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WO2020092877A1 - Modulation of mitochondrial biogenesis by increase in iron sulfur cluster activity - Google Patents

Modulation of mitochondrial biogenesis by increase in iron sulfur cluster activity Download PDF

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WO2020092877A1
WO2020092877A1 PCT/US2019/059345 US2019059345W WO2020092877A1 WO 2020092877 A1 WO2020092877 A1 WO 2020092877A1 US 2019059345 W US2019059345 W US 2019059345W WO 2020092877 A1 WO2020092877 A1 WO 2020092877A1
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cell
protein
cells
ampk
pgc
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Stefano Rivella
Ping LA
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The Children's Hospital Of Philadelphia
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G01N33/721—Haemoglobin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/22—Haematology

Definitions

  • the present disclosure relates generally to the fields of biology, medicine and pathoolgy. More particularly, it concerns alterations in mitochondrial biogenesis induced by drug and gene expression modulation regulation.
  • Mitochondrial biogenesis is the process by which cells increase mitochondrial mass. It was first described by John Holloszy in the l960s, when it was discovered that physical endurance training induced higher mitochondrial content levels, leading to greater glucose uptake by muscles. Mitochondrial biogenesis is activated by numerous different signals during times of cellular stress or in response to environmental stimuli, such as aerobic exercise.
  • mitochondrion The ability for a mitochondrion to self-replicate is rooted in its evolutionary history. It is commonly thought that mitochondria descend from cells that formed endosymbiotic relationships with a-protobacteria, they have their own genome for replication. However, recent evidence suggests that mitochondrial may have evolved without symbiosis. The mitochondrion is a key regulator of the metabolic activity of the cell and is also an important organelle in both production and degradation of free radicals. It is reckoned that higher mitochondrial copy number (or higher mitochondrial mass) is protective for the cell.
  • Mitochondrial diseases result from failures in the function of mitochondria. Because mitochondria are responsible for creating most of the energy needed to sustain life and support organ function, when they fail, less and less energy is generated within the cell, resulting in cell injury and even cell death. Ultimately, if this process is not stopped, whole organ systems begin to fail.
  • Symptoms can include seizures, strokes, severe developmental delays, inability to walk, talk, see, and digest food combined with a host of other complications. If three or more organ systems are involved, mitochondrial disease should be suspected.
  • Mitochondrial diseases the result from inherited or spontaneous mutations in mtDNA or nDNA which can lead to altered function of the proteins or RNAs residing in mitochondria. Because mitochondria perform so many different functions in different tissues, there are literally hundreds of different mitochondrial diseases. Each disorder produces a spectrum of abnormalities that can be confusing to both patients and physicians in early stages of diagnosis. Mitochondrial diseases are even more complex in adults because detectable changes in mtDNA occur as one ages and, conversely, the aging process itself may result from deteriorating mitochondrial function. Thus, there is a broad spectrum of metabolic, inherited and acquired disorders in which abnormal mitochondrial function has been postulated or demonstrated, yet treatments for these diseases are sorely lacking.
  • Fe-S clusters are essential cofactors for mitochondrial functions and are also synthesized within the mitochondria (Rouault and Maio, 2017). Fe-S clusters inhibit the expression of the iron importer transferrin receptor 1 (TfRl), which blocks the iron uptake required for mitochondria biogenesis (Zhang etal, 2014; Rensvold etal, 2013; Ishii etal, 2009). It is unclear however whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, if this negatively modulates TfRl expression and thus interfere with mitochondrial iron demand.
  • TfRl iron importer transferrin receptor 1
  • Fe-S clusters are synthesized in the mitochondria and cytosol by two different Fe- S cluster assembly machineries (Rouault, 2015; Braymer and Lill, 2017).
  • the early steps of cytosolic Fe-S cluster synthesis require the mitochondrial Fe-S cluster assembly machinery. This process is mediated by the mitochondrial transporter ABCB7 (Pondarre et al, 2006). Therefore, the mitochondrial assembly machinery is essential for all de novo Fe-S cluster synthesis.
  • IRP1 and IRP2 regulate iron metabolism by binding iron-responsive element (IRE) motifs.
  • IRPs bind to the 3’UTR IREs of TfRl mRNA, thereby stabilizing it and increasing iron import (Caspary et al, 1977).
  • IRPs binding to the 5’UTR IRE of 5'-Aminolevulinate Synthase 2 (ALAS2) the rate- limiting enzyme in the erythroid heme synthesis pathway abrogates ALAS2 translation and iron consumption (Duncan et al, 1999).
  • Iron and Fe-S cluster levels regulate IRE-binding activity of IRPs and thereby affect iron homeostasis.
  • iron and Fe-S clusters can associate with IRP1 and convert it into cytosolic aconitase (aka ACOl) while simultaneously losing the IRE-binding activity (Haile et al. , 1992). Therefore, increased iron and iron-sulfur cluster levels decrease IRE- binding activity of IRPs. This decreases iron intake by destabilizing TfRl mRNA while increasing iron consumption by de-repressing ALAS2 translation. In contrast, decreased iron and Fe-S clusters levels enhance iron uptake and decrease iron consumption, ultimately maintaining iron homeostasis.
  • Fe-S cluster synthesis modulates IRE-binding activity of IRP1 thereby orchestrating IRPl-targeted gene expression.
  • Genetic mutations in genes that controls Fe- S cluster synthesis cause Fe-S cluster insufficiency, ultimately disturbing mitochondria function, inhibiting erythroid heme synthesis and predisposing individuals to numerous diseases, including anemia and myelodysplastic syndromes (MDS) (Ajioka et al. , 2006; Bottomley and Fleming, 2014; Pondarre et al, 2007; Schmitz-Abe et al, 2015; Ye et al, 2010).
  • MDS myelodysplastic syndromes
  • Fe-S cluster synthesis coordinates with mitochondria biogenesis and whether this modulates iron uptake via changes on TfRl expression. This is also relevant in erythropoiesis, a process that requires active mitochondrial biogenesis and consumption of the majority of physiological iron (Muckenthaler et al, 2017).
  • a method of increasing b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization in an erythroid cell comprising contacting said cell with an agonist of AMPK or PCG-la.
  • the agonist may be an AMPK protein or expression construct coding therefore, PCG-la protein or expression construct coding therefore, metformin, resveratrol or AICAR.
  • the cell may be in a subject, such as a human subject. The subject may suffer from a hemoglobinopathy, such as b-thalessemia.
  • the method may further comprise contacting said cell with another agent that is involved in b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization, such as iron.
  • the cell may be contacted with said agonist more than once, such as on a chronic basis.
  • the agonist may not be AICAR.
  • the AMPK protein or PCG-la protein or AICAR may be administered directly to said cell.
  • the AMPK or PCG-la protein may be contacted with said cell by provision of an expression construct coding for the same.
  • the method may further comprise identifying said subject as being in need of increased b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization.
  • Also provided is a method of increasing mitochondrial biogenesis in a cell comprising contacting said cell with an agonist of AMPK or PCG-la.
  • the subject may suffer from a disease or disorder stemming from mitochondrial dysfunction, such as sideroblastic anemia.
  • the agonist may be an AMPK protein or expression construct coding therefore, PCG-la protein or expression construct coding therefore, metformin, resveratrol or AICAR.
  • the cell may be in a subject, such as a human subject.
  • the cell may be contacted with said agonist more than once, such as on a chronic basis.
  • the agonist may not be AICAR.
  • the cell may be a muscle cell, an adipocyte, an erythrocyte, or an epithelial cell.
  • the AMPK protein or PCG-la protein or AICAR may be administered directly to said cell.
  • the AMPK or PCG-la protein may be contacted with said cell by provision of an expression construct coding for the same.
  • the method may further comprise identifying said subject as being in need of increased mitochondrial biogenesis.
  • FIGS. 1A-E PGC-Ia stimulates Fe-S cluster synthesis.
  • 3T3-L1 cells were infected with retroviruses control or expressing Myc-PGC-la followed by puromycin selection. Puromycin-resistant cells were used for the following assays.
  • FIG. 1A Myc- PGC-la expression was verified by using Western blot assay and anti-Myc and anti-PGC- la antibodies.
  • FIG. 1B Mitochondria biogenesis was verified by Mitotrack Green and Deep Red staining and further FACS analysis. Mitotrack Green is proportional to mitochondrial mass, while Deep Red identifies alive mitochondria.
  • FIGS. 1A-E PGC-Ia stimulates Fe-S cluster synthesis.
  • 3T3-L1 cells were infected with retroviruses control or expressing Myc-PGC-la followed by puromycin selection. Puromycin-resistant cells were used for the following assays.
  • FIG. 1A Myc- PGC-la expression was
  • FIG. 1C ALAS1 protein and mRNA levels as well as heme contents were measured respectively by Western blot, qRT-PCR and heme assay kit.
  • FIG. 1D The activity and protein levels of aconitase were measured by In-Gel aconitase activity (the top panel) and Western blot assays (lower panels).
  • FIG. 1E Signals for the activity and protein levels of mitochondria aconitase (AC02) shown in FIG. 1D were quantified by ImageJ software. The quantity produced from AC02 aconitase activity was normalized by the number generated form AC02 protein levels. Subsequently, this ratio was compared to that obtained from control cells. #: a non-specific band.
  • FIGS. 2A-B PGC-Ia stimulates the gene expression of Fe-S cluster assembly machinery.
  • FIG. 2A 3T3-L1 cells were infected with virus control or expressing Myc- PGC-la followed by puromycin selection. Puromycin-resistant cells were analyzed for protein levels of genes involved in Fe-S cluster assembly.
  • FIG. 2B The same cells used in FIG. 2 A were analyzed for the mRNA levels of Fe-S cluster assembly machinery.
  • FIGS.3A-I Fe-S cluster synthesis coordinates with mitochondria biogenesis in the absence of PGC-Ia.
  • Wild-type (WT) and PGC-la-null (PGC-la-/-) prebrown adipocytes (day 0) were differentiated into matured brown adipocytes (day 6) by a drug treatment for 6 days.
  • FIG. 3A The activities and protein levels of aconitases were measured by In-Gel aconitase activity (top panel) and Western blot assays (bottom panels).
  • FIGS. 3B-C The same quantitative analysis used in FIG. 1E was applied for ACOl levels and activity (FIG. 3B).
  • FIG. 3C AC02 levels and activities were quantified and analyzed.
  • FIG. 3D The protein levels of genes responsible for Fe-S cluster synthesis were evaluated by Western blot assays.
  • FIG. 3E-I The genes analyzed in FIG. 3D were further evaluated for their mRNA levels by qRT-PCR assay.
  • FIGS. 4A-E Under enhanced mitochondria biogenesis in 3T3-L1 cells, Fe-S cluster synthesis is increased whereas TfRl protein levels are increased.
  • FIG. 4A Control and Myc-PGC-la-expressing 3T3-L1 cells were analyzed for IRE-binding activity (top panel) and IRPs protein levels (lower panels). The iron chelator DFO treatment was used as positive control, showing increased IRE-binding activity of IRPs due to IRP2 stabilization.
  • FIG. 4B Control and Myc-PGC-la-expressing 3T3-L1 cells were evaluated for TfRl mRNA (FIG. 4B) and protein (FIG. 4C) levels.
  • FIG. 4A Control and Myc-PGC-la-expressing 3T3-L1 cells were evaluated for TfRl mRNA (FIG. 4B) and protein (FIG. 4C) levels.
  • Control and Myc-PGC- la-expressing 3T3-L1 cells were treated with 0.05 pg/ml actinomycin D or cycloheximide for 24hrs followed by western blot assay.
  • FIG. 4E Cells were treated with 0.1 mM MG132 for 32 hrs and analyzed.
  • FIGS. 5A-D Under enhanced mitochondria biogenesis, IRP1 expression in brown adipocytes was stimulated and further increased TfRl expression.
  • FIG. 5A WT and PGC-la-/- prebrown adipocytes (day 0) were matured to brown adipocytes (day 6) by the drug treatment. These cells were analyzed for their IRP1 mRNA levels by qRT- PCR.
  • FIG. 5B The adipocytes used in A were analyzed for the IRE-binding activities (to panel) and protein levels of IRPs (lower panels).
  • FIGS. 5C-D The same cells used in FIG. 5B were analyzed for TfRl protein levels by western blot (FIG. 5C) and mRNA levels by qRT-PCR (FIG. 5D).
  • FIGS. 6A-E Fe-S cluster assembly coordinates with mitochondria biogenesis in erythroid cells.
  • FIG. 6A MEL cells were infected with lentiviruses control or expressing Myc-PGC-la. One day after infection, these cells were differentiated by the incubation with 50 mM HMBA for 5 days followed by the aconitase activity and western blot assays.
  • FIG. 6B Similarly, cells used in A were analyzed by qRT-PCR assays.
  • FIG. 6C MEL cells were infected with lentiviruses shRNA control or targeting murine PGC-la mRNA and selected with puromycin.
  • FIGS. 7A-E In erythroid cells, GATA1 expression coordinates with mitochondria biogenesis and potentially regulates TfRl gene expression.
  • FIG. 7A- B MEL cells control or expressing Myc-PGC-la were differentiated and then analyzed by IRE-binding activity (A, the top panel) and Western blot assays (FIG. 7A, the lower panels) and for TfRl mRNA levels by qRT-PCR (FIG. 7B).
  • FIGGS. 7C-D Control and PGC-la shRNA-targeted MEL cells were generated by lentiviral infection and puromycin selection. After differentiation, these cells were analyzed by IRE-binding activity (the top panel) and Western blot assays (FIG. 7C) and for TfRl mRNA levels by qRT-PCR (FIG. 7D).
  • FIG. 7E The model for different regulations on TfRl expression. Top: the canonical regulation mediated by Fe-S cluster synthesis; middle: the regulation mediated by increased IRP1 expression; botom: the regulation mediated by GATA1.
  • FIGS.8A-D Erythroid heme synthesis and hemoglobinization coordinate with mitochondria biogenesis.
  • FIG. 8A Control or Myc-PGC-la-expressing MEL cells were differentiated with HMBA incubation for 5 days, then pelleted and photographed (top panel of FIG. 8A) followed by western blot assays.
  • FIG. 8B The same cells used in FIG. 8A were analyzed by qRT-PCR.
  • FIG. 8C Control and PGC-la shRNA-targeted MEL cells were differentiated with HMBA treatment for 6 days, pelleted and photographed (top panel) followed by western blot assay.
  • FIG. 8D The same cells used in FIG. 8C were analyzed by qRT-PCR.
  • FIGS. 9A-D AMPK activation modulates the erythropoiesis in thalassemia and sideroblastic anemia.
  • FIG. 9A CD34 + cells were isolated from peripheral blood of a thalassemia patient and expanded for 10 to 13 days. These cells were further cultured in erythroid differentiation medium with 120 mM AICAR for different periods of time as indicated. Cells were harvested for ISCU protein levels analysis.
  • FIG. 9B CD34 + cells used in A were cultured in differentiation medium with different concentrations of AICAR as indicated. The medium was refreshed every 48hrs; after 96hrs, cells were analyzed for ALAS2, b-globin and a-globin levels.
  • FIG. 9C CD34 + cells were isolated from another thalassemia patient, cultured, differentiated and analyzed as described in B.
  • FIGS. 9B and 9C CD34 + cells were isolated from a sideroblastic anemia patient and expanded, After the expansion, these cells were switched in erythroid differentiation medium and cultured for 96 hrs with different concentrations of AICAR. Similarly, cells were harvested and analyzed for ALAS2, a-globin and b-globin protein levels.
  • FIG. SI. 3T3-L1 cells were infected with retroviruses control or expressing Myc- PGC-la followed by puromycin selection. Puromycin-resistant cells were analyzed for mRNA levels of genes involved in Fe-S cluster assembly.
  • FIGS. S2A-C HepG2 cells were infected with retroviruses control or expressing Myc-PGC-la and selected with puromycin. Puromycin-resistant HepG2 cells were analyzed by Western blot assay. # indicated nonspecific bands.
  • FIG. S2B The same cells used in FIG. S2A were analyzed for the protein levels and activities of ACOl and AC02.
  • FIG. S2C AC02 aconitase activity and protein levels, as shown in FIG. S2B, were quantified by Image! The AC02 aconitase activity was normalized by the AC02 protein levels. Subsequently, the ratio obtained from My c-PGC- la-expressing cells (the AC02 aconitase activity normalized by the AC02 protein levels) was compared to that achieved using control cells.
  • FIG. S3. The genes analyzed in FIG. 2H were further evaluated for the mRNA levels by qRT-PCR assay. Lighter bars stand for WT brown adipocytes while dark bars stand for PGC-la-/- brown adipocytes.
  • FIGS. S4A-E TfRl mRNA levels in control and My c-PGC- la- expressing 3T3-L1 cells were measured by qRT-PCR assay.
  • FIG. S4B TfRl protein levels in HepG2 cells control or expressing Myc-PGC-la were evaluated by Western blot analysis.
  • FIG. S4C Similar to FIG. S4B, TfRl mRNA levels in HepG2 cells were measured by qRT-PCR assay.
  • FIG. S4D TfRl mRNA levels were analyzed in prebrown (day 0) and brown (day 6) adipocytes by qRT-PCR assay.
  • FIG. S4E Similar to FIG. S4D, IRP 1 mRNA levels were analyzed.
  • FIGS. S5A-G MEL cells were infected with lentiviruses shRNA control or targeting murine PGC-la mRNA, selected with puromycin, and incubated with tomM HMBA for 7 days for differentiation. Cells were analyzied for PGC-la mRNA levesl by qRT-PCR assay.
  • FIGS. S5B-C Diffemtiated control and PGC-la shRNA-targeted MEL cells were analyzed by In-Gel aconitase activity and western blot assay (FIG. S5B) and by qRT-PCR for the mRNA levels of genes involved in Fe-S cluster assembly (FIG. S5C).
  • FIG. S5D mRNA levels of different genes as indicated were measured in differentiated control and My c-PGC- la-expressing MEL cells.
  • FIG. S5E Differentiated control and PGC-la shRNA-targeted cells were interrogated by IRE-binding activity (top panel) and Western blot assays.
  • FIGS. S5F-G TfRl mRNA levels were measured by qRT-PCR in differentiated control and PGC-la shRNA-targeted cells (FIG. S5F) and differentiated control and Myc-PGC-la-expressing cells (FIG. S5G).
  • FIGS. S6A-B FIGS. S6A-B.
  • Mitochondria are produced from the transcription and translation of genes both in the nuclear genome and in the mitochondrial genome.
  • the majority of mitochondrial protein comes from the nuclear genome, while the mitochondrial genome encodes parts of the electron transport chain along with mitochondrial rRNA and tRNA.
  • Mitochondrial biogenesis increases metabolic enzymes for glycolysis, oxidative phosphorylation and ultimately a greater mitochondrial metabolic capacity.
  • the cell may increase or decrease the number and size of mitochondria.
  • mitochondrial numbers and morphology vary according to cell type and context-specific demand, whereby the balance between mitochondrial fusion/fission regulates mitochondrial distribution, morphology, and function.
  • PGC-la a member of the peroxisome proliferator-activated receptor gamma (PGC) family of transcriptional coactivators, is the master regulator of mitochondrial biogenesis. It is known to co-activate nuclear respiratory factor 2 (NRF2/GABPA), and together with NRF-2 coactivates nuclear respiratory factor 1 (NRF1). The NRFs, in turn, activate the mitochondrial transcription factor A (tfam), which is directly responsible for transcribing nuclear-encoded mitochondrial proteins. This includes both structural mitochondrial proteins as well as those involved in mtDNA transcription, translation, and repair.
  • PGC- 1b a protein that is structurally similar to PGC-la, is also involved in regulating mitochondrial biogenesis, but differs in that it does not get increased in response to exercise.
  • AMP-activated kinase also regulates mitochondrial biogenesis by phosphorylating and activating PGC-la upon sensing an energy deficiency in muscle. In mice with reduced ATP/ AMP ratios that would occur during exercise, the energy depletion has been shown to correlate with AMPK activation. AMPK activation then continued to activate PGC- la and NRFs in these mice, and mitochondrial biogenesis was stimulated.
  • telomere loss has also been associated with decreased mitochondrial function.
  • Deficiency of telomerase reverse transcriptase (TERT) an enzyme that plays a role in preserving telomeres, has been correlated with activated p53, a protein that suppresses PGC-la.
  • telomeres and TERT have been associated with impaired mitochondrial biogenesis.
  • AMPK expression has also been shown to diminish with age, which may also contribute to suppressing mitochondrial biogenesis.
  • mitochondrial biogenesis is also evidence of mitochondrial biogenesis being involved in neurodegenerative disorders, cancer, metabolic syndrome, sarcopenia, cardiac pathophysiology as well as physiological processes like aging and erythropoiesis.
  • mitochondrial biogenesis As indicated above, a considerable number of proteins, transcription factors, upstream regulatory proteins and secondary mechanisms are involved in mitochondrial biogenesis. These molecules, including the main participating proteins (e.g., PGC-la and mtTFA), are candidates for therapeutic intervention in diverse disease.
  • the inventors explore the molecular regulation of mitochondrial biogenesis, and methods of exploiting regulatory pathways to alter mitochondrial function.
  • Iron-sulfur proteins are proteins characterized by the presence of iron-sulfur clusters containing sulfide-linked di-, tri-, and tetrairon centers in variable oxidation states. Iron-sulfur clusters are found in a variety of metalloproteins, such as the ferredoxins, as well as NADH dehydrogenase, hydrogenases, coenzyme Q - cytochrome c reductase, succinate - coenzyme Q reductase and nitrogenase. Iron-sulfur clusters are best known for their role in the oxidation-reduction reactions of mitochondrial electron transport. Both Complex I and Complex II of oxidative phosphorylation have multiple Fe-S clusters.
  • Fe-S proteins regulate gene expression. Fe-S proteins are vulnerable to attack by biogenic nitric oxide, forming dinitrosyl iron complexes. In most Fe-S proteins, the terminal ligands on Fe are thiolate, but exceptions exist.
  • Fe-S proteins In almost all Fe-S proteins, the Fe centers are tetrahedral and the terminal ligands are thiolato sulfur centers from cysteinyl residues.
  • the sulfide groups are either two- or three-coordinated. Three distinct kinds of Fe-S clusters with these features are most common.
  • the simplest polymetallic system the
  • the oxidized proteins contain two Fe 3+ ions, whereas the reduced proteins contain one Fe 3+ and one Fe 2+ ion. These species exist in two oxidation states, (Fe and Fe m Fe n .
  • a common motif features four iron ions and four sulfide ions placed at the vertices of a cubane-type cluster.
  • the Fe centers are typically further coordinated by cysteinyl ligands.
  • the [Fe 4 S 4 ] electron-transfer proteins ([Fe 4 S 4 ] ferredoxins) may be further subdivided into low-potential (bacterial-type) and high-potential (HiPIP) ferredoxins.
  • the cluster shuttles between [2Fe 3+ , 2Fe 2+ ] (Fe 4 S 4 2+ ) and [3Fe 3+ , Fe 2+ ] (Fe 4 S 4 3+ ).
  • the potentials for this redox couple range from 0.4 to 0.1 V.
  • the pair of oxidation states are [Fe 3+ , 3Fe 2+ ] (Fe 4 S 4 + ) and [2Fe 3+ , 2Fe 2+ ] (Fe 4 S 4 2+ ).
  • the potentials for this redox couple range from -0.3 to -0.7 V.
  • the two families of 4Fe- 4S clusters share the Fe 4 S 4 2+ oxidation state.
  • the difference in the redox couples is attributed to the degree of hydrogen bonding, which strongly modifies the basicity of the cysteinyl thiolate ligands.
  • a further redox couple which is still more reducing than the bacterial ferredoxins is implicated in the nitrogenase.
  • Some 4Fe-4S clusters bind substrates and are thus classified as enzyme cofactors.
  • the Fe-S cluster binds aconitate at the one Fe centre that lacks a thiolate ligand.
  • the cluster does not undergo redox but serves as a Lewis acid catalyst to convert citrate to isocitrate.
  • radical SAM enzymes the cluster binds and reduces S-adenosylmethionine to generate a radical, which is involved in many biosyntheses.
  • Proteins are also known to contain [FesSJ centres, which feature one iron less than the more common [Fe 4 S 4 ] cores. Three sulfide ions bridge two iron ions each, while the fourth sulfide bridges three iron ions. Their formal oxidation states may vary from
  • the [Fe 4 S 4 ] cluster can be reversibly converted by oxidation and loss of one iron ion to a
  • More complex polymetallic systems are common. Examples include both the 8Fe and the 7Fe clusters in nitrogenase. Carbon monoxide dehydrogenase and the [FeFe]- hydrogenase also feature unusual Fe-S clusters. A special 6 cysteine-coordinated [Fe 4 S 3 ] cluster was found in oxygen-tolerant membrane-bound [NiFe] hydrogenases.
  • the biosynthesis of the Fe-S clusters has been well studied.
  • the biogenesis of iron sulfur clusters has been studied most extensively in the bacteria E. coli and A. vinelandii and yeast S. cerevisiae.
  • At least three different biosynthetic systems have been identified so far, namely nif, suf, and isc systems, which were first identified in bacteria.
  • the nif system is responsible for the clusters in the enzyme nitrogenase.
  • the suf and isc systems are more general.
  • the yeast isc system is the best described.
  • Several proteins constitute the biosynthetic machinery via the isc pathway. The process occurs in two major steps: (1) the Fe/S cluster is assembled on a scaffold protein followed by (2) transfer of the preformed cluster to the recipient proteins.
  • the first step of this process occurs in the cytoplasm of prokaryotic organisms or in the mitochondria of eukaryotic organisms. In the higher organisms the clusters are therefore transported out of the mitochondrion to be incorporated into the extramitochondrial enzymes. These organisms also possess a set of proteins involved in the Fe/S clusters transport and incorporation processes that are not homologous to proteins found in prokaryotic systems. II. Mitochondrial Factors and Agents Affecting Such Factors
  • Peroxisome proliferator-activated receptor gamma coactivator l-alpha is a protein that in humans is encoded by the PPARGC1A gene.
  • PPARGC1A is also known as human accelerated region 20 (HAR20). It may, therefore, have played a key role in differentiating humans from apes.
  • PGC-la is the master regulator of mitochondrial biogenesis.
  • PGC-la is a transcriptional coactivator that regulates the genes involved in energy metabolism. It is the master regulator of mitochondrial biogenesis. This protein interacts with the nuclear receptor PPAR-g, which permits the interaction of this protein with multiple transcription factors. This protein can interact with, and regulate the activities of, cAMP response element-binding protein (CREB) and nuclear respiratory factors (NRFs). It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis, and is a major factor causing slow-twitch rather than fast-twitch muscle fiber types.
  • CREB cAMP response element-binding protein
  • NEFs nuclear respiratory factors
  • Endurance exercise has been shown to activate the PGC-la gene in human skeletal muscle.
  • Exercise-induced PGC-la in skeletal muscle increases autophagy and unfolded protein response.
  • PGC-la protein may be also involved in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity.
  • PGC-la is thought to be a master integrator of external signals. It is known to be activated by a host of factors, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), both formed endogenously in the cell as by-products of metabolism but upregulated during times of cellular stress. It is strongly induced by cold exposure, linking this environmental stimulus to adaptive thermogenesis. It is induced by endurance exercise and recent research has shown that PGC-la determines lactate metabolism, thus preventing high lactate levels in endurance athletes and making lactate as an energy source more efficient. It also is activated by AMP response element-binding (CREB) proteins, activated by an increase in cAMP following external cellular signals.
  • ROS reactive oxygen species
  • RNS reactive nitrogen species
  • Protein kinase B/Akt is thought to downregulate PGC-la, but upregulate its downstream effectors, NRF1 and NRF2.
  • Akt itself is activated by PIP3, often upregulated by PI3K after G-protein signals.
  • the Akt family is also known to activate pro-survival signals as well as metabolic activation.
  • SIRT1 binds and activates PGC-la through deacetylation inducing gluconeogenesis without affecting mitochondrial biogenesis.
  • PGC-la has been shown to exert positive feedback circuits on some of its upstream regulators.
  • PGC-la increases Akt (PKB) and Phospho-Akt (Ser 473 and Thr 308) levels in muscle.
  • PGC-la leads to calcineurin activation.
  • Akt and calcineurin are both activators of NF kappa B (p65). Through their activation PGC-la seems to activate NF kappa B. Increased activity of NF kappa B in muscle has recently been demonstrated following induction of PGC-la. The finding seems to be controversial. Other groups found that PGC- ls inhibit NF kappa B activity. The effect was demonstrated for PGC-l alpha and beta. PGC-la has also been shown to drive NAD biosynthesis to play a large role in renal protection in Acute Kidney Injury.
  • PPARGC1A has been implicated as a potential therapy for Parkinson's Disease conferring protective effects on mitochondrial metabolism.
  • brain-specific isoforms of PGC-l alpha have recently been identified which are likely to play a role in other neurodegenerative disorders such as Huntington's disease and Amyotrophic lateral sclerosis.
  • PGC-la and b has furthermore been implicated in M2 macrophage polarization by interaction with PPARy with upstream activation of STAT6.
  • An independent study confirmed the effect of PGC-l on polarisation of macrophages towards M2 via STAT6/PPAR gamma and furthermore demonstrated that PGC-l inhibits proinflammatory cytokine production.
  • PGC-la has been recently proposed to be responsible for b-aminoisobutyric acid secretion by exercising muscles.
  • the effect of b-aminoisobutyric acid in white fat includes the activation of thermogenic genes that prompt the browning of white adipose tissue and the consequent increase of background metabolism.
  • the b-aminoisobutyric acid could act as a messenger molecule of PGC-la and explain the effects of PGC-la increase in other tissues such as white fat.
  • 5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate- activated protein kinase is an enzyme (EC 2.7.11.31) that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low. It belongs to a highly conserved eukaryotic protein family and its orthologues are SNF1 and SnRKl in yeast and plants, respectively. It consists of three proteins (subunits) that together make a functional enzyme, conserved from yeast to humans. It is expressed in a number of tissues, including the liver, brain, and skeletal muscle.
  • the net effect of AMPK activation is stimulation of hepatic fatty acid oxidation, ketogenesis, stimulation of skeletal muscle fatty acid oxidation and glucose uptake, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipogenesis, activation of adipocyte lipolysis, and modulation of insulin secretion by pancreatic beta-cells.
  • AMPK is a heterotrimeric protein complex that is formed by a, b, and g subunits. Each of these three subunits takes on a specific role in both the stability and activity of AMPK.
  • the g subunit includes four particular Cystathionine beta synthase (CBS) domains giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio.
  • CBS Cystathionine beta synthase
  • the four CBS domains create two binding sites for AMP commonly referred to as Bateman domains. Binding of one AMP to a Bateman domain cooperatively increases the binding affinity of the second AMP to the other Bateman domain.
  • the g subunit undergoes a conformational change which exposes the catalytic domain found on the a subunit. It is in this catalytic domain where AMPK becomes activated when phosphorylation takes place at threonine- 172 by an upstream AMPK kinase (AMPKK).
  • AMPKK upstream AMPK kinase
  • the a, b, and g subunits can also be found in different isoforms: the g subunit can exist as either the g ⁇ , g2 or g3 isoform; the b subunit can exist as either the b ⁇ or b2 isoform; and the a subunit can exist as either the al or a2 isoform.
  • AMPK isosterically and by post-translational modification, which work together.
  • residue T172 of AMPK's a-subunit is phosphorylated AMPK is activated; access to that residue by phosphatases is blocked if AMP or ADP can block access for and ATP can displace AMP and ADP.
  • That residue is phosphorylated by at least three kinases (liver kinase Bl (LKB1) which works in a complex with STRAD and M025, Calcium/calmodulin-dependent protein kinase kinase II-(CAMKK2), and TGFb-activated kinase 1 (TAK1)) and is dephosphorylated by three phosphatases (protein phosphatase 2A (PP2A); protein phosphatase 2C (PP2C) and Mg2+-/Mn2+-dependent protein phosphatase 1E (PPM1E)).
  • P2A protein phosphatase 2A
  • P2C protein phosphatase 2C
  • PPM1E Mg2+-/Mn
  • AMPK is regulated allosterically mostly by competitive binding on its gamma subunit between ATP (which allows phosphatase access to T172) and AMP or ADP (each of which blocks access to phosphatases). It also appears that AMPK is a sensor of AMP/ ATP or ADP/ATP ratios and thus cell energy level. Regulation of AMPK by CaMKK2 requires a direct interaction of these two proteins via their kinase domains. The interaction of CaMKK2 with AMPK only involves the alpha and beta subunits of AMPK (AMPK gamma is absent from the CaMKK2 complex), thus rendering regulation of AMPK in this context to changes in calcium levels but not AMP or ADP.
  • AMPK may be inhibited or activated by various tissue-specific ubiquitinations.
  • AMPK phosphorylates acetyl-CoA carboxylase 1 (ACC1) or sterol regulatory element-binding protein lc (SREBPlc), it inhibits synthesis of fatty acids, cholesterol, and triglycerides, and activates fatty acid uptake and b-oxidation.
  • ACC1 acetyl-CoA carboxylase 1
  • SREBPlc sterol regulatory element-binding protein lc
  • AMPK stimulates glucose uptake in skeletal muscle by phosphorylating Rab- GTPase-activating protein TBC1D1, which ultimately induces fusion of GLUT4 vesicles with the plasma membrane.
  • AMPK stimulates glycolysis by activating phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2/3 and activating phosphorylation of glycogen phosphorylase, and it inhibits glycogen synthesis through inhibitory phosphorylation of glycogen synthase.
  • AMPK inhibits gluconeogenesis by inhibiting transcription factors including hepatocyte nuclear factor 4 (HNF4) and CREB regulated transcription coactivator 2 (CRTC2).
  • HNF4 hepatocyte nuclear factor 4
  • CRTC2 CREB regulated transcription coactivator 2
  • AMPK inhibits the energy-intensive protein biosynthesis process and can also force a switch from cap-dependent translation to cap-independent translation, which requires less energy, by phosphorylation of TSC2, RPTOR, transcription initiation factor 1A.66, and eEF2K.
  • TSC2 When TSC2 is activated it inhibits mTORCl .
  • mTORCl As a result of inhibition of mTORCl by AMPK, protein synthesis comes to a halt which results in inhibits a energy requiring pathway such as protein synthesis.
  • activation of AMPK signifies low energy charge of the cell, so all of the energy consuming pathways like protein synthesis are inhibited, and pathways that generate energy are activated to restore appropriate energy levels in the cell.
  • AMPK activates autophagy by directly and indirectly activating ULK1.
  • AMPK also appears to stimulate mitochondrial biogenesis by regulating PGC-la which in turn promotes gene transcription in mitochondria.
  • AMPK also activates anti-oxidant defenses.
  • AMPK allows the contracting muscle cells to adapt to the energy challenges by increasing expression of hexokinase II, translocation of GLUT4 to the plasma membrane, for glucose uptake, and by stimulating glycolysis. If bouts of exercise continue through a long-term training regimen, AMPK and other signals will facilitate contracting muscle adaptations by escorting muscle cell activity to a metabolic transition resulting in a fatty-acid oxidation approach to ATP generation as opposed to a glycolytic approach.
  • AMPK accomplishes this transition to the oxidative mode of metabolism by upregulating and activating oxidative enzymes such as hexokinase II, PPARalpha, PPARdelta, PGC-l, UCP-3, cytochrome C and TFAM.
  • oxidative enzymes such as hexokinase II, PPARalpha, PPARdelta, PGC-l, UCP-3, cytochrome C and TFAM.
  • AMPK activity increases with exercise and the LKB1/M025/STRAD complex is considered to be the major upstream AMPKK of the 5’-AMP-activated protein kinase phosphorylating the a subunit of AMPK at Thr-l72.
  • This fact is puzzling considering that although AMPK protein abundance has been shown to increase in skeletal tissue with endurance training, its level of activity has been shown to decrease with endurance training in both trained and untrained tissue.
  • the activity of AMPK immediately following a 2-hr bout of exercise of an endurance trained rat is unclear. It is possible that there exists a direct link between the observed decrease in AMPK activity in endurance trained skeletal muscle and the apparent decrease in the AMPK response to exercise with endurance training.
  • Maximum life span The C. elegans homolog of AMPK, aak-2, has been shown by Michael Ristow and colleagues to be required for extension of life span in states of glucose restriction mediating a process named mitohormesis.
  • Lipid metabolism One of the effects of exercise is an increase in fatty acid metabolism, which provides more energy for the cell.
  • One of the key pathways in AMPK’s regulation of fatty acid oxidation is the phosphorylation and inactivation of acetyl-CoA carboxylase.
  • Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT-l).
  • CPT-l transports fatty acids into the mitochondria for oxidation. Inactivation of ACC, therefore, results in increased fatty acid transport and subsequent oxidation.
  • MCD malonyl-CoA decarboxylase
  • HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from acetyl-CoA, into mevalonic acid, which then travels down several more metabolic steps to become cholesterol.
  • AMPK helps regulate fatty acid oxidation and cholesterol synthesis.
  • Glucose transport is a hormone which helps regulate glucose levels in the body. When blood glucose is high, insulin is released from the Islets of Langerhans. Insulin, among other things, will then facilitate the uptake of glucose into cells via increased expression and translocation of glucose transporter GLUT-4. Under conditions of exercise, however, blood sugar levels are not necessarily high, and insulin is not necessarily activated, yet muscles are still able to bring in glucose. AMPK seems to be responsible in part for this exercise-induced glucose uptake.
  • the concentration of GLUT-4 was increased in the plasma membrane, but decreased in the microsomal membranes, suggesting that exercise facilitates the translocation of vesicular GLUT-4 to the plasma membrane.
  • hexokinase phosphorylates a six-carbon sugar, most notably glucose, which is the first step in glycolysis.
  • glucose When glucose is transported into the cell it is phosphorylated by hexokinase. This phosphorylation keeps glucose from leaving the cell, and by changing the structure of glucose through phosphorylation, it decreases the concentration of glucose molecules, maintaining a gradient for more glucose to be transported into the cell.
  • Hexokinase II transcription is increased in both red and white skeletal muscle upon treatment with AICAR. With chronic injections of AICAR, total protein content of hexokinase II increases in rat skeletal muscle.
  • Mitochondria Mitochondrial enzymes, such as cytochrome c, succinate dehydrogenase, malate dehydrogenase, a-ketoglutarate dehydrogenase, and citrate synthase, increase in expression and activity in response to exercise.
  • AICAR stimulation of AMPK increases cytochrome c and d-aminolevulinate synthase (ALAS), a rate-limiting enzyme involved in the production of heme.
  • Malate dehydrogenase and succinate dehydrogenase also increase, as well as citrate synthase activity, in rats treated with AICAR injections.
  • LKB1 knockout mice there are decreases in cytochrome c and citrate synthase activity, even if the mice are "trained" by voluntary exercise.
  • Peroxisome proliferator-activated receptor gamma coactivator- la (PGC-la) is a transcriptional regulator for genes involved in fatty acid oxidation, gluconeogenesis, and is considered the master regulator for mitochondrial biogenesis.
  • NRF-l nuclear respiratory factor 1
  • MEF2 myocyte enhancer factor 2
  • HCF host cell factor
  • MEF2 and cAMP response element are essential for contraction- induced PGC-la promoter activity.
  • AMPK is required for increased PGC-la expression in skeletal muscle in response to creatine depletion.
  • LKB1 knockout mice show a decrease in PGC-la, as well as mitochondrial proteins.
  • AMPK and thyroid hormone regulate some similar processes. Knowing these similarities, Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid hormone. They found that all of the subunits of AMPK were increased in skeletal muscle, especially in the soleus and red quadriceps, with thyroid hormone treatment. There was also an increase in phospho-ACC, a marker of AMPK activity.
  • Glucose sensing system Loss of AMPK has been reported to alter the sensitivity of glucose sensing cells, through poorly defined mechanisms. Loss of the AMPKa2 subunit in pancreatic beta cells and hypothalamic neurons decreases the sensitivity of these cells to changes in extracellular glucose concentration. Moreover, exposure of rats to recurrent bouts of insulin induced hypoglycaemia/glucopenia, reduces the activation of AMPK within the hypothalamus, whilst also suppressing the counterregulatory response to hypoglycaemia. Pharmacological activation of AMPK by delivery of AMPK activating drug AICAR, directly into the hypothalamus can increase the counterregulatory response to hypoglycaemia.
  • AICAR 5-Aminoimidazole-4-carboxamide ribonucleotide
  • AICAR is an intermediate in the generation of inosine monophosphate.
  • AICAR is an analog of adenosine monophosphate (AMP) that is capable of stimulating AMP-dependent protein kinase (AMPK) activity.
  • AMPK AMP-dependent protein kinase
  • AICAR has been used clinically to treat and protect against cardiac ischemic injury. The drug was first used in the l980s as a method to preserve blood flow to the heart during surgery. Currently, the drug has also been shown as a potential treatment for diabetes by increasing the metabolic activity of tissues by changing the physical composition of muscle.
  • the nucleoside form of AICAR is an analog of adenosine that enters cardiac cells to inhibit adenosine kinase and adenosine deaminase. It enhances the rate of nucleotide re-synthesis increasing adenosine generation from adenosine monophosphate only during conditions of myocardial ischemia.
  • acadesine is phosphorylated to AICAR to activate AMPK without changing the levels of the nucleotides.
  • AICAR is able to enter the de novo synthesis pathway for adenosine synthesis to inhibit adenosine deaminase causing an increase in ATP levels and adenosine levels.
  • preconditioning A brief period of coronary arterial occlusion followed by reperfusion prior to prolonged ischemia is known as preconditioning. It has been shown that this is protective. Preconditioning preceded myocardial infarction, may delay cell death and allow for greater salvage of myocardium through reperfusion therapy.
  • AICAR has been shown to precondition the heart shortly before or during ischemia. AICAR triggers a preconditioned anti-inflammatory state by increasing NO production from endothelial nitric oxide synthase.
  • AICAR When AICAR is given 24 hours prior to reperfusion, it prevents post ischemic leukocyte-endothelial cell adhesive interactions with increased NO production. AICAR- dependent preconditioning is also mediated by an ATP-sensitive potassium channel and hemeoxygenase-dependent mechanism. It increases AMPK-dependent recruitment of ATP-sensitive K channels to the sarcolemma causing the action potential duration to shorten and preventing calcium overload during reperfusion. The decrease in calcium overload prevents inflammation activation by ROS. AICAR also increases AMPK- dependent glucose uptake through translocation of GLUT-4 which is beneficial for the heart during post-ischemic reperfusion.
  • AICAR The increase in glucose during AICAR preconditioning lengthens the period for preconditioning up to 2 hours in rabbits and 40 minutes in humans undergoing coronary ligation.
  • AICAR reduces the frequency and size of myocardial infarcts up to 25% in humans allowing improved blood flow to the heart.
  • the treatment has been shown to decrease the risk of an early death and improve recovery after surgery from an ischemic injury.
  • Metformin marketed under the trade name Glucophage among others, is the first- line medication for the treatment of type 2 diabetes, particularly in people who are overweight. It is also used in the treatment of polycystic ovary syndrome.
  • Metformin is taken orally and generally well tolerated. Common side effects include diarrhea, nausea, and abdominal pain. It has a low risk of causing low blood sugar. High blood lactic acid level is a concern if the medication is prescribed inappropriately or in overly large doses. It should not be used in those with significant liver disease or kidney problems. While no clear harm comes from use during pregnancy, insulin is generally preferred for gestational diabetes. Metformin is a biguanide antihyperglycemic agent. It works by decreasing glucose production by the liver and increasing the insulin sensitivity of body tissues. The most common adverse effect of metformin is gastrointestinal irritation, including diarrhea, cramps, nausea, vomiting, and increased flatulence; metformin is more commonly associated with gastrointestinal side effects than most other antidiabetic medications. The most serious potential side effect of metformin use is lactic acidosis; this complication is very rare, and the vast majority of these cases seem to be related to comorbid conditions, such as impaired liver or kidney function, rather than to the metformin itself.
  • metformin The molecular mechanism of metformin is not completely understood. Multiple potential mechanisms of action have been proposed: inhibition of the mitochondrial respiratory chain (complex I), activation of AMP-activated protein kinase (AMPK), inhibition of glucagon-induced elevation of cyclic adenosine monophosphate (cAMP) with reduced activation of protein kinase A (PKA), inhibition of mitochondrial glycerophosphate dehydrogenase, and an effect on gut microbiota. Ultimately, it decreases gluconeogenesis (liver glucose production). It also has an insulin-sensitizing effect with multiple actions on tissues including the liver, skeletal muscle, endothelium, adipose tissue, and the ovary. The average patient with type 2 diabetes has three times the normal rate of gluconeogenesis; metformin treatment reduces this by over one-third.
  • AMPK Activation of AMPK was required for metformin's inhibitory effect on liver glucose production.
  • AMPK is an enzyme that plays an important role in insulin signalling, whole body energy balance and the metabolism of glucose and fats.
  • AMPK Activation was required for an increase in the expression of small heterodimer partner, which in turn inhibited the expression of the hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase and glucose 6-phosphatase.
  • Metformin is frequently used in research along with AICA ribonucleotide as an AMPK agonist.
  • AMP cytosolic adenosine monophosphate
  • Increased cellular AMP has been proposed to explain the inhibition of glucagon-induced increase in cAMP and activation of PKA.
  • Metformin and other biguanides may antagonize the action of glucagon, thus reducing fasting glucose levels. Metformin also induces a profound shift in the faecal microbial community profile in diabetic mice and this may contribute to its mode of action possibly through an effect on glucagon-like peptide- 1 secretion.
  • metformin increases insulin sensitivity, enhances peripheral glucose uptake (by inducing the phosphorylation of GLUT4 enhancer factor), decreases insulin-induced suppression of fatty acid oxidation, and decreases absorption of glucose from the gastrointestinal tract. Increased peripheral use of glucose may be due to improved insulin binding to insulin receptors. The increase in insulin binding after metformin treatment has also been demonstrated in patients with NIDDM.
  • AMPK probably also plays a role in increased peripheral insulin sensitivity, as metformin administration increases AMPK activity in skeletal muscle. AMPK is known to cause GLUT4 deployment to the plasma membrane, resulting in insulin-independent glucose uptake. Some metabolic actions of metformin do appear to occur by AMPK- independent mechanisms.
  • Metformin hydrochloride (l,l-dimethylbiguanide hydrochloride) is freely-soluble in water, slightly soluble in ethanol, but almost insoluble in acetone, ether, or chloroform.
  • the pKa of metformin is 12.4.
  • the usual synthesis of metformin, originally described in 1922, involves the one-pot reaction of dimethylamine hydrochloride and 2-cyanoguanidine over heat.
  • Metformin has an oral bioavailability of 50-60% under fasting conditions and is absorbed slowly. Peak plasma concentrations (C max ) are reached within one to three hours of taking immediate-release metformin and four to eight hours with extended-release formulations. The plasma protein binding of metformin is negligible, as reflected by its very high apparent volume of distribution (300-1000 I after a single dose). Steady state is usually reached in one or two days.
  • Metformin has acid dissociation constant values (pKa) of 2.8 and 11.5, so exists very largely as the hydrophilic cationic species at physiological pH values.
  • the metformin pKa values make metformin a stronger base than most other basic medications with less than 0.01% nonionized in blood.
  • the lipid solubility of the nonionized species is slight as shown by its low logP value (log(lO) of the distribution coefficient of the nonionized form between octanol and water) of -1.43.
  • metformin The logP of metformin is less than that of phenformin (-0.84) because two methyl substituents on metformin impart lesser lipophilicity than the larger phenylethyl side chain in phenformin. More lipophilic derivatives of metformin are presently under investigation with the aim of producing prodrugs with superior oral absorption than metformin. Metformin is not metabolized. It is cleared from the body by tubular secretion and excreted unchanged in the urine; metformin is undetectable in blood plasma within 24 hours of a single oral dose. The average elimination half-life in plasma is 6.2 hours. Metformin is distributed to (and appears to accumulate in) red blood cells, with a much longer elimination half-life: 17.6 hours (reported as ranging from 18.5 to 31.5 hours in a single dose study of nondiabetics).
  • Resveratrol (3.5.4'-trihydroxy-/ra -stilbene) is a stilbenoid, a type of natural phenol, and a phytoalexin produced by several plants in response to injury or, when the plant is under attack by pathogens, such as bacteria or fungi.
  • Sources of resveratrol in food include the skin of grapes, blueberries, raspberries, mulberries, and peanuts.
  • Resveratrol has been identified as a pan-assay interference compound, which produces positive results in many different laboratory assays. Its ability for varied interactions may be due to direct effects on cell membranes.
  • resveratrol activates sirtuin 1, although this may be a downstream effect from its immediate biological target(s). It appears to signal through PGC-la, thereby affecting mitochondria. In cells treated with resveratrol, an increase is observed in the action of MnSOD (SOD2) and in GPER activity. In vitro, resveratrol was shown to act as an agonist of Peroxisome proliferator-activated receptor gamma, a nuclear receptor under pharmacological research as a potential treatment for type 2 diabetes.
  • One way of administering resveratrol in humans may be buccal delivery by direct absorption through the saliva.
  • Resveratrol is extensively metabolized in the body, with the liver and lungs as the major sites of its metabolism.
  • Resveratrol (3,5,4'-trihydroxystilbene) is a stilbenoid, a derivative of stilbene. It exists as two geometric isomers: cis- (Z) and trans- (A) with the trans- isomer shown in the top image.
  • the trans- and cv.v-resveratrol can be either free or bound to glucose.
  • the trans- form can undergo isomerization to the cis- form when exposed to ultraviolet irradiation, a process called photoisomerization:
  • Resveratrol is produced in plants by the action of the enzyme, resveratrol synthase.
  • the present disclosure in one aspect, relates to the production and formulation of mitochondrial biogenesis modulators as well as their delivery to cells, tissues or subjects.
  • recombinant production of proteins is well known and is therefore no described in detail here.
  • the discussion of nucleic acids and expression vectors, found below, is however incorporated in this discussion.
  • Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
  • purified protein as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally - obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • “purified” will refer to a protein composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term“substantially purified” is used, this designation will refer to a composition in which the protein forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • Various methods for quantifying the degree of purification of the protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a“-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low-pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • molecules are eluted from the column in decreasing size, so long as the shape is relatively constant.
  • Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl- D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present disclosure is discussed below.
  • cell permeability peptide also called a cell delivery peptide, or cell transduction domain
  • Such domains have been described in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007). Other examples are shown in Table 1, below.
  • NRARRNRRRVR 14 ALWMTLLKKVLKAAAKAALNAVL 36
  • KLAKLAKKLAKLAK 19 SMAQDIIS ⁇ GDLVKWIIQTVNXFTK 41
  • proteins are delivered to cells as a formulation that promotes entry of the proteins into a cell of interest.
  • lipid vehicles such as liposomes.
  • liposomes which are artificially prepared vesicles made of lipid bilayers have been used to delivery a variety of drugs.
  • Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains (like egg phosphatidylethanolamine) or other surfactants.
  • liposomes containing cationic or neutural lipids have been used in the formulation of drugs. Liposomes should not be confused with micelles and reverse micelles composed of monolayers, which also can be used for delivery.
  • a wide variety of commercial formulations for protein delivery are well known including PULSinTM, Lipodin-Pro, Carry-MaxR, Pro-DeliverlN, PromoFectin, Pro-Ject, ChariotTM Protein Delivery reagent, BioPORTERTM, and others.
  • Nanoparticles are generally considered to be particulate substances having a diameter of 100 nm or less. In contrast to liposomes, which are hollow, nanoparticles tend to be solid. Thus, the drug will be less entrapped and more either embedded in or coated on the nanoparticle. Nanoparticles can be made of metals including oxides, silica, polymers such as polymethyl methacrylate, and ceramics. Similarly, nanoshells are somewhat larger and encase the delivered substances with these same materials. Either nanoparticles or nanoshells permit sustained or controlled release of the peptide or mimetic and can stabilize it to the effects of in vivo environment.
  • expression cassettes are employed to express a peptide or protein, either for subsequent purification and delivery to a cell/subject, or for use directly in a genetic-based delivery approach.
  • Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells.
  • Elements designed to optimize messenger RNA stability and translatability in host cells also are defined.
  • the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide.
  • the term“expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i. e. , is under the control of a promoter.
  • A“promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • the phrase“under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.
  • An“expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase ( tk ) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
  • At least one module in each promoter functions to position the start site for RNA synthesis.
  • the best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.
  • viral promotes such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.
  • CMV human cytomegalovirus
  • SV40 early promoter the Rous sarcoma virus long terminal repeat
  • rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase
  • glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter with well-
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • a cDNA insert where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript.
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the disclosure, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals.
  • a terminator Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • IRES elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Samow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
  • the expression vector comprises a genetically engineered form of adenovirus.
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes.
  • the expression of the E2 region results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5 '-tripartite leader
  • recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
  • adenovirus generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh- Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA.
  • the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells.
  • the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells.
  • the preferred helper cell line is 293.
  • Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into l-liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present disclosure is replication defective and will not have an adenovirus El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the disclosure.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, as described by Karlsson et al. (1986), or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 12 plaque forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al 1990; Rich et al, 1993).
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990).
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants.
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively.
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
  • a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et ctl, 1975).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus etal, 1981).
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome.
  • new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).
  • viral vectors may be employed as expression constructs in the present disclosure.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990). In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell.
  • This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
  • a naked DNA expression construct into cells may involve particle bombardment.
  • This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987).
  • Several devices for accelerating small particles have been developed.
  • One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, /. e.. ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful.
  • Wong et al, (1980) demonstrated the feasibility of liposome- mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells.
  • Nicolau et al, (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection.
  • a reagent known as Lipofectamine 2000TM is widely used and commercially available.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-l) (Kato et ctl, 1991).
  • HMG-l nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-l.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang al, 1990
  • neoglycoprotein which recognizes the same receptor as ASOR, has been used as agene delivery vehicle (Ferkol etal, 1993; Perales etal., 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
  • the present disclosure provides for the treating of disorders relating to mitochondrial biogenesis and its disfunction, and to stimulating erythropoeisis.
  • the treatment may result in amelioration of any symptom of a given disease or disorder.
  • compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • Aqueous compositions of the present disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.
  • compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • these preparations are sterile and fluid to the extent that easy injectability exists.
  • Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • a coating such as lecithin
  • surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
  • the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the present disclosure generally may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups of the protein can also be
  • solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution generally is suitably buffered, and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
  • a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” l5th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • Fe-S clusters are essential cofactors for mitochondria functions, and mitochondria are required for Fe-S cluster synthesis. Additionally, mitochondria biogenesis demands cellular iron uptake, which is negatively regulated by Fe-S clusters. Fe-S clusters are synthesized in the mitochondria and cytosol by two different machineries. However, cytosolic Fe-S cluster synthesis necessitates the mitochondrial Fe- S cluster assembly machinery.
  • PGCla is a transcriptional coactivator and a master regulator of mitochondria biogenesis.
  • the inventors confirmed that overexpression of PGCla in adipocytes and hepatic cells stimulated mitochondria biogenesis, as measured by Mitotrack Green and Deep Red staining, which label total and alive mitochondria, respectively. They further measured Fe-S cluster synthesis by monitoring the gene expression of Fe-S cluster assembly machinery. By using RT-qPCR and Western Blot analyses, the inventors confirmed that PGC-la expression increases expression of ABCB7, ISCA1, ISCA2, ISD11, Nful and ISCU, components of the Fe-S assembly machinery, suggesting a coordination between mitochondria biogenesis and Fe-S cluster synthesis.
  • IRP1 and IRP2 Iron Regulatory Proteins (IRP1 and IRP2) control iron metabolism by binding to specific non-coding sequences within an mRNA, known as iron-responsive elements (IRE).
  • IRP1 acts as an aconitase (aka ACOl)
  • IRP2 is degraded by ubiquitination.
  • Aconitases represented by the cytosolic form ACOl and mitochondrial form AC02, catalyze the isomerization of citrate to isocitrate and require Fe-S clusters to be enzymatically active.
  • PGCla overexpression enhanced aconitase activity but not their protein levels, corroborating the notion that Fe-S cluster synthesis was increased.
  • the inventors evaluated the Fe-S cluster synthesis status during brown adipocyte maturation, which is characterized by enhanced mitochondria biogenesis and has been suggested to be PGC la- independent. They found that the synthesis of Fe-S cluster assembly machinery increased during maturation in both wild-type and PGC la-knockout brown adipocytes, indicating that Fe-S cluster synthesis coordinates with mitochondria biogenesis even in the absence of PGC la.
  • TfRl iron importer transferrin receptor 1
  • UTR untranslated region
  • IRP1 associates with Fe-S cluster and converted into ACOl
  • TfRl expression would decrease.
  • the inventors found that stimulated Fe-S cluster synthesis increased levels of TfRl, despite reduced IRP1 activity and destabilized TfRl mRNA. This suggests that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block iron uptake.
  • MEL murine erythroleukemia
  • Stimulated mitochondria biogenesis enhanced expression of the Fe-S cluster assembly machinery and Fe-S cluster synthesis in these cells.
  • TfRl protein levels were increased despite elevated Fe-S cluster synthesis and reduced IRP activity. They found increases in heme levels and the expression of aminolevulinic acid synthase 2 (ALAS2), the rate-limiting enzyme for erythroid heme synthesis.
  • ALAS2 mRNA contains IRE at the 5’UTR, binding of IRPs to the IRE inhibits translation while high Fe-S cluster levels lead to release.
  • Fe-S clusters are iron-containing prosthetic groups and enzymatic cofactors. They are strong oxidants when unbound yet essential in many processes like facilitating ATP production in mitochondria, promoting DNA, RNA and protein syntheses during cell proliferation and enhancing DNA repair in antioxidant defense.
  • Fe-S clusters are indispensable in erythropoiesis, where the majority of physiological iron is utilized and where Fe-S clusters are required for the heme synthesis.
  • Deficient Fe-S cluster synthesis predisposes individual to various diseases, such as cancer, metabolic and neurodegeneration diseases and blood disorders. However, it is unclear how Fe-S cluster synthesis is regulated and coordinates with environmental and developmental needs to prevent oxidative damage.
  • AMPK 5' AMP-activated protein kinase
  • the inventors found that the Fe-S cluster assembly enzyme (ISCU), a scaffold protein indispensable for Fe-S cluster biogenesis, contains putative AMPK phosphorylation motifs at serine (S) residues 14 and 29 (human numbering).
  • ISCU Fe-S cluster assembly enzyme
  • S serine residues 14 and 29
  • AMPK-mediated phosphorylation promoted ISCU binding to l4-3-3s, a family of proteins that, once associate with phosphorylated residues, modulates the stability and function of targeted proteins. Indeed, increased association with l4-3-3s stabilized ISCU proteins, corroborating the observation that AMPK promotes the activity of ISCU proteins.
  • the inventors extended their studies using A549 cells that do not have AMPK activity since they harbor a mutant LKB1 kinase, which is responsible for activating AMPK.
  • WT wild-type
  • KDM LKB1 kinase-dead mutant
  • the inventors then explored the effect of AMPK on Fe-S cluster synthesis in erythropoiesis by using the drug AICAR, an AMPK activator, in murine erythroleukemia (MEL) cells. They found that in MEL cells, AICAR treatment stabilized ISCU, increased Fe-S cluster levels and promoted the synthesis of the aminolevulinic acid synthase 2 (ALAS2) protein, which represents the rate-limiting enzyme in erythroid heme synthesis. Furthermore, this was associated with increased heme and globin chain synthesis, with a trend in increasing b-globin mRNA and proteins more than a-globin.
  • AICAR aminolevulinic acid synthase 2
  • the inventors further confirmed these observations in Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP-2) and CD34 + cells derived from peripheral blood isolated from both healthy individuals and B-thalassemic patients.
  • UDEP-2 Human Umbilical Cord Blood-Derived Erythroid Progenitor
  • CD34 + cells derived from peripheral blood isolated from both healthy individuals and B-thalassemic patients.
  • AMPK upregulation by AICAR administration not only increased ALAS2 expression and erythroid heme levels, but also enhanced the synthesis of both a- and B-globin chains, though with a preference for increasing b-globin levels. Analysis using specimens from thalassemic mice is in progress.
  • Human hepatocellular carcinoma cell line HepG2 and murine preadipocytes 3T3-L1 were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% (v/v) fetal calf serum, penicillin (100 units/ml) and streptomycin (100 pg/ml), under 5% CCh and at 37 °C.
  • Mouse prebrown adipocytes were generated a generous gift from Bruce Spiegelman (Harvard Medical School). They were cultured and differentiated as previously described (Uldry et al., 2006a).
  • Mouse erythroleukemia were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% (v/v) fetal calf serum, penicillin (100 units/ml) and streptomycin (100 pg/ml), under 5% CCh and at 37 °C.
  • RPMI Roswell Park Memorial Institute
  • HMBA hexamethylene bisacetamide
  • Aconitase activity assay In Gel assay was applied as described previously (Tong and Roualt, 2006; Ghosh et al, 2008). Briefly, cell lysis was separated by PAGE with Tris-borate buffer and then incubated with the solution containing cis-aconitic acid, isocitric dehydrogenase, phenazine methyl sulfate (PMS), NADP and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 37 °C for 10 minutes. After the incubation, gels were photographed.
  • Mitochondria biogenesis assays Cells were labeled with 100hM Mitotracker Green FM or Deep Red FM (Thermo fisher) and followed by fluorescence-activated cell sorting (FACS). FACS data were analyzed by using FlowJo7 software. Analysis of IRE-binding activities. IRE-binding activities were measured differently for FIGS. 3A-I and FIGS. 4A-E. For FIGS. 3A-I, IRE-binding activity was analyzed as previously described (Meyron-Holtz et al, 2004).
  • IRE sequence derived from ferritin heavy chain (FtH) mRNA was cloned and further labeled with phosphorus-32 radioactive isotope by using Riboprobe® Systems (Promega). Equal amount of total proteins was incubated with IRE probes and separated by PAGE followed by an exposure to X-ray films.
  • IRE-binding activities were measured by using LightShiftTM Chemiluminescent RNA EMSA Kit (Thermo Fisher) per manufacturer’s instruction.
  • the Myc epitope was fused at the N-terminus of human PGC-la cDNA and cloned in PMX-puro for retrovirus production and EGAWP for lentiviruses production. GFP was also introduced in these vectors as a control. Retroviruses was used on 3T3-L1 and HepG2 while lentiviruses were used on MEL cells.
  • PGC-la Peroxisome proliferator-activated receptor gamma coactivator-1 alpha
  • Myc-PGC-la Peroxisome proliferator-activated receptor gamma coactivator-1 alpha
  • Myc-PGC- la expression enhanced heme levels and the gene expression of ALAS1 (FIG. 1C), a rate- limiting enzyme in non-erythroid heme synthesis pathways (Handschin et al, 2005).
  • ALAS1 Fatty acid hydroxyase
  • Aconitases represented by the cytosolic form ACOl and mitochondrial form AC02, require Fe-S cluster to be enzymatically active. Their activity was used to monitor the status of Fe-S cluster synthesis in cytosol (ACOl) and mitochondria (AC02).
  • ACOl cytosol
  • AC02 mitochondria
  • Myc-PGC-la expression significantly increased ACOl activity despite limited increase in protein levels (FIG. 1D), indicating enhanced Fe-S cluster synthesis in the cytosol.
  • Myc-PGC-la expression increased both protein levels and activity.
  • simple overexpression of AC02 protein in 3T3-L1 did not increase its activity (data not shown).
  • the inventors quantified AC02 activity (shown by the blue color intensity in FIG. 1D, first panel from the top) and protein levels obtained by Western blot analysis (FIG. 1D, third panel from the top), then divided the former by the latter to normalize AC02 activity by its protein levels.
  • Myc-PGC-la overexpression increased AC02 activity by a factor of 2.7 (FIG. 1E), confirming that such an increase was due to an elevated Fe-S cluster synthesis in mitochondria.
  • Myc-PGC-la was also expressed in human hepatoma cell line HepG2 (FIG. S1A) and increased Fe-S clusters synthesis both in mitochondria and in cytosol (FIGS. S1B-S1C). Together, these data demonstrated that Fe-S cluster synthesis coordinates with PGC- la-enhanced mitochondria biogenesis.
  • Fe-S cluster synthesis coordinates with mitochondria biogenesis also in the absence of PGC-Ia.
  • PGC-la is the master regulator of mitochondria biogenesis
  • wild-type (WT) and PGC-la-null preadipocytes were differentiated to mature brown adipocytes by standard drug treatment for 6 days (Uldry et al. , 2006a).
  • PGC-la was induced only in wild-type cells while R ⁇ OIb, another member of the PGC-l family that is able to stimulate mitochondria biogenesis, was induced in both WT and PGC-la-null cells (FIGS. S2A- B).
  • PGC-la mRNA was detected in PGC-la-null cells due to the mRNA region targeted by the TaqMan probe not being eliminated and potentially transcribed in these cells Lin et al. , 2005).
  • expressions of uncoupling protein 1 (UCP1, a mitochondria marker), ALAS1 and heme levels were significantly induced in both of matured WT and PGC-la-null cells (FIGS. S2C-D), confirming that mitochondria biogenesis was significantly induced during matured brown adipocytes.
  • Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block mitochondria-required iron uptake.
  • enhanced mitochondria biogenesis stimulates heme and Fe-S cluster synthesis, two major iron-consuming pathways, the inventors explored how TfRl expression was regulated by mitochondria biogenesis- coordinated Fe-S cluster synthesis. Since enhanced Fe-S cluster availability might decrease the IRE-binding activity of IRP1 (Zhang et al. , 2014), the inventors analyzed IRPs/IRE activity. As shown in FIG.
  • TfRl regulation My c-PGC- la-expressing cells were incubated with either a proteasome inhibitor (MG132), a transcription inhibitor (Actinomycin D) or a translation inhibitor (cycloheximide). Incubation with MG132 reversed the increased TfRl protein levels while actinomycin D and cycloheximide had limited effect (FIGS. 4D-E). These data suggest that increased TfRl expression is primarily at the post-translational level. Possibly, increased ATP production due to the enhanced mitochondrial biogenesis potentiates TfRl folding and refolding to avoid degradation, though this requires further investigation.
  • MG132 proteasome inhibitor
  • Actinomycin D transcription inhibitor
  • cycloheximide translation inhibitor
  • Fe-S cluster assembly coordinates with mitochondria biogenesis but not iron uptake and ultimately modulates heme synthesis and hemoglobinization.
  • the regulation of iron metabolism, especially heme metabolism, are always different between erythroid and non-erythroid tissues (Ajioka el ah, 2006; Muckenthaler el al, 2017). Therefore, the inventors also investigated how mitochondria biogenesis, Fe-S cluster synthesis and iron uptake maintain homeostasis during erythropoiesis by using murine erythroleukemia (MEL) cells, an in vitro model of erythropoiesis (Cui et ah, 2014).
  • MEL murine erythroleukemia
  • Myc-PGC-la into MEL cells by viral transduction and differentiated these cells into erythrocytes by incubation with HMBA.
  • Myc-PGC-la expression stimulated the activity and expression of Fe-S cluster assembly machinery, as evidenced by increased ACOl and AC02 activities as well as increased mRNA and protein levels of genes involved in Fe-S cluster synthesis.
  • the inventors explored the impact of decreasing mitochondria biogenesis by lentiviral transduction of PGC-la shRNA (FIG. 6C).
  • FIG. 6D protein levels of ISD11, ISCU, ABCB7 and ISCA1/2 were decreased in PGC- la-knockdown cells vs. control.
  • Fe-S cluster synthesis was also inhibited, as demonstrated by decreased aconitase activities of ACOl and AC02 (FIG. 6D, top panel) and confirming a coordination between Fe-S cluster synthesis and mitochondria biogenesis in erythroid cells. Similar to non-erythroid cells, for some genes of the assembly machinery, the inventors did not observe the same trend between mRNA and protein levels changes, especially when PGC-la expression was suppressed (FIG. 6E). Of note, HMBA incubation did not interfere with Fe-S cluster synthesis coordinating with mitochondria biogenesis as similar effects were observed in undifferentiated Myc- PGC-la-expressing and PGC-la-shRNA-transduced MEL cells (FIGS. S4A-B). Nonetheless, these data demonstrated that Fe-S cluster synthesis also coordinates with mitochondria biogenesis in erythroid cells.
  • the inventors then examined how mitochondrial biogenesis and Fe-S cluster synthesis affect iron demand, namely TfRl expression in erythroid cells.
  • the IRE-binding activity was decreased in My c-PGC- la-expressing cells while IRPs levels remained similar.
  • the mRNA and protein levels of TfRl were both increased (FIGS. 7A-B), suggesting an IRE-binding and IRPs-independent mechanism.
  • Myc-PGC-la stimulated the expression of GATA1 (FIG.
  • ALAS2 contains a 5’UTR IRE, and its translation can be inhibited by IRPs binding.
  • Myc-PGC-la in MEL cells significantly stimulated ALAS2 levels and further hemoglobinization evidenced by a more reddish color of cell pellets (FIG. 8A).
  • HBA and HBB a- and b-globin
  • PGC-l a knockdown decreased the expression of ALAS2, HBB and HBA as well as hemoglobinization (FIGS. 8C-D).
  • PGC-la antagonizes the mitophagy that is required for the late stage of erythropoiesis (Lin et al. , 2005; Grosso et al, 2017)
  • Myc-PGC-la-expressing cells were treated with HMBA for a short period time, only 5 days to avoid this detrimental effect. Nonetheless, these data suggested that erythroid Fe-S cluster synthesis coordinates with mitochondria biogenesis without limiting iron uptake, but rather improving the synthesis of heme and hemoglobin.
  • mitochondrial Fe-S cluster synthesis happens in tandem with mitochondria biogenesis. This is not due to merely increased mitochondria mass, because not all the genes tested in our study demonstrated an increased expression level in Myc-PGC-la-expressing cells. For instance, protein levels of Nfsl were not increased by Myc-PGC-la expression though Nfsl is a mitochondria protein and required for Fe-S cluster synthesis (data not shown). This observation suggests that under enhanced mitochondria biogenesis, the increased expression of Fe-S cluster synthesis machinery is selective and not simply due to increased mitochondria mass.
  • PGC-la and PGC- I b are members of PGC-l family. As transcription activators, they functionally compensate one another by associating with transcription factors that play pivotal roles in stimulating mitochondria biogenesis (Uldry el ah, 2006b). By a bioinformatic analysis (UCSC genome browser), various binding sites for these transcription factors were identified in genes responsible for Fe-S cluster synthesis. For instance, FOXOl binding sites were predicted in ISCA1 gene and GR-alpha and GR- beta in ISCA2 genes, explaining an upregulated mRNA levels of these genes under enhanced mitochondria biogenesis (FIGS. 2A-B, 3A-I and 8A-D).
  • the gene expression could also be regulated on post-transcriptional level (Rensvold et al, 2013; Liu et al, 2017). Therefore, the expression of Fe-S cluster synthesis machinery could also be in accord with mitochondria biogenesis on post-transcriptional levels.
  • cytosolic Fe-S cluster synthesis also coordinates with mitochondria biogenesis. This could be due to the requirement of mitochondrial Fe-S cluster assembly machinery for cytosolic Fe-S cluster synthesis (Rouault and Maio, 2017). Interestingly, this coordination allows the IRE-binding activity of IRP1 and further ALAS2 expression as well as erythroid heme synthesis are in line with mitochondria biogenesis. Additionally, the inventors demonstrated that GATA1 expression also coordinates with mitochondria biogenesis, further supporting the positive correlation of mitochondria biogenesis and the gene expression of ALAS2, HBA and HBB that are all GATA1 targets (reference).
  • heme level also promotes the transcription and translation of HBB and HBA via the transcription inhibitor BTB Domain And CNC Homolog 1 (Bachl) and heme-regulated inhibitor (HRI), an EIF2A kinase (Chen, 2014; Tahara et al, 2004; Grosso et al, 2017). Therefore, our study demonstrated that erythropoiesis coordinates with mitochondria biogenesis on transcription and post-transcription levels and via Fe-S cluster synthesis as well as GATA1.
  • TfRl protein levels increase with mitochondria biogenesis (Resvold et al, 2013; Ishii et al, 2009; O’Hagan et al, 2009).
  • the inventors demonstrate that despite increased Fe-S cluster synthesis which coordinates with mitochondria biogenesis, TfRl expression was regulated with cell type-specific mechanisms to meet iron demand (FIG. 7E).
  • FOG. 7E iron demand
  • 3T3-L1 cells post- translationally stabilized TfRl protein possibly due to an increased mitochondrial ATP generation, which could facilitate TfRl folding and refolding.
  • IRP1 expression was induced and further upregulates TfRl expression.
  • IRP1 gene expression is upregulated.
  • AMPK activation also regulates disease- associated dyserythropoiesis.
  • thalassemia is a common genetic disorder found worldwide and caused by diminished b-globin gene expression due to a large spectrum of mutations in the globin gene locus.
  • Sideroblastic anemia is due to genetic mutations in genes involved in erythroid heme synthesis and consequently causes deficient heme production.
  • erythropoiesis was affected by AICAR treatment in Hbb mi+ mice that carry heterozygous B1/B2 globin gene deletion and are largely used as b-thalassemia intermedia disease model.
  • AICAR was not active once administered in vivo (data not shown). Therefore, we managed to isolate and differentiate erythroblasts isolated from the bone marrow and spleen, with or without AICAR treatments.
  • FIGS. S6A-B erythropoiesis from both bone marrow and spleen was enhanced with increased protein levels of hemoglobin and Alas2 compared to untreated control.
  • CD34 + cells from thalassemia and sideroblastic anemia patients, expanded and differentiated them to late erythrocytes.
  • cells were treated with different concentrations of AICAR.
  • the initial AICAR treatment significantly increased ISCU protein levels in a time-dependent manner (FIG. 9A).
  • ALAS2 and hemoglobin protein levels were all increased (FIG. 9B).
  • FIG. 9C This observation was further confirmed in differentiated CD34 + cells that were isolated from an additional thalassemia patient.
  • FIG. 9C we interrogated how erythropoiesis was regulated by AMPK activation in sideroblastic anemia.
  • ALAS2 and hemoglobin protein levels were both increased by AICAR treatment, suggesting that AMPK activation enhances erythropoiesis in an additional disease phenotype.
  • AMPK activation also stimulates the erythropoiesis in disease states and could potentially be explored for treatment of congenital anemias.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
  • Nicolas and Rubinstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (Eds.), Stoneham: Butterworth, 494-513, 1988. Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.
  • Palmiter et al. Cell, 29:701, 1982.
  • Palmiter et al. Nature, 300:611, 1982.
  • Rouault TA Nature reviews Molecular cell biology. 2015 Jan;l6(l):45-55.
  • Tahara et al The Journal of Biological Chemistry. 2004 Feb l3;279(7):5480-7. Tahara et al, Biochem Biophys Res Commun. 2004 Nov 5;324(l):77-85. Grosso et al, Blood Rev. 2017 Sep;3l(5):300-5.

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Abstract

The present disclosure relates to compositions and methods using the same to modulate mitochondrial biogenesis. The subject may suffer from a disease or disorder stemming from mitochondrial dysfunction, such as sideroblastic anemia. The agonist may be an AMPK protein or expression construct coding therefore, PCG- 1a protein or expression construct coding therefore, metformin, resveratrol or AICAR.

Description

DESCRIPTION
MODULATION OF MITOCHONDRIAL BIOGENESIS BY INCREASE IN IRON
SULFUR CLUSTER ACTIVITY
PRIORITY CLAIM
This application claims benefit of priority to U.S. Provisional Application Serial No. 62/754,606, filed November 2, 2018, the entire contents of which are hereby incorporated by reference.
BACKGROUND
1. Field
The present disclosure relates generally to the fields of biology, medicine and pathoolgy. More particularly, it concerns alterations in mitochondrial biogenesis induced by drug and gene expression modulation regulation.
2. Description of Related Art
Mitochondrial biogenesis is the process by which cells increase mitochondrial mass. It was first described by John Holloszy in the l960s, when it was discovered that physical endurance training induced higher mitochondrial content levels, leading to greater glucose uptake by muscles. Mitochondrial biogenesis is activated by numerous different signals during times of cellular stress or in response to environmental stimuli, such as aerobic exercise.
The ability for a mitochondrion to self-replicate is rooted in its evolutionary history. It is commonly thought that mitochondria descend from cells that formed endosymbiotic relationships with a-protobacteria, they have their own genome for replication. However, recent evidence suggests that mitochondrial may have evolved without symbiosis. The mitochondrion is a key regulator of the metabolic activity of the cell and is also an important organelle in both production and degradation of free radicals. It is reckoned that higher mitochondrial copy number (or higher mitochondrial mass) is protective for the cell.
Mitochondrial diseases result from failures in the function of mitochondria. Because mitochondria are responsible for creating most of the energy needed to sustain life and support organ function, when they fail, less and less energy is generated within the cell, resulting in cell injury and even cell death. Ultimately, if this process is not stopped, whole organ systems begin to fail.
The parts of the body, such as the heart, brain, muscles and lungs, requiring the greatest amounts of energy are the most affected. Symptoms can include seizures, strokes, severe developmental delays, inability to walk, talk, see, and digest food combined with a host of other complications. If three or more organ systems are involved, mitochondrial disease should be suspected.
Mitochondrial diseases the result from inherited or spontaneous mutations in mtDNA or nDNA which can lead to altered function of the proteins or RNAs residing in mitochondria. Because mitochondria perform so many different functions in different tissues, there are literally hundreds of different mitochondrial diseases. Each disorder produces a spectrum of abnormalities that can be confusing to both patients and physicians in early stages of diagnosis. Mitochondrial diseases are even more complex in adults because detectable changes in mtDNA occur as one ages and, conversely, the aging process itself may result from deteriorating mitochondrial function. Thus, there is a broad spectrum of metabolic, inherited and acquired disorders in which abnormal mitochondrial function has been postulated or demonstrated, yet treatments for these diseases are sorely lacking.
Fe-S clusters are essential cofactors for mitochondrial functions and are also synthesized within the mitochondria (Rouault and Maio, 2017). Fe-S clusters inhibit the expression of the iron importer transferrin receptor 1 (TfRl), which blocks the iron uptake required for mitochondria biogenesis (Zhang etal, 2014; Rensvold etal, 2013; Ishii etal, 2009). It is unclear however whether Fe-S cluster synthesis increases with mitochondria biogenesis and, in turn, if this negatively modulates TfRl expression and thus interfere with mitochondrial iron demand.
Fe-S clusters are synthesized in the mitochondria and cytosol by two different Fe- S cluster assembly machineries (Rouault, 2015; Braymer and Lill, 2017). However, the early steps of cytosolic Fe-S cluster synthesis require the mitochondrial Fe-S cluster assembly machinery. This process is mediated by the mitochondrial transporter ABCB7 (Pondarre et al, 2006). Therefore, the mitochondrial assembly machinery is essential for all de novo Fe-S cluster synthesis.
Two RNA-binding proteins IRP1 and IRP2 regulate iron metabolism by binding iron-responsive element (IRE) motifs. For instance, IRPs bind to the 3’UTR IREs of TfRl mRNA, thereby stabilizing it and increasing iron import (Caspary et al, 1977). In contrast, IRPs binding to the 5’UTR IRE of 5'-Aminolevulinate Synthase 2 (ALAS2), the rate- limiting enzyme in the erythroid heme synthesis pathway abrogates ALAS2 translation and iron consumption (Duncan et al, 1999). Iron and Fe-S cluster levels regulate IRE-binding activity of IRPs and thereby affect iron homeostasis. Increasing levels of iron trigger IRP2 protein degradation by a ubiquitination mediated process (Salahudeen et al. , 2009; V ashisht et al. , 2009). Additionally, Fe-S clusters can associate with IRP1 and convert it into cytosolic aconitase (aka ACOl) while simultaneously losing the IRE-binding activity (Haile et al. , 1992). Therefore, increased iron and iron-sulfur cluster levels decrease IRE- binding activity of IRPs. This decreases iron intake by destabilizing TfRl mRNA while increasing iron consumption by de-repressing ALAS2 translation. In contrast, decreased iron and Fe-S clusters levels enhance iron uptake and decrease iron consumption, ultimately maintaining iron homeostasis.
Fe-S cluster synthesis modulates IRE-binding activity of IRP1 thereby orchestrating IRPl-targeted gene expression. Genetic mutations in genes that controls Fe- S cluster synthesis cause Fe-S cluster insufficiency, ultimately disturbing mitochondria function, inhibiting erythroid heme synthesis and predisposing individuals to numerous diseases, including anemia and myelodysplastic syndromes (MDS) (Ajioka et al. , 2006; Bottomley and Fleming, 2014; Pondarre et al, 2007; Schmitz-Abe et al, 2015; Ye et al, 2010). Yet, it is unclear whether Fe-S cluster synthesis coordinates with mitochondria biogenesis and whether this modulates iron uptake via changes on TfRl expression. This is also relevant in erythropoiesis, a process that requires active mitochondrial biogenesis and consumption of the majority of physiological iron (Muckenthaler et al, 2017).
SUMMARY
Thus, in accordance with the present disclosure, there is provided a method of increasing b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization in an erythroid cell comprising contacting said cell with an agonist of AMPK or PCG-la. The agonist may be an AMPK protein or expression construct coding therefore, PCG-la protein or expression construct coding therefore, metformin, resveratrol or AICAR. The cell may be in a subject, such as a human subject. The subject may suffer from a hemoglobinopathy, such as b-thalessemia. The method may further comprise contacting said cell with another agent that is involved in b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization, such as iron. The cell may be contacted with said agonist more than once, such as on a chronic basis. The agonist may not be AICAR. The AMPK protein or PCG-la protein or AICAR may be administered directly to said cell. Alternatively, the AMPK or PCG-la protein may be contacted with said cell by provision of an expression construct coding for the same. The method may further comprise identifying said subject as being in need of increased b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization.
Also provided is a method of increasing mitochondrial biogenesis in a cell comprising contacting said cell with an agonist of AMPK or PCG-la. The subject may suffer from a disease or disorder stemming from mitochondrial dysfunction, such as sideroblastic anemia. The agonist may be an AMPK protein or expression construct coding therefore, PCG-la protein or expression construct coding therefore, metformin, resveratrol or AICAR. The cell may be in a subject, such as a human subject. The cell may be contacted with said agonist more than once, such as on a chronic basis. The agonist may not be AICAR. The cell may be a muscle cell, an adipocyte, an erythrocyte, or an epithelial cell. The AMPK protein or PCG-la protein or AICAR may be administered directly to said cell. Alternatively, the AMPK or PCG-la protein may be contacted with said cell by provision of an expression construct coding for the same. The method may further comprise identifying said subject as being in need of increased mitochondrial biogenesis.
It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
The use of the word “a” or“an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of“one or more,”“at least one,” and“one or more than one.” The word “about” means plus or minus 5% of the stated number.
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIGS. 1A-E. PGC-Ia stimulates Fe-S cluster synthesis. 3T3-L1 cells were infected with retroviruses control or expressing Myc-PGC-la followed by puromycin selection. Puromycin-resistant cells were used for the following assays. (FIG. 1A) Myc- PGC-la expression was verified by using Western blot assay and anti-Myc and anti-PGC- la antibodies. (FIG. 1B) Mitochondria biogenesis was verified by Mitotrack Green and Deep Red staining and further FACS analysis. Mitotrack Green is proportional to mitochondrial mass, while Deep Red identifies alive mitochondria. (FIG. 1C) ALAS1 protein and mRNA levels as well as heme contents were measured respectively by Western blot, qRT-PCR and heme assay kit. (FIG. 1D) The activity and protein levels of aconitase were measured by In-Gel aconitase activity (the top panel) and Western blot assays (lower panels). (FIG. 1E) Signals for the activity and protein levels of mitochondria aconitase (AC02) shown in FIG. 1D were quantified by ImageJ software. The quantity produced from AC02 aconitase activity was normalized by the number generated form AC02 protein levels. Subsequently, this ratio was compared to that obtained from control cells. #: a non-specific band.
FIGS. 2A-B. PGC-Ia stimulates the gene expression of Fe-S cluster assembly machinery. (FIG. 2A) 3T3-L1 cells were infected with virus control or expressing Myc- PGC-la followed by puromycin selection. Puromycin-resistant cells were analyzed for protein levels of genes involved in Fe-S cluster assembly. (FIG. 2B) The same cells used in FIG. 2 A were analyzed for the mRNA levels of Fe-S cluster assembly machinery.
FIGS.3A-I. Fe-S cluster synthesis coordinates with mitochondria biogenesis in the absence of PGC-Ia. Wild-type (WT) and PGC-la-null (PGC-la-/-) prebrown adipocytes (day 0) were differentiated into matured brown adipocytes (day 6) by a drug treatment for 6 days. (FIG. 3A) The activities and protein levels of aconitases were measured by In-Gel aconitase activity (top panel) and Western blot assays (bottom panels). (FIGS. 3B-C) The same quantitative analysis used in FIG. 1E was applied for ACOl levels and activity (FIG. 3B). In a similar fashion, AC02 levels and activities were quantified and analyzed (FIG. 3C). (FIG. 3D) The protein levels of genes responsible for Fe-S cluster synthesis were evaluated by Western blot assays. (FIGS. 3E-I) The genes analyzed in FIG. 3D were further evaluated for their mRNA levels by qRT-PCR assay.
FIGS. 4A-E. Under enhanced mitochondria biogenesis in 3T3-L1 cells, Fe-S cluster synthesis is increased whereas TfRl protein levels are increased. (FIG. 4A) Control and Myc-PGC-la-expressing 3T3-L1 cells were analyzed for IRE-binding activity (top panel) and IRPs protein levels (lower panels). The iron chelator DFO treatment was used as positive control, showing increased IRE-binding activity of IRPs due to IRP2 stabilization. (FIG. 4B) Control and Myc-PGC-la-expressing 3T3-L1 cells were evaluated for TfRl mRNA (FIG. 4B) and protein (FIG. 4C) levels. (FIG. 4D) Control and Myc-PGC- la-expressing 3T3-L1 cells were treated with 0.05 pg/ml actinomycin D or cycloheximide for 24hrs followed by western blot assay. (FIG. 4E) Cells were treated with 0.1 mM MG132 for 32 hrs and analyzed.
FIGS. 5A-D. Under enhanced mitochondria biogenesis, IRP1 expression in brown adipocytes was stimulated and further increased TfRl expression. (FIG. 5A) WT and PGC-la-/- prebrown adipocytes (day 0) were matured to brown adipocytes (day 6) by the drug treatment. These cells were analyzed for their IRP1 mRNA levels by qRT- PCR. (FIG. 5B) The adipocytes used in A were analyzed for the IRE-binding activities (to panel) and protein levels of IRPs (lower panels). (FIGS. 5C-D) The same cells used in FIG. 5B were analyzed for TfRl protein levels by western blot (FIG. 5C) and mRNA levels by qRT-PCR (FIG. 5D).
FIGS. 6A-E. Fe-S cluster assembly coordinates with mitochondria biogenesis in erythroid cells. (FIG. 6A) MEL cells were infected with lentiviruses control or expressing Myc-PGC-la. One day after infection, these cells were differentiated by the incubation with 50 mM HMBA for 5 days followed by the aconitase activity and western blot assays. (FIG. 6B) Similarly, cells used in A were analyzed by qRT-PCR assays. (FIG. 6C) MEL cells were infected with lentiviruses shRNA control or targeting murine PGC-la mRNA and selected with puromycin. Puromycin-resistant cells were differentiated with 50mM HMBA incubation for 6 days for differentiation. Cells were analyzed for PGC-la mRNA levels by qRT-PCR assay. (FIG. 6D) Cells used in FIG. 6C were analyzed by western blot analysis. (FIG. 6E) Similar to FIG. 6C and FIG. 6D, the same cells were analyzed by qRT-qPCR assay. FIGS. 7A-E. In erythroid cells, GATA1 expression coordinates with mitochondria biogenesis and potentially regulates TfRl gene expression. (FIGS. 7A- B) MEL cells control or expressing Myc-PGC-la were differentiated and then analyzed by IRE-binding activity (A, the top panel) and Western blot assays (FIG. 7A, the lower panels) and for TfRl mRNA levels by qRT-PCR (FIG. 7B). (FIGS. 7C-D) Control and PGC-la shRNA-targeted MEL cells were generated by lentiviral infection and puromycin selection. After differentiation, these cells were analyzed by IRE-binding activity (the top panel) and Western blot assays (FIG. 7C) and for TfRl mRNA levels by qRT-PCR (FIG. 7D). (FIG. 7E) The model for different regulations on TfRl expression. Top: the canonical regulation mediated by Fe-S cluster synthesis; middle: the regulation mediated by increased IRP1 expression; botom: the regulation mediated by GATA1.
FIGS.8A-D. Erythroid heme synthesis and hemoglobinization coordinate with mitochondria biogenesis. (FIG. 8A) Control or Myc-PGC-la-expressing MEL cells were differentiated with HMBA incubation for 5 days, then pelleted and photographed (top panel of FIG. 8A) followed by western blot assays. (FIG. 8B) The same cells used in FIG. 8A were analyzed by qRT-PCR. (FIG. 8C) Control and PGC-la shRNA-targeted MEL cells were differentiated with HMBA treatment for 6 days, pelleted and photographed (top panel) followed by western blot assay. (FIG. 8D) The same cells used in FIG. 8C were analyzed by qRT-PCR.
FIGS. 9A-D. AMPK activation modulates the erythropoiesis in thalassemia and sideroblastic anemia. (FIG. 9A) CD34+ cells were isolated from peripheral blood of a thalassemia patient and expanded for 10 to 13 days. These cells were further cultured in erythroid differentiation medium with 120 mM AICAR for different periods of time as indicated. Cells were harvested for ISCU protein levels analysis. (FIG. 9B) CD34+ cells used in A were cultured in differentiation medium with different concentrations of AICAR as indicated. The medium was refreshed every 48hrs; after 96hrs, cells were analyzed for ALAS2, b-globin and a-globin levels. (FIG. 9C) Similarly, CD34+ cells were isolated from another thalassemia patient, cultured, differentiated and analyzed as described in B. (FIG. 9D) As indicated with FIGS. 9B and 9C, CD34+ cells were isolated from a sideroblastic anemia patient and expanded, After the expansion, these cells were switched in erythroid differentiation medium and cultured for 96 hrs with different concentrations of AICAR. Similarly, cells were harvested and analyzed for ALAS2, a-globin and b-globin protein levels. FIG. SI. 3T3-L1 cells were infected with retroviruses control or expressing Myc- PGC-la followed by puromycin selection. Puromycin-resistant cells were analyzed for mRNA levels of genes involved in Fe-S cluster assembly.
FIGS. S2A-C. (FIG. S2A) HepG2 cells were infected with retroviruses control or expressing Myc-PGC-la and selected with puromycin. Puromycin-resistant HepG2 cells were analyzed by Western blot assay. # indicated nonspecific bands. (FIG. S2B) The same cells used in FIG. S2A were analyzed for the protein levels and activities of ACOl and AC02. (FIG. S2C) AC02 aconitase activity and protein levels, as shown in FIG. S2B, were quantified by Image! The AC02 aconitase activity was normalized by the AC02 protein levels. Subsequently, the ratio obtained from My c-PGC- la-expressing cells (the AC02 aconitase activity normalized by the AC02 protein levels) was compared to that achieved using control cells.
FIG. S3. The genes analyzed in FIG. 2H were further evaluated for the mRNA levels by qRT-PCR assay. Lighter bars stand for WT brown adipocytes while dark bars stand for PGC-la-/- brown adipocytes.
FIGS. S4A-E. (FIG. S4A) TfRl mRNA levels in control and My c-PGC- la- expressing 3T3-L1 cells were measured by qRT-PCR assay. (FIG. S4B) TfRl protein levels in HepG2 cells control or expressing Myc-PGC-la were evaluated by Western blot analysis. (FIG. S4C) Similar to FIG. S4B, TfRl mRNA levels in HepG2 cells were measured by qRT-PCR assay. (FIG. S4D) TfRl mRNA levels were analyzed in prebrown (day 0) and brown (day 6) adipocytes by qRT-PCR assay. (FIG. S4E) Similar to FIG. S4D, IRP 1 mRNA levels were analyzed.
FIGS. S5A-G. (FIG. S5A) MEL cells were infected with lentiviruses shRNA control or targeting murine PGC-la mRNA, selected with puromycin, and incubated with tomM HMBA for 7 days for differentiation. Cells were analyzied for PGC-la mRNA levesl by qRT-PCR assay. (FIGS. S5B-C) Diffemtiated control and PGC-la shRNA-targeted MEL cells were analyzed by In-Gel aconitase activity and western blot assay (FIG. S5B) and by qRT-PCR for the mRNA levels of genes involved in Fe-S cluster assembly (FIG. S5C). (FIG. S5D) mRNA levels of different genes as indicated were measured in differentiated control and My c-PGC- la-expressing MEL cells. (FIG. S5E) Differentiated control and PGC-la shRNA-targeted cells were interrogated by IRE-binding activity (top panel) and Western blot assays. (FIGS. S5F-G) TfRl mRNA levels were measured by qRT-PCR in differentiated control and PGC-la shRNA-targeted cells (FIG. S5F) and differentiated control and Myc-PGC-la-expressing cells (FIG. S5G). FIGS. S6A-B. (FIG. S6A) Mouse CD34+ cells were isolated from the bone marrow or (FIG. S6B) spleen of Hbbth3/+ mice, expanded and differentiated. During the 72hrs differentiation period, cells were also treated with AICAR. Differentiation medium combined with AICAR were refreshed every 48 hrs. In the end, cells were analyzed for Alas 2. a-globin and b-globin protein levels.
DFTATFFD DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Mitochondria are produced from the transcription and translation of genes both in the nuclear genome and in the mitochondrial genome. The majority of mitochondrial protein comes from the nuclear genome, while the mitochondrial genome encodes parts of the electron transport chain along with mitochondrial rRNA and tRNA. Mitochondrial biogenesis increases metabolic enzymes for glycolysis, oxidative phosphorylation and ultimately a greater mitochondrial metabolic capacity. However, depending on the energy substrates available and the REDOX state of the cell, the cell may increase or decrease the number and size of mitochondria. Critically, mitochondrial numbers and morphology vary according to cell type and context-specific demand, whereby the balance between mitochondrial fusion/fission regulates mitochondrial distribution, morphology, and function.
PGC-la, a member of the peroxisome proliferator-activated receptor gamma (PGC) family of transcriptional coactivators, is the master regulator of mitochondrial biogenesis. It is known to co-activate nuclear respiratory factor 2 (NRF2/GABPA), and together with NRF-2 coactivates nuclear respiratory factor 1 (NRF1). The NRFs, in turn, activate the mitochondrial transcription factor A (tfam), which is directly responsible for transcribing nuclear-encoded mitochondrial proteins. This includes both structural mitochondrial proteins as well as those involved in mtDNA transcription, translation, and repair. PGC- 1b, a protein that is structurally similar to PGC-la, is also involved in regulating mitochondrial biogenesis, but differs in that it does not get increased in response to exercise. While there have been significant increases in mitochondria found in tissues where PGC-la is overexpressed, as the cofactor interacts with these key transcription factors, knockout mice with disrupted PGC-la are still viable and show normal mitochondrial abundance. Thus, PGC-la is not required for normal development of mitochondria in mice, but when put under physiological stress, these mice exhibit diminished tolerance compared to mice with normal levels of PGC-la. Similarly, in knockout mice with disrupted PGC-l , the mice showed mostly normal levels of mitochondrial function with decreased ability to adapt to physiological stress. However, a double-knockout experiment of PGC-la/b created mice that died mostly within 24 hours by defects in mitochondrial maturation of cardiac tissue. These findings suggest that while both PGC-la and PGC- I b do not each solely establish a cell’s ability to perform mitochondrial biogenesis, together they are able to complement each other for optimal mitochondrial maturation and function during periods of physiological stress.
AMP-activated kinase (AMPK) also regulates mitochondrial biogenesis by phosphorylating and activating PGC-la upon sensing an energy deficiency in muscle. In mice with reduced ATP/ AMP ratios that would occur during exercise, the energy depletion has been shown to correlate with AMPK activation. AMPK activation then continued to activate PGC- la and NRFs in these mice, and mitochondrial biogenesis was stimulated.
The capacity for mitochondrial biogenesis has been shown to decrease with age, and such decreased mitochondrial function has been associated with diabetes and cardiovascular disease. Aging and disease can induce changes in the expression levels of proteins involved in the fission and fusion mechanisms of mitochondria, thus creating dysfunctional mitochondria. One hypothesis for the detrimental results of aging is associated with the loss of telomeres, the end segments of chromosomes that protect genetic information from degradation. Telomere loss has also been associated with decreased mitochondrial function. Deficiency of telomerase reverse transcriptase (TERT), an enzyme that plays a role in preserving telomeres, has been correlated with activated p53, a protein that suppresses PGC-la. Therefore, the loss of telomeres and TERT that comes with aging has been associated with impaired mitochondrial biogenesis. AMPK expression has also been shown to diminish with age, which may also contribute to suppressing mitochondrial biogenesis. There is also evidence of mitochondrial biogenesis being involved in neurodegenerative disorders, cancer, metabolic syndrome, sarcopenia, cardiac pathophysiology as well as physiological processes like aging and erythropoiesis.
As indicated above, a considerable number of proteins, transcription factors, upstream regulatory proteins and secondary mechanisms are involved in mitochondrial biogenesis. These molecules, including the main participating proteins (e.g., PGC-la and mtTFA), are candidates for therapeutic intervention in diverse disease. Here, the inventors explore the molecular regulation of mitochondrial biogenesis, and methods of exploiting regulatory pathways to alter mitochondrial function.
These and other aspects of the disclosure are described in detail below.
I. Fe-S Clusters
Iron-sulfur proteins are proteins characterized by the presence of iron-sulfur clusters containing sulfide-linked di-, tri-, and tetrairon centers in variable oxidation states. Iron-sulfur clusters are found in a variety of metalloproteins, such as the ferredoxins, as well as NADH dehydrogenase, hydrogenases, coenzyme Q - cytochrome c reductase, succinate - coenzyme Q reductase and nitrogenase. Iron-sulfur clusters are best known for their role in the oxidation-reduction reactions of mitochondrial electron transport. Both Complex I and Complex II of oxidative phosphorylation have multiple Fe-S clusters. They have many other functions including catalysis as illustrated by aconitase, generation of radicals as illustrated by SAM-dependent enzymes, and as sulfur donors in the biosynthesis of lipoic acid and biotin. Additionally, some Fe-S proteins regulate gene expression. Fe-S proteins are vulnerable to attack by biogenic nitric oxide, forming dinitrosyl iron complexes. In most Fe-S proteins, the terminal ligands on Fe are thiolate, but exceptions exist.
The prevalence of these proteins on the metabolic pathways of most organisms leads some scientists to theorize that iron-sulfur compounds had a significant role in the origin of life in the iron-sulfur world theory.
In almost all Fe-S proteins, the Fe centers are tetrahedral and the terminal ligands are thiolato sulfur centers from cysteinyl residues. The sulfide groups are either two- or three-coordinated. Three distinct kinds of Fe-S clusters with these features are most common.
The simplest polymetallic system, the | Fe^S^ | cluster, is constituted by two iron ions bridged by two sulfide ions and coordinated by four cysteinyl ligands (in Fe2S ferredoxins) or by two cysteines and two histidines (in Rieske proteins). The oxidized proteins contain two Fe3+ ions, whereas the reduced proteins contain one Fe3+ and one Fe2+ ion. These species exist in two oxidation states, (Fe and FemFen.
A common motif features four iron ions and four sulfide ions placed at the vertices of a cubane-type cluster. The Fe centers are typically further coordinated by cysteinyl ligands. The [Fe4S4] electron-transfer proteins ([Fe4S4] ferredoxins) may be further subdivided into low-potential (bacterial-type) and high-potential (HiPIP) ferredoxins.
In HiPIP, the cluster shuttles between [2Fe3+, 2Fe2+] (Fe4S4 2+) and [3Fe3+, Fe2+] (Fe4S4 3+). The potentials for this redox couple range from 0.4 to 0.1 V. In the bacterial ferredoxins, the pair of oxidation states are [Fe3+, 3Fe2+] (Fe4S4 +) and [2Fe3+, 2Fe2+] (Fe4S4 2+). The potentials for this redox couple range from -0.3 to -0.7 V. The two families of 4Fe- 4S clusters share the Fe4S4 2+ oxidation state. The difference in the redox couples is attributed to the degree of hydrogen bonding, which strongly modifies the basicity of the cysteinyl thiolate ligands. A further redox couple, which is still more reducing than the bacterial ferredoxins is implicated in the nitrogenase. Some 4Fe-4S clusters bind substrates and are thus classified as enzyme cofactors. In aconitase, the Fe-S cluster binds aconitate at the one Fe centre that lacks a thiolate ligand. The cluster does not undergo redox but serves as a Lewis acid catalyst to convert citrate to isocitrate. In radical SAM enzymes, the cluster binds and reduces S-adenosylmethionine to generate a radical, which is involved in many biosyntheses.
Proteins are also known to contain [FesSJ centres, which feature one iron less than the more common [Fe4S4] cores. Three sulfide ions bridge two iron ions each, while the fourth sulfide bridges three iron ions. Their formal oxidation states may vary from | Fe Sd (all-Fe3+ form) to | Fe S+p (all-Fe2+ form). In a number of iron-sulfur proteins, the [Fe4S4] cluster can be reversibly converted by oxidation and loss of one iron ion to a | Fe Sd cluster, e.g., the inactive form of aconitase possesses an | Fe Sd and is activated by addition of Fe2+ and reductant.
More complex polymetallic systems are common. Examples include both the 8Fe and the 7Fe clusters in nitrogenase. Carbon monoxide dehydrogenase and the [FeFe]- hydrogenase also feature unusual Fe-S clusters. A special 6 cysteine-coordinated [Fe4S3] cluster was found in oxygen-tolerant membrane-bound [NiFe] hydrogenases.
The biosynthesis of the Fe-S clusters has been well studied. The biogenesis of iron sulfur clusters has been studied most extensively in the bacteria E. coli and A. vinelandii and yeast S. cerevisiae. At least three different biosynthetic systems have been identified so far, namely nif, suf, and isc systems, which were first identified in bacteria. The nif system is responsible for the clusters in the enzyme nitrogenase. The suf and isc systems are more general. The yeast isc system is the best described. Several proteins constitute the biosynthetic machinery via the isc pathway. The process occurs in two major steps: (1) the Fe/S cluster is assembled on a scaffold protein followed by (2) transfer of the preformed cluster to the recipient proteins. The first step of this process occurs in the cytoplasm of prokaryotic organisms or in the mitochondria of eukaryotic organisms. In the higher organisms the clusters are therefore transported out of the mitochondrion to be incorporated into the extramitochondrial enzymes. These organisms also possess a set of proteins involved in the Fe/S clusters transport and incorporation processes that are not homologous to proteins found in prokaryotic systems. II. Mitochondrial Factors and Agents Affecting Such Factors
A. PGC-la
Peroxisome proliferator-activated receptor gamma coactivator l-alpha (PGC-la) is a protein that in humans is encoded by the PPARGC1A gene. PPARGC1A is also known as human accelerated region 20 (HAR20). It may, therefore, have played a key role in differentiating humans from apes. PGC-la is the master regulator of mitochondrial biogenesis.
PGC-la is a transcriptional coactivator that regulates the genes involved in energy metabolism. It is the master regulator of mitochondrial biogenesis. This protein interacts with the nuclear receptor PPAR-g, which permits the interaction of this protein with multiple transcription factors. This protein can interact with, and regulate the activities of, cAMP response element-binding protein (CREB) and nuclear respiratory factors (NRFs). It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis, and is a major factor causing slow-twitch rather than fast-twitch muscle fiber types.
Endurance exercise has been shown to activate the PGC-la gene in human skeletal muscle. Exercise-induced PGC-la in skeletal muscle increases autophagy and unfolded protein response. PGC-la protein may be also involved in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity.
PGC-la is thought to be a master integrator of external signals. It is known to be activated by a host of factors, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), both formed endogenously in the cell as by-products of metabolism but upregulated during times of cellular stress. It is strongly induced by cold exposure, linking this environmental stimulus to adaptive thermogenesis. It is induced by endurance exercise and recent research has shown that PGC-la determines lactate metabolism, thus preventing high lactate levels in endurance athletes and making lactate as an energy source more efficient. It also is activated by AMP response element-binding (CREB) proteins, activated by an increase in cAMP following external cellular signals. Protein kinase B/Akt is thought to downregulate PGC-la, but upregulate its downstream effectors, NRF1 and NRF2. Akt itself is activated by PIP3, often upregulated by PI3K after G-protein signals. The Akt family is also known to activate pro-survival signals as well as metabolic activation. SIRT1 binds and activates PGC-la through deacetylation inducing gluconeogenesis without affecting mitochondrial biogenesis. PGC-la has been shown to exert positive feedback circuits on some of its upstream regulators. PGC-la increases Akt (PKB) and Phospho-Akt (Ser 473 and Thr 308) levels in muscle. PGC-la leads to calcineurin activation. Akt and calcineurin are both activators of NF kappa B (p65). Through their activation PGC-la seems to activate NF kappa B. Increased activity of NF kappa B in muscle has recently been demonstrated following induction of PGC-la. The finding seems to be controversial. Other groups found that PGC- ls inhibit NF kappa B activity. The effect was demonstrated for PGC-l alpha and beta. PGC-la has also been shown to drive NAD biosynthesis to play a large role in renal protection in Acute Kidney Injury.
Recently PPARGC1A has been implicated as a potential therapy for Parkinson's Disease conferring protective effects on mitochondrial metabolism. Moreover, brain- specific isoforms of PGC-l alpha have recently been identified which are likely to play a role in other neurodegenerative disorders such as Huntington's disease and Amyotrophic lateral sclerosis.
PGC-la and b has furthermore been implicated in M2 macrophage polarization by interaction with PPARy with upstream activation of STAT6. An independent study confirmed the effect of PGC-l on polarisation of macrophages towards M2 via STAT6/PPAR gamma and furthermore demonstrated that PGC-l inhibits proinflammatory cytokine production.
PGC-la has been recently proposed to be responsible for b-aminoisobutyric acid secretion by exercising muscles. The effect of b-aminoisobutyric acid in white fat includes the activation of thermogenic genes that prompt the browning of white adipose tissue and the consequent increase of background metabolism. Hence, the b-aminoisobutyric acid could act as a messenger molecule of PGC-la and explain the effects of PGC-la increase in other tissues such as white fat.
B. AMPK
5' AMP-activated protein kinase or AMPK or 5' adenosine monophosphate- activated protein kinase is an enzyme (EC 2.7.11.31) that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and oxidation when cellular energy is low. It belongs to a highly conserved eukaryotic protein family and its orthologues are SNF1 and SnRKl in yeast and plants, respectively. It consists of three proteins (subunits) that together make a functional enzyme, conserved from yeast to humans. It is expressed in a number of tissues, including the liver, brain, and skeletal muscle. In response to binding AMP and ADP, the net effect of AMPK activation is stimulation of hepatic fatty acid oxidation, ketogenesis, stimulation of skeletal muscle fatty acid oxidation and glucose uptake, inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis, inhibition of adipocyte lipogenesis, activation of adipocyte lipolysis, and modulation of insulin secretion by pancreatic beta-cells.
AMPK is a heterotrimeric protein complex that is formed by a, b, and g subunits. Each of these three subunits takes on a specific role in both the stability and activity of AMPK. Specifically, the g subunit includes four particular Cystathionine beta synthase (CBS) domains giving AMPK its ability to sensitively detect shifts in the AMP:ATP ratio. The four CBS domains create two binding sites for AMP commonly referred to as Bateman domains. Binding of one AMP to a Bateman domain cooperatively increases the binding affinity of the second AMP to the other Bateman domain. As AMP binds both Bateman domains the g subunit undergoes a conformational change which exposes the catalytic domain found on the a subunit. It is in this catalytic domain where AMPK becomes activated when phosphorylation takes place at threonine- 172 by an upstream AMPK kinase (AMPKK). The a, b, and g subunits can also be found in different isoforms: the g subunit can exist as either the gΐ, g2 or g3 isoform; the b subunit can exist as either the bΐ or b2 isoform; and the a subunit can exist as either the al or a2 isoform. Although the most common isoforms expressed in most cells are the aΐ, bΐ, and gΐ isoforms, it has been demonstrated that the a2, b2, g2, and g3 isoforms are also expressed in cardiac and skeletal muscle.
Due to the presence of isoforms of its components, there are 12 versions of AMPK in mammals, each of which can have different tissue localizations, and different functions under different conditions. AMPK is regulated allosterically and by post-translational modification, which work together.
If residue T172 of AMPK's a-subunit is phosphorylated AMPK is activated; access to that residue by phosphatases is blocked if AMP or ADP can block access for and ATP can displace AMP and ADP. That residue is phosphorylated by at least three kinases (liver kinase Bl (LKB1) which works in a complex with STRAD and M025, Calcium/calmodulin-dependent protein kinase kinase II-(CAMKK2), and TGFb-activated kinase 1 (TAK1)) and is dephosphorylated by three phosphatases (protein phosphatase 2A (PP2A); protein phosphatase 2C (PP2C) and Mg2+-/Mn2+-dependent protein phosphatase 1E (PPM1E)). AMPK is regulated allosterically mostly by competitive binding on its gamma subunit between ATP (which allows phosphatase access to T172) and AMP or ADP (each of which blocks access to phosphatases). It also appears that AMPK is a sensor of AMP/ ATP or ADP/ATP ratios and thus cell energy level. Regulation of AMPK by CaMKK2 requires a direct interaction of these two proteins via their kinase domains. The interaction of CaMKK2 with AMPK only involves the alpha and beta subunits of AMPK (AMPK gamma is absent from the CaMKK2 complex), thus rendering regulation of AMPK in this context to changes in calcium levels but not AMP or ADP.
There are other mechanisms by which AMPK is inhibited by insulin, leptin, and diacylglycerol by inducing various other phosphorylations.
AMPK may be inhibited or activated by various tissue-specific ubiquitinations.
It is also regulated by several protein-protein interactions and may either by activated or inhibited by oxidative factors; the role of oxidation in regulating AMPK was controversial as of 2016.
When AMPK phosphorylates acetyl-CoA carboxylase 1 (ACC1) or sterol regulatory element-binding protein lc (SREBPlc), it inhibits synthesis of fatty acids, cholesterol, and triglycerides, and activates fatty acid uptake and b-oxidation.
AMPK stimulates glucose uptake in skeletal muscle by phosphorylating Rab- GTPase-activating protein TBC1D1, which ultimately induces fusion of GLUT4 vesicles with the plasma membrane. AMPK stimulates glycolysis by activating phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2/3 and activating phosphorylation of glycogen phosphorylase, and it inhibits glycogen synthesis through inhibitory phosphorylation of glycogen synthase. In the liver, AMPK inhibits gluconeogenesis by inhibiting transcription factors including hepatocyte nuclear factor 4 (HNF4) and CREB regulated transcription coactivator 2 (CRTC2).
AMPK inhibits the energy-intensive protein biosynthesis process and can also force a switch from cap-dependent translation to cap-independent translation, which requires less energy, by phosphorylation of TSC2, RPTOR, transcription initiation factor 1A.66, and eEF2K. When TSC2 is activated it inhibits mTORCl . As a result of inhibition of mTORCl by AMPK, protein synthesis comes to a halt which results in inhibits a energy requiring pathway such as protein synthesis. In results, activation of AMPK signifies low energy charge of the cell, so all of the energy consuming pathways like protein synthesis are inhibited, and pathways that generate energy are activated to restore appropriate energy levels in the cell. AMPK activates autophagy by directly and indirectly activating ULK1. AMPK also appears to stimulate mitochondrial biogenesis by regulating PGC-la which in turn promotes gene transcription in mitochondria. AMPK also activates anti-oxidant defenses.
Exercise/training. Many biochemical adaptations of skeletal muscle that take place during a single bout of exercise or an extended duration of training, such as increased mitochondrial biogenesis and capacity, increased muscle glycogen, and an increase in enzymes which specialize in glucose uptake in cells such as GLUT4 and hexokinase II are thought to be mediated in part by AMPK when it is activated. Additionally, recent discoveries can conceivably suggest a direct AMPK role in increasing blood supply to exercised/trained muscle cells by stimulating and stabilizing both vasculogenesis and angiogenesis. Taken together, these adaptations most likely transpire as a result of both temporary and maintained increases in AMPK activity brought about by increases in the AMP: ATP ratio during single bouts of exercise and long-term training.
During a single acute exercise bout, AMPK allows the contracting muscle cells to adapt to the energy challenges by increasing expression of hexokinase II, translocation of GLUT4 to the plasma membrane, for glucose uptake, and by stimulating glycolysis. If bouts of exercise continue through a long-term training regimen, AMPK and other signals will facilitate contracting muscle adaptations by escorting muscle cell activity to a metabolic transition resulting in a fatty-acid oxidation approach to ATP generation as opposed to a glycolytic approach. AMPK accomplishes this transition to the oxidative mode of metabolism by upregulating and activating oxidative enzymes such as hexokinase II, PPARalpha, PPARdelta, PGC-l, UCP-3, cytochrome C and TFAM.
AMPK activity increases with exercise and the LKB1/M025/STRAD complex is considered to be the major upstream AMPKK of the 5’-AMP-activated protein kinase phosphorylating the a subunit of AMPK at Thr-l72. This fact is puzzling considering that although AMPK protein abundance has been shown to increase in skeletal tissue with endurance training, its level of activity has been shown to decrease with endurance training in both trained and untrained tissue. Currently, the activity of AMPK immediately following a 2-hr bout of exercise of an endurance trained rat is unclear. It is possible that there exists a direct link between the observed decrease in AMPK activity in endurance trained skeletal muscle and the apparent decrease in the AMPK response to exercise with endurance training. Maximum life span. The C. elegans homolog of AMPK, aak-2, has been shown by Michael Ristow and colleagues to be required for extension of life span in states of glucose restriction mediating a process named mitohormesis.
Lipid metabolism. One of the effects of exercise is an increase in fatty acid metabolism, which provides more energy for the cell. One of the key pathways in AMPK’s regulation of fatty acid oxidation is the phosphorylation and inactivation of acetyl-CoA carboxylase. Acetyl-CoA carboxylase (ACC) converts acetyl-CoA to malonyl-CoA, an inhibitor of carnitine palmitoyltransferase 1 (CPT-l). CPT-l transports fatty acids into the mitochondria for oxidation. Inactivation of ACC, therefore, results in increased fatty acid transport and subsequent oxidation. It is also thought that the decrease in malonyl-CoA occurs as a result of malonyl-CoA decarboxylase (MCD), which may be regulated by AMPK. MCD is an antagonist to ACC, decarboxylating malonyl-CoA to acetyl-CoA, resulting in decreased malonyl-CoA and increased CPT-l and fatty acid oxidation. AMPK also plays an important role in lipid metabolism in the liver. It has long been known that hepatic ACC has been regulated in the liver by phosphorylation. AMPK also phosphorylates and inactivates 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), a key enzyme in cholesterol synthesis. HMGR converts 3-hydroxy-3-methylglutaryl-CoA, which is made from acetyl-CoA, into mevalonic acid, which then travels down several more metabolic steps to become cholesterol. AMPK, therefore, helps regulate fatty acid oxidation and cholesterol synthesis.
Glucose transport. Insulin is a hormone which helps regulate glucose levels in the body. When blood glucose is high, insulin is released from the Islets of Langerhans. Insulin, among other things, will then facilitate the uptake of glucose into cells via increased expression and translocation of glucose transporter GLUT-4. Under conditions of exercise, however, blood sugar levels are not necessarily high, and insulin is not necessarily activated, yet muscles are still able to bring in glucose. AMPK seems to be responsible in part for this exercise-induced glucose uptake. Researchers have observed that with exercise, the concentration of GLUT-4 was increased in the plasma membrane, but decreased in the microsomal membranes, suggesting that exercise facilitates the translocation of vesicular GLUT-4 to the plasma membrane. While acute exercise increases GLUT-4 translocation, endurance training will increase the total amount of GLUT-4 protein available. It has been shown that both electrical contraction and AICAR treatment increase AMPK activation, glucose uptake, and GLUT-4 translocation in perfused rat hindlimb muscle, linking exercise-induced glucose uptake to AMPK. Chronic AICAR injections, simulating some of the effects of endurance training, also increase the total amount of GLUT-4 protein in the muscle cell.
Two proteins are essential for the regulation of GLUT-4 expression at a transcriptional level - myocyte enhancer factor 2 (MEF2) and GLUT4 enhancer factor (GEF). Mutations in the DNA binding regions for either of these proteins results in ablation of transgene GLUT-4 expression. These results prompted a study in 2005 which showed that AMPK directly phosphorylates GEF, but it doesn’t seem to directly activate MEF2. AICAR treatment has been shown, however, to increase transport of both proteins into the nucleus, as well as increase the binding of both to the GLUT-4 promoter region.
There is another protein involved in carbohydrate metabolism that is worthy of mention along with GLUT-4. The enzyme hexokinase phosphorylates a six-carbon sugar, most notably glucose, which is the first step in glycolysis. When glucose is transported into the cell it is phosphorylated by hexokinase. This phosphorylation keeps glucose from leaving the cell, and by changing the structure of glucose through phosphorylation, it decreases the concentration of glucose molecules, maintaining a gradient for more glucose to be transported into the cell. Hexokinase II transcription is increased in both red and white skeletal muscle upon treatment with AICAR. With chronic injections of AICAR, total protein content of hexokinase II increases in rat skeletal muscle.
Mitochondria. Mitochondrial enzymes, such as cytochrome c, succinate dehydrogenase, malate dehydrogenase, a-ketoglutarate dehydrogenase, and citrate synthase, increase in expression and activity in response to exercise. AICAR stimulation of AMPK increases cytochrome c and d-aminolevulinate synthase (ALAS), a rate-limiting enzyme involved in the production of heme. Malate dehydrogenase and succinate dehydrogenase also increase, as well as citrate synthase activity, in rats treated with AICAR injections. Conversely, in LKB1 knockout mice, there are decreases in cytochrome c and citrate synthase activity, even if the mice are "trained" by voluntary exercise.
Peroxisome proliferator-activated receptor gamma coactivator- la (PGC-la) is a transcriptional regulator for genes involved in fatty acid oxidation, gluconeogenesis, and is considered the master regulator for mitochondrial biogenesis.
To do this, it enhances the activity of transcription factors like nuclear respiratory factor 1 (NRF-l), myocyte enhancer factor 2 (MEF2), host cell factor (HCF), and others. It also has a positive feedback loop, enhancing its own expression.
Both MEF2 and cAMP response element (CRE) are essential for contraction- induced PGC-la promoter activity. AMPK is required for increased PGC-la expression in skeletal muscle in response to creatine depletion. LKB1 knockout mice show a decrease in PGC-la, as well as mitochondrial proteins.
Thyroid hormone. AMPK and thyroid hormone regulate some similar processes. Knowing these similarities, Winder and Hardie et al. designed an experiment to see if AMPK was influenced by thyroid hormone. They found that all of the subunits of AMPK were increased in skeletal muscle, especially in the soleus and red quadriceps, with thyroid hormone treatment. There was also an increase in phospho-ACC, a marker of AMPK activity.
Glucose sensing system. Loss of AMPK has been reported to alter the sensitivity of glucose sensing cells, through poorly defined mechanisms. Loss of the AMPKa2 subunit in pancreatic beta cells and hypothalamic neurons decreases the sensitivity of these cells to changes in extracellular glucose concentration. Moreover, exposure of rats to recurrent bouts of insulin induced hypoglycaemia/glucopenia, reduces the activation of AMPK within the hypothalamus, whilst also suppressing the counterregulatory response to hypoglycaemia. Pharmacological activation of AMPK by delivery of AMPK activating drug AICAR, directly into the hypothalamus can increase the counterregulatory response to hypoglycaemia.
C. AICAR
5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) is an intermediate in the generation of inosine monophosphate. AICAR is an analog of adenosine monophosphate (AMP) that is capable of stimulating AMP-dependent protein kinase (AMPK) activity. AICAR has been used clinically to treat and protect against cardiac ischemic injury. The drug was first used in the l980s as a method to preserve blood flow to the heart during surgery. Currently, the drug has also been shown as a potential treatment for diabetes by increasing the metabolic activity of tissues by changing the physical composition of muscle.
The nucleoside form of AICAR, acadesine, is an analog of adenosine that enters cardiac cells to inhibit adenosine kinase and adenosine deaminase. It enhances the rate of nucleotide re-synthesis increasing adenosine generation from adenosine monophosphate only during conditions of myocardial ischemia. In cardiac myocytes, acadesine is phosphorylated to AICAR to activate AMPK without changing the levels of the nucleotides. AICAR is able to enter the de novo synthesis pathway for adenosine synthesis to inhibit adenosine deaminase causing an increase in ATP levels and adenosine levels. A brief period of coronary arterial occlusion followed by reperfusion prior to prolonged ischemia is known as preconditioning. It has been shown that this is protective. Preconditioning preceded myocardial infarction, may delay cell death and allow for greater salvage of myocardium through reperfusion therapy. AICAR has been shown to precondition the heart shortly before or during ischemia. AICAR triggers a preconditioned anti-inflammatory state by increasing NO production from endothelial nitric oxide synthase. When AICAR is given 24 hours prior to reperfusion, it prevents post ischemic leukocyte-endothelial cell adhesive interactions with increased NO production. AICAR- dependent preconditioning is also mediated by an ATP-sensitive potassium channel and hemeoxygenase-dependent mechanism. It increases AMPK-dependent recruitment of ATP-sensitive K channels to the sarcolemma causing the action potential duration to shorten and preventing calcium overload during reperfusion. The decrease in calcium overload prevents inflammation activation by ROS. AICAR also increases AMPK- dependent glucose uptake through translocation of GLUT-4 which is beneficial for the heart during post-ischemic reperfusion. The increase in glucose during AICAR preconditioning lengthens the period for preconditioning up to 2 hours in rabbits and 40 minutes in humans undergoing coronary ligation. As a result, AICAR reduces the frequency and size of myocardial infarcts up to 25% in humans allowing improved blood flow to the heart. As well, the treatment has been shown to decrease the risk of an early death and improve recovery after surgery from an ischemic injury.
D. Metformin
Metformin, marketed under the trade name Glucophage among others, is the first- line medication for the treatment of type 2 diabetes, particularly in people who are overweight. It is also used in the treatment of polycystic ovary syndrome.
Metformin is taken orally and generally well tolerated. Common side effects include diarrhea, nausea, and abdominal pain. It has a low risk of causing low blood sugar. High blood lactic acid level is a concern if the medication is prescribed inappropriately or in overly large doses. It should not be used in those with significant liver disease or kidney problems. While no clear harm comes from use during pregnancy, insulin is generally preferred for gestational diabetes. Metformin is a biguanide antihyperglycemic agent. It works by decreasing glucose production by the liver and increasing the insulin sensitivity of body tissues. The most common adverse effect of metformin is gastrointestinal irritation, including diarrhea, cramps, nausea, vomiting, and increased flatulence; metformin is more commonly associated with gastrointestinal side effects than most other antidiabetic medications. The most serious potential side effect of metformin use is lactic acidosis; this complication is very rare, and the vast majority of these cases seem to be related to comorbid conditions, such as impaired liver or kidney function, rather than to the metformin itself.
The molecular mechanism of metformin is not completely understood. Multiple potential mechanisms of action have been proposed: inhibition of the mitochondrial respiratory chain (complex I), activation of AMP-activated protein kinase (AMPK), inhibition of glucagon-induced elevation of cyclic adenosine monophosphate (cAMP) with reduced activation of protein kinase A (PKA), inhibition of mitochondrial glycerophosphate dehydrogenase, and an effect on gut microbiota. Ultimately, it decreases gluconeogenesis (liver glucose production). It also has an insulin-sensitizing effect with multiple actions on tissues including the liver, skeletal muscle, endothelium, adipose tissue, and the ovary. The average patient with type 2 diabetes has three times the normal rate of gluconeogenesis; metformin treatment reduces this by over one-third.
Activation of AMPK was required for metformin's inhibitory effect on liver glucose production. AMPK is an enzyme that plays an important role in insulin signalling, whole body energy balance and the metabolism of glucose and fats. AMPK Activation was required for an increase in the expression of small heterodimer partner, which in turn inhibited the expression of the hepatic gluconeogenic genes phosphoenolpyruvate carboxykinase and glucose 6-phosphatase. Metformin is frequently used in research along with AICA ribonucleotide as an AMPK agonist. The mechanism by which biguanides increase the activity of AMPK remains uncertain; however, metformin increases the concentration of cytosolic adenosine monophosphate (AMP) (as opposed to a change in total AMP or total AMP/adenosine triphosphate). Increased cellular AMP has been proposed to explain the inhibition of glucagon-induced increase in cAMP and activation of PKA. Metformin and other biguanides may antagonize the action of glucagon, thus reducing fasting glucose levels. Metformin also induces a profound shift in the faecal microbial community profile in diabetic mice and this may contribute to its mode of action possibly through an effect on glucagon-like peptide- 1 secretion.
In addition to suppressing hepatic glucose production, metformin increases insulin sensitivity, enhances peripheral glucose uptake (by inducing the phosphorylation of GLUT4 enhancer factor), decreases insulin-induced suppression of fatty acid oxidation, and decreases absorption of glucose from the gastrointestinal tract. Increased peripheral use of glucose may be due to improved insulin binding to insulin receptors. The increase in insulin binding after metformin treatment has also been demonstrated in patients with NIDDM.
AMPK probably also plays a role in increased peripheral insulin sensitivity, as metformin administration increases AMPK activity in skeletal muscle. AMPK is known to cause GLUT4 deployment to the plasma membrane, resulting in insulin-independent glucose uptake. Some metabolic actions of metformin do appear to occur by AMPK- independent mechanisms.
Metformin hydrochloride (l,l-dimethylbiguanide hydrochloride) is freely-soluble in water, slightly soluble in ethanol, but almost insoluble in acetone, ether, or chloroform. The pKa of metformin is 12.4. The usual synthesis of metformin, originally described in 1922, involves the one-pot reaction of dimethylamine hydrochloride and 2-cyanoguanidine over heat.
Metformin has an oral bioavailability of 50-60% under fasting conditions and is absorbed slowly. Peak plasma concentrations (Cmax) are reached within one to three hours of taking immediate-release metformin and four to eight hours with extended-release formulations. The plasma protein binding of metformin is negligible, as reflected by its very high apparent volume of distribution (300-1000 I after a single dose). Steady state is usually reached in one or two days.
Metformin has acid dissociation constant values (pKa) of 2.8 and 11.5, so exists very largely as the hydrophilic cationic species at physiological pH values. The metformin pKa values make metformin a stronger base than most other basic medications with less than 0.01% nonionized in blood. Furthermore, the lipid solubility of the nonionized species is slight as shown by its low logP value (log(lO) of the distribution coefficient of the nonionized form between octanol and water) of -1.43. These chemical parameters indicate low lipophilicity and, consequently, rapid passive diffusion of metformin through cell membranes is unlikely. As a result of its low lipid solubility it requires the transporter SLC22A1 in order for it to enter cells. The logP of metformin is less than that of phenformin (-0.84) because two methyl substituents on metformin impart lesser lipophilicity than the larger phenylethyl side chain in phenformin. More lipophilic derivatives of metformin are presently under investigation with the aim of producing prodrugs with superior oral absorption than metformin. Metformin is not metabolized. It is cleared from the body by tubular secretion and excreted unchanged in the urine; metformin is undetectable in blood plasma within 24 hours of a single oral dose. The average elimination half-life in plasma is 6.2 hours. Metformin is distributed to (and appears to accumulate in) red blood cells, with a much longer elimination half-life: 17.6 hours (reported as ranging from 18.5 to 31.5 hours in a single dose study of nondiabetics).
E. Resveratrol
Resveratrol (3.5.4'-trihydroxy-/ra -stilbene) is a stilbenoid, a type of natural phenol, and a phytoalexin produced by several plants in response to injury or, when the plant is under attack by pathogens, such as bacteria or fungi. Sources of resveratrol in food include the skin of grapes, blueberries, raspberries, mulberries, and peanuts.
Although commonly used as a dietary supplement and studied in laboratory models of human diseases, there is no high-quality evidence that resveratrol improves lifespan or has an effect on any human disease.
In a year long preliminary clinical trial in people with Alzheimer's disease, the most frequent adverse effects were diarrhea, weight loss, and nausea. A 2018 review of resveratrol effects on blood pressure found that some people had increased frequency of bowel movements and loose stools, and one person taking a 1000 mg daily dose developed an itchy rash.
Resveratrol has been identified as a pan-assay interference compound, which produces positive results in many different laboratory assays. Its ability for varied interactions may be due to direct effects on cell membranes.
As of 2015, many specific biological targets for resveratrol had been identified, including NQ02 (alone and in interaction with AKT1), GSTP1, estrogen receptor beta, CBR1, and integrin anb. It was unclear at that time if any or all of these were responsible for the observed effects in cells and model organisms.
In vitro studies indicate resveratrol activates sirtuin 1, although this may be a downstream effect from its immediate biological target(s). It appears to signal through PGC-la, thereby affecting mitochondria. In cells treated with resveratrol, an increase is observed in the action of MnSOD (SOD2) and in GPER activity. In vitro, resveratrol was shown to act as an agonist of Peroxisome proliferator-activated receptor gamma, a nuclear receptor under pharmacological research as a potential treatment for type 2 diabetes. One way of administering resveratrol in humans may be buccal delivery by direct absorption through the saliva. However, the viability of a buccal delivery method is unlikely due to the low aqueous solubility of the molecule. The bioavailability of resveratrol is about 0.5% due to extensive hepatic glucuronidation and sulfation.
Resveratrol is extensively metabolized in the body, with the liver and lungs as the major sites of its metabolism.
Resveratrol (3,5,4'-trihydroxystilbene) is a stilbenoid, a derivative of stilbene. It exists as two geometric isomers: cis- (Z) and trans- (A) with the trans- isomer shown in the top image. The trans- and cv.v-resveratrol can be either free or bound to glucose.
The trans- form can undergo isomerization to the cis- form when exposed to ultraviolet irradiation, a process called photoisomerization:
Figure imgf000028_0001
One study showed that ultraviolet irradiation to cis-resveratrol induces further photochemical reaction, producing a fluorescent molecule named“Resveratrone.”
'/ra/ v-resveratrol in the powder form was found to be stable under“accelerated stability” conditions of 75% humidity and 40 °C in the presence of air. The trans isomer is also stabilized by the presence of transport proteins. Resveratrol content also was stable in the skins of grapes and pomace taken after fermentation and stored for a long period. Ή- and 13C-NMR data for the four most common forms of resveratrols are reported in literature.
Resveratrol is produced in plants by the action of the enzyme, resveratrol synthase.
III. Protein/Peptide Delivery
The present disclosure, in one aspect, relates to the production and formulation of mitochondrial biogenesis modulators as well as their delivery to cells, tissues or subjects. In general, recombinant production of proteins is well known and is therefore no described in detail here. The discussion of nucleic acids and expression vectors, found below, is however incorporated in this discussion. A. Purification of Proteins
It will be desirable to purify proteins according to the present disclosure. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or even HPLC.
Certain aspects of the present disclosure concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide. The term “purified protein” as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally - obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
Generally,“purified” will refer to a protein composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term“substantially purified” is used, this designation will refer to a composition in which the protein forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of the protein will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a“-fold purification number.” The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity. Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater “-fold” purification than the same technique utilizing a low-pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi el al, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl- D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present disclosure is discussed below. B. Cell Permeability Peptides
The present disclosure contemplates the use of a cell permeability peptide (also called a cell delivery peptide, or cell transduction domain) linked to a protein or peptide of interest. Such domains have been described in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007). Other examples are shown in Table 1, below.
TABLE 1 - CDD/CTD PEPTIDES
SEQ ID SEQ ID NO: NO:
G ALFL GWL G A AG STMG AKKKRK 1 QAATATRGRSAASRPTERPRAPARS 23
ASRPRRPVE
V
RQIKIWF QNRRMKWKK 2 MGLGLHLLVLAAALQGAKSKRKV 24
RRMKWKK 3 AAVALLPAVLLALLAPAAANYKKP 25
KL
RRWRRWWRRWWRRWRR 4 MANLGYWLLALFVTMWTDVGLCK 26
KRPKP
RGGRLSYSRRRFSTSTGR 5 LGTYTQDFNKFHTFPQTAIGVGAP 27
Y GRKKRRQRRR 6 DPKGDPKGVTVTVTVTVTGKGDPX 28
PD
RKKRRQRRR 7 PPPPPPPPPPPPPP 29
YARAAARQARA 8 VRLPPPVRLPPPVRLPPP 30
RRRRRRRR 9 PRPLPPPRPG 31
KKKKKKKK 10 SVRRRPRPPYLPRPRPPPFFPPRLPPR 32
IPP
GWTLNSAGYLLGKINLKALAALA 11 TRSSRAGLQFPVGRVHRLLRK 33
KXIL
LLILLRRRIRKQANAHSK 12 GIGKFLHSAKKFGKAFVGEIMNS 34
SRRHHCRSKAKRSRHH 13 KWKLFKKIEK V GQNIRD GIIK AGP A 35
VAWGQATQIAK
NRARRNRRRVR 14 ALWMTLLKKVLKAAAKAALNAVL 36
VGANA
RQLRIAGRRLRGRSR 15 GIGAVLKVLTTGLPALISWIKRKRQ 37
Q
KLIKGRTPIKFGK 16 INLKALAALAKKIL 38
RRIPNRRPRR 17 GFFALIPKIISSPLPKTLLSAVGSALG 39
GSGGQE
KLALKLALKALKAALKLA 18 LAKWALKQGFAKLKS 40
KLAKLAKKLAKLAK 19 SMAQDIISΉGDLVKWIIQTVNXFTK 41
K
G ALFL GEL G A AG STN G AWSQPKK 20 LLGDFFRKSKEKIGKEFKRIVQRIKQ 42
RIKDFLANLVPRTES
KRKV
KETWWETWWTEW SQPKKKRK V 21 P AWRK AFRW AWRMLKK A A 43
LKKLLKKLLKKLLKKLLKKL 22 KLKLKLKLKLKLKLKLKL 44 C. Protein Delivery
In general, proteins are delivered to cells as a formulation that promotes entry of the proteins into a cell of interest. In a most basic form, lipid vehicles such as liposomes. For example, liposomes, which are artificially prepared vesicles made of lipid bilayers have been used to delivery a variety of drugs. Liposomes can be composed of naturally-derived phospholipids with mixed lipid chains (like egg phosphatidylethanolamine) or other surfactants. In particular, liposomes containing cationic or neutural lipids have been used in the formulation of drugs. Liposomes should not be confused with micelles and reverse micelles composed of monolayers, which also can be used for delivery.
A wide variety of commercial formulations for protein delivery are well known including PULSin™, Lipodin-Pro, Carry-MaxR, Pro-DeliverlN, PromoFectin, Pro-Ject, Chariot™ Protein Delivery reagent, BioPORTER™, and others.
Nanoparticles are generally considered to be particulate substances having a diameter of 100 nm or less. In contrast to liposomes, which are hollow, nanoparticles tend to be solid. Thus, the drug will be less entrapped and more either embedded in or coated on the nanoparticle. Nanoparticles can be made of metals including oxides, silica, polymers such as polymethyl methacrylate, and ceramics. Similarly, nanoshells are somewhat larger and encase the delivered substances with these same materials. Either nanoparticles or nanoshells permit sustained or controlled release of the peptide or mimetic and can stabilize it to the effects of in vivo environment.
IV. Nucleic Acid Delivery
As discussed above, in certain embodiments, expression cassettes are employed to express a peptide or protein, either for subsequent purification and delivery to a cell/subject, or for use directly in a genetic-based delivery approach. Expression requires that appropriate signals be provided in the vectors and include various regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression of the polypeptide. A. Regulatory Elements
Throughout this application, the term“expression cassette” is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and translated, i. e. , is under the control of a promoter. A“promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase“under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. An“expression vector” is meant to include expression cassettes comprised in a genetic construct that is capable of replication, and thus including one or more of origins of replication, transcription termination signals, poly-A regions, selectable markers, and multipurpose cloning sites.
The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase ( tk ) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.
At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription. In certain embodiments, viral promotes such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression of the gene product.
Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
Below is a list of promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct (Table 2 and Table 3). Additionally, any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene. Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000038_0002
Figure imgf000039_0001
Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the disclosure, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
B. Multigene Constructs and IRES
In certain embodiments of the disclosure, the use of internal ribosome binding sites
(IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Samow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker. C. Delivery of Expression Vectors
There are a number of ways in which expression vectors may introduced into cells. In certain embodiments of the disclosure, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein. In this context, expression does not require that the gene product be synthesized.
The expression vector comprises a genetically engineered form of adenovirus. Knowledge of the genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992). In contrast to retrovirus, the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication. The El region (El A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5 '-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.
In one system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh- Choudhury et al, 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete.
Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.
Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into l-liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the disclosure. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present disclosure. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
As stated above, the typical vector according to the present disclosure is replication defective and will not have an adenovirus El region. Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the disclosure. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors, as described by Karlsson et al. (1986), or in the E4 region where a helper cell line or helper virus complements the E4 defect.
Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109-1012 plaque forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991; Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec, 1991). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991; Stratford-Perricaudet et al 1990; Rich et al, 1993). Studies in administering recombinant adenovirus to different tissues include trachea instillation (Rosenfeld et al, 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al, 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle e/ al, 1993).
The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et ctl, 1975).
A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocy tes via sialogly coprotein receptors.
A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989).
There are certain limitations to the use of retrovirus vectors in all aspects of the present disclosure. For example, retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus etal, 1981). Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988; Hersdorffer et al, 1990).
Other viral vectors may be employed as expression constructs in the present disclosure. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990). In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle.
Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa el a/.. 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.
Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
In yet another embodiment of the disclosure, the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
In still another embodiment of the disclosure for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990; Zelenin et al, 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, /. e.. ex vivo treatment. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.
In a further embodiment of the disclosure, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful. Wong et al, (1980) demonstrated the feasibility of liposome- mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells. Nicolau et al, (1987) accomplished successful liposome-mediated gene transfer in rats after intravenous injection. A reagent known as Lipofectamine 2000™ is widely used and commercially available.
In certain embodiments of the disclosure, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-l) (Kato et ctl, 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-l. In that such expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present disclosure. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as agene delivery vehicle (Ferkol etal, 1993; Perales etal., 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
V. Methods of Treating Subjects
As discussed above, the present disclosure provides for the treating of disorders relating to mitochondrial biogenesis and its disfunction, and to stimulating erythropoeisis. The treatment may result in amelioration of any symptom of a given disease or disorder.
Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render peptides, proteins or delivery vectors stable and allow for uptake by target cells. Aqueous compositions of the present disclosure comprise an effective amount of the drug, vector or proteins, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present disclosure, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or cells of the compositions.
The active compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present disclosure may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Such compositions would normally be administered as pharmaceutically acceptable compositions, as described supra.
The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions of the present disclosure generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered, and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" l5th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
VI. Examples
The following examples are included to further illustrate various aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Example 1
As discussed above, Fe-S clusters are essential cofactors for mitochondria functions, and mitochondria are required for Fe-S cluster synthesis. Additionally, mitochondria biogenesis demands cellular iron uptake, which is negatively regulated by Fe-S clusters. Fe-S clusters are synthesized in the mitochondria and cytosol by two different machineries. However, cytosolic Fe-S cluster synthesis necessitates the mitochondrial Fe- S cluster assembly machinery.
PGCla is a transcriptional coactivator and a master regulator of mitochondria biogenesis. The inventors confirmed that overexpression of PGCla in adipocytes and hepatic cells stimulated mitochondria biogenesis, as measured by Mitotrack Green and Deep Red staining, which label total and alive mitochondria, respectively. They further measured Fe-S cluster synthesis by monitoring the gene expression of Fe-S cluster assembly machinery. By using RT-qPCR and Western Blot analyses, the inventors confirmed that PGC-la expression increases expression of ABCB7, ISCA1, ISCA2, ISD11, Nful and ISCU, components of the Fe-S assembly machinery, suggesting a coordination between mitochondria biogenesis and Fe-S cluster synthesis. Iron Regulatory Proteins (IRP1 and IRP2) control iron metabolism by binding to specific non-coding sequences within an mRNA, known as iron-responsive elements (IRE). In the absence of Fe-S clusters, IRP1 acts as an aconitase (aka ACOl), while IRP2 is degraded by ubiquitination. Aconitases, represented by the cytosolic form ACOl and mitochondrial form AC02, catalyze the isomerization of citrate to isocitrate and require Fe-S clusters to be enzymatically active. PGCla overexpression enhanced aconitase activity but not their protein levels, corroborating the notion that Fe-S cluster synthesis was increased.
To explore whether this coordination solely depends on PGC-la, the inventors evaluated the Fe-S cluster synthesis status during brown adipocyte maturation, which is characterized by enhanced mitochondria biogenesis and has been suggested to be PGC la- independent. They found that the synthesis of Fe-S cluster assembly machinery increased during maturation in both wild-type and PGC la-knockout brown adipocytes, indicating that Fe-S cluster synthesis coordinates with mitochondria biogenesis even in the absence of PGC la.
To explore the impact of Fe-S cluster synthesis on iron acquisition under enhanced mitochondria biogenesis, the inventors evaluated the expression of the iron importer transferrin receptor 1 (TfRl). TfRl mRNA contains IREs in the 3’ untranslated region (UTR). These 3’UTR IREs can be bound by IRPs and responsible for the subsequent stabilization of TfRl mRNA. Therefore, if IRP1 associates with Fe-S cluster and converted into ACOl, it is expected that TfRl expression would decrease. In contrast, the inventors found that stimulated Fe-S cluster synthesis increased levels of TfRl, despite reduced IRP1 activity and destabilized TfRl mRNA. This suggests that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block iron uptake.
Moreover, the inventors extended their work to erythropoiesis by using murine erythroleukemia (MEL) cells. Stimulated mitochondria biogenesis enhanced expression of the Fe-S cluster assembly machinery and Fe-S cluster synthesis in these cells. TfRl protein levels were increased despite elevated Fe-S cluster synthesis and reduced IRP activity. They found increases in heme levels and the expression of aminolevulinic acid synthase 2 (ALAS2), the rate-limiting enzyme for erythroid heme synthesis. Of note, the ALAS2 mRNA contains IRE at the 5’UTR, binding of IRPs to the IRE inhibits translation while high Fe-S cluster levels lead to release. Moreover, as a- and b-globin chain expression is stimulated by increased heme availability, the inventors also observed that mitochondria biogenesis was associated with increased synthesis of these two proteins and hemoglobinization. These data suggest that erythroid heme synthesis, hemoglobin expression and hemoglobinization coordinates with mitochondria biogenesis via Fe-S cluster.
In conclusion, these data show that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block cellular iron uptake, thus suggesting a potential unidentified iron regulator to ensure adequate iron for mitochondria biogenesis. Moreover, our work suggests a mechanism underlying the essential role of mitochondria biogenesis in erythropoiesis.
Example 2
As discussed above, iron-sulfur (Fe-S) clusters are iron-containing prosthetic groups and enzymatic cofactors. They are strong oxidants when unbound yet essential in many processes like facilitating ATP production in mitochondria, promoting DNA, RNA and protein syntheses during cell proliferation and enhancing DNA repair in antioxidant defense. In particular, Fe-S clusters are indispensable in erythropoiesis, where the majority of physiological iron is utilized and where Fe-S clusters are required for the heme synthesis. Deficient Fe-S cluster synthesis predisposes individual to various diseases, such as cancer, metabolic and neurodegeneration diseases and blood disorders. However, it is unclear how Fe-S cluster synthesis is regulated and coordinates with environmental and developmental needs to prevent oxidative damage.
The 5' AMP-activated protein kinase (AMPK) is a kinase activated by oxidative stress and energy starvation and critical for maintaining redox and energy homeostasis. In this study, the inventors investigated the role of AMPK on Fe-S clusters synthesis and function and extended our findings in normal and thalassemic erythroid cells.
Through bioinformatic analysis, the inventors found that the Fe-S cluster assembly enzyme (ISCU), a scaffold protein indispensable for Fe-S cluster biogenesis, contains putative AMPK phosphorylation motifs at serine (S) residues 14 and 29 (human numbering). Using the human cell line 293T, the inventors confirmed that AMPK phosphorylates ISCU, while point mutations in these residues prevented this activity. Moreover, AMPK-mediated phosphorylation promoted ISCU binding to l4-3-3s, a family of proteins that, once associate with phosphorylated residues, modulates the stability and function of targeted proteins. Indeed, increased association with l4-3-3s stabilized ISCU proteins, corroborating the observation that AMPK promotes the activity of ISCU proteins. The inventors extended their studies using A549 cells that do not have AMPK activity since they harbor a mutant LKB1 kinase, which is responsible for activating AMPK. By overexpression of wild-type (WT)-LKBl and LKB1 kinase-dead mutant (KDM), the inventors found that only WT-LKB1 restored AMPK activity, binding of ISCU to l4-3-3s and stability of ISCU. Moreover, under hydrogen peroxide incubation and glucose starvation, ISCU protein levels and Fe-S cluster synthesis were both increased only in the presence of LKB1-WT, but not in cells harboring KMD. LKB1-WT overexpressing cells also survived hydrogen peroxide incubation and glucose starvation better than those with KMD. Together, these data suggest that AMPK activation stabilizes ISCU protein and preserves Fe-S cluster synthesis to maintain a healthy redox and energy homeostasis.
The inventors then explored the effect of AMPK on Fe-S cluster synthesis in erythropoiesis by using the drug AICAR, an AMPK activator, in murine erythroleukemia (MEL) cells. They found that in MEL cells, AICAR treatment stabilized ISCU, increased Fe-S cluster levels and promoted the synthesis of the aminolevulinic acid synthase 2 (ALAS2) protein, which represents the rate-limiting enzyme in erythroid heme synthesis. Furthermore, this was associated with increased heme and globin chain synthesis, with a trend in increasing b-globin mRNA and proteins more than a-globin. The inventors further confirmed these observations in Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP-2) and CD34+ cells derived from peripheral blood isolated from both healthy individuals and B-thalassemic patients. In these cells, the inventors found that AMPK upregulation by AICAR administration not only increased ALAS2 expression and erythroid heme levels, but also enhanced the synthesis of both a- and B-globin chains, though with a preference for increasing b-globin levels. Analysis using specimens from thalassemic mice is in progress.
In conclusion, this work demonstrates that under redox and energetic stress, activated AMPK phosphorylates and stabilizes ISCU protein, thereby enhancing Fe-S cluster synthesis and maintaining their function. Moreover, AMPK activation with AICAR treatment increases erythroid heme synthesis and hemoglobin expression. Given that AMPK is the major kinase that responds to oxidative and energetic cues, our work provides a mechanistic explanation for how erythropoiesis responds to energy starvation and redox stress as well as a potential novel therapeutic target to treat blood and metabolic disorders. Example 3
Cell lines. Human hepatocellular carcinoma cell line HepG2 and murine preadipocytes 3T3-L1 were cultured in Dulbecco’s Modified Eagle’s Medium containing 10% (v/v) fetal calf serum, penicillin (100 units/ml) and streptomycin (100 pg/ml), under 5% CCh and at 37 °C. Mouse prebrown adipocytes were generated a generous gift from Bruce Spiegelman (Harvard Medical School). They were cultured and differentiated as previously described (Uldry et al., 2006a). Mouse erythroleukemia (MEL) were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% (v/v) fetal calf serum, penicillin (100 units/ml) and streptomycin (100 pg/ml), under 5% CCh and at 37 °C. For differentiation, MEL cells were treated with 50mM hexamethylene bisacetamide (HMBA) for different days as indicated in figure legends.
Western blot analysis. Cells were lysed in Radioimmunoprecipitation assay (RIP A) buffer with proteinase inhibitors. Protein concentrations were measured by using Pierce™ BCA Protein Assay Kit. The equal amount of total proteins was separated by SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was further incubated with different primary antibodies as indicated and followed by another incubation with HRP-conjugated secondary antibody to visualize the binding affinity of the primary antibody. Calnexin, GADPH or Actin was used as an internal control.
qRT-PCR assay. Total RNA was isolated by using RNeasy Mini kit (Qiagen), reverse-transcribed into cDNAs by using Superscript III Reverse Transcriptase (Thermo Fisher) and further analyzed by TaqMan Gene Expression Assays (Thermo Fisher). GADPH or ACTB mRNA levels were used as an internal control.
Aconitase activity assay. In Gel assay was applied as described previously (Tong and Roualt, 2006; Ghosh et al, 2008). Briefly, cell lysis was separated by PAGE with Tris-borate buffer and then incubated with the solution containing cis-aconitic acid, isocitric dehydrogenase, phenazine methyl sulfate (PMS), NADP and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 37 °C for 10 minutes. After the incubation, gels were photographed.
Analysis of intracellular heme levels. A colorimetric assay kit (Biovision Incorporated) was utilized per manufacturer's instructions.
Mitochondria biogenesis assays. Cells were labeled with 100hM Mitotracker Green FM or Deep Red FM (Thermo fisher) and followed by fluorescence-activated cell sorting (FACS). FACS data were analyzed by using FlowJo7 software. Analysis of IRE-binding activities. IRE-binding activities were measured differently for FIGS. 3A-I and FIGS. 4A-E. For FIGS. 3A-I, IRE-binding activity was analyzed as previously described (Meyron-Holtz et al, 2004). Briefly, to generate IRE probe, IRE sequence derived from ferritin heavy chain (FtH) mRNA was cloned and further labeled with phosphorus-32 radioactive isotope by using Riboprobe® Systems (Promega). Equal amount of total proteins was incubated with IRE probes and separated by PAGE followed by an exposure to X-ray films. For FIGS. 7A-E, IRE-binding activities were measured by using LightShift™ Chemiluminescent RNA EMSA Kit (Thermo Fisher) per manufacturer’s instruction.
Plasmid construction. The Myc epitope was fused at the N-terminus of human PGC-la cDNA and cloned in PMX-puro for retrovirus production and EGAWP for lentiviruses production. GFP was also introduced in these vectors as a control. Retroviruses was used on 3T3-L1 and HepG2 while lentiviruses were used on MEL cells.
Statistical analysis. Each experiment was repeated for a minimum of three times. Data was analyzed by standard Student T-test. Statistical significance was considered at p<0.05 vs corresponding controls or day 0.
Example 4
Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-la) induces Fe-S clusters synthesis. PGC-la is a transcriptional coactivator and plays a central role in governing mitochondrial biogenesis (Lin et al. , 2005). To test whether Fe-S cluster synthesis increases with mitochondria biogenesis, human PGC-la tagged with a Myc epitope (Myc-PGC-la) was expressed in preadipocytes 3T3-L1 (FIG. 1A). The Myc-PGC-la expression increased staining with Mitotrack Green and Deep Red (FIG. 1B), suggesting an increased mitochondria biogenesis. Moreover, Myc-PGC- la expression enhanced heme levels and the gene expression of ALAS1 (FIG. 1C), a rate- limiting enzyme in non-erythroid heme synthesis pathways (Handschin et al, 2005). Thus, these data confirmed that Myc-PGC-la functions similar to endogenous PGC-la.
To investigate whether mitochondria biogenesis affected Fe-S cluster synthesis, control and Myc-PGC-la-expressing 3T3-L1 cells were analyzed for aconitase activities. Aconitases, represented by the cytosolic form ACOl and mitochondrial form AC02, require Fe-S cluster to be enzymatically active. Their activity was used to monitor the status of Fe-S cluster synthesis in cytosol (ACOl) and mitochondria (AC02). By an in- Gel aconitase activity assay (Tong and Rouault, 2006), ACOl and AC02 proteins were separated by PAGE and then incubated with an aconitase substrate, resulting in a blue color whose intensity is proportional to aconitase activity. Myc-PGC-la expression significantly increased ACOl activity despite limited increase in protein levels (FIG. 1D), indicating enhanced Fe-S cluster synthesis in the cytosol. For AC02, Myc-PGC-la expression increased both protein levels and activity. However, simple overexpression of AC02 protein in 3T3-L1 did not increase its activity (data not shown). Moreover, the inventors quantified AC02 activity (shown by the blue color intensity in FIG. 1D, first panel from the top) and protein levels obtained by Western blot analysis (FIG. 1D, third panel from the top), then divided the former by the latter to normalize AC02 activity by its protein levels. Compared to control cells, Myc-PGC-la overexpression increased AC02 activity by a factor of 2.7 (FIG. 1E), confirming that such an increase was due to an elevated Fe-S cluster synthesis in mitochondria.
Since Fe-S cluster synthesis is stimulated under enhanced mitochondria biogenesis, the inventors next investigated whether this stimulation was due to increased gene expression of the Fe-S cluster assembly machinery. A subset of genes involved in this pathway were investigated. The protein levels of ISD11, ISCU and FXN, which belong to the mitochondrial core assembly machinery, were increased by Myc-PGC-la expression (FIG. 2A). This was observed also for ISCA1/2 and Nful, which belong to the mitochondrial Fe-S cluster delivery machinery, and for ABCB7 that is involved in Fe-S clusters mitochondrial export machinery and required for cytosolic Fe-S cluster synthesis (Rouault and Maio, 2017). For some genes such as ISCU and Nful, the mRNA level did not change significantly and proportionally to their protein levels (FIG. 2B), suggesting a post-transcriptional regulation and consistent with what have previously shown (Rensvold et al, 2013).
Similarly, Myc-PGC-la was also expressed in human hepatoma cell line HepG2 (FIG. S1A) and increased Fe-S clusters synthesis both in mitochondria and in cytosol (FIGS. S1B-S1C). Together, these data demonstrated that Fe-S cluster synthesis coordinates with PGC- la-enhanced mitochondria biogenesis.
Fe-S cluster synthesis coordinates with mitochondria biogenesis also in the absence of PGC-Ia. Although PGC-la is the master regulator of mitochondria biogenesis, the inventors explored if PGC-la was the only mediator and essential for Fe- S cluster synthesis coordinating with mitochondria biogenesis. Therefore, the inventors investigated how Fe-S cluster synthesis was regulated during brown adipocyte maturation, a process characterized by a significant increase in mitochondria biogenesis and heme synthesis (Seale et al, 2009). To this end, wild-type (WT) and PGC-la-null preadipocytes were differentiated to mature brown adipocytes by standard drug treatment for 6 days (Uldry et al. , 2006a). As expected, PGC-la was induced only in wild-type cells while RϋOIb, another member of the PGC-l family that is able to stimulate mitochondria biogenesis, was induced in both WT and PGC-la-null cells (FIGS. S2A- B). Of note, PGC-la mRNA was detected in PGC-la-null cells due to the mRNA region targeted by the TaqMan probe not being eliminated and potentially transcribed in these cells Lin et al. , 2005). Moreover, expressions of uncoupling protein 1 (UCP1, a mitochondria marker), ALAS1 and heme levels were significantly induced in both of matured WT and PGC-la-null cells (FIGS. S2C-D), confirming that mitochondria biogenesis was significantly induced during matured brown adipocytes.
To investigate whether Fe-S cluster synthesis increases with enhanced mitochondria biogenesis during brown adipocyte maturation, the protein amount and activity of ACOl and AC02 was measured in both WT and PGC-la-null cells. As shown in FIG. 3A, both protein levels and activities of ACOs were increased in mature brown adipocytes, regardless of PGC-la expression. Moreover, the quantitative ratio between aconitase activity and protein levels was enhanced by about 4-fold upon differentiation in cytosol and about 200-fold in mitochondria (FIGS. 3B-C). The protein levels of ISCU, ISD11, ISCA1/2 and Nful were all induced at day 6 of treatment in both of WT and PGC- la-null cells (FIG. 3D). Moreover, the mRNA levels of ISCA1 and ISCA2 genes were also induced (FIGS. 3E-F). As seen in 3T3-L1 cells (FIGS. 2A-B), the mRNA levels of several genes did not match to their protein levels changes (FIGS. 3G-I). Nevertheless, these data further demonstrated that Fe-S cluster synthesis coordinates with mitochondria biogenesis even in the absence of PGC-la.
Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block mitochondria-required iron uptake. As enhanced mitochondria biogenesis stimulates heme and Fe-S cluster synthesis, two major iron-consuming pathways, the inventors explored how TfRl expression was regulated by mitochondria biogenesis- coordinated Fe-S cluster synthesis. Since enhanced Fe-S cluster availability might decrease the IRE-binding activity of IRP1 (Zhang et al. , 2014), the inventors analyzed IRPs/IRE activity. As shown in FIG. 4A, the IRE-binding activity in Myc-PGC-la- expressing 3T3-L1 cells was reduced more than 50% compared to that in control cells, whereas IRP1 and IRP2 protein levels remained the same in control and Myc-PGC-la- expressing cells. Along with reduced IRE-binding activity, TfRl mRNA was decreased (FIG. 4B). However, in My c-PGC- la-expressing 3T3-L1 cells, TfRl protein levels were increased, suggesting enhanced iron intake (FIG. 4C). To better understand TfRl regulation, My c-PGC- la-expressing cells were incubated with either a proteasome inhibitor (MG132), a transcription inhibitor (Actinomycin D) or a translation inhibitor (cycloheximide). Incubation with MG132 reversed the increased TfRl protein levels while actinomycin D and cycloheximide had limited effect (FIGS. 4D-E). These data suggest that increased TfRl expression is primarily at the post-translational level. Possibly, increased ATP production due to the enhanced mitochondrial biogenesis potentiates TfRl folding and refolding to avoid degradation, though this requires further investigation. Nonetheless, these data demonstrate that Fe-S cluster synthesis coordinates with PGC-la-mediated mitochondria biogenesis but does not limit mitochondria- required iron uptake. These observations were also confirmed in HepG2 cells (FIG. S3), where TfRl protein levels were increased by PGC-l a expression despite decreased TfRl mRNA levels (FIG. S3) and the increased Fe-S cluster synthesis (FIG. Sl).
Moreover, the inventors investigated how iron uptake was modulated during the brown adipocyte maturation. Consistent with the increased IRPl/ACOl protein levels in matured brown adipocytes (FIG. 3 A), the inventors found that IRP1 mRNA levels were stimulated in matured WT and PGC- la-null brown adipocytes (FIG. 5 A). IRE-binding activities were significantly increased in differentiated brown adipocytes (FIG. 5B), despite the increased Fe-S cluster synthesis (FIGS. 3A). Consistent with this increased IRE-binding activity, TfRl mRNA and protein levels were increased as well (FIGS. 5C- D). These data suggest that in mature brown adipocytes, stimulated IRP1 expression increases apoIRPl levels, which in turn increases TfRl expression regardless of the status of Fe-S cluster synthesis. Although the mechanisms leading to increased TfRl levels may be different in 3T3-L1 vs. brown adipocytes, these data demonstrated that cells employ different means to overcome the negative effect of Fe-S cluster synthesis on TfRl expression and ultimately increase TfRl levels to meet mitochondria biogenesis- demanded iron intake.
In erythropoiesis, Fe-S cluster assembly coordinates with mitochondria biogenesis but not iron uptake and ultimately modulates heme synthesis and hemoglobinization. The regulation of iron metabolism, especially heme metabolism, are always different between erythroid and non-erythroid tissues (Ajioka el ah, 2006; Muckenthaler el al, 2017). Therefore, the inventors also investigated how mitochondria biogenesis, Fe-S cluster synthesis and iron uptake maintain homeostasis during erythropoiesis by using murine erythroleukemia (MEL) cells, an in vitro model of erythropoiesis (Cui et ah, 2014). First, the inventors introduced Myc-PGC-la into MEL cells by viral transduction and differentiated these cells into erythrocytes by incubation with HMBA. As shown in Figure 6A and 6B, Myc-PGC-la expression stimulated the activity and expression of Fe-S cluster assembly machinery, as evidenced by increased ACOl and AC02 activities as well as increased mRNA and protein levels of genes involved in Fe-S cluster synthesis. Next, the inventors explored the impact of decreasing mitochondria biogenesis by lentiviral transduction of PGC-la shRNA (FIG. 6C). As shown in FIG. 6D, protein levels of ISD11, ISCU, ABCB7 and ISCA1/2 were decreased in PGC- la-knockdown cells vs. control. Subsequently, Fe-S cluster synthesis was also inhibited, as demonstrated by decreased aconitase activities of ACOl and AC02 (FIG. 6D, top panel) and confirming a coordination between Fe-S cluster synthesis and mitochondria biogenesis in erythroid cells. Similar to non-erythroid cells, for some genes of the assembly machinery, the inventors did not observe the same trend between mRNA and protein levels changes, especially when PGC-la expression was suppressed (FIG. 6E). Of note, HMBA incubation did not interfere with Fe-S cluster synthesis coordinating with mitochondria biogenesis as similar effects were observed in undifferentiated Myc- PGC-la-expressing and PGC-la-shRNA-transduced MEL cells (FIGS. S4A-B). Nonetheless, these data demonstrated that Fe-S cluster synthesis also coordinates with mitochondria biogenesis in erythroid cells.
The inventors then examined how mitochondrial biogenesis and Fe-S cluster synthesis affect iron demand, namely TfRl expression in erythroid cells. As shown in FIG. 7 A, the IRE-binding activity was decreased in My c-PGC- la-expressing cells while IRPs levels remained similar. However, in contrast to this decreased IRE-binding activity, the mRNA and protein levels of TfRl were both increased (FIGS. 7A-B), suggesting an IRE-binding and IRPs-independent mechanism. Indeed, the inventors found that Myc-PGC-la stimulated the expression of GATA1 (FIG. 7A), a master regulator of erythropoiesis which has been shown to upregulate TfRl expression (Kaneko et al, 2012). Corresponding to the changes in My c-PGC- la-expressing MEL cells, the opposite variations were demonstrated in PGC-la-knockdown MEL cells, such as increased IRE-binding activity and decreased TfRl expression (FIGS. 7C-D). Again, the inventors observed a decreased GATA1 expression in PGC-la-knockdown MEL cells, suggesting that unlike the mechanisms employed by 3T3-L1 and brown adipocytes (FIG. 7E, top and middle), erythroid GATA1 expression was upregulated under enhanced mitochondria biogenesis. This upregulation of GATA1 overrides the negative effected of increased Fe-S cluster synthesis on TfRl expression to meet the iron demand (FIG. 7E, bottom).
Next, the inventors investigated how Fe-S cluster synthesis under enhanced mitochondria biogenesis regulates ALAS2 expression and potentially erythropoiesis. ALAS2 contains a 5’UTR IRE, and its translation can be inhibited by IRPs binding. Overexpression of Myc-PGC-la in MEL cells significantly stimulated ALAS2 levels and further hemoglobinization evidenced by a more reddish color of cell pellets (FIG. 8A). As heme stimulates the expression of a- and b-globin (HBA and HBB) (Chen, 2014; Tahara et al, 2004; Grosso et al, 2017), the inventors found that HBB and HBA expression was also stimulated. In contrast, PGC-l a knockdown decreased the expression of ALAS2, HBB and HBA as well as hemoglobinization (FIGS. 8C-D). Of note, because PGC-la antagonizes the mitophagy that is required for the late stage of erythropoiesis (Lin et al. , 2005; Grosso et al, 2017), Myc-PGC-la-expressing cells were treated with HMBA for a short period time, only 5 days to avoid this detrimental effect. Nonetheless, these data suggested that erythroid Fe-S cluster synthesis coordinates with mitochondria biogenesis without limiting iron uptake, but rather improving the synthesis of heme and hemoglobin.
TABLE 1 - Antibodies Used
Figure imgf000060_0001
Figure imgf000061_0001
Example 5
In this paper, the inventors demonstrated that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not block mitochondria biogenesis-required TfRl expression. In fact, TfRl expression is stimulated via alternative means to meet iron requirement for increased mitochondria biogenesis. Furthermore, ALAS2, HBB and HBA expression all coordinate with mitochondria biogenesis via Fe-S cluster synthesis and transcription factor GATA1, thereby providing a mechanism connecting mitochondria biogenesis and erythropoiesis.
Here, the inventors demonstrate that mitochondrial Fe-S cluster synthesis happens in tandem with mitochondria biogenesis. This is not due to merely increased mitochondria mass, because not all the genes tested in our study demonstrated an increased expression level in Myc-PGC-la-expressing cells. For instance, protein levels of Nfsl were not increased by Myc-PGC-la expression though Nfsl is a mitochondria protein and required for Fe-S cluster synthesis (data not shown). This observation suggests that under enhanced mitochondria biogenesis, the increased expression of Fe-S cluster synthesis machinery is selective and not simply due to increased mitochondria mass.
Moreover, PGC-la and PGC- I b are members of PGC-l family. As transcription activators, they functionally compensate one another by associating with transcription factors that play pivotal roles in stimulating mitochondria biogenesis (Uldry el ah, 2006b). By a bioinformatic analysis (UCSC genome browser), various binding sites for these transcription factors were identified in genes responsible for Fe-S cluster synthesis. For instance, FOXOl binding sites were predicted in ISCA1 gene and GR-alpha and GR- beta in ISCA2 genes, explaining an upregulated mRNA levels of these genes under enhanced mitochondria biogenesis (FIGS. 2A-B, 3A-I and 8A-D). Additionally, coordinating with mitochondria biogenesis, the gene expression could also be regulated on post-transcriptional level (Rensvold et al, 2013; Liu et al, 2017). Therefore, the expression of Fe-S cluster synthesis machinery could also be in accord with mitochondria biogenesis on post-transcriptional levels.
In this study, the inventors also demonstrated that cytosolic Fe-S cluster synthesis also coordinates with mitochondria biogenesis. This could be due to the requirement of mitochondrial Fe-S cluster assembly machinery for cytosolic Fe-S cluster synthesis (Rouault and Maio, 2017). Interestingly, this coordination allows the IRE-binding activity of IRP1 and further ALAS2 expression as well as erythroid heme synthesis are in line with mitochondria biogenesis. Additionally, the inventors demonstrated that GATA1 expression also coordinates with mitochondria biogenesis, further supporting the positive correlation of mitochondria biogenesis and the gene expression of ALAS2, HBA and HBB that are all GATA1 targets (reference). Moreover, heme level also promotes the transcription and translation of HBB and HBA via the transcription inhibitor BTB Domain And CNC Homolog 1 (Bachl) and heme-regulated inhibitor (HRI), an EIF2A kinase (Chen, 2014; Tahara et al, 2004; Grosso et al, 2017). Therefore, our study demonstrated that erythropoiesis coordinates with mitochondria biogenesis on transcription and post-transcription levels and via Fe-S cluster synthesis as well as GATA1.
As demonstrated previously, TfRl protein levels increase with mitochondria biogenesis (Resvold et al, 2013; Ishii et al, 2009; O’Hagan et al, 2009). Here, the inventors demonstrate that despite increased Fe-S cluster synthesis which coordinates with mitochondria biogenesis, TfRl expression was regulated with cell type-specific mechanisms to meet iron demand (FIG. 7E). For instance, 3T3-L1 cells post- translationally stabilized TfRl protein possibly due to an increased mitochondrial ATP generation, which could facilitate TfRl folding and refolding. In matured brown adipocytes, IRP1 expression was induced and further upregulates TfRl expression. Currently, it is unclear how IRP1 gene expression is upregulated. However, a bioinformatic analysis revealed multiple binding sites for transcription factors PPARs in IRP1 gene, which are all required for brown adipocytes maturation (Kajimura et al, 2010) and potentially responsible for the upregulation on IRP1 expression. In erythrocytes, GATA1 expression was increased under enhanced mitochondria biogenesis and could be responsible for stimulated TfRl expression. However, it is unclear how GATA1 gene expression is regulated in line with mitochondria biogenesis. This question warrants future studies to identify mechanisms, which might be exploited to correct dysfunctional mitochondria and iron metabolism (Rouault, 2016; Ginzburg and Rivella, 2011; Fleming, 2011; Chiang et al. , 2016).
In conclusion, these data indicate that Fe-S cluster synthesis coordinates with mitochondria biogenesis but does not limit mitochondria iron uptake. Ultimately, this work demonstrates that under enhanced mitochondria biogenesis, cells employ different routes to stimulate TfRl expression and ensure iron intake. It also suggests a mechanism underlying the essential role of mitochondria biogenesis in erythropoiesis. These data are important for devising new therapeutic modalities for a variety of dyserythropoietic diseases.
Example 6
The inventors further explored whether AMPK activation also regulates disease- associated dyserythropoiesis. thalassemia is a common genetic disorder found worldwide and caused by diminished b-globin gene expression due to a large spectrum of mutations in the globin gene locus. Sideroblastic anemia is due to genetic mutations in genes involved in erythroid heme synthesis and consequently causes deficient heme production.
The inventors initially explored how erythropoiesis was affected by AICAR treatment in Hbbmi+ mice that carry heterozygous B1/B2 globin gene deletion and are largely used as b-thalassemia intermedia disease model. Unfortunately AICAR was not active once administered in vivo (data not shown). Therefore, we managed to isolate and differentiate erythroblasts isolated from the bone marrow and spleen, with or without AICAR treatments. As shown in FIGS. S6A-B, erythropoiesis from both bone marrow and spleen was enhanced with increased protein levels of hemoglobin and Alas2 compared to untreated control.
Based on these promising results, we isolated CD34+ cells from thalassemia and sideroblastic anemia patients, expanded and differentiated them to late erythrocytes. During differentiation, cells were treated with different concentrations of AICAR. As shown in CD34+ cells isolated from a thalassemia patient, the initial AICAR treatment significantly increased ISCU protein levels in a time-dependent manner (FIG. 9A). At 96 hours of AICAR treatment, ALAS2 and hemoglobin protein levels were all increased (FIG. 9B). This observation was further confirmed in differentiated CD34+ cells that were isolated from an additional thalassemia patient (FIG. 9C). Last, we interrogated how erythropoiesis was regulated by AMPK activation in sideroblastic anemia. As shown in FIG. 9D, ALAS2 and hemoglobin protein levels were both increased by AICAR treatment, suggesting that AMPK activation enhances erythropoiesis in an additional disease phenotype. In summary, AMPK activation also stimulates the erythropoiesis in disease states and could potentially be explored for treatment of congenital anemias.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.
VII. References
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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Claims

WHAT IS CLAIMED IS:
1. A method of increasing b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization in an erythroid cell comprising contacting said cell with an agonist of AMPK or PCG-la.
2. The method of claim 1, wherein the agonist is an AMPK protein or expression construct coding therefore, PCG-la protein or expression construct coding therefore, metformin, resveratrol or AICAR.
3. The method of claim 1 , wherein the cell is in a subj ect, such as a human subj ect.
4. The method of claim 3, wherein said subject suffers from a hemoglobinopathy, such as b-thalessemia.
5. The method of claim 1, wherein said cell is contacted with said agonist more than once, such as on a chronic basis.
6. The method of claim 1, wherein the agonist is not AICAR.
7. The method of claim 1, further comprising contacting said cell with another agent that is involved in b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization, such as iron.
8. The method of claim 2, wherein the AMPK protein or PCG-la protein or AICAR are administered directly to said cell.
9. The method of claim 2, wherein the AMPK or PCG-la protein are contacted with said cell by provision of an expression construct coding for the same.
10. The method of claim 3, further comprising identifying said subject as being in need of increased b-globin levels, heme synthesis, hemoglobin synthesis and/or hemglobinization.
11. A method of increasing mitochondrial biogenesis in a cell comprising contacting said cell with an agonist of AMPK or PCG-la.
12. The method of claim 11, wherein the agonist is an AMPK protein or expression construct coding therefore, PCG-la protein or expression construct coding therefore, metformin, resveratrol or AICAR.
13. The method of claim 11, wherein the cell is in a subject, such as a human subject.
14. The method of claim 13, wherein said subject suffers from a disease or disorder stemming from mitochondrial dysfunction, such as sideroblastic anemia.
15. The method of claim 11, wherein said cell is contacted with said agonist more than once, such as on a chronic basis.
16. The method of claim 11, wherein the agonist is not AICAR.
17. The method of claim 11 , further comprising contacting said cell is a muscle cell, an adipocyte, an erythrocyte, or an epithelial cell.
18. The method of claim 12, wherein the AMPK protein or PCG-la protein or AICAR are administered directly to said cell.
19. The method of claim 21, wherein the AMPK or PCG-la protein are contacted with said cell by provision of an expression construct coding for the same.
20. The method of claim 13, further comprising identifying said subject as being in need of increased mitochondrial biogenesis.
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