WO2010150089A1 - Highly efficient systems for delivery of nucleic acids - Google Patents

Abstract

Embodiments of the present application provide methods of synthesis and applications of oligoelectrolyte carriers for delivery of cyclic or linear nucleic acids to cells. In some embodiments, the invention provides methods of growing and selecting transformants following transformation of yeast cells, animal cells, plant cells, fungal cells, and/or prokaryotic cells using the oligoelectrolyte delivery molecules. In other embodiments, the invention provides methods of improving transformation efficiency by modifying the composition of medium or transformation conditions. In other embodiments, the invention provides methods of improving transformation efficiency by changing the physical conditions using a heat/cold shock method. In other embodiments, the invention provides methods of improving transformation efficiency by changing the physical conditions using a freeze/thaw method.

Description

HIGHLY EFFICIENT SYSTEMS FOR DELIVERY OF NUCLEIC ACIDS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 61/220,910, filed June 26, 2009, entitled "HIGHLY EFFICIENT SYSTEMS FOR DELIVERY OF NUCLEIC ACIDS," which is incorporated herein in its entirety by reference as if fully set forth herein.

FIELD OF THE INVENTION

[0002] The field of invention relates to the synthesis and the use of polynucleotide delivery molecules as carriers for immobilization and delivery of nucleic acids into cells. In particular, the invention concerns and embodies the synthesis and use of oligoelectrolyte carriers for nucleic acid delivery and protection. The use of polynucleotide delivery molecules permits a highly efficient, simple, low-cost method of genetic transformation (transfection) of cells.

BACKGROUND

[0003] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Delivery of cyclic or linear DNA to cells is a ubiquitous procedure in both basic research and applied biotechnological studies. In particular, transformation of yeast cells is widely studied for potential use in pharmaceutical and agricultural applications. Commonly used methods of genetic transformation are based on use of chemical agents (specific ions) or electroporation. Application of chemical transformation is cheaper and more convenient compared to electroporation. However, the process is still time consuming and requires specially prepared competent cells. Electroporation, which also requires using specially prepared competent cells, provides higher efficiency of gene delivery as compared to chemical transformation. However, it is often accompanied by DNA damage and thus may create unwanted consequences in gene expression. Furthermore, in the instance when a large number of transformed cells must be analyzed (e.g. screening of homologous recombinant cells), both the chemical transformation method and the electroporation method have low efficiency of transformation for the majority of yeast species which may slow down research considerably. This may be further compounded through the use of cell types or species or strain of yeast that, while widely used in research, are specifically associated with a low transformation efficiency level (e.g. Yarrowia lipolitica, Dekkera bruxellensis (Brettanomyces bmxellensis), Phqffia rhodozyma (Xanthophyllomyces dendrorhous), Hansenula polymorpha, Candida lipolitica, etc.). Other cell types including several animal cells and plant cells have also been shown to be highly resistant to transformation. Gene-gun technology provides higher transformation efficiency, but the Gene-gun machine is very expensive and the sorption of nucleic acids on the surface of nanoparticles is time-consuming and does not work properly for short nucleic DNA or RNA sequences. One can use other techniques such as protoplast techniques, but they are not widely used because of difficulties and low efficiency of transformation. As a result, genetic manipulations to cells such as yeast cells can be very complicated, as well as both time consuming and expensive.

[0005] Thus, there is a need in the art for the development of novel efficient systems for gene delivery to cells and organisms, especially to strains of yeast.

SUMMARY

[0006] Some embodiments include a polynucleotide delivery molecule, which can have an oligoelectrolyte component with a backbone and 1 or more side chains and a polynucleotide molecule component bound to the oligoelectrolyte. The side chains can be adapted to penetrate a cell plasma membrane. The backbone of the oligoelectrolyte can include anionic groups and/or nonionic groups, and the side chains of the oligoelectrolyte can include cationic groups and/or nonionic groups. The side chains of the oligoelectrolyte can be grafted. In some embodiments, the oligoelectrolyte can include, for example, dimethylaminoethyl methacrylate (DMAEMA) and 5-(tertbutylperoxy)-5-methyl-l-hexen-3-yne (VEP). The sequences for delivery can be linear, circular, integrating, or nonintegrating, single-stranded, or double stranded sequences of nucleic acids.

[0007] Some embodiments include a method of constructing the polynucleotide delivery molecule by adding a monomer to another monomer to create a mixture, adding a solution of oligoperoxide metal complex (OMC), increasing the temperature, subjecting the mixture to Argon (Ar) flow, removing the solvent via vacuum distillation, dissolving the solvent in acetone, and subjecting the solvent in acetone to a multi-stage precipitation process to yield a precipitate. In some embodiments, the precipitate can be further dried until constant weight is achieved.

[0008] Some embodiments include a method of transforming a cell using the polynucleotide delivery molecule described in the preceding paragraphs. An effective amount of the polynucleotide delivery molecule can be administered to the cell to effectuate transformation, and the efficiency of transformation can be adjusted. In some embodiments, the cell can be an animal cell, a plant cell, a fungal cell, and/or a prokaryotic cell. The animal cell can be, but is not limited to, a mammalian cell, which can be, for example, a human cell. Merely by way of example, the human cell can be a 293T cell. In some embodiments, the polynucleotide delivery molecule can be administered in vivo. In some embodiments, the plant cell can be, but is not limited to, a protoplast. The prokaryotic cell can be of the species Escherichia coli , or of the species Streptomyces lividans, or other species. In some embodiments, the cell can be a yeast cell, such as, for example, of the species Hansenula polymorpha, Pichia pastoris, or Saccharomyces cerevisiae and the like.

[0009] Some embodiments include a method to effectuate stable transformation, which can include subjecting the cell to an incubation period in a transformation medium. In some embodiments, the efficiency of transformation can be adjusted by modifying the composition of the transformation medium. The transformation medium can contain, for example, dimethyl sulfoxide (DMSO) and CaCl₂. In some embodiments, the concentration of DMSO and/or CaCl₂ can be modified to change the transformation efficiency. In some embodiments, the transformation efficiency can be adjusted by subjecting cells to a heat/cold shock, and/or by freezing and thawing the cells. The cells can be subjected to cold shock only, or the cells can be subjected to heat shock only. The cells can be subjected to a heat shock followed by a cold shock, and/or a cold shock followed by a heat shock, or any combinations therof. The cells can be further frozen and thawed, which can also increase the transformation efficiency. [0010] Some embodiments include a polynucleotide delivery molecule, which can have an oligoelectrolyte component with a backbone and 1 or more side chains, which can be adapted to penetrate a cell plasma membrane, and a polynucleotide molecule bound to the oligoelectrolyte, and a pharmaceutically acceptable carrier component.

BRIEF DESCRIPTION OF THE FIGURES

[0011] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0012] Figure 1 depicts, in accordance with an embodiment herein, the general structure of an oligoelectrolyte carrier molecule and examples of fragments that can be the groups R2 or R3 (A), and monomeric components of the oligoelectrolytes, dimethyl aminoethyl methacrylate (DMAEMA) (B) and 5-tertbutylperoxy-5-methyl-l-hexene-3-yne (C), that can be polymerized to yield the polymeric oligoelectrolyte carriers.

[0013] Figure 2 depicts, in accordance with an embodiment herein, the structure of an example of an oligoelectrolyte as a carrier for immobilization and delivery and protection of nucleic acids.

[0014] Figure 3 depicts the structure of a commercially available branched polyethylenimine for cell transformation.

[0015] Figure 4 depicts, in accordance with an embodiment herein, the structure of an oligoperoxide metal complex (OMC).

[0016] Figure 5 depicts, in accordance with an embodiment herein, examples of results from three different methods of transformation after being plated on selective culture medium. Commercially available branched polyethylenimine showed 1.8 times lower efficiency of transformation compared to traditional Li/Acetate-based chemical transformation method, and 28 times lower efficiency of transformation compared to the inventors' system based on the oligoelectrolytes.

[0017] Figure 6 depicts, in accordance with an embodiment herein, a chart comparing transformation efficiency of Hansenula polymorpha NCYC 495 yeasts using classic Li/Ac method (Chem), the use of the inventors' system based on the oligoelectrolytes (Hare-1), and electroporation.

[0018] Figure 7 depicts, in accordance with an embodiment herein, the results of outside confirmation of transformation efficiency of the inventors' system (Harel). Hansenula polymorpha NCYC 495 yeasts were used and plasmid of 5.4 kbp. "Chem" represents classic Li/Ac based method for yeast transformation; "electro" represents electroporation.

[0019] Figure 8 depicts, in accordance with an embodiment herein, the results of transformation of Pichia pastoris GSl 15 yeasts using circular nonintegrating plasmid.

[0020] Figure 9 depicts, in accordance with an embodiment herein, the results of transformation of Pichia pastoris GSl 15 yeasts using linearized plasmid DNA..

[0021] Figure 10 depicts, in accordance with an embodiment herein, a chart comparing the number of colonies resulting from transformation of Saccharomyces cerevisiae yeasts using circular plasmid DNA.

[0022] Figure 11 depicts, in accordance with an embodiment herein, a chart comparing the number of stable transformants of Hansenula polymorpha NCY C 495 yeasts.

[0023] Figure 12 depicts, in accordance with an embodiment herein, the growth of transformants on solid selective medium.

[0024] Figure 13 depicts, in accordance with an embodiment herein, the growth of transformants on liquid selective medium.

[0025] Figure 14 depicts, in accordance with an embodiment herein, the effect of changes in the composition of medium on the transformation of Hansenula polymorpha NCYC 495 yeasts.

[0026] Figure 15 depicts, in accordance with an embodiment herein, the results of changing transformation procedure (such as, for example, using heat and/or cold shock) in order to optimize transformation of Hansenula polymorpha NCY C 495 yeasts.

[0027] Figure 16 depicts, in accordance with an embodiment herein, the results of changing transformation procedure (such as, for example, using heat and/or cold shock) in order to optimize transformation of Pichia pastoris GSl 15 yeasts. [0028] Figure 17 depicts, in accordance with an embodiment herein, the effect of destabilization of plasma membrane by freezing/thawing of the cells on Pichia pastoris GSl 15 yeast transformation.

[0029] Figure 18 depicts, in accordance with an embodiment herein, the results of transfection of human 293T cells using polyethylenimine (PEI) or the polymer electrolyte carriers.

[0030] Figure 19 depicts, in accordance with an embodiment herein, the results of DNA delivery to Streptomyces lividans cells using the fusion DNA delivery method or the polymer electrolyte carriers.

DETAILED DESCRIPTION

[0031] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et ah, Dictionary of Microbiology and Molecular Biology 3^rd ed., J. Wiley & Sons (New York, NY 2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5^th ed, J. Wiley & Sons (New York, NY 2001); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 3rd ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2001), provide one skilled in the art with a general guide to many of the terms used in the present application.

[0032] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods, materials, applications, and objects of application described.

[0033] As disclosed herein, the inventors tested novel branched oligoelectrolytes as carriers for delivery of nucleic acids to cells. These specially designed systems contain oligoelectrolytes possessing chemical groups for both plasma membrane binding and penetration and nucleic acids binding, and were used for the immobilization, protection and delivery of nucleic acids to cells. The cells can be of the species Hansenula polymorpha and other yeast species such as Pichia pastoris or Saccharomyces cerevisiae. An advantage of the novel oligoelectrolyte based systems for gene delivery is that, in contrast to using classical transformation procedures, specially prepared competent cells are not required. It is also much faster, cheaper, and more effective than electroporation and other currently known chemical transformation methods.

[0034] Notably, the oligoelectrolyte carriers are also highly effective in the transformation of Hansenula polymorpha, which are strongly resistant to genetic transformation, especially when using chemically-based transformation assays.

[0035] The oligoelectrolytes used for polyplex preparing were synthesized via solution radical polymerization initiated by surface-active oligoperoxide metal complexes as low temperature radical initiators providing efficient grafting of polymeric chains with specific length, functionality and reactivity. Compounds that can act as oligoelectrolyte carriers of nucleic acids are depicted in a general structure shown in Figure 1, where R2 and R3 are monomer links of backbone that can bear hydrophobic, hydrophilic, nonionic, electrolyte anionic or cationic groups. R2 and R3 can be repeating units of monomers such as, for example, those depicted in Figure 1. Rl can be cationic or cationic and nonionic simultaneously. R2 and R3 can be, but are not limited to, hydrocarbons such as alkylene. R2, and R3 can be, but are not limited to, polymer links of sodium styrene sulfonate, vinyl benzyl trimethyl ammonium chloride, polyacrylic acid, hydrolyzed copolymers of styrene and maleic anhydride, polyvinyl sulfonic acid, sulfonated polystyrene, sulfonated polyvinyl toluene, alkali metal salts of the foregoing acidic polymers, polyphosphoric acid, polyvinylsulfuric acid, polyvinylphosphonic acid, polyacrylic acid, and the like. Rl can be links of vinyl benzyl trimethyl ammonium chloride, polyethyleneimine, polyvinyl pyridine, and polydimethylaminoethyl methacrylate, quaternized polyethylene imine, quaternizedpoly (dimethylaminoethyl) methacrylate, polyvinyl methyl pyridinium chloride, polyallylamine, polyethyleneimine, polyvinylamine, polyvinylpyridine and the like. The oligoelectrolyte can have a backbone structure and side chains, which can give the carrier a branched structure that can allow it to bind the nucleic acid to be delivered and penetrate the cell membrane. The backbone can be linked to 1 or more side chains.

[0036] In some embodiments, the oligoelectrolytes are synthesized using monomers. The monomer can be dimethylaminoethyl methacrylate (DMAEMA) (Figure IB herein) and/or other hydrophilic cationic or nonionic groups, 5-tertbutylperoxy-5-methyl-l-hexene-3-yne (Figure 1C herein) and/or other hydrophobic groups. The polymeric oligoelectrolyte can be formed by attaching a plurality of units of the monomer of the invention to a polymeric substrate containing a plurality of functional groups reactive with the monomer. The polymeric substrate may be aliphatic or aromatic.

[0037] In various embodiments, the present invention provides a method of performing cell transformation by administering a polynucleotide bound to an oligoelectrolyte to the cell. In some embodiments, the present invention provides a method of cell transformation by administering a mixture of oligoelectrolyte and polynucleotide to a chilled pellet of cells which can be followed by incubation. In some embodiments, changing conditions of the transformation or destabilization of the cell membrane by chemical or physical influences can improve transformation efficiency. In some embodiments, the oligoelectrolyte includes a plasma penetration domain. In another embodiment, the oligoelectrolyte is synthesized by solution radical polymerization initiated by surface-active oligoperoxide metal complexes as low temperature radical initiators. In some embodiments, the oligoelectrolyte contains an anionic backbone and one or more cationic side chains. In some embodiments, the oligoelectrolyte is depicted in Figure 2 herein.

[0038] In some embodiments, the oligoelectrolyte is a polymer with dissociable ionic groups. In some embodiments, the ionic groups of the backbone impart electrolytic characteristics in forming salts and acids. In some embodiments, the oligoelectrolyte backbone can be polymers of sodium styrene sulfonate, and/or the grafted branch and backbone can be polymers of vinyl benzyl trimethyl ammonium chloride or other material of the same general type having an organic polymeric structure, which would be water insoluble film-forming material in the absence of the ionic groups. Merely by way of example, the backbone of polymers can be polyacrylic acid, hydrolyzed copolymers of styrene and maleic anhydride, polyvinyl sulfonic acid, sulfonated polystyrene, sulfonated polyvinyl toluene, polyphosphoric acid, polyvinylsulfuric acid, polyvinylphosphonic acid, polyacrylic acid, alkali metal salts of the foregoing acidic polymers, and the like. Merely by way of example, the side branches and backbone of polymers can be polyethyleneimine, polyvinyl pyridine, and polydimethylaminoethyl methacrylate, quaternized polyethylene imine, quaternized poly (dimethylaminoethyl) methacrylate, polyvinyl methyl pyridinium chloride, and the like. The ionic groups of backbone can be, but is not limited to, sulfonate, and the side branches can be quaternary ammonium and the like. In some embodiments, the ionic groups impart electrolytic characteristics in forming bases. The electrolyte side branches can be, but is not limited to, polyallylamine, polyethyleneimine, polyvinylamine, polyvinylpyridine, and similar polybases with groups capable of dissociation located on the chains or laterally. In some embodiments, the oligoelectrolytes can contain cationic, or both cationic and anionic, groups. In some embodiments, the oligoelectrolytes can contain nonionic groups. The oligoelectrolyte can be low molecular weight polyelectrolytes, polyions, or macromolecular polyelectrolytes. In some embodiments, the oligoelectrolyte can be of biological origin.

[0039] In some embodiments, the oligoelectrolyte is a strong electrolyte, which undergoes complete dissociation in solution for most pH values. In some embodiments, the oligoelectrolyte is a weak electrolyte, which undergoes partial dissociation at intermediate pH values, and has a dissociation constant (pKa or pKb) in the range of about 2 to about 10. In some embodiments, the oligoelectrolyte can be an ionomer in which the concentration of ionic groups is insufficient for water solubility, but the charges are sufficient for self-assembly.

[0040] In some embodiments, the cell is a yeast cell. In some embodiments, the yeast is

Hansenula Polymorpha. In some embodiments, the yeast is Pichia pastoris. In some embodiments, the yeast is Saccharomyces cerevisiae. In some embodiments, the cell is a plant cell, animal cell, fungal cell, or a prokaryotic cell.

[0041] In various embodiments, the present invention provides methods to improve the efficiency of transformation using the oligoelectrolyte carrier and modifications to the transformation conditions. In some embodiments, the modifications can include changes to the composition of the medium, such as, for example, changes in the concentration of DMSO and/or changes in the concentration of CaCl₂. In some embodiments, the modification can include a heat/cold shock method, which exposes the cell to a high temperature and/or a low temperature, or a combination of high or low temperature exposures in any order. The high temperature can be about 3O⁰C, or about 35 ⁰C, or about 40⁰C, or about 45⁰C, or about 50⁰C, or about 55°C, or about 60⁰C, or about 65°C, or about 70⁰C, or about 75°C, or about 80⁰C, or about 85⁰C, or about 9O⁰C, or about 95⁰C, or about 100⁰C, or about 105⁰C, or about 1 10⁰C. The low temperature can be below -40⁰C, or about -40⁰C, or about -35 ⁰C, or about -30⁰C, or about -25°C, or about -2O⁰C, or about -15°C, or about -10⁰C, or about -5°C, or about 0⁰C, or about 5⁰C, or about 10⁰C, or about 15°C, or about 20⁰C, or about 25⁰C.

[0042] In various embodiments, the present invention provides methods of synthesis and applications of oligoelectrolyte carriers for delivery of nucleic acids to prokaryotic cells, yeast cells, or plant cells, and includes methods and applications that can be useful in agriculture, energy, and biotechnology. Such methods and applications can be, but are not limited to, plant biology, eco-toxicology, aquaculture, biopharmaceutical production and processing.

[0043] In various embodiments, the present invention provides pharmaceutical compositions including a pharmaceutically acceptable excipient for nucleic acid delivery along with a therapeutically effective amount of oligoelectrolyte. "Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition (which can include nucleic acids) that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous

[0044] In various embodiments, the pharmaceutical compositions for nucleic acid delivery according to the invention may be formulated for delivery via any route of administration. "Route of administration" may refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, transmucosal, transdermal or parenteral. "Parenteral" refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders.

[0045] The pharmaceutical compositions for nucleic acid delivery according to the invention can also contain any pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting nucleic acids or other compounds of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier for nucleic acids delivery may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be "pharmaceutically acceptable" in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits. [0046] The pharmaceutical compositions for nucleic acid delivery according to the invention can also be encapsulated, tableted or prepared in an emulsion or syrup for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax.

[0047] The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.

[0048] The pharmaceutical compositions for nucleic acids delivery according to the invention may be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

[0049] Typical dosages of an effective branched oligoelectrolyte bound to polynucleotide can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models. Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity. Thus, the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.

One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0050] Embodiments of the present application are further illustrated by the following examples.

EXAMPLES

[0051] The following non-limiting examples are provided to further illustrate embodiments of the present application. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches discovered by the inventors to function well in the practice of the application, and thus can be considered to constitute examples of modes for its practice. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the application.

[0052] Example 1: Branched olioelectrolytes as carriers for delivery of nucleic acids -

Generally

[0053] The inventors have created novel branched oligoelectrolytes as carriers for delivery of both linear and cyclic nucleic acids to yeast cells. These specially designed systems contain oligoelectrolytes possessing chemical groups for binding and/or penetrating the plasma membrane and binding to nucleic acids, and were synthesized via solution radical polymerization initiated by surface-active oligoperoxide metal complexes as low temperature radical initiators providing efficient grafting of polymeric chains with specific length, functionality and reactivity (Figure 1 herein). These oligoelectrolytes were applied for immobilization and delivery of nucleic acids to Hansenula polymorpha and other yeast species, such as, for example, Pichia pastoris or Saccharomyces cerevisiae. One advantage of these novel oligoelectrolyte based systems for gene delivery is that its application, in contrast to using classical transformation procedures, does not require specially prepared and/or treated competent cells. It is also much faster than electroporation and chemical transformation methods. This delivery system demonstrated 5 to 60 times higher efficiency of yeast transformation compared to traditional chemical-based procedure, and 2 to 6 times higher efficiency compared to electroporation. Finally, the delivery of nucleic acids to yeast cells by branched oligoelectrolytes is much cheaper than other known methods that may require costly equipment.

[0054] Example 2: Attaching the oligoelectrolyte to a nucleic acid and then delivery to cell

[0055] The oligoelectrolyte used for polyplex preparing is a co-polymer of branched structure combining oligoelectrolytes of anionic type, as a backbone, and 1-3 grafted side chains of cationic type (Figure 1 herein). The combination of these chains in the designed molecule provides controlled solubility in a wide pH range, high surface activity, and ability to form interpolyelectrolyte complexes in water based systems.

[0056] The procedure of preparing the gene delivery system is based on adding a mixture of the oligoelectrolyte with the nucleic acid (to be delivered) to chilled pellet of exponentially growing yeast cells (OD₆₀₀ 0.4-0.6), and following heat/cold shock (41⁰C for 1 minute / O⁰C for 5 minutes). Afterwards, yeast cells are incubated for 30 minutes, normal culture medium added and yeast cells grown up for 1 hour under normal growing conditions that allowed selective marker protein to be synthesized. After that, yeast cells are plated on selective culture medium. No preliminary pretreatments, preparation of competent cells, or special equipment are required for performing the transformation procedure. Efficiency of transformation of Hansenula polymorpha (strain NCYC 495) yeast, when using the oligoelectrolyte based carriers, was 2 times higher than the efficiency of transformation of the yeast when using electroporation and 15 times higher than traditional Li/Acetate-based chemical transformation methods. Branched polyethylenimine (Sigma- Aldrich) used for the transfection of mammalian cells is the closest molecular analogue of utilized gene delivery systems. It showed 1.8 times lower efficiency of transformation compared to the traditional Li/Acetate-based chemical transformation method, and 28 times lower efficiency of transformation, compared to the inventors' oligoelectrolytes system. T-test was applied to prove statistical significance of the difference in yeast transformation efficiency observed using different methods of transformation.

[0057] Example 3: Advantages

[0058] In some embodiments, the invention relates to oligoelectrolyte based systems for delivery of nucleic acids to yeast cells. Principal advantages include: a) easy application, b) faster result achievement, c) no need of cell pretreatment or preparing competent cells, d) lower cost (does not require special costly equipment or reagents), and e) higher efficiency (compared to electroporation and chemical-based procedures of cell transformation). Specifically, the system is rapid (transformation procedure lasts for 30 hours), convenient (no need to use specially prepared competent cells: the procedure can include adding nucleic acid and novel branched oligoelectrolytes to the chilled pellet of yeast cells with or without subsequent heat/cold shock), and has significantly higher efficiency of transformation (1.8-2 to 60 times more efficient than any other known method). No costly equipment or reagents are needed in the case of using developed by us novel gene delivery systems. Additionally, the inventors' gene delivery polyplexes also proved to be efficient in special cases, when species of yeast that are known to give low efficiency of transformation under known transformation procedures are treated. For example, the developed nucleic acids delivery systems based on branched oligoelectrolytes were effective for Hansenula polymorpha and Pichia pastoris strains which are extremely resistant to genetic transformation, especially to chemically-based transformation assays.

[0059] These unique characteristics of nucleic acids delivery systems described herein can be used for genetic transformation of yeasts used in both basic research and applied biotechnology. In one embodiment, oligoelectrolyte based novel gene delivery systems can also be used for delivery of a nucleic acid into yeast.

[0060] Example 4: Table 1 - Characteristics of the oligoelectrolytes

[0061] The functional oligoelectrolyte can be a copolymer of branched or comb-like structures combining oligoelectrolytes of anionic type backbone and 1-3 grafted side chains of cationic type. The combination of these chains in the carrier molecule provides their controlled solubility in a wide pH range, high surface activity and ability to form and stabilize interpolyelectrolyte complexes and derived water based systems. Some characteristics of the branched oligoelectrolyte carriers are presented in Table 1 below:

Table 1. - Characteristics of the oligoelectrolytes

/ - average length of the blocks from corresponding monomer links, R - amount of the blocks from the same monomer links per 100 links in the copolymer.

[0062] Example 5: Synthesis of oligoelectrolytes

[0063] The oligoelectrolytes were synthesized via controlled radical polymerization initiated by the oligoperoxide metal complex (OMC) structure described herein in a polar organic media. The OMC can initiate controlled radical polymerization reactions.

[0064] A structure of the OMC is shown in Figure 4. In this example, the OMC is a coordinating complex of Cu²⁺ of oligoperoxide that is a copolymer of vinyl acetate, 5- terΛ>utylperoxy-5-methyl-l-hexene-3-yne and maleic anhydride, in which k = 22%, 1 = 34%, m = 42%, x = 2%, and [Cu] = 1.1%. The initial oligoperoxide and derived OMC were synthesized as previously described (A.S. Zaichenko et al., J Appl Pol Sci 67, 1061-1066 (1997); A.M. Toportseva et al. Chemistry 416, (1972); M.R. Vilenskaya et al. Khimicheskaya Promishlennost 7, 15-16 (1979)).

[0065] Synthesis of the branched oligoelectrolyte was carried out in accordance with the following technique: monomer mixture of dimethylaminoethyl methacrylate (DMAEMA) (from 17.13 to 28.18 g, optimal results achieved at 28.18 g) and 5-(terrt>utylperoxy)-5-methyl-l-hexen- 3-yne (VEP) (from 3.19 to 17.13 g, optimal results achieved at 3.19 g) were charged into round bottom glass reactor equipped with impeller mixer and backflow condenser; then solution of OMC (1.66 g) in alcohol or dimethyl formamide (67 g) was added at the stirring; the temperature was increased to 60⁰C, and the reactor was thermostated at that temperature for 8 hours under Ar flow; the monomer conversion was controlled using gravimetric technique for achievement of 60%; after the achievement of desired conversion at polymerization, the solvent was removed via vacuum distillation; final product was dissolved in acetone and thoroughly purified by multi-stage precipitation into hexane, next dried till constant weight under the vacuum.

[0066] Monomers: 5-(tertbutylperoxy)-5-methyl-l-hexen-3-yne was synthesized and characterized. After purification by vacuum distillation, its characteristics were: d₄ ²⁰=0.867 (ref. 0.8670); ri_d ²⁰=1.4480 (ref. 1.4482). Dimethylaminoethyl methacrylate (Aldrich) was used without additional purification.

[0067] Example 6: Transformation of yeast using circular nonintegrating plasmid

[0068] The polymeric oligoelectrolyte carriers are effective in delivering circular nonintegrating plasmid into yeast cells.

[0069] The procedure of preparing the gene delivery system using circular nonintegrating plasmid is based on growing Pichia pastoris yeast cells on the complete YPD (yeast extract, peptone, glucose) liquid medium to exponential growth (OD₆₀₀ 0.4-0.9) at 30⁰C (normal for P. pastoris growing conditions), pelleting the cells by centrifugation, suspending the pellet in complete medium and adding the polymer and 6.2 kbp circular plasmid DNA to the cell suspension. Afterward, yeast cells are incubated for 1 hour at 30⁰C or higher under normal growing conditions that allow selective marker protein to be synthesized, and yeast cells are plated with solid selective His-deficient medium. Following incubation for 5 days under standard conditions, the number of transformants colonies are counted and compared. No preliminary pretreatments, preparation of competent cells, or special equipment are required for performing the transformation procedure. Efficiency of transformation of Pichia pastoris GSl 15 yeast, when using the oligoelectrolyte based carriers, was 2 times higher than the efficiency of transformation of the yeast when using electroporation and 61 times higher than traditional Li/ Acetate-based chemical transformation methods (Figure 8 herein).

[0070] Example 7: Transformation of yeast with linearized plasmid DNA

[0071] The polymeric oligoelectrolyte carriers are effective in delivering linearized plasmid DNA into yeast cells. [0072] The procedure of preparing the gene delivery system using linearized plasmid DNA is based on growing Pichia pastoris GSl 15 yeast cells on the complete YPD liquid medium to exponential growth (OD₆oo 0.4-0.9) at 30⁰C, pelleting the cells by centrifugation, suspending the pellet in complete medium and adding the polymer and 6.2 kbp circular plasmid DNA to the cell suspension. Afterward, yeast cells are incubated for 1 hour at 30⁰C under normal growing conditions that allow selective marker protein to be synthesized, and yeast cells are plated with solid selective His-deficient medium. Following incubation for 5 days under standard conditions, the number of transformants colonies are counted and compared. No preliminary pretreatments, preparation of competent cells, or special equipment are required for performing the transformation procedure. Efficiency of transformation of Pichia pastoris GSl 15 yeast, when using the oligoelectrolyte based carriers with the linearized plasmid DNA, was 5 times higher than the efficiency of transformation of the yeast when using electroporation and 150 times higher than the results from traditional Li/Acetate-based chemical transformation methods (Figure 9 herein).

[0073] Example 8: Efficiency of transformation of Saccharomyces cerevisiae

[0074] The transformation efficiency of nucleic acid delivery into Saccharomyces cerevisiae yeast using different methods was compared.

[0075] The polymeric oligoelectrolytes provided 0.9 x 10⁵to 2.5 x 10⁵ colonies of transformants of Saccharomyces cerevisiae (strains BY4742 and B44742) per 1 ug of circular non-linearized plasmid DNA. The electroporation method resulted in 1.9 x 10⁵ to 4.6 x 10⁵ colonies and the chemical transformation yielded 1.2 x 10⁵ to 3.1 x 10⁵ colonies (Figure 10 herein). Although the efficiency level of the transformation method using polymeric oligoelectrolytes was slightly lower compared to other tested methods, the number of colonies that resulted from the method using polymeric oligoelectrolytes is sufficiently high for a majority of experiments. The method using polymeric oligoelectrolytes is much faster (1.5 to 2 hours) compared to the time required by electroporation method (9 hours) and chemical method (15 hours). The method using polymeric oligoelectrolytes is also much simpler than the other methods.

[0076] Example 9: Stable transformation [0077] The procedure of preparing stable transformants is based on growing yeast cells on the complete YPS medium (yeast extract, peptone, sucrose) to OD₆₀₀ 0.4-0.9, pelleting the cells by centrifugation, suspending the pellet in complete medium and adding the oligoelectrolyte carrier and 3.7 kbp linear DNA fragment to the cells suspension. Afterwards, yeast cells are incubated for 1 hour at 29⁰C under normal growing conditions that allow selective marker protein to be synthesized, and yeast cells are plated on the dishes with solid selective leucine-deficient medium. After 5 days, the number of transformants colonies is counted. The number of stable transformants of Hansenula polymorpha NCYC 495 yeast, when using the oligoelectrolyte based carriers, was 1.4 times higher than the transformation efficiency of using electroporation, and 8.5 times higher than the transformation efficiency of traditional Li/Acetate-based chemical transformation methods (Figure 11 herein). The presence of the inserted DNA vector in the yeast genome was confirmed by PCR analysis in all DNA-transformants obtained.

[0078] Example 10: Growth of transformants on solid selective medium

[0079] Transformation of cells using the polymeric oligoelectrolyte carriers results in a higher growth rate on solid selective medium.

[0080] Transformed Hansenula polymorpha yeasts were cultured on the dish with selective medium without leucine. The colonies with the fastest growth rates were identified and 10⁷ cells were used to make patches of yeasts (Figure 12A herein) on a new dish with solid selective leucine-deficient medium. Following 48 hours of incubation, the yeast transformants were plated on a dish with glutathione-deficient medium and grown for 4 days. The dish was then scanned and the density of the yeast cells patches on the replica dish was evaluated using GelPro 3.2 software. Compared to transformants obtained with electroporation or chemical transformation, 10% to 20% of the transformants obtained using oligoelectrolyte carriers displayed faster growth on the selective medium (Figure 12 herein). The increased growth rate of transformants suggests a lower toxicity of the oligoelectrolyte carriers and higher number of copies of incorporated DNA into the genome.

[0081]

[0082] Example 11: Growth of transformants on liquid selective medium [0083] Transformation of cells using the polymeric oligoelectrolyte carriers results in a higher growth rate on liquid selective medium.

[0084] Transformed Hansenula polymorpha yeasts were cultured on the dish with selective medium without leucine. The colonies with the fastest growth rates were identified and about 3 x 10⁴ cells of the colonies with the fastest growth rates were transferred into a tube with liquid selective glutathione-deficient medium. The yeasts were grown at standard conditions for 15 hours and the optical density of the yeast culture was measured. Transformants #4 and #5, which showed faster growth on the solid selective medium, also displayed higher growth in the liquid medium (Figure 13 herein).

[0085] Example 12: Optimization of transformation by altering the composition of medium

[0086] Polymer-dependent delivery of nucleic acid into yeast cells can be improved by altering transformation conditions, the composition of transformation medium, or both.

[0087] Hansenula polymorpha yeasts were grown in standard YPS medium at standard conditions to exponential growth (OD_60O 0.4 to 0.9). Cells were pelleted, resuspended in medium, and supplemented with the oligoelectrolyte carriers, CaCl₂, DMSO, and DNA (circular plasmid containing gene resistant to Zeocin antibiotic). Following 1-hour incubation at 37⁰C, cells were plated on the dish with selective Zeocin-containing medium and grown at 37 ⁰C for 5 days, after which the obtained colonies were counted (Figure 14 herein).

[0088] Introduction of DMSO into the transformation medium resulted in a 1.6 to 3.1 -fold increase in the number of Hansenula polymorpha transformants. For some species of yeasts, such as Hansenula polymorpha and Pichia pastoris, an increase in Ca²⁺ concentration can elevate the efficiency of nucleic acids delivery. The number of Hansenula polymorpha transformants was 1.3 to 2.8 times higher when the concentration of Ca²⁺ in the medium was increased (Figure 14 herein).

[0089] In order to determine the optimal concentrations Of CaCl₂ and DMSO, a range of concentrations Of CaCl₂ and DMSO was tested. The concentration of CaCl₂ tested ranged from 0.0000001M to 1.5M, and the concentration of DMSO tested ranged from 10^"7% to 60%. The optimal final concentrations that yielded the highest number of transformants of Hansenula polymorpha was 150 mM CaCl₂ and 2% DMSO. However, other changes in the transformation medium or growing conditions can lead to a shift in the optimal final concentration of CaCl₂ and DMSO. Changes in the transformation medium such as, for example, addition of other compounds affecting cell growth, cell viability, cell wall and/or membranes inside and outside of the cell, endosomes, lysosomes, Golgi apparatus and/or endoplasmic reticulum and the like can alter the effect on the transformation of yeasts when the oligoelectrolyte carriers are used.

[0090] Example 13: Optimization of transformation by use of the heat/cold shock method

[0091] Transformation conditions can be changed by modifying the physical conditions to which cells are exposed. The use of heat and/or cold shock elevates the efficiency of nucleic acid delivery into Hansenula polymorpha and Pichia pastoris yeast cells.

[0092] Hansenula polymorpha yeasts were grown in standard YPS complete medium at standard conditions. Cells were pelleted, resuspended in medium, and supplemented with the oligoelectrolyte carriers and circular plasmid DNA containing resistance gene to Zeocin antibiotic. Following 20-minute incubation on ice, the cells were then incubated at different temperatures for 5 minutes and at room temperature for 5 minutes. Complete YPS medium was added and cells were incubated for 1 hour at 37⁰C. After the cells were plated on the dish with solid Zeocin- containing medium and grown for 5 days, the number of colonies were counted (Figure 15 herein).

[0093] Pichia pastoris yeasts were grown in YPD complete medium at standard conditions. Cells were pelleted, resuspended in medium, and supplemented with the oligoelectrolyte carriers and circular plasmid DNA containing resistance gene to Zeocin antibiotic. Following a 20-minute incubation on ice, the cells were then incubated at different temperatures for 10 minutes and at room temperature for 5 minutes. Complete YPD medium was added and cells were incubated for 1 hour at 3O⁰C. After the cells were diluted 1 :5 with YPD, plated on the dish with solid Zeocin-containing medium and grown for 5 days, the number of colonies were counted (Figure 16 herein).

[0094] When used with the heat/cold shock method, the oligoelectrolyte carriers provided

0.5 to 10 times higher number of yeast transformants compared to the values in the absence of shock (Figure 15 herein). Optimal duration and the temperature of heat/cold shock can be different, and can change with the temperature resistance of the specific species of yeast. Optimal heat-shock condition for temperature resistant strains of Pichia pastoris was 7 to 10 minutes at 55 ⁰C (Figure 16 herein), while the optimal heat-shock condition for the growth of Hansenula polymorpha yeast transformants was 5 minutes at 45 ⁰C. The transformation efficiency can be altered using different combinations of heat shock and cold shock. The cold shock can be used without heat shock, or the heat shock can be used without cold shock. The cold shock can be used before heat shock, or the heat shock can be used before cold shock. One, two or more additional cold shock and/or heat shock treatments can be used in order to optimize delivery of nucleic acids into the cells.

[0095] Example 14: Improvement of yeast transformation by destabilizing the plasma membrane using a freeze/thaw method

[0096] The delivery of nucleic acid into cells can also be improved by inducing destabilization of the cell membrane.

[0097] Pichia pastoris yeasts were grown in standard YPD complete medium at standard conditions. Cells were pelleted, resuspended in medium, supplemented with oligoelectrolyte carriers and circular plasmid DNA containing resistance gene to Zeocin antibiotic, and frozen for 15 minutes at -70⁰C. Afterward, cells were thawed, supplemented with 1 ml of growing medium, incubated for 1 hour at 30⁰C and plated on a dish with solid Zeocin-containing medium. Colonies were counted after 5 days.

[0098] Freezing/thawing of the cells before, during or after adding the oligoelectrolyte carrier and nucleic acids to the cells led to 0.5 to 2.1-fold increase in the number of Pichia pastoris transformants (Figure 17 herein). Sonication also showed a 20% increase in the transformation efficiency when using the oligoelectrolyte carriers, compared to the use of oligoelectrolytes without sonication.

[0099] Example 15: Delivery of nucleic acids into mammalian cells

[00100] The oligoelectrolyte carriers can effectively deliver nucleic acid into mammalian cells. [00101] Human embryonic kidney cells of 293T cell line were grown to the confluency of about 65% in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin under standard conditions of 37°C and 5% CO₂. The medium in the culture dish was changed to fresh medium without FBS ("FBS-free medium").

[00102] Polymeric oligoelectrolyte carriers were dissolved in 0.01 umol to 100 mmol Tris-

HCl (with a pH of 7.4) and 0.01 umol to 100 mmol NaCl. The final concentration of the polymer oligoelectrolyte carriers was from 10^"9% to 1%. Next, 1.5 ug of plasmid DNA was added to the suspension and the mixture was incubated for 20 minutes at room temperature. The mixture was added to the 293T cells in FBS-free medium and the cells were cultured for the next 20 hours at 37°C in the presence of 5% CO₂. The plasmid contained green fluorescent protein (GFP) coding protein under constitutive eukaryotic promoter. The medium was removed and the transfected cells were detected using a fluorescent microscope. Compared to branched polyethylenimine, which is the closest commercially available analogue of the oligoelectrolyte carriers, the oligoelectrolyte carriers provide 1.83 times more transfected cells (Figure 18 herein).

[00103] Example 16: In vivo transfection

[00104] The polymeric oligoelectrolyte carriers can be effectively used to deliver nucleic acid into cells in vivo.

[00105] Polymeric oligoelectrolyte carriers were dissolved in 0.01 umol to 100 mmol Tris-

HCl (with a pH of 7.4) and 0.01 umol to 100 mmol NaCl. The final concentration of the polymer oligoelectrolyte carriers was from 10^"9% to 1%. Next, 1.5ug of plasmid DNA was added to the suspension and the mixture was incubated for 20 minutes at room temperature. The plasmid contained GFP coding protein under constitutive eukaryotic promoter. About 30 ul of the mixture containing 9 ug DNA was injected into a rat blood vessel, the rat was sacrificed 20 hours later, and the transfected cells were searched. GFP-positive transfected cells were found in the liver and lymph nodes. Optimization of transfection conditions and/or increase in the amount of mixture can provide better transfection results.

[00106] Example 17: Delivery of nucleic acids into prokaryotic cells

[00107] E. coli strains of BL-21 and DH5 were used in order to test nucleic acids delivery activity of the polymeric oligoelectrolyte carriers. [00108] Using the polymeric oligoelectrolyte carriers provides 20-600 lower transformation efficiency than known classic chemical method or electroporation. However, similar to the transformation of yeasts, the efficiency of E. coli transformation by means of the polymeric oligoelectrolyte carriers can be enhanced by changing the composition of the transformation medium and/or optimizing the transformation conditions. For example, application of heat/cold shock, addition of DMSO, or freeze/defreeze methods can induce 1.5-14 times elevation of transformation efficiency of E. coli when used with the polymeric oligoelectrolyte carriers. In addition, the transformants obtained by means of the polymeric oligoelectrolyte carriers can grow faster on the selective medium during the first 24 hours after transformation.

[00109] In contrast to the results obtained from using E. coli, in which the developed electrolyte carriers showed lower transformation efficiency, the electrolyte carriers showed good transformation efficiency for the delivery of DNA into the prokaryotic cells of Streptomyces genus when compared to the widely used fusion DNA delivery method (Figure 19 herein).

[00110] Example 18: Delivery of nucleic acids into plant cells

[00111] The polymeric oligoelectrolyte carriers can be used to deliver nucleic acids into plant cells.

[00112] Polymeric oligoelectrolyte carriers were tested for nucleic acid delivery into rose plant protoplasts. Doxorubicin was used to label double helix plasmid DNA and fluorescein isothiocyanate (FITC) was used to label the oligoelectrolyte carrier. Polymeric oligoelectrolyte carriers were dissolved in 0.01 umol to 100 mmol Tris-HCl (with a pH of 7.4) and 0.01 umol to 100 mmol NaCl. The final concentration of the polymer oligoelectrolyte carriers was in the range of 10^"9% to 1%. Next, 1.5 ug of plasmid DNA was added to the suspension and the mixture was incubated for 20 minutes at room temperature. Fast growing rose apex cells were isolated and the cell wall was removed. The plant cell protoplasts were incubated with the oligoelectrolyte/DNA mixture for 30 minutes and plated in the dish with 10 volumes of liquid Murashige and Skoog medium for additional 30 minutes. When the fluorescence inside of the cells was detected, some cells were both positive for FITC and doxorubicin fluorescence, suggesting delivery of the complex of the oligoelectrolyte carrier and DNA into plant protoplast cells. The transformation efficiency can be further improved by subjecting cells to a heat shock and/or a cold shock, and any combinations of heat and cold shocks in any order, and/or by changing the composition of the transformation medium. The transformation efficienct can also be enhanced by freezing and thawing the cells.

[00113] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.

[00114] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[00115] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[00116] In some embodiments, the numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the application are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.

[00117] In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[00118] Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[00119] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[00120] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A polynucleotide delivery molecule, comprising: an oligoelectrolyte adapted to penetrate a cell plasma membrane; and a polynucleotide molecule bound to the oligoelectrolyte.

2. The polynucleotide delivery molecule of claim 1, wherein the oligoelectrolyte comprises a backbone comprising anionic groups and/or nonionic groups.

3. The polynucleotide delivery molecule of claim 1, wherein the oligoelectrolyte comprises one or more side chains comprising cationic groups and/or nonionic groups.

4. The polynucleotide delivery molecule of claim 2, wherein the oligoelectrolyte has at least one grafted cationic side chain.

5. The polynucleotide delivery molecule of claim 2, wherein the oligoelectrolyte comprises dimethylaminoethyl methacrylate (DMAEMA) and 5-(ter/butylperoxy)-5-methyl-l-hexen- 3-yne (VEP).

6. A method of constructing the polynucleotide delivery molecule of claim 1, comprising: adding a monomer to another monomer to create a mixture; adding a solution of oligoperoxide metal complex (OMC); increasing the temperature; subjecting the mixture to Argon (Ar) flow; removing solvent via vacuum distillation; and dissolving the solvent in acetone and subjecting the solvent in acetone to a multistage precipitation process to yield a precipitate.

7. The method of claim 6, further comprising drying the precipitate until constant weight is achieved.

8. A method of transforming a cell, comprising: providing a polynucleotide delivery molecule, comprising an oligoelectrolyte adapted to penetrate a plasma membrane of the cell, and a polynucleotide molecule bound to the oligoelectrolyte; and administering an effective amount of the polynucleotide delivery molecule to the cell to effectuate transformation and adjusting the efficiency of transformation.

9. The method of claim 8, wherein the cell is an animal cell, a plant cell, a fungal cell, and/or a prokaryotic cell.

10. The method of claim 9, wherein the animal cell is a mammalian cell.

11. The method of claim 10, wherein the mammalian cell is a human cell.

12. The method of claim 11, wherein the human cell is a 293T cell.

13. The method of claim 10, further comprising using the polynucleotide delivery molecule in vivo.

14. The method of claim 9, wherein the plant cell is a cell with altered cell wall.

15. The method of claim 9, wherein the prokaryotic cell is of the species Escherichia coli.

16. The method of claim 9, wherein the prokaryotic cell is of the species Streptomyces lividans.

17. The method of claim 8, wherein the cell is a yeast cell.

18. The method of claim 17, wherein said yeast cell is of the species Hansenula polymorpha.

19. The method of claim 17, wherein said yeast cell is of the species Pichiapastoris.

20. The method of claim 17, wherein said yeast cell is of the species Saccharomyces cerevisiae.

21. The method of claim 8, wherein the transformation is a stable transformation.

22. The method of claim 8, wherein the cell is incubated in a transformation medium.

23. The method of claim 22, wherein adjusting the efficiency of transformation comprises modifying the composition of the transformation medium.

24. The method of claim 23, wherein the transformation medium comprises dimethyl sulfoxide (DMSO) and CaCl₂.

25. The method of claim 24, wherein the modification comprises changes in the concentration of DMSO and changes in the concentration OfCaCl₂.

26. The method of claim 8, wherein adjusting the efficiency of transformation comprises subjecting said cell to a heat shock, a cold shock, or one or more combinations thereof.

27. The method of claim 8, wherein adjusting the efficiency of transformation comprises freezing and thawing said cell.

28. A composition, comprising: a polynucleotide delivery molecule, comprising: an oligoelectrolyte adapted to penetrate a cell plasma membrane, and a polynucleotide molecule bound to the oligoelectrolyte; and a pharmaceutically acceptable carrier.

29. The composition of claim 28, wherein the oligoelectrolyte comprises a backbone comprising anionic groups and/or nonionic groups.

30. The composition of claim 28, wherein the side chains of the oligoelectrolyte comprises one or more side chains comprising cationic groups and/or nonionic groups.

31. The composition of claim 29, wherein the oligoelectrolyte has at least one grafted cationic side chain.

32. The composition of claim 29, wherein the oligoelectrolyte comprises dimethylaminoethyl methacrylate (DMAEMA) and 5-(tørtbutylperoxy)-5-methyl-l-hexen-3-yne (VEP).

33. The composition of claim 28, further comprising a pharmaceutically acceptable excipient.