Classifications

• • 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
  • 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
  • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
  • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors

Definitions

• This invention relates to gene therapy using a transporter system for delivering nucleic acid into a cell.
• Recombinant retroviral vectors have been used for delivery of genes to cells of living animals. Morgan et al., Annu . Rev. Biochem. , 62:191-217 (1993) .
• Retroviral vectors permanently integrate the transferred gene into the host chromosomal DNA.
• other virus have been used for gene delivery.
• Adeno- viruses have been developed as a means for gene transfer into epithelial derived tissues. Stratford-Perricaudet et al., Hum. Gene . Ther. , 1:241-256 (1990) ; Gilardi et al.
• Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce nonproliferating cells, as well as an ability to produce purified high titer virus.
• Recombinant adenoviral vectors have the advantage over retroviruses of being able to transduce nonproliferating cells, as well as an ability to produce purified high titer virus.
• receptor-mediated endocytosis Endocytosis is the process by which eucaryotic cells continually ingest segments of the plasma membrane in the form of small endocytotic vesicles.
• Endocytotic vesicles form in a variety of sizes and shapes and are usually enlarged by fusing with each other and/or with other intracellular vesicles. Stryer, Bioch . , Freeman and Co., New York (1988) . In most cells the great majority of endocytotic vesicles ultimately fuse with small vesicles called primary lysosomes to form secondary lysosomes which are specialized sites of intra-cellular digestion. Id. The lysosomes are acidic and contain a wide variety of degradative enzymes to digest the macromolecuiar contents of the vesicles. Silverstein et al . , Annu. .Rev. Biochem. , 46:669-722 (1977) ; Simianescu et al., J. Cell Biol . , 64:586-607 (1975) .
• Coated pits and vesicles provide a specialized pathway for taking up specific macromolecules from the extracellular fluid. This process is called receptor-mediated endocytosis.
• receptor-mediated endocytosis Goldstein et al. , Nature, 279:679-685 (1979) ; Pearse et al., Annu. .Rev. Biochem. , 50:85-101 (1981); Postan et al. , Annu . Rev. Physiol . , 43:239-250 (1981) .
• the macromolecules that bind to specific cell surface receptors are internalized via coated pits.
• Receptor-mediated endocytosis is a selective mechanism enabling cells to ingest large amounts of specific ligands without taking in correspondingly large amounts of extracellular fluid. Goldstein, supra .
• LDL low density lipoprotein
• Numerous studies have been performed involving LDL and the receptor-mediated endocytotic pathway.
• LDL many other cell surface receptors have been discovered to be associated with coated pits and receptor-mediated endocytosis.
• Pastan et al. Annu. Rev. Physiol., 43:239-250 (1981) .
• studies have analyzed the hormone insulin binding to cell surface receptors and entering the cell via coated pits. Stryer et al., Biochemistry, Freeman & Co., New York (1988) ; Alberts et al. , Molecular Biology of the Cell, Garland Publishing, New York (1983) .
• the asialoglycoprotein receptor has been used in targeting DNA to HepG2 cells in vitro and liver cells in vivo.
• u et al. J. Biol. Chem., 262:4429-4432 (1987); Wu et al . , Bio., 27:887-892 (1988) ; Wu et al. , J. Biol. Chem., 263:14620-14624 (1988) ; Wu et al. , J “ . Biol. Chem., 264:16985-16987 (1989) ; Wu et al. , J. Biol. Chem., 266:14338-14342 (1991) .
• Folate conjugated enzymes have been delivered into cells through this receptor system and retained activity for at least six hours. Leamon et al. , P. N.A. S. , 88:5572-5576 (1991) . Folate receptors have limited tissue distribution and are overexpressed in several malignant cell lines derived from many tissues. Weitman et al. , Cancer Res . , 52:3396-3401 (1992) ; Weitman et al. , Cancer Res . , 52:6708- 6711 (1992) ; Campbell, Cancer Res . , 51:5329-5338 (1991) ; Coney, Cancer Res . , 51:6125-6123 (1991) . Other studies have also used biotin or folate conjugated to proteins by biotinylation for protein delivery to the cell. Low et al., U.S. Patent 5,108,921.
• DNA or macromolecules Once DNA or macromolecules are targeted to a cell for delivery, the DNA or macromolecule must be released from the endosome to function as a therapeutic agent. If not, the delivery of DNA and macromolecule will be hindered by lysosomal degradation. Studies have analyzed the endosomal/lysosomal degradation process. It has been determined that organisms which are internalized via receptor-mediated endocytosis or receptor:ligand systems, like viruses and other microorganisms, escape lysosomal degradation in order to function. The entry mechanism of some viruses have been studied extensively. For some viruses outer membrane proteins have been demonstrated to be important for endosomal escape. Marsh et al. , Adv. Virus Res . , 36:107-151 (1989) .
• adenovirus has been coupled enzymatically to polylysine through the e-NH 2 of lysine and the ⁇ -carboxyl of glutamic acid. Wagner et al. , P. N.A . S. , 89:6099-03 (1992) . Chemical coupling of polylysine with the acidic residues of adenovirus also accomplishes the same objective.
• peptide sequences from other viruses have been used to achieve endosome rupture.
• a lytic peptide from influenza hemagglutinin has been used to augment gene transfer by transferrin-polylysine-DNA complexes.
• This virus ⁇ like genetic transfer vehicle has been shown to be functional in vi tro but 100-fold less effective than adenovirus, based on the delivery and expression of the luciferase reporter construct. Id.
• HIV human immunodeficiency virus
• U.S. Patent 5,149,782 (Chang et al. , issued September 22, 1992) .
• Peptide segments from HIV have been suggested to be useful as membrane blending agents to deliver nucleic acids.
• Td. These peptides are fusogenic and allow the associated nucleic acid or molecular conjugate to be inserted into the cellular plasma membrane.
• Id. These peptides are 10- 30 amino acids in length and are hydrophobic.
• the fusion proteins used contain repetitious Phe-X-Gly sequences, where X is a nonpolar amino acid residue. Id.
• listeriolysin toxin forms pores in membranes which contain cholesterol. These pores are large enough for macromolecules like immunoglobulins to pass. Ahnert- Hilger et al. , Mol . Cell Biol . , 31:63-90 (1989) ; Geoffroy et al., J " . Bacteriol . , 172:7301-7305 (1990) .
• nucleic acid transporter systems for enhanced delivery of nucleic acid into the cell.
• These particular transporter systems enhance delivery of nucleic acid into the cell by using synthetic lysis and nucleic acid binding molecules.
• the specific lysis agents are useful in disrupting the endosome thereby allowing the nucleic acid to avoid lysosomal degradation.
• the specific binding molecules are useful in delivering to the cell stabilized and condensed nucleic acid.
• these specific binding molecules are useful in delivering stabilized and condensed nucleic acid into the nucleus of the cell .
• These transporters can be used to treat diseases by enhancing delivery of specific nucleic acid to the appropriately targeted cells.
• the present invention takes advantage of lysis agents to avoid the problems of endosomal/lysosomal degradation in the delivery of nucleic acid to a cell.
• the present invention features use of a nucleic acid transporter system with nucleic acid binding complexes that includes.a specific lysis agent capable of releasing nucleic acid into the cellular interior from the endosome.
• the nucleic acid can be efficiently released without endosomal/lysosomal degradation. Once released into the cellular interior, the binding complexes help target the nucleic acid to the nucleus.
• the present invention also takes advantage of DNA binding molecules in order to increase DNA stability and DNA delivery to cells.
• the present invention features use of nucleic acid transporters with nucleic acid noncovalently bound to peptides capable of condensing the nucleic acid.
• These binding molecules provide smaller, or condensed, and more stable nucleic acid particles for delivery, thereby enhancing the transfection rates of the nucleic acid into the cell and into the nucleus.
• the present invention enhances delivery of nucleic acid by the nucleic acid transporter system.
• These components can be used alone, together or with other components of the nucleic acid transporter described below and disclosed in PCT publication WO 93/18759, Woo et al. , entitled "A DNA
• the transporter system together with the lysis and binding molecule, enhances the delivery of nucleic acid to specific cells by enhancing the release of stable, condensed nucleic acid from the endosome into the cellular interior.
• the present invention also features various nucleic acid binding complexes which contain a surface ligand and a nuclear ligand as well.
• the surface ligands are capable of binding to a cell surface receptor and entering a cell through cytosis ( e . g. , endocytosis, potocytosis, pinocy- tosis) .
• nucleic acid can be delivered using the nucleic acid transporter systems directly to the desired tissue.
• the nuclear ligands are capable of recognizing and trans ⁇ porting nucleic acid through the nuclear membrane to the nucleus of a cell. Such nuclear ligands help enhance the binding molecules' ability to target nucleic acid to the nucleus.
• the abilities of the above transporters to deliver nucleic acid to specific cells and to the nucleus also allows transgenic animal models to be used for the dissection of molecular carcinogenesis and disease, assessing potential chemical and physical carcinogens and tumor promoters, exploring model therapeutic avenues as well as livestock agricultural purposes.
• the above nucleic acid transporter system advantages allow methods for administration and treatment of various diseases.
• the above nucleic acid transporter systems can be used to transform cells to produce particular proteins, polypeptides, and/or RNA.
• the above nucleic acid transporter systems can be used in vi tro with tissue culture cells. In vi tro uses allow the role of various nucleic acids to be studied by targeting specific expression into specifically targeted tissue culture cells.
• a first aspect of the present invention features a nucleic acid transporter system for delivering nucleic acid into a cell .
• the nucleic acid transporter includes a nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and associated with a lysis agent.
• the transporter can also include an additional binding molecule noncovalently bound to the nucleic acid.
• the nucleic acid binding complex and/or the additional binding molecule may be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions.
• the lysis agent can be associated with the respective binding molecule by a spacer.
• lysis agent refers to a molecule, compound, protein or peptide which is capable of breaking down an endosomal membrane and freeing the contents into the cytoplasm of the cell.
• the lysis agent can work by: (1) a membrane fusion mechanism, i.e., fusogenic, whereby the lysis agent associates or fuses with the cell membrane to allow the endosomal contents to leak into the cytoplasm; (2) a membrane destabilization mechanism whereby the lysis agent disrupts the structural organization of the cell membrane thereby causing leakage through the endosome into the cytoplasm; or (3) other known or unknown mechanisms which cause endosomal lysis.
• lytic peptide refers to a chemical grouping which penetrates a membrane such that the structural organization and integrity of the membrane is lost. As a result of the presence of the lysis agent, the membrane undergoes lysis, fusion or both.
• a preferred lysis agent is the JTS-1 peptide or derivatives thereof.
• the amino acid sequence of JTS-1 lytic peptide is GLFEALLELLESLWELLLEA.
• the JTS- 1 lytic peptide and derivatives are designed as an a- helix, which contains a sequence of amino acids such that the side chains are distributed to yield a peptide with hydrophobic and hydrophilic sides.
• Such ⁇ -helixes are termed amphipathic or amphiphilic.
• the hydrophobic side contains highly apolar amino acid side chains, both neutral and non-neutral.
• the hydrophilic side contains an extensive number of glutamic acids but could also contain aspartic acid, as well as polar or basic amino acids.
• JTS-1 peptide would include any derivatives or modifications of the backbone thereof.
• the lytic peptide undergoes secondary structure changes at acidic pH resulting in the formation of oligomeric aggregates which possess selective lytic properties.
• parameters that are important for amphiphilic peptide lysis activity include the following.
• Hydrophobicity The peptide must have a high enough hydrophobicity of the hydrophobic face to interact with and penetrate phospholipid-cholesterol membranes, i.e., lipid binding per se is not sufficient. Red cell hemolysis assays give better indications of which peptides will have useful activity.
• Peptide aggregation The ability to aggregate plays an important role in lysis and transfection.
• pH sensitivity The amphiphilic peptide must be pH sensitive. Lysis activity can be controlled by the introduction of lysine, arginine and histidine residues into the hydrophilic face of JTS-1.
• Lipid membrane interaction The peptide must have a hydrophobic carboxyl terminal to permit interaction with lipid membranes, e . g. , tyrosine substitution for tryptophan greatly reduces activity.
• Peptide chain length The length must be greater than twelve residues in order to get stable helix formation and lipid membrane penetration and rupture.
• derivative refers to a peptide or compound produced or modified from another peptide or compound of a similar structure. This could be produced in one or more steps.
• modified or “modification” as used herein refers to a change in the structure of the compound or molecule. However, the activity of the derivative, modified compound or molecule is retained, enhanced, increased or similar to the activity of the parent compound or molecule. This would include the change of one amino acid in the sequence of the peptide or the introduction of one or more non- naturally occurring amino acids or other compounds. This includes a change in a chemical body, a change in a hydrogen placement, or any type of chemical variation.
• analog refers to a compound that resembles another structure, e . g. , JTS-1, K 8 , K N , or spermine (discussed below) . Analog is not necessarily an isomer.
• the JTS-1 peptide can be modified to change the L in position 2 to an F so as to have the structure GFFEALLELLESLWELLLEA. Such a change can increase the hydrophobicity of the peptide.
• increasing the length of the peptide would also be a modification, i . e . , GLFEALLELLESWELLLGLFE .
• a change in the S at position 12 to a K to modify JTS-1 to GLFEALLELLEKLWELLLEA can shift the pH optimum for lysis and enhance proteolysis.
• the above are only examples and meant to be nonlimiting.
• lysis agents include, but are not limited to, peptides of the Othromyxoviridae, Alphaviridae and Arenaviridae. Lysis agents also can include Pep24, Pep25, Pep26, (see PCT publication WO 93/18759, hereby incorporated by reference, including drawings) , any appropriate bacteria toxin, bacteria, adenovirus, para- influenza virus, herpes virus, retrovirus, hepatitis virus, or any appropriate lytic peptide or protein from a virus or bacteria. This includes use of any subfragments of the above which will provide endosomal escape activity.
• Particular bacterial toxins may include cytolytic toxins or active fragments from alveolysin, bifermentolysin, botulinolysin, capriciolysin, cereolysin 0, chauveolysin, histolyticolysin 0, ivanolysin, laterosporolysin, oedematolysin O, listeriolysin O, perfringolysin O, pneumolysin, sealigerolysin, septicolysin O, sordellilysin, streptolysin 0, tetanolysin or thuringolysin 0.
• the lysis agent can be a replication deficient virus.
• the term "replication deficient" refers to a virus lacking one or more of the necessary elements for replication.
• the lysis agent can also be the adenovirus of the structure F, Pep24, Pep25, or Pep26.
• bacteria toxins, listeriolysin or perfringolysin can be used. All of the above are disclosed in PCT publication WO 93/18759, which is hereby incorporated by reference, including drawings. The above are only examples and are nonlimiting. Lysis agents as used herein are pH sensitive. The pH optimum is determined by the sequence and the content of acidic and basic amino acid side chains.
• binding molecule refers to a molecule, compound, protein or peptide which is capable of stabilizing and condensing nucleic acid. This will include, but is not limited to, components which are capable of stabilizing and condensing nucleic acid by electrostatic binding, hydrophobic binding, hydrogen binding, intercalation or forming helical structures with the nucleic acid, including interaction in the major and/or minor grove of DNA.
• binding molecule can also be referred herein as condensing agent.
• the binding molecule is capable of noncovalently binding to nucleic acid.
• the binding molecule is also capable of associating with a surface ligand, a nuclear ligand, and/or a lysis agent.
• the term "associated with” as used herein refers to binding, attaching, connecting or linking molecules through covalent means or noncovalent means.
• covalent and noncovalent includes, but is not limited to, a binding molecule associated with a surface ligand, nuclear ligand and/or a lysis agent. In addition, it includes the association of a spacer (discussed below) with the above components.
• a preferred binding molecule is the peptide K 8 .
• the amino acid sequence of K 8 is YKAKKKKKKKKWK.
• the binding molecule is any peptide with the formula YKAK N WK, where N can be between 1-40. This formula or amino acid structure can be referred to as "K N " . This would include use of any subfragments of the above which provide nucleic acid stability and condensing characteristics.
• the above peptides can include lysine or arginine residues for electrostatic binding to nucleic acid. These positively charged amino acids help hold the nucleic acid intact .
• the binding molecule can also contain tyrosine which is useful in determining peptide concentration and iodination for tracking purposes in vi tro and in vivo . Tryptophan also increases the stability of interaction with the nucleic acid through intercalation. In addition, binding of the peptide to DNA quenches tryptophan fluorescence and allows the kinetics and thermodynamics of complex: formation to be determined.
• the binding molecule can also contain helix forming residues such as tryptophan, alanine, -leucine or glutamine.
• binding molecule can also include a stabilized cyclic version of K 8 or the general
• YKAK N WK structure K N Such a cyclic version can be formed by introducing a lactam or disulfide bridge. Likewise, dimers of K 8 or K N can also be used as a binding molecule.
• parameters that are important for binding molecules include the following.
• the peptide must contain sufficient lysine or arginine residues to permit ionic interaction with the DNA.
• the peptide must have sufficient length to form a stable helix, eleven or twelve residues, and condense the DNA to small particles, e . g. , K 4 forms larger particles than K 8 .
• the peptide helix that forms upon interaction with DNA can be stabilized by leucine zipper formation which gives a condensing agent less susceptible to ionic strength.
• the lysine or arginine sequence of the condensing peptide serves as an additional function as a nuclear localization sequence.
• the binding molecule can also include, but is not limited to, spermine, spermine derivative, spermidine, histones, polylysine, polyamines and cationic peptides.
• K 8 or K N this includes, but is not limited to, analogs, modifications or derivatives of the above compounds.
• spermine derivatives include compounds D, IV, VII, XXI, XXXIII, XXXVI, LIV, LVI, LXXXII, LXXXIV and CX as described in PCT publication WO 93/18759, hereby incorporated by reference, including drawings.
• the binding molecules such as K 8 , K N or spermine, whether associated with a surface ligand, nuclear ligand, lysis agent, or separate therefrom, can be different or similar binding molecules and bound at the same time, i.e., simultaneously and in various proportions.
• the binding molecule is a spermine derivative D, as shown in the above referenced publication.
• K 8 , K N , and spermine have advantages over poly-L-lysine as used for the binding molecule.
• the binding properties of K 8 , K N , or spermine have advantages over the binding properties of poly-L-lysine.
• the intranuclear K 8 , K N , or spermine concentration is approximately 3 to 10 mmol. This is higher than studies with poly-L-lysine, which suggest more efficient transfer of nucleic acid to the nucleus.
• the spacing of the amino groups of K 8 , K N or spermine is such that this naturally occurring polycation fits into the major groove of the DNA double helix with an exact fit.
• nucleic acid transporter system refers to a molecular complex which is capable of efficiently transporting nucleic acid through the cell membrane. This molecular complex is bound to nucleic acid noncovalently.
• nucleic acid transporter system In addition to nucleic acid, other macromolecules, including but not limited to, proteins, lipids and carbohydrates can also be delivered using the transporter system.
• the nucleic acid transporter system is capable of transporting nucleic acid in a stable and condensed state. It is also capable of releasing the noncovalently bound nucleic acid into the cellular interior. Furthermore, the nucleic acid transporter prevents degradation of the nucleic acid by endosomal lysis. In addition, although not necessary, the nucleic acid transporter system can also efficiently transport the nucleic acid through the nuclear membrane, as discussed below.
• the nucleic acid transporter system as described herein can contain, but is not limited to, six components. It comprises, consists or consists essentially of: (1) a nucleic acid or other macromolecule with a known primary sequence that contains the genetic information of interest or a known chemical composition; (2) an agent capable of stabilizing and condensing the nucleic acid or macromolecule in (1) above; (3) a lysis moiety that enables the transport of the entire complex from the cell surface directly into the cytoplasm of the cell; (4) a moiety that recognizes and binds to a cell surface receptor or antigen or is capable of entering a cell through cytosis; (5) a moiety that is capable of moving or initiating movement through a nuclear membrane; and/or (6) a nucleic acid or macromolecuiar molecule binding moiety capable of covalently binding the moieties of (3) , (4) and (5) , above.
• the term “delivery” refers to transportation of a molecule to a desired cell or any cell. Delivery can be to the cell surface, cell membrane, cell endosome, within the cell membrane, nucleus or within the nucleus, or any other desired area of the cell. Delivery includes not only transporting nucleic acid but also other macromolecules including, but not limited to, proteins, lipids, carbohydrates and various other molecules.
• the term “nucleic acid” as used herein refers to DNA or RNA. This would include naked DNA, a nucleic acid cassette, naked RNA, or nucleic acid contained in vectors or viruses. These are only examples and are not meant to be limiting.
• expression includes the efficient transcription by the cell of the transported gene or nucleic acid. Expression products may be proteins, polypeptides or RNA.
• the nucleic acid can be antisense RNA, oligonucleotides or ribozymes as well.
• proteins and polypeptides can be encoded by the nucleic acid.
• proteins or polypeptides which can be expressed include hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumor suppressors, cytokines, viral antigens, parasitic antigens and bacterial antigens.
• these compounds include proinsulin, insulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin growth factor binding proteins, epidermal growth factor, TGF- ⁇ , TGF-3, dermal growth factor (PDGF) , angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin) , matrix proteins (Type IV collagen, Type VII collagen, laminin) , oncogenes ⁇ ras, fos, myc, erb, src, sis, jun) , E6 or E7 transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8, IL-2, ⁇ , ⁇ , or ⁇ lFN, GMCSF, GCSF, viral capsid protein, and proteins from viral, bacterial and parasitic organisms.
• PDGF dermal growth factor
• angiogenesis factors acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin
• matrix proteins
• proteins or polypeptides which can be expressed include: phenylalanine hydroxylase, v-1-antitrypsin, cholesterol- 7 -hydroxylase, truncated apolipoprotein B, lipoprotein lipase, apolipoprotein E, apolipoprotein Al, LDL receptor, molecular variants of each, and combinations thereof.
• phenylalanine hydroxylase v-1-antitrypsin
• cholesterol- 7 -hydroxylase truncated apolipoprotein B
• lipoprotein lipase apolipoprotein E
• apolipoprotein Al apolipoprotein Al
• LDL receptor molecular variants of each, and combinations thereof.
• proteins belong to a wide variety of classes of proteins, and that other proteins within these classes can also be used. These are only examples and are not meant to be limiting in any way.
• the genetic material which is incorporated into the cells from the above nucleic acid transporter system includes (1) nucleic acid not normally found in the cells; (2) nucleic acid which is normally found in the cells but not expressed at physiological significant levels; (3) nucleic acid normally found in the cells and normally expressed at physiological desired levels; (4) other nucleic acid which can be modified for expression in cells; and (5) any combination of the above.
• nucleic acid binding complex refers to a complex which includes a binding molecule.
• the binding molecule as defined above, is capable of noncovalently binding to nucleic acid.
• the binding molecule is also capable of associating with a surface ligand, a nuclear ligand and/or a lysis agent.
• the binding complex can include a spacer which associates with the surface, nuclear or lysis agent to the binding molecule. Spacers are defined in more detail below.
• a second aspect of the present invention features a nucleic acid transporter system for delivery of a nucleic acid to a cell which includes a first nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and associated with a surface ligand.
• the transporter also includes a second nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and associated with a lysis agent.
• the transporter can also include an additional binding molecule noncovalently bound to the nucleic acid.
• binding complexes and/or binding molecules above can be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions.
• the binding molecules can be the same or different molecules.
• the surface ligand or lysis agent can be directly associated with the binding molecules or associated by a spacer, as defined below.
• surface ligand refers to a chemical compound or structure which will bind to a sur ⁇ face receptor of a cell.
• cell surface receptor refers to a specific chemical grouping on the surface of a cell for which the ligand can attach. Cell surface receptors can be specific for a particular cell, i.e., found predominantly in one cell rather than in another type of cell ( e . g. , LDL and asialoglycoprotein receptors are specific for hepatocytes) . The receptor facilitates the internalization of the ligand and attached molecules.
• a cell surface receptor includes, but is not limited to, a folate receptor, biotin receptor, lipoic acid receptor, low-density lipoprotein receptor, asialoglycoprotein receptor, insulin-like growth factor type II/cation-independent mannose-6-phosphate receptor, calcitonin gene-related peptide receptor, insulin-like growth factor I receptor, nicotinic acetylcholine receptor, hepatocyte growth factor receptor, endothelin receptor, bile acid receptor, bone morphogenetic protein receptor, cartilage induction factor receptor or glycosyl- phosphatidylinositol (GPI) -anchored proteins ( e . g. , ⁇ - andrenargic receptor, T-cell activating protein, Thy-1 protein, GPI-anchored 5' nucleotidase) .
• GPI glycosyl- phosphatidylinositol
• a receptor is a molecule to which a ligand binds specifically and with relatively high affinity. It is usually a protein or a glycoprotein, but may also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan or a glycocalyx.
• epitopes to which an antibody or its fragments binds is construed as a receptor since the antigen:antibody complex undergoes endocytosis.
• surface ligand includes anything which is capable of entering the cell through cytosis ( e . g. , endocytosis, potocytosis, pinocytosis) .
• ligand refers to a chemical compound or structure which will bind to a receptor. This includes but is not limited to ligands such as asialoorosomucoid, asialoglycoprotein, folate, lipoic acid, biotin, as well as those compounds listed in PCT publication WO 93/18759, hereby incorporated by reference.
• the ligand chosen will -depend on which receptor is being bound. Since different types of cells have different receptors, this provides a method of targeting nucleic acid to specific cell types, depending on which cell surface ligand is used. Thus, the preferred cell surface ligand may depend on the targeted cell type.
• a third aspect of the present invention features a nucleic acid transporter system for delivery of a nucleic acid into a cell which includes a first nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and associated with a surface ligand.
• the transporter also includes a second nucleic acid binding complex containing a binding molecule noncovalently bound to nucleic acid and associated with a nuclear ligand.
• the transporter also includes a third nucleic acid binding complex containing a binding molecule noncovalently bound to a nucleic acid and associated with a lysis agent.
• the transporter can include a fourth binding molecule noncovalently bound to said nucleic acid.
• the nucleic acid binding complexes and/or binding molecules can be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions.
• the binding molecules can be the same molecule or a combination of a different molecule as discussed above.
• the surface ligand, nuclear ligand, and lysis agent can be directly associated with the binding molecule or associated by a spacer as defined below.
• nuclear ligand refers to a ligand which will bind a nuclear receptor.
• nuclear receptor refers to a chemical grouping on the nuclear membrane which will bind a specific ligand and help transport the ligand through the nuclear membrane.
• Nuclear receptors can be, but are not limited to, those receptors which bind nuclear localization sequences.
• Nonlimiting examples of nuclear ligands include those disclosed in PCT publication WO 93/18759, hereby incorporated by reference.
• the surface ligand, the nuclear ligand and/or the lysis agent can be associated directly to the binding molecule or can be associated with the binding molecule via a spacer.
• spacer refers to a chemical structure which links two molecules to each other. The spacer normally binds each molecule on a different part of the spacer molecule.
• the spacer can be hydrophilic molecules comprised of about 6 to 30 carbon atoms. The spacer can also contain between 6 to 16 carbon atoms.
• the spacer can include, but is not limited to, a hydrophilic polymer of [ (gly) ( ser) j ] k wherein i ranges from 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20.
• the spacer and binding molecule compounds include, but are not limited to, those compounds disclosed in PCT publication WO 93/18759, hereby incorporated by reference.
• -CO-CH 2 -C CH-CO-NH-CH 2 -CH 2 -S-.
• the lysis agent is JTS-1, or derivative thereof, and the binding molecule K 8 , or derivative thereof.
• Still another embodiment of the present invention can include a surface, nuclear ligand and the lysis agent as disclosed herein, and a binding molecule of K 8 , K N or derivative thereof.
• the surface and nuclear ligand can be one of those disclosed herein, the lysis agent can be JTS-1 or derivative thereof, and the binding molecule can be K 8 , K N or derivative thereof.
• these embodiments can also include the use of spacers as described above.
• folate is used as the surface ligand and JTS-1 is used as the lysis agent.
• This transporter can deliver to the cytosol other macromolecules besides nucleic acid including, but not limited to, proteins, lipids and carbohydrates.
• the binding complexes of this aspect can be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions.
• the binding molecules can be the same or different and may attach to the ligands or lysis agents directly or by spacers as described above.
• a nucleic acid binding molecule K 8 , K N or derivative, can also be used in conjunction with either embodiment.
• the binding molecule can be noncovalently bound to the nucleic acid. More than one binding molecule can be noncovalently bound to the nucleic acid at the same time, i.e., simultaneously, and in various proportions.
• an asialoglycoprotein can be used as the surface agent, K 8 , K N or derivative as the binding molecule and JTS-1 or derivative, listeriolysin or perfringolysin as the lysis agent . Listeriolysin, perfringolysin or only a part of the toxins harboring the active subfragments need be used. Similarly, all microbial toxins and their active subfragments can be incorporated into the transporters of the present invention for endosomal escape.
• a fourth aspect of the present invention features the JTS-1 composition and derivative. As discussed above, these compositions are advantageous in that they have endosomal lysis properties. When used with the nucleic acid transporter system as described above, JTS-1 or derivatives enhance the expression of nucleic acid targeted to a cell. The JTS-1 compound and derivatives are described below in more detail .
• a fifth aspect of the present invention features the K 8 or K N compositions and derivatives.
• these binding molecules are advantageous in that they have nucleic acid condensing/stabilizing properties.
• K 8 or K N compositions and derivatives enhance the expression of nucleic acid targeted to a cell.
• the K 8 or K N compounds and derivatives are described below in more detail.
• nucleic acid transporter system described above containing a plurality of nucleic acid binding complexes with a binding molecule noncovalently bound to nucleic acid and attached to a surface ligand, a nuclear ligand or a lysis agent.
• binding molecule noncovalently bound to nucleic acid
• Spacers can be used to connect the surface ligand, nuclear ligand and/or lysis agent.
• a sixth related aspect of the present invention features a method of using the above described nucleic acid transporters for delivery of a nucleic acid or a molecule to a cell.
• Such use includes both in vivo and in vi tro uses. This would include cells transformed with the nucleic acid transporter system as described above for expression of nucleic acid targeted to the cell.
• the nucleic acid may include nucleic acid containing genetic material and coding for a variety of proteins, polypeptides or RNA.
• transformation is a mechanism of gene transfer which involves the uptake of nucleic acid by a cell or organism. It is a process or mechanism of inducing transient or permanent changes in the characteristics (expressed phenotype) of a cell. Such changes are by a mechanism of gene transfer whereby DNA or RNA is introduced into a cell in a form where it expresses a specific gene product or alters the expression or effect of endogenous gene products. Following entry into the cell, the transforming nucleic acid may recombine with that of the host. Such transformation is considered stable transformation in that the introduction of gene(s) into the chromosome of the targeted cell where it integrates and becomes a permanent component of the genetic material in that cell.
• the transforming nucleic acid may exist independently as a plasmid or a temperate phage, or by episomes.
• An episomal transformation is a variant of stable transformation in which the introduced gene is not incorporated in the host cell chromosomes but rather remains in a transcriptionally active state as an extrachromosomal element. Transformation can be performed by in vivo techniques as described below, or by ex vivo techniques in which cells are cotransfected with a nucleic acid transporter system containing nucleic acid and also containing a selectable marker. This selectable marker is used to select those cells which have become transformed.
• the transformed cells can produce a variety of compounds selected from proteins, polypeptides or RNA, including hormones, growth factors, enzymes, clotting factors, apolipoproteins, receptors, drugs, tumor anti ⁇ gens, viral antigens, parasitic antigens, and bacterial antigens. Other examples can be found above in the dis- cussion of nucleic acid.
• the product expressed by the transformed cell depends on the nucleic acid used. The above are only examples and are not meant to be limiting. These methods of use would include the steps of contacting a cell with a nucleic acid transporter system as described above for a sufficient time to transform the cell.
• Cell types of interest can include, but are not limited to, liver, muscle, lung, endothelium, bone, blood, joints and skin.
• the methods of use would also include a transgenic animal whose cells -contain the nucleic acid referenced above delivered via the nucleic acid transporter system. These cells include germ or somatic cells. Transgenic animal models can be used for dissection of molecular carcinogenesis and disease, assessing potential chemical and physical carcinogens and tumor promoters, exploring model therapeutic avenues and livestock agricultural purposes.
• the methods of use also include a method of treating humans, which is another aspect of the present invention.
• the method of treatment includes the steps of administering the nucleic acid transporters as described above so as to deliver a desired nucleic acid to a cell or tissue for the purposes of expression of the nucleic acid by the cell or tissue.
• Cell or tissue types of interest can include, but are not limited to, liver, muscle, lung, endothelium, joints, skin, bone and blood.
• the methods of treatment or use include methods for delivering nucleic acid into a hepatocyte by contacting a hepatocyte with the above referenced nucleic acid transporters.
• the surface ligand used with the nucleic acid transporter is one specific for recognition by hepatocyte receptors.
• the asialooro- somucoid protein is used as a cell surface ligand, K 8 , K N or a derivative as a binding molecule and JTS-1 or a derivative as a lysis agent.
• these methods of use also include delivery of nucleic acids using a transporter with JTS-1 and K 8 and no surface or nuclear ligands.
• hepatocyte refers to cells of the liver.
• An aspect of the methods of treatment or use includes a method for delivering nucleic acid to muscle cells by contacting the muscle cell with one of the above referenced nucleic acid transporter system.
• the surface ligand used is specific for receptors contained on the muscle cell.
• the surface ligand can be insulin-like growth factor-I.
• the binding molecule can be a K 8 , K N or a derivative and the lysis agent can be JTS-1 or a derivative.
• these methods of treatment or use also include delivery of nucleic acids using a transporter with JTS-1 and K 8 and no surface or nuclear ligands.
• the term "muscle cell” as used herein refers to cells associated with striated muscle, smooth muscle or cardiac muscle.
• Another aspect of the methods of treatment or use includes a method for delivering nucleic acid to bone- forming cells by contacting the bone-forming cell with the above referenced nucleic acid transporter system.
• the surface ligand used with the nucleic acid transporter system is specific for receptors associated with bone- forming cells.
• the surface ligands can include, but are not limited to, bone morphogenetic protein or cartilage induction factor.
• the binding molecule of the nucleic acid transporter can be K 8 , K N or a derivative, and the lysis agent JTS-1 or a derivative thereof.
• these methods of treatment or use also include delivery of nucleic acids using a transporter with JTS-1 and K 8 and no surface or nuclear ligands.
• the term "bone-forming cell” refers to those cells which promote bone growth.
• Nonlimiting examples include osteoblasts, stromal cells, inducible osteoprogenitor cells, determined osteoprogenitor cells, chondrocytes, as well as other cells capable of aiding bone formation.
• Another related aspect of the methods of treatment or use includes a method for delivering nucleic acid to a cell using the above referenced nucleic acid transporter system.
• the nucleic acid transporter system uses folate as a ligand.
• the nucleic acid transporter can use JTS-1 or a derivative as a lysis agent, and K 8 , K N or a derivative thereof as a binding molecule. This method targets cells which contain folate receptors, including, but not limited to, hepatocytes.
• Still another related aspect of the methods of treatment or use includes a method for delivering nucleic acid to synovialcytes or macrophages using the above referenced nucleic acid transporter system.
• the nucleic acid transporter system uses a ligand recognized by synovialcytes and/or macrophages.
• the nucleic acid transporter can use JTS-1 or a derivative as a lysis agent, and K 8 , K N or a derivative thereof as a binding molecule.
• this method of use also includes delivery of nucleic acids using a transporter with JTS-1 and K 8 and no surface or nuclear ligands.
• the term "synovialcytes" refers to cells associated with the joints or with the fluid space of the joints.
• the method of use also includes delivery using a nuclear ligand binding complex as well.
• nuclear transporters would help direct the nucleic acid to the nucleus.
• the above methods of use also include nucleic acid transporters with the binding molecule and the lysis agent, or a plurality thereof.
• the nucleic acid transporters of the above methods may be administered by various routes.
• administration refers to the route of introduction of the nucleic acid transporter or carrier of the transporter into the body.
• Administration may be intravenous, intramuscular, topical, olfactory or oral.
• Administration can be directly to a target tissue or through systemic delivery.
• administration may be by direct injection to the cells.
• administration may be intravenously, by hypospray or the use of PVP, an amorphous powder.
• Routes of administration include intramuscular, aerosol, oral, topical, systemic, olfactory, ocular, intraperitoneal and/or intratracheal.
• Figure 1 represents the JTS-1 amino acid sequence and ⁇ -helix structure.
• Figure 2 represents n-acyl tetrapeptides with membrane destabilizing activity.
• Figure 3 represents ⁇ -helical peptides with lytic activity.
• Figure 4 represents expression results of JTS-1 mediated expression in Skov-3, ML-3, Sol B, HCT-16 ⁇ or CIT-26 cells.
• Figure 5 represents expression results of JTS-1 mediated gene delivery.
• Figure 6 is a representation of K 8 peptides and various R group substitutions.
• Figure 7 is a representation of K 8 variations by changing side chain length and charged groups.
• Figure 8 is a representation of pseudopeptides substituted at core lysine sequences of K N to improve stability.
• Figure 9 is a schematic for formation of pegylated K N peptides.
• Figure 10 is a representation of transfection efficiency using K N peptides.
• Figure 11 is a schematic formula of JTS/K 8 conjugates.
• Figure 12 is a representation of transfection of C 2 C 12 myoblast cells with K 8 /JTS-l/DNA complexes.
• Figure 13 is a representation of transfection of 4Mbr- 5 bronchus cells with K 6 or K 7 /JTS-l/DNA complexes.
• Figure 14 is a representation of target ligands used to direct delivery of JTS/K 8 /DNA complex to the hepatocyte.
• Figure 15 is a representation of target ligands containing carbohydrates for uptake by asialoglycoprotein receptor.
• Figure 16 is a representation of target ligands for delivery of JTS/K 8 /DNA to cells with mannose or mannose-6- phosphate receptors.
• Figure 17 is a representation of RGD targeting ligands for delivery of JTS/K 8 /DNA to connective tissue, wounds and for healing.
• Figure 18 is a representation of ligands useful in delivery of JTS/K 8 /DNA to hepatocytes.
• nucleic acid transporter systems with lysis and/or binding molecules for delivery of nucleic acid to a cell. These examples are offered by way of illustration and are not intended to limit the invention in any manner.
• nucleic acid transporter systems that can be used to provide certain functionalities to the associated nucleic acid in the nucleic acid transporter system, and thus within a transformed cell or animal containing such associated nucleic acid.
• moieties of the nucleic acid transporter system can be identified as that containing the functional region providing the desirable properties of the nucleic acid transporter system. Such regions can be readily minimized using routine deletion, mutation, or modification techniques or their equivalent .
• fusogenic or membrane disruptive peptides were designed which would increase the rate of delivery of nucleic acid from the endosome to the cell and ensure that higher concentrations of the endocytosed nucleic acid would be released and not degraded in the endosomes.
• a number of fusogenic/lytic peptides have been previously described, including the amino terminal sequence of the vesicular stomatitis virus glycoprotein and the synthetic amphipathic peptide GALA. Ojcius et al., TIBS, 16:225-229 (1991) ; Doms et al . , Membrane Fusion, pp.
• These peptides are amphipathic membrane associating peptides. These amphipathic peptides were designed as an ⁇ -helix, containing a sequence of amino acids such that the side chains are distributed so that the peptide has a hydrophobic and hydrophilic side. The hydrophobic side contains highly apolar amino acid side chains, while the hydrophilic side contains an extensive number of glutamic acids.
• the amphipathic membrane associating peptides usually contain 21 amino acids or fewer.
• the design criteria requires that the amino acids have a high probability of forming amphiphilic species. This can be exhibited in the secondary structure of the membrane associating peptides, i.e., helices, turns, bends, loops, j ⁇ -sheets, and their oligomeric aggregates and other super secondary structures defined in the literature, e . g. , helix-turn-helix.
• the amino acids should have a high probability of being found in an ⁇ -helix and a low probability of forming a /3-sheet or turn structure. Leucine, lysine and glutamate are appropriate amino acids for such characteristics.
• lysine positioned on the lateral face of the ⁇ -helix and glutamate residues opposite leucine provide optimal charge distribution for lipid interaction.
• lysines and glutamates can be positioned to take advantage of potential helix stabilization. Helix dipole stabilization is optimized by removing the charge at the NH 2 and COOH-termini so NH 2 termini and COOH-terminal amides are useful. Such probabilities can be determined from secondary structural predictions or analogous methods to optimize secondary structural design. Unnatural amino acid which have been described for their propensity to induce helix structures in peptides are also used. The hydrophobic or lipophilic face has a great effect on lipid-peptide interactions.
• the lipophilic face is modeled after peptides known to interact with lipids.
• Hydrophobic and lipid interactive residues Al, Leu, Met, Val, Phe, Trp, Tyr, Cys, Pro
• an acid group and/or hydrophilic group Glu, Gin, His, Lys, Gly, Ser, Asp, Asn, Pro, Arg
• the lipophilic and hydrophilic faces can also contain residues which promote lipid interaction and/or induce endosomal lysis at acidic pH. Such an interaction is not limited to an ⁇ -helix promoting residue since glycine and serine positioned on the hydrophilic face have been shown to favorably influence activity as seen with the examples below.
• the JTS-1 peptide has a hydrophobic face which contains only strongly apolar amino acids, while the hydrophilic face is dominated by negatively charged glutamic acid residues at physiological pH values.
• the JTS-1 peptide uses the Gly-Leu-Phe sequence at amino acid positions 1-2-3, respectively, as a fusogenic or membrane disruptive sequence.
• Glu is added at amino acid position 4.
• Ser-Leu-Trp-Glu is used as a lipid binding site. The remaining sequences are arranged to provide the hydrophobic and hydrophilic face of JTS-1.
• the helical wheel of the amphipathic membrane associating peptide JTS- 1 can be found in Figure 1.
• This figure shows the division of the hydrophobic and hydrophilic faces within the JTS-1 helical structure.
• Amino acids 16, 9, 2, 13, 6, 17, 10, 3, 14, 7 and 18 form the hydrophobic face.
• Amino acids 5, 12, 1, 8, 15, 4 and 11 form the hydrophilic face.
• the following JTS peptides were constructed and characterized for lytic activity:
• n-acyl tetrapeptides with fusogenic or membrane destabilizing activity can be constructed. The structure of these is set forth in
• FIG. 3 shows the helical wheels of smaller fusogenic peptides.
• LLEKLLEWLE (number IV in Figure 3) is a shorter ⁇ -helical peptide with lytic properties.
• Leucine is used for hydrophobic properties and ⁇ -helical movement.
• Glutamic acid residues are used for lytic activity. These residues also have the propensity to form an ⁇ -helical structure at pH 4.0.
• a COOH-terminal amide is used to provide helix-dipole optimization. When in an cu-helical structure the hydrophobic face appears at positions 4, 7, 3, and 10.
• the peptide was lengthened to an 11-mer. Adding the additional amino acid to form the following peptide, Suc-GLFKLLEEWLE, allowed the activity of the three glutamic acids to be retained.
• the peptide was succinylated at the amino terminus to afford an i to i+4 salt bridge with lysine which is designed to stabilize the helix.
• JTS-1 peptides are synthesized by the solid phase method as developed by Merrifield et al., Solid Phase Peptide Synthesis, Academic Press (N.Y. 1980) .
• a modified polystyrene (Sparrow, J. Org. Chem . , 41:1350-1353 (1976)) and/or polyamide resin (Sparrow et al., Int. J. Peptide Prot . , 38:385-391 (1991)) with fast HBTU/HOBT coupling is used.
• HBTU/HOBT coupling is used. It should be noted that the following procedure was used to synthesize INF-7 as well.
• the procedures involved in solid phase synthesis include:
• the carboxyl terminal amino acid protected with the N- c butyloxycarbonyl group is esterified to bromomethyl-phenylacetic acid and coupled directly to aminomethyl-polystyrene resin or to a resin containing a long spacer chain between the point of attachment and the polystyrene backbone or directly to the amino-propyl polyamide resin.
• the above procedure is modified as follows to give a total program time of 45 minutes. Trifluoroacetic acid (100%) is used to deprotect the amino group in 6 minutes.
• the resulting salt is neutralized by the excess diisopropylethylamine used to activate the N- ⁇ utyloxycarbonyl amino acid with HBTU/HOBT in dimethylformamide (“DMF”) .
• DMF dimethylformamide
• the coupling reaction is allowed to proceed for 15 minutes and the resin washed extensively with DMF and dichlormethane (“DCM”) . These steps are then repeated until the sequence of interest has been synthesized.
• the peptide is cleaved from the solid support by treatment of 1 g of resin with 60 ml of anhydrous hydrogen fluoride containing 10% anisole and 1% ethanedithiol for 30 minutes at 0°C. In the case of peptides containing arginine, the cleavage is performed at -20°C for 3 hours.
• the hydrogen fluoride is evaporated under vacuum at 0°C and the peptide precipitated with ether.
• the peptide and resin are filtered off and washed with ether.
• the peptide is then extracted with trifluoroacetic ("TFA") (3 x 30 ml) and the TFA evaporated under vacuum.
• TFA trifluoroacetic
• the precipitate is suspended in 10 ml 1 M TRIS and 6 M GnHCl. An additional 30 ml of 6 M GnHCl is added to completely dissolve the peptide. The pH of the solution is adjusted to 8.0.
• the peptide is desalted on a column of BioGel P-2 equilibrated in 0.1 M ammonium bicarbonate for the lytic peptides.
• the peptide fractions are located by absorbance at 254 nm and 280 nm, pooled and lyophilized. The lyophilized peptide is dissolved in 25 ml of 6 M GnHCl for the lytic peptides. A 280 is used to determine peptide concentration.
• This solution is pumped onto a 2.5 x 25 cm Vydac C4 column (214TP152022; 300 A pore size) equilibrated in 0.01 M ammonium phosphate at a flow rate of 20 ml/min.
• Two buffers are used to elute the peptide, Buffer A (0.01 M ammonium phosphate in ddH 2 0, pH 6.7) and Buffer B (2- propanol 100%) .
• the peptide is eluted with a linear gradient between Buffer A and 30% Buffer B, and Buffer A and 50% Buffer B.
• the gradient program used is Buffer A to 50% Buffer B for 45 minutes, with retention time being 40-41 minutes.
• the peptide is detected by absorbance at 254 and 280 nm.
• the peptide containing fractions are pooled, the pH adjusted to 8.0 with ammonium hydroxide and desalted on a BioGel P-2 column equilibrated in 0.1 M ammonium bicarbonate.
• the peptide containing fractions are pooled and lyophilized.
• the peptide is dissolved in water after lyophilization. Diluted ammonium hydroxide is added to adjust the pH to 7.5 in order to completely dissolve the peptide.
• a single peak for JTS-1 occurs at the expected mass of 2302 amu.
• Secondary structure is determined by circular dichroic and FTIR spectroscopy. These standard methods are used to confirm the secondary structure of JTS-1.
• the lipid was resuspended in 100 mM calcein (adjusted to.pH 7.3 with sodium hydroxide) and sonicated with a probe sonicator for 20 minutes in an ice bath. Liposomes were separated from unentrapped calcein using a Sephadex G25 column. Calcein release was measured at 520 nm (excitation at 470 nm) using a fluorescence spectrophotometer. For leakage assays, liposomes were diluted 1000-fold in 150 mM NaCl, 15 mM sodium citrate, pH 7.0 or 5.0. Peptide was added at a concentration of 1 ⁇ g/ml. Fluorescence was measured before and 10 minutes after addition of peptide. 100% leakage was determined by adding Triton X-100 to a final concentration of 0.5%. The turbidity was monitored using apparent absorbance measurements at 400 nm on a visible-wavelength spectrophotometer. This experiment was performed at various pHs.
• Tween ⁇ O was used as a positive control.
• Tween ⁇ O is a surface active agent which concentrates at oil-water interfaces causing an emulsifying action. Such properties allow Tween ⁇ O to disrupt membranes of synthetic liposomes.
• PVP was used to see if there was any activity.
• PVP is an amorphous powder, is compatible with hydrophobic and hydrophilic residues. Although it can be used as a detergent, PVP has colloid protective properties. These properties allow PVP to act as a surface-active substance that prevents the dispersion of a suspension, i.e., liposomes, from coalescing by forming a thin layer on the surface of each particle.
• INF-7 showed pH dependent membrane activity on phosphatidylcholine liposomes.
• the activity of INF-7 was approximate 8-fold lower than that of JTS-1.
• the JTS peptides were studied for their ability to lyse erythrocytes.
• Erythrocytes were isolated by methods known in the art. Hemolysis assays were performed according to the literature. Erythrocyte lysis assays have been previously used to determine the membrane activity of bacterial toxins and membrane active peptides. Human erythrocytes were washed three times with phosphate buffered saline and were resuspended in 150 mM NaCl, 15 mM sodium citrate, pH 7.0 or 5.0, at a concentration of 7xl0 7 /ml.
• Peptides were diluted serially in 150 mM NaCl, 15 mM citrate pH, 7.0 or 5.0, in a 96-well plate. Next, 75 ⁇ l erythrocyte suspension was added to each well. The plates were incubated at pH 7.0 and pH 5.0 for 60 minutes at 37°C with occasional shaking. Unlysed erythrocytes were pelleted and the extent of hemolysis was determined visually. One hemolytic unit (HU) was defined as the amount of protein necessary to induce >50% hemolysis. All hemolysis assays were performed in duplicates.
• HU hemolytic unit
• JTS-1 was hemolyticly active at pH 5.0 and not at pH 7.0. At pH 5.0, the specific hemolytic activity was lxlO 7 erythrocytes lysed per ⁇ g of peptide. JTS-3 had a specific hemolytic activity at pH 5.0 of 8xl0 7 erythrocytes lysed per ⁇ g of peptide, whereas JTS-9 was 3x10 7 erythrocytes lysed per ⁇ g of peptide.
• INF-7 is the active region of HA 2 , influenza hemagglutinin, responsible for fusion of the influenza viral envelope with the plasma membrane of cells.
• INF-7 has the following amino acid sequence, GLFEAIEGFIENGWEGMID. Amino acid numbers 5, 16, 9, 2, 13, 6, 17, 10, 3 and 14 of the helical wheel form the hydrophobic face. Amino acid numbers 7, 18, 11, 4, 15, 8, 1 and 12 of the helical wheel form the hydrophilic face.
• Erythrocytes were isolated by methods well known in the art. Serial dilutions of INF-7 and JTS-1 peptides were incubated with washed human erythrocytes at pH 7.0 and pH 5.0. After one hour, the unlysed erythrocytes were pelleted. The concentration at which 50% lysis occurred was determined by visual reading. INF peptides showed no hemolytic activity at either pH 7.0 or pH 5.0. Just as above, JTS-1 was hemolyticly active at pH 5.0 and not at pH 7.0. At pH 5.0, the specific hemolytic activity was lxlO 7 erythrocytes lysed per ⁇ g peptide.
• Liposome membrane activity was measured by testing liposomal leakage. This assay measures the release of calcein, a fluorescent dye, from phosphatidylcholine
• PC fluorescence vesicles. Briefly, calcein is encapsulated into liposomes by well known procedures at a concentration where the fluorescence of the dye is greatly reduced (self-quenching) . When the liposomes are destroyed by the lysis agent, the fluorescent dye leaks out of the liposomes and is diluted in the incubation buffer. This causes a great increase of fluorescence (dequenching) which can be followed in a fluorescence spectrophotometer. Liposomes were incubated with monomeric forms of JTS- 1, JTS-3 and JTS-9 peptides at a concentration of 0.5 ⁇ g/ml in a sodium-citrate buffer with a pH ranging from 5.0 to 7.0.
• the fluorescence was determined.
• the fluorescence corresponding to 100% leakage was determined by complete lysis of the liposomes with a detergent (Triton X-100; final concentration 0.5%) and the values obtained were plotted as percentage leakage.
• INF-7 showed pH-dependent membrane activity on liposomes. In contrast to JTS-1, only very little hemolytic activity was observed. Furthermore, the JTS-9 peptide was shown to be more potent than the activity of JTS-1 and JTS-3.
• JTS-1 JTS-1 to mediate expression in cells in vi tro was tested using DNA complexes containing a CMV- / ⁇ -galactosidase expression vector with transferrin/poly-L- lysine and unmodified poly-L-lysine to create a positive particle.
• the cell lines tested were obtained from ATCC and cultured by well known methods in the art.
• the reaction was incubated for 24 hours at room temperature after which it was concentrated and resuspended in 2 M Guanidine-HCl, 50 mM HEPES, pH 7.3, and fractionated by gel filtration on a Superose 6 column with a Fast Protein Liquid Chromatography system ("FPLC”) .
• FPLC Fast Protein Liquid Chromatography system
• the DNA plasmid CMV/ ⁇ -gal containing the E. coli ⁇ - galactosidase gene under the control of the CMV enhancer and promoter was used as a reporter gene.
• the plasmid was isolated and purified by double CsCl banding. This plasmid has been thoroughly described in the art.
• unmodified poly-L-lysine was added to help create a positive particle.
• the JTS-1 peptides were added and bound to the DNA complex through ionic interactions.
• Conjugate/DNA complexes were prepared by diluting the conjugate in 150 ⁇ l of HBS (150 mM NaCl per 20 mM HEPES, pH 7.3) and diluting 6 ⁇ l of DNA, in 350 ⁇ l of HBS. The diluted DNA was added directly to the diluted conjugate while mixing. The reaction was allowed to incubate at room temperature for 30 minutes before analysis. Immediately following the incubation, all complexes were analyzed on 0.8% agarose gels and electrophoresed in TBE. Six ⁇ g of DNA complex was incubated with 3xl0 5 tissue culture cells.
• the complete media was removed and replaced with 1 ml of Low Glucose DMEM containing, 5 mM Ca 2+ and 2% fetal calf serum. After a 2 hour incubation at 37°C, 1.5 ml of complete media was added to the tissue culture cells and the incubation continued for 24 hours at 37°C.
• the cells are washed with IX PBS twice and incubated at 37°C for 30-60 minutes with the solution containing 0.2% X-gal, 10 mM sodium phosphate buffer, pH 7.0, 150 NaCl, 1 mM MgCl 2 , 3.3 mM K 4 Fe (CN) 6 3H 2 0 and 3.3 mM K 3 Fe (CN) 6 .
• the staining solution is removed and the cells washed with IX PBS twice.
• the stained cells can be identified under a phase-contrast microscope (40Ox) .
• ONPG can be used as a substrate with aliquots of cell extracts.
• Figures 4 and 5 show the expression results. With MCA-26 cells, up to 40% of the cells were stained blue; 3T3 cells, 30%; Sol 8 cells, 20%; 4MBR-5, 50%; 293 cells, 90%; human fibroblast, 30%; and SKOV3 cells, 1%. Without peptides no blue cells were observed.
• JTS-1 mediated expression was compared with an INF-7 peptide using the same procedures as above and transfecting Sol 8 myoblast cells. Up to 5% of the cells were positively stained blue where JTS-1 was part of the complex. In the absence of peptides, no cells were stained blue. With INF-7 peptides as part of the complex, only .a few blue cells ( ⁇ 0.01%) were observed.
• DNA complexes were made by condensing a CMV-S- galactosidase expression vector with transferrin/poly-L- lysine and unmodified poly-L-lysine to create a positive particle. The same procedures as described above were used. The lytic peptides were then added and were bound to the DNA complex through ionic interactions as discussed above. Various concentrations of DNA complex (5-20 ⁇ gm) and ratios of JTS-1 to transferrin-PLL (2.5-42.5 ⁇ gm) were incubated with 3xl0 5 Sol 8 myoblast cells. In addition, in one series of experiments 12-18 ⁇ gm of PLL was also added. After 24 hours, / S-galactosidase activity was determined.
• 125 I-LDL Human Fibroblast Uptake and Degradation of LDL/JTS Complexes
• 125 I-LDL was dissolved in dimethylsulfoxide and incubated with 10-fold excess of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide ("EDC") for 30 minutes at room temperature.
• EDC l-ethyl-3- (3-dimethylaminopropyl) carbodiimide
• a 30-fold excess was added to JTS peptides (89 KBg/ ⁇ g) in phosphate buffer and incubated for 4 hours at room temperature.
• the reaction was quenched with a 50-fold excess of ethanolamine.
• Free JTS was separated from the 125 I-LDL/JTS complex by passing the reaction mixture over a Sephadex G- 25 column equilibrated with phosphate buffered saline, at pH 7.4.
• a 10:1 molar ratio of JTS peptides bound to 125 I-LDL was incubated with human fibroblasts for 5 hours at 37°C. Fibroblasts were isolated and cultured by using methods well known in the art. JTS-1, JTS-3, JTS-8 and JTS-9 peptides were used for the uptake/degradation studies. As a control, 125 I-LDL without JTS peptides were also incubated with the same fibroblast and analyzed accordingly.
• HAEC HAEC were isolated and cultured using methods well known in the art. Binding and uptake of 125 I-LDL was not affected, while JTS-1 and JTS-8 peptides decreased degradation by 30% depending on the amount of JTS peptide used. To show that LDL uptake was specific for the JTS peptides, the experiment was repeated in the presence of 20-fold excess cold LDL. Under these conditions, more than 90% of the cellular uptake of 125 I-LDL-JTS was inhibited.
• the lysines i.e., "K”
• the tyrosine partially contributes to the A 280 and also allows for the iodination for tracking in vi tro . Tryptophan increases the stability of interaction with DNA through intercalation and also provides a fluorophore which quenches upon interaction with the DNA. When R in Figure 6 is tryptophan, smaller particles are obtained and improved transfection occurs.
• R group can also be substituted with other R groups to achieve the same effects. See Figure 6 for additional R groups for improving peptide activity.
• Alanine provides a linker that allows the tyrosine and nearest neighbor lysine residues to be wrapped around the DNA in a more helical fashion resulting in a more stable complex.
• K 8 activity is also useful.
• the K 8 peptide contains an octamer of polylysine.
• variations, as shown in Figure 7, of the side chain length and charged groups are made. Modifications introduced at the core cationic oligomer decrease or increase the number of methylenes placed between the side chain cationic group and the peptide backbone.
• Figure 7 shows the use of a peptide backbone spacer that varies the distance between cationic groups, where the cationic group is the ⁇ -amino group of the substituted amino acid, e . g. , for K 8 it would be the ⁇ -amino of lysine.
• NH 2 - and COOH- terminal substitutions and deletions can also be used to optimize DNA binding of the molecule.
• variation of a NH 2 or COOH terminal acyl group may yield enhanced activity.
• Functionalities of the charged or cationic groups include guanidinium, amine or imidazole.
• variations of the NH 2 - and COOH- termini include esters, acyl groups and amides, as well as deletions.
• amino charged groups can be substituted in the peptide backbone for condensing the associated nucleic acid.
• Pseudopeptides of the formula ⁇ [CH 2 NH] when substituted within the core lysine sequences (see Figure 8) improves stability and enhances electrostatic. interactions with the nucleic acid phosphates.
• Other possible substitutions using ⁇ [(CH 2 ) n X] where X is a heteroatom can help optimize the intermolecular ionic interactions important for condensing the nucleic acid.
• a 280 is used to determine peptide concentration. This is performed in water for K N peptides so that aggregation is not observed. The following molar extinction coefficient is used for K 8 - 6860.
• the peptide is purified by reversed phase HPLC.
• the peptide (50-100 mg in 5 ml of water) is diluted with 20 ml of 1% TFA and the solution pumped onto a 2.5 x 25 cm Vydac C18 column (218TP152022, 300 A pore size) equilibrated in 0.1% TFA (Buffer A) .
• the peptide is eluted with a linear gradient between 0.1% TFA and 0.1% TFA, 10% 2-propanol (Buffer B) at a flow rate of 20 ml/min. The gradient used is 100% A to 10% B for 30 minutes.
• the column is washed with 10% B to 90% B in 5 minutes, 90% B for 5 minutes, then 90% B to 100% A in 5 minutes.
• the retention time is 6.0 to 6.8.
• the peptide is detected by absorbance at 254 and 280 nm.
• the peptide containing fractions are pooled, frozen and lyophilized.
• the peptide is then dissolved in water.
• Purity is confirmed by analytical reversed phase HPLC.
• the purity and molecular weight is also determined by electrospray mass spectrometry ("ESMS”) .
• ESMS electrospray mass spectrometry
• ESMS electrospray mass spectrometry has been performed on K 8 peptides. A single peak for K 8 occurs at the expected mass of 1720 amu. Decomposition and purity is also determined by amino acid analysis of the peptides. Quantitation of the molar ratios of the peptide components determines peptide purity.
• Secondary structure is determined by circular dichroic and FTIR spectroscopy. These standard methods are used to confirm the secondary structure of K 8 and other K N peptides.
• HepG2 cells were incubated at 37°C for 24 hours with increasing concentrations of K 8 or poly-L-lysine (100 mer) , after which viable cells were counted.
• Poly-L-lysine concentrations of greater than 0.1 ⁇ M led to complete cell death of HepG2 cells.
• no cytotoxicity was observed for up to 100 ⁇ M of K 8 , the highest concentration tested. This indicates that K 8 is at least 1000-fold less toxic than poly-L-lysine for HepG2 cells.
• C 2 C 12 myotubes were transfected with K N /DNA complexes.
• DNA was added to each well in a volume containing 300 ⁇ l per well in Fisherbrand culture tubes.
• the K N peptide is added to each well with DNA and then vortexed before the two solutions mix. Samples then set for at least 30 minutes.
• JTS-1 peptide is to be added as in the studies below, then JTS-1 is added to each of the DNA-K N peptide samples and vortexed before the two solutions mix. The samples then set for at least 30 minutes.
• C 2 C 12 myotubes in DMEM containing 10% FBS are incubated for 30-50 minutes prior to transfection. Then, 300 ⁇ l of the solution complex is added to each well in a 24-well plate and incubated for 5 hours at 37°C. After 5 hours,
• Cell extracts are prepared by adding 100 ⁇ l lysis solution to each well (24 well-plate) . Cells are transferred to a microfuge tube and centrifuged for 10 minutes at 4°C to pellet any debris. Supernatant is transferred to a fresh microfuge tube and cell extracts are frozen at -70°C for future use.
• the cell extracts are diluted 50 times by ddH 2 0.
• Cell extracts in 5-20 ⁇ l aliquots are diluted so that the total volume is 20 ⁇ l.
• Reaction Buffer 200 ml
• Reaction Buffer 200 ml
• 300 ⁇ l of Accelerator is injected and the sample counted.
• the cells are harvested two days later and viewed for -Galactosidase activity. Chemiluminescent assays are then performed as discussed.
• Figure 10 is a graph showing the effects of K N peptide molecular weight on transfection efficiency in C 2 C 12 myotubes.
• K 8 provided the highest transfection efficiency in C 2 C 12 myotubes.
• K 8 also has nuclear targeting capabilities.
• the nuclear localization ligand containing peptide GYGPPKKKRKVEAPYKA(K) N WK was used to form a nuclear binding molecule by the same procedures as described above.
• the tyrosine can be used for incorporation of 125 I to quantify binding parameters and to determine stoichiometry of the DNA complex. Binding of the peptide to DNA quenches tryptophan fluorescence and allows the kinetics and thermodynamics of complex formation to be determined.
• the function of the EAP sequence is to extend the nuclear localization sequence, GYGPPKKKRKV, at right angles to the lysine backbone.
• the peptide is homogenous by reversed phase HPLC and has the expected molecular weight, determined by electrospray mass spectroscopy.
• JTS and K 8 can be associated by covalently linking JTS-1 and K 8 together to form a bifunctional condensing/endosomal peptide as depicted in Figure 11.
• JTS-1 peptides were combined with K 8 peptides at a concentration of 16 ⁇ M, along with l-ethyl-3- (3- dimethylaminopropyl) carbodiimide (EDC) at a final concentration of either 130 ⁇ M (low EDC) or 2600 ⁇ M (high EDC) in a final volume of 4 ml.
• EDC l-ethyl-3- (3- dimethylaminopropyl) carbodiimide
• JTS-1/K 8 complexes can be shown in Figure 11.
• the DNA used in complex formation was the plasmid pCMV/ ⁇ Gal, which contains the -galactosidase gene under the control of the CMV promoter. This complex was constructed as follows.
• the JTS-1/K 8 /DNA complexes were made by adding 10 ⁇ g of DNA, in 250 ⁇ l of HBS, to the JTS-l/K 8 conjugate in 250 ⁇ l, with continuous mixing, followed by incubation at room temperature for 30 minutes.
• the JTS-l/K 8 conjugate was synthesized and purified as described above. Sufficient JTS-1/K 8 conjugate to neutralize 75% of the charge on the DNA molecule was used. After complex formation, the complexes were either analyzed by electron microscopy or used with the cell line for analysis.
• LDL-receptor gene/K 8 /JTS-l complexes were also constructed as described above. LDL/K 8 /JTS-1 complexes were transfected with fibroblasts using the transfection procedures above. These complexes led to functional expression of LDL-receptor in Watanabe fibroblasts (see below) . DNA/K 8 /JTS-1 complexes mediated high levels of gene expression in a variety of cell lines (see below) .
• JTS-1 and K 8 can also be associated noncovalently.
• Nucleic acid, K 8 and JTS-1 were associated by calculating different ratios of DNA, K 8 and JTS-1 by only considering the negative charges of DNA (phosphate groups) and JTS-1 (5 ⁇ -carboxylic groups) , and the positive charges of K 8 (10e-amino groups) .
• Six ⁇ g of DNA in 500 ml 250 mM sucrose were mixed at a phosphate to amino group ratio of 1:2, 1:3 or 1:-4. After incubation for 30 minutes at room temperature, 7-38 mM JTS-1 was added to create either positively charged, neutral or negatively charged DNA/K 8 /JTS-1 complexes.
• Positively charged complexes had an overall +/- charge ratio of 0.66 to 0.7, neutral complexes a ratio of 1:1 and negatively charged complexes a ratio of 1:1.5.
• DNA/K 8 /INF-7 could also be prepared as above.
• Optimal transfections occur when using 2 ⁇ x ⁇ 6 and 0.75 ⁇ y ⁇ 2.
• the following is useful in characterizing the DNA/Kg/JTS-1 complex.
• the effects of pH on the particle size of 100 ⁇ g/ml pDNA/K 8 /JTS-l complex was examined.
• the complex was formed as discussed above. When the pH was dropped to below pH 7, JTS-1 caused the complex to precipitate. When the pH was above 9, K 8 deprotonates and loses its ability to bind DNA efficiently. Thus, the complex falls apart. Therefore, the optimal pH for the complex is between pH 7-9.
• Tween80 The Effects of Tween80 on the 100 ⁇ g/ml DNA/K S /JTS-1 Complex The following is useful in characterizing the DNA/K 8 /JTS-1 complex.
• the effects of Tween80 on particle size was examined. The complex was dissolved in water for injection ("WFI") and 5% mannitol. Tween ⁇ O did not enhance particle size.
• DNA/K 8 /JTS-1 complex The effects of filtration of peptide-DNA complexes on particle size were studied. DNA preparation in the range of 100-500 ⁇ g/ml were used.
• Results show large particles still exist after filtration of the K 8 /DNA/JTS-1 complex.
• the Effects of Centrifugation of Aggregates to Obtain Small Complexes of DNA/K e /JTS-l The following is useful in characterizing the DNA/K 8 /JTS-1 complex.
• the effect of centrifugation of aggregates was examined. Particle size can be reduced by using centrifugation at selective speeds ranging from 11,000 to 14,000 rpm.
• Figure 13 examines the transfection of 4Mbr-5 (monkey bronchis epithelial) cells using K 6 or K 7 peptides in conjunction with JTS-1.
• DNA complexes were made by condensing a CMV- / ⁇ -galactosidase expression vector with transferrin/K 6 or K 7 and unmodified K 6 or K 7 to create a positive particle.
• the lytic peptides were then added and were bound to the DNA complex through ionic interactions.
• Transfection experiments using the 4Mbr cells followed the procedures as discussed above. Between 3-12 ⁇ gs of DNA complex was incubated with 3xl0 5 4Mbr cells.
• the cells were stained with X-gal for ⁇ - galactosidase expression (see above) . Up to 50% of the cells were positively stained blue where 9 ⁇ g of DNA complex with K 7 was used, as compared to 40% using K 6 . In the absence of peptides, no cells were stained blue. 5 ⁇
• C 2 C 12 muscle cells were transfected with DNA/K 8 /JTS-1 complexes, using a 1/3/1 ratio. Up to 60% of the cells were positively stained blue using 20 mg of DNA complex. In the absence of peptides, no cells were stained blue. When the ratios were changed from l/3/l to l/6/l transfection rates dropped 2-fold.
• Transfection rates dropped as much as 75% when serum was added to the assay using 10 mg of DNA per well and the charge ratio was changed from 1/3/1 to l/6/l; with 20 mg of DNA under the same parameters, transfection rates dropped by 40%; 90% using 30 mg of DNA; and 60% using 40 mg of DNA complex. DNA complexed with lipofectamine showed no transfection rates at all. Lipofectamine was used as a control.
• DNA transfection efficiency can be correlated at least qualitatively with the following parameters: (1) the presence of the binding molecule, i.e., the condensing component; (2) the presence of a fusogenic component; and (3) DNA dose.
• DNA/K B /JTS-1 Mediated Gene Delivery Into HepG2 Cells DNA/K 8 /JTS-1 complexes were associated noncovalently by ionic interaction as described above. This procedure allowed the addition of more membrane active peptide per DNA complex.
• the different ratios of DNA, K 8 and JTS-1 were calculated by considering the negative charges of DNA (phosphate groups) and JTS-1 (carboxylic groups) , and the positive charges of K 8 (e-amino groups) .
• DNA was mixed with K 8 at a phosphate to amino group ratio of 1:2, 1:3 and 1:4.
• JTS-1 was added to form positively, neutral or negatively charged DNA/K 8 /JTS-1 complexes. These complexes had the tendency to form microaggregates slowly over time.
• the Photinus pyralis luciferase gene under the control of the early cytomegalovirus ("CMV") enhancer and promoter was used as a reporter gene.
• CMV early cytomegalovirus
• Receptor independent gene expression has been also reported for DNA/poly-L-lysine/influenza peptide complexes.
• the presence of a ligand like transferrin only made a 1.5- to 8-fold difference in gene expression.
• the effect of the ligand was cell type dependent.
• a dose response curve was performed. 1x10 s HepG2 cells were incubated with 0, 1, 3, 6, 9, 12, 15, 18 ⁇ g of DNA in complex form and 24 hours after gene delivery luciferase activity was determined. Incubation of cells with increasing amounts of DNA produced a non-linear response of gene expression. With 1 ⁇ g of DNA lxlO 3 light units/mg protein were achieved, with 3 ⁇ g of DNA lxlO 7 light units/mg protein were achieved, and with 6 to 15 ⁇ g of DNA lxlO 8 light units/mg protein were achieved. In fact, with 6 to 9 ⁇ g of DNA maximal levels of gene expression was achieved. No further increase was observed for higher DNA amounts.
• a plasmid containing a CMV-.E. coli -galactosidase expression cassette was used. Cells were incubated with positive DNA/K 8 /JTS-1 complexes and 24 hours after the gene delivery the cells were stained with X-gal. Twenty-five to 30% of cells were positive for 3-galactosidase expression. In contrast, no blue cells were observed in control cells.
• JTS-1 mediated gene delivery for DNA/K 8 complexes was compared with the gene transfer activity of INF-7. Positively, neutral and negatively charged complexes were formed similar to DNA/K 8 /JTS-1 complexes and 24 hours after gene delivery luciferase activity was determined. For every DNA/K 8 /INF-7 complex condition tested, the achieved level of gene expression was at least 1000-fold lower in direct comparison to DNA/K 8 /JTS-1 complexes. This difference in gene transfer activity corresponded well to the observed differences in the hemolytic activity of JTS- 1 and INF-7 using the hemolysis assay protocol described herein. However, the membrane activity of peptides is not the only factor which determines gene transfer activity. Single amino acid substitutions in the JTS-1 sequence which do not affect the membrane activity of the peptide will lead to considerable differences in gene transfer activity.
• HepG2 cells were infected with a recombinant adenovirus containing the same CMV-luciferase expression cassette (Adv/CMV-luc) as the plasmid.
• the adenovirus was grown in 293 cells and purified by double banding on CsCl gradients. The concentration of the virus was determined by ultraviolet spectrophotometric analysis and plaque assay, and the virus was stored in 10% (v/v) glycerol at -70°C. Adenovirus was thawed and used immediately for experiments.
• HepG2 cells were incubated with increasing M.O.I, of Adv/CMV-luc. Twenty-four hours after infection the cells were harvested and luciferase activity was determined. There was a linear increase in gene expression from a M.O.I, of 0.1 to 100. At an M.O.I, of 1000 no further increase was observed, due to cytopathic effects of the recombinant adenovirus. The maximal achieved level of gene expression was around 10 9 light units/mg protein. This was 10- to 50-fold higher than gene expression achieved with DNA/K 8 /JTS-1 complexes. This result indicates the potency of DNA/K 8 /JTS-1 complexes for the use of gene transfer in cultured cells.
• the observed difference between the recombinant adenovirus and DNA/K 8 /JTS-1 complexes can be due to a number of reasons. For example, after entry of adenovirus particles into the cytoplasm, they are translocated to the nuclear pore for efficient viral DNA delivery into the nucleus. This finding could be significant, since the incorporation of a nuclear localization sequence into DNA vectors increased gene expression 5- to 10-fold.
• DNA/Kp/JTS-1 Mediated Gene Delivery Into Mammalian Cells Cell lines from different species and organs were tested. Cells were incubated with positive DNA/K 8 /JTS-1 complexes as prepared above using the E. coli ⁇ - galactosidase as a reporter gene (see description above) . Twenty-four hours after gene delivery cells were stained with X-gal and the percentage of blue cells was determined. The transfection efficiencies in 14 cell lines varied between 1 and 50% with a mean of 23%. These results indicate that DNA/K 8 /JTS-1 complexes can be used to transduce a broad range of cell lines in vi tro . However, the efficiency varies from cell line to cell line as observed with other non-viral and viral delivery systems.
• the transduction efficiency of cells correlates well with expression levels of the specific receptor.
• receptor independent gene delivery the basic mechanism for cell type variation is poorly understood, but has been documented, especially for DNA/cationic liposome complexes.
• the type of cells tested included fibroblast, glioma, myoblast, colon carcinoma, hepatoma, ovarian cancer and embryonic kidney.
• Cell lines tested included 3T3, Watanabe, 9L, C6, C 2 C 12 , Sol ⁇ , MCA-26, HCT-116, ML3, HepG2, Skov3 and 293.
• LDL-receptor LDL-receptor
• Recombinant adenoviral vectors containing the LDL-R gene have been used to transiently correct the cholesterol levels in two animal models for hypercholesterolemia.
• Watanabe fibroblasts were incubated with DNA/K 8 /JTS- 1 complexes.
• Watanabe fibroblasts were derived from skin biopsies of Watanabe rabbits, which bear an inframe deletion of 12 nucleotides that eliminate four amino acids from the cysteine-rich ligand binding domain of the LDL-R. This deletion prevents LDL-R mediated uptake and degradation of LDL particles, resulting in dramatic increases of plasma cholesterol levels.
• the plasmid CMV- rbLDL-R containing the rabbit LDL receptor was constructed by digestion of the plasmid pAdLl/RSV-rbLDL-R with Xba I and Hind III. The isolated fragment was cloned into the plasmid pcDNA3, which contains a CMV expression cassette.
• DNA/K 8 /JTS-1 complexes were used to deliver the rabbit
• rbLDL-R LDL-R
• Figures 14-l ⁇ set forth various surface ligands that can be coupled to binding molecules, such as K 8 , or coupled to JTS-1 to direct delivery of the nucleic acid to a specific cell, see below.
• binding molecules such as K 8
• JTS-1 to direct delivery of the nucleic acid to a specific cell, see below.
• peptides containing carbohydrates for uptake via the asialoglycoprotein receptor were constructed ( Figures 14 and 15) .
• ligands in Figure 16 were coupled to
• JTS-1 or K 8 JTS-1 or K 8 .
• the following is a list of other receptor ligands coupled to K 8 or JTS-1 that have also been constructed and characterized.
• RGD targeting ligands can also be attached to K 8 peptides as set forth in Figure 17. Such a ligand is useful in delivery of therapeutic genes to connective tissue, wounds, and for healing.
• the lipids in Figure l ⁇ can be used for delivery to hepatocytes.
• One embodiment of the present invention includes cells transformed with nucleic acid associated with the nucleic acid transporter systems described above. Once the cells are transformed, the cells will express the protein, polypeptide or RNA encoded for by the nucleic acid. Cells included, but are not limited to, liver, muscle and skin. This is not intended to be limiting in any manner.
• the nucleic acid which contains the genetic material of interest is positionally and sequentially oriented within the host or vectors such that the nucleic acid can be transcribed into RNA and, when necessary, be translated into proteins or polypeptides in the transformed cells.
• a variety of proteins and polypeptides can be expressed by the sequence in the nucleic acid cassette in the trans ⁇ formed cells.
• These products may function as intracellu ⁇ lar or extracellular structural elements, ligands, hormones, neurotransmitters, growth regulating factors, apolipoproteins, enzymes, serum proteins, receptors, carriers for small molecular weight compounds, drugs, immunomodulators, oncogenes, tumor suppressors, toxins, tumor antigens, antigens, antisense inhibitors, triple strand forming inhibitors, ribozymes, or as a ligand recognizing specific structural determinants on cellular structures for the purpose of modifying their activity.
• Transformation can be done either by in vivo or ex vivo techniques.
• Transformation by ex vivo techniques includes co-transfecting the cells with DNA containing a selectable marker. This selectable marker is used to select those cells which have become transformed. Selectable markers are well known to those who are skilled in the art.
• one approach to gene therapy for hepatic diseases is to remove hepatocytes from an affected indi ⁇ vidual, genetically alter them in vi tro, and reimplant them into a receptive locus.
• the ex vivo approach includes the steps of harvesting hepatocytes, cultivating the hepatocytes, transducing or transfecting the hepato ⁇ cytes, and introducing the transfected hepatocytes into the affected individual.
• the hepatocytes may be obtained in a variety of ways. They may be taken from the individual who is to be later injected with the hepatocytes that have been transformed or they can be collected from other sources, transformed and then injected into the individual of interest.
• the ex vivo hepatocyte may be transformed by contacting the hepatocytes with media con- taining the nucleic acid transporter and maintaining the cultured hepatocytes in the media for sufficient time and under conditions appropriate for uptake and transformation of the hepatocytes.
• the hepatocytes may then be introduced into an orthotopic location (the body of the liver or the portal vasculature) or heterotopic locations by injection of cell suspensions into tissues.
• the cell suspension may contain: salts, buffers or nutrients to maintain viability of the cells; proteins to ensure cell stability; and factors to promote angiogenesis and growth of the implanted cells.
• harvested hepatocytes may be grown ex vivo on a matrix consisting of plastics, fibers or gelatinous materials which may be surgically implanted in an orthotopic or heterotopic location after transduction.
• This matrix may be impregnated with factors to promote angiogenesis and growth of the implanted cells. Cells can then be reimplanted.
• Administration refers to the route of introduction of the nucleic acid transporters into the body.
• Administration includes intravenous, intramuscular, topical, or oral methods of delivery. Administration can be directly to a target tissue or through systemic delivery.
• the present invention can be used for administering nucleic acid for expression of specific nucleic acid sequence in cells.
• Routes of administration include intramuscular, aerosol, olfactory, oral, topical, systemic, ocular, intraperitoneal and/or intratracheal.
• a preferred method of administering nucleic acid transporters is by intravenous delivery. Another preferred method of administration is by direct injection into the cells.
• PVP polyvinylpyrrolidone
• amorphous powder is a polyamide that forms complexes with a wide variety of substances and is chemically and physiologically inert.
• suitable PVP's are Plasdone-C ® 15, MW 10,000 and Plasdone-C ® 30, MW 50,000.
• administration may also be through an aerosol composition or liquid form into a nebulizer mist and thereby inhaled.
• the special delivery route of any selected vector construct will depend on the particular use for the nucleic acid associated with the nucleic acid transporter. In general, a specific delivery program for each nucleic acid transporter used will focus on uptake with regard to the particular targeted tissue, followed by demonstration of efficacy. Uptake studies will include uptake assays to evaluate cellular uptake of the nucleic acid and expression of the specific nucleic acid of choice. Such assays will also determine the localization of the target nucleic acid after ⁇ ptake, and establishing the requirements for maintenance of steady-state concentrations of expressed protein. Efficacy and cytotoxicity is then tested. Toxicity will not only include cell viability but also cell function.
• the chosen method of delivery should result in cytoplasmic accumulation and optimal dosing.
• the dosage will depend upon the disease and the route of administra- tion but should be between 1-1000 mg/kg of body weight/ day. This level is readily determinable by standard methods. It could be more or less depending on the optimal dosing.
• the duration of treatment will extend through the course of the disease symptoms, possibly continuously. The number of doses will depend upon disease delivery vehicle and efficacy data from clinical trials.
• the muscular dystrophies are a group of diseases that result in abnormal muscle development, due to many differ- ent reasons. These diseases can be treated by using the direct delivery of genes with the nucleic acid transporters of the present invention resulting in the production of normal gene product. Delivery to the muscle using the present invention is done to present genes that produce various antigens for vaccines against a multitude of infections of both viral and parasitic origin. The detrimental effects caused by aging can also be treated using the nucleic acid delivery system described herein. Since the injection of the growth hormone protein promotes growth and proliferation of muscle tissue, the growth hormone gene can be delivered to muscle, resulting in both muscle growth and development, which is decreased during the later portions of the aging process. Genes expressing other growth related factors can be delivered, such as Insulin Like Growth Factor-1 (IGF-1) . Furthermore, any number of different genes may be delivered by this method to the muscle tissue.
• IGF-1 Insulin Like Growth Factor-1
• IGF-1 can be used to deliver DNA to muscle, since it undergoes uptake into cells by receptor-mediated endocyto- sis.
• This polypeptide is 70 amino acids in length and is a member of the growth promoting polypeptides structurally related to insulin. It is involved in the regulation of tissue growth and cellular differentiation affecting the proliferation and metabolic activities of a wide variety of cell types, since the polypeptide has receptors on many types of tissue.
• the nucleic acid transporter delivery system of the present invention utilizes IGF-1 as a ligand for tissue-specific nucleic acid delivery to muscle.
• the advantage of the IGF- 1/nucleic acid delivery system is that the specificity and the efficiency of the delivery is greatly increased due to a great number of cells coming into contact with the ligand/nucleic acid complex with uptake through receptor- mediated endocytosis.
• Using the nucleic acid described above in the delivery systems of the present invention with the use of specific ligands for the delivery of nucleic acid to muscle cells provides treatment of diseases and abnormalities that affect muscle tissues.
• Factor IX can also be delivered to the muscle cells.
• DNA encoding Factor IX can be delivered using the nucleic acid transporters of the present invention.
• the nucleic acid transporter delivery system of the present invention utilizes nucleic acids encoding Factor IX to treat cells which are Factor IX deficient and are susceptible to disease and abnormalities due to such a deficiency.
• DNA encoding Factor IX can be coupled or associated with K 8 and JTS-1 as described above. The complex can then be delivered directly to muscle cells for expression. The preferred ratio of DNA to K 8 to JTS-1 is 1:3:1. Direct injection of the above complex is preferred.
• Use of the above nucleic acid delivery system of the present invention for the delivery of nucleic acid expressing Factor IX to muscle cells provides treatment of diseases and abnormalities that affect muscle tissues.
• the direct nucleic acid delivery system of the present invention can be used to deliver genes to cells that promote bone growth.
• the osteoblasts are the main bone forming cell in the body, but there are other cells that are capable of aiding in bone formation.
• the stromal cells of the bone marrow are the source of stem cells for osteoblasts.
• the stromal cells differentiate into a population of cells known as Inducible Osteoprogenitor Cells (IOPC) , which then under induction of growth factors, differentiate into Determined Osteoprogenitor Cells (DOPC) . It is this population of cells that mature directly into bone producing cells.
• the IOPCs are also found in muscle and soft connective tissues. Another cell involved in the bone formation process is the cartilage- producing cell known as the chondrocyte.
• Bone Morphogenetic Protein BMP
• CIF Cartilage Induction Factor
• osteoblasts are involved in bone production, genes that enhance osteoblast activity can be delivered directly to these cells. Genes can also be delivered to the IOPCs and the chondrocytes, which can differentiate into osteoblasts, leading to bone formation. BMP and CIF are the ligands that can be used to deliver genes to these cells. Genes delivered to these cells promote bone forma- tion or the proliferation of osteoblasts.
• the polypep ⁇ tide, IGF-1 stimulates growth in hypophysectomized rats which could be due to specific uptake of the polypeptide by osteoblasts or by the interaction of the polypeptide with chondrocytes, which result in the formation of osteoblasts.
• Other specific bone cell and growth factors can be used through the interaction with various cells involved in bone formation to promote osteogenesis.
• Nonlimiting examples of genes expressing the following growth factors which can be delivered to these cell types are Insulin, Insulin-Like Growth Factor-1, Insulin-Like Growth Factor-2, Epidermal Growth Factor, Transforming Growth Factor- ⁇ , Transforming Growth Factor- ⁇ , Platelet Derived Growth Factor, Acidic Fibroblast Growth Factor, Basic Fibroblast Growth Factor, Bone Derived Growth Factors, Bone Morphogenetic Protein, Cartilage Induction Factor, Estradiol, and Growth Hormone. All of these factors have a positive effect on the proliferation of osteoblasts, the related stem cells, and chondrocytes.
• BMP or CIF can be used as conjugates to deliver genes that express these growth factors to the target cells by the intravenous injection of the nucleic acid/Protein complexes of the present invention.
• Using the nucleic acid described above in the delivery systems of the present invention with the use of specific ligands for the delivery of nucleic acid to bone cells provides treatment of diseases and abnormalities that affect bone tissues.
• the inflammatory attack on joints in animal models and human diseases may be mediated, in part, by secretion of cytokines such as IL-1 and IL-6 which stimulate the local inflammatory response.
• the inflammatory reaction may be modified by local secretion of soluble fragments of the receptors for these ligands.
• the complex between the ligand and the soluble receptor prevents the ligand from binding to the receptor which is normally resident on the surface of cells, thus preventing the stimulation of the inflammatory effect.
• Therapy consists of the construction of a vector containing the soluble form of receptors for appropriate cytokines (for example, IL-1) , together with promoters capable of inducing high level expression in structures of the joint and a formulation which enables efficient uptake of this vector.
• This DNA is then used with the DNA transporters of the present invention.
• This DNA is injected into affected joints where the secretion of an inhibitor for IL-1 such as a soluble IL-1 receptor or natural IL-I inhibitor modifies the local inflammatory response and resulting arthritis.
• an inhibitor for IL-1 such as a soluble IL-1 receptor or natural IL-I inhibitor modifies the local inflammatory response and resulting arthritis.
• This method is useful in treating episodes of arth ⁇ ritis which characterize many "autoimmune" or "collagen vascular” diseases. This method can also prevent dis ⁇ abling injury of large joints by inflammatory arthritis.
• the present invention can also be used with the following method.
• Current therapy for severe arthritis involves the administration of pharmacological agents including steroids to depress the inflammatory response.
• Steroids can be administered systemically or locally by direct injection into the joint space.
• Steroids normally function by binding to receptors within the cytoplasm of cells. Formation of the steroid- receptor complex changes the structure of the receptor so that it becomes capable of translocating to the nucleus and binding to specific sequences within the genome of the cell and altering the expression of specific genes. Genetic modifications of the steroid receptor can be made which enable this receptor to bind naturally occurring steroids with higher affinity, or bind non-natural, synthetic steroids, such as RU486. Other modifications can be made to create steroid receptor which is "constitutively active" meaning that it is capable of binding to DNA and regulating gene expression in the absence of steroid in the same way that the natural steroid receptor regulates gene expression after treatment with natural or synthetic steroids.
• glucocorticoid steroids such as cortisone, hydrocortisone, prednisone, or dexamethasone which are the most important drugs available for the treatment of arthritis.
• One approach to treating arthritis is to introduce a vector in which the nucleic acid cassette expresses a genetically modified steroid receptor into cells of the joint, e . g. , a genetically modified steroid receptor which mimics the effect of glucocorticoids but does not require the presence of glucocorticoids for effect. This is termed the glucocortico-mimetic receptor.
• a constitutively active steroid receptor within cells of the joint which contains the DNA binding domain of a glucocorticoid receptor. This induces the therapeutic effects of steroids without the systemic toxicity of these drugs.
• steroid receptors which have a higher affinity for natural or synthetic glucocorticoids, such as RU466, can be introduced into the joint. These receptors exert an increased anti- inflammatory effect when stimulated by non-toxic concentrations of steroids or lower doses of pharmaco ⁇ logically administered steroids.
• consti ⁇ tution of a steroid receptor which is activated by a novel, normally-inert steroid enables the use of drugs which would affect only cells taking up this receptor. These strategies obtain a therapeutic effect from steroids on arthritis without the profound systemic complications associated with these drugs. Of particular importance is the ability to target these genes differentially to specific cell types (for example synovial cells versus lymphocytes) to affect the activity of these cells.
• These proteins are ligand activated transcription factors whose ligands can range from steroids to retinoids, fatty acids, vitamins, thyroid hormones and other presently unidentified small molecules. These compounds bind to receptors and either up-regulate or down-regulate transcription.
• the preferred receptor of the present invention is modification of the glucocorticoid receptor, i.e., the glucocorticoid-mimetic receptor.
• These receptors can be modified to allow them to bind various ligands whose structure differs from naturally occurring ligands, e . g. , RU466.
• small C-terminal alterations in amino acid sequence, including truncation result in altered affinity and altered function of the ligand.
• receptors can be customized to respond to ligands which do not activate the host cells own receptors.
• Steroid hormone receptors which may be mutated are any of those receptors which comprise the steroid hormone receptor super family, such as receptors including the estrogen, progesterone, glucocorticoid- ⁇ , glucocorticoid-,, mineral corticoid, androgen, thyroid hormone, retinoic acid, and Vitamin B3 receptors.
• DNA encoding for other mutated steroids such as those which are capable of only transrepression or of only transactivation are also within the scope of the above embodiment.
• Such steroids could be capable of responding to RU466 in order to activate transrepression.
• the present invention can also be used with the following method.
• Drugs which inhibit the enzyme prostaglandin synthase are important agents in the treatment of arthritis. This is due, in part, to the important role of certain prostaglandin in stimulating the local immune response.
• Salicylates are widely used drugs but can be administered in limited doses which are often inadequate for severe forms of arthritis.
• Gene transfer using the present invention is used to inhibit the action of prostaglandin synthase specifically in affected joints by the expression of an antisense RNA for prostaglandin synthase.
• RNA molecules are used for forming a triple helix in regulatory regions of genes expressing enzymes required for prostaglandin synthesis. Alterna ⁇ tively, RNA molecules are identified which bind the active site of enzymes required for prostaglandin synthesis and inhibit this activity.
• genes encoding enzymes which alter prostaglandin metabolism can be transferred into the joint. These have an important anti-inflammatory effect by altering the chemical composition or concentration of inflammatory prostaglandin.
• the present invention is useful for enhancing repair and regeneration of the joints.
• the regenerative capacity of the joint is limited by the fact that chondrocytes are not capable of remodelling and repairing cartilaginous tissues such as tendons and cartilage.
• collagen which is produced in response to injury is of a different type lacking the tensile strength of normal collagen.
• the injury collagen is not remodeled effectively by available collagenase.
• inappropriate expression of certain metalloproteinases is a component in the destruction of the joint.
• Gene transfer using promoters specific to chondro ⁇ cytes is used to express different collagens or appropriate collagenase for the purpose of improving the restoration of function in the joints and prevent scar formation.
• Gene transfer for these purposes is affected by direct introduction of DNA into the joint space where it comes into contact with chondrocytes and synovial cells. Further, the genes permeate into the environment of the joint where they are taken up by fibroblasts, myoblasts, and other constituents of periarticular tissue.
• Nucleic acid transporters of the present invention can also be used in reversing or arresting the progression of disease involving the lungs, such as lung cancer.
• One embodiment involves use of intravenous methods of adminis- tration to delivery nucleic acid encoding for a necessary molecule to treat disease in the lung.
• Nucleic acid transporters which express a necessary protein or RNA can be directly injected into the lungs or blood supply so as to travel directly to the lungs.
• an aerosol or a liquid in a nebulizer mist can also be used to administer the desired nucleic acid to the lungs.
• a dry powder form such as PVP discussed above, can be used to treat disease in the lung. The dry powder form is delivered by inhalation.

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• 1996
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