Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0047—Sonopheresis, i.e. ultrasonically-enhanced transdermal delivery, electroporation of a pharmacologically active agent
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
  • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0083—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the administration regime
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

• the present invention relates to methods of delivering nucleic acid molecules into cells and methods for measuring nucleic acid delivery into cells and the expression of the nucleic acids therein.
• BACKGROUND OF THE INVENTION A number of methods of delivering nucleic acid molecules, particularly plasmid DNA and other small fragments of nucleic acid, into cells have been developed. These methods are not ideal for delivery of larger nucleic acid molecules.
• methods of delivering nucleic acid molecules of increasing size and complexity, such as artificial chromosomes into cells. Methods are required for use with in vitro and in vivo procedures such as gene therapy and for production of transgenic animals and plants.
• Methods for delivery of large nucleic acid molecules into cells are provided.
• the methods which can be used to deliver nucleic acid molecules of any size, are suitable for delivery of larger nucleic acid molecules, such as natural and artificial chromosomes and fragments thereof, into cells.
• the methods are designed for in vitro, ex vivo and in vivo delivery of nucleic acid molecules for applications, including, but not limited to, delivery of nucleic acid molecules to cells for cell-based protein production, transgenic protein production and gene therapy.
• Methods of protein production in cells and in transgenic animals and plants, methods of introducing nucleic acid into cells to produce transgenic animals and plants, and methods for ex vivo and in vivo gene therapy are also provided.
• Methods provided herein are designed for delivering a large nucleic acid molecule into a cell, but may also be used to deliver smaller molecules.
• Some of the methods include the steps of exposing the nucleic acid molecule to a first delivery agent, typically an agent that increases contact between the nucleic acid molecule and the cell; and exposing the cell to a second delivery agent, which is generally different from the first agent, and is particularly an agent, such as energy, that enhances permeability of the cell.
• Selected delivery agents and combinations thereof are those that result in delivery of the nucleic acid into the cell to a greater extent than in absence of the agent or in the presence of one of the agents alone.
• the permeability enhancing agent is energy, such as electroporation or sonoporation, the cell is contacted therewith in the absence of the nucleic acid molecule.
• lipid agent particularly a dendrimer, such as SAINT-2 TM (1-methyl-4-(1 - octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride, also designated 1 -methyl-4-(19-cis,cis-hepatritiaconta-9,28-dienyl) pyridinium chloride), simultaneously with or sequentially with application of energy.
• SAINT-2 TM 1-methyl-4-(1 - octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride, also designated 1 -methyl-4-(19-cis,cis-hepatritiaconta-9,28-dienyl) pyridinium chloride
• the nucleic acid which is optionally treated with a delivery agent, is contacted with the so-treated cell.
• the selected delivery methods vary depending on the target cells
• nucleic acid molecules into which nucleic acid is delivered
• nucleic acid molecules include, but are not limited to, methods involving any of the following: mixing the nucleic acid molecule with a delivery agent, such as a cationic lipid that neutralizes the charge of the nucleic acid, and contacting the cell with the mixture of nucleic acid and delivery agent; contacting a cell with the nucleic acid molecule, and then contacting the cell with a delivery agent or contacting a cell with a delivery agent then contacting the cell with the nucleic acid molecule; contacting a cell in the absence of the nucleic acid molecule with a delivery agent, applying ultrasound or electrical energy to the cell contacted with the delivery agent, and contacting the cell with the nucleic acid molecule upon the conclusion of the application of the energy; applying ultrasound or electrical energy to a cell, and contacting the cell, upon conclusion of the application of the energy, with
• a delivery agent such as a cationic lipid that neutralizes the charge of the nucleic acid
• application of energy to the cells is done prior to introduction of the nucleic acid molecule.
• energy can be applied in the presence of the nucleic acid molecule can, for example, in instances when the integrity of the nucleic acid molecule is not compromised by application of energy in the presence of the nucleic acid molecule.
• nucleic acid molecules greater than about 0.5, 0.6. 0.7, 0.8, 0.9, 1 , 5, 10, 30, 50 and 100 megabase pairs may be delivered into cells using the methods provided herein.
• the methods may be used to deliver the large nucleic acid molecules into cells in vitro or in vivo.
• nucleic acid molecules may be delivered to cells directly in an animal subject.
• animals include, but are not limited to mammals.
• the animal subject may be a human or other primate, rodent, rabbit, dog, horse or monkey.
• Reagents can be administered locally or systemically (e.g., in the bloodstream) in the subject.
• local administration of the nucleic acids, and/or delivery agents may be into areas such as joints, the skin, tissues, tumors and organs.
• the nucleic acid molecules may be targeted to cells or tissues of interest.
• the delivery methods provided herein may also be used to deliver large nucleic acid molecules to a target cell in vitro which is then introduced into an animal subject, in particular human subjects, such as may be done, for example, in a method of ex vivo gene therapy.
• methods of in vivo and ex vivo gene therapy using the methods for delivering large nucleic acid molecules into cells as provided herein are also provided herein.
• the delivery agent is a cationic compound.
• Cationic compounds include, but are not limited to, a cationic lipid, a cationic polymer, a mixture of cationic lipids, a mixture of cationic polymers, a mixture of a cationic lipid and a cationic polymer and a mixture of a cationic lipid and a neutral lipid, polycationic lipids, non-liposomal forming lipids, activated dendrimers, ethanolic cationic lipids, cationic amphiphiles and pyridinium chloride surfactants.
• nucleic acid molecules that may be delivered into cells using the methods provided herein are artificial chromosomes, satellite DNA-based artificial chromosomes (SATACs, herein referred to as ACes) and natural chromosomes or fragments of any of these chromosomes.
• SATACs satellite DNA-based artificial chromosomes
• ACes natural chromosomes or fragments of any of these chromosomes.
• the ultrasound energy can be applied as one continuous pulse or two or more intermittent pulses.
• the intermittent pulses of the ultrasound energy can be applied for substantially the same length of time, at substantially the same energy level or can vary in energy level, the length of time applied, or energy level and the length of time applied.
• Ultrasound energy ranges and number of pulses can vary, from methods provided herein, according to the instrument selected and can be empirically determined. Typically, ultrasound will be applied for about 30 seconds to about 5 minutes. The power used is a function of the sonorporator used.
• the effects of the ultrasound energy may be enhanced by contacting a cell [in vitro) or administering to a subject (in vivo) a cavitation compound prior to the application of ultrasound energy.
• the provided methods may include the use of such cavitation compounds.
• electric fields When electric fields are employed in the methods provided herein, they are preferably applied to the cells in suspension for about 20 to 50 msec, but the timing and voltage is a function of the instrument used and the particular parameters.
• the electrical energy can be applied as one to five intermittent pulses. As noted, electrical field ranges and number of pulses can vary according to instrument specification and can be determined empirically.
• Methods are provided for generating transgenic animals, particularly non-human transgenic animals, by delivering large nucleic acid molecules into animal cells, in particular non-human animal cells, using delivery methods provided herein, and exposing the animal cells into which the large nucleic acid molecules are delivered to conditions whereby a transgenic animal develops therefrom.
• the methods for delivering large nucleic acid molecules into cells provided herein may also be used in methods of generating transplantable organs and tissues.
• Exemplary cells for use in methods of generating transgenic animals, particularly non-human transgenic animals, or transplantable organs include, but are not limited to, an embryonic stem cell, a nuclear transfer donor cell, a stem cell and a cell that is capable of the generation of a specific organ.
• the methods for delivering nucleic acid molecules into cells provided herein may also be used in methods of generating cellular protein production cell lines.
• nucleic acids into a cell Further provided are methods for monitoring delivery of nucleic acids into a cell. These methods permit the rapid and accurate measurement of nucleic acid transfer into cells, thus allowing for screening and optimizing the use of various delivery agents and protocols for delivery of any nucleic acid into any cell type, in vitro, ex vivo or in vivo. Further provided are methods to monitor delivery and expression of nucleic acids in a cell.
• labeled nucleic acid molecules such as DNA
• a detection method such as flow cytometry
• flow cytometry is then used to determine the number of cells containing the label as an indication of the ability of the delivery method to facilitate or effect delivery of the nucleic acid molecules.
• Other detection methods that may be used in place of or in addition to flow cytometry include, but are not limited to, fluorimetry, cell imaging, fluorescence spectroscopy and other such methods known to those of skill in the art for such detection and, as needed or desired, for quantitation.
• the nucleic acid molecule is an artificial chromosome labeled with a nucleoside or ribonucleoside analog, particularly a thymidine analog, such as iododeoxyuridine (IdU or IdUrd) and bromodeoxyuridine (BrdU), and the delivery agent is a cationic compound, which is used alone or in combination with energy.
• a nucleoside or ribonucleoside analog particularly a thymidine analog, such as iododeoxyuridine (IdU or IdUrd) and bromodeoxyuridine (BrdU)
• the delivery agent is a cationic compound, which is used alone or in combination with energy.
• the monitoring methods provided herein permit the rapid, simple and accurate detection of delivery of small numbers of nucleic acid molecules into cells. Such small numbers may be sufficient for purposes of transgenesis, gene therapy, cellular protein production and other goals of , gene transfer.
• the monitoring methods also make it possible to rapidly quantify differences in delivery efficiencies of differing delivery methods and thus facilitate the development and optimization of methods for the delivery of nucleic acid molecules, such as DNA, into cells. These methods can also be used to optimize transfection efficiencies into cells for which no delivery protocol has been established or which are not easily transfected. These methods also permit rapid screening of delivery protocols and agents for their ability to enhance or permit delivery of nucleic acid molecules, such as DNA, of any size into a cell.
• Methods are also provided that combine methods of monitoring nucleic acid molecule delivery with methods for monitoring expression of nucleic acid molecules. It is possible not only to assess the efficiency of delivery of nucleic acid molecules to cells, but also to monitor the subsequent expression of the delivered nucleic acid molecules in the same cell population. Thus, these methods also provide a method for the mapping of biological events between nucleic acid molecule delivery and early gene expression, using marker genes, such as, but are not limited to, fluorescent proteins, such as red, green or blue fluorescent proteins.
• nucleic acid molecules such as delivery of a chromosome and expression of genes encoded thereon, are monitored by IdU labeling of a nucleic acid molecule that contains sequences encoding a green fluorescent protein.
• the methods of monitoring delivery and expression of a nucleic acid molecule include the steps of: introducing labelled nucleic acid molecules that encode a reporter gene into cells; detecting labelled cells as an indication of delivery of the nucleic acid into a cell; and measuring the product of the reporter gene as an indication of DNA expression in the cell, whereby delivery and expression of nucleic acid molecules in the cell is detected or determined.
• the labelled cells can be detected, for example, by flow cytometry, fluorimetry, cell imaging or fluorescence spectroscopy.
• the label for example, can be iododeoxyuridine (IdU or IdUrd) or bromodeoxyuridine (BrdU), the reporter gene, for example, can be one that encodes fluorescent protein, enzyme, such as a luciferase, or antibody.
• the delivered nucleic acid molecules include, but are not limited to, RNA, including ribozymes, DNA, including naked DNA and chromosomes, plasmids, chromosome fragments, typically containing at least one gene or at least 1 Kb, naked DNA, or natural chromosomes.
• the method is exemplified herein by determining delivery and expression of artificial chromosome expression systems (ACes) .
• nucleic acid refers to a polynucleotide containing at least two covalently linked nucleotide or nucleotide analog subunits.
• a nucleic acid can be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or an analog of DNA or RNA.
• Nucleotide analogs are commercially available and methods of preparing polynucleotides containing such nucleotide analogs are known (Lin et al. (1 994) Nucl. Acids Res. 22:5220-5234; Jellinek et al. (1 995) Biochemistry 34: 1 1 363-1 1 372; Pagratis et al.
• the nucleic acid can be single-stranded, double-stranded, or a mixture thereof.
• the nucleic acid is double-stranded, or it is apparent from the context.
• nucleic acid refers to single-stranded and/or double- stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA. Also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives.
• PNA peptide nucleic acid
• DNA is meant to include all types and sizes of DNA molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.
• nucleotides include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.
• large nucleic acid molecules refers to a nucleic acid molecule of at least about 0.5 megabase pairs (Mbase) in size, greater than 0.5 Mbase, including nucleic acid molecules at least about 0.6. 0.7, 0.8, 0.9, 1 , 5, 10, 30, 50 and 100, 200, 300, 500 Mbase in size.
• Large nucleic acid molecules typically may be on the order of about 10 to about 450 or more Mbase, and may be of various sizes, such as, for example, from about 250 to about 400 Mbase, about 1 50 to about 200 Mbase, about 90 to about 1 20 Mbase, about 60 to about 1 00 Mbase and about 1 5 to 50 Mbase.
• large nucleic acid molecules include, but are not limited to, natural chromosomes and fragments thereof, especially mammalian chromosomes and fragments thereof which retain a centromere and telomeres, artificial chromosome expression systems
• ACes also called satellite DNA-based artificial chromosomes (SATACs); see U.S. Patent Nos. 6,025, 1 55 and 6,077,697), mammalian artificial chromosomes (MACs), plant artificial chromosomes, insect artificial chromosomes, avian artificial chromosomes and minichromosomes (see, e.g. , U.S. Patent Nos. 5,71 2, 1 34, 5,891 ,691 and 5,288,625).
• SATACs satellite DNA-based artificial chromosomes
• the large nucleic acid molecules may include a single copy of a desired nucleic acid fragment encoding a particular nucleotide sequence, such as a gene of interest, or may carry multiple copies thereof or multiple genes or different heterologous sequences of nucleotides. For example, ACes can carry 40 or even more copies of a gene of interest.
• Large nucleic acid molecules may be associated with proteins, for example chromosomal proteins, that typically function to regulate gene expression and/or participate in determining overall structure.
• an artificial chromosome is a nucleic acid molecule that can stably replicate and segregate alongside endogenous chromosomes in a cell. It has the capacity to act as a gene delivery vehicle by accommodating and expressing foreign genes contained therein.
• a mammalian artificial chromosome refers to chromosomes that have an active mammalian centromere(s) .
• Plant artificial chromosomes, insect artificial chromosomes and avian artificial chromosomes refer to chromosomes that include plant, insect and avian centromeres, respectively.
• a human artificial chromosome refers to chromosomes that include human centromeres. For exemplary artificial chromosomes, see, e.g. , U.S. Patent Nos.
• SATAC satellite DNA-based artificial chromosome
• ACes artificial chromosome expression system
• Foreign genes contained in these artificial chromosome expression systems can include, but are not limited to, nucleic acid that encodes traceable marker proteins (reporter genes), such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively), other reporter genes, such as /?-galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance.
• traceable marker proteins reporter genes
• reporter genes such as fluorescent proteins, such as green, blue or red fluorescent proteins (GFP, BFP and RFP, respectively
• reporter genes such as /?-galactosidase and proteins that confer drug resistance, such as a gene encoding hygromycin-resistance.
• heterologous DNA include, but are not limited to, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.
• heterologous and “foreign” with reference to nucleic acids are used interchangeably and refer to nucleic acid that does not occur naturally as part of a genome or cell in which it is present or which is found in a location(s) and/or in amounts in a genome or cell that differ from the location(s) and/or amounts in which it occurs in nature. It can be nucleic acid that is not endogenous to the cell and has been exogenously introduced into the cell.
• heterologous DNA include, but are not limited to, DNA that encodes a gene product or gene product(s) of interest introduced into cells, for example, for purposes of gene therapy, production of transgenic animals or for production of an encoded protein.
• heterologous DNA examples include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.
• delivery refers to the process by which exogenous nucleic acid molecules are transferred into a cell such that they are located inside the cell. Delivery of nucleic acids is a distinct process from expression of nucleic acids.
• expression refers to the process by which nucleic acid is translated into peptides or is transcribed into RNA, which, for example, may be translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, expression may, if an appropriate eukaryotic host cell or organism is selected, include splicing of the mRNA. For heterologous nucleic acid to be expressed in a host cell, it must initially be delivered into the cell and then, once in the cell, ultimately reside in the nucleus.
• cell recovery refers to a "total cell yield" after a specified time frame, which for purposes herein is twenty-four hours, and when used with reference to calculation of the clonal fraction
• cell recovery time refers to a time frame in order for a cell to equilibrate to new conditions.
• cell survival refers to cell viability after a cytotoxic event, such as a delivery procedure.
• control plating efficiency refers to the fraction of untreated cells, under standard optimal growth conditions for the particular cells, that survive a plating procedure.
• Plating efficiency refers to the fraction of treated cells that survive a plating procedure.
• clonal fraction is a measurement of cell recovery after delivery of exogenous nucleic acids into cells and the plating efficiency of the cells.
• transfer efficiency is the percentage of the total number of cells to which nucleic acids are delivered that contain delivered nucleic acid.
• transfection efficiency is the percentage of the total number of cells to which nucleic acids including a selectable marker are delivered that survive selection.
• index of potential transfection efficiency means the theoretical maximum transfection efficiency for a particular cell type under particular conditions, for example particular concentrations or amounts of particular delivery agents.
• the term "cell” is meant to include cells of all types, of eukaryotes and prokaryotes, including animals and plants.
• delivery agent refers to compositions, conditions or physical treatments to which cells and/or nucleic acids may be exposed in the process of transferring nucleic acids to cells in order to facilitate nucleic acid delivery into cells. Delivery agents include compositions, conditions and physical treatments that enhance contact of nucleic acids with cells and/or increase the permeability of cells to nucleic acids. In general, nucleic acids are not directly treated with energy, such as sonoporation.
• cationic compounds are compounds that have polar groups that are positively charged at or around physiological pH. These compounds facilitate delivery of nucleic acid molecules into cells; it is thought this is achieved by virtue of their ability to neutralize the electrical charge of nucleic acids.
• exemplary cationic compounds include, but are not limited to, cationic lipids or cationic polymers or mixtures thereof, with or without neutral lipids, polycationic lipids, non-liposomal forming lipids, ethanolic cationic lipids and cationic amphiphiles.
• Contemplated cationic compounds also include activated dendrimers, which are spherical cationic polyamidoamine polymers with a defined spherical architecture of charged amino groups which branch from a central core and which can interact with the negatively charged phosphate groups of nucleic acids (e.g., starburst dendrimers) .
• activated dendrimers which are spherical cationic polyamidoamine polymers with a defined spherical architecture of charged amino groups which branch from a central core and which can interact with the negatively charged phosphate groups of nucleic acids (e.g., starburst dendrimers) .
• Cationic compounds for use as delivery agents also include mixtures of cationic compounds that include peptides and protein fragments.
• the additional components may be non-covalently or covalently bound to the cationic compound or otherwise associated with the cationic compound.
• ultrasound energy is meant to include sound waves (for external application) and lithotripter-generated shock waves (for internal application).
• electrical energy is meant to include the application of electric fields to cells so as to open pores in membranes for the delivery of molecules into the cell, e.g., electroporation techniques.
• cavitation compound is meant to include contrast agents that are typically used with ultrasound imaging devices and includes gas encapsulated and nongaseous agents. These cavitation compounds enhance the efficiency of energy delivery of acoustic or shock waves.
• pharmaceutically acceptable refers to compounds, compositions and dosage forms that are suitable for administration to the subject without causing excessive toxicity, irritation, allergic response or other undesirable complication.
• embryonic stem cells are primitive, immature cells that are precursors to stem cells.
• stem cells are primitive, immature cells that are precursors to mature, tissue specific cells.
• nuclear transfer donor cells are cells that are the source of nuclei, which are transferred to enucleated oocytes during the process of nuclear transfer.
• subject refers to animals, plants, insects, and birds into which the large DNA molecules may be introduced. Included are higher organisms, such as mammals and birds, including humans, primates, rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chicken and others.
• administering to a subject is a procedure by which one or more delivery agents and/or large nucleic acid molecules, together or separately, are introduced into or applied onto a subject such that target cells which are present in the subject are eventually contacted with the agent and/or the large nucleic acid molecules.
• applying to a subject is a procedure by which target cells present in the subject are eventually contacted with energy such as ultrasound or electrical energy. Application is by any process by which energy may be applied.
• gene therapy involves the transfer or insertion of nucleic acid molecules, and, in particular, large nucleic acid molecules, into certain cells, which are also referred to as target cells, to produce specific gene products that are involved in correcting or modulating diseases or disorders.
• the nucleic acid is introduced into the selected target cells in a manner such that the nucleic acid is expressed and a product encoded thereby is produced.
• the nucleic acid may in some manner mediate expression of DNA that encodes a therapeutic product.
• This product may be a therapeutic compound, which is produced in therapeutically effective amounts or at a therapeutically useful time. It may also encode a product, such as a peptide or RNA, that in some manner mediates, directly or indirectly, expression of a therapeutic product. Expression of the nucleic acid by the target cells within an organism afflicted with a disease or disorder thereby provides a way to modulate the disease or disorder.
• the nucleic acid encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof.
• cells can be transfected in vitro, followed by introduction of the transfected cells into the body of a subject. This is often referred to as ex vivo gene therapy.
• the cells can be transfected directly in vivo within the body of a subject.
• a reporter gene includes any gene that expresses a detectable gene product, which may be RNA or protein. Preferred reporter genes are those that are readily detectable.
• reporter genes include, but are not limited to nucleic acid encoding a fluorescent protein, CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1 979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1 987), Mol. Cell. Biol. 7: 725-737); bacterial luciferase (Engebrecht and Silverman ( 1 984), PNAS V. 41 54-41 58; Baldwin et al. (1 984), Biochemistry 23: 3663-3667); and alkaline phosphatase (Toh et al.
• CAT chloramphenicol acetyl transferase
• a reporter gene construct is a DNA molecule that includes a reporter gene operatively linked to a transcriptional control sequence.
• the transcriptional control sequences include a promoter and other optional regulatory regions, such as enhancer sequences, that modulate the activity of the promoter, or control sequences that modulate the activity or efficiency of the RNA polymerase that recognizes the promoter, or control sequences that are recognized by effector molecules, including those that are specifically induced by interaction of an extracellular signal with a cell surface protein.
• modulation of the activity of the promoter may be effected by altering the RNA polymerase binding to the promoter region, or, alternatively, by interfering with initiation of transcription or elongation of the mRNA.
• transcriptional control elements Such sequences are herein collectively referred to as transcriptional control elements or sequences.
• the construct can include sequences of nucleotides that alter translation of the resulting mRNA, thereby altering the amount of reporter gene product.
• promoter refers to the region of DNA that is upstream with respect to the direction of transcription of the transcription initiation site. It includes the RNA polymerase binding and transcription imitation sites and any other regions, including, but not limited to repressor or activator protein binding sites, calcium or cAMP responsive sites, and any such sequences of nucleotides known to those of skill in the art to alter the amount of transcription from the promoter, either directly or indirectly.
• a promoter that is regulated or mediated by the activity of a cell surface protein is a promoter whose activity changes when a cell is exposed to a particular extracellular signal by virtue of the presence of cell surface proteins whose activities are affected by the extracellular protein.
• B. METHODS FOR THE DELIVERY OF DNA INTO CELLS A variety of methods for delivering nucleic acids, particularly large nucleic acid molecules, such as artificial chromosomes, including ACes (formerly designated SATACs), are provided. The methods generally involve exposing the nucleic acid molecule to an agent that increases contact between the nucleic acid molecule and the cell, and exposing the cell to a permeability enhancing agent.
• agents such as energy, which increase the permeability of a cell
• agents are applied before contacting the cell with a nucleic acid.
• large nucleic acid molecules are delivered using agents, including, but not limited to, delivery agents that enhance contact between the nucleic acid molecules and the cells and/or agents and treatments that increase cell permeability.
• the nucleic acid molecules are delivered using agents that enhance contact between the nucleic acid and cells by neutralizing the charge of the nucleic acid molecules, and also by using energy to increase permeability of the cells.
• the agents can be used individually and in various combinations and orders of application.
• energy such as sonoporation and electroporation, is not applied to cells after the nucleic acid molecule is added thereto.
• the method selected for delivering particular nucleic acid molecules, such as DNA, to targeted cells can depend on the particular nucleic acid molecule being transferred and the particular recipient cell.
• Preferred methods for particular nucleic acid molecules, such as DNA, and recipient cells are those that result in the greatest amount of nucleic acid molecules, such as DNA, transferred into the cell nucleus with an acceptable degree of cell survival.
• Suitable methods for delivery of particular pairings of nucleic acid molecules, such as DNA, and recipient cells can be determined using methods of monitoring nucleic acid molecules, such as DNA, delivery and methods of screening agents and conditions as provided herein or can be determined empirically using methods known to those of skill in the art.
• a method for detection of delivered nucleic acid is provided.
• This method which can be used for assessing delivery of any nucleic acid molecule, can be used as a rapid screening tool to optimize nucleic acid, e.g. , chromosome, transfer conditions.
• delivery methods can first be assessed for the ability to transfer nucleic acid molecules, such as DNA, into cells and to identify methods that provide a sufficient number of viable cells that express the transferred nucleic acid molecules, such as DNA. Once such methods are identified, they can be optimized using the delivery monitoring methods provided herein and then assessed for the ability to provide for expression of the transferred nucleic acid molecules. Delivery agents
• Delivery agents include compositions, conditions and physical treatments that enhance contact of nucleic acid molecules, such as DNA, with cells and/or increase the permeability of cells to nucleic acid molecules, such as DNA.
• Such agents include, but are not limited to, cationic compounds, peptides, proteins, energy, for example ultrasound energy and electric fields, and cavitation compounds.
• Delivery agents for use in the methods provided herein include compositions, conditions or physical treatments to which cells and/or nucleic acid molecules, such as DNA, can be exposed in the process of transferring nucleic acid molecules, such as DNA, to cells in order to facilitate nucleic acid molecules, such as DNA, delivery into cells.
• Cationic Compounds Cationic compounds for use in the methods provided herein are available commercially or can be synthesized by those of skill in the art. Any cationic compound can used for delivery of nucleic acid molecules, such as DNA, into a particular cell type using the provided methods. One of skill in the art by using the provided screening procedures can readily determine which of the cationic compounds are best suited for delivery of specific nucleic acid molecules, such as DNA, into a specific target cell type. (a) Cationic Lipids
• Cationic lipid reagents can be classified into two general categories based on the number of positive charges in the lipid headgroup; either a single positive charge or multiple positive charges, usually up to 5. Cationic lipids are often mixed with neutral lipids prior to use as delivery agents.
• Neutral lipids include, but are not limited to, lecithins; phospha- tidylethanolamine; phosphatidylethanolamines, such as DOPE (dioleoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidyl- ethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine) and distearoylphosphatidylethanolamine; phosphatidylcholine; phosphatidylcholines, such as DOPC
• DPPC dipalmitoylphosphatidylcholine
• POPC palmitoyloleoylphosphatidylcholine
• distearoyl- phosphatidylcholine fatty acid esters; glycerol esters; sphingolipids; cardiolipin; cerebrosides; and ceramides; and mixtures thereof.
• Neutral lipids also include cholesterol and other 3 ?OH-sterols.
• lipids contemplated herein include: phosphatidylglycerol; phosphatidylglycerols, such as DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), and distearoyl- phosphatidylglycerol; phosphatidylserine; phosphatidylserines, such as dioleoyl- or dipalmitoylphosphatidylserine and diphosphatidylglycerols.
• DOPG dioleoylphosphatidylglycerol
• DPPG dipalmitoylphosphatidylglycerol
• distearoyl- phosphatidylglycerol phosphatidylserine
• phosphatidylserines such as dioleoyl- or dipalmitoylphosphatidylserine and diphosphat
• cationic lipid compounds include, but are not limited to: Lipofectin (Life Technologies, Inc., Burlington, Ont.)(1 : 1 (w/w) formulation of the cationic lipid N-[1 -(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) and dioleoylphosphatidylethanol- amine (DOPE)); LipofectAMINE (Life Technologies, Burlington, Ont., see U.S. Patent No.
• Non-lipid cationic compounds include, but are not limited to
• SUPERFECTTM Qiagen, Inc., Mississauga, ON
• Activated dendrimer cationic polyme ⁇ charged amino groups
• CLONfectinTM Cationic amphiphile N-t-butyl-N'-tetradecyl-3-tetradecyl-aminopropionamidine
• Pyridinium amphiphiles are double-chained pyridinium compounds, which are essentially nontoxic toward cells and exhibit little cellular preference for the ability to transfect cells.
• pyridinium amphiphiles examples include the pyridinium chloride surfactants such as SAINT-2 (1 - methyl-4-(1 -octadec-9-enyl-nonadec-10-enylenyl) pyridinium chloride) (see, e.g. , van der Woude et al. (1 997) Proc. Natl. Acad. Sci. U.S.A. 94 ⁇ ⁇ 1 60).
• the pyridinium chloride surfactants are typically mixed with neutral helper lipid compounds, such as dioleoylphosphatidylethanolamine (DOPE), in a 1 : 1 molar ratio.
• DOPE dioleoylphosphatidylethanolamine
• Other Saint derivatives of different chain lengths, state of saturation and head groups can be made by those of skill in the art and are within the scope of the present methods.
• Delivery agents also include treatment or exposure of the cell and/or nucleic acid molecules, but generally the cells, to sources of energy, such as sound and electrical energy.
• sources of energy such as sound and electrical energy.
• the ultrasound source should be capable of providing frequency and energy outputs suitable for promoting transfection.
• the output device can generate ultrasound energy in the frequency range of 20 kHz to about 1 MHz.
• the power of the ultrasound energy can be, for example, in the range from about 0.05 w/cm 2 to 2 w/cm 2 , or from about 0.1 w/cm 2 to about 1 w/cm 2 .
• the ultrasound can be administered in one continuous pulse or can be administered as two or more intermittent pulses, which can be the same or can vary in time and intensity.
• Ultrasound energy can be applied to the body locally or ultrasound- based extracorporeal shock wave lithotripsy can be used for "in-depth” application.
• the ultrasound energy can be applied to the body of a subject using various ultrasound devices.
• ultrasound can be administered by direct contact using standard or specially made ultrasound imaging probes or ultrasound needles with or without the use of other medical devices, such as scopes, catheters and surgical tools, or through ultrasound baths with the tissue or organ partially or completely surrounded by a fluid medium.
• the source of ultrasound can be external to the subject's body, such as an ultrasound probe applied to the subject's skin which projects the ultrasound into the subject's body, or internal, such as a catheter having an ultrasound transducer which is placed inside the subject's body.
• Suitable ultrasound systems are known (see, e.g. , International PCT application No. WO 99/21 584 and U.S. Patent No. 5,676, 1 51 ) .
• the ultrasound can be applied to one or several organs or tissues simultaneously to promote nucleic acid molecule delivery to multiple areas of the subject's body.
• the ultrasound can be applied selectively to specific areas or tissues to promote selective uptake of the nucleic acid molecules, such as DNA.
• the transfection efficiency of the ultrasound can also be enhanced by using contrast reagents, which serve as artificial cavitation nuclei, such as Albunex (Molecular Biosystems, San Diego, CA), Imagent
• a contrast reagent can be introduced locally, such as a joint; introduced systematically, with the enhancement of cavitation efficiency by focusing lithotripter shock waves at a defined area; or by targeting a contrast reagent to a particular site and then enhancing cavitation efficiency by focusing lithotripter shock waves.
• Electroporation temporarily opens up pores in a cell's outer membrane by use of pulsed rotating electric fields.
• the methods provided herein can be used in the delivery of nucleic acids into any cells, including, but not limited to, any eukaryotic and prokaryotic cells.
• cells that can be used in the methods include, but are not limited to, cell lines, primary cells, primary cell lines, plant cells and animal cells, including stem cells and embryonic cells.
• fibroblasts including lung and skin fibroblasts, fibroblast-like cells, synoviocytes, fibroblast-like synoviocytes, stem cells, including embryonic and adult stem cells, such as mesenchymal stem cells, myoblasts, lymphoblasts, carcinoma and hepatoma cells are among the many cells into which nucleic acids, and in particular large nucleic acids and artificial chromosomes, can be delivered and monitored using the methods provided herein.
• Particular cells include mammalian cells, for example, A9 cells (mouse fibroblasts, HPRT " ; ATCC Accession no.
• CCL- 1 .4 CHO-S cells and DG44 cells (Chinese hamster ovary cells), V79 cells (Chinese hamster lung fibroblasts; ATCC Accession no. CCL-39), LMTK " cells (mouse fibroblasts; ATCC Accession No. CCL-1 .3), skin fibroblasts, L8 cells (rat myoblasts; ATCC Accession No. CRL-1 769), CCD1043 SK cells (human fibroblasts; ATCC Accession No. CRL-2056), adult-derived mesenchymal stem cells (e.g.
• the methods of delivery of nucleic acids into cells provided herein can be used in delivering nucleic acids into cells in order to treat a disease or disorder, e.g. , in gene therapy applications.
• the nucleic acid to be delivered into a cell may encode a therapeutic molecule, e.g. , a protein.
• successful gene therapy applications are complicated by a requirement that large nucleic acids be delivered into cells. It may also be desired to provide multiple copies of nucleic acid encoding one or more therapeutic molecules. Compounding the difficulties in gene therapy methods is the challenge that cells preferred for use in gene therapy applications are often not readily transfectable.
• nucleic acids which may be in the form of artificial chromosomes or fragments thereof, into cells as may be used in therapeutic applications.
• Cationic compounds and nucleic acid molecules, such as DNA can be added to cells in vitro either separately or mixed together and with or without the application of ultrasound or electrical energy. In general, if energy is applied, it is applied prior to contacting the cells with the nucleic acid molecule.
• nucleic acid molecules such as DNA
• cationic lipids/compounds can be added to a cell as described in the EXAMPLES.
• Parameters important for optimization of the delivery of nucleic acid molecules, such as DNA, into target cells will be apparent to those of skill in this art. These parameters include, for example, the cationic compound, cationic compound concentration, the nucleic acid molecules, such as DNA, the concentration of nucleic acid molecules, the cell growth medium, the cell culture conditions, the length of time cells are exposed to the cationic compound, the toxicity of the cationic compound to the target cell type, and the amount and time of use of ultrasound or electroporation among other parameters.
• the rapid screening method can provide direction as to what parameters may need to be adjusted to optimize delivery (see EXAMPLES). Alteration of culture conditions, time, reagent concentrations and other parameters, for use with different combinations of cationic compounds and target cell types and to optimize delivery, can be empirically determined. If ultrasound energy is required to be used to enhance transfection efficiency, it can be applied as described below and in the EXAMPLES. Electroporation can be performed as described below or by any suitable protocol known to those of skill in this art.
• nucleic acid molecules such as DNA
• contacting of cells with cationic compounds and nucleic acid molecules, such as DNA in separate and distinct steps can be generally carried out as described in the EXAMPLES.
• Those of skill in the art can readily vary the order of the application of the components to the target cell based on the disclosure herein.
• Ex Vivo Gene Therapy Delivery of nucleic acid molecules, such as DNA is carried out as described above in in vitro delivery. After selection has been completed, cells harboring the nucleic acid molecules, such as DNA, are introduced into the subject target by a variety of means, including injection, such as subcutaneous, intramuscular, intraperitoneal, intravascular and intralymphatic and intra-articular injection.
• the cells can be administered with or without the aid of medical devices such as arthroscopes, other scopes or various types of catheters.
• the cationic compound is first delivered to the target area (e.g. , tissue, organ, tumor or joint) . After waiting a suitable amount of time, the target area is then subjected to ultrasound frequency at a suitable energy level for a suitable time, which will be dependent on the equipment, tissue type and depth of the target area in the body. Alternatively, electrical energy is delivered to the target area.
• the nucleic acid molecule, such as DNA is then delivered to the same area.
• this procedure can be repeated so that the nucleic acid molecules, such as DNA, can be delivered via multiple injections over time or multiple administrations in different areas at the same time.
• the cationic compound mixed together with the nucleic acid molecules, such as DNA can be delivered to the target area.
• the target area can then be subjected to ultrasound frequency at a suitable energy level for a suitable time.
• ultrasound frequency Depending on the nucleic acid molecules, such as DNA, the in vivo location, the cationic compound used and other variables, it may not be necessary to use ultrasound or electroporation to achieve suitable transfer efficiency to cells at the target area.
• contrast reagents can be delivered to the target area to enhance transfer of the nucleic acid molecules, such as DNA.
• the nucleic acid molecules can be delivered to organs or tissues of the body such as skin, muscle, stomach, intestine, lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen, prostate and joints (for example the knee, elbow, shoulder, wrist, hip, finger, ankle and others) .
• Molecules can be delivered to, for example, primary cells and cell lines, such as fibroblast, muscle, stomach, intestine, lung, bladder, ovary, uterus, liver, kidney, pancreas, brain, heart, spleen, prostate to mimic in vivo systems.
• the cationic compounds and the nucleic acid molecules, such as DNA, separately or together can be delivered to the target area of the body by a variety of means, including injection (for example, subcutaneous, intramuscular, intraperitoneal, intravascular, intra-articular and intralymphatic injection), instillation, cannulation, slow infusion, topical application and any other mode of administration. They can be administered by any suitable mode, including systemically (for example by intravenous injection), locally, such as by delivery to a specific target area (tissue or area), using, for example, a catheter or by direct injection. They can be administered with or without the aid of medical devices such as arthroscopes, other scopes or various types of catheters.
• the cationic compounds can be administered also by coating a medical device, for example, a catheter, such as an angioplasty balloon catheter, with a cationic compound formulation. Coating may be achieved, for example, by dipping the medical device into a cationic lipid formulation or a mixture of a cationic compound formulation and a suitable solvent, for example, an aqueous-buffer, an aqueous solvent, ethanol, methylene chloride, chloroform and other suitable solvent. An amount of the formulation will naturally adhere to the surface of the device, which is subsequently administered to a subject, as appropriate. Alternatively, a lyophilized mixture of a cationic lipid formulation may be specifically bound to the surface of the device. Such binding techniques are known (see, e.g. , Ishihara et al. (1 993) Journal of Biomedical Materials Research 27: 1 309-1 314) .
• the cationic compounds and nucleic acid molecules can be formulated in pharmaceutically acceptable carriers, such as saline or other pharmaceutically acceptable solutions, for delivery in vivo.
• pharmaceutically acceptable carriers such as saline or other pharmaceutically acceptable solutions
• the nucleic acid molecules, such as DNA, and cationic compounds, regardless of the route of administration, are formulated into pharmaceutically acceptable dosage forms by standard methods known to those of skill in the art.
• the dosage level of the nucleic acid molecules may be varied to achieve optimal therapeutic response for a particular subject. This depends on a variety of factors including mode of administration, activity of the nucleic acid molecules, such as DNA, characteristics of the protein produced, the transfection efficiency of the target cells (their ability to take up the nucleic acid molecules, such as DNA), the route of administration, the location of the target cells and other factors
• the dosage to be administered and the particular mode of adminis- tration will vary depending upon such factors as the age, weight and the particular animal and region thereof to be treated, the particular nucleic acid molecule and cationic compound used, the therapeutic or diagnostic use contemplated, and the form of the formulation, for example, suspension, emulsion, or liposomal, as will be readily apparent to those skilled in the art.
• dosage is administered at lower levels and increased until the desirable therapeutic effect is achieved.
• the amount of cationic compound that is administered can vary and generally depends upon the amount of nucleic acid molecules, such as DNA, being administered.
• the weight ratio of cationic compound to nucleic acid molecules can be from about 1 : 1 to about 1 5: 1 , including, for example, a weight ratio of about 5: 1 to about 1 : 1 .
• the amount of cationic compound which is administered will vary from between about 0.1 milligram (mg) to about 1 gram (g).
• mg milligram
• g gram
• single dose ranging from 1 to 20 ml is administered as a single or repeated dose.
• compositions that contain cells selected for therapeutic treatment of a joint or rheumatoid arthritis, wherein the cells contain a large heterologous nucleic acid.
• the cells of such a composition may contain an artificial chromosome.
• the cells can contain an ACes.
• RA Rheumatoid arthritis
• RA is a chronic inflammatory disease characterized by joint inflammation and progressive cartilage and bone destruction.
• Treatment of RA is problematic with current strategies since relatively high systemic doses are necessary to achieve therapeutic levels of anti-rheumatic drugs in the joints.
• the available treatments are associated with significant untoward side effects.
• Gene therapy is thus a more efficient system for delivery of therapeutic molecules to the site of inflammation in the treatment of connective tissue diseases, rheumatic diseases and chronic erosive joint diseases such as RA, osteoarthritis, ankylosing spondylitis and juvenile chronic arthritis.
• synovium contains macrophage-like type A cells (presumably derived from macrophage/monocyte precursors and exhibiting phagocytic activity) and fibroblast-like type B cells (more fibroblast in appearance and associated with production of hyaluronic acid and other components of the joint fluid) .
• macrophage-like type A cells presumably derived from macrophage/monocyte precursors and exhibiting phagocytic activity
• fibroblast-like type B cells more fibroblast in appearance and associated with production of hyaluronic acid and other components of the joint fluid.
• Underlying the synovium is a sparsely cellular subsynovium which may be fibrous, adipose or areolar in nature.
• Fibroblast-like synoviocytes are distinguishable from normal fibroblast cells in the subintimal synovium by differential gene expression patterns. FLS have been shown to express high levels of uridine diphosphoglucose dehydrogenase (UDPGD), high levels of vascular cell adhesion molecule-1 (VCAM-1 ), intercellular adhesion molecule-1 (ICAM-1 ) as well as CD44 (hyaluronic acid receptor), fibronectin receptor and ⁇ 3 integrins. Sublining fibroblasts or fibroblasts from other sources do not express these markers or express them at lower levels [see, e.g., Edwards (1 995) Ann. Rheum. Dis. 54:395-397; Firestein (1 996) Arthritis Rheum. 35: 1 781 -1 790; Edwards (2000) Arthritis Res. 2:344-347].
• UDP uridine diphosphoglucose dehydrogenase
• VCAM-1 vascular cell adhesion molecule-1
• RA Disease progression in RA involves the thickening of the synovial lining due to the proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells (e.g. , lymphocytes, macrophages and mast cells).
• FLS fibroblast-like synoviocytes
• inflammatory cells e.g. , lymphocytes, macrophages and mast cells.
• the normal biology of synoviocytes is also altered in the pathological process of RA, including invasion and destruction of articular cartilage and bone.
• synoviocytes mediate the pathophysiological process of RA by expression of cell surface proteins involved in the recruitment and activation of lymphocytes and macrophages within the synovium.
• Proliferation of synovial cells leads to a pannus tissue that invades and overgrows cartilage, leading to bone destruction and destruction of joint structure and function.
• Proinflammatory cytokines for example, tumor necrosis factor- (TNF- ⁇ ) and interleukin-1 (IL-1 ) play key roles in inflammation and joint damage associated with RA. Pathological effects caused by these cytokines include leukocytic infiltration leading to synovial hyperplasia, cell activation, cartilage breakdown and inhibition of cartilage matrix synthesis.
• Nucleic acid transfer to rheumatoid synovial tissue may result in the production of mediators that inhibit inflammation or hyperplasia or provide toxic substances that specifically destroy the diseased synovium.
• Retroviral delivery of nucleic acid encoding interleukin-1 receptor antagonist (IL1 -RA) ex vivo and transduction of synoviocytes has been used in gene therapy of RA in humans to inhibit inflammation [see, e.g. , Evans (1 996) Human Gen. Ther. 7: 1 261 -1 280 and Del Vecchio et al.
• Adenoviral vectors have been proposed for delivery of nucleic acid encoding an IL-1 receptor antagonist to synoviocytes in in vivo transduction methods [see, e.g. , U.S. Patent No. 5,747,072 and PCT Application Publication No. WO 00/521 86].
• Artificial chromosomes provide advantages over virus-based systems for gene therapy. For example, artificial chromosome expression systems (ACes), and other artificial chromosomes as described in U.S. Patent Nos. 6,025, 1 55 and 6,077,697 and PCT Application No.
• WO97/401 83 serve as non-integrating, non-viral vectors with a large capacity for delivering large nucleic acids and/or multiple copies of a particular nucleotide sequence into cells, such as synoviocytes, both in vitro and in vivo.
• Such artificial chromosome systems offer further advantages in that they allow stable and predictable expression of genes producing single or multiple proteins over long periods of time.
• the methods provided herein may be used to introduce large nucleic acids, such as, for example, artificial chromosomes, into primary cells, such as, for example, synoviocytes (e.g. , fibroblast-like synoviocytes) and skin fibroblasts, and skeletal muscle fibroblast cell lines.
• a method for introducing heterologous nucleic acid into a synoviocyte by introducing in a chromosome, such as for example an artificial chromosome, into the synoviocyte.
• the artificial chromosome is an ACes.
• the synoviocyte can be, for example, a fibroblast-like synoviocyte.
• a particular method provided herein for introducing a large nucleic acid molecule into a synoviocyte includes steps of exposing the nucleic acid molecule to a delivery agent and contacting the synoviocyte with the nucleic acid molecule.
• the delivery agent is not energy.
• the large nucleic acid molecule is a chromosome.
• the nucleic acid can be an artificial chromosome, such as an ACes.
• the synoviocyte is a fibroblast-like synoviocyte. Any delivery agents, such as described herein, may be used in such methods.
• the delivery agent can be one that includes a cationic compound.
• a method for introducing a nucleic acid molecule into a synoviocyte that includes steps of exposing the nucleic acid molecule to a delivery agent, exposing the synoviocyte to a delivery agent and contacting the synoviocyte with the nucleic acid molecule, whereby the nucleic acid molecule is delivered into the synoviocyte, and wherein the steps are performed sequentially in any order or simultaneously.
• the delivery agent is energy, it is not applied to the nucleic acid molecule and it is not applied to the synoviocyte after contacting the synoviocyte with the nucleic acid molecule.
• the nucleic acid may be any nucleic acid.
• the nucleic acid is a large nucleic acid, chromosome, artificial chromosome or ACes.
• the synoviocyte is a fibroblast-like synoviocyte. Delivery agents, such as described herein, may be used in such methods.
• the delivery agent can be one that includes a cationic compound.
• Another method for delivering a nucleic acid molecule into a synoviocyte includes steps of contacting the synoviocyte in the presence or absence of the nucleic acid molecule with a delivery agent, and applying ultrasound energy or electrical energy to the synoviocyte, wherein the contacting and applying are performed sequentially or simultaneously, and then contacting the synoviocyte with the nucleic acid molecule, whereby the nucleic acid molecule is delivered into the synoviocyte.
• the nucleic acid may be any nucleic acid.
• the nucleic acid is a large nucleic acid, chromosome, artificial chromosome or ACes.
• the synoviocyte is a fibroblast-like synoviocyte.
• Numerous delivery agents, including agents such as those described herein, may be used in such methods.
• the delivery agent can be one that includes a cationic compound.
• the energy is ultrasound.
• nucleic acids in particular, large nucleic acids, such as chromosomes, including artificial chromosomes, e.g. , ACes, into primary cells, including synoviocytes and fibroblasts. These methods may be used in vitro and in vivo.
• synoviocyte comprising a large heterologous nucleic acid, a heterologous chromosome or portion thereof, or an artificial chromosome.
• the artificial chromosome is an ACes.
• Such synoviocytes include fibroblast-like synoviocytes.
• the synoviocytes may be from any species, including, but not limited to mammalian species.
• synoviocytes containing large nucleic acids, such as, for example, artificial chromosomes (e.g. , ACes) include primate synoviocytes, as well as rodent, rabbit, monkey, dog, horse and human synoviocytes.
• a method for treating or modulating a rheumatic disease process in a subject includes steps of introducing a large nucleic acid into the subject, wherein the large nucleic acid contains nucleic acid that is or that encodes an agent that modulates a rheumatic disease process.
• the nucleic acid can be or can encode a molecule that has an anti-rheumatic effect.
• Processes associated with rheumatic diseases are known in the art and are described herein.
• one such process is an inflammatory process that includes processes of cell activation, infiltration, proliferation and recruitment.
• the disease is rheumatoid arthritis.
• the nucleic acid may be, for example, a chromosome or portion thereof or an artificial chromosome, e.g., an ACes.
• the large nucleic acid is introduced into a site of inflammation in the subject.
• One possible site of inflammation is a joint.
• a method for treating a rheumatic disease in a subject in which a large nucleic acid is introduced into the subject wherein the large nucleic acid contains nucleic acid that is or that encodes a therapeutic agent.
• the nucleic acid can be or can encode a molecule that has an anti-rheumatic effect.
• the disease is rheumatoid arthritis.
• the nucleic acid may be, for example, a chromosome or portion thereof or an artificial chromosome, e.g. , an ACes.
• the large nucleic acid is introduced into a site of inflammation in the subject.
• One possible site of inflammation is a joint.
• the method may be practiced in any format, including ex vivo and in vivo formats.
• the nucleic acid can be introduced into a cell in vitro and then transferred into the subject.
• the nucleic acid can be introduced into a cell in vivo.
• the nucleic acid is introduced into a synoviocyte, which can be, for example, a fibroblast-like synoviocyte.
• the nucleic acid that is introduced can comprise any nucleic acid that is or that encodes a molecule that has an anti-rheumatic effect in the subject.
• the molecule may alter, counteract or diminish a process of the disease.
• the molecule may ameliorate symptoms of the disease.
• Molecules that provide anti-rheumatic effects in subjects with RA are known in the art [see, e.g. , Vervoordeldonk and Tak (2001 ) Best Prac. Res. Clin. Rheumatol. 75:771 -788 and WO 00/521 86].
• Such molecules include anti-inflammatory or immunomodulatory molecules.
• interleukin-1 receptor antagonists soluble interleukin-1 receptor, soluble tumor necrosis factor receptor, interferon- , interleukin-4, interleukin-10, interleukin-1 3, transforming growth factor ?, dominant negative IkappaB- kinase, FasL, Fas-associated death domain protein or CTLA-4 are among molecules that can have anti-rheumatic effects.
• nucleic acid molecule can be one that includes nucleic acid that is or encodes a candidate therapeutic agent.
• the method may include a step of determining if the nucleic acid molecule has any effects, and in particular any anti-rheumatic effects, on the animal.
• the disease is a rheumatic disease, such as, for example rheumatoid arthritis.
• the animal is any animal in which the disease may be modeled.
• the animal may be a mammal.
• the animal is a monkey, rodent, rabbit, dog, cat, horse, cow, pig or primate.
• the large nucleic acid may be, for example, a chromosome, or portion thereof, or an artificial chromosome, for example, an ACes.
• the nucleic acid molecule is in a synoviocyte, such as, for example, a fibroblast-like synoviocyte.
• the nucleic acid is introduced into a joint of the animal.
• the nucleic acid molecule may be introduced into the animal using in vitro or in vivo formats.
• the nucleic acid can be introduced into a cell in vitro and then be transferred into the animal.
• the nucleic acid is introduced into a cell in vivo.
• Animal models include, for example, animal models of RA.
• RA adjuvant-induced arthritis
• Such models include adjuvant-induced arthritis (AA) [see, e.g. , Kong et al. (1 999) Nature 4023:304-309] and collagen type II- induced arthritis [see, e.g. , Tak et al. ( 1 999) Rheumatology 33:362-369; Han et al. (1 998) Autoimmunity 28: ⁇ 97-208; Gerlag et al. (2000) J. Immunology 765: 1 652-1 658] .
• AA adjuvant-induced arthritis
• collagen type II- induced arthritis see, e.g. , Tak et al. ( 1 999) Rheumatology 33:362-369; Han et al. (1 998) Autoimmunity 28: ⁇ 97-208; Gerlag et al. (2000) J. Immunology 765: 1 652-1 658] .
• Microscopic and colony formation analysis methods that may be used in evaluating stable nucleic acid molecule delivery rely on manual visualization or measurement of nucleic acid molecules (e.g., a selectable marker gene) expression, which is a distinct process from delivery. Such methods are associated with time delays in obtaining an assessment of the delivery method.
• Microscopic techniques for visualizing chromosome or plasmid transfer using bromodeoxyuridine (BrdU) are time consuming, restricted by the large sample size required to detect low levels of transfer and limited by the necessity of manual scoring.
• Colony-forming transfection analysis may require four-to-six weeks to generate and evaluate marker- expressing transfection colonies.
• methods provided herein are based on rapid, auto- mated, sensitive and accurate analysis procedures, such as flow cytometry, and thus do not involve any time-consuming, laborious and error-prone steps, such as manual detection of individual transfected cells by microscopic techniques.
• the methods make possible the analysis of nucleic acid molecule delivery data within 48 hours after transfection.
• data collected by flow cytometry analysis is statistically superior due to the ease at which large numbers of events, e.g., nucleic acid molecule transfer, are collected.
• the positive values obtained in these methods are instrument derived and therefore not as susceptible to judgment errors.
• these methods provide for greater accuracy in assessing nucleic acid molecule delivery.
• microscopic analysis is limited by the time involved for scoring positive events and sample size is restrictive.
• nucleic acid molecules such as DNA
• a reporter gene expression product it is possible to measure absolute values of nucleic acid molecules transferred, within twenty-four hours, without being hindered by cell autofluorescence and by the problems of differentiating wild-type cells from cells expressing low levels of reporter gene products (see, e.g., Ropp et al. (1 995) Cytometry 21 :309-31 7).
• 1 Factors to consider in addressing delivery of nucleic acids Delivery of nucleic acids, including DNA, into cells is a process in which nucleic acids are transferred to the interior of a cell. Methods for the delivery of nucleic acids may be assessed in a variety of ways, including the following. a. Transfer Efficiency
• a delivery method may be assessed by determining the percentage of recipient cells in which the nucleic acids, including DNA, are present (i.e., the transfer efficiency) .
• the transfer efficiency i.e., the transfer efficiency
• additional factors beyond mere presence of the nucleic acid in recipient cells that should be considered. Included among these additional factors is cell viability.
• clonogenicity is the method of choice to measure viability. When the target cells population is non-dividing or slow growing, metabolic integrity can be monitored.
• Clonogenicity represents a measure of the survivability of cells with respect to a delivery procedure, growth conditions and cell manipulations (e.g., plating). It is important to assess clonogenicity to determine whether a delivery procedure results in a sufficient number of viable cells to achieve a desired number of cells containing the transferred nucleic acid. Clonogenicity may be expressed as a clonal fraction. The clonal fraction is an index that is calculated by multiplying two separate fractions and normalizing to a control plating efficiency correction factor
• CPE CPE
• the two separate fractions that are multiplied in this calculation are the fraction of cells that survive a delivery procedure (population cell yield) and the fraction of cells that survive a plating procedure.
• the calculation is thus as follows:
• Colonal Fraction # viable colonies after plating x # cells post-transfection # cells plated # cells transfected x CPE
• the values used in this calculation for the number of cells post- transfection (i.e., post-delivery) and the number of colonies post-plating is based on cell or colony numbers at certain times in the process.
• the value for the number of cells post-transfection is representative of the number of cells at a time after nucleic acid delivery that is sufficient for the delivery process to be completed. This time may be determined empirically. Typically this time ranges from 4-48 hours and generally is about one day after transfection.
• the value of the number of viable colonies post-plating is representative of the number of colonies at a time after nucleic acid delivery that is sufficient for the non- viable cells to be eliminated and the viable cells to be established as colonies. This time may be determined empirically. Typically this time ranges from that in which the average colony is made up of approximately 50 cells or generally is a time at which five cell cycles have passed.
• a correction factor is included to take into account the plating efficiency of control wells, which is the ratio determined by the number of colonies counted divided by the number cells initially plated (typically 600-1000 cells).
• the value of the correction factor typically ranges from about 0.7 to about 1 .2 and may be, for example, 0.9.
• the number of cells plated should remain constant at 1000
• reproductive or clonogenicity assays are not relevant. Less direct measurements of cell viability must be used to measure cell killing that monitor metabolic death rather than loss of reproductive capacity. These procedures include, for example: (1 ) membrane integrity as measured by dye exclusion, (2) inhibition of nucleic acid synthesis as measured by incorporation of nucleic acid precursors, (3) radioactive chromium release, and (4) MTT ASSAY (3-[4,5-dimethylthiazoI-2-yl]-2,5-diphenyl-tetrazolium bromide) . These methods are different from measurements of loss of proliferative capacity, as they reflect only immediate changes in metabolism, which can be reversed or delayed and hence lead to errors in estimation of cell viability. To minimize these errors, correlation of duplicate procedures is suggested. d. Potential Transfection Efficiency (PTE) and determination of Chromos Index (CI)
• the Chromos Index is an effective and rapid method to determine the Potential Transfection Efficiency of a proliferating population by using experimental values of % labeled nucleic acid, such as ACes, delivery to measure transfer efficiency and clonal fraction measured using a simplified clonogenicity assay.
• Chromos Index (CI) % labeled ACes delivery x estimated Clonal fraction x CF
• the values of the transfer efficiency and of the clonal fraction and viable fraction are calculated as described above.
• the correction factor (CF) takes into account sample size, sample time and control plating efficiency. If all these factors are constant for each variable i.e., sampling time and size then the correction factor will approach the inverse of the value for the C.P.E., i.e., such that the clonal fraction or transfer efficiency can still approach 100% even with a low CF, or in other words, if delivery and viability are 1 00%, then the maximum potential transfection efficiency will equal the plating efficiency of the control cells.
• the calculation of C.I. allows for determination of each variable optimization, with the goal being for parameters, such as transfer efficiency, clonal fraction, and CF to approach one (or 100%). If sample size or time varies for either clonal fraction or transfer efficiency, then CF represents the extrapolated value based on slope or rate of change. An application of this assessment is provided in the EXAMPLES.
• a stable transfection efficiency of about 1 % is in the range (1 - 100%) that is considered useful for the introduction of large nucleic acid molecules into target cells. It is possible, using methods provided herein, to predict which delivery methods have to be selected for achieving desired transfection efficiencies without having to grow transfectants for extended times under selective conditions and determine numbers of cells surviving selection marker expression. This analysis involves calculation of the Chromos Index (CI) which integrates a "biological" value (the clonal fraction) with a measurement of chromosomal "uptake” or transfer efficiency (percentage of cells containing delivered ACes) .
• CI Chromos Index
• nucleic acid molecules for transfer
• the nucleic acid molecules, such as DNA, to be delivered are labeled to allow for detection of the nucleic acid molecules in recipient cells after transfer into the cells.
• the nucleic acid molecules may be labeled by incorporation of nucleotide analogs. Any nucleic acid molecule analog that may be detected in a cell may be used in these methods.
• the analog is either directly detectable, such as by radioactivity, or may be detected upon binding of a detectable molecule to the analog that specifically recognizes the analog and distinguishes it from nucleotides that make up the endogenous nucleic acid molecules, such as DNA, within a recipient cell.
• Analogs that are directly detectable have intrinsic properties that allow them to be detected using standard analytical methods. Analogs may also be detectable upon binding to a detectable molecule, such as a labeled antibody that binds specifically to the analogs.
• the label on the antibody is one that may be detected using standard analytical methods.
• the antibody may be fluorescent and be detectable by flow cytometry or microscopy.
• the nucleic acid molecules, such as DNA, to be transferred is labeled with thymidine analogs, such as Iododeoxyuridine (IdUrd) or Bromodeoxyuridine (BrdU) .
• IdUrd is used to label the nucleic acid molecules, such as DNA, to be transferred.
• the transferred IdUrd-labeled nucleic acid molecules, such as DNA may be immunologically tagged using an FITC-conjugated anti-BrdU/ldUrd antibody and quantified by flow cytometry.
• the transfer of the labeled nucleic acid molecules, such as DNA, into recipient cells can be detected within hours after transfection.
• nucleic acid molecule such as DNA
• delivery conditions may have adverse effects on nucleic acid molecule structure.
• labeling techniques used in certain methods of monitoring nucleic acid molecules, such as DNA delivery may also impact nucleic acid molecules, such as DNA, structure and function.
• the effects of delivery conditions on nucleic acid molecules may be assessed in a variety of ways, including microscopic analysis.
• the stability of artificial chromosomes e.g. , ACes
• the chromosomes are exposed to the conditions of interest, e.g. , IdU labeling, and analyzed under a fluorescent microscope for the ability to remain intact and condensed after incorporation of nucleotide analogs.
• Methods of monitoring delivery of nucleic acid molecules delivery may also be combined with an assessment of nucleic acid molecule, such as DNA, expression in recipient cells to provide even further information concerning the overall process of nucleic acid molecule transfer for purposes of expression.
• reporter gene that encodes a readily detected product.
• reporter gene products include, but are not limited to green fluorescent proteins (GFP), Red Fluorescent protein (RFP), luciferases, and CAT.
• reporter gene products include, but are not limited, to ?-galactosidase and cell surface markers.
• artificial chromosomes such as ACes containing a GFP reporter gene, such as, but are not limited to, GFP coding sequences in combination with labeling of the ACes with DNA analogs, such as IdU
• delivery and expression can be rapidly and accurately monitored.
• the cells containing the ACes are split into two populations. One population is fixed and stained for IdU and analyzed by flow cytometry to determine percentage delivery. The other population is allowed to go through 4-5 cell divisions (approximately 72 hours), and the GFP fluorescence is measured as an indication of expression.
• Clontech, CA and is well known, see, e.g. , U.S. Patent Nos. 6,034,228, 6,037, 1 33, 5,985,577, 5,976,849, 5,965,396, 5,976,796, 5,843,884, 5,962,265, 5,965,396; see, also, U.S. Patent No. 4,937, 1 90) .
• This plasmid contains the internal ribosome entry site (IRES; Jackson (1 990) Trends Biochem. 75:477-483; Jang et al. (1 988) J. Virol. 52:2636-2643) of the encephalomyocarditis virus (ECMV) between the MCS and the enhanced green fluorescent protein (EGFP) coding region.
• ECMV encephalomyocarditis virus
• Plasmid plRES2- EGFP is designed for selection, by flow cytometry and other methods, of transiently transfected mammalian cells that express EGFP and the protein of interest. This vector can also be used to express EGFP alone or to obtain stably transfected cell lines without drug and clonal selection.
• Enhanced GFP is a mutant of GFP with a 35-fold increase in fluorescence. This variant has mutations of Ser to Thr at amino acid 65 and Phe to Leu at position 64 and is encoded by a gene with optimized human codons (see, e.g. , U.S. Patent No. 6,054,31 2).
• EGFP is a red- shifted variant of wild-type GFP (Yang et al. (1 996) Nucl. Acids Res. 24:4592-4593; Haas et al. (1 996) Curr. Biol. 5:31 5-324; Jackson et al. (1 990) Trends Biochem.
• EGFP encodes the GFPmutl variant (Jackson (1990) Trends Biochem. 15:411-483) which contains the double-amino-acid substitution of Phe-64 to Leu and Ser-65 I J MIA I CI
• the coding sequence of the EGFP gene contains more than 1 90 silent base changes which correspond to human codon-usage preferences (Jang et al. (1 988) J. Virol. 52:2636-2643) . Sequences flanking EGFP have been converted to a Kozak consensus translation initiation site (Huang et al. (1990) Nucleic Acids Res. 18: 937-947) to further increase the translation efficiency in eukaryotic cells.
• Plasmid pIRES-EGFP was derived from PIRESneo (originally called pCIN4) by replacing the neo gene downstream of the IRES sequence with the EGFP coding region.
• the IRES sequence permits translation of two open reading frames from one mRNA transcript.
• the expression cassette of pIRES-EGFP contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer followed by a multiple cloning site (MCS), a synthetic intron (IVS; Huang et al. (1 990) Nucleic Acids Res. 18: 937-947), the EMCV IRES followed by the EGFP coding region and the polyadenylation signal of bovine growth hormone.
• CMV human cytomegalovirus
• MCS multiple cloning site
• IVS synthetic intron
• CMV Human cytomegalovirus immediate early promoter
• Suitable host strains DH5a, HB101 , and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM 101 or XL1 -Blue.
• Selectable marker confers resistance to kanamycin (30 ⁇ g/ml) to E. coli hosts.
• E. coli replication origin pUC Copy number: — 500
• Plasmid incompatibility group pMB1 /ColE1 pCHEGFP2
• Plasmid pCHEGFP2 was constructed by deletion of the Nsi1 /Smal fragment from pIRES-EGFP. Plasmid pIRES-EGFP contains the coding sequence for a 2.1 kB Nru 1 /Xho fragment of pCHEGFP2 containing the CMV promoter, synthetic intron, EGFP coding sequence and bovine growth hormone polyadenylation signal. Digestion of pIRES-EGFP with Nru 1 and Sma 1 , yielded a 2.1 kb fragment.
• Cosmid pFK1 61 was obtained from Dr. Gyula Hadlaczky and contains a 9 kb Not ⁇ insert derived from a murine rDNA repeat (see clone 1 61 described in PCT Application Publication No. WO97/401 83 by
• This cosmid referred to as clone 1 61 contains sequence corresponding to nucleotides 1 0,232- 1 5,000 in SEQ ID NO. 1 6. It was produced by inserting fragments of the megachromosome (see, U.S. Patent No. 6,077,697 and International PCT application No. (WO 97/401 83); for example, H1 D3, which was deposited at the European Collection of Animal Cell Culture (ECACC) under Accession No. 96040929, is a mouse-hamster hybrid cell line carrying this megachromosome) into plasmid pWE1 5 (Stratagene, La Jolla, California) as follows.
• ECACC European Collection of Animal Cell Culture
• Plasmid DNA was isolated from colonies that survived growth on LB/Amp medium and was analyzed by Southern blot hybridization for the presence of DNA that hybridized to a pUC1 9 probe. This screening methodology assured that all clones, even clones lacking an insert but yet containing the pWE1 5 plasmid, would be detected.
• the clone was digested with Not ⁇ and Bam ⁇ and ligated with Not ⁇ I Bam HI -digested pBluescript KS (Stratagene, La Jolla, California) .
• Two fragments of the insert of clone no. 1 61 were obtained: a 0.2-kb and a 0.7-kb insert fragment.
• the same digest was ligated with 3a/r?HI-digested pUC1 9.
• Three fragments of the insert of clone no. 1 61 were obtained: a 0.6-kb, a 1 .8-kb and a 4.8-kb insert fragment.
• the insert corresponds to an internal section of the mouse ribosomal RNA gene (rDNA) repeat unit between positions 7551 -1 5670 as set forth in GENBANK accession no. X82564, which is provided as SEQ ID NO. 5.
• the sequence data obtained for the insert of clone no. 1 61 is set forth in SEQ ID NOS. 6-1 2.
• the individual subclones corresponded to the following positions in GENBANK accession no. X82564 (i.e., SEQ ID NO. 5) and in SEQ ID NOs. 6-1 2:
• sequence set forth in SEQ ID NOs. 6-1 2 diverges in some positions from the sequence presented in positions 7551 -15670 of GENBANK accession no. X82564. Such divergence may be attributable to random mutations between repeat units of rDNA.
• the rDNA insert from the clone was prepared by digesting the cosmid with Not ⁇ and Bgl ⁇ and was purified as described above. Growth and maintenance of bacterial stocks and purification of plasmids were performed using standard well known methods (see, e.g. , Sambrook et al.
• the murine A9 cell line was obtained from ATCC and cells were thawed and maintained as described below. Briefly, cells were plated at a density of 2X1 0 6 cells per 1 5 cm tissue culture dish (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) in growth medium containing of 90% DMEM (Canadian Life Technologies Burlington, ON) and 10% FBS (Can Sera, Rexdale ON), and were maintained at 37°C, 5% CO 2 . Cultures were routinely passaged when cells reached 70%-80% confluence.
• Sub culturing was carried out as follows: medium was removed by aspiration, 10 ml of 1 X trypsin-EDTA (Canadian Life Technologies Burlington, ON) was dispensed onto the cell monolayer and the dish gently swirled to distribute the trypsin-EDTA. Finally, the bulk of the trypsin-EDTA was removed by aspiration, and the dish placed at 37 °C for 5 minutes. To quench the trypsin-EDTA, 10 ml of growth medium was added to the dish, and the single cell suspension was transferred to a 50 ml conical tube. Cell counts were performed using a cell counting apparatus (Beckman-Coulter, Hialeah FL). The cells were diluted and re-plated as described above.
• cultures were harvested by treatment with trypsin-EDTA, counted and the cell suspension then centrifuged at 500Xg for 5 minutes in a swinging bucket centrifuge.
• the cell pellet was resuspended in freezing medium containing 90% DMEM, 20% FBS and 1 0% DMSO (Sigma-Aldrich, Oakville, ON) at a density of 1 X10 7 cells/ml.
• One ml aliquots of the cell suspension were then dispensed into cryo-vials (Nunc, Rochester NY), frozen over night in an isopropanol filled container (NUNC, Rochester NY) and placed at -70°C and then transferred to the gas phase of a liquid nitrogen freezer for long- term storage.
• A9 cells were transfected using the Ca 2 PO 4 co-precipitation method (see, e.g. , Graham et al. (1 978) Virology 52:456-457; Wigler et al.
• A9 cells were harvested 4 days post- transfection, resuspended in 10 ml of growth medium and sorted for GFP expressing populations using parameters described above.
• GFP positive cells were dispensed into a volume of 5-10 ml of growth medium supplemented with 1 X penicillin/streptomycin (Canadian Life Technologies Burlington, ON) while non-expressing cells were directed to waste.
• the expressing cells were further diluted to 50 ml using the same medium, plated onto 2X1 5 cm dishes and cultured as described in the previous section. When the sorted populations reached confluence they were resorted to enrich for GFP expressing cells.
• Fluorescence In-Situ Hybridization (FISH) screening was carried out on GFP enriched populations and single cell clones to detect amplification and/or artificial chromosome formation. Preparation of metaphase spreads and hybridizations were performed (see, Telenius et al. (1 999) Chromosome Res 7:3-7) . Probes used include pSAT 1 , which recognizes the mouse major repeat (see, e.g. , Wong et al. (1 988) Nucl. Acids Res. 16- ⁇ 645-1 1 661 ), pFK1 61 , which hybridizes to the mouse rDNA- containing regions and a PCR generated probe against the mouse minor repeat.
• heterologous nucleic acid that includes a selectable marker, e.g. , nucleic acid encoding a fluorescent protein or other protein that may be readily detected using flow cytometry-based methods or other methods, including, for example, fluorimetry, cell imaging or fluorescence spectroscopy, is introduced into a cell.
• a selectable marker e.g. , nucleic acid encoding a fluorescent protein or other protein that may be readily detected using flow cytometry-based methods or other methods, including, for example, fluorimetry, cell imaging or fluorescence spectroscopy
• rDNA and DNA encoding enhanced green fluorescent protein (EGFP) may be introduced into cells, e.g. , A9 cells.
• the transfected cells may be selected on the basis of properties detectable by flow cytometry- based methods, or other methods, including, for example, fluorimetry, cell imaging or fluorescence spectroscopy, e.g. , fluorescent properties.
• celis containing a fluorescent protein may be isolated from nontransfected cells using a fluorescence-activated cell sorter (FACS) . If the sorting is conducted prior to chromosomal analysis of the cells for the presence of artificial chromosomes, it provides a population of transfected cells that may be enriched for artificial chromosomes and thus facilitates any subsequent chromosomal analysis of the cells and identification and selection of cells containing an artificial chromosome, e.g. , ACes.
• FACS fluorescence-activated cell sorter
• the cells may be analyzed for indications of amplification of chromosomal segments, the presence of structures that may arise in connection with amplification and de novo artificial chromosome formation and/or the presence of artificial chromosomes, such as ACes.
• Analysis of the cells typically involves methods of visualizing chromosome structure, including, but not limited to, G- and C-banding and FISH analyses using techniques described herein and/or known to those of skill in the art.
• analyses can employ specific labelling of particular nucleic acids, such as satellite DNA sequences, heterochromatin, rDNA sequences and heterologous nucleic acid sequences, that may be subject to amplification.
• chromosomes During analysis of transfected cells, a change in chromosome number and/or the appearance of distinctive, for example, by increased segmentation arising from amplification of repeat units, chromosomal structures will also assist in identification of cells containing artificial chromosomes.
• Hoechst 35258 was excited with the primary UV laser beam, and excitation detected in FLI by using 420 nm hand-pass filter. Chromomycin A3 was excited by the second laser set at 458 nm and fluorescence detected in FL 4 by using a 475 nm long-pass filter. Both lasers had an output of 200 mW. Bivariate distributions (1 ,024 x 1024 channels) were accumulated during each sort. For all chromosome sorts, the sheath pressure was set at 30 lb/in 2 and a 50 ⁇ m diameter nozzle was installed. A drop delay profile was performed every morning and repeated after any major plug.
• Alignment of the instrument was performed daily by using 3.0 ⁇ m diameter Sphero rainbow beads (Spherotech, Libertyville, IL) . Alignment was considered optimized when a CV of 2.0% or less was achieved for FL1 and FL4.
• Condensing agents hexylene glycol, spermine and spermidine
• the sheath buffer contains 15 nM Tris HCI, 0.1 mM EDTA, 20 mM NaCl, 1 % hexylene glycol, 100 mM glycine, 20 M spermine and 50 ⁇ M spermidine.
• the sorted chromosomes were collected in 1 .5 ml screw-capped Eppendorf tubes at 4° C at a concentration of approximately 1 x 10 6 chromosomes/ml, which were then stored at 4° C.
• sorted chromosome samples were brought to 0.5% SDS, 50 mM EDTA and 100 ⁇ g/ml Proteinase K, then incubated for 18 hours at 50°C. 1 ⁇ l of a 20 mg/ml glycogen solution (Boehringer Mannheim) was added to each sample, followed by extraction with an equal volume of Phenol: Chloroform: Isoamyl Alcohol (25:24:1). After centrifugation at 21 ,000Xg for TO min, the aqueous phases were transferred to fresh microfuge tubes and were re-extracted as above.
• PCR was carried out on DNA prepared from sorted chromosome samples essentially as described (see, Co et al. (2000) Chromosome Research 3: 183-191 ) using primers sets specific for EGFP and RAPSYN. Briefly, 50 ⁇ l PCR reactions were carried out on genomic DNA equivalent to 10,000 or 1000 chromosomes in a solution containing 10 mM Tris-CI, pH 8.3, 50mM KCl, 200 ⁇ M dNTPs, 500 nM of forward and reverse primers, 1 .5 mM MgCI 2 , 1.25 units Taq polymerase (Ampli-Taq, Perkin- Elmer Cetus, CA). Separate reactions were carried out for each primer
• D coTisicn ⁇ utcr Dln c ⁇ -n set The reaction conditions were as follows: one cycle of 10 min. at 95 °C, then 35 cycles of 1 min. at 94°C, 1 min. at 55 °C, 1 min at 72°C, and finally one cycle of 10 min at 72°C. After completion the samples were held at 4°C until analyzed by agarose gel electrophoresis using the following primers (SEQ ID Nos.
• Vesicles were prepared at a lipid concentration of 700 nmoles/ml lipid (cationic lipid/DOPE 1 : 1 ) as follows.
• SAINT-2 350 nmol cationic lipid
• DOPE dioleoyl- phosphatidylethanolamine
• ChoPE Dioleoylphosphatidylethanolamine
• DOPE Avanti Polar Lipids, Alabaster, AL
• C s-unsaturated phosphatidylcholines are less effective.
• the solvent was evaporated under a stream of nitrogen (1 5 min/ 250 ⁇ l solvent at room temperature) .
• the remaining solvent was removed totally by drying the lipid for 1 5 min in an desiccator under high vacuum from a vacuum pump.
• To the dried mixture was added I ml ultrapure water. This was vortexed vigorously for about 5 min.
• the resulting solution was sonicated in an ultrasonication bath (Laboratory Supplies Inc. NY) until a clear solution was obtained.
• the resulting suspension contained a population of unilamellar vesicles with a size distribution between 50 to 100 nm.
• each individual manufacturer's protocol for complexing to naked DNA was followed, with the exception that the amount of transfection agent used was varied, to reflect the different amount and type of DNA present, as well as the different ionic strength of the complexing.
• One million ACes (in a volume of 800 ⁇ l) were typically combined with the transfection agent in a wide range of concentrations (between 5 times and 100 times the lowest manufacturers suggested concentration).
• the ACes/transfection mixture was allowed to complex for the time recommended by the manufacturer, in volumes ranging from 0.8 ml to 1 .9 ml; some manufacturers recommend adding media to the complexing reaction.
• the complexed mixture was then applied to the recipient cells and transfection allowed to proceed according to the manufacturer's protocol. Details on the various conditions used with different agents are presented in Table 1 .
• V79-4 cells Approximately 1 x 10 6 V79-4 cells were transfected with 1 x 10 6 IdUrd-labeled ACes complexed with a delivery agent (i.e., Lipofectamine PLUS and Lipofectamine or Superfect). The transfected cells were then fixed in ethanol. Fixed cells were denatured and exposed to FITC- conjugated antibody that specifically binds to BrdU/ldUrd-labeled nucleic acids.
• a delivery agent i.e., Lipofectamine PLUS and Lipofectamine or Superfect.
• the percentage of transfected cells containing IdUrd-labeled ACes was determined using flow cytometry and collecting FITC fluorescence. Data were accumulated to form bivariate channel distribution showing forward scatter versus green fluorescence (IdUrd-FITC). The fluorescence level at which cells were determined to be positive was established by visual inspection of the histogram of negative control cells such that the gate for the negative cells was set such that 1 % appeared in the positive region.
• the number of cells recovered at 24 hours post-transfection was determined by counting an aliquot using a Coulter Counter. To determine the control plating efficiency of a recipient cell line, the untreated cells were plated at 600-1 000 cells per 10 cm petri dish in growth medium and left stationary in a 5% CO 2 incubator at 37°C for approximately five cell cycles or until average colony was made up of 50 cells. At this point the number of viable colonies was determined. The treated cells were seeded at 1000 cells if the CPE is above 0.1 -0.2. If the CPE is low then the seeding density is increased to 5,000-50,000 cells per dish. EXAMPLE 5
• LM(tk-) ceils were grown at 37°C, 5% CO 2 , in DMEM with 4500 mg/L D-glucose, L-glutamine, pyridoxine hydrochloride and 10% Fetal Bovine Serum.
• the corner wells of a 1 2-well dish were seeded with 200,000 cells per well (this is to ensure no interference from the ultrasound waves from other wells) 24 hours before use.
• the GFP chromosomes were counted to verify approximately 1 X1 0 6 ACes per ml.
• the chromosomes were resuspended in the tube by flicking.
• Ten ⁇ l of chromosome suspension was removed and mixed with an equal volume of 30 mg/ml PI (propidium iodide) stain.
• Eight ⁇ l of the stained chromosomes was loaded onto a Petroff Hausser counting chamber and the chromosomes were counted.
• the medium was removed from the cells, and the cells were washed twice with HBSS (without phenol red, Gibco BRL) warmed to 37°C. 500 ⁇ l of the warmed HBSS was added to each well of cells (1 ⁇ l) LipofectAMINE (Gibco BRL) was added to each well. The plates were then sealed with parafilm tape and shaken gently at 20 rpm at room temperature for 30 minutes (Stagger plates - 10 minutes for ease of handling).
• Ultrasound gel (Other-Sonic Generic Ultra sound transmission gel, Pharmaceutical Innovations, Inc., Newark, NJ) was applied to the 2.5 cm sonoporator head. Ultrasound was applied with an ImaRX Sonoporator 100 at an output energy of 2.0 Watt/cm 2 , for 60 seconds, through the bottom of the plate of cells.
• one chromosome per seeded cell (2X10 5 ) or 200 ⁇ l GFP ACes in sheath buffer 15 nM Tris HCI, 0.1 mM EDTA, 20 mM NaCl, 1 % hexylene glycol, 100 mM glycine, 20 ⁇ M spermine and 50 ⁇ M spermidine) are added immediately to the well. (Repeat until all samples on the plate requiring ultrasound have been treated). The plate was then sealed once more with parafilm tape and shaken gently (20 rpm) for 1 hour at room temperature.
• HBSS Hanks balanced salt solution without Phenol Red (Gibco BRL, UK)
• 500 ⁇ l HBSS at 37°C was added per well, followed by 10 ⁇ l of the freshly prepared vesicle solution (prepared in Example 2) to yield a final concentration of 23.3 nmol/ml.
• the medium was removed from the cells, and the cells were washed twice with HBSS.
• 500 ⁇ l HBSS/lipid solution at 37 °C was added to each well.
• the HBSS/lipid solution was prepared by adding 1 ⁇ l ethanolic lipid solution (prepared as described above) to 500 ⁇ l HBSS under vigorous vortexing.
• the plates were then sealed with parafilm tape and shaken gently at room temperature for 30 min.
• ultrasound was applied at an output energy of 0.5 Watt/cm 2 for 60 sec through the bottom of the plate to the cells.
• the ultrasound was mediated by an ultrasound gel (Aquasonic 100, Parker, NJ) between transducer and plate.
• the ultrasound was applied with an ImaRx Sonoporator 100.
• one GFP chromosome per seeded cell (2 x 1 0 5 - 5 x 1 0 5 ) (prepared in Example 1 ) was added. The plate was then sealed again and shaken gently for 1 h at room temperature. After the incubation 1 ml medium (CHO-S-SFM 2 with 1 0% Fetal Calf Serum, 1 0000 ⁇ g/ml Penicillin and 10000 ⁇ g/ml Streptomycin Gibco BRL, Paisley, UK) was added to each well and the cells were incubated for 24 h at 37°C. The cells were then washed with medium, 1 ml medium was added, and the cells were incubated at 37 ° for another 24 h. Detection of expressed genes was then assayed by microscopy or detection of the transferred chromosome by FISH analysis.
• Hep-G2 cells were grown at 37 °C, 5% CO 2 , in DMEM with 4500 mg/l Glucose, with Pyridoxine/HCI, 1 0% Fetal Calf Serum, 10000 ⁇ g/ml Streptomycin and 1 000 ⁇ g/ml Penicillin. Between 2 x 10 5 and 5 x 1 0 5 cells were plated onto sterile glass slides in a 1 2 wells plate 24 hours before usage.
• IdUrd iododeoxyuridine
• fibroblast-like synoviocytes and rat skin fibroblasts were obtained from rats using standard methods [see, e.g. , Aupperle et al.
• Such methods include isolation of synoviocytes from rodent knees generally by removal of skin and muscle, followed by mincing of knee joint tissue. The minced tissue is then incubated with collagenase, filtered through nylon mesh and washed extensively. Cells can be cultured overnight, after which time non-adherent cells are removed. Adherent cells can be cultured and passaged by replating at a dilution when the cultures reach confluence.
• the cells were plated at 50,000-75,000 cells per 6-weII dish in media containing low glucose DMEM, 1 -glutamine, penicillin/streptomycin and 20% FBS. The cells were grown in a 5% CO 2 incubator at 37°C for 3-5 days until approximately 80% confluence or 500,000 cells per well.
• Transfection of cells with ACes One million IdUrd-labeled ACes were complexed with 2, 5 or 1 0 ⁇ l of Superfect (Qiagen) or Lipofectamine Plus (Life Technologies; Gibco) as follows. Complexing with Superfect was conducted for 1 0 minutes at room temperature.
• the indicated amounts of PLUS reagent were added to 1 million ACes and complexed at room temperature for 1 5 minutes.
• the indicated amounts of Lipofectamine were added into 200 ⁇ l of low glucose DMEM (no FBS) and combined with the ACes/PLUS complex for 1 5 minutes at room temperature.
• the complexed ACes were then added dropwise to the cells in 600 ⁇ l media (final volume of approximately 1 .4 ml). After 3 hrs at 37 °C in a 5% CO 2 incubator, a total volume of 3 ml of culture media (low glucose DMEM, l-glutamine, penicillin/streptomycin and 20% FBS) was added.
• the cells were trypsinized to form a single cell suspension, centrifuged to remove the supernatant and then fixed in cold 70% ethanol for a minimum of one hour. An aliquot of the fixed cells was saved for microscopic analysis.
• FITC-Conjugated antibody labeling of ACes Following transfection, the ACes were labeled with FITC- conjugated antibody that specifically binds to BrdU- or IdUrd-labeled nucleic acids and the cells were analyzed by FACs for FITC fluorescence and microscopic staining. Fixed cells were denatured in 2N HCI and 0.5% Triton-X for 30 minutes at room temperature. After denaturation, the cells were neutralized by a series of wash steps at 4°C. To minimize background staining, the sample was resuspended in PBS and 4% FBS or BSA and 0.1 % Triton-X (blocking buffer) for a minimum of 1 5 minutes.
• the delivery of intact ACes was detected within 24 to 48 hours post transfection.
• the number of cells recovered at 24 hours post- transfection was determined by counting an aliquot using a Coulter Counter.
• the cells were plated at 1000-10,000 cells per 10 cm petri dish in growth medium and left stationary in a 5% CO 2 incubator at 37°C for 10 days. At that point, the number of viable colonies was determined. The normalized plating efficiency was calculated as described herein.
• the percent delivery into fibroblast-like synoviocytes as determined by flow cytometry ranged from - 24% to — 66.3%.
• the normalized % plating efficiency was — 36% when 2 ⁇ l of Superfect was used and — 16% when 5 ⁇ l of Superfect was used. Higher doses of Superfect were associated with toxicity and multiple ACes per cell as compared to lower doses.
• Lipofectamine Plus was used as a delivery agent, the percent delivery into fibroblast-like synoviocytes as determined by flow cytometry ranged from — 1 1 % to — 27% with percent delivery increasing with increasing doses of agent.
• L8 and rat skin fibroblasts (RSF) that had been transfected with ACes were grown under hygromycin B selection and analyzed for lacZ expression. While in this example, a hygromycin selection gene was included in the ACes, there are numerous other selectable marker genes that may be used in connection with the transfer of heterologous nucleic acids into cells when it is desirable to include such genes. Such selection systems are known to those of skill in the art. A choice of selectable marker gene can, for instance, take into account the level of toxicity of the selection agent on the host cell for transfection. Identification of an appropriate selectable marker gene is routine employing the guidance provided herein.
• EXAMPLE 8 Ex vivo transfer of reporter genes into rat joints To examine transfer of a heterologous gene into an in vivo environment and expression of the gene in vivo, L8 cells transfected with ACes as described above were injected into the ankle joint of rats with adjuvant-induced arthritis. On day 0, adjuvant induction of arthritis was performed on Lewis rats. Methods for adjuvant induction of arthritis in animal models are known in the art [see, e.g. , Kong et al. (1 999) Nature 4023:304-309] .
• Lewis rats are immunized at the base of the tail with 1 mg Mycobacterium tuberculosis H37 RA (Difco, Detroit, Michigan) in 0.1 ml mineral oil on day 0. Paw swelling typically begins around day 1 0. On day 1 2, intra-articular injection of transfected L8 cells ( - 0.7 x
• Production cells lines (see Example 1 ) were grown in MEM medium
• Iododeoxyuridine or Bromodeoxyuridine was added directly to culture medium of the production cell line (CHO E4201 9) in the exponential phase of growth.
• Stock Iododeoxyuridine was made in tris base pH 10, and Bromodeoxyuridine stocks in PBS. Final concentrations of 0.05-1 ⁇ M for continuous label of 20-24 hours of 5-50 ⁇ M with 1 5 minute pulse. After 24 hours, exponentially growing cells were blocked in mitosis with colchicine (1 .0 ⁇ g/ml for 7 hours before harvest. Chromosomes were then isolated and stained with Hoechst 33258 (2.5 ⁇ g/ml) and chromomycin A3 (50 ⁇ g/ml).
• Condensing agents hexylene glycol, spermine, and spermidine were added to the sheath buffer to maintain condensed intact chromosome after sorting.
• IdU labeling index of sorted chromosomes was determined microscopically. An aliquot (2-10 ⁇ l) of sorted chromosomes was fixed in 0.2% formaldehyde solution for 5 minutes before being dried on clean microscopic slide. The microscope sample was fixed with 70% ethanol. The air-dried slide was denatured in coplin jar with 2N HCI for 30 minutes at room temperature and washed 2-3 times with PBS. Non specific binding was blocked with PBS and 4% BSA or serum for minimum of 1 0 minutes.
• V79-4 Chonese Hamster Lung fibroblast
• FBS Frequency B+ (Can Sera Rexdale ON) .
• the protocol was modified for use with LM (tk-) cell line by plating 500,000 cells. Lipid or dendrimer reagent was added to 1 X10 6 ACes sorted in — 800 ⁇ l sort buffer. Exemplary protocol variations are set forth in Table 1 . Chromosome and transfection agents were mixed gently. Complexes were added to cells drop-wise and plate swirled to mix.
• selection medium containing of DMEM and 10% FBS with 0.7 mg/ml hygromycin B, # 400051 (Calbiochem San Diego, CA) is added. Selection medium is changed every 2-3 days. This concentration of hygromycin B kills the wild type cells after selection for 7 days. At 10-1 4 days colonies were expanded and then screened by FISH for intact chromosome transfer and assayed for beta galactosidase expression. Table 1 : Delivery Transfection Protocols
• Cytometry (1 981 );6:385-393) was used except with some modifications at the neutralization step, the presence of detergent during denaturation and the composition of blocking buffer. Between each step samples are centrifuged at 300 g for 7-1 0 minutes and supernatant removed. Samples of 1 -2 million cells are fixed in 70% cold ethanol. Cells are then denatured in 1 -2 ml of 2N HCL plus 0.5% triton X for 30 minutes at room temperature. Sample undergoes 3-4 washes with cold DMEM until indictor is neutral. Final wash with cold DMEM plus 5% FBS.
• Blocking/permeabilization buffer containing PBS, 0.1 % triton X and 4% FBS is added for 1 0-1 5 minutes before pelleting sample by centrifugation.
• Percentage of transfected cells containing IdU labeled ACes was determined using a flow cytometry with an argon laser turned to 488 nm at 400 mW. FITC fluorescence was collected through a standard FITC

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