Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • 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
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

• Gene therapy is an approach to treating diseases based on modifying the expression of cellular genes toward a therapeutic goal. Gene therapy has been discussed in the context of treating lethal and disabling diseases although it also has a potential for disease prevention. A premise of gene therapy is based on correcting disease at its root.
• a nucleic acid may be coded to express the protein dystrophin that is missing or defective in Duchenne muscular dystrophy.
• the coded nucleic acid is delivered to a selected group or groups of cells and incorporated into those cell's genome or remain apart from the cell's genome. Subsequently, dystrophin is produced by the formerly deficient cells.
• Other examples of imperfect protein production that can be treated with gene therapy include the addition of the protein clotting factors that are missing in the hemophilias and enzymes that are defective in inborn errors of metabolism such as phenylketonuria (PKU).
• a delivered nucleic acid can also be therapeutic in acquired disorders such as neurodegeneratve disorders, cancer, heart disease, and infections.
• the nucleic acid has its therapeutic effect by entering the cell. Entry into the cell is required for the nucleic acid to produce the therapeutic protein, to block the production of a protein, or to decrease the amount of a RNA.
• Other therapeutic genes can be erythropoietin, vascular growth factors such as fibroblast growth factor (FGF) or vascular endothelial growth factor (NEGF).
• nucleic acid can be delivered to block gene expression.
• Such nucleic acids can be anti-sense by preventing translation of a messenger R ⁇ A or could block gene expression by preventing transcription of the gene. Preventing R ⁇ A translation and/or D ⁇ A transcription is considered preventing expression. Transcription can be blocked by the nucleic acid binding to the gene as a duplex or triplex. It could also block expression by binding to proteins that are involved in a particular cellular biochemical process.
• Nucleic acids may be delivered that recombine with genes.
• the nucleic acids may be DNA, RNA, hybrids and derivatives of natural nucleotides. Recombine is the mixing of the sequence of a delivered nucleic acid and the genetic code of a gene. Recombine includes changing the sequence of a gene.
• a challenge in gene therapy is to develop approaches for delivering genetic material to the appropriate cells of the patient in a way that is specific, efficient and safe. This problem of "drug delivery," where the gene is a drug, is particularly challenging. If genes are appropriately delivered they can potentially lead to a cure.
• a second challenge in gene therapy is to achieve sustained levels of gene expression following gene delivery. While efficacious levels of gene expression are observed shortly after delivery, these levels frequently drop below curative levels. A major focus of gene therapy research is therefore aimed at obtaining long term gene expression.
• nucleic acid is a term of art that refers to a string of at least two base-sugar- phosphate combinations.
• a polynucleotide is distinguished, here, from an oligonucleotide by containing more than 120 monomeric units.
• the term includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
• Nucleotides are the monomeric units of nucleic acid polymers.
• Anti-sense is a nucleic acid that interferes with the function of DNA and/or RNA. Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases.
• RNA may be in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, and ribozymes.
• DNA may be in form plasmid DNA, viral DNA, linear DNA, or chromosomal DNA or derivatives of these groups. In addition to these forms; DNA and RNA may be single, double, triple, or quadruple stranded.
• PNAs peptide nucleic acids
• phosphothionates and other variants of the phosphate backbone of native nucleic acids.
• DNA generally exists in either double stranded or single stranded form.
• double stranded DNA there are two anti-parallel strands of DNA that are bound to each other by complementary (guanine-cytosine or adenine-thymine pairing) binding by Watson-Crick binding between guanine-cytosine and adenine-thymidine pairs.
• Single stranded (ssDNA) DNA has one strand of DNA.
• a strand of DNA is one string of at least two base-sugar- phosphate combinations that are covalently bound to each other.
• Single stranded DNA can form loops or secondary structure within itself in which Watson-Crick pairing occurs between different parts of one strand.
• ssDNA refers to partially single stranded DNA in which the DNA has both double stranded and single stranded stretches (also referred to as partially single stranded DNA).
• ssDNA can have part that is not paired to another DNA strand and another part that is paired to another DNA strand.
• Single stranded DNA can be formed by a variety of methods.
• ssDNA can be formed by denaturing dsDNA. Denaturing means the process by which the two strands of dsDNA are separated so that the number of Watson-Crick pairings are reduced.
• dsDNA can be denatured by a variety of processes including heating, alkaline treatment, and addition of various chemicals.
• Single stranded DNA can also be produced from viruses.
• the virus can be a virus that infects prokaryotes or bacteria, i.e., bacteriophage.
• An example of such a bacteriophage is M13.
• ssDNA can also be obtained from viruses that infect eukaryotes such as plants and mammalian cells.
• ssDNA could be obtained from single stranded DNA viruses such as adeno-associated virus.
• ssDNA could also be produced by enzymatic reaction. This could be by DNases that digest one strand or by polymerases that synthesize DNA.
• the polymerase chain reaction PCR
• PCR polymerase chain reaction
• one of the primers contains a ligand that is used for purifying the one strand that contains the ligand.
• one of the two primers for PCR contains a biotin. After PCR, the DNA is denatured and the one strand with the biotin is purified using biotin-binding proteins such as streptavidin, neutravidin, or avidin. It could also be a biotin derivative that doesn't bind these proteins so strongly.
• ssDNA could also be produced by restriction enzyme digestion.
• plasmid DNA is digested by two different restriction enzymes and a polymerase (e.g. Klenow fragment of DNA polymerase) is used to add nucleotides at the end.
• the nucleotide could be modified with a ligand that enables one strand to be purified by affinity methods (e.g. using chromatography or magnetic beads or non-magnetic beads or centrifugation methods). Examples of these ligands are biotin, digoxigenin and dinitrophenol.
• ssDNA can also be synthesized via chemical means. This process is used routinely to synthesize oligonucleic acids, e.g., for use as primers in the PCR process. Single stranded nucleic acids of over 100 bases can be synthesized. Longer ssDNA can be prepared by chemically linking two or more oligomers.
• the ssDNA After delivery to the cell the ssDNA can be expressed which means it is transcribed and thus serves as a template for the production of RNA.
• the ssDNA may be converted to dsDNA after it is delivered to the cell.
• the ssDNA, or after conversion dsDNA may integrate into the host genome. Integration can occur in a random fashion, or in a specific location. Integration may influence the durability of expression by maintaining the transferred in the host cell for an indefinite period. Integration prevents the loss of non-integrated DNA during cell division. Integration may also affect the expression level of the transferred coding sequence, by influencing host cell effects upon regulatory sequences. This may proceed by altering the methylation status.
• the ssDNA could contain sequences from viruses that enhance either the efficiency of expression (e.g. the level of expression) or the stability of expression which means the length of time that the ssDNA is expressed after being delivered.
• viral sequences can be obtained from viruses such as AAN, adenoviral, or retroviral viruses.
• These viral sequences can be repeat sequences such as inverted terminal repeats (ITR's) or long terminal repeats (LTR's).
• Other sequences could include nuclear retention sequences or sequences that enhance intracellular transport of the D ⁇ A. ssD ⁇ A is more compact than dsD ⁇ A. It may therefore be advantageous for delivery to cells in vitro and especially in vivo.
• ssD ⁇ A For instance, transport of naked dsD ⁇ A to cells following intravascular delivery in mammals is restricted by small pores, therefore requiring high pressure for efficient transfection. Delivery is the smaller ssD ⁇ A may result in enhanced nucleic acid transfer, or require less stringent delivery means. This may be especially the case for nucleic acid complexes, where polymer - ssD ⁇ A complexes may be much smaller than their dsD ⁇ A counterparts.
• the nature of gene expression from ssDNA may proceed through conversion of ssDNA to dsDNA. An example of this process is observed following transduction of cells with the single stranded DNA adeno-associated virus (AAN).
• AAN single stranded DNA adeno-associated virus
• AAN encoded genes slowly increase over a period of days to weeks at which time the recombinant viral D ⁇ A is converted to dsD ⁇ A. Frequently, concatemers are formed, and the D ⁇ A may be integrated into the host genome. Expression is usually very long lasted. Delivery of (partially) ssD ⁇ A may follow a similar process. Long term expression of genes is often required for gene therapy purposes.
• Delivery of a nucleic acid means to transfer a nucleic acid from a container outside a mammal to within the outer cell membrane of a cell in the mammal.
• transfection is used herein, in general, as a substitute for the term delivery, or, more specifically, the transfer of a nucleic acid from directly outside a cell membrane to within the cell membrane.
• a delivered D ⁇ A can stay within the cytoplasm or nucleus apart from the endogenous genetic material. Alternatively, the D ⁇ A could recombine (become a part of) the endogenous genetic material. For example, D ⁇ A can insert into chromosomal D ⁇ A by either homologous or non-homologous recombination.
• nucleic acids can be delivered to cells in vivo (e.g., in a mammal) via direct injection of the nucleic acid in the target tissue, injection into the vasculature, injection of complexes of the nucleic acid with polymers, electroporation, iontophoresis, or particle bombardment.
• Polymers are used for drug delivery for a variety of therapeutic purposes. Polymers have also been used in research for the delivery of nucleic acids (polynucleotides and oligonucleotides) to cells with an eventual goal of providing therapeutic processes. Such processes have been termed gene therapy or anti-sense therapy.
• nucleic acid delivery to the cells is the use of DNA-polycation complexes. It has been shown that cationic proteins like histones and protamines or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents while small polycations like spermine are ineffective. The following are some important principles involving the mechanism by which polycations facilitate uptake of nucleic acid:
• Polycations provide attachment of nucleic acid to the cell surface.
• the polymer forms a cross-bridge between the polyanionic nucleic acids and the polyanionic surfaces of the cells.
• the main mechanism of DNA translocation to the intracellular space might be non-specific adsorptive endocytosis which may be more effective then liquid endocytosis or receptor-mediated endocytosis.
• polycations are a convenient linker for attaching specific ligands to nucleic acid and as result, nucleic acid - polycation complexes can be targeted to specific cell types.
• Polycations protect nucleic acid in complexes against nuclease degradation. This is important for both extra- and intracellular preservation of nucleic acid. Gene expression is also enabled or increased by preventing endosome acidification with NFLjCl or chloroquine. Polyethylenimine, which facilitates gene expression without additional treatments, probably disrupts endosomal function itself. Disruption of endosomal function has also been accomplished by linking to the polycation endosomal-disruptive agents such as fusion peptides or adenoviruses.
• Polycations can also facilitate nucleic acid condensation.
• the volume which one nucleic acid molecule occupies in a complex with polycations is drastically lower than the volume of a free nucleic acid molecule.
• the size of a nucleic acid/polymer complex is probably critical for gene delivery in vivo.
• nucleic acid needs to cross the endothelial barrier and reach the parenchymal cells of interest.
• the largest endothelia fenestrae holes in the endothelial barrier) occur in the liver and have an average diameter of 100 nm.
• the trans-epithelial pores in other organs are much smaller, for example, muscle endothelium can be described as a structure which has a large number of small pores with a radius of 4 nm, and a very low number of large pores with a radius of 20-30 nm.
• the size of the nucleic acid complexes is also important for the cellular uptake process. After binding to the cells the nucleic acid - polycation complex should be taken up by endocytosis. Since the endocytic vesicles have a homogenous internal diameter of about 100 nm in hepatocytes and are of similar size in other cell types, nucleic acid complexes smaller than 100 nm are preferred.
• Multivalent cations with a charge of three or higher have been shown to condense nucleic acid. These include spermidine, spermine, Co(NH 3 ) ⁇ 3+ ,Fe 3+ , and natural or synthetic polymers such as histone HI, protamine, polylysine, and polyethylenimine. Analysis has shown nucleic acid condensation to be favored when 90% or more of the charges along the sugar-phosphate backbone are neutralized.
• microaggregates form with short or long molecules and remain in suspension. Toroids, rods and small aggregates can be seen in such solution.
• Toroids have been considered an attractive form for gene delivery because they have the smallest size. While the size of DNA toroids produced within single preparations has been shown to vary considerably, toroid size is unaffected by the length of DNA being condensed. DNA molecules from 400 bp to genomic length produce toroids similar in size. Therefore one toroid can include from one to several DNA molecules. The kinetics of DNA collapse by polycations that resulted in toroids is very slow. For example DNA condensation by Co(NH 3 ) 6 Cl needs 2 hours at room temperature. The mechanism of DNA condensation is not clear. The electrostatic force between unperturbed helices arises primarily from a counterion fluctuation mechanism requiring multivalent cations and plays a major role in DNA condensation.
• T4 DNA complexes with polyarginine or histone can form two types of structures; an elongated structure with a long axis length of about 350 nm (like free DNA) and dense spherical particles. Both forms exist simultaneously in the same solution. The reason for the coexistence of the two forms can be explained as an uneven distribution of the polycation chains among the DNA molecules.
• the uneven distribution generates two thermodynamically favorable conformations.
• the electrophoretic mobility of nucleic acid - polycation complexes can change from negative to positive in excess of polycation. It is likely that large polycations don't completely align along nucleic acid but form polymer loops that interact with other nucleic acid molecules. The rapid aggregation and strong intermolecular forces between different nucleic acid molecules may prevent the slow adjustment between helices needed to form tightly packed orderly particles.
• preparation of polycation-condensed nucleic acid particles is of particular importance for gene therapy, more specifically, particle delivery such as the design of non-viral gene transfer vectors.
• particle delivery such as the design of non-viral gene transfer vectors.
• Optimal transfection activity in vitro and in vivo can require an excess of polycation molecules.
• the presence of a large excess of polycations may be toxic to cells and tissues.
• the non-specific binding of cationic particles to all cells forestalls cellular targeting. Positive charge also has an adverse influence on biodistribution of the complexes in vivo.
• nucleic acid - cation particles have been created to circumvent the nonspecific interactions of the nucleic acid - cation particle and the toxicity of cationic particles.
• modifications include attachment of steric stabilizers, e.g. polyethylene glycol, which inhibit nonspecific interactions between the cation and biological polyanions.
• steric stabilizers e.g. polyethylene glycol
• Another example is recharging the nucleic acid particle by the additions of polyanions which interact with the cationic particle, thereby lowering its surface charge, i.e. recharging of the DNA particle U.S. 09/328,975.
• Another example is cross-linking the polymers and thereby caging the complex U.S. 08/778,657, U.S. 09/000,692, U.S.
• Nucleic acid particles can be formed by the formation of chemical bonds and template polymerization U.S. 08/778,657, U.S. 09/000,692, U.S. 97/24089, U.S. 09/070299, and U.S. 09/464,871.
• a problem with these modifications is that they are most likely irreversible rendering the particle unable to interact with the cell to be transfected, and/or incapable of escaping from the lysosome once taken into a cell, and/or incapable of entering the nucleus once inside the cell.
• a method for formation of nucleic acid particles that is reversible under conditions found in the cell may allow for effective delivery of DNA.
• the conditions that cause the reversal of particle formation may be, but not limited to, the pH, ionic strength, oxidative or reductive conditions or agents, or enzymatic activity.
• PEG-AEPD pegylated comonomer
• D ⁇ A complexes formed from block co-polymers of polylysine and PEG MA Wolfert, EH Schacht, N Toncheva, K Ulbrich, O ⁇ azarova, LW Seymour. Human Gene Ther. 7:2123-2133, 1996.
• Blessing et al used bisthiol derivative of spermine and reaction of thiol-disulfide exchange to promote chain growth.
• Caging of polycation-condensed nucleic acid particles
• the stability of nucleic acid nanoassembhes based on nucleic acid condensation is generally low in vivo because they easily engage in polyion exchange reactions with strong polyanions.
• the process of exchange consists of two stages: 1) rapid formation of a triple nucleic acid- polycation-polyanion complex, 2) slow substitution of one same-charge polyion with another. At equilibrium conditions, the whole process eventually results in formation of a new binary complex and an excess of a third polyion.
• the presence of low molecular weight salt can greatly accelerate such exchange reactions, which often result in complete disassembly of condensed nucleic acid particles. Hence, it is desirable to obtain more colloidally stable structures where nucleic acid would stay in its condensed form in complex with corresponding polycation independently of environment conditions.
• DNA/pC DNA-polycation complex
• DNA/PLL DNA/PLL (1:3 charge ratio) formed in low salt 25 mM HEPES buffer and recharged with increasing amounts of various polyanions.
• the DNA particles were characterized after addition of a third polyion component to a DNA/polycation complex using a new DNA condensation assay (NS
• DNA polycation complexes can be both recharged and crosslinked or caged U.S. 08/778,657, U.S. 09/000,692, U.S. 97/24089, U.S. 09/070299, and U.S. 09/464,871.
• pH-sensitive lipids amphipathic compounds, and liposomes for drug and nucleic acid delivery
• DOTMA N-[l-(2J-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride
• a plethora of cationic lipids have been synthesized. Basically, all the cationic lipids are amphipathic compounds that contain a hydrophobic domain, a spacer, and positively-charged amine.
• the hydrophobic domains are typically hydrocarbon chains such as fatty acids derived from oleic or myristic acid.
• the hydrocarbon chains are often joined either by ether or ester bonds to a spacer such as glycerol.
• Quaternary amines often compose the cationic groups.
• the cationic lipids are mixed with a fusogenic lipid such as DOPE (dioleoyl phosphatidyl ethanolamine) to form liposomes.
• DOPE dioleoyl phosphatidyl ethanolamine
• the mixtures are mixed in chloroform that is then dried. Water is added to the dried lipid film and unilamellar liposomes form during sonication.
• Multilamellar cationic liposomes and cationic liposomes/nucleic acid complexes prepared by the reverse-phase evaporation method have also been used for transfection.
• Cationic liposomes have also been prepared by an ethanol injection technique.
• DOSPA the cationic lipid within the LipofectAMINE formulation (Life Technologies) contains a spermine linked via a amide bond and ethyl group to a trimethyl, quaternary amine [Hawley- Nelson, P, Ciccarone, V and Jessee, J. Lipofectamine reagent: A new, higher efficiency polycationic hposome transfection reagent. Focus 15:73-79, 1993].
• a French group has synthesized a series of cationic lipids such as DOGS (dioctadecylglycinespermine) that contain spermine [Remy, J-S, Sirlin, C, Nierling, P, et al. Gene transfer with a series of lipophilic D ⁇ A-binding molecules. Bioconjugate Chem. 5:647-654, 1994].
• DOGS dioctadecylglycinespermine
• D ⁇ A has also been transfected by lipophilic polylysines which contain dipalmotoylsuccinylglycerol chemically-bonded to low molecular weight (-3000 MW) polylysine [Zhou, X, Kilbanov, A and Huang, L.
• Lipophilic polylysines mediate efficient DNA transfection in mammalian cells. Biochim. Biophys. Acta 1065:8-14, 1991; Zhou, X and Huang, L. DNA transfection mediated by cationic liposomes containing lipopolylysine: Characterization and mechanism of action. Biochim. Biophys. Acta 1195-203, 1994]. Other studies have used adjuvants with the cationic liposomes. Transfection efficiency into Cos cells was increased when amphiphilic peptides derived from influenza virus hemagglutinin were added to DOTMA/DOPE liposomes [Kamata, H, Yagisawa, H, Takahashi, S, et al.
• Amphiphilic peptides enhance the efficiency of liposome-mediated DNA transfection. Nucleic Acids Res. 1994;22:536-537]. Cationic lipids have been combined with galactose ligands for targeting to the hepatocyte asialoglycoprotein receptor [Remy, J-S, Kichler, A, Mordvinov, N, et al. Targeted gene transfer into hepatoma cells with lipopolyamine-condensed D ⁇ A particles presenting galactose ligands: A stage toward artificial viruses. Proc. ⁇ atl. Acad. Sci. USA 1995 ;92: 1744-1748].
• Thiol-reactive phospholipids have also been incorporated into cationic lipid/pD ⁇ A complexes to enable cellular binding even when the net charge of the complex is not positive [Kichler, A, Remy, J-S, Boussif, O, et al. Efficient gene delivery with neutral complexes of lipospermine and thiol-reactive phospholipids. Biochem. Biophys. Res. Comm. 1995;209:444-450].
• D ⁇ A-dependent template process converted thiol-containing detergent possessing high critical micelle concentration into dimeric lipid-like molecule with apparently low water solubility (JP Behr and colleagues).
• Cationic liposomes may deliver nucleic acid either directly across the plasma membrane or via the endosome compartment. Regardless of its exact entry point, much of the nucleic acid within cationic liposomes does accumulate in the endosome compartment.
• Several approaches have been investigated to prevent loss of the foreign nucleic acid in the endosomal compartment by protecting it from hydrolytic digestion within the endosomes or enabling its escape from endosomes into the cytoplasm. They include the use of acidotropic (lysomotrophic), weak amines such as chloroquine that presumably prevent nucleic acid degradation by inhibiting endosomal acidification [Legendre, J. & Szoka, F.
• Niral fusion peptides or whole virus have been included to disrupt endosomes or promote fusion of liposomes with endosomes, and facilitate release of nucleic acid into the cytoplasm [Kamata, H., Yagisawa, H., Takahashi, S. & Hirata, H. Amphiphilic peptides enhance the efficiency of liposome- mediated DNA transfection. Nucleic Acids Res. 22, 536-537 (1994). Wagner, E., Curiel, D. & Cotten, M. Delivery of drugs, proteins and genes into cells using transferrin as a ligand for receptor-mediated endocytosis. Advanced Drug Delivery Reviews 14, 113-135 (1994)].
• phosphatidylethanolamine (PE) bilayers that are stabilized at non-acidic pH by the addition of lipids which contain a carboxylic acid group. Liposomes containing only PE are prone to the inverted hexagonal phase (H ⁇ ).
• H ⁇ inverted hexagonal phase
• the carboxylic acid's negative charge increases the size of the lipid head group at pH greater than the carboxylic acid's pK and thereby stabilizes the phosphatidylethanolamine bilayer.
• the uncharged or reduced charge species is unable to stabilize the phosphatidylethanolamine-rich bilayer.
• Anionic, pH-sensitive liposomes have delivered a variety of membrane-impermeant compounds including DNA. However, the negative charge of these pH-sensitive liposomes prevents them from efficiently taking up DNA and interacting with cells; thus decreasing their utility for transfection.
• pH-Sensitive Polymers for Drug and Nucleic Acid Delivery Polymers that pH-sensitive are have found broad application in the area of drug delivery exploiting various physiological and intracellular pH gradients for the purpose of controlled release of drugs (both low molecular weight and polymeric).
• pH sensitivity can be broadly defined as any change in polymer's physico-chemical properties over certain range of pH. More narrow definition demands significant changes in the polymer's ability to retain (release) a bioactive substance (drug) in a physiologically tolerated pH range (usually pH 5.5 - 8). pH-sensitivity presumes the presence of ionizable groups in the polymer (polyion).
• polyacids can be divided into three categories based on their ability to donate or accept protons in aqueous solutions: polyacids, polybases and polyampholytes.
• Use of pH-sensitive polyacids in drug delivery applications usually relies on their ability to become soluble with the pH increase (acid salt conversion), to form complex with other polymers over change of pH or undergo significant change in hydrophobicity/hydrophilicity balance. Combinations of all three above factors are also possible.
• Copolymers of polymethacrylic acid are known as polymers which are insoluble at lower pH but readily solubilized at higher pH, so they are used as enteric coatings designed to dissolve at higher intestinal pH (Z Hu et al. J. Drug Target., 7, 223, 1999).
• a typical example of pH-dependent complexation is copolymers of polyacrylate(graft)ethyleneglycol which can be formulated into various pH-sensitive hydrogels which exhibit pH-dependent swelling and drug release (F Madsen et al., Biomaterials, 20, 1701, 1999).
• Hydrophobically-modified N-isopropylacrylamide- methacrylic acid copolymer can render regular egg PC liposomes pH-sensitive by pH- dependent interaction of grafted aliphatic chains with lipid bilayer (O Meyer et al., FEBS Lett., 421, 61, 1998).
• Polymers with pH-mediated hydrophobicity can be used as endosomal disruptors for cytoplasmic drug delivery (Murthy, N., Robichaud, J.R., Tirrell, D.A., Stayton, P.S., Hoffinan, A.S. J. Controlled Release 61, 137, 1999).
• Polybases have found broad applications as agents for nucleic acid delivery in transfection gene therapy applications due to the fact they are readily interact with polyacids.
• a typical example is polyethyleneimine (PEI).
• PEI polyethyleneimine
• This polymer secures nucleic acid electrostatic adsorption on the cell surface followed by endocytosis of the whole complex. Cytoplasmic release of the nucleic acid occurs presumably via the so called “proton sponge” effect according to which pH-sensitivity of PEI is responsible for endosome rupture due to osmotic swelling during its acidification (O Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297, 1995).
• Cationic acrylates possess the similar activity (for example, poly-((2-dimethylamino)ethyl methacrylate) (P van de Wetering et al. J. Controlled Release 64, 193, 2000).
• polybases due to their polycationic nature pH-sensitive polybases have not found broad in vivo application so far, due to their acute systemic toxicity in vivo (JH Senior, Biochim. Biophys. Acta, 1070, 173, 1991).
• Milder polybases for example, linear PEI
• Membrane Active Compounds Many biologically active compounds, in particular large and/or charged compounds, are incapable of crossing biological membranes. In order for these compounds to enter cells they must either be taken up by the cells by endocytosis, into endosomes, or there must be a disruption of the cellular membrane to allow the compound to cross. In the case of endosomal entry, the endosomal membrane must be disrupted to allow for the entrance of the compound in the enterior of the cell. Therefore, either entry pathway into the cell requires a disruption of the cellular membrane. There exist compounds termed membrane active compounds that disrupt membranes. One can imagine that if the membrane active agent were operative in a certain time and place it would facilitate the transport of the biologically active compound across the biological membrane.
• a cellular transport step that has attracted attention for gene transfer is that of DNA release from intracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum.
• Intracellular compartments such as endosomes (early and late), lysosomes, phagosomes, vesicle, endoplasmic reticulum, golgi apparatus, trans golgi network (TGN), and sarcoplasmic reticulum.
• Release includes movement out of an intracellular compartment into cytoplasm or into an organelle such as the nucleus.
• a number of chemicals such as chloroquine, bafilomycin or Brefeldin Al have been used to disrupt or modify the trafficking of molecules through intracellular pathways. Chloroquine decreases the acidification of the endosomal and lysosom
• Brefeldin A an isoprenoid fungal metabolite, collapses reversibly the Golgi apparatus into the endoplasmic reticulum and the early endosomal compartment into the trans-Golgi network (TGN) to form tubules.
• Bafilomycin Al a macrolide antibiotic is a more specific inhibitor of endosomal acidification and vacuolar type H + -ATPase than chloroquine.
• Viruses Proteins and Peptides for Disruption of Endosomes and Endosomal Function Viruses such as adenovirus have been used to induce gene release from endosomes or other intracellular compartments (D. Curiel, Agarwal, S., Wagner, E., and Cotten, M. PNAS 88:8850, 1991). Rhinovirus has also been used for this purpose (W. Zauner et al. J. Nirology 69: 1085-92, 1995). Niral components such as influenza virus hemagglutinin subunit HA-2 analogs has also been used to induce endosomal release (E. Wagner et al. P ⁇ AS 89:7934, 1992).
• Amphipathic peptides resembling the ⁇ -terminal HA-2 sequence has been studied (K. Mechtler and E. Wagner, New J. Chem. 21:105-111, 1997). Parts of the pseudonmonas exotoxin and diptheria toxin have also been used for drug delivery (I. Pastan and D. FitzGerald. J. Biol. Chem. 264: 15157, 1989).
• Nucleic acid and gene delivery may involve the biological pH gradient that is active within organisms as a factor in delivering a polynucleotide to a cell.
• Different pathways that may be affected by the pH gradient include cellular transport mechanisms, endosomal disruption/breakdown, and particle disassembly (release of the DNA).
• Other gradients that can be useful in gene therapy research involve ionic gradients that are related to cells. For example, both Na + and K + have large concentration gradients that exist across the cell membrane.
• Systems containing metal-binding groups can utilize such gradients to influence delivery of a polynucleotide to a cell. Changes in the osmotic pressure in the endosome also have been used to disrupt membranes and allow for transport across membrane layer.
• Buffering of the endosome pH may cause these changes in osmotic pressure.
• the "proton sponge" effect of PEI O Boussif et al. Proc. Natl. Acad. Sci. USA 92, 7297, 1995
• certain polyanions Murthy, N., Robichaud, J.R., Tirrell, D.A., Stayton, P.S., Hoffinan, A.S. Journal of Controlled Release 1999, 61, 137
• This pressure increase results in membrane disruption and release of the contents of the endosome.
• both extra and intracellularly may be used to deliver the gene of interest either by aiding in the delivery to the cell or escape from intracellular compartments.
• Proteases found in serum, lysosome and cytoplasm, may be used to disrupt the particle and allow its interaction with the cell surface or cause it fracture the intracellular compartment, e.g. endosome or lysosome, allowing the gene to be released intracellularly.
• nucleic acid is a messenger RNA
• a ribosome translates the messenger RNA to produce a protein within the cytoplasm.
• nucleic acid is a DNA
• it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein.
• the nucleic acid contains sequences that are required for its transcription and translation. These include promoter and enhancer sequences that are required for initiation. DNA and thus the corresponding mRNA (transcribed from the DNA) contains introns that must be spliced, poly A addition sequences, and sequences required for the initiation and termination of its translation into protein. Therefore if a nucleic acid expresses its cognate protein, then it must have entered a cell.
• a therapeutic effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane or being secreted and dissociating from the cell where it can enter the general circulation and blood.
• Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e.g. alpha-antitrypsin) and other proteins that are present in the blood. Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or lipoprotein.
• the low density lipoprotein (LDL) receptor could be expressed in hepatocytes and lower blood cholesterol levels by removing excess LDL from the blood and thereby prevent atherosclerotic lesions that can cause strokes or myocardial infarction.
• Therapeutic proteins that stay within the cell can be enzymes that clear a circulating toxic metabolite as in phenylketonuria. They can also cause a cancer cell to be less proliferative or cancerous (e.g. less metastatic). A protein within a cell could also interfere with the replication of a virus.
• the present invention provides for the transfer of single stranded nucleic acids into cells within tissues in vitro, in situ and in vivo.
• Single stranded nucleic acids can be efficiently delivered to cells via a variety of non- viral gene transfer methods.
• the nature of single stranded nucleic acid may allow formation of smaller gene transfer units than would be possible for a similar double stranded nucleic acid, perhaps increasing delivery efficiency.
• Expression of a gene encoded by the single stranded nucleic acid may be prolonged, compared to the expression of the same gene encoded and delivered in the form of double stranded nucleic acid.
• a process for delivering a nucleic acid into a cell comprising selecting a nucleic acid for delivery wherein the nucleic acid is selected from the group consisting of single stranded DNA, partially single stranded DNA, double stranded heterogeneous nucleic acid, partially double stranded heterogeneous nucleic acid, partially multistranded DNA, partially multistranded heterogeneous nucleic acid. Then, transporting the nucleic acid to the cell by a means selected from the group consisting of microinjection, direct injection into an organ, intravascular injection. Finally, transfecting the cell.
• the cell may consist of a eukaryotic cell, a mammalian cell and a human cell.
• the nucleic acid may be linear and circular and may express a therapeutic gene.
• the nucleic acid may be non-viral and may be combined with a transfection reagent, polymer, and lipid.
• the nucleic acid may be transported to the cell by direct injection into an organ and by intravascular injection.
• a compound is described for transfecting an eukaryotic cell by microinjection, direct injection into an organ, or intravascular injection, comprising a molecule consisting of single stranded DNA.
• a compound is described for transfecting an eukaryotic cell into cell by microinjection, direct injection into an organ, or intravascular injection, comprising a molecule consisting of partially single stranded DNA.
• a compound for transfecting an eukaryotic cell by microinjection, direct injection into an organ, or intravascular injection, comprising a molecule selected from the group consisting of double stranded heterogeneous nucleic acid, and partially double stranded heterogeneous nucleic acid.
• a compound for transfecting an eukaryotic cell by microinjection, direct injection into an organ, or intravascular injection, comprising a molecule consisting of partially multistranded DNA, partially multistranded heterogeneous nucleic acid.
• nucleic acid is a teim of art that refers to a polymer containing at least two nucleotides.
• Nucleotides contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are the monomeric units of nucleic acid polymers. Nucleotides are linked together through the phosphate groups to form nucleic acid.
• a "polynucleotide” is distinguished here from an “oligonucleotide” by containing more than 100 monomeric units; oligonucleotides contain from 2 to 100 nucleotides.
• Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and other natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
• nucleic acid includes deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA").
• nucleic acid encompasses sequences that include any of the known base analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy- N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl- guanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino- methyl-2-thiouracil,
• DNA may be in the form of anti -sense, plasmid DNA, parts of a plasmid DNA, vectors (PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives of these groups.
• RNA may be in the form of oligonucleotide RNA, tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, (interfering) double stranded RNA, ribozymes, chimeric sequences, or derivatives of these groups.
• Anti-sense is a nucleic acid that interferes with the function of DNA and/or RNA. This may result in suppression of expression.
• Natural nucleic acids have a phosphate backbone; artificial nucleic acids may contain other types of backbones, nucleotides, or bases. These include PNAs (peptide nucleic acids), phosphothionates, and other variants of the phosphate backbone of native nucleic acids. Examples of modified nucleotides include methylation, mustard addition, aromatic nitrogen mustard addition, and LabellT modification.
• “Mustards” include nitrogen mustards and sulfur mustards. Mustards are molecules consisting of a nucleophile and a leaving group separated by an ethylene bridge. After internal attack of the nucleophile on the carbon bearing the leaving group a strained three membered group is formed. This strained ring (in the case of nitrogen mustards an aziridine ring is formed) is very susceptible to nucleophilic attack. Thus allowing mustards to alkylate weak nucleophiles such as nucleic acids.
• Nucleic acid may be single (“ssDNA”), double (“dsDNA”), triple (“tsDNA”), or quadruple (“ ⁇ sDNA”) stranded DNA, and single stranded RNA (“RNA”) or double stranded RNA (“dsRNA”).
• “Multistranded” nucleic acid contains two or more strands and can be either homogeneous as in double stranded DNA, or heterogeneous, as in DNA/RNA hybrids. Multistranded nucleic acid can be full length multistranded, or partially multistranded. It may further contain several regions with different numbers of nucleic acid strands. Partially single stranded DNA is considered a sub-group of ssDNA and contains one or more single stranded regions as well as one or more multiple stranded regions.
• Single stranded nucleic acids can be generated by a variety of means, including denaturation, separation, chemical synthesis, isolation from viruses, enzymatic reaction. "Denaturation” is the process in which multi-stranded nucleic acid is completely or partially separated into single stranded nucleic acids. This can proceed through heating, alkaline treatment, or the addition of chemicals such as chaotropic salts. A mixture of nucleic acids can be "separated” by physical means such as density gradient centrifugation, gel electrophoresis, or affinity purification.
• Affinity purification can be accomplished by incorporating a ligand in the nucleic acid (e.g., biotin), and using the corresponding ligate (e.g., strepavidin) bound to a matrix (e.g., magnetic beads) to specifically bind and purify this nucleic acid.
• a ligand in the nucleic acid (e.g., biotin), and using the corresponding ligate (e.g., strepavidin) bound to a matrix (e.g., magnetic beads) to specifically bind and purify this nucleic acid.
• “Chemical synthesis” refers to the process where a single stranded nucleic acid is formed by repetitively attaching a nucleotide to the end of an existing nucleic acid.
• the existing nucleic acid can be a single nucleotide.
• Single stranded oligonucleotides can be chemically linked together to form long nucleic acids.
• viruses can infect prokaryotes (e.g., M13, T7, lambda) or eukaryotes (e.g., adeno-associated virus [AAN], adenovirus, retrovirus, herpesvirus, Sindbis virus).
• AAN adeno-associated virus
• Isolation from single stranded D ⁇ A viruses will directly generate (partially) single stranded D ⁇ A.
• Enzymatic reaction refers to processes mediated by enzymes.
• One strand of a double stranded nucleic acid can be preferentially degraded into nucleotides using a nuclease.
• Many ribonucleases are known with specific activity profiles that can be used for such a process.
• R ⁇ ase H can be used to specifically degrade the R ⁇ A strand of an R ⁇ A-D ⁇ A double stranded hybrid nucleic acid, which in itself may have been formed by the enzymatic reaction of reverse transcriptase synthesizing the D ⁇ A stranded using the R ⁇ A strand as the template.
• a ribonuclease can specifically degrade the strand with the nick, generating a partially single stranded nucleic acid.
• a R ⁇ A or D ⁇ A dependent D ⁇ A polymerase can synthesize new D ⁇ A which can subsequently be isolated (e.g., by denaturation followed by separation).
• the polymerase chain reaction process can be used to generate nucleic acids. Formation of single stranded nucleic acid can be favored by adding one oligonucleotide primer in excess over the other primer ("asymmetric PCR"). Alternatively, one of the DNA strands formed in the PCR process may be separated from the other (e.g., by using a ligand in one of the primers).
• “Expression cassette” refers to a natural or recombinantly produced nucleic acid molecule which is capable of expressing protein(s).
• a DNA expression cassette typically includes a promoter (allowing transcription initiation), and a sequence encoding one or more proteins.
• the expression cassette may include trancriptional enhancers, non-coding sequences, splicing signals, transcription termination signals, and polyadenylation signals.
• An RNA expression cassette typically includes a translation initiation codon (allowing translation initiation), and a sequence encoding one or more proteins.
• the expression cassette may include translation termination signals, a polyadenosine sequence, internal ribosome entry sites (IRES), and non-coding sequences.
• a nucleic acid can be used to modify the genomic or extrachromosomal DNA sequences. This can be achieved by delivering a nucleic acid that is expressed. Alternatively, the nucleic acid can effect a change in the DNA or RNA sequence of the target cell. This can be achieved by homologous recombination, gene conversion, or other yet to be described mechanisms.
• the term "gene” generally refers to a nucleic acid sequence that comprises coding sequences necessary for the production of a therapeutic nucleic acid (e.g., ribozyme) or a polypeptide or precursor.
• the polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction) of the full-length polypeptide or fragment are retained.
• the term also encompasses the coding region of a gene and the including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb or more on either end such that the gene corresponds to the length of the full-length mRNA.
• the sequences that are located 5' of the coding region and which are present on the mRNA are referred to as "5' untranslated sequences".
• the sequences that are located 3' or downstream of the coding region and which are present on the mRNA are referred to as "3
• the term gene encompasses both cDNA and genomic forms of a gene.
• a genomic form or clone of a gene contains the coding region interrupted with "non- coding sequences" termed “introns” or “intervening regions” or “intervening sequences.”
• Introns are segments of a gene which are transcribed into nuclear RNA. Introns may contain regulatory elements such as enhancers.
• Introns are removed or "spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
• the mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.
• the term non-coding sequences also refers to other regions of a genomic form of a gene including, but not limited to, promoters, enhancers, transcription factor binding sites, polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, matrix attachment regions. These sequences may be present close to the coding region of the gene (within 10,000 nucleotide) or at distant sites (more than 10,000 nucleotides).
• Covalent modification of a gene may influence the rate of transcription (e.g., methylation of genomic DNA), the stability of mRNA (e.g., length of the 3' polyadenosine tail), rate of translation (e.g., 5' cap), nucleic acid repair, and immunogenicity.
• rate of transcription e.g., methylation of genomic DNA
• stability of mRNA e.g., length of the 3' polyadenosine tail
• rate of translation e.g., 5' cap
• nucleic acid repair e.g., 5' cap
• covalent modification of nucleic acid involves the action of LabellT reagents (Minis Corporation, Madison, WI).
• nucleic acid molecule encoding As used herein, the terms “nucleic acid molecule encoding.” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.
• an oligonucleotide having a nucleotide sequence encoding a gene means a nucleic acid sequence comprising the coding region of a gene or in other words the nucleic acid sequence which encodes a gene product.
• the coding region may be present in either a cDNA, genomic DNA or RNA form.
• the nucleic acid may be single-stranded, double-stranded, multistranded, partially single stranded, or partially multistranded.
• Suitable control elements such as, but not limited to, enhancers/promoters, splice junctions, and polyadenylation signals, may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and correct processing of the primary RNA transcript.
• the coding region utilized in the expression vectors may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, or a combination of both endogenous and exogenous control elements.
• isolated nucleic acid when used in relation to a nucleic acid, as in “an isolated nucleic acid” refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature.
• non-isolated nucleic acids are nucleic acids, such as DNA and RNA, found in the state they exist in nature.
• a given DNA sequence e.g., a gene
• RNA sequences such as a specific mRNA sequence encoding a specific protein
• isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
• the isolated nucleic acid may be present in single stranded, partially single stranded, multi stranded, or partially multi stranded form.
• RNA expression refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through "transcription" of a deoxyribonucleic gene (e.g., via the enzymatic action of an RNA polymerase), and for protein encoding genes, into protein through “translation” of mRNA.
• Gene expression can be regulated at many stages in the process.
• Up-regulation” or “activation” refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while “down-regulation” or “repression” refers to regulation that decrease production.
• Molecules e.g., transcription factors
• activators e.g., transcription factors
• naked nucleic acid and “naked polynucleotide” indicate that the nucleic acid or polynucleotide are not associated with a transfection reagent or other delivery vehicle that is required for the nucleic acid or polynucleotide to be delivered to the cardiac muscle cell.
• a '"transfection reagent is a compound or compounds used in the prior art that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and mediates their entry into cells. The transfection reagent also mediates the binding and internalization of oligonucleotides and polynucleotides into cells.
• transfection reagents include cationic liposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylenimine, and polylysine complexes. It has been shown that cationic proteins like histones and protamines, or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents, while small polycations like spermine are ineffective.
• the transfection reagent has a net positive charge that binds to the oligonucleotide 's or polynucleotide's negative charge.
• the transfection reagent mediates binding of oligonucleotides and polynucleotides to cells via its positive charge (that binds to the cell membrane's negative charge) or via ligands that bind to receptors in the cell.
• cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA or RNA.
• Polyethylenimine which facilitates gene transfer without additional treatments, probably disrupts endosomal function itself.
• Other vehicles are also used, in the prior art, to transfer genes into cells. These include complexing the nucleic acids on particles that are then accelerated into the cell. This is termed “Holistic” or “gun” techniques. Other methods include “electroporation.” in which a device is used to give an electric charge to cells. The charge increases the permeability of the cell.
• Intravascular refers to an intravascular route of administration that enables a polymer, oligonucleotide, or polynucleotide to be delivered to cells more evenly distributed and more efficiently than direct injections.
• Intravascular herein means within an internal tubular structure called a vessel that is connected to a tissue or organ within the body of an animal, including mammals.
• a bodily fluid flows to or from the body part.
• bodily fluid include blood, lymphatic fluid, or bile.
• vessels include arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, and bile ducts.
• the intravascular route includes delivery through the blood vessels such as an artery or a vein.
• “Intracoronary” refers to an intravascular route for delivery to the heart wherein the blood vessels are the coronary arteries and veins.
• Delivery of a nucleic acid means to transfer a nucleic acid from a container outside a mammal to near or within the outer cell membrane of a cell in the mammal.
• the term "transfection” is used herein, in general, as a substitute for the term “delivery.” or, more specifically, the transfer of a nucleic acid from directly outside a cell membrane to within the cell membrane.
• the nucleic acid is a primary RNA transcript that is processed into messenger RNA, a ribosome translates the messenger RNA to produce a protein within the cytoplasm.
• the nucleic acid is a DNA, it enters the nucleus where it is transcribed into a messenger RNA that is transported into the cytoplasm where it is translated into a protein.
• the nucleic acid contains sequences that are required for its transcription and translation. These include promoter and enhancer sequences that are required for initiation. DNA and thus the corresponding messenger RNA (transcribed from the DNA) contains poly A addition sequences, sequences required for the initiation and termination of its translation into protein, and may contain introns that must be spliced. Therefore if a nucleic acid expresses its cognate protein, then it must have entered a cell. A protein may subsequently be degraded into peptides, which may be presented to the immune system.
• a “therapeutic gene” refers herein to a nucleic acid that has a therapeutic effect upon transfection into a cell. This effect can be mediated by the nucleic acid itself (e.g., anti-sense nucleic acid), following transcription (e.g., anti-sense RNA, ribozymes, interfering dsRNA), or following expression into a protein.
• Protein refers herein to a linear series of greater than 2 amino acid residues connected one to another as in a polypeptide.
• a "therapeutic" effect of the protein in attenuating or preventing the disease state can be accomplished by the protein either staying within the cell, remaining attached to the cell in the membrane, or being secreted and dissociated from the cell where it can enter the general circulation and blood.
• Secreted proteins that can be therapeutic include hormones, cytokines, growth factors, clotting factors, anti-protease proteins (e g , alphal-antitrypsin), angiogemc proteins (e g , vascular endothelial growth factor, fibroblast growth factors), antiangiogenic protems (e g , endostatin, angiostatin), and other proteins that are present in the blood Proteins on the membrane can have a therapeutic effect by providing a receptor for the cell to take up a protein or hpoprotein Therapeutic proteins that stay within the cell (intracellular proteins) can be enzymes that clear a circulating toxic metabolite as m phenylketonu ⁇ a They can also cause a cancer cell to be less pro
• Vectors are nucleic acid molecules originating from a virus, a plasmid, or the cell of an organism into which another nucleic fragment of appropnate size can be integrated without loss of the vectors capacity for self-replication
• Vectors introduce nucleic acids into host cells, where it can be reproduced
• Examples are plasmids, cosmids, and yeast artificial chromosomes
• Vectors are often recombinant molecules containing nucleic acid sequences from several sources
• Vectors include viruses, for example adenovirus (an lcosahedral (20- sided) virus that contains DNA, there are over 40 different adenovirus vaneties, some of which cause respiratory disease), adeno-associated virus (AAV, a parvovirus that contains single stranded DNA), or retrovirus (any virus m the family Retroviridae that has RNA as its nucleic acid and uses the enzyme reverse transcnptase to copy its genome into the DNA and integrate into the host cell'
• a single stranded DNA resembles adeno-associated viral DNA wherein the viral structural codmg sequences may be (partially) replaced by therapeutic sequences (e g , an expression cassette with the human factor IX gene under transcnptional control of the human cytomegalovirus promoter)
• therapeutic sequences e g , an expression cassette with the human factor IX gene under transcnptional control of the human cytomegalovirus promoter
• sequences identical to the AAV inverted terminal repeat (ITR) are added to the ends of a single stranded nucleic acid
• sequences forming a structure similar to an AAV ITR double hairpin
• the ends of a smgle stranded nucleic acid can hybndize to itself, thus forming a short double stranded nucleic acid
• This double stranded element can be present at one or both ends of the nucleic acid
• the length of homology can be from 1 to 300 bp By
• a nucleic acid may be combined with a shorter single stranded nucleic acid, homologous to a part of this nucleic acid, thus forming a partially multistranded nucleic acid.
• a single stranded DNA containing an expression cassette is combined with another, shorter homologous single stranded nucleic acid, thus forming a partially double stranded DNA.
• Multiple different shorter single stranded DNA strands may be used, thus forming a DNA with multiple regions that are double stranded. These double stranded regions may be consecutive or spaced by single stranded regions. Regions that are triple or quadruple stranded may be formed as well.
• a single stranded nucleic acid is combined with a single stranded DNA containing an expression cassette wherein the single stranded nucleic acid is complementary to the end of the single stranded DNA.
• a nucleic acid is formed wherein one or both ends are double stranded.
• the nucleic acid is modified, prior or after combination with other nucleic acids, by methylation (for instance by using Sss I methylase), or chemical modification (for instance by aromatic nitrogen mustard addition of biotin or DNP).
• the modification is accomplished by using LabellT reagents (Minis Corporation, Madison, WI).
• transfection refers to the introduction of foreign DNA into eukaryotic cells.
• the nucleic acid could be used to produce a change in a cell that can be therapeutic.
• the delivery of nucleic acid for therapeutic and research purposes is commonly called “gene therapy.”
• the delivery of nucleic acid can lead to modification of the genetic material present in the target cell.
• stable transfection or “stably transfected” refers to the introduction and integration of foreign nucleic acid into the genome of the transfected cell.
• stable transfectant refers to a cell which has stably integrated foreign nucleic acid into the genomic DNA.
• transient transfection or “transiently transfected” refers to the introduction of foreign nucleic acid into a cell where the foreign nucleic acid does not integrate into the genome of the transfected cell.
• the foreign nucleic acid persists in the nucleus of the transfected cell.
• the foreign nucleic acid is subject to the regulatory controls that govern the expression of endogenous genes in the chromosomes.
• transient transfectant refers to a cell which has taken up foreign nucleic acid but has not integrated this nucleic acid.
• transfection A wide variety of methods have been described to enable transfection, including, but not limited to, calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, iontophoresis, and biolistics.
• naked nucleic acid indicates that the nucleic acid is not associated with a transfection reagent or other delivery vehicle that is required for the nucleic acid to be delivered to a cell.
• transfection reagent or “delivery vehicle” is a compound or compounds that bind(s) to or complex(es) with nucleic acids, or other desired compounds, and mediates their entry into cells.
• transfection reagents include, but are not limited to, cationic liposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylenimine, and polylysine complexes.
• the transfection reagent has a net positive charge that binds to the nucleic acid's negative charge.
• cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA or RNA.
• Two molecules are combined, to form a “complex” through a process called “complexation” or “complex formation.” if the are in contact with one another through “non-covalent” interactions such as, but not limited to, electrostatic interactions, hydrogen bonding interactions, and hydrophobic interactions.
• the charge, polarity, or sign of a compound refers to whether or not a compound has lost one or more electrons (positive charge, polarity, or sign) or gained one or more electrons (negative charge, polarity, or sign).
• An "interpolyelectrolyte complex” is a non-covalent interaction between polyelectrolytes of opposite charge.
• a molecule is "modified.” through a process called “modification.” by a second molecule if the two become bonded through a covalent bond. That is, the two molecules form a covalent bond between an atom form one molecule and an atom from the second molecule resulting in the formation of a new single molecule.
• a chemical "covalent bond” is an interaction, bond, between two atoms in which there is a sharing of electron density.
• Afferent blood vessels of organs are defined as vessels in which blood flows toward the organ or tissue under normal physiologic conditions.
• Efferent blood vessels are defined as vessels in which blood flows away from the organ or tissue under normal physiologic conditions.
• afferent vessels are known as coronary arteries, while efferent vessels are referred to as coronary veins.
• Permeability is defined herein as the propensity for macromolecules such as nucleic acids to move through vessel walls and enter the extravascular space.
• One measure of permeability is the rate at which macromolecules move through the vessel wall and out of the vessel.
• Another measure of permeability is the lack of force that resists the movement through the vessel wall and out of the vessel.
• Vessels contain elements that prevent macromolecules from leaving the intravascular space (internal cavity of the vessel). These elements include endothelial cells and connective material (e.g., collagen). High permeability indicates that there are fewer of these elements that can block the egress of macromolecules and that the spaces between these elements are larger and more numerous. In this context, high permeability enables a high percentage of nucleic acids being delivered to leave the intravascular space, while low permeability indicates that a low percentage of the nucleic acids will leave the intravascular space.
• the permeability of a blood vessel can be increased by increasing the intravascular hydrostatic pressure.
• the intravascular hydrostatic pressure is increased by rapidly (from 1 seconds to 30 minutes) injecting a nucleic acid in solution into the blood vessel, which increases the hydrostatic pressure.
• hydrostatic pressure is increased by obstructing the outflow of the injection solution from the tissue for a period of time sufficient to allow delivery of a nucleic acid. Obstructing means to block or impede the outflow of injection fluid, thereby transiently (reversibly) blocking the outflow of the blood.
• rapid injection may be combined with obstructing the outflow in yet another preferred embodiment.
• an afferent vessel supplying an organ is rapidly injected and the efferent vessel draining the tissue is blocked transiently (e.g., by ligation, or by an inflated intravascular balloon).
• the flow into and from the target area can be blocked by the application of a (blood pressure) cuff, for instance around the arm when targeting the lower arm or hand.
• the efferent vessel also called the venous outflow or tract
• draining outflow from the tissue is partially or totally clamped for a period of time sufficient to allow delivery of a nucleic acid.
• an efferent is injected and an afferent vessel is occluded.
• the intravascular pressure of a blood vessel is increased by increasing the osmotic pressure within the blood vessel.
• hypertonic solutions containing salts such as sodium chloride, sugars or polyols such as mannitol are used.
• “Hypertonic” means that the osmolality of the injection solution is greater than physiologic osmolality.
• “Isotonic” means that the osmolality of the injection solution is the same as the physiological osmolality (i.e., the tonicity or osmotic pressure of the solution is similar to that of blood).
• Hypertonic solutions have increased tonicity and osmotic pressure similar to the osmotic pressure of blood and cause cells to shrink.
• the permeability of the blood vessel can also be increased by a biologically-active molecule such as a protein or a simple chemical such as histamine that increases the permeability of the vessel by causing a change in function, activity, or shape of cells within the vessel wall such as the endothelial or smooth muscle cells.
• biologically active molecules that affect permeability interact with a specific receptor or enzyme or protein within the vascular cell to change the vessel's permeability.
• Biologically active molecules include vascular permeability factor (VPF) which is also known as vascular endothelial growth factor (VEGF).
• VPF vascular permeability factor
• VEGF vascular endothelial growth factor
• Another type of biologically active molecule can also increase permeability by changing the extracellular connective material.
• an enzyme could digest the extracellular material and increase the number and size of the holes of the connective material.
• Other biologically active molecules that may alter the permeability include ethylenediaminetetraacetic acid (EDTA), adenosine, papaverine, and nifedipine.
• EDTA ethylenediaminetetraacetic acid
• adenosine adenosine
• papaverine adenosine
• nifedipine nifedipine
• the liver and portal vein of mice are visualized through a ventral midline incision. Plasmid DNA in 1 ml of various solutions containing heparin to prevent clotting was injected into the portal vein using a needle over approximately 30 sec. At various times after the injection, the animals were sacrificed by cervical dislocation and the livers extracted for gene expression analysis (e.g., luciferase, ⁇ - galactosidase). Alternatively, if the gene product is secreted then blood is obtained from the retro-orbital venous sinus and the level of the secreted protein is assayed in the blood (e.g., human growth hormone, human factor IX, secreted alkaline phosphatase).
• gene expression analysis e.g., luciferase, ⁇ - galactosidase.
• the gene can produce an enzyme that corrects an abnormality in a disease state.
• the phenylalanine hydroxylase gene could be used to normalize the elevated phenylalanine blood levels in a genetic mouse model of phenylketonuria.
• a single stranded nucleic acid is delivered to a cell in culture (in vitro).
• the delivery of the single stranded nucleic acid is by means selected from the group, but not limited to, microinjection of the nucleic acid in the target cell, delivery of complexes of the nucleic acid with polymers, transfection, transformation, electroporation, iontophoresis, or particle bombardment.
• a single stranded nucleic acid is delivered to a cell in vivo.
• the cell is in a mammal.
• the delivery of the single stranded nucleic acid is by means selected from the group, but not limited to, direct injection of the nucleic acid in the target tissue, injection into the vasculature, microinjection, delivery of complexes of the nucleic acid with polymers, electroporation, iontophoresis, topical application, or particle bombardment.
• a partially single stranded nucleic acid is delivered to a cell in culture (in vitro).
• the delivery of the partially single stranded nucleic acid is by means selected from the group, but not limited to, microinjection of the nucleic acid in the target cell, delivery of complexes of the nucleic acid with polymers, transfection, transformation, electroporation, iontophoresis, or particle bombardment.
• a partially single stranded nucleic acid is delivered to a cell in vivo.
• the cell is in a mammal.
• the delivery of the partially single stranded nucleic acid is by means selected from the group, but not limited to, direct injection of the nucleic acid in the target tissue, injection into the vasculature, microinjection, delivery of complexes of the nucleic acid with polymers, electroporation, iontophoresis, topical application, or particle bombardment.
• single stranded nucleic acid is formed by means selected from the group, but not limited to, denaturation of multistranded nucleic acid, denaturation of multistranded nucleic acid followed by physical separation of the strands, isolation from single stranded viruses, enzymatic degradation of one strand of double stranded nucleic acid, asymmetric PCR, chemical synthesis.
• the single stranded nucleic acid is DNA.
• the single stranded nucleic acid contains one or two terminal ends with homologous regions such that one or more regions of double stranded or multistranded nucleic acid can be formed.
• these terminal regions are identical to the inverted terminal ends found in adeno-associated virus (human AAV-2).
• these terminal regions are identical to the inverted terminal ends selected from the group comprising of parvoviruses and dependoviruses.
• pCI-Luc + the plasmid vector pCI-Luc + , in which the luciferase gene is expressed under transcriptional control of the human cytomegalovirus promoter.
• 200-400 ⁇ g pCI-Luc + was diluted into 1.0 ml normal saline solution (final volume) in a 2 ml microcentrifuge tube. The tube was placed in boiling water (100 °C) for 8 minutes. Then, the tube was immediately transferred to a 0 °C water bath (mixture of ice and water) for 10 minutes. The tubes were inverted every two minutes. The denatured plasmid DNA solution was kept on ice until use (less then 30 minutes).
• Example 2 Generation of single stranded linear DNA by heat denaturation
• the plasmid DNA expression vector pCI-Luc + was linearized by digestion with the restriction enzyme BamH I. To this end, 600 ⁇ g of plasmid DNA (216 ⁇ l pCI-Luc + ) was mixed with 100 ⁇ l buffer K (Takara), 25 ⁇ l BamH I (Takara), and 659 ⁇ l water (total volume 1,000 ⁇ l). The mixture was incubated at 37 °C (water bath) for 42 hours. A small aliquot (2 ⁇ l) was removed and analyzed by standard agarose gel electtophoresis, demonstrating a single band of linearized plasmid DNA. The linearized plasmid DNA was subsequently heat denatured as described above: 8 minutes at 100 °C followed by 10 minutes at 0 °C. The denatured linear plasmid DNA solution was kept on ice until use (less then 30 minutes).
• Example 3 Luciferase expression in mouse liver following delivery of heat denatured plasmid DNA via the tail vein
• Plasmid DNA was delivered via the tail to 25 gram ICR mice (obtained from Harlan Sprague Dawley). Fifty ⁇ g of denatured pCI-Luc + (125 ⁇ l) was mixed with 2,375 ⁇ l Ringer solution and injected into the tail vein in approximately 7 seconds (8 mice). As a control, 50 ⁇ g of supercoiled pCI-Luc + was injected similarly (8 mice). Half the mice in each group were sacrificed 24 hours after injection, the other half after seven days. The liver was extracted, homogenized in 1.5 ml lysis buffer (Minis' luciferase assay kit), and analyzed for luciferase expression (see Table).
• Example 4 Luciferase expression in mouse liver following delivery of heat denatured linear plasmid DNA via the tail vein
• Plasmid DNA was delivered via the tail to 25 gram ICR mice (obtained from Harlan Sprague Dawley). Fifty ⁇ g of denatured and linearized pCI-Luc + (83J ⁇ l) was mixed with 2,417 ⁇ l Ringer solution and injected into the tail vein in approximately 7 seconds (8 mice). As a control, 50 ⁇ g of supercoiled pCI-Luc + was injected similarly (8 mice). Half the mice in each group were sacrificed 24 hours after injection, the other half after seven days. The liver was extracted, homogenized in 1 ml lysis buffer (Minis' luciferase assay kit), and analyzed for luciferase expression (see Table).
• Plasmid DNA was delivered via direct intramuscular injection to 25 gram ICR mice (obtained from Harlan Sprague Dawley). Twenty ⁇ g of denatured pCI-Luc + (100 ⁇ l in saline) was injected into the quadriceps in approximately 2 seconds (4 mice, both limbs). As a control, 20 ⁇ g of supercoiled pCI-Luc + was injected similarly (4 mice, both limbs). Half the mice in each group were sacrificed seven days after injection, the other half after 21 days. The liver was extracted, homogenized in 1 ml lysis buffer (Minis' luciferase assay kit), and analyzed for luciferase expression (see Table).
• Example 6 Luciferase expression in mouse skeletal muscle following delivery of heat denatured linear plasmid DNA Plasmid DNA was delivered via direct intramuscular injection to 25 gram ICR mice (obtained from Harlan Sprague Dawley). Twenty ⁇ g of denatured and linearized pCI-Luc + (33 J ⁇ l) was mixed with 66.7 ⁇ l saline solution and injected into the quadriceps in approximately 2 seconds (4 mice, both limbs). As a control, 20 ⁇ g of supercoiled pCI-Luc + was injected similarly (4 mice, both limbs). Half the mice in each group were sacrificed seven days after injection, the other half after 21 days. The liver was extracted, homogenized in 1 ml lysis buffer (Minis' luciferase assay kit), and analyzed for luciferase expression (see Table).
• M13 bacteriophage was grown according to standard methods as described in Maniatis et al. Briefly, 0.5 to 1 ml M13 phage supernatant (phage titer >10 12 ) was added to 2.5 ml of a JM109 bacteria culture in a 15 ml tube. After a 5 minute incubation, the mixture is added to 250 ml LB medium in a large culture flask (pre-warmed to 37 °C). The culture is incubated at 37 °C for 5 hours in a shaking incubator. Centrifuge in Sorvall SLA-3000 rotor, 8.5 K, 15 min., 4°C. Carefully pour the supernatant into a new bottle.
• the bacterial cell pellet may be stored at -20°C for preps of M13 RF DNA.
• the supernatant contains the Ml 3 phage particles. Filter the supernatant through a 0.8 ⁇ m filter. This will remove any remaining bacteria but the phage will filter through. At this point, the filtered supematant may be stored 4°C O/N.
• the single stranded DNA is isolated using a Qiagen Maxi Kit. Add 750 ⁇ l DNAse I (Roche) (2mg/ml) to each 250 ml supernatant. Incubate 37°C 30 min. This will remove any chromosomal or RF DNA that may be contaminating the supematant from dead bacteria. The single-stranded DNA is safely protected in the phage particle. Add 100 ml cold buffer Ml to each 250 ml supernatant. Mix well. Incubate on ice 60 min. This will precipitate the phage particles. Pellet the phage by centrifugation in the Sorvall SLA-3000 rotor, 8.5 K, 15 min. 4°C.
• step 13 After the above buffer is completely emptied, apply the lysed phage solution (the column can't hold more than 30 ml at once) to each column. After the liquid from step 13 is completely emptied, wash the column with 30 ml Buffer QC. When the QC buffer is completely emptied from the column, transfer the column to balance on top of a 30 ml Corex tube. These should have been baked to be endotoxin-free. Elute the DNA with 15 ml Buffer QF. This may be stored 4°C O/N. Precipitate the DNA from step 16 by adding 10.5 ml RT isopropanol. Parafilm the top of the tube and mix by inversion.
• Example 8 Luciferase expression in mouse liver following delivery of single stranded nucleic acid isolated from Ml 3 bacteriophage and delivered via the tail vein using TransIT In Vivo
• pCI-Luc + is a double stranded, circular, plasmid DNA expressing the firefly luciferase gene under control of the human cytomegalovirus promoter.
• the expression cassette of this vector was cloned into M13 bacteriophage mpl8 and mpl9 DNA.
• the resulting double stranded M13 vectors were transformed into JM109 bacteria, resulting in virus production. Both double stranded replicative DNA (RF) and single stranded DNA was isolated.
• RF double stranded replicative DNA
• the two M13 viruses either yield the template strand or the coding strand in a circular form.
• DNA was formulated with Minis TransIT In vivo gene delivery polymer prior to injection into the tail vein of ICR mice (3 mice per group).
• Example 9 Luciferase expression in mouse liver following delivery of single stranded nucleic acid isolated from M13 bacteriophage and delivered via the tail vein (naked DNA)
• pCI-Luc + is a double stranded, circular, plasmid DNA expressing the firefly luciferase gene under control of the human cytomegalovirus promoter.
• the expression cassette of this vector was cloned into M13 bacteriophage mpl8 and mpl9 DNA.
• the resulting double stranded M13 vectors were transformed into JM109 bacteria, resulting in virus production. Both double stranded replicative DNA (RF) and single stranded DNA was isolated.
• RF double stranded replicative DNA
• the two M13 viruses either yield the template strand or the coding strand in a circular form.
• DNA was diluted in Ringer's solution and injected into the tail vein of 25 gram ICR mice (2.5 ml solution in -5-7 seconds; 3 mice per group).
• Example 10 Luciferase expression in mouse skeletal muscle following intramuscular delivery of smgle stranded nucleic acid isolated from Ml 3 bacte ⁇ ophage (naked DNA)
• Double and single stranded DNA was prepared as desc ⁇ bed in the examples above DNA stocks were diluted in salme and injected into the quadnceps of ICR mice (200 ⁇ l solution, 5 mice per group, both legs) Equimolar amounts of each nucleic acid were injected (10 ⁇ g pCI-Luc + , 9 ⁇ g ssDNA) After three weeks, the mice were sacnficed, the quadnceps extracted and assayed for luciferase expression The results are presented in the Table above For the last group, the two ssDNA's were mixed together pnor to the injection These results indicate that single stranded DNA can be efficiently expressed in skeletal muscle in vivo
• Example 11 Size of nucleic acid particles formed with smgle stranded DNA isolated from Ml 3 bactenophage and complexed with poly(L-lys ⁇ ne)
• a drop of sample was applied on the surface of freshly cleaved mica discs and allowed to incubate for 10 min at room temperature. Excess liquid was removed by a stream of nitrogen gas. The sample was viewed using a Digital Instruments Nanoscope 3100 AFM instrument using Air Tapping Mode. The results showed that more than half the single stranded DNA complexes were smaller than 50 nm. A large percent of the larger complexes consisted of multiple single stranded DNA molecules, thus explaining the larger effective diameter measured by the ZetaPlus particle sizer.
• DNA-poly-L-lysine-peptide complexes were then added (200 ⁇ l) to a well containing 3T3 mouse embryonic fibroblast cells in Opti-MEM. After 4 hours, the medium was replaced with 90% Dulbecco's modified Eagle medium and 10% fetal bovine serum the cells were then allowed to incubate for 48 hours. The cells were then harvested and assayed for luciferase expression.
• the 2,2 -dimethylmaleic modification of the peptide melittin allows the peptide to complex with the cationic protein Histone HI and then cleave to release and reactivate in the lowered pH encountered by the complex in the cellular endosomal compartment. This caused a significant increase in luciferase expression over either unmodified melittin peptide or melittin peptide modified with succinic anhydride which allows complexing with Histone HI but will not cleave in lowered pH.
• Liposomes for each complex were formed by 5 minutes of bath sonication then purified in batch by addition of 250ul of DEAE sephadex A-25. DNA (25 ⁇ g, pCILuc)was then added to the supematant containing the purified liposomes of each complex. Transfections were carried out in 35 mm wells. 3T3 cells were grown in DMEM with 10% fetal bovine serum and were at approximately 60% confluency at the time of transfection. 50 ⁇ l of complex was added to each well. After an incubation of 48 hours, the cells were harvested and assayed for luciferase expression. Results:
• the 2 J -dimethylmaleic modification of DM-PAA-KL3 allows the polymer to complex with the cationic polymer L-cystine - l,4-bis(3-aminopropyl)piperazine copolymer and then cleavage of the 2,3 -dimethylmaleamic group to release and reactivate in the disulfide reducing environment encountered by the complex in the cell.
• Example G Transfection of 3T3 cells with complexes of pCI Luc pDNA/cationic polymers caged with compounds containing acid labile moieties. Several complexes were formed:
• the media containing the complexes was aspirated from the cells, and replaced with complete growth medium, DMEM with 10% fetal bovine serum. After an additional incubation of 48 hours, the cells were harvested and assayed for luciferase expression.
• the transfection results indicate that caging cationic pDNA complexes (PLL or Histone HI) with DTBP reduce the amount of expressed luciferase. Caging of the cationic pDNA complexes with MC211 results in an increased amount of expressed luciferase relative to the DTBP examples.
• Example Al Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ 1,4-Bis(3- aminopropyl)piperazine Glutaric Dialdehyde Copolymer (MC 140). Three complexes were prepared as follows:
• Complex I pDNA (pCI Luc, 50 ⁇ g) in 12.5 ml Ringers.
• Complex II pDNA (pCI Luc, 50 ⁇ g) was mixed with l,4-bis(3-aminopropyl)piperazine glutaric dialdehyde copolymer (50 ⁇ g) in 1.25 ml HEPES 25 mM, pH 8. This solution was then added to 11.25 ml Ringers.
• mice 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• Results indicate an increased level of pCI Luc DNA expression in pDNA / l,4-bis(3- aminopropy piperazine glutaric dialdehyde copolymer complexes over pCI Luc DNA/poly- L-lysine complexes. These results also indicate that the pDNA is being released from the pDNA / l,4-Bis(3-aminopropyl)piperazine-glutaric dialdehyde copolymer complexes, and is accessible for transcription.
• Example A2 Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ Imine Containing Copolymer' s.
• mice 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• Example A3 Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ Imine Containing Copolymer's.
• mice 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• mice 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• Results indicate an increased level of pCI Luc DNA expression in Complex III relative to Complex II indicating that when the acid labile homobifimctional amine reactive system (MC211) is used, more pDNA is accessible for transcription relative to the non-labile homobifimctional amine reactive system (DTBP). These results also indicate that the pDNA is being released from the pDNA / ketal containing copolymer complexes, and is accessible for transcription.
• MC211 acid labile homobifimctional amine reactive system
• DTBP non-labile homobifimctional amine reactive system
• Example A5 Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ Ketal Containing Copolymers.
• mice 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• results indicate that the acid labile homobifimctional amine reactive system (MC225) makes pDNA accessible for transcription. These results also indicate that the pDNA is being released from the pDNA / ketal containing copolymer complexes, and is accessible for transcription.
• Example A6 Mouse Tail Vein Injections of Complexes of pDNA (pCI Luc)/ Ketal Containing Copolymers.
• mice 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• Results indicate that the pDNA is being released from the pDNA / ketal containing copolymer complexes, and is accessible for transcription.
• Complex XI pDNA (pCI Luc, 50 ⁇ g)/MC369
• Complex XII pDNA (pCI Luc, 50 ⁇ g)/MC370 2.5 ml 1 of the complexes were injected into the tail vein of 25 gram ICR mice. After 24 hours, the mice were sacrificed, the liver extracted and homogenized, and luciferase activity determined in the homogenates.
• results indicate that the pDNA is being released from the pDNA / Silicon containing polycation complexes, and is accessible for transcription. Additionally, the results indicate that complex VIII (does not contain the silicon) is much less effective in the assay than is complex V. Additionally, the results indicate that upon the addition of a third layer, a polyanion (complex VI), the complex containing the silicon polymer allows for pDNA transcription.
• the complexes prepared from pCI Luc DNA and polymers containing acid labile moities are effective in direct muscle injections.
• the luciferase expression indicates that the pDNA is being released from the complex and is accessible for transcription.

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