WO2019108660A1 - Zinc finger moiety attached to a resin used to purify polynucleotide molecules - Google Patents

Abstract

The invention is directed to compositions and a process for purifying a polynucleotide of interest comprising a cis-element bound to a zinc finger moiety attached to a resin.

Description

ZINC FINGER MOIETY ATTACHED TO A RESIN

USED TO PURIFY POLYNUCLEOTIDE MOLECULES BACKGROUND OF THE INVENTION Field of the Invention

[0001] The invention relates to a resin comprising a zinc finger moiety, methods of using such resins to purify a polynucleotide molecule and kits comprising the zinc finger resins. Discussion of the Related Art

[0002] In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions

[0003] Development of gene therapy and DNA vaccine treatments has steadily increased over the past decade. The large-scale, economic purification of these polynucleotide therapeutics is increasingly becoming an important issue in biopharmaceutical industry.

[0004] Generally, nucleic acid based therapies are produced by culturing recombinant cells into which the nucleic acid is transferred and expanded. The nucleic acid is then purified from cell lysates that include impurities such as various medium- derived ingredients, cell by-products or the like. Substantial efforts are then undertaken to isolate and purify the nucleic acid from these impurities by processing the cell lysate through a combination of different modes of chromatography.

[0005] For example, current state of the art techniques for purifying DNA rely upon the use of laboratory scale centrifugation, extraction with organic solvents, and/or the use of animal-derived enzymes (lysozyme, RNase, Proteinase K). Final purification of DNA from a lysate is accomplished using methods that may include ultra-centrifugation, preparative gel electrophoresis, and column chromatography. [0006] However, these multi-level processing steps are expensive and time consuming and result in ever decreasing yields. Thus, there is a need for rapid, inexpensive and selective method of purifying nucleic acid molecules that result in higher yields, higher purity, and/or lower costs. SUMMARY OF THE INVENTION

[0007] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.

[0008] The invention relates to a simple and rapid method for purifying a polynucleotide molecule comprising a cis-element using a resin having an attached zinc finger moiety.

[0009] As described herein, one embodiment is a zinc finger resin comprising an attached zinc finger moiety comprising at least one zinc finger. The zinc finger is derived from a zinc finger family member, such as a) a ^^α-zinc finger family; b) a hormone- nuclear receptor family; c) a loop-sheet-helix family; or d) GAL4-type family. Specific examples of zinc fingers that can be used, include, but are not limited to a) a Cys2His2 zinc finger; b) a zinc finger described in Prosite PS50157; c) a zinc finger described in Prosite PS00028; and/or d) or a variant of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to (a)-(c).

[0010] In preferred embodiments, the zinc finger resin further comprises a solution comprising zinc. Such solutions can comprise, for example, zinc at a concentration of (a) 1 mM, 2mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19mM or 20mM; (b) between at least 1mM and at least 5 mM, between at least 1mM and at least 10 mM, between at least 1mM and at least 15 mM, or between at least 1mM and at least 20 mM; (c) between at least 5mM and at least 10 mM, between at least 5 mM and at least 15 mM, or between at least 5 mM and at least 20 mM; (d) between at least 10 mM and at least 15 mM, between at least 10 mM and at least 20 mM, or between at least 15 mM and at least 20 mM; or (e) any one of (a)-(d) wherein the concentration of zinc is provided by a solution of ZnCl2.

[0011] The zinc finger resin can further comprise a bound polynucleotide molecule, wherein said polynucleotide molecule comprises a cis-element capable of binding to zinc finger moiety. In some preferred embodiments, the cis-element endogenously exists in the polynucleotide sequence. In other preferred embodiments, the polynucleotide molecule comprises at least two cis-elements, at least three cis-elements, at least four cis- elements, or at least five cis-elements.

[0012] The resin can be any materials used to purify polynucleotide sequences, such as, for example, any of the following: a) Sepharose-6B; b) electrospun polymer nanofibers; c) agarose beads (e.g., Sepharose-6B); d) IMAC resins (using, for example, HIS fusion proteins or GSTransferase); e) SulfoLink; and/or f) purification fibers. In preferred embodiments, the electrospun polymer nanofibers are arranged as non-woven sheets stacked one on top of the other.

[0013] Also disclosed is a method of purifying a polynucleotide molecule, wherein the method comprises: a) adding to the zinc finger resin a solution, wherein said solution comprises a sufficient amount of zinc to enable the binding of the cis-element(s) to the zinc finger; b) adding to the zinc finger resin, a solution comprising the polynucleotide molecule, wherein said polynucleotide molecule comprises a cis-element(s) capable of binding to the attached zinc finger, c) adding to the zinc finger resin a second solution, wherein said second solution contains increased levels of salt and/or a zinc chelator sufficient to chelate zinc from the zinc finger to release the binding of the polynucleotide molecule containing the cis-element(s) from the zinc finger; and d) collecting the polynucleotide molecule released from the zinc finger resin. In preferred embodiments, the second solution comprises TPEN, DTPA, or chelex resin. In further preferred embodiments, the method further comprises a wash step between any one of steps a, b, c, or d.

[0014] Alternatively, the zinc finger resin can be generated using zinc finger moieties already in contact with zinc and in proper conformation. For example, the zinc finger moieties could be chemically synthesized, contacted with zinc to ensure proper zinc finger conformation and then attached to the resin. Alternatively, the zinc finger moieties can be recombinantly produced and then attached to the resin.

[0015] In further preferred embodiments, the polynucleotide molecule can be obtained from a cell-lysate. In preferred embodiments, the cell lysate can be derived from a bacterial cell, a mammalian cell, a vertebrate cell, a murine cell or a baculovirus cell. In further preferred embodiments, the cell-lysate is a crude cell lysate, a partially purified cell lysate, and an aqueous solution containing the extracted polynucleotide molecule acid by alkaline lysis. In further embodiments, the solution comprising the polynucleotide molecule is pre-treated to at least partially remove endotoxin. In preferred embodiments, the polynucleotide molecule is a plasmid DNA (pDNA).

[0016] Kits comprising the zinc finger resin as described herein are also contemplated as well as kits further comprising a cloning vector comprising the cis- element. BRIEF DESCRIPTION OF THE FIGURES

[0017] The objects and features of the invention can be better understood with reference to the following detailed description and accompanying drawings.

[0018] Figure 1A is a schematic diagram of both a single C2H2 zinc finger showing the binding of zinc between two amino acids of cysteine and two amino acids of histidine, along with a representative consequence sequence found N-terminal and C-terminal to the zinc binding domain. Figure 1A also shows a schematic of multiple C2H2 zinc fingers making up a zinc finger moiety as described herein.

[0019] Figure 1B is a 3-D conformational schematic of three separate zinc fingers binding to DNA in the presence of zinc.

[0020] Figure 2 is a schematic overview of an exemplified Purification Process starting from bacterial cell paste to bulk DS for an exemplified polynucleotide sequence (e.g., plasmid DNA).

[0021] Figure 3A-C shows the zinc fingers and cis-elements used in the described studies. The GAG-ZFP is SEQ ID NO:1, the GCT-ZFP is SEQ ID NO:2, the GCT-C20A is SEQ ID NO:3 and GCT-R91A is SEQ ID NO:4. [0022] Figure 4A shows a schematic of a microplate assay used to confirm binding and release of the ZFP to the cis-elements. Figure 4B is a schematic of how a zinc finger resin would be used to purify a polynucleotide comprising a cis-element.

[0023] Figure 5 shows the results of a microplate assay showing specificity and sensitivity of ZFP binding to their cis-elements and the effect of mutations on binding.

[0024] Figure 6 shows the results of the microplate assay testing the binding to cis-elements at varying salt concentrations.

[0025] Figure 7 shows the results of the microplate assay testing the binding of polynucleotides comprising single, double or triple cis-elements at varying salt concentrations. DETAILED DESCRIPTION DEFINITIONS

[0026] The following definitions are provided for specific terms which are used in the following written description.

[0027] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "zinc finger resin" or“zinc finger moiety” includes a plurality of resins or zinc fingers. The term "a polynucleotide molecule" includes a plurality of polynucleotide molecules.

[0028] As used herein, the term "comprising" is intended to mean, for example, that the zinc finger resin and methods using such resins include the recited elements, and can include other elements. "Consisting essentially of", when used shall mean excluding other elements of any essential significance to the combination. Thus, a method of using a zinc finger resin with consisting essentially of as described herein could include additional non-essential steps. "Consisting of" shall mean excluding additional essential and non-essential steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0029] The term "about" or "approximately" means within an acceptable range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5 fold, and more preferably within 2 fold, of a value. Unless otherwise stated, the term 'about' means within an acceptable error range for the particular value, such as ± 1-20%, preferably ± 1-10% and more preferably ±1-5%.

[0030] Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.

[0031] All percentages and ratios used herein are by weight of the total composition unless otherwise indicated herein. All temperatures are in degrees Celsius unless specified otherwise. All measurements made are at 25°C and normal pressure unless otherwise designated.

[0032] All ranges recited herein include the endpoints, including those that recite a range "between" two values. Terms such as "about," "generally," "substantially," "approximately" and the like are to be construed as modifying a term or value such that it is not an absolute, but does not read on the prior art. Such terms will be defined by the circumstances and the terms that they modify as those terms are understood by those of skill in the art. This includes, at very least, the degree of expected experimental error, technique error and instrument error for a given technique used to measure a value.

[0033] Where used herein, the term "and/or" when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present. For example, if a composition of the instant invention is described as containing characteristics A, B, and/or C, the composition can contain A feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0034] As used herein, the terms "polynucleotide",“polynucleotide molecule” and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes, for example, single-, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules (e.g., comprising modified bases, sugars, and/or internucleotide linkers).

[0035] The abbreviations used throughout the specification to refer to nucleic acids comprising specific nucleobase sequences are the conventional one-letter abbreviations. Thus, when included in a nucleic acid, the naturally occurring encoding nucleobases are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Also, unless specified otherwise, nucleic acid sequences that are represented as a series of one-letter abbreviations are presented in the 5'->3' direction.

[0036] The polynucleotide molecules that are purified through the disclosed process can be of any type of polynucleotides, so long as the polynucleotide comprises a cis-element(s) that is capable of being specifically bound by the zinc finger moiety. In preferred embodiments, at least two, three, four, or five cis-elements are used. In preferred embodiments, the polynucleotide molecule is a plasmid DNA. Examples of such plasmids include, but are not limited to prokaryotic and eukaryotic vectors, cloning and expression vectors, pBR322 and pUC vectors and their derivatives, etc., and to incorporate various origins of replication, for instance, prokaryotic origins of replication, such as pMB1 and ColE1, and eukaryotic origins of replication, such as those facilitating replication in yeast, fungi, insect, and mammalian cells (e.g., SV40 ori) and also to encompass numerous genetic elements to facilitate cloning and expression, such as selectable genes, polylinkers, promoters, enhancers, leader peptide sequences, introns, polyadenylation signals, etc. The selection of vectors, origins, and genetic elements will vary based on requirements and is well within the skill of workers in this art.

[0037] Similarly, a host can be chosen from among prokaryotes and eukaryotes, including bacterial, yeast, fungi, insect and mammalian cells. Preferred hosts are microbial cells, especially microorganisms like E. coli. Any suitable strain of E. coli is contemplated. [0038] Likewise, genes encoding diverse structural proteins (or peptides, polypeptides, glycoproteins, phosphoproteins, amidated proteins, etc.) may be inserted into the plasmid, which genes may constitute genomic DNA, cDNA, synthetic DNA, polynucleotide and oligonucleotide, etc. sequences so long as the plasmid also comprises a cis-element(s) capable of being bound by the zinc finger moiety. These sequences may be obtained using chemical synthesis or gene manipulation techniques (see Sambrook, Fritsch, Maniatis, Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989; and Current Protocols in Molecular Biology, Greene Publishing Assoc. & Wiley, 1987, both of which are expressly incorporated by reference herein) and, further, may be inserted into plasmids and the plasmids subsequently introduced into host cells using additional gene manipulation techniques. Culturing of a plasmid DNA-containing host may be carried out using known processes such as those disclosed herein, and are contemplated as including incubator, bioreactor, fermentor etc., according to batch fermentation, fed batch fermentation, continuous culture, Type I, II and III fermentation, aseptic fermentation, consortium fermentation, protected fermentation, etc. Fitting the conditions (e.g., medium, temperature, pH, hours, agitation, aeration, etc.) for culture to the circumstances is empirical and well within the skill of those in the art.

[0039] As used herein, "coding sequence" is a sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate expression control sequences. The boundaries of a coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, a prokaryotic sequence, cDNA from eukaryotic mRNA, a genomic DNA sequence from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

[0040] As used herein, two coding sequences "correspond" to each other if the sequences or their complementary sequences encode the same amino acid sequences.

[0041] As used herein, the term "peptide" refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds or by other bonds (e.g., as esters, ethers, and the like). [0042] As used herein, the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both D or L optical isomers, and amino acid analogs and peptidomimetics. A peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long (e.g., greater than about 10 amino acids), the peptide is commonly called a polypeptide or a protein. While the term "protein" encompasses the term "polypeptide", a "polypeptide" may be a less than full-length protein.

[0043] As used herein, unless specifically delineated otherwise, the three-letter amino acid abbreviations designate amino acids in the L-configuration. Amino acids in the D-configuration are preceded with a "D-." For example, Arg designates L-arginine and D-Arg designates D-arginine. Likewise, the capital one-letter abbreviations refer to amino acids in the L-configuration. Lower-case one-letter abbreviations designate amino acids in the D-configuration. For example, "R" designates L-arginine and "r" designates D- arginine.

[0044] Unless noted otherwise, when polypeptide sequences are presented as a series of one-letter and/or three-letter abbreviations, the sequences are presented in the N->C direction, in accordance with common practice.

[0045] As used herein, "genetically encoded amino acid" refers to L-isomers of the twenty amino acids that are defined by genetic codons. The genetically encoded amino acids are the L-isomers of glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine and lysine.

[0046] As used herein, "genetically non-encoded amino acid" refers to amino acids that are not defined by genetic codons. Genetically non-encoded amino acids include derivatives or analogs of the genetically-encoded amino acids that are capable of being enzymatically incorporated into nascent polypeptides using conventional expression systems, such as selenomethionine (SeMet) and selenocysteine (SeCys); isomers of the genetically-encoded amino acids that are not capable of being enzymatically incorporated into nascent polypeptides using conventional expression systems, such as D-isomers of the genetically-encoded amino acids; L- and D-isomers of naturally occurring α-amino acids that are not defined by genetic codons, such as α-aminoisobutyric acid (Aib); L- and D-isomers of synthetic α-amino acids that are not defined by genetic codons; and other amino acids such as α-amino acids, γ-amino acids, etc. In addition to the D-isomers of the genetically-encoded amino acids, exemplary common genetically non-encoded amino acids include, but are not limited to, norleucine (Nle), penicillamine (Pen), N- methylvaline (MeVal), homocysteine (hCys), homoserine (hSer), 2,3-diaminobutyric acid (Dab) and ornithine (Orn). Additional exemplary genetically non-encoded amino acids are found, for example, in Practical Handbook of Biochemistry and Molecular Biology, 1989, Fasman, Ed., CRC Press, Inc., Boca Raton, Fla., pp.3-76 and the various references cited therein.

[0047] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA transcribed from the genomic DNA.

[0048] As used herein, "under transcriptional control" or "operably linked" refers to expression (e.g., transcription or translation) of a polynucleotide sequence which is controlled by an appropriate juxtaposition of an expression control element and a coding sequence. In one aspect, a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription of that DNA sequence.

[0049] As used herein, "signal sequence" denotes the endoplasmic reticulum translocation sequence. This sequence encodes a signal peptide that communicates to a cell to direct a polypeptide to which it is linked (e.g., via a chemical bond) to an endoplasmic reticulum vesicular compartment, to enter an exocytic/endocytic organelle, to be delivered either to a cellular vesicular compartment, the cell surface or to secrete the polypeptide. This signal sequence is sometimes clipped off by the cell in the maturation of a protein. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0050] As used herein, "hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0051] As used herein, a polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) which has a certain percentage (for example, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%) of "sequence identity" to another sequence means that, when maximally aligned, using software programs routine in the art, that percentage of bases (or amino acids) are the same in comparing the two sequences.

[0052] Two sequences are "substantially homologous" or "substantially similar" when at least about 50%, at least about 60%, at least about 70%, at least about 75%, and preferably at least about 80%, and most preferably at least about 90 or 95% of the nucleotides match over the defined length of the DNA sequences. Similarly, two polypeptide sequences are "substantially homologous" or "substantially similar" when at least about 50%, at least about 60%, at least about 66%, at least about 70%, at least about 75%, and preferably at least about 80%, and most preferably at least about 90 or 95% of the amino acid residues of the polypeptide match over a defined length of the polypeptide sequence. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks. Substantially homologous nucleic acid sequences also can be identified in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. For example, stringent conditions can be: hybridization at 5xSSC and 50% formamide at 42°C, and washing at 0.1xSSC and 0.1% sodium dodecyl sulfate at 60°C.

[0053] The terms "percent (%) sequence similarity", "percent (%) sequence identity", and the like, generally refer to the degree of identity or correspondence between different nucleotide sequences of nucleic acid molecules or amino acid sequences of polypeptides that may or may not share a common evolutionary origin (see Reeck et al., supra). Sequence identity can be determined using any of a number of publicly available sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin), etc. [0054] To determine the percent identity between two amino acid sequences or two nucleic acid molecules, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions/total number of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are, or are about, of the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent sequence identity, typically exact matches are counted.

[0055] The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1990, 87:2264, modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 1993, 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al, J. Mol. Biol.1990; 215: 403. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to sequences of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to protein sequences of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al, Nucleic Acids Res. 1997, 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationship between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See ncbi.nlm.nih.gov/BLAST/ on the WorldWideWeb.

[0056] Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS 1988; 4: 1 1-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. [0057] In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 1970, 48:444-453), which has been incorporated into the GAP program in the GCG software package (Accelrys, Burlington, MA; available at accelrys.com on the WorldWideWeb), using either a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package using a NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70, or 80, and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that can be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is a sequence identity or homology limitation of the invention) is using a Blossum 62 scoring matrix with a gap open penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0058] Statistical analysis of the properties described herein may be carried out by standard tests, for example, t-tests, ANOVA, or Chi squared tests. Typically, statistical significance will be measured to a level of p=0.05 (5%), more preferably p=0.01, p=0.001, p=0.0001, p=0.000001

[0059] "Conservatively modified variants" are also contemplated. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka, et al., 1985, J. Biol. Chem. 260: 2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98).

[0060] The term "biologically active fragment", "biologically active form", "biologically active equivalent" of and "functional derivative" of a wild-type protein, possesses a biological activity that is at least substantially equal (e.g., not significantly different from) the biological activity of the wild type protein as measured using an assay suitable for detecting the activity. With respect to a zinc finger moiety, the biological activity is defined as the ability of the zinc finger moiety to bind to at least one, at least two, at least three, at least four, or at least five cis-element(s) in the presence of zinc and to release the cis-element(s) when zinc is removed.

[0061] An "antibody" is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies (e.g., bispecific antibodies). An "antibody combining site" is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, and those portions of an immunoglobulin molecule that contains the paratope, including Fab, Fab', F(ab')2 and F(v) portions, which portions are preferred for use in the therapeutic methods described herein.

[0062] An "epitope" is a structure, usually made up of a short peptide sequence or oligosaccharide that is specifically recognized or specifically bound by a component of the immune system. T-cell epitopes have generally been shown to be linear oligopeptides. Two epitopes correspond to each other if they can be specifically bound by the same antibody. Two epitopes correspond to each other if both are capable of binding to the same B cell receptor or to the same T cell receptor, and binding of one antibody to its epitope substantially prevents binding by the other epitope (e.g., less than about 30%, preferably, less than about 20%, and more preferably, less than about 10%, 5%, 1%, or about 0.1% of the other epitope binds). In the present invention, multiple epitopes can make up an antigen.

[0063] As used herein, the term "isolated" means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature. For example, with respect to an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0064] As used herein "purified" refers processing a polynucleotide molecule through the zinc finger resin described herein. [0065] As used herein“zinc finger moiety” refers to at least one zinc finger polypeptide capable of specifically binding to at least one, at least two, at least three, at least four, or at least five cis-element(s) in the presence of zinc and releasing the cis- element(s) when zinc is removed. Preferred examples of such zinc finger moieties are Cys2His2 zinc fingers. Additionally, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 zinc fingers can make up the zinc finger moiety. In preferred embodiments, zinc finger moieties are comprise sets of three zinc fingers, so that the zinc finger moiety comprises 3, 6, 9, 12, 15, etc zinc fingers.

[0066] As used herein,“zinc finger resin” is a purification media of any structure that comprises an attached zinc finger moiety that can be used to purify a polynucleotide molecule comprising at least one, at least two, at least three, at least four, or at least five cis-element(s).

[0067] The attachment of the zinc finger moiety can occur in any methods known to the skilled artisan, such as, for example, click chemistry, poly-His linkages, glutathione S-transferase (GST) or direct chemical coupling.

[0068] As used herein,“cis-element” is a short polynucleotide sequence that can be specifically bound by the zinc finger moiety. In preferred embodiments, the cis-element is between 5-20 nucleotides, between 5-10 nucleotides, between 10-15 nucleotides, between 15-20 nucleotides, between 20-25 nucleotides in length. In further preferred embodiments, at least two, at least three, at least four, or at least five cis-elements are part of the polynucleotide sequence. In further preferred embodiments, the polynucleotides comprise two cis-elements. Zinc-Finger Moiety

[0069] A zinc finger is a small protein structural motif that is characterized by the coordination of one or more zinc ions which stabilize the fold. Proteins that contain zinc fingers (zinc finger proteins; ZFPs) are classified into several different structural families, each with a unique three-dimensional architecture. Although a particular zinc finger protein's class is determined by this three-dimensional structure, the zinc finger can also be readily recognized based on the amino acid sequence of the protein. Zinc fingers are known to bind specific DNA and RNA sequences. Although engineering zinc fingers to have an affinity for a specific sequence is an area of active research, surprisingly, no one has yet to suggest the idea of directly attaching a zinc finger moiety to a resin to enable the purification of a polynucleotide molecule comprising a cis-element by the addition and then the removal of zinc (e.g., by either changing the concentration of salt or addition of a zinc chelator) from the purification media which then releases the polynucleotide from the zinc finger resin. More importantly and unexpectedly, the inventors found that at least two cis-elements are needed for optimal binding and recovery of a polynucleotide sequence.

[0070] One form of zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues. A systematic method has been used to classify zinc finger proteins. This method classifies zinc finger proteins into "fold groups" based on the overall shape of the protein backbone in the folded domain. Zinc finger proteins comprise well known, defined and characterized families of proteins. Information relating to zinc fingers proteins can be accessed in many different public data bases all of which are hereby incorporated by reference in the entirety.

Fol Sequence Motif C 2-Cys-X2,4-Cys-X12-His-X3,4,5-His Ga

Tr

Zin

Z

[0071] Zinc fingers can also be engineered using well known techniques to target desired genomic DNA sequences. See, Pabo, et al., "Design and Selection of Novel Cys2His2 Zinc Finger Proteins" Annual Review of Biochemistry, 70: 313–40 (2001); Jamieson AC, et al., "Drug Discovery with Engineered Zinc-Finger Proteins" Nature Reviews. Drug Discovery, 2(5): 361–8 (2003). The number of zinc fingers engineered to bind to a cis-element can be empirically determined; however, it is preferred that engineered zinc finger moieties comprise between 1 and 6, 1 and 5, 1 and 4, 2 and 6, 2 and 5, 2 and 4, 3 and 6, 3 and 5, 3 and 4 individual zinc fingers so that a specific cis-element ranging from 9 base pairs to 18 base pairs can be engineered into the sequence of the polynucleotide molecule. See, Liu Q, et al., "Design Of Polydactyl Zinc-Finger Proteins For Unique Addressing Within Complex Genomes" PNAS: 94(11): 5525–30 (1997). In further preferred embodiments, at least two, at least three, at least four, or at least five cis-elements are included in a polynucleotide sequence. In further preferred embodiments, the polynucleotide sequence comprises two, three, four, five, six, seven, eight, nine, or ten cis-elements. In even further preferred embodiments, the polynucleotide sequence comprises two or three cis-elements.

[0072] Engineered zinc finger moieties can be derived from any known zinc fingers. For example, the zinc finger domain of the murine transcription factor Zif268 or human transcription factor SP1 has routinely been used as a starting point. Zif268 has three individual zinc finger motifs that collectively bind a 9 bp sequence with high affinity. One approach to generating engineered zinc fingers is to combine smaller zinc finger "modules" of known specificity. The structure of the zinc finger protein Zif268 bound to DNA described in a publication over 25 years ago has been key to much of this work. This paper describes the concept of obtaining fingers for each of the 64 possible base pair triplets and then mixing and matching these fingers to design proteins with any desired sequence specificity. See, Pavletich NP, Pabo CO "Zinc Finger-DNA Recognition: Crystal Structure of a Zif268-DNA Complex at 2.1 A" Science, 252: 809–17 (1991). The most common modular assembly process involves combining separate zinc fingers that can each recognize a 3-basepair DNA sequence to generate 3-finger, 4-, 5-, or 6-finger moieties that recognize target sites ranging from 9 base pairs to 18 base pairs in length.

[0073] Alternatively, a different method uses 2-finger modules to generate zinc finger arrays with up to six individual zinc fingers. See, Shukla VK, et al., "Precise Genome Modification in the Crop Species Zea Mays Using Zinc-Finger Nucleases" Nature 459(7245):437–41 (2009). For example, phage display has been used to develop and characterize zinc finger domains that recognize most DNA triplet sequences. See, for example US2005/0084885 and US2007/0178499.

[0074] Even more preferably, modular assembly of zinc fingers that accounts for neighboring fingers has been reported to yield proteins with improved performance relative to standard modular assembly. See, Sander et al., "Selection-Free Zinc-Finger- Nuclease Engineering by Context-Dependent Assembly (Coda)" Nature Methods 8(1): 67–9 (2011).

[0075] Moreover, numerous selection methods have been used to generate zinc finger arrays capable of binding specific polynucleotide sequences. For example, phage display protocols have been used to select proteins that bound a given DNA target from a large pool of partially randomized zinc finger arrays. This technique often requires a multi-step process that generated a completely optimized 3-finger array by adding and optimizing a single zinc finger at a time was developed. See, Greisman HA and Pabo CO "A General Strategy For Selecting High-Affinity Zinc Finger Proteins For Diverse DNA Target Sites” Science 275(5300): 657–61 (1997). Alternatively, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells systems have been utilized. For example, novel 3-finger zinc finger arrays utilizing a bacterial two- hybrid system has been described. See, Maeder ML, et al.,“Rapid "Open-Source" Engineering of Customized Zinc-Finger Nucleases For Highly Efficient Gene Modification" Molecular Cell 31 (2):294–301 (2008). This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence.

[0076] Designing zinc fingers and their corresponding binding sequences are routine and web-based tools exist to aid in such design. See, for example, http://www.scripps.edu/barbas/zfdesign/zfdesignhome.php. Any of these described methods (as well as methods known in the art) can be used to generate zinc fingers to attach to a resin and used to purify a polynucleotide molecule comprising a cis-element as disclosed herein. [0077] Polypeptide variants of the wild-type zinc fingers are contemplated. For example, polypeptides at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98% or 99% identity to any known zinc finger that is capable of binding to a cis-element, as well as polynucleotides encoding these variants. Variants of the zinc fingers have the ability to function by specifically binding to a cis-element, although it doesn’t necessarily need to identical in sequence as the original cis-element from which the variant zinc finger was derived.

[0078] Polynucleotides encoding variants of the wild-type zinc fingers are contemplated. These polynucleotides can be at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, 96%, 97%, 98% or 99% identity to any known zinc finger that is capable of binding to a cis-element. Variants of the zinc fingers have the ability to function by specifically binding to a cis-element, although it doesn’t necessarily need to be identical in sequence as the original cis-element from which the variant zinc finger was derived.

[0079] Substantially similar genes encoding zinc fingers may be provided, e.g., genes with greater than about 50%, greater than about 70%, greater than 80%, greater than about 90%, and preferably, greater than about 95% identity to a known zinc finger gene or polypeptide sequence. Percent identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. In particular, preferred programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0080] "Conservatively modified variants" of zinc fingers also can be provided. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences of wild type zinc fingers, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer, et al., 1991, Nucleic Acid Res.19: 5081; Ohtsuka, et al., 1985, J. Biol. Chem.260: 2605-2608; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98).

[0081] Substantially similar zinc finger sequences may initially be identified by selecting a sequence which specifically hybridizes to a zinc finger domain sequence of interest under stringent hybridization conditions. Examples of stringent hybridization conditions include: incubation temperatures of about 25 degrees C to about 37 degrees C; hybridization buffer concentrations of about 6xSSC to about 10xSSC; formamide concentrations of about 0% to about 25%; and wash solutions of about 6xSSC. Examples of moderate hybridization conditions include: incubation temperatures of about 40 degrees C to about 50 degrees C.; buffer concentrations of about 9xSSC to about 2xSSC; formamide concentrations of about 30% to about 50%; and wash solutions of about 5xSSC to about 2xSSC. Examples of high stringency conditions include: incubation temperatures of about 55 degrees C to about 68 degrees C.; buffer concentrations of about 1xSSC to about 0.1xSSC; formamide concentrations of about 55% to about 75%; and wash solutions of about 1xSSC, 0.1xSSC, or deionized water. In general, hybridization incubation times are from 5 minutes to 24 hours, with 1, 2, or more washing steps, and wash incubation times are about 1, 2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be employed. Similarity can be verified by sequencing, but preferably, is also or alternatively, verified by function (e.g., ability to traffic to an endosomal compartment, and the like), using assays suitable for the particular domain in question.

[0082] Methods of constructing polynucleotides encoding zinc fingers are well known in the art, although the easiest approach to generate a zinc finger moiety comprising at least one zinc finger would be to synthesize the zinc finger chemically.

[0083] Alternatively, a polynucleotide sequence encoding the zinc finger moiety can be synthesized chemically or isolated by one of several approaches. The sequence to be synthesized can be designed with the appropriate codons for the desired amino acid sequence. In general, one will select preferred codons for the intended host in which the sequence will be used for expression. The complete sequence may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature 292: 756, 1981; Nambair, et al. Science 223: 1299, 1984; Jay, et al., J. Biol. Chem.259: 6311, 1984.

[0084] Alternatively, the zinc finger can be constructed using the polymerase chain reaction (M. A. Innis, et al., In PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990). For example, wild type zinc fingers can be isolated from publicly available clones known to contain them, but they may also be isolated from genomic DNA or cDNA libraries. These techniques are well known to those of skill in the art. Polynucleotides encoding the zinc fingers may be fused directly to each other (e.g., with no intervening sequences), or inserted into one another (e.g., where domain sequences are discontinuous), or may be separated by intervening sequences (e.g., such as linker sequences, such as for example, the amino acid sequences GPGPG or PMGLP).

[0085] The basic strategies for preparing oligonucleotide primers, probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. See, e.g., Sambrook, et al., 1989, supra; Perbal, 1984, supra. The construction of an appropriate genomic DNA or cDNA library is within the skill of the art. See, e.g., Perbal, 1984, supra. Alternatively, suitable DNA libraries or publicly available clones are available from suppliers of biological research materials, such as Clonetech and Stratagene, as well as from public depositories such as the American Type Culture Collection.

[0086] Once a clone containing the coding sequence for at least one zinc finger has been prepared or isolated, the sequence can be cloned into any suitable vector, preferably comprising an origin of replication for maintaining the sequence in a host cell.

[0087] In preferred embodiments, the polynucleotides encoding the zinc finger moiety or a polynucleotide comprising a cis-element also comprises an expression control sequence operably linked thereto to control expression of the polynucleotide sequence (e.g., transcription and/or translation) in the cell. Examples include plasmids, phages, autonomously replicating sequences (ARS), centromeres, and other sequences which are able to replicate or be replicated in vitro or in a host cell (e.g., such as a bacterial, yeast, or insect cell) and/or target cell (e.g., such as a mammalian cell). [0088] Expression control sequences include, but are not limited to, promoter sequences to bind RNA polymerase, enhancer sequences or negative regulatory elements to bind to transcriptional activators and repressors, respectively, and/or translation initiation sequences for ribosome binding. For example, a bacterial expression vector can include a promoter such as the lac promoter and for transcription initiation, the Shine- Dalgarno sequence and the start codon AUG (Sambrook, et al., 1989, supra). Similarly, a eukaryotic expression vector preferably includes a heterologous, homologous, or chimeric promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of a ribosome.

[0089] Expression control sequences may be obtained from naturally occurring genes or may be designed. Designed expression control sequences include, but are not limited to, mutated and/or chimeric expression control sequences or synthetic or cloned consensus sequences. Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).

[0090] In order to optimize expression and/or transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the vectors to eliminate extra, or alternative translation initiation codons or other sequences that may interfere with, or reduce, expression, either at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression. A wide variety of expression control sequences--sequences that control the expression of a polynucleotide sequence operatively linked to it--may be used in these vectors to express the polynucleotide sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma, adenovirus, herpes virus and other sequences known to control the expression of genes of mammalian cells, and various combinations thereof.

[0091] In one aspect, the polynucleotide sequence encoding the zinc finger moiety or a polynucleotide comprising a cis-element also comprises an origin of replication. Suitable origins therefore include, but are not limited to, those which function in bacterial cells (e.g., such as Escherichia sp., Salmonella sp., Proteus sp., Clostridium sp., Klebsiella sp., Bacillus sp., Streptomyces sp., and Pseudomonas sp.), yeast (e.g., such as Saccharomyces sp. or Pichia sp.), insect cells, and mammalian cells.

[0092] The polynucleotide may alternatively, or additionally, comprise sequences to facilitate integration of at least a portion of the polynucleotide into a target cell chromosome. For example, the polynucleotide may comprise regions of homology to target cell chromosomal DNA. In one aspect, the polynucleotide comprises two or more recombination sites which flank a nucleic acid sequence encoding the zinc finger moiety.

[0093] The polynucleotide comprising the zinc finger moiety or a polynucleotide comprising a cis-element may additionally comprise a detectable and/or selectable marker to verify that the polynucleotide has been successfully introduced in a target cell and/or can be expressed by the target cell. These markers can encode an activity, such as, but not limited to, production of RNA, peptide, or protein, or can provide a binding site for RNA, peptides, proteins, inorganic and organic compounds or compositions and the like.

[0094] Examples of detectable/selectable markers genes include, but are not limited to: polynucleotides that encode products which provide resistance against otherwise toxic compounds (e.g., antibiotics); polynucleotides that encode products which are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); polynucleotides that encode products which suppress the activity of a gene product; polynucleotides that encode products which can be readily identified (e.g., phenotypic markers such as beta-galactosidase, a fluorescent protein (GFP, CFP, YFG, BFP, RFP, EGFP, EYFP, EBFP, dsRed, mutated, modified, or enhanced forms thereof, and the like), and cell surface proteins); polynucleotides that bind products which are otherwise detrimental to cell survival and/or function; polynucleotides that otherwise inhibit the activity of other nucleic acid segments (e.g., antisense oligonucleotides); polynucleotides that bind products that modify a substrate (e.g., restriction endonucleases); polynucleotides that can be used to isolate or identify a desired molecule (e.g., segments encoding specific protein binding sites); primer sequences; polynucleotides, which when absent, directly or indirectly confer resistance or sensitivity to particular compounds; and/or polynucleotides that encode products which are toxic in recipient cells.

[0095] The cis-elements will be selected based on the binding specificity of the zinc finger moiety being used. In a preferred embodiment, the polynucleotide sequence that is to be purified by the disclosed invention is examined for the presence of an already existing cis-element that can be bound by a zinc finger moiety. If no such cis-element is present, a cis-element can be engineered, using standard molecular biology techniques well known to the skilled artisan, into the polynucleotide molecule. In such embodiments, the cis-element will be engineered in a manner so as not to disrupt the coding sequences of the polynucleotide molecule. Ideally, the cis-element will be located in a promoter, enhancer, and/or 3’UTR sequence of the polynucleotide molecule. Wherever the cis- element is located in the polynucleotide molecule, the function of the cis-element will be retained, which is to be bound by the zinc-finger moiety when attached to the zinc finger resin in the presence of zinc, and to be released when zinc is removed. As described herein, multiple cis-elements can exist and/or be engineered into the polynucleotide sequence, such as, for example at least one, at least two, at least three, at least four, or at least five cis-elements. Examples of Zinc Finger Resin

[0096] It specifically contemplated that any resin capable of linkage to a zinc finger moiety can be used to purify polynucleotides comprising cis-elements recognized by the specific zinc finger moiety. Preferred examples of such resins that can be used to a zinc finger moiety as described herein, include, but are not limited to purification columns, such as sepharose-based resins and agarose beads (e.g., Sepharose-6B, NHS-Activated Sepharose 4 Fast Flow, Sepharose 4B (e.g., EAH, ECH) CNCR-Activated Sepharose 4 Fast Flow or 4B, or Activated Thiol Sepharose 4B), IMAC resins (using, for example, HIS fusion proteins or GSTransferase), SulfoLink^TM(ThermoFisher) or, purification fibers, such as those disclosed in Harkick et al.,“Nanofiber Adsorbents for High Productivity Continuous Downstream Processing,” J. of Biotechnology 213 (2015) 74-82; US20140296464; and US20160288089.

[0097] The attachment of the zinc finger moiety to the resin can be accomplished by any standard linker/spacer technology, so long as the ability of the zinc finger moiety to bind to the cis-element is maintained. Preferred examples of such linkers/spacers include poly-His linkers, click technology, and/or GST-fusions. Purification Method

[0098] The specific interaction of cis-elements with their corresponding transcription factors has long been appreciated. Analytical techniques like Electrophoretic Mobility Shift Assays (EMSAs) and DNAse I Footprinting were used to elucidate specific DNA fragments and sequences that were bound by transcription factors. Subsequently, Kadonaga and Tjian (1986) used the cis-elements bound to a resin to specifically purify the transcription factor, Sp1 from a nuclear extract.

[0099] The present invention relies on the high specificity of binding of a zinc finger moiety comprising at least one zinc finger to a specific cis-element. By attaching the zinc finger moiety to a resin and then passing a solution comprising a polynucleotide of interest comprising a cis-element(s) over the resin in the presence of zinc, the zinc finger moiety is capable of specifically binding to the cis-element while allowing all other molecules in the solution to pass over/through the resin. After a couple of wash steps in the presence of zinc and/or salt, the polynucleotide can be elegantly released from the resin by the removal of zinc and/or increasing the salt concentration. An alternative approach might be to include a small amount of a Zn²⁺ chelating agent with an increase in salt. Examples of such chelating agents include, but are not limited to EDTA, EGTA, and/or 1, 10 phenanthroline. The resulting solution comprises a substantially purified polynucleotide. The resin can then be“regenerated” by passing a solution comprising zinc through the column to refold the zinc fingers. This regenerated column can then be used for additional purification processes.

[0100] This invention can be distinguished from what was described previously. Specifically, almost 15 years ago, a bifunctional protein-based affinity linker consisting of a zinc finger (ZF) DNA-binding protein was fused to glutathione S-transferase (GST-ZF). See, Woodgate et al.,“Protein-Mediated Isolation of Plasmid DNA by a Zinc Finger- Glutathione S-Transferase Affinity Linker,” Biotech and Bioengineering, Vol. 79, No. 4 (2002). Here, the ZF domain of the protein bound to a sequence, while the GST domain bound to a glutathione Sepharose^TM affinity matrix. The protein-pDNA complexes formed on the column could be recovered by competitive elution with reduced glutathione, however yield was recognized to be relatively low. See, Sousa et al., “Affinity Chromatography Approaches to Overcome the Challenges of Purifying Plasmid DNA,” Trends in Biotech Vol 26 No.9 pages 518-525 (2008). Importantly, and in contrast to the invention described herein, the eluted plasmid was contaminated with zinc-finger GST fusion protein. Specifically, because the Woodgate authors eluted the bound plasmid from the column by competing with glutathione, the resulting plasmid comprised a mixture of plasmid DNA and zinc-finger/glutathione fusion protein which would need further processing in order to separate the plasmid from the zinc-finger/glutathione fusion protein.

[0101] Moreover, this GST fusion protein process described in Woodgate used only one cis-element. However, as shown in the Examples, the inventors found at least two cis-elements are needed to for optimal purification. Specifically, when two cis-elements were used, substantially higher binding affinities were unexpectedly obtained as compared to the use of one cis element.

[0102] Thus, the present invention presents a simple, yet elegant way to purify plasmid (or any other polynucleotide sequence) comprising at least two, at least three, at least four, or at least five cis-elements. By relying on the ability to promote and reverse specific binding of the cis-elements to a zinc finger moiety, a polynucleotide of interest can readily be captured on, and then released from a zinc finger resin, by adjusting the concentration of zinc and/or salt. The resulting released plasmid is substantially free from contaminating proteins and/or polynucleotides not comprising cis-elements (such as chromosomal DNA).

[0103] Any polynucleotide sequence can be purified using the disclosed method, so long as the polynucleotide molecule contains the cis-elements capable of being specifically bound to a specific/selective zinc finger moiety when attached to a resin. Preferred examples of polynucleotide molecules include, but are not limited to, not limited to plasmid DNAs (pDNA), gene therapy vectors (e.g., in other aspects, adenovirus, retroviral vectors, poxviruses, vaccinia virus) DNA/RNA vaccines, and/or RNA molecules.

[0104] The purification method described herein can be scaled up or down. If scaled up, the purification method may result in the production of milligram, gram and kilogram quantities of the polynucleotide molecule. In one embodiment, the polynucleotide molecule is generated using standard recombinant cell lines as is well known in the art. The cell lines can be processed using standard techniques, such as by lysing the cells (e.g., bacteria, yeast, fungi, mammalian, insect or other cells) obtained through shake flask culture, bioreactor or fermenter propagation (generated in the laboratory, a pilot plant or an industrial scale centrifugation) to obtain a crude lysate and then processing the polynucleotide molecule contained in the crude lysate through the zinc finger resin as described herein.

[0105] Polynucleotide molecules can be expressed in a variety of host cell, including, but not limited to: prokaryotic cells (e.g., E. coli, Staphylococcus sp., Bacillus sp.); yeast cells (e.g., Saccharomyces sp.); insect cells; nematode cells; plant cells; amphibian cells (e.g., Xenopus); avian cells; and mammalian cells (e.g., human cells, mouse cells, mammalian cell lines, primary cultured mammalian cells, such as from dissected tissues).

[0106] To generate a crude lysate, a cell paste is preferably resuspended in a buffer using resuspension techniques known in the art to resuspend the cells. The cell paste can be freshly produced or obtained from a frozen sample. The resuspension buffer is not limited. In therapeutic applications, the buffer ideally will contain reagents generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). Such buffers include sodium acetate, potassium citrate, sodium phosphate monobasic or dibasic or a mixture, and the like, with sodium acetate being preferred. The selection of pH and ionic strength is well within the level of skill in the art.

[0107] The cells in the cell paste are then lysed, using lysis techniques known to the skilled artisan. In a preferred embodiment, the cells are lysed without resorting to animal- or otherwise-derived enzymes, such as lysozyme. In further preferred embodiments, lysis is carried out in a dilute base or a dilute base and a detergent. Neither the base nor the detergent are limited. It is preferred that they represent GRAS reagents. Such bases are sodium hydroxide, potassium hydroxide, sodium bicarbonate, etc., while such detergents constitute pharmaceutically acceptable nonionic surfactants, e.g., polysorbates (sold under the trademark Tween®), polyoxyethylene ethers sold by Union Carbide (Triton®), and the like. A NaOH and Tween® combination is advantageous, with a Tween® 80 being preferred. In even more preferred embodiments, especially when working with eukaryotic cells, a NaOH and SDS combination is used. Optimization of pH and ionic strength is well within the skill of the art. An alternative embodiment to alkaline lysis is mechanical breakage using, e.g., a French Press, a microfluidizer, and the like. [0108] Optionally, the lysate can be acidified at this stage, using acidification techniques, to facilitate subsequent removal of insoluble material resulting from cell lysis by reducing the viscosity created during alkaline treatment. Any acid may be employed. Preferably it is a GRAS ingredient, such as glacial acetic acid, phosphoric acid, citric acid, and the like. The matching of pH and ionic strength is empirical and well within the skill of workers in this art. The process cell lysate can then be clarified by centrifugation or passed through one or more depth filters to remove insoluble materials. Once clarified, the lysate can be applied directly to the resin as described herein or subjected to a pre- formulation step using TFF to ensure binding of the polynucleotide target to the zinc finger moiety attached to the resin.

Examples

[0109] Various embodiments of the invention have been described. The descriptions and examples are intended to be illustrative of the invention and not limiting. Indeed, it will be apparent to those of skill in the art that modifications may be made to the various embodiments of the invention described without departing from the spirit of the invention or scope of the appended claims set forth below.

[0110] All references cited herein are hereby incorporated by reference in their entireties. Example 1– Purification Protocol

[0111] Figure 2 is a schematic overview of an exemplified Purification Process starting from cell paste to bulk DS for an exemplified polynucleotide sequence (e.g., plasmid DNA). In this example, the step labeled“pDNAa Affinity chromatography” refers to a column comprising a zinc finger moiety attached to a resin used to isolate polynucleotides comprising the cis-elements (in this case, plasmid DNA). The following table provides exemplified solutions that can be used in the illustrated Purification Process.

Example 2– ZFP Binding and Release of Specific cis-Elements

[0112] C2H2 type zinc fingers were chosen as model proteins for pDNA purification due to their high specificity and high affinity. Moreover, they are most studied zinc fingers and can be designed de novo to bind selected nucleotide sequences. Each finger is comprised of approximately 30 amino acid residues and generally 3 fingers constitute a protein. In naturally occurring transcription factors, these fingers often appear as three in tandem to achieve functional binding affinity. Thus, a total of about 90 residues are needed to create three fingers, which readily can be chemically synthesized.

[0113] Figure 3A-C shows the different zinc finger proteins and corresponding cis-elements used in the following studies. The X at the N-terminus of the engineered zinc fingers denotes either a glutathione S-transferase (“GST“) tag when produced in E. coli as a recombinant protein or an azido-lysine when produced through chemical synthesis. The bolded letters shown in Figure 3A indicate mutations. The black highlighted with white lettering identifies the amino acid regions involved in zinc ion coordination, with the underlined amino acids (either cysteine or histidine) identifying the amino acids that contact zinc. The bolded, italicized and highlighted in grey residues denote significant differences between the GAG-ZFP and GCT-ZFP.

[0114] Using an online program called the Zinc Finger Tools (https://www.scripps.edu/barbas/zfdesign/zfdesignhome.php), a 92-residue protein was designed to recognize and bind the GGGGCGGAG cis-element (hereinafter called the “GAG cis-element”). This zinc finger used in the following studies was named“GAG-ZFP”. A second zinc finger protein was also generated which recognizes and binds to the GGGGCGGCT cis-element (hereinafter called the“GCT cis-element”) named“GCT-ZFP”. The different cis-elements used in the following studies are shown in Figure 3B and an example of how one would biotinylate a double stranded cis-element (in this example GAG) is shown in Figure 3C.

[0115] To confirm the necessity of the finger structure and the specificity of DNA binding, two mutations were made to the GCT-ZFP. Specifically, an alanine was replaced at position 20 (C20A mutation) in the first zinc finger (ZF) which would eliminate a key coordinator for Zn²⁺ ion, resulting a nonfunctioning ZF. A second mutation was made at R91 (R91A) within the third ZF, which would eliminate a key DNA binding-residue, leading to a lowered affinity for its specific cis-element. The amino acid sequences of these two mutants are also shown in Figure 3A. Other zinc fingers can be designed using zinc-finger consensus sequence framework and specificity rules. See, for example, Proc. Natl. Acad. Sci. U. S. A. 90, 2256–2260; Woodgate, J. et al. (2002) Protein-mediated isolation of plasmid DNA by a zinc finger-glutathione S-transferase affinity linker. Biotechnol. Bioeng.79, 450–456. Oligos used to demonstrate cis-element binding in the following studies were labeled at the 5’ end with Biotin (Figure 3C) for subsequent detection in a plate assay.

[0116] Figure 4A is a schematic of the microplate assay designed to test binding and release of a ZFP to its corresponding cis-element. In this exemplary designed microplate assay, a ZFP is produced as a fusion protein with GST and bound to a microplate using glutathione. The ZFP is then incubated with the different biotinylated cis-elements. The biotin labeled cis-elements can be detected using standard streptavidin HRP reaction.

[0117] As shown in Figure 4B, bound polynucleotide comprising the cis-element(s) can be released by changing salt concentration and/or by adding a zinc chelator (e.g., DTPA) to the solution, which elegantly allows for the purification of the polynucleotide without protein/ZFP contamination.

[0118] Figure 5 shows the results demonstrating the selectivity and sensitivity of the ZFPs to their corresponding cis-elements. The ZFPs (0.2 ug/ml) were coated in TBS- T/2 mg/ml BSA/90 μM ZnCl2 (100 ul per well) for 30min at room temperature, washed three times with TBS-T/0.1 mg/ml BSA/90 μM ZnCl2. 100 ul of oligo duplexes in TBS- T/0.1 mg/ml BSA/90 μM ZnCl2 were incubated per designated wells for 1 hour. The wells were washed 3x with TBS-T. 100 ul of streptavidin-HRP (0.2 ug/ml) per well. (Sigma cat# OR03L-200UG). Wash 3x with TBS-T and 100 ul of HRP reagents per well. (Supersignal Elisa Pico, Thermo Fisher/Pierce cat #37070).

[0119] The results demonstrated that immobilized ZFPs could bind the corresponding cis-elements in the nM concentration. The binding was specific with the GAG-ZFP unable to bind the GCT cis-element and GCT-ZFP unable to bind the GAG cis- element. Figure 5 also shows that proper conformation is required for efficient DNA binding. Mutation of residue C20A prevents the formation of the first zinc finger, resulting in the elimination of the DNA binding capacity. A second point mutation at a DNA-binding residue R91A also results in the loss of DNA binding without changing zinc finger structure. This data is supportive of the selectivity and sensitivity of this interaction.

[0120] Figure 6 shows the binding of the cis-elements is dependent upon salt concentration. The binding of DNA oligos (5nM) were evaluated in the presence of increasing concentrations of NaCl as shown. For GCT-ZFP and GCT cis-element (shown as a broken line), no binding was observed when the salt concentration was increased to 0.4M NaCl. For GAG ZFP and the corresponding GAG cis-element (shown as a solid line), a gradual decrease in binding was observed at NaCl concentration above 0.5 M and no binding at 2M. Assay conditions were the same as described previously. Oligo duplexes were incubated in buffers with increasing concentrations of NaCl (X axis). 0.2, 0.4, 0.6, 1.2, 2.0 Molar NaCl. These results demonstrate that GAG-ZFP and its corresponding GAG cis-element have a higher affinity and require more salt to release the cis-element. This standard assay can be used to determine the ideal salt concentration for binding and release for any ZFP and corresponding cis-element.

[0121] Figure 7 shows the results of the microplate assay testing the binding of GAG-ZFP and GCT-ZFP with a single (GAG-S or GCT-S), double (GAG-D or GCT-D) or triple (GAG-T or GCT-T) cis-element(s) at varying salt concentrations. The incubation of oligos and ZFPs was in 50 mM TrisHCl, pH 8.0, without NaCl. Increasing amount of NaCl was used to release the bound polynucleotide from the ZFP. Release of bound polynucleotide was directly impacted by the salt concentration depending on the particular ZFP and cis-element used. Elution of bound cis-elements with increasing NaCl concentration (0-600 mm) is shown on the X axis. As shown below, polynucleotides containing at least two cis-elements have the highest binding values and can be used to provide optimal purification of the polynucleotide.

Claims

What is Claimed 1. A zinc finger resin comprising an attached zinc finger moiety comprising at least one zinc finger.

2. The zinc finger resin of claim 1, wherein the zinc finger is derived from a zinc finger family member selected from:

a) a ^^α-zinc finger family;

b) a hormone-nuclear receptor family;

c) a loop-sheet-helix family; or

c) GAL4-type family. 3. The zinc finger resin of claim 2, wherein the zinc finger resin further comprises a solution comprising zinc. 4. The zinc finger resin of claim 3, wherein solution comprises zinc at a concentration of

(a) 1 mM, 2mM,

3 mM,

4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19mM or 20mM;

(b) between at least 1mM and at least 5 mM, between at least 1mM and at least 10 mM, between at least 1mM and at least 15 mM, or between at least 1mM and at least 20 mM;

(c) between at least 5mM and at least 10 mM, between at least 5 mM and at least 15 mM, or between at least 5 mM and at least 20 mM;

(d) between at least 10 mM and at least 15 mM, between at least 10 mM and at least 20 mM, or between at least 15 mM and at least 20 mM; or

(e) any one of (a)-(d) wherein the concentration of zinc is provided by a solution of ZnCl2.

5. The zinc finger resin of any one of claims 1-4, wherein the zinc finger is selected from:

a) a Cys2His2 zinc finger

b) a zinc finger described in Prosite PS50157;

c) a zinc finger described in Prosite PS00028; and/or

b) or a variant of at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identicial to (a)-(c).

6. The zinc finger resin of any one of claims 1-6 further comprising a bound polynucleotide molecule, wherein said polynucleotide molecule comprises a cis-element capable of binding to zinc finger moiety.

7. The zinc finger resin of claim 6, wherein the cis-element endogenously exists in the polynucleotide sequence.

8. The zinc finger resin of either claim 6 or 7, wherein the polynucleotide molecule comprises at least two, at least three, at least four, or at least five cis-elements.

9. The zinc finger resin of any one of claims 1-8, wherein the resin is selected from: a) Sepharose-6B

b) electrospun polymer nanofibers;

c) agarose beads (e.g., Sepharose-6B);

d) IMAC resins (using, for example, HIS fusion proteins or GSTransferase);

e) SulfoLink; and/or

f) purification fibers.

10. The zinc finger resin of claim 9, wherein the electrospun polymer nanofibers are arranged as non-woven sheets stacked one on top of the other.

11. A method of purifying a polynucleotide molecule, wherein the method comprises: a) adding to the zinc finger resin of any one of claims 1-10, a first solution comprising the polynucleotide molecule, wherein said polynucleotide molecule comprises a cis- element capable of binding to the attached zinc finger, b) adding to the zinc finger resin a second solution, wherein said second solution chelates zinc from the zinc finger to release the binding of the cis-element from the zinc finger; and

d) collecting the polynucleotide molecule released from the zinc finger resin.

12. The method of claim 11, wherein the polynucleotide molecule comprises at least two, at least three, at least four, or at least five cis-elements.

13. The method of claim 11 or 12, wherein the first solution also comprises zinc to enable the binding of the cis-element to the zinc finger.

14. The method of claim 11 or 12, wherein the zinc finger resin is pretreated with zinc to form zinc fingers.

15. The method of any one of claims 11-14, wherein the second solution comprises TPEN, DTPA, salt or chelex resin.

16. The method of any one of claims 11-15, wherein the method further comprises a wash step between steps any one of steps a, b, c, or d.

17. The method of claim 16, wherein the wash step is performed by tangential flow filtration.

18. The method of any one of claims 11-17, wherein the polynucleotide molecule is obtained from a cell-lysate.

19. The method of claim 18, wherein the cell-lysate is derived from a host cell selected from a bacterial cell, a mammalian cell, a vertebrate cell, a murine cell or a baculovirus cell.

20. The method of claim 18 or 19, wherein the cell-lysate is a crude cell lysate, a partially purified cell lysate, and an aqueous solution containing the extracted polynucleotide molecule acid by alkaline lysis.

21. The method of any one of claims 18-20, wherein the solution comprising the polynucleotide molecule is pre-treated to at least partially remove endotoxin.

22. The method of any one of claims 11-21, wherein the zinc finger affinity resin is regenerated.

23. The zinc finger affinity resin of any one of claims 1-10 or the method of any one of claims 11-22, wherein the polynucleotide molecule is a plasmid.

24. The zinc finger affinity resin of any one of claims 1-10 or the method of any one of claims 11-23, wherein the zinc finger moiety has at least 3 zinc fingers.

25. The zinc finger affinity resin of any one of claims 1-10 or the method of any one of claims 11-24, wherein the polynucleotide comprises at least 2 cis-elements.

26. A kit comprising the zinc finger resin of any one of claims 1-10.

27. The kit of claim 26 further comprising a cloning vector comprising the cis-element.