WO2017192761A1 - Compositions de propionyl-coa carboxylase et utilisations de celles-ci - Google Patents

Compositions de propionyl-coa carboxylase et utilisations de celles-ci Download PDF

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WO2017192761A1
WO2017192761A1 PCT/US2017/030904 US2017030904W WO2017192761A1 WO 2017192761 A1 WO2017192761 A1 WO 2017192761A1 US 2017030904 W US2017030904 W US 2017030904W WO 2017192761 A1 WO2017192761 A1 WO 2017192761A1
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pcc
subunit
pharmaceutical composition
pcca
pccb
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PCT/US2017/030904
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Jan P. Kraus
Tomas Majtan
Renata COLLARD
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The Regents Of The University Of Colorado, A Body Corporate
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01003—Propionyl-CoA carboxylase (6.4.1.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to compositions and methods for ameliorating deficits and deficiencies of propionyl-CoA carboxylase (PCC) including treating a spectrum of conditions such as propionic academia (PA), PA-related disorders, propionic aciduria, propionyl-CoA carboxylase deficiency, and/or ketotic glycinemia.
  • PCC propionyl-CoA carboxylase
  • Propionyl-CoA carboxylase is a complex mitochondrial matrix protein that catalyzes the conversion of propionyl-CoA to D-methylmalonyl-CoA in the mitochondrial matrix.
  • PCC is composed of nonidentical subunits, alpha ( ⁇ ) and beta ( ⁇ ).
  • Human PCC is an ⁇ 6 ⁇ 6 heterododecamer (PCCAB) that is about 800 kDa (See, Chloupkova et al., 2000 Mol Genet Metab.71:623-32, which is herein incorporated by reference in its entirety).
  • the 72 kDa ⁇ subunit and the 56 kDa ⁇ subunit are encoded by separate genes designated, PCCA, found on chromosome 13 (see, Lamhonwah et al., 1986 Proc. Nat. Acad. Sci.83:4864-8, which is herein incorporated by reference in its entirety), and PCCB, found on chromosome 3 (see, Kraus et al., 1986 Proc. Nat. Acad.
  • N-terminal leader sequences are proteolytically removed, and the mature enzyme is assembled.
  • the ⁇ -subunits form a central core hexameric core decorated on the outside by six non-interacting ⁇ - subunits.
  • Biotin, bicarbonate, and ATP have binding sites on the ⁇ -subunit while propionyl CoA binds to the ⁇ -subunit.
  • the crystal structure of a 780 kDa ⁇ 6 ⁇ 6 dodecamer of bacterial propionyl-CoA carboxylase has been determined to provide the three-dimensional structure of the enzyme. See, Huang et al., Nature.2010 Aug 19;466(7309):1001-5. doi:
  • PCCAB Human mature dodecamer
  • E. coli may be expressed in E. coli from a single plasmid.
  • E. coli covalently attaches the PCC cofactor biotin to produce a fully functional enzyme.
  • the molecular chaperone, GroES/EL is often co-expressed from a second plasmid to encourage proper PCCAB folding and assembly.
  • the alpha ( ⁇ ) subunit contains the sequence that accepts biotin (see, Kalousek et al., 1980 Journal of Biological Chemistry.255:60-5; Lamhonwah et al., 1987 Archives of Biochemistry & Biophysics.254:631-6; Leon-Del-Rio & Gravel 1994 Journal of Biological Chemistry.269:22964-8, which are herein incorporated by reference in their entireties) and binds CO 2 , Mg 2+ , ATP, and K + , which provide a means of regulation (see, Kalousek et al., 1980 Journal of Biological Chemistry.255:60-5).
  • the ⁇ subunit binds propionyl-CoA (Fenton et al., 2001 The Online Metabolic and Molecular Bases of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D., eds) pp.2165-2204, McGraw-Hill, Inc., New York, which is herein incorporated by reference in its entirety). Mutations in either gene may result in PCC deficiency to cause propionic acidemia (PA). To date, 81 and 86 mutations have been identified in the PCCA and PCCB genes, respectively, from PA patients.
  • PA propionic acidemia
  • Neonatal-onset PA the most common form of PA, is characterized by poor feeding, vomiting, and somnolence in the early days of life in a previously healthy infant, followed by lethargy, seizures, coma, and death.
  • Late-onset PA causes developmental regression, chronic vomiting, protein intolerance, failure to thrive, hypotonia, and occasionally basal ganglia infarction (resulting in dystonia and choreoathetosis) and cardiomyopathy (see, Shchelochkov et al., 2012 May 17 [Updated 2016 Oct 6] GeneReviews ® [Internet] (Pagon RA, Adam MP, Ardinger HH, et al., editors. Seattle (WA): University of Washington, Seattle; 1993-2017., which is herein incorporated by reference in its entirety).
  • PA has been estimated to be in the range of 1:35,000-1:70,000, which is similar to the incidence of methylmalonic acidemia (see, Saudubray et al., 1989 J Inherit Metab Dis.12:25-41; Chace et al., 2001 Clinical Chemistry 47:2040-44, which are herein incorporated by reference in their entireties). PA also results from a decrease in PCC activity from a lack of co-enzymes such as biotin. The incidence of PA carriers is about 5% in the Inuit population of Greenland, which is much higher than the incidence of most other autosomal recessive diseases (Ravn et al., 2000 Am J Hum Genet.67:203-6, which is hereby incorporated by reference in its entirety).
  • PA is a potentially life-threatening disease.
  • Enzyme replacement therapy (ERT) is a therapeutic approach in which the deficient enzyme is replaced by recombinant active protein. ERT would represent a major improvement in treatment of patients if the enzyme or its subunits could be imported into the mitochondrial matrix.
  • the invention provides compositions and methods for ameliorating deficits and deficiencies of propionyl-CoA carboxylase (PCC) including treating a spectrum of conditions such as propionic academia (PA), PA-related disorders, propionic aciduria, PCC deficiency, and/or ketotic glycinemia.
  • PCC propionyl-CoA carboxylase
  • the pharmaceutical compositions comprise one or more proteins or protein subunits which recapitulate the function of the PCC enzyme.
  • Such enzyme subunits include PCCA and/or PCCB.
  • the PCCA protein and/or PCCB protein comprises a mitochondrial leader sequence.
  • the PCCA protein and/or PCCB protein lack a mitochondrial leader sequence.
  • the PCCA protein and/or PCCB proteins are genetically engineered proteins or variants thereof.
  • the PCCA protein and/or PCCB protein is covalently linked to one or a plurality of cell penetrating peptides.
  • a cell penetration peptide is trans-activating transcriptional activator (TAT) or a tissue specific variant thereof.
  • the cell-penetrating peptide is chemically added post- translation to the assembled dodecamer.
  • the PCCA and/or PCCB proteins are produced recombinantly. As such, the PCCA and/or PCCB proteins may be produced in prokaryotic or eukaryotic cells, more specifically yeast, mammalian, or E. coli.
  • compositions comprising an isolated human PCCAB dodecamer having a post-translational modification comprising a cell penetrating peptide or mitochondria penetrating peptide.
  • the PCCAB dodecamer does not include a purification tag.
  • the PCCAB dodecamer comprises a PCCA subunit having the amino acid sequence of SEQ ID NO:41, or a fragment or a variant thereof sharing a sequence similarity of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% to SEQ ID NO:41.
  • the PCCAB dodecamer comprises a PCCA subunit having the amino acid sequence of SEQ ID NO:43, or a fragment or a variant thereof sharing a sequence similarity of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% to SEQ ID NO:43.
  • a nucleic acid sequence encoding the PCCAB is codon optimized for recombinant cell expression.
  • a nucleic acid sequence encoding the PCCA subunit is SEQ ID NO:40, or a fragment or a variant thereof sharing a sequence similarity of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% to SEQ ID NO:40.
  • a nucleic acid sequence encoding the PCCB subunit is SEQ ID NO:42, or a fragment or a variant thereof sharing a sequence similarity of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% to SEQ ID NO:42.
  • compositions for treating PCC-deficiency comprising a therapeutically effective amount of an isolated human PCC enzyme or a variant thereof, the PCC enzyme or the variant therefore comprising at least one member selected from the group consisting of an isolated propionyl-CoA carboxylase alpha chain protein (PCCA) subunit, and an isolated propionyl-CoA carboxylase beta chain protein (PCCB) subunit, each independently or in combination with a pharmaceutically acceptable carrier, diluent or excipient.
  • PCCA propionyl-CoA carboxylase alpha chain protein
  • PCCB isolated propionyl-CoA carboxylase beta chain protein
  • the PCCA subunit comprises the amino acid sequence of SEQ ID NO:2 or a fragment thereof. In certain embodiments, the PCCB subunit comprises the amino acid sequence of SEQ ID NO:4 or a fragment thereof. In certain embodiments, the PCCA subunit comprises a mitochondrial leader sequence. Alternatively, the PCCA subunit does not comprise a mitochondrial leader sequence. In certain embodiments, the PCCB subunit comprises a mitochondrial leader sequence. Alternatively, the PCCB subunit does not comprise a mitochondrial leader sequence. In certain embodiments, the PCCA subunit comprises at least one selected from the group consisting of SEQ ID NOs:48, 50, and 52.
  • the PCCB subunit comprises at least one sequence selected from SEQ ID NOs:49 and 51.
  • the PCCA subunit and/or the PCCB subunit comprises at least one mutation in a region of the protein relative to naturally occurring PCCA or PCCB to facilitate penetration of the PCCA subunit and/or the PCCB subunit into mitochondria.
  • the PCCA subunit is covalently linked to one or a plurality of cell penetrating peptides or mitochondria penetrating peptides.
  • the PCCB subunit is covalently linked to one or a plurality of cell penetrating peptides or mitochondria penetrating peptides.
  • the PCCA and the PCCB both are covalently linked to one or a plurality of cell penetrating peptides or mitochondria penetrating peptides.
  • the cell penetrating peptide or mitochondria penetrating peptide is derived from a protein selected from the group consisting of a trans-activating transcriptional activator (TAT), mitochondria penetrating proteins (MPP), antennapedia, herpes simplex virus type 1 protein VP22, penetratin, transportan, amphipathic protein MPG, Pep-1, MAP, SAP, PPTG1, poly-Arginine sequence, hCT, SynB, Pvec, and a tissue specific variant thereof.
  • TAT trans-activating transcriptional activator
  • MPP mitochondria penetrating proteins
  • antennapedia herpes simplex virus type 1 protein VP22
  • penetratin transportan
  • amphipathic protein MPG Pep-1
  • MAP MAP
  • SAP PPTG1
  • poly-Arginine sequence hCT
  • SynB SynB
  • Pvec a tissue specific variant thereof.
  • the PCCA subunit is produced recombinantly.
  • the PCCB subunit is produced
  • the PCCA subunit and/or the PCCB subunit are produced in prokaryotic cells.
  • the PCCA subunit and/or the PCCB subunit are produced in eukaryotic cells.
  • the prokaryotic cells are E. coli.
  • the eukaryotic cells are yeast or mammalian cells.
  • the pharmaceutical composition further comprises a His-tag at the C-terminal end and/or the N-terminal end of the PCCA subunit or the PCCB subunit.
  • the mitochondria penetrating peptide is an MPP1A or MPP2A.
  • the cell penetrating peptide is less the 100 amino acids, less than 50 amino acids, less than 30 amino acids, and less than 10 amino acids in length.
  • the pharmaceutical composition further comprises at least one
  • Various embodiments of the invention herein provide a method for reducing propionyl-CoA levels in a PCC deficient subject comprising administering a pharmaceutical composition described herein.
  • the pharmaceutical composition is administered by intravenous injection (IV), subcutaneous injection (SC), or intraperitoneal injection (IP).
  • IV intravenous injection
  • SC subcutaneous injection
  • IP intraperitoneal injection
  • the pharmaceutical composition is administered by
  • IP intraperitoneal injection
  • Various embodiments of the invention herein provide a method for elevating propionyl-CoA carboxylase, in a subject in need thereof comprising administering to said individual a pharmaceutical composition described herein.
  • Various embodiments of the invention herein provide a method for treating or ameliorating a disease, disorder, or condition in a subject, the disease, disorder, or condition being associated with elevated propionyl CoA, propionic acid, methylcitrate, beta-hydroxy- propionate, propionylglycine, tiglic acid, and/or ketones comprising administering to an individual in need thereof a pharmaceutically effective amount of a pharmaceutical composition described herein.
  • the subject presents with at least one symptom selected from the group consisting of poor feeding, vomiting, and somnolence, lethargy, seizures, coma, metabolic acidosis, anion gap, ketonuria, hypoglycemia, hyperammonemia, cytopenias, developmental regression, chronic vomiting, protein intolerance, failure to thrive, hypotonia, basal ganglia infarction, dystonia, choreoathetosis, and cardiomyopathy.
  • administering of the pharmaceutical composition occurs in a concentration of at least 0.5 ⁇ M, at least 1 ⁇ M, at least 2 ⁇ M, at least 5 ⁇ M, and at least 10 ⁇ M.
  • administering occurs at least once a day, at least twice a day, a least three times a day, and at least 4 times a day. In certain embodiments, administering is repeated for at least 2 days, at least 3 days, at least 4 days, at least 5 days, and at least 6 days.
  • Various embodiments of the invention herein provide a method of producing the PCCA subunit or the PCCB subunit described herein comprising recombinantly producing a PCCA precursor or a PCCB precursor including a cell penetrating peptide or mitochondria penetrating peptide.
  • the PCCA precursor is encoded by the nucleic acid sequence of SEQ ID NO:1.
  • the PCCB precursor is encoded by the nucleic acid sequence of SEQ ID NO:3.
  • Various embodiments of the invention herein provide a method of producing the pharmaceutical composition disclosed herein, the method comprising: transfecting cells with an expression vector encoding the PCCAB and at least one molecular chaperone to mediate formation of a PCCAB dodecamer, and conjugating a cell penetrating peptide or
  • the molecular chaperone is a GroEL or a GroES protein.
  • the PCCAB dodecamer comprises a PCCA subunit and a PCCB subunit.
  • the expression vector comprises a nucleic acid sequence of SEQ ID NO:40, or a fragment or a variant thereof sharing a sequence similarity of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% to SEQ ID NO:40 encoding the PCCA subunit.
  • the expression vector comprises a nucleic acid sequence of SEQ ID NO:42, or a fragment or a variant thereof sharing a sequence similarity of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% to SEQ ID NO:42 encoding the PCCB subunit.
  • the method further comprises formulating the pharmaceutical composition with at least one of a pharmaceutically acceptable salt or excipient.
  • the PCCA subunit comprises an amino acid sequence beginning at residue 52 of SEQ ID NO:2.
  • the PCCB subunit comprises an amino acid sequence beginning at residue 29 or SEQ ID NO:4.
  • Figure 1 shows non-limiting examples of PCC protein expression constructs. Further a C-terminal or N-terminal His-tag may be conjugated to the expression construct as shown for certain constructs in this figure.
  • Figure 2 shows images of a fluorescence immunostaining of MPP2A-PCCAB import into human PCCA deficient fibroblasts.
  • Panel A shows a merged image of panels B (nuclei), C (PCC), and D (mitochondria).
  • panel C medium gray
  • D mitochondria
  • PCC and mitochondrial stains co-localize, indicating import of PCC into the mitochondria of the patient fibroblast.
  • Figure 3 is a histogram plot showing PCC activity after import of a TAT or MPP2A conjugated PCCAB construct into isolated mouse mutant mitochondria.
  • Figure 4 shows a histogram plot of dose response of PCC activity across patient cell lines.
  • Figure 5 shows a graph of plasma C3/C2 ratios after in vivo IP administration of a conjugate of the invention.
  • Figure 6 shows a line graph of PCC activity in mouse plasma over time after in vivo IP administration of TAT-PCCAB conjugate of the invention.
  • Figure 7 shows a histogram plot of PCC activity in heart and liver after in vivo IP administration of a conjugate of the invention.
  • Figure 8 shows a line graph of C3/C2 ratios after 20 mg/kg TATPCCB injection IP in A138T mice.
  • Figure 9 shows oxygen consumption of isolated mitochondria during TAT-PCC conjugate import monitored over a 1.5-hour period. Substrate was added after oxygen was consumed at time points 37, 68, and 86 minutes.
  • Figure 10 shows a time course of PCC activity (0-20 min) demonstrated the susceptibility of PCC to trypsin.
  • the first group of samples (T0, T5, T10, and T20) were proteolyzed in the presence of a trypsin inhibitor.
  • trypsin was added to the PCC enzyme and the trypsin inhibitor was used to stop the reaction in 0-20 minutes (R0, R5, R10, and R20).
  • One unit of PCC activity is defined as pmol/min/mg protein.
  • Figure 11 shows a Western blot of PCC in mitochondrial lysates after import of increasing amounts of TAT-PCC into isolated mutant A138T PCCA mouse mitochondria and trypsin treatment for 25 minutes.
  • Lane M is the molecular weight marker Precision Plus Protein standards (Bio-Rad, Hercules, CA).
  • Lane E is 100ng of PCC enzyme; 1. WT mitochondria; 2. mutant mitochondria; 3. Import of 1 ⁇ M TAT-PCC; 4. Import of 5 ⁇ M TAT- PCC; and 5. Import of 10 ⁇ M TAT-PCC.
  • Figure 12 shows a graph of PCC activity in patient fibroblasts #3380 and #3383 having imported MPP1A-PCCAB, MPP2A-PCCAB, and TAT-PCCAB.
  • Figure 13 shows PCCA deficient cells grown on microscopy slides. Import of 1 ⁇ M TAT-PCCAB into the fibroblasts was performed for 1 hour at 37°C. After import, live cells were stained as follows: DAPI blue fluorescent dye (dark gray) was used to stain the nuclei in the far-left panel and MitoTracker CMXRos in the second from right panel.
  • compositions and methods of treatment for treating patients with PA or other PCC-deficiency related conditions for example, enzyme therapy.
  • PA also referred to as: propionyl-CoA carboxylase deficiency, PCC deficiency, ketotic glycinemia, hyperglycinemia with ketoacidosis and leukopenia, or ketotic hyperglycinemia
  • certain embodiments of the present invention provide a method for treating or ameliorating a disease, disorder, or condition in a subject, the disease, disorder, or condition being associated with elevation of at least one selected from the group of propionyl CoA, propionic acid, methylcitrate, beta-hydroxy-propionate, propionylglycine, tiglylglycine, and ketones, the method including a step of administering to the subject a pharmaceutically effective amount of a composition comprising PCC proteins.
  • compositions as administered to a patient by methods herein reduce or alleviate at least one symptom or clinical manifestation of the disease, eliminate the disease, alleviate secondary diseases resulting from the occurrence of the primary disease, and prevent incidence of the disease.
  • PCC is a biotin-dependent, mitochondrial matrix enzyme involved in organic acid metabolism in humans.
  • PA propionic acidemia
  • Embodiments of the invention address challenges of treatment of PA and other PCC-deficiency related conditions.
  • the resulting pharmaceutical compositions and methods of the invention exploit cell-penetrating proteins, such as TAT, to import assembled PCC or individual PCC subunits into cells, particularly mitochondria, to correct the propionyl-CoA carboxylase enzyme deficiency.
  • PCC makes up a multimeric mitochondrial biotin-dependent enzyme.
  • Human PCC enzyme is an D6E6 heterododecamer having a molecular weight of about 800 kDa.
  • the 72 kDa PCC-alpha subunit and the 56 kDa PCC-beta subunit are encoded by genes designated, PCCA and PCCB.
  • the D subunit contains biotin carboxylase and biotin carboxyl carrier protein domains.
  • the E subunit is responsible for carboxyltransferase activity of the enzyme.
  • the PCC protein includes, but is not limited to, purified PCCA and PCCB proteins, chemically cleaved and recombinantly produced PCCA and PCCB proteins, and isolated PCCA and PCCB proteins associated with other proteins or peptides.
  • an isolated human PCC peptide is a protein or peptide removed from its natural milieu (i.e., subject to human manipulation) and is combined with, for example, purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins.
  • the term "isolated” does not, in some cases, reflect the extent to which the protein has been purified.
  • compositions and conjugates may be designed and/or engineered.
  • Such starting sequences include the DNA sequence for propionyl Coenzyme A carboxylase alpha ( ⁇ ) subunit protein (PCCA), designated SEQ ID NO:1, and its amino acid sequence for full-length human PCCA, having 702 amino acid residues, designated SEQ ID NO:2.
  • the DNA sequence for propionyl Coenzyme A carboxylase beta subunit protein (PCCB) is designated SEQ ID NO:3
  • an its amino acid sequence for full-length human PCCB having 539 amino acid residues, is designated SEQ ID NO:4.
  • the invention provides a composition of matter comprising one or both of an isolated propionyl-CoA carboxylase alpha chain protein (PCCA) comprising the amino acid sequence of SEQ ID NO:2, and/or an isolated propionyl- CoA carboxylase beta chain protein (PCCB) comprising the amino acid sequence of SEQ ID NO:4 or functional fragments thereof.
  • PCCA propionyl-CoA carboxylase alpha chain protein
  • PCCB isolated propionyl- CoA carboxylase beta chain protein
  • Such functional fragments may represent the mature protein as well as any portion thereof.
  • Various embodiments provide PCC proteins or subunits conjugated to a cell penetrating peptide.
  • Cell-penetrating peptides and mitochondria penetrating peptides are short peptides (typically less than 30 amino acids) that facilitate cellular uptake of various molecules.
  • Cell penetrating and mitochondria penetrating peptides are tools for non-invasive cellular import of cargo and have been successfully applied for in vitro and in vivo delivery of therapeutic molecules, e.g., small chemical molecules, nucleic acids, proteins, peptides, liposomes, and particles.
  • ERT for mitochondrial enzymes requires transport of the cargo through the plasma membrane as well as through the outer and inner mitochondrial membranes.
  • delivery of protein is limited by their ability to penetrate the cell membrane.
  • a cell penetrating and mitochondria penetrating peptide can be linked to a molecule through covalent bonds or non-covalent bonds, and are coupled to the PCC peptides using standard methods of bioconjugation.
  • Mitochondria is made up of two membrane system. Whereas the mitochondrial outer membrane is similar to the plasma membrane in terms of protein to lipid constitution (1:1), there are no proteoglycans present on the surface of mitochondria although the phospholipid, cardiolipin, imparts a net negative charge to the membrane.
  • the inner mitochondrial membrane displays a higher protein to lipid ratio (3:1) compared to the plasma and outer mitochondria membranes (see, Gohil et al., J. Cell Biol. Vol.184 No.4469–472, 2009, which is herein incorporated by reference in its entirety).
  • mitochondrial proteins Apart of from proteins encoded by the mitochondrial genome most mitochondrial proteins need to be delivered into this organelle following their translation on cytoplasmic ribosomes. They are then transported with the help of translocases and chaperones.
  • the mitochondrial targeting sequence (MTS) is recognized by a receptor in the translocase of the outer membrane. After a protein arrives in the mitochondrial matrix, a protease removes its N-terminal matrix-targeting sequence.
  • HIV-1 trans-activating transcriptional activator (TAT) domain and its variants are used most frequently used for many different types of cargo. See, Frankel et al., Cell.1988 Dec 23;55(6):1189-93, which is herein incorporated by reference in its entirety.
  • the minimal peptide sequence of TAT protein responsible for cellular uptake is YGRKKRRQRRR (SEQ ID NO:5), which contains six arginine and two lysine residues and therefore possesses a high net positive charge at physiological pH levels.
  • the TAT domain has been used to deliver lipoamide dehydrogenase (LAD) to mitochondria in fibroblasts from patients suffering from LAD deficiency.
  • LAD lipoamide dehydrogenase
  • LAD is the third catalytic subunit (E3) of three multicomponent enzymatic in the mitochondrial matrix.
  • C6orf66 assembly factor that restores Complex I activity in patient cells was also successfully replaced. This demonstrated a possibility for repair of multicomponent complex proteins using a TAT fusion protein strategy (see, Marcus et al., Mol Med.2013; 19(1): 124–134, which is herein incorporated by reference in its entirety).
  • TAT-frataxin has also been announced as a drug candidate to increase lifespan and cardiac function in a conditional Friedreich's ataxia mouse model.
  • compositions herein include purified PCCAB conjugated to a cell penetrating peptide, such as TAT, or mitochondria-penetrating peptide (MPP) by maleimide- mediated thioether bond formation with available cysteine residue(s) of the PCCAB.
  • a cell penetrating peptide such as TAT
  • MPP mitochondria-penetrating peptide
  • a cell penetrating peptide is conjugated to PCCAB by amide bond formation between an NHS ester and lysine residues.
  • the TAT peptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO:5).
  • the TAT peptide has the amino acid sequence GRKKRRQRRRPQ (SEQ ID NO:6) or a fragment thereof.
  • the TAT peptide comprises the amino sequence
  • the TAT peptide comprises the amino acid sequence Maleoyl-beta- AGYGRKKRRQRRR (SEQ ID NO:21) or a fragment thereof, or the amino acid sequence GYGRKKRRQRRR (SEQ ID NO: 22) or a fragment thereof.
  • Additional examples of cell penetrating peptides known in the art include:
  • homeodomain transcription factors such as Antennapedia (RQIKIYFQNRRMKWKK, SEQ ID NO:7), herpes simplex virus type 1 protein VP22
  • GWTLNSAGYLLGKINLKALAALAKK IL SEQ ID NO:10
  • amphipathic proteins such as MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV,SEQ ID NO:11), Pep-1
  • GLFRALLRLLRSLWRLLLRA SEQ ID NO:15
  • peptides such as poly-Arginine sequences (e.g., RRRRRRRR, SEQ ID NO:16), hCT (LGTYTQDFNKTFPQTAIGVG AP, SEQ ID NO:17), SynB (RGGRLSYSRRRFSTSTGR, SEQ ID NO:18), and Pvec
  • LLC LLCILRRRIRKQAHAHSK, SEQ ID NO:19.
  • Cell penetrating proteins are discussed, for example, in: Fang et al., 2013 PLOS ONE 8(3):e57318; Ruoslahti et al., 2009 J Cell Biology 188(6):759-68; Foged & Nielsen, 2008 Expert Opin. Drug Deliv.5(1):105-17; and Treat et al., 2012 ACS Macro Lett.1(1):100-04, which are herein incorporated by reference in their entireties.
  • the MPP1A peptide comprises the amino acid sequence Cha-DArg-Cha-Lys-Cha-DArg-Cha-Lys (Cha-R-Cha-K-Cha-R-Cha-K) (SEQ ID NO:23).
  • the MPP2A peptide comprises the amino acid sequence Cha-DArg- Cha-Lys (Cha-R-Cha-K) (SEQ ID NO:24).
  • the MPP1A peptide consists essentially of the amino acid sequence Cha-DArg-Cha-Lys-Cha-DArg-Cha-Lys (Cha-R-Cha-K-Cha-R-Cha-K) (SEQ ID NO:23).
  • the MPP2A peptide consists essentially of the amino acid sequence Cha-DArg-Cha-Lys (Cha-R-Cha-K) (SEQ ID NO:24).
  • the MPP1A peptide is the amino acid sequence Cha- DArg-Cha-Lys-Cha-DArg-Cha-Lys (Cha-R-Cha-K-Cha-R-Cha-K) (SEQ ID NO:23).
  • the MPP2A peptide is the amino acid sequence Cha-DArg-Cha-Lys (Cha-R-Cha-K) (SEQ ID NO:24).
  • PCC derivatives are within the scope of the present invention.
  • PCC derivatives or variants include, but are not limited to, genetically engineered modifications including nucleic acid and/or amino acid modifications or chemical modifications.
  • modifications that mask potential immunogenic epitopes on the surface of a protein and/or hinder access to the protein for proteolytic enzymes are of interest.
  • modifications of interest are those that advantageously alter the physio-chemical properties of the PCC peptide, thus modifying its biodistribution, stability, and solubility without significantly detracting from its potency.
  • Such derivatives may be chemically modified PCC protein compositions in which PCC protein is linked to a polymer.
  • the polymer selected is typically water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as the physiological environment.
  • the polymer may be of any molecular weight, and may be branched or unbranched. Included within the scope of PCC protein polymers is a mixture of polymers. In specific embodiments, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.
  • cell penetrating peptides include homeodomain transcription factors such as Antennapedia, VP22, TAT, penetratin, and transportan; amphipathic molecules such as MPG, Pep-1, MAP, SAP and PPTG1; poly-Arginine sequences; hCT; SynB; and Pvec.
  • homeodomain transcription factors such as Antennapedia, VP22, TAT, penetratin, and transportan
  • amphipathic molecules such as MPG, Pep-1, MAP, SAP and PPTG1
  • poly-Arginine sequences hCT
  • SynB SynB
  • Pvec cell penetrating peptides
  • these ligands are antibodies, antigens, receptors and receptor ligands.
  • Manipulating the chemical formula of the lipid portion of the delivery vehicle can modulate the extracellular or intracellular targeting of the delivery vehicle.
  • liposomes of the present invention include those liposomes commonly used in, for example, protein delivery methods known to those of skill in the art.
  • a liposome with a protein of the examples herein provide nucleic acid and/or amino acid modifications or PCC chemical modifications that mask potential immunogenic epitopes on the surface of a protein and/or hinder access to the protein for proteolytic enzymes. Additional modifications analyzed herein include those that advantageously alter the physio-chemical properties of PCC, to modify biodistribution, stability, and solubility without significantly detracting from its potency. Further examples analyze the effect of chemical modifications to deliver the proteins to cells, and optimization of the TAT peptide sequence or other cell-penetrating peptides.
  • the amino acid sequence of the mitochondrial targeting leader corresponds to the first 51 amino acids of a full-length PCCA subunit (of SEQ ID NO:2) and the first 28 amino acids of a full-length PCCB subunit (of SEQ ID NO:4).
  • Both mature subunits may be modified covalently with TAT or mitochondrial targeting peptide.
  • the subunits including the leader sequences may then be expressed with TAT preceding the leader.
  • the PCCA protein and/or PCCB protein comprises a
  • the PCCA protein and/or PCCB protein lack a mitochondrial leader sequence.
  • the PCCA protein and/or PCCB proteins are genetically engineered proteins or variants thereof.
  • the PCCA protein and/or PCCB protein is covalently linked to one or a plurality of cell penetrating proteins.
  • a“cell penetrating protein” or“cell penetrating peptide” is an amino-acid based polypeptide which facilitates or fosters the transport of a biomolecule across any cell membrane.
  • a non-limiting example of such a cell penetration protein is trans-activating transcriptional activator (TAT) or a tissue specific variant thereof.
  • TAT transcriptional activator
  • the cell-penetrating protein is chemically added post-translation of the PCCA or PCCB peptide.
  • a PCC protein or conjugate of the present invention comprises an amino acid sequence that is less than 100% identical to SEQ ID NO:2 and/or SEQ ID NO:4, and in specific embodiments having 75% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 91% sequence identity, 92% sequence identity, 93% sequence identity, 94% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity, to SEQ ID NO:2 and/or SEQ ID NO:4.
  • the PCC protein derivative will have a single cell-penetrating or mitochondria-penetrating molecule at the amino terminus.
  • the PCC protein enzyme conjugated to the cell-penetrating or the mitochondria penetrating molecule provided by an embodiment has an average of about 1 to about 10, more particularly 2 to 5 and more particularly 3 to 5 cell-penetrating or mitochondria-penetrating molecules covalently attached to each PCC enzyme subunit in the composition.
  • the promoter sequence may be genetically engineered to improve the level of expression as compared to the native promoter.
  • Recombinant techniques useful for controlling the expression of nucleic acid molecules include, but are not limited to, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules to correspond to the codon usage of the host cell, and deletion of sequences that destabilize transcripts.
  • transcription control signals e.g., promoters, operators, enhancers
  • substitutions or modifications of translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • the PCCA and/or PCCB proteins are produced recombinantly.
  • the PCCA and/or PCCB proteins may be produced in prokaryotic or eukaryotic cells, more specifically yeast, mammalian, or E. coli cells.
  • Chromatographic separation comprises an ion exchange chromatography column for purification.
  • the ion exchange chromatography column is an anion exchanger.
  • anion exchange resins can be used, DEAE-cellulose, DEAE- cellulose DE 52, and DEAE-Sepharose-FF.
  • the anion exchange resin is DEAE-Sepharose-FF.
  • Additional chromatographic steps provided in certain embodiments of the methods of this invention for purifying PCC from a PCC-containing solution include use of a monomeric avidin column. Avidin columns are useful for non-denaturing affinity purification of biotinylated molecules.
  • Chromatography matrices useful in the method of the invention are materials capable of binding biochemical compounds, preferably proteins, nucleic acids, and/or endotoxins, wherein the affinity of said biochemical compounds to said chromatography matrix is influenced by the ion composition of the surrounding solution (buffer).
  • the method for purification comprises the step of homogenizing host cells, particularly recombinant cells and in certain embodiments, recombinant cells producing mammalian, preferably human, PCC proteins, wherein said recombinant construct encodes a PCC protein that is a naturally occurring or a genetically engineered variant thereof, and particularly wherein said construct has been optimized for recombinant cell expression.
  • said recombinant cells are prokaryotic cells, particularly bacterial cells or eukaryotic cells, particularly yeast or mammalian cells.
  • the bacterial cells are E.
  • coli cells and the PCC sequence has been engineered in the recombinant expression construct to be optimized for expression in said cells; a specific embodiment of such a nucleic acid sequence optimized for PCC expression in E. coli is set forth in the plasmid pPCCAB of the examples, which is also described in Kelson et al., 1996 Human Molecular Genetics.5:331-37.
  • cells are harvested, e.g. by centrifugation, and optionally stored at -80 ⁇ C.
  • Homogenization of host cells is performed by disrupting the cells using physical, chemical, or enzymatic means or by a combination thereof.
  • homogenization is performed by disrupting the cell wall of said bacterial host by sonication.
  • homogenizing is performed by destabilizing the bacterial cell wall of the host by exposure to a cell wall degrading enzyme such as lysozyme.
  • the methods of the invention can further comprise a clarified PCC homogenate, wherein cell debris is removed from the homogenate by either filtration or centrifugation.
  • clarifying is performed by centrifuging the homogenate at an effective rotational speed.
  • the centrifugation time depends inter alia on the volume of the homogenate, which is determined empirically to obtain a sufficiently solid pellet.
  • a combination of centrifugation and filtration can be performed on the homogenate.
  • Methods to measure protein expression levels of the PCC protein according to the invention include, but are not limited to Coomasie blue or silver staining of protein in a separation media, such as gel electrophoresis, Western blotting, immunocytochemistry, other immunologic-based assays; and assays based on a property of the protein including but not limited to, enzyme assays, ligand binding or interaction with other protein partners.
  • Various embodiments of the invention provide a method of correcting a PCC- deficiency related disease or condition in a cell including the steps of contacting the cell with a preparation of isolated human PCC at a concentration sufficient for the cell to take up a therapeutically effective amount of PCC, such that the preparation contains at least one selected from the group of an isolated propionyl-CoA carboxylase PCCA protein comprising a functional portion or variant of the amino acid sequence of SEQ ID NO:2 or an isolated propionyl-CoA carboxylase PCCB protein comprising a functional portion or variant of the amino acid sequence of SEQ ID NO:4.
  • compositions are superior to those in the art as they traffic efficiently to the mitochondria and have sufficient stability and duration of action to effect therapeutically relevant outcomes.
  • the present invention shows by confocal microscopy importation and colocalization of immunofluorescently labeled TAT-PCCAB with a mitochondria-specific marker, MITOTRACKER®.
  • the invention provides a method for treating PCC deficiency in an individual in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition of isolated human PCC to the individual in need thereof, wherein the isolated human PCC comprises one or both of an isolated propionyl-CoA carboxylase alpha chain protein (PCCA) conjugate comprising the amino acid sequence of SEQ ID NO:2 or functional fragment thereof, and/or an isolated propionyl-CoA carboxylase beta chain protein (PCCB) conjugate comprising the amino acid sequence of SEQ ID NO:4 or functional fragment thereof.
  • PCCA propionyl-CoA carboxylase alpha chain protein
  • PCCB isolated propionyl-CoA carboxylase beta chain protein
  • the invention provides pharmaceutical composition comprising a therapeutically effective amount of isolated human PCC wherein the PCC comprises one or both of an isolated propionyl-CoA carboxylase alpha chain protein (PCCA) comprising the amino acid sequence of SEQ ID NO:2 or functional fragment thereof, and/or an isolated propionyl-CoA carboxylase beta chain protein (PCCB) comprising the amino acid sequence of SEQ ID NO:4 or functional fragment thereof, and a pharmaceutically acceptable carrier, diluent or excipient.
  • PCCA propionyl-CoA carboxylase alpha chain protein
  • PCCB isolated propionyl-CoA carboxylase beta chain protein
  • the pharmaceutical composition is administered by intravenous injection (IV), subcutaneous injection (SQ), or intraperitoneal injection (IP).
  • IV intravenous injection
  • SQ subcutaneous injection
  • IP intraperitoneal injection
  • the pharmaceutical composition may comprise an amount of PCC protein wherein 0.01 mg/kg– 20 mg/kg is administered to an individual in need thereof.
  • the dose may be administered as a single daily dose, a weekly dose, a monthly dose, a yearly dose.
  • the dose is administered as a split dose whereby a total daily dose is divided into equal or unequal amounts and administered over the course of the same day.
  • the dose is 0.1 mg/kg to 0.5 mg/kg; 0.1 mg/kg to 2 mg/kg; about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 7 mg/kg; from 2-10 mg/kg; from 3-15 mg/kg; more than 10 mg/kg, more than 20 mg/kg.
  • the invention provides a method for treating or ameliorating a disease, disorder, or condition, associated with elevated propionyl CoA, propionic acid, methylcitrate, beta-hydroxy-propionate, propionylglycine, tiglic acid, and ketones comprising administering to an individual in need thereof a pharmaceutically effective amount of a pharmaceutical composition of PCC.
  • the disease, disorder, or condition associated with elevated propionyl CoA, propionic acid, methylcitrate, beta-hydroxy-propionate, propionylglycine, tiglic acid, and ketones is poor feeding, vomiting, and somnolence, lethargy, seizures, coma, metabolic acidosis, anion gap, ketonuria, hypoglycemia, hyperammonemia, cytopenias, developmental regression, chronic vomiting, protein intolerance, failure to thrive, hypotonia, basal ganglia infarction, dystonia, choreoathetosis, and cardiomyopathy.
  • the pharmaceutical composition is administered by intravenous injection, subcutaneous injection, or intraperitoneal injection.
  • the invention provides a method for treating or ameliorating a disease, disorder, or condition, associated with elevated propionyl CoA, propionic acid, methylcitrate, beta-hydroxy-propionate, propionylglycine, tiglylglycine, and ketones comprising administering to an individual in need thereof a pharmaceutically effective amount of a pharmaceutical composition of PCC.
  • the disease, disorder, or condition associated with elevated propionyl CoA, propionic acid, methylcitrate, beta-hydroxy-propionate, propionylglycine, tiglylglycine, and ketones is poor feeding, vomiting, and somnolence, lethargy, seizures, coma, metabolic acidosis, anion gap, ketonuria, hypoglycemia, hyperammonemia, cytopenias, developmental regression, chronic vomiting, protein intolerance, failure to thrive, hypotonia, basal ganglia infarction, dystonia, choreoathetosis, and cardiomyopathy.
  • compositions of the present invention may be formulated in any manner suitable for delivery.
  • the formulation may be, but is not limited to, nanoparticles, poly(lactic- co-glycolic acid)(PLGA) microspheres, lipidoids, lipoplex, liposome, polymers,
  • carbohydrates including simple sugars, cationic lipids and combinations thereof.
  • the formulation is a nanoparticle which may comprise at least one lipid.
  • the lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids.
  • the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.
  • Formulation may be in standard saline solutions or any suitable buffer.
  • Activity refers to the condition in which things are happening or being done. Compositions of the invention may have activity and this activity may involve one or more biological events.
  • Administered in combination means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.
  • Amelioration As used herein, the term “amelioration” or“ameliorating” refers to a lessening of severity of at least one indicator of a condition or disease. For example, in the context of neurodegeneration disorder, amelioration includes the reduction of neuron loss.
  • animal refers to any member of the animal kingdom. In some embodiments,“animal” refers to humans at any stage of development. In some embodiments,“animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
  • Antibody As used herein, the term "antibody” is referred to in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies formed from at least two intact antibodies), and antibody fragments (e.g., diabodies) so long as they exhibit a desired biological activity (e.g.,“functional”). Antibodies are primarily amino acid based molecules but may also comprise one or more modifications (including, but not limited to the addition of sugar moieties, fluorescent moieties, chemical tags, etc.).
  • Non-limiting examples of antibodies or fragments thereof include V H and V L domains, scFvs, Fab, Fab’, F(ab’)2, Fv fragments, diabodies, linear antibodies, single chain antibody molecules, multispecific antibodies, bispecific antibodies, intrabodies, monoclonal antibodies, polyclonal antibodies, humanized antibodies, codon-optimized antibodies, tandem scFv antibodies, bispecific T-cell engagers, mAb2 antibodies, chimeric antigen receptors (CAR), tetravalent bispecific antibodies, biosynthetic antibodies, native antibodies, miniaturized antibodies, unibodies, maxibodies, antibodies to senescent cells, antibodies to conformers, antibodies to disease specific epitopes, or antibodies to innate defense molecules.
  • V H and V L domains scFvs, Fab, Fab’, F(ab’)2, Fv fragments, diabodies, linear antibodies, single chain antibody molecules, multispecific antibodies, bispecific antibodies, intrabodies, monoclonal
  • the term“approximately” or“about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value.
  • the term“approximately” or“about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context and except where such number would exceed 100% of a possible value.
  • the terms“associated with,” “conjugated,”“linked,”“attached,” and“tethered,” when used with respect to two or more moieties means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
  • An“association” need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the “associated” entities remain physically associated. Conjugation may be via covalent linkage.
  • Bacterial cell refers to bacteria that produces a mammalian, preferably human, PCC protein inter alia using recombinant genetic methods including progeny of said recombinant cell.
  • the PCC protein is a naturally occurring or a genetically engineered variant.
  • Bifunctional refers to any substance, molecule or moiety which is capable of or maintains at least two functions.
  • the functions may affect the same outcome or a different outcome.
  • the structure that produces the function may be the same or different.
  • Biocompatible As used herein, the term“biocompatible” means compatible with living cells, tissues, organs, or systems posing little to no risk of injury, toxicity, or rejection by the immune system.
  • biologically active refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • Complementary and substantially complementary refers to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands. Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil rather than thymine is the base that is considered to be
  • complementarity or 100% complementarity refers to the situation in which each nucleotide unit of one polynucleotide strand can form hydrogen bonds with a nucleotide unit of a second polynucleotide strand.
  • Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands can form hydrogen bonds with each other. For example, for two 20-mers, if only two base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 10% complementarity. In the same example, if 18 base pairs on each strand can form hydrogen bonds with each other, the polynucleotide strands exhibit 90% complementarity.
  • Compounds of the present disclosure include all of the isotopes of the atoms occurring in the intermediate or final compounds.“Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei.
  • isotopes of hydrogen include tritium and deuterium.
  • the compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
  • conditionally active refers to a mutant or variant of a wild-type polypeptide, wherein the mutant or variant is more or less active at physiological conditions than the parent polypeptide. Further, the conditionally active polypeptide may have increased or decreased activity at aberrant conditions as compared to the parent polypeptide. A conditionally active polypeptide may be reversibly or irreversibly inactivated at normal physiological conditions or aberrant conditions.
  • conserved refers to nucleotides or amino acid residues of a polynucleotide sequence or polypeptide sequence, respectively, that are those that occur unaltered in the same position of two or more sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
  • two or more sequences are said to be“completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be“highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be“highly conserved” if they are about 70% identical, about 80% identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another.
  • two or more sequences are said to be “conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another. Conservation of sequence may apply to the entire length of a polynucleotide or polypeptide or may apply to a portion, region, or feature thereof.
  • control elements refers to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like, which provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed, and/or translated in an appropriate host cell.
  • Controlled Release refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome.
  • Delivery refers to the act or manner of delivering a particle, compound, substance, entity, moiety, cargo, or payload.
  • Delivery Agent refers to any substance which facilitates, at least in part, the in vivo delivery of compound or pharmaceutical composition to targeted cells.
  • Detectable label refers to one or more markers, signals, or moieties which are attached, incorporated, or associated with another entity that is readily detected by methods known in the art including radiography, fluorescence, chemiluminescence, enzymatic activity, absorbance, and the like.
  • Detectable labels include radioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots, and the like. Detectable labels may be located at any position in the peptides or proteins disclosed herein. They may be within the amino acids, the peptides, or proteins, and located at the N- or C- termini.
  • Digest As used herein, the term“digest” means to break apart into smaller pieces or components. When referring to polypeptides or proteins, digestion results in the production of peptides.
  • Disease refers to deviation from the normal health of a patient and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., infection, gene mutation, genetic defect, etc.) has occurred, yet symptoms are not yet manifested (e.g., a predisease condition).
  • a deviation e.g., infection, gene mutation, genetic defect, etc.
  • Dosing regimen As used herein, a“dosing regimen” is a schedule of
  • embodiments of the invention are“engineered” when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type, or native molecule.
  • an effective amount of an agent is an amount sufficient to effect beneficial or desired results, for example, clinical results, and, as such, an“effective amount” depends upon the context in which it is being applied.
  • an effective amount of an agent is, for example, an amount sufficient to achieve treatment, as defined herein, of PCC deficiency, as compared to the response obtained without administration of the agent.
  • an“epitope” refers to a surface or region on a molecule that is capable of interacting with a biomolecule.
  • a protein may contain one or more amino acids, e.g., an epitope, which interacts with an antibody, e.g., a biomolecule.
  • an epitope when referring to a protein or protein module, may comprise a linear stretch of amino acids or a three-dimensional structure formed by folded amino acid chains.
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5 ⁇ cap formation, and/or 3 ⁇ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.
  • Feature refers to a characteristic, a property, or a distinctive element.
  • a“formulation” includes at least one pharmaceutical compound or active agent and a delivery agent.
  • fragment refers to a portion.
  • fragments of proteins may comprise polypeptides obtained by digesting full-length protein isolated from cultured cells.
  • a“functional” biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • Gene expression refers to the process by which a nucleic acid sequence undergoes successful transcription and in most instances translation to produce a protein or peptide.
  • measurement of“gene expression” this should be understood to mean that measurements may be of the nucleic acid product of transcription, e.g., RNA or mRNA or of the amino acid product of translation, e.g., polypeptides or peptides. Methods of measuring the amount or levels of RNA, mRNA, polypeptides and peptides are well known in the art.
  • homology refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar to each other.
  • the term“homologous” necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences).
  • two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% for at least one stretch of at least about 20 amino acids.
  • homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4–5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4–5 uniquely specified amino acids.
  • two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical for at least one stretch of at least about 20 amino acids.
  • Heterologous region refers to a region which would not be considered a homologous region.
  • homologous region refers to a region which is similar in position, structure, evolution origin, character, form or function.
  • Identity refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); which is incorporated herein by reference in its entirety. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
  • Inhibit expression of a gene means to cause a reduction in the amount of an expression product of the gene.
  • the expression product can be an RNA transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene.
  • a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
  • in vivo refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).
  • Isolated refers to a substance or entity that has been separated from at least some of the components with which it was associated (whether in nature or in an experimental setting). Isolated substances may have varying levels of purity in reference to the substances from which they have been associated. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
  • isolated agents are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • a substance is“pure” if it is substantially free of other components.
  • Substantially isolated By“substantially isolated” is meant that a substance is substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the substance of the present disclosure. Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
  • Linker refers to a molecule or group of molecules which connects two molecules, such as a V H chain and V L chain of an antibody.
  • a linker may be a nucleic acid sequence connecting two nucleic acid sequences encoding two different polypeptides.
  • a linker may be amino acid based.
  • the linker may or may not be translated.
  • the linker may be a cleavable linker.
  • Modified refers to a changed state or structure of a molecule of the invention. Molecules may be modified in many ways including chemically, structurally, and functionally.
  • Naturally Occurring As used herein,“naturally occurring” or“wild-type” or “native” means existing in nature without artificial aid, or without involvement of the hand of man.
  • Non-human vertebrate As used herein, a“non-human vertebrate” includes all vertebrates except Homo sapiens, including wild and domesticated species. Examples of non- human vertebrates include, but are not limited to, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep water buffalo, and yak.
  • mammals such as alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep water buffalo, and yak.
  • Open reading frame As used herein,“open reading frame” or“ORF” refers to a sequence which does not contain a stop codon in a given reading frame.
  • Operably linked refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties, or the like.
  • patient refers to a subject who may seek or be in need of treatment, requires treatment, is receiving treatment, will receive treatment, or a subject who is under care by a trained professional for a particular disease or condition.
  • Peptide As used herein,“peptide” is less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • composition“pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient.
  • Excipients may include, for example: anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration.
  • anti-adherents antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration.
  • excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,
  • a pharmaceutically acceptable carrier may be a controlled release formulation that slowly releases the pharmaceutical composition into a patient or culture.
  • compositions described herein also includes pharmaceutically acceptable salts of the compounds described herein.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid).
  • examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, ole
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two;
  • nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p.1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.
  • Pharmacokinetic refers to any one or more properties of a molecule or compound as it relates to the determination of the fate of substances administered to a living organism. Pharmacokinetics is divided into several areas including the extent and rate of absorption, distribution, metabolism and excretion. This is commonly referred to as ADME where: (A) Absorption is the process of a substance entering the blood circulation; (D) Distribution is the dispersion or dissemination of substances throughout the fluids and tissues of the body; (M) Metabolism (or Biotransformation) is the irreversible transformation of parent compounds into daughter metabolites; and (E) Excretion (or Elimination) refers to the elimination of the substances from the body. In rare cases, some drugs irreversibly accumulate in body tissue.
  • Physicochemical As used herein,“physicochemical” means of or relating to a physical and/or chemical property.
  • Preventing refers to partially or completely delaying onset of an infection, disease, disorder and/or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular infection, disease, disorder, and/or condition; partially or completely delaying progression from an infection, a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the infection, the disease, disorder, and/or condition.
  • Prophylactic refers to a therapeutic or course of action used to prevent the spread of disease.
  • Prophylaxis As used herein, a“prophylaxis” refers to a measure taken to maintain health and prevent the spread of disease.
  • Protein of interest As used herein, the terms“proteins of interest” or“desired proteins” include those provided herein and fragments, mutants, variants, and alterations thereof.
  • Purified As used herein,“purify,”“purified,”“purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.“Purified” refers to the state of being pure.“Purification” refers to the process of making pure.
  • Recombinant cell refers to suitable cells (including progeny of such cells) from any species (prokaryotic or eukaryotic) into which a recombinant expression construct capable of expressing a nucleic acid encoding PCC peptide has been introduced.
  • the construct is preferably a human PCC protein or genetically engineered variant thereof.
  • Recombinant expression construct refers to a nucleic acid having a nucleotide sequence of a mammalian, preferably human, PCC protein, and sequences sufficient to direct the synthesis of PCC protein in cultures of cells into which the recombinant expression construct is introduced and the progeny thereof.
  • Region refers to a zone or general area.
  • a region when referring to a protein or protein module, a region may comprise a linear sequence of amino acids along the protein or protein module or may comprise a three- dimensional area, an epitope, and/or a cluster of epitopes.
  • regions comprise terminal regions.
  • terminal region refers to regions located at the ends or termini of a given agent.
  • terminal regions may comprise N- and/or C-termini. N-termini refer to the end of a protein comprising an amino acid with a free amino group.
  • N- and/or C-terminal regions refer to the end of a protein comprising an amino acid with a free carboxyl group.
  • N- and/or C-terminal regions may therefore comprise the N- and/or C-termini as well as surrounding amino acids.
  • N- and/or C-terminal regions comprise from about 3 amino acid to about 30 amino acids, from about 5 amino acids to about 40 amino acids, from about 10 amino acids to about 50 amino acids, from about 20 amino acids to about 100 amino acids and/or at least 100 amino acids.
  • N-terminal regions may comprise any length of amino acids that includes the N-terminus, but does not include the C-terminus.
  • C-terminal regions may comprise any length of amino acids, which include the C-terminus, but do not comprise the N-terminus.
  • a region when referring to a polynucleotide, a region may comprise a linear sequence of nucleic acids along the polynucleotide or may comprise a three dimensional area, secondary structure, or tertiary structure. In some embodiments, regions comprise terminal regions. As used herein, the term“terminal region” refers to regions located at the ends or termini of a given agent.
  • terminal regions may comprise 5’ and 3’ termini.
  • 5’ termini refer to the end of a polynucleotide comprising a nucleic acid with a free phosphate group.
  • 3’ termini refer to the end of a polynucleotide comprising a nucleic acid with a free hydroxyl group.5’ and 3’ regions may there for comprise the 5’ and 3’ termini as well as surrounding nucleic acids.
  • 5’ and 3’ terminal regions comprise from about 9 nucleic acids to about 90 nucleic acids, from about 15 nucleic acids to about 120 nucleic acids, from about 30 nucleic acids to about 150 nucleic acids, from about 60 nucleic acids to about 300 nucleic acids and/or at least 300 nucleic acids.
  • 5’ regions may comprise any length of nucleic acids that includes the 5’ terminus, but does not include the 3’ terminus.
  • 3’ regions may comprise any length of nucleic acids, which include the 3’ terminus, but does not comprise the 5’ terminus.
  • RNA or RNA molecule refers to a polymer of ribonucleotides
  • ribonucleic acid molecule refers to a polymer of ribonucleotides
  • DNA or “DNA molecule” or“deoxyribonucleic acid molecule” refers to a polymer of
  • DNA and RNA can be synthesized naturally, e.g., by DNA replication and transcription of DNA, respectively; or be chemically synthesized.
  • DNA and RNA can be single-stranded (i.e., ssRNA or ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • mRNA or“messenger RNA”, as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more polypeptide chains.
  • sample refers to a subset of its tissues, cells or component parts (e.g. body fluids, including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).
  • body fluids including but not limited to blood, mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen).
  • a sample further may include a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs.
  • a sample further refers to a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecule.
  • Signal Sequences As used herein, the phrase“signal sequences” refers to a sequence which can direct the transport or localization of a protein.
  • Similarity refers to the overall relatedness between polymeric molecules, e.g. between polynucleotide molecules (e.g. DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of percent similarity of polymeric molecules to one another can be performed in the same manner as a calculation of percent identity, except that calculation of percent similarity takes into account conservative substitutions as is understood in the art.
  • Stable refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
  • Stabilized As used herein, the term“stabilize”,“stabilized,”“stabilized region” means to make or become stable.
  • Subject refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
  • the term“substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term“substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Substantially equal As used herein as it relates to time differences between doses, the term means plus/minus 2%.
  • Susceptible to An individual who is“susceptible to” a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition but harbors a propensity to develop a disease or its symptoms.
  • an individual who is susceptible to a disease, disorder, and/or condition may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition.
  • a genetic mutation associated with development of the disease, disorder, and/or condition for example, cancer
  • a genetic polymorphism associated with development of the disease, disorder, and/or condition
  • increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition (4) habits and/
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
  • Synthetic means produced, prepared, and/or manufactured by the hand of man. Synthesis of polynucleotides or polypeptides or other molecules of the present invention may be chemical or enzymatic.
  • Targeted cells refers to any one or more cells of interest.
  • the cells may be found in vitro, in vivo, in situ or in the tissue or organ of an organism.
  • the organism may be an animal, preferably a mammal, more preferably a human and most preferably a patient.
  • Therapeutic Agent refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
  • therapeutically effective amount means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • a therapeutically effective amount is provided in a single dose.
  • a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses.
  • a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.
  • therapeutically effective outcome means an outcome that is sufficient in a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition.
  • Total daily dose As used herein, a“total daily dose” is an amount given or prescribed in a 24 hr period. It may be administered as a single unit dose.
  • transfection refers to methods to introduce exogenous nucleic acids into a cell. Methods of transfection include, but are not limited to, chemical methods, physical treatments, and cationic lipids or mixtures.
  • Treating refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and/or condition.
  • “treating” cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be
  • Unmodified refers to any substance, compound or molecule prior to being changed in any way. Unmodified may, but does not always, refer to the wild type or native form of a biomolecule. Molecules may undergo a series of modifications whereby each modified molecule may serve as the“unmodified” starting molecule for a subsequent modification.
  • Vector As used herein, a“vector” is any molecule or moiety which transports, transduces or otherwise acts as a carrier of a heterologous molecule. Vectors of the present invention may be produced recombinantly.
  • sequences may comprise any one or more of the following sequences: a polynucleotide sequence encoding a polypeptide or multi-polypeptide, whose sequence may be wild-type or modified from wild- type and which sequence may encode full-length or partial sequence of a protein, protein domain, or one or more subunits of a protein; a polynucleotide comprising a modulatory or regulatory nucleic acid which sequence may be wild-type or modified from wild-type; and a transgene that may or may not be modified from wild-type sequence .
  • sequences may serve as either the“donor” sequence of one or more codons (at the nucleic acid level) or amino acids (at the polypeptide level) or“acceptor” sequences of one or more codons (at the nucleic acid level) or amino acids (at the polypeptide level).
  • Skin fibroblast cultured cells used were from two patients bearing mutations in either the PCCA or the PCCB subunit as well as from a wild type healthy control.
  • the cells were grown in a humidified atmosphere with 5% CO2 at 37°C and maintained in Minimum Essential Medium (HyClone, Logan, UT) supplemented with 15% of Fetal clone III serum (HyClone, Logan, UT) 100ug/ml penicillin and 100ug/ml streptomycin, 100 ⁇ M MEM non- essential amino acids (HyClone, Logan, UT).
  • Minimum Essential Medium HyClone, Logan, UT
  • Fetal clone III serum HyClone, Logan, UT
  • penicillin and 100ug/ml streptomycin 100 ⁇ M MEM non- essential amino acids
  • Mitochondria were obtained from the liver of A138T mice (PCC deficient) using a differential centrifugation protocol. The liver from these mice have 2% of wild type PCC activity. Freshly dissected livers were minced finely before using a motor driven TEFLONTM and glass Potter Elvehjem homogenizer, 6-9 strokes at 1000RPM.
  • the homogenization buffer contained 220mM D-mannitol, 70mM sucrose, 2mM Hepes pH 7.4 and 0.5mg/mL BSA (HMS+).
  • the first centrifugation of a 15% homogenate in HMS+ buffer was performed for 1 minute at 3000g Beckman Avanti J-25) at 4°C to remove nuclei and cell debris. The supernatant was centrifuged 2 minutes for 18,750g at 4°C in order to obtain the mitochondria pellet. The resulting pellet was resuspended in HMS+ buffer and 0.035% digitonin. Digitonin improves mitochondrial recovery by selectively disrupting lipid membranes enriched in sterols to improve purity of the mitochondrial preparations and increase the yield. After 5 minutes of centrifugation at 18,900g the mitochondria were washed 3 times in HMS buffer without BSA before the import. Alternatively, after 5 minutes of centrifugation at 12,500 RPM, the mitochondria were washed twice in HMS buffer without BSA to prepare for import.
  • the mitochondrial pellet was resuspended in 2.5mL of respiration medium MiR05 0.5 mM EGTA, 3 mM MgCl2*6H2O, 60 mM potassium lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM Sucrose, and 1 g/l fatty acid free BSA.
  • An Oroboros Oxygraph 2K was used to measure Oxygen consumption was measured in a medium containing the actively respirating mitochondria. Trypsin was applied to one of the mitochondria samples in a ratio of 1:100 trypsin/enzyme after incubation for 5 minutes of import and another sample after 25 minutes of import at 37°C. The mitochondrial pellet was resuspended in 2.5 mL of respiration medium: MiR050.5 mM EGTA, 3 mM MgCl 2 *6H 2 O, 60 mM potassium lactobionate, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM Sucrose, and 1 g/L fatty acid free BSA. Measurement of oxygen consumption in isolated mitochondria was in a closed chamber for approximately 1 hour. When oxygen was completely consumed, the chamber was opened and the process was repeated 3 times.
  • conjugated TAT-PCC, MPP1A- PCC, MPP2A-PCC was used for import into mitochondria and fibroblasts.
  • maleoyl-beta-Ala-TAT Karlin
  • a neutral buffer like PBS a neutral buffer like PBS.
  • the reaction of peptide maleolyl—beta-Ala-TAT with 1 ⁇ M PCC was performed overnight in 20 mM Hepes, pH 7.0, 500mM KCl at a ratio of 2:1 (TAT:PCC).
  • the excess peptide was removed on a Biorad Bio-Spin 6 column, for import into mitochondria equilibrated in HMS buffer (220mM D-mannitol, 70mM sucrose, 2mM Hepes pH 7.4) or PBS for import into fibroblasts.
  • the mitochondrial pellet was resuspended in fresh lysis buffer (50mM TrisHCl pH 8.0, 1mM DTT, 1mM EDTA pH 8.0, inhibitors (Sigma P8340). Use 3x the volume of the buffer as compared to the volume of mitochondrial pellet.
  • the mitochondria were homogenized in the lysis buffer by pipetting. Sonication was performed using the following settings: twice for 10 seconds at power 3, pulse 1 second on and 0.5 sec off using a microtip probe. The supernatant was collected in a fresh tube after pelleting in a centrifuge (20,000g, 4C) for 15 min. Protein concentration was determined by Bradford assay.
  • TAT-PCC The import of TAT-PCC at the desired concentration was performed at 27°C for 30 minutes.
  • MPP2A-PCC conjugate the reaction was performed at 27°C for 1 hour.
  • the excess peptide was removed on a Biorad Bio-Spin 6 column equilibrated with HMS buffer.
  • the mitochondria were centrifuged for 5 minutes for 12,500 RPM in 4°C.
  • the reaction mixture contained 50mM Tris-HCl, pH 8.0, 2mM ATP, 125mM KCl, 10mM MgCl2, 3mM propionyl-CoA, 0.5mg/ml bovine serum albumin, PCC enzyme (0.1 ⁇ g of purified PCC, 150 ⁇ g for mitochondria lysates and fibroblast lysates), and 10 mM [14C]bicarbonate in a final volume of 50 ⁇ l, and was incubated at 37 °C for 15 minutes. The reaction was terminated by adding 50 ⁇ l of 10% trichloroacetic acid.
  • the mixture was centrifuged at 13,000 g for 5 minutes and 50 ⁇ l of supernatant was dried in a scintillation vial in a heating block at 80°C for 50 minutes.
  • the dry residue was dissolved in 0.15ml of H2O, and 4ml of OPTI-Fluor scintillation fluid (PerkinElmer Life Sciences) was added.
  • the samples were counted in a Beckman LS 3801 scintillation counter. A blank containing the assay mixture without propionyl-CoA was subtracted.
  • Protein content was determined by a Bradford assay using bovine serum albumin as a standard.
  • One unit of PCC activity is defined as 1pmol of bicarbonate per minute per mg of protein at 37°C.
  • Proteins were resolved on 10% sodium dodecyl sulfate polyacrylamide electrophoresis gels and transferred onto Immun-Blot PVDF membrane (Bio-Rad). For all mitochondrial lysates samples were loaded with 50 ⁇ g of protein/lane or 100ng/lane of purified PCC. Western blot analysis was performed using anti-PCCA (Abcam, Cambridge, UK) and anti-PCCB (Abcam, Cambridge, UK) antibodies at 1:1000 dilution to identify the proteins.
  • Polyclonal rabbit Abcam ab154254 Anti-PCCA recognized quantities higher than 100ng in the correct size for PCCA 72kDa, and had a slightly stronger signal then Polyclonal mouse Abcam ab89784 Anti-PCCA but showed a second band around 50kDA.
  • the fluorescent imaging system TyphoonTM with Alexa 647 as a secondary antibody detected, similarly as on chemiluminescent developed Western blot, a second band around 50kDA.
  • polyclonal mouse PCCA Abnova H00005095-B01P and polyclonal mouse PCCB Abcam ab70416 were used for immunostaining.
  • polyclonal mouse PCCA Abnova H00005095-B01P, polyclonal mouse PCCB Abcam ab70416, polyclonal rabbit PCCA Abcam ab154254, polyclonal mouse PCCA Abcam ab89784, or polyclonal rabbit PCCAB Krauslab positively recognizes PCCA or PCCB in both the chemiluminescent and the fluorescent detection systems may be used.
  • Fibroblast cells were grown in 150cm 2 flasks. When the cells reached 90% confluency the medium was removed with PBS and replaced by 1 ⁇ M TAT-PCC, MPP1A- PCC, or MPP2A-PCC diluted in PBS at a concentration of 0.13mg/mL. After 1 hour incubation at 37°C, the cells were washed with PBS, trypsinized, pelleted and kept at -80°C until use.
  • the cell pellet was resuspended in the lysis buffer (50mM Tris HCl pH 8.0, 1mM DTT, 1mM EDTA pH 8.0, protease inhibitors (Sigma P8340). Three times the volume of the buffer as compared to the volume of cell pellet was used. Cells were homogenized in the lysis buffer by pipetting. The cells were sonicated twice for 10 seconds at power 3 and pulsed for 1 second on/0.5 sec off using a microtip probe. The supernatant was collected after a 15 minute centrifugation at 20,000 g and 4°C. Protein concentration was determined by the Bradford assay using 20x or 40x dilutions.
  • Fibroblast cells were grown in a complete MEM media on 8-chamber tissue culture slides (Falcon) to 70% confluency and incubated with 1 ⁇ M TAT-PCC for 1.5 hours. The cells were then washed with PBS before staining. First, Mitotracker Red CMXRos was used to stain mitochondria live cells. Next, cells were fixed with 4% formaldehyde for 10 ⁇ minutes and permeabilized by methanol. The cells were blocked with 2% BSA + 5% goat serum in PBS for 30 minutes at room temperature, then were washed 3-5 times with PBS.
  • Ab89784 anti-PCCA mouse or anti-PCCB were used as the primary antibodies, and anti-mouse IgG ATTO 488 (Sigma) was used as a secondary fluorescent antibody.
  • DAPI staining was used to visualize the nuclei.
  • the cells were washed a final time with PBS.
  • a mounting medium was added to the cells, and a coverslip was sealed with nail polish over each chamber on the slide.
  • the cells were washed a final time with PBS.
  • a mounting medium was added to the cells, and a coverslip was sealed with nail polish over each chamber on the slide.
  • Cell penetrating proteins or peptides linked or conjugated to the constructs and expressed in the vectors described include those detailed in Table 3.
  • PCC subunit may include a cell penetrating peptide (CPP) region.
  • CPP cell penetrating peptide
  • a leader sequence spans the region 230-376 and a His tag spans the region 2171-2134.
  • pET28-C-TATprePCCB SEQ ID NO:26
  • a leader sequence spans the region 230-307 and a His tag spans the region 1850-1867.
  • pET47- NP-TAT-prePCCA SEQ ID NO:27
  • the leader sequence spans the region 249-395, a His tag spans the region 165-182, and a 3C cleavage sequence spans the region 192-214.
  • pET47-NP-TAT-prePCCB SEQ ID NO:28
  • the leader sequence spans the region 249-326
  • a His tag spans the region 165-182
  • a 3C cleavage sequence spans the region 192-214.
  • region 210-212 of SEQ ID NO:27 or SEQ ID NO:28 was mutated from GGA (Gly) to GGG (Gly) to change the SanDI restriction site to a ApaI restriction site.
  • GGC Gly codon mutated to GGT (Gly) codon to remove the ApaI restriction site.
  • a ribosome binding site spans the region 58-63, and an S-tag spans 3810-3854.
  • a mitochondrial leader sequence spans the region of 743-748 and the mature PCCA chain spans the region of 64-740 of SEQ ID NO:33.
  • a mitochondrial leader sequence spans the region of 40-551, a 6x His tag spans the region of 554-559, and the mature PCCB chain spans the region of 40-551 fo SEQ ID NO:35.
  • a mitochondrial leader sequence spans the region of 31-79
  • a 6x His tag spans the region of 3-8
  • a HRV3C protease binding site spans the region of 12-19
  • the mature PCCA chain spans the region of 80-757 of SEQ ID NO:37.
  • a 6x His tag spans the region of 3-8
  • a HRV3C protease binding site spans the region of 12-19
  • a TAT peptide spans the region of 20-28
  • a mitochondrial leader sequence spans the region of 31-56
  • the mature PCCB chains spans the region of 57-268 of SEQ ID NO:39.
  • PCCAB i.e. native PCC dodecamer consisting of 6 PCCA and 6 PCCB subunits
  • ERT enzyme replacement therapy
  • Plasmid constructs were developed for expression of propionyl-CoA carboxylase enzyme in E. coli.
  • constructs were codon optimized for expression in E. coli.
  • Parental nucleic acid constructs which were codon optimized include those of SEQ ID NO:1 or 3 or portions thereof. These were codon optimized with or without the encoded mitochondrial targeting leader amino acid sequence, and individual PCC subunits with and without a cell-penetrating peptide, such as trans-activating transcriptional activator (TAT) peptide, e.g., YGRKKRRQRRR (SEQ ID NO:5).
  • TAT trans-activating transcriptional activator
  • GENSCRIPT® produced the codon-optimized sequence used herein.
  • the sequence was optimized for each subunit i.e. including a penetrating peptide (TAT) sequence, a mitochondrial leader sequence, and the coding sequence itself.
  • TAT penetrating peptide
  • constructs were prepared for both: ready-to-use (similar to individual subunits constructs) and post-purification modification with TAT. These constructs did not contain an additional purification tag.
  • doubly transformed cells were selected on LB media containing 50 mg/ml ampicillin and 50 mg/ml chloramphenicol. Bacterial cultures grown to confluence overnight were diluted 1/100 and used to inoculate 0.5 L aliquots of LB media which were grown with shaker aeration at 37 ° C in the presence of ampicillin (300 mg/ml),
  • the lysate was sonicated twice for 5 minutes at 50% duty with a power setting of 3–4 using a model W225 sonicator (Heat-Ultrasonics, Inc.). Cell lysates were cleared by centrifugation at 15,000 g for 15 minutes and the supernatant (soluble fraction) was collected. The pellet (insoluble fraction) was resuspended in the original volume of Laemmli sample buffer and dissolved by boiling for 5 min.
  • the DEAE fractogel resin was significantly improved. Phosphate concentration in elution buffer (to 100mM). The yield from DEAE capture step was in an acceptable range. Capture column step using Fractogel DEAE resin was significantly improved after the change in the size of the column.
  • PCC proteins or subunits were linked or conjugated to a molecule that permits cell entry of the PCC protein.
  • This example identifies peptide candidates for addition of an N-terminal maleimide suitable to prepare a complex with PCC enzyme. Synthesis of the peptides was performed by GenScript.
  • TAT comprises the amino acid sequence Maleoyl-beta-Ala-Gly-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg (SEQ ID NO:21).
  • MPP1A comprises the amino acid sequence, Cha (cyclohexylalanine)-DArg-Cha- Lys-Cha-DArg-Cha-Lys (SEQ ID NO:23) and MPP2A comprises the amino acid sequence Cha-DArg-Cha-Lys (SEQ ID NO:24). Success of import into mitochondria and cells was evaluated by enzyme activity measurement and Western blot.
  • Maleoyl-beta-Ala-TAT was dissolved in neutral buffer, i.e., PBS. The mixture was reacted with PCC overnight in room temperature. Maleoyl-beta-Ala-TAT was present in excess of PCC in a ratio of 2:1. Unconjugated peptide and conjugate were separated for further analysis.
  • the conjugate of PCC and TAT was prepared in 140uL volume for GE Spin Trap.
  • a sample of one of the reaction mixtures produced in Example 5 was suspended by vortexing.
  • the bottom closure of the column was removed, and the column was placed in an appropriately sized collection tube.
  • the storage solution was removed by centrifugation for 1 minute at 800g using Beckman GS-15R swinging bucket rotor F2402H.
  • the column was equilibrated by addition of 400 ⁇ l equilibration buffer PBS and centrifugation for 1 minute at 800g. This step was repeated four additional times. After each centrifugation, the flow- through was discarded and the collection tube replaced. A new clean collection tube was used for sample collection.
  • the sample 100–180 ⁇ l was applied slowly to the middle of the packed bed.
  • PCCA or PCCB conjugated to TAT a sequence encoding a precursor of the PCCA or PCCB subunit was cloned into an expression plasmid preceded by the TAT peptide sequence.
  • the expressed protein was reconstituted from inclusion bodies and added to the MEM medium in which hamster cells were grown on a microscope slide. The cells were fixed and stained with anti- PCCA or anti-PCCB antibody. Subsequently, a secondary fluorescent antibody was used. Presence of PCC was detected throughout the cells.
  • Inclusion bodies pET47-NP-TATprePCCA 5.5mg construct (55ml) and pET47- NP-TATprePCCB 6.8mg construct (68ml) were extracted and purified according to the Novagen protocol and solubilized in N-Lauroylsarcosine to a 0.06% final concentration.
  • Table 9 contains the different conditions for chemical refold of PCCA and PCCB for 4 different constructs.
  • Samples 3, 5, 7-8, 10, and 12 were dialyzed in HMS.
  • Hepes- mannitol-sucrose buffer, which is known in the art, were used during mitochondrial import.
  • Chloroform/methanol precipitation was used to purify C-TATprePCCA by precipitating 205 ⁇ g and 102.5 ⁇ g in a total volume of 100 ⁇ l 6M guanidine buffer (added to 100 ⁇ l). The obtained pellet was dissolved in 205/102.5 ⁇ l of SDS sample buffer to an assumed concentration 1mg/ml. Sonication was necessary to disperse the pellet. After boiling, samples were separated on SDS-PAGE.
  • Typhoon scan detected the fluorescent signal of Alexa 647 and FITC.
  • the FITC signal detected in the native gel was not as strong as the signal in SDS-PAGE. Therefore, after import and uptake by mitochondria, TAT was likely hidden in a complex protein.
  • the Typhoon scan was performed with the red laser settings of 633nm, 670 short pass filter, 600 PMT
  • TAT and MPP proteins were then conjugated to PCC or a subunit thereof and prepared for import into mitochondria and human fibroblasts.
  • VDAC voltage-dependent anion channels
  • PCC mitochondria-penetrating peptides
  • TAT peptide replacement therapy
  • Import of various forms of PCC subunits, with and without mitochondrial leader peptides, and/or with and without cell-penetrating peptides were examined in: isolated rat liver mitochondria, human control samples, human PA patient derived cells, and PA mouse models. Pharmacokinetic and pharmacodynamic characterization of the PCC import and levels of toxic metabolites characteristic of PA disease were also analyzed.
  • MPPs were developed as mitochondrial transporters, as they are synthetic cell- permeable peptides that are able to enter mitochondria. Efficient uptake of MPPs was observed in a variety of cell types, and organelle specificity is attained with sequences that possess specific chemical properties. MPPs are cationic and lipophilic; this combination of characteristics facilitates permeation of the hydrophobic mitochondrial membrane (see, Horton et al, Chemistry & Biology, 15, 375-382, 2008, which is herein incorporated by reference in its entirety).
  • PCC proteins and subunits were conjugated to three distinct peptides suitable for cargo delivery: TAT, MPP1A and MPP2A.
  • MPP1A, MPP2A, or TAT were each conjugated to PCC, PCC subunits, or subregions of the PCCA or PCCB subunits were imported into mitochondria and fibroblasts.
  • a 2:1 ratio of MPPs to PCC, PCC subunits or subregions of the PCCA or PCCB subunits was combined in an overnight reaction.
  • trypsin was used in all import experiments to digest any adsorbed PCC to the outside of the outer mitochondrial membrane to ensure that the mitochondrial lysate represented only PCC that had been imported to the inside of the organelle, the susceptibility of the enzyme to digestion by trypsin was first assessed.
  • Example 9 Import of 3 ⁇ M TAT and MPP2A conjugates into isolated mitochondria [000277] Selected MPP peptides and TAT were tested for their ability to deliver propionyl- CoA carboxylase into mitochondria. PCC activity was measured in mitochondrial lysates. The effectiveness of different amounts of PCCAB for import into mitochondria were compared.
  • Lysates of mitochondria from livers of mutant A138T mice and lysates of mitochondria from livers of mutant A138T mice after import of TAT-PCC, MPP1A-PCC, and MPP2A-PCC were compared to control PCC enzyme on a Western blot.
  • Primary antibodies for Western blot were Abcam anti-PCCA 80784 and anti-PCCB 70416 diluted 1:1000.
  • results of the Western blot provided evidence that lysate of mutant A138T mouse liver contained PCC, especially the PCC alpha subunit.
  • the amount of PCC in A138T lysates after the import of TAT-PCC and MPP2A-PCC into the isolated mitochondria was observed to be significantly greater than the amount of PCC in the control A138T-MITO mitochondria lysate.
  • TAT-PCC conjugate had been previously observed to be stable during the freeze thaw process.
  • 1 ⁇ M TAT-PCC was incubated with isolated mutant A138T PCC-/- mouse liver mitochondria at 27°C for 30 minutes followed by trypsinization for time periods of 5 and 25 minutes at 37°C.
  • trypsinization was measured in mitochondrial lysates of wild type liver without performing the import.
  • Table 10 shows PCC activity of mitochondrial lysates after import of TAT-PCC for 30 minutes in samples: 1. WT mitochondria, 2.
  • A138T PCC-/- mouse mitochondria 3. Import of 1 ⁇ M TAT-PCCAB into A138T PCC-/- mouse mitochondria, 4. Import of 1 ⁇ M TAT-PCCAB into A138T PCC- /- mouse mitochondria followed by 5 minutes trypsinization, and 5. Import of 1 ⁇ M TAT- PCCAB into A138T PCC-/- mouse mitochondria and 25 minutes of trypsin.
  • the mutant liver mitochondrial lysate activity was significantly lower, i.e., about 8% of wild type activity. After TAT-PCCAB delivery into mutant mitochondria, the PCC activity reached the level of the wild type liver mitochondria PCC activity. The length of trypsinization did not affect the activity.
  • PCC alpha and beta subunits were observed in both 5 minutes and 25 minutes trypsin treated samples indicating that the PCC enzyme was inside of the mitochondria protected from the protease.
  • PCC activity was measured in control A138T mitochondria and the three mitochondria lysates in which import was performed. Import of PCCAB into mitochondria was performed in each lysate for 30 minutes at 27°C. After the import, trypsin in a ratio 1:100 was added and one sample that had been incubated 5 minutes, was added to a different sample that had been incubated 25 minutes at 37°C. Specific activities in each 10 ⁇ L of the four samples were analyzed.
  • PCC was detected in wild type mitochondria.
  • the second import which was allowed to incubate for a longer time was the most efficient of the import samples, and activity was highest of the four samples. Therefore, a longer import time may improve the import.
  • PCC activity increased more than 10-fold in mitochondria after PCCAB import compared to specific activity of PCC in the control mitochondrial lysate.
  • PCC activity after trypsin treatment was observed to remain similar to activity levels in samples not treated with trypsin.
  • Lane E represents PCC FITC-TAT, the wild-type control.
  • PCCA e.g. NP-TAT prePCCA
  • PCCB e.g. C-TAT prePCCB
  • the solution of inclusion bodies resuspended in N-lauroyl sarcosyl 0.06% were diluted 20x from starting concentration 20 mg/ml to achieve the approximately 1 ⁇ M import.
  • trypsin was added to the suspension in a ratio 1:100 for 25 minutes, and the trypsin reaction was stopped with trypsin inhibitor from soybean in ratio 1:1. Lysates were prepared as previously described.
  • Samples were stained with MITOTRACKER® CMX 2000x dye specific to mitochondria.
  • the primary antibody was polyclonal mouse Anti PCCA Abcam Ab89784 antibody 200x, and the secondary antibody was AttoM4881000x. Samples were also stained with DAPI, which is specific to DNA.
  • Fibroblast cell line #3380 after TAT-prePCCA import was stained with DAPI, MITOTRACKER® CMX, and Anti PCCA antibody Abcam Ab89784.
  • the samples were stained as follows: Stain Mitotracker only; Stain Anti PCCA antibody only; and DAPI, MITOTRACKER® CMX and Anti PCCA antibody Abcam Ab89784.
  • PCC activity was measured in cell lysates and immunofluorescence was observed for these fibroblast cells.
  • PCC activity in fibroblasts after import was measured in the cell lysate following import of 3 ⁇ M MPP1A-PCCAB and MPP2A-PCCAB into patient fibroblasts #3380 patient.
  • Lysis buffer containing 50mM TrisHCl pH 8.0, 1 mM DTT, 1 mM EDTA pH 8.0, inhibitors (Sigma P8340) was commercially obtained. Three times the volume of the buffer as compared to the volume of cell pellet was used. Cells in the lysis buffer was homogenized by pipetting. The suspension was sonicated twice for 10 seconds at power 3 pulsing 1 second on, 0.5 second off using a microtip. Cells were spun in a cooled microcentrifuge (20,000 g, 4°C) for 15 minutes. Supernatant was transferred into a fresh tube. Protein concentration was measured by Bradford assay using 20x or 40x dilutions.
  • 3 ⁇ M MPP2A-PCCAB and 3 ⁇ M MPP1A-PCCAB were separately incubated with cells for 1.5 hour in 37°C.
  • immunofluorescence was performed. A control was created by staining of patient fibroblast #3380, PCCA deficient cells, without import.
  • Results from immunofluorescence after import of MPP2A-PCC into patient fibroblasts #3380 provided a strong green fluorescent stain of PCC.
  • the side to side view with MITOTRACKER® staining provided comparison of the stains.
  • An overlay of the results indicated that some green and red areas were superimposing, which is evidence of import into mitochondria.
  • PCC was not detectable in the #3380 patient fibroblast cells, as expected. See Figure 2.
  • Example 13 Staining to confirm import in patient fibroblasts [000312] Pure PCC was linked to a FITC-labeled TAT peptide. Fibroblasts were grown in complete MEM media.1 ⁇ M PCC was imported into #3380 fibroblasts (two mutations in PCCA) for 1 hour at 37°C as described in Example 1.
  • Maleoyl-beta-Ala-TAT (KeraFAST ® ) was combined with the purified ⁇ 6 ⁇ 6 PCC.
  • Two different PA patient fibroblast cell lines were transfected with the PCC and TAT protein mixture. Each cell line reproducibly demonstrated enzyme activity in the range of full restoration of enzymatic activity of normal control fibroblasts to about ten times the activity of normal control fibroblasts.
  • the reaction of PCC enzyme with maleoyl-beta-Ala-TAT was incubated overnight. One reaction was used for the import as is, and the second was purified on a spin column G-25 to remove excess of maleoyl-beta-Ala-TAT peptide.
  • PCC activity was measured in cell lysate. PCC activity was analyzed in normal control fibroblast #5142 and in patient fibroblasts #3380 and #3383, and in each type of fibroblasts after import of conjugate TAT-PCCAB.
  • the sample or dilution buffer for the blank were added at a volume of 15 ⁇ l, and the mixture was transferred at 37°C.
  • the combined mixture was incubated for 2 minutes prior to starting the reaction.
  • the adjusted 14 C sodium bicarbonate, which starts the reaction, was added in a volume of 10 ⁇ l.
  • the reaction was incubated at 37°C for 15 minutes under the hood.
  • the reaction was stopped by mixing with 50 ⁇ l of ice-cold 10% TCA, and further transfer to ice.
  • the tubes were centrifuged at max speed for 5 minutes in the hood.50 ⁇ l of the supernatant was transferred into a labeled glass scintillation vial. Unreacted CO2 was evaporated in a dry block at 80°C for 20-30 minutes.
  • Each sample was combined with a reaction mixture containing 50mM Tris-HCl, pH 8.0, 2mM ATP, 250mM KCl, 10mM MgCl 2 , 3mM propionyl-CoA, 0.5mg/ml bovine serum albumin, PCC enzyme (150 ⁇ g of cell lysate), and 10mM [14C] bicarbonate for a final volume of 50uL, and incubated at 37°C for 15 minutes. The reaction was terminated with 50 ⁇ L of 10% trichloroacetic acid.
  • the mixture was centrifuged at 13,000g for 5 minutes, and 50 ⁇ L of supernatant was dried in a scintillation vial in a heating block at 80 °C for 30 minutes.
  • the dry residue was dissolved in 0.15ml of H 2 O, and 4ml of OPTI-Fluor scintillation fluid (PerkinElmer Life Sciences) was added.
  • the samples were counted by a Beckman LS 3801 scintillation counter. A blank containing the assay mixture without propionyl-CoA was subtracted from the measured values.
  • the 1 ⁇ M import had a specific activity of about 4000 ⁇ mol/min/mg
  • the 5 ⁇ M import had a specific acitivty of about 13,500
  • PCC import into fibroblast cells from propionic acidemia patients One patient was homozygous for two different mutations in the PCCA subunit and the second one was PCCB deficient. Fibroblast cells were incubated for 1 hour at 37°C with different amounts of TAT-PCC. The cells were harvested with 0.25% trypsin (ratio 1:1 protease/protein). The PCC activity was tested in cell lysates.
  • Table 13 shows C, control cell line; 1, PCCA deficient fibroblast lysate; 2– 4, Import of 1 ⁇ M, 5 ⁇ M, and 10 ⁇ M TAT-PCC into PCCA deficient cells for one hour; 5, PCCB deficient fibroblasts; and 6 -8, import of 1 ⁇ M, 5 ⁇ M, and 10 ⁇ M TAT-PCC into PCCB deficient cells for one hour.
  • a wild type mouse control and liver from PCC-/- A138T mice were analyzed to confirm that TAT-PCCAB was successfully imported into cells’ mitochondria and affects metabolite levels in mouse plasma.
  • the import was performed for two hours into isolated mitochondria from the PCC deficient mice only. Trypsin was applied to one of the mitochondrial samples in a ratio of 2:1 trypsin/enzyme. Each type of sample was analyzed using Western blot and PCC activity was measured.
  • PCC was observed in the sample of wild type mouse mitochondrial lysate. Import of PCC was also observed; therefore, PCCA is potentially only found in this sample, as PCC was not detected in the other samples.
  • TLCK was used to inhibit trypsin action.
  • A138T mice have elevated levels of propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy, which is similar to levels of these compounds in PA patients.
  • PCC-/- mice Blood from PCC-/- mice was collected in anti-coagulant treated tubes. After centrifugation for 10 minutes at 200g, the plasma was transferred to a clean tube. Stability of PCC in plasma was measured at time points 0, 20, 40, 60, 90, and 180 minutes, remaining at 37°C.
  • Plasma was placed in a 37°C water bath for 10 minutes to preincubate.
  • PCCAB enzyme in 20 mM Hepes buffer pH 7.4, 10mM KCl (18 mg/ml) was added to the plasma to a final dilution of 180x to reach an enzyme concentration of 100 ng/ ⁇ L.
  • PCC activity decreases gradually in time, about 10% decrease was observed in PCC enzyme activity after 1 hour incubation and up to 23% decrease in PCC activity after 3 hours of incubation, PCCAB was diluted to a concentration of 100ng/ ⁇ L in plasma at 37°C. Results were confirmed by Western blot showing that PCC enzyme appeared to be relatively stable over 3 hours, 77% of PCC activity is still measured after 3 hours in plasma at 37°C. There were no degradation products observed on Western blot.
  • PCC activity was observed to decrease gradually over time. An about 10% decrease in PCC enzyme activity was observed after 1 hour of incubation and an up to 23% decrease in PCC activity was observed after 3 hours of incubation. [000340] PCC enzyme was observed to be relatively stable over the course of 3 hours, as 77% of PCC activity is measured after 3 hours in plasma at 37oC. Further, no degradation of products was observed on the Western blot.
  • PCC enzyme was observed to be stable in plasma at 37°C. Results indicate that PCC activity decreases gradually over time. For example, an about 40% decrease in PCC activity after 24 hours of incubation was observed.
  • mice There were 3 groups of mice each consisting of 4 animals: 1 untreated/uninjected control group, and 2 treated/injected groups each receiving either 10 mg/kg or 20 mg/kg.
  • the untreated control group was bled throughout a day to find out diurnal variation of metabolites and establish the best timing for injecting/bleeding. Bleedings of the treated mice were performed 15 minutes after the first injection (T1), 24 hours after the first injection and prior to the second injection (T24), 24 hours after the second injection (T48), and 72 hours after the last injection (T96).
  • Metabolic marker MC (methylcitrate) did not show significant variation throughout the day. Both treated groups were observed to experience a decline in C3/C2 ratio for 96 hours with no additional effect observed for the group receiving 20 mg/kg. Therefore, 10 mg/kg was determined to be a sufficient dosage for examples herein.
  • C3/C2 ratio in healthy heterozygous mice is 0.1 ⁇ M, respectively.
  • PA-relevant metabolites were not significantly altered in plasma of the A138T PA mouse model.
  • a slight trend in lowering both C3/C2 ratio and MC plasma levels was observed but does not seem relevant due to relatively high variation in the trend.
  • Similar to PCC activity measurement a 2 times higher dose did not result in a 2-fold decrease in C3/C2 ratio.
  • PCCA A138T mice were split into 4 groups each consisting of 4 or 2 animals: 4 mice were injected by IV and 3 groups of 2 mice each were injected IV, IP, or SQ with TAT- PCCAB. The first injection was administered at 13:00, and a second injection was administered 24 hours later. For bleeding the IV injected mice (total of 6), mice were sub- divided into 2 groups of 2+1 and bled as follows: Group 1 - 15 minutes, 24 hours, 48 hours, 72 hours, 96 hours, and 168 hours after the first injection; Group 2– 4 hours, 28 hours, 48 hours, 72 hours; 96 hours, and 168 hours after the first injection. The IP and SQ injected mice were bled as follows: 4 hours, 24 hours, 48 hours, 72 hours, 96 hours, and 168 hours after the first injection.
  • C3/C2 ratio was observed to be significantly decreased only in the IP-injected group. C3/C2 levels were altered for less than 24 hours which indicates that a higher dosage and/or increased administration occurrence would prolong the desired effect.
  • Acylcarnitines (C3 and C2) were measured in all plasma samples collected from mice treated with 2 doses of TAT-PCCAB (20 mg/kg) administered via IV, SQ, or IP.
  • IP administration of TAT-PCCAB resulted in a significant improvement of C3/C2 ratio 4 hours after the first injection.
  • the effect was no longer present.
  • PCCA A138T mice were split into 2 groups each consisting of 3-4 animals (depending on enzyme availability) with injections made by two different individuals. Two injections were administered 3 hours apart at 7:00 and 10:00. Plasma samples were collected as follows: before the 2nd injection at 10:00 (T3), 6 hours after the first injection at 13:00 (T6) and 8 hours after the first injection at 15:00 (T8). Urine sample were collected 6 hours after the first injection at 13:00. After the final bleeding, mice were sacrificed and flushed with PBS. The liver, heart, and brain were harvested and frozen in liquid nitrogen for PCC activity measurement in tissues.
  • hypomorphic PCCA mice i.e. knock-out for mouse PCCA, but carrying transgene for human PCCA A138T mutant
  • Two IP injections were administered 3 hours apart: at 7:00 (Injection A) and 3 hours later at 10:00 (Injection B). Dose was the same as used in previous examples: 20 mg/kg.
  • Mice were split into two groups: injected by two different individuals. Plasma samples were collected as follows: before the 2nd injection at 10:00 (T3), 6 hours after the first injection at 13:00 (T6), and 8 hours after the first injection at 15:00 (T8).
  • mice were sacrificed, flushed with PBS and liver, heart and brain were harvested and frozen in liquid nitrogen. Metabolites in plasma and PCC activity were measured in plasma as well as tissue homogenates. The results are shown in the graphs of Figures 5-7.
  • Examples herein analyze changes in pharmokinetics (PK) and pharmacodynamics (PD) of metabolites after a single IP injection.
  • TAT-PCCAB The length of the effect of IP administered TAT-PCCAB and PK of TAT-PCCAB importation into the cells was analyzed to determine the length of time that TAT-PCCAB persists in the mice following IP administration.
  • PCCA A138T mice were split into 2 sub- groups each consisting of 4 animals to split bleedings and maximize the number of timepoints over the course of a day.
  • a single dose of TAT-PCCAB was administered IP at 8:00. Plasma samples were collected as follows: Group A– 2 hours (T2), 4 hours (T4) and 8 hours (T8) after injection; Group B– 3 hours (T3), 6 hours (T6), and 9 hours (T9) after injection. All mice were also bled 24 hours (T24) after injection.
  • Control group C (injected IP with PBS only) was bled at the same intervals. Mice were sacrificed, and each liver was harvested. The groups are organized in Tables 19-21 below. Table 19 provides details of Group A having plasma samples taken at T2, T4, and T8 after injection.
  • Table 20 provides details of Group B having plasma samples taken at T3, T6, and T9 after injection.
  • Table 21 provides details of the control group (Group C) that was injected with PBS only.
  • articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
  • any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

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Abstract

L'invention concerne des compositions de propionyl-CoA carboxylase humaine recombinée (PCC). L'invention concerne également le dodécamère PCCAB et des sous-unités, comprenant du PCCA et du PCCB, conjugués à des peptides de pénétration cellulaire et de pénétration des mitochondries, ainsi que des compositions pharmaceutiques de ceux-ci. L'invention concerne également des méthodes permettant de traiter des affections telles qu'une acidémie propionique (PA), une acidurie propionique, un déficit en propionyl-CoA carboxylase et une glycinémie avec cétose à l'aide des compositions de l'invention.
PCT/US2017/030904 2016-05-03 2017-05-03 Compositions de propionyl-coa carboxylase et utilisations de celles-ci WO2017192761A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO2020264353A1 (fr) * 2019-06-27 2020-12-30 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Gènes synthétiques pour le traitement de l'acidémie propionique provoquée par des mutations de propionyl-coa carboxylase alpha
US20220025369A1 (en) * 2016-05-04 2022-01-27 Curevac Ag Rna encoding a therapeutic protein
EP3861131A4 (fr) * 2018-10-01 2022-06-15 Ultragenyx Pharmaceutical Inc. Thérapie génique pour le traitement de l'acidémie propionique

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