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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env

Definitions

• the present invention in some embodiments thereof, relates to a method of editing DNA in a cell and constructs capable of same.
• the lentivirus genome derived from immunodeficiency viruses has been split into multiple fragments to minimize the potential formation of replication-competent viruses.
• immunodeficiency viruses such as human immunodeficiency virus-1 (HIV-1)
• HIV-1 human immunodeficiency virus-1
• Such vectors target heterogeneous cell populations and lead to semi-random insertion of transgenes into cellular chromosomes. Somatic gene transfer thus results in a complex mosaic with coexistence of initially millions of cell clones that are distinguished not only by the site and number of transgene insertions, but potentially also by their intrinsic developmental potential.
• the transgene sequence including its epigenetic modulation and the level of transgene expression may differ between individual gene-modified cells. Owing to the multifaceted nature of this selection process, presently available approaches do not allow reliable predictions of potential consequences of insertional mutagenesis.
• ZF-ATFs Zinc finger artificial transcription factors
• PZF polydactyl zinc finger
• Cys2His2 ZF domains typically bind three- contiguous DNA bases per ZF and are highly modular. These features allow construction of more complex PZF fusion proteins, which in principle can recognize unique sites within a complex genome.
• ANN and recently also CNN target sites where N represents any of the four bases, the most optimal zinc finger designs have been characterized, making ZF-ATF construction possible for almost any target site. More recently Zinc-Finger-Recombinase chimeras were also demonstrated. While being efficient in cases where the targeting is specific, the design of the above proteins remains very challenging and is currently expensive and lengthy process of trial-and- error with no guarantee of a successful outcome.
• the RDOs applied in the first gene correction experiments were all comprised of
• DNA and RNA in a complementary hairpin configuration contain a "mutator" region of 5 DNA nucleotides complementary to the target sequence, except for the targeted point mutation flanked by two blocks of 2 prime-O-methyl RNA residues of ten nucleotides each, also complementary to the target locus.
• the 2'-0-methylation serves as protection from nucleases present in the cell nuclei.
• a nick is embedded in the duplex by extending the 3' end of the oligonucleotide to form a 5-bp-long GC clamp bridged by four T residues (a T-loop) at the end.
• the nick is provided as a swivel to permit topological intertwining of the RDO into the target double stranded DNA. It is recessed within the duplex to enhance thermal stability and nuclease resistance.
• modified ssDNA oligonucleotides can be used to modify genomic DNA.
• the modified ssDNA oligonucleotides comprise nuclease resistant residues, preferably at or near the termini of the oligonucleotides.
• U.S. Patent Application No. 20070122822 teaches compositions and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration. These methods and compositions involve cells with altered levels or activities of a protein from the RAD52 epistasis group.
• International Patent Application WO 1990/014092 teaches single-stranded oligonucleotides for site-directed modification in mammalian cells.
• International Patent Application WO 1990/014092 teaches that the oligonucleotides comprise sequences which have substantial homology with the site of insertion, but may differ by one or more bases, usually not more than about 10.
• an isolated polynucleotide comprising a single-stranded DNA polynucleotide encoding a regulatory site, the regulatory site being longer than 15 bases and being flanked by two targeting sequences, wherein the two targeting sequences hybridize with a continuous sequence of mammalian genomic DNA and wherein each of the targeting sequence is at least 10 bases.
• a method of inserting a nucleic acid sequence of interest into genomic DNA in a cell comprising introducing at least one single-stranded DNA polynucleotide into the cell, wherein the at least one single-stranded DNA polynucleotide comprises the nucleic acid sequence of interest flanked by two targeting sequences, wherein the two targeting sequences hybridize under physiological conditions with a continuous sequence of the genomic DNA and wherein the nucleic acid sequence of interest is longer than 15 bases, thereby inserting the nucleic acid sequence into genomic DNA of a cell.
• a method of inserting a nucleic acid of interest into genomic DNA of a population of cells comprising:
• a method of deleting a target sequence from genomic DNA in a cell comprising:
• a method of treating a trinucleotide repeat-associated disease in a subject in need thereof comprising:
• the regulatory site comprises a recombination site.
• the isolated polynucleotide is attached to a targeting moiety.
• the recombination site is selected from the group consisting of a hix site, an att site, a lox site and a frt site.
• the lox site comprises a sequence as set forth in SEQ ID NOs: 9-15.
• each of the two targeting sequences is 20 bases.
• At least one of the two targeting sequences comprises a modified base.
• the continuous sequence of genomic DNA repeats at least twenty times in a single chromosome of the cell.
• the at least one single- stranded DNA polynucleotide comprises at least two identical single-stranded DNA polynucleotides.
• the at least one single- stranded DNA polynucleotide comprises two non-identical single-stranded DNA polynucleotides.
• the two non-identical single- stranded DNA polynucleotides comprise non-identical nucleic acid sequences of interest.
• the single-stranded DNA polynucleotide comprises non-identical targeting sequences.
• the genomic DNA is selected from the group consisting of nuclear DNA, mitochondrial DNA and chloroplast DNA.
• the nucleic acid sequence of interest comprises at least one stem-loop structure.
• the nucleic acid sequence of interest comprises less than 50 bases.
• the nucleic acid sequence of interest encodes a recombination site.
• At least one of the two targeting sequences is greater than 10 bases.
• At least one of the two targeting sequences is 20 bases.
• At least one of the two targeting sequences comprises a modified base.
• the cell is a eukaryotic cell.
• the cell is a mammalian cell, According to some embodiments of the invention, the cell is a plant cell,
• the cell is an intact cell, According to some embodiments of the invention, the mammalian cell is a human cell.
• the cell is a stem cell.
• the stem cell is an embryonic stem cell.
• the cell is a diseased cell.
• the method is effected in vivo.
• the method is effected ex vivo.
• the single stranded DNA polynucleotide is associated with a cell-targeting moiety.
• step (a) is affected prior to step (b). According to some embodiments of the invention, step (a) is effected concomitant to step (b).
• the genomic DNA is selected from the group consisting of nuclear DNA, mitochondrial DNA and chloroplast DNA.
• the recombinase enzyme is selected from the group consisting of Hin invertase, ⁇ integrase, Cre and Flp.
• the nucleic acid of interest encodes at least one detectable moiety.
• the method further comprises selecting cells which comprise the nucleic acid of interest.
• the method further comprises selecting at least one of the plurality of cells, prior to step (b) wherein the at least one of the plurality of cells comprises at least two recombinase sites integrated into a single chromosome thereof.
• the trinucleotide repeat-associated disease is Huntington's chorea.
• the method further comprises selecting at least one of the cells, prior to step (b) wherein the at least one of the plurality of cells comprises at least two recombinase sites integrated into a single chromosome thereof.
• the cells comprise neuronal cells.
• the cells comprise stem cells.
• stem cells Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE DRAWINGS
• FIGs. 1A-C are schemes and photographs illustrating fragment incorporation into a 40bp sequence (which was repeated 22 times) in HeLa cells according to an embodiment of the present invention.
• A Schematic representation of the genomic regions containing multiple repeats, the primers used in this study and the fragment containing LoxP (SEQ ID NO: 1) which was incorporated.
• B Schematic representation of the expected PCR products
• C Experimental PCR products following transfection with the fragment that contains loxP (SEQ ID NO: 1), or GFP plasmid into genomic DNA of HeLa cells.
• FIG. 2A is a photograph of the experimental PCR products following transfection with the fragment that contains loxP (SEQ ID NO: 1), or GFP plasmid into genomic DNA of HeLa cells (identical to Figure 1C).
• FIG. 2B is a sequence (SEQ ID NO: 2) of the PCR product following transfection with the fragment (SEQ ID NO: 1). The band was excised from gel, ligated to pGEM-t-easy, grown in bacteria and sequenced. LoxP incorporation sites are highlighted in green.
• FIGs. 3A-D are photographs illustrating fragment (SEQ ID NO: 1) in different cell lines and under different conditions.
• N stands for "No Primers", "O” for OFI.
• A Standard procedure as in Figure 2A.
• B Same as ( Figure 2A) after 6 cell divisions.
• C Same as ( Figure 2A) in HI line of human ESCs.
• D Same as ( Figure 2A) after irradiation (60Rad) of the HeLa cells prior to the transfection of the fragment.
• FIG. 4 are photographs and schematic representations illustrating the dependence of the efficiency of fragment incorporation on the insert. Fragments with different inserts but same flanking region (targeted to the 22 repeat loci) were transfected to HeLa cells. Shown here are PCR results for the incorporation detection (top), the secondary structure and sequence, respectively SEQ ID NOs: 3, 4, 1, and 5) of each of the constructs (middle) and its free energy (bottom).
• FIGs. 5A-B are photographs illustrating the contribution of genomic location on the efficiency of fragment incorporation.
• A fragment with a single loxP insert was incorporated into two different 22 repeats of a 40bp sequence: rpt22-l(SEQ ID NO: 1) and rpt22-2 (SEQ ID NO: 6).
• B fragment with a single lox66 insert was incorporated into 3 different genomic repeats: rpt22 (22 repeats, SEQ ID NO: 4), rpt 5188 (5188 repeats, SEQ ID NO: 7) and rpt5192 (5192 repeats, SEQ ID NO: 8).
• FIG. 6A is a schematic representation of a vector plasmid according to one embodiment of the present invention.
• FIG. 6B is a photograph of FACS positive cells expressing the plasmid 38 days following transfection.
• FIG. 7 is a schematic representation of the incorporation of the fragments of embodiments of the present invention.
• Dividing cells are transfected with the fragment. Cells are allowed to grow for over two passages to verify stable incorporation into the genome.
• the same cells are then transfected with a plasmid carrying the Cre recombinase and a second plasmid carrying the gene of interest, loxP site and selection marker.
• the subpopulation of cells that by now contains the intragenic loxP site will integrate the second Plasmid (3) and start expressing the selection marker;
• FIGs. 8A-C are schematic representations of constructs designed to identify cells that underwent genomic incorporation of the plasmid.
• A The tet-off system. tetR protein binds to tetO sequences only upon introduction of Dox, thus leading to the repression of the downstream gene.
• B Schematics of a modified system expressing the selection marker only upon its integration into the genome. A mutant loxRE site is inserted between an upstream tetO sequences and a downstream selection marker (eGFP). In the presence of tetR (introduced on a different plasmid) and Dox, the expression of eGFP from the plasmid is prevented by the repressor.
• C Integration of the plasmid into the genome displaces the repressor-bound tetO sequence (and two mutated loxP sites, LE and RE) downstream to the selection marker, which is now driven by fully active, CMV promoter.
• FIGs. 9A-B are schematic representations illustrating the mechanism of Cre/loxP-mediated plasmid integration into the genome.
• A The standard recombination model, exhibiting equilibrium between integration and excision reactions with a strong preference for excision.
• B Integration with mutant lox sites. Integration creates two different lox sites (WT and full-mutant) which can rarely be excised, thus tilting the equilibrium almost solely towards integration.
• FIGs. 10A-B are schematic representations for identifying and analyzing fragment incorporation into hES cells.
• the vector plasmid carries two different fluorophore genes - GFP and TFP - downstream of a constitutive PGK promoter. The GFP gene is separated from its promoter by a lox site. Successful recombination into genomic lox site would therefore eliminate the GFP promoter and will turn off the expression of GFP.
• the vector plasmid carries two different fluorophore genes - GFP and TFP - downstream of a constitutive PGK promoter. The TFP gene is separated from its promoter by a lox site. Successful recombination into genomic lox site would therefore eliminate the TFP promoter and will turn off the expression of TFP. Genetically modified cells will be GFP+/TFP- while unmodified cells will express both GFP and TFP.
• FIGs. 11A-G are schematic representations and images illustrating the transformation of cells with exemplary fragments according to Figure 10B
• A Schematic representation of the processes leading to transformation of cells.
• FIGs. 12A-B are schematic representations illustrating Random intergenic recombinations between wild-type loxP sites.
• Cre recombinase any number of recombinations can occur leading to unwanted effects such as deletions of part of chromosomes and combining two or more chromosomes into one. Lines denote chromosomes and triangles denote lox sites.
• A The lox sites after fragment introductions but before the introduction of Cre. Blue arrows indicate possible lox recombination pairs
• B Some of the possible ill effects following Cre introduction.
• FIGs. 13A-E are schematic representations of mutant lox sites that only allow plasmid integration.
• A Wild type lox sites that allow any recombination.
• B Insertion only lox mutants that allow only plasmid integration but not excision. The mutation is located at the flanks of the lox sequence.
• C lox Mutants that can not recombine with themselves, but are able to undergo recombination with another lox site. The mutation is located in the center (spacer) part of the lox sequence.
• D Combined mutant that can not recombine with itself, and is not excitable
• E More detailed representation and sequence of the double-mutant lox site from (D).
• FIGs. 14A-F are schematic representations and photographs illustrating the effectiveness of the mammalian genome targeting approach of embodiments of the present invention.
• Figures 14A-B Schematics of targeting repeated (14A) or unique genomic sequences (14B), either within genes or outside genes.
• Figure 14C Schematics of detection of exemplary fragment incorporation in (14B) using PCR.
• 14D Successful incorporation of exemplary fragments into the Pdxl intron of Hela cells, as determined by PCR bands.
• 14E Sequence-based verification of successful targeting of the Pdxl locus - SEQ ID NO: 27.
• 14F Schematics of a 5-day protocol resulting in fragments incorporation (day 1), its exchanges with an arbitrary plasmid (day 3), and selection of modified cells by antibiotic or by FACS.
• FIGs. 15A-C are photographs illustrating fragment-dependent survival of Hela cells under G418 selection.
• Hela cells were targeted as shown in Figure 14F and were exposed for 1 week to lmg/ml of G418. Resulting colonies were stained with methylene blue. Both the number and size of surviving colonies were clearly dependent on the dosage of fragments in the first step of the incorporation, indicating that the subsequent incorporation of the neo plasmid (2 nd step) was indeed mediated by successful incorporation of the fragment in the preceding step. Note that all three cases were transfected with the same amount of plasmid (3 ⁇ g/10 6 cells) carrying neo.
• FIGs. 16A-B are images of a colony of human iPS cells that were derived from a Huntington's patient and were targeted with OFI directed against the 180 tri-nucleotide repeats in the Huntington gene. The incorporated fragment was then exchanged with a plasmid carrying GFP. Green colonies were FACS-sorted and expanded using standard techniques of growing human embryonic stem cells. Shown are a phase contrast ( Figure 16A), and a fluorescent image ( Figure 16B) of a single colony.
• FIGs.l7A-B illustrate OFI-mediated disruption of CXCR4 and protection from HIV-1 infection in human cells.
• the HIV virus surface receptor gpl20 binds CD4 and CXCR4 in order to infect cells. Once Infected the HIV inserts its genome into the target cell genome at a random position. The cell then continues to produce virions.
• a mixture of OFIs can be used to disrupt both the integrated viral sequence and the endogenous co-receptor CXCR4. This allows establishing a virus-free population of T- cells or T-cell precursors which are resistant to HIV infection due to their lacking of the CXCR4 co-receptor.
• B Example of targeting of the CXCR4 gene using a single OFI.
• the depicted sequence (SEQ ID NO: 36) is part of the second exon of CXCR4 gene with OFI-mediated lox66 incorporation.
• the incorporation causes frameshift rendering the gene inactive.
• the lox66 sequence can be further used to recombine a sequence of interest, such as selection marker, into this genomic location.
• Lane 1 of the gel is a ladder.
• Lane 2 is a PCR of the OFI-treated cells using genomic primers to verify genomic DNA integrity.
• Lane 3 is a PCR of the OFI-treated cells with genomic forward primer and lox66 reverse primer which works only if the lox66 sequence was successfully incorporated into the genomic locus.
• Lane 4 is a PCR performed on non- treated, wild-type genomic DNA with genomic forward primer and lox66 reverse primer. Lane 3 produces a band of the correct size while the negative control Lane4 is empty as expected.
• the present invention in some embodiments thereof, relates to a method of editing DNA in a cell and constructs capable of same.
• RNA/DNA oligonucleotides RDOs
• ssODNs modified single-stranded oligodeoxyribonucleotides
• the present inventors have devised ssDNA inserts designed to achieve a dramatic improvement in the ability to modify the mammalian genome. Specifically, the present inventors designed oligonucleotides such that a 34 base pair LoxP fragment was stably inserted into the cell genome at a targeted site ( Figures 2A-B, 3A-D and 14B-E). Subsequent transfection with a plasmid encoding a CRE recombinase enabled the exchange of the initial insert with a (co-transfected) DNA sequence of choice ( Figures 6B, 15A-C).
• a method for targeted insertion of a nucleic acid sequence of interest into DNA in a cell.
• the method comprises introduction of a single-stranded DNA polynucleotide into the cell, wherein the single-stranded DNA polynucleotide comprises an exogenous nucleic acid sequence of interest flanked by two targeting sequences, wherein the two targeting sequences hybridize under physiological conditions with a continuous sequence of the genomic DNA and wherein the exogenous nucleic acid sequence of interest is longer than 15 bases, thereby inserting the nucleic acid sequence into genomic DNA of a cell.
• Cells into which the nucleic acid sequence of interest may be inserted are typically eukaryotic cells, including for example mammalian cells (e.g. human) and plant cells.
• the mammalian cells are stem cells, such as embryonic stem cells or mesenchymal stem cells.
• the present invention contemplates insertion of the nucleic acid sequence of interest into induced pluripotent cells (iPS) cells. Such cells are further described herein below.
• iPS induced pluripotent cells
• the cells may be isolated e.g. part of a cell line or primary cells.
• the cells may be comprised in a tissue, organ or organism.
• the cells are diseased cells.
• the cells are proliferating.
• the cells are intact cells (i.e. not part of a cell extract).
• the genomic DNA into which the nucleic acid of interest is inserted includes nuclear DNA and organellar DNA, such as mitochondrial DNA and chloroplast DNA.
• the nucleic acid of interest may be inserted into any part of the DNA - e.g. in the regulatory portion of a gene, in the promoter region, in the 3 Or 5' non- translated region of a gene, in the coding region of a gene, in the junction between an intron and an exon, or in a transformed region, i.e. non-native DNA.
• the insertion site may be a unique sequence of the genome or may be repeated multiple times.
• the nucleic acid of interest may be inserted into the genome in a region which is devoid of genes, regardless of the chromatin state. Specifically, the method applies to both open and closed chromatin regions.
• the present invention also contemplates inserting more than one type of single stranded polynucleotide into the cell at the same time.
• the present invention contemplates inserting an identical sequence of interest into the genome of a cell using non-identical targeting sequences (see Example 6 herein below). Additionally, or alternatively, the present invention contemplates inserting non-identical sequences of interest into the genome using identical targeting sequences.
• the nucleic acid of interest is inserted into a region of genomic DNA which repeats at least 5 times in a single chromosome. According to another embodiment, the nucleic acid of interest is inserted into a region of genomic DNA which repeats at least 10 times in a single chromosome. According to yet another embodiment, the nucleic acid of interest is inserted into a region of genomic DNA which repeats at least 20 times in a single chromosome.
• the method is affected by introducing a single-stranded DNA polynucleotide comprising the nucleic acid sequence of interest flanked by two targeting sequences into the cell, wherein the two targeting sequences hybridize under physiological conditions with a continuous sequence of the genomic DNA and wherein the nucleic acid sequence of interest is longer than 15 bases.
• Polynucleotides designed according to the teachings of the present invention can be generated according to any polynucleotide/oligonucleotide synthesis method known in the art such as enzymatic synthesis or solid phase synthesis.
• Equipment and reagents for executing solid-phase synthesis are commercially available from, for example, Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the capabilities of one skilled in the art and can be accomplished via established methodologies as detailed in, for example, "Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.
• the single-stranded targeting DNA polynucleotides are typically greater than 60 nucleotides in length (combined length). According to one embodiment, the single- stranded targeting DNA polynucleotide is about 80 nucleotides in length. According to one embodiment, the single-stranded targeting DNA polynucleotide is about 90 nucleotides in length. According to one embodiment, the single-stranded targeting DNA polynucleotide is about 100 nucleotides in length. According to yet another embodiment, the single-stranded targeting DNA polynucleotide is between about 60 nucleotides in length and 400 nucleotides in length.
• the nucleic acid sequence of interest is at least 10 nucleotides in length, at least 15 nucleotides in length, at least 20 nucleotides in length, at least 25 nucleotides in length or at least 30 nucleotides in length.
• the nucleic acid sequence of interest is no longer than 50 nucleotides in length.
• the nucleic acid sequence of interest comprises at least one stem loop structure.
• Analysis of the sequence of a particular nucleic acid enables the determination of its structure. Determination of structure of a particular nucleic acid can be aided by the use of specific computer programs, such as the Mfold web server for nucleic acid folding and hybridization prediction (Nucleic Acids Res. 31 (13), 3406-15, 2003) and Vienna RNA webservers.
• the nucleic acid sequence of interest is selected such that it avoids identification by the DNA repair machinery, which might otherwise operate to excise and/or degrade the inserted sequence. This protection may increase the efficiency of insertion.
• the nucleic acid sequence of interest encodes a regulatory site.
• regulatory sites include, but are not limited to recombination sites, lac Operator sites (e.g. SEQ ID NO: 16), tet Operator sites (e.g. SEQ ID NO: 17), a UAS site (e.g. SEQ ID NO: 18), and any other type of binding site for any type of gene or another molecule (e.g. transcription factor, chromatin modifying enzyme, co-repressor, co-activator, RNA molecule, DNA molecule, etc).
• lac Operator sites e.g. SEQ ID NO: 16
• tet Operator sites e.g. SEQ ID NO: 17
• UAS site e.g. SEQ ID NO: 18
• any other type of binding site for any type of gene or another molecule e.g. transcription factor, chromatin modifying enzyme, co-repressor, co-activator, RNA molecule, DNA molecule, etc.
• DNA-binding proteins and/or other types of molecules
• nucleic acid sequences of interest that comprise at least one stem loop structure are recombination sites and mutations thereof, including, but not limited to hix sites (e.g. SEQ ID NO: 19,20), att sites (e.g. SEQ ID NO: 21, 22), lox sites (e.g. SEQ ID NOs: 9-15) and frt sites (e.g. SEQ ID NO: 23).
• hix sites e.g. SEQ ID NO: 19,20
• att sites e.g. SEQ ID NO: 21, 22
• lox sites e.g. SEQ ID NOs: 9-15
• frt sites e.g. SEQ ID NO: 23
• the two targeting sequences on either side of the nucleic acid sequence of interest are designed such that they are capable of hybridizing under physiological conditions with a continuous sequence of the genomic DNA.
• the targeting sequences are designed such that they hybridize with the lagging strand of the genomic DNA (i.e. the targeting sequences comprise antisense oligonucleotides)
• physiological conditions refer to the conditions present in cells, tissue or a whole organism or body.
• the physiological conditions used by the present invention include a temperature between 34-40 °C, more preferably, a temperature between 35-38 °C, more preferably, a temperature between 36 and 37.5 °C, most preferably, a temperature between 37 to 37.5 °C; salt concentrations (e.g., sodium chloride NaCl) between 0.8-1 %, more preferably, about 0.9 ; and/or pH values in the range of 6.5-8, more preferably, 6.5-7.5, most preferably, pH of 7-7.5.
• At least one of the two targeting sequences are greater than 10 nucleotides in length - for example about 20 nucleotides in length. According to another embodiment, each of the two targeting sequences is greater than 10 nucleotides in length - for example about 20 nucleotides in length.
• At least one of the two targeting sequences comprises a modified base or linkage.
• the modification may be at the ends of the single-stranded targeting DNA polynucleotide, however the present invention also contemplates modification along a portion or even the entire length of the single- stranded targeting DNA polynucleotide.
• modified ssDNA oligonucleotides can be used to modify genomic DNA.
• the modified ssDNA oligonucleotides comprise nuclease resistant residues (e.g. 2'-0-methyl analogs or locked nucleotide analogs), preferably at or near the termini of the oligonucleotides.
• WO 2003/076574 incorporated herein by reference, teaches single- stranded oligonucleotides for site-directed modification in plant cells, whereby the oligonucleotides are modified to comprise at least one cationic phosphoramidate internucleoside linkage.
• Methods for preparing cationic oligonucleotides having such linkages are found in Dagle & Weeks, Nucl. Acids Res. 24: 2143-2149,1996 ; Dagle et al., Nucl. Acids Res 28: 2153-2157,2000 ; US 6,274, 313 and US 5,734,040 the disclosures of which are incorporated herein by reference.
• the cationic phosphoramidate may be incorporated during oligonucleotide synthesis via oxidative amidation.
• the cationic phosphoramidate have both a primary and a tertiary amine.
• Positively charged internucleoside linkages can be generated by using a diethylethylenediamine such as N, N-diethylethylenediamine (DEED) and methoxyehtylamine phosphoramidates.
• polynucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5' phosphodiester linkage.
• Additional modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'- amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5 -3' or 2'-5' to 5'-2'.
• Various salts, mixed salts and free acid forms can also be used.
• modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
• morpholino linkages formed in part from the sugar portion of a nucleoside
• siloxane backbones sulfide, sulfoxide and sulfone backbones
• formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
• alkene containing backbones sulfamate backbones
• sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH2 component parts, as disclosed in U.S. Pat. Nos.
• oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
• the base units are maintained for complementation with the appropriate polynucleotide target.
• An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA).
• PNA peptide nucleic acid
• a PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
• the bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
• Oligonucleotides of the present invention may also include base modifications or substitutions.
• "unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
• Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2- thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyI and other 5-substit
• 5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
• 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. [Sanghvi YS et al. (1993) Antisense Research and Applications, CRC Press, Boca Raton 276-278] and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
• the polynucleotides of the present invention may be attached (either directly, or non-directly) to a targeting moiety.
• the targeting moiety may be a cell targeting moiety or an intracellular targeting moiety (e.g. nuclear targeting moiety).
• the present invention contemplates attaching the polynucleotide of the present invention to an antibody or fragment thereof, a receptor ligand or a glycosaminoglycan.
• the polynucleotides of the present invention may be attached (either directly, or non- directly) to a detectable moiety - e.g. a fluorophore.
• non-covalent association of the polynucleotide of the present invention with a targeting moiety may be effected via a nucleic acid carrier which is associated with both the polynucleotide of the present invention and the targeting moiety.
• a nucleic acid carrier which is associated with both the polynucleotide of the present invention and the targeting moiety.
• Covalent association of molecules such as the nucleic acid carrier, the targeting moiety, and the polynucleotide of the present invention may be achieved using any of various chemical and biological methods well known to the ordinarily skilled artisan.
• chemical methods for general guidance regarding the practice of such chemical methods, refer, for example, to the extensive guidelines provided by The American Chemical Society (http://worldwidewebdotchemistrydotorg/portal/Chemistry).
• One of ordinary skill in the art, such as, for example, a chemist will possess the required expertise for practicing chemical techniques suitable for covalently associating molecules for practicing the present invention.
• Introducing the polynucleotide of the present invention into genomic DNA in a cell can be effected using any method known in the art. Depending on the location of the cell, the polynucleotides may be introduced in vivo, ex vivo or in vitro. Selection of a particular method of introduction is typically dependent on the cell type being targeted.
• the polynucleotide of the present invention may be incorporated into its genome using calcium phosphate method, electroporation or via a nucleic acid carrier.
• a nucleic acid carrier which prevents chemical interaction of the polynucleotide of the present invention with a non cytoplasmic environment, such as serum in particular, can serve to prevent, in-vivo and in-vitro, degradation of the DNA molecule by the non cytoplasmic environment.
• nucleic acid carriers that may be used to transfect the polynucleotides of the present invention into mammalian cells include, but are not limited to liposomes, cationic lipids and synthetic polymers such as polyethylenimine (PEI) or poly-L-lysine.
• PEI polyethylenimine
• Exemplary cationic lipids that may be used as carriers include, but are not limited to l,2-Dilauroyl-sn-Glicero-3-Phosphoethanolamine (DLPE) and 1,2-Dilauroyl-sn- Glicero-3-Glycerol (DLPG), dioleoyl-l,2-diacyl-3-trimethylammonium-propane (DOTAP, at 18:1; 14:0; 16:0, 18:0) and N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethlylammonium chloride (DOTMA); dimethyldioctadecylammonium (DDAB); l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (Ethyl PC , at 12:0; 14:0; 16:0; 18:0; 18:1; 16:0-18:1); l,2-di-(9Z
• the polynucleotides of the present invention may be introduced into plants by one or more techniques typically used for direct DNA delivery into cells. Such protocols may vary depending on the type of plant or plant cell, i. e. monocot or dicot, targeted for gene modification. Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606, direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U. S. Patent No. 4,945,050; WO 91/10725 and McCabe et al. (1988) Biotechnology 6: 923-926).
• one embodiment of this aspect of the present invention comprises inserting a recombinase site into the genome of a cell, thus allowing the subsequent or concomitant insertion of an additional nucleic acid of interest.
• This may be effected by a recombinase enzyme-mediated site-specific integration of transfected DNA into the chromosomally positioned recombinase site.
• Site-specific recombination is a process involving reciprocal exchange between specific nucleic acid sites (referred to as target sites) catalyzed by specialized proteins known as site-specific recombinases.
• Site-specific recombinases recognize specific DNA sequences, and in the presence of specific recombination sites they catalyze the recombination of DNA strands.
• recombinases can catalyze excision or inversion of a DNA fragment according to the orientation of their specific target sites. Recombination between directly oriented sites leads to excision of the DNA between them, whereas recombination between inverted target sites causes inversion of the DNA between them.
• the lambda integrase family of site-specific recombination systems consists of more than 100 different members. Among the most prominent of these are lambda Int, Cre/lox, FLP/FRT, R/RS, and Gin/gix.
• Recombinases such as Cre, FLP, R and Gin catalyze DNA recombination between their respective DNA substrates or target sites, loxP, FRT, RS and gix. These recombination systems use a common reaction pathway to carry out very different biological functions. They utilize a single polypeptide recombinase capable of recognizing a small DNA sequence without requiring any accessory factors.
• Cre/lox, FLP/FRT, R/RS, Gin/gix carry out a freely reversible reaction, whereas lamda-Int requires additional factors to carry out the reverse reaction.
• the cells are transfected with a mixture of two polynucleotides (plasmids): (i) a polynucleotide encoding the recombinase enzyme (e.g. CRE recombinase) and (ii) a polynucleotide comprising the nucleic acid of interest and an additional recombinase site (e.g. loxP site).
• a polynucleotide encoding the recombinase enzyme e.g. CRE recombinase
• a polynucleotide comprising the nucleic acid of interest and an additional recombinase site (e.g. loxP site).
• Exemplary recombinase enzymes include, but are not limited to Hin invertase, ⁇ integrase, Cre and Flp, mutated forms thereof, temperature sensitive forms thereof as well as self destructing forms thereof (i.e. plasmids with delayed expression of RNAi against the recombinase.
• the nucleic of interest may encode a selectable marker or detectable moiety, such that selection can be applied to acquire a homogenous cell population, or so that the success rate of integration may be followed, or even to visualize the number of sites that were incorporated in specific cells (e.g. by swapping the recombination site with a cascade of DNA sequences (denoted "tagging cascade") that can be bound by specific proteins carrying a visible tag, such as the lacO/lacR-GFP system or the UAS/GAL4-GFP system).
• the polynucleotide (ii) may encode a selectable marker in addition to the nucleic acid of interest.
• a selectable marker encodes a product that the host cell cannot make, such that the cell acquires resistance to a specific compound or is able to survive under specific conditions.
• the marker may code for an antibiotic resistance factor.
• antibiotic resistance markers include, but are not limited to, those coding for proteins that impart resistance to kanamycin, spectomycin, puromycin, neomycin, geneticin (G418), ampicillin, tetracycline, and chlorampenicol.
• the selectable marker may code for proteins that confer resistance to herbicides, such as chlorsulfuron or phosphinotricin acetyltransferase.
• selectable markers include the thymidine kinase (tk) and the adenine phosphoribosyltransferase (apr) genes, which enable selection in tk ' and apr " cells, respectively, and the dihydrofloate reductase (dhfr) genes that confer resistance to methotrexate or trimethoprim.
• tk thymidine kinase
• apr adenine phosphoribosyltransferase
• dhfr dihydrofloate reductase
• Example 3 One approach is described in Example 3, herein below. Another approach is illustrated in Figures 8A-C.
• the Tet-Off system carries a tetR repressor which shuts the transcription from the plasmid by binding to a tetO sequences in the presence of Doxycycline (Dox). If the tetO sequences are located upstream of a promoter of a gene, then in the presence of tetR and Dox, the gene is silenced ( Figure 8A). Upon plasmid integration, it undergoes linearization at the lox site, placing the repressor site away from the region to be expressed ( Figure 8C).
• a plasmid may be engineered with at least one tetO sequence upstream of a gene encoding a detectable moiety (e.g. eGFP gene) driven by a constitutive promoter (e.g. CMV promoter).
• a constitutive promoter e.g. CMV promoter
• a lox site may be placed ( Figure 15B).
• the expression of eGFP from this plasmid is repressed by tetR in the presence of Dox.
• the tetO sequences will move far downstream of the CMV promoter, thus removing the inhibition on the expression of eGFP (Figure 8C).
• Figures 10A Yet another approach is illustrated in Figures 10A.
• the plasmid has two different fluorophores (e.g. a GFP and Tomato - TFP -) under a constitutive promoter (e.g. CMV promoter or a PGK promoter which was shown to work in ES cells (among others)).
• the GFP is separated from its promoter by the lox site.
• Successful recombination of the plasmid into the genomic lox site separates the GFP from its promoter thereby turning of the green fluorescence while retaining the red fluorescence.
• the cells that have incorporated the plasmid are then sorted for TFP positive and GFP negative signals.
• insertion of more than one recombinase site into a single chromosome of a cell allows for a deletion of a portion of that chromosome between the two recombinase sites.
• a method of deleting a target sequence from genomic DNA in a cell comprising:
• step (b) can be effected following step (a) or alternatively concomitant with step (a).
• cells may be selected that comprise more than one inserted recombinase site in a particular chromosome - e.g. by PCR or other such technique, or by insertion of a tagging cascade (as described above).
• the targeting sequences flanking the recombinase site have to target sites which repeat at least once along the length of the chromosome.
• a cocktail of targeting sites, each being homologous to a different locus can be used in order to integrate a single nucleic acid of interest into multiple sites that are different in sequence.
• any number of recombinations can occur leading to unwanted (or alternatively desired) effects such as deletions of part of chromosomes and combining two or more chromosomes into one.
• the recombinase site which is selected possesses two main qualities: (a) Uni-directionality; which means that the incorporation reaction would be preferred to the excision reaction ( Figures 9A-B and Figure 13B) (b) Inability to recombine with itself; this way the genomic mutant recombination sites would be unable to recombine among themselves while still being able to recombine with plasmid based recombination sites ( Figure 13C).
• loxP mutants SEQ ID NO: 14, 15
• Such mutants should allow efficient recombination of all and any plasmids carrying complementary lox into the genome whilst at the same time avoid all non-specific by-products, such as those illustrated in Figures 12A-B.
• the targeting sequences are engineered such that they are capable of hybridizing to a repeating motif.
• a method of treating a trinucleotide repeat-associated disease in a subject in need thereof comprising:
• step (b) may be effected following or concomitant to step (a), as described earlier for other aspects of the present invention.
• the present invention also contemplates using other single-stranded DNA polynucleotides of the present invention (which do not encode a recombination site) in order to disrupt the trinucleotide repeating motif of the genomic DNA of the patient.
• Diseases associated with repeating DNA sequences are described for example in Seminars in Pediatric Neurology, Volume 14, Issue 1, March 2007, Pages 26-33, incorporated herein by reference.
• the trinucleotide repeat-associated disease is Huntington's chorea.
• the cells are neuronal cells derived from the subject.
• the cells are pluripotent cells (e.g. induced pluripotent cells) derived from the subject.
• pluripotent stem cells e.g. induced pluripotent cells
• Methods of inducing pluripotent stem cells are known in the art - see for example U.S. Patent Application 20090047263.
• Example 7 of the Examples section herein below demonstrates successful incorporation of a fragment using the methods described herein into in iPS cell derived from a Huntington's patient.
• the cells are typically re-differentiated towards a particular phenotype - e.g. neuronal phenotype.
• a particular phenotype e.g. neuronal phenotype.
• Methods of differentiating cells towards a neuronal phenotype are known in the art, including those methods used for differentiating embryonic or mesenchymal stem cells towards a neuronal phenotype.
• the polynucleotide of interest incorporated into the iPS cells can also be exchanged (by recombination) with another sequence carrying a fluorescent reporter and/or a selection gene downstream of a promoter specific to the desired cell type. This could be used to isolate the manipulated cells after they have re-differentiated to the desired type of tissue.
• the cells are typically transplanted back into the patient at the appropriate site.
• treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
• the term "subject” includes mammals, preferably human beings at any age which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology. According to one embodiment of the invention the described method can be used to target specific genomic locations in order to allow controlled incorporation of the gene of interest for example in order to treat enzyme deficiencies.
• the described method can be used to overexpress a gene in human cells thus causing differentiation or transdifferentiation of the cells into a different cell type.
• primary liver cells can be extracted from a patient with Diabetes, grown in culture, and a PDX1 gene (under strong inducible promoter) may be introduced thereto using methods described herein.
• a PDX1 gene under strong inducible promoter
• the cells would transdifferentiate into ⁇ -cells which can produce insulin in response to blood glucose levels. These cells can then be reintroduced to the patient thus aiding in the treatment of Diabetes.
• the promoter region of the endogenous gene e.g. a master regulator gene such as PDX1 gene
• PDX1 down-regulate
• the methods described herein can be used in order to clean HIV-infected T-cell progenitors from the HIV virus by cutting out the incorporated HIV genome (Figure 16).
• This is currently impossible to do in any other way since the HIV incorporates randomly into the genome; its size doesn't allow the usage of homologous recombination to target it, and Zinc-Fingers are inapplicable because of the high HIV mutation rate which renders Zinc-Finger technology inapplicable.
• polynucleotides of the present invention may be introduced to the subject per se, or as part of a pharmaceutical composition.
• a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
• active ingredient refers to the polynucleotides accountable for the biological effect.
• physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not abrogate the biological activity and properties of the administered compound.
• An adjuvant is included under these phrases.
• excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
• excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
• Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
• neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
• molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
• pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
• the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
• each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
• tissue refers to part of an organism consisting of an aggregate of cells having a similar structure and/or a common function. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
• compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
• compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
• the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions and/or in biocompatible gels, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
• the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
• Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
• Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
• Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
• disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
• Dragee cores are provided with suitable coatings.
• suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
• Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
• compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
• the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
• the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
• stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
• compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• a suitable propellant e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
• Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
• the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
• compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
• the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
• a suitable vehicle e.g., sterile, pyrogen-free water based solution
• compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
• compositions suitable for use in context of the present invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., Huntington's Chorea) or prolong the survival of the subject being treated.
• a disorder e.g., Huntington's Chorea
• the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
• a dose can be formulated in animal models to achieve a desired concentration or titer: Such information can be used to more accurately determine useful doses in humans.
• Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
• the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
• the dosage may vary depending upon the dosage form employed and the route of administration utilized.
• the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
• Dosage amount and interval may be adjusted individually to provide levels of the active ingredient that are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC).
• MEC minimum effective concentration
• the MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.
• dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
• compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
• compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.
• Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• target site A 40 b.p. target site was selected which repeated 22 times in a 27kb region of chromosome 1 (chrl:2576961-2603453). This region is non-conserved and not recognized as a simple repeat. It is devoid of genes but is flanked with active genes both immediately upstream (MMELl) and downstream (ACTRT2), both of which were reported to be expressed in HeLa cells.
• MMELl immediately upstream
• ACTRT2 downstream sequence used to target this repeat was SEQ ID NO: 1.
• Transfection of cells with the fragments of the present invention Several approaches were used to transfect the DNA in order to rule out influence of the transfection step. These include electroporation and using chemical agents such as PEI, JetPEI, MIRUS Transit, FuGENE HD.
• the DNA was extracted by lysis followed by phenol-chloroform extraction;
• the DNA was extracted using Sigma Genelute mammalian genomic DNA miniprep kit.
• Fibroblast also showed loxP incorporation (not shown).
• a fragment with a single loxP insert was incorporated into two different 22 repeats of a 40bp sequence: rpt22-l(SEQ ID NO: 1) and rpt22-2 (SEQ ID NO: 6). Additionally, a fragment with a single lox 66 insert was incorporated into 3 different genomic repeats: rpt22 (22 repeats, SEQ ID NO: 4), rpt 5188 (5188 repeats, SEQ ID NO: 7) and rpt5192 (5192 repeats, SEQ ID NO: 8).
• Fragments and plasmids (i) A vector plasmid carrying the eGFP gene under a CMV promoter ( Figure 6A). (ii) a fragment comprising a sequence as set forth in SEQ ID NO: 4.
• Transfection Briefly, cells were transfected on day 1 with the fragment (SEQ ID NO: 4) via electroporation. On day 3 (48 hours later), the same cells were transfected again with mixture of plasmids carrying, respectively, eGFP and the Cre recombinase gene.
• FACS analysis FACS was performed on FACS ARIA. Only cells with GFP levels above background were collected and re -plated.
• FACS was performed on day 7 (4 days following eGFP and Cre recominase trasfection) to collect only the GFP positive cells, which at this point indicate the cells that successfully underwent transfection. At this stage there were 70 % GFP positive cells overall. These cells were passaged (1:10 dilution each ⁇ 3 days) for two more weeks without selection, and re-sorted by FACS on day 22. By that time, -0.5 % of the cells were GFP positive. The cells were then passaged for 16 more days (without selection) and imaged on day 38.
• Figure 6B shows that almost all the cells were still expressing the eGFP gene after 38 days, a period much longer than that of plasmid retention in mammalian cells indicating that the plasmid was retained in the genome of the cell.
• a "cocktail" of 5 fragments (SEQ ID NOs: 28-32) were used each with complementary sequences to different regions located close-by within 1.5 kbase of the Pdxl intron ( Figure 14B). Inserting the loxP site into the intron allowed for its subsequent exchange with a fluorescent protein (XFP) flanked by sequences that promote its splicing into the Pd l gene as an additional exon, see for example [Frenkel- Morgenstern M., et al., Nucleic Acids Research, October 9, 2009. Vol 38(Database issue 2010):D508-D512]).
• XFP fluorescent protein
• the bands were extracted from the gel, cloned using pgem-t- easy vector, and the colonies sequenced. As illustrated in Figure 14E, correct insertion of the loxP site into the intronic region of Pdxl was found. Finally, the exchange of the loxP site with a plasmid carrying eGFP driven by a PGK promoter and G418 resistance gene (neo) driven by SV40 promoter was tested. Both the selection and fluorescent markers were stably exchanged into the Hela cells following transient transfection of the plasmid together with the Cre recombinase ( Figure 14F). The entire procedure of OFI incorporation and exchange takes no more than 5 days.
• the present inventors targeted human induced pluripotent cells (hiPSCs) containing abnormally long tri-nucleotide repeats (-180 repeats) of the Huntington gene.
• iPCSs Skin cells from a Huntington patient were induced to pluripotency using the method described by Takahashi et al., j.cell.2007.11.019.
• GFP was successfully incorporated into the induced pluripotent stem cells.
• CXCR4 the co-receptor mediating HIV infection
• a single OFI was used to target CXCR4 gene - SEQ ID NO: 36 ( Figure 17B).
• This gene is a co-receptor for HIV X4 strain entry into T- Cells, and without which the infection is impossible (Establishment of HTV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases Nature Biotechnology 26, 808 - 816 (2008) Published online: 29 June 2008 doi:10.1038/nbtl410).
• the cells were transfected with the OFI and allowed to proliferate for 2 days. Genomic DNA was extracted. PCR analysis of the genomic DNA (using the primers set forth in SEQ ID NOs: 38 and 39), showed correct single incorporation of lox66 site into desired locus ( Figure 17B).

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