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
  • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
• • 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
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/15—Nucleic acids forming more than 2 strands, e.g. TFOs

Definitions

• nucleic acid transfer is a technique underlying all major biotechnology applications and increasing the efficiency of nucleic acid transfer is a very important issue for the development of these applications.
• the efficiency of nucleic acid transfer depends on many factors including the ability of nucleic acids to reach the target cell, their ability to cross the plasma membrane and their ability to be transported within the cell to the nucleus.
• nucleic acid transfer stems from the fact that genetic information is often little or not directed at the target organ for which it is intended. Furthermore, once the nucleic acid has entered the target cell, it must still be directed to the nucleus in order to be expressed there. In addition, in the case of transfer of nucleic acids into differentiated or quiescent cells, the nucleus is limited by a nuclear envelope which constitutes an additional barrier to the passage of these nucleic acids.
• Recombinant viruses used as vectors have advanced and effective mechanisms to guide nucleic acids to the nucleus.
• viral vectors have certain drawbacks inherent in their viral nature, which unfortunately cannot be totally excluded.
• Another strategy therefore consists either in transfecting naked DNA, or in using non-viral agents capable of promoting the transfer of DNA into eukaryotic cells.
• non-viral vectors do not have sub-cellular or nuclear targeting signals.
• the passage of naked DNA, or in combination with a non-viral agent, from the cytoplasm to the nucleus is for example a step which has a very low efficiency (Zabner et al., 1995).
• Transfection vectors comprising a synthetic polypeptide coupled by electrostatic interactions to a DNA sequence have also been described in patent application WO 95/31557, said polypeptide consisting of a polymer chain of basic amino acids, an NLS peptide, and a hinge region which connects the NLS peptide to the polymer chain and makes it possible to avoid steric interactions.
• this type of construction poses a stability problem because the interactions involved between DNA and the targeting signal are of an electrostatic nature.
• Such a vector has the advantage of being able to direct double-stranded DNA to specific cells or cellular compartments by virtue of the targeting signal, without genetic expression being inhibited.
• the Applicant has indeed shown that, thanks to the formation of stable site-specific triple helices, it is now possible to link a targeting signal to a double stranded DNA in a site-specific manner. Consequently, it is possible to fix the targeting signal outside the expression cassette for the gene to be transferred.
• the Applicant has thus shown that genetic expression in the cell is not inhibited despite the chemical modification of DNA.
• the presence of a triple helix as a means for binding the targeting signal to the DNA is particularly advantageous since it makes it possible to maintain a DNA size suitable for transfection.
• the vector obtained also has the advantage of incorporating targeting signals which are very stably linked to the double stranded DNA, in particular when the oligonucleotide capable of forming the triple helix is modified by the presence of a alkylating agent.
• Another advantage of the invention is that it makes it possible to couple the DNA to be transferred to targeting signals the number and nature of which are both controlled. Indeed, it is possible to control the number of targeting signals linked to each double-stranded DNA molecule by introducing an adapted number of specific sequences conducive to the formation of triple helices on said double-stranded DNA molecule. Of the Likewise, it is possible to introduce on the same double-stranded DNA molecule several oligonucleotides linked to different targeting signals (intracellular and / or extracellular), and in this case it is also possible to determine the proportions beforehand. respective. In addition, these different targeting signals can be fixed to the double-stranded DNA molecules in a more or less stable manner depending on whether the triple helix is formed with or without covalent bond (i.e. with or without the use of an alkylating agent).
• a first object of the invention therefore relates to a vector useful in transfection capable of targeting a specific cell and / or cell compartment. More particularly, the vector according to the invention comprises a double-stranded DNA molecule and at least one oligonucleotide coupled to a targeting signal and capable of forming, by hybridization, a triple helix with a specific sequence present on said double-stranded DNA molecule .
• This double stranded DNA can be in linear or circular form.
• the double-stranded DNA may be in a supercoiled or relaxed state.
• the DNA molecule is circular in shape and in a supercoiled conformation.
• Double stranded DNA can also carry an origin of functional or non-functional replication in the target cell, one or more marker genes, transcription or replication regulatory sequences, genes of interest therapeutic, antisense sequences modified or not, regions of binding to other cellular components, etc.
• the double-stranded DNA comprises an expression cassette consisting of one or more genes of interest under the control of one or more promoters and a transcriptional terminator active in the target cells.
• the expression “gene expression cassette” means a DNA fragment which can be inserted into a vector at specific restriction sites.
• the DNA fragment comprises a nucleic acid sequence coding for an RNA or a polypeptide of interest and further comprises the sequences necessary for expression (enhancer (s), promoter (s), polyadenylation sequences, etc.). ) of said sequence.
• the cassette and restriction sites are designed to ensure insertion of the expression cassette into a reading frame suitable for transcription and translation.
• the term “gene of therapeutic interest” in particular means any gene coding for a protein product having a therapeutic effect.
• the therapeutic product thus coded can in particular be a protein or a peptide.
• This protein product can be homologous with respect to the target cell (that is to say a product which is normally expressed in the target cell when the latter presents no pathology).
• the expression of a protein makes it possible for example to compensate for an insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or else to overexpress said protein.
• the gene of therapeutic interest can also code for a mutant of a cellular protein, having increased stability, modified activity, etc.
• the protein product can also be heterologous with respect to the target cell.
• an expressed protein can for example supplement or provide a deficient activity in the cell, allowing it to fight a pathology, or stimulate an immune response.
• therapeutic products within the meaning of the present invention, there may be mentioned more particularly enzymes, blood derivatives, hormones, lymphokines [interleukins, interferons, TNF, etc. (FR 92/03120)], growth factors, neurotransmitters or their synthetic precursors or enzymes, trophic factors [BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc., dystrophin or a minidystrophin (FR 91/1 1947)] , the CFTR protein associated with cystic fibrosis, tumor suppressor genes [p53, Rb, RaplA, DCC, k-rev, etc.
• trophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc., dystrophin or a minidystrophin (FR 91
• the DNA of therapeutic interest can also be an antisense gene or sequence, the expression of which in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNA.
• Such sequences can, for example, be transcribed in the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308.
• the genes of therapeutic interest also include the sequences encoding ribozymes, which are capable of selectively destroying Target RNAs (EP 321 201), or the sequences encoding single chain intracellular antibodies such as for example the ScFvs.
• deoxyribonucleic acid may also contain one or more genes coding for an antigenic peptide, capable of generating an immune response in humans or animals.
• the invention therefore makes it possible to produce either vaccines or immunotherapeutic treatments applied to humans or animals, in particular against microorganisms, viruses or cancers.
• These may in particular be antigenic peptides specific for the Epstein Barr virus, the HIV virus, the hepatitis B virus (EP 185 573), the pseudo-rabies virus, the "syncitia forming virus", influenza virus, cytomegalovirus (CMV), other viruses or tumor-specific (EP 259 212).
• promoters of the El A, MLP, CMV, RSV, etc. genes can be modified by adding activation, regulation sequences, etc. It can also be a promoter, inducible or repressible.
• a triple helix corresponds to the binding of an oligonucleotide modified or not on double stranded DNA by hydrogen bonds called "Hoogsteen" between the bases of the third strand and those of the double helix region. These pairings occur in the large groove of the double helix and are specific to the sequence considered [Frank-Kamenetski, MD, Triplex DNA Structures, Ann. Rev. Biochem., 1995, 64, pp. 65-95].
• the specific double helix sequence may in particular be a hornopuric-homopyrimide sequence.
• triple helices with a third homopyrimide strand this is parallel with respect to the purine strand, and the formation of the triple helix is dependent on the pH: an acid pH lower than six allows the protonation of the cytosines and the formation of a additional hydrogen bond stabilizing the CG * C + triplet.
• the oligonucleotides used in the present invention are oligonucleotides which hybridize directly with double stranded DNA. These oligonucleotides can contain the following bases: - thymidine (T), which is capable of forming triplets with the A.T doublets of double stranded DNA (Rajagopal et al, Biochem 28 (1989) 7859),
• A - adenine
• oligonucleotide and the specific sequence present on the DNA are complementary.
• an oligonucleotide and a specific sequence which are perfectly complementary are used for the vector according to the invention. It may in particular be a poly-CTT oligonucleotide and a specific poly-GAA sequence.
• This sequence forms a triple helix with the oligonucleotides: 5'-AAGGAGAGGAGGGAGGGAA-3 '(SEQ LD N ° 4) or 5'-TTGGTGTGGTGGGTGGGTT-3' (SEQ LD N ° 5).
• the oligonucleotide binds in an antiparallel orientation to the polypuric strand.
• These triple helices are only stable in the presence of Mg2 + as has been specified previously (Vasquez et al., Biochemistry, 1995, 34, 7243-7251; Beal and Dervan, Science, 1991, 251, 1360-1363).
• the specific sequence may be a sequence naturally occurring on double-stranded DNA, or a synthetic or naturally occurring sequence artificially introduced therein. It is particularly advantageous to use an oligonucleotide capable of forming a triple helix with a sequence present 10
• the vectors according to the invention with unmodified plasmids, in particular commercial plasmids of the pUC, pBR322, pSV type, etc.
• plasmids in particular commercial plasmids of the pUC, pBR322, pSV type, etc.
• the natural homopuric-homopyrimide sequences present on the DNA double strand there may be mentioned a sequence comprising all or part of the sequence 5'-CTTCCCGAAGGGAGAAAGG-3 '(SEQ LD No. 6) present in the origin of replication ColEl of E. coli.
• the oligonucleotide forming the triple helix has the sequence: 5'-GAAGGGTTCTTCCCTCTTTCC-3 '(SEQ LD No.
• the oligonucleotide used can be natural (composed of natural bases, unmodified) or chemically modified.
• the oligonucleotide can advantageously exhibit certain chemical modifications making it possible to increase its resistance, its protection with respect to nucleases, its affinity with respect to the specific sequence or still making it possible to bring other properties. Additional (J. Goodchild, Conjugates of Oligonucleotides and Modified Oligonucleotides: A 11
• oligonucleotide is also understood to mean any chain of nucleosides having undergone a modification of the skeleton.
• oligonucleotides which are capable of forming triple helices with DNA (Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), as well as oligonucleotides having formacetal or methylphosphonate skeletons (Matteucci et al., J. Am. Chem. Soc, 1991, 113. 7767-7768).
• oligonucleotides synthesized with nucleotide anomers which also form triple helices with DNA (Le Doan et al., Nucleic Acids Res., 1987, L5, 7749-7760).
• Another modification of the skeleton is the phosphoramidate bond.
• ribonucleotides 2'-0-methylribose, phosphotriester, ... (Sun and Hélène, Curr. Opinion Struct. Biol., 1 16. 3143-3144 ).
• the phosphorus backbone can finally be replaced by a polyamide backbone as in the PNA (Peptide Nucleic Acid), which can also form triple helices (Nielsen et al., Science, 1991, 254, 1497-1500; Kim et al., J . Am. Chem. Soc, 1993, 115.
• the length of the oligonucleotide used in the process of the invention is at least 3 bases, and preferably between 5 and 30 bases.
• An oligonucleotide with a length of between 10 and 30 bases is advantageously used.
• the length can of course be adapted on a case-by-case basis by a person skilled in the art as a function of the selectivity and the stability of the interaction sought.
• oligonucleotides according to the invention can be synthesized by any known technique. In particular, they can be prepared using nucleic acid synthesizers. Any other method known to those skilled in the art can also be used. 13
• targeting signal is understood to mean targeting molecules of various kinds. In most cases these are known peptides for targeting. They can be used to interact with a component of the extracellular matrix, a receptor of the plasma membrane, to target an intracellular compartment or to improve the intracellular trafficking of DNA, during the non-viral transfer of genes in gene therapy.
• targeting signals can include, for example, growth factors (EGF, PDGF, TGFb, NGF, IGF I, FGF), cytokines (LL-1, LL-2, TNF, Interferon, CSF), hormones (insulin, growth hormone, prolactin, glucagon, thyroid hormone, steroid hormones), sugars that recognize lectins, immunoglobulins, ScFv, transferrin, lipoproteins, vitamins such as vitamin B12, peptide hormones or neuropeptides (tachykinins , neurotensin, VIP, endothelin, CGRP, CCK, ...), or any motif recognized by integrins, for example the RGD peptide, or by other proteins extrinsic from the cell membrane.
• growth factors EGF, PDGF, TGFb, NGF, IGF I, FGF
• cytokines LL-1, LL-2, TNF, Interferon, CSF
• hormones insulin, growth hormone, prolactin,
• Whole proteins can be used, or peptide sequences derived from these proteins, or peptides which bind to their receptor and are obtained by the "phage display” technique or by combinatorial synthesis.
• karyopherins for example.
• the role of these sequences is to direct DNA inside the nucleus, where it is then immediately available to the transcription machinery, and can be expressed.
• “Mixed” targeting signals that is to say signals which can be used for both intracellular and extracellular targeting, also fall within the scope of the present invention. Mention may for example be made of sugars which target lectins which are located on the cell membrane but also at the level of the nuclear pores. Targeting with these sugars therefore concerns both extracellular targeting and nuclear import.
• oligonucleotide modified by a terminal thiol, amino or carboxyl group in the 3 ′ or 5 ′ position
• These couplings are formed by establishment of disulfide, thioether, ester, amide, or amine bonds between the oligonucleotide and the targeting signal
• Any other known method skilled in the art can be used, such as bifunctional coupling reagents for example
• compositions comprising a vector as defined above
• cationic lipids are in particular monocationic lipids (DOTMA Lipofectin®), certain cationic detergents (DDAB), lipopolyamines and in particular dioctadecylamidoglycyl spermine (DOGS) or 5-carboxyspermylamide of palmitoylphosphatidylethanolamine (DPPES)
• DOTMA Lipofectin® monocationic lipids
• DDAB certain cationic detergents
• DOGS dioctadecylamidoglycyl spermine
• DPES 5-carboxyspermylamide of palmitoylphosphatidylethanolamine
• Another advantageous family of lipopolyamines is represented by the compounds described in patent application WO 97/18185 incorporated herein by reference, for example in patent application EP 394 1 1 1.
• Many other cationic lipids have been developed and can be used with the vectors according to the invention
• cationic polymers of the polylysine and DEAE dextran type are also advantageous. It is also possible to use the polymers of polyethylene imine (PEI) and of polypropylene imine (PPI) which are commercially accessible and can be prepared according to the process described in patent application WO 96/02655.
• PEI polyethylene imine
• PPI polypropylene imine
• any synthetic agent known to transfect the nucleic acid can be associated with the vectors according to the invention 16
• the neutral lipids used in the context of the present invention are lipids with 2 fatty chains.
• natural or synthetic lipids are used, z itterionic or devoid of ionic charge under physiological conditions. They can be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE), oleoyl-palmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoyl phosphatidylethanolamines as well as their N-methylated derivatives 1 to 3 times, phospol glycols , glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as in particular sphingo yelins) or alternatively asialogangliosides (such as in particular asialoGMl and GM2).
• DOPE dioleoylphosphatidylethanolamine
• the vectors of the invention may, by way of illustration, be used for the in vitro, ex vivo or in vivo transfection of DNA coding for proteins or polypeptides.
• compositions according to the invention can be formulated for administration by topical, cutaneous, oral route, rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc.
• the compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, especially for a direct injection at the level of the desired organ, or for topical administration (on the skin and / or mucosa).
• Figure 3 schematic representation of the plasmid pXL2813. 20
• Figure 4 schematic representation of the plasmid pXL2652.
• Figure 5 Analysis on 15% polyacrylamide gel of the formation of triple helices between the plasmid pXL2813 and the oligonucleotide-peptide chimera Pso-GA ⁇ 9 -NLS.
• Figure 6 In vitro expression of the transgene ( ⁇ -galactosidase) for tests carried out with the plasmid pXL2813 alone, the vector pXL2813-Pso-GA ⁇ 9 -NLS, and without plasmid.
• Figure 7 Characterization of the peptide part of the Pso-GAi9-NLS oligonucleotide-peptide chimera by interaction with importine 60-GST (analysis on 15% polyacrylamide gel).
• Figure 8 Analysis on 15% polyacrylamide gel of the oligonucleotide-peptide chimera (GA19-NLS) by proteolytic action of trypsin.
• Figure 9 Graphical representation of the kinetics of formation of triple helices (% of triple helix sites occupied as a function of time) between the plasmid pXL2813 and the chimera GA ⁇ 9 -NLS.
• Figure 10 Characterization of the peptide part of the oligonucleotide-peptide GA 19 -NLS chimera by interaction with importine 60-GST.
• Figure 1 1 schematic representation of the plasmid pXL2997.
• Figure 12 Graphic representation of the kinetics of triple helix formation (% of triple helix sites occupied as a function of time) between the plasmid pXL2997 and the chimera pim-NLS.
• Figure 13 histogram representing the ⁇ -galactosidase activity in vivo in human lung tumors H 1299 of the plasmids pXL2813 (indicated Bgal in the figure) and ⁇ XL2813-Pso-GA ⁇ 9 -NLS (indicated NLS-Bgal in the figure) in RLU ("Relative Light Unit") per tumor.
• the transfection was carried out using electrotransfer techniques as described in applications WO99 / 01157 and WO 99/01 158.
• the oligonucleotides used are either the sequence 5'- AAGGAGAGGAGGGAGGGAA-3 '(SEQ LD N ° 4) with a length of 19 bases and referred to as "GA ⁇ 9 " in the rest of the text, or sequence 5 '- GGGGAGGGGGAGG-3' (SEQ LD N ° 15) with a length of 13 bases and referenced under the name "pim" in the rest of the text (because it is the sequence of the protooncogene pim-1).
• oligonucleotides noted GA1 9 -SH or pim-SH have the same sequences as GA19 and pim respectively and a thiol group at the 5 'end, with a six-carbon spacer between the thiol and the phosphate at the 5' end. Oligonucleotides noted 23
• Pso-GA ⁇ 9 -SH have a thiol group at the 3 'end (SH), and in addition, a psoralen at the 5' end (Pso), with a spacer of six carbons between the psoralen and the phosphate of l 'end 5'.
• the oligonucleotides noted Pso-GA ⁇ 9 do not have a thiol group.
• Peptides The peptides used for the couplings are synthesized by a solid phase automaton. They contain :
• N-terminal lysine is chemically modified: it contains a maleimide group on the ⁇ carbon and a protective group 9- 24
• the oligonucleotide-peptide chimera is purified by reverse-phase high pressure liquid chromatography on a Vydac C8 column containing spheroidal silica with a diameter of 5 ⁇ m and a porosity of 300 ⁇ . Using a 0.1 M triethylammonium acetate buffer (TEAA) and a gradient of acetonitrile passing from 5% to 50% in 35 minutes. The products are detected at 260 nm. 25
• TEAA triethylammonium acetate buffer
• the plasmid used to study the formation of triple helices with the chimeras GA ⁇ 9 - peptide is called pXL2813 (7257 bp, see FIG. 3).
• This plasmid expresses the ⁇ -galactosidase gene under the control of the strong promoter of the early genes of Cytomegalovirus (CMV), as well as the gene for resistance to Ampicillin.
• CMV Cytomegalovirus
• pXL2652 (7391 bp and the representation of which is shown diagrammatically in FIG. 4) which expresses the gene for ⁇ -Galactosidase under the control of the strong promoter of the early genes of Cytomegalovirus (CMV), as well as the gene resistance to Ampicillin.
• CMV Cytomegalovirus
• This promoter comes from pCDNA3, the LacZ gene and its polyA come from pCHl 10, and the rest comes from pGL2.
• sequences were cloned upstream of the promoter between the unique cleavage sites of the Muni and Xmal enzymes.
• sequences were cloned upstream of the promoter between the unique cleavage sites of the Muni and Xmal enzymes.
• the two complementary oligonucleotides containing the sequence to be cloned 6651 (5'- AATTGATTCCTCTCCTCCCTCCCTTAC-3 ') and 6652 (3'- 26
• CTAAGGAGAGGAGGGAGGGAATGGG-5 ' were heated for 5 minutes at 95 ° C, then hybridized, allowing the temperature to drop slowly.
• the plasmid pXL2652 was then digested with the enzymes Muni and Xmal, for 2 hours at 37 ° C., and the products of this double digestion were separated by electrophoresis on 1% agarose gel and staining with ethidium bromide.
• the fragment of interest for the following cloning was eluted according to the Jetsorb protocol (Genomed) and 200 ng of this fragment were linked to 10 ng of the mixture of oligonucleotides hybridized by T4 ligase, for 16 hours at 16 ° C. .
• E.Coli competent bacteria of the DH5 ⁇ strain were transformed by electroporation with the reaction product, spread on Petri dishes containing LB medium and Ampicillin. The clones resistant to Ampicillin were selected and the DNA was extracted by alkaline lysis and analyzed on 1% agarose gel. A clone corresponding, in size, to the expected product was sequenced.
• This method is based on the principle of exclusion chromatography of solutions containing the plasmid and the oligonucleotide capable of forming a triple helix.
• the exclusion columns used (Columns, Linkers 6, Boehringer Mannheim) are composed of sepharose beads and have 194 base pairs as exclusion limit, which makes it possible to retain in the column the oligonucleotides not paired with the plasmids.
• the oligonucleotides are radioactively labeled at their 3 ′ end by the Terminal Transferase using dATP ⁇ - ⁇ S.
• the protocol used comes from Amersham: 10 pmol of oligonucleotides are incubated for 2 hours at 37 ° C, with 50 ⁇ Ci of dATP ⁇ ⁇ S in the presence of 10 units of Terminal Transferase, in a volume of 50 ⁇ l of buffer containing cocodylate sodium.
• the percentage of labeled oligonucleotides is evaluated according to the following method: l ⁇ l of a sample diluted to 1/100 of the solution after labeling is deposited on Whatman DE81 paper, in duplicate. One of the two papers is washed twice for 5 minutes with 2xSSC, for 30 seconds with water and for 2 minutes with ethanol. The radioactivities of the two papers are compared. The radioactivity of the washed paper corresponds to the 35s actually incorporated.
• triple helices takes place in a volume of 35 ⁇ l.
• the sepharose columns are balanced, before use, with the reaction buffer and centrifuged at 2200 rpm for 4 minutes, to compact them. 25 ⁇ l of the reaction medium are deposited on the columns and these are centrifuged under the same conditions as above. 25 ⁇ l of 28
• reaction buffer are then deposited on the columns which are again centrifuged. The eluate is recovered.
• cpm deposit
• cpm eluate
• % of oligonucleotides elected cpm (eluate) / [5 x cpm (deposit)] xl00.
• the percentage of plasmids which are effectively eluted during the experiments is evaluated by estimating the optical density at 260 nm of the eluate and that of the deposit, which makes it possible to calculate the percentage of oligonucleotides attached to all of the plasmids:
• % of fixed oligonucleotides [% eluted oligonucleotides /% eluted plasmids x 100.
• the importin 60 subunit used to study the interaction with the oligonucleotide-peptide conjugates is of murine origin and fused with Glutathione S-Transferase (GST).
• GST Glutathione S-Transferase
• the sequence of importin 60 was cloned into a vector pGEX-2T to merge it with GST.
• the recombinant protein was produced in Escherichia coli [Imamoto, N. et al., In vivo evidence for involvement ofa 58kDa component of nuclearpore-targeting complex in nuclear protein import, The EMBO Journal, 1995, 14 (15), pp. 3617-3626].
• the recombinant proteins are incubated in the presence of sepharose beads covered with glutathione groups (Pharmacia Biotech) 1 ⁇ g of recombinant protein is used for 10 ⁇ l of beads. After an incubation of 30 minutes, at room temperature, in 500 ⁇ l of fixing buffer, the mixture is centrifuged at 2000 G for 30 seconds, and the supernatant is removed. The beads are washed five times by resuspension in 500 ⁇ l of fixing buffer and centrifugation, as described above. The beads are resuspended in fixing buffer to obtain a suspension containing 50% of beads coated with recombinant proteins.
• oligonucleotide or oligonucleotide-peptide 60 ⁇ l of the suspension containing 50% of beads covered with recombinant proteins are incubated with 2 ⁇ g of oligonucleotide or oligonucleotide-peptide, in a volume of 500 ⁇ l of fixing buffer. After an incubation of 30 minutes, at room temperature, the mixture is centrifuged at 2000 G for 30 seconds, and the supernatant is removed. 30 ⁇ l of the supernatant are collected to analyze the fraction which is not fixed on the beads. The beads are washed five times by resuspension in 500 ⁇ l of fixing buffer and centrifugation, as described above.
• the nucleic acids are revealed by silver staining using a Biorad kit [Rexach, M. and G. Blobel, Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins, Cell, 1995, 83, pp. 683-692].
• the cell type used is NLH3T3 (ATCC CRL-1658). They are mouse fibroblasts. These cells are cultured in a modified Dulbecco medium, with glucose 4.5 g / 1 (DMEM - Gibco), glutamine 2 mM, penicillin (100 U / ml) and streptomycin (100 ⁇ g / ml) , and 10% fetal calf serum (Gibco). They are incubated at 37 ° C in an oven with 5% C0 2 .
• the wells of a 24-well plate are seeded with 50,000 cells per well.
• the vectors are diluted in 150 mM NaCl and mixed with a cationic lipid (the compound of condensed formula H2N (CH2) 3NH (CH2) 4NH (CH2) 3 NHCH 2 COGlyN [(CH 2 ) i7CH3] 2 described in the patent application WO 97/18185 under number (6)), diluted in 150 mM NaCl.
• the mixture is made with 6 nmol of lipid per microgram of plasmid. This mixture is diluted 1/10 in culture medium without serum and deposited on the cells. After incubation at 37 ° C in an oven at 5% CO 2 for two hours, 10% fetal calf serum is added.
• the cells are washed twice with PBS and lysed with 250 ⁇ l of lysis buffer (Promega).
• the ⁇ -galactosidase is quantified according to the “Lumigal ⁇ -Galactosidase genetic reporter system” protocol (Clontech).
• the activity is measured on a Lumat LB9501 luminometer (Berthold).
• the amount of protein is measured with the BCA kit (Pierce).
• This example illustrates the possibility of coupling the oligonucleotide Pso-GA ⁇ -SH to the maleimide-NLS peptide.
• the oligonucleotide Pso-GA ⁇ 9 -SH of sequence 5'-AAGGAGAGGAGGGAGGGAA-3 ', with a thiol group at the 3' end, was coupled to the NLS peptide which carries a maleimide group at its N-terminal end according to the described method 31
• HPLC high pressure liquid chromatography
• the oligonucleotide-peptide chimera (Pso-GA ⁇ 9 -NLS) was analyzed by electrophoresis in polyacrylamide gel after proteolytic action of trypsin which makes it possible to demonstrate the presence of the peptide part of the chimera, after migration on polyacrylamide gel denaturing and staining of nucleic acids with silver (as indicated in the section “Materials and methods” under the section “Analysis of chimeras by trypsin digestion”).
• the chimera Pso-GA ⁇ 9 -NLS exhibits a delayed electrophoretic migration compared to the oligonucleotide Pso-GA ! 9 -SH, and the product of the proteolytic digestion is visualized at an intermediate level between the migration levels of Pso-GA ⁇ - NLS and Pso-GA ⁇ 9 -SH, as shown in Figure 2.
• the chimera Pso-GAi 9 -NLS therefore contains a peptide part accessible to trypsin.
• This example illustrates the formation of triple helices between the plasmid pXL2813 and the chimera Pso-GA ⁇ 9 -NLS which is modified by a photoactivatable alkylating agent.
• This example also indicates what is the proportion of plasmids modified according to the excess in molarity of oligonucleotides relative to the plasmid.
• the plasmid pXL2813 represented in FIG. 3, contains the complementary homopuric sequence of GA ⁇ 9 which is capable of forming a triple helix with the oligonucleotides GA ⁇ 9 , Pso-GA ⁇ 9 , Pso-GA ⁇ 9 -SH or Pso-GA] 9 - NLS. Divalent cations, such as Mg 2+ , stabilize these triple helices.
• oligonucleotide and the plasmid are mixed in a buffer containing 100 mM MgCl 2 .
• the excess in molarity of oligonucleotide relative to the plasmid varies from 0 to 200.
• the expression of the transgene increases following the modification made by the covalent attachment of a triple helix upstream of the promoter.
• the purpose of this example is to verify that expression in vivo is not inhibited by the presence of a targeting signal associated with the plasmid via a triple helix.
• the plasmid pXL2813-Pso-GA 19 -NLS expresses the transgene in vivo at a level greater than or equal to that obtained with the plasmid pXL2813 unmodified (see FIG. 13). 35
• This example illustrates the possibility of coupling the oligonucleotide GA19-SH to the maleimide-NLS peptide.
• the chimera is not modified by a photoactivatable alkylating agent.
• the oligonucleotide GA 19 -SH of sequence 5'-AAGGAGAGGAGGGAGGGAA-3 '(SEQ LD No. 4), with a thiol group at the 5' end, was coupled to the maleimide-NLS peptide which has a maleimide group with its N-terminal end under the same conditions as for the oligonucleotide Pso-GA ⁇ 9 -SH (see example 1).
• HPLC high pressure liquid chromatography
• This example illustrates the possibility of forming triple helices between the plasmid pXL2813 and the chimera GA i9 -NLS in the absence of an alkylating agent.
• FIG. 9 represents the kinetics of formation of the triple helices.
• This example illustrates the characterization of the peptide part of the chimera GA19-NLS.
• the peptide sequence used, the NLS signal of the SV40 T antigen, is recognized by receptors of the ⁇ karyopherin family, as already mentioned in example 4.
• the raurin equivalent, called importin 60, fused to a glutathione S-transferase group, was used to characterize the oligonucleotide-peptide conjugates. It was operated as described in "Materials and Methods" under the section "Interactions with importines".
• the pellet of beads (containing the elements interacting with the importins) is separated from the supernatant.
• Example 9 This example illustrates the possibility of coupling the oligonucleotide 'pim-SH to the maleimide-NLS peptide.
• the oligonucleotide pim-SH of sequence 5'-GGGGAGGGGGAGAG-3 '(SEQ LD No. 15), with a thiol group at the 5' end, was coupled to the maleimide-NLS peptide which has a maleimide group at its N-terminal end under the same conditions as for the oligonucleotide GA 19 -SH (see example 5).
• Example 10 This example illustrates the possibility of forming triple helices between the plasmid pXL2997 and the chimera pim-NLS in the absence of an alkylating agent.
• FIG. 12 represents the kinetics of formation of the triple helices.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Biotechnology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biomedical Technology (AREA)
• General Health & Medical Sciences (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Biophysics (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• Animal Behavior & Ethology (AREA)
• Epidemiology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (5)

Publications (1)

Family

ID=26234216

Family Applications (1)

Country Status (12)

Families Citing this family (6)

Family Cites Families (3)

• 1999
  • 1999-03-19 IL IL13862499A patent/IL138624A0/xx unknown
  • 1999-03-19 JP JP2000538027A patent/JP2002507429A/ja not_active Withdrawn
  • 1999-03-19 CA CA002323831A patent/CA2323831A1/fr not_active Abandoned
  • 1999-03-19 CN CN998039314A patent/CN1292827A/zh active Pending
  • 1999-03-19 BR BR9909044-9A patent/BR9909044A/pt not_active IP Right Cessation
  • 1999-03-19 HU HU0102413A patent/HUP0102413A3/hu unknown
  • 1999-03-19 EP EP99910417A patent/EP1066396A1/fr not_active Withdrawn
  • 1999-03-19 KR KR1020007010485A patent/KR20010074456A/ko not_active Application Discontinuation
  • 1999-03-19 WO PCT/FR1999/000643 patent/WO1999049067A1/fr not_active Application Discontinuation
  • 1999-03-19 PL PL99342944A patent/PL342944A1/xx not_active Application Discontinuation
• 2000
  • 2000-05-12 AU AU34062/00A patent/AU758406B2/en not_active Ceased
  • 2000-09-18 NO NO20004648A patent/NO20004648L/no not_active Application Discontinuation

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events