WO2006077456A1 - Composition de therapie genique a administration par voie orale dans laquelle est utilise un agent de transport comportant un groupe amine et un polymere polycationique - Google Patents

Composition de therapie genique a administration par voie orale dans laquelle est utilise un agent de transport comportant un groupe amine et un polymere polycationique Download PDF

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Publication number
WO2006077456A1
WO2006077456A1 PCT/IB2005/001172 IB2005001172W WO2006077456A1 WO 2006077456 A1 WO2006077456 A1 WO 2006077456A1 IB 2005001172 W IB2005001172 W IB 2005001172W WO 2006077456 A1 WO2006077456 A1 WO 2006077456A1
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moiety
accordance
therapeutic agent
dna
insulin
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PCT/IB2005/001172
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English (en)
Inventor
Gonzalo Hortelano
Gomez Vargas
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Neox, Inc.
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Priority to PCT/IB2005/001172 priority Critical patent/WO2006077456A1/fr
Publication of WO2006077456A1 publication Critical patent/WO2006077456A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/27—Growth hormone [GH], i.e. somatotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
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    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0031—Rectum, anus
    • A—HUMAN NECESSITIES
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration

Definitions

  • This invention relates to the administration of an active agent to an organism via oral administration; particularly to the efficacious administration of an active/therapeutic agent coupled to a transporting agent , thereby enabling widespread distribution, systemic expression and sustained delivery of said active agent via oral administration whereby the utilities of organ regeneration, insulin delivery, antibody delivery, delivery of factors for symptomatic reversal of hemophilia, delivery of DNA plasmid including the cDNA for Alpha 1 Antitrypsin, and delivery of human growth hormone are enabled .
  • Gene therapy offers an alternative to the currently available treatment modalities for a variety of 5 conditions , particularly genetic and acquired disorders affecting a range of cells and tissues .
  • Several in vivo gene therapy protocols based on viral vectors are known, albeit several safety related issues exist .
  • Oral gene delivery has been attempted with little success , largely due to the extensive degradation of DNA in the stomach and gastrointestinal tract .
  • Attempts at oral gene therapy via the use of liposomal formulations as a protectant has met with limited success , in that the efficiency of delivery is relatively low .
  • Quong et al in an article entitled "DNA Protection form Extracapsular Nucleases , within Chitosan or PoIy-L- lysine-coated Alginate Beads" (Biotechnology and Bioengineering, Vol . 60, No . 1 , 10/98 , pages 124-134 ) discloses immobilization of DNA within an alginate matrix using either an internal or external source of calcium followed by membrane coating with chitosan or poly-L- lysine ( PLL) .
  • PLL poly-L- lysine
  • Ward et al (Blood, 15 April 2001 , Volume 97 , Number 8 , Pages 2221-2229 ) is directed toward intravenous forms of gene therapy capable of systemic circulation .
  • Wender et al ( PNAS , 11/21/2000 , vol . 97 , no . 24 , 13003- 13008 ) discloses polyguanidine peptoid derivatives which preserve the 1 , 4-backbone spacing of side chains of arginine oligomers to be efficient molecular transporters as evidenced by cellular uptake . While it is suggested that these peptoids could serve as effective transporters for the molecular delivery of drugs, drug candidates , and agents into cells , the reference is nevertheless silent as to the concept of oral delivery via this route , and does not disclose the formation of a complex between the active ingredient, e . g . DNA or a drug, and the polyguanidine peptoid derivatives .
  • Hortelano et al disclose delivery of Human Factor IX by use of encapsulated recombinant myoblasts .
  • Droplets of an alginate-cell mixture were collected in a calcium chloride solution . Upon contact, the droplets gelled . Subsequently, the outer alginate layer was cross-linked with poly-L-lysine hydrobromide (PLL) and then with another layer of alginate . The remaining free alginate core was then dissolved via sodium citrate to yield microcapsules with an alginate-PLL-alginate membrane containing cells . Similar technology is disclosed in Awrey et al, Biotechnology and Bioengineering, Vol . 52 , Pp .
  • the composition comprises entrapped or encapsulated microorganisms capable of removing the undesired chemicals or amino acids .
  • the capsules may comprise a variety of polymers , elastomers , and the like, inclusive of which are chitosan-alginate and alginate-polylysine-alginate compounds .
  • U . S . Patent No . 6, 177 , 274 is directed toward a compound for targeted gene delivery consisting of polyethylene glycol (PEG) grafted poly (L-lysine ) and a targeting moiety .
  • the polymeric gene carriers of this invention are capable of forming stable and soluble complexes with nucleic acids , which are in turn able to efficiently transform cells .
  • the reference fails to suggest or disclose a complex including DNA, nor the use of such a complex for oral delivery thereof .
  • U . S . Patent No . 6, 258 , 789 is directed towards a method of delivering a secreted protein into the bloodstream of a mammalian subj ect .
  • intestinal epithelial cells of a mammalian subj ect are genetically altered to operatively incorporate a gene which expresses a protein which has a desired effect .
  • the method of the invention comprises administration of a formulation containing DNA to the gastrointestinal tract , preferably by an oral route .
  • the expressed recombinant protein is secreted directly into the bloodstream.
  • the method of the invention to provide for short term, e . g . two to three days , delivery of gene products to the bloodstream.
  • U . S . Patent No . 6, 255 , 289 discloses a method for the genetic alteration of secretory gland cells , particularly pancreatic and salivary gland cells, to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subj ect .
  • the expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein .
  • the transformed secretory gland cells provide long term ' therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein .
  • U . S . Patent No . 6, 225, 290 discloses a process wherein the intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect .
  • Intestinal cell transformation is accomplished by administration of a formulation composed primarily of naked DNA .
  • Oral or other intragastrointestinal routes of administration provide a method of administration, while the use of naked nucleic acid avoids the complications associated with use of viral vectors to accomplish gene therapy .
  • the expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein .
  • the transformed intestinal epithelial cells provide short or possibly long term therapeutic cures (e . g .
  • U . S . Patent No . 5 , 837 , 693 is directed to intravenous hormone polypeptide delivery by salivary gland expression .
  • Secretory gland cells are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subj ect .
  • the expressed protein may be secreted directly into the gastrointestinal tract and/or blood stream.
  • the transformed secretory gland cells may provide therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein .
  • U . S . Patent No . 5 , 885 , 971 is directed toward gene therapy by secretory gland expression .
  • Secretory gland cells particularly pancreatic and salivary gland cells , are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subj ect .
  • the expressed protein may be secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein .
  • the transformed secretory gland cells provide long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein .
  • U . S . Patent No . 6, 004 , 944 is directed to protein delivery via secretory gland expression .
  • Secretory gland cells particularly pancreatic, hepatic, and salivary gland cells , are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect on a mammalian subj ect .
  • the expressed protein may be secreted directly into the bloodstream to obtain therapeutic levels of the protein thereby treating the patient in need of the protein .
  • the transformed secretory gland cells may provide long term or short term therapies for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein .
  • U . S . Patent No . 6, 008 , 336 relates to compacted nucleic acids and their delivery to cells .
  • Nucleic acids are compacted, substantially without aggregation, to facilitate their uptake by target cells of an organism to which the compacted material is administered .
  • the nucleic acids may achieve a clinical effect as a result of gene expression, hybridization to endogenous nucleic acids whose expression is undesired, or site-specific integration so that a target gene is replaced, modified or deleted .
  • the targeting may be enhanced by means of a target cell-binding moiety .
  • the nucleic acid is preferably compacted to a condensed state .
  • nucleic acid complexes are consisting essentially of a single double-stranded cDNA molecule and one or more polylysine molecules , wherein said cDNA molecule encodes at least one functional protein, wherein said complex is compacted to a diameter which is less than double the theoretical minimum diameter of a complex of said single cDNA molecule and a sufficient number of polylysine molecules to provide a charge ratio of 1 : 1 , in the form of a condensed sphere, wherein the nucleic acid complexes are associated with a lipid .
  • U . S . Patent No . 6, 287 , 817 discloses a protein conj ugate consisting of antibody directed at the plgR and Ai AT which can be transported specifically from the basolateral surface of epithelial cells to the apical surface .
  • This approach provides the ability to deliver a therapeutic protein directly to the apical surface of the epithelium, by targeting the plgR with an appropriate ligand .
  • U . S . Patent No . 6, 261 , 787 sets forth a bifunctional molecule consisting of a therapeutic molecule and a ligand which specifically binds a transcytotic receptor; said bifunctional molecule can be transported specifically from the basolateral surface of epithelial cells to the apical surface .
  • This approach provides the ability to deliver a therapeutic molecule directly to the apical surface of the epithelium, by targeting the transcytotic receptor with an appropriate ligand .
  • U . S . Patent No . 5 , 877 , 302 is directed toward compacted nucleic acids and their delivery to cells .
  • Nucleic acids are compacted, substantially without aggregation, to facilitate their uptake by target cells of an organism to which the compacted material is administered .
  • the nucleic acids may achieve a clinical effect as a result of gene expression, hybridization to endogenous nucleic acids whose expression is undesired, or site-specific integration so that a target gene is replaced, modified or deleted .
  • the targeting may be enhanced by means of a target cell-binding moiety, e . g . polylysine .
  • the nucleic acid is preferably compacted to a condensed state .
  • Patent No . 6, 159, 502 relates to an oral delivery system for microparticles .
  • the complexes of the invention comprise a microparticle coupled to at least one carrier, the carrier being capable of enabling the complex to be transported to the circulation or lymphatic drainage system via the mucosal epithelium of the host, and the microparticle entrapping or encapsulating, or being capable of entrapping or encapsulating, the substance (s ) .
  • Suitable carriers are mucosal binding proteins , bacterial adhesins , viral adhesins , toxin binding subunits , lectins , Vitamin Bi 2 and analogues or derivatives of Vitamin B 12 possessing binding activity to Castle ' s intrinsic factor .
  • This invention differs from the instant disclosure in requiring entrapment or encapsulation, which neither insures nor enables the widespread distribution, systemic expression, or sustained delivery which are novel features of the instantly disclosed invention .
  • U . S . Patent No . 6 , 011 , 018 discloses regulated transcription of targeted genes and other biological events . Dimerization and oligomerization of proteins are general biological control mechanisms that contribute to the activation of cell membrane receptors , transcription factors , vesicle fusion proteins , and other classes of intra- and extracellular proteins .
  • the patentees have developed a general procedure for the regulated ( inducible ) dimerization or oligomerization of intracellular proteins . In principle , any two target proteins can be induced to associate by treating the cells or organisms that harbor them with cell permeable , synthetic ligands .
  • an orally deliverable composition capable of achieving : a) widespread delivery and distribution of an active agent, e . g . a therapeutic agent such as DNA, an antibody, a homeobox gene in a manner to effect virtual organ regeneration, delivery of hormones such as insulin and human growth hormone, proteins such as Factor IX and enzymes such as Alpha 1 antitrypsin, to essentially all cells of the targeted subject b) an ability to provide a sustained (e . g .
  • non- transient expression of the active agent (either ubiquitously or in a tissue specific manner) , from a single administration, via cellular uptake in virtually all organs and cellular systems throughout the entire body, and c) without eliciting an unwanted immune response .
  • the present invention is directed toward a composition and non-invasive process for administration of an active, e . g . a therapeutic agent .
  • an active e . g . a therapeutic agent .
  • the terms therapeutic agent and active agent will be used synonymously .
  • the invention discloses a composition for use in the administration of oral gene therapy and a process for its production and use .
  • Various obstacles have prevented an efficient oral gene therapy protocol .
  • the primary obstacle has been the extensive degradation of ingested DNA . Protecting this otherwise naked DNA from destruction when placed in the ⁇ gastrointestinal tract, for example via the use of chitosan, collagen, alginate or the like, enables limited absorption of DNA via the gastrointestinal tract, albeit with limited scope of delivery and poor expression .
  • DNA requires a protective covering .
  • alginate is a means of providing protection in the gastrointestinal tract .
  • a transporting agent is required, which is capable of transporting the DNA via natural pathways , and without eliciting an unwanted or undesirable immunogenic response during transport .
  • the transporting agent in its broadest sense , may be any compound containing an amine group that is capable of coupling with the DNA (or other therapeutic agent ) in a manner effective to produce efficacious and widespread distribution and cellular uptake subsequent to passage via said natural gastrointestinal pathway .
  • Such coupling of the therapeutic agent and transporting agent thereby enables efficacious and widespread absorption, distribution and expression thereof .
  • the transporting agent is preferably a polypeptide or a modification thereof, e . g . of an amino acid, but may be any compound having an amine group and an acidic group which will effectively enable in vivo distribution .
  • the transporting agent is necessary in order to achieve efficient and widespread distribution of the therapeutic product, e . g . DNA in vivo.
  • the instantly disclosed formulations will couple DNA to the amino compound, e . g . via electrostatic binding, covalent binding, or the like , while protecting the DNA from degradation in the gastrointestinal tract, e . g . with an alginate or equivalent protective compound .
  • Such a formulation may be illustratively exemplified as an alginate cross-linked with poly-L-lysine, such as in the form of a microcapsule . While the instant inventors have shown that limited expression is possible by merely protecting DNA in the GI tract via the use of gelatin or alginate, without PLL, or even via the administration of naked DNA, the effectivity is clearly much lower, and therefore inclusion of a protective agent and a transporting agent (e . g . alginate/PLL) is most preferred,.
  • a protective agent and a transporting agent e . g . alginate/PLL
  • DNA is first mixed with alginate or a compound having similar properties in affording GI tract protection for the DNA, then the capsules are physically formed with DNA-alginate inside , and later the transporting agent, e . g . PLL, is added to cross-link the alginate beads , in a manner such that conjugation or coupling between the transporting agent and DNA occurs , although the transport agent does not specifically encapsulate the therapeutic agent .
  • the transporting agent e . g . PLL
  • our experiments indicate that there is no widespread distribution or delivery nor is there systemic or sustained expression . This evidences the theory that an interaction or coupling of the transporting agent and therapeutic agent occurs within the capsules , thereby explaining the efficacy of the instantly disclosed microcapsules in the distribution of DNA to all major organs .
  • tissue-specific expression of therapeutic genes can be achieved by using tissue-specific genetic regulatory elements (promoters ) that restrict gene expression to specific organs . Via the judicious use of promoters , the degree of expression may be tailored to meet specific needs . For example, via the use of ⁇ -Actin, a ubiquitous promoter, widespread expression is achieved . Alternatively, use of tissue specific genetic regulatory elements (promoters ) , illustrated, but not limited to albumin promoter ( liver expression) , muscle creatine kinase (MCK) for muscle expression, and keratinocyte ( skin expression) provide the ability to express protein in a particularly desired portion of the body .
  • tissue-specific genetic regulatory elements promoter
  • albumin promoter liver expression
  • MCK muscle creatine kinase
  • keratinocyte skin expression
  • a complete transcriptional unit e . g . DNA and RNA
  • components which enable a complete transcriptional unit within the cells , e . g . inteins and exteins , which delivery may be achieved to virtually all cells of an organism, via an oral pathway .
  • inherited genetic diseases e . g . hemophilia, Duschenne Muscular Dystrophy, Cystic Fibrosis , diabetes , etc .
  • acquired diseases for infectious diseases , for which both prevention and treatment are
  • FIGURE 1 is a graph of glucose levels in blood in mice treated with pdx-1 DNA
  • FIGURE 2 is a graph of glucose levels in plasma in mice treated with pdx-1 DNA
  • FIGURE 3 is an immunohistochemistry slide of liver tissue having a pancreas related tissue structure ;
  • FIGURE 4 is an immunohistochemistry slide which confirms the secretion of insulin in liver tissue of pdx-1 DNA treated mice ; .
  • FIGURE 5 is a Western blot which evidences secretion of product that reacted with human IgG antibodies ;
  • FIGURE 6 is a graph which evidences that oral administration of DNA coding for antibodies can be used to deliver antibodies in a treated host ;
  • FIGURE 7 is a graph which evidences the effect on clotting time (Activated Partial Thromboplastin Time (APTT) ) of orally administered FIX gene which was taken up by the treated mice ;
  • APTT Activity Partial Thromboplastin Time
  • FIGURE 8 represents results of a PCR technique used to amplify DNA sequences specific to the GFP plasmid administered orally, the results of which persisted for greater than 400 days ;
  • FIGURE 9 is a graph of ⁇ l-antitrypsin plasma concentration of mice treated orally with DNA nanoparticles containing various amounts of a DNA plasmid including the cDNA for human ⁇ l-antitrypsin;
  • FIGURE 10 is a graph of human growth hormone (hGH) concentration of mice treated orally with DNA nanoparticles containing a DNA plasmid including the cDNA for hGH;
  • FIGURE 11 is a graph showing blood glucose levels in mice administered insulin DNA
  • FIGURE 12 is a graph showing plasma glucose levels in mice administered insulin DNA
  • FIGURE 13 is an immunohistochemistry slide of studies using anti-insulin antibody to confirm the secretion of insulin in liver tissue in mice treated orally with insulin DNA .
  • the primary obj ective of this invention is the oral administration of a transporting agent , exemplified as , but not limited to, an amino acid carrier, e . g . poly-1- lysine , polyarginine and polyornithine , for the purpose of carrying a compound, which although not limited to DNA, will nevertheless be exemplified as such for purposes of illustration herein, through the gastrointestinal tract and enabling its widespread distribution and systemic and sustained expression throughout the body .
  • a transporting agent exemplified as , but not limited to, an amino acid carrier, e . g . poly-1- lysine , polyarginine and polyornithine , for the purpose of carrying a compound, which although not limited to DNA, will nevertheless be exemplified as such for purposes of illustration herein, through the gastrointestinal tract and enabling its widespread distribution and systemic and sustained expression throughout the body .
  • the compound e . g . DNA
  • it should be protected from enzyme degradation and low pH as it passes through the stomach and small intestine .
  • this is accomplished via the use of protective compounds , illustrative of which are alginate, gelatin (which is mainly collagen) and the like .
  • alginate , gelatine and collagen in protecting the key formulation ( DNA-amino acid complex) through the stomach is very important to ensure DNA integrity ( thereby facilitating the achievement of delivery efficacy) , but can also be accomplished with alternative formulations such as chitosan, methacrylate, or alternatively, one or more of the conventional oral delivery systems used by the pharmaceutical industry, e . g . degradable capsules , gels , etc .
  • the present inventors have determined that straight uncoupled ("naked") DNA, if adequately protected with gelatin (collagen) or the like, is also taken through the intestinal wall and may be expressed in certain tissues , but not all of the tissues , as is possible via use of the teachings of the instant invention .
  • it is important to distinguish that such cases a ) the efficacy of the delivery and expression of naked DNA is extremely low and b) it is not long lasting, which is in agreement with attempts to perfect the oral delivery of DNA described in the prior art .
  • the instant inventors have achieved limited success absent effective coupling to a transporting agent , this remains a non- preferred embodiment of the instant invention .
  • rectal delivery is indeed contemplated by the instant inventors as an alternative route for administration via the gastrointestinal pathway .
  • Any transport agent is deemed to be useful in the context of the instant invention provided it couples with a therapeutic agent in a manner effective to produce efficacious and widespread distribution and cellular uptake subsequent to passage via said natural gastrointestinal pathway .
  • Alternative transport agents contemplated as being useful within the context of this invention may include, but are not limited to, amino acids having an altered electrical charge , chemically modified compounds or amino acids , or synthesized molecules having the requisite functional groupings to make advantageous use of the natural transport pathways described herein .
  • Mathiowitz et al utilized polyanhydrides of a combination of fumaric and sebacic acids to encapsulate a plasmid DNA ( ⁇ -galactosidase ) .
  • quantification of ⁇ -galactosidase activity in tissue extracts showed no significant activity in stomach or liver, but measurable activity within the intestine . This is indicative of an inability of the Mathiowitz technology to evidence transport through the intestine so as to enable delivery and/or expression in other organs .
  • Prior artisans have used DNA bound to PLL, but it has not been effective in delivering genes into animals because they failed to recognize the importance of oral delivery .
  • Prior artisans have used orally administered DNA protected with chitosan, but failed to bind DNA to a transporting and distribution agent, such as polypeptides , thus failing to produce widespread distribution .
  • Prior artisans have also used oral delivery of DNA (oligonucleotides-short segments of DNA-not including a whole gene or genetic regulatory sequences ) , enclosed in alginate-PLL microcapsules , with the intent of retrieving DNA from feces and thereby determining if DNA had mutated through the intestine .
  • DNA oligonucleotides-short segments of DNA-not including a whole gene or genetic regulatory sequences
  • RNA which has commercial interest owing to its ability to inactivate the transcription/translation of unwanted proteins
  • siRNA interference RNA - as an approach to inhibit gene expression
  • ribozymes which are defined as catalytic RNA having the ability to recognize, bind and cleave a specific sequence of cellular RNA such as that of a virus , which could be delivered as a means of treating infectious diseases , such as AIDS .
  • those molecules useful as transporting agents will exhibit the ability to form charged molecules , e . g . positive or negative side chains , so as to enable binding, e . g . conj ugation, of the active agent with the transporting agent .
  • DNA plasmids containing a cDNA coding for a transgene and appropriate genetic regulatory elements such as a promoter is performed as follows .
  • a volume of 300 ⁇ l of a solution containing DNA plasmid at a concentration of 1 mg/ml is mixed with 0.6 ml of 1.5% potassium alginate (Kelmar, Kelco Inc . , Chicago, USA) in a syringe and extruded through a 27 G needle with a syringe pump (39.3 ml/h) .
  • An air-jet concentric to the needle created fine droplets of the DNA/alginate mixture that are collected in a 1.1% CaCl 2 solution .
  • the alginate/DNA droplets gel .
  • the microcapsules are extruded, they are subjected to the washes as indicated in the list below .
  • the outer alginate layer is chemically cross-linked with poly-L-lysine hydrobromide ( PLL, Sigma, St . Louis , USA) with Mr in a 15, 000 - 30, 000 range for 6 minutes, and then with another layer of alginate . Finally, the remaining free alginate core may be dissolved with sodium citrate for 3 minutes , to yield microcapsules with an alginate-PLL- alginate membrane containing DNA inside .
  • the standard microcapsule protocol uses a 6 minutes citrate wash . With 3 minutes of citrate we increase the concentration of alginate left in the capsule core . This procedure appears to have an effect on the coupling of DNA. Washes (unless stated otherwise, washing steps are performed with no incubation time in between) :
  • Microcapsules are finally mixed with a 1 : 1 volume of a 50% gelatin solution to obtain a homogeneous mixture that can be administered .
  • DNA-alginate-PLL particles are finally mixed with a 1 : 1 volume of a 50% gelatin solution to obtain a homogeneous mixture that can be administered .
  • DNA-PLL-alginate particles A volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of 3% calcium alginate, and mixed at 4°C for 3 hours with gentle agitation . A volume of 50 ⁇ l of poly-L-Lysine is added . The mixture is vortexed for 30 seconds and mixed at 4°C for one additional hour with gentle agitation . Finally, 50 ⁇ l of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered .
  • DNA-PLL-alginate particles A volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of 3% calcium alginate, and mixed at 4°C for 3 hours with gentle agitation . A volume of 50 ⁇ l of poly-L-Lysine is added . The mixture is vortexed for 30 seconds and mixed at 4°C for one additional hour with gentle agitation
  • DNA-PLL-Alginate microcapsules An exemplary, but non-limiting example of forming DNA-PLL-Alginate microcapsules , a volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of poly-L-Lysine , and mixed at 4°C for 3 hours with gentle agitation . A volume of 50 ⁇ l of 3% calcium alginate is added . The mixture is vortexed for 30 seconds and mixed at 4°C for one additional hour with gentle agitation . Finally, 50 ⁇ l of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered .
  • DNA-ornithine-alginate particles DNA-ornithine-alginate particles
  • DNA-arginine-alginate particles A volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of poly-L-Ornithine . The mixture is vortexed for 30 seconds and mixed at 4°C for 3 hours with gentle agitation . A volume of 50 ⁇ l of 3% calcium alginate is added and mixed at 4°C for one additional hour with gentle agitation . Finally, 50 ⁇ l of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered .
  • DNA-arginine-alginate particles A volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of poly-L-Ornithine . The mixture is vortexed for 30 seconds and mixed at 4°C for 3 hours with gentle agitation . A volume of 50 ⁇ l of 3% calcium alginate is added and mixed at 4°C for one additional hour with gentle agit
  • a volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of poly-L-Arginine .
  • the mixture is vortexed for 30 seconds and mixed at 4°C for 3 hours with gentle agitation .
  • a volume of 50 ⁇ l of 3% calcium alginate is added and mixed at 4°C for one additional hour with gentle agitation .
  • 50 ⁇ l of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered .
  • a volume of 100 ⁇ l of DNA plasmid at a concentration of 1 mg/ml is mixed with 50 ⁇ l of a 50% gelatin solution, and mixed thoroughly to obtain a homogeneous mixture that can be administered .
  • the formulations of the instant invention may also be manufactured as nanoparticles or macroparticles of a variety of sizes , in combination with amphiphilic compounds , or the like , so as to deliver a compound such as ' DNA coupled to a (polyamine or polypeptide ) amino acid .
  • lysine , arginine and ornithine are illustrated herein as exemplary transporting agents
  • other compounds and/or compositions having at least the requisite functional groups and if required, an appropriate charge may also function as transporting agents in a similar fashion .
  • compositions of the instant invention afford the compositions of the instant invention the added utility of controllable trangene expression in vivo.
  • Tissue-specific expression of therapeutic genes can be achieved by using tissue-specific genetic regulatory elements that restrict gene expression to specific tissues , as known to those skilled in the art . Via the judicious use of such promoters , the degree of expression may be tailored to meet specific needs .
  • ⁇ -Actin a ubiquitous promoter, expression in multiple tissues is achieved .
  • tissue specific genetic regulatory elements illustrated, but not limited to albumin promoter ( liver expression) , muscle creatine kinase (MCK) for muscle expression, and keratinocyte ( skin expression) provide the ability to express a transgene in a particularly desired location, e . g . a specific portion of the body, specific organ, or specific cell or tissue type .
  • a therapeutic agent includes any nucleic acids which is introduced into a host in order to instigate a desirable biological effect .
  • Such genetic materials may include, but are not limited to DNA, RNA, siRNA, Ribozyme, Antisense, Hybrids , either Single or Double stranded, or combinations thereof .
  • a desirable biological effect may include , but is not limited to, gene expression, gene inhibition, and gene correction .
  • Said biological effect may include , but is not limited to, those effects which are directly related to the cellular uptake of a therapeutic agent following oral delivery, e . g . FIX DNA which leads to FIX production .
  • Said biological effect may directly occur as a result of said cellular uptake , as a result of systemic expression, or alternatively targeted expression which is understood to include expression specifically directed to a particular organ, system or a targeted cell or group of cells .
  • Said biological effect is exemplified by, but not limited to, modulation of a disease state, wherein expression of a therapeutic agent modifies the onset, course, manifestation or severity of the disease state .
  • systemic expression is understood to mean measurable cellular uptake of a therapeutic agent within cells , inclusive of, but not limited to both the epithelial cells and basement membrane cells found in organs such as the intestine, liver, kidney, heart, lungs and the like .
  • sustained expression or sustained delivery is understood to mean measurable expression of a therapeutic agent sufficient to instigate a desirable biological effect, as a result of a single administration, which effect is detectable for a sustained period of time .
  • Actual expression may be intracellular or extracellular .
  • widespread distribution is understood to mean distribution of a therapeutic agent to essentially all organs (as evidenced and exemplified in Tables 1 and 2 and the accompanying figures ) , including but not limited to the central nervous system, in particular to the brain, heart and bone marrow; such distribution effected, for example , via the basal membrane of the intestinal epithelium and beyond to multiple organ sites .
  • transdifferentiating moiety is understood to mean the combination of a nucleic acid sequence coding for a homeogene operably linked to a promoter which can be tissue specific or ubiquitous .
  • insulin producing moiety is understood to mean the combination of an insulin encoding DNA, e . g . a DNA which induces a cell to encode insulin, operably linked to a promoter which can be tissue specific or ubiquitous .
  • therapeutic agent producing moiety is understood to mean either the combination of a DNA (e . g .
  • a DNA which instigates a cell to produce a therapeutic agent operably linked to a promoter which can be tissue specific or ubiquitous or alternatively, when it is desired to inhibit the production of a particular agent in order to alleviate a condition, DNA alone .
  • a promoter which can be tissue specific or ubiquitous or alternatively, when it is desired to inhibit the production of a particular agent in order to alleviate a condition, DNA alone .
  • the inclusion of a promoter is not required, and the DNA alone is classified as the "therapeutic agent producing moiety" wherein inhibition of a deleterious moiety is interchangeable or equivalent to production of a beneficial moiety .
  • therapeutic agent is understood to mean an agent required by a mammal to ameliorate a particular physiological condition .
  • the instant invention is directed toward the formation of a distributable moiety, which moiety is formed by the coupling of a transporting agent and at least one genetic material in a manner effective to provide, via a natural gastrointestinal pathway (e . g . orally or rectally) , for widespread distribution, systemic expression and sustained delivery of said material .
  • Said genetic material may, for example, be a complete transcriptional unit, which is broadly defined as the combination of at least a particular portion of DNA coding for a therapeutic agent for which expression is desired, in combination with a promoter sufficient to provide expression, subsequent to intracellular absorption, of the desired therapeutic agent .
  • Said agent may comprise any expressed entity which exhibits therapeutic value , and may include , but is not limited to, proteins , antibodies , DNA, RNA, or particular portions or fragments thereof .
  • Proteins useful as therapeutic agents refers to recombinant or naturally occurring proteins , whether human or animal, useful for prophylactic, therapeutic or diagnostic application .
  • the therapeutic agent can be natural, synthetic, semi-synthetic or derivatives thereof .
  • compositions of present invention may include but are not limited to hormones , cytokines, hematopoietic factors , growth factors , antiobesity factors, trophic factors , anti-inflammatory factors, and enzymes .
  • hormones cytokines, hematopoietic factors , growth factors , antiobesity factors, trophic factors , anti-inflammatory factors, and enzymes .
  • Such proteins would include but are not limited to interferons ( see, U . S . Pat . Nos . 5 , 372 , 808 , 5 , 541, 293 4 , 897 , 471, and 4 , 695, 623 hereby incorporated by reference including drawings ) , interleukins ( see , U . S . Pat . No . 5, 075, 222 , hereby incorporated by reference including drawings ) , erythropoietins ( see, U . S . Pat . Nos .
  • biologically active agents can also include but are not limited to anti-obesity related products , insulin, gastrin, prolactin, adrenocorticotropic hormone (ACTH) , thyroid stimulating hormone (TSH) , luteinizing hormone (LH) , follicle stimulating hormone ( FSH) , human chorionic gonadotropin (HCG) , motilin, interferons (alpha, beta, gamma) , interleukins ( IL-I to IL-12) , tumor necrosis factor (TNF) , tumor necrosis factor-binding protein (TNF-bp) , brain derived neurotrophic factor (BDNF) , glial derived neurotrophic factor (GDNF) , neurotrophic factor 3 (NT3) , fibroblast growth factors ( FGF) , neurotrophic growth factor (NGF) , bone growth factors such as osteoprotegerin (OPG) , insulin-like growth factors ( IGFs ) , macro
  • a promoter for the expression of the transgene is considered to be mandatory in order to successfully accomplish the systemic expression which is a hallmark of the present invention, a promoter is not required when the goal is inhibition of the production of an existing therapeutic product (i . e . hepatitis virus or HIV genes in humans ) . Additionally, use of a tissue specific, as opposed to a ubiquitous promoter provides a degree of freedom in tailoring the degree of systemic expression achieved . Furthermore, delivery of antisense nucleic acids (RNA and/or DNA) or ribozymes can be accomplished without including a promoter .
  • RNA and/or DNA antisense nucleic acids
  • ribozymes can be accomplished without including a promoter .
  • Another application contemplated by the present technology in which a complete transcriptional unit is not required, has to do with judicious utilization of inteins and exteins in order to achieve a type of gene therapy .
  • Inteins are insertion sequences embedded within a precursor protein, and they are capable of protein splicing that removes the intein sequence and at the same time ligates the flanking polypeptides (termed exteins ) .
  • the therapeutic gene can be split into 2 distinct entities that are administered separately via the instantly disclosed technique .
  • Inteins have been utilized to produce a functional protein, following the splitting of the gene in 2 parts , that were expressed separately . After the two proteins are made (translation) , the intein portions are removed (by themselves ) , and the adj acent extein portions (one at the end of a first part of the gene and the second at the beginning of second part of the gene part) are j oined together to form a full functional protein . The incorporation of a promoter within one portion will nevertheless be in order for both parts of the protein to be expressed .
  • vectors such as Adenoassociated- virus (AAV) form concatamers inside the infected cells .
  • AAV Adenoassociated- virus
  • the vector multiplies itself to create a series of copies of the vector that are placed one after the other .
  • One can exploit this fact using the instantly disclosed transport agent technology, to split a gene in half, and express both portions separately in two vectors . I f one then transports and introduces both vectors inside the same cell, both vectors can come together physically, and the full promoter-gene context can be re-established inside the cell .
  • Zhou et al "Concatamerization Of Adeno-Associated Virus Circular
  • Acidic - aspartic acid glutamic acid Basic - arginine , histidine, lysine Hydroxylic - serine, threonine Sulphur-containing - cysteine, methionine Amidic ( containing amide group) - asparagine , glutamine Peptides :
  • Two individual amino acids can be linked to form a larger molecule , with the loss of a water molecule as a by- product of the reaction .
  • the newly created C-N bond between the two separate amino acids is called a peptide bond .
  • the term ' peptide bond ' implies the existence of the peptide group which is commonly written in text as - CONH- ;
  • Dipeptide two molecules linked by a peptide bond become what is called a dipeptide ;
  • Polypeptide a chain of molecules linked by peptide bonds ;
  • Proteins made up of one or more polypeptide chains , each of which consists of amino acids which have been mentioned earlier .
  • a protein may be formed of a single polypeptide chain, or it may consist of several such chains held together by weak molecular bonds .
  • the R groups of the amino acid subunits determine the final shape of the protein and its chemical properties ; whereby an extraordinary variety of proteins are produced .
  • more than 150 other amino acids have been found in nature, including some that have the carboxyl and amino groups attached to separate carbon atoms . These unusually structured amino acids are most often found in fungi and higher plants . Any having the requisite functional groupings, and which are capable of being coupled to the therapeutic agent of choice are contemplated for use within the instant invention .
  • DNA Deoxyribonucleic acid
  • DNA is understood to mean a long polymer of nucleotides j oined by phosphate groups
  • DNA is the genetic material that provides the blueprint for the proteins that each different cell will produce in its lifetime . It consists of a double stranded helix consisting of a five-sided sugar (deoxyribose ) without a free hydroxyl group, a phosphate group linking the two nucleotides , and a nitrogenous base .
  • RNA Ribonucleic acid
  • Ribonucleic acid is understood to mean a long polymer of ribose (a five-sided sugar with a free hydroxyl group) and nitrogenous bases linked via phosphate groups . It is complementary to one of the DNA strands and forms the proteins that are specified by the cell .
  • Zwitterions is understood to mean amino acids in a form of neutrality where the carboxyl group and amino group are ready to donate and accept protons , respectively .
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • a therapeutic agent e . g . DNA or RNA may be generally distributed throughout an organism via oral administration, thereby eliciting a detectable alteration .
  • This detectable alteration may be broadly directed toward all cells of the organism, thereby effecting a cure for a disease, or enhancement of a particular characteristic .
  • the detectable alterations may be limited to expression in particularly determined locations , thereby providing a safe and effective means for oral administration of chemical or genetic modifiers , whose locus of activity is particularly controlled .
  • the amino acids that form charged side chains in solution are lysine, arginine, histidine, aspartic acid, and glutamic acid . While aspartic acid and glutamic acid release their protons to become negatively charged in normal human physiologic conditions , lysine and arginine gain protons in solution to become positively charged . Histidine is unique because it can form either basic or acidic side chains since the pKa of the compound is close to the pH of the body . As the pH begins to exceed the pKa of the molecule, the equilibrium between its neutral and acidic forms begins to favor the acidic form (deprotonated form) of the amino acid side chain . In other words , a proton is more likely to be released into solution .
  • histidine In the case of histidine, a proton can be released to expose a basic NH2 group when the pH rises above its pKa ( 6 ) . However, histidine can become positively charged under conditions where the pH falls below 6. Because histidine is able to act as an acid or a base in relatively neutral conditions , it is found in the active sites of many enzymes that require a certain pH to catalyze reactions , and is contemplated as being useful in the instant invention .
  • Amino acids can be polar or non-polar .
  • Polar amino acids have R groups that do not ionize in solution but are quite soluble in water due to their polar character . They are also known as hydrophilic, or "water loving" amino acids . These include serine , threonine , asparagine , glutamine , tyrosine, and cysteine .
  • the honpolar amino acids include glycine, alanine , valine , leucine, * isoleucine , methionine , proline, phenylalanine and tryptophan .
  • Nonpolar amino acids are soluble in nonpolar environments such as cell membranes and are called hydrophobic molecules because of their "water fearing" properties . These compounds are contemplated for use where a charge may be induced or wherein the therapeutic agent is caused to be charged so as to initiate a coupling effect .
  • Homeo box genes exemplified by, albeit not limited to pdx-1 , are master genes that trigger the activation of other genes .
  • Homeo box genes are active during embryogenesis and are key in the differentiation of tissues and in the development of organs .
  • Homeo box genes are typically silenced in adult individuals , and are not expressed .
  • Pdx-1 is a mouse homeo box known to be important in the development of pancreatic tissue . Expression of pdx-1 using viral vectors have been used in diabetic mice to induce the transdifferentiation of liver cells into insulin-producing cells . No oral delivery of pdx-1 has been heretofore accomplished .
  • Pdx-1 is a murine gene . Treatment of humans with this approach would require a human gene .
  • the human homologue to pdx-1 is insulin promoter factor 1 , homeodomain transcription factor ( IPFl ) .
  • IPFl homeodomain transcription factor
  • Such gene can be used in humans suffering from diabetes to regenerate pancreatic tissue .
  • C57BL/ 6 mice were rendered diabetic by the inj ection of 250mg streptozotocin, a drug that targets and destroy pancreatic islets .
  • Half of the mice were also administered 100 micrograms of pdx-1 DNA operably linked to a ⁇ -actin promoter orally . The other half of the mice did not receive any additional treatment, and were kept as control .
  • mice The levels of glucose in mice ( Fig . 1 ) were determined by a hand-held glucose meter .
  • Mice treated with pdx-1 DNA showed initially an increase in glucose that was reduced by day 20 post-treatment .
  • the glycemia of the treated mice was thereafter normalised .
  • glycemia of control mice increased due to the streptozotocin treatment, and all mice were dead by day 21. Therefore, the oral administration of pdx-1 DNA achieved a reduction in the glucose levels .
  • FIG. 3 Immunohistochemistry was performed on treated mice, 90 days after treatment . Mice were sacrificed, liver removed, and insulin in the sections detected with anti insulin antibodies .
  • This figure depicts a liver tissue with a tissue structure related to pancreas, not liver, that is producing insulin . Therefore, the treatment of pdx-1 DNA caused the generation of new pancreatic tissue .
  • MAbs monoclonal antibodies
  • autoimmune diseases such as arthritis and lupus
  • infectious diseases such as HIV
  • delivery of antibody protein is technically challenging, and very expensive . What is lacking in the art is a method to achieve circulating levels of antibodies following the administration of DNA coding for antibodies .
  • C57BL/6 mice were orally administered 100 micrograms of DNA coding for a humanized IgG recognising human tumor cells , operably linked to a CMV promoter, and genetically altered to facilitate extracellular secretion of the IgG .
  • the control mice did not receive any treatment .
  • Murine cells transfected with the expression vector showed by western blot a secretable product that reacted with anti human IgG antibodies , and had a separation pattern comparable to that of human IgG .
  • Mice were bled on day 20 post-treatment .
  • IgG molecules were purified from the plasma of mice using a protein G column . Purified IgG was detected in an ELISA test using anti-human IgG antibody as capture and labeled anti-human IgG antibody as detector . A standard made of serial dilutions of human IgG was used for quantification .
  • mice had detectable levels of circulating human IgG above the control background, indicating that oral administration of DNA coding for antibodies can be used to deliver antibodies in a treated host .
  • This invention has applications in the delivery of antibodies for the treatment of diseases such as cancer, infectious diseases and autoimmune diseases . In addition, it can be used as a method for the production of antibodies in animals for medical, biotechnology and veterinary applications Delivery of Factor IX
  • a plasmid containing the human factor IX gene such that FIX secretion is restricted to the liver was administered to hemophilic mice, by feeding each mouse 100 micrograms of DNA formulation . Circulating hFIX was detected in the blood of treated mice .
  • PCR technique ( Figure 8 ) was used to amplify DNA sequences specific to the GFP plasmid administered orally . A positive signal was evident in all organs tested from mice sacrificed 400 days after a single oral administration of DNA, indicating that oral DNA is distributed to all organs in the body . Until now, this achievement was only possible by creating a transgenic animal . Production of Therapeutic Agent
  • mice Groups of mice ( Figure 9) were treated orally with DNA nanoparticles containing various amounts of a DNA plasmid including the cDNA for human ⁇ l-antitrypsin . Mice were bled at regular intervals , and the concentration of human ⁇ l-antitrypsin in the blood of the treated mice was measured by ELISA . All mice had high levels of therapeutic product above the normal physiological concentration that was dose dependent and persistent . These findings further strengthen our claim that this invention can be used to produce human therapeutics in mammals, for example in the treatment of emphysema and cystic fibrosis . Delivery of Human Growth Hormone
  • mice treated orally with DNA nanoparticles containing a DNA plasmid including the cDNA for human growth hormone (hGH) , demonstrated sustained concentrations of hGH which persisted for at least 120 days .
  • hGH human growth hormone
  • mice were determined by a hand-held glucose meter .
  • Mice treated with insulin DNA showed glucose levels that were basically normalized by day 20 post-treatment .
  • the glycemia of the treated mice was thereafter normalised .
  • glycemia of control mice increased due to the streptozotocin treatment, and all mice were dead by day 21. Therefore, the oral administration of insulin DNA achieved a reduction in the glucose levels .

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Abstract

La présente invention concerne un procédé destiné à l'administration par voie orale d'une composition comprenant un acide nucléique, un agent de transport comportant un groupe amine, tel que de la poly-L-lysine ou de la poly-L-arginine, et un polymère polycationique, permettant une large diffusion, une expression systémique et une libération prolongée dudit acide nucléique exprimant un produit destiné au traitement d'un mammifère nécessitant un traitement de ce type. Un mode de réalisation de l'invention concerne l'expression d'un pdx-1, homogène, permettant d'obtenir de l'insuline, pour le traitement d'un mammifère diabétique.
PCT/IB2005/001172 2005-01-18 2005-01-18 Composition de therapie genique a administration par voie orale dans laquelle est utilise un agent de transport comportant un groupe amine et un polymere polycationique WO2006077456A1 (fr)

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WO2004054620A1 (fr) * 2002-12-18 2004-07-01 Neox, Inc. Induction de tolerance par administration par voie orale d'un agent therapeutique couple a un agent de transport

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WO2004009123A2 (fr) * 2002-07-18 2004-01-29 Neox, Inc. Administration par voie orale d'un agent therapeutique couple a un agent de transport
WO2004054620A1 (fr) * 2002-12-18 2004-07-01 Neox, Inc. Induction de tolerance par administration par voie orale d'un agent therapeutique couple a un agent de transport

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