WO2000011217A1 - Composes et procedes permettant d'ameliorer l'apport d'un polynucleotide libre - Google Patents

Composes et procedes permettant d'ameliorer l'apport d'un polynucleotide libre Download PDF

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WO2000011217A1
WO2000011217A1 PCT/US1999/018726 US9918726W WO0011217A1 WO 2000011217 A1 WO2000011217 A1 WO 2000011217A1 US 9918726 W US9918726 W US 9918726W WO 0011217 A1 WO0011217 A1 WO 0011217A1
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tissues
polynucleotide
group
cells
transfection
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PCT/US1999/018726
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Robert W. Malone
Jill G. Malone
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University Of Maryland, Baltimore
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Priority to CA002341017A priority Critical patent/CA2341017A1/fr
Priority to AU55684/99A priority patent/AU5568499A/en
Priority to EP99942265A priority patent/EP1119642A1/fr
Publication of WO2000011217A1 publication Critical patent/WO2000011217A1/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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the field of the invention generally relates to the use of transfection enhancing agents to enhance the polynucleotide transfer and expression associated with direct delivery of 'free' polynucleotide preparations to target cells or tissues.
  • the field of the invention more specifically relates to nuclease inhibitors which prevent the extracellular and mtracellular cleavage and degradation of the free polynucleotide.
  • the invention relates to the use of direct competitive nuclease inhibitors, such as au ⁇ n t ⁇ carboxyhc acid (ATA), a nuclease and apoptosis inhibitor, to enhance respiratory, mucosal, ' and skm tissue transfection.
  • ATA au ⁇ n t ⁇ carboxyhc acid
  • the invention allows for simple and safe alternatives to biological vector-based delivery and transfection techniques.
  • the invention allows for both in vitro and in vivo transfection.
  • Utilities include but are not limited to the engineering of cultured prokaryotic and eukaryotic cells, bioreactor protein production, polynucleotide therapies for use m conjunction with organ transplantation, angioplasty, skm transplantation, and other clinical treatments which will benefit from genetic therapies.
  • Bio vector systems utilize a biological organism, such as a virus or a bacteria, to effect the delivery and transfer of a polynucleotide of interest to target tissues within a host organism.
  • Synthetic vector systems rely on endogenous cellular pathways (such as endocytosis) to effect delivery and transfer of the polynucleotide of interest.
  • the synthetic vector preparation may be as simple as a plasmid or further include a polynucleotide complexed to colloidal materials (such as catiomc lipids and liposomes).
  • colloidal materials such as catiomc lipids and liposomes.
  • Biological vector polynucleotide transfer systems particularly adenovirus and adeno associated virus (AAV) vectors, are most widely used for pre-clmical studies.
  • AAV adeno associated virus
  • Synthetic vector polynucleotide transfer systems particularly hposomal preparations, are safer and more amenable to large scale manufacture and distribution.
  • Biologic clearance of polynucleotide delivery preparations in vitro and in vivo is a critical determinant of transfection or transduction efficiency and one that affects all polynucleotide delivery systems.
  • the biological clearance may occur before the polynucleotide preparation has reached the target tissue or cell, via extracellular nucleases. Alternatively, the clearance may occur after the polynucleotide preparation has entered the cell but before it has entered the nucleus or acted on the mtracellular target, via mtracellular nucleases.
  • hpidic delivery systems and viral packaging provide protection from endolytic degradataion by endogenous nucleases
  • 'free' polynucleotides are susceptible to mactivation via endo- or exonucleolytic cleavage (by both extra- and mtracellular nucleases).
  • endo- or exonucleolytic cleavage by both extra- and mtracellular nucleases.
  • cell damage such as needle injection of significant volumes
  • release of nucleases from damaged cells can compound this effect.
  • viral vector systems are often subject to rapid nonspecific (e.g., complement) and specific (e.g., antibody neutralization, CTL) immunologic clearance
  • hpoplexes are efficiently cleared by the reticuloendothehal system, and may also be subject to complement- mediated effects.
  • the invention herein overcomes the problem of biologic clearance of free polynucleotide preparations, thereby enhancing the delivery and expression of the polynucleotide of interest.
  • the invention involves the use of nuclease inhibitors to prevent the nucleolytic degradation of free polynucleotides.
  • the invention finds utility for both in vitro and in vivo transfection
  • agents which indirectly inhibit nuclease activity by reducing the effective concentration of enzymatically required divalent cations are known m the art. Examples include ethylenediammetetraacetic acid (EDTA) and citrate
  • EDTA ethylenediammetetraacetic acid
  • the invention herein specifically utilizes agents which directly inhibit extracellular and mtracelluar nucleases.
  • agents include various competitive inhibitors including nucleotide analogs and au ⁇ n t ⁇ carboxyhc acid (ATA).
  • the method utilizes the inhibition of extracellular and mtracellular nuclease activity.
  • This method finds utility both in vitro and in vivo transfection
  • the invention relates to transfection enhancing agents that allow for improved transfer of free polynucleotide preparations to targeted tissues of a host organism, thereby increasing the level of expression of the polynucleotide of interest in situ.
  • the method involves the administration of transfection enhancing agents m the form of direct competitive nuclease inhibitors combination with free polynucleotide preparations, * the combination allowing for more efficient transfection of a polynucleotide of interest.
  • the competitive nuclease inhibitor is aurm tricarboxyhc acid or a functional derivative thereof.
  • the polynucleotide preparations of the present invention may be used to enhance the transfer of free polynucleotide preparations to eukaryotic or prokaryotic cells m culture, thereby enhancing the level of expression of the polynucleotide (or polynucleotides) of interest.
  • the polynucleotide preparations of the present invention may be used for gene therapy m general, more specifically for delivering exogenous copies of a therapeutic gene or polynucleotide to a specific cellular or tissue target in vivo.
  • Enhanced polynucleotide delivery also finds utility not only m vaccine therapies, such as polynucleotide vaccines, mucosal and mtradermal polynucleotide vaccines, but also genetic therapies for inborn metabolic diseases, such as cystic fibrosis, and expression of immunomodulatory agents, such as cytokines or costimulatory molecules, and delivery of such therapeutic polynucleotides into cells of the immune system including antigen presenting cells.
  • the examples desc ⁇ bed m detail herein confirm that a diverse range of species including mu ⁇ ne, rat and macaque respiratory tissues may be transfected by simple direct application of plasmid.
  • the examples further confirm that rodent, primate and human tissues express DNAse activity, that this activity may be inhibited, and that inhibition of such activity enhances the transfection of respiratory tissues.
  • ATA co-admmistration enhances this transfection activity.
  • Figure 1 Depicts assays preformed using lavage fluid obtained from 4 different mice and demonstrates the presence of significant levels of nuclease activity in the mu ⁇ ne lung lavage fluid.
  • Figures 2A-2B Depicts the degradation of mtratracheally administered plasmid, demonstrating the effect of ATA on this process in vitro.
  • Figure 2A depicts the results associated with lavage fluid.
  • Figure 2B depicts the results associated with tissue extract. See Table 1 for the details of the protocol.
  • Figures 3A-3B Depicts the experimental results of the Balb-C mouse lung transfection resulting from co-admmistration of ATA and pND2Lux plasmid.
  • Figure 3 A depicts the ratio ATA to free DNA at each dosage, from 0.1 [g/g to 6 [g/g.
  • Figure 3 A also shows the negative results associated with doses of EDTA and sodium citrate.
  • Figure 3B depicts the same expe ⁇ mental results from Figure 3 A associated with doses of ATA, further including error bars.
  • Figure 4- Depicts the optimization of efficacy of ATA plasmid administration , showing the mean and standard deviation of total relative units (less background) obtained from groups of three similarly aged animals treated under the same conditions with same DNA at the same time. Each mouse was treated with 100 micrograms of pND2Lux in 100 microhters of water via mtratracheal installation using the methods desc ⁇ bed m detail below.
  • Figure 5 Depicts the transfection enhancing activity associated with c-admimstration of free plasmid with DNAse inhibitors ATA, EDTA, and citrate.
  • ATA co-admmistration markedly augmented the levels of reporter protein expression detected after mtratracheal administration of plasmid.
  • EDTA nor citrate significantly enhanced the level of expression.
  • administration with EDTA was associated with significant toxicity.
  • Figure 6 Depicts a map of the expression vector (pND) constructed to contain the following elements: (1) the CMV immediate early promoter (HCMV LEI), (2) the CMV IE1 mtron, (3) a cloning site, and (4) the RNA termmator/polyadenylation site from bovme growth hormone (BGH) These elements are contained m a pUC19 rephcon.
  • HCMV LEI CMV immediate early promoter
  • BGH bovme growth hormone
  • Figure 7 Depicts the results, m terms of luciferase expression, of mtradermal injection of luciferase DNA into rat skm, both with and without ATA.
  • Figure 8 Depicts the results, m terms of luciferase expression, of mtradermal transfection of luciferase DNA into mouse skm, both with and without ATA.
  • Figure 9 Depicts the histology of treated mouse lung tissue obtained from control and treated mice. Images include representative low (4X) and intermediate (20X) and high (40X) power fields of hematoxilyn and eosm stained lung sections. Tissues were fixed, embedded, sectioned, and stained two days after treatment yet prior to staining. Note the absence of polymorphonuclear cells indicating a lack of acute inflammatory response, the presence of mtact respiratory epithelium lining conducting airway, and the lack of significant fluid or inflammatory cell infiltrates withm the parenchyma.
  • Figures 10A-10C Depicts the levels of DNAse activity observed in murme (10A), macaque (10B), and human (10C) lung fluids and that ATA inhibits lung DNAse activity in vitro.
  • the rate of clearance is frequently an important determinant of in vivo drug activity.
  • multiple mechanisms may contribute to clearance of extracellular polynucleotides.
  • these include nuclease-mediated degradation, mucous entrapment with mucocihary clearance, phagocytic clearance, and absorption and distribution to non-respiratory tissues
  • nuclease activity in tissues and fluids was demonstrated in vitro by incubating plasmid with lung extracts and lavage fluids and in vivo m various animal systems by direct mtradermal, intramuscular and mtratracheal plasmid administration and electrophoretic analysis of lavage or tissue recovered.
  • mtracellular (predominantly cytoplasmic) nucleases also contribute to mtracellular clearance of transfected polynucleotides.
  • the focus of the instant invention is that addition of nuclease inhibitors to applied plasmid significantly improves transfection by reducing nuclease-mediated clearance.
  • Murme, rat and primate models were used for the proof of principle experiments described herein. It was discovered that co-admmistration of plasmid with the nuclease inhibitor ATA markedly boosted subsequent reporter protein expression relative to free DNA treatment.
  • Efficacy may be defined in terms of reporter protein expression, or may be defined in the context of clinical response (which in turn reflects the level of protein expression necessary to generate a response). Toxicity includes both short term deleterious effects (atelectasis, enhanced vascular permeability, or non-therapeutic inflammation, apoptosis, necrosis), as well as long term risks (fibrosis, msertional mutagenesis/carcinogenesis, cellular cytotoxicity and/or autoimmumty).
  • the clinical utility of any polynucleotide delivery system will be a function of the intended application and the ratio of efficacy and toxicity of the system.
  • Such medicines may provide treatments for inborn errors of metabolism such as cystic fibrosis, acute treatments for various pulmonary disorders such as latrogemc pulmonary fibrosis, and prophylactic treatments such as mucosal and parenteral polynucleotide vaccination.
  • simple and safe gene delivery methods may be particularly useful for analysis of gene function and for validation of potential pharmaceutical targets.
  • tissues may be genetically modified by administration of 'free' or 'naked' polynucleotides [Balasubramaniam et al., Gene Therapy: 3(2): 163-172 (1996) Malone et al.. Advances. Challenges And Applications for Self-Assembling Systems. 4.1.1 (1996); Meyer et al., Gene Ther. 2(7):450- 460 (1995), Tsan et al, Am J Physiol. 268 (6Pt 1) 1052-L1056 (1995); Zabner et al, Chn Invest.
  • the invention employs the co-admmistration of transfection enhancing agents with free polynucleotide preparations, the enhancing agent acting as a protective agent, preventing the nucleolytic degradation of the polynucleotide by the endogenous nucleases.
  • the transfection enhancing agents contemplated by the instant invention are nuclease inhibitors, preferably direct competitive nuclease inhibitors. Although the agent need not be a specific nuclease inhibitor, it is preferable that the agent have activity against those nucleases found in the target cells and tissues of the host organism.
  • Nucleases are enzymes which break the linkages (phosphodiesters) between nucleic acids in a polynucleotide chain. Nucleases are found both mtracellular (e.g. cytoplasmic nucleases) and extracellular (e.g. circulatory or systemic nucleases). The instant invention is primarily concerned with nucleases that are endogenous to the target system, both mtra- and extracellular Nucleases may be specific to RNA or DNA, to internal or external sites.
  • RNAses or ⁇ bonucleases are nucleases that catalyze the breaking of some linkages between nucleotide m RNA DNAses or deoxy ⁇ bonucleases are nucleases that hydrolyze the interior bonds of deoxy ⁇ bonucleotides and strings them together into o gonucleotides or polynucleotides
  • pancreatic DNAse I yields di- and oligo- nucleotide 5- phosphates
  • pancreatic DNAse II yields 3-phosphates.
  • Restriction enzymes or restriction 'endonucleases' are proteins that recognize specific, short polynucleotides and cuts DNA at those sites. Bacteria contain over 400 such enzymes that recognize and cut over 100 different DNA sequences.
  • Nucleases that cleave a polynucleotide substrate at the internal sites (the phosphodi ester bonds) in the polynucleotide sequence are referred to as 'endonucleases' 'Endo ⁇ bonucleases' are endonucleases that specifically hydrolyze the interior bonds of a ⁇ bonucleotide.
  • endonuclease SI a nuclease enzyme from the fungi Asperg ⁇ lus oryzae which cuts the phosphodiester between nucleotides m smgle-stranded DNA or RNA, producing individual nucleotide molecules
  • Nucleases that cleave nucleotides sequentially from the free ends of a linear nucleic substrate are referred to as 'exonucleases'.
  • exonucleases include but are not limited to exonuclease III, an exonuclease which removes nucleotide one at a time from the 5 '-end of duplex DNA which does not have a phosphorylated 3 '-end, exonuclease VII, an exonuclease which makes ohgonucleotide by cleaving chunks of nucleotide off of both ends of smgle-stranded DNA, and exonuclease lambda, an exonuclease that removes nucleotide from the 5' end of duplex DNA which have 5 '-phosphate groups attached to them.
  • nuclease inhibitors are effective transfection enhancing agents for direct plasmid transfection of tissues, more particularly respiratory or mucosal tissues.
  • the experiments described below include tests performed with direct and indirect nuclease ' inhibitors.
  • ATA is a direct competitive nuclease inhibitor
  • both EDTA and citrate indirectly inhibit nucleases by sequestering the divalent cation cofactors required for nuclease activity
  • ATA is a potent direct transfection enhancing agent while EDTA and citrate did not augment free DNA transfection.
  • nuclease inhibiting pharmaceuticals are subject to rapid systemic absorption and distribution. While not wishing to be bound by theory, it is possible that differences m the concentration and distribution of target hgands can account for differences m in vivo transfection enhancing activity In general, the absorption of proteins across respiratory epithelium is much less efficient than the diffusion of divalent cations. Therefore, the nuclease activity of respiratory fluids treated with ATA will be influenced by the ATA/nuclease binding constant and the concentration of ATA and nuclease within the respiratory space. In contrast, divalent cations are present at relatively high concentrations in both extracellular fluid and withm cells.
  • nuclease inhibitors examples include but are not limited to NuiA, a protein produced by Anabaena sp. [Muro-Pastor AM, J Mol Biol. 268(3):589-98 (May 9 1997)] and DMI-2, an acid nuclease inhibitor and polyketide metabolite of Streptomyces sp. strain 560 [Ross GF, et al., Gene Ther. 5(9): 1244-50 (Sep 1998)].
  • synthetic nuclease inhibitors examples include but are not limited to diethyl pyrocarbonate [Zs dely A, et al., Acta Biochim Biophvs Acad Sci Hung. 5(4):423-34 (1970)].
  • the nuclease inhibitor is preferably a direct competitive nuclease inhibitor.
  • competitive nuclease inhibitors include but are not limited to ac ⁇ dme dimers [Malvy C et al, Chem Biol Interact. 73(2-3):249-60 (1990)]; actm and actm derivatives [Macanovic M, et al., Clm Exp Immunol.
  • RNAse inhibitors include but are not limited to bromopyruvic acid, an mactivator of bovine pancreatic ⁇ bonuclease A [Wang MH, et al., Biochem J. 320 ( Pt 1): 187- 92 (Nov 15, 1996)]; retmoids, inhibitors of ⁇ bonuclease P activity [Papadimou E, et al., J Biol Chem.
  • DNAse inhibitors include but are not limited to 5838-DNI (or 1 ,4,4a,5, 12, 12a-hexahydro-4,4a, 11 , 12a-tetrahydroxy-3,8-d ⁇ methoxy-9- methoxycarbonyl- 10- methyl-l,5,12-t ⁇ oxo naphthacene), a competitive inhibitor of porcine spleen DNAse II produced by Streptomyces sp. Strain No. A-5838 and having a structure similar to tetracenomycm C [Uyeda et al., J Enzvme Inhib.
  • Other direct competitive nuclease inhibitors include polyclonal antibodies capable of binding to nucleases or fragments thereof [Crespeau et al., C R Acad Sci III. 317(9): 819-23 (Sep 1994)] Such antibody preparations are available from a variety of commercial sources including but not limited to Genentech, Worthmgton laboratories, and Sigma- Ald ⁇ ch. Such antibodies can be prepared by immunizing a mammal with a preparation of a nuclease. Methods for accomplishing such immunizations are well known m the art. Monoclonal antibodies (or fragments thereof) can also be produced by immunizing splenocytes with activated APF (by modifying the procedures of Kohler et al.
  • Antibody-based DNA inhibitors can be adapted to incorporate human protein sequences ('humanized antibody') and thereby be used in humans without generating an immune response
  • the nuclease inhibitor is ATA or a functional derivative * thereof.
  • ATA is a t ⁇ phenylmethane-de ⁇ vitive dye first synthesized in 1892, and initially prepared as a pure compound m 1949.
  • the molecular mass of the polycarboxylated ATA molecule is 473 daltons, and it can polymerize into larger complexes of up to 6,000 Da [Guo et al., Thromb Res 71(l):77-88 (1993)]. It has been reported that ATA does not permeate mtact cell membranes [Api ⁇ on et al., Sprmger-Verlag. 3:327-340 (1975)], and so presumably either must bind to extracellular factors such as nucleases or interact with cell membrane-associated molecules thereby altering mtracellular events.
  • ATA inhibits many endonucleases including DNAse I, RNAse A, SI nuclease, exonuclease III, and a variety of restriction endonucleases [Halhck et al., Nucleic Acids Res, 4(9):3055-64 (1977)]
  • ATA-mediated inhibition of pancreatic ⁇ bonuclease A/ nucleic acid complex formation has been investigated by proton magnetic resonance spectroscopy, and these experiments indicate that the mechanism of RNAse inhibition involves competition between the nucleic acid and polymeric ATA for binding in the active site of the protein [Gonzalez et al., Biochemistry. 19(18):4299-4303 (1980)].
  • many investigators have also employed the agent to inhibit apoptosis in cultured cells and tissues in vivo.
  • ATA anti-apoptotic activity of ATA is somewhat controversial, in part due to the wide variety of activities attributed to the compound. These activities include inhibition of topoisomerase II [Catchpoole et al , Anticancer Res. 14(3A):853-856 (1994)], induction of erbB4 phosphorylation [Okada et al., Biochem Biophvs Res Commun. 230(2):266-269 (1997)], activation of the Jak2-Stat5 signaling athway [Rui et al., Biol Chem. 273(5273):352- 354 (1998)], and inhibition of mu- and m-calpam activities [Posner et al., Biochem Mol Biol Int.
  • Topoisomerase II mediates chromatin condensation during apoptosis, and ATA inhibits the action of the protein at about 0.2 micromolar concentrations, levels lower than those usually employed to inhibit apoptosis.
  • ATA acts to inhibit extracellular nuclease activity, and apparently also acts to inhibit various pathways associated with apoptosis As both mechanisms can impact on the levels of reporter gene expression observed after direct administration of free plasmid, augmentation of reporter gene expression by ATA co-admmistration may be mediated by either or both mechanisms. Therefore, ATA can augment direct transfection of tissues by nuclease inhibition and/or by inhibition of apoptosis.
  • a 'functional derivative' of ATA is a compound which possesses a biological activity (either functional or structural) that is substantially similar to a biological activity of ATA, for example inhibiting the activity of endogenous nucleases and/or inhibiting apoptosis.
  • the term 'functional derivative' is intended to include the 'fragments,' 'variants,' 'analogues,' or 'chemical derivatives' of the compound.
  • a 'fragment' of a compound such as ATA is meant to refer to any chemical subset of the molecule.
  • a 'variant' of a compound such as ATA is meant to refer to a compound substantially similar in structure and function to either the entire compound, or to a fragment thereof.
  • a compound is said to be 'substantially similar' to another compound if both compounds have substantially similar structures or if both compounds possess a similar biological activity.
  • two compounds possess a similar activity they are considered variants as that term is used herein even if the structure of one of the compounds is not found in the other
  • An 'analogue' or agent which mimics the function of a compound such as ATA is meant to refer to a compound substantially similar in function but not in structure to either the entire compound or to a fragment thereof.
  • a compound is said to be a 'chemical derivative' of another molecule when it contains additional chemical moieties not normally a part of the compound. Such moieties may improve the compound's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed in Remington 's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art. Analogues of ATA or agents which mimic the function of ATA can be used as transfection enhancing agents as well, inhibiting the activity of endogenous nucleases or apoptosis.
  • polynucleotide preparations of the instant invention are referred to as 'free' or 'naked'.
  • 'Free polynucleot ⁇ de(s)' refers to DNA or RNA and can include sense and antisense strands as appropriate to the goals of the therapy practiced according to the invention
  • Polynucleotide in this context may include ohgonucleotides and ⁇ bozymes.
  • 'Free' in this context means polynucleotides which are not complexed to colloidal materials (including hposomal preparations), or contained withm a vector which would cause integration of the polynucleotide into the host genome.
  • the free polynucleotide preparations of the instant invention are intended for direct delivery to target tissue, additional vector and vehicle components are not required.
  • the free polynucleotide preparation may comprise one or more expression cassette and necessarily includes at least one polynucleotide of interest.
  • the polynucleotide of interest is operably linked to the requisite transcription and translation elements required for expression of the polynucleotide of interest in eukaryotic organisms in situ.
  • an expression cassette is composed of a promoter region, a transc ⁇ ptional initiation site, a ⁇ bosome binding site (RBS), an open reading frame (orf) encoding a protein (or fragment thereof), with or without sites for RNA splicing (only in eukaryotes), a translational stop codon, a transc ⁇ ptional terminator and post-transc ⁇ ptional poly-adenosme processing sites (only m eukaryotes) (Wormmgton, Curr. Opin. Cell Biol.. 5:950-954 (1993); Rezmkoff et al, Maximizing Gene Expression. Eds., Butterworths, Stoneham, Mass. (1986)).
  • the particular expression cassette employed the present invention is not critical thereto, and can be selected from the many commercially available cassettes. Examples include but are not limited to pCEP4 and pRc/RSV (Invitrogen Corporation, San Diego, CA); pXTl, pSG5, pPbac and pMbac (Stratagene, La Jolla, CA); pPUR, pEGFP-1, pND and pMAM (ClonTech, Palo Alto, CA); and pSV ⁇ -gal (Promega Corporation, Madison, WI). Alternatively, the cassette may be synthesized either de novo or by adaptation of a publicly or commercially available expression system.
  • reporter genes include but are not limited to beta-galactosidase, green fluorescent protein, and luciferase.
  • An enhanced green fluorescent protein gene is commercially available and can be amplified from a commercial vector (pEGFP-1, Clonetech, Palo Alto, CA) incorporating Sal I and BamH I sites into the primers.
  • the first 28 ammo acids of the protein are from Drosophila Alcohol Dehydrogenase followed by the fused E. coh ⁇ -galactosidase sequences. The insect sequences are reported to give higher expression m mammalian cells presumably by providing eukaryotic translation initiation signals.
  • the individual elements withm the expression cassette can be derived from multiple sources and may be selected to confer specificity in sites of action or longevity of the cassettes in the host cell or target tissue. Such manipulation of the expression cassette can be done by any standard molecular biology approach.
  • These expression cassettes usually are in the form of plasmids, and contain various promoters well-known to be useful for driving expression of genes in animal cells, such as the viral derived SV40, CMV and, RSV promoters or eukaryotic derived ⁇ -casem, uteroglobm, ⁇ - actm or tyrosmase promoters.
  • the particular promoter is not critical to the present invention, except in the case where the object is to obtain expression m only targeted cell types or tissues.
  • the promoter is selected to be one which is only active in the targeted cell type or tissue.
  • tissue specific promoters include, but are not limited to the tyrosmase promoter which is active m lung and spleen cells, but not testes, brain, heart, liver or kidney [Vile et al, Cane. Res.. 54:6228-6234 (1994)]; the mvoluce ⁇ n promoter which is only active m differentiating keratmocytes of the squamous epithelia [Carroll et al, J. Cell Sci., 103:925-930 (1992)]; and the uteroglobm promoter which is active m lung and endomet ⁇ um [Helftenbem et al, Annal. N.Y. Acad. Sci.. 622:69-79 (1991)].
  • tissue/ cell specific enhancer sequences can be used to control expression.
  • tissue specific expression is to use a hormone responsive element (HRE) to specify which cell lineages a promoter will be active in, for example, the MMTV promoter requires the binding of a hormone receptor, such as progesterone receptor, to an upstream HRE before it is activated [Beato, FASEJB J., 5:2044- 2051 (1991); and Truss et al, J Steroid Biochem. Mol. Biol , 41 :241-248 (1992)]
  • HRE hormone responsive element
  • Additional genetic elements may be included on the expression cassette m order to modify its behavior mside the host cell [Hodgson, Bio/Technology. 13:222-225 (1995)].
  • Such elements include viral genome components such as the DNA genome of a recombinant adenovirus or the self-rephcatmg "rephcon" RNA of an alphavirus such as semhki forest or smdbus virus.
  • Additional elements include but are not limited to mammalian artificial chromosome elements or elements from the autonomous replicating circular minichromosomes, such as found in D1F1 colorectal cancer cells, to allow stable non-mtegrated retention of the expression cassette [Huxley et al, Bio/Technology.
  • additional genetic elements may also include substantial regions of viral genomes, so that integration and or autonomous replication of the polynucleotide of interest will be enabled by the viral sequence elements
  • inclusion of the AAV ITR sequences together with the rep protein ORF m the expression cassette can provide integration.
  • the instant invention focuses on direct polynucleotide delivery.
  • This technique is often referred to as naked DNA injection, direct injection or free DNA injection.
  • the administration route is not critical to the instant invention.
  • the approach generally involves the introduction of a polynucleotide preparation encoding a polynucleotide of interest which is then expressed withm cells of the host organism.
  • the instant invention involves the direct application of high concentration polynucleotide preparations to target tissues.
  • the findings of the instant invention are particularly applicable to those techniques utilizing naked or free polynucleotide. Parameters such as formulation, dosage and delivery means are all standardly optimized using routine techniques and are well withm the purview of those skilled m the art.
  • polynucleotides to animal tissues in vivo can be achieved by dermal administration, intramuscular injection, transmucosal and transepithehal delivery.
  • the preferred routes of administration to the respiratory tract will be by inhalation or insufflation. Routes of administration to other mucosal tissues will vary according to their location.
  • the parenteral routes of administration is possible in limited cases though not generally preferred as the crux of free polynucleotide delivery is minimal mvasivity.
  • the delivery technique in vitro or in vivo may involve direct injection, particle bombardment, jet injection systems, electroporation and catiomc liposomes.
  • the use of liposomes for delivery of the free polynucleotides of the invention is not preferred as such delivery mechanisms frequently result m reduced levels of expression
  • 'Dermal' administration refers to routes of administration which apply the free polynucleot ⁇ de(s) on, to or through skm.
  • Dermal routes include epidermal, mtradermal and subcutaneous injections as well as transdermal transmission.
  • the transdermal transmission may be active (e.g. delivery driven by iontophoresis) or passive (e.g., delivery driven by diffusion alone).
  • Iontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously through unbroken skm for periods of several days or more. Use of this method allows for controlled transmission of pharmaceutical compositions m relatively great concentrations, permits infusion of combination drugs and allows for contemporaneous use of an absorption promoter.
  • the introduction of the polynucleotide preparation can be accomplished by simple intramuscular (IM) or mtradermal (ID) injections using needles, the combination of needle or other injection methods together with electroporation, as well as by propelling DNA-coated gold particles through various tissues, preferentially the dermis.
  • IM intramuscular
  • ID mtradermal
  • electroporation electroporation
  • DNA-coated gold particles through various tissues, preferentially the dermis.
  • the level of expression of the polynucleotide of interest leads to surp ⁇ smgly strong immune responses [Fynan EF et al.,
  • the instant invention allows for direct delivery of polynucleotides to mucosal sites to be revisited
  • the means of introduction will vary according to the location of the point of entry.
  • mtranasal administration means are most preferred. These means include inhalation of aerosol suspensions or insufflation of the naked polynucleotide or mixtures thereof.
  • Suppositories and topical preparations will also be suitable for introduction to certain mucosa, such as genital and ocular sites.
  • vaginal delivery of free polynucleotides are vaginal sandwich-type rings and pessa ⁇ es.
  • Respiratory-associated epithelia may be accessed using drug delivery systems which are based on oro- and/or nasopharyngeal administration.
  • drug delivery systems which are based on oro- and/or nasopharyngeal administration.
  • ontogeny, differentiation, function, and pathologies of cells lining respiratory tissues vary, this large, continuous, arbo ⁇ zed epithelial surface shares the common feature of being exposed to inhaled gases and particulates. Physicians are able to select from a wide range of well developed technologies for administering drugs to this surface via the naso- and oropharynx.
  • compositions of free polynucleotides and mixtures of polynucleotides may be placed into a pharmaceutically acceptable suspension, solution or emulsion.
  • the particular pharmaceutically acceptable carrier or diluents employed is not critical to the present invention and will necessarily vary with the administration route, particular host organism and target tissue.
  • the polynucleotide may be dissolved in water, salme or an endotoxm-free mjectable PBS.
  • the concentration preferably ranges from 0.1 to 2 mg/ml (w/v), more preferably 1 mg/ml (w/v)
  • the polynucleotide may be injected in 25% (w/v) sucrose.
  • diluents include a phosphate buffered salme, citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame
  • carriers include proteins, e.g., as found in skim milk, sugars, e.g., sucrose, or polyvmylpyrrolidone (PVP). Typically these carriers would be used at a concentration of about 0.1-90% (w/v) but preferably at a range of 1-10% (w/v).
  • Suitable mediums include salme and may include hposomal preparations (for those embodiments which do not rely on antigen presenting cells for delivery of the polynucleotides into target tissue) More specifically, pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and mjectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replemshers, electrolyte replemshers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelatmg agents, and inert gases and the like Further, a composition of free polynucleotides may be lyophihzed using means well known in the art, for subsequent reconstitution and use according to the invention.
  • delivery of the polynucleotide is preferably facilitated without need for injection by use of detergents, absorption promoters, chemical irritants (such as keratmolytic agents), or mechanical lr ⁇ tants.
  • Detergents and absorption promoters which facilitate uptake of small molecules other than ' genes are well known in the art and may, without undue experimentation, be adapted for use in facilitating uptake of genes.
  • Another substantially nonmvasive approach to introducing the free polynucleotides is by transdermal transmission (preferably iontophoresis) which has been used with success for transdermal transmission of peptides.
  • the amount and concentration of the polynucleotide to be administered will vary depending on the species of the subject, as well as the desired response and the disease or condition that is being treated. Generally, it is expected that up to 100-200 ⁇ g of DNA can be administered in a single dosage, although as little as about 0.3 ⁇ g of DNA administered through skm or mucosa can induce long lasting immune responses. For purposes of the mvention, however, it is sufficient that the free polynucleotides be supplied at a dosage sufficient to cause expression of the polynucleotide of interest carried by the polynucleotide.
  • Dose-response experiments can be used to efficacy, toxicity, and effective dose of a particular enhancing agent or polynucleotide preparation. Such experiments were used to define an effective dose for ATA as an adjuvant for direct transfection of respiratory tissues. Histologic evaluation of treated lung tissue was subsequently performed. As virtually all medicines exhibit toxicity at some dose, an attempt was made to define the upper end of the dosmg range by identifying a toxic dose for mtratracheal ATA administration. ATA is often employed for inhibition of cultured cell apoptosis, but relatively few publications describe in vivo applications.
  • the polynucleotide preparations herein are specifically designed for direct delivery of the polynucleotide of interest to a particular host organism or target tissue or cell.
  • the polynucleotide preparations may be used to enhance polynucleotide transfer to cells and/or tissues both in vivo and in vitro.
  • the target cell and/or tissue is not critical to the invention.
  • the target tissues include all eukaryotic cells as well as prokaryotic cells in which mtracellular nuclease activity reduces transfection activity.
  • the cells may be present m the intact animal, a primary cell culture, explant culture or a transformed cell line.
  • Certain cells or tissues contemplated by the instant invention include but are not limited to embryos and embryonic tissues, fetal tissues, oocytes, embryonic or pleu ⁇ potent tissue or cells.
  • the particular cells and tissue source are ⁇ f the cells is not critical to the present invention.
  • Injection of uncomplexed polynucleotides results in transfection of cells within a wide variety of tissues, including skeletal muscle, cardiac muscle, liver, tumors, skm, thyroid, thymus, synovium, and bram [Wolff, Nat Med. 3(8)-849-854 (1990); Conry, Int J Biochem Cell Bio. 27:633-45 (1995); Acsadi, Biol Chem.
  • the host organism employed m the present invention is not critical thereto and includes all organisms withm the kingdom ammalia, such as those of the families mammalia, pisces, avian, reptiha.
  • Preferred animals are mammals, such as humans, bovmes, ovmes, porcmes, felines, buffalos, canines, goats, equmes, donkeys, deer, and primates. The most preferred animal is a human.
  • tissues are preferred targets for expression of the polynucleotide of interest.
  • particular tissues of interest include but are not limited to skeletal muscle, cardiac muscle, vascular endothehum, liver, tumors, skm, thyroid, thymus, synovium, and bram.
  • preferred target tissues are those which have a high level of DNAse activity, those that secrete large amounts of DNAse, or those that exist m an environment having a high level of DNAse activity.
  • Such tissues include but are not limited to hepatic, pancreatic, mucosal and respiratory tissues.
  • mucosal tissue or mucosal associated tissues contemplated include oral tissues, ocular tissues, gastro-mtestmal tissues, gut-associated lymphoid tissues, bronchial-associated lymphoid tissues, nasal-associated lymphoid tissues, genital-associated lymphoid tissues, Waldeyer's ⁇ ng, Peyer's patches, and tonsils.
  • Particular respiratory tissues or respiratory associated tissues contemplated include oropharyngeal mucosa, nasopharyngeal mucosa, conducting airway epithelium, and pulmonary parenchyma.
  • Respiratory tract tissues are subject to a variety of pathologies, provide a first line of defense against many environmental toxms and pathogens, are selectively permeable to a range of molecules including biopolymers, and are largely associated with a rich vascular bed. Therefore, technologies for genetic modification of respiratory tissues may be used to develop treatments for inborn errors of metabolism (such as Cystic Fibrosis), to treat systemic disease via absorption to or from the circulatory compartment or to modify either pathologic or beneficial immune responses (asthma, mucosal immunity).
  • 'polynucleotide of interest' means a polynucleotide sequence encoding a protein or fragment thereof or anti-sense RNA or catalytic RNA, which is endogenous or foreign to the particular host species.
  • the term 'recombinant polynucleotide' refers to a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin which is distinct m form, linkage or association from the form, linkage, or association m which the polynucleotide exists in nature.
  • the term 'recombinant DNA' refers to a DNA molecule produced by operatively linking two DNA segments.
  • a recombinant DNA molecule is a hybrid molecule comprising at least two nucleotide sequences not normally found together m nature.
  • Recombinant DNA molecules not having a common biological origin i.e., evolutiona ⁇ ly different
  • 'purified polynucleotide' refers to a polynucleotide which is essentially free, I e., containing less than about 50%, preferably less than about 70%, even more preferably less than about 90% of polypeptides with which the polynucleotide is naturally associated.
  • the polynucleotide preparation delivers a sequence specific polynucleotide of interest into a target cell or tissue.
  • the polynucleotide of interest may encode for a gene, vaccine antigen, an immunoregulatory agent, or a therapeutic agent.
  • the * polynucleotide of interest may be either a foreign gene or a endogenous gene.
  • the polynucleotide of interest need not encode a protein Rather, the polynucleotide of interest may be a therapeutic polynucleotide or one which will affect the biology of a cell, tissue or host; such polynucleotides find particular utility in the field of gene discovery (e.g., identification and functional characterization of new genes) and rational drug design (e.g., identification and validation genetic targets for pharmaceutical manipulation).
  • 'foreign gene' means a polynucleotide encoding a protein or fragment thereof or anti-sense RNA or catalytic RNA, which is foreign to the recipient animal cell or tissue, such as a vaccine antigen, immunoregulatory agent, or therapeutic agent.
  • An 'endogenous gene' means a polynucleotide encoding a protein or part thereof or anti-sense RNA or catalytic RNA which is naturally present in the recipient animal cell or tissue.
  • the polynucleotide of interest encodes a vaccine antigen.
  • the term 'vaccine antigen' refers to an agent capable of stimulating the immune system of a living organism, inducing the production of an increased level of antibodies, the production of a cellular immune response, or the activation other immune responsive cells involved in the immune response pathway against said antigen.
  • the vaccine antigen expression may be performed to elicit an immune response and/or to induce tolerance to the encoded antigen.
  • expression of antigens in cells which lack co-stimulatory molecule expression can enable the development of tolerance to the antigen.
  • the vaccine antigen may be a protein or antigemc fragment thereof from viral pathogens, bacterial pathogens, and parasitic pathogens.
  • the vaccine antigen may be a synthetic polynucleotide, constructed using recombinant DNA methods, which encode antigens or parts thereof from viral, bacterial, parasitic pathogens. These pathogens can be infectious m humans, domestic animals or wild animal hosts.
  • the antigen can be any molecule that is expressed by any viral, bacterial, parasitic pathogen prior to or during entry into, colonization of, or replication in their animal host.
  • the viral pathogens from which the viral antigens are derived, include, but are not limited to, Orthomyxoviruses, such as influenza virus, Retroviruses, such as RSV and SIV, Herpesviruses, such as EBV; CMV or herpes simplex virus, Lentiviruses, such as human immunodeficiency virus; Rhabdoviruses, such as rabies; Picornoviruses, such as pohovirus; Poxviruses, such as vaccinia; Rotavirus; and Parvoviruses.
  • Orthomyxoviruses such as influenza virus, Retroviruses, such as RSV and SIV, Herpesviruses, such as EBV; CMV or herpes simplex virus, Lentiviruses, such as human immunodeficiency virus; Rhabdoviruses, such as rabies; Picornoviruses, such as pohovirus; Poxviruses, such as vaccinia; Rotavirus; and Par
  • protective antigens of viral pathogens include the human immunodeficiency virus antigens Nef, p24, gpl20, gp41, Tat, Rev, and Pol et al, Nature, 313:277-280 (1985)) and T cell and B cell epitopes of gpl20(Palker et al, J. Immunol., 142:3612-3619 (1989)); the hepatitis B surface antigen (Wu et al, Proc. Natl. Acad. Sci., USA, 86:4726-4730 (1989)); rotavirus antigens, such as VP4 (Mackow et al, Proc. Natl. Acad.
  • influenza virus antigens such as hemagglutmm or nucleoprotem (Robinson et al., Supra; Webster et al, Supra) and herpes simplex virus thymidme kmase (Whitley et al, In: New Generation Vaccines, pages 825-854).
  • the bacterial pathogens, from which the bacterial antigens are derived include but are not limited to, Mycobacterium spp., Hehcobacter pylon, Salmonella spp., Shigella spp., E.
  • protective antigens of bacterial pathogens include the Shigella sonnei form 1 antigen (Formal et al, Infect. Immun., 34:746-750 (1981)); the O-antigen of V. cholerae Inaba strain 569B (Forrest et al, J. Infect. Dis., 159:145-146 (1989); protective antigens of enterotoxigemc E. coh, such as the CFA I fimb ⁇ al antigen (Yamamoto et al, Infect.
  • the parasitic pathogens from which the parasitic antigens are derived, include but are not limited to, Plasmodium spp., Trypanosome spp., Giardia spp., Boophilus spp., Babesia spp., Entamoeba spp., Eime ⁇ a spp., Leishmania spp , Schistosome spp., Brugia spp., Fascida spp., Dirofila ⁇ a spp., Wuchere ⁇ a spp., and Onchocerea spp.
  • protective antigens of parasitic pathogens include the circumsporozoite antigens of Plasmodium spp. (Sadoff et al, Science, 240:336-337 (1988)), such as the circumsporozoite antigen of P. bergern or the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium spp. (Spetzler et al, Int. J. Pept. Prot. Res., 43:351- 358 (1994)); the galactose specific lectin of Entamoeba histolytica (Mann et al, Proc. Natl. Acad.
  • the polynucleotide of interest can encode a therapeutic agent to animal cells or animal tissue.
  • the polynucleotide can encode tumor-specific, transplant, or autoimmune antigens or parts thereof.
  • the polynucleotide can encode synthetic genes, which encode tumor-specific, transplant, or autoimmune antigens or parts thereof.
  • tumor specific antigens include but are not limited to prostate specific antigen (Gattuso et al, Human Pathol., 26:123-126 (1995)), TAG-72 and CEA (Guadagm et al, Int. J. Biol. Markers, 9:53-60 (1994)), MAGE-1 and yrosmase (Couhe et al, J. Immunothera., 14:104-109 (1993)).
  • mice immunization with non- malignant cells expressing a tumor antigen provides a vaccine effect, and also helps the animal mount an immune response to clear malignant tumor cells displaying the same antigen (Koeppen et al, Anal. N.Y Acad. Sci., 690:244-255 (1993)).
  • transplant antigens examples include the CD3 receptor on T cells (Alegre et al, Digest. Dis. Sci., 40:58-64 (1995)). Treatment with an antibody to CD3 receptor has been shown to rapidly clear circulating T cells and reverse most rejection episodes (Alegre et al, supra).
  • autoimmune antigens examples include IAS ⁇ -cham (Topham et al, Proc Natl Acad. Sci., USA, 91.8005-8009 (1994)). Vaccination of mice with an 18 ammo acid peptide from IAS ⁇ -cham has been demonstrated to provide protection and treatment to mice with experimental autoimmune encephalomyehtis (Topham et al, supra).
  • the polynucleotide of interest can encode immunoregulatory molecules.
  • immunoregulatory molecules include, but are not limited to, growth factors, such as M-CSF, GM-CSF; and cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 or IFN-.gamma Delivery of cytokines expression cassettes to tumor tissue has been shown to stimulate potent systemic immunity and enhanced tumor antigen presentation without producing a systemic cytokine toxicity (Golumbek et al, Cane. Res.,
  • the polynucleotide of interest may encode an antisense RNA or catalytic RNA.
  • the RNA can be targeted against any molecule present withm the recipient cell or likely to be present withm the recipient cell. These include but are not limited to RNA species encoding cell regulatory molecules, such as ⁇ nterlukm-6 (Mahieu et al, Blood, 84:3758-3765 (1994)), oncogenes such as ras (Kashani-Sabet et al, Antisen. Res. Devel., 2:3-15 (1992)), causitive agents of cancer such as human papillomavirus (Steele et al, Cane.
  • RNAs can also be targeted at non-transcribed DNA sequences, such as promoter or enhancer regions, or to any other molecule present m the recipient cells, such as but not limited to, enzymes involved in DNA synthesis or tRNA molecules [Scanlon et al, Proc. Natl. Acad. Sci. USA. 88:10591- 10595 (1991); and Baier et al, Mol. Immunol.. 31:923-932 (1994)].
  • a plasmid was administered m combination with pharmaceuticals which indirectly or directly inhibit nucleases. It was observed that DNAse activity in lung fluids significantly contributes to the respiratory clearance of extracellular plasmid..
  • a plasmid employing the human CMV IE promoter/enhancer to drive expression of the P pyrahs luciferase reporter protein was mtratracheally administered into mouse lung +/- the nuclease/apoptosis inhibitor aurm t ⁇ carboxylic acid (ATA). Lavage samples and tissue extracts were used to characterize nuclease activity. ATA dose escalation experiments were performed using lung homogenate reporter protein assays to characterize transfection.
  • toxicity was assessed histologically. Reporter protein levels detected m lung extracts were markedly enhanced (50 to 65 fold) by treatment with the nuclease/apoptosis inhibitor au ⁇ n t ⁇ carboxylic acid (ATA), but not with EDTA or sodium citrate. Histological features of tissues treated +/- optimized ATA were indistinguishable. It is conceivable that the transfection enhancing activity of ATA involves inhibition of nuclease activity and/or interference with apoptosis. The identification and pharmaceutical inhibition of pathways which limit the tissue transfection activity of 'free' plasmid can enhance the efficiency of direct DNA delivery systems.
  • ATA nuclease/apoptosis inhibitor au ⁇ n t ⁇ carboxylic acid
  • Plasmids and Chemical Reagents The P pyralis luciferase cDNA was subcloned into the plasmid pND2/CMV. This plasmid was transformed into competent E. coh DH5-a cells, amplified m terrific broth, and prepared by alkaline lysis with the isolation of covalently closed circular plasmid DNA using two rounds of CsCl-EtBr gradient ultracent ⁇ fugation. The plasmid DNA was subsequently treated with DNAse-free RNAse, phenol/chloroform extracted, and purified by precipitation from an ethanol/sodium acetate solution. DNA purity was determined by agarose gel electrophoresis and optical density (OD 260/280 greater than or equal to 1.8). The resulting plasmid DNA is referred to as pND2Lux. See Figure 6 for plasmid map.
  • DOTAP was prepared neat as a dry thm film (1 mM), followed by resuspension sterile water (1 ml, 1 mM) with vortex mixing and somcation at the time of use.
  • Au ⁇ n t ⁇ carbarboxyic acid (Sigma A-0885) was used to test the hypothesis that ATA might affect DNA transfection m both the m-vivo and m-vitro systems. The doses and treatment groups are described below.
  • the amplified DNA was inserted into pND using the same sites.
  • the product was tested for activity by transfectmg NIH 3T3 cells and staining for enzyme activity
  • the ⁇ - galactosidase Expression Vector may further be utilized.
  • a hybrid ⁇ -galactosidase gene was amplified from a commercial vector (pCMV ⁇ , Clonetech, Palo Alto, CA) incorporating Sal I and BamH I sites into the primers.
  • the first 28 ammo acids are from Drosophila Alcohol Dehydrogenase followed by the fused E. coh ⁇ -galactosidase sequences.
  • the insect sequences are reported to give higher expression m mammalian cells presumably by providing eukaryotic translation initiation signals.
  • the amplified DNA was inserted into pND using the same sites.
  • the product was tested for activity by transfectmg NIH 3T3 cells and stammg for enzyme activity.
  • the human respiratory epithelial cell line, 16HBE14o (Dieter Grunert, UCSF) was cultured on flasks coated with fibronectm (Collaborative Research-Bectm Dickinson), vitrogen (Collagen Corp), basal media (Biofluids, Inc.) and bovme serum albumen (Biofluids, Inc.) as previously described [Gruenert, 1990 #7; Cozens, 1994 #8]. Cells were cultured on twenty four well tissue culture plates (Corning) coated with the above mixture. Culture media was * Eagle's modified essential medium (MEM) supplemented with 10% fetal bovme serum.
  • MEM Eagle's modified essential medium
  • 16HBE14o cells were plated at a density of 5 x 10 4 cells/ml 24 hours p ⁇ or to transfection and cells were transfected as subconfluent monolayer.
  • NIH3T3 (ATCC CRL 1658) were grown in Dulbecco's Modified Eagle's Medium and 10% calf serum. NIH3T3 were also plated at a density of 5 x 10 4 cells/ml m 24 well plates 24 hours p ⁇ or to transfection and cells were transfected as subconfluent monolayer.
  • the treatment groups of aurmt ⁇ carbarboxyic acid were 80 ⁇ g/ml, 40 ⁇ g/ml, 10 ⁇ g/ml, 2 ⁇ g/ml, 1 ⁇ g/ml, 0.5 ⁇ g/ml, 0.25 ⁇ g/ml, and 0.0125 ⁇ g/ml. Each treatment group was repeated in four wells. All experiments were repeated twice and both NIH3T3 and 16HBE14o cell lines were used. In vitro transfections using NIH3T3 and 16HBE14o cell lines yielded no significant differences between groups that received the DNA with ATA/DOT AP versus those groups that just received the DNA/DOTAP complexes at ATA does which were comparable to those given to mice .
  • the ATA was toxic and killed cells which greatly reduced the number of relative light units.
  • the examples described herein shows a 57% increase over free DNA transfections with doses of ATA that were virtually nontoxic to the lung as shown by H&E staining.
  • Health assessments were also used to determine the health status mice for up to 48 hours after transfection. All mice were healthy with no physical or behavioral side effects observed.
  • mice were sacrificed and the lungs were lavaged with 300 microliters of phosphate buffered salme. 10 microliters of this salme wash was then mixed with 750 ng of plasmid DNA m PBS to yield a total of 20 microliters. Samples were incubated for 1/2 hour to 2 hours at 37 °C, and 1% mimgel agarose TBE gel electrophoresis performed. The data summarized m the Figure 1 is representative of assays performed using lavage fluid obtained from 4 different mice, and demonstrates the presence of significant levels of nuclease activity in murme lung lavage fluid.
  • ATA-Mediated Inhibition of Nuclease Activity To detect degradation of mtratracheally administered plasmid, and to assess the effect of ATA on this process in vitro, lavage samples were prepared as described above, and a whole lung tissue extract was prepared by homogemzation of lung m PBS followed by clarification via cent ⁇ fugation. As a positive control for DNAse activity, dornase alpha (Pulmozyme, Genentech, So. SF, CA) was obtained as a lmg/ml solution. Optimal conditions for degradation of plasmid were determined by mini gel analysis.
  • Example II In vivo Transfection -Mice Five 7 week old female Balb-C mice (Charles River) were used for the in vivo expe ⁇ ments. All mice were anesthetized with a mixture of ketamme (22 mg/kg), xylazme (2.5 mg/kg) and acepromazme 0.75 mg/kg) IP p ⁇ or to both mtratracheal or intramuscular (IM) injections.
  • Intratracheal injections were performed by making a 1 cm medial cut through the skm at the ventral site of the neck The musculature and salivary gland were teased apart using blunt dissection to expose the trachea. With the trachea visualized, a 30.5 gauge needle with a 1 cc tuberculin synnge was placed through the rings of the trachea toward the bronchi. DNA and DNAse inhibitors with DNAse inhibitors (see below) for a total volume of 100 ⁇ l in water for injection were administered into the lung. After injection, the salivary gland was placed back over the trachea, and the superficial neck wound was closed with staples using a 9 mm autochp appher (Bectm-Dickmson).
  • Intratracheal treatment groups consisted of four mice and each group received the following treatments: 8 ⁇ g/g, 6 ⁇ g/g, 4 ⁇ g/g, 2 ⁇ g/g, 1 ⁇ g/g, 0.5 ⁇ g/g, 0.4 ⁇ g/g, 0.3 ⁇ g/g, 0.2 ⁇ g/g, or 0.1 ⁇ g/g of ATA, 50 mm or 100 mm EDTA, 50 mm or 100 mm sodium citrate with the 100 ⁇ g of DNA. All experiments had a DNA alone group and water for injection only as a control group. All experiments were repeated at least once. The results are shown in Figures 3A and 3B.
  • Intramuscular injections were performed by injecting 50 ⁇ l of DNA or DNA ATA complexes into the quadriceps muscle. Intramuscular treatment groups consisted of four mice. All groups received pND2Lux with or without 6 ⁇ g/g ATA.
  • mice were euthanized using C02 at 48 hours and the tracheal /lung block or quadriceps muscle were dissected out, and assayed as described below.
  • Relative luciferase activity was determined using the Enhanced Luciferase Assay Kit and Monohght 2010 luminometer (Analytical Luminescence Laborato ⁇ es, San Diego, CA). For tissue culture, this was accomplished by directly applying 330 ⁇ l of concentrated luciferase lysis buffer to each well and placing the cells on ice for 30 minutes. For in vivo expe ⁇ ments, each lung/trachea block or quadricep muscle was homogenized in 1 ml of lysis buffer (diluted 1 :3 in distilled water). Samples were allowed to sit on wet ice for thirty minutes.
  • luciferase light emissions from 20 ⁇ l of the cell lysate were measured over a 10 second period, and results expressed as a function of the total lysate volume.
  • luciferase light emissions from 40 ⁇ l of the lysate were measured over a 10 second pe ⁇ od, and results expressed as a function of the total lysate volume
  • mice lung tissue was treated with ATA +/- plasmid and routine histologic analyses were performed High pulmonary doses of ATA (> 2 ⁇ g/g body weight) were associated with significant mortality, and expression of significant levels of TNF- ⁇ after mtratracheal 'free' plasmid administration have been reported by Tsan et al Hum Gene Ther.
  • Example III In vivo Transfection - Macaque Extending the experiment from murme to nonhuman primate respiratory tissues, the activity of 'free' plasmid DNA +/- ATA in macaque respiratory tissues using male rhesus macaques was tested Initially, the lavage fluid is tested for DNAse activity +/- ATA by incubation with plasmid after which gel electrophoresis is performed as desc ⁇ bed above with mu ⁇ ne lung lavage samples After a 70 day quarantine, the animals are treated using three general techniques For direct administration of lipid DNA complexes to pulmonary parenchyma, macaques are anesthetized (ketamme, veterinarian assisted) and a pediat ⁇ c bronchoscope will be passed per nasum with direct visualization of the bronchial tree Typically, plasmid preparations (3ml/Kg macaque weight) or related treatments (plasmid/ ATA mixtures etc ) are instilled directly via the bronchoscope into the right lower lobe The left
  • Tissue samples were either formalin fixed, processed and stained (H&E), were frozen in OCT for in situ hybridization, or were homogenized and assayed for luciferase activity.
  • Typical levels of luciferase expression are summarized below in Table 2, and key findings from these experiments include 1) lack of histologically apparent acute toxicity with 'free' plasmid administration with or without ATA, and 2) substantially higher levels of luciferase activity m tissues treated with 'free' plasmid and ATA.
  • relative luciferase activity is determined using the Enhanced Luciferase Assay Kit and a Monohght 2010 lummometer (both from Analytical Luminescence Laboratories, San Diego, CA) Typically the tissue sample is weighed and 2 volumes of lx luciferase lysis buffer (final concentration 0.1M KP0 4 pH 7.8, 1% T ⁇ ton X-100, ImM DTT, 2mM EDTA) is added and the sample is stored on water ice. Samples are then homogenized to uniformity using a Branson sonifier (typically 30 seconds to one mmute).
  • a Branson sonifier typically 30 seconds to one mmute.
  • Luciferase light emissions from 31 microliters of the lysate are measured over a 10 second pe ⁇ od, and results are expressed as a function of total protein, total tissue mass, and total DNA dose.
  • XGal histochemistry is performed for the detection of beta-galactosidase.
  • Fixation of cryosections consists of treatment with paraformaldehyde/pipes (4C, 5 minutes) followed by washing (IX PBS with 2mM MgCl 2 , 5 minutes, 4C, repeat twice) and permeabihzation (lx PBS, 2mM MgCl 2 , 0.01% sodium deoxycholate, 0.02% NP40, 5 minutes, 4C).
  • the sections are then stained for 2 to 8 hours with Xgal (5-bromo-4-chloro-3-mdoyl b-D-galactopyranoside, lmg/ml) m 30mM K 3 Fe(CN) 6 , 30mM K 4 Fe(CN) 6 .3H 2 0, 2mM MgCl 2 0.01% sodium deoxycholate, 0.02% NP40 m IX PBS. (37C) After staining, the sections are again washed in PBS, lightly counter stained with eosm, and cover slipped for analysis.
  • Enhanced green fluorescent protein activity is assessed using wet tissue obtained either at necropsy or via biopsy.
  • wet tissue is compressed between glass slides and viewed with an inverted Nikon eclipse microscope (TE 200) equipped with 4, 10, 40 ' and 60x objectives and an appropriate light source and filters. Images are captured using a Dage 330 3 chip color digital camera and a Scion CG-3 color capture board operated from a Macintosh 7200 under the control of Scion Image software (vl .62). This system enables frame averaging and compilation, providing substantial low light sensitivity.
  • Example IV Intradermal Injection - Mouse. Rat, and Macaque
  • the experiment was further extended from murme to primate, testing direct mtradermal injection on macaques.
  • the control solution was identical to that described for the mice and rat protocols.
  • the test solution contained 100 ⁇ g of pND21ux plasmid and 50 ⁇ g of ATA in 100 ⁇ l water.
  • Ten macaques were included m the control group and twelve were mcluded m the test group.
  • the results are shown below in Table 3. On average, the test group showed an 11 fold enhancement of reporter gene expression.
  • Statistical analysis of the results shows p ranges from 0.048 (one tail) to 0.095 (two tail).
  • Example V ATA Enhancing Immune Response in Vivo
  • a test antigen beta-galactosidase
  • mice were mnoculated with 100 micrograms of pND2beta (encoding beta-galactosidase) via intradermal, mfranasal, intramuscular and mtratracheal routes using the same techniques described above.
  • ATA dose was 1.0 microgram/gram animal weight.
  • Beta- galactosidase assays showed increased IgG and IgA responses to infradermal (ID), infranasal (IN), intramuscular (IM) and intratracheal (IT) pND2beta plasmid innoculations.
  • (+) indicates increased antibody response relative to pND2betas plasmid without added cofactors
  • Example VI DNAse Activity in Lung Fluid
  • Lung lavage fluid was obtained from mice, macaques and humans.
  • the mouse experiments involved 4 test animals (A, B, C, and D) and one plasmid control.
  • the macaque experiments involved 3 test animals (J788, J740, and J628) and one plasmid control.
  • the human experiments involved six test patients (A, B, C, D, E, and F) and one plasmid control.
  • Two hundred nanograms of lung lavage protein was incubated at 37 ° C with 1 ⁇ g of pND2Lux in a 1 mM solution of MnCl 2 for 2 hours. Varying amounts of ATA were added (1 or 4 ⁇ g). Mass of protein was determined by Bradford Assay and normalized for all samples.

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Abstract

La découverte de procédés pharmaceutiquement définis, non toxiques et simples pour modifier génétiquement des cellules et des tissus permet de développer des variétés de médecines moléculaires. L'administration d'un polynucléotide libre, direct ou nu est un procédé d'apport de polynucléotide pharmaceutiquement défini, simple, et apparemment sûr. Des essais cliniques effectués sur des souris, des macaques et des hommes ont montré une transaction de différents tissus, tels que des tissus respiratoires, après l'application directe d'un polynucléotide libre. Toutefois, une transfection directe d'ADN est relativement inefficace en comparaison de beaucoup de systèmes de transfection. Cette invention concerne des agents d'amélioration de transfection qui augmentent l'activité de transfection d'un polynucléotide libre, ce qui facilite le développement d'alternatives simples et sûres à la transaction de tissu, et plus particulièrement à la transaction de tissu respiratoire. Les expériences décrites dans les spécifications montrent que des nucléases, à la fois extra- et intracellulaires, présents dans de nombreux fluides biologiques, tel qu'un fluide respiratoire, accélèrent le dégagement de plasmide biologiquement actif à partir du tissu, et que la co-administration d'un inhibiteur de nucléase avec un polynucleotide libre a pour effet une amélioration marquée de l'expression du polynucléotide considéré. Ces découvertes justifient l'invention d'un système d'apport de polynucléotide amélioré, et d'une technologie de transfection à base de plamide libre.
PCT/US1999/018726 1998-08-19 1999-08-19 Composes et procedes permettant d'ameliorer l'apport d'un polynucleotide libre WO2000011217A1 (fr)

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CA002341017A CA2341017A1 (fr) 1998-08-19 1999-08-19 Composes et procedes permettant d'ameliorer l'apport d'un polynucleotide libre
AU55684/99A AU5568499A (en) 1998-08-19 1999-08-19 Compounds and methods for enhancing delivery of free polynucleotide
EP99942265A EP1119642A1 (fr) 1998-08-19 1999-08-19 Composes et procedes permettant d'ameliorer l'apport d'un polynucleotide libre

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004042061A1 (fr) * 2002-11-08 2004-05-21 Klaus Strebhardt Composition pharmaceutique de suppression de l'expression non souhaitee d'un gene

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5830730A (en) * 1997-05-08 1998-11-03 The Regents Of The University Of California Enhanced adenovirus-assisted transfection composition and method

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5830730A (en) * 1997-05-08 1998-11-03 The Regents Of The University Of California Enhanced adenovirus-assisted transfection composition and method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROSS G.F. ET AL: "Enhanced reporter gene expression in cells tranfected in the presence of DMI-2, an acid nuclease inhibitor", GENE THERAPY, vol. 5, 1998, pages 1244 - 1250, XP002922028 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004042061A1 (fr) * 2002-11-08 2004-05-21 Klaus Strebhardt Composition pharmaceutique de suppression de l'expression non souhaitee d'un gene

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