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WO1999007723A1 - Vehicule de fixation d'apport d'acide nucleique - Google Patents

Vehicule de fixation d'apport d'acide nucleique Download PDF

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Publication number
WO1999007723A1
WO1999007723A1 PCT/US1998/013710 US9813710W WO9907723A1 WO 1999007723 A1 WO1999007723 A1 WO 1999007723A1 US 9813710 W US9813710 W US 9813710W WO 9907723 A1 WO9907723 A1 WO 9907723A1
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Prior art keywords
nucleic acid
peptide
vehicle
gly
utarve
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Inventor
Laure Aurelian
Michael Kulka
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University of Maryland Baltimore
University of Maryland College Park
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University of Maryland Baltimore
University of Maryland College Park
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Priority to AU81798/98A priority Critical patent/AU8179898A/en
Publication of WO1999007723A1 publication Critical patent/WO1999007723A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a nucleic acid uptake and release vehicle comprising a protein wherein a ligand and an endosomolytic peptide are fused to provide a vehicle which can be utilized to enhance the intracellular delivery of moieties consisting of nucleic acids oligonucleotides or modified nucleic acid or oligonucleotides.
  • the invention may be modified by the incorporation of amino acids or peptides to provide additional sites of nucleic acid attachment and to impart flexibility to the secondary structure
  • the invention may be further modified by the incorporation of a nuclear localization signal.
  • the vehicle can also contain a polylysylleucyl peptide so as to provide additional nucleic acid attachment sites; as well as a nuclear localization signal peptide so as to enhance intranuclear localization of the nucleic acid.
  • BACKGROUND OF THE INVENTION Genes have been used to treat certain diseases.
  • anti-sense oligonucleotides and modified oligonucleotides have been used to treat diseases, such as viral infections, cancer, and genetic defects, so as to inhibit genes that are known to be associated with these diseases.
  • diseases such as viral infections, cancer, and genetic defects
  • nucleic acid molecules into cells at the levels required for such therapy.
  • An object of the present invention is to provide a nucleic acid uptake and release vehicle.
  • Another object of the present invention is to provide a DNA construct encoding a nucleic acid uptake and release vehicle. Still another object of the invention is to provide a method of intracellular delivery of therapeutic nucleic acid molecules using the nucleic acid uptake and release vehicle.
  • UTARVE nucleic acid uptake and release vehicle
  • UTARVE may be modified by the incorporation of amino acids or peptides to provide additional sites of nucleic acid attachment and to impart flexibility to the secondary structure
  • UTARVE may also be modified by the incorporation of a nuclear localization signal.
  • UTARVE nucleic acid uptake and release vehicle
  • the UTARVE also comprises: (iii) a polylysylleucyl (KL) m peptide. In still a more preferred embodiment, the UTARVE also comprises:
  • the above-described objects of the present invention have been met by a method of intracellular delivery of therapeutic nucleic acid molecules comprising contacting cells with a UTARVE-nucleic acid complex.
  • Figure 1 shows HSV-1 growth inhibition in Vero cells, where the Vero cells were exposed to the UTARVE- 1E4.5S A complex (•) or the UTARVE-IE1TI complex (o), and infected with HSV-1.
  • FIG. 2 shows HSV-1 growth inhibition in Vero cells, where the Vero cells were exposed to the UTARVE-1E4,5SA complex (open bar) or uncomplexed 1E4,5SA (solid bar), and infected with HS V- 1.
  • the present invention relates to a UTARVE comprising: (i) an adenovirus (Ad) penton base protein; and
  • an influenza virus hemagglutinin endosomolytic peptide (Gly n -HA-Gly n ).
  • the UTARVE targets the cell surface, and causes endosomolysis and increases membrane permeability for the nucleic acid molecules.
  • the Ad penton base protein contains a receptor binding site motif (RGD) for attachment to the ubiquitous cell receptors - integrins (Bai et al, J Virol., 67:5198-5205
  • peernton base protein refers to the entire Ad penton base protein or fragments thereof which include at least amino acids 1-354 which contain the receptor binding motif.
  • the particular adenovirus from which the Ad penton base protein sequence is derived is not critical to the present invention. Examples of such adenoviruses include Ad2, Ad5 and Ad3.
  • the amino acid/nucleic acid sequences encoding Ad penton base proteins are well- known in the art. For example, the amino acid/nucleic acid sequence of the Ad2 penton base protein is described by Roberts et al ( J Biol.
  • influenza HA endosomolytic peptide provides the endosomolytic function necessary for intracellular release of the UTARVE after its receptor mediated endocytosis (Wagner et al, Proc. Natl. Acad. Sci., USA, 89:7934-7938, 1992).
  • the expression "influenza HA endosomolytic peptide” refers to the wild-type sequence, as well as mutants thereof which retain the endosomolytic function, e.g., a mutant wherein Gly 4 is mutated to Glu 4 ; and a mutant with, e.g., 2-5 additional amino acids at the amino or carboxy terminus.
  • amino acid sequence encoding the wild-type HA endosomolytic peptide, as well as the mutant thereof wherein Gly 4 is mutated to Glu 4 are well-known in the art (Wharton et al, J. Gen. Virol, 69:1847-1857, 1988); Wagner et al, Proc. Natl. Acad. Sci., USA, 89:7934-7938, 1992; and Plank et al, J. Biol. Chem., 269:12918-12924, 1994).
  • the glycine (Gly) residues flanking the HA endosomolytic peptide permit formation of ⁇ -helices, and, e.g., impart flexibility to the secondary structure at the HA-penton base protein junction.
  • the number of Gly residues flanking the endosomolytic HA peptide, i.e., the value of n, is not critical to the present invention, but generally ranges from between 0 to 12, preferably 1 to 8.
  • the UTARVE also comprises:
  • (iii) a polylysylleucyl (KL) m peptide (iii) a polylysylleucyl (KL) m peptide.
  • the (KL) m peptide provides additional sites for complexing with the nucleic acid molecule.
  • the K residues (Lys) interact with the nucleic acid, while the L residues (Leu) decrease potential stearic hindrance resulting from adjacent charged Lys residues.
  • the value of m is not critical to the present invention, but generally represents from 1 to 300 alternating lysine (K) and leucine (L) residues, preferably from 3 to 100 alternating lysine (K) and leucine (L) residues.
  • (KL) m can be used as the basis for generating tandem repeats of (KL) m in an intermediate vector prior to, e.g., insertion adjacent to the penton base protein coding sequence, e.g., the Ad2 penton base protein coding sequence in pETllaHA/PB described in Example 1 below.
  • the penton base protein coding sequence e.g., the Ad2 penton base protein coding sequence in pETllaHA/PB described in Example 1 below.
  • the UTARVE also comprises: (iv) a nuclear localization signal peptide.
  • the particular nuclear localization signal (NLS) peptide employed is not critical to the present invention.
  • NLS peptides include the NLS peptide of the SV40 large T antigen (Van Dermonne et al, TIBS, 21:59-64, 1996);and those described by Boulikas, Critical Reviews in Eukaryotic Gene Expression, 3:193-227, 1993), the NLS peptides listed in said references are incorporated by reference herein in their entirety.
  • NLS peptide coding sequences can be introduced into DNA encoding a UTARVE by, for example, preparing a dsDNA encoding the NLS peptide of SV40 large T antigen using the following sense and anti-sense primers, respectively.
  • Lys Val Glu Asp Pro (SEQ ID NO:3), flanked by sequences for restriction endonuclease cleavage by Bgll (italic) and Bell (underlined).
  • This sequence includes the phosphorylation site for casein kinase II (CKII) which is required for optimal NLS function (Gams et al, Oncogene, 9:2961-2968, 1994).
  • CKII casein kinase II
  • the double-stranded 87 nucleotide DNA molecule can be inserted into a plasmid encoding (KL) m , i.e., p(KL) m , e.g., p(KL) 10 .
  • KL plasmid encoding
  • p(KL) m plasmid encoding
  • p(KL) 10 plasmid encoding
  • such a sequence can be inserted downstream of KL at the BcR site or upstream at the BgRl site or both. This results in one or two NLS sequences per UTARVE molecule.
  • the orientation of the elements in UTARVE is not critical to the present invention.
  • UTARVE can contain the elements covalently linked via peptide bonds in the following N-terminal to C-terminal (5' to 3' in the DNA) orientation shown in the Table below:
  • Ad penton base protein (Gly n -HA-Gly n ) peptide; Gly n -HA-Gly n peptide: Ad penton base protein;
  • Ad penton base protein Gly n -HA-Gly n peptide:(KL) m peptide;
  • Ad penton base protein (KL) m peptide: Gly n -HA-Gly n peptide;
  • Gly n -HA-Gly n peptide Ad penton base protein: (KL) m peptide;
  • Gly n -HA-Gly n peptide (KL) m peptide: Ad penton base protein; (KL) m peptide: Ad penton base protein Gly n -HA-Gly n peptide;
  • Ad penton base protein Gly n -HA-Gly n peptide: NLS peptide;
  • Ad penton base protein NLS peptide: Gly n -HA-Gly n ;
  • Gly n -HA-Gly n peptide Ad penton base protein: NLS peptide; Gly n -HA-Gly n peptide: NLS peptide: Ad penton base protein;
  • NLS peptide Ad penton base protein: Gly n -HA-Gly n peptide;
  • Ad penton base protein Gly n -HA-Gly n peptide:(KL) m peptide: NLS peptide;
  • Ad penton base protein Gly n -HA-Gly n peptide: NLS peptide: (KL) m peptide
  • Ad penton base protein NLS peptide: Gly n -HA-Gly n peptide: (KL) m peptide
  • Gly n -HA-Gly n peptide (KL) m peptide
  • Ad penton base protein (KL) m peptide: Gly n -HA-Gly n peptide: NLS peptide;
  • Ad penton base protein (KL) m peptide: NLS peptide: Gly n -HA-Gly n peptide;
  • Ad penton base protein NLS peptide:(KL) m peptide: Gly n -HA-Gly n peptide;
  • Gly n -HA-Gly n peptide Ad penton base protein: (KL) m peptide: NLS peptide; Gly n -HA-Gly n peptide: Ad penton base protein: NLS peptide: (KL) m peptide;
  • NLS peptide Ad penton base protein: (KL) m peptide
  • Gly n -HA-Gly n peptide (KL) m peptide: Ad penton base protein: NLS peptide;
  • Gly n -HA-Gly n peptide (KL) m peptide: NLS peptide: Ad penton base protein;
  • Gly n -HA-Gly n peptide NLS peptide:(KL) m peptide: Ad penton base protein; (KL) m peptide: Ad penton base protein: Gly n -HA-Gly n peptide: NLS peptide
  • (KL) m peptide Ad penton base protein: NLS peptide: Gly n -HA-Gly n peptide; (KL) m peptide: NLS peptide: Ad penton base peptide: Gly n -HA-Gly n peptide; (KL) m peptide: Gly n -HA-Gly n peptide: Ad penton base protein: NLS peptide; (KL) m peptide: Gly n -HA-Gly n peptide: NLS peptide : Ad penton base protein; (KL) m peptide: NLS peptide: Gly n -HA-Gly n peptide: Ad penton base protein; (KL) m peptide: NLS peptide: Gly n -HA-Gly n peptide: Ad penton base protein; NLS peptide: Ad penton base protein: Gly n -HA-Gly n
  • NLS peptide Ad penton base protein: (KL) m peptide: Gly n -HA-Gly n peptide; NLS peptide: Gly n -HA-Gly n peptide: Ad penton base protein: (KL) m peptide; NLS peptide: Gly n -HA-Gly n peptide:(KL) m peptide: Ad penton base peptide; NLS peptide:(KL) m peptide: Ad penton base protein: Gly n -HA-Gly n peptide; and NLS peptide: (KL) m peptide: Gly n -HA-Gly n peptide: Ad penton base protein.
  • UTARVE The mechanism of action of UTARVE involves increased intracellular delivery (uptake) of the nucleic acid molecules, and increased release of the nucleic acid molecules from endocytic-like vesicles in which they are entrapped during intracellular delivery.
  • the UTARVE is useful for enhancing the intracellular delivery of nucleic acid molecules, such as single or double stranded anti-sense or sense oligonucleotides for inhibition of gene expression, or single or double stranded sense oligonucleotides for gene delivery in gene therapy.
  • nucleic acid molecule is not critical to the present invention.
  • examples of such include eukaryotic or viral gene expression vectors under complete, partial or no eukaryotic or non-eukaryotic regulatory control, which express RNA transcripts that are anti- sense to viral or cellular RNA transcripts.
  • anti-sense oligonucleotides are useful to inhibit, reduce or alter expression from the viral or cellular transcripts associated with or critical to the infection/disease state and/or process.
  • pMK53 which expresses anti-sense HSV-2 ICP10 mRNA under the control of a viral gene promoter, such as the
  • Cytomegalovirus immediate early (CMV, IE) promoter has been used to inhibit ICP10 expression and induce apoptosis in cells which express both HSV-2 ICP10, as well as the cellular homolog of ICP10 (Smith et al, Virus Genes, 5:215-226, 1991).
  • anti-sense oligonucleotide employed is not critical to the present invention.
  • anti-sense oligonucleotides include:
  • Antisense IE45SA, IE1T1, IE3T1, IE4T1, which have been used to inhibit expression of HSV-1 immediate early genes IE4, IE1, IE3 and IE4, respectively, as well as HSV-1 growth (Kulka et al, Proc. Natl. Acad. Sci., USA, 86:6868-6872, 1989; and Kulka et al, Antimicrob. Agents Chemother., 38:675-680, 1994).
  • These sense oligonucleotides are useful in order to inhibit, reduce or alter protein- protein interactions or protein-nucleic acid interactions or nucleic acid-nucleic acid interactions, or expression from the viral or cellular transcripts or gene promoters associated with or critical to the infection/disease state and/or process.
  • sense oligonucleotide employed is not critical to the present invention.
  • sense oligonucleotides include:
  • Sense tissue factor which has been used to overexpress tissue factor in order to determine the role of angiogenesis in tumor growth (Zhang, J. Clin. Invest., 92:1320-1327, 1994);
  • eukaryotic or viral gene expression vectors under complete, partial or no eukaryotic or non-eukaryotic regulatory control, which express catalytic RNAs or ribozymes which cleave covalent bonds in target RNA are useful in order to inhibit, reduce or alter expression from the viral or cellular transcripts associated with or critical to the infection/disease state and/or process, can be employed in the present invention as the nucleic acid molecule. Examples of such include:
  • Anti-ras ribozyme which has been used to cleave the activated H-ras oncogene and thereby alter the malignant phenotype of an invasive human bladder cancer cell line (Eastham et al, J.UroL, 156:1186-1188, 1996).
  • the UTARVE-nucleic acid complexes are used as anti- viral agents, e.g., for the treatment of herpesvirus infections, as well as other human virus infections which can cause disease including the following virus families: Picornaviridae, e.g., Coxsackie virus; Togaviridae, e.g., Eastern equine encephalitis virus; Flaviviridae, e.g., St.
  • Picornaviridae e.g., Coxsackie virus
  • Togaviridae e.g., Eastern equine encephalitis virus
  • Flaviviridae e.g., St.
  • Coronaviridae e.g., Coronavirus
  • Rhabdoviridae e.g., Rabies virus
  • Filoviridae e.g., Ebola virus
  • Paramyxoviridae e.g., Respiratory syncytial virus
  • orthomyxoviridae e.g., Influenza virus
  • Bunyaviridae e.g., Rift valley fever virus
  • Arenaviridae e.g., Lassa fever virus
  • Reoviridae e.g., Human rotavirus
  • Calciviridae e.g., Norwalk virus
  • Retroviridae e.g., HIV
  • Hepadnaviridae e.g., Hepatitis B virus
  • Parvoviridae e.g., Human parvovirus B-19
  • Papovaviridae e.g., Human papillomavirus
  • Adenoviridae e.g., Human adenovirus
  • Poxviridae e.g., Vaccinia virus.
  • the UTARVE complexes are used as anti-viral agents for the treatment of animal virus infections that can cause disease including the following virus families: Picornaviridae, e.g., Foot-and-mouth disease virus; Togaviridae, e.g., Eastern equine encephalitis virus; Flaviviridae, e.g., Tick-borne encephalitis virus; Coronaviridae, e.g., Feline infectious peritonitis virus; Rhabdoviridae, e.g., Rabies virus; Filoviridae, e.g., Ebola virus; Paramyxoviridae, e.g., Canine distemper virus; orthomyxoviridae, e.g., Influenza viruses swine, horse and fowl; Bunyaviridae, e.g., Rift valley fever virus; Arenaviridae, e.g., LCM virus; Reoviridae, e
  • virus families
  • the UTARVE complexes can be used in the treatment of genetic diseases, such as hemophilia, adenosine deaminase deficiency, beta- thalassemia, diabetes and cystic fibrosis.
  • the UTARVE complexes can be used in the treatment of cancers, such as breast cancer, cervical cancer, lung cancer, bladder cancer, prostate cancer and acute lymphocytic leukemia (ALL) for which potentially involved genes have been identified.
  • cancers such as breast cancer, cervical cancer, lung cancer, bladder cancer, prostate cancer and acute lymphocytic leukemia (ALL) for which potentially involved genes have been identified.
  • ALL acute lymphocytic leukemia
  • the UTARVE complexes may also contain oligonucleotides, DNA, or expression vectors designed to inhibit, complement or replace the defective, aberrantly regulated and/or dysfunctional gene(s) associated with or critical to the targeted genetic disorder, or to the development and/or maintenance of the neoplastic state.
  • UTARVE complexes with non-integrin cell receptors, the endosomolysis following UTARVE-complex uptake and the efficient release of nucleic acid molecules which are covalently bound to the lysine residues in the Ad penton base protein or in the (KL) m peptide, represent important issues/mechanisms relevant to the successful delivery of nucleic acid molecules to target cell/tissues.
  • UTARVE design and versatility enables variation of its construction in order to address these important issues.
  • protein ligands or sugars or other moieties which can bind specifically to cell surface receptors may be cross-linked to UTARVE so as to alter/increase the binding specificity and uptake of the UTARVE-nucleic acid complex with the target cell/tissue.
  • Cross-linking can be effected by any one of a variety of commercially available cross-linking/derivatizing agents and procedures, e.g.: (1) BMH, which is reactive with sulfhydryl groups (Chen et al, J.Biol. Chem.,
  • protein ligands examples include transferrin, cholera toxin B subunit, and epidermal growth factor.
  • sugars containing mannose and/or galactose include complex sugars containing mannose and/or galactose, and their derivatives. Complexation of nucleic acid molecules to UTARVEs is accomplished so that:
  • the complex can also be obtained by ionic bonding (for DNA vectors) or covalent bonding (for oligonucleotides) to the lysine residues in the (KL) m peptide or in the Ad penton base protein in the UTARVE.
  • the nucleic acid molecule of interest is reacted with purified UTARVE in PBS over a range of molarity calculated to ionically bind, i.e., neutralize from 10%-50% of the negative charges on the nucleic acid molecule.
  • a quantity of poly-L-lysine sufficient to neutralize the remainder of the charges can be added to achieve electroneutrality of the input nucleic acid molecule.
  • the oligonucleotides can be derivatized such that they have a thiol (SH) group positioned on the alkyl side chain located on the 5' terminal nucleotide, and then conjugated to NH 2 groups of lysine in the (KL) m peptide or in the Ad penton base protein as described by Orgel et al, Nucl. Acids Res., 16:3671 (1988); and King et al, Biochem., 17:1499-1506 (1978), resulting in the attachment of the oligonucleotides to the UTARVE through disulfide, -S-S-, bridges.
  • SH thiol
  • the primary means for intracellular release of oligonucleotides from the UTARVE is provided by the phosphodiester bond located between the nucleotides of the oligonucleotide that are susceptible to hydrolysis by endonucleases inside of the cell (Levis, J, Ph.D. Dissertation, The Johns Hopkins University, 1995).
  • Cleavage of the disulfide bond occurs after internalization and acidification of the UTARVE-S-S-oligonucleotide containing endosomal vesicle, thereby providing an alternative means for intracellular release of the oligonucleotide from the carrier.
  • oligonucleotides to UTARVE can be used, e.g., linkage chemistry based on an oligonucleotide derivative that has a carboxyl group attached to an alkyl linker arm on the 5' terminal nucleotide.
  • the carboxyl moiety can be covalently attached to the NH 2 groups of lysine as described by Salter et al, EE5S Letters, 20:302-306 (1972).
  • the conjugation of oligonucleotides to lysine residues requires the use of the activating agent ⁇ DAC [l-ethyl-3- (3-dimethylaminopropyl) carbodiimide] that reacts with the carboxyl group on the oligonucleotide derivative to form an urea-type intermediate which is then susceptible to nucleophilic attack by the NH 2 groups of pL.
  • the end product of this conjugation reaction is the attachment of the oligonucleotide to UTARVE through an amide, -NH-CO-, i.e., peptide, linkage. This amide bond is not susceptible to cleavage under conditions that might reduce the -S-S- bond. Intracellular release of the oligonucleotide will rely on the cleavage of phosphodiester bonds in the nucleic acid molecule.
  • Another conjugation method that can be employed involves making use of aminoxy functional groups positioned on the 5' terminal nucleotide of the oligonucleotide. This involves reaction of the NH2 groups of lysine with glyoxylic acid or the n- hydroxyphthalimide ester of glyoxylic acid to derivatize the lysine. The derivatized moieties are then conjugated to the oligonucleotide derivative through formation of an oxime bond, as described by Rose et al, J.Am. Chem. Soc. 116:30-33 (1994). The oxime linkage is stable over the pH range of 2-7, and the pH in the endosomal vesicles is in the acidic range. Thus, release of the oligonucleotide from the complex will rely on the cleavage of the phosphodiester bond located between the first and second nucleotide of the oligonucleotide (Levis, supra).
  • the.native backbone, phosphodiester, of the oligonucleotides can be replaced with a synthetic backbone, e.g., methylphosphonate, phosphorothioate, phosphodithioate, phosphoramidate; a mixed backbone, e.g., native and/or synthetic; oligoribonucleotides and their 2' modified derivatives, e.g., 2'-O-methylriboside phosphodiesters, 2 '-O-methylriboside methylphosphonates, or alternate 2 '-O-methylriboside methylphosphonates/phosphodiesters (alt-mr-OMP) which are 5', 3' and/or internally derivatized to contain a photofluor, e.g., BODIPY, and/or any cross- linking moieties, e.g.,
  • the nucleic acid molecule is modified such that the phosphodiester backbone is replaced with alt-mr-OMP or phosphorothioate backbones.
  • the oligonucleotides are resistant to intracellular nucleases, but one phosphodiester bond remains located between the first and second nucleotide that is susceptible to hydrolysis by endonucleases inside the cell (Levis, supra)
  • the above-described objects of the present invention have been met by a method of intracellular delivery of therapeutic nucleic acid molecules comprising contacting cells with a UTARVE-nucleic acid complex.
  • ⁇ -galactosidase expression DNA construct pCMV ⁇ : CMVIE promoter/E. coli ⁇ -galactosidase gene cassette, Clontech, Palo Alto, CA
  • ⁇ -galactosidase expression provides the enzymatic marker necessary to determine the efficiency of DNA transfection efficiency by
  • UTARVE UTARVE- ⁇ -galactosidase nucleic acid complexes under conditions which examine a range of UTARVE concentrations and times of incubation.
  • cells are screened for ⁇ -galactosidase expression by 5-bromo- 4-chloro-3-indolyl- ⁇ D-galactoside (X-GAL) as described by Nielsen et al, Proc. Natl. Acad. Sci, USA, 80:5198-5202 (1983). Cell staining is observed by phase contrast light microscopy, and the transfection efficiency calculated as the percentage X-GAL stained cells.
  • X-GAL 5-bromo- 4-chloro-3-indolyl- ⁇ D-galactoside
  • limiting dilutions of the complexes can be evaluated for the capacity to transfer detectable levels of fluorescence using UTARVE-nucleic acid molecule-BODIPY, or other photofluors, e.g., fluorescein.
  • logarithmic dilutions of the complexes in 2.0% (v/v) fetal calf serum/DMEM are applied to 5.0 x 10 5 cells (e.g., HeLa, Vero, or MRC5 cells) selected for the presence of different levels of receptors for the UTARVE on the cell surface.
  • various molar ratios of complex components are evaluated for the capacity to mediate transfer of nucleic acid molecules.
  • Intracellular localization can be determined by double immunofluorescence. Staining with fluorescein-labeled antibodies to coated vesicle proteins (viz. clathrin) can be carried out to determine if co-localization occurs. This can be carried out immediately after cell treatment, i.e., 1, 5, 10, 30, 60 mins, since endosomolysis mediated by viral proteins occurs almost upon delivery.
  • fluorescence e.g., red in cells given only UTARVE-nucleic acid complex and yellow in cells given the complex and stained with fluorescein-labeled anti-clathrin antibody, will be originally punctate, and become diffusely distributed throughout the cell at later times post infection.
  • Staining with fluorescein-conjugated HA antibody can also be carried out to: (i) establish whether green fluorescence (UTARVE) is dissociated from red fluorescence (nucleic acid molecules), and (ii) define the kinetics of dissociation.
  • UTARVE green fluorescence
  • red fluorescence nucleic acid molecules
  • the ability of the nucleic acid molecules, delivered as a UTARVE-nucleic acid complex, to inhibit expression of the target gene, and the biological activity thereof, can be determined with unlabeled nucleic acid molecules.
  • the results are then compared to recombinant adenovirus co-exposure in order to determine whether UTARVE is a superior delivery method. That is, cells are treated with the UTARVE-nucleic acid complex and transfected with the respective transcription unit, e.g., IE110 DNA or infected with HSV-1.
  • the parameters are those which achieve optimal intracellular bioavailability. Gene expression and function alteration is determined 16-72 hrs later. Activity is expressed as the IC 50 and IC 90 of the complexed nucleic acid molecules.
  • UTARVE-nucleic acid complex can be administered topically either proximal and/or distal to the site of disease, to skin, mucous membranes and or eye in the absence or presence of creams/ointments/lipid carriers, e.g., polyethylene glycol or liposomes, designed to facilitate complex uptake and/or stability at the site of topical application and/or disease.
  • the UTARVE-nucleic acid complex can also be administered intradermally either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and or stability at the site of injection and/or disease.
  • the UTARVE-nucleic acid complex can also be administered subcutaneously either proximal and or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the UTARVE-nucleic acid complex can also be administered intramuscularly either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the UTARVE-nucleic acid complex can also be administered intravenously by injection and/or infused intravenously either proximal and/or distal to the site of disease in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and or stability at the site of injection and/or disease.
  • the UTARVE-nucleic acid complex can also be administered nasally or orally by inhalation and/or ingestion either proximal or distal to the site of disease either contained within (or not) biodegradable capsules in the presence of buffered physiologic saline and/or other solutions containing (or not) lipid carriers designed to facilitate complex uptake and/or stability at the tissue/organ of administration and/or disease.
  • the UTARVE-nucleic acid complex can also be administered ex vivo to cell cultures and/or tissues by incubation in cultures/growth media containing (or not) lipid carriers, e.g., liposomes, designed to facilitate complex uptake and/or stability at the site of injection and/or disease.
  • the amount of UTARVE-nucleic acid complex to be administered will vary depending upon the age, weight, sex, and species of the subject (cells), as well as the disease to be treated and the nucleic acid molecule to be used. However, typically, the UTARVE- nucleic acid complex will be administered in an amount of from about 0.1 nM to 100 ⁇ M, preferably from about 0.1 nM to 10 ⁇ M.
  • a prototype UTARVE was constructed by assembly of DNA sequences encoding: (i) the adenovirus type 2 penton base protein, which contains the RGD motif that binds the ubiquitous cell surface receptors-integrin, and is involved in receptor mediated uptake and endosomolysis, and (ii) an endosomolytic peptide derived from influenza virus HA protein.
  • the HA peptide was used to increase the endosomolytic activity of the purified penton base protein.
  • a dsDNA encoding the HA endosomolytic peptide (20 amino acids) plus 5' and 3' flanking sequences (Gly) were generated by PCR amplification of a primary single-stranded 70 nucleotide (nt) DNA sequence.
  • the Gly residues permit formation of a- helices flanking the HA peptide (Gly 2 -HA-Gly 2 ), thereby imparting flexibility to the secondary structure at the HA-penton base junction.
  • Gly 2 -HA-Gly 2 was cloned into pETl laPB, a bacterial expression vector which contains the entire Ad2 penton base protein under the control of a T7 phage promoter, by blunt-end ligation in the Nhel site
  • PETllaHA/PB located upstream of the penton base protein initiator Met.
  • the resulting plasmid, PETllaHA/PB codes for a chimeric protein that begins with Gly 2 -HA-Gly 2 (initiator Met is encoded by vector), followed by the penton base protein coding sequence. Positive clones were identified by sequencing.
  • a dsDNA sequence top strand: 5'- GAGGTGGTGGTGG-3' (SEQ ID NO:5) encoding Gly 2 -Gly 2 (no HA) was cloned into pETllaPB by blunt-end ligation in the Nhel site (mung bean nuclease treated) located upstream of the penton base protein initiator Met.
  • the resulting plasmid pETl la ⁇ HA/PB codes for a chimeric protein that begins with Gly 2 -Gly 2 (initiator Met is encoded by vector), followed by the penton base protein coding sequence. Positive clones were identified by sequencing.
  • the Ad2 Penton Base Protein pETllaPB was constructed as described by Bai et al, J. Virol., 67:5198-5205 (1993).
  • pETl 1 aPB contains the entire Ad2 penton base protein under the control of the T7 phage promoter.
  • the Ad2 penton base protein reading frame begins pETl laPB, which also encodes a four-amino acid extension (Met-Ala-Ser-Thr) at the N-terminal of the Ad2 penton base protein, and continues with the first amino acid (Met) of the Ad2 penton base protein sequence. All of the signals for transcriptional and translational regulation of the Ad2 penton base protein gene are present within pETllaPB.
  • This plasmid also contains an ampicillin resistance gene for positive (bacterial transformation) selection, as well as sequences important for its growth and replication in E. coll only E. coli strains, such as BL21(DE3) (Studier et al, Methods Enzymoi, 185:60-89, 1990), which contain integrated copies of T7 RNA polymerase, are suitable for expression, isolation and purification of the Ad2 penton base protein after transformation with PETllaPB.
  • a DNA molecule encoding the influenza HA endosomolytic peptide was first produced, and then cloned into PETllaPB.
  • a dsDNA molecule was prepared which encodes the 20 amino acid HA endosomolytic peptide plus, 5' and 3' flanking sequences that provide the nucleotides suitable for cloning into pETllaPB, as well as encoding for glycine residues that flank HA (Gly 2 -HA-Gly 2 ).
  • the dsDN-A was generated by PCR amplification of the -following primary single-stranded 70 bp DNA sequence:
  • primers each contain five 5' terminal nucleotides designed to encode for 2 glycines upon ligation into the Nhel site of pETllaPB.
  • PETllaPB was then digested with Nhel to cleave at a site located between the second and third amino acids (Ala and Ser) of the Ad2 penton base protein 4 amino acid extension (Met-Ala-Ser-Thr) in pETllaPB and blunt ended with mung bean nuclease.
  • the PCR amplified 70 bp fragment was ligated to the digested pETllaPB, to give rise to plasmid pETllaHA/PB.
  • pETllaHA/PB encodes for a fusion protein that begins with a start Met followed by Gly 2 -HA-Gly 2 , a Thr residue and the Ad2 penton base protein coding sequence.
  • E. coli strain DH5 ⁇ was transformed with PETllaHA/PB, and growth selected on
  • Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Munl(an isoschizomer of Mfel) restriction enzyme analysis. Plasmids containing both the Gly 2 -HA-Gly 2 and Ad2 penton base protein sequence gain an additional Muni restriction site (present in the HA sequence).
  • flanking glycine residues may be varied according to the length of the primary nucleotide sequences which flank the HA encoding region.
  • the Gly 2 -HA-Gly 2 sequence may be extended at the 5' and 3' ends by PCR amplification using sense and anti-sense primers that contain terminal sequences which encode ' for additional glycines.
  • the strategy used to clone Gly n -HA-Gly n sequences (where n > 2) into pETllaPB, and confirm the success of the cloning is similar to that used for Gly 2 - HA-Gly 2 .
  • Another prototype UTARVE was constructed to include 2 (KL) 10 units.
  • the PCR fragment generated from Seq ID 9-11 were digested with BgRl and BcR and ligated into p(KL) 10 that had been digested with BcR, which cuts only once in p(KL) 10 .
  • BgR and BcR digestion creates compatible cohesive ends which may be ligated to each other, but which cannot be digested with either enzyme after their ligation.
  • 2 or more tandem repeats of polylysylleucyl peptides (KL) ]0 can be constructed that are suitable for cloning in-frame into either the BamHI site or an Fspl site (engineered by site-directed mutagenesis) in the Ad2 penton base protein coding sequence of PETl laHA/PB.
  • DNA molecule encoding (KL) ]0 was prepared using the following sense and anti-sense primers, respectively:
  • CTGATCAGAATTCATCG-3' (SEQ ID NO: 11), which encodes for 10 polylysylleucyl repeat sequences (identified in brackets) flanked by sequences for restriction endonuclease cleavage by EcoRI (bold), BqRl (italics) and BcR (underlined).
  • EcoRI symbold
  • BqRl italics
  • BcR underlined
  • the dsDNA was digested with EcoRI, and ligated into the EcoRI site of EcoRI-digested pUC18 DNA.
  • the resulting recombinant clones were screened by restriction analysis to identify those that contain a single copy of (KL) 10 inserted into pUC18.
  • This screening is based on the fact that the parental vector PUC18 does not contain restriction sites for either BgRl or BcR, and therefore confirmation of single copy insertion is accomplished by jSg/II/ite/I-digestion of (KL) 10 with subsequent gel electrophoresis.
  • the presence of a 73 bp band indicates single copy (KL) ]0 insertion, whereas band of 73 bp and 226 bp indicate the insertion of multiples of
  • (KL) 10 inserts.
  • the single repeat construct was designated p(KL) 10 .
  • pETllaHA/PB was modified by site-directed mutagenesis to create a Fspl site at the end of the Ad2 penton base protein coding sequence (following amino acid 571).
  • This new Fspl site allows for the cloning of the polylysylleucyl peptide encoding sequences in- frame into the HA/penton base protein truncated (BamHl site, Ad2 penton base protein amino acid 419) or full-length (Fspl site, Ad2 penton base protein amino acid 571).
  • pETl laHA/PB was digested with BamHl and Hindlll, and the resulting 1.4 kb BamHl/ Hindlll fragment, which includes the carboxyl half of the Ad2 penton base protein, was cloned directionally into M13mpl8, and subjected to site-directed mutagenesis using the following primer:
  • the resulting mutated 1.4 kb BamHl/Hindlll fragment was religated into BamHl/Hindl ⁇ l-digested pETl 1 aHA/PB so as to replace the wild-type BamHl/Hindlll sequence with the mutated sequence.
  • the resulting construct is designated pETllaHA/PBmut.
  • an Fspl site was generated at the carboxyl-terminus of the Ad2 penton base protein coding sequence, and (ii) two novel stop codons in-frame with the Ad2 penton base protein sequence were generated immediately after the engineered Fspl site. With the exception of the 2 newly generated in-frame stop codons, all of the other signal sequences for transcription translation initiation and termination of the HA/Ad2 penton base protein (with or without in-frame expression of polylysylleucyl sequences) remained functional and intact as described for pETllaPB.
  • the (KL) 10 repeat sequences were cloned into pETllaHA PBmut at a position 3' adjacent to amino acid 419 (truncated) of the Ad2 penton base protein (BamHl site).
  • the 2 (KL) ]0 repeat encoding fragment was then cloned in-frame with the Ad2 penton base protein coding sequence by directional ligation into the BamHl/ Fspl-digested pETllaHA PBmut.
  • Directional ligation occurs because BamHl and BgRl are compatible cohesive ends and as such, ensure the correct orientation of the 2 (KL) 10 sequences upon ligation into pETl laHA/PBmut.
  • (KL) 10 encoding fragment (KL) 10 sequences cloned into PETllaHA/PBmut in an inverse (incorrect) orientation will regenerate a Bel site such that subsequent digestion with BamHl (site in Ad2 penton base protein coding sequence) and BcR will generate a 460 bp band as identified by gel electrophoresis. (KL) ]0 sequences cloned in the correct (in-frame) orientation will not generate the 460 bp band after BamHl/BcR digestion. D. Isolation and Purification of UTARVE
  • the UTARVEs were transformed in E. coli strain BL21(DE3), which contains integrated copies of T7 RNA polymerase, and the UTARVE protein produced, isolated and purified as described by Bai et al, J. Virol, 67:5198-5205 (1993).
  • the insoluble fraction containing UTARVE was collected by centrifugation and resuspended in buffer comprising 20 mM Tris-HCl (pH 7.5), 1.0 mM EDTA and 0.1 % (w/v) Nonidet P-40. After three additional cycles of washing, the final pellet was resuspended in a small volume of 6.0 M urea, diluted with 9 volumes of buffer comprising 50 mM K 2 PO 4 (pH 10.7), 50 mM NaCl, 1.0 mM EDTA, and dialyzed against phosphate buffered saline (PBS) and then 50 mM phosphate buffer (pH 7.5). The resulting dialyzate was sterilized by filtration and stored at 4°C or frozen at -70°C.
  • the UTARVE vectors are stable for at least 1 month at 4°C.
  • UTARVE protein was confirmed by SDS-PAGE and Coomassie staining. Only one protein band was observed, representing the purified UTARVE. A similar band was seen for UTARVE ⁇ HA.
  • the prototype UTARVE lacking the (KL) m , but having the HA endosomolytic peptide obtained in Example 1 was mixed with non-ionic oligonucleotides that have a deoxy- methylphosphonate backbone, and: (i) are complementary to the translation initiation site of the immediate early (IE) gene 1 (IE1TI) of Herpes simplex virus-1 (HSV-1); (ii) complementary to the splice acceptor junction of HSV-1 IE pre-mRNAs 4 and
  • IE1T1 is complementary to the translation initiation site for the HSV-1 IE1 gene that codes for a major trans-activating protein designated IE110 or ICPO.
  • IE1TI has been shown to inhibit expression of IE110 and significantly reduce HSV-1 growth. Its inhibitory activity for virus growth synergizes with that of IE4,5SA (Kulka et al, Antimicrobial Agents Chemother., 38:675-680, 1993; Kulka et al, Antiviral Res., 20:115-130, 1994).
  • IE4,5SA has been shown to inhibit splicing of the target mRNA, as well as HSV-1 protein and DNA synthesis, and it significantly reduces viral growth in vitro, and in infected animals (Kulka et al, Proc. Natl .Acad.Sci., tJ&4,86:6868-6872, 1989; Kulka et al, Antimicro.
  • lElTlmuI is complementary to the translation initiation site of HSV-1 gene IE1, but has two inverted central nucleotides. It does not inhibit IE110 synthesis or HSV-1 growth (Kulka et al, Antimicrobial Agents Chemother., 38:675-680, 1993; and Kulka et al, Antiviral Res. 20:115-130, 1994).
  • IE4,5SA/wwl does not inhibit splicing of IE pre-mRNA 4,5 splicing or HSV-1 growth (Kulka et al, Proc. Natl. Acad. Sci., USA, 86:6868-6872, 1989; Kulka et al, Antimicro. Agents Chemother., 38:675-680, 1993; and Kulka et al, Antiviral Res., 20:115-130, 1994).
  • alt-mr-IE4,5SA has the same sequence as IE4,5SA, but the backbone consists of alternate 2 '-O-methylriboside methylphosphonate and phosphodiester groups.
  • alt-mr-IE4,5SA77wl has the same sequence as IE4,5SA ⁇ wwl, but the backbone consists of alternate 2' -O-methylriboside methylphosphonate and phosphodiester groups.
  • IE1TI is as follows: 5'-GCGGGGCTCCAT-3' (SEQ ID NO: 13).
  • sequence of IE4,5SA and alt-mr-IE4,5SA is as follows: 5'-TTCCTCCTGCGG-3' (SEQ ID NO: 14).
  • the sequence of lElTIwwl is as follows: 5'-GCGGGCGTCCAT-3' (SEQ ID NO: 15).
  • the sequence of IE4,5SA/ /l and alt-mr-IE4,5SA ⁇ m/l is as follows: 5'- TTCCCTCTGCGG-3' (SEQ ID NO: 16).
  • Methylphosphonate non-ionic oligonucleotides are synthesized so as to contain 3'5'- deoxy-methylphosphonate groups (d-MP) in place of all of the negatively-charged phosphodiester groups (Ts'o et al, Ann. N.Y. Acad. Sci., 660:159-177, 1992), and synthesis was carried out by solid phase techniques, as described by Miller et al, In: Oligonucleotides and their analogues, Ed. Eckstein, Oxford University Press, Oxford pages 137-154, 1991).
  • d-MP 3'5'- deoxy-methylphosphonate groups
  • OMPs oligonucleotides
  • Ionic alt-mr-OMP oligonucleotides were synthesized so as to contain alternating phosphodiester and 2'-O-methlyriboside methylphosphonate groups using solid phase techniques as described by Miller et al (1991), supra.
  • the OMPs were cystamine derivatized, as described by Miller, In: Immun. Method ofEnzymology, Ed. Lalley et al, 211 :54-64 (1992), so as to have a thiol (SH) group positioned on the alkyl side chain located on the 5 '-terminal nucleotide of the OMPs.
  • the resulting OMPs were conjugated as described by Orgel et al, Nucleic Acids Res.,
  • the primary means for intracellular release of OMPs from the prototype UTARVE is provided by the phosphodiester bond located between the first and second nucleotides of the OMP that is susceptible to hydrolysis by endonucleases inside the cell (Levis, supra).
  • each (KL) 10 unit contains 10 lysine residues, a total of 20 lysine residues are available for co ' mplexation to the OMP.
  • Complexation of the OMPs to the 2 (KL) 10 lysine residues to saturation yields 20 OMP per UTARVE. Based on these values, one may convert a given molar concentration of OMP to an approximation of either the number of UTARVE-OMP complexes/cell or the number of OMPs/cell within any assay.
  • a 1.0 nM concentration of the OMP achieved an IC 50 , which is equivalent to 7.5 x 10 6 UTARVE-OMP complexes/cell or 1.5 x 10 6 OMP molecules/cell.
  • a 100 nM concentration of OMPs in the prototype complex achieved an IC 50 , which is equivalent to approximately 7.5 x 10 6 UTARVE-OMP complexes/cell or 1.5 x 10 8 OMP molecules/cell.
  • IE1TI functions in the cytoplasm, at translation, while IE4,5SA functions in the nucleus, at splicing, (Kulka et al, Proc. Natl. Acad. Sci., USA, 86:6868-
  • the UTARVE-IE4,5SA complex inhibited virus growth, as determined by plaque reduction relative to untreated cells, at all three concentrations. At these concentrations uncomplexed IE4,5SA (solid bar) did not inhibit virus growth. The IC 50 of the uncomplexed IE4,5SA was 25 ⁇ M, i.e., 2500-fold higher.
  • IE4,5SA was bound to the photofluor BODIPY, and the resulting product was complexed to the prototype UTARVE to saturate the lysine sites as described above. 10 7 Vero cells were then exposed to 50 ⁇ M of the resulting complex for 24 hrs, fixed with acetone and examined for intracellular fluorescence. It was compared to the fluorescence of similarly treated cells exposed to BODIPY-IE4,5SA that was not complexed to the prototype UTARVE.
  • the cells treated with the UTARVE-IE4,5SA-BODIPY complex showed a high level of cellular fluorescence distributed throughout the cytoplasm and the nuclei. Approximately 90% of the cells were positive. This compares to the relatively low levels of nuclear and small punctate fluorescence seen in approximately 40% of the cells exposed to IE4,5SA-
  • IE4.5SA IE4.5SA
  • UTARVE-alt-mr-IE4 5SA for 2 hrs, and infected with 5.0 pfu/cell of HSV-1 (strain F). HSV-1 titers were determined 24 hrs later by plaque assay.
  • the IC 50 for UTARVE-alt-mr-IE4,5SA was 1.0 nM, as compared to 100 nM for uncomplexed alt-mr- IE4,55A and 25 nM for UTARVE ⁇ HA-alt-mr-IE4,5SA.
  • UTARVE delivery increases intracellular oligonucleotide levels and antiviral activity
  • HA endosomolytic peptide component of UTARVE contributes significantly to the antiviral activity presumably because it enhances the endosomolytic activity of the penton base protein.
  • the antiviral activity seen with UTARVE complexed OMP is well within the therapeutically significant doses (also in diploid cells). Indeed, IC 50 values for acyclovir are HSV strain dependent and range between 0.3-3.0 ⁇ M.
  • the UTARVE-OMP complex is not toxic as evidenced by the finding that actin expression was not inhibited as determined by immunoblotting, and toxicity was not observed in dye release assays.
  • UTARVE design allows one to achieve this goal. For example, a length of 20 (KL) 10 units in UTARVE will increase the number of OMP molecules/UTARVE particle to 200, as compared to 20 for the second prototype UTARVE. Using the results achieved with the prototype UTARVE as an example, this increase enables one to attain similar inhibitory OMP concentrations with 90% less UTARVE particles than those used in the prototype experiment.
  • the second prototype UTARVE was complexed to an antisense expression vector for cyclin E, a protein which is involved in the regulation of the cell cycle.
  • Coverslip cultures of breast cancer cells (MD-MBA-157) were treated with unconjugated or UTARVE conjugated antisense expression vector for cyclin E (48 hrs) and pulsed (1 hr) with 100 nM bromodeoxy uridine (BudR) and processed for immunofluorescent detection of incorporated BudR.
  • the percent of DNA synthesizing nuclei was scored against the total nuclei counter-stained with Hoechst 33258 (Molecular Probes Inc., Eugene, OR).

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Abstract

Ce véhicule de fixation d'apport d'acide nucléique, qui comporte une protéine d'adénovirus à base penton ainsi qu'un peptide endosome d'hémagglutinine du virus de la grippe, peut éventuellement comporter un peptide polylysylleucyle destiné à assurer des sites supplémentaires aux fins de la fixation d'acide nucléique ainsi qu'un peptide signal de localisation nucléaire facilitant la localisation intranucléaire de l'acide nucléique apporté. Il est également possible d'utiliser ce véhicule pour accroître l'apport intracellulaire de molécules d'acide nucléique.
PCT/US1998/013710 1997-08-07 1998-07-02 Vehicule de fixation d'apport d'acide nucleique Ceased WO1999007723A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000045850A3 (fr) * 1999-02-06 2001-07-05 Aurx Inc Vehicule servant a administrer un medicament
EP3252068A2 (fr) 2009-10-12 2017-12-06 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro

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WO1994004696A1 (fr) * 1992-08-25 1994-03-03 Miles Inc. Apport nucleaire de macromolecules facilite par un signal de translocation
WO1995028494A1 (fr) * 1994-04-15 1995-10-26 Targeted Genetics Corporation Proteine de fusion d'apport de gene
US5547932A (en) * 1991-09-30 1996-08-20 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
US5661025A (en) * 1992-04-03 1997-08-26 Univ California Self-assembling polynucleotide delivery system comprising dendrimer polycations
US5736392A (en) * 1995-06-07 1998-04-07 Life Technologies, Inc. Peptide-enhanced cationic lipid transfections

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US5547932A (en) * 1991-09-30 1996-08-20 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
US5661025A (en) * 1992-04-03 1997-08-26 Univ California Self-assembling polynucleotide delivery system comprising dendrimer polycations
WO1994004696A1 (fr) * 1992-08-25 1994-03-03 Miles Inc. Apport nucleaire de macromolecules facilite par un signal de translocation
WO1995028494A1 (fr) * 1994-04-15 1995-10-26 Targeted Genetics Corporation Proteine de fusion d'apport de gene
US5736392A (en) * 1995-06-07 1998-04-07 Life Technologies, Inc. Peptide-enhanced cationic lipid transfections

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ELLISON K.E., ET AL.: "FUSIGENIC LIPOSOME-MEDIATED DNA TRANSFER INTO CARDIAC MYOCYTES.", JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY., ACADEMIC PRESS, GB, vol. 28., 1 January 1996 (1996-01-01), GB, pages 1385 - 1399., XP002914073, ISSN: 0022-2828, DOI: 10.1006/jmcc.1996.0130 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000045850A3 (fr) * 1999-02-06 2001-07-05 Aurx Inc Vehicule servant a administrer un medicament
EP3252068A2 (fr) 2009-10-12 2017-12-06 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro
EP4089169A1 (fr) 2009-10-12 2022-11-16 Larry J. Smith Procédés et compositions permettant de moduler l'expression génique à l'aide de médicaments à base d'oligonucléotides administrés in vivo ou in vitro

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