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WO2007018744A2 - Biosynthese sans cellule d'acide nucleique - Google Patents

Biosynthese sans cellule d'acide nucleique Download PDF

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Publication number
WO2007018744A2
WO2007018744A2 PCT/US2006/023439 US2006023439W WO2007018744A2 WO 2007018744 A2 WO2007018744 A2 WO 2007018744A2 US 2006023439 W US2006023439 W US 2006023439W WO 2007018744 A2 WO2007018744 A2 WO 2007018744A2
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Prior art keywords
therapeutic
dna
composition according
research
sequence
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PCT/US2006/023439
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English (en)
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WO2007018744A3 (fr
Inventor
Yin Chen
Frederic Kendirgi
Frank Vazquez
Malcolm Skolnick
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Cytogenix, Inc.
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Priority claimed from PCT/US2005/045028 external-priority patent/WO2006063355A2/fr
Application filed by Cytogenix, Inc. filed Critical Cytogenix, Inc.
Publication of WO2007018744A2 publication Critical patent/WO2007018744A2/fr
Publication of WO2007018744A3 publication Critical patent/WO2007018744A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features

Definitions

  • the invention relates to a process for making research and high quality nucleic acids in a cell-free system, products made using this process, the use of these products in research and therapeutic applications, and an apparatus designed for making large batches of these products.
  • Typical plasmid purification procedures from bacteria and other cell sources include methods that use organic, mutagenic and toxic compounds including phenol, ethidium bromide and cesium chloride, and enzymes such as lysozyme, proteinase K and RNase A. All of these can constitute potential health hazards if injected as contaminants in a DNA-based therapeutic. Such procedures also carry a potential risk of incorporating unintended contaminating transposons and other foreign episomal DNA into the plasmid. There is also the potential for contamination by residual host cell nucleic acids, other cellular proteins and endotoxins. Such impurities can minimize the efficiency of DNA uptake and can lead to dose-related toxicity. To remove these impurities, accepted purification methods often use multiple chromatographic steps, including anion exchange, affinity, and size-exclusion. These purification procedures are costly.
  • Phi29 DNA polymerase is a highly processive, strand displacement polymerase.
  • Phi29 DNA polymerase can reliably reproduce DNA strands greater than 70 kilobases long (the full length of Phi29 genome) and can be used in DNA sequencing, DNA amplification, and for synthesis of DNA greater than 10 kilobases long.
  • modified forms of the enzyme including an exonuclease-deficient form which is used to reduce the enzyme's inherent ability to remove the labeled bases useful in sequencing reactions.
  • modified polymerases produce copies with lower overall fidelity and are primarily useful for only sequencing-type reactions where lower fidelity is preferred.
  • Random primers as well as sequence specific primers have been used, offering flexibility for use of the process in multiple applications.
  • sequence specific primers designed to nest on newly synthesized DNA can be used to initiate secondary synthesis of already amplified product. Amplification can occur exponentially, providing an isothermal alternative to PCR for the detection of multiple targets simultaneously. Long terminal repeats have been used to facilitate these nested amplifications and to quickly identify the presence of a particular sequence in a sample.
  • the use of multiple primers enables the process to be used to identify specific target sequences, as well as for the synthesis and detection of address tags and oligonucleotides.
  • DNA polymerases have also been used to improve the performance of some of these common research applications, including sequencing, cloning, mapping, genotyping, probe generation and diagnostic screening.
  • DNA polymerases lacking the normal corrective 3',5'-exonuclease activity have been designed and used to improve the rate of incorporation of labeled nucleotides for improving the efficiency of incorporation of labeled nucleotides during sequencing reactions.
  • Phi29 DNA polymerase can produce large amounts of high fidelity nucleic acid in a relatively short period of time without thermal cycling and with an extremely low error rate of about 4 x 10-6.
  • Pol III bacterial DNA polymerases III
  • I bacterial DNA polymerases III
  • Pol I can also be used in RCA.
  • Different enzymes confer different advantages to the system.
  • Pol III reportedly has a clamp-like activity that provides an increased rate of DNA synthesis (about 700-800 nucleotides per second) which may be optimized by adding helicases or stabilizing proteins.
  • Pol I can be used to amplify templates smaller than 100 bp because it uses predominantly single stranded templates; small circular templates can be readily formed without steric hindrance which is often associated with extremely short double-stranded templates.
  • a modified Pol I comprising a sequence derived from T7 DNA polymerase has been shown to increase its efficiency up to 500-fold while reducing its ability to discriminate between deoxy- and dideoxynucleotides and conferring an advantage for sequencing applications.
  • Phi29 DNA polymerase can use either ss or ds templates, but Pol I can only use a ss template. Phi29 polymerase also recognizes both RNA and DNA templates and therefore has more flexibility for use in RCA and other similar amplification reactions.
  • RCA has been adapted to produce RNA or DNA oligonucleotides (28-74 nucleotides long) using a small single- stranded circular DNA template.
  • MIDGE vectors minimalistic, immunogenically defined gene expression vectors
  • Vaccine, 22: 1709-1716, 2004 which minimally contain sequences needed for eukaryotic gene expression and induction of an immune response.
  • end modification of the MIDGE expression cassettes to have hairpin loops increases their longevity and expression efficiency.
  • the MIDGE vectors must first be cut out of bacterially grown plasmid and then modified; this is a complex, time consuming, and labor intensive process.
  • the appropriate host encodes for an inducible recombinase needed for the recombination event which can be induced after sufficient plasmid is produced inside the growing cell culture.
  • the intracellular recombination generates two separate minicircles: one with the expression cassette and the other with bacterial genetic materials. This system again uses bacterially grown plasmid DNA which carries with it the problem of purifying away bacteria associated contaminants.
  • GMP Good Manufacturing Practice
  • Plasmid DNA produced in large-scale facilities should be free of contaminating genomic DNA ( ⁇ 10 ng/dose), host proteins ( ⁇ 10 ng/dose), RNA (non- detectable on 0.8% agarpse gel), and endotoxins ( ⁇ 1 Unit/kg body weight, or ⁇ 0.1 EU/ug plasmid).
  • the plasmid should be sterile and, in present practice, preferably in supercoiled form that can be more efficiently expressed.
  • purification reagents such as ethidium bromide, chloroform, phenol, lysozyme, proteinase K, RNase A, and any potential contaminants that may leach from the purification columns such as quaternary amines from anion exchangers.
  • Any DNA therapeutic requires a high level of purity with such minimal, almost non-detectable levels of impurities.
  • DNA products derived from bacterially grown plasmid are costly to produce because of the need to initially grow huge bacterial cultures in fermentation tanks and then to purify the product to eliminate contaminating bacterial cell products including proteins, DNA, RNA, toxins and endotoxins to meet the high standards of purity.
  • Accepted purification methods primarily use multiple chromatographic procedures including a combination ofanion exchange, affinity, and size-exclusion chromatography purification steps. It is significant that the purification methods needed for therapeutic applications requires specialized equipment, expensive resins, extensive housing facilities and time.
  • high quality DNA can be made in a cell-free system, virtually free of bacteria contaminants, and optimally free of flanking bacterial gene coding sequences which can minimize or silence gene expression when used for expression inside a target cell.
  • the cell-free system herein is a rapid method that produces a cleaner end product suitable for therapeutic applications, with less effort and expense, which can be designed to be more efficiently expressed in a target cell.
  • the end product can be easily adapted for use as a DNA therapeutic due to affordable manufacture and lower levels of bacterial cell components and toxins.
  • the invention herein includes (1) a method optimized for the cell-free production of high quality nucleic acid, which may comprise an expression cassette, clean enough for use in gene therapy, DNA vaccines or other therapeutic applications and which may be free of unnecessary plasmid replication sequences; (2) a cell-free method for the production of DNA for any research or therapeutic purpose that is essentially free of inherent bacterial cell contaminants and/or bacterial toxins; (3) DNA vaccines comprising DNA made in a cell-free system which by virtue of the cell- free system of production, carry virtually "no" bacterial cell contaminants and "no” bacterial toxins; and (4) and apparatus for the large scale manufacture of DNA using the disclosed cell- free system.
  • One aspect of the invention relates to optimizing a cell free DNA amplification system for large-scale (e.g., > 1 mg) nucleic acid production, using streamlined expression cassette templates having a sequence of interest, sequence specific or random primers, high-fidelity polymerases, and a minimalistic buffer system.
  • This system can be used to produce large amounts of nucleic acids, in small volumes, in short periods of time, with the need for only minimal and inexpensive purification procedures.
  • the system can produce high-quality therapeutic grade nucleic acids for any basic analytical or research purpose, but also for therapeutic use.
  • the current invention combines several techniques for the purpose of affordably producing large amounts of high-quality nucleic acid for therapeutic, diagnostic and research applications.
  • the method of the invention can produce 250-300 times more nucleic acids than what is produced in a comparable volume of bacteria culture.
  • a standard plasmid can be replicated quickly and affordably in a cell-free system, useful for both research and therapeutic applications.
  • the advantage of producing a plasmid using this cell-free system is that the end product DNA is essentially free of any bacterial cell components and bacterial toxin.
  • typical bacterially grown plasmid can be purified to acceptable levels for FDA applications as a therapeutic, these purification procedures are costly as well as time consuming, and the final product still has minimal levels of all sorts of bacterial contaminants that are not present in the end product of the current cell-free system. Many unknown contaminants may remain in even the highly purified DNA preparations that use traditional bacterially grown plasmid.
  • DNA produced in a cell-free system is in a well defined environment and minimizes this risk significantly.
  • a template may simply be a circular expression cassette containing a sequence of interest flanked by genetic elements needed for expression and processing of the expressed product in a host (promoter, polyA, etc.).
  • Streamlined templates having no extra genetic sequences offer multiple benefits: they eliminate any extraneous sequence that may silence the expression of the sequence of interest; the smaller constructs are more compact and can be more efficiently taken up by the target cell, leading to higher transfection efficiency; and they are more cost effective due to production of a larger quantity of an expression cassette with less material, a statistical increase in fidelity of the final product and no need for extensive purification.
  • Sequence-specific primers are more efficient and economical in large scale amplifications but require pre-planned sequence analysis and primer synthesis.
  • Primer sizes may range from four to greater than twenty nucleotides, and they may comprise modified bases and/or backbones for increased affinity, stability and prolonged storage.
  • a specific primer with phosphorothioate end-modification may be used to produce a large amount (about 1.5 mg in 1 ml) of nucleic acid.
  • the amplification step can use any specific polymerase providing buffer and temperature conditions are adjusted to accommodate the specific needs of that polymerase.
  • Some embodiments use a thermocycling polymerase requiring multiple denaturation and annealing steps (ex, when using a high temperature taq-like polymerase).
  • Others use processive, strand-displacing polymerases such as Phi29-like polymerases, to efficiently amplify templates without thermal cycling.
  • Preferred embodiments use Plii29 or Phi29-like polymerases, but other polymerases such as Pol I, Pol III, and T7 DNA polymerase, and their derivatives can also be used.
  • the invention can also use other modified or chimeric polymerases designed to improve efficiency and/or fidelity.
  • the nucleic acid product may be further processed in a manner to facilitate its intended use.
  • Research purposes including detection, identification or sequencing, would typically only require shorter linear units (delivery unit) of the concatamer which may be attained by either restriction enzyme digestion or by physical or chemical methods such as shearing or induced cleavage at specific, photolabile nucleotide.
  • Cellular transfections may be accomplished with a variety of forms, but higher efficiencies of uptake are typically attained with circular or supercoiled nucleic acid.
  • linear forms can be used to produce a greater immune response than the comparable plasmid when used to effectuate immunity in an animal system.
  • a preferred embodiment includes the use of a linear product made in the cell-free system as the active component of a DNA based therapeutic. Another embodiment incoiporates a subsequent ligation step using DNA ligase to make circular nucleic acids (CNAs) from the linear forms. Another embodiment uses a recombinase or a similar enzyme to circularize the delivery unit into CNAs. Another includes the use of a DNA gyrase to supercoil the circular product to produce supercoiled CNA (sCNA).
  • CNAs circular nucleic acids
  • the product is intended for expression in eukaryotic cells, uptake by the cell is critical, whether in research (cell and culture) applications or in therapeutic applications.
  • Transfections can be accomplished using circular, supercoiled CNA or specially designed linear forms which may be stabilized with modifications in the internal base and/or the ends of the linear unit.
  • Such modifications include: blunting the ends by filling in with a Klenow fragment-like enzyme; phosphorothioating the ends of linear strands with appropriately modified bases; incorporating other modified bases either during the amplification process or following digestion of the concatamer, which stabilize or minimize degradation of the linear in vivo; and designing the expression cassette to comprise stabilizing sequences which facilitate rapid uptake and/or prolong longevity of expression of the cassette once inside the cell (Kay, M.A. et al., Molec. Ther. 3(3): 403-410, Mar. 2001).
  • the linear can be stabilized during the amplification reaction by the random incorporation of chemically or structurally modified primers during replication.
  • Such modification may incorporate components that are known to stimulate the immune response in a manner similar to the actions of an adjuvant.
  • the degree of modification or processing following the cell free amplification step is dependent upon the intended use for the product.
  • the final processed product is then purified in order to eliminate reagents, contaminants, and/or any alternative forms of the product.
  • Different forms of the product may include linear fragments, open circles, covalently closed circles comprising monomers, dimers, trimers, etc., as well as supercoiled circles.
  • the intended form is dependent upon the specific application and may alternate between any of the aforementioned forms.
  • the product can be subjected to chromatography, ultra filtration, dialysis, nucleic acid precipitation, or any other appropriate method known in the field. Those embodiments incorporating gel filtration and/or dialysis can provide high quality products for therapeutic applications. All forms of the DNA product made according to the method of the current invention are referred to herein as synDNA.
  • FIG.l shows multiple mechanisms for generating useful templates.
  • templates may also be produced by plasmid modification (restriction enzyme digestion with subsequent ligation, or intraplasmid recombination), PCR amplification, chemical synthesis, or cDNA synthesis.
  • FIG.2 shows a cell-free amplification process using a polymerase to synthesize a concatamer from the circular template.
  • the concatamer can be later processed into smaller fragments, which may comprise at least one intact expression cassette having a sequence of interest.
  • the end product may be used as short linear units (DUs), circularized nucleic acids (CNAs), or supercoiled circular nucleic acids (sCNAs).
  • DUs short linear units
  • CNAs circularized nucleic acids
  • sCNAs supercoiled circular nucleic acids
  • FIG.3 shows an embodiment that separately amplifies the forward (A) and reverse (B) strands of a double-stranded template in separate reaction vessels.
  • Each strand is separately amplified using a strand-specific primer and circularized into single-stranded circles.
  • a second oligonucleotide comprising a sequence for a restriction site (ORl or OR2) is then annealed to a predesigned site in the single-stranded concatamer, whereby short segments of double-stranded templates are generated to enable digestion by a restriction enzyme. Following digestion but prior to denaturation, the double-stranded ends are circularized using a DNA ligase.
  • the oligonucleotide is denatured to from single-stranded circles, which are then combined with the complementary single-stranded circles to form double-stranded circles that comprise only monomers. This method minimizes the formation of dimers, trimers and other multimer byproducts.
  • FIG.4 depicts the scale-up of the cell-free amplification process.
  • the process involves sequential addition of template, primer, buffer components and enzymes at the designated times and shifting to the designated temperatures. This provides an efficient method for producing large amounts of product in a short period of time. Diluting the reaction volume prior to ligation favors the formation of monomeric circular product. Dilution, ligation and gyrase reactions are all optional.
  • FIG.5 depicts the design for an automated amplification apparatus.
  • A represents a model where large numbers of individual reactions comprising volumes of less than 1 ml can be used to amplify numerous individual templates simultaneously as for diagnostic purposes;
  • B shows the use of a single vessel enabling the synthesis of large quantities of a single DNA product.
  • FIG.6 schematically summarizes various mixing strategies for viscous reaction mixtures: (A) propeller-like mixing vessel; (B) perforated disk mixing vessel; (C) recycling mixing vessel using a peristaltic pump; (1) adjustable automated control and port for calibrated addition of reagents held in (2).
  • the adjustable control (1) enables controlled mixing of reagent with a small stream of reaction mixture and supports the overall mixing of the reaction mixture by depositing the reagent modified reaction mixture back into the chamber at a position opposite the outlet port. Continued pumping without reagent facilitates thorough mixing.
  • FIG.7 depicts a process for intra-molecular ligation. Following amplification and digestion of DNA in vessel (B), the reaction mixture is added slowly to a second vessel (A) containing a ligation cocktail. Slow addition of the DNA into vessel (A) provides sufficient dilution of the DNA to facilitate monomeric circular nucleic acid (CNA) formation.
  • CNA monomeric circular nucleic acid
  • FIG.8 shows results of IgG antibody titers against gpl ⁇ O produced in Balb/c mice after immunization with a plasmid, a short expression cassette (synDNA) produced in accordance with the invention, and a control solution. These results clearly show that the synthetic DNA is effective in inducing immune responses in mice.
  • FIG.9 shows results from immunization of rabbits using a plasmid or a synDNA (expression cassette), containing a sequence for the Hepatitis B vims small surface antigen (HBs(S)).
  • FIG.10 shows immunization results following the injection of BALBc mice against influenza HlNl virus 8 weeks post-immunization; 2 weeks post last boost. Animals injected 3x, at weeks 0, 2 and 6.
  • FIG.11 shows immunization results following the injection of BALBc mice with a control plasmid or synDNA containing a sequence for a smallpox gene, B5R.
  • FIG.12 shows the expression of luciferase in mouse muscle at 24, 72 and 144 hours post-injection. Each mouse received a single injection of 50 ⁇ g of DNA (linear cell-free synDNA or intact plasmid DNA) in one leg. There was no significant difference (higher or lower) in expression of the luciferase enzyme over time between the linear Luc-SynDNA of the current invention and the standard circular luciferase plasmid DNA.
  • FIG.13 shows stability of the linear SynDNA made using two different modified primers (methyl phosphonate (MP) and phosphorothioate (P)).
  • MP methyl phosphonate
  • P phosphorothioate
  • the invention includes methods for making cell-free nucleic acid, products made by this method which have fewer contaminants than traditional bacterially grown products, and an apparatus for producing large amounts of high-quality nucleic acids. Methods of the invention use a cell-free system to produce therapeutically useful and minimally contaminated nucleic acid products (Fig. 2).
  • composition' typically refers to a carrier (buffer or delivery vehicle) mixed with an active effector molecule which, in the context of this invention, is a high-quality nucleic acid molecule.
  • Delivery includes methods to administer an active compound or effector molecule to the target cell or organ, and may include injection (intramuscular, intravenous, intradermal), oral compositions, aerosol sprays, eyedrops, suppositories, topical ointments, skin patches and soaks, as well as surgically implanted devices.
  • Delivery vehicle in the context of this invention means any earner suitable for transporting a nucleic acid effector molecule or sequence of interest to a site within the host (cell, animal, human, plant) which may or may not improve the uptake of the effector molecule by the target cell.
  • Some delivery vehicles may target the effector molecule to a particular cell or organ, or may diminish uptake by the cell in order to improve extracellular effector action.
  • Typical delivery vehicles include viral packaging systems, topical ointments, aerosols, liposomes, microsomes, polymers, nanotubules, cell penetrating or receptor adhering peptides, and various oral carriers, but may be as simple as saline buffer or water.
  • Expression cassettes mean any combination of nucleic acid sequences that comprise the proper promoter, enhancer and/or termination sequences needed for expression of a particular sequence of interest.
  • the invention is adaptable for the use of multiple expression cassettes where each cassette may contain a different sequence of interest.
  • High quality in the context of this invention primarily refers to nucleic acid products that have only defined components as contaminants and only trace levels of bacterial cell components, endotoxin or other bacterial toxins (contributed only by the addition of purified enzymes used in the cell-free process, which are themselves purified from the end synDNA product). Without bacterial cell components in the end product, the DNA so produced is easily and affordably purified for therapeutic and critical research applications where contaminating bacterial components can interfere with the efficiency and efficacy of the DNA application.
  • Nucleic acid may be DNA or RNA, or an analog (e.g., phosphorothioate analog). Nucleic acids or oligonucleotides may also include modified bases (ex: phosphorothioates, morpholinos, methyl phosphonates, or other mimetic molecules), backbones, and/or ends. Synthetic backbones may include phosphorothioate (Pt), peptide nucleic acid (PNA), locked nucleic acid (LNA), xylose nucleic acid (XNA), or analogs thereof that confer stability and/or other advantages to the nucleic acids.
  • Pt phosphorothioate
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • XNA xylose nucleic acid
  • Plasmid replication sequences for this invention include origins of replication, antibiotic resistance genes, other marker or selection genes, and other bacterial specific sequences required for plasmid replication inside a prokaryotic cell.
  • Protective response means a beneficial response that a host elicits to counter a disease stale caused by either a genetic aberration, an environmental inducer causing an aberrant expression pattern, or by a pathogen or toxic agent.
  • Reporter construct refers to a nucleic acid sequence useful for tagging and labeling which may compise antibiotic resistance genes and other common reporter or marker sequences including, but not limited to at least partial sequences of the B-galactosidase (LacZ), luciferase (Luc), secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP), and chloramphenicol acetyltransferase (CAT) genes.
  • LacZ B-galactosidase
  • Luc luciferase
  • SEAP secreted alkaline phosphatase
  • GFP green fluorescent protein
  • CAT chloramphenicol acetyltransferase
  • Sequence of interest in the context of this invention, means any nucleic acid sequence that is sufficient to elicit a cellular response in the targeted environment.
  • the sequence can be as small as a typical oligonucleotide which may be as small as seven nucleotides in length, or as large as a polycistronic message comprising several genes, or genomic segment containing both exons and introns for the production of an unprocessed precursor protein.
  • An SOI may be a nucleic acid, oligo or oligonucleotide with or without chemical modification which may contain a potential therapeutic SOI or a reporter or marker sequence for research purposes.
  • compositions as used in the context of this invention includes as an active component, at least one nucleic acid, oligo or oligonucleotide molecule which may be chemically modified.
  • the active component will by virtue of the sequence used, be capable of acting prophylactically by eliciting a protective response (including cellular and/or an immune responses), remedial or growth inhibitory response inside an organism and may be applied in the appropriate composition for useful gene therapy, vaccinations, pathogen inhibition and other disease states when the composition is administered to a living organism.
  • Research compositions for this invention refer to any composition comprising at least one sequence of interest useful for research and/or pre-clinical purposes.
  • a research composition can be used for any traditional research application which uses plasmids, viral and other similar molecular constructs, and can be effectively used as a tool to compare the efficacy of synDNA products produced according this invention to other traditional molecular transfer tools including plasmids and viruses.
  • the process uses a polymerase to synthesize a concatamer from a circular template.
  • the concatamer may be processed into smaller fragments, which may comprise at least one intact expression cassette.
  • the synthesized product may be used as short linear units or these may be further processed to produce circularized nucleic acids (CNAs) or supercoiled circular nucleic acids (sCNAs).
  • CNAs circularized nucleic acids
  • sCNAs supercoiled circular nucleic acids
  • the method can be adapted to use either DNA or RNA templates.
  • the reactions starting with RNA templates would include a reverse transcriptase, such as the avian myeloblastosis virus reverse transcriptase, to make a cDNA template.
  • a reverse transcriptase such as the avian myeloblastosis virus reverse transcriptase
  • Any method known in the art may be used to prepare a circular template for use in a method of the invention, as shown in FIG. 2. Some of these methods will be described in detail later with reference to FIG. 1.
  • Single-stranded binding proteins can be used to stabilize the templates and improve efficiencies of the amplifications for some polymerases. Additional enzymes can also be included in the amplification reaction to repair mistakes. Protein mediated error correction enzymes, such as the mutation splicing protein (MutS), can also effectively improve a polymerase's overall fidelity and may be used during or after the amplification reaction (Carr, P., et al., Nuc Ac Res 32(20): el 62, 2004).
  • MotS mutation splicing protein
  • the DNA polymerases used in a method of the invention may be any known prokaryotic, fungal, viral, bacteriophage, plant or eukaryotic DNA polymerases and may include holoenzymes and any functional portions of the holoenzymes or any modified polymerase that can effectuate the synthesis of a nucleic acid molecule.
  • Useful DNA polymerases include: bacteriophage phi29 DNA polymerase, other phi29-like polymerase (such as phage M2 DNA polymerase, phage B 103 DNA polymerase, or phage GA-I DNA polymerase), phage phi-PRDl polymerase, VENT DNA polymerase, DEEP VENT DNA polymerase, KlenTaq DNA polymerase, DNA polymerase I, Kl enow fragment of DNA polymerase I, DNA polymerase III holoenzyme, T5 DNA polymerase, T4 DNA polymerase holoenzyme, T7 DNA polymerase, Bst polymerase, rBST DNA polymerase, N29 DNA polymerase, TopoTaq DNA polymerase, and ThermoPhiTM DNA polymerase.
  • Preferred embodiments of the invention use Phi29 polymerase, Phi29-like polymerase, or other high-fidelity polymerases (e.g., hybrid
  • Preferred embodiments of the invention use processive, strand-displacing polymerase to amplify DNA under conditions for high fidelity base incorporation.
  • a high fidelity "DNA polymerase” is one that under recommended conditions, has an error incorporation rate equal to or lower than those (1.5 x 10-5 - 5.7 x 10-5) associated with commonly used thennostable PCR polymerases, such as Vent DNA Polymerase, KlenTaq DNA Polymerase, or T7 DNA Polymerase. Additional enzymes may be included in the reaction to minimize misincorporation events including protein mediated error correction enzymes, such as MutS, which effectively improves polymerase fidelity either during or following the polymerase reaction (Carr, P. et al, Nuc Ac Res 32(20):el62, 2004).
  • RNA polymerase has an error incorporation rate equal to or lower than those of common RNA polymerases (Promega Technical Information).
  • RNA polymerases useful in this invention include T7 RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, and their modified or chimeric versions.
  • the circular template is replicated by a polymerase in the presence of deoxyribonucleoside triphosphates (dNTPs), ribonucleoside triphosphates (NTPs), or modified counterparts, forming a long concatamer comprising tandem repeats of the template.
  • the concatamers are subsequently cleaved, e.g., by restriction enzyme cleavage or physical shearing, into smaller fragments referred to as "short expression cassettes" (SECs).
  • SECs short expression cassettes
  • An SEC contains a sequence of interest and may optionally contain eukaryotic expression sequences (or cassettes).
  • Preferred embodiments use SECs that comprise at least one eukaryotic expression cassette.
  • an SEC of the invention consists solely of a sequence of interest flanked by the intended eukaryotic sequences, but no bacterial genetic material.
  • the "short expression cassette" may include: a eukaryotic promoter recognized by the targeted cell; the sequence of interest which may be an intact gene, a gene fragment, or a specific sequence of interest (SOI); and a transcription termination sequence.
  • the short expression cassette may be flanked by additional sequences to facilitate ligation (e.g., making CAN) or to stabilize a linear fragment.
  • the expression cassette together with the desired flanking sequences, comprises a "delivery unit" (DU), and does not contain unnecessary genetic material which is solely used for selection and replication of a plasmid produced in bacterial culture.
  • unnecessary plasmid replication gene sequences include but are not limited to origins of replication, marker genes, and plasmid selection genes.
  • Enzymatic or chemical methods can be used to improve the homogeneity of the final products by eliminating DU with mismatched nucleotides resulting from errors in polymerization.
  • enzymes used in mutation detection such as resolvases, T4 Endonuclease VII, or T7 Endonuclease I
  • other enzymes used to detect gene mutations or polymorphism and in high- throughput screening of point mutations such as TILLING
  • TILLING high- throughput screening of point mutations
  • FIG. 1 shows three commonly used methods for generating useful circular templates that include at least one sequence of interest (SEC or DU).
  • SEC sequence of interest
  • One method involves enzymatic modification of an existing plasmid, whereby the DU including the eukaryotic expression cassette is selectively excised from a plasmid by restriction endonuclease digestion.
  • the DU is free of the origin of replication or selectable marker genes, such as an antibiotic resistance mediator, which can silence expression of the SOI in vivo.
  • a preferred embodiment of the invention uses a template comprising an intact eukaryotic expression cassette with flanking sequences on either side of the cassette (Fig. 1) to enable circularization of the linear SEC into a CNA.
  • the template can be any single- or double- stranded nucleic acid (DNA or RNA), which is converted into a circular template and includes plasmid as well as minicircle DNA. Pre-ligation reactions may be carried out as in the case of using padlock probes (Baner, J., et al., Nuc Ac Res 26(22): 5073-78, 1998).
  • Double-stranded templates may need to be denatured initially to optimize the polymerase reaction depending upon the polymerase used. In such reactions, both the forward and reverse strands can be simultaneously amplified in the same reaction. Subsequent processing may then require the addition of a restriction endonuclease, a ligase, and/or a gyrase. The products may then be purified to yield DUs for therapeutic applications.
  • a second method for making the templates involves PCR amplification from a larger DNA template using specified oligonucleotides that flank the specific expression cassette to produce relatively short DUs for circularization.
  • a third method shown in FIG. 1 involves chemical synthesis of oligonucleotides (oligos) to make a single nucleic acid strand or complementary strands that are then circularized to produce a template containing a DU or expression cassette.
  • oligos oligonucleotides
  • the template may be freely suspended in solution or bound to a support, such as a chromosome or protein, or a solid support such as glass or polystyrene beads.
  • a support such as a chromosome or protein, or a solid support such as glass or polystyrene beads.
  • Each strand of a double-stranded template may be separately amplified using appropriately designed primers to produce single stranded concatamers of DUs.
  • the separately amplified concatamers are individually mixed with oligos containing specific restriction sites and cleaved with the restriction enzymes.
  • the temporarily double-stranded ends of these fragments are ligated to form circular single-stranded products (Dahl, F., et al., PNAS 101(13): 4548-53, 2004).
  • the advantage of this method is that the single-stranded circles of each reaction can then be combined to form a single class of double-stranded monomeric circles, thus avoiding the need to purify the monomers away from other multimeric fo ⁇ ns of the reaction.
  • the monomeric circles can then be supercoiled with a DNA-gyrase or a similar enzyme to improve the efficiency of uptake and expression of the expression cassette.
  • primers that specifically bind at designated sites to initiate concatamer synthesis.
  • the primers can comprise any of the different variations of "nucleic acid" to improve stability, and may be of various lengths where the length is determined by the annealing temperatures of the DNA polymerase used.
  • the primer sequences may comprise random or specific sequences, may be designed to have specific sequence alterations, or may include tags or detection sequences that are non-complementary to the template in order to facilitate manipulation or analysis of the amplified sequences. For example, in one embodiment, random hexamers are used to effectively amplify a DU, which upon processing and transfection into cells, would produce the desired effects.
  • Sequence-specific primers as short as a tetramer, may be used to effectively amplify a specific DU.
  • the polymerases, restriction endonucleases, ligases, and other enzymes as used in this invention constitute soluble forms of the enzymes.
  • solid phase amplification reactions or solid phase processing reactions including restriction digestion, ligation and supercoiling reactions may also be employed to streamline the amplification process.
  • fusion proteins comprising optimal regions of different enzymes (especially polymerases) which are designed to improve fidelity, efficiency, and processing or the final product may be used.
  • Recombinant forms of the enzymes containing one or more affinity tags such as 6XHis, S-Tag, Calmodulin-binding peptide, Protein A and others
  • affinity tags such as 6XHis, S-Tag, Calmodulin-binding peptide, Protein A and others
  • the advantage of using tagged enzymes is that they can be readily eliminated from the final product using affinity chromatography. Following purification, the recovered enzymes, immobilized on a solid matrix through the tag moiety, may be used in subsequent enzymatic reactions.
  • the concatamer is cleaved into short expression cassettes (SECs) comprising at least one DU, where a single SEC may comprise multiple copies of a DU and may be designed as such in order to optimize delivery and expression.
  • SECs short expression cassettes
  • the linear SECs may be directly administered as the linear fragments, circularized fragments (CNA), or supercoiled circularized fragments (sCNA) to facilitate uptake by the target cell.
  • CNA circularized fragments
  • sCNA supercoiled circularized fragments
  • Processing of the SEC can include any one or more of the following: additional cutting of the SEC with other physical or enzymatic methods; filling in or processing the ends of the SEC either by enzymatic cleavage, as with Klenow, or by chemical methods; internally ligating the two ends of the SEC to produce a circularized CNA; supercoiling the CNA with gyrase-type enzymes including topoisomerase type II; enzymatically or chemically treating any of the forms to have modified internal bases or modified ends; ligating two or more SECs together; or ligating an SEC to a specific ligand to produce a functional conjugate.
  • ligand as defined in the context of this invention includes: a nucleic acid, including DNA, RNA, PNA, LNA or modifications thereof; peptides, either to facilitate targeting and cellular uptake or to increase therapeutic efficacy; polypeptides that may be enzymatically active and/or physically functional; aptamers, nucleic acids that recognize, bind and modify a protein's function; bio-physical tags, including fluorescent, magnetic, and radiolabeled components; as well as polymers which facilitate either stabilization of the nucleic acid, or targeting of the product to the intended cell or tissue.
  • Therapeutic applications that can be successfully administered using DNA produced by the invention include several approaches to DNA therapy, including antibody production and gene silencing.
  • antibodies can be produced in vivo following successful administration of appropriate expression cassettes designed to prevent or treat a disease caused by a pathogen, such as influenza,HiV, hepatitis or smallpox viruses.
  • a pathogen such as influenza,HiV, hepatitis or smallpox viruses.
  • the sequence encoding the influenza haemagglutinin (HA) protein under the control of an eukaryotic promoter may be used to elicit a humoral and cellular immune response in animals targeted by influenza A virus.
  • the expression of a sequence encoding a truncated Human Immunodeficiency Virus (HTV) envelope protein can successfully induce an effective immunogenic response against HFV in mice.
  • HTV Human Immunodeficiency Virus
  • the amplified nucleic acid of this invention can be shown to induce immune response in several viral vaccine animal models, such as HIV, influenza, hepatitis and smallpox, indicating that linear form DNA can be delivered with or without carriers such as polyethyl-eneimine (PEI) both in vivo and in vitro and can be biologically active in an animal.
  • PKI polyethyl-eneimine
  • the amplified nucleic acid of this invention can also be shown to mediate targeted gene silencing in vivo.
  • Herpes Simplex Virus which causes painful blisters and sores on various parts of the body
  • Herpes Zoster which causes chicken pox (initial infection) and shingles (upon recurrence)
  • ICP4 and ICP47 are members of the same family of viruses which require the expression of both ICP4 and ICP47 proteins to effectuate a viral infection.
  • amplified SECs expressing antisense oligos specific for ICP4 or ICP47 may be used to modulate these protein expression in vivo and can minimize further proliferation of the virus.
  • ICP47 functions to inhibit the major histocompatibility complex (MHC) presentation pathway, which is critical for shielding the virus from host immunogenic attack.
  • MHC major histocompatibility complex
  • the gene product of ICP47 binds to a transporter protein involved in the presentation of antigens on the outside of an infected cell, thus blocking the major histocompatibility complex (MHC) class I antigen presentation pathway. Consequently, the HSV-infected cells are masked from immune recognition by cytotoxic T-lymphocytes. Thus, ICP47 plays an essential role in HSV-infection.
  • Transfecting the lung cancer cell line, A549, with an ICP47 SEC amplified according to this invention can effectively express antisense sequences and block production of the ICP47 protein as assayed by Western blot analysis.
  • ICP's infected cell proteins
  • Other gene silencing targets include the respiratory viruses such as the rhinoviruses, coronavirus, adenovirus, influenza and para-influenza viruses, which are frequently associated with both upper and lower respiratory tract infections including the common cold, pneumonia, asthma, and chronic obstructive pulmonary disease (COPD).
  • the human rhinovirus (HRV) has a single- stranded RNA genome that is approximately 7.2 kb in size with a single-open-reading frame that encodes for a capsid coat protein, an RNA polymerase and two viral proteases. Upon infection, the viral proteins effectively redirect the host machinery to manufacture thousands of viral particles which are eventually released when the cell lyses.
  • I intercellular adhesion molecule I
  • SEC intercellular adhesion molecule I
  • ICAM-I intercellular adhesion molecule I
  • Other useful strategies for combating respiratory diseases include in vivo expression of antisense-like molecules (antisense, aptamers, triplex forming molecules, and similar molecules) to block activities of essential proteins that mediate infection, such as viral proteases that are required to process viral particles.
  • SECs may include using the SECs to block mediators (e.g., bradykinin, prostaglandins, tachykinins, histamine, and various cytokines) of pathogen-induced tissue responses, or to block the cellular receptors that effectuate the physiological effect caused by these mediators.
  • mediators e.g., bradykinin, prostaglandins, tachykinins, histamine, and various cytokines
  • HPVs human papilloma viruses
  • genital HPVs can be passed from one person to another through sexual intercourse as well as through oral or anal sex.
  • Virus-infected cervical cells can transition from an initial benign wart, into premalignant cells and eventually develop into a carcinoma.
  • Cervical cancer is probably one of the best known examples of how infection with a virus can lead to cancer. In humans and animals, cell division is primarily regulated by Rb and p53.
  • the E6 and E7 proteins of HPV can attach directly to Rb and/or p53, inhibit the tumor suppressor effects of the proteins and cause the infected cells to reproduce without control (Didelot, C. et al., Intl J Oncology 23:81-87, 2003). While the virus serves only as the initiating event, over time some of the wildly growing cells develop permanent changes in their genetic structure that cannot be repaired. By expressing antisense-like constructs designed to block E6 and E7, viral infections would be rendered ineffective. [0085] Other types of HPV infections may manifest themselves as warts on or around the genitals and anus of both men and women and are also valid candidates for therapeutic antisense- like expression using the nucleic acid produced by this invention.
  • the amplification reaction of the invention can also be used to amplify either an intact plasmid comprising bacterial sequences, or a modified version of the plasmid to exclude these sequences.
  • a single-stranded DNA expression vector, pssXE which includes: 1) a Mouse Moloney leukemia viral reverse transcriptase (MoMuLV RT) gene coding for a truncated but fully active RT; 2) a primer binding site (PBS) with flanking regions essential for reverse transcription initiation by MoMuLV RT; 3) a target gene coding sequence for the production of an antisense, an aptamer, a DNA enzyme, or a sequence that induces triplex formation; and 4) a stem- loop structure designed for the termination of the reverse transcription reaction, as an intact expression cassette, can be effectively amplified according to the invention.
  • MoMuLV RT Mouse Moloney leukemia viral reverse transcriptase
  • PBS primer binding site
  • the amplified products can be transfected and used to effectively silence mammalian, viral, and bacterial genes.
  • the transfected RT Upon expression inside the cell, the transfected RT subsequently uses an endogenous host tRNA (e.g., tRNAPro or tRNAVal) as a primer to bind to a primer binding site (PBS) at the 3' end of the RNA transcript and initiates ssDNA synthesis.
  • PBS primer binding site
  • ssDNA may be released when the mRNA template is degraded by RNase H or the RNase H activity of RT.
  • Delivery of the nucleic acid can be accomplished by simple injection of a naked nucleic acid in stabilizing buffer into the targeted recipient.
  • Embodiments of the invention may also use delivery vectors or other delivery vehicles which help target and delivery of the nucleic acid into the cell (Dias, N. Molec Cancer Ther 1 : 347-355, 2002).
  • Some embodiments use a viral vector system which may be an attenuated virus system, a viral packaging system that includes few or no immunogenic protein (Srivastava, LK. and Liu, M.A. Ann Intern Med. 138: 550-559, 2003).
  • inventions include the use of neutral or cationic liposomes which either encapsulate the nucleic acids or bind the nucleic acid by electrostatic interactions. These embodiments may also use helper molecules (e.g., chloroquine or l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine) to prevent sequestering of the delivered nucleic acid in the endosomal compartments.
  • helper molecules e.g., chloroquine or l,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine
  • Some of the commercially available liposomal vectors include Lipofectin, Eufectins, Cytofectin and Lipofectamine.
  • Other methods of delivery include covalent coupling of the nucleic acids to cationic peptides (delivery vehicles), which may modulate the permeability of plasma membrane by physical interactions, receptor- or transporter-mediated mechanisms. Such coupling increases the effectiveness of the delivered nucleic acid which is delivered directly into the cytoplasm and is readily transported to the nucleus for expression (Luo, D. and Saltzman, W.M. Nature Biotech 18: 33-37, 2000). Still other embodiments use cationic polymers which interact electrostatically with the therapeutic nucleic acid to deliver nucleic acid to the cell.
  • Cationic polymers include poly-L-lysine (PLL), polyethylene glycol (PEG), PEG-block-PLL-dendrimers, polyamidoamine (PAMAM) dendrimers, polyalkylcyano-acrylate nanoparticles, and polyethyl- eneimine (PEI) and its conjugates (such as mannose-PEI, transferin-PEI, linear PEI).
  • PLL poly-L-lysine
  • PEG polyethylene glycol
  • PAMAM polyamidoamine
  • PEI polyethyl- eneimine
  • conjugates such as mannose-PEI, transferin-PEI, linear PEI.
  • Aerosol delivery is a noninvasive mode of delivery to airway epithelium and pulmonary surfaces.
  • formulations comprising the delivery vehicle, PEI, and a nucleic acid can effectuate high level airway or pulmonary transfection upon delivery by nebulization.
  • This application of PEI-nucleic acid complexes can effectuate higher levels of gene expression than many cationic lipid formulations, and exhibits a remarkably high efficiency (nearly 100%) of transfection into cells of the airway epithelium and lung parenchyma.
  • repeated aerosol administrations of PEI-based formulations are associated with very low toxicity.
  • TNF- ⁇ tumor necrosis factor alpha
  • IL-l ⁇ interleukin 1 beta
  • a frequent problem of using bacterially produced plasmid DNA results from exposure of the host to unmethylated motifs inherent in bacterially processed DNA. Unmethylated DNA can induce a CpG-mediated cytokine response and the induction of pro-inflammatory cytokines which is a serious problem associated with lung toxicity and reduced efficiency of therapeutic applications. Consequently, the use of bacterially produced DNA has severely hampered many of the current gene therapy approaches used to date.
  • Masking of the CpG response by PEI can facilitate the sustained expression of genes that are delivered via PEI-gene aerosol and, thus, the sustained therapeutic response achieved.
  • PEI-based aerosols can be extremely effective delivery systems for DNA therapeutics to lung and airway epithelium.
  • Some of the embodiments also use long-term release systems.
  • Biocompatible controlled-release polymers such as poly(D,L-lactide-co-glycolide) (PLGA) microspheres and poly- (ethylene-co-vinyl acetate (EVAc) matrices can effectuate a controlled, adjustable and predictable release of the bioactive nucleic acid for up to several months, and both components have been approved for therapeutic use by the U.S. Food and Drug Administration.
  • Electroporation may be efficient for transferring therapeutics to skin cells, corneal endothelium and other tissues including muscle.
  • Pressure-mediated or hydrodynamic injection can effectuate up to 50% efficiency in mammalian systems.
  • Other methods include ultrasonic nebulization for delivery of DNA-lipid complexes in many different types of cells, including plants, and particle bombardment is also useful for plants.
  • these physical delivery systems constitute additional delivery vehicles for effectuating the uptake of a therapeutic nucleic acid into a cell.
  • Scale-up of the cell free amplification process may be performed using a semi- or fully- automated platform, where sequential additions of salts, enzymes and nucleic acids, together with temperature and incubation times, can be tightly controlled for optimal efficiency (Fig. 4).
  • scale-up can be accomplished by increasing the number of reactions while keeping each reaction volume relatively small ( ⁇ 1 ml) whereby the template(s) can be amplified simultaneously using multi-well plates in standard or custom built platforms (Fig. 5A).
  • scale-up may involve larger volumes (e.g., 10 liters) to generate large quantities (kg amounts) of a single nucleic acid product in a single run using a fermenter-like vessel under environmental controls (Fig. 5B). Larger volumes may be used to produce larger yields of product.
  • Multiple platforms of mixed capacities can be arranged in parallel within a confined space and can function in a coordinate manner as part of a larger bio-manufacturing facility that can meet various amplification scale requirements.
  • the production of large amounts of nucleic acid in a small volume presents the problem of mixing reagents into a highly viscous reaction mixture.
  • the invention includes a reaction vessel that can be either a hardened pre-formed container or a flexible container such as a self contained plastic bag.
  • the reaction vessel and all components that come in contact with the reaction mixture are clean, sterile and free of any contaminating nucleic acid sequences.
  • the hardened pre-formed container contents are preferably mixed by a device that is contained inside the reaction vessel, but may involve a re-circulating, device.
  • the flexible vessel is preferably mixed by a re-circulating mechanism which could include the use of a peristaltic-like pump, or may incorporate an external mechanical device such as an automated squeezing apparatus or a low-energy pulsation device that avoids shearing of the nucleic acid product.
  • a re-circulating mechanism which could include the use of a peristaltic-like pump, or may incorporate an external mechanical device such as an automated squeezing apparatus or a low-energy pulsation device that avoids shearing of the nucleic acid product.
  • Internal devices can use several different mechanisms including propeller-like stirring devices with electronically controlled speeds and automated timing (FIG. 6A), or controlled liquid displacement processes using a perforated disk fixed to a shaft running from top to bottom within the reaction vessel's inner diameter (FIG. 6B). The disks are raised and lowered at various speeds within the liquid to provide adequate mixing of the reaction mixture.
  • Both of these mixing [ chambers can be equipped with a dispensing device which may comprise a small tube attached to the shaft of each mixer which delivers various stock components, which are chambered separately outside the mixing vessel, into the reaction mixture using a peristaltic pump to control the precise and sequential delivery of the various reagents.
  • Another embodiment implements a system where a steady constant flow of the reaction mixture is pumped from and then back into the chamber.
  • an outlet located at the bottom of the chamber enables a small stream of fluid to be combined with an added reagent and then channeled back through an entry port located at the top of the same reaction chamber to effectuate mixing (Fig. 6C).
  • Peristaltic pumps and intake valves control and monitor the dispensing of various solutes and enzymes during the recycling process (Fig. 6C).
  • Yet another embodiment utilizes the thixotropic nature of the DNA mixture, wherein the mixture is cylindrically configured into an elongated form.
  • Thixotropic compounds can change viscosity according to the degree of shear force applied to the compound. Typically, an increase in the shear force can decrease a thixotropic compound's viscosity. Once the shear force is removed, such a compound will begin to regress to its original viscosity.
  • the container holding the viscous reaction mixture has evenly spaced pores through which necessary chemicals are injected for processing.
  • Elongation of the viscous reaction mixture through the small diameter cylinders therefore changes the viscosity sufficiently to promote localized mixing with reagents which are slowly infused into the small diameter cylinders and into the less viscous reaction mixture for a sufficiently long period in which to effectuate mixing.
  • the apparatus preferably includes one or more inline real-time monitoring of all relevant physical and biochemical parameters to verify product stability and maintain quality control and quality assurance, which are necessary to maintain certified good manufacturing practice (cGMP) required for a product acceptable for therapeutic applications.
  • This may include a computer or similar means for monitoring viscosity, nucleic acid concentration, solution turbidity; conductivity; pH; temperature; protein content; endotoxin, bioburden, and/or chemical contaminants arising from degradable components of the system.
  • Processing of the linear SEC into a circular form requires that the ligation step favor an intramolecular (self-adhering) reaction over an intermolecular reaction.
  • Traditional dilution of the final amplification product can be used to manipulate the molar ratio to favor intramolecular ligation.
  • Preferred embodiments minimize the overall reaction volume by mixing small amounts of the reaction mixture into a ligation cocktail containing the enzyme and buffer components.
  • the amplified product is added into a small stream of reaction mixture as shown in Fig. 6C, using very slow or pulsating pump rates.
  • FIG. 7 Other embodiments dispense the amplified reaction mixture drop-wise into a second vessel containing the ligation cocktail to achieve dilution without generating large volumes of ligated reaction mixture. Sufficient time is allowed between each aliquot addition to optimize the intramolecular ligation process for each new aliquot dispensed. Once ligation of the aliquot is complete, the circular DNA is no longer substrate for the enzyme and becomes part of the dilution mix. A second aliquot is then dispensed, and the cycle repeats until all the amplified DNA is dispensed and ligated. This process allows intramolecular ligation to occur without large dilutions of the initial amplification reaction and can incorporate multiple dispensing chambers to allow for simultaneous aliquots to be ligated and to minimize processing time.
  • Final purification of the product can be streamlined by using permeable membrane- based methods during the reaction process. These membranes permit low molecular weight molecules (salts, unincorporated primers, dNTPs, NTPs and other small molecules) in the amplified DNA reaction mixture to diffuse away while retaining the product. A modification of the hemodialysis process can be used to allow the selective retention of the amplified DNA over other reaction components.
  • the amplification reaction is pumped from the vessel to a filter comprising membranes with specific molecular weight cut-offs. The DNA is at least partially purified when the smaller reagents diffuse from the reaction across the membrane of these small capillaries.
  • Purified DNA is then either pooled, evaluated for quality and/or dispensed for end-use applications, or directly aliquoted and stored for analysis at a later time.
  • Other embodiments utilize an ultrafiltration purification step which comprises a low-pressure membrane separation process to partition high molecular weight compounds from a feed stream to achieve the desired purification of the final RCA products.
  • the final product may be analyzed by traditional methods for size, form, contamination, and expression capacity. Gel electrophoresis, sequencing, and biochemical or HPLC analysis is routine. Expression of the final product is tested by transfection into appropriate cells, using standard techniques such as calcium phosphate treatments, electroporation or related techniques.
  • Administration of the amplified product as a therapeutic compound may include but is not limited to topical applications, intravenous, intramuscular and intra-tissue injections, nasal applications, suppository applications, injections using implanted reservoirs and/or pumps such as Omaya reservoirs, eye-drop applications, orally administered pharmaceuticals, and delivery using ultrasound techniques.
  • all such mechanisms constitute a "delivery vehicle.”
  • Traditional delivery vehicles including liposome-mediated or polymer-based transport vehicles as well as a wide variety of capsule or protein-targeting vehicles, and appropriate aerosol carriers for respiratory administration can also be used effectively.
  • pSV- ⁇ -Galactosidase (LacZ) vector (Promega Corp. Madison, WI, USA) was partially digested with EcoR I and Pst I. A fragment of about 4.2 kb containing the CMV promoter, LacZ ORF and SV40 small T antigen termination sequences (LacZ- DU) [SEQ ID NO: 1] was isolated, blunt ended with T4 DNA polymerase and cloned into the Smal site of pGEMTM-7Zf(+) (Promega Corp. Madison, WI, USA) creating the pGEM-LacZ-DU vector. The LacZ-DU was subsequently excised from pGEM-LacZ-DU with Xbal, gel purified, and circularized using T4 DNA ligase (New England Biolabs, Beverly, MA, USA) as per manufacturer recommendations.
  • LacZ-DU was amplified from the pVAXTM200-GW/lacZ vector (Invitrogen Carlsbad, CA) using forward (5'-CGGGATCCGACTCTTCGCGATG TAC-3') and reverse (5'-CGGGATCCCAGCATGCCTGC-S ') primers containing the BamHI endonuclease recognition site. LacZ-DU was amplified in 50 ⁇ l reactions with 200 ng of each primer 10 ng pVAXTM200-GW/lacZ vector; 0.2 mM dNTPs; Ix Herculase buffer and 2.5 U HerculaseTM polymerase (Stratagene, La Jolla, CA, USA).
  • Amplification was carried out in a RoboCycler Gradient 40 (Stratagene, La Jolla, CA, USA) under the following conditions: 2 min at 94oC; 5 cycles (30 sec 92oC; 30 sec 40oC, 5 min 72oC); 25 cycles (30 sec 92oC; 30 sec 55oC, 5 min 72oC) and 10 min 72oC.
  • the ⁇ 4.2kb product was digested with BamHI, gel purified and circularized with T4 DNA ligase.
  • the phi29 DNA polymerase was heat inactivated (5 min; 65oC) and the amplified LacZ-DU concatamer was ethanol/salt precipitated and digested with the appropriate endonuclease (Xbal or BamHI) as recommended by the enzyme manufacturer.
  • Phi29 DNA polymerase (1OU, New England Biolabs, Beverly, MA, USA); 1 mM dNTPs; 5% glycerol; 0.7 U yeast inorganic pyrophosphatase (Sigma, St.Louis, MO, USA) and lOO ⁇ g/ml BSA were added. Amplification was carried out at 30oC in 50 mM Tris-HCl pH 7.5; 10 mM MgC12; 10 mM (NH4)2SO4, 4 mM DTT for 16 hr.
  • the phi29 DNA polymerase was heat inactivated (10 min; 65 oC) and the amplified LacZ-DU concatamer was ethanol/salt precipitated and digested with the appropriate endonuclease (Xba T) as recommended by the enzyme manufacturer.
  • LacZ-DU was amplified using a sequence defined pentamer (5'GpGpApApA-3') which anneals to LacZ-DU at 19 different sites: 8 on the reverse strand at positions 465, 889, 1326; 1695, 2580, 3666 and 3912; 11 on the forward strand at positions 80, 119, 191 , 602, 750, 912, 2871, 3239, 3606, 3815.
  • sequence defined pentamer 5'GpGpApApA-3'
  • [0111] i) Amplification using a polymerase cocktail. Using the same conditions as described in section 1 -e, LacZ-plasmid was amplified in the presence of phi29 DNA polymerase and T4 DNA polymerase at ratios ranging from 10:3 to 3:10 (Phi29 enzyme unit:T4 enzyme unit). Optimal amplifications conditions were also shown to work for other templates, i.e. Luciferase DU.
  • the pGL3 vector (Promega Corp. Madison, WI, USA) was digested with Sal I and Xho 1. A fragment of about 2.17 kb containing the SV40 promoter, Luciferase ORF and SV40 small T antigen termination sequences (Luc-DU) [SEQ ID NO: 3] was isolated, purified and re-circularized using T4 DNA ligase (Invitrogen, Carlsbad, CA) as recommended.
  • Phi29 DNA polymerase (1OU, New England Biolabs, Beverly, MA); 1 mM dNTPs (25/25/25/25); 5% glycerol; 0.7 U yeast inorganic pyrophosphatase (Sigma, St.Louis, MO, USA) and lOO ⁇ g/ml BSA were added. Amplification was carried out in 25 ⁇ l reaction at 30oC in 50 mM Tris-HCl pH 7.5; 10 mM MgC12; 10 mM (NH4)2SO4, 4 mM DTT for 16 Iu-.
  • the phi29 DNA polymerase was heat inactivated (10 min; 65oC) and the amplified Luc-DU concatamers were ethanol/salt precipitated and digested with endonuclease (BamH I) as recommended by the enzyme manufacturer.
  • EXAMPLE 3 Expression of Amplified c ⁇ -gal and Luciferase synDNA in human cells.
  • DMEM Dulbecco's modified Eagle's medium
  • Hyclone Logan, UT
  • penicillin 100 ⁇ g/ml streptomycin
  • DNA solution was then mixed with 7 ⁇ l of GenePORTER 2 reagent pre-diluted in 50 ⁇ l of serum/antibiotics-free DMEM and incubated at room temperature for an additional 5 min. Meanwhile, A549 cells were washed with PBS and topped with 0.9 ml of serum/antibiotic-free DMEM to which the DNA/GenePORTER solution was subsequently added. Following 4 hr incubation in normal growth environment, the cells were washed with PBS and transfection medium was replaced with normal growth medium supplemented with 10 ⁇ l/m of Booster 3 (Gene Therapy System, San Diego, CA).
  • Booster 3 Gene Therapy System, San Diego, CA
  • a) Amplification buffer Glycerol concentration - Two amplification reactions using 10 ng of Luc-DU template each were set up as described in EXAMPLE 2. In one, addition of glycerol was omitted and replaced with water. Following amplification, DNA was ethanol/salt precipitated and subsequently digested with the appropriate restriction enzyme prior to spectrophotometric quantification at 260 and 280 nm wave lengths. In reactions where glycerol concentration was less than 4% w/v (carry over from the phi29 DNA polymerase and inorganic pyrophosphatase stock solutions) a 5.65% increase in amplification efficiency was observed.
  • dNTP Deoxyribonucleoside triphosphate
  • d) Customization of dNTP ratio to template Amplification reactions containing 578 nM Luc-DU template were prepared essentially as described in EXAMPLE 2. dATP, dCTP, dGTP and dTTP were individually added to the amplification mix to a final concentration of 9 mM. The ratio of each dNTP with respect to the entire pool was tailored such as to reflect the composition of the luciferase template DNA unit i.e. 27.2% A, 22.3% C, 24.2% G and 26.3% T. Following amplification, DNA was ethanol/salt precipitated and subsequently digested with the appropriate restriction enzyme. Nucleic acid concentrations were determined spectrophoto-metrically at 260 and 280 nm wavelength. About a 2,780-fold amplification was recorded using 578 nM template under the amplification conditions delineated above.
  • Phi29 DNA polymerase concentration e) Phi29 DNA polymerase concentration. Amplification reactions were prepared as described above in which various phi29 DNA polymerase (New England Biolabs) concentrations ranging from 1 to 20 U/578 nM DNA template were tested in the presence of 9 mM dNTPs. Following amplification, DNA was digested with the appropriate restriction enzyme and nucleic acid concentrations were determined spectrophotometrically. 1 U of phi29 polymerase/578 nM was sufficient to produce a 290-fold amplification, while 20 U of phi29 DNA polymerase amplified 10 ng of template DNA 3,985 times.
  • phi29 DNA polymerase New England Biolabs
  • LacZ-plasmid and Luc-DU were amplified in reactions containing half the total enzyme concentration (including Phi29 DNA polymerase, T4 DNA polymerase and Inorganic pyrophosphatase) and 289nM DNA template.
  • the amplification was carried out for 16 hr at 32oC. Following heat inactivation of the polymerases and subsequent endonuclease digestion of the amplification product, the DNA yields and quality were determined as described above.
  • T4 DNA ligase Following restriction enzyme digestion of cell free amplified DNA, heat inactivation of the restriction enzyme and ethanol/salt precipitation of the DNA, the intramolecular ligation (self-ligation) of linear DU was performed in 138 ⁇ l and 690 ⁇ l reactions respectively containing 700 fmol of DNA in Ix ligation buffer (5% PEG-8000; 50 mM Tris-HCl pH 7.5; 10 mM MgC12; 1 mM DTT; 1 mM ATP). Various amounts of T4 DNA ligase (Invitrogen Carlsbad, CA) were then added (0.6-1.5 U) and ligations were carried out at 14oC for at least 1 lir. Ligation efficiency was subsequently visually determined by agarose gel electrophoresis of said DNA. 290 ⁇ U of T4 DNA ligase per fmol DNA in 690 ⁇ l reactions was deemed sufficient for driving the synthesis of monomeric circular DU.
  • T4 DNA ligation products were ethanol/salt precipitated and resuspended in 20 ⁇ l of Plasmid SafeTM DNase buffer (Epicenter) containing 5U of ATP-dependent DNase as per manufacturer recommendations. Following 30 min incubation at 37oC, DNase enzyme was heat inactivated at 65oC for 20 min. Reaction efficiency was visually determined by agarose gel electrophoresis revealing the presence of only circular dsDNA which can be re-digested to linear form with appropriate restriction enzymes.
  • EXAMPLE 7 Expression of Luciferase synDNA in mouse lung (tail vein injection + carrier).
  • Luc-DUs were prepared including: linear form, phosphorothioate modified linear form, circular form, circular form treated with Plasmid-SafeTM ATP-dependent DNase (Epicenter, Madison, Wisconsin). 1 ⁇ g of various forms of Luc-DUs were complexed with MAA-PEI at an N:P ratio of 15:1 in PBS at a Final volume of 200 ⁇ L/mouse. Each group comprising 5 BALB/c mice was injected via tail vein without anesthesia with a single form of MAA-PEI-Luc-DU. Mouse lungs were harvested 24 hours following injection and homogenized in luciferase assay buffer. Luciferase gene expression was measured using Bright-GloTM kits from Promega according to the manufacturer's instructions.
  • a eukaryotic cassette expressing a modified form of human immunodeficiency virus (HIV-I) envelope protein gpl 60 (gpl45 ⁇ CFl; Chakrabarti et al, J. Virol. 2002; 76: 5357-68; Kong et al., J. Virol. 2003; 77: 12764-72) [SEQ ID NO: 5] was used as template to generate large quantities of linear gpl45 ⁇ CFl-DU expression cassette as described in Example 2.
  • HAV-I human immunodeficiency virus
  • mice Groups of 5 mice were used for each DNA type in addition to a control group injected with saline only.
  • the serum from each blood sample was then used in Enzyme-linked immunosorbant assays (ELISA) to assess the IgG antibody titers against g ⁇ l60.
  • ELISA Enzyme-linked immunosorbant assays
  • 96 well microtiter plates were coated with a solution of 12.5 ng/ ⁇ L of purified recombinant HIV-I IIIB gpl60 (Advanced Biotechnologies Inc.) in 50 mM carbonate buffer pH 9.5.
  • the wells were subsequently washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked with a solution of 3% BSA in PBS-T.
  • the amplification product is purified by gel filtration chromatography using Sephacryl SF-1000 (GEHC). Briefly, DNA is added onto 1.7 m x 1.5 cm Econo-column (Bio-Rad) and eluted with 10 mM Tris Ph 8, 150 mM NaCl, 5 mM EDTA at a flow rate of 1 mL/3.6 min. The DNA content of each elution fraction is monitored by agarose gel electrophoresis and the desired fractions are pooled. The fractions are subsequently concentrated using Centriplus 300 cartridges (Millipore Corp.) are recommended by manufacturer.
  • GEHC Sephacryl SF-1000
  • Each production lot is assigned an identification number and undergoes a series of test to determine DNA concentration, purity and integrity.
  • DNA concentration is determined my photometric absorbance reading at 260 nm and/or fluorometry using the Quant-iTTM PicoGreen® dsDNA Assay Kit (Invitrogen, Corp.). DNA purity is determined using several methods. Photometric A260/280 ratio, real time PCR (Genomic DNA contamination); HPLC (RNA contamination); micro-BCA test (Protein content, Pierce kit) and LAL test (Endotoxin content, Cambrex kit). In addition a bioburden test is carried out to confirm the sterility of the end product. Each set of test needs to comply with the specification set by the therapeutic industry.
  • EXAMPLE 11 Genetic immunization in rabbits against Hepatitis B virus (HBV).
  • HBV Hepatitis B virus
  • Groups of 3 NZ female albino rabbits were immunized via bilateral (hind limb) intramuscular injections on days 0, 28 and 56 with either a total dose of 400 ⁇ g of the plasmid each time or the gene equivalent quantity of cell-free amplified linear DNA.
  • FIG. 9 shows ELISA assay absorbance readings for sera taken from 3 rabbits immunized with either HBs(S) supercoiled plasmid or cell-free HBVs(S)-DU linear DNA for days 28 and 63 (normalized for day 0).
  • EXAMPLE 12 Genetic immunization in mice against influenza HlNl virus.
  • Five BALB/c mice were utilized in each experiment. All animal experiments were approved by the Institutional Review Board for Animal Studies (Baylor College of Medicine; BCM).
  • Influenza A/Puerto Rico/8/34 was obtained from the Respiratory Pathogens Research Unit, BCM.
  • the influenza virus comprises a family of related viruses with slightly different lipid coat proteins on the outer surface. Two of the better characterized variable coat proteins involved in epidemics and pandemics of flu comprise hemaglutinin (HA; at least 15 types) and neuraminidase (NA; at least 9 types).
  • the HlNl is one of the earlier characterized viral forms and is used widely in researching influenza.
  • DNA immunization was conducted as described above using 50 ⁇ g of plasmid DNA or a gene equivalent amount of cell-free DNA in PBS.
  • the influenza hemaglutinin open reading frame from viral strain A/PR8/34 (HA) was isolated from pCAG-HA-WPRE plasmid (Garg et al, 2004, J. Immunol. 173(l):550-8) and subcloned into pCMV-MCS (Stratagene) giving pCMV-HA.
  • the CMV-HA expression cassette devoid of plasmid backbone (HA-DU) [SEQ ID NO: 9] was amplified as described in Example 2. Animals were given 3 injections at weeks 0, 2 and 6. Five different experiments were conducted.
  • mice were immunized with: (1) 50 ⁇ g of pCMV-HA; (2) 32 ⁇ g of HA-DU; (3) a mixture of 16 ⁇ g of HA-DU and 25 ⁇ g of plasmid DNA devoid of any expression cassette (Empty Vector, pEV); (4) a mixture of 10.6 ⁇ g of HA-DU and two cytokine-expressing plasmids (i.e. 16.7 ⁇ g of pCMVi-GMCSF and 16.7 ⁇ g of pCAGGSIL12) (Orson et al., 2006, J.
  • a eukaryotic cassette expressing the B5R gene encoding a type I membrane glycoprotein essential for the formation of the extracellular virion envelope; Hooper J.W. et al. 2004; J Virol. 78(9): 4433-4443) was used as template to generate large quantities of linear B5R- DU expression cassette [SEQ ID NO: 11] as described above.
  • Groups of 5, 4 and 3 Balb/c mice were immunized via intramuscular injections with: 100 ⁇ g of B5R plasmid, 34 ⁇ g of B5R-DU (gene equivalent quantity), and 100 ⁇ g of pVax empty vector (na ⁇ ve), respectively.
  • a) Protein ELISA Serum samples are isolated from orbital bleeds. The blood is spun down at 1100 rpm for 5 minutes, and the serum carefully removed and stored at 4 0 C until ready for analysis. Nunc 96-well plates are coated with B5R protein (Viral Genomix) (1 microgram/well) in PBS at 4°C overnight. After three washes with PBS, nonspecific binding sites are blocked with 1% BSA in PBS solution for 2 hours at room temperature. Duplicate samples are loaded into the appropriate wells in a dilution series, and incubated for 2 hours at room temperature. After washing, anti -murine IgG, conjugated with horseradish peroxidase is added at a dilution of 1 :1000. Using TMB as a substrate, bound antibody is measured in an ELISA reader at 405 nm.
  • IFN ⁇ ELISPOT 96-well plates (Millipore) are coated with anti-mouse IFN- ⁇ capture antibody (MabTech, Sweden) and incubated overnight at 4 0 C. The plates are then washed and blocked for 2 hours with RlO media (blocked prior to loading of cells). Approximately 2 x 105 splenocytes from immunized mice are added to the ELISPOT plates and stimulated overnight at 37°C, 5% CO2, either in the presence of RPMIl 640 (negative control), Concanavilin A (positive control), or B5R peptides.
  • EXAMPLE 14 Methods of Delivery for immunization using synDNA products.
  • a) Aerosol Delivery Standard conditions for aerosol include a 10: 1 N:P ratio (N: PEI nitrogen; P: DNA phosphate) in water with a final concentration of DNA at 2 mg/10 mL for nebulization. Maximal gene expression is normally observed at 48h for the gene constructs used herein. Mice are enclosed in a standard mouse cage wherein each mouse is place in an individual wire mesh enclosure. The nebulization uses a standard clinical jet nebulizer with compressed 5% CO2 in air as the gas source at 10 L/min. The procedure is complete in about 25-30 minutes. Intermittent exposure to the aerosol for shorter or longer periods of time as well as the use of variable amounts of DNA can be adjusted as the particular construct demands.
  • Standard conditions for intravenous (IV) delivery of DNA include the use of 1 ug of DNA complexed with MAA-PEI at an N:P ratio of 15:1 in PBS at a final volume of 200 uL/mouse. Maximal gene expression is normally observed between 24 and 48 h. Mice are injected via tail vein without anesthesia, usually over 20-30 seconds, although the time of injection is not critical to gene expression.
  • EXAMPLE 15 Influenza virus challenge and determination of pulmonary vims levels.
  • the A/PR8/34 influenza virus strain was grown in MDCK cells. The medium and cells from the infected flasks were harvested when the infected monolayers exhibited approximately 90% virus-induced cytopathic effects (CPE). The medium was clarified by centrifugation and then filtered through a 0.4 ⁇ m filter. It was then stored at -70 0 C in 1 or 2 ml aliquots. A sample of each harvest was titered to determine its tissue culture infectious doses (TCID50), median mouse infectious (MID50) and median mouse lethal (MLD50) dose.
  • TCID50 tissue culture infectious doses
  • MID50 median mouse infectious
  • MLD50 median mouse lethal
  • mice were lightly anesthetized with Isoflurane (Abbot Laboratories, North Chicago) and 50 ⁇ L of medium containing approximately 100 MID50 of the A/PR8/34 virus was instilled into the nares of each mouse using a pipetting aid. Mice were sacrificed 4 days later; lungs were harvested, homogenized, serially diluted and tested for the flu virus (FV) levels. After 4 days of incubation at 37 0 C, the plates were removed from the incubator and a 0.5% suspension of chicken red blood cells (rbc) in PBS was added to each well.
  • rbc chicken red blood cells
  • rbc in the tissue control wells formed a tight button
  • the hemagglutination pattern in each well was read and recorded.
  • Wells with a tight button of rbc were considered to be negative for FV, while those with a diffuse hemagglutination pattern were recorded as positive for virus.
  • Virus titers were recorded as the loglO of the reciprocal of the last dilution in which virus replication (CPE) was evident. Titers are shown as TCID50/lung (loglO). Lower values represent greater protection by the immunizing agent.
  • HA plasmid provides minimal protection alone but significant protection when injected with the two cytokine expression plasmids.
  • HA linear synDNA provides significant protection in the presence of a carrier plasmid (blank or cytokine expression). HIV plasmid provides minimal protection while the HIV linear synDNA had a slightly better protective effect.
  • EXAMPLE 16 Effect of time on expression of Luc-synDNA in vivo.
  • Linear Luc-DU synDNA was prepared using phosphorothioate primers according to the method of the current invention and supercoiled luciferase plasmid was prepared by growth in bacteria and purified using a Qiagen endofree kit; both were injected as naked DNA following dilution in sterile saline into the anterior tibialis muscle of mice (BALB/c). Each mouse received a single injection of 50 ⁇ g DNA in one leg. Groups of 5 mice were used for each DNA types. 24, 72 and 144 hr post-injection mice were sacrificed and tibialis muscles were dissected, ground in saline and processed for luciferase activity assay.
  • Luc-DU reporter construct can be used as an experimental tool for testing the effectiveness and applicability of synDNA in existing research systems.
  • Our in vitro amplification results using the LacZ-DU construct worked in vitro in a manner similar to Luc-DU so it would be expected to express in vivo in a similar manner.
  • any well characterized reporter gene such as secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP), and chloramphenicol acetyltransferase (CAT) would be as useful as the Luc-DU to be used as a testing tool for the use of synDNA in any ongoing research application.
  • SEAP secreted alkaline phosphatase
  • GFP green fluorescent protein
  • CAT chloramphenicol acetyltransferase
  • Phi29 DNA polymerase (10 U, Fermentas, MD, USA); Ix Phi 29 reaction buffer [0.1% Tween-20; 33 mM TrisOAc pH 7.9; 10 mM Mg(OAc)2; 66 mM KOAc; 1 mM DTT as supplied by Fermentas]; 6 mM dNTP (Stratagene, CA, USA); 0.7 U yeast inorganic pyrophosphatase (Sigma, St.Louis, MO, USA); 3 U T4 DNA polymerase (Fermentas, MD, USA) and 100 ⁇ g/ml BSA were added. Amplification was carried out at 30oC for 16 hr.
  • phi29 DNA polymerase was heat inactivated (20 min; 65oC), the amplified DNA concatamer digested with Xhol (New England Biolabs, MA) for 4 hr at 37oC, and heat inactivated at 65oC for 20 min.
  • the intensity of the 7.1 kb band was determined using ImageJ software (NIH).
  • the decrease in 7.1 kb band intensity over time was determined using Microsoft Excel 2003 software ( Figure 13B). This shows that the phosphorothioate amplified DNA (P-synDNA) is about '1.5 times more sensitive to exonuclease degradation than the methyl phosphonate-synDNA (MP-synDNA).
  • the primary advantage of this technology is that it offers a method for rapidly making high quality DNA with almost no bacterial cell components or bacterial toxin (endotoxins).
  • This cell-free amplification process can be streamlined for efficiency by the optional removal of unnecessary flanking sequences from the plasmid prior to amplification which can reduce the effectiveness of the DNA used as a target effector.
  • the end product that is produced has lower levels of known and potentially unknown toxins which are common to bacterially grown plasmid preparations and their end products; purification requirements are reduced and costs are minimized.
  • the method is faster, cleaner and less cumbersome to use.
  • the end products can be easily adapted for use in DNA based therapeutics as vaccines, gene therapeutics, or as tools for down-regulating gene expression (triplex, antisense) or protein activity (aptamer).

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Abstract

La présente invention concerne un processus permettant de produire un ADN de haute qualité dans un système sans cellule, exempt d'impuretés bactériennes et exempt de manière optimale de séquences codantes pour des gènes bactériens qui peuvent minimiser ou rendre silencieux une expression de gène lorsqu'elle est utilisée pour l'expression à l'intérieur d'une cellule cible. Ce système sans cellule est un procédé rapide qui fournit un produit final hautement fidèle, plus propre convenant pour des applications thérapeutiques avec moins d'efforts et de dépenses et, qui peut être adapté à l'amplification de matrices de type plasmide manquant de séquences de gènes plasmide bactériennes non nécessaires. Ceci permet d'accroître l'efficacité du système et d'accroître l'efficacité du vecteur d'expression de produit final. Ce produit final peut être facilement utilisé comme ADN thérapeutique du fait de la faible incidence des composants de cellule bactérienne et des toxines bactériennes. Cette invention concerne aussi un procédé de production d'ADN pour quelque recherche ou quelque but thérapeutique que ce soit, qui est sensiblement exempt d'impuretés de cellule bactérienne, ainsi qu'une thérapeutique de produit fini comprenant des vaccins ADN.
PCT/US2006/023439 2005-08-03 2006-06-14 Biosynthese sans cellule d'acide nucleique WO2007018744A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010005365A1 (fr) * 2008-07-09 2010-01-14 General Electric Company Produit d'amplification par cercle roulant non traité
US8921072B2 (en) 2008-09-02 2014-12-30 General Electric Compnay Methods to generate DNA mini-circles
US9125845B2 (en) 2008-07-09 2015-09-08 General Electric Company DNA vaccines, uses for unprocessed rolling circle amplification product and methods for making the same
US11324839B2 (en) 2019-09-18 2022-05-10 Intergalactic Therapeutics, Inc. b Synthetic DNA vectors and methods of use

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US6875619B2 (en) * 1999-11-12 2005-04-05 Motorola, Inc. Microfluidic devices comprising biochannels
GB0315160D0 (en) * 2003-06-28 2003-08-06 Royal Holloway University Of L In vitro amplification of DNA
WO2006063355A2 (fr) * 2004-12-11 2006-06-15 Cytogenix , Inc. Biosynthese acellulaire d'acide nucleique de haute qualite et utilisations correspondantes

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010005365A1 (fr) * 2008-07-09 2010-01-14 General Electric Company Produit d'amplification par cercle roulant non traité
JP2011527570A (ja) * 2008-07-09 2011-11-04 ゼネラル・エレクトリック・カンパニイ 未処理ｒｃａ産物
US9125845B2 (en) 2008-07-09 2015-09-08 General Electric Company DNA vaccines, uses for unprocessed rolling circle amplification product and methods for making the same
US8921072B2 (en) 2008-09-02 2014-12-30 General Electric Compnay Methods to generate DNA mini-circles
US11324839B2 (en) 2019-09-18 2022-05-10 Intergalactic Therapeutics, Inc. b Synthetic DNA vectors and methods of use
US11602569B2 (en) 2019-09-18 2023-03-14 Intergalactic Therapeutics, Inc. Synthetic DNA vectors and methods of use
US11684680B2 (en) 2019-09-18 2023-06-27 Intergalactic Therapeutics, Inc. Synthetic DNA vectors and methods of use
US11766490B2 (en) 2019-09-18 2023-09-26 Intergalactic Therapeutics, Inc. Synthetic DNA vectors and methods of use
GB2606844B (en) * 2019-09-18 2025-08-06 Aldevron Llc Synthetic DNA vectors and methods of use

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