WO2006107832A2

WO2006107832A2 - Methods and kits for isolating nucleic acids

Info

Publication number: WO2006107832A2
Application number: PCT/US2006/012226
Authority: WO
Inventors: Joseph Pearlberg
Original assignee: President And Fellows Of Harvard College
Priority date: 2005-04-05
Filing date: 2006-04-04
Publication date: 2006-10-12
Also published as: WO2006107832A3

Abstract

Methods are provided for isolation of a large plurality of nucleic acids in a high throughput procedure. Nucleic acids are absorbed to an immobilized hyperlipidemic agent, for example, cholestyramine. An improved later step is provided for collecting the plurality of nucleic acids on a filter plate and rinsing with an organic solvent, for example, isopropanol, prior to dissolving in an aqueous buffer. The high yield nucleic acids obtained by the methods herein provide compositions for vaccination and immunization.

Description

METHODS AND KITS FOR ISOLATING NUCLEIC ACIDS.

Technical Field Methods are provided for isolation of a large plurality of nucleic acids in a high throughput procedure in which the nucleic acids are absorbed to an immobilized hyperlipidemic agent

Government Support The invention was supported in part by^" funding from the National Institutes of

Health grant CA 060636. The government has certain rights in the invention.

Background As is generally true of cloning methods, particularly those methods used for cloning done in high throughput scale (for example, hundreds or thousands of genes at a time), the success rate depends on the efficiency of each step and materials used in these steps. Such steps and materials generally common to cloning procedures include PCR amplification, the quality of the competent E. coli used for bacterial transformation, and the cloning reaction itself. An improvement in the efficiency of any one of these procedures would improve the success rate of cloning, and other processes that involve nucleic acid enzymological reactions.

The nucleic acids for plasmids or other structures such as viral DNA, for the purpose of transfection into mammalian cells must be of very high purity, hi particular, endotoxins present in almost all Gram negative bacteria such as Escherichia coli and other strains commonly used in laboratories and found as contaminants of food and water, are known to be inhibitory to transfections, and are toxic to many mammalian cells and to animals that are recipients of DNA vaccines. Two methods of DNA purification, centrifugation through a cesium chloride gradiant, and anion exchange chromatography, have been employed to generate DNA of sufficient purity for transfection. Adapting these methods to high throughput, for example, 96 well format for microtiter plates and robotic handling, has been challenging.

In addition, nucleic acids can be used directly for delivery of a vehicle encoding amino acids sequences of peptide and proteins for immunizations of subjects, including humans, the so-called "naked DNA" approach. DNA used for such a purpose must meet a rigorious standard of purity. In this case any method which provides an improvement in yield and purity contributes to the speed and decreases the cost both of research efforts, and more importantly, of responding to potential viral epidemics involving new antigenic determinants, such as influenza strains. Improved processes that are capable of efficient scale up both of overal yield quantity and for high throughput screens of large numbers of independent sample, are needed for obtaining highly purified nucleic acids.

Summary

An embodiment of the invention provides a method for purifying at least one nucleic acid, comprising contacting the nucleic acid with a hypolipidemic agent; the agent binds to the nucleic acid to form a resulting complex; and then the method involves eluting the nucleic acid, and the nucleic acid eluted is purified. The term, "hypolipidemic agent" refers to the effect of a class of therapeutic agents following administration to a subject, the effect being to reduce a lipid burden in a subject, e.g., to lower blood cholesterol levels. An alternative term, "hyperlipidemic agent" has appeared in the literature in reference to the same class of agents, referring to the condition being treated. The word "hypolipidemic" is used herein.

Accordingly, in certain embodiments of the method for purifying at least one nucleic acid, contacting the nucleic acid is contacting a cleared cell lysate. The method is designed to be suitable for high throughput screening, so in general, the phrase, "at least one nucleic acid" as used herein can be a plurality of nucleic acids, for example, 96, or 384, or even a larger number of different nucleic acids in a multi- well format, or immobilized on beads, or chips, or slides, or micro-cantilevers. Once the nucleic acid binds to the agent to form a complex, eluting from the complex is further contacting the complex with an aqueous solvent of high salt concentration. Alternatively, the method can also be used for individual large scale "maxi- preparations" involving a lysate made from at least about 1 mg, about 10 mg, about 100 mg, about 1.0 g or about 1O g of starting biological material such as cells or viruses.

High salt is about 2 M salt, for example, 2 M NaCl, or 2 M KCl, or any standard salt at a concentration of at least about 1.0 M, or about 1.2 M, or about 1.5 M. Prior to eluting the nucleic acid the complex is rinsed with an aqueous solvent of intermediate salt concentration. For example, the nucleic acid is rinsed with at least about 0.5 M, or about 0.6 M, or about 0.7 M salt solution, or less than about 1.2 M or 1.0 M salt solution. The rinse removes impurities from the complex of the nucleic acid and the hypolipidemic agent.

The hypolipidemic agent comprises at least one positively charged functional group such as an ammonium group, the function being in general that it binds bile acids. In general, the nucleic acid is an RNA. Alternatively, the nucleic acid is a DNA. The nucleic acid is single stranded. Alternatively, the nucleic acid is double stranded. In certain embodiments, the nucleic acid is chromosomal DNA. In certain embodiments, the nucleic acid is plasmid DNA. Alternatively, in certain embodiments, the nucleic acid is viral nucleic acid. In general, prior to contacting, the hypolipidemic agent is immobilized. For example, the hypolipidemic agent is immobilized on a bead, a resin, or a micro-titer plate surface. Alternatively, the hypolipidemic agent is immobilized on the bead which is magnetic.

In embodiments herein, the hypolipidemic agent is selected from the group of at least one of a statin, a colestipol, a guggulsterone, and a cholestyramine. In examples shown herein, the hypolipidemic agent is cholestyramine. The cholestyramine is attached to a resin, the cholestyramine-resin being obtained from Sigma, Corp., St. Louis, MO, catalog number C4650. For example, the resin is a polymer comprising a styrene-divinylbenzene copolymer backbone with attached trimethylbenzylammonium groups. In certain embodiments, the polymer contains 4 gm cholestyramine resin per 9 gm total powder (QUESTRAN ). In certain embodiments, the polymer contains 4 gm cholestyramine resin per 5 gm total powder (QUESTRAN LIGHT^®). The agents are commercially available, and are used as therapeutic agents for lowering or removal of bile acids and cholesterol, and less commonly, removal of clostridial toxins. While toxins have an affinity for the resin, the affinity of the nucleic acid is greater, and the wash step described above solubilizes the toxins if any are present in the nucleic acid, and the nucleic acid remains attached to the resin.

Also provided herein is a method for purifying a plurality of plasmid preparations, the method comprising contacting a plurality of cell extracts containing plasmids with a cholestyramine resin, such that the resin binds to the plasmid; and eluting the plasmid from the resin with an aqueous high salt solution, so that eluted plasmid is purified. Further, after contacting and prior to eluting, the method involves centrifuging the resin and the bound plasmid with the resin. Centrifuging causes the resin to precipitate, and the nucleic acid is separated by being bound to the resin, from the majority of impurities found in the cell extract. Any of these methods in certain embodiments involve, after eluting, filtering a plurality of plasmids or nucleic acids on a filter plate and rinsing with the nucleic acids on the filter with isopropanol, and then dissolving the plasmids or nucleic acids in an aqueous buffer.

Any of these methods in various embodiments further comprises transfecting a cell with the eluted nucleic acid or plasmid. For example, these methods can further comprise cloning a gene into any of the nucleic acid or plasmid eluted, wherein the gene encodes an amino acid sequence of an immunogen and the gene is operably cloned at a location in the nucleic acid for protein expression, to produce a resulting expression vector; and contacting a subject with the resulting expression vector, wherein the subject expresses the gene in vivo, and produces antibodies to the immunogen.

Yet another embodiment provided herein is a kit for purifying nucleic acids comprising a hypolipidemic agent immobilized to a resin, a container, and instructions for use in isolating nucleic acid. Accordingly, the agent is selected from a statin, a colestipol, a guggulsterone, and a cholestyramine. In general, the agent is cholestyramine. The kit, in some embodiments, further comprises a DNA vector. The kit, in some embodiments, further comprises a bacterial recipient. The kit, in some embodiments, further comprises buffers and reagents for DNA isolation and analysis. The kit, in some embodiments, further comprises a multi-well filter plate. Yet another embodiment of the invention provided here is an article of manufacture for isolation of a plurality of nucleic acids, comprising a hypolipidemic agent attached to a surface, the hypolipidemic agent having a plurality of separable, i.e., addressable locations or "spots". In certain embodiments, the surface is a glass or a plastic. For example, the surface is a glass slide. Alternatively, the surface is a multi-well plastic plate.

Also provided herein is a composition comprising a nucleic acid made by any of the methods or the kits described above. For example, a composition is provided comprising a nucleic acid having a nucleotide sequence encoding an amino acid sequence wherein the amino acid sequence is expressed in a cell, and elicits antibody production in a subject, such that the nucleic acid is purified by a method above, for example, by binding to cholestyramine. In an embodiment, the amino acid sequence is an antigen of a pathogen. Alternatively, the amino acid sequence is a binding partner of an IgE molecule and elicits IgG production. The pathogen is a virus, for example, wherein the pathogen is an influenza, an HIV, a smallpox, a SARS, a Herpes, or a hepatitis. The influenza can be an avian influenza, i.e., bird flu.

Also provided, in a method for isolation of a nucleic acid, is the improvement to the method comprising absorbing the nucleic acid to a hyperlipidemic agent. The agent is generally cholestyramine. Also provided, in a method for isolation of a nucleic acid from a plurality of high salt solutions, is the improvement comprising precipitating the nucleic acids with isopropanol and collecting precipitated nucleic acid on a multi-well filter plate.

Brief Description of the Drawings

Fig. 1 is a photograph of a 96 well agarose electrophoresis block containing replicates of a clone of a plasmid carrying a gene capable of expressing green fluorescent protein, each of the plurality of preparations of the clone prepared by the methods herein using binding to cholestyramine resin as described in the Examples. Slots on the right side of the figure carry one kBase markers (New England Biolabs, Beverly, MA). The photograph shows that among the plurality of plasmid preparations from a plurality of different cultures, there is a great degree of uniformity of purity, size and yield.

Fig. 2 is a photograph of a 96 well agarose electrophoresis block containing replicates of the same clone of a plasmid as Fig. 1, however each of the plurality of preparations of the clone prepared by the methods using compounds other than cholestyramine. Slots on the right side of the figure carry a one kBase marker (New England Biolabs, Beverly, MA). The photograph shows that the plasmid preparations fail to have uniform purity, size or yield, and that yield is generally inferior to, i.e., less than those in Fig. 1.

Fig. 3 is a set of three photographs that show recipient cells following transfection with 100 ng of the plasmid DNA into 293 T cells, the DNA purified by each of three different methods. The same plasmid vector, which for comparison purposes was prepared by each of the three methods, expresses green fluorescent protein (GFP) as a marker, so that the successful transfectant cells among the recipient cells in the figures can be distinguished by extent of fluorescent glowing compared to the non-transfectant background cells.

Fig. 3A shows cells transfected with 100 ng of miniprep DNA.

Fig. 3 B shows cells transfected with 100 ng of Qiagen purified maxiprep DNA.

Fig. 3C shows cells transfected with 100 ng of cholestyramine-purified DNA by the methods herein.

Detailed Description of Embodiments

The methods of the invention herein arise from the surprising discovery that anti-hyperlipidemic agents, termed hypolipidemic agents herein, particularly cholestyramine, can bind to DNA with sufficient affinity and specificity that they are useful for DNA purification and isolation away from other cell components.

Examples herein were motivated by the need for an inexpensive 96 well high throughput format for producing DNA from a plurality of cultures. While such kits may be obtained commercially, for example, a kit having an anion exchange preparation for purification of plasmid DNA for transfection, available for example from Qiagen and a small number of other companies, these can be prohibitively expensive (costing hundreds of dollars for a single 96 well plate). The proprietary contents cannot be duplicated in the laboratory, as methods for manufacture their resin are not available.

Cholestyramine resin is known as an agent administered to a subject for the use to lower cholesterol. It does so by binding bile acids in the gut. This chemical has a less commonly known therapeutic use, which is, to bind a toxin produced by the Gram positive spore forming pathogen Clostridium difficile in dire cases of C. difficile enterocolitis. It is commercially available from Sigma Corp. This resin and this agent have not previously been used as reagents to purify any nucleic acid.

Pharmaceutical Compositions In one aspect of the present invention, pharmaceutical compositions are provided, wherein these compositions comprise a nucleic acid prepared by the methods herein for purposes of vaccination, and optionally comprise a pharmaceutically acceptable carrier. In certain embodiments, these compositions optionally further comprise one or more additional therapeutic agents. In certain embodiments, the additional therapeutic agent or agents are selected from the group consisting of growth factors, anti-inflammatory agents, vasopressor agents, collagenase inhibitors, topical steroids, matrix metalloproteinase inhibitors, ascorbates, angiotensin II, angiotensin III, calreticulin, tetracyclines, fibronectin, collagen, thrombospondin, transforming growth factors (TGF), keratinocyte growth factor (KGF), fibroblast growth factor (FGF), insulin-like growth factors (IGF), epidermal growth factor (EGF), platelet derived growth factor (PDGF), neu differentiation factor (NDF), hepatocyte growth factor (HGF), B vitamins such as biotin, and hyaluronic acid. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, PA, 1995 discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, saffiower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Therapeutically Effective Dose

In yet another aspect, according to the methods of treatment of the present invention, the treatment for vaccination to immunize a subject, as described herein, by contacting for example the subject with a pharmaceutical composition comprising a nucleic acid encoding the immunogen, as described herein. Thus, the invention provides methods for the immunizing a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the encoding nucleic acid administered to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. It will be appreciated that this encompasses administering an inventive pharmaceutical as a therapeutic measure to inhibit or even prevent development of a viral disease, for example, influenza, SARS, HIV, Herpes, measles, chicken pox, mumps, rubella, polio, hepatitis A, B or C, or a bacterial disease (for example, anthrax caused by Bacillus anthracis or a bacterial pneumonia as caused by Streptococcus pneumoniae, Haemophilus influenzae, Chlamydia trachomatis, Mycoplasma pneumoniae, Legionella pneumophila, or whooping cough caused by Bordetella pertussis) or other conditions as described herein, or as a prophylactic measure to minimize development of the disease (for example, after a known exposure to a patient suffering from the disease). The nucleic acid as prepared herein encodes an immunogen from at least of one of the above pathogens, in a vector such that it is operably expressed following transfection into a cell. Alternatively, the nucleic acid encodes an antigen capable of remediating an allergic condition, such as a protein produced by a dust mite (North American House Dust Mite, Dermatophagoides farinae Hughes; European House Dust Mite, Dermatophagoides pteronyssinus), a peanut (Arachis spp.), or a salivary protein of a dog or a cat.

In certain embodiments of the present invention a "therapeutically effective amount" of the pharmaceutical composition is that amount effective for stimulating development of antibodies against a pathogen, or reducing the presence of IgE specific for an allergen, or other conditions. The compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for treating the condition. Thus, the expression "amount effective for promoting an development of immune antibodies", or another condition such as an allergic reaction, as used herein, refers to a sufficient amount of composition to stimulate immunity, or to inhibit or even prevent allergic reactions. The exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active agent(s) or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, e.g., extent of infection from prior exposure, history of the condition; age, weight and gender of the patient; diet, time and frequency of administration; drug combinations; reaction sensitivities; and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered several times a day, every day, 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular composition. The active agents of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression "dosage unit form" as used herein refers to a physically discrete unit of active agent appropriate for the patient to be treated. It will be understood, however, that the total daily usage of the compositions of the present invention is to be decided by the attending physician within the scope of sound medical judgment. For any active agent, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. A therapeutically effective dose refers to that amount of active agent that ameliorates the symptoms or condition. Therapeutic efficacy and toxicity of active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose is therapeutically effective in 50% of the population) and LD50 (the dose is lethal to 50% of the population). While it is not anticipated that the nucleic acid vaccines described herein will exhibit toxicity at standard doses, the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred and are expected for the vaccines herein. Data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. Administration of Pharmaceutical Compositions

After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions of this invention can be administered to humans and other mammals topically such as transdermally (resuspended into suitable preparations from powders, or as ointments, or drops), i.e., as applied directly to the skin. Alternative and additional cutaneous routes as exemplified by oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, bucal, or nasal, depending on the severity of the condition being treated, are envisioned. Liquid dosage forms for administration include buffers and solubilizing agents, preferred diluents such as water, preservatives such as thymosol, and one or more biopolymers or polymers for conditioning the solution, such as polyethylene glycol, hydroxypropylmethylcellulose, sodium hyaluronate, sodium polyacrylate or tamarind gum. Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active agent is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. For example, an antiviral or antibacterial vaccine accompanied by ocular or cutaneous infections may be treated with aqueous drops, a mist, an emulsion, or a cream. Administration may be therapeutic or it may be prophylactic. Prophylactic formulations may be present or applied to the site of potential wounds, or to sources of wounds, such as contact lenses, contact lens cleaning and rinsing solutions, containers for contact lens storage or transport, devices for contact lens handling, eye drops, surgical irrigation solutions, ear drops, eye patches, and cosmetics for the eye area, including creams, lotions, mascara, eyeliner, and eyeshadow. The invention includes transdermal devices, surgical devices, audiological devices or products which contain disclosed compositions (e.g., gauze bandages or strips), and methods of making or using such devices or products. These devices may be coated with, impregnated with, bonded to or otherwise treated with a disclosed composition.

The ointments, pastes, creams, and gels may contain, in addition to an active agent of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the agents of this invention, excipients such as talc, silicic acid, aluminum hydroxide, calcium silicates, polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlled delivery of the active ingredients to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. In order to prolong the effect of an active agent, it is often desirable to slow the absorption of the agent from subcutaneous or intramuscular injection. Delayed absorption of a parenterally administered active agent may be accomplished by dissolving or suspending the agent in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the agent in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of active agent to polymer and the nature of the particular polymer employed, the rate of active agent release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the agent in liposomes or microemulsions that are compatible with body tissues.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the active agent(s) of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active agent(s).

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active agent(s) may be admixed with at least one inert diluent such as sucrose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active agent(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Uses of Pharmaceutical Compositions

As discussed above and described in greater detail in examples in the Appendices, administered nucleic acid vaccines are useful as stimulators of antibody development, or as inhibitors of allergic reaction, by intracellular synthesis of an immunogen or allergen or portion thereof (which is a potent and specific ligand of the appropriate MHC protein involved in immune response or allergic response). In general, it is believed that these nucleic acid preparations will be clinically useful in retarding or even preventing a number of autoimmune conditions including multiple sclerosis, myasthenia gravis, Hashimoto's disease, systemic lupus erythematosis, uveitis, Guillain-Barre syndrome, Grave's disease, idiopathic myxedema, autoimmune oophoritis, chronic immune thrombocytopenic purpura, colitis, diabetes, psoriasis, pemphigus vulgaris, and rheumatoid arthritis. It is believed that the nucleic acid preparations will be clinically useful in retarding or even preventing a number of infectious diseases, including viral diseases and bacterial diseases. . It is believed that the nucleic acid preparations will be clinically useful in retarding or even preventing a number of allergies, including allergies to antigens of plant origin and animal origin. In each case of use of the nucleic acids, the nucleic acids comprise a nucleotide sequence encoding an appropriate amino acid sequence which is at least 6- 7 amino acids in length, or at least 15 amino acids in length, or 25 amino acids in length, capable of being operably expressed in a cell of a recipient subject or patient, and capable of interacting with the appropriate MHC protein of the recipient.

It will be appreciated that the therapeutic nucleic acids encompassed by the present invention are not limited to treating the above conditions in humans, but may be used to treat these conditions in any mammal including but not limited to farm animals including mammals such as bovine, canine, feline, caprine, ovine, porcine, murine, and equine species, and zoo animals such as elephants and exotic feline and equine species. When treating a given species, it is preferred, but not required, that the amino acid sequence is appropriate to the amino acid sequence of pathogen or immunogen or antigen as it occurs naturally in the species.

EXAMPLES General Methods

AU manipulations were performed at room temperature (about 25 degrees C), and generally known routine procedures described in standard method texts were followed. See, for example, Short Protocols in Molecular Biology, Third Edition, Ausubel, F.M., et al, Ed., New York: J.Wiley & Sons, 1995; Ausubel, F.M., et al. Current Protocols in Molecular Biology. J. Wiley & Sons, New York, 1997; and Sambrook, J., E.F. Fritsch, and T. Maniatis. Molecular Cloning: a Laboratory Manual (3rd. edition). Cold Spring Harbor Laboratory Press, Plainview, N.Y., 2000. Preparation of DNA from bacteria for high throughput plasmid production

In general a bacterial culture is grown in a culture volume from 0.2 to 2 mL roughly) overnight and the bacteria are pelleted by centrifugation. The supernatant is decanted, and to the pellet containing the bacteria, the following are added: 150μL of Solution I, and the bacteria are resuspended; 150 μL of Solution II, and the contents are mixed gently; and 150 μL Solution III, and the contents are mixed gently. Formulae for preparation of the three solutions are shown in Table 1 and are essentially as described in the Maniatis et al.

Table 1. Preparation of Solutions for Production of DNA from bacteria

Solution I - Keep at 4°C

To make stock RNAseA: add 1 gram RNAse A (Sigma R.4875) to 1OmL deionized H₂O. Boil 5 minutes, aliquot and store frozen.

Solution II - store at room temperature

Solution III - store at 4°C

Titrate the pH to 4.8 using concentrated HCl, fill to volume indicated. The lysate is cleared by transfer of each to a filter plate followed by centriguation spinning through the filter plate. This cleared lysate is then added to lOmg of cholestyramine, as follows. A stock solution contains 100mg/mL of the slurry of the cholestyramine resin in water; an aliquot of volume 100 μL of this slurry is added to each well of a 96 well plate to make a plate having 96 affinity chromatography wells. The plate is centrifuged for 4 minutes at 50xg at room temperature, to collect the DNA bound to the resin as a pellet in each well. The pelleted DNA is washed by conventional buffers, for example, 250 μL of

5OmM MOPS (3-[N-Morpholino]propanesulphonic acid), pH7 and NaCl (low salt, for example in the range of 50OmM to 1.25mM). Alternative buffers can be used for the wash, for example, buffers comprising ethanol or isopropanol or nonionic detergents). Following the wash, the DNA is again collected by centrifugation at 50xg for 4 minutes. The DNA is then eluted in high salt, a solution of 150 μL of 2M NaCl, 1OmM Tris, pH8.5. While NaCl at 2M is used, it is envisioned that any salt at a high concentration will elute a nucleic acid from the cholestyramine resin.

To remove the DNA from the high salt, the eluate is added 100 μL isopropanol and the mixture is loaded onto a filter plate 0.45 micron (or 0.2 micron or 0.1 micron), and centrifuged 750xg for 4 minutes. The DNA collected on the surface of the filter plate is washed with 80% ethanol, and the DNA is again collected by centrifugation at 750xg for 4 minutes. The DNA is then eluted by dissolving in 150 μL TE buffer added to the well, for example, in 10 mM Tris (pH 7.5 to 8.0) with or without ImM EDTA. Dissolved DNA is collected by removal from the well with a pipet, or by filtering by centrifugation into a fresh set of wells.

Plasmids were tested for purity as shown in Fig. 1, for endotoxin contamination, and for biological function measured as transfection efficiency. In tests of transfection efficiency, plasmids isolated by the methods herein are as efficient as those prepared by a procedure used for production from large volumes of cell cultures and many steps of purification, for example, the Maxiprep DNA method available from Qiagen.

Example 1. Use of cholestyramine resin for high throughput isolation of nucleic acids Accordingly, more than a 15 resins commercially available anion exchange resins were obtained, for example, from Sigma Corp., St. Louis, MO, and from ResinTech, West Berlin, NJ. and tested for chromatographic properties with respect to absorption of DNA from a plasmid grown in Escherichia coli. Fig. 1 shows data obtained with replicate bacterial cultures (plasmid grown in

Escherichia coli) each culture having the same plasmid, purified by absorption onto commercially available Cholestyramine resin. Data show that the amount and purity, as well as uniformity in size (single band) from each culture was high, and results were replicable. These data compared favorably to the 4 columns on the right side of Fig. 2, which show data with Qiagen's anion exchange resin, as a positive control. Comparison with the data in Fig. 1 show that the cholestyramine resin (Sigma) is superior to the anion exchange resin.

Examples 2-15. Results with additional commercially available resins Further, the following were also tested herein as part of the program to obtain novel methods for isolation of DNA. Data shown in Fig. 2 indicated that in terms of yield these were found herein to be inferior to cholestyramine resin of Example 1 (Sigma):

Example 2. DEAE Sepharose

Example 3. Q sepharose

Example 4. silica-polyethyleneimine

Example 5. Amberlyst A 26

Example 6. Amberlite IRA 400 Example 7. Amberlite IRA 900Amberlyst A21

Example 8. Amberjet 4200

Example 9. Amberlyte IRA 67

Example 10. Resintech SBG2 RTl -10953

Example 11. Resintech WBG30B 16610 Example 12. Resintech P8972 SIR-700

Example 13. Resintech P8494 WBG30-OH

Example 14. Biorad Macroprep High Q

Example 15. Biorad Macroprep DEAE Data from Examples 2-15 were observations on yield and purity of DNA from cell extracts which were mixed with each of the resins. These data indicated low yields, so that Fig. 2 was overexposed, in order to obtain sufficient data to visualize any plasmid that might have absorbed to the resin in each preparation. It is a conclusion from the above examples that, surprisingly, cholestyramine resin specifically absorbs DNA or another type of nucleic acid with sufficient specificity, affinity and reversibility to be of use as a reagent for purification for research purposes and for purification for use as a vaccine. However other resins tested herein were inferior to that of cholestyramine.

Example 16. Downstream processing of nucleic acids subsequent to binding to cholestyramine

After the DNA was eluted from the cholestyramine resin in high salt, it was necessary prior to use that the high salt must be removed from the DNA. The salt is inhibitory for many of the purposes to which the DNA is purified.

In existing protocols such as the QIAGEN 96 well ultraprep (which is the state of the art 96 well anion exchange tranfection quality kit), several additional steps are required to get rid of the salt, for example, binding DNA to a plate with silica resin, then washing and finally eluting the nucleic acid in a buffer having a low salt concentration (a suitable buffer is TE, which as is commonly known in the art is Tris- HCl 1OmM, EDTA 1 mM, pH 7.4, 7.5, 7.9 or 8.0), after which the DNA is then suitable for use.

A downstream processing protocol is provided herein for removing the high salt from the DNA after eluting from cholestyramine. This protocol is an improvement, as it requires fewer steps that those described above, and produces a yield of equal or greater amount of DNA.

In general in the method provided herein, the DNA is eluted from the cholestyramine using a high salt buffer, then 0.7 volumes of isopropanol is added to the eluate to precipitate the DNA. Each sample is passed through an individual well of a 96 well filter plate (having a pore size, for example, of 0.45 micron, 0.2 micron or 0.1 micron), such that the filter captures the precipitated DNA. The DNA on the surface of the filter is then washed with 80% ethanol in water, and DNA is then eluted by dissolving in low salt buffer such as TE. The DNA is collected by filtering and centrifugation.

Example 17. Cholestyramine isolation has increased purity as analyzed by transfection efficiency Data shown in Fig. 3 indicates that plasmid prepared by the methods herein are much more efficient than two other methods, as indicated by increased yield of transfected cells per 100 ng of DNA. Successful transfectants express GFP and appear as bright cells. The photograph in Fig. 3 C shows a much larger number of cells and a greater proportion of successful transfectants obtained with DNA isolated by the cholestyramine-resin method provided herein, than shown in the photographs in Figs. 3A and 3B. Clearly the cholestyramine-resin method provided yields DNA having much less cytotoxic contaminating and transfection-inhibiting materials.

Claims

What is claimed is:

1. A method for purifying at least one nucleic acid, comprising contacting the nucleic acid with a hypolipidemic agent, wherein the agent binds to the nucleic acid to form a resulting complex; and eluting the nucleic acid, wherein nucleic acid eluted is purified.

2. The method according to claim 1, wherein contacting the nucleic acid is contacting a cleared cell lysate.

3. The method according to claim 1, wherein the at least one nucleic acid is a plurality of nucleic acids.

4. The method according to claim 1, wherein eluting from the complex is contacting the complex with an aqueous solvent of high salt concentration.

5. The method according to claim 4, wherein prior to eluting the nucleic acid the complex is rinsed with an aqueous solvent of intermediate salt concentration.

6. The method according toclaim 1, wherein the hypolipidemic agent comprises at least one positively charged functional group that binds bile acids.

7. The method according to claim I₅ wherein the nucleic acid is an RNA.

8. The method according to claim 1, wherein the nucleic acid is a DNA.

9. The method according to claim 1, wherein the nucleic acid is single stranded.

10. The method according to claim 1, wherein the nucleic acid is double stranded.

11. The method according to claim 1, wherein the nucleic acid is chromosomal DNA.

12. The method according to claim 1, wherein the nucleic acid is plasmid DNA.

13. The method according to claim 1, wherein the nucleic acid is viral nucleic acid.

14. The method according to claim 1, wherein prior to contacting, the hypolipidemic agent is immobilized.

15. The method according to claim 14, wherein the hypolipidemic agent is immobilized on a bead, a resin, a slide, a chip, a micro-cantilever, an inner surface of a centrifuge tube, or a micro-titer plate surface.

16. The method according to claim 15, wherein the bead is magnetic.

17. The method according to claim 1, wherein hypolipidemic agent is selected from a statin, a colestipol, a guggulsterone, and a cholestyramine.

18. The method according to claim 1, wherein the hypolipidemic agent is cholestyramine.

19. The method according to claim 18, wherein the cholestyramine is attached to a resin.

20. The method according to claim 19, wherein the resin is a polymer comprising a styrene-divinylbenzene copolymer backbone with attached trimethylbenzylammonium groups.

21. The method according to claim 20, wherein the polymer contains 4 gm cholestyramine resin per 9 gm total powder (QUESTRAN^®).

22. The method according to claim 20, wherein the polymer contains 4 gm cholestyramine resin per 5 gm total powder (QUESTRAN LIGHT^®).

23. A method for purifying a plurality of plasmid preparations comprising contacting a plurality of cell extracts containing plasmids with a cholestyramine resin, wherein the resin binds to the plasmid; and eluting the plasmid from the resin with an aqueous high salt solution, wherein eluted plasmid is purified.

24. The method according to claim 23, further comprising after contacting and prior to eluting, centrifuging the resin and bound plasmid.

25. The method according to any of claims 1-24, further comprising after eluting, filtering a plurality of plasmids or nucleic acids on a filter plate and rinsing with isopropanol and dissolving plasmids or nucleic acids in an aqueous buffer.

26. The method according to any of claims 1 -25, further comprising transfecting a cell with the eluted nucleic acid or plasmid.

27. The method according to any of claims 1 -26, further comprising: cloning a gene into any of the nucleic acid or plasmid eluted, wherein the gene encodes an amino acid sequence of an immunogen and the gene is operably cloned at a location in the nucleic acid for protein expression, to produce a resulting expression vector; and contacting a subject with the resulting expression vector, wherein the subject expresses the gene and produces antibodies to the immunogen.

28. A kit for purifying nucleic acids comprising a hypolipidemic agent immobilized to a solid surface such as a resin, a container, and instructions for use with a cell or viral extract.

29. The kit according to claim 28, wherein the agent is selected from a statin, a colestipol, a guggulsterone, and a cholestyramine.

30. The kit according to claim 28, wherein the agent is cholestyramine.

31. The kit according to any of claims 28-30, further comprising a DNA vector.

32. The kit according to any of claims 28-31 , further comprising a bacterial recipient.

33. The kit according to any of claims 28-32, further comprising buffers and reagents for DNA isolation and analysis.

34. The kit according to any of claims 28-33, further comprising a multi-well filter plate.

35. An article of manufacture for isolation of a nucleic acid, comprising a hypolipidemic agent attached to a surface.

36. The article according to claim 35, wherein the nucleic acid is a plurality of nucleic acids having a corresponding plurality of separable locations.

37. The article according to claim 35, wherein the surface is a glass, a metal or a plastic.

38. The article according to claim 37, wherein the surface is a metal chip, a metal micro-cantilever, or a glass slide.

39. The article according to claim 37, wherein the surface is a multi-well plastic plate or an inner surface of a plastic centrifuge tube.

40. A composition comprising a nucleic acid made by any of the methods herein.

41. The composition according to claim 40, comprising a nucleotide sequence encoding an amino acid sequence wherein the amino acid sequence is expressed in a cell, and elicits antibody production in a subject.

42. The composition according to claim 40, wherein the amino acid sequence is an antigen of a pathogen.

43. The composition according to claim 40, wherein the amino acid sequence is a binding partner of an IgE molecule and elicits IgG production.

44. The composition according to claim 42, wherein the pathogen is a virus.

45. The composition according to claim 44, wherein the pathogen is selected of the group consisting of an influenza, an HIV, a smallpox, a SARS, a Herpes, and a hepatitis.

46. The composition according to claim 45, wherein the influenza is a bird flu.

47. In a method for isolation of a nucleic acid, the improvement comprising absorbing the nucleic acid to a hyperlipidemic agent.

48. The method according to claim 47, wherein the agent is cholestyramine.

49. The method according to either of claims 47-48, wherein the nucleic acid is present in an extract of a biological material comprising at least about 1 mg, 10 mg, 100 mg, 1 g, or about 1O g of a tissue or cells of a cell line, or virions of a virus strain.

50. In a method for isolation of a nucleic acid from a plurality of high salt solutions, the improvement comprising precipitating the nucleic acids with isopropanol and collecting precipitated nucleic acid on a multi-well filter plate.