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WO2001011960A1 - Systeme de livraison genique specifique de muqueuse a base d'exotoxine bacterienne modifiee - Google Patents

Systeme de livraison genique specifique de muqueuse a base d'exotoxine bacterienne modifiee Download PDF

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Publication number
WO2001011960A1
WO2001011960A1 PCT/US2000/022715 US0022715W WO0111960A1 WO 2001011960 A1 WO2001011960 A1 WO 2001011960A1 US 0022715 W US0022715 W US 0022715W WO 0111960 A1 WO0111960 A1 WO 0111960A1
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Prior art keywords
nucleic acid
dna
exotoxin
delivery system
gene
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Lisa M. Welter
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AGRIVAX Inc
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AGRIVAX Inc
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention consists of a novel nucleic acid delivery system that will specifically facilitate targeted delivery of nucleic acid.
  • This novel system makes use of recombinant toxin protein components modified to contain a nucleic acid binding moiety resulting in the targeted delivery of nucleic acid to mucosal surfaces.
  • the invention provides methods for therapeutic and prophylactic treatment using the delivery system.
  • coli heat-labile enterotoxin (LT) and cholera toxin (CT) are related members of the ADP-ribosylating exotoxin (bARE) family, which also include Bordetella pertussis-de ⁇ ved pertussis toxin (PT), Pseudomonas aeruginosa exotoxin A (ETA), and Coryne bacterium diphtheria-de ⁇ ved diphtheria toxin Krueger et al (1995).
  • PT Bordetella pertussis-de ⁇ ved pertussis toxin
  • ETA Pseudomonas aeruginosa exotoxin A
  • Both LT and CT have five identical B subunits that form a pentamer which facilitates binding to cell surface ganglioside receptors (GM1).
  • LT exhibits a lectin- like binding capacity that results in the binding to a broader range of receptors on mammalian cells for LT than for CT, which binds only GM1 (Angstrom et al (1994); Clements et al (1980); Holgrem (1994)).
  • CT and LT have an enzymatic active A subunit that enters the cell and catalyzes the ADP-ribosylation of guanine nucleotide binding proteins of the adenylate cyclase complex, resulting in activation of adenylate cyclase and increased intracellular cyclic AMP (cAMP).
  • Both CTA and LTA are proteolytically cleaved into an enzymatically active Al subunit and an A2 linker fragment that is inserted into the central pore of the B pentamer
  • the structure of these proteins has been characterized at the molecular level (Sixma et al (1993)
  • LT and CT are potent mucosal adjuvants (Williams et al (1999), Freytag et al (1999)) Some degree of A subunit enzyme activity is required for oral adjuvant function (Sixma et al (1993)) While ADP-ribosyltransferase activity enhances adjuvanticity, it is also responsible for toxicity Mutant LT and CT molecules have been constructed with altered A subunits, resulting in reduced ADP ribosylation activity and reduced toxicity, yet some maintained their adjuvant function The effect of CT and LT on the immune responses, include antigen presentation, cytokine production, with inhibitory as well as enhancing effects (Williams et al (1999), Matousek et al (1998)]
  • LT and CT have been engineered to carry protein or peptide molecules through chemical coupling or as chimeric fusion proteins (Williams et al (1999), Cardenas et al (1993), Lipscombe et al (1991), Loregian (1999), O'Doed et al (1999), Bagdasarian et al (1999))
  • Both CT and LT modified to remove their toxicity while maintaining their adjuvanticity have been demonstrated to enhance immune responses to a wide variety of co-administered antigens (Freytag et al (1999), Bagdasarian et al (1999))
  • Receptor-mediated gene transfer has been shown to be successful in introducing transgenes into suitable recipient cells, both in vitro and in vivo
  • This procedure involves linking the DNA to a polycatiomc protein (usually poly-L-lysine) containing a covalently attached hgand, which is selected to target a specific receptor on the surface of the tissue of interest
  • the gene is taken up by the tissue, transported to the nucleus of the cell and expressed for varying times
  • the overall level of expression of the transgene in the target tissue is dependent on several factors the stability of the DNA-car ⁇ er complex, the presence and number of specific receptors on the surface of the targeted cell, the receptor-carrier gand interaction, endocytosis and transport of the complex to the nucleus, and the efficiency of gene transcription in the nuclei of the target cells
  • Wu, et al , U S Pat No 5,166,320 discloses tissue-specific delivery of DNA using a conjugate of a polynucleic acid binding agent (such as polylys
  • Stomp, et al , U S Pat No 5, 122,466 and McCabe, et al , U S Pat No 5, 120,657 disclose attaching DNA to a metal pellet by covalently attaching polylysine to the material and then allowing DNA to be complexed to it The resulting product is then used for ballistic transformation of a cell
  • Stomp, et al , column 7, lines 29- 37 and McCabe, et al , column 7, lines 49-65 Wagner, et al., Proc. Natl Acad. Sci. USA, 88:4255-4259 (1991) disclose complexing a transferrin-polylysine conjugate with DNA for delivering DNA to cells via receptor mediated endocytosis.
  • Wagner et al. teach that it is important that there be sufficient polycation in the mixture to ensure compaction of plasmid DNA into toroidal structures of 80-100 nm diameter, which, they speculate, facilitate the endocytic event.
  • the current invention describes an exotoxin engineered to contain a nucleic acid binding moiety such as polylysine or protamine for use as a nucleic acid carrier and delivery system.
  • the current invention utilizes the general principle of coupling nucleic acid to a protein carrier to facilitate gene delivery, but in the described system the carrier protein will consist of recombinant exotoxin complexes engineered to carry the nucleic acid and target delivery to the mucosal surfaces.
  • Such a carrier system for delivery of nucleic acid makes use of exotoxin's specific ability to bind to a broad range of cells of the mucosal surfaces.
  • the described invention is unique in that it does not use covalent linking to attach ligand to the poly cationic protein.
  • the exotoxin variant of the present invention is capable of binding DNA while maintaining its receptor binding capabilities.
  • the novel nucleic acid delivery system of the present invention will specifically facilitate delivery of nucleic acid by utilizing recombinant exotoxin protein components modified to contain a nucleic acid binding moiety resulting in the targeted delivery of genes to the specific cell target (i.e. mucosal membranes).
  • This system consists of two primary components: 1) a modified recombinant exotoxin variant and 2) a nucleic acid containing the gene(s) to be delivered.
  • the exotoxin is engineered to carry exogenous nucleic acid by addition of a nucleic acid affinity domain (NAAD) and/or condensing motif of polylysine, protamine or similar polycationic amino acid sequences, or nucleic acid binding motifs.
  • NAAD nucleic acid affinity domain
  • exotoxin proteins are engineered such that they retain their functional ability to self assemble and bind their natural receptor target (i.e. ganglioside receptor, GM1)
  • GM1 natural receptor target
  • the system thus utilizes components of exotoxins to enhance the specificity and efficiency of nucleic acid delivery, retaining some of the best properties of exotoxins as mucosal carriers, while avoiding problems inherent in biological agents and enabling pharmaceutical quality control over the final preparations.
  • the system offers universal application for use in a variety of animal species including man, the receptor is found on cells in most animal species, as well as the system's added flexibility of complexing any combination or variety of nucleic acids carrying a gene of interest.
  • any exotoxin targeting moiety can be modified to bind and deliver the nucleic acid, including, but not limited to, members of the bacterial ADP-ribosylating exotoxin (bARE) family which include Cholera toxin (CT), E.coli heat-labile enterotoxin (LT), Bordetella pertussis-derived pertussis toxin (PT), Pseudomonas aeriiginosa exotoxin A (ETA) and Corynebacterium diphtheria-derived diphtheria toxin, etc.
  • CT Cholera toxin
  • LT E.coli heat-labile enterotoxin
  • PT Bordetella pertussis-derived pertussis toxin
  • ETA Pseudomonas aeriiginosa exotoxin A
  • Corynebacterium diphtheria-derived diphtheria toxin etc.
  • any nucleic acid binding moiety can be used to modify the exotoxin, including, but not limited to polylysine, protamine and other polycationic amino acid sequences, DNA binding motifs from transcription factors and other nucleoproteins, nucleic acid condensing molecules, and molecular conjugates.
  • Variants of the nucleic acid delivery molecule enhanced for specific receptor targeted delivery and nucleic acid delivery can be generated by applying DNA shuffling techniques familiar to those in the art (for a review see Sedlack (2000); Licking (1999)).
  • This procedure may be applied to human gene therapy.
  • the major advantages of this method are (i) the ease of preparation of the DNA complex; (ii) the ability to target genes to mucosal specific tissues ; and (iii) the relative safety of the complex, since it is devoid of infectious viral DNA.
  • This procedure has may also be applied to nucleic acid vaccine delivery.
  • the major advantages of this method are (i) the ease of preparation of the DNA complex;
  • Utility of this invention includes, but is not limited to delivery of therapeutic or prophylactic genes, vaccines, or maker genes to cells and tissues by ex vivo or in cell culture
  • This invention can be applied to induce an immune response to proteins encoded by the delivered genes, or the commercial production of proteins encoded by the delivered genes, or for the treatment of diseases such as cancer, infectious diseases, hereditary diseases, autoimmune diseases, allergic diseases etc , but its use is not limited to these diseases or to any specific disease state
  • This mucosal nucleic acid delivery system offers the following advantages over recombinant live vectors 1) capacity to transport nucleic acid, 2) the lack of size restriction and therefore the potential capacity to carry any size gene or combination of genes, 3) ease of constructing vectors carrying different antigenic genes, 4) the enhanced ability to enter cells and deliver nucleic acid, 5) elimination of costly production and purification of high titer recombinant viruses, 6) lack of viral genes and therefore virulence or toxicity associated with viral based systems, 7) elimination of potential interference with pre-existing immunity to the vaccine earner, 8) the elimination of problems associated with needle injections, and 9) the targeting of nucleic acid vaccine or gene delivery to the mucosal surfaces
  • Advantages over naked DNA vaccines include the potential to eliminate problems associated with needle injections, facilitation of uptake via the mucosal surfaces, targeting vaccine or therapeutic gene delivery to the mucosal surfaces, efficient internalization, and gene expression
  • Figures 1 A and B show western blot and PAGE analysis of purified recombinant LTBpLh proteins expressed in E. coli
  • Figure 1A shows a Coomassie stain of the PAGE analysis of LTBpLh fractions eluted from the Talon affinity column
  • Lane 1 contains fraction 1
  • lane 2 contains fraction 2
  • lane 3 contains
  • Figure IB shows reactivity of the LTBpLh with anti-his antibody
  • Lane 1 contains pRSETB no insert cell lysate and lane 2 contains pRSET-LTBpLh cell lysate
  • Figure 2 shows PAGE analysis of recombinant LTBpLh and wild type LTB proteins Both are able to assemble into pentamer size complexes (Lanes 2 and 9, respectively)
  • a monomer LTBpLh and wild type LTB proteins (wt LTB) show migration on denaturing SDS-PAGE corresponding to expected size lanes 1 and 8, respectively
  • Lanes 3, 4, and 5, show the effect of DNA on pentamer formation of LTBpLh in the presence of 0 4, 3, or 12 micrograms of GFP plasmid DNA, respectively
  • Lanes 6 and 7 show the effect of 50 ⁇ g of protamine sulfate on LTBpLh complex formation in the presence of 12 micrograms of DNA (lane 6) or absence of DNA (lane 7)
  • Lane 9 shows pentamer formation by wt LTB in the absence of DNA, and lanes 10 and 1 1 in the presence of 12 micrograms of DNA Lanes 11 and 12 are in the presence of 50 micrograms of Protamine Sulfate
  • Figure 3 shows PAGE analysis of recombinant LTBpLh treated with EKMax to remove vector derived sequences
  • Lanes 1 and 2 represent boiled samples and unboiled samples of LTBpLh, respectively
  • Lanes 3 and 4 represent boiled and unboiled LTBpLh-treated with enterokinase M represents molecular weight markers
  • Figure 4 shows DNA binding analysis of recombinant LTBpLh and wild type
  • Figure 5 shows quantitation of GM1 binding by ELISA analysis of recombinant LTBpLh and wild type LTB
  • Figures 6A-D show the effect of DNA (Fig 6 A), or Protamine Sulfate (Fig 6B) or varying concentrations of DNA, Protamine sulfate, and LTBpLh concentrations (Figs 6C and 6D) on GM1 bind by ELISA analysis of recombinant LTBpLh and wild type LTB
  • Figure 7 shows that recombinant LTBpLh enhances cellular DNA uptake and transfection of Yl cells The number of GFP fluorescent cells was assayed by UV microscopy
  • Figures 8A-D show that recombinant LTBpLh are able to target DNA delivery to Yl cells (Fig 8 A) whereas wt LTB did not (Fig 8B) Addition of protamine t sulfate further enhanced delivery by LTBpLh (Fig 8C) as compared to protamine sulfate alone (Fig 8D)
  • Figure 9 is the sequence of LTBpL (SEQ ID NO 9), an LTB fusion protein with a polylysine DNA binding moiety (bold) followed by a termination codon (asterisk)
  • Figure 10 is the sequence of LTBpLh (SEQ ID NO 10), a LTB fusion protein with a hinge region (boxed in capital letters) and a polylysine DNA binding moiety (bold), followed by a termination codon (asterisk)
  • Figure 11 is the sequence of LTB-P (SEQ ID NO 11), an LTB fusion protein with the DNA binding moiety protamine (bold) followed by a termination codon (asterisk)
  • Figure 12 is the sequence of LTB-Ph (SEQ ID NO 12), an LTB fusion protein with a hinge region (boxed in capital letters) and a DNA binding moiety protamine (bold) followed by a termination codon (asterisk)
  • Exotoxin which can be used to make the exotoxin variant of the present invention are preferably exotoxins which are ligands of receptors which are primarily found on mucosal cells, particularly if such receptors are substantially specific to mucosal cells
  • enterotoxins bind not only to intestinal cells, but usually bind to other mucosal cells as well, such as within the respiratory system
  • Preferred examples of exotoxins in accordance with the present invention are members of the bacterial ADP-ribosylating exotoxin (bARE) family which include, but are not limited to, Cholera toxin (CT), E.coli heat-labile enterotoxin (LT), Bordetella pertussis-de ⁇ ved pertussis toxin (PT), Pseudomonas aeruginosa exotoxin A (ETA) and Corynebacterium d ⁇ htheria-de ⁇ ved diphtheria to
  • CT Cholera toxin
  • LT E
  • the "Mucosal Cell Binding Moiety (MCBM)" is a moiety of the exotoxin which binds specifically to an accessible structure (the "receptor") of the intended mucosal cells It is not necessary that it be absolutely specific for those cells, however, it must be sufficiently specific for the conjugate to be therapeutically effective There is no absolute minimum affinity which the MCBM must have for an accessible structure of the mucosal cell, however, its cross-reactivity with other cells should be minimum
  • the MCBM may interact with a lectin, for which there is a cognate carbohydrate structure on the cell surface
  • the MCBM may be a hgand which is specifically bound by any of the known receptors of the exotoxin bARE family or those yet to be defined carried by the mucosal cells
  • One class of ligands of interest are carbohydrates, especially mono- and oligosaccharides Suitable ligands include galactose, lactose and mannose For example it has been shown that
  • Exotoxin variants of the present invention are preferably derived from exotoxin proteins as defined above, or at least the MCBM thereof, through the fusion of a nucleic acid affinity domain to the precursor polypeptide
  • the nucleic acid affinity domain can be operably linked to the precursor polypeptide, such as LTB, at the amino-terminus, the carboxy-terminus or at any suitable position within the precursor polypeptide sequence
  • nucleic acid affinity domain (NADD) is chosen such that it posses an affinity for the nucleic acid to be delivered in the sub-millimolar range
  • nucleic acid affinity domains can be present on a single exotoxin variant The choice of nucleic acid affinity domain can be determined by one skilled in the art
  • NAAD nucleic acid affinity domain
  • the NAAD is a polycation. Its positively charged groups bind ionically to the negatively charged DNA, and the resulting charge neutralization reduces nucleic acid-solvent interactions.
  • NAAD examples include, but are not limited to polycationic domains such as polylysine, polyhistidine, polyarginine, other mixed sequences composed primarily of Arg-Lys-His mixed polymers, polyornithine, histones, avidin, and protamines.
  • NAAD also include domains with homology to know NAAD including helix-turn-helix, leucine zipper, zinc finger, helix-loop-helix, single stranded DNA binding motifs, and RNA binding motifs. Other domains know to bind nucleic acids or those yet to be found are also included.
  • Nucleic acid affinity domains can be readily screened by one skilled in the art for their ability to bind nucleic acids through an electrophoretic gel mobility shift assay or nucleic acid filter binding assays.
  • a second desired feature for the addition of a nucleic acid affinity domain to the exotoxin is that the resulting exotoxin variant must remain structurally functional. For example, it must remain competent for receptor binding and entry into the cell.
  • Variants with suitable NAAD can be screened for their structural and functional integrity, for example, by screening for pentamer formation of the LTB using native polyacrylamide gel electrophoresis as outlined in Example 3, or any other technique known in the art, including sedimentation studies, by screening for receptor binding activity as described Example 3.
  • Exotoxin variants include any derivatives of the variant modified by mutation for enhancement of functional activity.
  • variants can be modified through mutation for enhancement of receptor binding and targeting and or enhanced for nucleic acid binding and delivery.
  • Techniques used to introduce such mutations are known to those skilled in the art for examples, the use of polymerase chain reaction or random mutagenesis to introduce mutations. Examples include point mutations that result in altered codon usage, amino acid substitutions, additions, and or deletions such that the alteration results in a variants of the exotoxin with enhanced or altered structural or functional properties.
  • An example of such mutations includes the substitution of various receptor binding domains.
  • nucleic acid material which may be delivered by the exotoxin variant of the present invention may be any molecule containing nucleic acid.
  • the nucleic acid may be a DNA, RNA, or a DNA or RNA derivative such as a derivative resistant to degradation in vivo, as discussed below.
  • references to DNA apply, mutatis mutandis, to other nucleic acids as well, unless clearly forbidden by the context.
  • the nucleic acid may be single or double stranded.
  • the bases may be the "normal” bases adenine (A), guanine (G), thymidine (T), cytosine (C) and uracil (U), or abnormal bases such as those listed in 37 CFR ⁇ 1.822 (p) (1).
  • the nucleic acid molecule can be synthetic, cDNA, or of genomic origin, or a combination thereof.
  • the gene may be one which occurs in nature, a non-naturally occurring gene which nonetheless encodes a naturally occurring polypeptide, or a gene which encodes a recognizable mutant of such a polypeptide.
  • DNA can include non-transcribed and transcribed regions (such as 5' and 3' non-coding regions, introns and exons) or cDNA and mRNA molecules contain sequences corresponding to transcribed regions.
  • the genetic material may also be a hybrid of the above types of material, or a hybrid with a protein or proteins.
  • the "nucleic acid material” may be prepared by any desired procedure.
  • DNA may be produced by amplification reaction (such as polymerase chain reaction (PCR)), or DNA or RNA may be produced by oligonucleotide synthesis and or ligation of smaller fragments.
  • PCR polymerase chain reaction
  • the nucleic acid material comprises an expressible gene which is functional in the target cell.
  • the gene may encode vaccine antigens or the genes may encode enzymes or factors involved in specific metabolic defects, receptors, or membrane transporters.
  • the coding sequence must be operably linked to a promoter sequence functional in the target cell.
  • Two DNA sequences are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation in the region sequence to direct the transcription of the desired gene sequence, or (2) interfere with the ability of the gene sequence to be transcribed by the promoter region sequence
  • a promoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcription of that DNA sequence
  • a nucleic acid molecule, such as DNA is said to be "capable of expressing" a mRNA if it contains nucleotide sequences which contain transc ⁇ ptional regulatory information and such sequences are "operably linked” to nucleotide sequences which encode the RNA
  • the non-coding region 3' to the gene sequence coding for the desired RNA product may be obtained This region may be retained for its transc ⁇ ptional termination regulatory sequences, such as those which provide for termination and polyadenylation Thus, by retaining the 3 '-region naturally contiguous to the coding sequence, the transc ⁇ ptional termination signals may be provided Where the transc ⁇ ptional termination signals are not satisfactorily functional in the expression host cell, then a 3' region functional in the host cell may be substituted
  • the promoter may be an "ubiquitous" promoter active in essentially all cells of the host orgamsm, e g , for mammals, the beta-actin promoter, or it may be a promoter whose expression is more or less specific to the target cell or a promoter native to a gene which is naturally expressed in the target cell may be used for this purpose
  • promoters include albumin, metallothionem, surfactant, apoE, pyruvate kinase, LDL receptor HMG CoA reductase or any promoter which has been isolated, cloned and shown to have an appropriate pattern of tissue specific expression and regulation by factors (hormones, diet, heavy metals, etc ) required to control the transcription of the gene in the target tissue
  • a broad variety of viral promoters can be used, these include MMTV, SV-40 and CMV
  • An "expression vector” is a vector which (due to the presence of appropriate transc ⁇ ptional and/or translational control sequences) is capable
  • the nucleic acid may comprise sequences homologous to genetic material of the target cell, whereby it may insert itself ("integrate") into the genome by homologous recombination, thereby displacing a coding or control sequence of a gene, or deleting a gene altogether
  • the nucleic acid molecule is "antisense" to a genomic or other DNA sequence of the target organism (including viruses and other pathogens) or to a messenger RNA transcribed in cells of the organisms, which hybridizes sufficiently thereto to inhibit the transcription of the target genomic DNA or the translation of the target messenger RNA
  • the efficiency of such hybridization is a function of the length and structure of the hybridizing sequences The longer the sequence and the closer the complementarily to perfection, the stronger the interaction As the number of base pair mismatches increases, the hybridization efficiency will fall off
  • the GC content of the packaging sequence DNA or the antisense RNA will also affect the hybridization efficiency due to the additional hydrogen bond present in a GC base pair compared to an AT (or AU) base pair
  • a target sequence richer in GC content is preferable as a target It is desirable to avoid antisense sequences which would form secondary structure due to intramolecular hybridization, since this would render the antisense nucleic acid less active or inactive for its intended purpose
  • nucleic acid molecule may be an analogue of DNA or RNA
  • the present invention is not limited to use of any particular DNA or RNA analogue, provided it is capable of fulfilling its therapeutic purpose, has adequate resistance to nucleases, and adequate bioavailabi ty and cell take-up DNA or RNA may be made more resistant to in vivo degradation by enzymes, e g , nucleases, by modifying internucleoside linkages (e g , methylphosphonates or phosphorothioates) or by incorporating modified nucleosides (e g , 2'0-methyl ⁇ bose or 1 '-alpha-anomers)
  • Exotoxin variants can be prepared by a number of known techniques, including peptide synthesis, chemical or enzymatic gation of peptides, and preferably by recombinant expression from host cells
  • the present invention includes nucleic acid molecules encoding the exotoxin protein variants of the invention, vectors, host cells, and production methods
  • a nucleic acid molecule according to the invention encodes, or is complementary to, an exotoxin protein variant of the invention
  • a complementary nucleotide sequence is capable of forming Watson-C ⁇ ck bonds with its complement, in which adenine pairs with thymine or uracil and guamne pairs with cytosine
  • a double-stranded nucleic acid molecule encodes one of the exotoxin protein va ⁇ ants, whereas a single-stranded DNA or RNA molecule is either the coding (sense) or the noncoding (anti-sense) strand
  • nucleic acid molecules related to each exotoxin protein va ⁇ ant More specifically, because several different codons encode the same ammo acid, a large number of different nucleic acid molecules encode (or are complementary to a nucleic acid encoding) the same exotoxin protein va ⁇ ant
  • silent mutations within the scope of the present invention include, e g , mutations that create or destroy restriction endonuclease sites to facilitate construction of a desired vector and mutations that enhance expression of the encoded exotoxin protein variant Examples of the latter include nucleotide substitutions designed to reduce formation of 5' stem and loop structures in transcribed mRNA or to provide codons that are more readily transcribed by the selected host (e g , the well-known preference codons for E coli or yeast
  • a nucleic acid molecule of the present invention can be incorporated into a vector for propagation and/or expression in a host cell
  • Such vectors typically contain a replication sequence capable of effecting replication of the vector in a suitable host cell (l e , an origin of replication) as well as sequences encoding a selectable marker, such as antibiotic resistance gene
  • the vector can replicate and function independently of the host genome or integrate into the host genome
  • Vector design depends, among other things, on the intended use and host cell for the vector, and the design of a vector of the invention for a particular use and host cell is within the level of skill in the art
  • the vector includes one or more control sequences capable of effecting and/or enhancing expression of an operably linked exotoxin variant coding sequence
  • Control sequences that are suitable for expression in prokaryotes for example, include a promoter sequence, an operator sequence, and a ⁇ bosome binding site Control sequences for the expression in eukaryotic
  • An exotoxin variant expression vector can also include other sequences, for example, nucleic acid sequences encoding a signal sequence or an amp fiable gene A signal sequence directs the secretion of a polypeptide fused thereto from a cell expressing the protein
  • nucleic acid encoding a signal sequence is linked to the exotoxin vanant coding sequence so as to preserve the reading frame of exotoxin va ⁇ ant coding sequence
  • the vectors of the invention most commonly encode a purification domain that is operably linked to the expressed protein, resulting in the expression of a fusion protein.
  • Suitable purification domains are hexahistidine sequences that bind metal affinity columns; glutathione-S-transferase, that binds to glutathione; protein A (or derivative or fragments thereof), that binds IgG molecules; maltose binding protein, that binds maltose or any variety of other protein or protein domains that can bind to an affinity support with an association constant (Ka) of >10 5 M "1 .
  • the purification domain is most frequently linked to the exotoxin variant through a linker region.
  • the linker region most often encodes a substrate sequence for a sequence specific protease to allow the elution of the fusion protein during purification.
  • the linker sequence may also contain any other cleavable peptide sequence as would be known by one skilled in the art, including sequences susceptible to chemically induced cleavage.
  • a vector of the present invention is produced by linking desired elements by ligation at convenient restriction site. If such sites do not exist, suitable sites can be introduced by standard mutagenesis (e.g., site-directed or cassette mutagenesis) or synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.
  • suitable sites can be introduced by standard mutagenesis (e.g., site-directed or cassette mutagenesis) or synthetic oligonucleotide adapters or linkers can be used in accordance with conventional practice.
  • the present invention also provides a host cell containing a vector for this invention.
  • host cells are available for propagation and/or expression. Examples include prokaryotic cells (such as E. coli and strains of Bacillus, Pseudomonas, and other bacteria), yeast or fungal cells (including S. cerevesiae and P. pastoris), insect cells, plant cells, and phage, as well as higher eukaryotic cells (such as human embryonic kidney cells and other mammalian cells).
  • Host cells according to the invention include cells in culture and cells present in living organisms, such as transgenic plants and animals.
  • a preferred embodiment of the present invention utilizes E. coli as a host cell for expression of the exotoxin protein variant.
  • a vector of the present invention is introduced into a cell by any convenient method, which will vary depending on the vector-host system employed.
  • a vector is introduced into a host cell by transformation (also known as transfection) or infection with a virus (i.e., phage) bearing the vector.
  • transformation also known as transfection
  • virus i.e., phage bearing the vector.
  • Known methods, suitable for the host-vector system employed, acceptable for the use in the invention include:
  • exotoxin variants recombinantly host cells containing the exotoxin variant expression vector are prepared and cultured under conditions suitable for cell growth and for expression of the exotoxin variant
  • the culture media contains appropriate nutrients and growth factors for the host cell employed
  • the nutrients and growthfactors are, in many cases, well known or can be readily determined empirically by those skilled in the art
  • the culture conditions should allow transcription, translation, and protein transport between cellular components
  • Factors that affect these processes are well-known and include DNA/RNA copy number, factors that stabilize DNA, nutrients, supplements, and transcriptional inducers or repressors present in the culture medium, temperature, pH and osmolality of the culture, and cell density
  • the adjustment of these factors to promote expression in a particular vector-host cell system is within the level of skill in the art
  • the cell culture procedure employed in the production of the exotoxin variant of the present invention can be any of a number of well known procedures for large- or small-scale production of proteins These include, but are not limited to, the use of a shaker flask, fermentor, a fluidized bed bioreactor, a roller bottle culture system, and a stirred tank bioreactor system
  • the protein can be produced in a batch, fed-batch, or continuous mode
  • Methods for recovery of the recombinant exotoxin proteins produced as described above are well-known and vary depending on the expression system used For example, if the exotoxin variant contains a signal sequence the recombinant protein is recovered from the culture media or the periplasm The recombinant exotoxin protein is secreted into the periplasmic space as a mature protein Alternatively, the recombinant exotoxin protein can also be expressed intracellularly and recovered from cell lysates
  • the recombinant exotoxin can be purified from the culture media or a cell lysate by any method capable of separating the recombinant exotoxin protein from
  • the exotoxin variant is separated from the host cell and /or culture media components that would interfere with the intended use of the exotoxin variant.
  • the culture medium or cell lysate is usually centrifuged or filtered to remove cellular debris.
  • the supernatant is then typically concentrated or diluted to a desired volume or diafiltered into a suitable buffer to condition the preparation for further purification.
  • the exotoxin variant is further purified using well-known techniques. The techniques chosen will vary depending on the properties of the exotoxin variant. If, for example, the exotoxin variant is expressed as a fusion protein containing an affinity domain, purification typically includes the use of an affinity column containing the cognate binding partner. For instance, the exotoxin variant fused with the hexahistidine or similar metal affinity tags can be purified by fractionation on an immobilized metal affinity column.
  • the following exemplary procedures can be used or adapted for purifying the exotoxin variant of the invention: fractionation on an immuno affinity column, fractionation on an ion-exchange column, fractionation on a immobilized D-galactose column, ammonium sulfate or ethanol precipitation, reverse phase HPLC chromatography on silica, isoelectric focusing, SDS-PAGE or gel filtration.
  • this recombinant variant represents the minimal components necessary to achieve efficient targeted delivery of exogenous material to cells expressing the specific receptor that binds the ligand.
  • the delivery system is assembled from the exotoxin variant and the nucleic acid to be delivered. The components are mixed to form functional complexes. It is preferable to mix the components at low temperature with the therapeutic material of interest.
  • the material to be delivered can be any nucleic acid material that would be desired to be delivered to the target cell. Any nucleic acid of interest can be chosen for inclusion in the nucleic acid delivery system of the invention. Nucleic acid includes those described under "Nucleic Acid" section of this specification.
  • the nucleic acid may be a marker gene which includes but is not limited to ⁇ -lactamase, green fluorescent protein, luciferase and selectable markers such as the neomycin resistance gene, the ⁇ -galactosidase (lacZ) gene, and the chloramphenicol transferase (CAT) gene
  • the nucleic acid material of interest can also include therapeutic genes If the targeted cell or tissue-type has a defective gene, for instance as a result of a hereditary condition, a functional copy of the gene may be included in the nucleic acid material Suitable genes for this approach include, but are not limited to, tumor necrosis factor (TNF) genes, such as TNF-alpha, genes encoding interferons such as Interferon-alpha, Interferon-beta, Interferon-gamma, genes encoding interleukins such as IL-1, IL-l ⁇ , and Interleukins 2 throughl5, genes encoding G-CSF, M-CSF, and
  • the nucleic acid material can also encode proteins that catalyze the conversion of prodrugs with reduced toxicity to cytotoxic drugs within a cell or tissue-type of interest
  • An example of such an enzyme is the herpes simplex virus thymidine kinase Cells expressing this enzyme are susceptible to the drug ganciclovir
  • the nucleic acid material can also encode proteins with systemic or long range effects, such as hormones
  • An example is endostatin, which inhibits angiogenesis and has proven effective for tumor regression
  • the nucleic acid material can also encode proteins that stimulate an immune response
  • Such genes could be used as prophylactic or therapeutic vaccines They include nucleic acid vaccines that are capable of inducing an appropriate immune response in a host subject such that administration results in amelioration or prevention of the disease condition Examples include for prevention or treatment of infectious diseases or for the prevention or treatment of autoimmune or allergy diseases It is particularly useful to produce such vaccines in mucosal cells Protection against disease includes amelioration to the symptoms of the disease, decrease in mortality and morbidity, or decrease in susceptibility to the infectious organism An example would be genes encoding pathogen derived proteins that are capable of inducing an appropriate immune response in a host subject such that results in reducing or preventing infection
  • Such genes can be derived from bacteria, viruses, fungi, and parasites or infectious organisms
  • relevant vaccines include, but are not limited to, influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoid combined with pertussis vaccine, hepatitis A vaccines hepatitis B vaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, measles
  • 2Z vaccines rubella vaccine, mumps vaccine, mixed vaccine of measles, mumps and rubella, papillomavirus vaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio vaccine, malaria vaccine, varicella vaccine, gonorrhea vaccine, HIV vaccines schistosomiasis vaccine, rota vaccine, mycoplasma vaccine pneumococcal vaccine, meningococcal vaccine, and others
  • Influenza vaccine a vaccine comprising genes encoding the whole or part of hemagglutmm, neuramimdase, nucleoprotein and matrix protein which are obtainable from purified Influenza virus by genetic engineering techniques or chemical synthesis
  • Pertussis vaccine a vaccine comprising genes encoding the whole or a part of pertussis toxin, hemagglutmm and K-agglutin which are obtained from bacterial cells of Bordetella pertussis by genetic engineering techniques or chemical synthesis, Diphtheria and tetan
  • Another example is the use as a therapeutic vaccine which would include genes encoding autoimmune antigens or allergens that are capable of inducing an appropriate immune response in a host subject such that administration results in amelioration or prevention of the disease condition
  • Autoimmune antigens include antigens specific for autoimmune diseases whereas allergen antigens, include antigens involved in eliciting specific allergic reactions
  • Examples include genes which encode proteins that are known to protect against autoimmune diseases, to treat autoimmune diseases, or to prevent or treat allergic reactions Protection against disease includes amelioration to the symptoms of the autoimmune disease, decrease in mortality and morbidity, or decrease in sensitivity to the antigen
  • Example of such genes include but is not limited to genes which encode insulin or glutamate decarboxylase (GAD) or heat shock protein (HSP) for the treatment of diabetes mellitus type I, genes which encode myehn basic protein (MBP) or myelin proteo pid protein (PLP) or mye n-oligodendrocyte glycoprotein (MOG) for the treatment
  • the present invention also provides for methods of treating patients with exotoxin-nucleic acid complexes (ENAC) that provides protection against cancer, hereditary diseases, infectious diseases, autoimmune disease and allergy disease, comprising administering ENAC composition including a nucleic acid encoding a relevant gene having an amount of ENAC sufficient to protect the patient against the specific disease to which the nucleic acid is directed
  • ENAC exotoxin-nucleic acid complexes
  • the methods and compositions are directed towards treating and protecting humans as well as animals, including domestic animals Indeed, any multicellular organism into which it may be desirable to introduce exogenous nucleic acid is a potential subject for the present invention
  • the multicellular organism may be a plant or an animal, preferably the latter
  • the animal is preferably a vertebrate animal, and more preferably a higher vertebrate, l e , a mammal or bird, the former being especially preferred Among mammals, preferred subjects are human and other primates, laboratory animals such as mice, rats, rabbits and hamsters, pet animals such as dogs and cats, and farm animals such as horses, cows, goats, pigs and sheep It will be noted that these animals come from four orders of class Mammalia P ⁇ mata, Rodenta, Carnivora and Artiodactyla
  • the mode of administration may be, by way of example and not by way of limitation, by an intradermal, intramuscular, lntrape ⁇ toneal, intravenous, subcutaneous, transdermal, epidural, pulmonary, oral, nasal, gastric, intestinal, rectal, vaginal, or urethral route
  • the route of administration is a mucosal route of administration, I e , through a mucosal membrane or surface, such as an oral, nasal, gastric, intestinal, rectal, vaginal or urethral route
  • the mucosal route of admimstration is through oral or nasal membrane or any other means of administration capable of delivering the ENAC to the target cell of interest can be used
  • the route and site of administration may be chosen to enhance targeting
  • the DNA-complex may be administered m aerosol form
  • Another example to target cells in the digestive tract involves administering the DNA-complex by feeding Polymers of positively charged amino acids are known to act as nuclear localization signals (NLS)
  • 'Targeting is the administration of the nucleic acid in such a manner that it enters the target cells, i e , the mucosal cells, in amounts effective to achieve the clinical purpose
  • the amount of nucleic acid administered is such that it is effective to achieve the clinical purpose
  • DNA and RNA are capable of replication in the nucleus of the target cell, and in consequence the ultimate level of the nucleic acid in the cell may increase after uptake
  • the nucleic acid acts as a template, and thus high levels of protein expression can be achieved, even if the number of copies of the nucleic acid in the cell is low Nonetheless, it is desirable to deliver high concentrations of DNA to increase the number of target cells which take up the DNA and the number of DNA molecules taken up by each cell
  • SEQ ID 3 was designed containing an alternate NAAD 5' [CGG CCG CTC GAG CTA ACG CCT CCT GCG GCC TCC TCT CCT GCG ACG CCG GGA CAC CCT GGG GCG ACG GCG CCT GCG GAC ACG GCG GCT GGC TCT GCG TCT TCT GGG CAT CGA GCTCGG TAC CCG GGG ATCATG GTT TTT CAT ACT GAT TGC CGC 3']
  • This primer contains the identical sequence as SEQ ID 2 desrcibed above with the following exception, CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT (residues 16-45 of SEQ ID NO 2) was replaced by 93 nucleotides which encode a 31 amino acid nucleoprotamine (MPRRRRASRRVRRRRRRRPRVSRRRRRGGRRRR) (SEQ ID NO 8) which was fused in frame to the 3' terminus
  • the chime ⁇ c LTB proteins can be derived using 3' primers that contain no spacer (hinge) regions which have the following sequences 3' Primer SEQ ID 4 [5' CGG CCG CTC GAG CTA CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT CTT GTT TTT CAT ACT GAT TGC CGC 3'] which is identical to SEQ ID 2 with the exception that the 24 nucleotides which encode hinge region (HDPRVPSS) (SEQ ID NO 6) have been deleted which results in the 30 nucleotides encoding the 10 polylysine NAAD sequence being fused in frame to the 3' end of the eltB gene sequence (the resulting construct being designated LTBpL and having the sequence shown in Fig 9), or 3' Primer SEQ ID 5 [5' CGG CCG CTC GAG CTA ACG CCT CCT GCG GCC TCC TCT CCT GCG ACG CCG GGA CAC CCT GGG GCG ACG
  • IX LTBpLh protein expression was IPTG induced in E. coli BL21 cells (Novagen) and the modified recombinant enterotoxin protein was purified by Talon (Clonetech) column affinity chromatography per manufacture's protocol
  • Talon Chromassie and Western analysis of whole cell lysates (using anti-HIS) indicated LTBpLh was expressed as an apparent molecular weight of 13 kDa band (predicted size 20 kDa)
  • Initial attempts to purify LTBpLh using the Talon system indicated the protein seemed to be insoluble as it remained with the membrane pellet after sonication of the cells as analyzed by PAGE and Coomassie staining indicating that the protein appeared to form inclusion bodies
  • Others have reported that alteration of the carboxyl-terminus of LTB changes assembly and secretion properties in E.
  • EKMax Invitrogen
  • EKMax Invitrogen
  • the EK-Max treated sample had several bands of similar intensity with apparent molecular weights of 14, 10, 8 kDa in both the boiled and unboiled fractions representing the cleavage products of the vector derived sequence and the LTBpLh and an additional lower smear implying some degradation was occurring.
  • the unboiled EKMax treated LTBpLh sample was able to form the predicted pentamer complex ( Figure 3 lane 4).
  • Nucleic acid gel mobility shift experiment 2 Binding of DNA
  • LTBpLh had a higher binding reactivity than wild type LTB proteins at concentration between 50 to 0 78 micrograms of protein However, this diminished more sharply as compared to the wild type LTB when tittered below 0 2 micrograms or less as shown in Figure 5
  • plasmid DNA or a PCR dervived DNA between 0 0078 ⁇ g and 3 ⁇ g encoding the Green Fluorescent Protein marker gene (pGFP) under the control of a viral promoter (for example CMV) was conjugated to varying concentrations of either wild type LTB or LTBpLh between 0 195 ⁇ g and 50 ⁇ g in duplicate samples and incubated at room temperature for 30 minutes
  • the enterotoxin-DNA complex can be mixed with free polylysine or protamine to increase the condensing of the DNA This may also result in the protection of the DNA from degradation
  • varying concentration of protamine sulfate (PS) between 0 488 ⁇ g and 50 ⁇ g (Sigma Chemical) were added to one set of samples and incubated an additional 15 minutes
  • PS protamine sulfate
  • EXAMPLE 5 Testing the ability of recombinant enterotoxins to target delivery of nucleic acids in vivo. Testing the ability of recombinant enterotoxin-DNA complexes for targeting and immunogenicity using DNA encoding a sporozoite attachment factors (pBKCMV- SAP (United States patent 5,861, 160).) or GP900 (United States patent 6,015,882). We have previously shown that the intramuscular immunization of both pigs and mice with DNA encoding a coccidiosis sporozoite attachment factor (SAP) results in immune response to the SAP.
  • SAP coccidiosis sporozoite attachment factor
  • Animals will be divided into six groups representing five different routes of vaccine administration, either oral; nasal; transcutaneous; intraperitoneal; or intramuscular, and a control group receiving no treatment. Each group will be subdivided into three groups. These subdivided groups represent the three different vaccine combinations to be administered consisting of either DNA alone, DNA conjugated to the modified LTBpLh, DNA conjugated to the modified LTBpLh plus protamine sulfate.
  • Optimum complex formation conditions as defined by the Yl cell transformation protocol are used to form the enterotoxin nucleic acid complexes.
  • Vaccine will be administered orally and or intranasally (10 ⁇ l total dose starting at 10 ⁇ g of LTBpLh:5 ⁇ g of DNA with or without 32 ⁇ g protamine sulfate) four times at one week intervals. Serum samples will be collected prior to vaccination and on days 7, 14, 21 and 35 (end of test). Sera samples are tested for reactivity with the sporozoite attachment factors and LTB protein by ELISA and/or western blot analysis. Western blot analysis is described in the previous example.
  • ELISA titers are determined as follows, the amount of anti-LT or anti-SAP antibody is quantitated by analyzing two-fold serial dilutions of serum in 96-well microtiter plates coated with either purified recombinant LTB or SAP proteins followed by subsequent detection with anti-mouse conjugated to horseradish peroxidase (Sigma Chemical) Substrate reactions are stopped by the addition of 1 N H 2 SO and optical densities at 450 nm are determined using a Bio-Tek microtiter reader End point titers (reciprocal dilution of O D > 0 2) are statistically compared between the vaccinated groups Briefly, 96 well microtiter plates are coated with 1 tol 5 ⁇ g/well of recombinant protein for LTB or SAP in carbonate buffer Plates are incubated overnight and stored at 4°C Prior to use, plates are washed three times with carbonate buffer followed by two washes with PBS-0 05% Tween and tapped dry

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Abstract

La présente invention concerne un système de livraison d'acide nucléique à base de variantes protéiques d'exotocines recombinantes purifiées. L'invention concerne plus particulièrement une variante d'exotoxine purifiée incluant un domaine de ciblage de récepteur et un domaine d'affinité d'acide nucléique fonctionnellement reliés au domaine protéique de l'exotoxine. Pour le transfert du matériel d'acide nucléique, le domaine d'affinité d'acide nucléique est de préférence un domaine polycationique tel qu'une polylysine. L'invention concerne également, d'une part des procédés permettant de transférer sur des tissus étudiés, tels que des cellules de muqueuse, des matériaux d'acides nucléiques, en utilisant le système de livraison de l'invention, et d'autre part des procédés permettant un traitement thérapeutique par utilisation du système de livraison.
PCT/US2000/022715 1999-08-18 2000-08-18 Systeme de livraison genique specifique de muqueuse a base d'exotoxine bacterienne modifiee Ceased WO2001011960A1 (fr)

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Publication number Priority date Publication date Assignee Title
US8420352B2 (en) 2009-08-27 2013-04-16 Synaptic Research, Llc Protein delivery system to generate pluripotent stem (iPS) cells or tissue specific cells

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Publication number Priority date Publication date Assignee Title
WO1998021344A1 (fr) * 1996-11-12 1998-05-22 Michigan State University Vaccins ltb chimeres

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WO1998021344A1 (fr) * 1996-11-12 1998-05-22 Michigan State University Vaccins ltb chimeres

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8420352B2 (en) 2009-08-27 2013-04-16 Synaptic Research, Llc Protein delivery system to generate pluripotent stem (iPS) cells or tissue specific cells
US9102921B2 (en) 2009-08-27 2015-08-11 Synaptic Research, Llc Protein delivery system to generate induced pluripotent stem (iPS) cells or tissue-specific cells

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