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WO2011156831A1 - Formation d'acides nucléiques à l'intérieur d'une capsule polymère - Google Patents

Formation d'acides nucléiques à l'intérieur d'une capsule polymère Download PDF

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Publication number
WO2011156831A1
WO2011156831A1 PCT/AU2010/000756 AU2010000756W WO2011156831A1 WO 2011156831 A1 WO2011156831 A1 WO 2011156831A1 AU 2010000756 W AU2010000756 W AU 2010000756W WO 2011156831 A1 WO2011156831 A1 WO 2011156831A1
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Prior art keywords
capsule
polymeric
capsules
nucleic acid
rna
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Kim Wark
Frank Caruso
Andrew Price
Alexander N. Zelikin
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Commonwealth Scientific and Industrial Research Organization CSIRO
University of Melbourne
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Commonwealth Scientific and Industrial Research Organization CSIRO
University of Melbourne
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • C12N2310/12—Type of nucleic acid catalytic nucleic acids, e.g. ribozymes
    • C12N2310/127—DNAzymes
    • 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
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • 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
    • C12N2330/00—Production
    • C12N2330/30—Production chemically synthesised
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell

Definitions

  • the present invention relates to a method for producing a nucleic acid molecule within a polymeric capsule where the nucleic acid molecule is synthesised de novo.
  • the invention further relates to polymeric capsules produced by the method and to the use of these capsules to deliver the nucleic acid to a cell.
  • the present inventors have used micrometer-sized, polymeric capsules in encapsulated de novo RNA synthesis and subsequent cellular internalization of the capsules carrying the RNA.
  • the capsules act as both microreactors and drug carriers.
  • polymeric capsules also allow real-time monitoring of RNA synthesis and, therefore, precise control over the
  • the method developed by the present invention also provides for de novo synthesis of DNA within capsules, enabling DNA directed RNA silencing.
  • enzyme-catalyzed synthesis of the nucleic acid within the confines of a drug delivery vehicle has the potential to i) minimize the handling of RNA, bypassing isolation and purification steps, ii) protect the RNA until it reaches its site of action and iii) increase the dosage of therapeutic molecule administered.
  • the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule, mixing the capsule with a polymerase and nucleotides, wherein the polymerase and the nucleotides can diffuse into the capsule, to form a reactant mixture, incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.
  • the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule and a polymerase, mixing the capsule with nucleotides which can diffuse into the capsule and optionally a polymerase which can diffuse into the capsule, to form a reactant mixture, and incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.
  • the present invention provides a polymeric capsule comprising a nucleic acid molecule wherein the capsule is produced according to the method of the first or second aspect of the present invention.
  • the present invention provides a method of delivering a nucleic acid to a cell comprising contacting the cell with a polymeric capsule of the third aspect of the present invention.
  • Double-stranded DNA (dsDNA) amplicons are electrostatically absorbed onto positively charged silica colloids (SiO+) at subsaturation coverage followed by electrostatic adsorption of thiol-modified polymethacrylic acid (PMASH).
  • dsDNA Double-stranded DNA
  • PMASH thiol-modified polymethacrylic acid
  • pH 4.0 hydrogen bonding poly-N-vinylpyrrolidone (PVPON) and PMASH are layered onto the colloids via the layer-by-layer (LbL) technique. After layering, PMASH is crosslinked at the free thiols and the silica cores are dissolved with buffered hydrofluoric acid
  • RNA polymerase T7Pol
  • FIG. 1 Internalization of RNA-loaded 1.35-pm diameter PMA polymeric capsules by mammalian cells as imaged by CLSM.
  • the cell membranes are stained red and the RNA encapsulated in PMA polymeric capsules is labeled with a green fluorophore.
  • the cell lines are (a) Chinese hamster ovary (CHO) cells and (b) Ramos B lymphocytes.
  • the present inventors have succeeded in synthesising RNA and DNA within polymeric capsules.
  • the method results in the production of polymeric capsules which include multiple copies of RNA or DNA molecules.
  • the polymeric capsules can then be used to deliver their nucleic acid "payload" to cells.
  • RNA within the confines of a polymeric capsule represents a robust technique for the controlled capsule synthesis of RNA, en route to a drug-delivery platform for RNA therapeutics.
  • the protection which the capsule provides to the synthesised RNA is of particular value given the susceptibility of RNA to
  • RNA polymerase poly(methacrylic acid)
  • DNA has been encapsulated into poly(methacrylic acid) (PMA) polymeric capsules through LbL assembly (Zelikin et al, ACS Nano, 2007, 1 , 63).
  • PMA poly(methacrylic acid)
  • the PMA also referred to as PMASH
  • PMASH polymeric capsules entrap DNA through a combination of size exclusion and electrostatic repulsion.
  • T7Pol T7 RNA polymerase
  • PCR can be conducted within the capsule resulting in a capsule which includes multiple copies of the DNA template.
  • PCR is conducted using DNA primers, a thermostable DNA polymerase such as Taq polymerase and deoxyribonucleotides.
  • the reaction mixture including the capsule containing the DNA template is the thermocycled.
  • the capsules may also have provided therein the required polymerase. In such an arrangement contacting the polymeric capsule with the polymerase is obviously optional.
  • the system can be further improved by attaching targeting molecules to the surface of the capsule which recognize molecular markers on target cells or tissues.
  • RNAi silencing for example depends on which step in the silencing pathway the RNA exerts its action.
  • capsules with DNA delivered directly to cells have the potential to make RNAi continuously which alleviates the transiency commonly seen in current RNA delivery systems.
  • the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule, mixing the capsule with a polymerase and nucleotides, wherein the polymerase and the nucleotides can diffuse into the capsule, to form a reactant mixture, incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.
  • the present invention provides a method of producing a nucleic acid molecule within a polymeric capsule, the method comprising providing a polymeric capsule having entrapped therein a DNA molecule and a polymerase, mixing the capsule with nucleotides which can diffuse into the capsule and optionally a polymerase which can diffuse into the capsule, to form a reactant mixture, and incubating the reactant mixture for a time and under conditions such as to allow the de novo synthesis of a nucleic acid molecule within the capsule.
  • nucleotides are ribonucleotides and the RNA synthesised is single stranded RNA, hairpin RNA or double stranded RNA.
  • the synthesised RNA may be a therapeutic molecule selected from the group consisting of microRNA (miRNA), hairpin RNA, shRNA siRNA and ribozymes.
  • miRNA microRNA
  • hairpin RNA hairpin RNA
  • shRNA siRNA shRNA siRNA
  • ribozymes ribozymes
  • the polymerase may be any of a wide range of polymerase known in the art including RNA Poll, PolII, PolIII, T7 RNA polymerase and DNA polymerases
  • the entrapped DNA will comprise at least one promoter.
  • the entrapped DNA comprises a single T7 promoter and the polymerase is T7 RNA polymerase.
  • the present invention is particularly advantageous for the production and delivery of miRNA.
  • miRNA is typically readily degraded, however, in the present invention the synthesised miRNA is protected during synthesis and delivery by the polymeric capsule.
  • the entrapped DNA will typically include two promoters. These may be in sequence or opposed promoters such that two RNA strands which will hybridise are produced. Where it is desired to synthesise two or more RNA forms and or-sequences, two or more different entrapped DNAs can be used. The resultant R As may or may not hybridise.
  • the method of the present invention may also be used to synthesise DNA within the capsule.
  • the reactant mixture will further comprises DNA primers and the polymerase will be a DNA polymerase.
  • the synthesis of DNA basically means conducting PCR within the capsule.
  • the reactant mixture is thermocycled so as to allow amplification of the DNA within the capsule
  • the synthesised DNA may be a sequence which encodes a protein of interest, a therapeutic RNA such as hairpin RNA, shRNA, siRNA or miRNA, or may itself be a functional molecule such as a DNAzyme.
  • a therapeutic RNA such as hairpin RNA, shRNA, siRNA or miRNA
  • a functional molecule such as a DNAzyme.
  • the polymeric capsule is prepared by a layer-by-layer methodology around the DNA template which is adsorbed onto positively charged silica colloids.
  • the polymeric capsule may have a charged outer surface.
  • the charge may be positive or negative.
  • the charge is negative.
  • the polymeric shell of the polymeric capsule does not comprise a lipid.
  • the polymeric shell of the polymeric capsule does not comprise a surfactant.
  • the polymeric capsule can be made from any suitable material. In one
  • the polymeric capsule includes a polymer ' selected from the group consisting of chain growth polymers, step growth polymers, polyelectrolytes, and polysaccharides.
  • the polymeric capsule includes poly(methacrylic acid) crosslinked with disulfide linkages.
  • the polymeric capsule includes poly(Iysine) and poly(glutamic acid).
  • the polymeric capsule includes
  • the polymeric capsule is biodegradable.
  • the polymeric capsule is substantially resistant to degradation in an extra-cellular environment and degrades in an intra-cellular environment.
  • Suitable polymers for use in layer-by-layer- systems are described in US 7101575 and WO 2005/032512.
  • one or more of the layers is internally cross-linked.
  • two or more of the layers are cross-linked to one another.
  • the cross- linking is achieved by the reaction of cross-linking groups incorporated within a polymer of the layer or layers.
  • the percentage of cross-linking groups incorporated within the polymer is from 10 to 25 mol%, preferably 15 to 20 mol%, more preferably 17 to 19 mol%, even more preferably 18%.
  • the cross-linking group is a thiol and the cross-link is a disulphide group.
  • the cross-linking group may be maleimide.
  • the polymeric capsule is a polymeric shell comprising layers thiolated poly(methacrylic acid) PMASH wherein the PMASH layers are cross-linked by disulphide bonds.
  • the outer layer of the polymeric capsule is PMASH-
  • polymeric capsules formed of alternating layers of poly(styrene sulfonate) (PSS) and poly(allylamine) hydrochloride (PAH) may be formed by a similar layer-by- layer methodology.
  • the polymeric capsule is a polymeric shell comprising alternating layers of poly(styrene sulfonate) (PSS) and poly(allylamine) (PAH)hydrochloride.
  • PSS poly(styrene sulfonate)
  • PAH poly(allylamine)hydrochloride.
  • the polymeric capsule may comprise one or more targeting molecules associated with the surface of the polymeric capsule.
  • the one or more targeting molecules target the polymeric capsule to a specific molecule expressed either on the surface of a target cell or tissue.
  • the targeting molecule can be an antibody or a portion thereof or a peptide.
  • suitable antibodies include W02, which recognises the A-beta protein involved in 20 Alzheimer's disease, anti -transferrin receptors, anti-human insulin receptor, and antiepidermal growth factor receptor.
  • suitable antibodies included those that recognise proteins such as clusterin, insulin-like growth factor binding protein, cell adhesion protein to name but a few.
  • association of the targeting molecule with the outer surface of the polymeric capsule may be carried out by any suitable means.
  • the association may be due to electrostatic forces between the outer surface and the targeting molecule.
  • the association may be by means of a covalent bond between the targeting molecule and the outer surface.
  • examples include using classic amine chemistry.
  • the targeting molecule can be modified by placing a free thiol through the introduction of a cysteine residue thus facilitating conjugation to maleimide residue on the surface of the capsules.
  • the polymeric capsules may be administered in the form of a composition comprising a pharmaceutically acceptable diluent, excipient or carrier.
  • the composition may be administered in any way known in the art.
  • the composition may be administered parenterally either subcutaneously, intramuscularly or intravenously, or alternatively nasally.
  • the present invention provides a polymeric capsule comprising a nucleic acid molecule wherein the capsule is produced according to the method of the first or second aspect of the present invention.
  • the present invention provides a method of delivering a nucleic acid to a cell comprising contacting the cell with a polymeric capsule of the third aspect of the present invention.
  • Poly(methacrylic acid), MW 15 KDa was purchased from Polysciences (USA). Alexa Fluor 488 and 633 maleimides, RPMI-1640 + L-Glutamine, DMEM, L- Glutamine, Opti- MEM 1 reduced serum medium, MEM non essential amino acids, sodium pyruvate, trypsin, penicillin and streptomycin were purchased from Invitrogen. Fetal bovine serum (FBS) was from JRH. Methods
  • Flow cytometry measurements were performed using Partec CyFlow Space flow cytometer with an absolute volume counting capability using 488 nm and 633 nm excitation wavelengths.
  • the capsules and cells were imaged using an Olympus 1X71 Digital Wide field fluorescence microscope and a Leica confocal laser scanning microscope.
  • the PMA polymeric capsules were fluorescently labelled with Alexa Fluor 633 using a 1 g/L solution of Alexa Fluor 633 maleimide in DMSO which was added to a dispersion of PMA polymeric capsules in 50 mM MES buffer, pH 6, to a final DMSO content of 5-10 vol %.
  • approximately 1010 - 101 1 500 nm PMA polymeric capsules were labelled using 10 - 50 ⁇ g of the dye.
  • the fluorescent polymer being in excess to that released from the capsules, effectively infiltrates into the PMA polymeric capsules to give rise to highly fluorescent capsules.
  • This procedure has proven effective for labelling of the PMA polymeric capsules with sizes as low as 300 nm and allowed for reliable and reproducible counting of the capsules on a CyFlow Partee Space flow cytometer with absolute volume counting triggering on Alexa Fluor 488 fluorescence.
  • CHO-K1 WT (ATCC: CCL61), NR6 (a 3T3 derivative), HEK293T (ATCC: CRL 1 1268), M17 (ATCC: CRL-2267), P388D1 (ATCC: CCL-.46) and RAMOS (ATCC: CRL-1596) cell lines were maintained according to the American Type Culture Collection (ATCC) protocols.
  • RAMOS cells were grown in suspension at 37°C and 5 % C0 2 . All other cells were cultured as monolayers at 37°C and 5% C0 2 .
  • CHO and HEK293T cells were grown in RPMI-1640 + L-glutamine supplemented with 5% FBS and antibiotics (penicillin 500U/ml, streptomycin 50 ⁇ g/ml).
  • P388D1 and RAMOS cells were grown in RPMI-1640 + L-glutamine supplemented with 10% FBS and penicillin 50 U/ml; streptomycin 50 ⁇ g/ml.
  • NR6 fibroblasts were grown in DMEM supplemented with 2mM L-glutamine, 5% FBS and penicillin 500U/ml and streptomycin 50 g/ml.
  • Ml 7 cells were grown in Opti-MEM 1 reduced serum medium supplemented with non essential amino acids, sodium pyruvate, 10% FBS and penicillin 500 U/ml, and streptomycin 50 ug/ml.
  • PMA polymeric capsules Uptake. Cells were seeded into 96-well plates (Nunc) at a density of 4 ⁇ 10 4 cells per well in complete growth medium and allowed to attach overnight at 37°C and 5% C0 2 . After this time, PMA polymeric capsules in PBS were added to the cells to give a final well volume of 290 ⁇ and incubated for 24 h at 37°C, 5% C0 2 , with shaking at 90 rpm. Prior to analysis, the cells were washed twice with pre- warmed PBS, trypsinized and stored on ice. Cells were then analyzed using a CyFlow Partec flow cytometer. Raw data were analyzed using Flow Jo software with a built-in population comparison algorithm.
  • CellTiter-Glo® Cell Viability Assay Cells were seeded into 96-well opaque tissue culture plates (PerkinElmer) at a density of 4 x 10 4 cells/well in 100 ⁇ growth medium and allowed to attach overnight at 37°C and 5% C0 2 . The cells were then exposed to 500 nm PMA polymeric capsules at various capsule: cell ratios and incubated for 24 h. The viability of the cells was assessed using CellTiter-Glo® luminescent cell viability kit from Promega Corporation (Madison, WI) according to the manufacturer's instructions. The assay detected the amount of bioluminescent ATP present, which was directly proportional to the number of viable cells. Luminescence was measured on a FLUOstar Optima (BMG LABTECH).
  • Assembly of PMA polymeric capsules relies on the hydrogen bonding-assisted sequential deposition of PMA and poly(vinylpyrrolidone) onto sacrificial template particles. It has been previously shown that this assembly proceeds without aggregation of the.particles, which makes it a convenient starting platform to obtain polymer capsules of different compositions (single component PMA18 or PVPON20 hydrogel capsules). Single component disulfide stabilized PMA polymeric capsules as used in this work were obtained using thiol modified PMA, conversion of thiols into bridging disulfide linkages within the assembled multilayered film, removal of the core particles, and finally increasing pH to release PVPON.
  • This assembly was routinely performed using 500 nm and 1 ⁇ sized particles without aggregation which ensures a high yield (i.e. minimal particle loss during assembly) and produces a population of capsules monodisperse in size.
  • To assemble PMA polymeric capsules we used 300 nm, 500 nm and 1 ⁇ commercial monodisperse silica template particles. The resulting single component hydrogel capsules were larger in size compared to the parent core particles due to the characteristic hydrogel swelling. Herein, capsules sizes are quoted as those of the respective template particles. To ensure accurate and reproducible dosage, the capsules were counted using a flow cytometer with an absolute volume counting function using fluorescence parameter as a trigger.
  • Capsule cytotoxity an essential aspect of drug delivery, was assessed in five different cells lines including kidney (HEK), muscle (NR6), ovary (CHO), neuron (Ml 7) and macrophage (P388D1) over a 24 h period using CellTiter-Glo® cell viability assay.
  • a range of capsule to cell ratios was tested using 500 nm PMA polymeric capsules (Fig. 2).
  • Cell viability was not significantly affected in any of the cell lines, even at a ratio of 1000 capsules to 1 cell, whereas all cell lines treated with 25 % DMSO viability was reduced to less than 20%.
  • the morphology of CHO cells was not affected following treatment with 500 nm capsules as observed by fluorescence microscopy.
  • PSS 4-styrenesulfonate
  • PAH poly(allylamine hydrochloride)
  • BSA bovine serum albumin
  • PAH alginate/polylysine, etc
  • PES 4-styrenesulfonate
  • PAH poly(allylamine hydrochloride)
  • BSA bovine serum albumin
  • PAH alginate/polylysine
  • others demonstrate that polyelectrolyte capsules can be non-toxic to cells (e.g. PSS/PAH capsules taken in a 100: 1 ratio to dendritic cells and macrophages) which may be reflective of different length of incubation with cells, capsule. concentration, cell specific effects and possibly batch-to-batch variation in the capsules.
  • PMA polymeric capsules have consistently proven to be non-toxic at all concentrations and to all cell types tested.
  • RAMOS B cells which exhibited a greater uptake level at the 3 h time point compared with 24 h for both capsule sizes.
  • the latter observatio suggests either that RAMOS has a different uptake mechanism/s or physiological conditions specific to its cell membrane which differs from the other epithelial cell lines tested.
  • Ml 7 cells exhibited a lower level of uptake for the 1 ⁇ capsules, it doe not imply that the uptake of PMA polymeric capsules by these cells is restricted to smaller capsules, as these cells successfully internalized 1 ⁇ PMA polymeric capsules at higher capsule to cell ratios.
  • Poly(methacrylic acid, sodium salt) (PMA), Mw 15,000, was purchased from Polysciences (USA), and poly(vinylpyrrolidone) (PVPON), Mw 10,000, dithiothreitol (DTT), and spermidine hydrochloride were purchased from Sigma- Aldrich and used as received.
  • 1,8-bis-Maleimidodiethyleneglycol (BM(PEG)2) and 1,4 bismaleimidyl-2,3-dihydroxybutane (BMDB) were purchased from Thermo Scientific.
  • Short oligonucleotides were purchased from Geneworks and pPCR-script Amp SK(+) plasmid was purchased from Stratagene. Taq DNA polymerase (recombinant), DNase I (recombinant), Vent polymerase (recombinant), T7 RNA polymerase
  • T4 polynucleotide kinase T4 polynucleotide kinase, low range ssRNA ladder, and PCR buffer were obtained from New England Biosciences.
  • QIAquick PCR purification kit was purchased from Qiagen. Alexa Fluor 633 maleimide, Alexa Fluor 488 UTP, Sybr Green II gel stain, and RNaseOUT were purchased from Invitrogen.
  • High- purity water with a resistivity greater than 18 ⁇ cm was obtained from an in-line Millipore RiOs/Origin system (MilliQ water) and treated with Diethyl pyrocarbonate (DEPC) prior to use.
  • MEPC Diethyl pyrocarbonate
  • Thiol-modified PMA with 15 mole% of thiol groups (PMASH) was synthesized via carbodiimide chemistry, as described in detail previously.1 Methods.
  • Flow cytometry was performed on a Becton Dickson FACS calibur flow cytometer using an excitation wavelength of 488 ran. Particles and cells were imaged on anOIympus 1X71 digital wide-field fluorescence microscope with a fluorescein filter cube and a Leica time-correlated single-photon-counting confocal fluorescence microscope. UV-Vis measurements were performed on a Thermo Scientific NanoDrop-1000 spectrophotometer. PC temperature cycling was carried out in an Applied Biosystems GeneAmp PCR System 9700.
  • PCR amplicons were synthesized by PCR using a mixture containing 200 ⁇ dNTPs, 10 pmol primers, 2.5 U Taq DNA polymerase, and 100 ng of dsDNA template in a total volume of 20 ⁇ , PCR buffer (20 mM Tris-HCl pH 8.4, 50 mM KC1, 1.5 mM MgC12). Cycling conditions were 5 min at 94 °C, 30 cycles of 94 °C for 30 s, 60 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 5 min.
  • the amplicons synthesized are listed in Table 1 below.
  • +T7DNA and -T7DNA are -800 bp amplicons with or without the 5' T7 promoter sequence on the sense strands respectively.
  • +T7DNA2 is -T7DNA modified to contain the T7 promoter sequence, enabling transcription of -T7DNA.
  • Samples were purified using the QIAquick PCR cleanup kit. A portion of the samples were separated on 1% agarose gel in TAE buffer (40 mM
  • the particles After being washed three times in 25 mM sodium acetate buffer (pH 4), the particles were suspended in a 1.0 g L-1 solution of PVPON (Mw 10,000) for 15 min. PMASH and PVPON were added sequentially until 8 layers (3.25 ⁇ particles) or 6 layers ( 1.1 1 ⁇ particles) had been deposited, after which time the PMASH layers were crosslinked by suspending the particles in a 0.5 g L-l solution of either BM(PEG)2 or BMDB in 50 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH 6) for 16 h.
  • MES 2-morpholinoethanesulfonic acid
  • the particles were washed with fresh buffer and dispersed in 50 ⁇ , of 25 mM sodium acetate buffer (pH 4), to which 100 uL of 2 M HF/8 M NH4F (pH ⁇ 5) was added.
  • the resulting capsules were washed by centrifugation (4500g for 5 min) and dispersed into 10 mM Tris- HC1, 50 ⁇ EDTA, pH 8, which was repeated until the pH of the supernatant was equal to the pH of the fresh buffer solution.
  • the capsules were suspended in 50 ⁇ ⁇ buffer.
  • the capsule concentration was ascertained by counting a small portion of the capsule solution with flow cytometry. Labeling of the capsules was achieved by reaction of 1 ⁇ g of Alexa Fluor 633-maleimide with the capsules suspended in 50 mM MES, pH 6.
  • Ramos cells were harvested and washed twice with pre-warmed phosphate buffered saline solution. CHO cells were washed twice and then trypsinized and harvested. The outer cell membranes were stained using FM 4-64 FX dye according to the manufacturer's instructions. See also the Supporting Information for detailed experimental procedures.
  • PMA polymeric capsules have been developed as colloidal drug carriers and were shown to be effectively internalized by cancerous cells and white blood cells in human blood without any reduction in cell viability from the capsules in the absence of drugs.
  • PMA polymeric capsules with de novo synthesized RNA were internalized by other mammalian cells ( Figure2).
  • Figure2 This aspect, combined with the ability to synthesize a controlled amount of RNA within the capsule interior, establishes a unique biomedical platform en route to the cellular delivery of RNA therapeutics.
  • the complete synthetic nature of the PMA polymeric capsules provides opportunities for engineering its structure, allowing such features as controlled degradability and cellular targeting.
  • Approximately 107 capsules were added to a 50 pi transcription reaction supplemented with 0.1 nmol of fluorescently labeled uridine5 '-triphosphate (UTP, green) and the mixture was incubated at 37 °C.1131 A concentration of 50 units of T7Pol in the 50 ⁇ reaction was sufficient to enable diffusion of enzyme into the core of the PMA polymeric capsules and initiate transcription.
  • Confocal laser scanning microscopy (CLSM) was used to image , fluorescently labeled polymeric capsules (red) following 3 h of incubation.
  • RNA was clearly visible in the interior of the PMA polymeric capsules containing DNA with the T7 promoter sequence, however, no fluorescence was observed in the PMA polymeric capsules containing DNA without the T7 promoter sequence.
  • An identical reaction was performed using 1.35 ⁇ 0.15 p.m PMA polymeric capsules, each containing— 500 copies of +T7DNA.
  • Flow cytometry was used to measure the progress of the reaction within the PMA polymeric capsules (Fig. lc), allowing real-time monitoring of RNA synthesis. Synthesis was slow in the first hour but then increased to a linear rate of synthesis between— 1.5 h and 5 h, and finally leveled off over the next 24 h. The initial slow rate of synthesis possibly reflects the time required for the T7Pol to diffuse into the capsules and bind to the T7 promoter sequences.
  • RNA was recovered from the PMA polymeric .capsules.
  • the capsules were first treated with a large excess of DNase I, an endonuclease to degrade the DNA template, as confirmed by gel electrophoresis (data not shown).
  • Recovered RNA was subjected to non- denaturing gel electrophoresis on a 1.5% agarose gel which confirmed that the products of the encapsulated reactions were the same size (758 bases) as the positive control reaction (P) performed in the absence of capsules.
  • Encapsulated reactions that were incubated for longer time periods produced more RNA, verifying the flow cytometry measurements. As expected, no product was observed for the negative-control capsules (N) containing - T7DNA, which were incubated for 5 h.
  • RNA transcript of +T7DNA P
  • T RNA transcript
  • the probe hybridized to the RNA transcript indicating the RNA synthesized was the expected sequence. This is important as binding is necessary for its further use as a reaction component (e.g., reverse transcription) or as a biologically active therapeutic.
  • the amount of RNA synthesized in the capsules at 5 h of incubation for 2 x 10 capsules was estimated to be 200 ng or an encapsulated RNA concentration of 775 p,g ml . "1 .
  • PMA polymeric capsules have been developed as colloidal drug carriers and were shown to be effectively internalized by cancerous cells and white blood cells in human blood without any reduction in cell viability from the capsules in the absence of drugs.
  • PMA polymeric capsules with de novo synthesized RNA were internalized by other mammalian cells ( Figure2).
  • Figure2 This aspect, combined with the ability to synthesize a controlled amount of RNA within the capsule interior, establishes a unique biomedical platform en route to the cellular delivery of RNA therapeutics.
  • the complete synthetic nature of the PMA polymeric capsules provides opportunities for engineering its structure, allowing such features as controlled degradability and cellular targeting.

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Abstract

La présente invention concerne un procédé de production d'une molécule d'acide nucléique à l'intérieur d'une capsule polymère. Le procédé comprend l'apport d'une capsule polymère comprenant une molécule d'ADN piégée en elle et le mélange de la capsule avec une polymérase et des nucléotides, la polymérase et les nucléotides pouvant diffuser à l'intérieur de la capsule afin de former un mélange réactif. Le mélange réactif est ensuite incubé pendant une durée et dans des conditions telles qu'elles permettent la synthèse de novo d'une molécule d'acide nucléique à l'intérieur de la capsule. Le procédé est particulièrement utile pour la préparation d'ARN encapsulés, tels que des miARN.
PCT/AU2010/000756 2010-06-17 2010-06-17 Formation d'acides nucléiques à l'intérieur d'une capsule polymère Ceased WO2011156831A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2288526A1 (fr) * 1997-05-14 2001-05-01 Itaru Urabe Capsule uniforme pour la synthese d'un biopolymere et methode de fabrication de cette capsule
WO2003078659A2 (fr) * 2002-03-20 2003-09-25 Innovativebio.Biz Microcapsules encapsulant un melange de reaction d'amplification de l'acide nucleique et utilisation de celles-ci en tant que compartiments de reaction pour des reactions paralleles
WO2010017349A1 (fr) * 2008-08-06 2010-02-11 Genome Corporation Microcapsules et procédés d’utilisation pour amplification et séquençage

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2288526A1 (fr) * 1997-05-14 2001-05-01 Itaru Urabe Capsule uniforme pour la synthese d'un biopolymere et methode de fabrication de cette capsule
WO2003078659A2 (fr) * 2002-03-20 2003-09-25 Innovativebio.Biz Microcapsules encapsulant un melange de reaction d'amplification de l'acide nucleique et utilisation de celles-ci en tant que compartiments de reaction pour des reactions paralleles
WO2010017349A1 (fr) * 2008-08-06 2010-02-11 Genome Corporation Microcapsules et procédés d’utilisation pour amplification et séquençage

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MAK, W.C. ET AL.: "Diffusion controlled and temperature stable microcapsule reaction compartments for high-throughput microcapsule-PCR", ADVANCED FUNCTIONAL MATERIALS, vol. 18, 2008, pages 2930 - 2937 *
PRICE, A.D. ET AL.: "A Biomolecular ''Ship-in-a-Bottle'': continuous RNA synthesis within hollow polymer hydrogel assemblies", ADVANCED MATERIALS, vol. 22, February 2010 (2010-02-01), pages 720 - 723 *
ZELIKIN A.N. ET AL.: "A general approach for DNA encapsulation in degradable polymer microcapsules.", ACS NANO., vol. 1, no. 1, 2007, pages 63 - 69 *

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