recycling pathway. In endocytosis, endocytic vesicles form at the plasma membrane and fuse to form early endosomes. These mature and become late endosomes where intraluminal vesicles bud off into an intra-vesicular lumen. Instead of fusing with the lysosome, these multivesicular bodies directly fuse with the plasma membrane and release exosomes into the extracellular space. Exosome biogenesis, protein cargo sorting, and release involve the endosomal sorting complex required for transport (ESCRT complex) and other associated proteins such as Alix and Tsg101. In contrast, microvesicles, are produced directly through the outward budding and fission of membrane vesicles from the plasma membrane, and hence, their surface markers are largely dependent on the composition of the membrane of origin. Further, they tend to constitute a larger and more heterogeneous population of extracellular vesicles, ranging from 150 to 1000 nm in diameter. However, both types of vesicles have been shown to deliver functional mRNA, miRNA and proteins to recipient cells.
In some embodiments, the polynucleotides are delivered to the somatic cells, or the donor cells for EVs, intracellularly via a gene gun, a microparticle or nanoparticle suitable for such delivery, transfection by electroporation, three-dimensional nanochannel electroporation, a tissue nanotransfection device, a liposome suitable for such delivery, or a deep-topical tissue nanoelectroinjection device. In some embodiments, a viral vector can be used. However, in other embodiments, the polynucleotides are not delivered virally.
Electroporation is a technique in which an electrical field is applied to cells in order to increase permeability of the cell membrane, allowing cargo (e.g., reprogramming factors) to be introduced into cells. Electroporation is a common technique for introducing foreign DNA into cells.
Tissue nanotransfection allows for direct cytosolic delivery of cargo (e.g., reprogramming factors) into cells by applying a highly intense and focused electric field through arrayed nanochannels, which benignly nanoporates the juxtaposing tissue cell members, and electrophoretically drives cargo into the cells.
In one embodiment, the disclosed compositions are administered in a dose equivalent to parenteral administration of about 0.1 ng to about 100 g per kg of body weight, about 10 ng to about 50 g per kg of body weight, about 100 ng to about 1 g per kg of body weight, from about 1 μg to about 100 mg per kg of body weight, from about 1 μg to about 50 mg per kg of body weight, from about 1 mg to about 500 mg per kg of body weight; and from about 1 mg to about 50 mg per kg of body weight. Alternatively, the amount of the disclosed compositions administered to achieve a therapeutic effective dose is about 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, 10 μg, 100 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 500 mg per kg of body weight or greater.
In some embodiments, the disclosed compositions and methods are used to create a vasculature that can serve as a scaffolding structure. This scaffolding structure can then be used, for example, to aid in the repair of nerve tissue. Applications of this include peripheral nerve injuries, and pathological/injurious insults to the central nervous system such as traumatic brain injury or stroke. In some embodiments, the created vasculature can be used to nourish composite tissue transplants, or any tissue graft.
In some embodiments, the disclosed compositions and methods are used to convert “unwanted” tissue (e.g., fat, scar tissue) into vasculature. Such newly formed vasculature is expected to “resorb” under non-ischemic conditions.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Low back pain affects 70-85% of the world's population and is the leading cause of disability worldwide with over $100 billion in medical expenses in the U.S.A Alone. Intervertebral Disc degeneration is a main contributor of low back pain but current therapies do not target the underlying disease.
Such treatments include surgical interventions and medication which can result in non-unions, nerve injuries, ect. Some proposed treatments include tissue engineering, cell therapy, and even injectable hydrogel constructs. However, those treatments are under desired by clinicians and lack mechanical integrity of the IVD.
A native healthy IVD is gelatinous in the middle (Nucleus Pulposus) with surrounding fibers (Annulus Fibrosus). It's the largest avascular organ in the body. However, with aging, increased mechanical loads, and unknown disease pathologies, the disc degenerates. This degeneration has shown to cause pain due to pressure on the spinal cord along with unwarranted neurovascular invasion.
The overarching goal of this technology is to use the combination of transcription factor and TNT/Evs to revert diseased intervertebral disc cells to a healthy phenotype (
Methods
Transcription factor plasmid expansion via transformation into DH5a E. coli cells, selectivity via ampicillin resistance and plasmid DNA isolated
NP cells isolated from IVD tissue of human patients (n=5) undergoing spinal surgery and cadaveric tissue and expanded in monolayer until 80% confluent.
FOX Family transcription factor or non-transcription factor containing vector (SHAM) transfected in NP cells via bulk electroporation Neon™ Transfection System MPK5000 (V=1425 Volts, t=30 msec, 1 Pulse). Cells expanded and seeded in 2% Agarose Gels (Ø=8 mm, H=4 mm).
Gels taken down for analysis at Day 0, Week 2, and week 4 and analyzed for: cell viability (Live/Dead Assay Calcein/Ethidium), gene expression (qPCR) and Glycosaminoglycan (GAG) content (Dimethyl methylene Blue Assay (DMMB) normalized to DNA (Hoechst Assay).
Mann Whitney statistical tests used to evaluate significance at α=0.05.
Thompson Grading is a standard grade for disc degeneration where 1=healthy and 5=degenerate. Autopsy samples are graded as they do not come directly from diseased tissue. Surgical samples are diseased tissue removed from patients during routine spinal surgery. Table 2 shows human surgical and autopsy NP cells expanded 2 weeks.
Results
This study demonstrates: (i) the ability to transfect degenerate cells using bulk electroporation, (ii) transfected cell maintained viability over 4 weeks of culture in 3D constructs, (iii) introduction of FOX family gene into the cytosolic environment of the cell induces proteoglycan (GAG) production critical for IVD function and (iv) inhibition of inflammatory and neuron growth factor.
Low back pain (LBP) is the leading cause of disability worldwide with an associated socioeconomic burden of over $100 billion annually in the U.S alone [Katz, J. et al, 2006]. Intervertebral disc (IVD) degeneration is a major contributor to LBP and is characterized by decreases in cellularity and proteoglycan synthesis, upregulation of matrix degrading enzymes (MMPs), and increases in pro-inflammatory factors with neurovascular invasion[Rodriques-Pinto R. et al, 2014; Freemont A. J. et al, 2009]. Current treatment strategies are highly invasive and fail to target the underlying pathology or promote tissue repair. Pro-anabolic approaches have been proposed which includes gene therapy through viral infection, but this has raised safety concerns due to mutagenesis and unwarranted immune responses. To avoid such safety risks, electroporation of plasmids carrying DNA for transcription factors can be introduced into endogenous cells without alteration of native DNA to stimulate IVD repair. The transcription factor, Brachyury (BrachT), is expressed in the developing notochord and is associated with maintaining a healthy immature nucleus pulposus (NP) phenotype [Vujovic, S. et al, 2006; Tang, R. et al, 2018]. As disclosed herein, delivery of BrachT into degenerate human IVD cells can reprogram diseased NP cells into healthy cells with increased proteoglycan and decreased inflammatory, catabolic and pain associated factors which are critical for maintaining the structure and function of the healthy IVD. Thus, the overall objective of this study was to examine the effects of BrachT transfection on human NP cell phenotype and function.
Methods
BrachT transcription factor plasmids (OriGene Tech, Cat: SC303281) were expanded via transformation into DH5a E. coli cells with ampicillin resistance and plasmid DNA isolated for downstream electroporation. Human NP cells were isolated from non-degenerate (ND) cadaveric IVDs from autopsy (n=5, 19-58 y.o) or from the painful-degenerate (PD) IVD tissue of human patients with back pain (n=5, 19-70 y.o, IRB: 2015H0385) undergoing microdiscectomy (2 mg/mL Pronase-1 hour, 2 mg/mL Collagenase 11-4 hours). NP cells were expanded in monolayer (p2) until 80% confluent before bulk electroporation with empty plasmids (SHAM) or BrachT plasmids via Neon™ Transfection System MPK5000 (V=1425 Volts, t=30 msec, 1 Pulse). Successful transfection was verified with RT-qPCR at 48 hours. Transfected cells were then expanded in disc cell media (High glucose DMEM, 10% FBS, 1% P/S, 50 μg/ml ascorbic acid fresh) and seeded in 2% 3D agarose gel constructs at 20E6 cells/mL. Dependent variables were examined at day 0, week 2 and week 4 for cell viability (Calcein/Ethidium staining), extracellular matrix, phenotypic marker and inflammatory/catabolic gene expression (RT-qPCR) and proteoglycan/GAG content (Dimethylmethylene Blue Assay with DNA/Hoechst normalization). Non-parametric statistical tests were used (Mann-Whitney Tests, α=0.05).
Results
Cell viability remained high for all groups and BrachT gene expression was maintained over 4 weeks in both ND and PD cells with a decline in PD cells at week 4 only (fold change >100).
Expression of NP marker KRT19 was significantly increased in BrachT transfected PD cells at 2 weeks compared to SHAM controls while ND cells showed significant increases at all time-points (
Expression of matrix protein ACAN was increased at 2 weeks in transfected ND and PD cells (significant for PD cells) with significant decreases at 4 weeks for both groups (
Expression of MMP13 was significantly decreased in transfected ND cells at all time points and demonstrated a significant decrease at week 4 for transfected PD cells (
Pro-inflammatory cytokines IL-1β (
Nerve growth factor (NGF) showed a significant decrease in expression at week 2 for transfected PD cells but significant decreases at 2 and 4 weeks in transfected ND cells (
PD cells demonstrated a significant increase in GAG content in BrachT transfected groups at 2 weeks compared to their respective SHAM and this was observed to a lesser extent at week 4 (
These results demonstrate that human NP cells can be successfully transfected with transcription factor BrachT and reprogrammed to a healthy NP phenotype with up-regulation of key phenotypic markers, enhanced proteoglycan synthesis and down-regulation of inflammatory, catabolic and pain-related markers. High expression of BrachT was maintained over 4 weeks in 3D culture without any detrimental effects on cell viability. ND cells transfected with BrachT demonstrated increases in gene expression for healthy NP marker KRT19, decreases in MMP13 suggesting a decrease in catabolism, decreases in pro-inflammatory and pain genes IL-1β, IL-6, and NGF which all suggest reprogramming towards a ‘healthier’ IVD phenotype. While similar effects were observed in PD cells this was considered more temporal with peak anabolic effects observed at 2 weeks. Temporal effects suggest further optimization of the delivery system as bulk electroporation involves disruption of the cellular membrane and is less efficient compared to techniques such as engineered vesicles or tissue nanotransfection [Gallego-Perez et al, 2017]. The same temporal effects are seen at week 2 in GAG content with significant increases in GAG compared to the SHAM group at 2 weeks only. In conclusion, this study demonstrated the potential of BrachT to promote a healthy IVD phenotype via transfection into human ND and PD NP cells with increased GAG accumulation.
This is the first study to demonstrate successful reprogramming of diseased human NP cells into healthy NP cells using non-viral transfection of transcription factor BrachT. Further development of this treatment in conjunction with novel, minimally invasive tissue nanotransfection methods has high potential as a regenerative strategy for the treatment of LBP and other musculoskeletal diseases.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Methods:
FOXF1 plasmids were expanded and transfected into NP as in cells with culture media collected at 48 hours and extracellular vesicles isolated (Total exosome Isolation Kit). Exosomes were introduced to separate NP cells in monolayer with FOXF1 gene expression assessed at 2 and 7 days. Non-parametric statistical tests were used (Mann-Whitney Tests, α=0.05).
Results: When human EVs labelled with membrane dye PKH26 (red) were incubated with human NP cells in-vitro, uptake of labelled EVs was observed for SHAM and FOXF1 groups (
Tagged EVs showed high efficiency in microscopic images and high expression of FOXF1 gene packaged within EVs. Significant upregulation of FOXF1 in FOXF1 EV treated cells implies successful transfection of NP cells using generated EVs. This study demonstrated the potential of FOXF1 to promote a healthy IVD phenotype via transfection into degenerate human NP cells using EVs as a delivery mechanism.
Method: EVs were generated as before and injected into mice lumbar intervertebral disc in-vivo and accessed for 7 days (N=3). In an ongoing study, mice discs were punctured and will be accessed biweekly over 12 and 24 weeks.
Results: Viability staining of Mouse disc showed no cytotoxicity compared to non-injected control discs and injected discs showed upregulation of FOXF1 along with healthy NP marker brachyury (
Conclusion: This experiment shows the non-cytotoxic effects of transcription factor delivery via EVs and that the transcription factor successfully integrates into the intervertebral disc space along with upregulation of a healthy marker that was not Injected into the disc. Furthermore, ongoing studies show behavioral differences between injured untreated mice compared to foxfl treated mice as seen in
This application claims benefit of U.S. Provisional Application No. 62/782,734, filed Dec. 20, 2018, which is hereby incorporated herein by reference in its entirety.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US2019/067448 | 12/19/2019 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
62782734 | Dec 2018 | US |