Animal Health – Claudio Passananti, Nicoletta Corbi, Maria Grazia Di Certo, Elisabetta Mattei, Cinzia PISANI, Georgios STRIMPAKOS, Siro LUVISETTO, Consiglio Nazionale delle Richerche CNR
Abstract for “Compositions of treatment for muscular dystrophy”
“The present invention includes modified human transcription factor capable of increasing the expression of utrophin, recombinant Adeno-associated vectors to deliver the modified human transcription factor, and methods for treating muscle diseases including Duchenne muscular dystrophy.”
Background for “Compositions of treatment for muscular dystrophy”
“The invention concerns compositions that include modified human transcription factor, recombinant Adeno-associated virus vectors (AAVs), comprising modified human transcript factors, and methods to treat muscular defects.”
“Duchenne’s muscle dystrophy (DMD), is a severe Xlinked, degenerative condition characterized by the absence or dystrophin cytoskeletal protein. Dystrophin protein is responsible for stabilizing the sarcolemma, i.e. the cell membrane of muscle cells. It links the intracellular cytoskeletal system to the extracellular matrix. Dystrophin is absent, and muscle contraction can mechanically stress the cell membrane, causing progressive damage to myofibers. Initial damage to skeletal muscles is primarily caused by the disease. However, the damage spreads to the cardiac muscle, and often death occurs from cardiac failure. DMD is one the most common genetic disorders, with an estimated 1 in 3600 male births affected each year. DMD is a debilitating condition that gradually worsens over the 25-year lifespan of those who are affected. Despite years of research, there is no cure for muscular dystrophies, including DMD. The current therapies only manage symptoms.
“Designing new therapies for DMD and related dystrophinopathies has focused on functionally salvaging the missing or defective dystrophin proteins. Therefore, compositions that increase utrophin expression are needed that are less immunogenic and more effective therapeutically.
“The invention includes a modified transcription element that contains at least one first, two second, and three zinc fingermotifs. The transcription factor can increase utrophin expression in cardiac or skeletal muscle tissue. The transcription factor may also include a fourth, fifth, sixth, seventh, eighth, or ninth zinc finger motif in some embodiments. Each zinc finger motif in some embodiments includes an alpha-helix. In some embodiments, transcription factors are derived from genomically encoded human transcription factors. Zif268 is the genomically encoded transcription factor in some embodiments.
“In one embodiment, the first motif of zinc can contain a sequence with at least 80 sequence identity for SEQID NO:48. The second motif of zinc can contain a sequence with at least 80% identity to the SEQID NO:49 and the third motif of zinc can contain a sequence with at least 80% identity to the SEQID NO:50.
“In the second embodiment, the first motif of the zinc fingers can include the SEQID NO:48, while the second can include the SEQID NO:49 and the third can include the SEQID NO:50.”
“In a third embodiment, a transcription factor may include a sequence with at least 95% sequence ID to SEQ ID No: 38. The transcription factor can contain: i. a first zilfinger motif that includes an alpha helix and an Arg sequence at position 1, a Glu residue in position 3, and a Glu residue in position 6, and an Asn sequence at location 8, and ii. a second zinc-finger motif including an Alpha-helix that contains a Ser at the position 1 and Arg at 6 and Arg at 5 and Arg at positions 6, an Arg at 6, an Asn at 8 and an Asn at 9
“In a fourth embodiment, a first zinc finger motif may include a sequence with at least 80 sequence identity. The second zinc finger motif could include a series with at least 80% of sequence identity. SEQID NO:52 can also be used. Third zinc finger motif: A sequence that has at least 80% identity to the SEQID NO:53 can be used.
“In a fifth embodiment the first zinc finger motif may include SEQID NO:51, while the second can include the SEQID NO:52 and the third can include the SEQID NO:53.”
“In a sixth embodiment, a transcription factor may include a sequence with at least 95% sequence ID to SEQ ID No: 39. The transcription factor may include: i. a first zinc-finger motif that includes an alpha helix and an Asn residue. Position 3, a Val residue 5 and position 6, and an Asn residue 8 at positions 3 and 6. Position ii. A second zinc-finger motif that contains an alpha helix and an Asp residue. Position?1, Arg at 6 and an Asn at 6, an Asn at 8.
“Some embodiments of the invention allow the transcription factor to include an amino acid sequence SEQ ID No: 38 or 39. Another embodiment of the invention states that the transcription factor is not immunogenic when it is expressed in humans. Another embodiment of the invention is capable to increase muscle contractile strength. This can be measured in in vivo or ex vivo assays. A further embodiment of the transcription factor can increase muscle endurance. This is done using a forced exercise test. Some aspects of this embodiment increase muscle endurance by at least 10% relative to reference.
“The invention also includes a recombinant Adeno-associated Vector (AAV), which allows expression of a gene within skeletal or cardiac muscles tissue. It includes a muscle-specific promor and any of these transcription factors. The gene may be expressed in both skeletal and cardiac muscles tissue, depending on the embodiment. Some embodiments of the muscle-specific inducer are constitutively expressed during differentiation. Certain embodiments include alpha-actin and cardiac troponin B, myosin light chains 2A, myosin lightchain 2A, and skeletal beta-actin.
“In one embodiment, the vector may have a serotype from any of the following: AAV1, AAV2, and AAV3, as well as AAV4, AAV5, or AAV6, AAV7, or AAV8. The vector may be muscle tropic in some instances. The vector may have an AAV6 or 8 serotype. Another embodiment of the vector may include at least one element from the following groups: an inverted terminal repeat (or AAV6), a cap signal (or AAV8), an intron donor site, an Intron splice acceptor site, an epitope tags, a nuclear sequence, and a polyadenylation consensus sequencing. Another embodiment of the vector contains the sequence of SEQID NO:86 and SEQID NO:87. The transcription factor binds specifically to the promoter of utrophin gene in any embodiment of the invention. promoter. The human or mouse utrophin?A is used in certain aspects of this embodiment. Promoter contains the sequence of SEQID NO:9, 10 or 11 or SEQID NO:40.
“The invention also includes a composition that contains any of the above vectors and a pharmaceutically approved carrier. The invention also includes a method for treating a muscle disorder in a subject who is in need of it, which involves administering an effective amount the previous composition. The invention also includes a method for treating a muscular disease. This method involves contacting the utrophin genes of muscle cells with any of the above modified transcription factors. The contacting can result in an increase or decrease in muscle endurance, depending on the embodiment.
“In some embodiments the composition can be administered locally or systemically. Other embodiments of the composition can be administered intramuscularly or intravenously, subcutaneously or intraperitoneally. The treatment can increase muscle endurance or muscle contractile force in any of the methods described above. The invention can treat any type of muscle disease. This includes Duchenne’s Muscular Dystrophy and Becker’s Muscular Dystrophy.
“DEFINITIONS”
“Administering” is the term used herein. “Administering” is a way of administering a dose of a composition to a subject (e.g., a composition containing a recombinant vector AAV vector encoding a polynucleotide encoding an fusion protein or modified transcript factor capable of increasing the expression of utrophin). You can administer the compositions described in this article by any of the following routes: parenteral (e.g. intravenous, intraperitoneal), dermal or transdermal, oral, buccal, sublingual and perilingual; nasal, rectal; topical; and oral. These compositions can be administered either locally or systemically using the methods described. Depending on the treatment being used and the component of the composition, the preferred method of administration may vary.
“?Amino acid sequence? As used herein, an oligopeptide or peptide or polypeptide or protein sequence and fragments or portions thereof as well as naturally occurring or synthetic molecules.
“By ?cardiac muscle? The form of striated tissue found in the heart that is controlled by the autonomic nervous systems, i.e. it is involuntarily controlled, is called “cardiac muscle”. Cardiomyocytes and myocardiocytes are the cells that make up cardiac muscle. The heart is a large organ made up mainly of cardiac muscle and connective tissues.
“By modified transcription factor?” A transcription factor which is substantially derived form a genomically-encoded transcription gene. Modified transcription factors can also be obtained from transcription factors that are known to the art, such as zinc finger transcription factors. A transcription factor, such as a human transcription factors, can have its zinc finger motifs replaced, modified or engineered to alter the target sequence it recognizes and binds. Modified transcription factors that are derived from human transcription factor genes are called?modified human transcript factors? herein. Zif268, also known by the?Early Growth Response Protein 1?, is an example human zinc finger transcription factor. ?Egr1,? ?Egr1? Zif268 encodes a protein that belongs to the EGR family, which includes three-zinc finger-containing proteins of C2H2-type. Zif268 functions as a nuclear protein and transcriptional regulator. Zif268’s principal isoform contains 543 amino acid with a molecular mass of 57.5kDa. It binds to the sequence 5?-GCGTGGGCG-3. (SEQ ID NO. 44). SEQ ID NO. 41 shows an exemplary sequence of amino acids for human Zif268. You can find an exemplary sequence of mRNA for human Zif268 under NCBI Accession No. NM_001964 and in SEQ ID N:19.
“As used herein.?conservative variants? Or?conservative modified versions? A particular sequence is a sequence of amino acids encoded using nucleic acids that encode identical or essentially the same amino acid sequences. Or where the nucleic Acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy in the genetic code, many functionally identical nucleic acid sequences may encode any given protein. These nucleic acids variations can be called silent variations. They are one type of conservatively modified variants. Any one with skill will be able to recognize that any codon in a nucleic acids (except AUG which is the codon for methionine) may be modified using standard techniques to produce a functionally identical molecule. Each silent variant of a nucleic acids that encodes a specific peptide is implicitly included in the amino acid sequence. One of the most common modifications is conservatively modified versions. These variations result in the substitution or deletion of an amino acid by a chemically identical amino acid. The art is well-versed in the use of conservative substitution tables that provide functionally identical amino acids. Each of the following six groups contains amino acids that can be used as conservative substitutes: 1) Alanine, Serine, and Threonine; 2) Aspartic Acid (D), Glutamic (E); 3) Asparagine [N], Glutamine [Q]); 4) Arginine(R), Lysine (10), 5) Isoleucine (1), Leucine (5), Methionine (6M), Valine (10); 6) Phenylalanine (7), Tyrosine (W), and Tyrosine (Y), and Tryptophan (W);
“By ?contact? “By?contact? as used herein, is intended to permit or promote a state or combination of at least two elements. The fusion protein or modified transcript factor of the invention can be used to contact the utrophin genes. This binds specifically to a target sequence within the utrophin?A?. This will increase the expression of utrophin.
As used herein, “a?deletion?” refers to a change of either amino acid sequence or nucleotide sequence that results in one or more amino acids or nucleotide symposia being absent.
“By DNA binding element?” An element capable of binding DNA. A DNA binding element can bind to a specific sequence or response element of a particular gene, such as in a promoter. Examples of DNA binding elements include, but are not limited: domains that contain zinc finger motif, helix/turn helix domains and domains that contain winged helix motifs, winged leucine zipper domains and domains that include winged helix turns helix domains. HMG box domains and domains for Wor3 and immunoglobulin domains. TAL effector DNA binding domains. RNA-guided DNAbinding domains. ZFP51. The ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZFP1 and JZZZZZZZZZZZZZZZZZZZ.
“By an effective amount of a compound? The amount of a compound administered to improve, inhibit or alleviate a condition or symptom of disorder or disease. Treatment is achieved if there is any improvement in the subject. A sufficient amount to treat is a dose that decreases, inhibits or prevents one or more of the symptoms of DMD. It is also a dose that lessens or stops the severity or duration of symptoms. The effective dosage of the pharmaceutical composition to treat DMD depends on the method of administration, as well as the patient’s age, weight and general health. The appropriate dosage and frequency can be determined by a physician or researcher.
“expression vectors” is the term used herein. “expression vectors” or?expressionplasmids, as well as similar terms, are DNA sequences required to clone copies of genes and/or translate their mRNAs in a suitable host. These vectors can be used for the expression of viral, prokaryotic or eukaryotic gene in a variety hosts, including bacteria, E.coli, blue-green alga, plant cells and insect cells as well as fungal and yeast cells.
“For the purposes of this article, the term “fusion protein” is used. A compound that contains all or part of the amino acids sequences of more than one protein. These modified transcription factors, such as modified human transcription factors, are not included in the definition of fusion protein. A fusion protein may include a DNA binding element, such as Jazz, Bagly or UtroUp, and one or more transcriptional activation factors (e.g. Vp16 or CJ7 or SP1, or Gal4). The terms “protein” and “polypeptide?” are interchangeable. The terms?protein? und?polypeptide are interchangeable herein. These terms are interchangeable herein. The term “portion” is used interchangeably herein. includes any region of a polypeptide, such as a fragment (e.g., a cleavage product or a recombinantly-produced fragment) or an element or a domain (a region of a polypeptide having an activity, such as, e.g., enzymatic activity or antigen- or DNA-binding capacity) that contains fewer amino acids than the full-length polypeptide (e.g., 5%, 10%, 12%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% fewer). One or more linkers may be included in a fusion protein to connect the amino acid sequences. The terms “portion” and “fragment” are well-known to those who have studied the art. The terms?portion? und?fragment are also used interchangeably in the art. These words can also be used interchangeably in this article.
“By ?host,? ?subject,? or ?patient,? Any organism is meant, including a mammal, primate, human, dog, cat and cow, as well as any animal such a fish or bird. “A host can also refer to a domestic animal (e.g. a farm animal), or a companion animal, such as a pet.
“By ?immunogenicity? “Immunogenicity” refers to the ability of a specific substance (e.g. an antigen, epitope or protein) to trigger an immune response in the human or animal’s body. A cell-mediated or humoral immune response can be included in an immune response. Treatment with therapeutic proteins, such as artificial transcription factors, is generally not recommended because patients may develop or have antibodies that bind to the therapeutic protein. This can lead to the inactivation of therapeutic proteins and sometimes adverse side effects. The level of immunogenicity can be affected by many factors, including the delivery route, the delivery vehicle, the dose regiment, the dosage regiment, aggregation and innate immune system activation, molecular structure, protein structure, degradationability and the ability of a protein to interface with humoral (B) and cellular (T) responses.
“Increasing muscle contractile strength” means: This refers to increasing the force exerted by a muscle contraction by at least 5% or 10%, 15% or 20%, 25% or 40%, 50% or 60%, 70% or 80%, 90% or 100%, 150% or 3-fold, 4-fold or 5-fold, respectively, as compared with reference. In vivo, ex situ or ex vivo measurements of the force generated by a muscle are possible. For example, ex vivo or in-situ analysis of the contractile profiles of one intact limb muscle (e.g. The extensor digitalorum longus, abdominal muscles, and the tibialis anterior muscle are used for ex-vivo assays. Grip force analysis, downhill treadmill exercise (manual muscle testing), myometry (e.g. Myometer to assess upper and lower extremity strength, including evaluations of knee flexors, extensors as well as elbow flexors. Shoulder abductors can also be evaluated. Assays for sustained maximum voluntary contraction (MVC), or any of the other assays described by Hakim et. al., Methods Mol. Biol. 709: 75-89, 2011; Sharma et al., Neurology 45: 306-310, 1995; and McDonald et al., Muscle Nerve 48: 343-356, 2013.”
“Increased muscle endurance” means: It refers to increasing the endurance of a muscle group or muscle (e.g. a decrease of at least 1%, 10%, 15% or 20%), over a prolonged period of time (e.g. hours (e.g. 1, 2, 3, 4, 6, 7, 8, 9, 10, hours), days (e.g. 1, 2, 3, 5, 6, 7, 7, 8, 9, 10), weeks (e.g. 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, or 12 months. 1, 2, 3, 5, 6, 7, 8, 10, 11 or 12 months. Assays that measure muscle endurance include treadmill exercise, 6-minute walk test (6MWT), and timed function tests (e.g. time taken for a person to stand up from a supine or to walk 10 m or climb/descend 4 standard-sized steps). These tests can be used to determine muscular endurance or strength in mice. Disord. 22(2):170-182, 2012) and at accelerated speeds (see Di Certo et. al., Hum. Mol. Genet. 19:752-760, 2010 or Strimpakos et al., J. Cell. Phys. 229:1283-1291 (2014). Voluntary wheel exercise, grip strength, the hang wire, inverted grid, and the rotarod tests or any of the assays described by McDonald et al. Muscle Nerve 48: 343-356 (2013).
“Increasing the utrophin expression?” This is to increase utrophin gene expression and/or protein activity compared with a normal or positive tissue or cell. An expression product can be anRNA transcribed directly from the gene (e.g. An mRNA or a polypeptide derived from an mRNA from the gene can be used as an expression product. An increase in mRNA levels will usually result in an increase of the polypeptides translated from it. The level of expression and/or activity may be determined using standard techniques for detecting and measuring mRNA or protein, including but not limited to RT-PCR, Western blotting, enzyme-linked immunosorbant assay (ELISA), immunohistochemistry, immunofluorescence, and mass spectrometry.”
“An ?insertion? “An?insertion? or?addition? As used herein, it refers to any change in an amino or nucleotide sequence that results in the addition of one (or more) amino acids or nucleotide atoms.
“The terms ‘linker,? ?linker region,? or ?linker domain,? These terms are used to describe elements located between adjacent polynucleotide and polypeptide sequences. When constructing a cloning vector, or fusion protein, you can introduce stretches of polynucleotide sequence or polypeptide sequence. Chemical conjugation can also introduce linkers, such as?click-chemistry?-based approaches. A linker region may be used to introduce cloning sites in the nucleotide sequence or a flexible component or region-creating region between two proteins domains or create an affinity tag for specific molecular interactions. As a result of recombinant nuclear acid production, a linker region can be added to a fusion proteins.
“By ?muscle-specific promoter? Any sequence of DNA that promotes the expression of a particular gene in muscle tissue is called a “muscle-specific promoter”. A muscle-specific promoter will typically be found upstream from the transcription initiation site for a gene. A muscle-specific promoter, for example, may increase the expression of a gene within a muscle by at least 2-fold and 3-fold, 4-folds, 5-folds, 10-folds, 15-folds, 20-folds, 30-folds, 50-folds, 50-folds, 100-folds, or more than a non-muscle tissue. Some muscle-specific genes are expressed during differentiation. Examples of muscle-specific promoters are: alpha-actin and cardiac troponin C. Myosin light chains 2A, skeletal beta -actin. CK6. Dystrophin. Synthetic C5-12 (Syn), Myf5, MyoD1, MyoD1, Pax7. See, for example, U.S. Patent Application 2011/0212529, McCarthy et al., Skeletal Muscle 2:8, 2012; and Wang et al., Gene Ther. 15:1489-1499 (2008), the entire of which are incorporated by reference.
“By ?muscular dystrophy? “Muscular dystrophy” refers to a group of diseases that affect the muscles and cause impairments in movement. Muscular dystrophies can cause progressive weakness, defects in muscle proteins, death of tissue and cells, as well as a loss of muscle function. Dystrophinopathy is a term that describes a variety of muscle diseases, including dystrophinopathy. This includes dystrophinopathy which refers to a range of muscle diseases where there is not enough dystrophin protein in the muscles cells. This causes instability in the structure and membrane of the muscle cells. Dystrophinopathies can also include Duchenne’s muscular disorder (DMD), Becker’s muscle dystrophy, and Benign pseudohypertrophic mild dystrophy (BMD). DMD and BMD can be X-linked recessive disorders caused by mutations of the dystrophin gene. This gene encodes the protein dystrophin. DMD is more severe that BMD. This is because in DMD there is no dystrophin protein produced, while in BMD dystrophin is. Congenital muscular disorders include facioscapulohumeral muscle dystrophy and limb-girdle muscle dystrophy.
“?Nucleic acid sequence? “?Nucleic acid sequence” is the term used herein to refer to an oligonucleotide or nucleotide or polynucleotide and any fragments or portions thereof.
“As used herein the term ‘operable linkage? Or?operable linkage? A physical or functional combination of components described above that allows them to work in their intended way. For example, two DNA sequences that are operably linked mean that they are arranged (cis- or trans) so that at least one sequence can exert a physiological influence on the other. A muscle specific promoter, for example, can be operably linked to a gene in order to promote the gene’s muscle-specific transcription.
“By ?pharmaceutical composition? “Pharmaceutical composition” means any composition that contains a therapeutically and biologically active agent. This includes any composition that contains a nucleic acid molecule that encodes all of the fusion protein, which is a transcriptional activation molecule, and a DNA binding element. It can also include any composition that can be administered to a subject. You can administer the compositions described in this article by any route you choose, including parenteral (e.g. intravenous or transdermal), dermal, transdermal and ocular, buccal, sublingual. perilingual. nasal, rectal. topical. These compositions can be administered either locally or systemically using the methods described. Parenteral is a term that refers to the administration of the compositions described herein. The term “parenteral” as used herein means subcutaneous, supracutaneous, intermuscular, intraperitoneal and intramuscular injections, as well any other infusion techniques. Sterile injectable compositions can be either a solution or suspension in an acceptable non-toxic solvent or diluent. These solutions include, among others, 1,3-butanediol and mannitol. Fixed oils can also be used as solvents or suspending media (e.g. synthetic mono- and diglycerides). Injectables can be prepared using fatty acids such as oleic acid or its glyceride derivatives. Natural pharmaceutically acceptable oils such as castor oil and olive oil, polyoxyethylated, are also useful. These oil solutions and suspensions can also contain long-chain alcohol diluents or dispersants such as carboxymethylcellulose or similar dispersing agents. You can also use other commonly used surfactants such as Tweens or Spans, or similar emulsifying agent or bioavailability boosters, that are used to make pharmaceutically acceptable solids, liquids, and other dosage forms. These formulations can all be prepared using well-known, accepted art methods. For example, Remington: Science and Practice of Pharmacy (21st edition). ), ed. A. R. Gennaro Lippincott Williams & Wilkins 2005 and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick Informa Healthcare 2006, which are each hereby incorporated by reference.
“pharmaceutically acceptable excipient, carrier or adjuvant” A diluent or excipient, carrier, adjuvant, or adjuvant that is physiologically acceptable for the subject and retains the therapeutic properties of its pharmaceutical composition is considered to be acceptable. Formulations can be prepared for injection in either liquid or solid forms. Acceptable carriers, excipients, or stabilizers for intravenous administration are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN?, PLURONICS?, or polyethylene glycol (PEG). Water, saline and dextrose are all acceptable excipients. These compositions can also contain non-toxic auxiliary substances like pH buffering agents, wetting agents, pH buffering, and so on. One skilled in the art can also identify other physiologically acceptable excipients or carriers and their formulations.
“By?recombinant-adenoassociated vector (AAV),” A recombinantly generated virus or viral particle is one that contains a polynucleotide. It can be delivered to a host cell in vivo, ex-vivo, or in vitro. AAV is a human parvovirus that is non-pathogenic and is used in gene transfer in mammals. AAV’s genome is single-stranded DNA. It contains inverted terminal repeats at both ends of DNA strands and two open reading frames, rep and cap. These are responsible for coding replication and capsid protein. The native rep and cap genes can be replaced by a foreign polynucleotide. You can make AAVs with many different serotype capsids that have different transduction profiles. for different tissue types. There are many AAV serotypes, including AAV1, AAV2, and AAV3, AAV4, AV5, AAV6, AV7, AV8, AAV9, as well as AAVrh10. Triple transfection of subconfluent HeK293 cells can produce AAV vectors. AAV cis-plasmid contains the gene of interest and AAV trans-plasmid contains AAV rep, cap and other genes. An adenovirus helper virus plasmid is, for instance, pDF6. SEQ ID Nos. 1 or 2, or SEQID NOs. 62-65, can all be used to produce recombinant Adeno-associated Vectors (e.g. viral particles that contain nucleotide sequences that enable muscle-specific expressions of artificial transcription factors). Herein, viral particles are described by the expression vector that was used to produce them and their serotype. The viral particle mAAV8Vp16Jazz, for example, has a capsid serotype 8, and is produced using the expression vector mAAVVp16Jazz.
“By ?reference? Any sample, standard or level used for comparison purposes. A “normal reference sample” is a sample taken from the same subject before the onset of a disorder (e.g., a muscular dystrophy), if it has been successfully treated for that condition or not. A?normal reference sample? can be any sample taken from the same subject before the onset of the disorder (e.g., muscular dystrophy), or from a patient who has been treated successfully for the disorder or disease. What is meant by “reference level”? A reference sample is a number or value that has been calculated from another. A normal reference level or standard can be any value or number that is derived from a normal subject and that matches a sample of the subject by at least one criteria: age, weight or disease stage. A “positive reference”? A sample, standard or value is a sample or standard derived from a subject who is known to have an illness (e.g., muscular dystrophy). It is matched to a sample by at least one of these criteria: weight, age, disease stage and overall health.
“By ?skeletal muscle? The form of striated tissue that is controlled by the somatic nervous systems, i.e. it is voluntary controlled, is called skeletal muscle. Multiple bundles of muscle fibers are called muscle. They are held together by connective tissues. Tendons can be used to attach skeletal muscles to bone. Skeletal muscles can be attached to bone by tendons. The following skeletal muscles are examples: the diaphragm; extensor digitalorum longus; tibialis posterior, gastrocnemius; soleus.
“By ?specifically binds? “By?specifically binds?” A molecule (e.g. an artificial transcription factor) that recognizes and binds another molecular (e.g. a polynucleotide) but does not substantially recognize or bind other molecules. An artificial transcription factor binds to a specific DNA sequence in the utrophin A? The promoter does not bind to other polynucleotide combinations. The dissociation constant (Kd) can be used to represent the binding affinity of a molecule for its partner Y. The term “specific binding” is also known as: The term?specific binding? refers to a specific binding. Is it?specific for? A molecule can be shown to be specific for a particular molecule (e.g., polynucleotide, polypeptide) by having a Kd of the molecule it binds of at most 10/3 M, 5/6 M and 10/7 M. ?Binding affinity? “Binding affinity” generally refers to how strong the sum of all noncovalent interactions between a single binding location of a molecule (e.g. a protein fusion) and its binding partner.
“Substantially the same” is a term that refers to sequences that are substantially identical. When used in this context with regard to the comparison between a sequence and a reference sequence, it applies to sequences that are at most about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or at the very least about 99% sequence identity to their reference sequence. Standard methods and algorithms can be used to determine percent identity between sequences, including BLASTN (NCBI, Schaffer et. al., Nucleic Acids Res. 29:2994-3005, 2001), BLASTX (NCBI; Schaffer et al., Nucleic Acids Res. 29:2994-3005 2001, ALIGN(GCG, Accelrys), FASTA (Pearson et al. Proc. Natl. Acad. Sci. U.S.A. 85.2444-2448 (88) programs that may use default settings. The invention includes amino acid sequences that, in various examples, are identical to reference sequences.
“Substitution” as it is used herein refers to the substitution of one or more nucleotides or amino acids by other amino acids, or nucleotides.
“By ?transcriptional activation element? A polypeptide capable of initiating transcription is a polypeptide that can make an RNA transcript (e.g. an mRNA) using a DNA template. This is done by RNA polymerase. A transcriptional activation element could include a trans-activation region of a transcription factor protein, or other domains that directly or indirectly promote transcription. You can also find transcriptional activation elements that are polypeptides or fragments of them. These include transcriptional coactivators, transcriptional coregulators (e.g. TAF9 and MED15), transcriptional coactivators (e.g. CBP/p300), transcriptional coactivators (e.g. transcriptional keygulators, transcriptional de-acetylases or histonemethylases), chromatin remodelers, kinases or DNA methylases to promote transcription. Transcriptional activation element may recruit transcriptional inhibitors or proteins that promote transcription. To bring a transcriptional activation element into close proximity to DNA, it usually requires a DNA binding element. Examples of transcriptional activation elements are: hydrophobic or acidic activation domains (e.g. from Gal4 and Gcn4, respectively), nine-aminoacid transactivation Domains (9aaTAD), e.g. from p53 and Vp16. MLL, E2A and NF?B), p65, SP1, Zif268, the trans-activation domain (CJ7) derived from human Che-1/AATF (see, for example, BMC Mol. Biol. 14:3, 2013). For example, see US Patent Application Publication No. 2007/0020627 and Blancafort et al., Mol. Pharmacol. 66(8): 1361-137, 2004. Transcriptional activation element can also be derived or include physiological regulators of Utrophin expression such as GABP, NFAT? and GABP?. SEQ ID No:37 shows an example sequence of Vp16 amino acids, while SEQ ID No:75 shows an exemplary sequence of CJ7 amino acids.
“By ?transcription factor? A protein that binds with specific DNA sequences and controls transcription of DNA to messenger RNA (mRNA) is called a “transcription factor”. This activity can be performed by transcription factors either alone or in combination with other proteins. They promote or block the recruitment of RNA Polymerase. By ?artificial transcription factor? Any transcription factor that is not found in nature is referred to as an “artificial transcription factor”. A fusion protein, or modified transcription factor can be an artificial transcription factor.
“By ?treating? “Treating” is the act of administering a pharmaceutical composition according to the invention for therapeutic and/or prophylactic purposes. For example, prophylactic treatment can be given to a patient who is not yet sick but is at risk for a specific muscle disease or defect. For example, DMD treatment can be given to a patient who is already experiencing symptoms. According to the claims and embodiments, treating refers to the administration of medication to a subject for either therapeutic or prophylactic reasons. Sometimes, treatment can be used to treat a disorder (e.g. DMD) or a symptom. This may reduce the severity or progression of the disorder or decrease the frequency or progression of symptoms by, for example, 1% or 2%, 10%, 20% or 30%. One can use electromyography (EMG), genetic testing, muscle biopsy, serum Creatine Kinase levels, muscular strength tests (e.g. manual muscle testing), and range-of motion (ROM) tests like the six-minute walk test to measure symptoms of DMD. DMD can cause symptoms that range from mild to severe. It is possible to have DMD symptoms, depending on the location of the disease and the causative mutation. A successful treatment will show an improvement or absence in one or more of the symptoms of muscular dystrophy.
“By ?utrophin ?A? promoter? or ?utrophin promoter ?A?? The promoter region located at 5? The utrophin gene is located in the upstream region. The utrophin A? The promoter is located in an unmethylated CpG islands and is active within muscle cells. This is contrary to the utrophin B? This promoter is located immediately upstream from the large second exon utrophin. It is active in endothelial cell cells. A sequence that is representative of human utrophin?A? SEQ ID No:20 defines the promoter. It can also include sequences with SEQ ID NOS:9, 10, 12 or 14. A sequence that is representative of mouse utrophin?A? SEQ ID No:33 defines the promoter or may include sequences with SEQ ID NOs 11-13 or 15.
“By ?zinc finger motif? This is a protein structural motif. Zinc finger motifs are a group of motifs that coordinate with one or more zincions to stabilize the fold. A zinc finger DNA binding domain can contain 1, 2, 3, 4, 5, 6 7, 8, 9, and more zinc finger motifs, arranged in tandem arrays that can bind to the major groove of DNA. The Cys2-His2 zinc fingers, which are composed of 28-31 amino acid that fold into a???, is a well-known zinc finger motif. structure. The Cys2-His2 type zinc fingers motif typically has a sequence of the form X3-Cys-X2-4-Cys-X12-His-X3-6-His-X4, wherein X is any amino acid (e.g. X2-4 is an oligopeptide of 2-4 amino acids. The sequence variation of 28-31 amino acid sequences of the zinc finger polypeptide is quite wide. Invariant are only the consensus histidine and consensus cysteine residues that are bound to the central Zinc atom. Three to five of the remaining residues are highly conserved while there is considerable variation among them. The alpha-helix for each motif (often called?recognition Helix?) The sequence-specific contact of a motif’s alpha-helix to DNA bases can be made. Reactants from one recognition helix usually contact three base pairs of DNA. Modifications to the positions of key amino acids (e.g. positions?1, +3, or +6) in the zinc finger alpha helix can alter the DNA binding target specificity for a zinc-finger motif. See, for example, Corbi et al., Biochem. Cell. Biol. 82:428-436, 2004; Klug, Q. Rev. Biophys. 43:1-21, 2010; Choo et al., Curr. Opin. Struct. Biol. 7:117-125, 1997; Pabo et al., Annu. Rev. Biochem. 70:313-340 2001; Segal et. al., Curr. Opin. Biotechnol. 12:632-637 2001; and Sera. Drug Delivery Rev. 61:513-526, 2009. Hugo Gene Nomenclature Commission (HGNC) has a list of human CystHist type zinc fingers proteins. Cys2-His2 type zinc finger proteins often contain an effector domain located N-terminally to the zinc finger region, such as the KRAB (Kruppel-Associated-Box), SCAN (SRE-ZBP, CTfin51, AW-1 and Number18 cDNA) and BTB (Broad-Complex, Tramtrack and Bric-a-bric) effector domains. Another example of zinc finger motifs are the Gag knuckle and Treble clef.
“Other features or advantages of the invention will become apparent from the detailed description and the claims.”
Dystrophinopathies, including DMD and BMD, are caused by dystrophin protein deficiency in muscle cells. Utrophin can be considered dystrophin’s autosomal homologue, as the two proteins share many of the same structural and functional motifs. Dystrophin (and utrophin) function as connecting proteins between the cell membrane, cytoskeletal actin and the extracellular matrix via proteins collectively known as DAPs (dystrophin-associated proteins). Utrophin is most abundant in the fetus, and its expression is decreased after delivery. Dystrophin is more prevalent after birth. Adults have utrophin that is localized at the neuromuscular junction. Dystrophin, however, is found along the length of the Sarcolemma. Transgenic mdx mice have been used as a model animal for DMD. They show that utrophin overexpression is associated with redistribution across the entire sarcolemma. This results in a marked improvement of dystrophic phenotype.
The present invention aims to up-regulate, i.e. increase, the dystrophin-related genes utrophin expression in DMD patients’ muscle to functionally rescue (i.e. to complement) dystrophin function. You can do this by contacting the Utrophin gene using a modified human transcription element capable of increasing utrophin. The modified transcription factors of this invention have the advantage of reducing immunogenicity after being introduced to a subject. The present invention provides muscle-specific, recombinant human transcription factor vectors that can promote the expression of modified human transcription factors in both skeletal and cardiac muscle. This is useful in the treatment of DMD, which has severe skeletal and cardiac muscle disorders. The vectors of this invention have shown that AAV delivery of modified human transcription factor (recombinant) significantly improves dystrophic phenotype in mice suffering from mdx dystrophin deficiency. This includes improvements in both muscle strength and endurance. The compositions and methods described in the invention can be used to treat DMD, particularly muscle defects.
“AAV Vectors & Compositions”
“In some embodiments of the invention, a nucleic acids sequence or fragment encoding a gene (e.g., an Artificial transcription Factor (e.g. a Fusion Protein or Modified transcription Factor) capable of increasing Utrophin expression is delivered to muscle cells via a viral vector. Many viruses are known and readily available in the art. Preferably, the artificial transcription factors are delivered to both cardiac and skeletal muscle. The therapeutic vector should be non-toxic and minimally immunogenic. It should also be easy to create and deliver DNA into target cells. The viral vector can be a recombinant Adeno-associated Vector (AAV) in certain embodiments. Other embodiments of the invention provide a therapeutic composition that includes an AAV including an artificial transcription factor, such as a fusion protein, or modified transcription factor, under the control a muscle-specific promor.
AAVs are a popular method of exogenous DNA delivery. They are relatively safe, efficient, and easy to optimize for specific purposes. There are more than 30 AAV types that naturally occur. There are many natural variants of the AAV capsid that allow identification and use an AAV with specific properties for muscle cells. Conventional molecular biology techniques can be used to engineer AAV viruses. This allows for optimization of these particles for cell-specific delivery of nucleic acids sequences, for immunogenicity minimization, tuning stability, particle lifetime, and efficient degradation. Human serotype 2, which was the first AAV to be isolated from human or nonhuman primates (NHP), is a gene transfer vector. It has been extensively used in gene transfer experiments in various target tissues and animal models. Other AAV serotypes include AAV1, AAV3, and AAV4, as well as AAV5, AAV6, the AAV7, AAV8, and AAV9, among others. For a detailed discussion on AAV serotypes, see International Patent Application WO 2005/033321.
“In many embodiments, artificial transcription factors (or other genes) can be expressed in muscle cells by delivery via recombinantly-engineered AAVs. These AAVs may contain nucleic acids sequences encoding the artificial transcription factors or genes that have been operably linked with muscle-specific promoters. These AAVs can be used to treat muscle defects in certain preferred embodiments.
US Patent Application Publication No. 2013/0136729 describes muscle-specific expression vectors. It also describes the combination AAV9 with muscle-specific CK6 enhancer as a highly efficient system for systemically carrying genes into ischemic skeletal muscles. The combination of AAV9 with the cardiac troponinC promoter is described in this publication as a system that is especially effective in cardiomyocytes, skeletal myocytes and those with ischemic injury. International Patent Application (WO 2005/118611) describes microutrophin delivery via adenoassociated vectors in the mouse model of mdx dystrophic. It also includes incorporation of muscle-active promoters such as dystrophin promoters for skeletal?-actin, myosin light chains 2A promoters and muscular creatinekinase kinase promoters.
“Expression vectors to induce exogenous genes, such as the zinc finger transcription factors, in muscle and non-muscle tissues are also described in U.S. Pat. No. No. 8,304,235 describes the artificial transcription factor (UTroUp) (encoding a 6-zinc finger repeat that is designed to specifically bind to the utrophin promoterA) fused to the transcriptional activationdomain?Vp16). From the Herpes virus under the control regulatory sequences of Cytomegalovirus (CMV), and its expression via the eukaryotic vector, pRK5(Clontech), Italian Patent Application (RM2005A000493). This describes the artificial transcription factor Vp16Jazz and its expression via the vector vector pMEX. It is controlled by the muscle-specific promoter/enhancer region of the murine meosin light chain gene (MLC). These expression vectors allow the exogenous gene to be expressed in either skeletal or cardiac muscle tissues. However, it is not possible to express them both. This invention allows exogenous genes to be expressed in both the skeletal and cardiac muscles.
The cap proteins (including vp1,vp2, and vp3) are desirable AAV fragments that can be assembled into vectors. Also, hypervariable regions and the rep proteins (rep78, rep68 and rep52), and sequences encoding these proteins are important. These fragments can be used in many vector systems and host cell types. These fragments can be used alone or together with fragments from other AAV serotypes or AAV viral sequences. Artificial AAV serotypes can include AAVs with a non-naturally occuring capsid protein, as described herein. An artificial capsid can be created using any suitable technique. It may be made from a selected AAV sequence, such as a fragment from a vp1 capid protein, in combination with heterologous sequences that may be obtained from different AAV serotypes, non-contiguous parts of the same AAV Serotype, from non-AAV virus sources, or from non-viral sources. A pseudotyped AAV capid, an AAV capsid chimeric, an AAV capsid recombinant, or a humanized AAV capid are all possible options. AAV capsid. The invention makes use of pseudotyped vectors in which the capsid from one AAV is replaced by a heterologous proteins capsid.
“In one embodiment, vectors that are useful in compositions or methods described herein include sequences that encode a selected AAV serotype capid, e.g. an AAV8 capsid or a fragment thereof. Another embodiment contains sequences that encode a select AAV serotype rep proteins, such as the AAV8 rep protein or a fragment thereof. These vectors can contain AAV rep and cap proteins. Vectors that contain both AAV rep or cap can have both AAV rep or cap sequences. However, they do not need to be both of the same serotype origin. Other embodiments of the vectors that are useful in compositions or methods described herein include sequences encoding AAV6 capsids, or a fragment thereof. Some embodiments include sequences that encode an AAV6 rep protein or a fragment thereof. In some other embodiments, both the AAV rep or cap sequences are included in vectors.
Vectors can also be used in which rep sequences come from AAV serotypes that are different from the one providing the cap sequences. One embodiment expresses the rep and cap sequences from different sources (e.g. separate vectors or a host cell and vector). These rep sequences can be fused together in frame to cap sequences from a different AAV serotype in another embodiment to create a chimeric vector. No. No. 7,282,199, which can be incorporated herein by reference.”
A suitable recombinant AAV can be created by cultivating a host cell that contains the following: a nucleic acids sequence encoding the AAV serotype capsid proteins, or a fragment thereof; a functional rep genes; a transgene sequence comprising AAV inverted termin repeats (ITRs), and a muscle-specific promor sequence; sufficient helper functions to allow packaging the transgene into the AAV capsid proteins. Trans may be used to provide the components that must be grown in the host cell to pack an AAV minigene into an AAV capid. A stable host cell may also contain the components required to package an AAV minigene in an AAV capsid. The present invention allows for the production of AAV vectors by triple transfection of subconfluent HeK293 cells with three plasmids. AAV cis-plasmid contains the gene of interest, AAV Trans-plasmid contains AAV rep, cap, and an Adenovirus Helper plasmid such as pDF6.
“A stable host cell could contain the necessary component(s) when it is under the control an inducible inducer. The constitutive promoter may control the necessary component. In the discussion below, we provide examples of inducible and suitable constitutive promoters. Another alternative is to have a select stable host cell contain certain components that are under the control a constitutive and other components that are under the control a one or more of the inducible promotors. A stable host cell can be created from 293 cells, which contain E1 helper function under the control a constitutive inducer. However, it may also contain the rep and/or caps proteins under the control inducible promoters. Another method of creating stable host cells is to use one skilled in the art.
“The transgene and any rep sequences, caps sequences, or helper functions necessary for the production of the recombinant aAV of the invention can be delivered to the packaging host cells in the form of any gene element that transfers the sequences. Any suitable method can be used to deliver the selected genetic element, including those described in this document. Anyone skilled in nucleic acids manipulation will be familiar with the methods that were used to create any embodiment of this invention. These include genetic engineering, synthetic techniques, and recombinant technology. Sambrook et. al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press. Cold Spring Harbor, N.Y. Similar methods for generating recombinant AAV viruses are well-known and it is not the limitation of the invention to choose a suitable method. See, e.g., Fisher et al., J. Virol. 70:520-532 1992, and U.S. Pat. No. No. 5,478,745, and other. These publications are included herein as a reference.”
“Unless otherwise stated, the AAVITRs and other selected components of AAVs described herein may be easily selected from any AAV serotype including AAV1, AAV2, and AAV3, and AAV4, and AAV5, and AAV6, and AAV7, and AAV8, and AAV9, and AAV10, as well as other unknown and known AAV serotypes. These ITRs and other AAV components can be easily isolated using techniques that are available to skilled art practitioners from any AAV serotype. These AAV can be obtained from commercial, academic, or public sources (e.g. American Type Culture Collection Manassas Va.). Alternately, AAV sequences can be obtained by synthetic or other suitable methods, using published sequences, such as those found in literature, PubMed, GenBank, and the like.
“A transgene according to the invention includes, for example, an artificial transcript factor (e.g. a fusion factor or modified transcription factor), nucleic acid sequence (e.g. Vp16 -Jazz or Vp16 -Bagly or Vp16 -CJ7 -UtroUp or JZif1) or any other gene that is desired to be delivered directly to muscles as described above and its regulatory sequences and 5? 3. AAV inverted terminal repetitions (ITRs) One desirable embodiment uses the ITRs from AAV serotype 8. You can also choose ITRs from other serotypes. This transgene is then packaged in a capsid protein, and delivered to a chosen host cell.
The regulatory sequences are conventional control elements that can be linked to the transgene in such a way as to permit its transcription, translation, and/or expression in cells transfected or infected by the invention’s virus. The expression control sequences include sequences which regulate transcription initiation, termination and promoter, as well as RNA processing signals like splicing (polyA) signals and splicing (polyA); sequences to stabilize cytoplasmic mRNA; splicing and polyadenylation signals (i.e. Kozak consensus sequence); and sequences that increase protein stability. They also contain sequences that confer nuclear location (e.g. nuclear localization sequences). When desired, they can enhance the secretion. There are many expression control sequences that have been developed and can be used. Many species are suitable for poly A sequencing, including humans, SV-40 and bovines.
“Regulators useful in the constructs described in the present invention could also include an intron. This intron is desirable to be located between the promoter/enhancer and the gene. A 100 bp intron sequence, derived from SV-40 is referred to as SD?SA. It is a mini-intron splice donater/splice taker. The preferred embodiment of the intron includes the first intron from the human alpha-actin genes, which also includes the splice donor sites. It also includes part of both the second intron and third exons of the human betaglobin genes, which include the splice acceptance site.
“In other embodiments, recombinant AAV Vectors may contain additional functional elements such as cap signals, sequences for epitope tags (e.g. haemmaglutinin, myc tag and maltose binding protein (MBP), tag, green fluorescent pro (GFP) or other fluorescent proteins). Multicloning sites, and/or multicloning locations that contain multiple restriction enzyme cutting sites. This allows it to insert any gene coding a particular protein into the vector. The sequence of amino acids for an exemplary myctag is shown in SEQ ID No:75.
“The promoter that will be used in the recombinant AAV can be chosen from a variety of constitutive, induceable, cell-type or tissue-specific promoters that are capable of expressing the transgene in the desired muscles cells. The promoter must be muscle-specific in a preferred embodiment. Particularly preferred embodiments have the promoter specific to expression of the transgene within skeletal and cardiac muscles cells.
“The promoter(s), used in the present invention, may be derived any species. Due to limitations in the AAV vector’s size, one embodiment of the promoter is small, less than 1000 bp. Another embodiment has a promoter that is less than 400 bp. The invention includes, but is not limited to, the retroviral Rous sarcomavirus (RSV), LTR promoter (optionally containing the RSV enhancer), cytomegalovirus, (CMV), promoter (optionally containing the CMV enhancer), SV40 promoter and the dihydrofolate reductase promor, as well as the EF1 promoter from Invitrogen, the phosphoglycerol kinase kinaserol kinasa promoter.
“Inducible promotors which can be used with the present invention can regulate gene expression. They can be controlled by exogenously supplied chemicals, environmental factors like temperature or the presence of a certain physiological state, such as acute phase, a specific differentiation state of the cells, or replicating cells only. Inducible systems and inducers are available from many commercial sources including Clontech, Invitrogen and Ariad. There are many other systems that have been described, and they can all be easily selected by an artist with skill in the arts. Examples of inducible promoters regulated by exogenously supplied compounds, include, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin-inducible system. Others types of inducible inducers that may be useful in this context include those that are controlled by a specific physiological condition, such as temperature, acute phase, or in replicating cell only. You can use any type of inducible promotor that is specifically targeted for the target cell type.
Enhancer sequences are another useful regulatory sequence. The invention includes enhancer sequences that are useful for the invention, such as the IRBP enhancer and the immediate early cytomegalovirus enhancementr. One derived from the immunoglobulin gene, SV40 enhancer, and the cis-acting elements identified in the mouse proximal promor. The art also includes other enhancer sequences.
Preferential embodiments include a muscle-specific inducer. A muscle-specific promoter, for example, may increase the expression of a gene within a muscle by at least 2-fold to 3-fold, 4-fold and 5-fold, 10-fold to 20-fold, 15-fold to 20-fold, 30-fold or 50-fold compared with a non-muscle tissue. Examples of muscle-specific promoters are: alpha-actin and cardiac troponin C. Myosin light chains 2A, CK6, dystrophin and muscular creatine kinase. See, for example, U.S. Patent Application 2011/0212529, McCarthy et al., Skeletal Muscle 2:8, 2012; and Wang et al., Gene Ther. 15:1489-1499, 2008.”
“The selection of these and other common regulatory elements is a conventional procedure and there are many sequences. See, e.g. Sambrook et.al., supra, and the references cited therein. Ausubel et.al., Current Protocols in Molecular Biology John Wiley & Sons New York 1989. Not all vectors or expression control sequences are equally capable of expressing all transgenes. One skilled in the art can make a selection from these expression control sequences, or other ones, without departing completely from the scope and intent of the invention.”
“Expression Vectors to Produce Muscle-Specific Recombinant (AAV)”
“The invention contains expression vectors that allow the production of recombinant AAV which are muscle-specific (e.g. viral particles that contain a polynucleotide sequencing comprising a muscle specific promoter and an artificial transcription factors (e.g. a fusion protein, modified transcription factor). The expression vectors SEQ ID Nos. 1 or 2, or SEQ Id NOs. 62-65, can be used in one embodiment to create the recombinant AV. The expression vector in a particular embodiment includes the nucleotide sequence SEQ ID NO.1. This embodiment refers to the expression vector as “muscle AAV?” (?mAAV?). Below are the functional elements of mAAV.
“Located at the 5?” End is the Left Inverted Terminal Repeat sequence of the Adeno-associated Virus (L-ITR), while the 3? End is the Right Inverted Terminal Repeat sequence of the Adeno-associatedvirus. These inverted terminal repeated sequences are well-documented in the literature. They are located in SEQ ID NO.1 between the nucleotide positions 1 bp – 141 bp, (L-ITR), and 2899 bp – 3040 bp(R-ITR).
“Between nucleotide position 156 bp & 2219 bp SEQ ID No:1 there is also transcriptional regulatory area which includes the promoter for the human alpha -actin genes, part the first introns of the human human beta-globin genes, and part the third exons of human beta-globin genes, including the site of splice acceptance.”
“Between nucleotide position 2219 and 2264bp of SEQID NO:1 there’s also a polylinker or multiple cloning station that contains a multitude of restriction enzyme cutting site. This allows you to insert any gene coding for a particular protein into the expression vector.”
“In addition to essential functional elements mentioned above, there may be additional functional elements such as a cap signal or sequence coding for tags (for example Myctag), a polyadenylation consensus, etc.”
“The table below shows the main restriction sites, the polylinker and regulatory region, as well as the elements of the SEQ ID No:1 mAAV expression vector.
“TABLE?1\nFunctional?elements?of?mAAV?expression?vector\nPosition\nMain?functional?elements (bp)\nMain?restriction 2?NotI?sites ?149\nsites SEQ?ID?NO:?27?GCGGCCGC 2899\n1?MluI?site\nSEQ?ID?NO:?28?ACGCGT ?155\nPolylinker SEQ?ID?NO:?7 2219-2264\nATCGATGGGAATTCCGGGATCCGGTCGACCGTACGTACAAGATCT\nRegulatory?region Alpha-actin?promoter?and?part?of?the?intron ?156-2219\ncomprising?the?beta-globin?gene?acceptor\nsplice?donor?site:?SEQ?ID?NO:?29?GCCCAGGTAGGG 1606\nsplice?acceptor?site:?SEQ?ID?NO:?30?CCCACAGCTCCT 2154\nElements?of?the Left-ITR ?? ?1-141\nAAV?vector Right-ITR 2899-3040”
“As mentioned above, the expression vector for the invention can accommodate any sequence coding to any gene of interest. This includes, for example, a gene that codes for a reporter protein, or a gene that codes for a protein to specifically express in skeletal and heart muscle tissue. Artificial transcription factors that increase the expression of the utrophin genes can be used to express specific proteins in skeletal or cardiac muscle tissue.
“The mAAV vector described herein can also be used to create recombinant AAV. This includes viral particles that contain a polynucleotide that contains a muscle-specific enhancer and an artificial transcription factor. The recombinant AAV virus particles are made up of polynucleotides which are portions the mAAV expression sequences. For example, the viral particles can include a polynucleotide sequence defined by SEQ ID NO:83-87, corresponding to portions of mAAV-Vp16-Jazz, mAAV-Vp16-Bagly, mAAV-Vp16-CJ7-UtroUp, mAAV-JZif1, and mAAV-JZif2, respectively. Herein, viral particles are described by the expression vector that was used to produce them and their serotype. The viral particles known as mAAV8/Vp16/Jazz and the mAAV6/Vp16/Jazz viruses have a capsid type 8 and 6 respectively. They can be made using the mAAV/Vp16/Jazz expression vector, methods described herein, or those that are well-known in the art.
“DNA Binding Elements”
“The DNA binding elements of the invention are included in the fusion proteins and modified transcription factors. Preferential embodiments of DNA binding elements include zinc finger motifs which specifically bind to specific DNA sequences. The DNA binding elements in the preferred embodiments specifically bind to sequences within the utrophin?A? promoter. The DNA binding element in the present invention can be isolated or derived from known zinc finger motifs. The preferred zinc finger motif is one that is derived from a Cys2-His2 zinc finger. The zinc finger DNA binding element is possible to be produced or derived from a wild-type zinc finger-containing polypeptide, either by truncation, expansion, or as a variant or wild-type-derived polypeptide through a process called site directed mutationagenesis. U.S. Pat. Nos. 6,242,568, 6,140,466, and 6,140.081. The term “truncated” refers to a shortened version of the original. The term?truncated? refers to a Zinc Finger-nucleotide-binding polypeptide with fewer than the original number of zinc fingers motifs or that has been stripped of undesirable sequences. A polypeptide that has only one to three zinc fingers might be a truncated zinc finger-nucleotide bound protein TFIIIA. This protein naturally contains nine zinc finger motifs. An expansion refers to a polypeptide with zinc fingers that has additional zinc finger motifs. For example, TFIIIA could be extended to 12 fingers with the addition of 3 zinc finger motifs.
“In addition, a Zinc Finger DNA binding element could include zinc finger motifs of more than one wild type protein, which can result in a hybrid? zinc finger polypeptide. Mutagenized is a term that refers to a zinc finger polypeptide. The term “mutagenized” refers to a zinc-finger polypeptide that was obtained using any of the methods known for random or site-directed mutation of the DNA encoding it. TFIIIA, Zif268 are two examples of zinc finger proteins that can easily be truncated or expanded according to the invention to alter the function a zinc finger nucleotide-binding motif. Experts in the art are familiar with other zinc finger-containing nucleotide-binding polypeptides.
“A DNA binding element according to the present invention usually comprises a number of DNA binding domains. The preferred DNA binding domains are zinc finger motifs. Preferably, the DNA-binding element contains between 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these motifs. More preferably, 2 to 6 motifs are preferred and most preferably 3 motifs. Each DNA binding domain is operably linked. One embodiment links the DNA binding domains directly or bonds them together using well-known peptide linkages. Another embodiment links the DNA binding domains using a peptide linking containing between 5 and 50 amino acids. The linker should contain between 5 and 40 amino acids residues, but more preferably between 5 and 30 amino acid residues. It is even better if it contains 5 to 15 amino amino acid residues. Linkers should be flexible. Exemplary linkers can be found in the US Patent Application Publication No. 2007/0020627.”
The DNA binding elements in the invention may be either naturally occurring or not. The art is well-versed in the use of naturally occurring zinc finger DNA binding domains. Preferably, at least one DNA binding area of a current DNA binding element is not naturally occurring. Each of the DNA binding zinc fingers motifs is preferably designed to specifically bind nucleotide targets sequences according to the formula 5-NNN-3?. N is any nucleotide, i.e. A, C or G. These DNA binding domains are well-known in the art. See, e.g., U.S. Pat. Nos. Nos. 6,242,568, 6,140.466 and 6,140.081. Is there a recognized?recognitioncode? A known?recognition code? that links the amino acids of a single motif of zinc fingers to the associated DNA target can be used as a guide in the design of DNA binding elements according to the invention. See, for example, Corbi et al., Biochem Cell Biol. 82:428-36, 2004; Klug, Q. Rev. Biophys. 31(1):1-21, 2010; Pabo et al., Annu. Rev. Biochem. 70:313-340, 2001; Segal et al., Curr. Opin. Biotechnol. 12(6):632-637, 2001; Klug, Annu. Rev. Biochem. 79:213-231 in 2010, and Bhakta et. al., Methods Mol. Biol. 649:3-30, 2010. This code can be used to create modular assemblies of DNA binding elements. For example, it can combine three distinct zinc finger motifs, each capable of recognizing a 3 bp sequence, to produce a three-zinc-finger DNA binding element, which can recognize a specific 9 bp target location. Screening methods and selection strategies can also be used to identify zinc fingers sequences that bind specifically to a particular DNA sequence. These include phage display, yeast two-hybrid system, and bacterial one and two-hybrid system, as well as other methods that are known to those skilled in the art (see Maeder et. al., Mol. Cell. 31:294-301, 2008). Combinations of zinc finger motif motifs that bind with specific DNA sequences can either be obtained commercially or by using the tools provided to you by the Zinc Finger Consortium.
“Structural information regarding known zinc finger motifs can help to design DNA binding elements that bind with a desired sequence. The structure of a three-finger polypeptide-DNA complex, which was derived from Zif268 (also called Krox-24), has been solved using X-ray crystallography. (See Pavletich and al., Science 252:809-817 1991). The fingers contain an anti-parallel beta turn, a finger tip area, and a short amphipathic helix that, in the case Zif268, is bound in the major groove. The structure of each finger domain is also stabilized by the conserved hydrophobic amino acid and zinc coordination by histidine and cysteine residues. Zif268’s crystal structure indicates that DNA recognition is performed by specific histidines (non-zinc-coordinating His residues). The hydrogen bonding of DNA guanines is performed by the charged amino acids located immediately before the alpha-helix, at helix positions 2, 3 and 6. Modifications in the alpha-helix or close to it are more likely than modifications in the framework regions (e.g. beta turns, linker regions) to alter DNA binding specificity. However, they can or might not affect structural integrity (e.g. “Proper folding” of the protein.
The zinc finger DNA binding motif is typically composed of a unique (contiguous sequence 7 amino acids residues) within an alpha-helix. This largely determines the binding specificity to a target molecule. Although the heptameric sequence is possible to be found anywhere in the?-helical domain, it is preferable that the heptamer extends from position?1 through position 6, as the residues are traditionally numbered in this art. One or more modifications to the key amino acids positions (?1,+3, and +6) can allow it to bind the desired sequence of DNA. One or more modifications can be made to the zinc finger alpha helix at other positions to allow it to bind the desired sequence of DNA. Other embodiments allow for modifications to residues that are not part of the heptamer to make the protein bind to the desired sequence. Any?-sheet or framework sequence can be used to form a zinc finger motif. Some embodiments do not modify the?-sheet or framework sequences during modification of a zinc finger domain to bind with a target sequence. A zinc finger motif in a DNA binding element (e.g. in a fusion protein, modified human transcription factor), may have 50-55%, 60%, 70%, 75% and 99% sequence identities to a known Zinc Finger motif. There are only a few amino acid residues that can be modified from the existing zinc finger motif. These modifications can be found in the alpha helix or the amino acid residues around the alpha helix. Modification is not recommended for residues that affect structural integrity or proper 3D protein folding (e.g. zinc coordination). Experts in the field are familiar with methods to determine whether a DNA-binding element can bind to a specific DNA sequence. These include electrophoretic mobility shift (EMSA), chromatin immuneprecipitation(ChIP) and DNase I protectionassays.
“Shown in Table 2, is a comparison between the sequences of zinc finger motif sequences used for the Jazz, Bagly and UtroUp DNA binding components. The invention’s zinc finger motifs have at least 50% sequence identity with any of the zinc fingers motifs listed in Table 2 or SEQ ID NOs 54-60. The invention’s zinc finger motifs have less than 15 amino acids substitutions than any of the zinc fingers motifs in Table 2 or SEQ ID NOs.54-60.
“TABLE?2\nComparison?of?zinc?finger? (ZF)?motif?sequences?of?selected?DNA?binding?elements\nDNA\nBinding # ZF\nElement Motifs ZF?Motif?Sequences DNA?target?sequence\nJazz 3 ZF1:?CPVESCDRRFSRSDELTRHIRIH? (SEQ?ID?NO:?54) GCTGCTGCG\nZF2:?CRICMRNFSSRDVLRRHNRTH? (SEQ?ID?NO:?55) (SEQ?ID?NO:?9)\nZF3:?CDICGRKFASRDVLRRHNRIH? (SEQ?ID?NO:?56)\nBagly 4 ZF1:?CPVESCDRRFSRSDELTRHIRIH? (SEQ?ID?NO:?54) CGGGCTGCTGCG\nZF2:?CRICMRNFSSRDVLRRHNRTH?SEQ?ID?NO:?55) (SEQ?ID?NO:?12)\nZF3:?CDICGRKFASRDVLRRHNRIH? (SEQ?ID?NO:?56) (human)\nZF4:?CAECGKAFVESSKLKRHQLVH? (SEQ?ID?NO:?57) CCGGCTGCTGCG\n(SEQ?ID?NO:?13)\n(mouse)\nUtroUp 6 ZF1:?CPVESCDRRFSRSDNLVRHIRIH? (SEQ?ID?NO:?58) GCTGCTGCGGG-\nZF2:?CRICMRNFSRSDHLTTHNRTH? (SEQ?ID?NO:?59) CTGGGAG\nZF3:?CDICGRKFADPGHLVRHNRIH? (SEQ?ID?NO:?60) (SEQ?ID?NO:?14)\nZF4:?CPVESCDRRFSRSDELTRHIRIH?(SEQ?ID?NO:?61)\nZF5:?CRICMRNFSSRDVLRRHNRTH?(SEQ?ID?NO:?55)\nZF6:?CDICGRKFASRDVLRRHNRIH? (SEQ?ID?NO:?56)\n\nFusion Proteins”
“The fusion proteins discussed herein typically contain between 30 and 40, 50, 60 or 70, 80, 90 to 100, 200, 300 to 400, 500 to 600, 700, 800, 700, 900, 900, 1000, or more amino acids. People of ordinary skill in this field will understand that polypeptides are also possible to be prepared using other methods, such as recombinant techniques or synthesis. Sambrook et. al., supra, provides examples of proper cloning, sequencing, and instructions that can be used to guide persons with skill through many cloning activities. Information about products from manufacturers of experimental equipment and biological reagents, such as SIGMA Chemical Company (Saint Louis), Mo. ), and New England BioLabs, Ipswich (Mass.) They also offer useful information in biological methods. This brief description provides an overview of different recombinant polypeptide manufacturing methods applicable to certain embodiments.
“The polypeptides discussed herein are derived mainly from transcriptional activation and DNA binding elements. It is known the nucleotide sequences of many transcriptional activator and DNA binding elements. The nucleic acids sequences of many transcriptional activator elements and DNA binding elements are known. The transcriptional activation element for the fusion protein can be selected from any of the following: a group that includes but is not limited to the acidic or hydrophobic activation sites (e.g. from Gal4 and Gcn4), nine-amino?acid transactivationdomains (9aaTAD), e.g. from p53 and Vp16, E2A and NFIL6 and NF?B), p65, SP1, Zif268, Vp65, Vp65, MLL and the human Che-1/AATF. Some embodiments use physiological regulators of the expression of utrophin, such as GABP?, NFAT?, and GABP??. A fusion protein may have multiple transcriptional activation element, such as Vp16 or CJ7 in some embodiments.
Summary for “Compositions of treatment for muscular dystrophy”
“The invention concerns compositions that include modified human transcription factor, recombinant Adeno-associated virus vectors (AAVs), comprising modified human transcript factors, and methods to treat muscular defects.”
“Duchenne’s muscle dystrophy (DMD), is a severe Xlinked, degenerative condition characterized by the absence or dystrophin cytoskeletal protein. Dystrophin protein is responsible for stabilizing the sarcolemma, i.e. the cell membrane of muscle cells. It links the intracellular cytoskeletal system to the extracellular matrix. Dystrophin is absent, and muscle contraction can mechanically stress the cell membrane, causing progressive damage to myofibers. Initial damage to skeletal muscles is primarily caused by the disease. However, the damage spreads to the cardiac muscle, and often death occurs from cardiac failure. DMD is one the most common genetic disorders, with an estimated 1 in 3600 male births affected each year. DMD is a debilitating condition that gradually worsens over the 25-year lifespan of those who are affected. Despite years of research, there is no cure for muscular dystrophies, including DMD. The current therapies only manage symptoms.
“Designing new therapies for DMD and related dystrophinopathies has focused on functionally salvaging the missing or defective dystrophin proteins. Therefore, compositions that increase utrophin expression are needed that are less immunogenic and more effective therapeutically.
“The invention includes a modified transcription element that contains at least one first, two second, and three zinc fingermotifs. The transcription factor can increase utrophin expression in cardiac or skeletal muscle tissue. The transcription factor may also include a fourth, fifth, sixth, seventh, eighth, or ninth zinc finger motif in some embodiments. Each zinc finger motif in some embodiments includes an alpha-helix. In some embodiments, transcription factors are derived from genomically encoded human transcription factors. Zif268 is the genomically encoded transcription factor in some embodiments.
“In one embodiment, the first motif of zinc can contain a sequence with at least 80 sequence identity for SEQID NO:48. The second motif of zinc can contain a sequence with at least 80% identity to the SEQID NO:49 and the third motif of zinc can contain a sequence with at least 80% identity to the SEQID NO:50.
“In the second embodiment, the first motif of the zinc fingers can include the SEQID NO:48, while the second can include the SEQID NO:49 and the third can include the SEQID NO:50.”
“In a third embodiment, a transcription factor may include a sequence with at least 95% sequence ID to SEQ ID No: 38. The transcription factor can contain: i. a first zilfinger motif that includes an alpha helix and an Arg sequence at position 1, a Glu residue in position 3, and a Glu residue in position 6, and an Asn sequence at location 8, and ii. a second zinc-finger motif including an Alpha-helix that contains a Ser at the position 1 and Arg at 6 and Arg at 5 and Arg at positions 6, an Arg at 6, an Asn at 8 and an Asn at 9
“In a fourth embodiment, a first zinc finger motif may include a sequence with at least 80 sequence identity. The second zinc finger motif could include a series with at least 80% of sequence identity. SEQID NO:52 can also be used. Third zinc finger motif: A sequence that has at least 80% identity to the SEQID NO:53 can be used.
“In a fifth embodiment the first zinc finger motif may include SEQID NO:51, while the second can include the SEQID NO:52 and the third can include the SEQID NO:53.”
“In a sixth embodiment, a transcription factor may include a sequence with at least 95% sequence ID to SEQ ID No: 39. The transcription factor may include: i. a first zinc-finger motif that includes an alpha helix and an Asn residue. Position 3, a Val residue 5 and position 6, and an Asn residue 8 at positions 3 and 6. Position ii. A second zinc-finger motif that contains an alpha helix and an Asp residue. Position?1, Arg at 6 and an Asn at 6, an Asn at 8.
“Some embodiments of the invention allow the transcription factor to include an amino acid sequence SEQ ID No: 38 or 39. Another embodiment of the invention states that the transcription factor is not immunogenic when it is expressed in humans. Another embodiment of the invention is capable to increase muscle contractile strength. This can be measured in in vivo or ex vivo assays. A further embodiment of the transcription factor can increase muscle endurance. This is done using a forced exercise test. Some aspects of this embodiment increase muscle endurance by at least 10% relative to reference.
“The invention also includes a recombinant Adeno-associated Vector (AAV), which allows expression of a gene within skeletal or cardiac muscles tissue. It includes a muscle-specific promor and any of these transcription factors. The gene may be expressed in both skeletal and cardiac muscles tissue, depending on the embodiment. Some embodiments of the muscle-specific inducer are constitutively expressed during differentiation. Certain embodiments include alpha-actin and cardiac troponin B, myosin light chains 2A, myosin lightchain 2A, and skeletal beta-actin.
“In one embodiment, the vector may have a serotype from any of the following: AAV1, AAV2, and AAV3, as well as AAV4, AAV5, or AAV6, AAV7, or AAV8. The vector may be muscle tropic in some instances. The vector may have an AAV6 or 8 serotype. Another embodiment of the vector may include at least one element from the following groups: an inverted terminal repeat (or AAV6), a cap signal (or AAV8), an intron donor site, an Intron splice acceptor site, an epitope tags, a nuclear sequence, and a polyadenylation consensus sequencing. Another embodiment of the vector contains the sequence of SEQID NO:86 and SEQID NO:87. The transcription factor binds specifically to the promoter of utrophin gene in any embodiment of the invention. promoter. The human or mouse utrophin?A is used in certain aspects of this embodiment. Promoter contains the sequence of SEQID NO:9, 10 or 11 or SEQID NO:40.
“The invention also includes a composition that contains any of the above vectors and a pharmaceutically approved carrier. The invention also includes a method for treating a muscle disorder in a subject who is in need of it, which involves administering an effective amount the previous composition. The invention also includes a method for treating a muscular disease. This method involves contacting the utrophin genes of muscle cells with any of the above modified transcription factors. The contacting can result in an increase or decrease in muscle endurance, depending on the embodiment.
“In some embodiments the composition can be administered locally or systemically. Other embodiments of the composition can be administered intramuscularly or intravenously, subcutaneously or intraperitoneally. The treatment can increase muscle endurance or muscle contractile force in any of the methods described above. The invention can treat any type of muscle disease. This includes Duchenne’s Muscular Dystrophy and Becker’s Muscular Dystrophy.
“DEFINITIONS”
“Administering” is the term used herein. “Administering” is a way of administering a dose of a composition to a subject (e.g., a composition containing a recombinant vector AAV vector encoding a polynucleotide encoding an fusion protein or modified transcript factor capable of increasing the expression of utrophin). You can administer the compositions described in this article by any of the following routes: parenteral (e.g. intravenous, intraperitoneal), dermal or transdermal, oral, buccal, sublingual and perilingual; nasal, rectal; topical; and oral. These compositions can be administered either locally or systemically using the methods described. Depending on the treatment being used and the component of the composition, the preferred method of administration may vary.
“?Amino acid sequence? As used herein, an oligopeptide or peptide or polypeptide or protein sequence and fragments or portions thereof as well as naturally occurring or synthetic molecules.
“By ?cardiac muscle? The form of striated tissue found in the heart that is controlled by the autonomic nervous systems, i.e. it is involuntarily controlled, is called “cardiac muscle”. Cardiomyocytes and myocardiocytes are the cells that make up cardiac muscle. The heart is a large organ made up mainly of cardiac muscle and connective tissues.
“By modified transcription factor?” A transcription factor which is substantially derived form a genomically-encoded transcription gene. Modified transcription factors can also be obtained from transcription factors that are known to the art, such as zinc finger transcription factors. A transcription factor, such as a human transcription factors, can have its zinc finger motifs replaced, modified or engineered to alter the target sequence it recognizes and binds. Modified transcription factors that are derived from human transcription factor genes are called?modified human transcript factors? herein. Zif268, also known by the?Early Growth Response Protein 1?, is an example human zinc finger transcription factor. ?Egr1,? ?Egr1? Zif268 encodes a protein that belongs to the EGR family, which includes three-zinc finger-containing proteins of C2H2-type. Zif268 functions as a nuclear protein and transcriptional regulator. Zif268’s principal isoform contains 543 amino acid with a molecular mass of 57.5kDa. It binds to the sequence 5?-GCGTGGGCG-3. (SEQ ID NO. 44). SEQ ID NO. 41 shows an exemplary sequence of amino acids for human Zif268. You can find an exemplary sequence of mRNA for human Zif268 under NCBI Accession No. NM_001964 and in SEQ ID N:19.
“As used herein.?conservative variants? Or?conservative modified versions? A particular sequence is a sequence of amino acids encoded using nucleic acids that encode identical or essentially the same amino acid sequences. Or where the nucleic Acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy in the genetic code, many functionally identical nucleic acid sequences may encode any given protein. These nucleic acids variations can be called silent variations. They are one type of conservatively modified variants. Any one with skill will be able to recognize that any codon in a nucleic acids (except AUG which is the codon for methionine) may be modified using standard techniques to produce a functionally identical molecule. Each silent variant of a nucleic acids that encodes a specific peptide is implicitly included in the amino acid sequence. One of the most common modifications is conservatively modified versions. These variations result in the substitution or deletion of an amino acid by a chemically identical amino acid. The art is well-versed in the use of conservative substitution tables that provide functionally identical amino acids. Each of the following six groups contains amino acids that can be used as conservative substitutes: 1) Alanine, Serine, and Threonine; 2) Aspartic Acid (D), Glutamic (E); 3) Asparagine [N], Glutamine [Q]); 4) Arginine(R), Lysine (10), 5) Isoleucine (1), Leucine (5), Methionine (6M), Valine (10); 6) Phenylalanine (7), Tyrosine (W), and Tyrosine (Y), and Tryptophan (W);
“By ?contact? “By?contact? as used herein, is intended to permit or promote a state or combination of at least two elements. The fusion protein or modified transcript factor of the invention can be used to contact the utrophin genes. This binds specifically to a target sequence within the utrophin?A?. This will increase the expression of utrophin.
As used herein, “a?deletion?” refers to a change of either amino acid sequence or nucleotide sequence that results in one or more amino acids or nucleotide symposia being absent.
“By DNA binding element?” An element capable of binding DNA. A DNA binding element can bind to a specific sequence or response element of a particular gene, such as in a promoter. Examples of DNA binding elements include, but are not limited: domains that contain zinc finger motif, helix/turn helix domains and domains that contain winged helix motifs, winged leucine zipper domains and domains that include winged helix turns helix domains. HMG box domains and domains for Wor3 and immunoglobulin domains. TAL effector DNA binding domains. RNA-guided DNAbinding domains. ZFP51. The ZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZFP1 and JZZZZZZZZZZZZZZZZZZZ.
“By an effective amount of a compound? The amount of a compound administered to improve, inhibit or alleviate a condition or symptom of disorder or disease. Treatment is achieved if there is any improvement in the subject. A sufficient amount to treat is a dose that decreases, inhibits or prevents one or more of the symptoms of DMD. It is also a dose that lessens or stops the severity or duration of symptoms. The effective dosage of the pharmaceutical composition to treat DMD depends on the method of administration, as well as the patient’s age, weight and general health. The appropriate dosage and frequency can be determined by a physician or researcher.
“expression vectors” is the term used herein. “expression vectors” or?expressionplasmids, as well as similar terms, are DNA sequences required to clone copies of genes and/or translate their mRNAs in a suitable host. These vectors can be used for the expression of viral, prokaryotic or eukaryotic gene in a variety hosts, including bacteria, E.coli, blue-green alga, plant cells and insect cells as well as fungal and yeast cells.
“For the purposes of this article, the term “fusion protein” is used. A compound that contains all or part of the amino acids sequences of more than one protein. These modified transcription factors, such as modified human transcription factors, are not included in the definition of fusion protein. A fusion protein may include a DNA binding element, such as Jazz, Bagly or UtroUp, and one or more transcriptional activation factors (e.g. Vp16 or CJ7 or SP1, or Gal4). The terms “protein” and “polypeptide?” are interchangeable. The terms?protein? und?polypeptide are interchangeable herein. These terms are interchangeable herein. The term “portion” is used interchangeably herein. includes any region of a polypeptide, such as a fragment (e.g., a cleavage product or a recombinantly-produced fragment) or an element or a domain (a region of a polypeptide having an activity, such as, e.g., enzymatic activity or antigen- or DNA-binding capacity) that contains fewer amino acids than the full-length polypeptide (e.g., 5%, 10%, 12%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% fewer). One or more linkers may be included in a fusion protein to connect the amino acid sequences. The terms “portion” and “fragment” are well-known to those who have studied the art. The terms?portion? und?fragment are also used interchangeably in the art. These words can also be used interchangeably in this article.
“By ?host,? ?subject,? or ?patient,? Any organism is meant, including a mammal, primate, human, dog, cat and cow, as well as any animal such a fish or bird. “A host can also refer to a domestic animal (e.g. a farm animal), or a companion animal, such as a pet.
“By ?immunogenicity? “Immunogenicity” refers to the ability of a specific substance (e.g. an antigen, epitope or protein) to trigger an immune response in the human or animal’s body. A cell-mediated or humoral immune response can be included in an immune response. Treatment with therapeutic proteins, such as artificial transcription factors, is generally not recommended because patients may develop or have antibodies that bind to the therapeutic protein. This can lead to the inactivation of therapeutic proteins and sometimes adverse side effects. The level of immunogenicity can be affected by many factors, including the delivery route, the delivery vehicle, the dose regiment, the dosage regiment, aggregation and innate immune system activation, molecular structure, protein structure, degradationability and the ability of a protein to interface with humoral (B) and cellular (T) responses.
“Increasing muscle contractile strength” means: This refers to increasing the force exerted by a muscle contraction by at least 5% or 10%, 15% or 20%, 25% or 40%, 50% or 60%, 70% or 80%, 90% or 100%, 150% or 3-fold, 4-fold or 5-fold, respectively, as compared with reference. In vivo, ex situ or ex vivo measurements of the force generated by a muscle are possible. For example, ex vivo or in-situ analysis of the contractile profiles of one intact limb muscle (e.g. The extensor digitalorum longus, abdominal muscles, and the tibialis anterior muscle are used for ex-vivo assays. Grip force analysis, downhill treadmill exercise (manual muscle testing), myometry (e.g. Myometer to assess upper and lower extremity strength, including evaluations of knee flexors, extensors as well as elbow flexors. Shoulder abductors can also be evaluated. Assays for sustained maximum voluntary contraction (MVC), or any of the other assays described by Hakim et. al., Methods Mol. Biol. 709: 75-89, 2011; Sharma et al., Neurology 45: 306-310, 1995; and McDonald et al., Muscle Nerve 48: 343-356, 2013.”
“Increased muscle endurance” means: It refers to increasing the endurance of a muscle group or muscle (e.g. a decrease of at least 1%, 10%, 15% or 20%), over a prolonged period of time (e.g. hours (e.g. 1, 2, 3, 4, 6, 7, 8, 9, 10, hours), days (e.g. 1, 2, 3, 5, 6, 7, 7, 8, 9, 10), weeks (e.g. 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, or 12 months. 1, 2, 3, 5, 6, 7, 8, 10, 11 or 12 months. Assays that measure muscle endurance include treadmill exercise, 6-minute walk test (6MWT), and timed function tests (e.g. time taken for a person to stand up from a supine or to walk 10 m or climb/descend 4 standard-sized steps). These tests can be used to determine muscular endurance or strength in mice. Disord. 22(2):170-182, 2012) and at accelerated speeds (see Di Certo et. al., Hum. Mol. Genet. 19:752-760, 2010 or Strimpakos et al., J. Cell. Phys. 229:1283-1291 (2014). Voluntary wheel exercise, grip strength, the hang wire, inverted grid, and the rotarod tests or any of the assays described by McDonald et al. Muscle Nerve 48: 343-356 (2013).
“Increasing the utrophin expression?” This is to increase utrophin gene expression and/or protein activity compared with a normal or positive tissue or cell. An expression product can be anRNA transcribed directly from the gene (e.g. An mRNA or a polypeptide derived from an mRNA from the gene can be used as an expression product. An increase in mRNA levels will usually result in an increase of the polypeptides translated from it. The level of expression and/or activity may be determined using standard techniques for detecting and measuring mRNA or protein, including but not limited to RT-PCR, Western blotting, enzyme-linked immunosorbant assay (ELISA), immunohistochemistry, immunofluorescence, and mass spectrometry.”
“An ?insertion? “An?insertion? or?addition? As used herein, it refers to any change in an amino or nucleotide sequence that results in the addition of one (or more) amino acids or nucleotide atoms.
“The terms ‘linker,? ?linker region,? or ?linker domain,? These terms are used to describe elements located between adjacent polynucleotide and polypeptide sequences. When constructing a cloning vector, or fusion protein, you can introduce stretches of polynucleotide sequence or polypeptide sequence. Chemical conjugation can also introduce linkers, such as?click-chemistry?-based approaches. A linker region may be used to introduce cloning sites in the nucleotide sequence or a flexible component or region-creating region between two proteins domains or create an affinity tag for specific molecular interactions. As a result of recombinant nuclear acid production, a linker region can be added to a fusion proteins.
“By ?muscle-specific promoter? Any sequence of DNA that promotes the expression of a particular gene in muscle tissue is called a “muscle-specific promoter”. A muscle-specific promoter will typically be found upstream from the transcription initiation site for a gene. A muscle-specific promoter, for example, may increase the expression of a gene within a muscle by at least 2-fold and 3-fold, 4-folds, 5-folds, 10-folds, 15-folds, 20-folds, 30-folds, 50-folds, 50-folds, 100-folds, or more than a non-muscle tissue. Some muscle-specific genes are expressed during differentiation. Examples of muscle-specific promoters are: alpha-actin and cardiac troponin C. Myosin light chains 2A, skeletal beta -actin. CK6. Dystrophin. Synthetic C5-12 (Syn), Myf5, MyoD1, MyoD1, Pax7. See, for example, U.S. Patent Application 2011/0212529, McCarthy et al., Skeletal Muscle 2:8, 2012; and Wang et al., Gene Ther. 15:1489-1499 (2008), the entire of which are incorporated by reference.
“By ?muscular dystrophy? “Muscular dystrophy” refers to a group of diseases that affect the muscles and cause impairments in movement. Muscular dystrophies can cause progressive weakness, defects in muscle proteins, death of tissue and cells, as well as a loss of muscle function. Dystrophinopathy is a term that describes a variety of muscle diseases, including dystrophinopathy. This includes dystrophinopathy which refers to a range of muscle diseases where there is not enough dystrophin protein in the muscles cells. This causes instability in the structure and membrane of the muscle cells. Dystrophinopathies can also include Duchenne’s muscular disorder (DMD), Becker’s muscle dystrophy, and Benign pseudohypertrophic mild dystrophy (BMD). DMD and BMD can be X-linked recessive disorders caused by mutations of the dystrophin gene. This gene encodes the protein dystrophin. DMD is more severe that BMD. This is because in DMD there is no dystrophin protein produced, while in BMD dystrophin is. Congenital muscular disorders include facioscapulohumeral muscle dystrophy and limb-girdle muscle dystrophy.
“?Nucleic acid sequence? “?Nucleic acid sequence” is the term used herein to refer to an oligonucleotide or nucleotide or polynucleotide and any fragments or portions thereof.
“As used herein the term ‘operable linkage? Or?operable linkage? A physical or functional combination of components described above that allows them to work in their intended way. For example, two DNA sequences that are operably linked mean that they are arranged (cis- or trans) so that at least one sequence can exert a physiological influence on the other. A muscle specific promoter, for example, can be operably linked to a gene in order to promote the gene’s muscle-specific transcription.
“By ?pharmaceutical composition? “Pharmaceutical composition” means any composition that contains a therapeutically and biologically active agent. This includes any composition that contains a nucleic acid molecule that encodes all of the fusion protein, which is a transcriptional activation molecule, and a DNA binding element. It can also include any composition that can be administered to a subject. You can administer the compositions described in this article by any route you choose, including parenteral (e.g. intravenous or transdermal), dermal, transdermal and ocular, buccal, sublingual. perilingual. nasal, rectal. topical. These compositions can be administered either locally or systemically using the methods described. Parenteral is a term that refers to the administration of the compositions described herein. The term “parenteral” as used herein means subcutaneous, supracutaneous, intermuscular, intraperitoneal and intramuscular injections, as well any other infusion techniques. Sterile injectable compositions can be either a solution or suspension in an acceptable non-toxic solvent or diluent. These solutions include, among others, 1,3-butanediol and mannitol. Fixed oils can also be used as solvents or suspending media (e.g. synthetic mono- and diglycerides). Injectables can be prepared using fatty acids such as oleic acid or its glyceride derivatives. Natural pharmaceutically acceptable oils such as castor oil and olive oil, polyoxyethylated, are also useful. These oil solutions and suspensions can also contain long-chain alcohol diluents or dispersants such as carboxymethylcellulose or similar dispersing agents. You can also use other commonly used surfactants such as Tweens or Spans, or similar emulsifying agent or bioavailability boosters, that are used to make pharmaceutically acceptable solids, liquids, and other dosage forms. These formulations can all be prepared using well-known, accepted art methods. For example, Remington: Science and Practice of Pharmacy (21st edition). ), ed. A. R. Gennaro Lippincott Williams & Wilkins 2005 and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick Informa Healthcare 2006, which are each hereby incorporated by reference.
“pharmaceutically acceptable excipient, carrier or adjuvant” A diluent or excipient, carrier, adjuvant, or adjuvant that is physiologically acceptable for the subject and retains the therapeutic properties of its pharmaceutical composition is considered to be acceptable. Formulations can be prepared for injection in either liquid or solid forms. Acceptable carriers, excipients, or stabilizers for intravenous administration are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN?, PLURONICS?, or polyethylene glycol (PEG). Water, saline and dextrose are all acceptable excipients. These compositions can also contain non-toxic auxiliary substances like pH buffering agents, wetting agents, pH buffering, and so on. One skilled in the art can also identify other physiologically acceptable excipients or carriers and their formulations.
“By?recombinant-adenoassociated vector (AAV),” A recombinantly generated virus or viral particle is one that contains a polynucleotide. It can be delivered to a host cell in vivo, ex-vivo, or in vitro. AAV is a human parvovirus that is non-pathogenic and is used in gene transfer in mammals. AAV’s genome is single-stranded DNA. It contains inverted terminal repeats at both ends of DNA strands and two open reading frames, rep and cap. These are responsible for coding replication and capsid protein. The native rep and cap genes can be replaced by a foreign polynucleotide. You can make AAVs with many different serotype capsids that have different transduction profiles. for different tissue types. There are many AAV serotypes, including AAV1, AAV2, and AAV3, AAV4, AV5, AAV6, AV7, AV8, AAV9, as well as AAVrh10. Triple transfection of subconfluent HeK293 cells can produce AAV vectors. AAV cis-plasmid contains the gene of interest and AAV trans-plasmid contains AAV rep, cap and other genes. An adenovirus helper virus plasmid is, for instance, pDF6. SEQ ID Nos. 1 or 2, or SEQID NOs. 62-65, can all be used to produce recombinant Adeno-associated Vectors (e.g. viral particles that contain nucleotide sequences that enable muscle-specific expressions of artificial transcription factors). Herein, viral particles are described by the expression vector that was used to produce them and their serotype. The viral particle mAAV8Vp16Jazz, for example, has a capsid serotype 8, and is produced using the expression vector mAAVVp16Jazz.
“By ?reference? Any sample, standard or level used for comparison purposes. A “normal reference sample” is a sample taken from the same subject before the onset of a disorder (e.g., a muscular dystrophy), if it has been successfully treated for that condition or not. A?normal reference sample? can be any sample taken from the same subject before the onset of the disorder (e.g., muscular dystrophy), or from a patient who has been treated successfully for the disorder or disease. What is meant by “reference level”? A reference sample is a number or value that has been calculated from another. A normal reference level or standard can be any value or number that is derived from a normal subject and that matches a sample of the subject by at least one criteria: age, weight or disease stage. A “positive reference”? A sample, standard or value is a sample or standard derived from a subject who is known to have an illness (e.g., muscular dystrophy). It is matched to a sample by at least one of these criteria: weight, age, disease stage and overall health.
“By ?skeletal muscle? The form of striated tissue that is controlled by the somatic nervous systems, i.e. it is voluntary controlled, is called skeletal muscle. Multiple bundles of muscle fibers are called muscle. They are held together by connective tissues. Tendons can be used to attach skeletal muscles to bone. Skeletal muscles can be attached to bone by tendons. The following skeletal muscles are examples: the diaphragm; extensor digitalorum longus; tibialis posterior, gastrocnemius; soleus.
“By ?specifically binds? “By?specifically binds?” A molecule (e.g. an artificial transcription factor) that recognizes and binds another molecular (e.g. a polynucleotide) but does not substantially recognize or bind other molecules. An artificial transcription factor binds to a specific DNA sequence in the utrophin A? The promoter does not bind to other polynucleotide combinations. The dissociation constant (Kd) can be used to represent the binding affinity of a molecule for its partner Y. The term “specific binding” is also known as: The term?specific binding? refers to a specific binding. Is it?specific for? A molecule can be shown to be specific for a particular molecule (e.g., polynucleotide, polypeptide) by having a Kd of the molecule it binds of at most 10/3 M, 5/6 M and 10/7 M. ?Binding affinity? “Binding affinity” generally refers to how strong the sum of all noncovalent interactions between a single binding location of a molecule (e.g. a protein fusion) and its binding partner.
“Substantially the same” is a term that refers to sequences that are substantially identical. When used in this context with regard to the comparison between a sequence and a reference sequence, it applies to sequences that are at most about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, or at the very least about 99% sequence identity to their reference sequence. Standard methods and algorithms can be used to determine percent identity between sequences, including BLASTN (NCBI, Schaffer et. al., Nucleic Acids Res. 29:2994-3005, 2001), BLASTX (NCBI; Schaffer et al., Nucleic Acids Res. 29:2994-3005 2001, ALIGN(GCG, Accelrys), FASTA (Pearson et al. Proc. Natl. Acad. Sci. U.S.A. 85.2444-2448 (88) programs that may use default settings. The invention includes amino acid sequences that, in various examples, are identical to reference sequences.
“Substitution” as it is used herein refers to the substitution of one or more nucleotides or amino acids by other amino acids, or nucleotides.
“By ?transcriptional activation element? A polypeptide capable of initiating transcription is a polypeptide that can make an RNA transcript (e.g. an mRNA) using a DNA template. This is done by RNA polymerase. A transcriptional activation element could include a trans-activation region of a transcription factor protein, or other domains that directly or indirectly promote transcription. You can also find transcriptional activation elements that are polypeptides or fragments of them. These include transcriptional coactivators, transcriptional coregulators (e.g. TAF9 and MED15), transcriptional coactivators (e.g. CBP/p300), transcriptional coactivators (e.g. transcriptional keygulators, transcriptional de-acetylases or histonemethylases), chromatin remodelers, kinases or DNA methylases to promote transcription. Transcriptional activation element may recruit transcriptional inhibitors or proteins that promote transcription. To bring a transcriptional activation element into close proximity to DNA, it usually requires a DNA binding element. Examples of transcriptional activation elements are: hydrophobic or acidic activation domains (e.g. from Gal4 and Gcn4, respectively), nine-aminoacid transactivation Domains (9aaTAD), e.g. from p53 and Vp16. MLL, E2A and NF?B), p65, SP1, Zif268, the trans-activation domain (CJ7) derived from human Che-1/AATF (see, for example, BMC Mol. Biol. 14:3, 2013). For example, see US Patent Application Publication No. 2007/0020627 and Blancafort et al., Mol. Pharmacol. 66(8): 1361-137, 2004. Transcriptional activation element can also be derived or include physiological regulators of Utrophin expression such as GABP, NFAT? and GABP?. SEQ ID No:37 shows an example sequence of Vp16 amino acids, while SEQ ID No:75 shows an exemplary sequence of CJ7 amino acids.
“By ?transcription factor? A protein that binds with specific DNA sequences and controls transcription of DNA to messenger RNA (mRNA) is called a “transcription factor”. This activity can be performed by transcription factors either alone or in combination with other proteins. They promote or block the recruitment of RNA Polymerase. By ?artificial transcription factor? Any transcription factor that is not found in nature is referred to as an “artificial transcription factor”. A fusion protein, or modified transcription factor can be an artificial transcription factor.
“By ?treating? “Treating” is the act of administering a pharmaceutical composition according to the invention for therapeutic and/or prophylactic purposes. For example, prophylactic treatment can be given to a patient who is not yet sick but is at risk for a specific muscle disease or defect. For example, DMD treatment can be given to a patient who is already experiencing symptoms. According to the claims and embodiments, treating refers to the administration of medication to a subject for either therapeutic or prophylactic reasons. Sometimes, treatment can be used to treat a disorder (e.g. DMD) or a symptom. This may reduce the severity or progression of the disorder or decrease the frequency or progression of symptoms by, for example, 1% or 2%, 10%, 20% or 30%. One can use electromyography (EMG), genetic testing, muscle biopsy, serum Creatine Kinase levels, muscular strength tests (e.g. manual muscle testing), and range-of motion (ROM) tests like the six-minute walk test to measure symptoms of DMD. DMD can cause symptoms that range from mild to severe. It is possible to have DMD symptoms, depending on the location of the disease and the causative mutation. A successful treatment will show an improvement or absence in one or more of the symptoms of muscular dystrophy.
“By ?utrophin ?A? promoter? or ?utrophin promoter ?A?? The promoter region located at 5? The utrophin gene is located in the upstream region. The utrophin A? The promoter is located in an unmethylated CpG islands and is active within muscle cells. This is contrary to the utrophin B? This promoter is located immediately upstream from the large second exon utrophin. It is active in endothelial cell cells. A sequence that is representative of human utrophin?A? SEQ ID No:20 defines the promoter. It can also include sequences with SEQ ID NOS:9, 10, 12 or 14. A sequence that is representative of mouse utrophin?A? SEQ ID No:33 defines the promoter or may include sequences with SEQ ID NOs 11-13 or 15.
“By ?zinc finger motif? This is a protein structural motif. Zinc finger motifs are a group of motifs that coordinate with one or more zincions to stabilize the fold. A zinc finger DNA binding domain can contain 1, 2, 3, 4, 5, 6 7, 8, 9, and more zinc finger motifs, arranged in tandem arrays that can bind to the major groove of DNA. The Cys2-His2 zinc fingers, which are composed of 28-31 amino acid that fold into a???, is a well-known zinc finger motif. structure. The Cys2-His2 type zinc fingers motif typically has a sequence of the form X3-Cys-X2-4-Cys-X12-His-X3-6-His-X4, wherein X is any amino acid (e.g. X2-4 is an oligopeptide of 2-4 amino acids. The sequence variation of 28-31 amino acid sequences of the zinc finger polypeptide is quite wide. Invariant are only the consensus histidine and consensus cysteine residues that are bound to the central Zinc atom. Three to five of the remaining residues are highly conserved while there is considerable variation among them. The alpha-helix for each motif (often called?recognition Helix?) The sequence-specific contact of a motif’s alpha-helix to DNA bases can be made. Reactants from one recognition helix usually contact three base pairs of DNA. Modifications to the positions of key amino acids (e.g. positions?1, +3, or +6) in the zinc finger alpha helix can alter the DNA binding target specificity for a zinc-finger motif. See, for example, Corbi et al., Biochem. Cell. Biol. 82:428-436, 2004; Klug, Q. Rev. Biophys. 43:1-21, 2010; Choo et al., Curr. Opin. Struct. Biol. 7:117-125, 1997; Pabo et al., Annu. Rev. Biochem. 70:313-340 2001; Segal et. al., Curr. Opin. Biotechnol. 12:632-637 2001; and Sera. Drug Delivery Rev. 61:513-526, 2009. Hugo Gene Nomenclature Commission (HGNC) has a list of human CystHist type zinc fingers proteins. Cys2-His2 type zinc finger proteins often contain an effector domain located N-terminally to the zinc finger region, such as the KRAB (Kruppel-Associated-Box), SCAN (SRE-ZBP, CTfin51, AW-1 and Number18 cDNA) and BTB (Broad-Complex, Tramtrack and Bric-a-bric) effector domains. Another example of zinc finger motifs are the Gag knuckle and Treble clef.
“Other features or advantages of the invention will become apparent from the detailed description and the claims.”
Dystrophinopathies, including DMD and BMD, are caused by dystrophin protein deficiency in muscle cells. Utrophin can be considered dystrophin’s autosomal homologue, as the two proteins share many of the same structural and functional motifs. Dystrophin (and utrophin) function as connecting proteins between the cell membrane, cytoskeletal actin and the extracellular matrix via proteins collectively known as DAPs (dystrophin-associated proteins). Utrophin is most abundant in the fetus, and its expression is decreased after delivery. Dystrophin is more prevalent after birth. Adults have utrophin that is localized at the neuromuscular junction. Dystrophin, however, is found along the length of the Sarcolemma. Transgenic mdx mice have been used as a model animal for DMD. They show that utrophin overexpression is associated with redistribution across the entire sarcolemma. This results in a marked improvement of dystrophic phenotype.
The present invention aims to up-regulate, i.e. increase, the dystrophin-related genes utrophin expression in DMD patients’ muscle to functionally rescue (i.e. to complement) dystrophin function. You can do this by contacting the Utrophin gene using a modified human transcription element capable of increasing utrophin. The modified transcription factors of this invention have the advantage of reducing immunogenicity after being introduced to a subject. The present invention provides muscle-specific, recombinant human transcription factor vectors that can promote the expression of modified human transcription factors in both skeletal and cardiac muscle. This is useful in the treatment of DMD, which has severe skeletal and cardiac muscle disorders. The vectors of this invention have shown that AAV delivery of modified human transcription factor (recombinant) significantly improves dystrophic phenotype in mice suffering from mdx dystrophin deficiency. This includes improvements in both muscle strength and endurance. The compositions and methods described in the invention can be used to treat DMD, particularly muscle defects.
“AAV Vectors & Compositions”
“In some embodiments of the invention, a nucleic acids sequence or fragment encoding a gene (e.g., an Artificial transcription Factor (e.g. a Fusion Protein or Modified transcription Factor) capable of increasing Utrophin expression is delivered to muscle cells via a viral vector. Many viruses are known and readily available in the art. Preferably, the artificial transcription factors are delivered to both cardiac and skeletal muscle. The therapeutic vector should be non-toxic and minimally immunogenic. It should also be easy to create and deliver DNA into target cells. The viral vector can be a recombinant Adeno-associated Vector (AAV) in certain embodiments. Other embodiments of the invention provide a therapeutic composition that includes an AAV including an artificial transcription factor, such as a fusion protein, or modified transcription factor, under the control a muscle-specific promor.
AAVs are a popular method of exogenous DNA delivery. They are relatively safe, efficient, and easy to optimize for specific purposes. There are more than 30 AAV types that naturally occur. There are many natural variants of the AAV capsid that allow identification and use an AAV with specific properties for muscle cells. Conventional molecular biology techniques can be used to engineer AAV viruses. This allows for optimization of these particles for cell-specific delivery of nucleic acids sequences, for immunogenicity minimization, tuning stability, particle lifetime, and efficient degradation. Human serotype 2, which was the first AAV to be isolated from human or nonhuman primates (NHP), is a gene transfer vector. It has been extensively used in gene transfer experiments in various target tissues and animal models. Other AAV serotypes include AAV1, AAV3, and AAV4, as well as AAV5, AAV6, the AAV7, AAV8, and AAV9, among others. For a detailed discussion on AAV serotypes, see International Patent Application WO 2005/033321.
“In many embodiments, artificial transcription factors (or other genes) can be expressed in muscle cells by delivery via recombinantly-engineered AAVs. These AAVs may contain nucleic acids sequences encoding the artificial transcription factors or genes that have been operably linked with muscle-specific promoters. These AAVs can be used to treat muscle defects in certain preferred embodiments.
US Patent Application Publication No. 2013/0136729 describes muscle-specific expression vectors. It also describes the combination AAV9 with muscle-specific CK6 enhancer as a highly efficient system for systemically carrying genes into ischemic skeletal muscles. The combination of AAV9 with the cardiac troponinC promoter is described in this publication as a system that is especially effective in cardiomyocytes, skeletal myocytes and those with ischemic injury. International Patent Application (WO 2005/118611) describes microutrophin delivery via adenoassociated vectors in the mouse model of mdx dystrophic. It also includes incorporation of muscle-active promoters such as dystrophin promoters for skeletal?-actin, myosin light chains 2A promoters and muscular creatinekinase kinase promoters.
“Expression vectors to induce exogenous genes, such as the zinc finger transcription factors, in muscle and non-muscle tissues are also described in U.S. Pat. No. No. 8,304,235 describes the artificial transcription factor (UTroUp) (encoding a 6-zinc finger repeat that is designed to specifically bind to the utrophin promoterA) fused to the transcriptional activationdomain?Vp16). From the Herpes virus under the control regulatory sequences of Cytomegalovirus (CMV), and its expression via the eukaryotic vector, pRK5(Clontech), Italian Patent Application (RM2005A000493). This describes the artificial transcription factor Vp16Jazz and its expression via the vector vector pMEX. It is controlled by the muscle-specific promoter/enhancer region of the murine meosin light chain gene (MLC). These expression vectors allow the exogenous gene to be expressed in either skeletal or cardiac muscle tissues. However, it is not possible to express them both. This invention allows exogenous genes to be expressed in both the skeletal and cardiac muscles.
The cap proteins (including vp1,vp2, and vp3) are desirable AAV fragments that can be assembled into vectors. Also, hypervariable regions and the rep proteins (rep78, rep68 and rep52), and sequences encoding these proteins are important. These fragments can be used in many vector systems and host cell types. These fragments can be used alone or together with fragments from other AAV serotypes or AAV viral sequences. Artificial AAV serotypes can include AAVs with a non-naturally occuring capsid protein, as described herein. An artificial capsid can be created using any suitable technique. It may be made from a selected AAV sequence, such as a fragment from a vp1 capid protein, in combination with heterologous sequences that may be obtained from different AAV serotypes, non-contiguous parts of the same AAV Serotype, from non-AAV virus sources, or from non-viral sources. A pseudotyped AAV capid, an AAV capsid chimeric, an AAV capsid recombinant, or a humanized AAV capid are all possible options. AAV capsid. The invention makes use of pseudotyped vectors in which the capsid from one AAV is replaced by a heterologous proteins capsid.
“In one embodiment, vectors that are useful in compositions or methods described herein include sequences that encode a selected AAV serotype capid, e.g. an AAV8 capsid or a fragment thereof. Another embodiment contains sequences that encode a select AAV serotype rep proteins, such as the AAV8 rep protein or a fragment thereof. These vectors can contain AAV rep and cap proteins. Vectors that contain both AAV rep or cap can have both AAV rep or cap sequences. However, they do not need to be both of the same serotype origin. Other embodiments of the vectors that are useful in compositions or methods described herein include sequences encoding AAV6 capsids, or a fragment thereof. Some embodiments include sequences that encode an AAV6 rep protein or a fragment thereof. In some other embodiments, both the AAV rep or cap sequences are included in vectors.
Vectors can also be used in which rep sequences come from AAV serotypes that are different from the one providing the cap sequences. One embodiment expresses the rep and cap sequences from different sources (e.g. separate vectors or a host cell and vector). These rep sequences can be fused together in frame to cap sequences from a different AAV serotype in another embodiment to create a chimeric vector. No. No. 7,282,199, which can be incorporated herein by reference.”
A suitable recombinant AAV can be created by cultivating a host cell that contains the following: a nucleic acids sequence encoding the AAV serotype capsid proteins, or a fragment thereof; a functional rep genes; a transgene sequence comprising AAV inverted termin repeats (ITRs), and a muscle-specific promor sequence; sufficient helper functions to allow packaging the transgene into the AAV capsid proteins. Trans may be used to provide the components that must be grown in the host cell to pack an AAV minigene into an AAV capid. A stable host cell may also contain the components required to package an AAV minigene in an AAV capsid. The present invention allows for the production of AAV vectors by triple transfection of subconfluent HeK293 cells with three plasmids. AAV cis-plasmid contains the gene of interest, AAV Trans-plasmid contains AAV rep, cap, and an Adenovirus Helper plasmid such as pDF6.
“A stable host cell could contain the necessary component(s) when it is under the control an inducible inducer. The constitutive promoter may control the necessary component. In the discussion below, we provide examples of inducible and suitable constitutive promoters. Another alternative is to have a select stable host cell contain certain components that are under the control a constitutive and other components that are under the control a one or more of the inducible promotors. A stable host cell can be created from 293 cells, which contain E1 helper function under the control a constitutive inducer. However, it may also contain the rep and/or caps proteins under the control inducible promoters. Another method of creating stable host cells is to use one skilled in the art.
“The transgene and any rep sequences, caps sequences, or helper functions necessary for the production of the recombinant aAV of the invention can be delivered to the packaging host cells in the form of any gene element that transfers the sequences. Any suitable method can be used to deliver the selected genetic element, including those described in this document. Anyone skilled in nucleic acids manipulation will be familiar with the methods that were used to create any embodiment of this invention. These include genetic engineering, synthetic techniques, and recombinant technology. Sambrook et. al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press. Cold Spring Harbor, N.Y. Similar methods for generating recombinant AAV viruses are well-known and it is not the limitation of the invention to choose a suitable method. See, e.g., Fisher et al., J. Virol. 70:520-532 1992, and U.S. Pat. No. No. 5,478,745, and other. These publications are included herein as a reference.”
“Unless otherwise stated, the AAVITRs and other selected components of AAVs described herein may be easily selected from any AAV serotype including AAV1, AAV2, and AAV3, and AAV4, and AAV5, and AAV6, and AAV7, and AAV8, and AAV9, and AAV10, as well as other unknown and known AAV serotypes. These ITRs and other AAV components can be easily isolated using techniques that are available to skilled art practitioners from any AAV serotype. These AAV can be obtained from commercial, academic, or public sources (e.g. American Type Culture Collection Manassas Va.). Alternately, AAV sequences can be obtained by synthetic or other suitable methods, using published sequences, such as those found in literature, PubMed, GenBank, and the like.
“A transgene according to the invention includes, for example, an artificial transcript factor (e.g. a fusion factor or modified transcription factor), nucleic acid sequence (e.g. Vp16 -Jazz or Vp16 -Bagly or Vp16 -CJ7 -UtroUp or JZif1) or any other gene that is desired to be delivered directly to muscles as described above and its regulatory sequences and 5? 3. AAV inverted terminal repetitions (ITRs) One desirable embodiment uses the ITRs from AAV serotype 8. You can also choose ITRs from other serotypes. This transgene is then packaged in a capsid protein, and delivered to a chosen host cell.
The regulatory sequences are conventional control elements that can be linked to the transgene in such a way as to permit its transcription, translation, and/or expression in cells transfected or infected by the invention’s virus. The expression control sequences include sequences which regulate transcription initiation, termination and promoter, as well as RNA processing signals like splicing (polyA) signals and splicing (polyA); sequences to stabilize cytoplasmic mRNA; splicing and polyadenylation signals (i.e. Kozak consensus sequence); and sequences that increase protein stability. They also contain sequences that confer nuclear location (e.g. nuclear localization sequences). When desired, they can enhance the secretion. There are many expression control sequences that have been developed and can be used. Many species are suitable for poly A sequencing, including humans, SV-40 and bovines.
“Regulators useful in the constructs described in the present invention could also include an intron. This intron is desirable to be located between the promoter/enhancer and the gene. A 100 bp intron sequence, derived from SV-40 is referred to as SD?SA. It is a mini-intron splice donater/splice taker. The preferred embodiment of the intron includes the first intron from the human alpha-actin genes, which also includes the splice donor sites. It also includes part of both the second intron and third exons of the human betaglobin genes, which include the splice acceptance site.
“In other embodiments, recombinant AAV Vectors may contain additional functional elements such as cap signals, sequences for epitope tags (e.g. haemmaglutinin, myc tag and maltose binding protein (MBP), tag, green fluorescent pro (GFP) or other fluorescent proteins). Multicloning sites, and/or multicloning locations that contain multiple restriction enzyme cutting sites. This allows it to insert any gene coding a particular protein into the vector. The sequence of amino acids for an exemplary myctag is shown in SEQ ID No:75.
“The promoter that will be used in the recombinant AAV can be chosen from a variety of constitutive, induceable, cell-type or tissue-specific promoters that are capable of expressing the transgene in the desired muscles cells. The promoter must be muscle-specific in a preferred embodiment. Particularly preferred embodiments have the promoter specific to expression of the transgene within skeletal and cardiac muscles cells.
“The promoter(s), used in the present invention, may be derived any species. Due to limitations in the AAV vector’s size, one embodiment of the promoter is small, less than 1000 bp. Another embodiment has a promoter that is less than 400 bp. The invention includes, but is not limited to, the retroviral Rous sarcomavirus (RSV), LTR promoter (optionally containing the RSV enhancer), cytomegalovirus, (CMV), promoter (optionally containing the CMV enhancer), SV40 promoter and the dihydrofolate reductase promor, as well as the EF1 promoter from Invitrogen, the phosphoglycerol kinase kinaserol kinasa promoter.
“Inducible promotors which can be used with the present invention can regulate gene expression. They can be controlled by exogenously supplied chemicals, environmental factors like temperature or the presence of a certain physiological state, such as acute phase, a specific differentiation state of the cells, or replicating cells only. Inducible systems and inducers are available from many commercial sources including Clontech, Invitrogen and Ariad. There are many other systems that have been described, and they can all be easily selected by an artist with skill in the arts. Examples of inducible promoters regulated by exogenously supplied compounds, include, the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system and the rapamycin-inducible system. Others types of inducible inducers that may be useful in this context include those that are controlled by a specific physiological condition, such as temperature, acute phase, or in replicating cell only. You can use any type of inducible promotor that is specifically targeted for the target cell type.
Enhancer sequences are another useful regulatory sequence. The invention includes enhancer sequences that are useful for the invention, such as the IRBP enhancer and the immediate early cytomegalovirus enhancementr. One derived from the immunoglobulin gene, SV40 enhancer, and the cis-acting elements identified in the mouse proximal promor. The art also includes other enhancer sequences.
Preferential embodiments include a muscle-specific inducer. A muscle-specific promoter, for example, may increase the expression of a gene within a muscle by at least 2-fold to 3-fold, 4-fold and 5-fold, 10-fold to 20-fold, 15-fold to 20-fold, 30-fold or 50-fold compared with a non-muscle tissue. Examples of muscle-specific promoters are: alpha-actin and cardiac troponin C. Myosin light chains 2A, CK6, dystrophin and muscular creatine kinase. See, for example, U.S. Patent Application 2011/0212529, McCarthy et al., Skeletal Muscle 2:8, 2012; and Wang et al., Gene Ther. 15:1489-1499, 2008.”
“The selection of these and other common regulatory elements is a conventional procedure and there are many sequences. See, e.g. Sambrook et.al., supra, and the references cited therein. Ausubel et.al., Current Protocols in Molecular Biology John Wiley & Sons New York 1989. Not all vectors or expression control sequences are equally capable of expressing all transgenes. One skilled in the art can make a selection from these expression control sequences, or other ones, without departing completely from the scope and intent of the invention.”
“Expression Vectors to Produce Muscle-Specific Recombinant (AAV)”
“The invention contains expression vectors that allow the production of recombinant AAV which are muscle-specific (e.g. viral particles that contain a polynucleotide sequencing comprising a muscle specific promoter and an artificial transcription factors (e.g. a fusion protein, modified transcription factor). The expression vectors SEQ ID Nos. 1 or 2, or SEQ Id NOs. 62-65, can be used in one embodiment to create the recombinant AV. The expression vector in a particular embodiment includes the nucleotide sequence SEQ ID NO.1. This embodiment refers to the expression vector as “muscle AAV?” (?mAAV?). Below are the functional elements of mAAV.
“Located at the 5?” End is the Left Inverted Terminal Repeat sequence of the Adeno-associated Virus (L-ITR), while the 3? End is the Right Inverted Terminal Repeat sequence of the Adeno-associatedvirus. These inverted terminal repeated sequences are well-documented in the literature. They are located in SEQ ID NO.1 between the nucleotide positions 1 bp – 141 bp, (L-ITR), and 2899 bp – 3040 bp(R-ITR).
“Between nucleotide position 156 bp & 2219 bp SEQ ID No:1 there is also transcriptional regulatory area which includes the promoter for the human alpha -actin genes, part the first introns of the human human beta-globin genes, and part the third exons of human beta-globin genes, including the site of splice acceptance.”
“Between nucleotide position 2219 and 2264bp of SEQID NO:1 there’s also a polylinker or multiple cloning station that contains a multitude of restriction enzyme cutting site. This allows you to insert any gene coding for a particular protein into the expression vector.”
“In addition to essential functional elements mentioned above, there may be additional functional elements such as a cap signal or sequence coding for tags (for example Myctag), a polyadenylation consensus, etc.”
“The table below shows the main restriction sites, the polylinker and regulatory region, as well as the elements of the SEQ ID No:1 mAAV expression vector.
“TABLE?1\nFunctional?elements?of?mAAV?expression?vector\nPosition\nMain?functional?elements (bp)\nMain?restriction 2?NotI?sites ?149\nsites SEQ?ID?NO:?27?GCGGCCGC 2899\n1?MluI?site\nSEQ?ID?NO:?28?ACGCGT ?155\nPolylinker SEQ?ID?NO:?7 2219-2264\nATCGATGGGAATTCCGGGATCCGGTCGACCGTACGTACAAGATCT\nRegulatory?region Alpha-actin?promoter?and?part?of?the?intron ?156-2219\ncomprising?the?beta-globin?gene?acceptor\nsplice?donor?site:?SEQ?ID?NO:?29?GCCCAGGTAGGG 1606\nsplice?acceptor?site:?SEQ?ID?NO:?30?CCCACAGCTCCT 2154\nElements?of?the Left-ITR ?? ?1-141\nAAV?vector Right-ITR 2899-3040”
“As mentioned above, the expression vector for the invention can accommodate any sequence coding to any gene of interest. This includes, for example, a gene that codes for a reporter protein, or a gene that codes for a protein to specifically express in skeletal and heart muscle tissue. Artificial transcription factors that increase the expression of the utrophin genes can be used to express specific proteins in skeletal or cardiac muscle tissue.
“The mAAV vector described herein can also be used to create recombinant AAV. This includes viral particles that contain a polynucleotide that contains a muscle-specific enhancer and an artificial transcription factor. The recombinant AAV virus particles are made up of polynucleotides which are portions the mAAV expression sequences. For example, the viral particles can include a polynucleotide sequence defined by SEQ ID NO:83-87, corresponding to portions of mAAV-Vp16-Jazz, mAAV-Vp16-Bagly, mAAV-Vp16-CJ7-UtroUp, mAAV-JZif1, and mAAV-JZif2, respectively. Herein, viral particles are described by the expression vector that was used to produce them and their serotype. The viral particles known as mAAV8/Vp16/Jazz and the mAAV6/Vp16/Jazz viruses have a capsid type 8 and 6 respectively. They can be made using the mAAV/Vp16/Jazz expression vector, methods described herein, or those that are well-known in the art.
“DNA Binding Elements”
“The DNA binding elements of the invention are included in the fusion proteins and modified transcription factors. Preferential embodiments of DNA binding elements include zinc finger motifs which specifically bind to specific DNA sequences. The DNA binding elements in the preferred embodiments specifically bind to sequences within the utrophin?A? promoter. The DNA binding element in the present invention can be isolated or derived from known zinc finger motifs. The preferred zinc finger motif is one that is derived from a Cys2-His2 zinc finger. The zinc finger DNA binding element is possible to be produced or derived from a wild-type zinc finger-containing polypeptide, either by truncation, expansion, or as a variant or wild-type-derived polypeptide through a process called site directed mutationagenesis. U.S. Pat. Nos. 6,242,568, 6,140,466, and 6,140.081. The term “truncated” refers to a shortened version of the original. The term?truncated? refers to a Zinc Finger-nucleotide-binding polypeptide with fewer than the original number of zinc fingers motifs or that has been stripped of undesirable sequences. A polypeptide that has only one to three zinc fingers might be a truncated zinc finger-nucleotide bound protein TFIIIA. This protein naturally contains nine zinc finger motifs. An expansion refers to a polypeptide with zinc fingers that has additional zinc finger motifs. For example, TFIIIA could be extended to 12 fingers with the addition of 3 zinc finger motifs.
“In addition, a Zinc Finger DNA binding element could include zinc finger motifs of more than one wild type protein, which can result in a hybrid? zinc finger polypeptide. Mutagenized is a term that refers to a zinc finger polypeptide. The term “mutagenized” refers to a zinc-finger polypeptide that was obtained using any of the methods known for random or site-directed mutation of the DNA encoding it. TFIIIA, Zif268 are two examples of zinc finger proteins that can easily be truncated or expanded according to the invention to alter the function a zinc finger nucleotide-binding motif. Experts in the art are familiar with other zinc finger-containing nucleotide-binding polypeptides.
“A DNA binding element according to the present invention usually comprises a number of DNA binding domains. The preferred DNA binding domains are zinc finger motifs. Preferably, the DNA-binding element contains between 2, 3, 4, 5, 6, 7, 8, 9 or 10 of these motifs. More preferably, 2 to 6 motifs are preferred and most preferably 3 motifs. Each DNA binding domain is operably linked. One embodiment links the DNA binding domains directly or bonds them together using well-known peptide linkages. Another embodiment links the DNA binding domains using a peptide linking containing between 5 and 50 amino acids. The linker should contain between 5 and 40 amino acids residues, but more preferably between 5 and 30 amino acid residues. It is even better if it contains 5 to 15 amino amino acid residues. Linkers should be flexible. Exemplary linkers can be found in the US Patent Application Publication No. 2007/0020627.”
The DNA binding elements in the invention may be either naturally occurring or not. The art is well-versed in the use of naturally occurring zinc finger DNA binding domains. Preferably, at least one DNA binding area of a current DNA binding element is not naturally occurring. Each of the DNA binding zinc fingers motifs is preferably designed to specifically bind nucleotide targets sequences according to the formula 5-NNN-3?. N is any nucleotide, i.e. A, C or G. These DNA binding domains are well-known in the art. See, e.g., U.S. Pat. Nos. Nos. 6,242,568, 6,140.466 and 6,140.081. Is there a recognized?recognitioncode? A known?recognition code? that links the amino acids of a single motif of zinc fingers to the associated DNA target can be used as a guide in the design of DNA binding elements according to the invention. See, for example, Corbi et al., Biochem Cell Biol. 82:428-36, 2004; Klug, Q. Rev. Biophys. 31(1):1-21, 2010; Pabo et al., Annu. Rev. Biochem. 70:313-340, 2001; Segal et al., Curr. Opin. Biotechnol. 12(6):632-637, 2001; Klug, Annu. Rev. Biochem. 79:213-231 in 2010, and Bhakta et. al., Methods Mol. Biol. 649:3-30, 2010. This code can be used to create modular assemblies of DNA binding elements. For example, it can combine three distinct zinc finger motifs, each capable of recognizing a 3 bp sequence, to produce a three-zinc-finger DNA binding element, which can recognize a specific 9 bp target location. Screening methods and selection strategies can also be used to identify zinc fingers sequences that bind specifically to a particular DNA sequence. These include phage display, yeast two-hybrid system, and bacterial one and two-hybrid system, as well as other methods that are known to those skilled in the art (see Maeder et. al., Mol. Cell. 31:294-301, 2008). Combinations of zinc finger motif motifs that bind with specific DNA sequences can either be obtained commercially or by using the tools provided to you by the Zinc Finger Consortium.
“Structural information regarding known zinc finger motifs can help to design DNA binding elements that bind with a desired sequence. The structure of a three-finger polypeptide-DNA complex, which was derived from Zif268 (also called Krox-24), has been solved using X-ray crystallography. (See Pavletich and al., Science 252:809-817 1991). The fingers contain an anti-parallel beta turn, a finger tip area, and a short amphipathic helix that, in the case Zif268, is bound in the major groove. The structure of each finger domain is also stabilized by the conserved hydrophobic amino acid and zinc coordination by histidine and cysteine residues. Zif268’s crystal structure indicates that DNA recognition is performed by specific histidines (non-zinc-coordinating His residues). The hydrogen bonding of DNA guanines is performed by the charged amino acids located immediately before the alpha-helix, at helix positions 2, 3 and 6. Modifications in the alpha-helix or close to it are more likely than modifications in the framework regions (e.g. beta turns, linker regions) to alter DNA binding specificity. However, they can or might not affect structural integrity (e.g. “Proper folding” of the protein.
The zinc finger DNA binding motif is typically composed of a unique (contiguous sequence 7 amino acids residues) within an alpha-helix. This largely determines the binding specificity to a target molecule. Although the heptameric sequence is possible to be found anywhere in the?-helical domain, it is preferable that the heptamer extends from position?1 through position 6, as the residues are traditionally numbered in this art. One or more modifications to the key amino acids positions (?1,+3, and +6) can allow it to bind the desired sequence of DNA. One or more modifications can be made to the zinc finger alpha helix at other positions to allow it to bind the desired sequence of DNA. Other embodiments allow for modifications to residues that are not part of the heptamer to make the protein bind to the desired sequence. Any?-sheet or framework sequence can be used to form a zinc finger motif. Some embodiments do not modify the?-sheet or framework sequences during modification of a zinc finger domain to bind with a target sequence. A zinc finger motif in a DNA binding element (e.g. in a fusion protein, modified human transcription factor), may have 50-55%, 60%, 70%, 75% and 99% sequence identities to a known Zinc Finger motif. There are only a few amino acid residues that can be modified from the existing zinc finger motif. These modifications can be found in the alpha helix or the amino acid residues around the alpha helix. Modification is not recommended for residues that affect structural integrity or proper 3D protein folding (e.g. zinc coordination). Experts in the field are familiar with methods to determine whether a DNA-binding element can bind to a specific DNA sequence. These include electrophoretic mobility shift (EMSA), chromatin immuneprecipitation(ChIP) and DNase I protectionassays.
“Shown in Table 2, is a comparison between the sequences of zinc finger motif sequences used for the Jazz, Bagly and UtroUp DNA binding components. The invention’s zinc finger motifs have at least 50% sequence identity with any of the zinc fingers motifs listed in Table 2 or SEQ ID NOs 54-60. The invention’s zinc finger motifs have less than 15 amino acids substitutions than any of the zinc fingers motifs in Table 2 or SEQ ID NOs.54-60.
“TABLE?2\nComparison?of?zinc?finger? (ZF)?motif?sequences?of?selected?DNA?binding?elements\nDNA\nBinding # ZF\nElement Motifs ZF?Motif?Sequences DNA?target?sequence\nJazz 3 ZF1:?CPVESCDRRFSRSDELTRHIRIH? (SEQ?ID?NO:?54) GCTGCTGCG\nZF2:?CRICMRNFSSRDVLRRHNRTH? (SEQ?ID?NO:?55) (SEQ?ID?NO:?9)\nZF3:?CDICGRKFASRDVLRRHNRIH? (SEQ?ID?NO:?56)\nBagly 4 ZF1:?CPVESCDRRFSRSDELTRHIRIH? (SEQ?ID?NO:?54) CGGGCTGCTGCG\nZF2:?CRICMRNFSSRDVLRRHNRTH?SEQ?ID?NO:?55) (SEQ?ID?NO:?12)\nZF3:?CDICGRKFASRDVLRRHNRIH? (SEQ?ID?NO:?56) (human)\nZF4:?CAECGKAFVESSKLKRHQLVH? (SEQ?ID?NO:?57) CCGGCTGCTGCG\n(SEQ?ID?NO:?13)\n(mouse)\nUtroUp 6 ZF1:?CPVESCDRRFSRSDNLVRHIRIH? (SEQ?ID?NO:?58) GCTGCTGCGGG-\nZF2:?CRICMRNFSRSDHLTTHNRTH? (SEQ?ID?NO:?59) CTGGGAG\nZF3:?CDICGRKFADPGHLVRHNRIH? (SEQ?ID?NO:?60) (SEQ?ID?NO:?14)\nZF4:?CPVESCDRRFSRSDELTRHIRIH?(SEQ?ID?NO:?61)\nZF5:?CRICMRNFSSRDVLRRHNRTH?(SEQ?ID?NO:?55)\nZF6:?CDICGRKFASRDVLRRHNRIH? (SEQ?ID?NO:?56)\n\nFusion Proteins”
“The fusion proteins discussed herein typically contain between 30 and 40, 50, 60 or 70, 80, 90 to 100, 200, 300 to 400, 500 to 600, 700, 800, 700, 900, 900, 1000, or more amino acids. People of ordinary skill in this field will understand that polypeptides are also possible to be prepared using other methods, such as recombinant techniques or synthesis. Sambrook et. al., supra, provides examples of proper cloning, sequencing, and instructions that can be used to guide persons with skill through many cloning activities. Information about products from manufacturers of experimental equipment and biological reagents, such as SIGMA Chemical Company (Saint Louis), Mo. ), and New England BioLabs, Ipswich (Mass.) They also offer useful information in biological methods. This brief description provides an overview of different recombinant polypeptide manufacturing methods applicable to certain embodiments.
“The polypeptides discussed herein are derived mainly from transcriptional activation and DNA binding elements. It is known the nucleotide sequences of many transcriptional activator and DNA binding elements. The nucleic acids sequences of many transcriptional activator elements and DNA binding elements are known. The transcriptional activation element for the fusion protein can be selected from any of the following: a group that includes but is not limited to the acidic or hydrophobic activation sites (e.g. from Gal4 and Gcn4), nine-amino?acid transactivationdomains (9aaTAD), e.g. from p53 and Vp16, E2A and NFIL6 and NF?B), p65, SP1, Zif268, Vp65, Vp65, MLL and the human Che-1/AATF. Some embodiments use physiological regulators of the expression of utrophin, such as GABP?, NFAT?, and GABP??. A fusion protein may have multiple transcriptional activation element, such as Vp16 or CJ7 in some embodiments.
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