CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of application serial number 10/199,914 filed on July 18,2002, currently pending and incorporated herein by reference.

FIELD OF THE INVENTION The instant invention relates to the induction of tolerance and the abrogation of a pre-existing immune response by the oral administration of a composition to a host animal; particularly to the induction of tolerance and the abrogation of a pre-existing immune response by the administration of a genetic agent coupled to a transporting agent; and most particularly to the induction of tolerance and the abrogation of a pre-existing immune response by the oral administration of a genetic material, e. g. a DNA construct, coupled to a polypeptide carrier whereby widespread distribution, systemic or organ specific expression and sustained delivery may be accomplished.

BACKGROUND OF THE INVENTION Animals are protected by an immune system, an effective arsenal of proteins and cells that can neutralize and destroy foreign cells, viruses and macromolecules. (The Immunoassay Handbook, Edited by David Wild, Stockton Press, page 3,1994). The repertoire of specificities are randomly generateS : by the immune cells (B and T lymphocytes) and asa result the immune system is tremendously diverse. Many of the specificities generated are specific for self- components. Normally, an organism establishes self-tolerance mechanisms to distinguish between self and non-self

determinants so as to avoid auto-reactivity. However, self- tolerance often breaks down and the immune system becomes detrimental to an organism. Many diseases have been identified in which there is auto-immunity due to copious production of auto-antibodies and auto-reactive T cells.

Examples of such diseases include rheumatoid arthritis, pemphigus vulgaris, glomerulonephritis, pernicious anemia, thyroiditis and systemic lupus erythematosus.

An immune response can be detrimental in the treatment of Hemophilia A. Hemophilia A has been treated by administration of commercially available Factor VIII to raise the Factor VIII concentration in the blood to normal levels. However, the immune system often generates inhibitors (antibodies) that attach and neutralize the commercial Factor VIII rendering it non-functional (see US 5,543, 145).

The transplantation of organs and tissues to replace diseased organs is currently an important medical therapy. An immune response is often an impediment to successful transplantation. When tissues containing nucleated cells are transplanted, T-cell responses to the highly polymorphic MHC (major histocompatibility complex) molecules almost always trigger a response against the grafted organ. Matching the MHC type of donor and recipient increases the success rate of grafts, but perfect matching is possible only when donor and recipient are related and, in these cases, genetic differences at other loci still trigger rejection.

Currently, several different immunosuppressive agents, including both drugs and antibodies, have been used clinically in auto-immune disease and graft rejection.

Several examples are methotrexate, azathiopurine,

cyclophosphamide, prednisone, cyclosporine A, FK506 (tacrolimus), anti-lymphocyte globulin (ALG) and anti- thymocyte globulin (ATG).

However, the presently developed immunosuppressant drugs have a common problem in that cells that are not related to immune response or normal cells are all affected by the drugs. This causes serious side effects that cannot be avoided, such as the development of infections in immunosupressed patients (see US 6,458, 934 for a discussion of methods to suppress the immune response). The potential exists for limited therapeutic use of antibodies as immunosupressants since antibodies can also be formed to the therapeutic antibody and thus clear the therapeutic antibody from the circulation eliminating its effectiveness.

Prior artisans have searched for ways to manipulate the immune system and/or induce tolerance in order to suppress unwanted immune responses in such conditions as auto-immune disorders, therapy resistant hemophilia and graft rejection.

Tolerance refers to the absence or the prevention of an immune response. Particularly, tolerance refers to the state of a host that does not respond immunologically to an antigen, whereas under normal circumstances the host responds. Even more particularly, tolerance to a foreign antigen or tissue (or self-antigen or tissue) is a state wherein an otherwise normal immune system is specifically unable to respond aggressively to that antigen or tissue, which it therefore treats like a normal body component, yet at the same time it can respond to aggressively to foreign or diseased antigens or tissues to which it has not specifically been made tolerant by natural processes of self-tolerance or by a therapeutic tolerance induction.

Common testing for tolerance requires a demonstration that the tolerant individual fails to become immune to the specific antigen or tissue when one or more attempts to immunize are made at a later time, when the same individual can be shown to respond to an unrelated antigen or tissue.

Tolerance can occur through several mechanisms for example: a) ignorance, a state wherein the immune system ignores the antigen so as not to elicit an immune response and b) inhibition, a state wherein the antigen is modulated so as to not recognize the given antigen.

The instant inventors have discovered that their method for oral delivery of genetic material (which is disclosed and claimed in the parent application 10/199,914 filed on July 18,2002) leads to long-term expression of human (exogenous) transgenes in animals without the development of antibodies to the human transgenes.

Additionally, the instant inventors have shown that their method for oral delivery of genetic material can eliminate a pre-existing immune response even in the presence of stimulation by the specific antigen. Thus, the instant invention provides a method for the induction of tolerance by the use of oral gene therapy that can be beneficial in the therapy of conditions described above, such as auto- immune disorders, graft rejection, and therapy resistant hemophilia.

Gene therapy offers an alternative to the currently available treatment modalities for a variety of conditions, particularly genetic and acquired disorders affecting a range of cells and tissues. There exist ex vivo approaches based upon the implantation of autologous genetically-modified cells. 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.

Additionally, germ line transmission of DNA is a concern in gene therapy protocols (E. Marshall Science 294: 1640 2001 and E. Marshall Science 294: 2268 2001). There are the potential dangers of passing on the new trait to future human generations or introducing a particular transgene into the environment.

Although various methods have been attempted, with an eye toward distribution of DNA via oral administration, what has eluded prior artisans is a process and a device which enables widespread distribution of DNA throughout all organs and tissues via oral administration, whereby persistent and efficient protein expression is accomplished.

DESCRIPTION OF THE PRIOR ART Quong et al., in an article entitled ADNA Protection from Extracapsular Nucleases, within Chitosan or Poly-L-lysine-coated Alginate Beads@ (Biotechnology and Bioengineering, Vol. 60, No. 1,10/98, pages 124-134,1998) 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). The work carried out by Quong et al. concluded that PLL coating provides enhanced protection of DNA against DNase in vitro when compared to uncoated beads.

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.

Complexes of poly (L-lysine) (PLL) have been targeted to various cell lines in vitro by covalent attachment of targeting ligands to the PLL, resulting in transgene expression. Ward characterizes these complexes as having little use in vivo since they have poor circulatory half- lives. Ward further theorizes that since complexes activate human complement in vitro and stimulate the immune system, this most likely accounts for their poor half-life in vivo.

Thus, this work fails to disclose any form of widespread transgene distribution or expression (of proteins, antibodies or the like coded products) via this methodology.

Rothbard et al. (Nature Medicine, Volume 6, Number 11, November 2000, Pp. 1253-1257) discloses the conjugation of arginine and cyclosporin-A to form a compound useful in traversing the stratum corneum and thereby entering the epidermis. The disclosed process is useful in forming a conjugate which, unlike cyclosporin-A alone, is capable of reaching dermal T lymphocytes and inhibiting cutaneous inflammation. The reference fails to teach or suggest the conjugation of DNA to arginine, nor does it in any way contemplate oral ingestion of a conjugated arginine of any kind.

Wender et al. (PNAS USA, 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 pe-ptoIds~~~-couId 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.

One of the instant inventors is co-author of a series of articles related to gene therapy. In an article in Human Gene Therapy, (6: 165-175 (February 1995) Al-Hendy et al.) nonautologous somatic gene therapy via the use of encapsulated myoblasts secreting mouse growth hormone to growth hormone deficient Snell dwarf mice is disclosed.

Immunoprotective alginate-poly-l-lysine-alginate microcapsules were used to protect recombinant allogeneic cells from rejection subsequent to their implantation. Oral gene therapy is neither contemplated nor suggested.

In Blood, Vol. 87, No. 12, June 15,1996, Pp.

5095-5103, 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. 472-484 (1996), Peirone et al., Encapsulation of Various Recombinant mammalian Cell types in different alginate microcapsules, Journal of Biomedical Materials Research 42 (4): 587-596, 1998), and in Haemophilia (2001), 7, 207-214. The references neither disclose nor suggest the use of immuno- isolation devices for the delivery of gene therapy via an oral route.

However, the Hortelano et al. reference (Blood 87 (12): 5095-5103 1996) mentioned above is particularly important with regard to the induction of tolerance. In the experiment discussed in this article, immunocompetent mice were implanted with alginate microcapsules enclosing recombinant myoblasts continuously secreting human factor IX (hFIX). These mice have detectable hFIX in their circulation for up to two weeks. Starting at day 14 an increasing concentration of anti-hFIX antibodies were detected in the plasma of treated mice, which can explain the transient delivery of the hFIX. Evidence of this rationale is shown in Hortelano et al. (Haemophilia 7: 207-214 2001) wherein immunodeficient mice treated with the same protocol as described in the Blood reference noted above had sustained levels of hFIX for at least 77 days.

In an article by Chang et al., Tibtech/Trends in Biotechnology, 17 (2); February 1999, entitled AThe in Vivo Delivery of Heterologous Proteins by Microencapsulated Recombinant Cells@ the use of microencapsulated E-Coli engineered to express Klebsiella aerogens urease gene was administered orally. It is disclosed that passage of the live bacteria via the gastrointestinal tract was found to permit the clearance of urea, thereby lowering the plasma urea levels. This disclosure is not suggestive of the use of oral gene therapy to result in widespread dissemination of DNA via an oral pathway.

Brown et al.,"Preliminary Characterization of Novel Amino Acid Based Polymeric Vesicles as Gene and Drug Delivery Agents" (Bioconjugate Chem. 2000, 11,880-891) teaches formation of an amphiphilic polymer matrix using poly-L-lysine with polyethylene glycol modification, as a means of gene delivery to a cell in vivo. The disclosure is directed toward transfer of DNA into live cells when

incorporated within PLL-PEG vesicles. The disclosure fails to teach oral administration, nor the combination of an GI tract protector, such as alginate, in combination with a polypeptide suitable for use as a DNA transporting agent in accordance with the teachings of the instant invention.

Leong et al.,"Oral Gene Delivery With Chitosan- DNA Nanoparticles Generates Immunologic Protection In A Murine Model Of Peanut Allergy" (Nature Medicine, Volume 5, Number 4, April 1999, Pp 387-391) discloses chitosan/DNA nanoparticles synthesized by complexing plasmid DNA with chitosan for oral ingestion to treat allergic response to peanut antigen. The reference fails to show widespread distribution, in that staining only showed gene expression in the stomach and small intestine.

U. S. Patent No. 6,217, 859 discloses a composition for oral administration to a patient for removal of undesirable chemicals or amino acids caused by disease. 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 invent-ion a-re--e-apabl-e--of---fo-r-mi-ng--s-t-abl-e-and---solubl-e 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 subject. In the disclosed method, intestinal epithelial cells of a mammalian subject 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. Of particular interest is the use of 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 subject. 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. short term being up to about 2-4 days, while long-term, via incorporation in intestinal villi is theorized to possibly last for weeks or months) for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein. It is noted, however, that the expression is limited to within the gastrointestinal tract, thus relegating distribution of the expressed entity to the bloodstream, where immunogenic response and resulting neutralization of said entity via the immune system becomes problematic.

U. S. Patent No. 5,837, 693 is directed to intravenous hormone polypeptide delivery by salivary 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 subject. 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 subject. 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 subject. 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. In one embodiment, 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 conjugate consisting of antibody directed at the pIgR 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 pIgR 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.

U. S. Patent No. 6,159, 502 relates to an oral delivery system for microparticles. There are disclosed complexes and compositions for oral delivery of a substance or substances to the circulation or lymphatic drainage system of a host. 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). Examples of suitable carriers are mucosal binding proteins, bacterial adhesins, viral adhesins, toxin binding subunits, lectins, Vitamin B12 and analogues or derivatives of Vitamin B12 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. Regulated intracellular protein association with these cell permeable, synthetic ligands are deemed to offer new capabilities in biological research and medicine, in particular, in gene therapy. Using gene transfer techniques to introduce these artificial receptors, it is indicated that one may turn on or off the signaling pathways that lead to the overexpression of therapeutic proteins by administering orally active"dimerizers"or"de-dimerizers", respectively.

Since cells from different recipients can be configured to have the pathway overexpress different therapeutic proteins for use in a variety of disorders, the dimerizers have the potential to serve as"universal drugs". They can also be viewed as cell permeable, organic replacements for therapeutic antisense agents or for proteins that would otherwise require intravenous injection or intracellular expression (e. g. , the LDL receptor or the CFTR protein). what is lacking in the art is an orally deliverable composition capable of achieving: a) widespread delivery and distribution of a therapeutic agent such as DNA, to essentially all cells of the targeted subject b) an ability to provide a sustained (e. g. non-transient)

expression of a therapeutic moiety by said therapeutic 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 instant inventors have developed a composition and a methodology to accomplish a, b, and c as noted above in the instant paragraph; said composition and methodology is claimed in the parent application 10/199,914, filed on July 18,2002, which is incorporated herein by reference. While working on these concepts, the instant inventors concluded that the generally disclosed concept could be utilized for the induction of tolerance to a foreign transgene or a self- antigen in an animal host and can also provide a process to eliminate a pre-existing immune response to a foreign transgene or a self-antigen in an animal host that can be beneficial in the therapy of conditions described above, such as auto-immune disorders, graft rejection, and therapy resistant hemophilia.

SUMMARY OF THE INVENTION The instant invention is directed toward a method for the induction of tolerance to a foreign transgene or a self antigen in an animal host by administration of a composition to an animal host via a natural gastrointestinal pathway.

Additionally, the method of the instant invention can also be used to eliminate a pre-existing immune response in an animal host. More particularly, the invention discloses a method useful for both eliciting tolerance and a method for abrogation of a pre-existing immune response using oral gene therapy.

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.

In order to achieve maximum distribution and efficacy via oral administration, it has been determined that DNA requires a protective covering. For example, alginate is a means of providing protection in the gastrointestinal tract. Additionally, 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. In a particularly preferred embodiment, 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. Thus, in a preferred embodiment, the instantly disclosed formulations will couple DNA to the amino compound, e. g. via electrostatic binding, 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 nanoparticle. 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.

In order to make DNA microcapsules, 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. Absent the presence of 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 ß 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.

The instant inventors have shown that their method for oral delivery of DNA leads to long-term expression of transgenes in animal hosts without the development of antibodies to the transgenes, thus they have achieved a method for the induction of tolerance in an animal host.

Additionally, the instant inventors have shown that by using their method of oral delivery of DNA, transgene expression in the presence of antigen stimulation can be achieved in an animal host that has previously developed an immune response to the given antigen, thus they have achieved a method of abrogation of a pre-existing immune response. Using these methods it is possible to circumvent the disadvantages of conventional methods of immunosuppression mentioned above in the Background of the Invention section.

Accordingly, it is an objective of the instant invention to provide a method for the induction of tolerance in an animal host (or a method for abrogation of a pre- existing immune response) by systemic delivery of a complete transcriptional unit, e. g. DNA and RNA, or components which enable a complete transcriptional unit within the cells, e. g. FIX cDNA coupled to a suitable promoter and polyadenylation signal, to virtually all cells of an organism, via an oral pathway.

It is a further objective of the instant invention to provide a method for the induction of tolerance in an

animal host (or a method for abrogation of a pre-existing immune response) by oral delivery of DNA wherein the expression of the DNA is controllable (e. g. ubiquitous or tissue specific) via a complete transcriptional unit in conjunction with judicious promoter selection.

It is still a further objective of the instant invention to provide a method for the induction of tolerance in an animal host (or a method for abrogation of an immune response) by delivery of DNA and RNA to a variety of organs, including but not limited to heart, muscle, lungs, skin, kidney, liver, brain and spleen, in conjunction with appropriate expression of the DNA and RNA, as desired.

Other objectives and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying drawings wherein are set forth, by way of illustration and example, certain embodiments of this invention. The drawings constitute a part of this specification and include exemplary embodiments of the present invention and illustrate various objects and features thereof.

BRIEF DESCRIPTION OF THE FIGURES In the accompanying drawings, which illustrate an exemplary embodiment of the present invention: FIGURE 1 is a fluorescent micrograph illustrating expression in the Liver; FIGURE 2 is a fluorescent micrograph illustrating expression in the Kidney; FIGURE 3 is a fluorescent micrograph illustrating expression in the Lung;

FIGURE 4 is a fluorescent micrograph illustrating expression in the Heart; FIGURE 5 is a fluorescent micrograph illustrating expression in the Muscle; FIGURE 6 is a fluorescent micrograph illustrating expression in the Skin; FIGURE 7 is a fluorescent micrograph illustrating expression in the Vessels; FIGURE 8 represents a graphical analysis of an in vitro assay of Activated Partial Thromboplastin Time (APTT); FIGURE 9 shows GFP DNA expression by PCR analysis in organs of mice fed GFP DNA and sacrificed on day 42 post injestion; FIGURE 10 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Duodenum; FIGURE 11 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Jejunum; FIGURE 12 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Ileum; FIGURE 13 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Colon ; FIGURE 14 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Liver;

FIGURE 15 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Spleen; FIGURE 16 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Kidney; FIGURE 17 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Lung; FIGURE 18 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Heart; FIGURE 19 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Muscle; FIGURE 20 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Pancreas; FIGURE 21 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Brain; FIGURE 22 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Gonads; -F-I-GuR-E-2-3--ls----a---f-luo-rescent microgr : aph_illustrating expression utilizing Arginine/Ornithine transport agents in the Skin;

FIGURE 24 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Vessels; FIGURE 25 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Bone Marrow; FIGURE 26 is a graphical representation showing the levels of hGH in treated mice; FIGURE 27 illustrates that anti-hGH antibodies were not detected post hGH production; FIGURE 28 is a fluorescent micrograph illustrating tissue specific expression in the liver utilizing an albumin promoter; FIGURE 29 is bar graph comparing the level of hGH achieved using alternative technologies; FIGURE 30 is a graphical analysis over time of hGH levels achieved using alternative technologies; FIGURE 31 is illustrative of the presence of hGH in various organs achieved using alternative technologies; FIGURE 32 depicts weight gain attributable to hGH levels achieved using alternative technologies; FIGURE 33 is a schematic representation of the construct used for ubiquitous expression of Factor IX; <BR> <BR> <BR> --FTGURE--34-is----a---schemati-c----repre-sent-ation-of--th e construct-- used for ubiquitous expression of GFP; FIGURE 35 is a graphical analysis of hGH in mice fed hGH DNA orally;

FIGURE 36 is a graphical analysis of the levels of hFIX in immunized C57B1/6 mice; FIGURE 37 is a graphical analysis of anti-hFIX antibody reduction in mice (tolerance) by oral administration of DNA; FIGURE 38 is a graphical representation of hFIX in mice with pre-existing immunity; and FIGURE 39 is a graphical representation of the levels of hFIX in Balb/c mice treated with oral DNA.

DEFINITIONS The following list defines terms and phrases used throughout the instant specification.

As used herein, the term"transgene"refers to a nucleotide sequence which is artificially incorporated into the genome of a cell, either integrated or episomal, and is used to modify the cell for the purpose of achieving a genetic pool distinct from the"normal"or"wild-type" state. A transgene can be obtained from either the same cell (for example, but not limited to, a mutated gene) as the cell into which it is incorporated or it can be obtained from any other source; for example, but not limited to, a different organism or an artificial sequence.

As used herein, the term"exogenous gene"refers to a transgene that has been obtained from a different organism or cell other than the organism or cell into which it has been incorporated.

The terms"transgene"and"exogenous gene"are used interchangeably herein.

As used herein, the term"wild-type"refers to the natural organism or cell or to the natural state of an organism or cell which is unmodified by man.

As used herein, the term"natural gastrointestinal pathway"refers to the pathway taken by ingested nutrients in a human or in an animal.

As used herein, the term"genetic material"can refer to DNA, RNA, ribozymes, antisense RNA, hybrids of DNA/RNA, either single or double stranded, or combinations thereof.

As used herein, the term"protective agent"refers to an agent that protects genetic material from destruction as it passes through a natural gastrointestinal pathway, for example, but not limited to, collagen, chitosan, alginate and the like.

As used herein, the term"transporting agent" refers to a compound containing an amine group that is capable of coupling with the genetic material in a manner effective to produce widespread distribution and cellular uptake subsequent to passage via a natural gastrointestinal pathway, for example, but not limited to, a polypeptide.

As used herein, the abbreviation"PLL"refers to poly-L-lysine.

As used herein, the term"tolerance"refers to the state of a host that does not respond immunologically to an under normal circumstances the host would respond to the antigen. A host that is tolerant of a particular antigen does not mount an immune response upon stimulation with that antigen.

As used herein, the phrase"pre-existing immune response"refers to the state of a host that is in the process of responding to stimulation by an antigen. When the experiments described in the instant specification refer to "a pre-existing immune response"it is meant the treatment (DNA formulation administered orally) is applied after the host has begun to respond to an antigen as evidenced by the presence of circulating antibodies.

As used herein, the phrase"reduced immune response"or"reducing an immune response"refers to an event (for example, a treatment or method) that results in sustained expression of a transgene together with a decrease in antibody titre.

DETAILED DESCRIPTION OF THE INVENTION The primary objective of this invention is the induction of tolerance or the elimination of a pre-existing immune response by oral administration of a transporting agent, exemplified as, but not limited to an amino acid carrier, e. g. poly-l-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.

In order for the compound, e. g. DNA to be distributable via the gastrointestinal tract with the highest possible degree of efficacy, it should be protected from enzyme degradation and low pH as it passes through the stomach and small intestine. In a preferred embodiment, this is accomplished via the use of protective compounds,

illustrative of which are alginate, gelatin (which is mainly collagen) and the like.

The role of alginate, gelatin 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 (Anaked@) DNA, if adequately protected with gelatin (collagen) or the like, is also taken through the intestinal wall and expressed in certain tissues, but not all of the tissues. However, it is important to distinguish that in this case: 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. Thus, while the instant inventors have achieved limited success absent effective coupling to a transporting agent, this remains a non-preferred embodiment of the instant invention. Additionally, while the preferred, and most efficacious gastrointestinal route is via oral delivery, rectal delivery is indeed contemplated by the instant inventors as an alternative route for administration via the gastrointestinal pathway.

As we have noted above, the encapsulation of DNA in alginate-poly-L-lysine microcapsules has already been described, however prior artisans failed to appreciate the importance of coupling the therapeutic agent with the

transport agent, e. g. via electrostatic binding, in a manner effective to produce efficacious and widespread distribution and cellular uptake subsequent to passage via said natural gastrointestinal pathway. While we have exemplified an embodiment which utilizes electrostatic binding, preferably via the use of positively charged amino acids which bind to a negatively charged therapeutic agent such as DNA, alternative binding techniques are contemplated for use in the instant invention. 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.

Prior artisans such as Aggarwal et al. (Canadian Journal of Veterinary Research, 1999,63 : 148-152) and Mathiowitz et al., (Nature, Vol 386, March 1997, Pp. 410- 414) teach the use of biodegradable and biologically adhesive microspheres respectively, as a means for oral drug delivery of genetic material containing agents such as DNA.

Neither of these artisans recognized or pursued the use of a transport agent as outlined by the instant invention, nor ---did they recognize the value of coupling a therapeutic agent thereto so as to facilitate the widespread, systemic and sustained delivery and expression which are hallmarks of the instant inventive concept. In contrast, while not achieving the desirable distribution, delivery, efficacy or

expression, the prior artisans nevertheless required encapsulation of the therapeutic agent, a requirement which is overcome via the instantly taught invention.

Mathiowitz et al. utilized polyanhydrides of a combination of fumaric and sebacic acids to encapsulate a plasmid DNA (ß-galactosidase). However, as evidenced in Figure 5 of the article, quantification of ß-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.

In order to determine the relative effectiveness of the Aggarwal embodiments a comparative study was performed between a formulation in accordance with the instant invention (alginate-PLL-DNA) nanoparticles (hereafter referred to as alginate formulations) and the alginate-PLL microcapsules made by internal gelation as described in Aggarwal and hereafter referred to as Canola capsules (made using canola oil).

A single dose of 100 micrograms of a DNA plasmid containing the human growth hormone cDNA in an alginate-DNA- PLL nanoparticles in accordance with the instant invention was administered orally to C57BL/6 mice. A second group of mice (n=3) received the same plasmid in canola capsules.

Note that these mice each received 300 micrograms of DNA, <BR> --ra-t-her-than---t-he--1-00--mi-erog-rams-g-i-ve-n-in-t-he- ålginate __ formulation (three times more DNA). A control group of mice received nothing.

Mice were bled on days 0,3 and 5 (so as to compare expression up to day 5, thus reproducing the results as determined by Aggarwal et al.).

The level of human growth hormone (hGH) in mouse serum on day 5 following the treatment was determined by ELISA (UBI Inc. , NY).

Mice receiving alginate formulation had comparatively high levels of hGH in the serum. In contrast, hGH was not detected on day 5 in mice receiving canola capsules, even though mice receiving this formulation were administered three times more DNA than mice receiving the alginate formulation. As expected, control mice did not have detectable hGH in serum.

These data, as seen in Fig. 29 show that the efficacy of alginate formulation is much higher than canola capsules.

Now referring to Fig. 30, this graph depicts the level of hGH in mouse serum on days 3 and 5.

Mice administered Canola capsules had very modest but detectable hGH on day 3. However, this delivery was transient, and hGH was undetectable on day 5. This is consistent with the paper by Aggarwal et al. , where it is necessary to feed mice daily for three days in order to detect circulating hGH on day 5. The transient nature of hGH delivery is consistent with the uptake of DNA by the intestine, rather than the distribution of DNA systemically, <BR> <BR> <BR> <BR> as taught by the instant invention.<BR> <P>In contrast, mice administered alginate formulation showed high hGH levels on day 3, that continue to increase on day 5. This is consistent with all our

previous data, indicating that the alginate formulation leads to sustained, not transient, gene expression.

Thus, the uptake and expression of DNA is different with both formulations. The different trend of hGH delivery with both formulations would suggest that both formulations are taken by different routes and/or mechanism (s).

With reference to Fig. 31, on day 5, mice were sacrificed and the presence of hGH in the various organs was determined. High levels of hGH were recorded in the organs described in this graph in mice receiving alginate DNA formulation. In contrast, none of the mice receiving canola capsules had detectable hGH in any of the above organs, even though these mice received three times more DNA than the former group.

These results are consistent with our previous data showing wide systemic distribution of DNA in major organs following administration of alginate formulation.

These results are also consistent with the lack of systemic distribution of DNA using formulations described in the prior art. Finally, these results also highlight the obvious difference in efficacy between both formulations.

As further evidence of the efficacy of delivery in accordance with the present invention, a comparison of weight gain due to the presence of efficacious levels of hGH was determined and is the subject of Figure 32.

~ growth hormone induces weight gain. However, gene therapy experiments delivering hGH have only demonstrated weight gain after very high levels of hGH are delivered (efficacious levels).

All mice were weighed on day 0, before treatment, and during the 5 days of the experiment. Mice that were fed canola capsules did not gain more weight than the control mice (p<0.145). In contrast, mice that were fed alginate formulation gained weight amounting to a 109.7% increase on day 5. The difference in weight gain between mice fed alginate formulation and mice receiving canola capsules was statistically significant (p<0.05).

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, albeit not coupled or conjugated to the transporting agent (as is required by the instant invention), with the intent of retrieving DNA from feces and thereby determining if DNA had mutated through the intestine. These artisans failed to recognize or suggest whether DNA could be taken up by the intestine and expressed, and therefore failed to recognize the instantly disclosed product or process of oral gene delivery. Oral delivery of DNA for widespread distribution, in conjunction with systemic and sustained expression of therapeutics has thus not heretofore_been_achieved. __ _ _ __ Furthermore, in addition to DNA, it is contemplated to similarly transport additional therapeutic agents, non-limiting examples of which are RNA, which has commercial interest owing to its ability to inactivate the

transcription/translation of unwanted proteins; and 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.

DNA MICROCAPSULES: In the formation of the various species of the invention as hereafter described, it is understood that 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. conjugation, of the active agent with the transporting agent.

DNA Microcapsules-Example 1 In a particular, albeit non-limiting embodiment, formation of DNA plasmids containing a cDNA coding for a transgene and appropriate genetic regulatory elements such as a promoter is performed as follows. A suspension of DNA is mixed with 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% CaCl2 solution. Upon contact, the alginate/DNA droplets gel. After the microcapsules are extruded, they are subjected to the washes as indicated in the li-s-t--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): - 1. 1% calcium chloride - 0, 55% calcium chloride - 0. 28% calcium chloride - 0. 1% CHES (2- (Cyclohexylamino) ethanesulfonic acid) for about 3 minutes - 1. 1% calcium chloride - 0. 05% PLL for about 6 minutes - 0. 1% CHES (2- (Cyclohexylamino) ethanesulfonic acid) - 1. 1% calcium chloride - 0. 9% sodium chloride - 0. 03% potassium alginate for about 4 minutes - 0. 9% sodium chloride - 0. 055 M sodium citrate for about 3 minutes (standard microcapsule protocol is 6 minutes) - 0. 9% sodium chloride DNA Microcapsules-Example 2

A volume of 300 ßl of DNA plasmid at a concentration of 1 g/jUl is mixed with 6 ml of 1. 5% calcium alginate.

Alginate beads are cross-linked with, e. g. Poly-L-Lysine (PLL) resulting in microcapsules containing DNA-alginate in the inside. Microcapsules are subsequently mixed with a 1: 1 volume of a 50% gelatin solution to obtain a homogeneous mixture that can be administered.

DNA-alginate-PLL particles: A volume of 100 gl of DNA plasmid at a concentration of 1 gg/gl is mixed with 50 ßl of 3% calcium alginate, and mixed at 4°C for 3 hours with gentle agitation. A volume of 50 ßl 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 gl of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered.

DNA-PLL-alginate particles: In an exemplary, but non-limiting example of forming DNA-PLL-Alginate microcapsules, a volume of 100 1 of DNA plasmid at a concentration of 1 ßg/l is mixed with 50 gl of poly-L-Lysine, and mixed at 4°C for 3 hours with gentle agitation. A volume of 50 ßl 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 ßl of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered.

DNA-ornithine-alginate particles: A volume of 100 Al of DNA plasmid at a concentration of 1 Ag/gl is mixed with 50 gl 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 Al of 3% calcium alginate is added and mixed at 4°C for one additional hour with gentle agitation. Finally, 50 Al 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 ßl of DNA plasmid at a concentration of 1 ßg/l is mixed with 50 Al 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 ßl of 3% calcium alginate is added and mixed at 4°C for one additional hour with gentle agitation. Finally, 50 gl of a 50% gelatin solution is added to the mixture to obtain a homogeneous mixture that can be administered.

Naked DNA in collagen: A volume of 100 ßl of DNA plasmid at a concentration of 1 AgIAl is mixed with 50 Al 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 an amino acid.

Although 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.

The inclusion of particular genetic regulatory elements (promoters), afford the compositions of the instant invention the added utility of controllable 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.

Via the judicious use of such promoters, the degree of expression may be tailored to meet specific needs.

For example, via the use of P-Actin, a ubiquitous promoter, widespread expression is achieved. Alternatively, use of 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 protein in a particularly desired location, e. g. a specific portion of the body, specific organ, or specific cell or tissue type.

In accordance with the present invention administration of the DNA formulation can be either oral or rectal and can be administered to a"wild-type"or a transgenic animal, for example, the animal to which the formulation is administered can be genetically altered (transgenic, either classically or in accordance with the teachings of this invention) at the time of administration in order that the formulation can correct the previous genetic alteration, or alternatively provide further modification. The formulation can also be administered to a "wild-typen animal, for example, an animal that is genetically unmodified by man at the time of administration.

In accordance with the present invention a therapeutic agent includes any genetic material 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, Ribozyme, Antisense, Hybrids, either Single or Double stranded, or combinations thereof.

In accordance with the present invention 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.

In accordance with the present invention systemic expression is understood to mean measurable cellular uptake of a therapeutic agent within cells, inclusive of, but not limited to cells of the epithelial, connective, nervous and musculo-skeletal tissues, found in various organs throughout the body.

In accordance with the present invention, sustained expression or sustained delivery is understood to --mean measu-r--able---exp-r-e-ss--i-on--of--a-therapeutic--ag-ent suf ficient to instigate a desirable biological effect, as a result of a single administration, which effect is detectable for a minimum of 40 days. The protein encoded by the therapeutic agent may be intracellular or extracellular.

In accordance with the present invention 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.

In its preferred embodiments, 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 and other genetic regulatory elements 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.

While the use of 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 mandatory 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 may 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 adjacent 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 joined 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.

Additionally, some vectors, such as Adenoassociated-virus (AAV) form concatamers inside the infected cells. In the process 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. If 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. Alternatively, as shown by Zhou et al, "Concatamerization Of Adeno-Associated Virus Circular Genomes Occurs Through Intermolecular Recombination" (J Virology 1999 Nov; 73 (11) : 9468-77), one could place the promoter in one vector, and the transgene in a second vector, that are administered separately.

The following listing of amino acids, their derivatives, and related compounds, are non-limiting illustrative examples of compounds containing the requisite structure deemed necessary for widespread distribution of DNA in vivo.

Amino acids and derivatives: Aliphatic-alanine, glycine, isoleucine, leucine, proline, valine Aromatic-phenylalanine, tryptophan, tyrosine Acidic-aspartic acid, glutamic acid Basic-arginine, histidine, lysine -Hydrox-ylic- serine, threon-ine 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.

It is known that when a living cell makes protein, the carboxyl group of one amino acid is linked to the amino group of another to form a peptide bond. The carboxyl group of the second amino acid is similarly linked to the amino group of a third, and so on, until a long chain is produced, called a polypeptide. 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. In addition to the amino acids that form proteins, more than 150 other amino acids -have- have been-found-in-nature,-ncludirLg-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.

As used herein, the term Deoxyribonucleic acid (DNA) is understood to mean a long polymer of nucleotides joined 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.

As used herein, the term Ribonucleic acid (RNA) 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.

As used herein the term 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.

The evolution and mutation of proteins can be realized through changes in deoxyribonucleic acid (DNA).

DNA is translated to proteins via ribonucleic acid (RNA).

Although every cell. contains an identical copy of DNA with complete instructions for all types of body tissues, only certain proteins are produced by each cell type. In this way, cells of different tissues can perform diverse tasks through the production of unique proteins. In accordance with the teachings of the present invention, 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.

Alternatively, by judicious use of organ or tissue specific promoters, 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. 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 nonpolar 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.

Examples Biodistribution Of Oral DNA Which Expresses Green Fluorescent Protein (GFP) Single administration of alginate/PLL GFP DNA nanoparticles in mice (n=3) was carried out. The vector used for ubiquitous expression of GFP is illustrated in Figure 34. Three formulations were tested: 1) DNA alginate/PLL microcapsules (Capsules); 2) Alginate/DNA/PLL nanoparticles (Alginate); and 3) PLL/DNA/alginate nanoparticles (PLL).

9 mice were treated, and were sacrificed on Day 42. Tissue samples from all are designated as Figures 1-7. Figure 1 is a fluorescent micrograph illustrating expression in the Liver; Figure 2 is a fluorescent micrograph illustrating expression in the Kidney; Figure 3 is a fluorescent micrograph illustrating

expression in the Lung; Figure 4 is a fluorescent micrograph illustrating expression in the Heart; Figure 5 is a fluorescent micrograph illustrating expression in the Muscle; Figure 6 is a fluorescent micrograph illustrating expression in the Skin; and Figure 7 is a fluorescent micrograph illustrating expression in the Vessels.

GFP (green fluorescent protein) is intracellular and stays in the cell where it is produced. As is readily apparent by reviewing the accompanying figures and as summarized in the Table 1, fluorescent microscopy detects virtually all cells in all major organs examined as being green (wherein"green"is represented in Figures 1-7,10-25, and 28 by the white contrast against the black background).

Table 1 Tissue Liver Kidney Lung Heart Muscle Brain Skiri Vesse opta Capsules +++ ++ ++ +++ +++ ++ +++ +I--I- 'Alginate +++ ++ ++ +++ +++ ++ +++ ++ PLL +++ ++ ++ +++ +++ ++ +++ + Tissue Bone Spleen Pancreas Duodenum J'ejunun Tleu Colori Gonads marrow Capsules ++ ++ ++ ++ +++ +++ + ++ +++.. ++.. 4-44 + + 4+ + ++ ++ 4-44.. + +

This indicates that DNA, in the form of microcapsules conjugated with the transporting agent (PLL) and internalized within a capsule comprising cross-linked alginate/transporting agent goes through the intestine and is transported to all major organs where it enters the cells <BR> <BR> <BR> <BR> --and---is effie-i-en-t-l-y-exp-r-essed.-----This--is-.-ir__contradi_st inction to prior art encapsulated DNA, wherein the PLL acted as a structural element which prevented/reduced diffusion of DNA.

As a validation of the technique, analysis of tissue samples was performed utilizing polymerase chain reaction (PCR) as an amplification technique.

At day 42 post-treatment, the mice were sacrificed. DNA from various tissues was amplified by PCR, and showed that orally administered DNA is found in every major organ examined (Table 2). This finding further confirms that DNA administered orally is taken to all organs, where it enters cells.

Table 2 PCR of GFP DNA in tissues (day 42) Tissue L, iver, Kidney I ; iyg Heart Muscle Brain Skin Positive Positive Positive Positive Positive Positive Positive Tissue Spleen Pancreas Duodenurri Jejunum Ileum Colori Vessel avorta Positive Positive Positive Positive Positive Positive Positive Note: PCR in bone marrow and gonads were not conducted.

Example: To determine the importance of alginate and PLL for efficient expression of oral DNA the following experiment was carried out.

A single administration of alginate/PLL hFIX DNA nanoparticles was given to mice (n=3). Three formulations were teste alginate L nanopar ic es control), alginate/DNA nanoparticles (no PLL), and PLL/DNA nanoparticles (no alginate).

At day 3,7 and 14 post-treatment, mice were bled.

Control mice had hFIX in blood (approx. 70 ng/ml). None of the mice with no alginate or with no PLL had detectable hFIX (sensitivity 3 ng/ml). Thus, it was concluded that both alginate and PLL are needed to insure widespread DNA distribution and subsequent protein expression. While not wishing to be bound to a particular theory of operation, it appears that alginate protects DNA in the GI tract, and PLL helps distribute DNA into all organs.

Example using HFIX: To determine the degree of expression obtainable, additional experimentation was conducted to demonstrate Human factor IX (FIX) delivery.

A single administration of alginate/PLL FIX DNA nanoparticles was carried out in hemophilic mice.

APTT (Blood clotting time test) was done to determine correction of the disease in the treated hemophilic mice.

As further illustrated in Figure 8, treated hemophilia mice demonstrated a normalized bleeding pattern for at least 180 days.

Now referring to Figure 9, amplification of data via PCR was performed on tissue samples harvested from a plurality of organs on day 42 post ingestion of alginate/PLL GFP DNA nanoparticles. All organ samples demonstrated a positive presence of GFP via PCR analysis. This data is additionally set forth in Table 2 above.

Further experimentation was conducted to validate the efficacy of distribution and expression using alternative transport agents. Poly-ornithine and poly- arginine were conjugated with DNA coding for GFP and

alginate and formulated into nanoparticles. The nanoparticles were administered to mice (n=3) in a manner as earlier described. At day 10, the mice were sacrificed and fluorescent micrographs were taken (Figures 10-25). Figure 10 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Duodenum; Figure 11 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Jejunum; Figure 12 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Ileum; Figure 13 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Colon; Figure 14 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Liver; Figure 15 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Spleen; Figure 16 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Kidney; Figure 17 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Lung; Figure 18 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Heart; Figure 19 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Muscle; Figure 20 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Pancreas; Figure 21 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Brain; Figure 22 is a fluorescent micrograph illustrating expression utilizing

Arginine/Ornithine transport agents in the Gonads; Figure 23 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Skin; Figure 24 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Vessels; and Figure 25 is a fluorescent micrograph illustrating expression utilizing Arginine/Ornithine transport agents in the Bone Marrow.

The figures illustrate that DNA which is coded for the production of green fluorescent protein was distributed throughout all organs and tissues, and successful protein expression has occurred.

Example-Delivery of human growth hormone in mice Sustained delivery of human growth hormone (hGH) by gene therapy is very challenging. The main reason is that the antigenic nature of hGH elicits a strong antibody response in immunocompetent mice. As a result, hGH delivery reported in the literature is consistently modest (1-3 ng/ml) and transient in nature (lasts for days).

Alginate-PLL-hGH DNA nanoparticles were prepared as described in protocols and mixed with Jell-O.

Adult immunocompetent C57BL/6 mice (20 weeks of age) were fed 100 ig of DNA nanoparticles orally (n=3). Mice were bled regularly. The concentration of hGH was determined by ELISA (UBI Inc). The presence of antibodies against hGH was determined by ELISA.

Treated mice had high levels of hGH (peak of-50 ng/ml). More importantly, hGH delivery persisted for at least 120 days (Figure 26). Furthermore, anti-hGH antibodies were not detected (Figures 27 and 35). This data indicates that this technology can deliver sustained levels of

therapeutic products such as hGH, without eliciting an antibody response.

Example-Delivery of a therapeutic product in a tissue-specific in mice Tissue specific delivery of hFIX Day 85 post-treatment A plasmid containing the human factor IX cDNA under the control of the albumin promoter was administered to hemophilic mice, by feeding each mouse 100 micrograms of DNA in alginate-PLL nanoparticle formulation.

The albumin promoter is specific for liver.

'FIX was detected in the blood of treated mice.

Immunohistochemistry (hFIX present in the various tissues was detected using antibodies specific to hFIX) showed that expression of hFIX in treated mice was restricted to the liver, and was not expressed in other tissues as illustrated in Figure 28.

This validates the achievement of tissue-specific expression of a transgene following oral administration of DNA.

Experimental Protocol: Alginate-PLL-hFIX DNA nanoparticles were prepared as described in protocols and mixed with Jell-O. The human factor IX (hFIX) DNA was cloned in a plasmid such that the -expression_of_hFIX_was placed under the control_of the albumin promoter. The albumin promoter is liver-specific.

Therefore, expression of hFIX is only expected in liver cells, while cells from other organs harboring this plasmid would not be able to secrete hFIX. Alternatively,

ubiquitous expression may be achieved with the use of the 3-actin promoter as illustrated in Figure 33. Adult immunocompetent C57BL/6 mice (20 weeks of age) were each fed 100 zig of DNA nanoparticles orally (n=3). Mice were bled regularly, and the concentration of hFIX in plasma determined by ELISA (Affinity Biologicals). All treated mice had therapeutic levels of hFIX in blood, while no antibodies were detected.

In order to further evidence that oral gene delivery can both tolerize and eliminate a pre-existing immune response the following experiments were conducted.

Balb/c mice were treated with a single oral dose of 100ug human factor IX DNA formulation. By day 3 post- treatment all treated mice had therapeutic levels of plasma hFIX. This level persisted for at least 49 days (Figure 39).

In contrast control mice did not have any detectable plasma hFIX. Although balb/c mice develop strong immune responses to human transgenes, no antibodies to hFIX were detected in any of the treated mice. Normally, mice that mount an immune response against a particular product recognize and eliminate that product from the body, thus no circulating hFIX is expected to be seen in the treated mice. Factor IX (Christmas factor) is a plasma serine protease necessary for effective blood coagulation. Individuals having absent or defective Factor IX suffer from hemophilia B and are characterized by excessive bleeding. Addition of a functional Factor IX protein can reverse this genetic defect. Normally, when mice are transfected with the human transgene for factor IX (hFIX) they mount an immune response (Hortelano et al. Blood 87 (12) : 5095-5103 1996). C57BL/6 mice were immunized with recombinant hFIX protein according to standard immunization protocol. As expected, mice developed

a high titre of anti-hFIX antibodies. At this time, half of the mice were given a single oral dose of FIX DNA formulation. The other half of the mice were not treated. In spite of the pre-existing immune response to hFIX, all treated mice developed circulating therapeutic levels of hFIX that persisted for at least 28 days. Anti-hFIX antibody titre remained high in the untreated mice, but was somewhat reduced in treated mice. These results indicate that oral DNA formulations can lead to transgene persistence even in the presence of constant transgene stimulation. A repeat run of this experiment gave similar results.

A group of immunocompetent C57BL/6 mice were implanted with alginate microcapsules enclosing recombinant C2C12 myoblasts that continuously secrete human factor IX.

Circulating levels of hFIX were observed in the treated mice starting on day 3 post-treatment. Detectable hFIX was observed for up to two weeks. At this time, circulating levels of hFIX disappeared and anti-hFIX antibodies were increasingly detectable in the treated mice. At this point, when no hFIX was detected and antibody titre was extremely high (up to 1: 1,000. 000) mice were treated with alginate- poly-L-lysine hFIX DNA nanoparticles. Each mouse was administered a single dose of 100ug of hFIX DNA. Mice were then bled at regular intervals and plasma was collected and stored. The concentration of hFIX antigen and anti-hFIX antibodies were measured using ELISA techniques. Treated mice had sustained circulating levels of hFIX (Figure 36 and 38). The titre of anti-hFIX antibodies was reduced in all -treated mice_and_reached undetectable levels in 315 mice (Figure 37). The control group of mice that was not treated with the oral DNA formulation continued to have high anti- hFIX antibody titre and no detectable circulating hFIX.

Taken together these experiments show that oral delivery of

DNA leads to sustained transgene expression even in the presence of a strong pre-existing immune response. Oral delivery can also reduce the pre-existing immune response.

The technology of the instant invention can induce tolerance in a host, thus may have implications in the treatment of immune disorders and organ transplantations.

In summary, the instant inventors have confirmed that orally administered DNA is effectively taken up through the intestine and distributed throughout the body, when protected as it traverses the GI tract by alginate (or any similar agent), and if the DNA is conjugated to a polypeptide (such as PLL). Formulations with no protective coating or no polypeptide evidenced minimal distribution, and very low efficacy and/protein expression. Although not wishing to be limited to any particular theory of operation, it is theorized that DNA is transported to all organs through a natural amino acid distribution mechanism with high efficiency. The DNA enters virtually all cells in all major organs examined and the coded therapeutic product is produced in the various tissues. The inclusion of promoters, either ubiquitous or tissue specific, enable precise control of protein expression.

Delivery is sustained long-term (for at least 180 days). The therapeutic product may be secreted by the cells into the circulation (in the case of secretable products).

Alternatively, non-secretable proteins will remain in the cells where they are produced.

-----------A-1--l--på-tents--and-publicat : ións_mentioned il_this specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was

specifically and individually indicated to be incorporated by reference.

It is to be understood that while a certain form of the invention is illustrated, it is not to be limited to the specific form or arrangement of parts herein described and shown. It will be apparent to those skilled in the art that various changes may be made without departing from the scope of the invention and the invention is not to be considered limited to what is shown and described in the specification. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The oligonucleotides, peptides, polypeptides, biologically related compounds, methods, procedures and techniques described herein are presently representative of the preferred embodiments, are intended to be exemplary and are not intended as limitations on the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the following claims.