# US 8,541,550 B2 · Methods and compositions for delivering polynucleotides

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- Number: US 8,541,550 B2
- Document ID: US8541550B2
- Status: Granted
- Kind: B2
- Application number: 13/112,705
- Filed: 2011-08-19
- Published: 2011-12-08
- Granted: 2013-09-24
- Earliest claimed: 2003-10-24
- Assignees: Gencia Corporation
- Applicants: Shaharyar Khan
- Inventors: Shaharyar Khan
- Web page: https://patentgrep.com/patent/US8541550B2

## CPC

- C12N 15/85: for animal cells
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 1/02: Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
- A61P 1/04: for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 5/06: of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 11/06: Antiasthmatics
- A61P 13/12: of the kidneys
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/04: Centrally acting analgesics, e.g. opioids
- A61P 25/08: Antiepileptics; Anticonvulsants
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/02: Ophthalmic agents
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/08: for leprosy
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/62: DNA sequences coding for fusion proteins
- C07K 2319/07: containing a mitochondrial localisation signal
- C07K 2319/09: containing a nuclear localisation signal
- C07K 2319/10: containing a tag for extracellular membrane crossing, e.g. TAT or VP22
- C07K 2319/81: containing a Zn-finger domain for DNA binding
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Methods and compositions for delivering polynucleotides are provided. One embodiment provides a non-viral vector comprising a recombinant polynucleotide-binding protein comprising a protein transduction domain operably linked to a targeting signal. Methods for modifying the genome of non-nuclear organelles are also provided.

## Claims

### Claim 1

A transcription factor A-mitochondrial (TFAM) fusion protein comprising a protein transduction domain, a mitochondrial localization signal, and a mature transcription factor A-mitochondrial (TFAM),
wherein the protein transduction domain comprises a plurality of amino acid residues having a net positive charge under physiological conditions.

### Claim 2 (depends on 1)

The fusion protein of claim 1, wherein the mitochondrial localization signal comprises a mitochondrial localization signal sequence of a protein selected from the group consisting of hexokinase I, amine oxidase (flavin-containing) A, hexokinase IV, pancreatic beta cell form, peripheral benzodiazepine receptor-related protein, metaxin 2, putative mitochondrial outer membrane protein import receptor (hTOM), glutathione transferase, voltage-dependent anion channel 2 (outer mitochondrial membrane protein porin), cytochrome b5, peripheral benzodiazepine receptor, germ cell kinase anchor S-AKAP84, A kinase anchor protein, carnitine O-palmitoyltransferase I precursor, hexokinase II, amine oxidase (flavin-containing) B, long-chain-fatty-acid—CoA ligase 2, long-chain-fatty-acid—CoA ligase 1 (palmitoyl-CoA ligase), voltage-dependent anion channel 1, metaxin 1, Human putative outer mitochondrial membrane 34 kDa translocase hTOM34, voltage-dependent anion channel 4 (outer mitochondrial membrane protein porin), cytochrome-b5 reductase, voltage-dependent anion channel 3 (outer mitochondrial membrane protein porin), Mitochondrial import receptor subunit TOM20 homolog (Mitochondrial 20 kd outer membrane protein) (Outer mitochondrial membrane receptor TOM20), tumorous imaginal discs homolog precursor (HTID-1) electron transfer flavoprotein alpha chain precursor (mouse), electron transfer flavoprotein beta chain (mouse), dihydrolipoamide dehydrogenase precursor (human), nucleoside-triphosphate—adenylate kinase 3 (mouse), citrate synthase (mitochondrial), carbamoyl-phosphate synthetase 1, malic enzyme complex (mitochondrial) (mouse), propionyl-CoA carboxylase alpha chain precursor (mouse), propionyl-CoA carboxylase alpha chain precursor, carbonyl reductase (NADPH) (mouse), Mitochondrial processing peptidase beta subunit precursor (beta-mpp) (ubiquinol-cytochrome c reductase complexcore protein I), acyl-CoA dehydrogenase precursor—short-chain-specific, electron transfer flavoprotein alpha chain precursor, dihydrolipoamide S-(2-methylpropanoyl)transferase precursor, heat shock protein 60 precursor, Mitochondrial processing peptidase alpha chain precursor, isovaleryl-CoA dehydrogenase precursor, 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) E1-beta chain precursor, oxoglutarate dehydrogenase (lipoamide) precursor, malate dehydrogenase (NAD+) precursor (mitochondrial), ferredoxin—NADP+ reductase, long form, precursor, long-chain-acyl-CoA dehydrogenase (LCAD), aldehyde dehydrogenase (NAD+) 5 precursor (mitochondrial), peptidylprolyl isomerase 3 precursor, Carbamoyl-phosphate synthase, arginine-specific, small chain precursor (arginine-specific carbamoyl-phosphate synthetase, glutamine chain) (cps-a), D-beta-hydroxybutyrate dehydrogenase precursor (3-hydroxybutyrate dehydrogenase), hydroxymethylglutaryl-CoA lyase, pyruvate carboxylase, propionyl-CoA carboxylase beta chain precursor, glutamate dehydrogenase (NAD(P)+) 2 precursor, chaperonin-10,short/branched chain acyl-CoA dehydrogenase precursor, dodecenoyl-CoA Delta-isomerase precursor (mitochondrial), Acyl-CoA dehydrogenase, medium-chain specific precursor (MCAD), tryptophanyl-tRNA synthetase 2 (mitochondrial) (human), mitochondrial seryl-tRNA synthetase (cDNA FLJ20450 FIS, CLONE KAT05607) (human), long-chain specific precursor (LCAD), citrate synthase (mitochondrial), phenylalanine-tRNA synthetase (FARS1) mRNA, nuclear gene encoding mitochondrial protein (human), ATP synthase oligomycin sensitivity conferral protein precursor (mitochondrial), adrenodoxin precursor, cytochrome c, Probable leucyl-tRNA synthetase (mitochondrial), Pyruvate dehydrogenase (E2) dihydrolipoamide acetyltransferase, 10 KD heat shock protein mitochondrial (hsp10) (10K chaperonin) (mouse), Chaperonin homolog hsp60 precursor (heat shock protein 60) (hsp-60), aldehyde dehydrogenase (NAD+) 2 precursor (mitochondrial), glutamate dehydrogenase (NAD(P)+) precursor, dihydrolipoamide dehydrogenase precursor, pyruvate dehydrogenase (lipoamide) alpha chain precursor, pyruvate dehydrogenase (lipoamide) beta chain precursor, pyruvate dehydrogenase (lipoamide) alpha chain precursor, testis-specific (E1), 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) alpha chain precursor, malate dehydrogenase precursor (mitochondrial), superoxide dismutase (Mn) precursor, enoyl-CoA hydratase, mitochondrial (short chain enoyl-CoA hydratase (SCEH)), 4-aminobutyrate aminotransferase, mitochondrial precursor (gamma-amino-N-butyrate-transaminase) (GABA transaminase), glutaryl-CoA dehydrogenase precursor (GCD) (human), Glutaryl-CoA dehydrogenase precursor (GCD) (mouse), Probable 3-hydroxyacyl-CoA dehydrogenase F54C8.1, Probable 3-hydroxyacyl-CoA dehydrogenase B0272.3, heat shock protein 60 precursor, hydroxymethylglutaryl-CoA lyase precursor (HG-CoA lyase) (HL) (3-hydroxy-3-methylglutarate-CoA lyase), 2-oxoglutarate dehydrogenase E1 component, aldehyde dehydrogenase (NAD+) 2 precursor (mitochondrial), short-chain specific precursor (SCAD) (butyryl-CoA dehydrogenase), acyl-CoA dehydrogenase precursor, medium-chain-specific, pyruvate dehydrogenase kinase isoform 1 (human), pyruvate dehydrogenase kinase isoform 2 (human), pyruvate dehydrogenase kinase isoform 3 (human), long-chain-fatty-acid beta-oxidation multienzyme complex alpha chain precursor (mitochondrial), long-chain-fatty-acid beta-oxidation multienzyme complex beta chain precursor (mitochondrial), pyruvate carboxylase precursor, 3-hydroxyacyl-CoA dehydrogenase, short chain-specific, precursor, nucleoside-triphosphate—adenylate kinase 3, dimethylglycine dehydrogenase, mitochondrial precursor (ME2GLYDH) (human), malate dehydrogenase mitochondrial precursor, mitochondrial processing peptidase beta subunit precursor (beta-MPP) (P-52), 2-oxoglutarate dehydrogenase E1component (alpha-ketoglutarate dehydrogenase), Probable pyruvate dehydrogenase E1component-alpha subunit precursor (PDHE1-a), ornithine carbamoyltransferase precursor, alanine—glyoxylate aminotransferase, Delta-1-pyrroline-5-carboxylate dehydrogenase precursor (P5C dehydrogenase), VAR1-mitochondrial ribosomal protein, mitochondrial processing peptidase alpha subunit precursor (alpha-MPP) (P-55) (HA1523), pyruvate dehydrogenase kinase isoform 4 (human), thiosulfate sulfurtransferase, aspartate transaminase precursor (mitochondrial), methylmalonyl-CoA mutase alpha chain precursor, NADH dehydrogenase (ubiquinone) 40K chain, pyruvate dehydrogenase (lipoamide), dihydrolipoamide S-acetyltransferase heart-human, probable DNA-directed RNA polymerase-mitochondrion plasmid maranhar (SGC3), electron transfer flavoprotein beta chain, 3,2-trans-enoyl-CoA isomerase, mitochondrial precursor (dodecenoyl-CoA delta-isomerase), 3-methyl-2-oxobutanoate dehydrogenase (lipoamide) beta chain, methylmalonyl-CoA mutase precursor (MCM), hypothetical protein t05g5.6, citrate (si)-synthase (mitochondrial), Lon proteinase homolog, citrate synthase homolog, heat shock protein 10, malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+) precursor (mitochondrial), ferredoxin—NADP+reductase precursor, dihydrolipoamide transacylase precursor, branched chain alpha-ketoacid dehydrogenase chain E1-alpha precursor, Succinyl-CoA:3-ketoacid-coenzyme A transferase precursor (succinyl CoA:3-oxoacid CoA-transferase) (OXCT), 5-aminolevulinate synthase 2,5-aminolevulinate synthase 1 precursor, Probable leucyl-tRNA synthetase, mitochondrial precursor (Leucine—tRNA ligase) (Leurs) (KIAA0028), 5-aminolevulinate synthase mitochondrial precursor (erythroid-specific) (ALAS-E), leucine—tRNA ligase precursor (mitochondrial), tyrosine—tRNA ligase precursor (mitochondrial), Probable tryptophanyl-tRNA synthetase (mitochondrial) (tryptophan—tRNA ligase) (TRPRS), thiosulfate sulfurtransferase, mitochondrial intermediate peptidase, pyruvate dehydrogenase complex protein X subunit precursor, aspartate transaminase precursor (mitochondrial), ornithine—oxo-acid transaminase precursor, serine—pyruvate aminotransferase (SPT) (alanine—glyoxylate aminotransferase) (AGT), ornithine—oxo-acid transaminase precursor, dihydrolipoamide S-acetyltransferase precursor, GCV3-glycine decarboxylase, subunit H, PIM1- ATP-dependent protease (mitochondrial), MRPL16-ribosomal protein of the large subunit (mitochondrial), PET112- required to maintain rho+mitochondrial DNA, MRP21- Mitochondrial ribosomal protein, CBP6-apo-cytochrome B pre-mRNA processing protein, MRPL36- ribosomal protein YmL36 precursor (mitochondrial), MRPS9- ribosomal protein S9 precursor (mitochondrial), MCX1- C1pX homologue in mitochondria, MRPS5- ribosomal protein S5 (mitochondrial), MRPL37- ribosomal protein YmL37 (mitochondrial), MRPL27- ribosomal protein YmL27 precursor (mitochondrial), MRPL32- ribosomal protein YmL32 (mitochondrial), asn-tRNA synthetase (mitochondrial), RIM1 - ssDNA-binding protein (mitochondrial), IMG1 - ribosomal protein (mitochondrial), MRPL11- ribosomal protein of the large subunit (mitochondrial), KGD2-2-oxoglutarate dehydrogenase complex E2 component, MSS116- RNA helicase of the DEAD box family (mitochondrial), MRPL28- ribosomal protein of the large subunit (YmL28) (mitochondrial), LPD1- dihydrolipoamide dehydrogenase precursor, succinate-CoA ligase beta subunit, superoxide dismutase (Mn) precusor mitochondrial (SOD2), MAM33- mitochondrial acidic matrix protein, MRPL49- ribosomal protein YmL49 (mitochondrial), RSM7- similarity to bacterial, chloroplast and mitochondrial ribosomal protein S7, NFU1 - iron homeostasis, ISA1-mitochondrial protein required for normal iron metabolism, REX2- putative 3′-5′ exonuclease, COQ5 - ubiquinone biosynthesis, methyltransferase, ECM40 - acetylornithine acetyltransferase, ABF2 - high mobility group protein, HMI1- mitochondrial DNA helicase, GLO4-glyoxalase II (hydroxyacylglutathione hydrolase), LSC1- succinate-CoA ligase alpha subunit, MRP51- strong similarity to S.kluyveri hypothetical protein, ISU1 - protein with similarity to iron-sulfur cluster nitrogen fixation proteins, YAH1- similarity to adrenodoxin and ferrodoxin, FUM1-fumarate hydratase, MSF1 - phenylalanine—tRNA ligase alpha chain (mitochondrial), and ISA2- mitochondrial protein required for iron metabolism.

### Claim 3 (depends on 1)

The fusion protein of claim 1, wherein the mitochondrial localization signal comprises the mitochondrial localization signal of a superoxide dismutase (Mn).

### Claim 4 (depends on 1)

The fusion protein of claim 1, wherein the mitochondrial localization signal comprises amino acids 1-24 of a human superoxide dismutase (Mn).

### Claim 5 (depends on 1)

The fusion protein of claim 1, wherein the protein transduction domain comprises 8-15 amino acid residues.

### Claim 6 (depends on 1)

The fusion protein of claim 1, wherein the protein transduction domain comprises amino acids 48-60 of the HIV-1 TAT protein, the third helix of Antennapedia homeodomain, a poly-arginine region, 11 arginine residues, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:207, or a combination thereof.

### Claim 7 (depends on 1)

The fusion protein of claim 1, wherein the mature TFAM comprises amino acids 56-259 of SEQ ID NO:210.

### Claim 8

A transcription factor A-mitochondrial (TFAM) fusion protein comprising a protein transduction domain, a mitochondrial localization signal, and a transcription factor A-mitochondrial (TFAM) polypeptide comprising amino acids 60-130 and 165-231 of SEQ ID NO:210.

### Claim 9 (depends on 8)

The fusion protein of claim 8, wherein the TFAM polypeptide comprises amino acids 56-259 of SEQ ID NO:210.