WO2000046384A1 - Vehicule de fixation d'apport d'acide nucleique - Google Patents
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- WO2000046384A1 WO2000046384A1 PCT/US2000/003074 US0003074W WO0046384A1 WO 2000046384 A1 WO2000046384 A1 WO 2000046384A1 US 0003074 W US0003074 W US 0003074W WO 0046384 A1 WO0046384 A1 WO 0046384A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a nucleic acid uptake and release vehicle comprising a fusion protein wherein a ligand and a hemagglutinin endosomolytic peptide are covalently bonded by an amide linkage 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.
- a preferred embodiment comprises an adenovirus penton base protein; and an influenza virus hemagglutinin endosomolytic peptide covalently bonded by an amide linkage that can be utilized to enhance the intracellular delivery of nucleic acid molecules, e.g., DNA vectors encoding sense or anti- sense oligonucleotides, as well as a DNA construct encoding the same, and a method of use of the same for intracellular delivery of nucleic acid molecules.
- Other suitable preferred ligands include epidermal growth factor.
- 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.
- 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.
- 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 nucleic acid uptake and release vehicle
- a ligand for a receptor and a hemagglutinin endosomolytic peptide are covalently bonded by an amide linkage 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.
- 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:
- the UTARVE also comprises:
- the above-described objects of the present invention have been met by a DNA construct encoding UTARVE.
- 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- 1E4.5SA complex (•) or the UTARVE-IE1TI complex (o), and infected with HSV-1.
- Figure 2 shows HSV- 1 growth inhibition in Vero cells, where the Vero cells were exposed to the
- the invention comprises a ligand for a receptor covalently bonded by an amide linkage to an influenza virus hamaggluntin endosomolytic peptide.
- the present invention relates to a UTARVE comprising: (i) an adenovirus (Ad) penton base protein covalently bonded by an amide linkage to
- 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 (1993).
- RGD receptor binding site motif
- 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.
- Suitable ligands or base proteins include transferrin, cholera toxin B subunit, and epidermal growth factor.
- 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. ScL, 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,
- 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:
- 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:
- NLS nuclear localization signal
- 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. 5'-AGATCTCATCGGACGACG-3' (SEQ ID NO: 1 ); and
- 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 Bell site or upstream at the Bgl ⁇ l 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. For example,
- 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 Table 1 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 Glygroint al.
- 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 Glygroin-HA-Gly n peptide: NLS peptide;
- Ad penton base protein NLS peptide: Gly n -HA-Gly ⁇ ;
- 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;
- NLS peptide Ad penton base protein: Gly n -HA-Gly n peptide: (KL) m peptide; 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 ⁇ 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).
- CMV Cytomegalovirus immediate early
- anti-sense oligonucleotide employed is not critical to the present invention.
- anti-sense oligonucleotides include:
- Antisense AKT 2 which has been used to inhibit AKT 2 expression and tumorigenicity in PANCI cells (Chang et al, Proc. Acad. Natl. ScL, USA, 93:3636-3641, 1996);
- 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. ScL, USA, 86:6868-6872, 1989; and Kulka et al,
- 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: (1) 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); and (2) Sense HIV- 1 protease, which has been used in cultured cells to induce cleavage of BCL- 2, and thereby cause apoptosis (Stack, Proc. Natl. Acad. ScL, USA, 93:9571-9576, 1996).
- 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-HPVl 8 ribozyme which has been used to alter HPV18 mRNA expression through cleavage (of HPV mRNA), and thereby alter the growth rates and properties of selected cell lines (Chen et al, Cancer Gene Ther., 3:18-23, 1996); and
- 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
- 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. Louis encephalitis virus; Coronaviridae, e.g.,
- Coronavirus cortavivirus
- 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 parvovirusB-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;
- 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.g., African horse sickness virus
- Calciviridae e.g.., Feline calciviruses
- Retroviridae e.g., Avian sarcoma and leukosis viruses
- Hepadnaviridae e.g., Hepatitis Belike virus
- Parvoviridae e.g., Canine parvovirus B-19
- Papovaviridae e.g., Bovine papillomavirus
- the UTARVE complexes can be used in the treatment of genetic diseases, such as hemophilia, adenosine deaminase deficiency, beta-thalassemia, diabetes and cystic fibrosis. Additionally, 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.
- genetic diseases such as hemophilia, adenosine deaminase deficiency, beta-thalassemia, diabetes and cystic fibrosis.
- 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 design and versatility enables variation of its construction in order to address these important issues. This includes:
- 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.:
- the particular protein ligand employed is not critical to the present invention.
- protein ligands include transferrin, cholera toxin B subunit, penton base protein, and epidermal growth factor.
- the particular sugar employed is not critical to the present invention. Examples of such sugars 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 amide 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.,
- 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, FEBS Letters, 20:302-306 (1972).
- the conjugation of oligonucleotides to lysine residues requires the use of the activating agent EDAC [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.
- EDAC l-ethyl-3- (3- dimethylaminopropyl) carbodiimide
- 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.
- 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'-0-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., ps
- a photofluor e.g., BODIPY
- any cross-linking moieties e.g., p
- 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.
- the complexation and cellular delivery of nucleic acid molecules by UTARVE to target cells can be conducted using a ⁇ -galactosidase expression DNA construct (pCMV ⁇ : CMVIE promoter/E. coli ⁇ -galactosidase gene cassette, Clontech, Palo Alto, CA), and evaluated on the basis of marker gene expression, ⁇ -galactosidase expression provides the enzymatic marker necessary to determine the efficiency of DNA transfection efficiency by UTARVE.
- pCMV ⁇ CMVIE promoter/E. coli ⁇ -galactosidase gene cassette, Clontech, Palo Alto, CA
- 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
- 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:
- nucleic acid molecules and (ii) define the kinetics of dissociation.
- 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.
- the 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.
- creams/ointments/lipid carriers e.g., polyethylene glycol or liposomes
- 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.
- the UTARVE-anti-sense oligonucleotide IE4,5SA complex described in Example 3 below, which has an alt- mr-OMP backbone was administered in an amount of from about 0.1 to 50 nM.
- 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.
- the sequence for 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 (mung bean nuclease treated) 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), and is followed by and covalently bonded by an amide linkage to the penton base protein coding sequence. Positive clones were identified by sequencing.
- a dsD ⁇ A sequence top strand: 5'-GAGGTGGTGGTGG-3' (SEQ ID ⁇ O: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 pETl laPB was constructed as described by Bai etal, J. Virol, 67:5198-5205 (1993).
- pETllaPB contains the entire Ad2 penton base protein under the control of the T7 phage promoter.
- the Ad2 penton base protein reading frame begins pETl 1 aPB, 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.
- Ad2 penton base protein gene All of the signals for transcriptional and translational regulation of the Ad2 penton base protein gene are present within pET 1 laPB .
- 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. coli.
- E. coli strains such as BL21(DE3) (Studier et al, Methods En ⁇ ymol, 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 pETl laPB, as well as encoding for glycine residues that flank HA (Gly 2 -HA-Gly 2 ).
- the dsDNA was generated by PCR amplification of the following primary single-stranded 70 bp DNA sequence: 5' -GAGGTGGACTCTTCGAAGCAATTGCAGGTTTAATCGAAAACGGCTGGGA AGGCATGATCGACGGTGGTGG-3' (SEQ IDNO:6), using the following sense and anti-sense primers, respectively: 5'-GAGGTGGACTCTTCGAAGCA-3' (SEQ ID NO:7); and 5' -
- 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 Nbel 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.
- 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). Positive clones were subjected to double-stranded sequencing using the PCR primers described- above to confirm in-frame ligation of Gly 2 -HA-Gly 2 sequence to the Ad2 penton base protein sequence 15 in pETllaHA/PB.
- 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- 1 1 were digested with Bglll and Bell and ligated into p(KL) 10 that had been digested with Bell, which cuts only once in p(KL) 10 .
- BgU and Bell digestion creates compatible cohesive ends which may be ligated to each other, but which cannot be digested with either enzyme after their ligation.
- the strategy and methods described above to create 2 tandem copies of (KL) 10 in pUC18 may be repeated in a sequential fashion to generate an increasingly larger number of tandem (KL) 10 repeats in PUC 18.
- the overall strategy of this method is to permit generation of (KL) 10 repeats the polylysylleucyl codons of which are "in- frame" from one repeat to the next.
- 2 or more tandem repeats of polylysylleucyl peptides (KL) 10 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) 10 was prepared using the following sense and anti-sense primers, respectively:
- 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 BgUl or Bell, and therefore confirmation of single copy insertion is accomplished by i?g/II/.9c/I-digestion of (KL) 10 with subsequent gel electrophoresis.
- the presence of a 73 bp band indicates single copy (KL) 10 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 BamHllHindlll 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: 5' -AGGATGGACTATTATGCGCAAAA-3 ' (SEQ ID NO: 12)
- the resulting mutated 1.4 kb BamHllHindlll fragment was religated into BamHUHindl ⁇ l- ⁇ igested pETl laHA/PB so as to replace the wild-type BamHllHindlll 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).
- pETllaHA/PBmut was subjected to Fspl digestion followed by BamHl digestion and electrophoretic gel purification to remove the BamHIIFspl Ad2 penton base protein fragment encoding amino acids 420 to 571.
- a plasmid containing the 2 (KL) 10 repeat sequence was digested with Bell, treated with Klenow I so as to blunt-end this site, subjected to a second digestion with Bglll and the 2 (KL) 10 sequences isolated, and purified by electrophoretic gel purification.
- the 2 (KL) 10 repeat encoding fragment was then cloned in-frame with the Ad2 penton base protein coding sequence by directional ligation into the Ti wHI/ESTJl-digested pETllaHA/PBmut.
- Directional ligation occurs because BamHl and Bglll are compatible cohesive ends and as such, ensure the correct orientation of the 2 (KL), 0 sequences upon ligation into pETl laHA/PBmut.
- These (KL) 10 repeats could also be cloned into pETl laHA/PBmut at a position 3' adjacent to amino acid 571 (full length) of the Ad2 penton base protein (Fspl site).
- the plasmid containing the 2 (KL) 10 sequences is digested with Bglll and BcR treated with Klenow I so as to blunt-end these sites, and the (KL) 10 encoding fragment isolated and purified.
- pETl laHA/PBmut is then digested with Fspl, and ligated to the blunt-ended Bgl ⁇ llBcll (KL) 10 encoding fragment.
- 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 P0 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 18 in Example 1 was mixed with non-ionic oligonucleotides that have a deoxy-methylphosphonate backbone, and:
- HSV-1 Herpes simplex virus- 1
- IE ITT Herpes simplex virus- 1
- HSV-1 Herpes simplex virus- 1
- IE4,5SA complementary to the translation initiation site of IE1TI in which the two central nucleotides were inverted (lElTlmul); (iv) complementary to the splice acceptor junction of HSV-1 IE pre-mRNAs 4 and 5 in which the two central nucleotides were inverted (lE4,5SAmul); or have an alternate 2'-0-methyl riboside methylphosphonate/phosphodiester backbone, and are:
- 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.
- IE1 TI has been shown to inhibit expression of IE110 and significantly reduce HSV- 1 growth. Its inhibitory activity for virus growth synergizes with that ofIE4,5SA (Kulka et al, Antimicrobial Agents Chemother., 38:675-680, 1993; Kulka etal, 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
- lElTlmul 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,
- IE4,5SAmwl does not inhibit splicing of IE pre-mRNA 4,5 splicing or HSV- 1 growth (Kulka et al, Proc. Natl. Acad. ScL, 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 '-0- methylriboside methylphosphonate and phosphodiester groups.
- alt-mr-IE4,5SA ⁇ WMl has the same sequence as LE4,5SA»2wl, butthe backbone consists of alternate 2' -O-methylriboside methylphosphonate and phosphodiester groups.
- IE1TI The sequence of 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: 13).
- lElTlmul The sequence of lElTlmul is as follows: 5'-GCGGGCGTCCAT-3' (SEQ ID NO: 15).
- sequence of IE4,5SAr ⁇ wl and alt-mr-IE4,5SAw «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.
- Ionic alt-mr-OMP oligonucleotides were synthesized so as to contain alternating phosphodiester and 2'-0-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 of Enzymology, Ed. Lalley et al, 21 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.
- SH thiol
- the resulting OMPs were conjugated as described by Orgel et al, Nucleic Acids Res., 16:3671 (1988); and King et al, Biochem., J : 1499- 1506 (1978), resulting in the attachment of the OMPs to the prototype UTARVE through disulfide, -S-S-, bridges.
- 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).
- (KL) 10 unit contains 10 lysine residues, a total of 20 lysine residues are available for complexation 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.
- both UTARVE-IEITI (•) and UTARVE-IE4,5SA (o) complexes inhibit HSV-1 growth, as determined by plaque reduction relative to untreated cells, with IC 50 values of approximately 1.0 nM and IC 90 values of approximately 100 nM.
- the complexed controls UTARVE-IEITI (•) and UTARVE-IE4,5SA (o)
- IE1TI functions in the cytoplasm, at translation, while IE4,5SA functions in the nucleus, at splicing, (Kulka et al, Proc. Natl. Acad. ScL, USA, 86:6868-6872, 1989; Kulka et al, Antimicrob. Agents Chemother., 38:675-680, 1994).
- the finding that similar results are obtained with both OMPs indicates that the UTARVE complexed OMP can reach the intracellular site of its target, be it cytoplasmic or intranuclear.
- 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-BODIPY that was not complexed to the prototype
- UTARVE A single large/dense endocytic-like vesicle evidenced good levels of fluorescence.
- Vero cells which have only an intermediate level of penton base protein receptors, wereexposedto 0.1-50nMalt-mr-IE4,5SA,UTARVE ⁇ HA-alt-mr-IE4.5SA, orUTARVE-alt- mr-IE4. 5SA for 2 hrs, and infected with 5.0 pfu/cell of HSV-1 (strain F). HSV-1 titers were determined
- 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.
- 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).
- UTARVE was constructed by assembly of DNA sequences encoding: (i) the adenovirus type 2 penton base protein (PBP), which binds the cell surface receptors- integrin and (ii) an endosomolytic peptide derived from influenza virus HA.
- PBP adenovirus type 2 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 alpha-helices flanking the HA peptide (Gly 2 -HA-Gly 2 ), thereby imparting flexibility to the secondary structure at the HA-penton base junction.
- the sequence for Gly 2 -HA-Gly 2 was first cloned into pGEMT (Promega, Madison, WI), a PCR cloning vector.
- 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 expression vector and penton base protein coding sequences. Positive clones were identified by sequencing.
- pETl laHA/PB was subjected to collapse ligation at the Ndel located upstream of the penton base protein initiator Met.
- the resulting plasmid pETl 1 a ⁇ HA/PB codes for a chimeric protein that begins with an initiator Met, Ala, Ser and Thr all encoded by vector followed by the penton base protein coding sequence. Positive clones were identified by sequencing.
- Ad2 Penton Base Protein 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. coli.
- a DNA molecule encoding the influenza HA peptide was synthesized and then cloned into pETllaPB.
- dsDNA molecule which encodes the 20 amino acid HA plus, 5' and 3' flanking sequences that provide the nucleotides suitable for cloning into either pETl 1 a or pETllaPB, as well as encoding for HA flanking glycine residues (Gly 2 -HA-Gly 2 ).
- the dsDNA was generated by PCR amplification of the following primary single-stranded 70 bp DNA sequence for HA: 5' -GAGGTGGACTCTTCGAAGCAATTGCAGGTTTAATCGAAAACGGCTGGGA AGGCATGATCGACGGTGGTGG-3' (SEQ ID NO: 17), using the following sense and anti-sense primers, respectively: 5'-CATATGGGAGGTGGACTCTTCGAAGCA-3 * (SEQ ID NO: 18); and 5'
- primers each contain seven 5' terminal nucleotides designed to encode for 2 glycines upon ligation into the Ndel site of pETllaPB.
- the dsDNA was ligated into pGEMT according to manufacturer's (Promega, Madison, WI) recommended procedures and transformed into E. coli DH5 ⁇ (Life Technologies) followed by growth selected on culture media containing ampicillin and 5-bromo-4- chloro-3-indoyl- ⁇ -D-galactose (X-GAL). Growth selection of pGEMT transformed DH5 on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media.
- Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Ndel and Mfel restriction enzyme analysis. Positive clones (pGEMT/HA) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
- the Gly 2 -HA-Gly 2 encoding ds DNA sequence was removed from pGEMT by Ndel restriction digestion and cloned into Ndel digested pETl laPB to give plasmid pETl laHA/PB.
- the Ndel cleavage site in pETl laPB overlaps the vector encoded initiation (Met) codon.
- pETl laHA/PB encodes for a fusion protein that begins with a start Met followed by I) Gly 2 -HA-Gly 2 , ii) His, Met, Ala, Ser and Thr residues encoded by the expression vector and iii) the Ad2 penton base protein coding sequence.
- E. coli DH5 ⁇ was transformed with PETl laHA/PB, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Ndel restriction enzyme analysis. Positive recombinants were subjected to a secondary screening using Mfel and Nhel restriction analysis in order to confirm the correct orientation of Gly 2 -HA-Gly 2 sequence within pETl laHA/PB. The Mfel restriction site is present in the
- HA encoding sequence and the Nhel site is located 5' adjacent to the AD2 PBP sequence.
- Positive clones were subjected to double-stranded sequencing using the universal forward primers described-above to confirm in-frame ligation of Gly 2 -HA-Gly 2 sequence to the Ad2 penton base protein sequence in pETllaHA/PB.
- Another UTARVE was constructed to contain a DNA molecule encoding Gly 2 -HA-Gly 2 followed by a multiple cloning site (MCS) cloned into pETl la.
- MCS multiple cloning site
- the Ndel fragment encoding Gly 2 -HA-Gly 2 from pGEMT/HA was cloned into the Ndel site of pETl la to give plasmid pETl laHA.
- This plasmid was further modified through restriction digestion with BamHl and Nhel, which cut downstream of the Gly 2 -HA-Gly 2 encoding sequence, both sites made blunt-ended followed by a collapse ligation of these sites.
- the unique Hindlll site in this plasmid was deleted through a subsequent modification involving digestion with Hindlll followed by mung bean nuclease treatment and blunt-end ligation of this site to generate plasmid pETl laHA+.
- This cloning strategy creates a single site (BamHl) downstream of Gly 2 -HA-Gly 2 for ligation/insertion of the MCS.
- a ds DNA molecule was prepared which contains endonuclease cleavage sites for several enzymes including Bglll, Xhol, Ecll36III, Pmll, Ascl, Ncol, Sail, Bsrgl and BamHl as well as encoding for translational stop codons in all three reading frames.
- the ds DNA was generated by PCR amplification of the following primary single-stranded 54 bp DNA sequence 5' - CTCGAGCTCACGTGGCGCGCCATGGTCGACTGTACAGGATCCTAACTAGGTAAG-3' (SEQ ID 20) using the following sense and antisense primers, respectively 5'- A G A T C T C T C G A G C T C A C G T G G C G C 3 ' ( S E Q I D 2 1 ) a n d 5 ' -
- AGATCTCTTACCTAGTTAGGATCCTG-3' (SEQ ID 22).
- the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- ⁇ -D-galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media.
- Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using BamHl restriction enzyme analysis which cuts only once (in the MCS) in a positive recombinant. Positive clones (pGEMT/MCS) were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert. The MCS was removed from pGEMT/MCS and cloned into the unique BamHl site of pETl laHA+ to create plasmid pETl laHAmcs.
- 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 pGEMT, pETllaPB and pETl laHAmcs and confirm the success of the cloning is similar to that used for Gly 2 -HA-Gly 2 .
- DNA molecule encoding (KL) 10 was prepared using the following sense and anti-sense primers, respectively:
- CTTAAGCTCAAGCTTAAGCTC1AGATCTGAATTC-3' (SEQ ID NO:25), which encodes for 10 polylysylleucyl repeat sequences (identified in brackets) flanked by sequences for restriction endonuclease cleavage by Sail (bold), BamHl (italics), Bglll (underlined) and Hindlll (underlined within brackets).
- the dsDNA was ligated into pGEMT, transformed and grown as above. Growth selection of pGEMT (no insert) transformed DH5 on this culture media yields blue colonies.
- pGEMT/KL were subjected to ds DNA sequencing using universal primer which targets vector sequences upstream of the insert.
- the KL 10 encoding fragment was removed from pGEMT/KL by Sall/Bglll digestion and cloned into the Sall/BamHI sites in the MCS of pETl laHAmcs to give plasmid pETl laHA/KL. Transformation into DH5 ⁇ and growth selection on Amp containing media followed by Hindlll restriction analysis was used to identify positive clones. Final confirmation through ds DNA sequencing provided nucleotide sequence confirmation. Cloning the Sall/Bglll fragment into pETl laHAmcs affects a BsrGI to Acc65I change downstream of the Sail site in the MCS.
- the KL 10 encoding fragment may be cloned into pETl 1 aHAmcs as a BamHI/Bglll fragment thereby retaining the Bsrgl site within the MCS.
- the utility of this cloning strategy is to provide the potential for i) establishing an alternate cloning site within the MCS and ii) generating translational frame-dependent expression of a Cys codon (present in a BerGI but not Acc65I recognition sequence) upstream of the translational stop codons.
- a Cys amino acid can provide an alternative to Lys as a site for derivatization/complexation of drug moieties.
- pETl laHA/PBP/KL was created to contain DNA sequences encoding in frame for Gly 2 -HA-Gly 2 , PBP and KL, 0 .
- This DNA sequence was constructed by cloning the PBP sequence from pETl laPBP in pETl laHA/KL using a two-step strategy. In the first step the 1.6kb
- Mfel/Ascl fragment of pETl laPB which encodes the carboxyl region of Gly 2 -HA-Gly 2 and the coding sequence of PBP from amino acids 1 through 507, was cloned directionally into Mfel/Ascl digested pETl laHA/KL to create plasmid pETl laHA/PBpart/KL.
- Recombinants were growth selected on Amp containing media and positive recombinants identified by restriction analysis with BamHl wherein positives recombinant plasmids are cut twice with this enzyme.
- a ds DNA PBP sequence encoding amino acids 495 to 571 was amplified by PCR using the following sense and antisense primers 5'-CGTGTTCAATCGCTTTCCCGAGAA-3' (SEQ ID26) and 5'- GTCGACAAAAGTGCGGCTCGATAGGACG-3' ( SEQ ID 27), respectively.
- the antisense primer contains six 5' terminal nucleotides designed to create a Sail restriction site 3' adjacent to the codon for PBP amino acid 571 with concomitant elimination of the translational stop codon.
- the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 ⁇ and growth selected on culture media containing ampicillin and 5-Bromo-4- chloro-3-indoyl- ⁇ -D-galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Ascl restriction enzyme analysis.
- PGEMT/PBterm Positive clones
- pGEMT/PBterm Positive clones
- PGEMT/PBterm was digested with Ascl and Sail and the 204bp fragment encoding PBP amino acids 507 to 571 cloned into the Ascl/Sall sites of pETl laHA/PBpart/KL to generate pETl laHA/PB/KL.
- E. Coli DH5 ⁇ was transformed with this recombinant and growth selected on Amp containing culture media. Plasmid DNA is extracted from isolated colonies, and subjected to screening (for positive recombinants) using BamHl restriction enzyme analysis.
- pETl laHA/PB/KL encodes for a fusion protein that begins with a Met followed by I)Gly 2 -HA-Gly 2 , ii) His, Met, Ala, Ser, and Thr residues encoded by vector sequences, iii) the Ad2 penton base protein, iv) Val, Asp, Gly, Asn, Gly and Ser encoded by the
- a dsDNA molecule was prepared which encodes for Ala followed by the first 25 amino acids of EGF plus 5' flanking sequences that provide the nucleotides suitable for cloning into the appropriate site in vectors such as pETl laHAmcs or pETHA/KL and 3' flanking sequences that provide for cloning (ligation) adjacent with the second EGF fragment (below).
- the dsDNA was generated by PCR amplification of the following primary single-stranded 73 bp DNA sequence: 5'-GCCAACTCAGATTCAGAATGTCCACTGTCACACGATGGCTACTGCCTCCATGACGGAG TGTGCATGTATATCG-3' (SEQ ID NO:28), using the following sense and annti-sense primers, respectively: 5'-CTCGAGGCCAACTCAGATTCAGAATG-3' (SEQ ID NO:29); and
- the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 ⁇ and growth selected on culture media containing ampicillin and 5-Bromo-4-chloro-3-indoyl- ⁇ -D-galactose (X-GAL). Growth selection of pGEMT transformed DH5 ⁇ on this culture media yields blue colonies. Disruption of lacZ expression in pGEMT via insertion/ligation of a PCR product can result in the formation of white colonies on this media. Plasmid DNA was extracted from white colonies (putative positive recombinants) and subjected to secondary screening using Xhol and Pstl restriction enzyme analysis. Positive clones (pGEMT/EGFl) were subjected to ds DNA sequencing using universal primer which targetsvector sequences upstream of the insert.
- a dsDNA molecule was prepared which encodes for amino acids 25 to 53 of EGF plus 5' flanking sequences that provide the nucleotides suitable for cloning (ligation) adjacent to the first EGF fragment (above) and 3' flanking sequences that provide for cloning inot the appropriate site in vectors such as pETl laHAmcs or pETl laHA/KL.
- the dsDNA was generated by PCR amplification of the following primary single-stranded 83 bp DNA sequence:
- the dsDNA was ligated into pGEMT according to manufacturer's recommended procedures and protocols and transformed into DH5 ⁇ and growth selected on culture media containing ampicillin and
- the ds DNA sequence the second EGF fragment was removed from pGEMT/EGF2 by Stul/Sall restriction digestion and cloned into the Stul/Sall sites of pGEMT/EGF 1.
- the resulting recombinant plasmid pGEMT/EGF contains a novel DNA sequence which encodes for a native EGF protein plus a 5' Ala amino acid.
- E. coli DH5 ⁇ was transformed with pGEMT/EGF, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Xhol/Ndel restriction enzyme analysis. Positive recombinants were subjected to a secondary screening using Xhol/Stul and Xhol/Sall restricition analysis in order to screen for the presence of one complete copy of EGF encoding DNA sequence within the vector. Positive clones were subjected to double-stranded sequencing using the universal forward primers described-above to confirm the nucleotide sequence of the EGF encoding DNA.
- pETl laHA/EGF Another protype UTARVE, pETl laHA/EGF was created to contain a DNA sequence which encodes inframe for Gly 2 -HA-Gly 2 and EGF.
- the Ala-EGF encoding dsDNA sequence was removed from pGEMT/EGF by Xhol/Sall restriction digestion and ligated into the Xhol/Sall sites of pETl laHAmcs to give pETl laHA/EGF.
- E. coli DH1 was transformed with putative recombinant, pETl laHA/EGF, and growth selected on Amp containing culture media. Plasmid DNA was extracted from isolated colonies, and subjected to initial screening (for positive recombinants) using Xhol/Sall restriction enzyme analysis.
- pETl laHA/EGF encodea for a fusion protein that gegins with Met followed by i) Gly 2 -HA-Gly 2 , (ii) His, Met, Gly, Leu, Asp and Glu encoded by vector and MCS sequences, iii) Ala followed the EGF protein and iv) Val, Asp, Cys, Thr, Glu and Ser encoded by MCS.
- pETl laHA/EGF/KL Another protype UTARVE, pETl laHA/EGF/KL was created to contain DNA sequences encoding for Gly 2 -HA-Gly 2 inframe with EGF followed by L 10 .
- the L 10 encoding fragment was removed from pGEMT/KL by Sall/Bglll digestion and cloned into the Sall/BamHI sites in the MCS of pETl laHA/EGF to give pETl laHA/EGF/KL.
- the Ala-EGF containing Xhol/Sall fragment from pGEMT/EGF could be cloned into Xhol/Sall sites of pET 1 1 aHA/KL to generate the same recombinant plasmid.
- pETl laHA/EGF/KL encodes for a fusion protein that begins with Met followed by i) Gly 2 -HA-Gly 2 ii) His, Met, Gly, Leu, Asp, and Glu encoded by vector and MCS sequences, iii) Ala followed by EGF protein, iv) Val, Asp, Gly, Asn, Gly and Ser, v) KL 10 and vi) end terminal amino acids Arg and Ser.
- the UTARVEs were transformed in E. coil strain BL21(DE3 and the UTARVE protein produced, isolated and purified as described by Bai et al, J. Virol, 67:5198-5205 (1993).
- 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 P0 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).
- PBS phosphate buffered saline
- the resulting dialyzate was sterilized by filtration and stored at 4°C or frozen at -70°C.
- the purity of the 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.
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PCT/US2000/003074 WO2000046384A1 (fr) | 1999-02-08 | 2000-02-08 | Vehicule de fixation d'apport d'acide nucleique |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU3223900A (fr) |
WO (1) | WO2000046384A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000045850A2 (fr) * | 1999-02-06 | 2000-08-10 | Aurx Inc. | Vehicule servant a administrer un medicament |
WO2008118013A3 (fr) * | 2007-03-23 | 2009-02-19 | To Bbb Holding B V | Administration intracellulaire ciblée d'agents antiviraux |
LU92353A1 (en) * | 2014-01-14 | 2015-07-15 | Univ Muenster Wilhelms | Antibody-mediated delivery of RNAI |
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 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995028494A1 (fr) * | 1994-04-15 | 1995-10-26 | Targeted Genetics Corporation | Proteine de fusion d'apport de gene |
-
2000
- 2000-02-08 AU AU32239/00A patent/AU3223900A/en not_active Abandoned
- 2000-02-08 WO PCT/US2000/003074 patent/WO2000046384A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995028494A1 (fr) * | 1994-04-15 | 1995-10-26 | Targeted Genetics Corporation | Proteine de fusion d'apport de gene |
Non-Patent Citations (2)
Title |
---|
BONGARTZ ET. AL.: "Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide.", NUCLEIC ACIDS RESEARCH, vol. 22, no. 22, November 1994 (1994-11-01), pages 4681 - 4688, XP002928364 * |
WICKHAM ET. AL.: "Intergrins a nu beta3 and a nu beta5 promote adenovirus internalization but not virus attachment.", CELL, vol. 73, April 1993 (1993-04-01), pages 309 - 319, XP002928363 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000045850A2 (fr) * | 1999-02-06 | 2000-08-10 | Aurx Inc. | Vehicule servant a administrer un medicament |
WO2000045850A3 (fr) * | 1999-02-06 | 2001-07-05 | Aurx Inc | Vehicule servant a administrer un medicament |
WO2008118013A3 (fr) * | 2007-03-23 | 2009-02-19 | To Bbb Holding B V | Administration intracellulaire ciblée d'agents antiviraux |
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 |
LU92353A1 (en) * | 2014-01-14 | 2015-07-15 | Univ Muenster Wilhelms | Antibody-mediated delivery of RNAI |
Also Published As
Publication number | Publication date |
---|---|
WO2000046384A9 (fr) | 2001-09-07 |
AU3223900A (en) | 2000-08-25 |
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