US 7,863,017 B2Grant
TAT-utrophin as a protein therapy for dystrophinopathies
Issue Date:2011-01-04
•35 Claims
•12 Drawing Sheets
Abstract
Disclosed is a fusion protein including a full-length TAT-utrophin or an anti-dystrophinopathic fragment thereof, a method of treating dystrophinopathies (including Duchenne muscular dystrophy) using the fusion protein, a pharmaceutical composition for treating dystrophinopathies in mammals comprising the fusion protein, and nucleic acid constructs for expressing the fusion protein.
Metadata
Assignee
- Wisconsin Alumni Research Foundation
Inventors
- James M. Ervasti
- Kevin J. Sonnemann
Application Information
Application Number:US 11/998,798
Filing Date:2007-11-30
Priority Date:2006-12-01
Art Unit:1649
Classifications
IPC:
C07K19/00C12N15/62
Patent Drawings (12 sheets)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] Priority is hereby claimed to provisional application Ser. No. 60/868,119, filed Dec. 1, 2006, which is incorporated herein by reference.
FEDERAL FUNDING STATEMENT
[0002] This invention was made with United States government support awarded by the following agency: NIH Grant AR042423. The United States goverment has certain rights in this invention.
FIELD OF THE INVENTION
[0003] The invention is directed to a fusion protein comprising a full-length TAT-utrophin or an anti-dystrophinopathic fragment thereof, a method of treating dystrophinopathies (including Duchenne muscular dystrophy) using the fusion protein and a pharmaceutical composition for treating dystrophinopathies in mammals comprising the fusion protein.
BACKGROUND
[0004] Duchenne muscular dystrophy (DMD) is the most prevalent and severe form of human muscular dystrophy. DMD occurs with an incidence of 1 in 4000 male births. Onset of DMD is typically between 3 and 6 years of age with skeletal muscle weakness preferentially affecting the large proximal muscle groups. The disease is invariably progressive, leading to loss of ambulation by 11 to 13 years, and death typically in the 20's. Significant laboratory findings include grossly elevated serum CK-MM levels. Skeletal muscle biopsy samples reveal a dystrophic pattern of muscle degeneration and regeneration with fiber-size variation, increased central nuclei, and progressive interstitial fibrosis.
[0005] Becker muscular dystrophy (BMD) was long considered to be a potentially allelic disorder because of its clinical similarities to DMD and a common pattern of X-linked inheritance. The shared genetic basis for DMD and BMD was confirmed after the identification of the protein dystrophin; both DMD and BMD patients were shown to have dystrophin gene mutations. Typically, patients with DMD lack any detectable dystrophin expression in their skeletal muscles, and this is correlated with deletion mutations that disrupt the translational reading frame or point mutations that create stop codons. In contrast, muscle from patients with BMD contains mutated dystrophins having an altered size and/or reduced abundance secondary to deletion mutations that maintain the reading frame.
[0006] While clinical descriptions of DMD date back to the 1850's, over 100 years passed before evidence suggested that the muscle cell plasma membrane, or sarcolemma, is compromised in DMD muscle. The molecular basis for DMD and its associated sarcolemmal instability became more clear with landmark studies published in the mid-to-late 1980's which identified the gene encoding dystrophin as being defective in DMD (O'Brien and Kunkel, 2001). The DMD locus spans over 2.5 million bases distinguishing it as the largest gene in the human genome. The array of transcripts expressed from the DMD gene is complex due to the presence of multiple promoters and alternative splicing. The largest transcripts encode dystrophin, a four-domain protein with a predicted molecular weight of 427,000. Dystrophin is the predominant DMD transcript expressed in striated muscle. DMD gene mutations, deletions, or duplications most frequently result in a loss of dystrophin expression in muscle of patients afflicted with DMD. Based on its localization to the cytoplasmic face of the sarcolemma, and its sequence similarity with domains/motifs common to proteins of the actin-based cytoskeleton, dystrophin was hypothesized early on to play a mechanical role in anchoring the sarcolemma to the underlying cytoskeleton It has also been hypothesized that dystrophin plays a role in protecting the sarcolemma against stress imposed during muscle contraction or stretch.
[0007] Biochemical studies aimed at confirming the hypothesized structure and function of dystrophin revealed its tight association with a multi-subunit complex, the so-named dystrophin-glycoprotein complex. See FIG. 1 , which is a schematic representation showing the sarcolemma and the interaction of dystrophin with the other elements of the dystrophin-glycoprotein complex. Through its cysteine-rich and C-terminal domains, dystrophin in striated muscle interacts with the integral membrane dystroglycan sub-complex and the sarcoglycan/sarcospan sub-complex, as well as the subsarcolemmal dystrobrevins and syntrophins (Cohn and Campbell, 2000; Blake et al., 2002). The N-terminal domain and a portion of middle rod domain of dystrophin act in concert to effect an extensive lateral association with actin filaments in vitro (Rybakova et al., 1996) and in vivo (Rybakova et al., 2000; Warner et al., 2002; Rybakova and Ervasti, 1997; Amann et al., 1998; Amann et al., 1999).
[0008] Utrophin is a widely expressed autosomal gene product with high sequence similarity to dystrophin (Tinsley et al., 1992). Utrophin is distributed throughout the sarcolemma in fetal and regenerating muscle, but is down-regulated in normal adult muscle and is restricted to the myotendinous and neuromuscular junctions (Blake et al., 1996). Because utrophin and dystrophin bind the same complement of proteins (Matsumura et al., 1992; Kramarcy et al., 1994; Winder et al., 1995), it was hypothesized that utrophin may be capable of compensating for dystrophin deficiency. Indeed, continued utrophin expression in adult mdx mice partially attenuates the phenotype associated with dystrophin deficiency. In short, mice lacking both dystrophin and utrophin exhibit a more severe phenotype similar to that seen in human DMD patients (Deconinck et al., 1997a; Grady et al., 1997). Moreover, transgenic overexpression of full-length utrophin completely rescued the dystrophic phenotype in mdx mice (Tinsley et al., 1998).
[0009] Methods to express and purify full-length utrophin using a baculovirus system has been demonstrated (Rybakova et al., 2002 and 2006). It has also been shown that purified recombinant utrophin is a soluble, rod-shaped monomer with the expected molecular weight of 400,000 Da. Recombinant utrophin-bound actin filaments display an affinity (Kd=0.2 μM) similar to that measured for purified dystrophin-glycoprotein complex (Rybakova et al., 2002). Recombinant utrophin-bound F-actin displays a stoichiometry of 1 utrophin per 14 actin monomers, which implies a more extensive lateral association with actin filaments than anticipated from studies with isolated fragments, but a less extensive lateral association than the 1 per 24 stoichiometry measured for purified recombinant dystrophin (Rybakova et al., 2006). Like the dystrophin-glycoprotein complex, recombinant utrophin protected actin filaments from forced depolymerization in a concentration-dependent manner that saturated at molar ratios equal to or greater than 1 utrophin per 14 actin monomers. Also different from purified dystrophin-glycoprotein complex, the binding of recombinant utrophin to actin filaments was completely insensitive to increasing ionic strength up to 0.8 M. These results (Rybakova et al., 2002) (Rybakova et al., 2006) indicate that dystrophin and utrophin both bind laterally alongside actin filaments through contributions by the spectrin-like repeats of the rod domain, but that the rod domain epitopes involved differ between the two proteins. Utrophin appears to bind laterally along actin filaments through a contribution of the first 10 acidic spectrin-like repeats (Rybakova et al., 2002) rather than a cluster of basic repeats as employed by dystrophin (Rybakova et al., 1996; Amann et al., 1998); (Rybakova et al., 2006).
[0010] Most viruses, including the human immunodeficiency viruses (HIV), encode proteins for regulating genome transcription. In HIV, the tat gene plays a role in driving the transcription of the HIV genetic code. The tat gene encodes a small nuclear protein of from 86 to 101 amino acids, depending upon the viral strain. Both the tat gene and its encoded protein, TAT, are known. The protein itself is designated TAT, for “transactivator protein.” The typical HIV-1 laboratory strains HXB2 and NL4-3 express an 86 amino acid-long TAT protein, while other HIV strains express a 101 amino acid-long TAT protein. See, for example, Kuppuswamy et al., 1989.
[0011] Despite all that is now known, and despite continuing efforts by many laboratories around the world (Gregorevic and Chamberlain, 2003), there is presently no cure or effective treatment to alleviate the devastating progression of DMD.
SUMMARY OF THE INVENTION
[0012] The primary object of the present invention is a method of treating dystrophinopathies in mammals, including humans. The method comprises administering an anti-dystrophinopathic-effective amount of a chimeric protein (i.e., a fusion protein) encoding TAT-utrophin. The chimeric protein is administered in an amount effective to transduce skeletal muscle cells and thereby to correct the pathologies associated with dystrophin deficiency. The chimeric protein may comprise a full-length TAT protein (e.g., 86 amino acids long or 101 amino acids long) or a fragment thereof, such as the HIV-1 TAT protein transduction sequence (see SEQ. ID. NO: 5). Similarly, the chimeric protein may comprise a full-length utrophin protein or an anti-dystrophinopathic-effective fragment thereof. (For purposes of brevity, both full-length and fragmented versions of the chimeric protein will be referred to herein as the “TAT-utrophin chimeric (or fusion) protein.”) Utrophin fragments can be evaluated for their anti-dystrophinopathic effects by transgenically over-expressing the putative anti-dystrophinopathic fragment in mdx mice in the same fashion as Tinsley et al., 1998 and observing whether the dystrophic phenotype in the mdx mice is ameliorated or eliminated. Alternatively, the TAT-utrophin chimeras can be tested on mdx mice as described herein below for their anti-dystrophinopathic efficacy.
[0013] The invention is also directed to a baculovirus construct that drives the expression of the TAT-utrophin chimeric protein, the chimeric protein encoding TAT-utrophin itself, as well as a pharmaceutical composition for treating dystrophinopathies that comprises an anti-dystrophinopathic amount of the TAT-utrophin chimeric protein in combination with a pharmaceutically suitable carrier.
[0014] Thus, one version of the invention is directed to a fusion protein comprising a first protein region which is effective to transduce the fusion protein into mammalian muscle cells. The first protein region preferably comprises an HIV TAT protein or a transduction-effective fragment thereof. The first protein region is operationally linked to a second protein region comprising a full-length utrophin protein or an anti-dystrophinopathic fragment thereof. Also included within the invention are pharmaceutically suitable salts of the fusion proteins.
[0015] Another version of the invention is directed to a nucleic acid construct (vector) that drives the expression of the above-noted fusion protein when the construct is transformed into a suitable host or disposed in a suitable cell-free expression system. Many cell-free expression systems are commercially available. For example, Promega (Madison, Wis.) markets a suitable cell-free expression system under the registered trademark “TNT.” Promega's “TNT”®-brand systems are single-tube, coupled transcription/translation reactions for eukaryotic cell-free protein expression. To use these systems, 0.2 to 2.0 μg of circular plasmid DNA containing a T7, T3 or SP6 promoter, or a PCR-generated fragment containing a T7 promoter, is added to an aliquot of the “TNT”®-brand Quick Master Mix and incubated in a 50 μl reaction volume for 60 minutes at 30° C. Other cell-free systems are offered commercially by Qiagen (Valencia, Calif.), Invitrogen (Carlsbad, Calif.), and others.
[0016] The transformed host itself is also encompassed within the scope of the present invention.
[0017] Another version of the invention is directed to a pharmaceutical composition for treating dystrophinopathies in mammals, including humans. The pharmaceutical composition comprises a fusion protein as noted previously, or a pharmaceutically suitable salt thereof, in an anti-dystrophinopathic amount, in combination with a pharmaceutically suitable carrier.
[0018] Yet another version of the invention is directed to a method of treating dystrophinopathies, including DMD, in mammals. The method comprises administering to a mammalian subject in need thereof an anti-dystrophinopathic amount of an isolated fusion protein or a pharmaceutically suitable salt thereof, wherein the fusion protein comprises a first region which is effective to transduce the fusion protein into mammalian muscle cells. The first region is operationally linked to a second region comprising a full-length utrophin protein or an anti-dystrophinopathic fragment thereof.
[0019] As described herein, the present inventors have expressed full-length utrophin in a baculovirus system and have shown that the expressed protein can be purified as a highly soluble monomer. The monomer has actin-binding activities similar to those measured for recombinant dystrophin and purified dystrophin glycoprotein complex. The invention also encompasses a baculovirus expression construct (i.e. a “bacmid”) that encodes full-length mouse utrophin fused with an amino-terminal peptide corresponding to the protein transduction domain of the HIV TAT protein. TAT-utrophin expresses to high levels in insect cells, is fully soluble, and can be rapidly purified by affinity chromatography.
[0020] Transduction of TAT-utrophin into the skeletal muscle of dystrophin-deficient mdx mice corrects the dystrophic phenotype displayed by the mdx mice.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 is a schematic diagram of the dystrophin-glycoprotein complex.
[0022] FIG. 2 is a graph depicting the relative lengths and actin-binding properties (Kd and Bmax) of serially-deleted constructs of utrophin.
[0023] FIGS. 3A , 3B, 3C, and 3D compare in various terms the actin-binding properties of recombinant dystrophin versus the actin-binding properties of utrophin. FIG. 3A shows parallel gels containing (moving from left to right) a Coomassie blue-stained gel loaded with recombinant utrophin (rUTR) and recombinant dystrophin (rDYS), western blots stained with rabbit 31 antibodies (Rab31) specific for dystrophin, and DRP2 antibodies specific against utrophin. FIG. 3B is a graph depicting F-actin co-sedimentation data for rDYS (lower trace) and rUTR (upper trace); the X-axis plots concentration in μM, the Y-axis plots bound rDYS and rUTR (mol/mol actin). FIG. 3C is a graph depicting the effect of dystrophin/utrophin on depolymerization of actin filaments containing PRODAN-labeled monomers (-●-=actin, -□-=rDYS, -▪-=rUTR). FIG. 3D is a graph depicting the relative lengths and actin-binding properties (Kd and Bmax) of the serially-deleted constructs.
[0024] FIG. 4 is a graph depicting the quantitation of dystrophin and utrophin levels in skeletal muscle. The abundance of dystrophin and utrophin was measured in wild-type, mdx, and “Fiona” transgenic mdx mice overexpressing full-length utrophin by quantitative western blot analysis using recombinant dystrophin and utrophin as standards. Values are expressed as percent of total muscle protein and percent of dystrophin abundance in wild-type muscle.
[0025] FIG. 5 is a gel showing the expression and purification of TAT-utrophin in the baculovirus system. See the examples for lane assignments.
[0026] FIGS. 6A and 6B depict uptake and membrane localization of TAT-utrophin in mdx muscle. FIG. 6A is a western blot that shows increased utrophin immuno-reactivity in several tissues of an mdx mouse after 6 intraperitoneal injections of TAT-UTR (+) compared to PBS-injected controls (−). FIG. 6B depicts the results of immunofluorescence analysis, which shows increased sarcolemmal HA-tag and DRP2 immunoreactivity in the TAT-UTR-treated animal (upper-left and upper-right panels, respectively) as compared to the sarcolemmal HA-tag and DRP2 immunoreactivity in PBS-injected controls (lower-left and lower-right panels, respectively).
[0027] FIGS. 7A , 7B, 7C, 7D, 7E, and 7F are a series of photographs showing greatly reduced histopathology in TAT-utrophin-treated mdx muscle versus controls. FIGS. 7A , 7B, and 7C depict TAT-utrophin-treated mdx muscle (TA and quadriceps), while FIGS. 7D , 7E, and 7F depict PBS-treated mdx muscle (TA and quadriceps). Haematoxylin and eosin stained sections revealed decreased numbers of centrally nucleated fibers and less fibrosis in TAT-UTR treated compared to PBS-injected mdx muscle.
[0028] FIGS. 8A and 8B are graphs depicting the increased susceptibility of mdx muscles to eccentric contraction. Shown are tracings of maximal force versus time obtained during the first (upper trace) and fifth (lower trace) eccentric contraction imposed on isolated EDL muscle from wild-type (WT) and dystrophin-deficient mdx mice. Note the greater force drop in mdx muscle versus WT muscle as previously reported by Petrof et al. (1993) and Moens et al. (1993).
[0029] FIG. 9A is a schematic representation of mini- and micro-TAT-utrophin constructs according to the present invention. FIG. 9B is a Coomassie Blue-stained protein gel of the truncated TAT-utrophin constructs depicted schematically in FIG. 9A .
[0030] FIG. 10 is a map of the pFastBac1-brand plasmid, available commercially from Invitrogen.
[0031] FIG. 11 is a histogram depicting the dose-dependent ability of TAT-utrophin to increase the specific force of muscle tissue in mdx mice treated with the TAT-utrophin.
[0032] FIG. 12 is a histogram depicting the dose-dependent ability of TAT-utrophin to decrease contraction-induced injury of muscle tissue in mdx mice treated with the TAT-utrophin.
[0033] FIG. 13A depicts the results of immunofluorescence analysis on 10 μm thick cryosections from PBS- or TAT-utrophin-injected quadriceps from mdx mice. Primary antibodies to utrophin (NCL-DRP2; Utr), β-dystroglycan (NCL-b-DG; β-DG), α-sarcoglycan (NCL-a-SARC; α-SG), and γ-sarcoglycan (NCL-g-SARC; γ-SG) demonstrated peripherally localized dystrophin complex members in the TAT-utrophin-treated mice. FIG. 3B is a histogram depicting serum activity levels of the muscle enzyme creatine kinase from PBS- or TAT-utrophin-injected quadriceps from mdx mice. Creatine kinase levels were reduced 50% in 38 day-old TAT-utrophin-treated mice as compared to PBS-injected controls. (*) denotes p<0.05.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and Abbreviations:
[0034] The following abbreviations and definitions are used throughout the specification and claims. Any terms not explicitly defined herein are to be given their accepted meanings in the fields of molecular biology, physiology, and/or biochemistry.
[0035] Affinity tag: Any moiety (typically a small oligopeptide) that can be affixed to a protein (by any means) which allows the resulting fused entity to be isolated by affinity chromatography.
[0036] Anti-dystrophinopathic fragment: a fragment of a full-length utrophin protein that functions to ameliorate dystrophinopathic symptoms when administered as part of the fusion protein described herein. Explicitly included within this definition are the utrophin fragments shown in SEQ. ID. NOS: 10-25 in the attached Sequence List. (The “delta” nomenclature used in the Sequence List reflects the number of deleted repeats. Thus, the construct “murine TAT-UTR delta 4-21” encodes a murine TAT-utrophin fusion protein deleted for repeats 4-21.) It is preferred that the fragment be no more than 75% of the mass of the full-length utrophin protein, more preferred that the fragment be no more than 50% of the mass of the full-length utrophin protein, and still more preferred that the fragment be no more than 25% of the mass of the full-length utrophin protein.
[0037] Bacmid: baculovirus shuttle vector.
[0038] BMD: Becker muscular dystrophy.
[0039] DMD: Duchenne muscular dystrophy.
[0040] Dystrophinopathy: All pathological conditions in mammals, including humans, due in full or in part to mutations in the gene(s) encoding the protein dystrophin (both now known or discovered in the future). Explicitly included within the definition of “dystrophinopathy” are BMD, DMD, EDMD, SBMA, XLDCM, elevated serum creatine kinase, and the like.
[0041] EDL: extensor digitorum longus muscle.
[0042] EDMD: Emery-Dreifuss muscular dystrophy.
[0043] “FLAG”-brand polypeptide: Generally, any polypeptide having the sequence DYKDDDDK (SEQ. ID. NO: 1), or a fragment thereof, such as the tetrapeptide DYKD (SEQ. ID. NO. 1), which can be used for isolating fusion proteins via affinity chromatography. The terms “FLAG” and “ANTI-FLAG” are registered trademarks of Sigma-Aldrich Biotechnology LP (St. Louis, Mo.). “FLAG”-brand polypeptides are available commercially from Sigma-Aldrich. See also Chubet & Brizzard (1996) “Vectors for expression and secretion of FLAG epitope-tagged proteins in mammalian cells,” Biotechniques 20(1):136-141.
[0044] HIV-TAT or TAT: Human immunodeficiency virus transactivator protein. “Tat” is short for “transactivator,” a regulatory gene that accelerates the production of more HIV virus. “TAT” designates the protein, while “tat” designates the corresponding gene that encodes the TAT protein. In its native milieu, the TAT protein binds to the start of a new HIV RNA strand. Once bound, TAT encourages the transcription of the remainder of the HIV genetic code. TAT from HIV is a protein containing from 86 to 101 amino acids, depending upon the strain of HIV. The 86 amino acid-long sequence of HIV-1 TAT is shown in SEQ. ID. NO: 2. The entire genomic sequence of the HIV-1 virus, including the tat gene (at nts 5377-5591 and 7925-7970), is shown in SEQ. ID. NO: 3. See Gaynor, R. B. (1995) Regulation of HIV-1 gene expression by the transactivator protein Tat. Curr Top Microbiol Immunol 193, 51-77. See also GenBank Accession No. AF033819 for a fully annotated version of the HIV-1 genomic sequence.
[0045] LGMD: Limb-Girdle muscular dystrophy.
[0046] mdx Mice: A strain of mice arising from a spontaneous mutation (mdx) in inbred C57BL mice. The mutation is X chromosome-linked and produces viable homozygous animals that lack the muscle protein dystrophin. Mdx mice have high serum levels of muscle enzymes, and possess histological lesions similar to human muscular dystrophy. The histological features, linkage, and map position of mdx make these mice a widely utilized animal model for Duchenne muscular dystrophy. Mdx mice can be purchased from several commercial suppliers, including The Jackson Laboratory, Bar Harbor, Me. (sold under the registered trademark “JAX”).
[0047] Operationally linked: when referring to two or more regions of a protein or a nucleotide sequence, “operationally linked” means the two regions are physically linked either directly or indirectly via intervening amino acid residues, nucleotide bases, or any other type of linking moiety.
[0048] PBS: phosphate-buffered saline.
[0049] PCR: polymerase chain reaction.
[0050] Pharmaceutically-suitable salt: any acid or base addition salt whose counter-ions are non-toxic to the patient in pharmaceutical doses of the salts so that the beneficial inhibitory effects inherent in the free base or free acid are not vitiated by side effects ascribable to the counter-ions. A host of pharmaceutically-suitable salts are well known in the art. For basic active ingredients, all acid addition salts are useful as sources of the free base form even if the particular salt, per se, is desired only as an intermediate product as, for example, when the salt is formed only for purposes of purification, and identification, or when it is used as intermediate in preparing a pharmaceutically-suitable salt by ion exchange procedures. Pharmaceutically-suitable salts include, without limitation, those derived from mineral acids and organic acids, explicitly including hydrohalides, e.g., hydrochlorides and hydrobromides, sulphates, phosphates, nitrates, sulphamates, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene bis-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane sulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates, quinates, and the like. Base addition salts include those derived from alkali or alkaline earth metal bases or conventional organic bases, such as triethylamine, pyridine, piperidine, morpholine, N methylmorpholine, and the like.
[0051] SBMA: Spinal bulbar muscular atrophy (also known as Kennedy's disease).
[0052] TA: tibialis anterior muscle.
[0053] Transduction: in general, the transfer of DNA from one cell to another; typically transduction is mediated via a bacteriophage, but any means of transferring the DNA from its original source to its ultimate destination are included within the term “transduction” as used herein.
[0054] UTR or UTRN: utrophin. The nucleotide sequence for the human utrophin gene and the corresponding amino acid sequence for the encoded human utrophin protein are shown in SEQ ID NOS: 6 and 7, respectively; the nucleotide sequence for the murine utrophin gene and the corresponding amino acid sequence for the murine utrophin protein are shown in SEQ. ID. NOS: 8 and 9, respectively.
[0055] WT: wild-type.
[0056] XLDCM: X-linked dilated cardiomyopathy.
[0057] A first version of the invention is directed to a TAT-utrophin fusion protein (TAT-UTR), and use of the TAT-UTR fusion protein to treat dystrophinopathies in mammals, including humans. To demonstrate the efficacy of the TAT-UTR to treat dystrophinopathies in mammals, the mdx mouse is used as a model to demonstrate that TAT-UTR is imported into striated muscle cells and that the TAT-UTR fusion protein eliminates or significantly reduces the dystrophic phenotype in mdx mice.
[0058] Thus, in this first version of the invention, purified TAT-utrophin is injected into dystrophin-deficient mdx mice in an anti-dystrophic-effective amount. The mdx mouse model serves to demonstrate efficacy in all mammals, including humans. Measurements are then taken to assess the extent to which the TAT-utrophin is transduced into striated muscle cells in vivo. The localization of the TAT-utrophin is then assessed to determine how much of the TAT-utrophin is localized to the sarcolemma. (As shown in FIG. 1 , natural dystrophin exerts its biological effect in close conjunction with the sarcolemma.) Measurements are also taken to determine whether the TAT-UTR fusion protein becomes stably associated with other dystrophin-associated proteins. The progress of mdx mice treated with the TAT-UTR is then followed to measure the improvement of several well-established parameters of the dystrophic phenotype, such as specific force and force drop in the muscles of the treated mice versus the control mice.
[0059] A second version of the invention is directed to mini- and micro-TAT-UTR constructs and methods of using these constructs to treat dystrophinopathies in mammals, including humans. Thus, the invention also encompasses truncated mini- and micro-TAT-utrophin constructs and the use of these truncated versions of the protein to treat dystrophinopathies. Reducing the physical size of the fusion protein results in improved protein transduction in vivo. Two representative truncated constructs are described herein. These truncated fusion proteins are designed to retain full activity for all known binding partners of utrophin, but with a 40 to 50% reduction in the mass of the protein. A third construct is designed to mimic the structure of the most extensively truncated, fully-functional dystrophin micro-gene.
[0060] Using TAT-UTR as a protein-based therapy for treating dystrophinopathies is a relatively low-cost, low-risk, but high-return approach to treating these currently intractable and fatal conditions. At present, there simply is no effective treatment available to treat prevalent dystrophinopathies such as DMD.
[0061] The present invention includes a series of utrophin constructs encoding the amino-terminal, actin-binding domain alone (UTRN), or the amino-terminal domain plus 4, 7, 10, or 11 spectrin-like repeats. FIG. 2 depicts the relative lengths of these constructs and their binding characteristics. As shown in FIG. 2 , the constructs are designated herein as UTRN-R3, UTRN-R6, UTRN-R9, and UTRN-R10, respectively. Interestingly, the UTRN-R10 protein bound to actin filaments with essentially the same properties as full-length recombinant utrophin (rUTR), which suggests UTRN-R10 encodes the complete actin-binding region of utrophin (see FIG. 2 ). The UTRN-R9, UTRN-R6, and UTRN-R3 proteins each bound to actin filaments with progressively lower affinity and stoichiometry as compared to full-length utrophin and UTRN-R10. (See FIG. 2 .) These results demonstrate that the first ten (10) spectrin-like repeats of utrophin dramatically enhance the F-actin binding affinity and lateral association of the amino-terminal domain and provide a molecular basis for the greater effectiveness of full-length utrophin in rescuing dystrophin-deficient muscle as compared to a utrophin mini-gene deleted for spectrin-like repeats 4-19.
[0062] The present inventors have also expressed and characterized full-length mouse dystrophin. Recombinant dystrophin binds to actin filaments with a Kd of 0.4 μM and Bmax of 1 dystrophin molecule per 24 actin monomers (see FIG. 3D , second construct), which is remarkably close to the actin-binding properties of purified dystrophin-glycoprotein complex (Rybakova et al., 1996). In direct comparisons (see FIGS. 3A , 3B, and 3C), dystrophin and utrophin differed only in their extent of lateral association with actin filaments (1-to-24 for dystrophin and 1-to-14 for utrophin), and in the effect of increasing ionic strength on actin filament binding. These results strongly suggest that dystrophin and utrophin are both actin-binding proteins, but that the molecular epitopes important for filament binding differ between the two proteins.
[0063] While transgenic utrophin overexpression rescued all known phenotypes associated with dystrophin-deficiency in mdx mice (Tinsley et al., 1998), there remains a widespread perception that utrophin levels must greatly exceed the amount of dystrophin expressed in normal muscle in order to cause full rescue from the dystrophinopathic phenotype exhibited by mdx mice. This perception is based, at least in part, on an early quantitative estimate (Hoffman et al., 1987) of dystrophin abundance in normal muscle (0.002% of total muscle protein) and the present inventors' own measurements of utrophin expression (Rybakova et al., 2002) in normal (0.0006%) and mdx muscle (0.0013%), as well as in the Fiona line of transgenic mdx mice that overexpress utrophin to levels (0.014%) that fully corrected the mdx phenotype. (See Tinsley et al., 1998). From these measurements, it can reasonably be concluded that up to 7-fold greater levels of utrophin (0.014%/0.002%) may be necessary to compensate for dystrophin deficiency.
[0064] However, the early measurements of dystrophin levels in normal muscle used a relatively small recombinant protein fragment (Hoffman et al., 1987). While state-of-the-art at that time, the much smaller protein fragment used as the standard likely transferred to nitrocellulose more efficiently than the full-length dystrophin protein. Thus, it is possible that the previous measurements (Hoffman et al., 1987) may have significantly underestimated the abundance of dystrophin in normal muscle. Therefore, the abundance of dystrophin in normal skeletal muscle has now been measured by quantitative western blotting using full-length recombinant mouse dystrophin as the standard and iodinated secondary antibody as previously described for utrophin (Rybakova et al., 2002). The measurements (see the table shown in FIG. 4 ) suggest that the abundance of dystrophin in normal muscle is 10-times greater (0.021±0.003%, n=7)(Rybakova et al., 2006) than previously reported (Hoffman et al., 1987). The new measurements more closely agree with the measured abundance of dystrophin (Ohlendieck et al., 1991) in highly purified sarcolemma vesicles (2% of sarcolemmal protein) and with quantitative estimates that sarcolemmal proteins comprise 1% of total muscle protein based on the density of sodium channels in total homogenates (0.09 pmol/mg total protein) and in purified sarcolemmal vesicles (8 pmol/mg sarcolemmal protein) from rat skeletal muscle (Barchi and Weigele, 1979).
[0065] Most importantly, however, these data indicate that utrophin can fully rescue the mdx phenotype (Tinsley et al., 1998) when expressed to levels approaching that of dystrophin in normal muscle (0.014%/0.02%=70%).
[0066] The present invention is thus a method of using recombinant utrophin as a protein-based therapy for treating dystrophinopathies in general and DMD in particular. The present method uses TAT-utrophin chimeric (i.e., fusion) proteins. The TAT portion of the chimeric protein serves to mobilize the protein (i.e., transduce the protein) into muscle cells. The UTR portion of the chimeric protein serves to ameliorate or to eliminate the dystrophic condition.
[0067] One distinct benefit of the invention is that utrophin itself is not toxic. Therefore, the TAT-UTR fusion proteins can be administered in relatively high doses, thereby making it easier to transduce therapeutically effective amounts of the TAT-UTR fusion protein into muscle cells. Ubiquitous transgenic over-expression of utrophin itself caused no toxicity in a broad range of tissues (Fisher et al., 2001). Thus, in the present invention, an 11 kb full-length mammalian utrophin cDNA (mouse) (Guo et al., 1996) was cloned in-frame into the bacterial expression vector pTAT (Nagahara et al., 1998), which was kindly provided by Dr. Steven Dowdy (University of California, San Diego). A Kozak consensus sequence and a “FLAG”-brand type epitope were engineered in-frame 5′ to TAT-utrophin by PCR.
[0068] The FLAG-TAT-utrophin construct was inserted into the pFastBac 1 donor plasmid (purchased commercially from Invitrogen, Carlsbad, Calif.). A map of the pFastBac1 donor plasmid is shown in FIG. 10 and the complete sequence of pFastBac1 is presented in SEQ. ID. NO: 4. Subsequent transformation into DHIOBac cells (purchased commercially from Invitrogen, catalog no. 18290-015) allowed for site-specific transposition into bMON14272 bacmid DNA. (The bMON14272 bacmid, along with the helper plasmid pMON7124, are included with the DHIOBac cells sold by Invitrogen. See Invitrogen's catalog no. 10359-016, and the product literature for Invitrogen's “BAC-TO-BAC”®-brand baculovirus expression system.)
[0069] Colonies containing recombinant bacmid DNA were identified by blue/white screening and high titer viral stocks were used to infect Sf21 insect cells (Spodoptera frugiperda) for protein expression. (Sf21 cells are available commercially from a number of international suppliers, including Orbigen Inc., San Diego Calif., and Gentaur, Brussels, Belgium.) Infected Sf21 cells were harvested 72 h post-infection and TAT-utrophin was purified from cell lysates using “ANTI-FLAG”-brand M2 affinity resin (obtained commercially from Sigma-Aldrich, St. Louis, Mo.). The gel depicted in FIG. 5 shows that FLAG-TAT-utrophin is expressed as a fully soluble protein and can be easily purified by “ANTI-FLAG” M2 affinity chromatography. Thus, sufficient TAT-utrophin can easily be prepared to perform a host of experiments. Moving from left-to-right, the lanes of the gel in FIG. 5 depict the cell lysate prior to chromatography, the M2 column void volume, and the M2 column wash. The lanes numbered 1-6 then depict the elution of the M2 column to obtain the resulting fusion protein.
[0070] To assess whether TAT-utrophin is measurably transduced into skeletal muscle, a 2.5 week-old mdx mouse received six intraperitoneal injections of TAT-utrophin (20 mg/kg in sterile PBS) administered biweekly. As a control, a littermate mdx mouse was sham-injected with sterile PBS in parallel. At age six weeks, both mice were euthanized, perfused with PBS, and muscle tissue was excised for western blot, immunofluorescence and histological analyses. Western blot analysis of lysates from several tissues showed increased utrophin immunoreactivity in the TAT-utrophin-treated mdx mouse compared to the PBS-injected animal. See FIG. 6A , which is a gel depicting the utrophin immunoreactivity of the treated mouse versus the untreated mouse in several different tissue types. Importantly, immunofluorescence analysis of muscle cryosections revealed both increased HA-tag and DRP2 immunoreactivity localized to the sarcolemma of muscle from the animal treated with TAT-utrophin. See FIG. 6B , where the two upper panels depict immunoreactivity in the treated mouse and the two lower panels depict immunoreactivity in the untreated mouse.
[0071] Most strikingly, light microscopic analysis of haematoxylin and eosin-stained muscle cryosections showed dramatically decreased fibrosis and numbers of centrally nucleated myofibers in the TAT-utrophin treated animal compared to PBS-injected control. Compare FIGS. 7A , 7B, and 7C (which are photos of tibialis anterior (“TA”) and quadriceps (“QUAD”) muscle fibers from treated mice) to FIGS. 7D , 7E, and 7F (which are corresponding photos from untreated mice). In the quadriceps, the percentage of centrally nucleated fibers was 48% in the PBS-injected control, but only 24% in the TAT-utrophin-treated animal. The combined data of FIGS. 6A , 6B, 7A, 7B, 7C, 7D, 7E, and 7F show that TAT-utrophin effectively transduced skeletal muscle cells in vivo, correctly localized to the sarcolemma, and improved the histopathology of dystrophin-deficient mdx muscle.
[0072] Of course, recovery of muscle function is the ultimate criterion for evaluating the efficacy of any therapy for dystrophinopathies. Several studies have demonstrated that specific force production by mdx muscle is significantly decreased. It has also been shown that mdx muscle is hypersensitive to lengthening and eccentric contraction (Petrof et al., 1993; Moens et al., 1993). Therefore, these parameters were measured in sham- and TAT-utrophin treated mdx mice. (Kind thanks are extended to Dr. Richard L. Moss for his aid in conducting these tests.) FIGS. 8A and 8B provide data demonstrating that the eccentric contraction protocol described in Petrof et al. (1993) and Moens et al. (1993) can be performed and that these tests performed by the present inventors reproduced the key findings of Petrof et al. (1993) and Moens et al. (1993).
[0073] Regarding the key utility of the present invention, the Examples presented below clearly demonstrate that dystrophinopathic mammals treated according to the present invention show a significantly increased specific force produced by their muscles as compared to untreated dystrophinopathic mammals, as well as a significantly decreased force drop. See Example 3 and FIGS. 11 and 12 . Thus, the utility of the compounds, compositions, and methods of the present invention is to ameliorate the disabling effects of dystrophinopathic conditions in mammals, including DMD in humans.
[0074] As indicated above, the invention includes pharmaceutical compositions comprising the fusion protein(s) described herein and/or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier. The compositions may also include other therapeutically active substances in addition to the fusion protein and/or salt thereof. The pharmaceutical compositions of the invention comprise an amount of the fusion protein and/or a pharmaceutically suitable salt thereof that is effective to ameliorate dystrophinopathic symptoms in a mammal suffering from a dystrophinopathy. In a pharmaceutical composition of the invention, the carrier must be pharmaceutically acceptable in the sense of being compatible with other ingredients in the particular composition and not deleterious to the recipient thereof. The compositions include those suitable for oral, topical, rectal or parenteral (including subcutaneous, intramuscular, intraperitoneal, intradermal and intravenous) administration. Parenteral administration, either via the intramuscular or the intraperitoneal routes, is preferred.
[0075] In a particular version of the invention, the pharmaceutical compositions comprise the active ingredient (the fusion protein or a salt thereof) presented in a unit dosage form. The term “unit dosage” or “unit dose” is denoted to mean a predetermined amount of the active ingredient sufficient to be effective for treating dystrophinopathy. Preferred unit dosage formulations are those containing a daily dose, daily sub-dose, or an appropriate fraction thereof, of the administered active ingredient.
[0076] The pharmaceutical compositions may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid or solid carrier and then, if necessary, shaping the product into the desired unit dosage form.
[0077] Compositions of the present invention suitable for oral administration may be presented as discrete unit dosages, e.g., as capsules, cachets, tablets, boluses, lozenges and the like, each containing a predetermined amount of the active ingredient; as a powder or granules; or in liquid form, e.g., as a collyrium, suspension, solution, syrup, elixir, emulsion, dispersion and the like.
[0078] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active compound in a free-flowing form, e.g., a powder or granules, optionally mixed with accessory ingredients or excipients, e.g., binders, lubricants, inert diluents, surface active or dispersing agents. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered active compound with any suitable carrier.
[0079] Compositions suitable for parenteral administration conveniently comprise a sterile injectable preparation of the active ingredient in, for example, a solution which is preferably isotonic with the blood of the recipient. Useful formulations also comprise concentrated solutions or solids containing the active ingredient which upon dilution with an appropriate solvent give a solution suitable for parenteral administration. The parenteral compositions include aqueous and non-aqueous formulations which may contain conventional adjuvants such as buffers, bacteriostats, sugars, thickening agents and the like. The compositions may be presented in unit dose or multi-dose containers, for example, sealed ampules and vials.
[0080] Compositions suitable for topical or local application (including ophthamological administration) comprise the active ingredient formulated into pharmaceutically-acceptable topical vehicles by conventional methodologies. Common formulations include drops, collyriums, aerosol sprays, lotions, gels, ointments, plasters, shampoos, transferosomes, liposomes and the like.
[0081] Compositions suitable for inhalation administration, wherein the carrier is a solid, include a micronized powder or liquid formulation having a particle size in the range of from about 5 μm or less to about 500 μm, for rapid inhalation through the nasal or oral passage from a conventional inhalation squeeze or spray container. Suitable liquid nasal compositions include conventional nasal sprays, nasal drops and the like, of aqueous solutions of the active ingredient and optional adjuvants.
[0082] In addition to the aforementioned ingredients, the compositions of this invention may further include one or more optional accessory ingredients(s) utilized in the art of pharmaceutical formulations, e.g., diluents, buffers, flavoring agents, colorants, binders, surfactants, thickeners, lubricants, suspending agents, preservatives (including antioxidants), and the like.
[0083] The amount of active ingredient required to be effective for any specific dystrphinopathy in any specific patient will, of course, vary with the individual mammal being treated and is ultimately at the discretion of the medical or veterinary practitioner. The factors to be considered include the species and sex of the mammal, the dystrophinopathic condition being treated, the route of administration, the nature of the formulation, the mammal's body weight, surface area, age and general condition, and the particular compound to be administered.
[0084] In general, the pharmaceutical compositions of this invention contain from about 0.5 to about 500 mg and, preferably, from about 5 to about 350 mg of the active ingredient, preferably in a unit dosage form, for each of the indicated activities. However, a suitable effective dose is in the range of about 0.1 to about 200 mg/kg body weight per day, preferably in the range of about 1 to about 100 mg/kg per day, calculated as the non-salt form of the fusion protein. The total daily dose may be given as a single dose, multiple doses, e.g., two to six times per day, or by intravenous or parenteral infusion for a selected duration. Dosages above or below the range cited above are within the scope of the present invention and may be administered to the individual patient if desired and necessary. In topical formulations, the subject compounds are preferably utilized at concentrations of from about 0.1% to about 5.0% by weight.
EXAMPLES
[0085] The following Examples are presented solely to provide a more complete description of the invention disclosed and claimed herein. The Examples do not limit the scope of the invention claimed herein in any fashion.
Example 1
—Expression, Purification of TAT-Utrophin; General Protocols:
[0086] 1.a. Expression and Purification of TAT-Utrophin. High titer stocks of recombinant baculovirus encoding the “FLAG”-tagged TAT-utrophin chimera were used to infect Sf21 insect cells for protein expression by a shaker culture procedure described in the manufacturer's instructions. Infected Sf21 cells were harvested 72 h post-infection and resuspended in 10 ml of 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and a cocktail of protease inhibitors. The soluble lysate was circulated over a 2 ml “ANTI-FLAG” M2 agarose column (Sigma-Aldrich). The column was washed extensively with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl and bound protein eluted with the same buffer containing 100 μg/ml “FLAG”-brand peptide (Sigma-Aldrich). Purified protein was concentrated in a Centricon 100 column (Amicon) and quantified with the Bio-Rad DC Protein Assay Kit using BSA as standard. The typical yield of pure utrophin was 700 μg when only five 177 cm2 plates of cell monolayer were used as a starting material. The protocols can be easily scaled up as needed.
[0087] Quality control analysis. The data indicate that TAT-utrophin is abundantly expressed in a highly soluble form that can be readily purified by “ANTI-FLAG” affinity chromatography (see FIG. 5 ). It is critical to note that including the TAT sequence within the fusion protein has no adverse effect on utrophin structure/function. The purified TAT-utrophin is to be analyzed by gel permeation chromatography (Rybakova and Ervasti, 1997), velocity sedimentation analysis (Ervasti et al., 1991) and electron microscopy after rotary shadowing (Rybakova et al., 2002). These analyses yield quantitative measures for the native molecular weight, dimensions, shape, oligomeric/aggregative state as well as an assessment of proper folding.
[0088] The F-actin binding properties of TAT-utrophin are measured using the established high-speed co-sedimentation assay (see FIG. 3B ) and binding data is analyzed by nonlinear regression analysis. These experiments will yield both the apparent Kd and Bmax of recombinant protein binding to F-actin. See FIG. 3D . The ability of different proteins to protect actin filaments from depolymerization is measured by monitoring the time-dependent decay in fluorescence of preformed filaments seeded with PRODAN-labeled (i.e., 6-propionyl-2-(N,N-dimethyl)aminonaphthalene-labeled) monomers at Cys374 (Marriott et al., 1988; Miyata et al., 1997) as shown in FIG. 3C . All data is compared to those measured for recombinant utrophin performed in parallel.
[0089] More specifically, an 11 kb full-length murine utrophin cDNA was subcloned in-frame into the bacterial expression vector pTAT to generate PTAT-Utr. To prepare for eventual expression and purification of TAT-Utrophin in Sf21 insect cells using a baculovirus expression system, a Kozak consensus sequence and FLAG-epitope were engineered in-frame at the extreme 5′ end of TAT-Utr using PCR primers KJS36 (5′ gcggccgcacaccatggactacaaggacaacgatgacaaggctacggccgcaagaaac-3′) (SEQ. ID. NO: 26) (FLAG-epitope is underlined) and KJS32 (5′-ggagatgcacagcaacagtttcaggacttagg-3′) (SEQ. ID. NO: 27). This FLAG-TAT-utrophin construct was inserted into the bacmid donor plasmid pFastBac1 (Invitrogen, Carlsbad, Calif.) and subsequently transformed into DH10BAC (Invitrogen) bacterial cells to allow for site-specific transposition into bacmid DNA. Recombinant bacmid DNA was purified and used to transfect Sf21 cell monolayers in order to generate recombinant baculovirus. Recombinant virus infection of Sf21 monolayers and recombinant protein purification using anti-FLAG M2 affinity resin (Sigma, St. Louis, Mo.) was performed as per the manufacturer's instructions.
[0090] Elution fractions were pooled, dialyzed against phosphate buffered saline (PBS) overnight, and concentrated using a Centricon 100 (Millipore, Concord, Mass.). The purified protein was sterilized for injection by passage through a 0.22 μm filter and injected into the intraperitoneal cavity of mdx mice at a concentration of 0.5 to 1.0 mg/ml. The pure protein was stable for up to 4 days when kept on wet ice at 4° C. (assessed by a lack of degradation on Coomassie blue stained SDS-polyacrylamide gels), so a single protein preparation was utilized for up to 2 injections when possible. Otherwise, protein was prepared fresh for each injection.
[0091] 1.b. Treatment Time Course. Pairs of female C57Bl/10ScSn-Dmdmdx/J (The Jackson Laboratory, Bar Harbor, Me.) littermates were treated in parallel, one of which received a dose of 20 μg TAT-utrophin/g body weight while the control mouse received equal volume injections of sterile PBS. A total of 6 biweekly injections were administered over three weeks, beginning at 18 days and culminating at 35 days of age. Three days after the final injection, serum and tissue were collected for creatine kinase, western blot, immunofluorescence, histological, and physiological analyses. Animals were housed and treated in accordance with the standards set by the University of Wisconsin Institutional Animal and Care and Use Committee.
[0092] 1.c. Protein extracts and Western Blotting. Tissues were dissected from freshly killed mice and snap frozen in liquid nitrogen. Frozen tissue was pulverized with a liquid nitrogen-cooled mortar and pestle and solubilized in 1% SDS, 5 mM EGTA, and a cocktail of protease inhibitors. Samples were incubated for 2 minutes at 100° C. and centrifuged 2 min at 12000×g. The supernatant protein concentration was determined with the Bio-Rad DC protein assay kit using bovine serum albumin as standard. Equal amounts of protein was separated by SDS-PAGE and transferred to nitrocellulose. Western blot analysis of utrophin levels was performed with rabbit polyclonal antibody 103 raised against the carboxyl-terminus of utrophin (generously provided by Dr. Stanley Froehner, University of Washington) diluted 1:250 in BLOTTO (i.e., bovine lacto transfer technique optimizer, a blocking reagent made from nonfat dry milk and PBS) (5% milk in PBS, pH 7.5) and anti-FLAG monoclonal antibody M2 (Sigma) diluted 1:1000 in BLOTTO. (BLOTTO blocking reagents are also commercially available from, for example, Thermo-Fisher Scientific, Waltham, Mass., catalog no. PI-37530.)
[0093] 1.d. Histological and Morphometric Analysis. Individual muscles were dissected from freshly killed mice, coated with “O.C.T.” matrix solution (“TissueTek”®-brand, Sakura Finetek, Torrance, Calif.; O.C.T. refers to “optimum cutting temperature,” a specimen matrix formulation comprising water-soluble glycols and resins for cryostat sectioning at temperatures of −10° C. and below), and rapidly frozen in liquid nitrogen-cooled isopentane. Ten (10) μm thick cryosections were cut on a Leica CM3050 cryostat, allowed to dry, and stained with hematoxylin and eosin-phloxine. Sections cut from the mid-belly of both the tibialis anterior and quadriceps were selected for histological assessment. Images were collected on a Zeiss Axiovert 25 microscope and compiled into montages of entire sections in CorelDraw 10 and exported to Scion Image (Scion Corporation, Frederick, Maryland) for morphometric analyses. The percentage of centrally nucleated fibers and fiber diameters were determined from one muscle of each mouse, with every fiber scored for CNF analysis and ˜700 fiber diameters measured per muscle section. A Student's t-test was used to compare average CNF values and average fiber diameter. To determine statistical significance of fiber diameter variability, a student's t-test was performed on the standard deviations of individual muscle sections.
[0094] 1.e. Immunofluorescence. 10 μm thick cryosections were fixed in 4% paraformaldehyde for 10 minutes, washed 3×10 minutes in PBS, and blocked in 5% goat serum for 30 minutes. Primary antibodies were applied in 5% goat serum overnight at 4° C. and washed off 3×10 minutes in PBS. “ALEXA”®-brand 488- or 568-conjugated secondary antibodies (Invitrogen) were incubated for 30 min before a final 3×10 minute wash cycle. Coverslips were applied with a drop of Anti-Fade Reagent (Molecular Probes) and confocal images obtained using a Bio-Rad MRC 1000 scan head mounted transversely to an inverted Nikon Diaphot 200 microscope at the Keck Center for Biological Imaging. Primary monoclonal antibodies used were anti-HA tag HA. 11 (BABCO, Berkeley, Calif.) 1:1000; anti-utrophin DRP2 (Novacastra, Newcastle upon Tyne, UK) 1: 10; anti-β-dystroglycan b-DG (Novacastra) 1:1000; anti-α-sarcoglycan (NCL-a-SARC; α-SG), (Novacastra) 1:1000; and anti-γ-sarcoglycan g-SARC (Novacastra) 1:1000.
[0095] 1.f. Contractile Properties. All mechanical properties were adapted from Petrof et al. After rapid PBS perfusion, the extensor digitorum longus (EDL) muscles were quickly dissected tendon to tendon and immersed in an O2-saturated Ringer's solution (135 mM NaCl, 4 mM KCl, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, and 1.8 mM CaCl2, pH 7.4) at 25° C. Suture silk (4-0) was used to attach one tendon to a rigid support and the other to a dual lever force transducer (Aurora Scientific, Ontario, Canada) and the entire apparatus was immersed in oxygenated Ringer's solution and allowed to equilibrate for 5 minutes. Muscles were stimulated through two platinum plate electrodes on either side of the muscle. A range of twitch stimulations were performed to determine Lo, the muscle length at which maximal twitch force was produced. After 5 minutes of recovery, the EDL was maximally activated to determine maximal tetanic tension. Data were normalized against cross-sectional area of each individual muscle.
[0096] Protection against mechanical injury was assessed by subjecting the muscle to a series of five eccentric contractions (ECC). Each ECC consisted of maximally activating the muscle for 700 ms, with a stretch of 0.5 Lo/s over the final 200 ms to result in a total stretch of 0.1 Lo. Five minutes of recovery time was allowed between each ECC. Force drop was calculated as (ECC1-ECC5)/ECC1. Data were compared using ANOVA followed by a Tukey post hoc test.
[0097] 1.g. Serum CK Analysis. Retro-orbital bleeds were performed on anesthetized mice using heparinized capillary tubes. Approximately 100 μl of blood was obtained per mouse, centrifuged at 5000 rpm and the serum layer removed and stored at —80° C. for analysis. Creatine kinase levels were determined using Vitros CK DT slides (Ortho-Clinical Diagnostics, Raritan, N.J.) and analyzed using a Kodak Ektachem DT60 Analyzer as per the manufacturer's instructions. Data were collected in Units/ml and compared using a Student's T-test.
Example 2
—Effect of TAT-Utrophin on the Dystrophic Phenotype of mdx Mice:
[0098] In this Example, purified TAT-utrophin is injected into dystrophin-deficient mdx mice. The mice are then examined to assess the extent to which the TAT-utrophin is transduced into striated muscle cells in vivo. The extent of uptake is measured, and the amount of TAT-utrophin localized to the sarcolemma is determined. Optionally, it may also be determined whether the TAT-utrophin becomes stably associated with other dystrophin-associated proteins. The quantitative improvement of several well-established parameters of the dystrophic phenotype is then measured in mdx mice treated with TAT-utrophin and compared to untreated controls and placebo groups.
[0099] Administration of TAT-utrophin—Purified TAT-utrophin is dialyzed against phosphate-buffered saline and sterilized by passage through a Millex-GP 0.22 μm filter. Assuming 100% protein transduction specifically into skeletal muscle, a minimal dose of 11 μg TAT-utrophin per gram body weight is believed to compensate for dystrophin deficiency. Of course, it is likely that TAT-utrophin will distribute to all tissues and transduction efficiency will almost certainly be less than complete. Therefore, TAT-utrophin is preferably administered via intraperitoneal injection at several different concentrations ranging from 1-5 mg/ml and total injection volumes of 0.1-0.5 ml.
[0100] Measurement of TAT-Utrophin Uptake and Cellular Location - TAT-utrophin uptake into skeletal muscle and cellular localization is assessed by two methods. In the first method, mice are deeply anesthetized with avertin, the chest wall is opened, and the animals are infused for 20 minutes with phosphate-buffered saline through the left ventricle with an outflow path from the right atrium. Skeletal muscles are then dissected and used in the preparation of KCl-washed skeletal muscle membranes (Ohlendieck et al., 1991), or immediately snap-frozen in liquid nitrogen to prepare SDS total protein lysates (Rybakova et al., 2002). Both preparations are analyzed for TAT-utrophin content by quantitative western blot analysis using “ANTI-FLAG”-brand M2 antibody (Sigma-Aldrich) detected with 125I-goat anti-mouse IgG and the signals quantitated by phosphor autoradiography. Analysis of total protein lysates and KCl-washed membranes provides a measure of the fraction of TAT-utrophin stably associated with the sarcolemma. The absolute utrophin content in SDS muscle lysates of TAT-utrophin-treated mice is also quantitatively compared to the utrophin content of sham-treated mdx mice and transgenic mdx mice expressing full-length utrophin (Fiona) to levels that rescue all known phenotypes of mdx mice. These comparisons provide a quantitative assessment of the TAT-utrophin uptake relative to a fully-rescued transgenic animal model.
[0101] In the second method, anesthetized animals are infused for 2 minutes with PBS followed by a 20 minute infusion of 2% paraformaldehyde in PBS. Various skeletal muscles are dissected, post-fixed for 5 minutes in 2% paraformaldehyde, and frozen in liquid nitrogen-cooled isopentane. From 8 μm cryo-sections, both the uptake and cellular location of TAT-utrophin is assessed using confocal immunofluorescence microscopy.
[0102] The KCl-washed membranes, SDS lysates and cryosections prepared from TAT-utrophin-treated mdx mice are also used to detect alterations in the abundance and sarcolemmal localization of other proteins within the dystrophin-glycoprotein complex including α- and β-dystroglycan, α-, β-, γ- and δ-sarcoglycans, syntrophin and α-dystrobrevins. Relative protein abundance can be assessed by quantitative western blot analysis of total muscle SDS extracts (Rybakova et al., 2002), while cellular localization and organization can be assessed by immunofluorescence analysis of both longitudinal and transverse cryosections and mechanically peeled sarcolemma (Rybakova et al., 2000).
[0103] Assessment of costamere structure and function—To assess whether TAT-utrophin treatment can restore mechanical coupling between the sarcolemma and costameric γ-actin, confocal immunofluorescence microscopy analysis is performed on mechanically peeled sarcolemma (Rybakova et al., 2000) from sham and TAT-utrophin-treated mdx mice. Paraformaldehyde-fixed sarcolemma are blocked for 2 h at 4° C. with 5% serum in PBS and incubated with the appropriate primary antibodies overnight at 4° C. The specimens are washed with PBS, incubated with fluorescent secondary antibody for 30 min at 37° C., rinsed and sealed under coverslips in an anti-fade solution.
[0104] Assessment of Dystrophic Phenotype—Skeletal and cardiac muscle of dystrophin-deficient mdx mice exhibits several histologic and physiologic defects in common with patients suffering from Duchenne muscular dystrophy. Most notable (and easily measured) are a dramatic elevation in centrally nucleated fibers of irregular size resulting from muscle fiber necrosis/regeneration and elevated serum creatine kinase levels due to sarcolemmal instability.
[0105] For histologic analysis, 8 μm cryosections of skeletal muscle from control, sham-injected, and TAT-utrophin-injected mdx mice are stained with haematoxylin and eosin and the percentage of central nuclei and mean fiber diameter measured. Histological analyses are also performed on several different muscles to compare the effects of TAT-utrophin on different fiber types, and muscles experiencing different work loads and activities. Measurement of these parameters in C57BL/10 control and sham-injected mdx mice provides a baseline and elevated values for normal and dystrophic muscle, respectively. While the number of centrally-nucleated fibers is already quite high (˜40%) in 4 week-old mdx mice (Warner et al., 2002), this parameter doubles yet again by 10-12 weeks of age (Warner et al., 2002). Therefore, it is possible to measure a decrease in the percentage of centrally nucleated fibers in mdx mice treated for 2 months with TAT-utrophin compared to sham-treated mice.
[0106] To assess for sarcolemmal damage, quantitative colorimetric analysis of serum creatine kinase levels is performed using CK DT slides (Ortho-Clinical Diagnostics) measured with a Kodak Ektachem DT 60 Analyzer. A minimum of 5 animals in each treatment regime are measured at several time points post-injection. Evans blue infiltration is also assessed, which has been shown to accumulate significantly in dystrophin deficient mdx cardiac and skeletal muscle (Straub et al., 1997). Evans blue dye in sterile PBS is injected into the tail veins of control and knockout littermates and the animals sacrificed 3-6 h after dye administration. After skinning, the animals are visually inspected for macroscopic dye uptake by a blue coloration of limb muscles. 100% of mdx mice and 0% of control mice exhibit indication of membrane damage by this technique (Straub et al., 1997). In addition, 8 μm cryosections are examined by immunofluorescence microscopy to quantitate the fraction of muscle cells infiltrated by Evans blue (Straub et al., 1997).
[0107] Assessment of contractile function—Several studies have demonstrated that specific force production by mdx muscle is significantly decreased and hypersensitive to lengthening, or eccentric contraction (Petrof et al., 1993; Moens et al., 1993). Thus, the measure isometric twitch and tetanic tension in intact muscles from sham- and TAT-utrophin treated mdx mice are measured. The EDL muscle is dissected tendon to tendon and allowed to equilibrate in oxygenated mammalian Ringers' solution (Eddinger et al., 1986), and then tied into a dual mode force transducer (Aurora Scientific). The muscle length (Lo) at which maximal twitch tension is obtained is determined with a single pulse at a stimulation frequency of 2500 Hz at increasing muscle lengths. After a 10 minute wait, the muscle undergoes a series of 5 eccentric contractions (ECC) with the maximal tetanic tension measured for each round. The ECC protocol involves stimulation at 150 Hz at Lo for 500 msec followed by lengthening the muscle by 0.5 Lo/sec for 200 msec before relaxing at a rate of 0.5 Lo/sec for 200 msec. This protocol results in a stretch equal to 10% Lo. There is a 5 minute wait in between each ECC to allow the muscle to recover. All measurements are recorded and analyzed using Dynamic Muscle Control and Analysis Software (Aurora Scientific).
Example 3
—Generation of Mini- and Micro-TAT-Utrophin Constructs:
[0108] In parallel with the experiments described in Example 1, the invention also encompasses fusion proteins wherein the utrophin portion of the fusion protein has been truncated (to lower the molecular weight of the fusion protein), without deleteriously impacting the anti-dystrophinopathic activity of the fusion protein. Thus, the invention encompasses truncated, but fully functional mini- and micro-TAT-utrophin constructs. It is hoped that reducing the size of the chimera leads to improved protein uptake.
[0109] Bacmid construction—Mini- and micro-TAT-utrophin constructs are generated with the “BAC-TO-BAC”-brand expression system (anvitrogen), which has been used to express full-length mouse utrophin (Rybakova et al., 2002), dystrophin (see FIG. 3A ), and numerous truncation constructs (see FIG. 2 ). Briefly, all expression constructs are PCR-amplified from the TAT-utrophin construct using PfuUltra high-fidelity DNA polymerase (Stratagene) to incorporate an amino-terminal “FLAG”-brand type purification tag (DYKDDDDK) (SEQ. ID. NO: 1) followed by the HIV TAT protein transduction sequence (YGRKKRRQRRR) (SEQ. ID. NO: 5). The HIV TAT protein transduction sequence is preferred. However, any sequence that functions to transduce the fusion protein into mammalian muscle cells may be used in its place. The mini- and micro-constructs planned or actually made are shown schematically in FIG. 9 . Preferably, the constructs all contain intact cysteine-rich and carboxy-terminal domains to ensure optimal β-dystroglycan binding activity (Ishikawa-Sakurai et al., 2004).
[0110] Based on actin-binding studies of serially-truncated utrophin constructs performed by the present inventors (data not shown), it is expected that TAT-UTRΔR11-22 should have near-optimal actin filament binding activity, but with a 40% reduction in molecular weight (237,000) compared to full-length utrophin (394,000). TAT-UTRΔR7-22, which is less than half the molecular weight of full-length utrophin (189,000) will also be evaluated, but at the expense of diminished actin-binding activity. TAT-UTRΔR4-21 will also be generated and tested. This construct is expected to bind actin with the lowest affinity. It is an attractive compound for incorporation into a pharmaceutical composition because based on its small size (42% of full-length utrophin), and in light of the success of the analogous dystrophin micro-gene to rescue the mdx phenotype (Harper et al., 2002).
[0111] pFASTBAC1 donor plasmids carrying each new TAT construct is transformed into DHIOBAC for site-specific transposition into bMON14272 bacmid DNA. Colonies containing recombinant bacmid DNA are identified by blue-white screening and high titer viral stocks produced for infection of Sf21 insect cells for protein expression. Protein purification, quality control and transduction efficacy are performed as described earlier.
Example 4
—Dose-Dependent Amelioration of Dystrophin-Deficient Phenotype:
[0112] To determine whether the ability of TAT-utrophin to ameliorate the dystrophin-deficient phenotype is dose-dependent, the protective effects of increased dosages of TAT-utrophin were assessed on the dystrophin-deficient mdx mouse. An initial dosage of 20 μg protein/g mouse body weight was arbitrarily designated as a dosage of “1×.” A study was then performed in which littermate mdx mice were injected with 1× (20 μg protein/g body weight), 2× (40 μg protein/g body weight), and 5× (100 μg protein/g body weight) levels of TAT-utrophin. The timeline of treatment was consistent with the original 1× studies (see above) in which 2.5 week-old mdx mice received six intraperitoneal injections at the indicated dosage. The doses were administered bi-weekly. As controls, littermate mdx mice received sterile PBS injections in parallel. At six weeks of age, treated and control mice were euthanized and assessed for several functional and histological parameters of dystrophin deficiency.
[0113] Of note, the 5×-treated mdx mouse demonstrated an approximately 45% increase in specific force generation over PBS-injected mice (see FIG. 11 , which depicts the results for PBS-treated versus TAT-utrophin-treated mice). Specific force is an index of maximal force generated by a muscle normalized against the cross-sectional area of the muscle; mdx muscle typically generates approximately 25-30% less specific force than wild-type mice (Petrof et al., 1993).
[0114] Additionally, the treated mdx mice exhibited a dose-dependent improvement in protection against contraction-induced injury (see FIG. 12 , which depicts the results for PBS-treated versus TAT-utrophin-treated mice). Contraction-induced injury is a parameter quantified by the drop in maximal force generation after five (5) consecutive damaging eccentric contractions. Wild-type force drop values are typically 15-25%, while the corresponding mdx values range from 60-80% (Petrof et al., 1993). As shown in FIG. 12 , the 5×-treated mdx mice had a force drop value in the range of 20%, which is well within the range for non-mdx, wild-type mice. In contrast, the PBS-treated mdx mice had a force drop value typical of mdx mice, an approximately 65% drop.
Example 5
—Reduction of Serum Creatine Kinase in TAT-Utrophin-treated Mice:
[0115] To assess whether the protective effects of TAT-utrophin were mitigated through restoration of dystrophin complex members to the sarcolemma, immunofluorescence analyses were carried out on cryosections from TAT-utrophin and PBS-injected quadriceps. While no signal was observed on cryosections from PBS-treated muscle stained for the transmembrane glycoproteins β-dystroglycan, α-sarcoglycan, and γ-sarcoglycan, each antibody probe revealed intense staining along the periphery of muscle cells from TAT-utrophin-treated mice (FIG. 13A ). Sarcolemmal integrity was also assessed by measuring serum levels of the muscle-specific enzyme creatine kinase (CK), which are typically elevated ˜20 fold in mdx mice. TAT-utrophin-treated mice demonstrated a 50% reduction in serum CK activity compared to PBS-injected controls. See FIG. 13B . These results strongly indicate that TAT-utrophin not only restored dystrophin complex members to the sarcolemma but also partially protected against membrane instability.
[0116] The significance of these Examples is that they show that the TAT-utrophin constructs function to ameliorate dystrophinopathy in a dose-dependent fashion. The Examples also show the now best-known combination of transduction efficiency, size, and pharmacological activity to rescue phenotypically dystrophic mammals.
REFERENCES
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Claims
Certificate of Correction present
The claims shown below may not include correction changes. Use the corrected PDF for authoritative text.
What is claimed is:
1. An isolated fusion protein comprising:
a first protein region comprising a human immunodeficiency virus transactivator protein (HIV-TAT) or a transduction-effective fragment thereof which is effective to transduce the fusion protein into mammalian muscle cells, operationally linked to;
a second protein region comprising a full-length utrophin protein or an anti-dystrophinopathic fragment thereof.
2. The isolated fusion protein of claim 1, further comprising an affinity tag operationally linked to the fusion protein.
3. The isolated fusion protein of claim 2, wherein the affinity tag comprises an amino acid sequence DYKDDDDK (SEQ. ID. NO: 1) or a fragment thereof.
4. The isolated fusion protein of claim 2, wherein the affinity tag comprises an amino acid sequence DYKD (SEQ. ID. NO: 28).
5. The isolated fusion protein of claim 1, wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain domain plus 4, 7, 10, or 11 spectrin-like repeats.
6. The isolated fusion protein of claim 1, wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
7. The isolated fusion protein of claim 1, which is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 11, 13, 15, 17, 19, 21, 23, and 25.
8. The isolated fusion protein of claim 1, wherein the first protein region is an amino acid sequence as shown in SEQ. ID. NO: 2.
9. The isolated fusion protein of claim 8, wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain domain plus 4, 7, 10, or 11 spectrin-like repeats.
10. The isolated fusion protein of claim 8, wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7and 9.
11. The isolated fusion protein of claim 1, wherein the first protein region is an amino acid sequence as shown in SEQ. ID. NO: 5: YGRKKRRQRRR.
12. The isolated fusion protein of claim 11, wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain domain plus 4, 7, 10, or 11 spectrin-like repeats.
13. The isolated fusion protein of claim 11, wherein the second protein region is an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7and 9.
14. The isolated fusion protein of claim 1, wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising no more than 75% of the mass of the full-length utrophin protein.
15. The isolated fusion protein of claim 1, wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising no more than 50% of the mass of the full-length utrophin protein.
16. The isolated fusion protein of claim 1, wherein the second protein region is an anti-dystrophinopathic utrophin fragment comprising no more than 25% of the mass of the full-length utrophin protein.
17. Pharmaceutically suitable salts of the isolated fusion protein recited in claim 1.
18. A pharmaceutical composition for treating dystrophinopathies in mammals, including humans, comprising:
an isolated fusion protein or a pharmaceutically suitable salt thereof as recited in any one of claims 1-7 and 8-17, in combination with a pharmaceutically suitable carrier.
19. A method of treating dystrophinopathies in mammals, the method comprising administering to a mammalian subject in need thereof an anti-dystrophinopathic amount of an isolated fusion protein or a pharmaceutically suitable salt thereof as recited in any one of claims 1-7 and 8-17.
20. An isolated nucleic acid expression construct encoding a fusion protein, the nucleic acid expression construct comprising:
a first nucleic acid region that encodes a first protein region of the fusion protein, wherein the first protein region comprises a human immunodeficiency virus transactivator protein (HIV-TAT) or a transduction-effective fragment thereof which is effective to transduce the fusion protein into mammalian muscle cells, operationally linked to;
a second nucleic acid region that encodes a second protein region of the fusion protein, wherein the second protein region comprises a full-length utrophin protein or an anti-dystrophinopathic fragment thereof;
wherein the expression construct drives expression of the fusion protein when transformed into a suitable host cell or disposed into a suitable cell-free expression system.
21. The isolated nucleic acid expression construct of claim 2, further comprising a third nucleic acid region that encodes an affinity tag that is operationally linked to the fusion protein.
22. The isolated nucleic acid expression construct of claim 20, wherein the third nucleic acid region encodes an amino acid sequence DYKDDDDK (SEQ. ID. NO: 1) or a fragment thereof.
23. The isolated nucleic acid expression construct of claim 20, wherein the third nucleic acid region encodes an amino acid sequence DYKD (SEQ. ID. NO: 28).
24. The isolated nucleic acid expression construct of claim 20, wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain domain plus 4, 7, 10, or 11 spectrin-like repeats.
25. The isolated nucleic acid expression construct of claim 20, wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
26. The isolated nucleic acid expression construct of claim 20, which is a nucleic acid sequence selected from the group consisting of SEQ. ID. NOS: 10, 12, 14, 16, 18, 20, 22, and 24.
27. The isolated nucleic acid expression construct of claim 20, wherein the first nucleic acid region encodes an amino acid sequence as shown in SEQ. ID. NO: 2.
28. The isolated nucleic acid expression construct of claim 27, wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain domain plus 4, 7, 10, or 11 spectrin-like repeats.
29. The isolated nucleic acid expression construct of claim 27, wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
30. The isolated nucleic acid expression construct of claim 20, wherein the first nucleic acid region encodes an amino acid sequence as shown in SEQ. ID. NO: 5: YGRKKRRQRRR.
31. The isolated nucleic acid expression construct of claim 30, wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising an amino-terminal actin-binding domain of the utrophin protein, or the amino-terminal actin-binding domain domain plus 4, 7, 10, or 11 spectrin-like repeats.
32. The isolated nucleic acid expression construct of claim 30, wherein the second nucleic acid region encodes an amino acid sequence selected from the group consisting of SEQ. ID. NOS: 7 and 9.
33. The isolated nucleic acid expression construct of claim 20, wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising no more than 75% of the mass of the full-length utrophin protein.
34. The isolated nucleic acid expression construct of claim 20, wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising no more than 50% of the mass of the full-length utrophin protein.
35. The isolated nucleic acid expression construct of claim 20, wherein the second nucleic acid region encodes an anti-dystrophinopathic utrophin fragment comprising no more than 25% of the mass of the full-length utrophin protein.
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| US2005/0069985(A1) | 2005-03-01 | Kaufman | Applicant |
| US2005/0158281(A1) | 2005-07-01 | Chamberlain et al. | Examiner |
| US2006/0073586(A1) | 2006-04-01 | Xiao | Applicant |
Non-Patent Literature (30)
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- Amann et al. (1998). A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction. J. Biol. Chem. 273:28419-28423.Applicant
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- Deconinck et al. (1997a). Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90:717-727.Applicant
- Deconinck et al. (1997b). Expression of truncated utrophin leads to major functional improvements in dystrophin-deficient muscles of mice. Nature Med. 3:1216-1221.Applicant
- Eddinger et al. (1986). Mechanical and histochemical characterization of skeletal muscles from senescent rats. Am. J. Physiol. 251:C421-C430.Applicant
- Ervasti et al. (1991). Purification of dystrophin from skeletal muscle. J. Biol. Chem. 266:9161-9165.Applicant
- Gregorevic et al. (2003). Gene therapy for muscular dystrophy—a review of promising progress. Expert. Opin. Biol. Ther. 3:803-814.Applicant
- Guo et al. (1996). Cloning and expression of full length mouse utrophin: The differential association of utrophin and dystrophin with AChR clusters. FEBS Lett. 398:259-264.Applicant
- Hoffman et al. (1987). Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51:919-928.Applicant
- Ishikawa-Sakurai et al. (2004). ZZ domain is essentially required for the physiological binding of dystrophin and utrophin to beta-dystroglycan. Hum. Mol. Genet. 13:693-702.Applicant
- Kramarcy et al. (1994). Association of utrophin and multiple dystrophin short forms with the mammalian Mr 58,000 dystrophin-associated protein (syntrophin). J. Biol. Chem. 269:2870-2876.Applicant
- Kuppuswamy et al. (1989). Multiple functional domains of Tat, the trans-activator of HIV-1, defined by mutational analysis. Nucleic Acids Research, 17(9):3551-3561.Applicant
- Lindsay, M.A. (2002). Peptide-mediated cell delivery: application in protein target validation. Curr. Opin. Pharmacol. 2:587-594.Applicant
- Marriott et al. (1988). Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate. Biochemistry 27:6214-6220.Applicant
- Matsumura et al. (1992). Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle. Nature 360:588-591.Applicant
- Miyata et al. (1997). Cooperative association of actin protomers and crosslinked actin oligomers in filaments at low ionic strength. J. Biochem. (Tokyo) 121:527-533.Applicant
- Moens et al. (1993). Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch. J. Muscle Res. Cell Motil. 14:446-451.Applicant
- Petrof et al. (1993). Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc. Natl. Acad. Sci. U.S.A. 90:3710-3714.Applicant
- Rybakova et al. (1996). A new model for the interaction of dystrophin with F-actin. J. Cell Biol. 135:661-672.Applicant
- Rybakova et al. (1997). Dystrophin-glycoprotein complex is monomeric and stabilizes actin filaments in vitro through a lateral association. J. Biol. Chem. 272:28771-28778.Applicant
- Rybakova et al. (2006) Dystrophin and utrophin bind actin through distinct modes of contact. J. Biol Chem. 281 (15): 9996-10001.Applicant
- Snyder et al. (2004). Cell penetrating peptides in drug delivery. Pharm. Res. 21:389-393.Applicant
- Tinsley et al. (1998). Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nature Med. 4:1441-1444.Applicant
- Tinsley et al. (1992). Primary structure of dystrophin-related protein. Nature 360:591-593.Applicant
- Winder et al. (1995). Utrophin actin binding domain: analysis of actin binding and cellular targeting. J. Cell Sci. 108:63-71.Applicant