WO2005023312A2 - Procedes - Google Patents

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Publication number
WO2005023312A2
WO2005023312A2 PCT/GB2004/003765 GB2004003765W WO2005023312A2 WO 2005023312 A2 WO2005023312 A2 WO 2005023312A2 GB 2004003765 W GB2004003765 W GB 2004003765W WO 2005023312 A2 WO2005023312 A2 WO 2005023312A2
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Prior art keywords
nucleic acid
cell
acid molecule
previous
transfection
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PCT/GB2004/003765
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English (en)
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WO2005023312A3 (fr
Inventor
Anthony Nigel Warrens
Daxin Chen
Anthony Dorling
Robert Ian Lechler
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Imperial College Innovations Limited
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Publication of WO2005023312A2 publication Critical patent/WO2005023312A2/fr
Publication of WO2005023312A3 publication Critical patent/WO2005023312A3/fr

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    • 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
    • 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
    • A61K48/0033—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 the non-active part being non-polymeric
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a method for improving the efficiency of transfection of nucleic acid into a cell. Such a method is of use in, for example, gene therapy.
  • nucleic acids The introduction of foreign nucleic acids into a cell is central to many methods of both molecular biology and gene therapy.
  • the direct introduction of nucleic acids into a cell is called transfection.
  • nucleic acids There are various methods by which nucleic acids may be transfected into a cell: co- preciptiation with calcium phosphate, incorporation into lipid vesicles (liposomes), and the exposure of cells to a brief electrical pulse that transiently opens pores in the plasma membrane (electroporation) are a few of the more common methods.
  • transduction An alternative, indirect, means of introducing foreign nucleic acids into a cell is via viruses, called transduction. This can be achieved using, for example, bacterial, animal or plant viruses. For animal cells, for example, retroviruses are particularly useful in this respect, since their life cycle involves the stable integration of viral DNA into the genome of infected cells.
  • Gene therapy can be broadly defined as the transfer of genetic material into a cell to transiently or permanently alter the cellular phenotype. Gene therapy can be performed by the transfer of genetic material into target cells in vitro and then administration of the modified cells to an animal or patient. Alternatively, in vivo gene therapy is accomplished by direct transfer of genetic material into target cells while the target cell remains present in an animal or patient.
  • Gene therapy can be used as a form of therapy, for example to alleviate a genetically-mediated disorder such as SCID or cystic f ⁇ brosis.
  • gene therapy can also be used to introduce a desirable trait into an organism, for example the genetic manipulation of a non-human animal to modulate post- transplant inflammation as a step towards the use of the animal for organ xenotransplantati on.
  • germ line and somatic Two basic types of gene therapy can be applied to humans and animals: germ line and somatic.
  • the goal of germ-line gene therapy is the more ambitious: to introduce transgenic cells into the germ line as well as into the somatic cell population. Not only should this therapy correct a disease or introduce a trait into the human or animal treated, but some gametes could also carry the introduced genetic material. No human germ-line gene therapy has been performed to date.
  • Somatic gene therapy focuses only on the body excluding the germ line cells (soma).
  • the approach is to attempt to correct a disease phenotype or introduce a desirable trait by treating some somatic cells in the human or animal.
  • the method typically proceeds by removing some cells from a human or animal and making these cells transgenic through the introduction of copies of desired genetic material.
  • the transgenic cells are then reintiOduced into the patient's body, where they provide normal gene function.
  • somatic gene therapy as a treatment for single gene inherited diseases and some acquired conditions, such as certain types of cancer, represents one of the most important technical advances in medicine.
  • X-linked immunodeficiencies or chronic granulomatous disease (CGD)
  • CCD chronic granulomatous disease
  • ADA-SCID adenosine deaminase dependent severe combined immunodeficiency
  • T-cells including the genes required by the patients are not immortal, requiring the therapy to be repeated at regular intervals.
  • attempts to effect a permanent correction for example by gene transfer into pluripotent haematopoietic stem cells (PHSC), have thus far been unsuccessful.
  • PHSC pluripotent haematopoietic stem cells
  • Gene therapy is also particularly useful for modulating inflammatory or immunological responses of a tissue or organ intended for transplantation.
  • the pig is generally regarded as likely to be the preferred donor animal in xenotransplantation. Although many hurdles remain to be cleared, it would be useful to be able to manipulate porcine endothelium genetically, among other reasons, to test approaches in the modulation of inflammation. However, as a non-dividing cell, it is less easy to manipulate.
  • targeting techniques have allowed the localisation of gene expression to specific vascular beds, taking advantage of organ-specific receptors (Reynolds et al (2000) Mol Ther 2, 572; Takei et al (1999) Transpl Proceedings 31, 790), such as angiotensin-converting enzyme in pulmonary vascular endothelium or cell type- specific promoters to limit the expression of transfected genes (Reynolds et al (2001) Nat Biotechnol 19, 838)
  • Retroviral vectors offer a very high efficiency of chromosomal integration and are suited to gene therapy strategies where it is a requirement that the therapeutic gene should be stably transmitted to future progeny of the target cell. Retroviral vectors introduce foreign nucleic acids into a cell via transduction.
  • Recombinant retroviruses have been used in many clinical gene therapy protocols for ex vivo transduction of cultured T lymphocytes, fibroblasts, keratinocytes, hepatocytes, haemopoietic stem cells and neoplastic cells.
  • transfective methods of gene transfer offer a number of advantages over transductive methods.
  • Transfective methods may also be more appropriate for non-dividing cells than, for example, retroviruses
  • Transfective methods are, in general, less immunogenic than transduction.
  • the transfective vectors are easier to prepare.
  • transfective methods have a number of advantages over transductive methods, transfective methods all appear to be associated with a lower efficiency of introduction of foreign nucleic acids into a cell than transduction, and they confer a shorter duration of expression of the foreign nucleic acids in the transformed cell.
  • much work has been devoted to improving the efficiency transfective methods of introducing foreign nucleic acids into a cell. (Nishikawa et al (2001) Hum Gene Ther 12, 861).
  • Transfective gene transfer methods are characterised in that genetic materials are directly transferred into cells.
  • many approaches have been used to induce cell uptake of DNA. These include the injection of naked DNA, the administration of DNA-coated gold particles, electroporation, and the administration of DNA delivered as a complex with cationic lipid or cationic polymers. Modifications of the latter group have been developed to allow the escape of DNA from the endosome. These modifications include the use of fusogenic lipids or peptides or techniques to buffer the pH drop in late endosomes. In addition, other techniques have been developed to allow the targeting of vectors to specific tissues.
  • organ-specific receptors such as angiotensin- converting enzyme in pulmonary vascular endothelium (Reynolds et al (2000) Mol Ther 2, 572) or the use of cell type-specific promoters to limit the expression of transfected genes (Reynolds et al (2001) Nat Biotech 19, 838).
  • organ-specific receptors such as angiotensin- converting enzyme in pulmonary vascular endothelium
  • cell type-specific promoters to limit the expression of transfected genes
  • a first aspect of the invention is a method for improving the efficiency of nucleic acid transfection of a cell, the method comprising binding an agent in a minor groove of the nucleic acid molecule to be transfected.
  • a second aspect of the invention is the use of an agent which binds in a minor groove of a nucleic acid molecule in a method according to the first aspect of the invention.
  • a third aspect of the invention is a pharmaceutical composition
  • a nucleic acid molecule having an agent bound to a minor groove of said nucleic acid molecule and a pharmaceutically acceptable carrier is a pharmaceutical composition
  • the method of the first aspect of the invention increases the transfection efficiency of a nucleic acid molecule into a cell, as can be seen from the accompanying examples.
  • the method of the invention may proceed by incubating a nucleic acid molecule with an agent which binds to a minor groove of the nucleic acid molecule. While the accompanying examples demonstrate that the agent may be bound to the nucleic acid molecule prior to transfection into a cell, the agent may also be bound to the nucleic acid molecule during the transfection process.
  • Any excess of agent which does not bind to the nucleic acid molecule may be removed from the reaction mixture prior to transfection, or the excess may be left in the reaction mixture and included in the transfection process.
  • 'transfection we mean the direct introduction of nucleic acids into a cell using the various methods outlined above, for example co-preciptiation with calcium phosphate, incorporation into lipid vesicles (liposomes), electroporation, injection of naked nucleic acid molecules, the administration of nucleic acid-coated gold particles, and the administration of nucleic acid molecules as a complex with cationic polymers. Further methods of transfection will be appreciated by a person skilled in the art.
  • the method of the invention allows for the efficiency of transfection of nucleic acid into a cell to be increased by 0.1 fold, 0.5 fold, 1 fold, 2 fold, 5 fold, 10 fold 13 fold, 15 fold, 20 fold, 25 fold 50 fold, or 100 fold or more relative to that of the same nucleic acid molecule not subjected to the method of the invention.
  • binding an agent' we mean that a nucleic acid molecule is exposed to the agent and a quantity of the agent binds to the molecule.
  • the agent may covalently bind to the nucleic acid molecule by, for example, crosslinking the agent to atoms in the minor groove of the molecule .
  • the binding is non-covalent, for example ionic or electrostatic, van der Waals or hydrogen bonding.
  • non-covalent bonds are, as appreciated by a person skilled in the art, readily reversible and, hence, the agent can be displaced in the cell once the nucleic acid has been transfected.
  • the method of the first aspect of the invention comprises binding an agent to a minor groove of a nucleic acid molecule.
  • the structure of helical nucleic acids includes the presence of two kinds of grooves, called the minor groove (6 angstroms wide) and the major groove (12 angstroms wide). They arise because the glycosidic bonds of a base pair of a intramolecular and intermolecular helical nucleic acid are not diametrically opposite each other.
  • the minor groove contains the pyrimidine 0-2 and the purine N-3 of the base pair, and the major groove is on the opposite side of the pair.
  • the major groove is slightly deeper than the minor one.
  • Each groove is lined by potential hydrogen-bond donor and acceptor atoms and hence can act to allow agents to bind in the groove.
  • Nucleic acids exist naturally in two forms: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA is usually found in the form of a double helix, the genome of some viruses consist of single- stranded DNA. Within the cell there are usually several forms of the RNA, often present as single strands. However, a single stranded nucleic acid may generate double-helical regions. An intramolecular duplex region can form within a single-stranded molecule that contains two complementary sequences if these sequences base pair with one another. Or a single-stranded molecule may base pair with an independent, complementary single-stranded molecule to form an intermolecular duplex. Base pairing between independent complementary single strands is not restricted to DNA-RNA or RNA-RNA interactions, but can also occur between a DNA molecule and an RNA molecule.
  • both single stranded and double stranded nucleic acid molecules can have a linear structure, or the molecule can form a helix, and both structures are possible within the same molecule. It is considered that the nucleic acid molecule contains helical structures when bound to the agent.
  • An embodiment of the first, second or third aspects of the invention is wherein the nucleic acid molecule comprises a gene therapy vector.
  • Gene therapy vectors known in the art may be useful in the practise of these aspects of the invention.
  • suitable vectors, gene therapy constructs and delivery systems which may be adapted in relation to the present invention, including systems in which expression of the encoded polypeptide is under the control of an inducible promoter, are described in, for example WO01/18038.
  • Vectors, constructs, tissue-specific promoters and routes of administration for gene therapy aspects of this invention are known to a person of skill in the art, and are described for example in WO 01/18038, which is incorporated herein by reference.
  • the method of the invention may be used to improve the efficiency of gene therapy vector transfection into a cell.
  • such a vector may be of particular use in, for example, the genetic transformation of cells both in vivo and ex vivo.
  • a further embodiment of the first, second or third aspects of the invention is wherein the nucleic acid molecule comprises an oligonucleotide.
  • Oligonucleotides are well known to those skilled in the art may comprise for example, a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides in total.
  • oligonucleotides can comprise a sequence of nucleotide residues extending to 20, 25, 30, 40 or 50 nucleotides in total.
  • Oligonucleotides may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.
  • the method of the invention may be used to improve the efficiency of oligonucleotide transfection into a cell.
  • a method of RNA interference may be of particular use in a method of RNA interference.
  • both single stranded and double stranded nucleic acid molecules can form a helix.
  • An embodiment of the first, second or third aspect of the inventions is wherein the nucleic acid molecule is single stranded.
  • a further embodiment of the first, second or third aspect of the inventions is wherein the nucleic acid molecule is double stranded.
  • Both single and double stranded nucleic acid molecules can be used in the invention. Each form has different properties which may mediate different functions in the cell following transfection.
  • double stranded nucleic acid molecules can be used in gene transfer methods, while single stranded nucleic acids can be used in RNA interference.
  • nucleic acids can also form triplexes and quadruplexes with additional strands of nucleic acid.
  • a DNA triplex is formed when pyrimidine or purine bases occupy the major groove of the DNA double Helix forming
  • TFO triplex forming oligonucleotides
  • Intramolecular triplexes are the major elements of H-DNAs, unusual DNA structures, which are formed in homopurine-homopyrimidine regions of supercoiled DNAs.
  • TFOs are promising gene-drugs, which can be used in an anti-gene strategy, that attempt to modulate gene activity in vivo.
  • a further embodiment of the first, second or third aspects of the inventions is wherein the nucleic acid molecule is a triplex.
  • a further embodiment of the first, second or third aspects of the invention is wherein the nucleic acid molecule comprises DNA.
  • a further embodiment of the first, second or third aspects of the invention is wherein the nucleic acid molecule comprises RNA.
  • a further embodiment of the first, second or third aspects of the invention is wherein the nucleic acid molecule comprises a DNA/RNA hybrid.
  • the method of the first aspect of the invention comprises binding an agent to the minor groove of a nucleic acid molecule.
  • agents which bind to the grooves of nucleic acids have attracted much interest because of the diverse functions they can mediate, for example antimircobial or anti-tumor.
  • the agents can also be used as tools with which to study proteins which bind to nucleic acids as they can block the protein binding to the groove. It will be appreciated that agents which bind to the groove of a nucleic acid molecule may be toxic or non-toxic to a cell.
  • Agents that bind to a groove of a nucleic acid molecule can have a preference for A/T base pairs or G/C base pairs.
  • an embodiment of the first, second or third aspects of the invention is wherein the agent binds preferentially to A/T rich regions of the nucleic acid molecule.
  • agents are the minor groove binding agents distamycin, netropsin, Hoechst 33258 and 4,6-diamidino-2-phenylindole (DAPI).
  • DAPI 4,6-diamidino-2-phenylindole
  • a particular embodiment of the first, second or third aspects of the invention is wherein the agent is DAPI.
  • a further embodiment of the first, second or third aspects of the invention is wherein the agent binds preferentially to C/G rich regions of the nucleic acid molecule.
  • agents are the synthetic oligopeptides described in Forrow et al supra.
  • a fourth aspect of the invention is a method for delivery of a gene therapy vector to a cell comprising: i) performing a method according to any of the preceding claims on a gene therapy vector; and, ii) transfecting the treated gene therapy vector into a cell.
  • Gene therapy and gene therapy vectors has been discussed extensively above. As has already been noted, the method of the invention may be used to improve the efficiency of gene therapy vector transfection into a cell. As can be seem from the accompanying examples, such a vector may be of particular use in, for example, the genetic transformation of cells both in vivo and ex vivo.
  • An embodiment of the fourth aspect of the invention is wherein the gene therapy vector is transfected into a cell by liposomal transfection.
  • Liposomal transfection reagents are lipids which are able to bind to nucleic acids by their positive charge.
  • the liposome/nucleic acid complex fuses with the cell membrane and the nucleic acid is introduced into the cell where it may be translated in the cytoplasm or pass to the nucleus for possible incorporation into the genome.
  • Examples of liposomal transfection reagents which may be used in this aspect of the invention include the Escort Transfection ReagentTM (Sigma, MO, USA), or the CLONfectinTM transfection system from Clontech (www . clontech. com) .
  • a fifth aspect of the invention is the use of a gene therapy vector prepared according to the method of any of the previous claims in a method of gene therapy.
  • a sixth aspect of the invention is a method for delivery of a RNA interference molecule to a cell comprising: i) performing a method according to any of the preceding claims on a RNA interference molecule; and, ii) transfecting the treated RNA interference molecule into a cell.
  • RNA interference we also include antisense methods of regulating endogenous gene expression.
  • Antisense oligonucleotides are single-stranded nucleic acids, which can specifically bind to a complementary nucleic acid sequence. By binding to the appropriate target sequence, an RNA-RNA, a DNA-DNA, or RNA- DNA duplex is formed. These nucleic acids are often termed "antisense" because they are complementary to the sense or coding strand of the gene. Recently, formation of a triple helix has proven possible where the oligonucleotide is bound to a DNA duplex. It was found that oligonucleotides could recognise sequences in the major groove of the DNA double helix. A triple helix was formed thereby.
  • the above oligonucleotides can inhibit the function of the target nucleic acid. This could, for example, be a result of blocking the transcription, processing, poly(A)addition, replication, translation, or promoting inhibitory mechanisms of the cells, such as promoting RNA degradation.
  • Antisense oligonucleotides are prepared in the laboratory and then introduced into cells, for example by microinjection or uptake from the cell culture medium into the cells, or they are expressed in cells after transfection with plasmids or retroviruses or other vectors carrying an antisense gene.
  • Antisense oligonucleotides were first discovered to inhibit viral replication or expression in cell culture for Rous sarcoma virus, vesicular stomatitis virus, herpes simplex virus type 1, simian virus and influenza virus. Since then, inhibition of mRNA translation by antisense oligonucleotides has been studied extensively in cell-free systems including rabbit reticulocyte lysates and wheat germ extracts.
  • the cap, 5' untranslated region, and poly(A) signal lie within the sequence repeated at the ends of retrovirus RNA (R region) and the oligonucleotides complementary to these may bind twice to the RNA.
  • antisense oligonucleotides are 15 to 35 bases in length.
  • 20-mer oligonucleotides have been shown to inhibit the expression of the epidermal growth factor receptor mRNA (Witters et al, Breast Cancer Res Treat 53:41-50 (1999)) and 25-mer oligonucleotides have been shown to decrease the expression of adrenocorticotropic hormone by greater than 90% (Frankel et al, J Neurosurg 91 :261-7 (1999)).
  • An embodiment of the sixth aspect of the invention is wherein the RNA interference molecule is transfected into a cell by liposomal transfection.
  • suitable transfection reagents are provided above.
  • a seventh aspect of the invention is the use of a RNA interference molecule prepared according to the method of any of the previous claims in a method of RNA interference.
  • An embodiment of the fourth, fifth, sixth or seventh aspects of the invention is wherein the gene therapy vector or RNA interference molecule comprises nucleic acid capable of modulating tissue inflammation.
  • transplantation of tissues to replace diseased organs is now an important medical therapy.
  • adaptive immune responses to the grafted tissues are the major impediment to successful transplantation.
  • Rejection is caused by immune responses to alloantigens on the graft, which are proteins that vary from individual to individual within a species, and are thus perceived as foreign by the recipient.
  • Transplant rejection is also a major obstacle in the path of xenotransplantation.
  • One method which may be of use in overcoming this is the genetic manipulation animals to modulate tissue inflammation prior to or post transplantation.
  • nucleic acids which may be used to modulate tissue inflammation include genes which encode anticoagulants, for example tissue factor pathway inhibitor, hirudin, anti-thrombin, tick anticoagulant peptide (TAP), heparin, snake venom anticoagulant peptides such as the 231 amino acid protein C activator purified from the venom of the snake Agkistrodon contortrix, genes encoding molecules which regulate apoptosis or immunological tolerance, for example FAS ligand, TNF- related apoptosis-inducing ligand, tweak, or genes involved in anti-fibrotic or anti-inflammatory responses, for example matrix mellatoproteinases or inhibitors of transforming growth factor ⁇ .
  • anticoagulants for example tissue factor pathway inhibitor, hirudin, anti-thrombin, tick anticoagulant peptide (TAP), heparin
  • snake venom anticoagulant peptides such as the 231 amino acid protein C activator purified from the venom of the snake Agkistrodon cont
  • the gene therapy vector or RNA interference molecule of this embodiment of the invention may be of particular use in modulating inflammation in a tissue, both prior to or post transplantation of the tissue.
  • a further embodiment of the fourth, fifth, sixth or seventh aspects of the invention is wherein the gene therapy vector or RNA interference molecule comprises nucleic acid capable of modulating restenosis following angioplasty.
  • Angioplasty is a method of treating blockage or narrowing of a blood vessel or heart valve by inserting a balloon into the constriction to reopen it.
  • the technique is used to treat a narrowed artery in a heart or limb.
  • this treatment can cause smooth muscle cell proliferation in the blood vessel leading to stenosis or restenosis.
  • nucleic acids which may be used to modulate restenosis following angioplasty include genes which encode transforming growth factor ⁇ as well as nucleic acids useful for modulating tissue inflammation, as discussed above.
  • the gene therapy vector or RNA interference molecule of this embodiment of the invention may be of particular use in modulating restenosis, both during and following angioplasty.
  • the pig is regarded as likely to be the preferred donor animal in xenotransplantation.
  • the endothelium is an attractive target for gene therapy for those interested in modulating inflammatory or immunological responses.
  • the tissue is porcine endothelium.
  • the method of the first aspect of the invention comprises transfecting a nucleic acid molecule into a cell.
  • the cell may be any prokaryotic or eukaryonic cell.
  • the cell is a eukaryotic cell.
  • the cell may be a unicellular organism, for example, yeast or Pichia pastoris, or the cell may be part of a multicellular organism.
  • the cell is part of an multicellular organism.
  • the cell may be of any cell type, for example an endothelial cell, smooth muscle cell, germ cell, mucosal epithelian cell such as gut and bronchial/pulmonary epithelium, neuronal cell, stem cell or an embryonic cell.
  • the method may be particularly useful with non-dividing cells.
  • a further embodiment of any of the previous aspects of the invention is wherein the cell is an endothelial cell.
  • endothelial cells are attractive targets for gene therapy for those interested in modulating inflammatory or immunological responses.
  • a further embodiment of any of the previous aspects of the invention is wherein the cell is a smooth muscle cell.
  • smooth muscle cells are targets for gene therapy, for example, to control restenosis-related cell proliferation following angioplasty.
  • tissue or organs include the endothelium, muscle (smooth and skeletal), fibroblasts, neural tissue, foetal tissue, heart, liver, lung, pancreas, islets, skin, small bowel, cornea, cartilage, bone, or kidney.
  • tissue is the endothelium.
  • the tissue or organ may be human or animal, for example porcine.
  • the tissue or organ may be useful as an allograft, ie for transplant to a member of the same species, or as a xenograft.
  • the tissue or organ may be treated ex vivo or in vivo.
  • a further embodiment is wherein the tissue or organ is ex vivo.
  • a further embodiment is wherein the tissue or organ is present in an animal.
  • an animal examples include a human, pig, sheep, cow, rabbit, mouse, rat, guinea pig, hamster, goat, dog, cat or horse.
  • An eighth aspect of the invention is a kit of parts comprising an agent which binds to a minor groove of a nucleic acid molecule and a nucleic acid molecule to be transfected into a cell and, optionally, a system to allow liposomal transfection of a cell.
  • An embodiment of the eighth aspect of the invention is wherein the agent binds preferentially to A/T rich regions of nucleic acid, for example DAPI, or binds preferentially to or G/C rich regions of the minor groove of the nucleic acid, and the nucleic acid molecule is a single stranded or double stranded or triplex gene vector or oligonucleotide comprising DNA or RNA or a DNA/RNA hybrid.
  • a ninth aspect of the invention is the use of a pharmaceutical composition according to any of the preceding aspects of the invention or a kit of parts according to the eighth aspect of the invention in the manufacture of a medicament for the treatment of a patient in need of gene therapy.
  • a method of treatment of a patient in need of gene therapy comprising administering to the patient a pharmaceutical composition according to any of the preceding aspects of the invention or the use of a kit of parts according to the eighth aspect of the invention.
  • a tenth aspect of the invention is the use of a pharmaceutical composition according to any of the preceding aspects of the invention or a kit of parts according to the eighth aspect of the invention in the manufacture of a medicament for modulating the inflammation of a tissue.
  • a method of modulating the inflammation of a tissue of a patient comprising administering to the tissue or patient a pharmaceutical composition according to any of the preceding aspects of the invention or the use of a kit of parts according to the eighth aspect of the invention.
  • An eleventh aspect of the invention is the use of a pharmaceutical composition according to any of the preceding aspects of the invention or a kit of parts according to the eighth aspect of the invention in the manufacture of a medicament for the treatment of restenosis following angioplasty.
  • a method of treatment of restenosis following angioplasty comprising administering to the patient a pharmaceutical composition according to any of the preceding aspects of the invention or the use of a kit of parts according to the eighth aspect of the invention.
  • compositions or pharmaceutical composition or formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection.
  • the treatment may consist of a single dose or a plurality of doses over a period of time.
  • a compound Whilst it is possible for a compound to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be "acceptable” in the sense of being compatible with the compound and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free- flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • a binder eg povidone, gelatin, hydroxypropylmethyl cellulose
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • FIG. 1 EscortTM-mediated pGL3-lucif erase gene transfer into pig vascular cells is increased by the presence of increasing amounts of DAPI. Varying concentrations of DAPI were included in a mixture of EscortTM and DNA and used in in vitro transfection studies of porcine vascular endothelial and smooth muscle cells. Identical numbers of cells were seeded in each well and grown to 80% confluence before transfection. The success of transfection was determined by the degree of luciferase activity in a chemiluminescence assay. The reproducibility of replicates was such that the error bars indicating standard deviation are obscured by the data symbol for all but one data point. This experiment was performed three times.
  • Figure 2 DAPI EscortTM-mediated pGL3-luciferase gene transfer into pig vascular cells is increased with increasing time of exposure.
  • Figure 3 DAPI/EscortTM-mediated pGL3-luciferase gene transfer is effective even in the presence of low levels of foetal calf serum.
  • EscortTM/hirudin-CD4 was used in in vitro transfections with either transferrin or DAPI and level of surface hirudin expression determined by flow cytometry. EscortTM/hirudin-CD4 alone and EscortTM/ pGL3 control vector ("empty vector”) were used as negative controls. The percentage of cells positive, relative to that seen on the flow cytogram of cells exposed to EscortTM/ pGL3 control vector ("empty vector”) is recorded. This experiment was performed three times.
  • Figure 5 Lack of cytotoxicity induced by DAPI or Escort.
  • Figure 6 Southern blot analysis of transfected endothelial cells.
  • Porcine endothelial cells were transfected and genomic DNA was isolated and probed for the integration of luciferase cDNA.
  • Figure 7 Serum protection assay.
  • Figure 8 Ex vivo transfection of carotid artery.
  • Isolated porcine common carotid arteries were exposed ex vivo to pGL3- luciferase/EscortTM which had been supplemented with either transferrin or DAPI for a period of 48 hours. Arteries were then stained with either or both of anti-CD31 or anti-luciferase. In addition, sections were stained with DAPI as a nuclear counterstain. Staining with a control Texas Red conjugated antibody was used and proved negative (data not shown). This experiment was performed three times. (Magnification lOOx).
  • Figure 9 In vivo transfection of carotid artery.
  • Example 1 Efficient gene transfer into porcine vascular cells
  • the pig is generally regarded as likely to be the preferred donor animal in xenotransplantation. Although many hurdles remain to be cleared, it would be useful to be able to manipulate porcine endothelium genetically, among other reasons, to test approaches in the modulation of inflammation. However, as a non-dividing cell, it is less easy to manipulate.
  • the endothelium is an attractive target for gene therapy for those interested in modulating inflammatory or immunological responses.
  • In vitro strategies have employed non-viral (7) or viral (2) DNA transfection techniques to successfully introduce genetic material into endothelial cells.
  • a smaller number of studies have described in vivo strategies which have resulted in successful gene expression.
  • this has involved the administration of vectors into the peripheral circulation.
  • targeting techniques have allowed the localisation of gene expression to specific vascular beds, taking advantage of organ-specific receptors (3, 4), such as angiotensin-converting enzyme in pulmonary vascular endothelium or cell type-specific promoters to limit the expression of transfected genes (5).
  • Porcine endothelial cells were isolated from fresh pig aortas by gentle scraping. Cells were seeded into 2% gelatin-coated tissue culture flasks in DMEM (Gibco BRL, Paisley, UK) supplemented with 20% heat-inactivated foetal calf serum (FCS) (Globepharm, Surrey, UK), containing 25 IU/mL penicillin, 25 ⁇ g/mL streptomycin, 2 mM L-glutamine. Cells were stained with anti-CD31 to confirm that they were ECs.
  • FCS foetal calf serum
  • Porcine aortic vascular smooth muscle cells were obtained from pig aortas by mincing with fine dissecting scissors (Aesculap, Sheffield, UK) after the adventitia was removed and digested with 20 mL of digestion solution (0.125 mg/mL elastase, 0.25 mg/mL soybean trypsin inhibitor, 10 mg/mL collagenase I, 2.0 mg/mL crystallized bovine albumin and 15 mM HEPES (all Sigma)) at 37°C for 45 minutes.
  • digestion solution 0.125 mg/mL elastase, 0.25 mg/mL soybean trypsin inhibitor, 10 mg/mL collagenase I, 2.0 mg/mL crystallized bovine albumin and 15 mM HEPES (all Sigma)
  • the cellular digests were filtered through sterile 100 ⁇ M nylon mesh (Becton Dickinson Labware, NJ, USA), centrifuged at 1,000 rpm for 10 minutes and washed twice in DMEM containing 10% FCS and cultured in DMEM containing 25 IU/mL penicillin, 25 ⁇ g/mL streptomycin, 2 mM L-glutamine and 10% heat-inactivated FCS in 5% C0 2 at 37 ° C. That they were VSMCs was confirmed by staining with anti- human ⁇ -actin.
  • DNA 1 ⁇ g DNA was added to either 10 ⁇ g 4',6'- diamidino-2-phenylindole (DAPI) (Sigma, MO, USA) or 30 ⁇ g bovine transferrin (Sigma, MO, USA) (the latter as previously described (77)) and made up to 100 ⁇ L using DMEM without supplements, mixed by pipetting and incubated at room temperature for 15 minutes.
  • DAPI 4',6'- diamidino-2-phenylindole
  • bovine transferrin the latter as previously described (77)
  • This solution was then mixed with 15 ⁇ g Escort Transfection ReagentTM (Sigma, MO, USA), a liposome formulation of the cationic lipid N-[l(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTAP) and dioleoyl phosphatidylathanolamine (DOPE) present at a ratio of 1 : 1 (w/w), in 100 ⁇ L FCS-free DMEM (without supplements) for a further 30 minutes at room temperature before the mixture was added to cells.
  • DOTAP l(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride
  • DOPE dioleoyl phosphatidylathanolamine
  • Identical numbers of cells were seeded at 2 x 10 ⁇ cells per well in six-well plates and growth arrested at 80% confluence with medium containing 0.2% FCS for 2 days before transfection. On the day of transfection, cells were washed twice in serum-free DMEM and incubated for two hours with 800 ⁇ L DMEM without antibiotics (but supplemented with L-glutamine because we had previously demonstrated that it had no effect on transfection efficiency - data not shown). Cultures were transfected in triplicate with DNA, prepared as outlined above. After various periods of exposure, the medium was replaced with 2 mL of DMEM (with supplements, as outlined above) containing 10% FCS. Cells were cultured for 48 hours before assaying for luciferase activity.
  • Luciferase activity was detected by the manufacturers' standard protocol and luciferase assay kit (Promega, Southampton, UK): briefly, after transfection, cells were washed twice with PBS buffer, lysed with lx cell culture lysis reagent, transferred to a microcentrifuge tube and then centrifuged briefly at 12,000 g to pellet the cell debris. 20 ⁇ L of cell extract was mixed with 100 ⁇ L of luciferase assay buffer, containing luciferase substrate, allowing ATP-dependent oxidation of beetle luciferin, and then the mixture was placed in a luminometer.
  • the expression vector, pH ⁇ -actin, containing the anticoagulant gene, hirudin, linked to domains 3 and 4 of human CD4 sequence, (77) was also transfected into pig endothelial cells using the same methods as described above. The expression of hirudin at the surface of the cells was examined by flow cytometric analysis.
  • LDH-release cytotoxicity assay A Promega Cytotoxicity kit was used to quantity the activity of lactate dehydrogenase (LDH) in solution after cells had been exposed to the various components of the transfection system. Briefly, 48 hours after the transfection of primary porcine ECs with pGL3 control vector with or without DAPI and EscortTM, supernatants were collected to assess LDH released due to cell death. Intracellular LDH activity was also measured at each stage by using freeze-thawing to lyse the transfected cells. 50 ⁇ L of "substrate mix" was added to each well and incubated for 30 minutes at room temperature. Optical density values were recorded on a Titertek microplate reader at 492 nm after the addition of "stop solution”.
  • LDH lactate dehydrogenase
  • Genomic DNA was isolated by using a Nucleon extraction and purification kit (Amersham Life Science, UK). Briefly, after 48 hours, 10 7 transfected cells were collected following treatment with trypsin-EDTA (Gibco, UK) and washed with PBS by three times. Cells were lysed in cell lysis buffers provided in the kit, treated with 15 ⁇ L of RNase A solution (50 ⁇ g/mL) at 37°C for 30 minutes. DNA was extracted by adding 2 mL chloroform and 500 ⁇ L of Nucleon resin after deproteinisation with 300 ⁇ L of sodium perchlorate solution, precipitated by 1 mL of cold absolute ethanol and dissolved in Tris-EDTA buffer.
  • the isolated genomic DNA was quantified using a spectrophotometer (GeneQuant; Pharmacia Biotech, UK). 10 ⁇ g of DNA was digested with Xbal and Ncol (15 U/ ⁇ g D ⁇ A each) (Roche Diagnostics, Mannheim, Germany) at 37°C for 5 hours. The digests were loaded onto a 0.8% agarose gel (Gibco-BRL, Paisley, UK) and resolved by a gel electrophoresis (BIO-RAD, USA) at 0.5 V/cm for 10 hours. The DNA fragments were then transferred overnight at room temperature in the presence of 20x SSC to a positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) after being denatured by 1.5 M NaCl/0.5 M NaOH).
  • the DNA was cross-linked to the nylon membrane using a UV Stratalinker (UVItec, Cambridge, UK). Southern blotting and chemiluminescence detection of the luciferase gene were performed by using a DIG Southern Starter Kit (Roche Diagnostics GmbH, Mannheim, Germany). Simply, hybridisation was carried out at 42°C in a Techne- hybridiser HB-ID oven overnight with a 656 bp luciferase DNA sequence as a probe and washed as recommended by the manufacturer. The membrane was then incubated with dig-specific antibody covalently coupled to alkaline phosphatase.
  • the luciferase probe was developed by alkaline phosphatase metabolising cdp-star, a highly sensitive chemiluminescence substrate (provided with the kit) and visualized following exposure to hyperfilm ECL (Amersham Pharmacia, UK).
  • Isolated carotid arteries were cut into 0.5 cm segments and washed 3 times with PBS and then transfected using the same method used to transfect cultured cell lines, described in the previous section. The long-term culture of arteries has been described previously (18). The transfected arteries were collected, washed with PBS X 3, then cryosectioned and stained with anti- luciferase and CD31.
  • the transfectant solution (1 ⁇ g/mL DNA with or without 5 ⁇ l/mL EscortTM and 10 ⁇ g/mL DAPI gently mixed with serum- free DMEM, as described above) was directly instilled into the isolated vessel segment using a syringe pump at the speed of 50 mL/hour over 30 minutes.
  • a syringe pump at the speed of 50 mL/hour over 30 minutes.
  • Pigs were anaesthetised as above between 2 and 30 days following transfection. Vessels were identified, dissected out and removed. The animals were then sacrificed. Arteries were divided into three or four serial segments, each approximately 3mm in length and embedded in CRYO-M- BED (BDH, UK), an embedding compound, and frozen immediately in isopentane (BDH) using liquid nitrogen. For immunocytochemical staining, 5 ⁇ m cryosections were cut, airdried for one hour at room temperature, rinsed three times with PBS for five minutes each, fixed for 30 minutes in methanol at -20°C and stored dessicated at -20°C until use.
  • CRYO-M- BED CRYO-M- BED
  • BDH isopentane
  • Frozen sections were equilibrated to room temperature for 30 minutes before staining. They were rinsed briefly 3 times in PBS. 0.2% Triton X 100 (Sigma, MO, USA) was added to permeabilize the tissues for 30 minutes. Sections were drained thoroughly with moist filter paper. Tissues were blocked for one hour with 5% horse serum (Sigma, MO, USA) in PBS and again drained thoroughly. 50 ⁇ L of directly conjugated anti-luciferase- FITC or anti-luciferase-Texas Red antibody (1:50) was added and the slides were incubated in darkness overnight at 4°C. The slides were then washed three times in PBS and mounted in a 1 :1 mixture of PBS:glycerol (BDH, UK). Analyses were performed by fluorescent microscopy.
  • transfection efficiency Number of transfected cells counted in endothelium ⁇ total number of cells counted in endothelium x 100%.
  • Figure 9 illustrates expression of transgene in animals sacrificed 48 hours following transfection. In other experiments, we demonstrated expression in animals at several time points up to and including 21 days following transfection, although expression declined with time (data not shown). The possibility of the transfected cells being adherent CD31 + leucocytes was excluded by determining that the luciferase-expressing cells were negative for CD 14 and CD41 (data not shown). Discussion
  • Non-viral gene transfer is, in general, less immunogenic than viral gene transfer.
  • the non- viral vectors are easier to prepare.
  • Many approaches have been used to induce the uptake of DNA include the injection of naked DNA, the administration of DNA-coated gold particles, electroporation, and the administration of DNA delivered as a complex with cationic lipid or cationic polymers. Modifications of the latter group have been developed to allow the escape of DNA from the endosome. These modifications include the use of fusogenic lipids or peptides or techniques to buffer the pH drop in late endosomes.
  • organ-specific receptors such as angiotensin-converting enzyme in pulmonary vascular endothelium (3) or the use of cell type-specific promoters to limit the expression of transfected genes (5).
  • Approaches from both viral and non-viral approaches may be combined to generate what proves empirically to be the most effective and appropriate mechanism in a given situation.
  • FCS foetal calf serum

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Abstract

L'invention concerne un procédé destiné à augmenter l'efficacité de la transfection d'acides nucléiques d'une cellule. Ce procédé consiste à fixer un agent dans un sillon mineur de la molécule d'acide nucléique à transfecter. L'invention concerne également l'utilisation d'un agent se fixant dans un sillon mineur d'une molécule d'acide nucléique pour améliorer l'efficacité de la transfection d'acides nucléiques d'une cellule. L'invention concerne encore une composition pharmaceutique contenant une molécule d'acide nucléique comprenant un agent fixé dans un sillon mineur de ladite molécule, et un excipient pharmaceutiquement acceptable.
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WO2006066001A2 (fr) * 2004-12-17 2006-06-22 Nitto Denko Corporation Surface solide comprenant un polymere cationique degradable immobilise afin de transfecter des cellules eucaryotes
WO2020188302A1 (fr) * 2019-03-21 2020-09-24 The University Of Nottingham Transfection améliorée
WO2021118927A1 (fr) * 2019-12-13 2021-06-17 Insideoutbio, Inc. Méthodes et compositions pour l'administration ciblée d'agents thérapeutiques par l'acide nucléique

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006066001A2 (fr) * 2004-12-17 2006-06-22 Nitto Denko Corporation Surface solide comprenant un polymere cationique degradable immobilise afin de transfecter des cellules eucaryotes
WO2006066001A3 (fr) * 2004-12-17 2006-09-08 Nitto Denko Corp Surface solide comprenant un polymere cationique degradable immobilise afin de transfecter des cellules eucaryotes
WO2020188302A1 (fr) * 2019-03-21 2020-09-24 The University Of Nottingham Transfection améliorée
CN114072182A (zh) * 2019-03-21 2022-02-18 诺丁汉大学 增强转染
WO2021118927A1 (fr) * 2019-12-13 2021-06-17 Insideoutbio, Inc. Méthodes et compositions pour l'administration ciblée d'agents thérapeutiques par l'acide nucléique

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