WO2013103964A1 - Méthodes et compositions de traitement de protéinopathies - Google Patents

Méthodes et compositions de traitement de protéinopathies Download PDF

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WO2013103964A1
WO2013103964A1 PCT/US2013/020535 US2013020535W WO2013103964A1 WO 2013103964 A1 WO2013103964 A1 WO 2013103964A1 US 2013020535 W US2013020535 W US 2013020535W WO 2013103964 A1 WO2013103964 A1 WO 2013103964A1
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amyloidosis
mitochondrial
disease
subject
tfam
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PCT/US2013/020535
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English (en)
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Shaharyar Khan
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Gencia Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the field of the invention generally relates to proteinopathies and compositions and methods for limiting the progression of, delaying, or treating proteinopathies in a subject.
  • Proteinopathy refers to a disease, most typically a neurodegenerative disease, caused by a malformed protein. Mutations in a number of genes result in accumulation of protein aggregates, for example, ⁇ , ABri, ADan, superoxide dismutase, a-synuclein, huntingtin, ataxins, and neuroserpin, triggering the pathological cascade that can lead to many phenotypically distinct neurodegenerative diseases (Forman, et al, Nat. Med., 10: 1055-1063 (2004); Ross and Poirier, Nat.
  • AD Alzheimer's disease
  • NFTs neurofibrillary tangles
  • LOAD late-onset AD
  • sporadic AD the complex and more common form of the disease.
  • the data supports a "dual pathway” model of causality, whereby Abeta and tau can be linked by separate mechanisms driven by a common upstream driver (Small and Duff, Neuron, 60(4): 534-542 (2008)).
  • Brain imaging of plaque burden is an intense research field, and a number of imaging modalities is currently being developed or used.
  • compositions and methods for treating or preventing amyloid beta expression, deposition, aggregation or plaque formation in a subject, and compositions and methods for a disease or disorder characterized by increased amyloid beta expression, deposition, aggregation or plaque are provided.
  • the methods typically include administering to a subject in need thereof one of the disclosed compositions in an effective amount to reduce, delay, or inhibit the level, formation, or production of amyloid beta, particularly amyloid beta 42, in the subject compared to a control.
  • the composition is a TFAM or a TFAM fusion protein such as SEQ ID NO:26.
  • the TFAM or fusion protein thereof is administered to the subject in an effective amount to reduce the ratio of amyloid beta42/amyloid beta40 in the subject compared to the control. In some embodiments, administration of the TFAM or fusion protein reduces amyloid beta 42- mediated inhibition of mitochondrial respiration in the subject compared to the control.
  • the compositions and methods are effective to treat a number of amyloid beta related diseases, including non-Alzheimer's diseases.
  • compositions and methods for treating or preventing tau expression, tau phosphorylation, or pathologies associated with the aggregation of tau protein in the brain and compositions and method for treating or preventing a disease or disorder characterized by increased tau expression, increased tau phosphorylation, or pathologies associated with the aggregation of tau protein in the brain are also disclosed.
  • the methods typically include administering to a subject in need thereof an effective amount of one or more of the disclosed compositions to reduce, delay, or inhibit the expression of or phosphorylation of tau in the subject compared to a control.
  • the compositions and methods are effective to treat a number of tauopathies, including non-Alzheimer's tauopathies.
  • Methods of treating or preventing proteinopathies and amyloidosis are also disclosed.
  • the methods can include administering to a subject in need thereof an effective amount of one or more of the disclosed compositions to reduce, delay, or inhibit the expression or accumulation of one or more misfolded proteins.
  • the methods can be used to treat or alleviate one or more symptoms of a proteinopathy and amyloidosis.
  • compositions for use with the disclosed methods typically include a mitochondrial DNA-binding polypeptide, preferably a
  • mitochondrial transcription factor or mitochondrial DNA-binding fragment or fusion protein thereof.
  • mitochondrial DNA-binding polypeptides include, but are not limited to, mitochondrial transcription factors such as transcription factor A, mitochondrial (TFAM), transcription factor B l, mitochondrial (TFB 1M), transcription factor B2, mitochondrial (TFB2M), Polymerase (RNA) Mitochondrial (DNA directed) (POLRMT); and functional fragments, variants, and fusion polypeptides thereof.
  • Exemplary fusion polypeptides include a mitochondrial DNA binding polypeptide, a protein transduction domain, and a targeting signal or targeting domain.
  • Suitable mitochondrial DNA-binding polypeptides include a mature transcription factor A - mitochondria (TFAM) polypeptide, or a mitochondrial DNA binding fragment thereof.
  • the targeting signal is an amino acid sequence that targets the fusion protein to the mitochondria, for example a mitochondrial localization signal.
  • the mitochondrial DNA binding polypeptide is TFAM polypeptide having at least 95% sequence identity to SEQ ID NO:26 or a mitochondrial DNA binding fragment thereof.
  • Figure 1 is a bar graph showing the level of ATP in the brain of db/db mice as determined by an ex vivo luciferase luminescence assay following administration of a low dose (0.2 mg/kg) of rhTFAM, a high dose (1.2 mg/kg) of rhTFAM or vehicle control once a week for 12 weeks.
  • the rhTFAM doses are approximately 0.2, 0.4, 0.8 and 1.2 mg/kg respectively for an average mouse with a body weight of about 30 grams.
  • Figure 2 is a bar graph showing optical density measurements of immunoblotting using an antibody cocktail for the various subunits of Complexes I-V (electron transport chain subunits) of brain lysates from vehicle control and rhTFAM treated db/db mice.
  • I-V electron transport chain subunits
  • Figure 3A is a bar graph showing expression of total tau normalized in to beta-actin in vehicle control and rhTFAM treated db/db mice.
  • Figure 3B is a bar graph showing expression of phospho-Serine 396
  • Figure 4 is a bar graph showing optical density measurements of immunoblotting using an antibody cocktail for the various subunits of Complexes I-V (electron transport chain subunits) of brain lysates from vehicle control and rhTFAM treated aged Tg2576 mice.
  • Figure 5 is a bar graph showing optical density measurements of immunoblotting using antibody for Beta-amyloid (1-42) of brain lysates from vehicle control and rhTFAM treated aged Tg2576 mice (normalized to beta-actin).
  • Figure 6 is a bar graph showing the results of beta-amyloid ELISA of brain lysates from vehicle control and rhTFAM treated aged Tg2576 mice as a ratio of ap42:ap40.
  • FIG 7 is a line graph showing the oxygen consumption rates (OCR) as a % deviation from control for control and abeta treated cells (oligomeric or fibrilar form) treated with vehicle control or rhTFAM at time point "compound.”
  • OCR oxygen consumption rates
  • FCCP maximal respiration at time point
  • rotenone rotenone
  • Figure 8 is a line graph showing the oxygen consumption rates (OCR) as a % deviation from control for abeta treated cells (oligomeric or fibrilar form) treated with vehicle control or rhTFAM at time point
  • FCCP maximal respiration at time point
  • rotenone rotenone
  • proteinopathy refers to any disease caused by a malformed protein. Some proteinopathies are neurodegenerative disorders.
  • amyloids are insoluble fibrous protein aggregates sharing specific structural traits.
  • amyloidosis or “amyloid related diseases” refers to a variety of conditions wherein amyloid proteins are abnormally deposited in organs or tissues and cause harm.
  • tauopathy reference to a neurodegenerative disease associated with the pathological aggregation of tau protein in the human brain.
  • mice and rats refer to a mammal, including, but not limited to, humans, rodents, such as mice and rats, and other animals.
  • treating includes inhibiting, alleviating, limiting the progression of or eliminating one or more symptoms, conditions, or comorbidities of the proteinopathy, amyloidosis, tauopathy, or other amyloid beta-related disease relative to a control.
  • polypeptides includes proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R),
  • Variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Modifications and changes can be made in the structure of the polypeptides disclosed and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide's biological functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties.
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a polypeptide is generally understood in the art. It is known that certain amino acids can be substituted for other amino acids having a similar hydropathic index or score and still result in a polypeptide with similar biological activity. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8);
  • tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments.
  • the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ⁇ 1); threonine (-0.4); alanine (-0.5); histidine (- 0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
  • isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide. In such changes, the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gin, His), (Asp: Glu, Cys, Ser), (Gin: Asn), (Glu: Asp), (Gly: Ala), (His: Asn,
  • embodiments of this disclosure thus contemplate functional or biological equivalents of a polypeptide as set forth above.
  • embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.
  • Identity is a relationship between two or more polypeptide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences.
  • Identity can also mean the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide.
  • Identity and similarity can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing:
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics
  • a polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy -terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide.
  • low stringency refers to conditions that permit a polynucleotide or polypeptide to bind to another substance with little or no sequence specificity.
  • purified and like terms relate to the isolation of a molecule or compound in a form that is substantially free (at least 60% free, preferably 75% free, and most preferably 90% free) from other components normally associated with the molecule or compound in a native environment.
  • isolated is meant to describe a compound of interest (e.g., nucleic acids, polypeptides, etc.) that is in an environment different from that in which the compound naturally occurs, e.g., separated from its natural milieu such as by concentrating a peptide to a concentration at which it is not found in nature.
  • isolated is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. Isolated nucleic acids or polypeptides are at least 60% free, preferably 75% free, and most preferably 90% free from other associated components.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water and emulsions such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • “Operably linked” refers to a juxtaposition wherein the components are configured so as to perform their usual function.
  • control sequences or promoters operably linked to a coding sequence are capable of effecting the expression of the coding sequence
  • an organelle localization sequence operably linked to protein will assist the linked protein to be localized at the specific organelle.
  • "Localization Signal or Sequence or Domain” or “Targeting Signal or Sequence or Domain” are used interchangeably and refer to a signal that directs a molecule to a specific cell, tissue, organelle, intracellular region or cell state.
  • the signal can be polynucleotide, polypeptide, or carbohydrate moiety or can be an organic or inorganic compound sufficient to direct an attached molecule to a desired location.
  • Exemplary targeting signals include mitochondrial localization signals from the precursor proteins list in U.S. Patent No. 8,039,587, and cell targeting signals known in the art such as those in Wagner et al, Adv Gen, 53 :333-354 (2005) the disclosures of which are specifically incorporated by reference herein in their entirety. It will be appreciated that the entire sequence need not be included, and modifications including truncations of these sequences are within the scope of the disclosure provided the sequences operate to direct a linked molecule to a specific cell type.
  • Targeting signals of the present disclosure can have 80 to 100% sequence identity to the mitochondrial localize signal or cell targeting signal sequences.
  • One class of suitable targeting signals includes those that do not interact with the targeted cell in a receptor: ligand mechanism.
  • targeting signals include signals having or conferring a net charge, for example a positive charge. Positively charged signals can be used to target negatively charged cell types such as neurons and muscle. Negatively charged signals can be used to target positively charged cells.
  • Tropism refers to the propensity of a molecule to be attracted to a specific cell, cell type or cell state.
  • tropism can refer to the way in which different viruses and pathogens have evolved to preferentially target to specific host species, or specific cell types within those species.
  • the propensity for a molecule to be attracted to a specific cell, cell type or cell state can be accomplished by means of a targeting signal.
  • Cell Type is a manner of grouping or classifying cells in the art.
  • the term cell type refers to the grouping of cells based on their biological character determined in part through common biological function, location, morphology, structure, expression of polypeptides, nucleotides or metabolites.
  • Cell State refers to the condition of a cell type. Cells are dynamic throughout their life and can achieve various states of differentiation, function, morphology and structure. As used herein, cell state refers to a specific cell type throughout its lifetime.
  • Cell surface marker refers to any molecule such as moiety, peptide, protein, carbohydrate, nucleic acid, antibody, antigen, and/or metabolite presented on the surface or in the vicinity of a cell sufficient to identify the cell as unique in either type or state.
  • compositions disclosed herein can reduce the level, formation, or production of amyloid proteins over time, reduce the expression of or phosphorylation of tau over time, reduce the level of amyloid- mediated inhibition of mitochondrial maximal respiration, increase ATP production in the brain, and increase the level of electron transport chain protein expression in the brain.
  • administration of a recombinant protein including a protein transduction domain, a mitochondrial localization signal, and a TFAM polypeptide reduced the level of pathogenic amyloid beta 42 (Abeta42) in a mouse model, reduced tau expression and tau hyperphosphorylation in a mouse model, and reduced Abeta42 inhibition of mitochondrial respiration in an in vitro assay for maximal mitochondrial respiration.
  • the recombinant protein also increased ATP production and electron transport chain protein expression in the brains of mice. Taken together, these results are consistent with a model in which the recombinant protein drives an increase in mitochondrial function which reduces or inhibits the formation of Abeta42 and reduces or inhibits tau expression and phosphorylation.
  • the model places mitochondrial dysfunction upstream of amyloid production, plaque formation, and tau hyperphosphorylation in proteinopathy, amyloidosis, and tauopathy disease pathways. It is therefore believed that the compositions disclosed herein are effective to treat a number of proteinopathies, amyloidosis, and tauopathies as discussed in more detail below.
  • compositions and methods can be used to limit the progression of, reduce, delay, or inhibit the level, formation, or production of amyloid proteins in subject over time; limit the progression of, reduce, delay, or inhibit the expression of or phosphorylation of tau in a subject over time, limit the progression of, reduce, delay, or inhibit the level of amyloid- mediated inhibition of mitochondrial maximal respiration in a subject;
  • compositions are administered in an effective amount to reduce the expression or
  • the subject has been diagnosed or is likely to be diagnosed with a proteinopathy, amyloidosis, a tauopathy or a disorder, condition, symptom or comorbidity thereof.
  • the disclosed methods typically include administering to the subject an effective amount of the disclosed compositions to limit the progression of, reduce, delay, or inhibit the level, formation, or production of amyloid proteins, for example beta amyloid 42, in subject over time; limit the progression of, reduce, delay, or inhibit the expression of or phosphorylation of tau in a subject over time, limit the progression of, reduce, delay, or inhibit the level of amyloid-mediated inhibition of mitochondrial maximal respiration in a subject; increase brain ATP production in the brain of a subject; increase the level of electron transport chain protein expression in the brain of a subject; or a combinations thereof.
  • amyloid proteins for example beta amyloid 42
  • compositions and methods can be used to treat one or more proteinopathies, amyloidosis, tauopathies or disorders, conditions, symptoms or comorbidities thereof.
  • the subject is a human subject.
  • the subject can be male or female.
  • proteinopathies, amyloidosis, and tauopathies occur with higher frequency in adult or elderly subjects relative to younger age groups, however other age groups can also be affected by these diseases and conditions. Therefore, newborns, infants, children, adolescents, adults, and elderly can be treated for a proteinopathy or amyloidosis using the compositions and methods disclosed herein. In some preferred embodiments the subject is elderly.
  • the composition is administered in an effective amount to treat one or more one or more symptoms or
  • compositions and methods limit the progression of, reduce, delay, or inhibit the level, formation, or production of amyloids; limit the progression of, reduce, delay, or inhibit the expression of or phosphorylation of tau; limit the progression of, reduce, delay, or inhibit the level of amyloid-mediated inhibition of mitochondrial maximal respiration; increase brain ATP production in the brain; increase the level of electron transport chain protein expression in the brain; or a combinations thereof in a treated subject relative to an untreated subject.
  • compositions and methods limit the progression of, reduce, decrease, or ameliorate one or more symptoms, characteristics or comorbidities of a proteinopathy, amyloidosis, or a tauopathy; reverse the progression of one or more symptoms, characteristics or comorbidities of a proteinopathy, amyloidosis, or a tauopathy; halt the progression of one or more symptoms, characteristics or comorbidities of a proteinopathy, amyloidosis, or a tauopathy; limit the progression of the occurrence of one or more symptoms, characteristics or comorbidities of an a proteinopathy, amyloidosis, or a tauopathy; inhibit the rate of development of one or more symptoms, characteristics or comorbidities or combinations thereof of a proteinopathy, amyloidosis, or a tauopathy in a treated subject relative to an untreated subject.
  • the untreated subject can be the treated subject prior to initiation of treatment, or a matched subject not receiving the treatment.
  • compositions for use with the disclosed methods are discussed in more detail below and typically include a mitochondrial DNA- binding polypeptide, preferably a mitochondrial transcription factor, such as transcription factor A- mitochondrial (TFAM) or mitochondrial DNA- binding fragment thereof.
  • the mitochondrial DNA- binding polypeptide can be a recombinant fusion protein including a mitochondrial DNA-binding polypeptide, a protein transduction domain, and optionally one or more targeting signals.
  • the recombinant protein is a transcription factor A- mitochondrial (TFAM) fusion protein including a protein transduction domain, and a mitochondrial localization signal.
  • Exemplary fusion proteins containing a mitochondrial transcription factor polypeptide are disclosed in U.S. Patent Nos. 8,039,587, 8,062,891, 8, 133,733, and U.S. Published Application Nos. 2009/0123468, 2009/0208478, and 2006/021 1647 all of which are specifically incorporated by reference herein in their entireties.
  • the disclosed compositions inhibit or reduce mitochondrial dysfunction, cause an increase in mitochondrial number, increase mitochondrial respiration, increase mitochondrial Electron Transport Chain (ETC) activity, increased oxidative phosphorylation, increased oxygen consumption, increased ATP production, or combinations thereof relative to a control.
  • ETC Electron Transport Chain
  • compositions that reduce oxidative stress or oxidative damage in the subject compared to a control.
  • compositions disclosed herein can be used to prevent, reduce, delay, or inhibit the formation or aggregation of misfolded proteins; prevent, reduce, delay, or inhibit the level, formation, or production of amyloid proteins, such as amyloid beta, in a subject over time; prevent, reduce, delay, or inhibit the expression of or phosphorylation of tau in a subject over time, prevent, reduce, delay, or inhibit the level of amyloid- mediated inhibition of mitochondrial maximal respiration in a subject; increase brain ATP production in the brain of a subject; increase the level of electron transport chain protein expression in the brain of a subject; and combinations thereof.
  • the compositions are particularly useful for treating a subject with, or likely to develop, a proteinopathy, amyloidosis, or a tauopathy.
  • Methods of treating or preventing proteinopathies and amyloidosis are disclosed.
  • the methods can include administering to a subject in need thereof an effective amount of one or more of the disclosed compositions to reduce, delay, or inhibit the expression or accumulation of one or more misfolded proteins.
  • compositions can be administrated to a subject in an effective amount to treat a proteinopathy, or symptom, characteristic or comorbidity thereof.
  • Proteinopathies include, but are not limited to, Alzheimer's disease, cerebral ⁇ -amyloid angiopathy, Retinal ganglion cell degeneration in glaucoma, Prion diseases, Parkinson's disease and other synucleinopathies, Tauopathies, Frontotemporal lobar degeneration (FTLD), FTLD-FUS, Amyotrophic lateral sclerosis (ALS), Huntington's disease and other triplet repeat disorders, Familial British dementia, Familial Danish dementia, Hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHWA-I), CADASIL, Alexander disease, Seipinopathies, Familial amyloidotic neuropathy, Senile systemic amyloidosis, Serpinopathies, AL (light chain) amyloidosis (primary systemic amyloidosis), AH (heavy chain
  • the subject has a mutation in a gene, such as the ⁇ , ABri, ADan, superoxide dismutase, a-synuclein, huntingtin, ataxins, or neuroserpin genes, which could lead to accumulation of malformed protein or protein aggregates which could trigger a pathological cascade leading to clinical manifestation of a proteinopathy.
  • a gene such as the ⁇ , ABri, ADan, superoxide dismutase, a-synuclein, huntingtin, ataxins, or neuroserpin genes, which could lead to accumulation of malformed protein or protein aggregates which could trigger a pathological cascade leading to clinical manifestation of a proteinopathy.
  • the subject may or may not be exhibiting physical symptoms of the proteinopathy at the time treatment is initiated.
  • compositions can also be administrated to a subject in an effective amount to treat amyloidosis, or symptom, characteristic or comorbidity thereof.
  • the amyloidosis is caused by the amyloid protein beta amyloid ( ⁇ ), medin (AMed), Apolipoprotein AI (AApoAl), atrial natriuretic factor (AANF), Cystatin (ACys), IAPP
  • AIAPP beta 2 microglobulin
  • ⁇ 2 ⁇ Transthyretin
  • GRR Gelsolin
  • AGel Lysozyme
  • huntingtin keratoepithelin
  • ACal alpha-synuclein
  • Prolactin APro
  • S-IBM serum amyloid A
  • AL immunoglobulin light chain AL
  • ArP PrPSc
  • Amyloidosis includes, but is not limited to, diseases such as
  • Alzheimer's disease (beta amyloid), aortic medial amyloid (Medin), atherosclerosis (Apolipoprotein AI), cardiac arrhythmias and isolated atrial amyloidosis (atrial natriuretic factor),cerebral amyloid angiopathy (beta amyloid), cerebral amyloid angiopathy - Icelandic type (Cystatin), diabetes mellitus type 2 (IAPP- Amylin), dialysis related amyloidosis (beta 2 microglobulin), familial amyloid polyneuropathy, (transthyretin), finnish amyloidosis (gelsolin), hereditary non-neuropathic systemic amyloidosis (lysozyme), Huntington's disease, (Huntingtin), lattice corneal dystrophy (keratoepithelin), medullary carcinoma of the thyroid (calcitonin), multiple myeloma (paraprotein), Parkinson's disease (alpha
  • immunoglobulin light chain AL primary cutaneous amyloidosis, AA amyloidosis, senile amyloid of atria of heart, familial visceral amyloidosis, Cerebral amyloid angiopathy (British-type and Danish-type), medullary carcinoma of the thyroid, familial corneal amyloidosis, prion disease systemic amyloidosis, leptomeningeal amyloidosis, haemodialysis-associated amyloidosis, and transmissible spongiform encephalopathies (PrPSc).
  • AL immunoglobulin light chain AL
  • AA amyloidosis AA amyloidosis
  • senile amyloid of atria of heart familial visceral amyloidosis
  • Cerebral amyloid angiopathy (British-type and Danish-type)
  • medullary carcinoma of the thyroid familial corneal amyloid
  • transmissible spongiform encephalopathies include, but are not limited to, human diseases such as Creutzfeld Jakob Disease, variant Creutzfeld Jakob Disease, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), kuru and Alpers' syndrome, and non- human diseases such as bovine spongiform encephalopathy (BSE, commonly known as mad cow disease) in cattle, chronic wasting disease (CWD) in elk and deer, and scrapie in sheep, transmissible mink encephalopathy, feline spongiform encephalopathy and ungulate spongiform encephalopathy.
  • human diseases such as Creutzfeld Jakob Disease, variant Creutzfeld Jakob Disease, Gerstmann-Straussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI), kuru and Alpers' syndrome
  • non- human diseases such as bovine spongiform encephalopathy (BSE, commonly known
  • compositions and methods to treat a disease characterized by increased amyloid beta expression, deposition, aggregation or plaque formation can include administering to a subject in need thereof a composition including an effective amount of a recombinant transcription factor A - mitochondrial (TFAM) or fusion protein thereof to reduce, delay, or inhibit the level, formation, or production of amyloid beta in the subject compared to a control.
  • TFAM recombinant transcription factor A - mitochondrial
  • Abeta-related diseases and disorders include, but are not limited to, Alzheimer's disease, cerebral amyloid angiopathy (also known as congophilic angiopathy), Lewy body dementia, retinal ganglion cell degeneration (such as in glaucoma), sporadic inclusion body myositis (sIBM) and hereditary inclusion body myopathy (hIBM).
  • cerebral amyloid angiopathy also known as congophilic angiopathy
  • Lewy body dementia also known as congophilic angiopathy
  • retinal ganglion cell degeneration such as in glaucoma
  • sIBM sporadic inclusion body myositis
  • hIBM hereditary inclusion body myopathy
  • the abeta-related disease or diseases treated using the disclosed method is not Alzheimer's disease or Lewy body dementia.
  • Abeta is formed after sequential cleavage of the amyloid precursor protein (APP), a transmembrane glycoprotein of undetermined function.
  • APP amyloid precursor protein
  • APP can be processed by ⁇ -, ⁇ - and ⁇ -secretases;
  • Abeta protein is generated by successive action of the ⁇ and ⁇ secretases.
  • the ⁇ secretase which produces the C-terminal end of the Abeta peptide, cleaves within the transmembrane region of APP and can generate a number of isoforms of between 36 and 43 amino acid residues in length.
  • the most common isoforms are Abeta40 and Abeta42; the longer form is typically produced by cleavage that occurs in the endoplasmic reticulum, while the shorter form is produced by cleavage in the trans-Golgi network.
  • the Abeta40 form is the more common of the two, but Abeta42 is the more fibrillogenic and is thus associated with disease states.
  • the Examples below show that administration of a recombinant TFAM fusion protein reduced the expression or accumulation of Abeta42, and reduce the ratio of Abetal-42:Abetal-40, in a mouse model over time.
  • compositions are administered in an effective amount to reduce the expression or accumulation of Abeta42, or to reduce the ratio of Abeta 1-42 :Abeta 1-40, or a combination thereof.
  • the compositions are used to reduce, prevent or delay the amyloid beta expression, deposition, aggregation or plaque formation in a subject in need thereof.
  • the method typically includes administering to the subject a composition including an effective amount of a recombinant transcription factor A - mitochondrial (TFAM) or fusion protein thereof to reduce, delay, or inhibit the level, formation, or production of amyloid beta in the subject compared to a control.
  • TFAM recombinant transcription factor A - mitochondrial
  • the method can include treating subjects that have not yet been diagnosed with a specific disease or disorder.
  • compositions and methods can also be used to treat diseases characterized by increased tau expression, increased tau
  • a method of treating a disease or disorder characterized by increased tau expression, increased tau phosphorylation, or pathologies associated with the aggregation of tau protein in the brain can include administering to a subject in need thereof a composition including an effective amount of a recombinant transcription factor A - mitochondrial (TFAM) protein to reduce, delay, or inhibit the expression of or
  • tauopathies and conditions associated therewith include, but are not limited to Alzheimer's disease, Argyrophilic grain disease (AGD), Chronic Traumatic Encephalopathy (CTE), Dementia pugilistica (chronic traumatic encephalopathy), frontotemporal dementia,
  • frontotemporal lobar degeneration gangliocytoma, Ganglioglioma, gangliocytoma, Lytico-Bodig disease (Parkinson-dementia complex of Guam), meningioangiomatosis, Frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), Pick's disease, Progressive supranuclear palsy, subacute sclerosing panencephalitis,tangle-predominant dementia, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, lipofuscinosis, corticobasal degeneration.
  • the taupathy is a non-Alzheimer's tauopathy.
  • Non-Alzheimer's tauopathies are sometimes grouped together as "Pick's complex.”
  • the compositions are used to reduce, prevent or delay tau expression, tau phosphorylation, or pathologies associated with the aggregation of tau protein in the brain.
  • the method can include administering to the subject an effective amount of a recombinant transcription factor A - mitochondrial (TFAM) protein to reduce, delay, or inhibit the expression of or phosphorylation of tau in the subject compared to a control.
  • TFAM transcription factor A - mitochondrial
  • the method can include treating subjects that have not yet been diagnosed with a specific disease or disorder.
  • Combination therapies include administering the composition containing an effective amount of the fusion proteins disclosed herein in combination with one or more second therapeutic agents.
  • the composition itself can include a combination of a polynucleotide-binding polypeptide and one or more second therapeutic agents.
  • a first composition including a polynucleotide-binding polypeptide is co-administered with one or more additional compositions including one or more second therapeutic agents.
  • Preferred polynucleotide- binding polypeptides include mitochondrial transcriptions factors such as TFAM.
  • the second therapeutic agent can be a conventional therapeutic agent for treating a proteinopathy, amyloidosis, or a tauopathy.
  • the second agent can determined based on the disease to be treated. For example, if the disease is Alzheimer's disease, the compositions disclosed herein can be coadministered with a conventional Alzheimer's disease treatment such as ⁇ 42 immunization (Wisniewski and Konietzko, Lancet Neurol, 7:805-811 (2008)), tarenflurbil (FlurizanTM, Myriad Pharmaceuticals) which is believed to act by decreasing the production of ⁇ 42 (Aisen, Lancet Neurol, 7:468- 469 (2008)), and tramiprosate (AlzhemedTM, Neurochem Inc.) which was designed to bind to beta amyloid peptide and prevent it from reacting with glycosaminoglycans (Aisen et al., Curr. Alzheimer Res., 4:473-478 (2007)).
  • ⁇ 42 immunization Wi
  • compositions can be administered prophylactically,
  • the composition can be administered during a period before, during, or after onset of one or more symptoms of the disorders disclosed herein.
  • the composition is administered with one or more additional therapeutic agents as part of a co-therapy, one or more second treatments, or combinations thereof.
  • compositions may be administered in a physiologically acceptable carrier to a host.
  • Preferred methods of administration include systemic or direct administration to a cell.
  • the compositions can be administered to a cell or patient, as is generally known in the art for protein therapies.
  • One embodiment provides a pharmaceutical composition including a recombinant mitochondrial DNA-binding protein, for example a fusion protein.
  • the fusion protein preferably contains polynucleotide- binding domain of a mitochondrial DNA-binding protein, a targeting domain, and a protein transduction domain and a pharmaceutically acceptable carrier or excipient.
  • the polynucleotide-binding polypeptide includes TFAM or a fragment thereof capable of binding a polynucleotide.
  • the composition typically includes an effective amount of the fusion protein to prevent, reduce, delay, or inhibit the level, formation, or production of amyloid proteins in subject; reduce the expression or accumulation of Abeta42 in subject; reduce the ratio of Abeta42/Abeta40 in a subject in subject; prevent, reduce, delay, or inhibit the expression of tau or phosphorylation of tau in a subject; limit the progression of, reduce, delay, or inhibit the level of amyloid-mediated inhibition of mitochondrial maximal respiration in a subject; increase brain ATP production in the brain of a subject; increase the level of electron transport chain protein expression in the brain of a subject; or a combination thereof.
  • compositions can be combined in admixture with a
  • Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 17th edition, Osol, A. Ed. (198)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients or stabilizers 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; 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, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as Tween ® , Pluronics ® or PEG.
  • buffers such as phosphate, citrate and other organic acids
  • antioxidants including ascorbic acid
  • parenteral administration is characterized by administering a pharmaceutical composition through a physical breach of a subject's tissue.
  • Parenteral administration includes administering by injection, through a surgical incision, or through a tissue-penetrating non- surgical wound, and the like.
  • parenteral administration includes subcutaneous, intraperitoneal, intravenous, intraarterial, intramuscular, intrasternal injection, and kidney dialytic infusion techniques.
  • Parenteral formulations can include the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative.
  • Parenteral administration formulations include suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, reconstitutable dry (i.e. powder or granular) formulations, and implantable sustained-release or biodegradable formulations. Such formulations may also include one or more additional ingredients including suspending, stabilizing, or dispersing agents.
  • Parenteral formulations may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • Parenteral formulations may also include dispersing agents, wetting agents, or suspending agents described herein. Methods for preparing these types of formulations are known.
  • Sterile injectable formulations may be prepared using non-toxic parenterally-acceptable diluents or solvents, such as water, 1,3 -butane diol, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic monoglycerides or diglycerides.
  • Other parentally - administrable formulations include microcrystalline forms, liposomal preparations, and biodegradable polymer systems.
  • Compositions for sustained release or implantation may include pharmaceutically acceptable polymeric or hydrophobic materials such as emulsions, ion exchange resins, sparingly soluble polymers, and sparingly soluble salts.
  • compositions may be prepared, packaged, or sold in a buccal formulation.
  • Such formulations may be in the form of tablets, powders, aerosols, atomized solutions, suspensions, or lozenges made using known methods, and may contain from about 0.1% to about 20% (w/w) active ingredient with the balance of the formulation containing an orally dissolvable or degradable composition and/or one or more additional ingredients as described herein.
  • powdered or aerosolized formulations have an average particle or droplet size ranging from about 0.1 nanometers to about 200 nanometers when dispersed.
  • the composition can include one or more additional ingredients.
  • additional ingredients include: excipients, surface active agents, dispersing agents, inert diluents, granulating agents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents, preservatives, physiologically degradable compositions (e.g., gelatin), aqueous vehicles, aqueous solvents, oily vehicles and oily solvents, suspending agents, dispersing agents, wetting agents, emulsifying agents, demulcents, buffers, salts, thickening agents, fillers, emulsifying agents, antioxidants, antibiotics, antifungal agents, stabilizing agents, and pharmaceutically acceptable polymeric or hydrophobic materials.
  • Other additional ingredients which may be included in the pharmaceutical compositions are known. Suitable additional ingredients are described in Remington's Pharmaceutical Sciences, 17 th ed. Mack Publishing Co., Genaro, ed., Easton, Pa. (1985).
  • Dosages and desired concentrations of the polynucleotide-binding polypeptide disclosed herein in pharmaceutical compositions of the present disclosure may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al, Eds., Pergamon Press, New York 1989, pp. 42- 96.
  • composition can be administered intravenously in a wide dosing range from about 0.01 milligram per kilogram body weight (mg/kg) to about 10 mg/kg, alternatively about 0.01 milligram per kilogram body weight (mg/kg) to about 1.0 mg/kg, depending on patient's age and physical state, as well as dosing regimen and schedule.
  • the dose can be administered in separate administrations of 2, 3, 4, 5 or 6 doses.
  • the dose can be administered every day, every two days, every three days, every four days, every five days, every six days, every seven days, once every two weeks, or once a month.
  • the composition is lyophilized in 20 mM glutamate, 10 mg/mL trehalose, 30 mg/mL mannitol, pH 4.5 and
  • composition is lyophilized in 20 mM histidine, 10 mg/mL trehalose, 30 mg/mL mannitol, pH 6.5 and reconstituted in sterile water prior to use.
  • composition is dissolved in 20mM histidine, 150mM NaCl pH 6.5 and kept frozen prior to use.
  • compositions for use in the disclosed methods are also provided.
  • the disclosed compositions cause an increase in mitochondrial number, can increase mitochondrial respiration relative to a control, or both.
  • the composition typically includes an effective amount of a mitochondrial DNA-binding polypeptide.
  • mitochondrial DNA-binding polypeptides include, but are not limited to, mitochondrial transcription factors such as transcription factor A, mitochondrial (TFAM) having GenBank Accession No. mitochondrial NM_003201; transcription factor B l, mitochondrial (TFB 1M) having GenBank Accession No.
  • AF151833 transcription factor B2, mitochondrial (TFB2M) having
  • GenBank Accession No. AK026835 Polymerase (RNA) Mitochondrial (DNA directed) (POLRMT) having GenBank Accession No. NM_005035; and functional fragments, variants, and fusion polypeptides thereof.
  • the composition includes a recombinant fusion protein including a polynucleotide-binding polypeptide, a protein transduction domain, and optionally one or more targeting signals.
  • the disclosed compositions cause an increase in mitochondrial number, an increase in mitochondrial respiration, an increase mitochondrial Electron Transport Chain (ETC) activity, increased oxidative
  • the composition reduces oxidative stress.
  • Exemplary fusion proteins containing a mitochondrial transcription factor polypeptide are disclosed in U.S. Patent Nos. 8,039,587, 8,062,891, 8,133,733, and U.S. Published Application Nos. 2009/0123468,
  • compositions for use in the methods disclosed herein include an effective amount of a mitochondrial DNA-binding polypeptide optionally having a PTD and optionally having one or more targeting signals or domains.
  • the mitochondrial DNA-binding polypeptide is a polypeptide known to bind or package a mtDNA.
  • the mitochondrial DNA-binding polypeptide is a recombinant polypeptide.
  • the recombinant polypeptide can be used as a therapeutic agent either alone or in combination with a polynucleotide, or any other active agent.
  • the polynucleotide-binding domain includes mature TFAM, a functional fragment of TFAM, or a variant thereof.
  • the polynucleotide-binding polypeptide includes at least a portion of a member of the high mobility group (HMG) of proteins effective to bind a polynucleotide, for example an HMG box domain.
  • HMG high mobility group
  • TFAM refers to TFAM after it has been post-translationally modified and is in the form that is active in the mitochondrion.
  • a mature TFAM is one in which the endogenous mitochondrial signal sequence has been cleaved.
  • TFAM mitochondrial transcription factor A
  • variant TFAM can have 80%, 85%, 90%, 95%, 99% or greater sequence identity with a reference TFAM, for example naturally occurring TFAM having GenBank Accession No. NM_003201.
  • the variant TFAM has 80%, 85%, 90%, 95%, 99% or greater sequence identity with a reference TFAM.
  • the variant TFAM has 80%, 85%, 90%, 95%, 99% or greater sequence identity over the full-length of mature human TFAM.
  • TFAM is a member of the high mobility group (HMG) of proteins having two HMG-box domains. TFAM as well as other HMG proteins bind, wrap, bend, and unwind DNA.
  • HMG high mobility group
  • embodiments of the present disclosure include polynucleotide binding polypeptides including one or more polynucleotide binding regions of the HMG family of proteins, and optionally induce a structural change in the polynucleotide when the polypeptide binds or becomes associated with the polynucleotide.
  • the polynucleotide-binding polypeptide is full- length TFAM polypeptide, or variant therefore.
  • a preferred TFAM polypeptide has at least 80, 85, 90, 95, 99, or 100 percent sequence identity to the full-length TFAM precursor
  • the preprotein sequence includes a signal peptide as known as an "amino-terminal signal", or a "presequence” that facilitates translocation from the cytosol through the mitochondrial translocation machinery in the outer membrane called the Tom complex (Translocator outer membrane) as well as the machinery in the inner membrane called the Tim complex (Translocator Inner Membrane).
  • the signal sequence is cleaved by a protease such as MPP.
  • a mitochondrial protein with the signal sequence cleaved or removed can be referred to as a "mature" protein.
  • the polynucleotide-binding polypeptide is a mature TFAM polypeptide, or variant thereof.
  • the cleavable mitochondrial targeting sequence of a TFAM preprotein is amino acid residue 1 of SEQ ID NO: 1 to amino acid residue 42 of SEQ ID NO: 1, MAFLRSMWGV LSALGRSGAE LCTGCGSRLR SPFSFVYLPR WF (SEQ ID NO: 2).
  • a preferred TFAM polypeptide has at least 80, 85, 90, 95, 99, or 100 percent sequence identity to the mature TFAM sequence
  • the polynucleotide-binding polypeptide is functional fragment of TFAM, or variant therefore.
  • Functional fragments can be effective when administered alone, or can be effective when administered in combination with a polynucleotide.
  • Functional fragments of TFAM can include, but are not limited to, a fragment of full-length TFAM sufficient to bind non-specifically to a polynucleotide, a fragment of full- length TFAM sufficient to bind specifically to the mtDNA light strand promoter (LSP), the mtDNA heavy strand promoter 1 (HSP1), the mtDNA heavy stand promoter 2 (HSP2), or combinations thereof, a fragment of full- length TFAM sufficient to induce mitochondrial transcription, a fragment of full-length TFAM sufficient to induce oxidative phosphorylation, a fragment of full-length TFAM sufficient to induce mitochondrial biogenesis, and combinations thereof.
  • LSP mtDNA light strand promoter
  • HSP1 mtDNA heavy strand
  • mature TFAM includes four domains, a first HMG box (also referred to herein as HMG box 1), followed by a linker region (also referred to herein as linker), followed by a second HMG box (also referred to herein as HMG box 2), followed by a C-terminal tail.
  • Functional fragments of TFAM typically include one or more domains of mature TFAM, or a variant thereof.
  • the functional fragment includes one or more HMG box 1 domains of TFAM, one or more linker domains of TFAM, one or more HMG box 2 domains of TFAM, one or more C-terminal tail domains of TFAM, or combinations thereof.
  • the domains can be arranged in the same orientation of the domains of endogenous TFAM, or they can be rearranged so they are in a different order or orientation than the domains found in endogenous TFAM protein.
  • the functional fragment includes a first HMG box domain, and second HMG box domain linked to the first HMG box domain with a linker, typically a peptide linker.
  • the linker can be the endogenous linker domain of TFAM, or a heterologous linker that allows the first and the second HMG box domains to maintain their functional activity. Deletion studies characterizing the activity of different domains and hybrid constructs of TFAM are known in the art and characterized for example in Dairaghi, et al, J. Mol.
  • a functional fragment is one or more domains of TFAM according to SEQ ID NO: 3.
  • an HMG box 1 of TFAM can be a polypeptide including the sequence from amino acid residue 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of SEQ ID NO: 3 to amino acid residue 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 of SEQ ID NO: 3, or a variant thereof with 80, 85, 90, 95, 99, or greater than 99 percent sequence identity to the corresponding fragment of SEQ ID NO: 3.
  • a linker region of TFAM can be a polypeptide including the sequence from amino acid residue 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85 of SEQ ID NO: 3 to amino acid residue 105, 106, 107, 108, 109, 1 10, 1 11, 112, 113, 114, or 115 of SEQ ID NO: 3, or a variant thereof with 80, 85, 90, 95, 99, or greater than 99 percent sequence identity to the corresponding fragment of SEQ ID NO: 3.
  • An HMG box 2 of TFAM can be a polypeptide including the sequence from amino acid residue 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 11 1, 112, 113, 114, or 115 of SEQ ID NO: 3 to amino acid residue 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,
  • a C-terminal tail of TFAM can be a polypeptide including the
  • TFAM include one or more conservative amino acid substitutions relative to the corresponding reference sequence, for example SEQ ID NO:3, or a
  • One embodiment provides a TFAM polypeptide having one or more serine residues at positions 1, 2 and 13 SEQ ID NO:3
  • a preferred embodiment provides a TFAM polypeptide having serine 13 of SEQ ID NO:3 substituted for an alanine or threonine.
  • the variant TFAM polypeptides have improved mtDNA binding in the presence of glucose or elevated glucose levels.
  • Selected model organisms that have TFAM sequences that are useful in the compositions and methods disclosed herein include, but are not limited to those disclosed in Table 1 :
  • D. melanogaster ref:NP_524415.1 - mitochondrial transcription 34 % / 183 aa factor A [Drosophila melanogaster] (see ProtEST )
  • TFBIM Transcription Factor Bl, Mitochondrial
  • the polynucleotide-binding polypeptide can be transcription factor Bl, mitochondrial (TFBIM).
  • TFBIM transcription factor Bl
  • a preferred TFBIM has GenBank Accession No. AF 151833.
  • TFB1 is part of the complex involved in mitochondrial transcription. The process of transcription initiation in mitochondria involves three types of proteins: the mitochondrial RNA polymerase (POLRMT), mitochondrial transcription factor A (TFAM), and
  • TFBIM has about 1/10 the transcriptional activity of TFB2M, and both TFBs are also related to rRNA
  • TFBIM can bind S-adenosylmethionine and methylate mitochondrial 12S rRNA. Additionally, TFB IM and TFB2M can bind single-stranded nucleic acids.
  • a preferred TFBIM polypeptide has at least 80, 85, 90, 95, 99, or
  • the polynucleotide-binding polypeptide includes TFB2M.
  • TFB2M polypeptide has GenBank Accession No. AK026835.
  • TFB2M also possesses a Rossmann- fold making it part of the NAD-binding protein family.
  • TFB2M levels modulate mtDNA copy number and levels of mitochondrial transcripts as would be expected of a mitochondrial transcription factor. It is appreciated by those skilled in the art that increased activity of mitochondria causes an increase in mitochondrial biogenesis.
  • a preferred TFB2M polypeptide has at least 80, 85, 90, 95, 99, or
  • RNA Polymerase
  • POLRMT DNA directed
  • POLRMT polynucleotide-binding polypeptide that can be used to modulate mitochondrial biological activity
  • the POLRMT polypeptide has GenBank Accession No. NM_005035.
  • POLRMT is a mitochondrial RNA polymerase similar in structure to phage RNA polymerases. Unlike phage polymerases, POLRMT contains two pentatricopeptide repeat (PPR) domains involved in regulating mitochondrial transcripts. It is appreciated by those skilled in the art that deletion of regulatory domains enables constitutive function.
  • PPR pentatricopeptide repeat
  • a preferred POLRMT polypeptide has at least 80, 85, 90, 95, 99, or 100 percent sequence identity to
  • the polynucleotide-binding polypeptide is a non-TFAM HMG domain.
  • the HMG domain includes a global fold of three helices stabilized in an " L-shaped' configuration by two hydrophobic cores.
  • the high mobility group chromosomal proteins HMGl or HMG2 which are common to all eukaryotes, bind DNA in a non- sequence-specific fashion, for example to promote chromatin function and gene regulation. They can interact directly with nucleosomes and are believed to be modulators of chromatin structure. They are also important in activating a number of regulators of gene expression, including p53, Hox transcription factors and steroid hormone receptors, by increasing their affinity for DNA.
  • HMG proteins include HMG- 1/2, HMG-I(Y) and HMG- 14/17.
  • the HMG-l/2-box proteins can be further distinguished into three subfamilies according to the number of HMG domains present in the protein, their specific of sequence recognition and their evolutionary relationship.
  • the first group contains chromosomal proteins bound to DNA with no sequence specificity (class I, HMG1 and HMG2), the second contains ribosomal and mitochondrial transcription factors which show sequence specificity in the presence of another associating factor when bound with DNA (class II, yeast ARS binding protein ABF-2, UBF and mitochondrial transcription factor mtTF-1), and the third contains gene-specific
  • HMG1/2- box DNA binding domain is about 75 to about 80 amino acids and contains highly conserved proline, aromatic and basic residues. Common properties of HMG domain proteins include interaction with the minor groove of the DNA helix, binding to irregular DNA structure, and the capacity to modulate DNA structure by bending.
  • SOX SRY-type HMG box proteins have critical functions in a number of developmental processes, including sex determination, skeleton formation, pre-B and T cell development and neural induction.
  • SOX9 plays a direct role during chondrogenesis by binding and activating the chondrocyte- specific enhancer of the Col2al gene. Loss of SOX9 gene function leads to the genetic condition known as Campomelic Dysplsia (CD), a form of dwarfism characterized by extreme skeletal malformation, and one in which three-quarters of XY individual are either intersexes or exhibit male to female sex reversal.
  • CD Campomelic Dysplsia
  • the preferred DNA-binding site of SOX9 have been defined to be AGAACAATGG (SEQ ID NO: 6), which contains the SOX core-binding element (SCBE), AACAAT, flanking 5' AG and 3' GG nucleotides enhance binding by SOX9.
  • the recombinant polynucleotide-binding polypeptide has at least one HMG box domain, generally at least two, more particularly 2-5 HMG box domains.
  • the HMG box domain can bind to an AT rich DNA sequence, for example, using a large surface on the concave face of the protein, to bind the minor groove of the DNA. This binding bends the DNA helix axis away from the site of contact. The first and second helices contact the DNA, their N-termini fitting into the minor groove whereas helix 3 is primarily exposed to solvent. Partial intercalation of aliphatic and aromatic residues in helix 2 occurs in the minor groove.
  • the polynucleotide-binding polypeptide can have at least one polynucleotide binding domain, typically two or more polynucleotide binding domains.
  • the polynucleotide binding domains can be the same or different.
  • the polynucleotide-binding polypeptide can include at least one HMG box in combination with one or more DNA binding domains selected from the group consisting of an HMG box, homeodomain and POU domain; zinc finger domain such as C2H2 and C2C2; amphipathic helix domain such as leucine zipper and helix-loop-helix domains; and histone folds.
  • the polynucleotide binding domain can be specific for a specific polynucleotide sequence, or preferably non- specifically binds to a polynucleotide.
  • the polynucleotide- binding polypeptide can have more a combination of at least one
  • polynucleotide binding domain that binds in a sequence specific manner and at least one polynucleotide binding-domain that binds DNA non-specifically. f. Helix-turn-helix
  • Certain embodiments provide polynucleotide-binding polypeptides having a helix-turn-helix motif or at least a polynucleotide binding region of a helix-turn-helix protein.
  • Helix-turn-helix proteins have a similar structure to bacterial regulatory proteins such as the 1 repressor and cro proteins, the lac repressor and so on which bind as dimers and their binding sites are palindromic. They contain 3 helical regions separated by short turns which is why they are called helix-turn-helix proteins.
  • One protein helix (helix 3) in each subunit of the dimer occupies the major groove of two successive turns of the DNA helix.
  • polynucleotide-binding polypeptides can form dimers or other multi- component complexes, and have 1 to 3 helices.
  • the polynucleotide-binding polypeptide includes a homeodomain or a portion of a homeodomain protein.
  • Homeodomain proteins bind to a sequence of 180 base pairs initially identified in a group of genes called homeotic genes. Accordingly, the sequence was called the homeobox. The 180 bp corresponds to 60 amino acids in the corresponding protein. This protein domain is called the homeodomain.
  • Homeodomain-containing proteins have since been identified in a wide range of organisms including vertebrates and plants. The homeodomain shows a high degree of sequence conservation.
  • the homeodomain contains 4 a helical regions. Helices II and III are connected by 3 amino acids comprising a turn. This region has a very similar structure to helices II and III of bacterial DNA binding proteins.
  • Zinc finger proteins have a domain with the general structure: Phe (sometimes Tyr) - Cys - 2 to 4 amino acids - Cys - 3 amino acids - Phe
  • Another type of zinc finger which binds zinc between 2 pairs of cysteines has been found in a range of DNA binding proteins.
  • the general structure of this type of zinc finger is: Cys - 2 amino acids - Cys - 13 amino acids - Cys - 2 amino acids - Cys.
  • This is called a C2C2 zinc finger. It is found in a group of proteins known as the steroid receptor superfamily, each of which has 2 C2C2 zinc fingers.
  • Another embodiment provides a modified polynucleotide-binding polypeptide having a leucine zipper or at least a portion of a leucine zipper protein.
  • the first leucine zipper protein was identified from extracts of liver cells, and it was called C/EBP because it is an enhancer binding protein and it was originally thought to bind to the CAAT promoter proximal sequence. C/EBP will only bind to DNA as a dimer. The region of the protein where the two monomers join to make the dimer is called the dimerization domain. This lies towards the C-terminal end of the protein.
  • the amino acid sequence was examined it was found that a leucine residue occurs every seventh amino acid over a stretch of 35 amino acids. If this region were to form an a helix then all of these leucines would align on one face of the helix.
  • leucine has a hydrophobic side chain
  • one face of the helix is very hydrophobic.
  • the opposite face has amino acids with charged side chains which are hydrophilic.
  • the combination of hydrophobic and hydrophilic characteristics gives the molecule is amphipathic moniker.
  • Adjacent to the leucine zipper region is a region of 20-30 amino acids which is rich in the basic (positively charged) amino acids lysine and arginine. This is the DNA binding domain - often referred to as the bZIP domain - the basic region of the leucine zipper.
  • C/EBP is thought to bind to DNA by these bZIP regions wrapping round the DNA helix
  • the leucine zipper - bZIP structure has been found in a range of other proteins including the products of the jun and fas oncogenes.
  • C/EBP binds to DNA as a homodimer of identical subunits
  • fos cannot form homodimers at all and jun/jun homodimers tend to be unstable.
  • fos/jun heterodimers are much more stable.
  • AP 1 binds to a variety of promoters and enhancers and activates transcription.
  • the consensus API binding site is TGACTCA which is palindromic
  • Helix-loop-helix proteins are similar to leucine zippers in that they form dimers via amphipathic helices. They were first discovered as a class of proteins when a region of similarity was noticed between two enhancer binding proteins called E47 and E12. This conserved region has the potential to form two amphipathic separated by a loop hence helix-loop-helix.
  • a DNA binding domain again rich in basic amino acids and referred to as the bHLH domain.
  • the modified polynucleotide-binding polypeptide includes a histone polypeptide, a fragment of a histone polypeptide, or at least one histone fold.
  • Histone folds exist in histone polypeptides monomers assembled into dimers.
  • Histone polypeptides include H2A, H2B, H3, and H4 which can form heterodimers H2A-2B and H3-H4. It will be appreciated that histone-like polypeptides can also be used in the disclosed compositions and methods.
  • Histone-like polypeptides include, but are not limited to, HMf or the histone from Methanothermous fervidus, other archaeal histones known in the art, and histone-fold containing polypeptides such as MJ1647, CBF, TAFII or transcription factor IID, SPT3, and Drl-DRAP (Sanderman, K., et al., Cell. Mol. Life Sci.
  • the polynucleotide-binding polypeptide is fusion protein modified to include a protein transduction domain (PTD).
  • PTD protein transduction domain
  • a "protein transduction domain” or PTD refers to a polypeptide, polynucleotide, carbohydrate, organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • a PTD attached to another molecule facilitates the molecule traversing membranes, for example going from extracellular space to intracellular space, or cytosol to within an organelle.
  • the protein transduction domain is a polypeptide.
  • a protein transduction domain can be a polypeptide including positively charged amino acids.
  • PTDs Protein transduction domains (PTD), also known as a cell penetrating peptides (CPP), are typically polypeptides including positively charged amino acids.
  • PTDs are known in the art, and include but are not limited to small regions of proteins that are able to cross a cell membrane in a receptor-independent mechanism (Kabouridis, P., Trends in Biotechnology (1 1):498-503 (2003)). Although several PTDs have been documented, the two most commonly employed PTDs are derived from TAT (Frankel and Pabo, Cell, 55(6): 1 189-93(1988)) protein of HIV and
  • Exemplary protein transduction domains include polypeptides with 1 1 Arginine residues, or positively charged polypeptides or polynucleotides having 8-15 residues, preferably 9-11 residues.
  • the Antennapedia homeodomain is 68 amino acid residues long and contains four alpha helices.
  • Penetratin is an active domain of this protein which consists of a 16 amino acid sequence derived from the third helix of Antennapedia.
  • TAT protein consists of 86 amino acids and is involved in the replication of HIV- 1.
  • the TAT PTD consists of an 11 amino acid sequence domain (residues 47 to 57; YGRKKRRQRR R (SEQ ID NO:7)) of the parent protein that appears to be critical for uptake. Additionally, the basic domain Tat(49-57) or RKKRRQRRR (SEQ ID NO: 8) has been shown to be a PTD.
  • TAT has been favored for fusion to proteins of interest for cellular import.
  • modifications to TAT including substitutions of Glutatmine to Alanine, i.e., Q- ⁇ A, have demonstrated an increase in cellular uptake anywhere from 90% (Wender et al, Proc Natl Acad Sci US A., 97(24): 13003-8 (2000)) to up to 33 fold in mammalian cells. (Ho et al, Cancer Res., 61(2):474-7 (2001)).
  • PTDs can include a sequence of multiple arginine residues, referred to herein as poly-arginine or poly-ARG.
  • sequence of arginine residues is consecutive.
  • sequence of arginine residues is non-consecutive.
  • a poly- ARG can include at least 7 arginine residues, more preferably at least 8 arginine residues, most preferably at least 1 1 arginine residues.
  • the poly-ARG includes between 7 and 15 arginine residues, more preferably between 8 and 15 arginine residues. In some embodiments the poly-ARG includes between 7 and 15, more preferably between 8 and 15 consecutive arginine residues.
  • An example of a poly-ARG is RRRRRRR (SEQ ID NO:9). Additional exemplary PTDs include but are not limited to; RRQRRTSKLM KR (SEQ ID NO: 10);
  • WEAKLAKALA KALAKHLAKA LAKALKCEA (SEQ ID NO: 12); and RQIKIWFQNR RMKWKK (SEQ ID NO: 13).
  • polynucleotide-binding polypeptide includes an endosomal escape sequence that enhances escape of the polypeptide-binding protein from
  • the endosomal escape sequence is part of, or consecutive with, the protein transduction domain. In some embodiments, the endosomal escape sequence is non-consecutive with the protein transduction domain. In some embodiments the endosomal escape sequence includes a portion of the hemagglutinin peptide from influenza (HA).
  • HA hemagglutinin peptide from influenza
  • GDIMGEWG NEIFGAIAGF LG (SEQ ID NO: 14).
  • the polynucleotide-binding polypeptide is modified to include one or more targeting signals or domains.
  • the targeting signal can include a sequence of monomers that facilitates in vivo localization of the molecule.
  • the monomers can be amino acids, nucleotide or nucleoside bases, or sugar groups such as glucose, galactose, and the like which form carbohydrate targeting signals.
  • Targeting signals or sequences can be specific for a host, tissue, organ, cell, organelle, non-nuclear organelle, or cellular compartment.
  • the polynucleotide-binding polypeptide includes both a cell-specific targeting domain and an organelle specific targeting domain to enhance delivery of the polypeptide to a subcellular organelle of a specific cells type.
  • the polynucleotide-binding polypeptide is modified to target a subcellular organelle.
  • Targeting of the disclosed polypeptides to organelles can be accomplished by modifying the disclosed compositions to contain specific organelle targeting signals. These sequences can target organelles, either specifically or non-specifically.
  • the interaction of the targeting signal with the organelle does not occur through a traditional receptor: ligand interaction.
  • the eukaryotic cell comprises a number of discrete membrane bound compartments, or organelles.
  • the structure and function of each organelle is largely determined by its unique complement of constituent polypeptides. However, the vast majority of these polypeptides begin their synthesis in the cytoplasm. Thus organelle biogenesis and upkeep require that newly synthesized proteins can be accurately targeted to their appropriate compartment. This is often accomplished by amino-terminal signaling sequences, as well as post-translational modifications and secondary structure.
  • Organelles can have single or multiple membranes and exist in both plant and animal cells. Depending on the function of the organelle, the organelle can consist of specific components such as proteins and cofactors. The polypeptides delivered to the organelle can enhance or contribute to the functioning of the organelle. Some organelles, such as mitochondria and chloroplasts, contain their own genome. Nucleic acids are replicated, transcribed, and translated within these organelles. Proteins are imported and metabolites are exported. Thus, there is an exchange of material across the membranes of organelles. Exemplary organelles include the nucleus, mitochondrion, chloroplast, lysosome, peroxisome, Golgi, endoplasmic reticulum, and nucleolus. Synthetic organelles can be formed from lipids and can contain specific proteins within the lipid membranes. Additionally, the content of synthetic organelles can be manipulated to contain
  • polynucleotide-binding polypeptides that specifically target mitochondria.
  • Mitochondria contain the molecular machinery for the conversion of energy from the breakdown of glucose into adenosine triphosphate (ATP). The energy stored in the high energy phosphate bonds of ATP is then available to power cellular functions. Mitochondria are mostly protein, but some lipid, DNA and RNA are present. These generally spherical organelles have an outer membrane surrounding an inner membrane that folds (cristae) into a scaffolding for oxidative phosphorylation and electron transport enzymes. Most mitochondria have flat shelf-like cristae, but those in steroid secreting cells may have tubular cristae. The mitochondrial matrix contains the enzymes of the citric acid cycle, fatty acid oxidation and mitochondrial nucleic acids.
  • Mitochondrial DNA is double stranded and circular.
  • Mitochondrial RNA comes in the three standard varieties; ribosomal, messenger and transfer, but each is specific to the mitochondria. Some protein synthesis occurs in the mitochondria on mitochondrial ribosomes that are different than cytoplasmic ribosomes. Other mitochondrial proteins are made on cytoplasmic ribosomes with a signal peptide that directs them to the mitochondria.
  • the metabolic activity of the cell is related to the number of cristae and the number of mitochondria within a cell. Cells with high metabolic activity, such as heart muscle, have many well developed mitochondria. New mitochondria are formed from preexisting mitochondria when they grow and divide.
  • the inner membranes of mitochondria contain a family of proteins of related sequence and structure that transport various metabolites across the membrane.
  • Their amino acid sequences have a tripartite structure, made up of three related sequences about 100 amino acids in length.
  • the repeats of one carrier are related to those present in the others and several characteristic sequence features are conserved throughout the family.
  • Mitochondrial targeting agents generally consist of a leader sequence of highly positively charged amino acids. This allows the protein to be targeted to the highly negatively charged mitochondria. Unlike
  • the mitochondrial localization signal of some embodiments is drawn to mitochondria because of charge. Therefore, in some embodiments, the mitochondrial targeting agent is a protein transduction domain including but not limited to the protein transduction domains discussed in detail above.
  • Mitochondrial targeting agents also include short peptide sequences (Yousif, et al, Chembiochem., 10(13):2131 (2009), for example mitochondrial transporters-synthetic cell-permeable peptides, also known as mitochondria-penetrating peptides (MPPs), that are able to enter mitochondria.
  • MPPs are typically cationic, but also lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane.
  • MPPs can include alternating cationic and hydrophobic residues (Horton, et al, Chem Biol, 15(4):375-82 (2008)).
  • Some MPPs include delocalized lipophilic cations (DLCs) in the peptide sequence instead of, or in addition to natural cationic amino acids (Kelley, et al, Pharm. Res., 201 1 Aug 1 1 [Epub ahead of print]).
  • DLCs delocalized lipophilic cations
  • Other variants can be based on an oligomeric carbohydrate scaffold, for example attaching guanidinium moieties due to their delocalized cationic form (Yousif, et al, Chembiochem., 10(13):2131 (2009).
  • Mitochondrial targeting agents also include mitochondrial localization signals or mitochondrial targeting signals.
  • Many mitochondrial proteins are synthesized as cytosolic precursor proteins containing a leader sequence, also known as a presequence, or peptide signal sequence.
  • cytosolic chaperones deliver the precursor protein to
  • GIP General Import Pore
  • TOM Translocase of Outer Membrane
  • the precursor protein is translocated through TOM, and the intermembrane space by small TIMs to the TIM23 or 22 (Translocase of Inner Membrane) at the inner membrane.
  • the targeting sequence is cleaved off by mtHsp70.
  • a protein in order to enter the mitochondria, a protein generally must interact with the mitochondrial import machinery, consisting of the Tim and Tom complexes (Translocase of the Inner/Outer
  • Mitochondrial Membrane With regard to the mitochondrial targeting signal, the positive charge draws the linked protein to the complexes and continues to draw the protein into the mitochondria.
  • the Tim and Tom complexes allow the proteins to cross the membranes. Accordingly, one embodiment of the present disclosure delivers compositions of the present disclosure to the inner mitochondrial space utilizing a positively charged targeting signal and the mitochondrial import machinery.
  • PTD-linked compounds containing a mitochondrial localization signal do not seem to utilize the TOM/TIM complex for entry into the mitochondrial matrix, see Del Gaizo et al. Mol Genet Metab. 80(1- 2): 170-80 (2003).
  • the N-terminal region of the proteins can be used to target molecules to the mitochondrion.
  • sequences are known in the art, see for example, U.S. Patent No. 8,039,587, which is specifically incorporated by reference herein.
  • the identification of the specific sequences necessary for translocation of a linked compound into a mitochondrion can be determined using predictive software known to those skilled in the art, including the tools located at http://ihg.gsf.de/ihg/mitoprot.html. Using the software the predicted sequence from Etfa that can be used to target the disclosed composition is
  • the mitochondrial targeting agent is the mitochondrial localization signal of a mangano-superoxide dismutase (also referred to herein as "SOD2" and “Mn-SOD” and "superoxide dismutase
  • mitochondrial targeting signal includes the amino acid sequence
  • the composition is preferentially delivered to the mitochondrial using a mitochondrial delivery vehicle, such as a lipid raft, mitochondrially targeted nanoparticle, or mitochondriotropic liposome.
  • a mitochondrial delivery vehicle such as a lipid raft, mitochondrially targeted nanoparticle, or mitochondriotropic liposome.
  • one or more polynucleotide-binding polypeptides can be associated with, encapsulated within, dispersed in or on, or covalently attached to the mitochondrial delivery vehicle.
  • polynucleotide-binding polypeptides are encapsulated, coupled to, or otherwise associated with mitochondriotropic liposomes.
  • Mitochondriotrophic liposomes are cationic liposomes that can be used to deliver an encapsulated agent to the mitochondria of a cell.
  • Mitochondriotropic liposomes are known in the art. See, for example, U.S. Patent Application Publication No. US 2008/0095834 to Weissig, et al, which is specifically incorporated by reference herein in its entirety.
  • Mitochondriotropic liposomes are liposomes which contain a hydrophobized amphiphilic delocalized cation, such as a triphenylphosphonium or a quinolinium moiety, incorporated into or conjugate to the lipid membrane of the liposome.
  • a hydrophobized amphiphilic delocalized cation such as a triphenylphosphonium or a quinolinium moiety
  • polynucleotide-binding polypeptides are encapsulated within, dispersed in, associated with, or conjugated to a nanoparticle functionalized with one or more mitochondrial targeting agents.
  • the nanoparticle may contain one or be functionalized with one or more lipophilic cations or polypeptide targeting agents.
  • the nanoparticles may be formed from one or more polymers, copolymers, or polymer blends.
  • the one or more polymers, copolymers, or polymer blends are biodegradable.
  • suitable polymers include, but are not limited to, polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polycaprolactones; poly(orthoesters); polyanhydrides; poly(phosphazenes); poly(hydroxyalkanoates); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides;
  • polyesters poly(dioxanones); poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals ; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes;
  • poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), and copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), polyvinylpyrrolidone), poly(hydroxy alkylmethacrylamide), poly(hydroxy
  • the mitochondrial targeting agents are polypeptides that are covalently linked to the surface of the nanoparticle after particle formulation.
  • the mitochondrial targeting agents are lipophilic cations that are covalently bound to the particle surface.
  • a cationic polymer is incorporated into the particle to target the particle to the mitochondrion.
  • Polynucleotide-binding polypeptides can also be targeted to the mitochondria using lipid rafts or other synthetic vesicle compositions. See, for example, U.S. Patent Application Publication No. US 2007/0275924 to Khan, et al, which is specifically incorporated by reference herein in its entirety.
  • the lipid raft compositions can include cholesterol, and one or more lipids selected from the group consisting of sphingomylein, gangliosides, phosphatidylethanolamine, phosphatidylserine,
  • lipid rafts can be prepared and loaded with one or more polynucleotide-binding polypeptides using methods known in the art. See, for example, U.S. Patent No.
  • a preferred polynucleotide-binding polypeptide that targets mitochondria has at least 80, 85, 90, 95, 99 or 100 percent sequence identity to
  • ARRRRRRRRR RRMAFLRSMW GVLSALGRSG AELCTGCGSR LRSPFSFVYL PRWFSSVLAS CPKKPVSSYL RFSKEQLPIF KAQNPDAKTT ELIRRIAQRW RELPDSKKKI YQDAYRAEWQ VYKEEISRFK EQLTPSQIMS LEKEIMDKHL KRKAMTKKKE LTLLGKPKRP RSAYNVYVAE RFQEAKGDSP QEKLKTVKEN WKNLSDSEKE LYIQHAKEDE TRYHNEMKSW EEQMIEVGRK DLLRR IKKQ RKYGAEEC (SEQ ID NO:23).
  • Another embodiment provides a nucleic acid encoding the polypeptide according to SEQ ID NO:22 is
  • sequence encoding the protein transduction domain is underlined, and the sequence encoding the mitochondrial localization signal is double underline.
  • Still another embodiment provides a nucleic acid having at least 80, 85, 90, 95, 99 or more percent sequence identity to SEQ ID NO:24
  • Another preferred polynucleotide-binding polypeptides that targets mitochondria has at least 80, 85, 90, 95, 97, 99, or 100 percent sequence identity to
  • the recombinant polypeptide is encoded by a nucleic acid having at least 80, 85, 90, 95, 97, 99, or 100% sequence identity to
  • the mitochondrial targeting signal, domain, or agent does not permanently damage the mitochondrion, for example the mitochondrial membrane, or otherwise impair mitochondrial function,
  • proteins of interest disclosed herein can be modified to target a specific cell type or population of cells.
  • the proteins of interest can be modified with galactosyl- terminating macromolecules to target the polypeptide of interest to the liver or to liver cells.
  • the modified polypeptide of interest selectively enters hepatocytes after interaction of the carrier galactose residues with the asialoglycoprotein receptor present in large amounts and high affinity only on these cells.
  • the targeting signal binds to its ligand or receptor which is located on the surface of a target cell such as to bring the composition and cell membranes sufficiently close to each other to allow penetration of the composition into the cell.
  • the targeting molecule is selected from the group consisting of an antibody or antigen binding fragment thereof, an antibody domain, an antigen, a T-cell receptor, a cell surface receptor, a cell surface adhesion molecule, a major histocompatibility locus protein, a viral envelope protein and a peptide selected by phage display that binds specifically to a defined cell.
  • Targeting a polypeptide of interest to specific cells can be accomplished by modifying the polypeptide of interest to express specific cell and tissue targeting signals. These sequences target specific cells and tissues. In some embodiments the interaction of the targeting signal with the cell does not occur through a traditional receptor: ligand interaction.
  • the eukaryotic cell comprises a number of distinct cell surface molecules. The structure and function of each molecule can be specific to the origin, expression, character and structure of the cell. Determining the unique cell surface complement of molecules of a specific cell type can be determined using techniques well known in the art.
  • compositions are provided that enable the addition of cell surface antigen specific antibodies to the composition for targeting the delivery of polynucleotide-binding polypeptide.
  • Exemplary cell surface antigens are disclosed in Wagner et al, Adv Gen, 53 :333-354 (2005) which is specifically incorporated by reference herein in its entirety.
  • peptidyl hormones can be used a targeting moieties to target delivery to those cells which possess receptors for such hormones.
  • Chemokines and cytokines can similarly be employed as targeting signals to target delivery of the complex to their target cells.
  • a variety of technologies have been developed to identify genes that are preferentially expressed in certain cells or cell states and one of skill in the art can employ such technology to identify targeting signals which are preferentially or uniquely expressed on the target tissue of interest.
  • the targeting signal is directed to cells of the nervous system, including the brain and peripheral nervous system.
  • Cells in the brain include several types and states and possess unique cell surface molecules specific for the type.
  • cell types and states can be further characterized and grouped by the presentation of common cell surface molecules.
  • the targeting signal is directed to specific neurotransmitter receptors expressed on the surface of cells of the nervous system.
  • the distribution of neurotransmitter receptors is well known in the art and one so skilled can direct the compositions described by using neurotransmitter receptor specific antibodies as targeting signals.
  • the targeting signal consists of a neurotransmitter or ligand capable of specifically binding to a neurotransmitter receptor.
  • the targeting signal is specific to cells of the nervous system which may include astrocytes, microglia, neurons, oligodendrites and Schwann cells. These cells can be further divided by their function, location, shape, neurotransmitter class and pathological state. Cells of the nervous system can also be identified by their state of differentiation, for example stem cells. Exemplary markers specific for these cell types and states are well known in the art and include, but are not limited to CD 133 and Neurosphere.
  • Another embodiment provides an antibody or antigen binding fragment thereof bound to the disclosed proteins of interest acting as the targeting signal.
  • the antibodies or antigen binding fragment thereof are useful for directing the vector to a cell type or cell state.
  • the polypeptide of interest possesses an antibody binding domain, for example from proteins known to bind antibodies such as Protein A and Protein G from Staphylococcus aureus.
  • some embodiments include the amino acids sequence HDEAQQNAFY QVLNMPNLNA DQRNGFIQSL KDDPSQSANV
  • the polynucleotide-binding protein has at least 80, 85, 90, 95, 99, or 100 percent sequence identity to
  • the antibody is polyclonal, monoclonal, linear, humanized, chimeric or a fragment thereof.
  • Representative antibody fragments are those fragments that bind the antibody binding portion of the non-viral vector and include Fab, Fab', F(ab'), Fv diabodies, linear antibodies, single chain antibodies and bispecific antibodies known in the art.
  • the targeting domain includes all or part of an antibody that directs the vector to the desired target cell type or cell state.
  • Antibodies can be monoclonal or polyclonal, but are preferably monoclonal.
  • antibodies are derived from human genes and are specific for cell surface markers, and are produced to reduce potential immunogenicity to a human host as is known in the art.
  • transgenic mice which contain the entire human immunoglobulin gene cluster are capable of producing "human" antibodies can be utilized.
  • fragments of such human antibodies are employed as targeting signals.
  • single chain antibodies modeled on human antibodies are prepared in prokaryotic culture.
  • the polypeptide of interest is itself a fusion protein.
  • the fusion protein can include, for example, a polynucleotide- binding polypeptide, a protein transduction domain, and optionally one or more targeting signals.
  • a preferred polypeptide of interest is SEQ ID NO:26.
  • Other exemplary fusion proteins containing a mitochondrial transcription factor polypeptide that are suitable for use as a polypeptide of interest are disclosed in U.S. Patent Nos. 8,039,587, 8,062,891, 8,133,733, and U.S. Published Application Nos. 2009/0123468, 2009/0208478, and 2006/021 1647 all of which are specifically incorporated by reference herein in their entireties.
  • the fusion protein can optionally include additional sequences or moieties, including, but not limited to linkers and purification tags.
  • the purification tag is a polypeptide.
  • Polypeptide purification tags are known in the art and include, but are not limited to His tags which typically include six or more, typically consecutive, histidine residues; FLAG tags, which typically include the sequence DYKDDDDK (SEQ ID NO:32); haemagglutinin (HA) for example, YPYDVP (SEQ ID NO:33); MYC tag for example ILKKATAYIL (SEQ ID NO:34) or EQKLISEEDL (SEQ ID NO:35).
  • Methods of using purification tags to facilitate protein purification are known in the art and include, for example, a chromatography step wherein the tag reversibly binds to a chromatography resin.
  • Purifications tags can be N-terminal or C-terminal to the fusion protein.
  • the purification tags N-terminal to the fusion protein are typically separated from the polypeptide of interest at the time of the cleavage in vivo. Therefore, purification tags N-terminal to the fusion protein can be used to remove the fusion protein from a cellular lysate following expression and extraction of the expression or solubility enhancing amino acid sequence, but cannot be used to remove the polypeptide of interest.
  • Purification tags C- terminal to the fusion protein can be used to remove the polypeptide of interest from a cellular lysate following expression of the fusion protein, but cannot be used to remove the expression or solubility enhancing amino acid sequence.
  • Purification tags that are C-terminal to the expression or solubility enhancing amino acid sequence can be N-terminal to, C-terminal to, or incorporated within the sequence of the polypeptide of interest.
  • to fusion protein includes one or more linkers or spacers.
  • linker or spacer is one or more polypeptides.
  • the linker includes a glycine-glutamic acid di-amino acid sequence. The linkers can be used to link or connect two domains, regions, or sequences of the fusion protein.
  • Molecular biology techniques have developed so that therapeutic proteins can be genetically engineered to be expressed by microorganisms.
  • the gram negative bacterium, Escherichia coli is a versatile and valuable organism for the expression of therapeutic proteins.
  • many proteins with therapeutic or commercial uses can be produced by recombinant organisms, the yield and quality of the expressed protein are variable due to many factors.
  • heterologous protein expression by genetically engineered organisms can be affected by the size and source of the protein to be expressed, the presence of an affinity tag linked to the protein to be expressed, codon biasing, the strain of the microorganism, the culture conditions of microorganism, and the in vivo degradation of the expressed protein.
  • Exemplary expression or solubility enhancing amino acid sequences include maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and a small ubiquitin-related modifier (SUMO).
  • MBP maltose-binding protein
  • GST glutathione S-transferase
  • TRX thioredoxin
  • NUS A ubiquitin
  • Ub ubiquitin
  • SUMO small ubiquitin-related modifier
  • the compositions disclosed herein include expression or solubility enhancing amino acid sequence.
  • the expression or solubility enhancing amino acid sequence is cleaved prior administration of the composition to a subject in need thereof.
  • the expression or solubility enhancing amino acid sequence can be cleaved in the recombinant expression system, or after the expressed protein in purified.
  • the expression or solubility enhancing is a ULP1 or SUMO sequence.
  • Recombinant protein expression systems that incorporate the SUMO protein (“SUMO fusion systems”) have been shown to increase efficiency and reduce defective expression of recombinant proteins in E. coli., see for example Malakhov, et al, J. Struct. Fund.
  • Genomics 5: 75-86 (2004), U.S. Patent No. 7,060,461, and U.S. Patent No. 6,872,551.
  • SUMO fusion systems enhance expression and solubility of certain proteins, including severe acute respiratory syndrome coronavirus (SARS-CoV) 3CL protease, nucleocapsid, and membrane proteins (Zuo et al., J. Struct. Funct. Genomics, 6: 103-1 11 (2005)).
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • PTD-TFAM TFAM
  • rhTFAM a fusion protein with a protein transduction domain, a mitochondrial localization signal, and a TFAM polypeptide.
  • Example 1 rhTFAM increases brain ATP in aged db/db mice
  • db/db mice exhibit metabolic abnormalities due to a defect in leptin signaling resulting from a point mutation in the gene for the leptin receptor.
  • the leptin receptor is highly expressed in the hypothalamus and mice with a defect in this protein are unable to regulate their energy stores appropriately.
  • Plasma insulin levels become elevated very early in life (10-14 days of age), and the affected animals are obese by the time they are 3-4 weeks old. Blood glucose levels begin rising at this time. The animals are insulin resistant, hypertriglyceridemic, and have impaired glucose tolerance.
  • db/db mice Although commonly used to study diabetes, db/db mice also display cognitive impairment and amyloid buildup in brain, as well as tau hyperphosporylation (Kim, et al, Endocrinology, 150(12):5294-301 (2009)), which are hallmarks of amyloid beta-related diseases and tauopathies such as Alzheimer's disease.
  • mice Fifty (50) 10 week old BKS.Cg-Dock7m +/+ Leprdb/J (642) (referred to herein as db/db) male mice were ear notched for identification using the standard mouse ID format. Mice were housed at a density of 5 per cage in polycarbonate cages which were both individually and positively ventilated. Bed-o'cobs® corn cob bedding was provided and was changed every two weeks or as needed. The animal room was lighted entirely with artificial fluorescent lighting on controlled 12 hr light/dark cycle (6 a.m. to 6 p.m. light). The normal temperature and relative humidity ranges in the animal rooms were maintained at 22 ⁇ 4°C and 50 ⁇ 15%, respectively. The animal room was set for 15 air exchanges per hour. Filtered tap water acidified to a pH of 2.8 to 3.2 was provided ad libitum. LabDiet 5K0Q was provided ad libitum.
  • mice After a 2 week acclimation, the mice were grouped by mean body weight per cage.
  • mice were dosed at lOOuL via IV tail vein once a week for 12 weeks according to Table 2:
  • mice from each group were sacrificed, and brain, heart, liver, kidney and skeletal muscle were flash frozen.
  • the CellTiter-Glo® Luminescent Cell Viability Assay kit was used for ATP assay following the manufacturer's instruction. Briefly, the assay buffer and substrate were equilibrated to room temperature, and the buffer was transferred to and gently mixed with the substrate to obtain a homogeneous solution. After a 30 min equilibration of the plate to room temperature, 100 ⁇ of the assay reagent was added into each well containing proteins and the content was mixed for 2 min. After 10 min incubation at room temperature, the luminescence was read on a PHERAstar FS Reader (BMG Labtech Cary, NC).
  • Db/db mice are commonly used to study diabetes. To measure energy production and availability in the brains of these mice, the brain tissue was harvested, and ATP was measured as described. As shown in Figure 1, brain ATP was increased by as much as 30% over vehicle treated animals. Optical density measurements of immunoblots probing for electron transport chain proteins show a significant increase of 70% in electron transport chain subunits in rhTFAM treated db/db mice at the 1.5mg/kg dose (Figure 2)
  • Example 2 rhTFAM reduces tau hyperphosporylation in aged db/db mice
  • db/db mice Twelve-week-old db/db mice were treated with rhTFAM (.3 mg/kg and 1.5 mg/kg) or vehicle alone once a week for 12 weeks as discussed above.
  • rhTFAM rhTFAM
  • vehicle alone once a week for 12 weeks as discussed above.
  • brains were isolated and subjected to analysis of mitochondrial function and tau status.
  • Total tau and phosphorylated tau were detected by immunoblotting.
  • the phospho-Serine 396 epitope was detected as a measure of tau phosphorylation state.
  • Formalin fixed db/db brain sections were also probed for total tau to determine changes in aggregation state using an anti-tau antibody, and an anti-alexa 488 secondary antibody.
  • AD Alzheimer's disease
  • Aged (24 week) db/db mice have been reported as a model of tau hyperphosporylation (Kim, et al., Endocrinology, 150(12):5294-301 (2009).
  • Aged db/db mice produce robust tau hyperphosprylation commensurate with hyperglycemia and cognitive dysfunction.
  • Abnormally phosphorylated tau is a major building block of neurofibrillary tangles, a classic neuropathological characteristic of AD.
  • Example 1 above was paralleled by a decrease in the expression of total tau, indicating a relationship between bioenergetics and tau expression in db/db mice ( Figure 3A).
  • the phospho-Serine 396 epitope was also detected as a measure of tau phosphorylation state.
  • rhTFAM proved to be safe and well-tolerated at once weekly dosing for 12 weeks, and improved mitochondrial electron transport chain function in aged db/db mice.
  • Mitochondrial ETC proteins were elevated 70% over vehicle control mice.
  • brain ATP was significantly elevated indicating improved bioenergetics following rhTFAM treatment.
  • Assessment of total tau protein showed a significant decrease in protein levels as well as a reduction in the phosphorylation state of Serine396.
  • Example 3 rhTFAM decreases Abeta42 protein levels and reduces the ratio of Abeta42/40 in aged Tg2576 mice
  • Tg2576 mice are a well know animal model for studying amyloidosis and Alzheimer's disease (see, for example, Kim, et al, "The Preventive and Therapeutic Effects of Intravenous Human Adipose-Derived Stem Cells in Alzheimer's Disease Mice", PLoS One, 7(9): e45757 (2012)).
  • Tg2576 mice overexpress a mutant form of amyloid precursor protein (APP), referred to as APPK670/671L, which is linked to early onset familial Alzheimer's disease (AD). They develop amyloid plaques and progressive cognitive deficits.
  • APP amyloid precursor protein
  • IV intravenous
  • brain lysates were subjected to SDS-PAGE and transferred for immunoblotting using an antibody cocktail for the various subunits of Complexes I-V (Mitosciences).
  • Beta-amyloid (1-42) and beta-actin were detected using antibodies from Abeam (ab39377 and ab8226 respectively).
  • Beta-amyloid ELISA (Invitrogen) was conducted according to the manufacturer's protocol. Frozen brain samples ( ⁇ 100mg) were homogenized and incubated in 5M Guanidine buffer for several hours. Samples were diluted 1 :50 with dilution buffer (5% BSA, 0.03% Tween-20, lxDPBS). Samples were diluted 1 :2 or 1 : 10 in ELISA sample diluent and analyzed according to manufacturer's protocol (Invitrogen, Cat# KHB3441 ( ⁇ 42) and Cat#KHB3481 ( ⁇ 40) respectively). The concentration of Ab was calculated based on an internal standard curve, and the ratio of a 42:a 40 was determined.
  • Beta-amyloid ELISA assay was also conducted. rhTFAM treatment produced a decrease in Abeta 1-42 evident by the decreased ratio of the 1- 42/1-40 peptides ( Figure 6).
  • rhTFAM proved to be safe and well-tolerated at once weekly dosing for 8 weeks, and improved mitochondrial electron transport chain function in aged Tg2576 mice. Mitochondrial ETC proteins were elevated 50% over vehicle control mice indicating improved. Assessment of Abetal-42 protein showed a significant decrease of 70% in protein levels as well as a reduction in the ratio of Abeta 1-42/ 1-40. In conclusion, improving mitochondrial function using rhTFAM prevents the formation of one of the hallmarks of Alzheimer's disease as well as significantly improving brain metabolism and bioenergetics.
  • Examples 1-3 indicate that rhTFAM is effective for reversing the decline in brain energy metabolism as well as reducing two neuropathological hallmarks of Alzheimer's disease: amyloid beta (Tg2576) and tau (aged db/db).
  • FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone
  • Oligomycin was used in some experiments at a concentration of 1 ⁇ g/mL. Astrocytes, Pan 02 and HepG2 cell lines were tested.

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Abstract

La présente invention concerne des méthodes et des compositions de traitement ou de prévention d'une maladie ou d'un trouble caractérisé par l'augmentation de l'expression de la bêta-amyloïde, le dépôt, l'agrégation ou la formation de plaque, ou d'une maladie ou d'un trouble caractérisé par une augmentation de l'expression de tau, une augmentation de la phosphorylation de tau, ou des pathologies associées à l'agrégation de protéine tau dans le cerveau. L'invention a également trait à des méthodes et des compositions de traitement de protéinopathies ou d'amyloïdose. Lesdites méthodes comprennent habituellement l'administration, à un sujet en ayant besoin, d'une composition comportant une quantité efficace d'un facteur de transcription A recombinant - mitochondrial (TFAM) ou de sa protéine de fusion.
PCT/US2013/020535 2012-01-06 2013-01-07 Méthodes et compositions de traitement de protéinopathies WO2013103964A1 (fr)

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US201261660328P 2012-06-15 2012-06-15
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WO2015089375A1 (fr) * 2013-12-13 2015-06-18 The General Hospital Corporation Espèce tau soluble de poids moléculaire élevé (hmw) et ses applications
WO2015106098A1 (fr) * 2014-01-09 2015-07-16 University Of South Florida Protéine précurseur de l'amyloïde (app) basée sur des peptides inhibiteurs de la b-secrétase, et procédés d'utilisation

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US20190117799A1 (en) 2016-04-01 2019-04-25 The Brigham And Women's Hospital, Inc. Stimuli-responsive nanoparticles for biomedical applications
US20200085758A1 (en) 2016-12-16 2020-03-19 The Brigham And Women's Hospital, Inc. Co-delivery of nucleic acids for simultaneous suppression and expression of target genes

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013188873A1 (fr) * 2012-06-15 2013-12-19 Gencia Corporation Compositions et procédés permettant d'améliorer les réponses immunitaires
WO2015089375A1 (fr) * 2013-12-13 2015-06-18 The General Hospital Corporation Espèce tau soluble de poids moléculaire élevé (hmw) et ses applications
WO2015106098A1 (fr) * 2014-01-09 2015-07-16 University Of South Florida Protéine précurseur de l'amyloïde (app) basée sur des peptides inhibiteurs de la b-secrétase, et procédés d'utilisation
US9926354B2 (en) 2014-01-09 2018-03-27 University Of South Florida Amyloid precursor protein (APP) based Ã#-secretase inhibitor peptides, and methods of use

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