WO2018204603A1 - Compositions de propionyl-coa carboxylase et leurs utilisations - Google Patents

Compositions de propionyl-coa carboxylase et leurs utilisations Download PDF

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WO2018204603A1
WO2018204603A1 PCT/US2018/030826 US2018030826W WO2018204603A1 WO 2018204603 A1 WO2018204603 A1 WO 2018204603A1 US 2018030826 W US2018030826 W US 2018030826W WO 2018204603 A1 WO2018204603 A1 WO 2018204603A1
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pcc
pccab
tat
mitochondria
seq
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PCT/US2018/030826
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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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    • 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
    • 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
    • A61K38/51—Lyases (4)

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 acidemia (PA), PA-related disorders, propionic aciduria, propionyl- CoA carboxylase deficiency, and or ketone 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 (a) 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 hereby incorporated by reference herein in its entirety).
  • the 72 kDa a subunit and the 56 kDa ⁇ subunit (see, Gravel et ., ⁇ 9 0 Archives of Biochemistry &
  • N-terminal leader sequences are proteolytically removed, and the mature enzyme is assembled.
  • the ⁇ -subunits (also referred to as PCCB) form a central core hexameric core decorated on the outside by six non-interacting a- subunits.
  • Biotin, bicarbonate, and ATP have binding sites on the a-subunit (also referred to as PCCA) while propionyl CoA binds to the ⁇ -subunit.
  • the crystal structure of a 780 kDa ⁇ 6 ⁇ 6 dodecamer of bacterial PCC has been determined to provide the three-dimensional structure of the enzyme. See, Huang et al., Nature. 2010 Aug 19;466(7309): 1001-5, which is hereby incorporated by reference in its entirety.
  • PCCAB Human mature dodecamer
  • E. coll from a single plasmid.
  • E. colt 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 a-subunit contains the sequence that accepts biotin (see, Kalousek et al., 1980 Journal of Biological Chemistry. 255:60-5; Lamhonwah et aL, ⁇ 9%1 Archives of Biochemistry & Biophysics. 254:631-6; Leon-Del-Rio & Gravel 1994 Journal of Biological Chemistry.
  • Propionic acidemia is an autosomal recessive disorder in which a defective form of PCC results in the accumulation of propionic acid, propionyl-CoA, 3-hydroxypropionate, propionyl carnitine, and methyl citrate, primarily in mitochondria of hepatocytes.
  • 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. The condition is frequently accompanied by metabolic acidosis with anion gap, ketonuria, hypoglycemia, hyperammonemia, and cytopenias. 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]
  • PA GeneReviews ® [Internet] (Pagon RA, Adam MP, Ardinger HH, et al., editors. Seattle (WA): University of Washington, Seattle; 1993-2017, which is hereby incorporated by reference herein in its entirety).
  • the incidence of 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 hereby incorporated by reference in their entireties).
  • PA also results from a decrease in PCC activity from a lack of co-enzymes such as biotin.
  • PA carriers 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 (see, Ravn et al., 2000 Am J Hum Genet. 67:203-6, which is hereby incorporated by reference in its entirety).
  • Biochemically, patients with PA have elevated levels of propionyl CoA, propionic acid, methylcitrate, beta-hydroxy-propionate, propionylglycine, tiglylglycine, and ketones. Ketones, such as butanone, may also be found in urine of these patients (Menkes et al., 1966 The Journal of pediatrics. 69:413-21).
  • PA is a potentially life-threatening disease.
  • 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.
  • Various embodiments of the invention herein provide a method for reducing propionyl-CoA levels in a PCC deficient subject comprising administering a pharmaceutical composition comprising an isolated human PCCAB dodecamer conjugated to a cell penetrating peptide or mitochondria penetrating peptide.
  • Various embodiments of the invention herein provide a method for reducing the ratio of propionyl-carnitine (C3) and acetyl-carnitine (C2) in a PCC deficient subject comprising administering a pharmaceutical composition comprising an isolated human PCCAB dodecamer conjugated to a cell penetrating peptide or mitochondria penetrating peptide.
  • Various embodiments of the invention herein provide a method for reducing propionyl-carnitine (C3) levels of in a PCC deficient subject comprising administering a pharmaceutical composition comprising an isolated human PCCAB dodecamer conjugated to a cell penetrating peptide or mitochondria penetrating peptide.
  • the PCCAB dodecamer comprises a PCC A subunit comprising the amino acid sequence of SEQ ID NO:41, or a fragment or a variant thereof sharing a sequence similarity with SEQ ID NO:41 of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • the PCCAB dodecamer comprises a PCCB subunit having the amino acid sequence of SEQ ID NO:43, or a fragment or a variant thereof sharing a sequence similarity with SEQ ID NO:43 of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • a nucleic acid sequence encoding the PCCAB dodecamer 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 with SEQ ID NO:40 of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • a nucleic acid sequence encoding the PCCB subunit is SEQ ID NO:42, or a fragment or a variant thereof sharing a sequence similarity with SEQ ID NO:42 of at least 99%, at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
  • the pharmaceutical composition is administered by intravenous injection (TV), subcutaneous injection (SC), or intraperitoneal injection (IP).
  • the pharmaceutical composition is formulated in a dosage within a range of about 20mg kg to about 30mg/kg, about 25mg/kg to about 35mg kg, about 30mg/kg to about 40mg/kg, or about 35mg kg to about 45mg/kg.
  • administering occurs at least at least twice a day, a least three times a day, or at least 4 times a day. In certain embodiments of the method, administering occurs consecutively for more than one day. In certain embodiments of the method,
  • administering occurs consecutively for at least 4 days. In certain embodiments of the method, administering occurs at more than one dose. In certain embodiments, the method further comprises administering an additional dose of the pharmaceutical composition every other day. In certain embodiments of the method, administering the additional dose occurs about 4 hours after administering the pharmaceutical composition. In certain embodiments of the method, the additional dose is the same as a dose of the pharmaceutical composition. In certain embodiments of the method, the dose of the pharmaceutical composition and the additional dose are about 20mg/kg. In certain embodiments of the method, the additional dose is greater than a dose of the pharmaceutical composition. In certain embodiments of the method, the additional dose is about 40mg kg and the dose of the pharmaceutical composition is about 20mg/kg.
  • Various embodiments of the invention herein provide a method of producing a TAT- PCCAB conjugate comprising: providing a nucleic acid sequence codon-optimized for expression in a recombinant cell system; co-expressing PCCAB with a molecular chaperone protein; purifying PCCAB; and conjugating PCCAB to at least one cell penetrating peptide after purifying, Ihereby producing a TAT-PCCAB conjugate
  • FIG. 1 shows non-limiting examples of PCC protein expression constructs.
  • a C- terminal or N-terminal His-tag may be conjugated to the expression construct as shown for certain constructs in Figure 1.
  • MTS represents a mitochondrial targeting sequence
  • 'TAT represents the cell-penetrating peptide from the transactivator of transcription of human immunodeficiency virus (HIV).
  • FIG. 2 is a graph showing PCC activity after import of a TAT or mitochondria penetrating peptide 2A (MPP2A) conjugated PCCAB construct at 3 ⁇ into isolated A138T mutant mouse mitochondria
  • Figure 3 shows oxygen consumption of isolated mitochondria during TAT-PCCAB conjugate import monitored over a 1.5-hour period.
  • FIG. 4 shows a graph of PCC activity in patient fibroblast cell lines 3380 and 3383 after import of mitochondria penetrating peptide 1 A (MPP 1 A)-PCCAB, MPP2 A-PCCAB, and TAT-PCCAB at 3 ⁇ .
  • MPP 1 A mitochondria penetrating peptide 1 A
  • compositions and methods of treatment for treating patients with propionic acidemia (PA) or other PCC-deficiency related conditions for example, enzyme therapy.
  • PA propionic acidemia
  • PCC deficiency 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 (also referred to as PCCAB) enzyme is an ⁇ heterododecamer having a molecular weight of about 800 kDa.
  • the 72 kDa PCC-alpha (PCCA) subunit and the 56 kDa PCC-beta (PCCB) subunit are encoded by genes designated, PCCA (ENSG00000175198) and PCCB
  • the a-subunit contains biotin carboxylase and biotin carboxyl carrier protein domains.
  • the ⁇ -subunit (PCCB) 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.
  • TAT Transactivator of transcription
  • HTV human deficiency virus
  • the TAT protein from human immunodeficiency virus type 1 is a potent viral transactivator that is essential for viral replication.
  • the TAT protein has been frequently studied for its unique ability to penetrate cell membranes.
  • the TAT dodecapeptide also used in the Examples herein, facilitates cellular uptake of different types of cargo and has been classified as a cell penetrating peptides.
  • the exact mechanism of how the TAT-protein complex enters the cells or mitochondria is not fully understood. See, Palm-Apergi et al. 2012. Mol Ther 20:695- 697 and Madani et al. 2011. J Biophys 2011:414729, which are both hereby incorporated by reference in their entireties.
  • 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 hereby 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.
  • LAD dehydrogenase
  • LAD lipoamide dehydrogenase
  • E3 catalytic subunit of three multicomponent enzymatic in the mitochondrial matrix.
  • C6orf66 assembly factor that restores Complex I activity in patient cells was also successfully replaced.
  • a successful delivery of TAT-MTS-coupled mitochondrial enzyme into patient cells was also reported for the NAD dehydrogenase complex I assembly factor
  • Mitochondrial complex I deficiency is a disorder caused by mutations in
  • NDUFAF4 A recombinant protein containing the TAT-MTS-NDUFAF4 WT was efficiently taken up by patient-derived NDUFAF4-deficient cells, resulting in a significant increase in complex I activity and improved mitochondrial function. See, Marcus et al. Mol Med 19:124 - 134, which is hereby incorporated by reference herein in its entirety. 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 hereby incorporated by reference in its entirety).
  • the native PCCAB dodecamer of 780 kDa was imported into both the isolated PCC- deficient mouse liver mitochondria and the patient fibroblasts.
  • the native PCC has dimensions of 155 A by 155 A by 170 A, which is hereby incorporated by reference herein in its entirety.
  • TAT-MTS-LAD 58.1 kDa. See, Rapoport et al. 2008. Mol Ther 16:691- 697, which is hereby incorporated by reference herein in its entirely.
  • the A138T mouse model of PA accumulates propionylcarnitine in plasma hence, it has a substantially elevated C3/C2 ratio.
  • compositions and conjugates may be designed and/or engineered.
  • Such starting sequences include the DNA sequence for PCC alpha (a) subunit protein (PCCA), designated SEQ ID NO: 1, and the amino acid sequence for full-length human PCCA, having 702 amino acid residues, designated SEQ ID NO:2.
  • the DNA sequence for PCC beta ( ⁇ ) subunit protein (PCCB) is designated SEQ ID NO:3, and 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 PCCAB 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 peptides and MPPs 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.
  • 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
  • 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 MPP can be linked to a molecule through covalent bonds or non-covalent bonds and are coupled to the
  • 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
  • mitochondrial targeting sequence 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.
  • the PCCAB, PCCA, 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- translationally or post-purification of the PCCAB, PCCA, or PCCB peptide.
  • PCC-deficient patients generally have only one subunit affected (either PCCA or PCCB), expression of an individual subunit for ERT development was explored.
  • single subunit PCCA or PCCB import has been described (see, Damavandi et ai.,Mol Genet Metab Rep. 2016 Sep; 8: 51-60, which is hereby incorporated by reference in its entirely).
  • the import of PCC heterododecamer ( ⁇ 6 ⁇ 6), PCCAB, using the TAT transduction domain was analyzed herein.
  • TAT may be conjugated either simultaneously with translation or post-translationally or post-purification.
  • TAT-PCCAB may be synthesized.
  • a 6x His tag on either a C-terminus or an N-terminus was added to purify the protein from an insoluble fraction (inclusion bodies).
  • the 6x His tag may be used with 1MAC chemistry for purification under native or denaturing conditions.
  • 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 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.
  • 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 peptide at the amino terminus.
  • the PCC protein enzyme conjugated to the cell-penetrating or the mitochondria penetrating peptide 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 peptides covalently attached to each PCC enzyme subunit in the composition.
  • the cell penetrating peptide is a TAT peptide.
  • the TAT peptide comprises the amino acid sequence YGRKKRRQRRR (SEQ ID NO:5) or a fragment thereof.
  • 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.
  • homeodomain transcription factors such as Antennapedia (RQIKIYFQNRRM WKK, 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
  • LLHLRRRIRKQAHAHSK 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. DrugDeliv. 5(1):105-17; and Treat et al., 2012 ACSMacro Lett. l(l):100-04, which are hereby 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.
  • Other 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.
  • ligands to improve delivery of the pharmaceutical composition 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.
  • Use of recombinant DNA technologies to improve control of expression of transfected nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within the host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational or post-purification modifications was explored in the examples. Additionally, the promoter sequence may be genetically engineered to improve the level of expression as compared to the native promoter.
  • 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
  • translational control signals e.g., ribosome binding sites, Shine-Dalgarno sequences
  • the vector is at least one from Table 5. In certain embodiments, the vector is at least one from Table 5.
  • pET28-C-TATprePCCA SEQ ID NO:25
  • 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 ihe 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 die 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 Apal restriction site.
  • GGC (Gly) codon mutated to GGT (Gly) codon to remove the Apal restriction site In certain embodiments of pETDSl-PCCAB (SEQ ID NO: 29), 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 of SEQ ID NO:35.
  • a mitochondria] 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. £0067 ⁇
  • the PCCA and/or PCCB proteins are produced
  • PCCA and/or PCCB proteins may be produced in prokaryotic or eukaryotic cells, more specifically yeast, mammalian, or E coli cells. These constructs for individual subunits yielded protein for transport, i.e. already expressed as a single polypeptide with a penetrating peptide (TAT) and mitochondria-targeting leader.
  • 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.
  • co-expression of molecular chaperones with PCCAB in forms of TAT conjugated precursors or as mature polypeptides in a preferred expression host (SE1 ) was used.
  • co-expression of GroEL ES was observed to result in 3-4-fold higher PCC specific activity.
  • expression of PCC from the pETDSl-PCCAB construct along with pGro7 in SE1 is used for purification of native PCCAB. Chromatographic separation
  • 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 monomelic 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).
  • Controlling the ion composition of said solution allows to use the chromatography materials of the invention either in subtractive mode (PCC passes through said chromatography matrix and at least certain contaminants bind to said chromatography matrix) or, preferably, in adsorptive mode (PCC binds to the chromatography matrix).
  • 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.
  • a specific embodiment of such a nucleic acid sequence optimized for PCC expression in K 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 was 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 PCCAB, PCCA, or PCCB 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 may 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 Coomassie 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.
  • ERT PHARMACEUTICAL COMPOSITIONS AND METHODS OF USE £0078 ⁇ PA is a devastating disease with only dietary management treatment.
  • ERT for PA or for any mitochondrial disease would constitute an amazing achievement.
  • the use of ERT has been studied in various metabolic enzyme deficiencies, such as Gaucher' s, Hurler's, Fabry's, Pompe's, homocystinuria, PKU, glycogen storage disease type II, and mucopolysaccharidosis I and VI, and in Maroteaux-Lamy syndrome to reverse the pathogenesis of the chief clinical manifestations of these diseases. See, Amalfitano et al. 2001. Genet Med 3:132-138; Bublil et al. 2016.
  • 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® CMX 2000x dye MITOTRACKER® CMX 2000x dye.
  • PCCAB imported into the cells by the processes described herein provided reproducible data demonstrating that the diagnostic C3/C2 ratio (propionyl-/acetyl- carnitine) in cell extracts of the PCC-treated cells significantly decreased in comparison to untreated controls.
  • 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 pharmaceutical composition further comprises a
  • 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
  • O.Olmg/kg - 20mg/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, or 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. lmg/kg to O.Smg/kg; 0. lmg/kg to 2mg/kg; about 3mg/kg, about 4mg/kg, about 5mg/kg, about 7mg/kg; from 2-10mg/kg; from 3-15mg/kg; more than lOmg/kg, more than 20mg/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
  • compositions of the present invention may be formulated in any manner suitable for delivery.
  • the formulation may be, but is not limited to, nanoparticles, poly0actic-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.
  • 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, IS, 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.
  • 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.
  • animals include, but are not limited to, mammals, birds,
  • 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 VH and VL 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.
  • VH and VL domains include VH and VL domains, scFvs, Fab, Fab', F(ab') 2 , Fv fragments, diabodies, linear antibodies, single chain antibody molecules, multispecific antibodies
  • association 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.
  • Btfunctional 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 As used herein, the term
  • 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 complementary to adenosine. However, when a U is denoted in the context of the present invention, the ability to substitute a T is implied, unless otherwise stated. Perfect 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. [0119] In some embodiments, two or more sequences are said to be "completely conserved” if they are 100% identical to one another.
  • 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. In some embodiments, 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.
  • 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 affect 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,
  • 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 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 is a schedule of administration or physician determined regimen of treatment, prophylaxis, or palliative care.
  • Engineered As used herein, 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.
  • Effective Amount As used herein, the term "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. For example, in the context of administering an agent that treats PCC deficiency, 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.
  • 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 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.
  • 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.
  • 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 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.
  • 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. For example, 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.,
  • 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 P AMI 20 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.
  • 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. etal, 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 VH chain and VL 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.
  • H e phrase “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.
  • compositions described 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, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C,
  • “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).
  • suitable organic acid examples 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.
  • 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; generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17* 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 means of or relating to a physical and/or chemical property.
  • the term "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. 'Turification” refers to the process of making pure.
  • Recombinant cell refers to suitable cells (including progeny of such cells) from any species (prokar otic 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. When referring to polynucleotides, terminal regions may comprise 5' and 3' termini. 5' termini refer to H e 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 IS nucleic acids to about 120 nucleic acids, from about 30 nucleic acids to about ISO 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; the term ' ⁇ " or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleoudes.
  • 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 ahomogenate, 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.
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and humans
  • 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%.
  • Substantially simultaneously As used herein and as it relates to plurality of doses, the term means within 2 seconds.
  • 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.
  • an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
  • 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 canonic 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 administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • 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 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-polypepude, 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).
  • ccdA antidote gene with its own regulatory elements from Delphi Genetic's pStabyl.2 vector was PCR amplified and inserted into a Sphl site of Novagen's pET-DUET-1, producing pETDSl or pETDS2 based on direction of insertion. pETDSl was used for further cloning.
  • E. coli SE1(DE3) cells carrying ampicillin-resistant PCC expression vector pETDSl-PCCAB and chloramphenicol-resistant GroEL/ES expression plasmid pGro7 were grown in Luria-Bertani (LB) medium supplemented with 30 ⁇ D-biotin.
  • GroEL ES molecular chaperone was induced by adding 2mg/ml L- arabinose when cells reached an optical density at 600nm of 0.7 to 0.8. About 20 minutes later, lmM IPTG (isopropyl-P-D-thiogalactopyranoside) was added to induce the expression of PCC, followed by an additional 16 hours at 37°C.
  • SEPHADEX® spin column (GE Healthcare, Chicago, IL), formulated into 20mM HEPES (pH 7.4)-100mM KC1, divided into aliquots, and stored at about 80°C.
  • the purification yield of about 99% pure PCC from 6 liters of culture was usually between 30 and lOOmg.
  • doubly transformed cells were selected on LB media containing 50mg/ml ampicillin and 50mg/ml chloramphenicol.
  • Bacterial cultures grown to confluence overnight were diluted 1/100 and used to inoculate 0.5L aliquots of LB media which were grown with shaker aeration at 37°C in the presence of ampicillin (300mg/ml), chloramphenicol (30mg/ml), and biotin (5mM) to a turbidity of about 0.4 at 600nm prior to induction with lmM IPTG (BRL).
  • the induced cells were allowed to grow for 2-24 hours before collection.
  • Cells were harvested on ice, collected by centrifugation (10,000 g for 10 minutes), washed with phosphate-buffered saline (PBS), and resuspended in lOOmM Tris-HCl, pH 7.5, ImM EDTA, 0. ImM DTT, and lmg/ml lysozyme followed by stirring for 1 hour at 4°C.
  • 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 H e 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 minutes.
  • the conditions for the capture column from the previous example with slightly adjusted conditions for an affinity column (PIERCETM Monomelic Avidin with sepharose).
  • PIERCETM Monomelic Avidin with sepharose was slightly adjusted conditions for an affinity column (PIERCETM Monomelic Avidin with sepharose).
  • a different formulation buffer was used for the final protein elution on a G25 SEPHADEX® spin column.
  • PCC enzyme activity was analyzed for the above fractions gathered by various techniques. Table 3 provides the amount of recovery of PCC enzyme activity from common protein purification techniques utilizing a capture column. For this purification, PCC enzyme activity was measured in each fraction to determine the percent of enzyme recovered as compared to the wild type.
  • TAT-PCC For import into mitochondria and fibroblasts, conjugated TAT-PCC, MPP1 A-PCC, MPP2A-PCC was used.
  • a maleolyl-P-Ala-TAT also known as maleolyl-beta-Ala-TAT
  • erafast targeting accessible cysteine residues was used for conjugation with PCCAB to prepare TAT-PCCAB.
  • maleoyl-P-Ala-TAT Kerafast was dissolved in a neutral buffer like PBS.
  • the reaction of peptide maleolyl-beta-Ala-TAT with ⁇ PCC was performed overnight in 20mM Hepes, pH 7.0, 500mM KC1 at a ratio of 2:1 (TAT:PCCAB).
  • the excess peptide was removed on a Bio-SpinTM 6 column (Bio-Rad), for import into mitochondria equilibrated in HMS buffer (220mM D-mannitol, 70mM sucrose, 2mM Hepes pH 7.4) or PBS for import into fibroblasts.
  • Conjugation reaction samples larger than 0.5ml were processed through a column using a G25 SEPHADEX® resin (GE Healthcare, Chicago, IL).
  • the TAT-PCCAB conjugate was stable during the freeze-thaw process.
  • the mixture was reacted with PCCAB overnight in room temperature. Unconjugated peptide and conjugate were separated for further analysis.
  • Conjugation of MPP1A and MPP2A was performed using a similar protocol.
  • reaction mixtures were used to produce conjugated TAT- PCCAB.
  • One mixture was used for import as is, and the second mixture was purified using a Spin column (PD SpinTrap G-2S preparation).
  • Table 4 provides the components of each reaction mixture: ⁇ PCC concentration and a 2x excess of TAT peptide (KERAFAST® Maleoyl-P-Ala-TAT, catalog number: EAA001).
  • the FITC label was visible by scanning with a TYPHOONTM scan.
  • the fluorescent antibody ALEXA FLUOR® 647 dye was used as a secondary antibody for Mouse anti-PCCA 1:1000 (Abnova) and Mouse anti-PCCB ABCAM® 70416 1:1000.
  • a red laser of 633nm was used to detect ALEXA FLUOR® 647 dye and a green laser of 532nm was used to detect the FITC label.
  • 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.
  • Isolated 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 O.Smg/mL bovine serum albumin (BSA) (HMS+).
  • BSA bovine serum albumin
  • the first centrifugation of a 15% homogenate in HMS+ buffer was performed for 1 minute at 3000g using BECKMAN COULTERTM Avanti J-25 centrifuge) 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% digjtonin. Digitonin improves mitochondrial recovery by selectively disrupting lipid membranes enriched in sterols to improve purity of the mitochondrial preparations and increase the yield.
  • Digitonin improves mitochondrial recovery by selectively disrupting lipid membranes enriched in sterols to improve purity of the mitochondrial preparation
  • 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.
  • TAT-PCC The import of TAT-PCC at the desired concentration was performed at 27°C for 30 minutes. Trypsin was used at a protease/protein ratio of 1:20 (wt/wt) for 5 or 30 min, and the reaction was stopped with soybean trypsin inhibitor at a ratio of 1 : 1 (wt/wt) with trypsin. The trypsin reaction was stopped with trypsin inhibitor from soybean in ratio 1:1. The excess peptide was removed on a BIO-SPINTM 6 column (Bio-Rad) equilibrated with HMS buffer.
  • PCC The activity of PCC was assessed indirectly by measuring the incorporation of a label from [l- 14 C]propionate into cellular macromolecules, which is hereby incorporated by reference in its entirety.
  • Control and patient fibroblasts were grown on six-well plates (Corning). The import of 5 ⁇ TAT-PCC conjugates or incubation with PBS was performed at 80% confluence for 1 hour at 37°C. Subsequently, the fibroblasts were incubated for 18 hours in MEM supplemented with 15% fetal bovine serum (Fetal Clone III) and ⁇ [l- 14 C]propionate (MD Biochemical), diluted wilh unlabeled propionate to give a final specific activity of ⁇ / ⁇ . At the end of the incubation, the cells were harvested with trypsin, and the cellular
  • Polyclonal rabbit ABCAM® abl 54254 Anti-PCCA antibody recognized quantities higher than lOOng 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 TYPHOONTM fluorescent imaging system (GE Life Sciences) used with ALEXA FLUOR® 647 dye as a secondary antibody, similarly as on a chemiluminescent developed western blot, detected a second band around 50kDA.
  • SUPERSIGNALTM West Pico (THERMOSCIENCETM) chemiluminescence substrate developed membrane polyclonal mouse Anti-PCCB ABCAM® ab70416, polyclonal mouse anti-lOOOx antibody to identify H e PCCB 58kDa subunit after 2 minutes of exposure in a quantity higher than lOOng. Quantities of purified PCC enzyme higher than 175ng were visible in scans at a size corresponding to 58kDa.
  • a TYPHOONTM scan with ALEXA FLUOR® 647 dye conjugated to a secondary antibody provided a low signal for 50ng.
  • Polyclonal mouse PCCA (Abnova Catalog number H0000S09S-B01P) and polyclonal mouse PCCB ABCAM® ab70416 were used for immunostaining.
  • polyclonal mouse PCCA (Abnova Catalog H00005095-B01P)
  • polyclonal mouse PCCB ABCAM® ab70416, polyclonal rabbit PCCA ABCAM® abl54254, polyclonal mouse PCCA ABCAM® ab89784, or polyclonal rabbit PCCAB Krauslab (positively identifies PCCA or PCCB in both the chemiluminescent and the fluorescent detection systems) may be used.
  • Skin fibroblast cultured cells used were from two patients bearing mutations (e.g. A138T) in either the PCCA (cell line 3380) or the PCCB subunit (cell line 3383) as well as from a wild type healthy control (cell line 5142).
  • the cells were grown in a humidified atmosphere with 5% CO2 at 37°C and maintained in ⁇ Minimum Essential Medium (MEM) (HyClone, Logan, UT) supplemented with 15% of FETALCLONETM ⁇ serum (HyClone, Logan, UT) ⁇ penicillin and ⁇ streptomycin, and non-essential amino acids (HyClone, Logan, UT).
  • MEM Minimum Essential Medium
  • PCC was diluted to a final concentration of 0. lmg/ml in A138T mouse plasma, followed by incubation for the indicated times. The incubation was terminated by mixing 18 ⁇ 1 of the reaction mixture with 2 ⁇ 1 of protease inhibitor cocktail (Sigma; catalog no. 8340) on ice, and the PCC activity was determined. The results are an average of two measurements + the standard errors of the mean (SEM).
  • Fibroblast cells were grown in 150cm 2 flasks. When the cells reached 80-90% confluency the medium was removed with PBS and replaced by ⁇ TAT-PCC, MPP1 A-PCC, or MPP2A-PCC diluted in PBS at a concentration of 0.13mg/mL.
  • Each TAT-PCCAB conjugate is added to 5mL of complete MEM media and applied to a 150cm 2 flask with patient 3380 fibroblast cells 80-90% confluent. Cells are incubated for 1 hour at 37°C. After import, cells were harvested using 0.25% trypsin. The pellet of cells was washed 3x in PBS, each time resuspended in 20mL PBS and centrifuged at 800g for 10 minutes, then transferred to a small Eppendorf tube. Cells were stored at -80°C until use.
  • Lysis buffer containing 50mM TrisHCl pH 8.0, lmM DTT, lmM EDTA pH 8.0, inhibitors SIGMA-ALDRICH® P8340 protease inhibitor cocktail
  • Three times the volume of the buffer as compared to the volume of the 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 (20,000g, 4°C) for 15 minutes. Supernatant was transferred into a fresh tube. Protein concentration was measured by Bradford assay using 20x or 40x dilutions.
  • the cell pellet was resuspended in the lysis buffer (50mM Tris HC1 pH 8.0, lmM DTT, lmM EDTA pH 8.0) and protease inhibitors (SIGMA-ALDRICH® P8340 protease inhibitor cocktail). 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 20,000g and 4°C. Protein concentration was determined by the Bradford assay using 20x or 40x dilutions.
  • the activit in fibroblast lysate extracts was measured for 150 ⁇ g of protein in a ⁇ assay. Protein concentration was calculated using a Bradford protein assay. The reaction was terminated by adding 50 ⁇ 1 of 10% trichloroacetic acid. The mixture was centrifuged at 13,000g for 5 minutes and 50 ⁇ 1 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 H20, and 4ml of OPTI-FLUOR® scintillation fluid (PerkinElmer Life Sciences) was added. The samples were counted in a BECKMAN COULTER® LS 3801 scintillation counter. A blank containing the assay mixture without propionyl-CoA was subtracted.
  • Cells were homogenized in the lysis buffer by pipetting. The cell suspension was sonicated twice for 10 seconds at power 3, pulse 1 second on, 1 second off using a microtip. Volumes were too small to use a power setting greater than 3. Cells were centrifuged in a cooled microcentrifuge at 20,000g and 4°C for IS minutes. The supernatant was transferred to a fresh tube. Twenty ⁇ of the 2x Reaction mixture was combined with the cells. The 30mM
  • propionylCoA final 3mM was added at a volume of 5 ⁇ 1, or 5 ⁇ 1 of water was added for a blank.
  • Protein content was determined by a Bradford assay using bovine serum albumin (BSA) as a standard.
  • BSA bovine serum albumin
  • U PCC activity
  • each sample was combined with a reaction mixture containing 50mM Tris-HCl, pH 8.0, 2mM ATP, 125mM KC1, lOmM MgC12, 3mM propionyl-CoA, 0.5mg/ml BSA, PCC enzyme (O. ⁇ g of purified PCC, 150 ⁇ g for mitochondria ly sates and fibroblast lysates), and lOmM [14C] sodium bicarbonate in a final volume of 50 ⁇ 1 and was incubated at 37°C for 15 minutes. The reaction was terminated with 50 ⁇ , of 10% trichloroacetic acid.
  • the mixture was centrifuged at 13,000g for 5 minutes, and 50 ⁇ , of supernatant and unreacted CO2 was evaporated in a dry block at 80°C for 20-30 minutes.
  • the dry residue was dissolved in 0.15ml of H20, and 4ml of OPTI-FLUOR® scintillation fluid (PerkinElmer Life Sciences) was added.
  • 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 ⁇ .
  • the reaction was incubated at 37°C for 15 minutes under the hood.
  • the reaction was stopped by mixing with 50 ⁇ 1 of ice-cold 10% TCA.
  • the mixture was centrifuged at 13,000g for 5 min, and 50 ⁇ 1 of the supernatant was dried in a scintillation vial in a heating block at 80°C for 50 min.
  • the dry residue was dissolved in 0.15 ml of H2O, and 4 ml of OPTI-FLUOR® scintillation fluid (Perkin-Elmer 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.
  • One unit of PCC activity is defined as 1 pmol of product per min at 37°C per mg of protein.
  • the tubes were centrifuged at max speed for 5 minutes in the hood. 50 ⁇ 1 of the supernatant was transferred into a labeled glass scintillation vial.
  • the dry pellet was dissolved in 150 ⁇ 1 of ddH 2 0. Scintillation liquid was added at a volume of
  • Fibroblast cells were grown in a complete MEM on 8-chamber tissue culture slides (Falcon) to 70% confluency and incubated with ⁇ TAT-PCC for 1.5 hours. The cells were then washed with PBS before staining. First, MTTOTRACKER® Red CMXRos dye was used to stain mitochondria in live cells. Next, cells were fixed with 4% formaldehyde for 10 minutes and permeabilized by methanol. The cells were blocked with 2% BSA and 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 antibodies were used as the primary antibodies, and anti-mouse IgG Atto 488 antibody (SIGMA- ALDRICH®) was used as a secondary fluorescent antibody.
  • SIGMA- ALDRICH® anti-mouse IgG Atto 488 antibody
  • 4',6-diamidino-2-phenylindole (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.
  • mice retained 2% of the PCC enzyme activity of the wild type and survive to adulthood. Further, A138T mice had elevated levels of propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy, which was similar to levels of these compounds in PA patients. The mice were bred, maintained, and genotyped as described in Guenzel et al. Liver PCC activity in A138T mouse was 2.2% of the WT PCC activity. The A138T human cDNA produces 9.4% of the PCC activity in transfected fibroblasts. (See, Clavero et al. 2002. Biochim Biophys Acta 1588:119 -125).
  • a single-use lancet for submandibular bleeding was used for blood collection into Capiject T-MLHG lithium heparin (12.5 IU) tubes with gel (Terumo). Tubes were then centrifuged at l,200g for 10 minutes, followed by collection of plasma into 1.5-ml tubes and storage at -80°C.
  • Plasma was placed in a 37°C water bath for 10 minutes to pre-incubate.
  • PCCAB enzyme in 20mM Hepes buffer pH 7.4, lOmM KC1 (18mg/ml) was added to the plasma to a final dilution of 180x to reach an enzyme concentration of 100 ng/ ⁇ ..
  • a lOSng/ ⁇ PCC to 330 ⁇ 1_, plasma + 2 ⁇ . of PCC (18mg/mL) sample was prepared, then the 2 ⁇ 1 of protease inhibitor further diluted the sample.
  • the plasma and enzyme mixture was incubated in water bath at 37°C. At each timepoint: 0, 20, 40, 60, 90, 180 minutes, a sample of 28 ⁇ . of plasma was added to 2 ⁇ , of protease inhibitor incubated on ice (protease inhibitor cocktail (SIGMA-ALDRICH® P8340 protease inhibitor cocktail) to prevent proteolytic degradation.
  • protease inhibitor cocktail SIGMA-ALDRICH® P8340 protease inhibitor cocktail
  • a western blot and PCC enzyme activity assay were performed on a sample from each timepoint and a sample of 18mg/mL PCCAB. The western blot was performed with anti-PCCAB antibody purified, diluted 200x and secondary anti-rabbit diluted SOOOx to analyze the presence of PCCAB at each timepoint.
  • a sample having a concentration of lOOng/ ⁇ . of PCC was diluted lOx. A ⁇ . aliquot was used in each assay as lOOng of enzyme was needed for detection by
  • Cell penetrating proteins or peptides linked or conjugated to the constructs and expressed in the vectors described in Table 5 include those detailed in Table 6.
  • PCC enzyme and enzyme subunit conjugates were produced in the vectors provided in Table 5 for the studies disclosed herein is given in Table 7.
  • the vector name is also given in Table 7.
  • a mitochondrial leader sequence spans amino acid positions 743-748 and the mature PCCA chain spans amino acid positions 64-740 of SEQ ID NO:33.
  • a mitochondrial leader sequence spans amino acid positions 40-551, a 6x His tag spans amino acid positions 554-559, and the mature PCCB chain spans amino acid positions 40-551 of SEQ ID NO:35.
  • a mitochondrial leader sequence spans amino acid positions 31-79, a 6x His tag spans amino acid positions 3-8, a HRV3C protease binding site amino acid positions 12-19, and the mature PCCA chain spans amino acid positions 80-757 of SEQ ID NO:37.
  • a 6x His tag spans amino acid positions 3- 8
  • aHRV3C protease binding site spans amino acid positions 12-19
  • a TAT peptide spans amino acid positions 20-28
  • a mitochondrial leader sequence spans the region of 31-56
  • the mature PCCB chains spans amino acid positions 57-268 of SEQ ID NO:39.
  • PCC was conjugated with fluorescein isothiocyanate (FITC)-labeled TAT peptide ( erafast) to follow the fluorescent label on a western blot using a TYPHOONTM fluorescent imaging system GE Healthcare).
  • FITC fluorescein isothiocyanate
  • PCCA subunits were detected with anti-PCCA antibody, which detects the unmodified PCCA subunit in PCCAB, as well as the TAT-modified PCCA subunit in both TAT-PCCA and FITC-TAT-PCCA.
  • PCCAB i.e. native PCC dodecamer consisting of 6 PCCA and 6 PCCB subunits
  • ERT enzyme replacement therapy
  • GenScript Biotech 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
  • 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
  • constructs for individual subunits yielded protein for transport, i.e. already expressed as a single polypeptide with a penetrating peptide (TAT) and mitochondria-targeting leader.
  • TAT penetrating peptide
  • Example 5 Production of Conjugates
  • 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 Biotech.
  • 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-D Arg-Cha-Ly s (SEQ ID NO:23) and MPP2A comprises the amino acid sequence Cha- DArg-Cha-Lys (SEQ ID NO:24).
  • PCC proteins and subunits were conjugated to three distinct peptides suitable for cargo delivery: TAT, MPP1A and MPP2A.
  • 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 PCCAB, subunit, or sub-region that had been imported to the inside of the organelle, the susceptibility of the enzyme to digestion by trypsin was first assessed.
  • results of the western blot provided evidence that lysate of mutant Al 38T mouse liver contained PCC, especially the PCCA (alpha) subunit.
  • the amount of PCCAB in A138T lysates after the import of TAT-PCCAB and MPP2A-PCCAB into the isolated mitochondria was observed to be significantly greater than the amount of PCCAB in the control A138T mitochondria lysate.
  • TAT-PCCAB conjugate used here had been previously observed to be stable during the freeze/thaw process.
  • ⁇ TAT-PCCAB was incubated with isolated mutant A138T PCC-/- mouse liver mitochondria at 27°C for 30 minutes followed by trypsinization for time periods of 5 or 30 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. Wild type mitochondria, 2.
  • A138T PCC-/- mouse mitochondria 3. Import of 1 ⁇ TAT-PCCAB into A138T PCC-/- mouse mitochondria, 4. Import of 1 ⁇ TAT-PCCAB into A138T PCC-/- mouse mitochondria followed by 5 minutes trypsinization, and 5. Import of 1 ⁇ TAT-PCCAB into A138T PCC-/- mouse mitochondria and 30 minutes of trypsinization.
  • PCC enzyme activity was significantly lower, i.e., about 8% of wild type activity, in the mutant liver mitochondrial lysate. After TAT-PCCAB delivery into mutant mitochondria, the PCC activity reached the level of the wild type liver mitochondria PCC activity. The length of tiypsinization was not observed to affect the activity. PCC activity increased more than 10- fold in mitochondria after PCCAB import compared to specific activity of PCC in the control mitochondrial lysate. PCC activit after trypsin treatment was observed to remain similar to activity levels in samples not treated with trypsin.
  • PCC activity was measured in control A138T mouse mitochondria and the three mitochondria lysates in which import was performed. PCC was also 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 the PCC enzyme activity was the highest of the four samples. Therefore, a longer incubation time for import may improve efficiency of the import.
  • VDAC voltage-dependent anion channels
  • TAT-PCCAB The effects of increasing concentrations of TAT-PCCAB during import into isolated mutant mitochondria were analyzed. Concentrations of ⁇ , 2 ⁇ , and 5 ⁇ of TAT-PCC were introduced into an isolated mutant A138T mouse mitochondria, then the mitochondria were subjected to trypsin treatment as described in Example 1.
  • N-Lauroyl sarcosyl was used to dissolve mitochondria and structures of the mitochondria were not distinguishable after staining.
  • the staining for PCCA was positive, but an overlay of staining for PCCB was unclear.
  • PCC enzyme was observed to be stable in plasma at 37°C. Results indicate that PCC activity decreased gradually over time. For example, an about 40% decrease in PCC activity after 24 hours of incubation was observed. About one-third of its activity was observed to be intact by 72 hours. This experiment showed that injecting PCC into circulation will not result in immediate degradation, thus enabling its transport through the bloodstream to target tissues.
  • the samples were stained as follows: Stain MITOTRACKER® CMX 2000x dye only; Stain Anti- PCCA antibody only; and DAPI, MITOTRACKER® CMX 2000x dye only and Anti-PCCA antibody ABCAM® Ab89784.
  • PCC enzyme activity in fibroblasts after import was measured in the cell lysate following import of 3 ⁇ MPP1A-PCCAB and MPP2A-PCCAB into patient fibroblast cell lines 3380 and 3383.
  • the patient fibroblast cell lines 3380 and 3383 were observed to have a very low PCC activity as shown in Figure 4.
  • PCC activity was observed to increase about 14-fold compared to PCC activity in the mutant A138T mitochondria lysate.
  • a significant increase of PCC enzyme activity in cell lysates was observed after the import of 3 ⁇ TAT-PCCAB conjugate, similar to additional results gathered herein.
  • Patient fibroblast cell line 3380 was transfected with the PCCAB and TAT protein mixture.
  • 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 G25 SEPHADEX® spin column to remove excess of maleoyl-beta-Ala-TAT peptide.
  • PCC enzyme activity results are shown in Table 13.
  • 'Blank represents the buffer without added enzyme or reaction mixture.
  • PCCAB represents the enzyme without a cell-penetrating peptide and that has not been imported to mitochondria
  • CE prepared RC represents the crude extract of resting wild type fibroblasts.
  • PCCAB+TAT represents a reaction mixture that was not purified using a G25 SEPHADEX® spin column to remove excess TAT peptide.
  • PCCAB TAT G25 represents a reaction mixture that was purified using a G25 SEPHADEX® spin column to remove excess TAT peptide.
  • 3380 TAT+PCCAB represents lysate after import of TAT-PCCAB reaction mixture that was not purified using a G25
  • TAT PCCAB G25 represents lysate after import of TAT-PCCAB reaction mixture that was purified using a G25 SEPHADEX® spin column to remove excess TAT peptide.
  • 3380 patient represents lysates from patient fibroblast cell line 3380 without import of TAT-PCCAB.
  • 5142 control fibroblast represents lysate from a wild type patient fibroblast cell line.
  • 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.
  • Patient fibroblasts from cell line 3380 (3380 patient) were observed to have low PCC activity. After import of ⁇ TAT-PCCAB conjugate, the activity of PCC was 34 times higher than in the control sample without treatment (3380 TAT+PCCAB) and doubled compared to the control fibroblast cell line (5142 control fibroblast).
  • PCC was imported into fibroblast cells from propionic acidemia (PA) patients.
  • the import was performed for 1 hour at different concentrations: ⁇ , 5 ⁇ , and 10 ⁇ , of PCCAB using the technique in Example 1 for the import of ⁇ TAT-PCCAB into patient fibroblast cell lines.
  • Patient fibroblast cell line 3380 had a mutation in the PCCA subunit and cell line 3383 had a mutation in the PCCB subunit, and as a consequence both cell lines have low PCC activity.
  • the ⁇ import had a specific activity of 3825.93 pmol/min/mg
  • the 5 ⁇ import had a specific activity of about 13378.79 pmol/min/mg
  • the ⁇ had a specific activity of about 17164.47 pmol/min/mg.
  • the ⁇ import had a specific activity of 2074.47 pmol/min/mg
  • the 5 ⁇ import had a specific activity of about 9211.81 pmol min/mg
  • the ⁇ had a specific activity of about 13775.69 pmol/min/mg.
  • Both PCCA and PCCB deficient skin fibroblasts had less than 3% of control fibroblast activity.
  • the conjugated TAT-PCCAB was successfully imported into patient cells with either defective PCCA or PCCB.
  • the activity at the highest concentration, ⁇ TAT- PCCAB, in the incubation mixture exceeded the control activity 11- and 9-fold for the PCCA and PCCB deficient cells, respectively.
  • a wild type mouse control and liver samples from PCC-/- A138T mice were compared to confirm that TAT-PCCAB was successfully imported into cells' mitochondria and affected metabolite levels in mouse plasma. Each type of sample was analyzed using western blot and PCC activity was measured. PCCAB was observed in the sample of wild type mouse mitochondrial lysate. Import of PCCAB was also observed; therefore, PCCA is potentially only found in these samples, as PCCAB was not detected in the other samples. Tosyl phenylalanyl chloromethyl ketone (TLCK) was used to inhibit trypsin action.
  • TLCK Tosyl phenylalanyl chloromethyl ketone
  • PCCA alpha (PCCA) subunit was missing in the lysate of mutant mitochondrial fraction A138T PCC-/- mouse.
  • PCC alpha (PCCA) and beta (PCCB) subunit in the same mitochondria remained present after ⁇ PCCAB import.
  • PCC alpha (PCCA) and beta (PCCB) subunit were present in the samples treated for 25 minutes with trypsin indicating that PCCAB was inside of the mitochondria and was protected until preparation of ly sate. Activity was analyzed as described in Example 1. Results are provided below in Table IS.
  • PCC enzyme activity decreased gradually in time. A decrease of about 10% was observed in PCC enzyme activity after 1 hour incubation, and a decrease of up to 23% was observed in PCC enzyme activity after 3 hours of incubation.
  • PCCAB was diluted to a concentration of lOOng/ ⁇ . in plasma at 37°C. Results were confirmed by western blot showing that PCCAB appeared to be relatively stable in the course of 3 hours, e.g., 77% of PCC activity was still measured after 3 hours in plasma at 37°C. There were no degradation products observed on western blot.
  • Example 18 Activity in vivo after IV. IP and SO administration
  • 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.
  • 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.
  • MC methylcitrate
  • Example 19 Activity sustainabilitv in vivo after IP administration
  • 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.
  • 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: 20mg/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). After the final bleeding, 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.
  • Table 17 provides C3/C2 ratios in plasma after in vivo IP administration of TAT- PCCAB at a dose of 20mg kg.
  • Table 18 provides the PCC activity in mouse plasma over time after in vivo IP administration of TAT-PCCAB conjugate at a dose of 20mg/kg.
  • Table 19 provides the specific activity of PCC in heart and liver tissue homogenates after in vivo IP administration of TAT-PCCAB at a dose of 20mg/kg.
  • Injection A was observed to result in the highest level of PCC specific activity compared to the specific activity measured in wild type mice, untreated mice, and mice treated with Injection B.
  • Example 20- Import studies in vivo Diurnal variation
  • hypomorphic PCCA mice knock-out for mouse PCCA and containing the transgene for human PCCA A138T mutant; further abbreviated PCCA A138T were bled at time TO before a first injection for metabolites, for example, two days prior to injection or immediately prior to the injection.
  • 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 lOmg/kg or 20mg/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 IS minutes after the first injection (Tl), 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).
  • Examples herein analyze changes in pharmacokinetics (PK) and pharmacodynamics (PD) of metabolites after a single IP injection.
  • 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 described in Tables 22-24 below. Table 22 provides details of Group A having plasma samples taken at T2, T4, and T8 after injection.
  • Table 23 provides amounts and ratios of propionylCoAcarnitine (C3) and acetylCoAcarnitine (C2) for mice in Group A. Table 23. Changes in C3 and C2 amounts and ratios over 24 hours in Group A
  • Table 25 provides details of Group B having plasma samples taken at T3, T6, and T9 after injection.
  • Table 26 provides amounts and ratios of propionylCoAcarnitine (C3) and acetylCoAcarnitine (C2) for mice in Group B.
  • Table 28 provides details of the control group (Group C) that was injected with PBS
  • Table 29 provides amounts and ratios of propionylCoAcarnitine (C3) and acetylCoAcarnitine (C2) for mice in Group C.
  • the TAT-PCCAB was administered IP twice a day at 20mg/kg, once in the morning at 8:00 and 8 hours later at 16:00 for 4 days. On the fifth day, only the morning injection was administered. Plasma samples were collected once a day before the second daily injection. On fifth day, 8 hours after the last morning injection, mice were bled, sacrificed, perfused with PBS and had their livers harvested for PCC activity measurement. Mice were at least 2 months old, and most were females because previous screenings have shown that females have higher C3/C2 ratio than males. The mean of the C3/C2 ratios for the treated and PBS-treated group and the standard error of the mean (SEM) values are shown in Table 31.
  • hypomorphic PCCA mice i.e. knock-out for mouse PCCA but carrying transgene for human PCCA A138T mutant
  • TAT-PCCAB was concentrated to 8.6mg/ml and was formulated into 20mM HEPES pH7.5, 150mM NaCl on a G25 SEPHADEXTM spin column, concentrated on Amicon YM10 and filter sterilized (PVDF, 0.22 ⁇ m).
  • TAT-PCCAB was administered IP once a day 20mg/kg (dilute enzyme 2. lSx to 4mg/ml in a filter-sterilized formulation buffer) in the morning at 8:00 for 4 days.
  • Plasma samples were collected once a day, 8 hours after the injection at 16:00. Half of the mice were female, and all were at least 2 months old.
  • mice were 3 groups of mice each consisted of 4 animals.
  • Table 32 provides the mean of each group of mice treated with either TAT-PCCAB or Buffer and the untreated control mice.
  • Example 25 Multiple IP injections of 30mg/kg and 40mg/kg
  • mice There were 2 sub-groups of mice each consisting of 4 animals: TAT-PCCAB-treated and buffer-injected controls.
  • the TAT-PCCAB was administered IP once a day at 8:00 for 4 days at a dose of 30mg/kg. In addition, on day 2 and day 4, an extra second dose of 40mg/kg was administered 4 hours after the morning injection at noon. Plasma samples were collected once a day at 8 hours after the first morning injection at 16:00.
  • FITC-TAT-PCCAB was formulated to a concentration of 5.5mg/ml in 20mM HEPES pH7.5, 150mM NaCl on a G25 SEPHADEXTM spin column, concentrated on Amicon YM10, filter sterilized (PVDF, 0.22 ⁇ ), and modified O/N.
  • TAT-PCCAB was formulated in 20mM HEPES pH7.5, 150mM NaCl on a G25 SEPHADEXTM spin column concentrated on Amicon YM10 and filter sterilized (PVDF, 0.22um) to a concentration of 7.2mg/ml and modified O/N.
  • the C3/C2 ratios were calculated for each mouse at the designated timepoints. The mean C3/C2 ratios for each group are shown in Table 33.
  • hypomorphic PCCA mice i.e. knockout for mouse PCCA but carrying transgene for human PCCA A138T mutant
  • the TAT-PCCAB was administered IP at a dose of 20mg/kg (enzyme was diluted 1.8x to 4mg/ml in a filter-sterilized formulation buffer).
  • TAT- PCCAB was also formulated in 20mM HEPES pH7.5, 150mM NaCl on a G25TM spin column to a concentration of 7.2mg/ml, modified O/N, and filter sterilized (PVDF, 0.22um) on Amicon YM10.
  • mice were not bled before the injection for metabolites at time -1, and this example used the values in Example 23.
  • the TAT-PCCAB was administered once a day at 8:00 for 4 days. In addition, on day 2 and day 4, an extra second dose was administered 4 hours after the morning injection at noon. Plasma samples were collected once a day at 8 hours after the first morning injection at 16:00. Table 34 provides the mean C3/C2 ratios for each group.
  • 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 recombinante (PCC), des sous-unités PCC conjuguées, comprenant PCCA et PCCB, et des compositions pharmaceutiques de ces dernières. L'invention concerne également des méthodes permettant de traiter des états pathologiques tels qu'une acidémie propionique (PA), une acidurie propionique, un déficit en propionyl-CoA carboxylase et une glycinémie cétosique à l'aide des compositions selon l'invention.
PCT/US2018/030826 2017-05-03 2018-05-03 Compositions de propionyl-coa carboxylase et leurs utilisations WO2018204603A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2016179138A1 (fr) * 2015-05-03 2016-11-10 The Regents Of The University Of Colorado Compositions de propionyl-coa carboxylase et utilisations de celles-ci

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* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 20 May 2005 (2005-05-20), "Homo sapiens propionyl Coenzyme A carboxylase, alpha polypeptide, mRNA (cDNA clone MGC:5056 IMAGE:3048998), complete cds.", XP055545708, retrieved from NCBI Database accession no. BC000140 *
DATABASE UniProtKB 13 August 1987 (1987-08-13), "PROPIONYL-COA CARBOXYLASE ALPHA CHAIN (EC 6.4.1.3) (PCCASE) (PROPANOYL-COA: CARBON DIOXIDE LIGASE) (GENE NAME: PCCA) (FRAGMENT)", XP055545693, retrieved from UniProt Database accession no. P05165 *

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