EP0831771A2

EP0831771A2 - Oral delivery of gene constructs

Info

Publication number: EP0831771A2
Application number: EP96918287A
Authority: EP
Inventors: Peter E. Daddona; Gamil G. De Chadarevian; Joseph A. Fix; Phyllis I. Gardner; Howard B. Rosen
Original assignee: Alza Corp
Current assignee: Alza Corp
Priority date: 1995-06-07
Filing date: 1996-06-07
Publication date: 1998-04-01
Also published as: KR19990022184A; AU6097896A; JPH11507340A; CA2221323A1; WO1996040081A3; WO1996040081A2

Abstract

The present invention is a method for the delivery of a gene construct to a targeted portion in the gastrointestinal tract. Delivery of the gene construct occurs as a bolus dose that will cause the expression of a therapeutically effective amount of a therapeutic protein at the target site. The invention is also directed to a device for delivery of the gene construct at the target site.

Description

ORAL DELIVERY OF GENE CONSTRUCTS

FIELD OF THE INVENTION

The present invention is related to orally delivered gene therapy vectors. More particularly, it is related to oral delivery of gene constructs to a target site in the gastrointestinal tract to accomplish mucosal or systemic immunization or expression of a therapeutic protein for local or systemic use.

BACKGROUND OF THE INVENTION

Gene therapy has been suggested for treating a wide variety of genetic and acquired diseases. Somatic gene therapy has previously focused on transduction of bone marrow stem cells and lymphocytes derived from the circulation and solid tissues. Many delivery methods have been described for gene therapy including nasal, pulmonary and intra-muscular methods. The Gl system has been noted as advantageous as a target for gene therapy in that it is readily accessible by endoscope, it contains progenitor cells in the crypts which are immortal, and it is capable of sustained proliferation in vivo (F. Ledley, J. Pediatric Gastroenterology and Nutrition. 14:328-337(1992). Gene therapy can be effected in two basic ways: by in vivo delivery and by ex vivo delivery followed by return of cells to the patient (M. Morsy et al, JAMA. 270:10:2338-2345 (November 17, 1993). US Patent No. 5,399,346 to French et al. describes the use of gene treated antigen-specific lymphocytes for delivering a gene product to a target site or as a systemic therapeutic. Delivery of the lymphocytes is accomplished via a central venous catheter. Liposomes and DNA-lipid complexes have also been suggested as an alternative to in vivo delivery of gene products via viral vectors. These complexes can be added to cells in vitro, injected parenterally

SUBSTITUTE SHEET or aerosolized for pulmonary delivery to achieve transfection of cells and tissue in vivo (D. Lasic et al, Science. 267:1275-1276 (March 3, 1995).

Intestinal delivery of vectors has also been described. H-Soriano- Brϋcher. AGA Abstracts. Gastroenterology 100:5.2 (May 1991) suggests that somatic gene therapy into the intestinal tract may be easier than other tissues due to its accessibility. The authors describe the use of a non-replicating retrovirus vector to deliver a reporter gene into a rat ileum. PCT/US92/07029 describes adenovirus mediated transfer of therapeutic genes to the Gl tract for production of a therapeutic protein for systemic and/or local use. The viral vectors are delivered orally by means of an enteric coated capsule. PCT/US94/04589 describes the use of a DNA binding protein for gene transfer into cells. Lactoferrin is an iron binding protein that binds both to DNA and to specific receptors on target cells. The lactoferrin-DNA complex binds to the lactoferrin receptor in the small bowel and enters the cells by receptor mediated endocytosis. A lactoferrin-DNA complex thus can be used for oral gene delivery where formulated with a milk lipid micelle.

Difficulties of gene transfer through the intestinal epithelium have been studied. Sweeter et al, Proc Natl Acad Sci. USA 85:9611-9615 (December 1988) describes the intestinal epithelium as a continuously proliferating mass of cells and reports that the analysis of expression of the hGH reporter in transgenic mice indicates regional differences in gene expression of the epithelium that result both from cell-specific and geographic factors. Jones et al, J Biol Chem 265:24, 14684-14690 (1990) indicates that intestinal targeted gene therapy can be achieved and is desirable in view of the ease with which the small bowel may be accessed and its susceptibility to viral infection. Sandberg et al, Human Gene Thera^py 5:323-329 (1994) further notes the attraction of the intestinal epithelium as a site for somatic gene therapy. The reference suggests that intestinal stem cells in the crypts of Lieberkuhn are the ideal targets for gene therapy and that access to these cells can be

B I improved by distending the intestine, thereby shortening the villi and widening the intervillus space, and removing the protective mucous layer in vivo.

Although each of these references recognizes the intestine to be a preferred site for somatic gene therapy, there continues to be a need for delivering vectors at a programmed time and site in the intestine.

Osmotic dispensing devices for delivery of therapeutically active agents are well known in the art. Such devices use an expansion means to deliver an agent to an environment of use over a period of hours, days or months. The expansion means absorbs liquid, expands, and acts to drive out beneficial agent formulation from the interior of the device in a controlled, usually constant manner. The osmotic expansion means is used to controllably deliver the agent, usually relatively slowly, and over a period of time. Thus, these devices are not generally used to delay the initial release of the agent, followed by the rapid release, or substantially simultaneous introduction, of all of the agent or all of the dosage form(s) containing the agent into the environment of use at one time (see e.g. US Patent Nos. 3,845,770 and 3,916,899 which are incorporated by reference herein). US Patent No. 5,342,624, which is incorporated by reference herein, discloses an oral delivery capsule having two pieces, one of which is a plug made of a water-sensitive material. A delay in the initial release of an agent occurs when the plug takes up water and results in the plug swelling or expanding and eventually separating from the body of the capsule to allow release of the agent contained within. Unfortunately, the swelling and eventual release of the plug causes the volume inside the capsule to expand, which creates a vacuum within the capsule that causes fluid from the fluid environment to enter the capsule and come into contact with the active agent. When the active agent is sensitive to fluids, as are the viral and nonviral vectors useful for gene therapy, such contact can cause agglomeration or inactivation of the agent. Additionally, the fluid taken up by the swelling

SUBSTITUTE SHEET water-sensitive plug also can come into contact with fluid-sensitive active agents.

SUMMARY OF THE INVENTION

The invention is directed to a method for providing human gene therapy. The method involves oral administration of a gene construct or vector that is delivered as a bolus dose to a target site in the gastrointestinal tract. The present invention is also directed to a fluid-imbibing dispensing device for the delivery of a gene construct to the gastrointestinal tract for gene therapy. The device comprises a housing having two separable pieces. The first separable piece defines a water-impermeable container that contains a gene construct formulation. An expansion agent in the device causes separation of the pieces of the housing after exposure to the environment of use to effect delivery of the gene construct to the target site in the Gl tract.

DESCRIPTION OF THE DRAWINGS

The drawings are not drawn to scale, but are set forth to illustrate various embodiments of the invention. Like numbers refer to like structures. FIG. 1 is a cross-sectional view of one embodiment of the device of the present invention, the device being in closed or prepared form prior to placement in the environment of use. FIG. 2 is the device of FIG. 1 in operation after activation by placement in the environment of use, showing the device opened to release the gene construct to the environment.

FIG. 3 is a cross-sectional view of a further embodiment of the device of the present invention, the device being in closed or prepared form prior to placement in the environment of use. FIG. 4 is the device of FIG. 3 in operation after activation by placement in the environment of use, showing the device opened to release the gene construct to the environment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method and device for delivering a gene construct to a targeted site in the gastrointestinal (Gl) tract. Delivery of the gene construct formulation, once begun, is quickly completed. The delivery device is designed to simultaneously introduce all of the gene construct formulation to a target site in the Gl tract in a bolus dose after entry of the device into the Gl tract.

Definitions By the term "gene therapy" is intended the delivery of gene constructs or vectors to a target site to accomplish mucosal or systemic immunization or expression of a therapeutic protein for local or systemic use.

By the term "bolus dose" we intend that essentially all of the gene construct formulation in a device is released in a short period of time of one hour or less and usually within 45 minutes, preferably in less than about 30 minutes. By "essentially all" of the gene construct formulation is intended greater than about 85%, preferably above 95% and usually greater than 98% of the gene construct formulation contained in a device.

As used herein, the terms "therapeutically effective amount" or "therapeutically effective rate" refer to the amount or administration rate of a gene such that the expression level results in the desired therapeutic, often beneficial result.

By "therapeutic protein" is intended a protein or polypeptide that provides some pharmacologic, often beneficial effect. This effect may be localized in the gastrointestinal tract or may be systemic. Examples of these agents that are useful for systemic administration include but are not limited to c*_! antitrypsin, factor VIII, factor IX and other coagulation factors, growth hormone, insulin and other peptide hormones, adrenal cortical stimulating hormone, thyroid stimulating hormone and other pituitary hormones, interferon α, β and δ, erythropoietin, growth factors, tissue plasminogen activator, CD4, interleukin-1 receptor antagonist, tumor necrosis factor receptor, and recombinant antibodies and antibody fragments and the like. Examples of therapeutic proteins that provide localized gene therapy of the Gl tract include, but are not limited to pancreatic enzymes, lactase, cytokines, interleukin-1 receptor antagonist, tumor necrosis factor receptor, recombinant antibodies and antibody fragments, tumor suppressor proteins, cytotoxic proteins and the like. These proteins are useful for the treatment of a variety of conditions including but not limited to hemophilia and other blood disorders, growth disorders, diabetes, leukemia, hepatitis, renal failure, HIV infection, hereditary diseases such as cerebrosidase deficiency and adenosine deaminase deficiency, hypertension, septic shock, autoimmune diseases such as multiple sclerosis, Graves disease, systemic lupus erythematosus and rheumatoid arthritis, shock and wasting disorders, cystic fibrosis, lactose intolerance, Crohn's diseases, inflammatory bowel disease, and gastrointestinal and other cancers. A further beneficial effect, the induction of oral tolerance for treatment of autoimmune diseases, allergies and prophylaxis of contact hypersensitivity, may be accomplished by gene production of recombinant peptides, and oral immunization may be accomplished by gene production of recombinant viral, parasitic, bacterial or other antigens.

With reference to the construction of the devices, "impermeable" is meant that the particular portion of the device is impermeable to fluids and ingredients contained in the device and in the intestinal environment. By "semipermeable" is meant that the housing is permeable to fluid but impermeable to other ingredients contained in the device or in the intestinal environment.

The terms "vector" and "gene construct" are used interchangeably herein and refer to combinations of genetic elements that encode a therapeutic protein with elements that regulate the level of expression of the product. By "viral vector" is intended the packaging of a recombinant gene within a viral or viral-related particle. Viral function is then used to infect and integrate the gene into the target cell. The gene will then express the therapeutic protein. Viral vectors that are useful in the devices of the invention include but are not limited to retroviruses, adenoviruses, adeno- associated viruses, vaccinia and herpes simplex viruses. In contrast, by "nonviral vector" is intended a recombinant gene that may or may not be associated with chemical or biological compounds that stabilize or facilitate cellular uptake of the DNA. In one such example a recombinant DNA molecule with a promoter and polyadenylation signal is incorporated into a DNA backbone. Other examples include the use of cationic and neutral lipids complexed with the DNA molecules and the addition of proteins or chimeric proteins that confer receptor-mediated uptake of DNA (see eg, MA Morsy et al, JAMA. 270:19, 2338-2345 (November 17, 1993) and FD Ledley, J Pediatric Gastroenterology and Nutrition 14:328-337 (1992) which are incorporated by reference herein).

The term "gene construct formulation" or "vector formulation" intends the viral or nonviral vector optionally in combination with pharmaceutically acceptable carriers and additional inert materials. It is to be understood that more than one type of viral or nonviral vector may be incorporated into the gene construct formulation in a device of this invention, and that the use of the term "vector" or "gene construct" in no way excludes the use of two or more such agents.

The amount of viral or non-viral vector employed in the delivery device will be that amount necessary to cause the expression of a therapeutically effective amount of the therapeutic protein to achieve the desired result. In practice, this will vary widely depending upon the particular agent, the site of delivery, the severity of the condition, and the desired therapeutic effect. Thus, it is not practical to define a particular range for the amount of gene construct or vector incorporated into the device.

The pharmaceutically acceptable carrier useful herein may comprise more than one ingredient, such as, for example, buffers, viscosity regulating vehicles, surfactants, dyes, permeation enhancers, proteinase inhibitors, mucolytic agents or other formulation ingredients and additives such as those that facilitate uptake of the agents by the targetted cells or that promote rapid release of the gene construct from the delivery device, as are known in the art. The carrier may contain more than one gene construct.

The present invention involves delivery of a gene construct to a targeted portion of the Gl tract. Potential portions of the Gl tract that may be targeted include but are not limited to the distal small intestine, the M cells, the Peyer's Patches and the upper colon. Such targeting will promote transduction of gastrointestinal epithelial cells or M cells at the delivery site. In order to achieve targeted delivery, devices such as those described below may be taken orally. These devices will retain the gene construct formulation such that delivery into the stomach and upper intestine will be prevented and a bolus dose will be delivered at the target site.

FIG. 1 depicts in cross-sectional view one embodiment of a delivery device for delivering a gene construct according to the present invention. The device is shown in closed or prepared form prior to placement in the environment of use. Dispensing device 1 comprises a housing 12 formed of a first wall section 14 and a second wall section 16. First wall section 14 and second wall section 16 are in reversibly sliding telescopic arrangement with each other. Housing 12 surrounds and defines an internal compartment 18. First wall section 14 surrounds that portion of internal compartment 18 that contains the gene construct formulation 20. Second wall section 16

SUBSTITUTE SHEET surrounds that portion of internal compartment 18 that contains an expansion agent 22 for expanding and for occupying space in compartment 18. Second wall section 16 also contains a piston 24 which is positioned between the agent formulation 20 and the expansion agent 22. Piston 24, may comprise a composition that is substantially impermeable to the passage of fluid, so that it restricts the passage of fluid present in the expansion means into that area of compartment 18 that contains the gene construct formulation. It operates to essentially maintain the integrity of the gene construct formulation and the expansion agent. Additionally, and importantly, piston 24 acts to insure that the expanding driving force generated by the expansion agent 22 is applied directly against the first wall section 14 to effect the separation of the two wall sections. Thus, piston 24 must be of sufficient strength, thickness and rigidity to transfer the driving force against first wall section 14.

First wall section 14 and second wall section 16 at their open ends are close in size and they form a friction fit there between. The friction generated is sufficient to maintain the two wall sections together prior to activation of the expansion means but not so great as to keep the two wall sections from sliding apart once an expanding driving force is exerted. Where additional friction is desired, protrusions or other means may be present on one or the other of the contacting surfaces of the first and second wall sections. First wall section 14 and second wall section 16 can be telescoped completely into a closed and continuous external walled position. The open end of first wall section 14 is adapted to fit within second wall section 16.

In operation, as the expansion agent 22 absorbs and imbibes fluid through second wall section 16 from the environment of use, it expands and pushes against piston 24, causing the piston to slide inside compartment 18. Piston 24 pushes against first wall section 14 to cause the first wall section to slide apart from second wall section 16 as the expansion agent 22 continues to expand. This causes the two wall sections to become separated and the gene construct formulation 20 to be exposed to the environment of use, as

SUBSTITUTE SHEET illustrated by arrows 28 in FIG. 2. These types of devices, referred to as Chronset® devices (ALZA Corporation, Palo Alto, CA) and are further described, for example, in US Patent No. 5,223,265 which is incorporated herein by reference. FIG. 2 illustrates the dispensing device 1 of FIG. 1 in operation after activation of the device by placement in the environment of use. FIG. 2 shows device 1 opened to release all of the gene construct formulation 20 to the environment substantially at the same time. First wall section 14 has been separated from second wall section 16 by the expanding driving force of the expansion agent 22, which has expanded in size as a result of imbibing fluid from the environment. The arrows 28 in FIG. 2 indicate the exiting of the gene construct formulation 20 from internal compartment 18 through the open end of first wall section 14, which is now in communication with the environment. First wall section 14 may comprise a composition that is semipermeable, that is, it is permeable to fluid but impermeable to the gene construct and other ingredients contained in dispensing device 1, or it may, alternatively, comprise a composition that is impermeable to the exchange of fluid, agent and other ingredients. When the gene construct formulation is sensitive to fluid from an exterior fluid present in the environment of use, it is preferred that first wall section 14 be substantially impermeable to the ingress of the external fluid to serve as a means for substantially protecting the gene construct formulation 20 until it is expelled from housing 12 and delivered into the environment of use. Because expansion agent 22 operates by the imbibition of external fluid, second wall section 16 in at least a portion that is adjacent to expansion agent 22 must be semipermeable; that is, it is permeable to the passage of fluid while being substantially impermeable to the passage of other ingredients contained in dispensing device 1. Wall sections 14 and 16 optionally comprise additional ingredients such as, for example, a plasticizer. Impermeable and semipermeable compositions suitable for use in wall sections 14 or 16, as well as suitable additives, are known in the art, examples of which are disclosed in U.S. Pat. 4,874,388, which is incorporated herein by reference.

Housing 12, comprising wall sections 14 and 16, is nontoxic, biologically inert, nonallergenic and nonirritating to body tissue, and it maintains its physical and chemical integrity; that is, housing 12 does not erode or degrade in the environment of use during the dispensing period. It is within the scope of the invention that the housing be insoluble only during the period of intended use and can thereafter dissolve away in the environment of use. Thus, a dispenser is contemplated which is unaffected by its environment, solubility-wise, at the site of use or which, alternatively, is only slightly soluble during the period of intended use, such that once its contents have been removed, it will dissolve or erode away leaving no objectionable residue or empty container at the site of use.

The expansion agent or expandable driving member 22, operable for separating the first and second wall sections to release the gene construct formulation 20 from the dispensing device of the invention, is nontoxic, nonallergenic and biologically inert. Expansion agent 22 comp ses, in one embodiment, an osmopolymer. Osmopolymers interact with water and aqueous biological fluids and swell or expand to ah equilibrium state. Osmopolymers exhibit the ability to swell in fluid and to retain a significant portion of the imbibed and absorbed fluid within the polymer structure. The expansion agent 22 in another embodiment comprises an osmagent.

Osmagents are known also as osmotically effective solutes and compounds. Osmagents that can be used for the purpose of this invention include inorganic and organic compounds that exhibit an osmotic pressure gradient across a semipermeable, i.e. a fluid-permeable, wall. The expansion agent 22 in yet another embodiment comprises an osmagent dispersed within an

SUBSTITUTE SHEET osmopolymer. The expansion agent 22 can comprise a tablet or a layer, or a plurality of tablets or layers, or it can be pressed into second wall section 16. The osmagent or osmopolymer can be in any suitable form such as particles, crystals, pellets, granules, and the like, when pressed into a tablet layer and into wall section 16. Osmagents and osmopolymers are known to the art and are described in, for example, U.S. Pat. Nos. 3,865,108, 4,002,173, 4,207,893, 4,327,725 and 4,612,008.

Piston 24, present in certain embodiments of the invention between the active agent formulation and the expansion agent transmits the force generated by the expansion agent against the first wall section 14 and maintains the separate identity of the gene construct formulation and the expansion agent, and substantially restricts the passage of fluid between the gene construct formulation and the expansion agent. Representative materials useful as a piston 24 are known to the art in, for example, U.S. Pat. No. 4,874,388.

FIG. 3 illustrates another embodiment of a dispensing device for delivering the a gene construct according to the present invention. As illustrated in this figure, dispensing device 30 has a housing 32 with an orifice 34 and a plug 36. The housing 32 has a permeable or semipermeable portion 38 that contains an expansion agent 40 and an impermeable portion 44 that contains the gene construct formulation 20. An impermeable piston 42 separates the expansion agent 40 and the gene construct formulation 20. As shown in FIG. 4, the expansion agent 40 will begin to expand in the fluid environment, the cylindrical plug 36 and housing 32 will separate and the gene construct formulation 20 will be delivered to the target site in the Gl tract. These type of devices referred to as Pulsincap™ devices (R.P. Scherer Co., Swindon, England) are further described in EPO 0384642 which is herein incorporated by reference.

The total delay time prior to separation of the dispensing device and delivery of the gene construct formulation is usually in the range of from about 1 to 24 hours, preferably between about 2 and 15 hours and often in the 4 to 8 hour range and can be controlled by a number of means. For example, the rate of fluid imbibition into the expansion agent can be controlled by the particular choice of semipermeable membrane. The rate of expansion of the expansion agent can be controlled by the choice of composition of the expansion agent. The distance of overlap between the open end portions of the first and second wall sections can determine the period of time required for the two sections to separate. Combinations of such means may be used. Such control means are known in the art and can be determined without undue experimentation.

Although a particular device has been described that will deliver a gene construct to a particular location in the gastrointestinal tract, other devices known in the art to have the capabilities of delivering a bolus dose to a target site in the Gl tract can also be used. One example of such a device is the Pulsincap™ that comprises an impermeable capsule and a hydrogel plug. The plug is designed to swell and dislodge from the device after a particular time period, thereby delivering its contents in a bolus following a predetermined delay. See, e.g., European Patent No. 0384642, which is incorporated by reference herein. Another example of a device that will deliver a bolus dose following a particular delay is the Time-Clock® described in U.S. Patent No. 5,310,558 which is incorporated by reference herein. In this device, a core containing the active agent is coated by a hydrophobic layer. The hydrophobic layer is programmed to disintegrate after a particular amount of time and thereafter release the active agent. Other devices have been designed that release a bolus dose upon, for example, activation of a radio-frequency signal (see, e.g., U.S. Patent No. 3,894,538, incorporated by reference herein), and as a result of a heating by ultrasonic waves (see e.g., U.S. Patent No. 4,507,115, which is incorporated by reference herein). The bolus delivery described in the present application can be effected by the above-described drug delivery devices as well as any other devices that are now known, or hereafter developed, to have the same capabilities.

For proper delivery of the gene construct, the dispensing device must deliver the gene construct formulation to a particular portion of the Gl tract. Thus, it is necessary for the device to pass through particular portions of the Gl tract to another prior to delivering the gene construct. In some cases an enteric coating may be applied over at least that portion of the housing of the dispensing device that is comprised of a semipermeable membrane adjacent to the fluid-activated expansion means. Enteric coatings will remain intact in the stomach but will rapidly dissolve once they arrive at the small intestine, thereafter allowing fluid to be imbibed to activate the dispensing device. Enteric coatings are well known in the art and are discussed at, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA; and Polymers for Controlled Drug Delivery, Chapter 3, CRC Press, 1991. As noted above, the gene constructs may be prepared from viral or nonviral vectors. Where the gene construct is prepared from a viral vector, the formulation is prepared by generating specific constructs containing the desired genetic material. The replication region of the viral genome is deleted and replaced with a therapeutic gene. The replication-defective virus is then propagated, recovered after cell lysis, purified and then concentrated. Where the gene construct is prepared from a nonviral vector, the formulation may include the therapeutic gene alone or the gene may be incorporated, for example, into a liposome or a cationic lipid complex. In order to prepare the device shown in FIGs. 1 and 2 standard manufacturing techniques may be used. First housing 14 (the vessel) and second housing 16 (the cap) may be separately molded or extruded to the desired shape. Possible semipermeable materials from which the second housing 16 may be prepared include, for example, water flux enhanced Hytrel® polyester elastomers (Du Pont), cellulose esters, water flux enhanced ethylene-vinyl acetate copolymers, semipermeable membranes made by blending a rigid polymer with water-soluble low molecular weight compounds, and other semipermeable materials known in the art. Impermeable materials from which the first housing 14 may be prepared include, for example, polyethylene, polystyrene, ethylene-vinyl acetate copolymers, Hytrel® polyester elastomers (Du Pont) and other impermeable materials known in the art.

The device can be assembled as follows. Gene construct formulation 20 is placed in first wall section 14 at its end opposite the end that is initially open. A "bilayer osmotic plug" composed of piston 24 and expansion agent 22 is prepared in a shape that will fit within second wall section 16. The piston 24 and expansion agent 22 are compressed into a tablet on a rotary bilayer tablet press. The bilayer osmotic plug is placed within the second wall section 16 and the assembly is placed over the end of the filled first housing so that piston 24 is adjacent to the gene construct formulation 20. When the device of the invention has the configuration shown in FIGs.

3 and 4, again, standard manufacturing techniques may be used. The housing 32 may be prepared as a hollow cylinder that is closed at one end. The cylinder may be a water permeable material as described above and coated with an impermeable coating in the portion that contains the gene construct formulation 20. The plug 36 may be a water-swellable material. The device is assembled by preparing an osmotic plug as described above from the expansion agent 40 and the piston 42. This plug is placed into the closed end of the hollow cylinder, with the expansion agent 40 adjacent the closed end of the cylinder. The gene construct formulation is then placed into the housing 32 adjacent the piston 42. The plug 36 is then placed into the open end 34 of the housing 32.

The above description has been given for ease of understanding only. No unnecessary limitations should be understood therefrom, as modifications will be obvious to those skilled in the art.

SUBSTITUTE SHEET The following examples are illustrative of the present invention. They are not to be construed as limitations of the scope of the invention. Variations and equivalents of these examples will be apparent to one skilled in the art in light of the present disclosure, the drawings and the claims herein.

EXAMPLE 1

A delivery device for delivering a gene construct formulation according to the invention is prepared as follows. The osmotic engine portion of the device is a compressed bilayer tablet composed of a 150 mg polymeric osmotic formulation (expansion agent) and a 50 mg wax-based barrier (piston).

The polymeric osmotic formulation has a composition of 60 wt% polyethylene oxide (Polyox® 303, Union Carbide), 29 wt% sodium chloride, 5 wt% polyacrylic acid (Carbomer® 934P, B.F. Goodrich), 5 wt% hydroxypropylmethylcellulose E-5, and 1 wt% ferric oxide. During preparation, each of the above components is screened through a 40 mesh screen, and the sized components are added to a mixing vessel in the appropriate proportions. The dry components are mixed thoroughly for 10 minutes; then, SDA 3A ethanol is slowly added while mixing continues until a wet mass is formed. The wet mass is then screened through a 20 mesh screen, and the wet granules are allowed to air dry for 18 hours. After drying, the granules are passed once more through a 20 mesh screen.

The wax barrier has a composition of 95 wt% microcrystalline wax (MF-2JH Durawax®, Astor Wax Corp.) and 5 wt% gelatin (Type A, 275-300 bloom). During preparation, each component is screened through a 40 mesh screen before being added in the correct weight ratio to a mixing vessel. The dry materials are mixed thoroughly for 10 minutes; then, purified water is slowly added to the mixture while stirring continues. After a wet mass forms, the mixture is passed through a 20 mesh screen, and the granules are oven-

SUBSTTTUTE SHEET dried at 40°C for 24 hours. After the granules are dried, they are re-screened through a 20 mesh screen.

The osmotic formulation and the wax barrier formulation are compressed in a hydraulic or rotary press into a cylindrical bilayer tablet. The osmotic face of the tablet is convex, to conform to the shape of the delivery device, while the barrier face of the tablet is flat. Tabletting is conducted to produce a clean, distinct interface between the two layers.

To prepare the vessel portion (first wall section) of the device, 70 wt% cellulose acetate 320 and 30 wt% polypropylene glycol are thoroughly mixed together and are then added to the hopper of a screw extruder. The polymeric mixture is heated at 127°C as it is extruded through the heated barrel of the extruder and into a mold for the vessel. The polymer mixture is allowed to cool after injection into the mold, after which the vessel is removed from the opened mold. The cap portion (second wall section) of the device is prepared in the same manner as the vessel, the composition of the cap being 70 wt% cellulose acetate 320 and 30 wt% polypropylene glycol. The heated polymeric mixture is injected into a mold for the cap and allowed to cool, and the finished cap is then ejected. To assemble the delivery device, the desired gene construct formulation is placed into a completed vessel by manual or automated fill mechanisms. The osmotic engine bilayer tablet is placed into a completed cap with the convex osmotic layer pointed into the closed end of the cap and the barrier layer exposed toward the cap opening. The open end of the filled vessel is fitted inside the open end of the cap, and the two pieces are compressed together until cap, osmotic bilayer tablet and vessel fit together tightly.

SUBSTITUTE SHEET EXAMPLE 2

A delivery device is prepared as in Example 1. The assembled device is then coated with approx. 20 mg of a methacrylic acid copolymer enteric coating (Eudragit® L 100-55, Rohm Pharma).

EXAMPLE 3

A delivery device according to the present invention containing an adenovirus vector formulation is prepared as follows. The E1 region of the adenovirus genome is deleted and replaced with the α1-antitrypsin expression cassette as described in Crystal, PCT/US92/07029, which is incorporated herein by reference. This replication-defective virus is propagated in the human kidney cell line 293 which supplies the E1 products in trans. Virus is recovered after cells lysis, purified using cesium chloride gradients, and concentrated. The adenovirus gene construct is then incorporated into the delivery device described in Example 1.

EXAMPLE 4

Delivery devices according to the present invention containing non¬ viral vector formulations are prepared follows. Liposomes are prepared by the dehydration-rehydration techniques described by Gregoriadis and Panagiotidi (Immunol. Lett. 1989, 20:237-240). Distearolyphosphatidylcholine, hydrogenated phosphatidylse ne and cholesterol in a molar ratio of 1 :1 :2 are dissolved in chloroform/methanol (9:1 v/v). Following rotary evaporation, the dried lipid film is rehydrated with the aqueous solution of gene construct Factor VIII expression plasmid as described in Eaton et al., Biochemistry 25, 8343-8347, which is incorporated by reference herein, with vortexing for 10 minutes at 50-55°C. The

SUBSTITUTE SHEET preparation is lyophilized. A portion of the preparation is rehydrated with 0.9% NaCl. The liposome/construct formulations, one in lyophilized form and one in rehydrated form are loaded into separate delivery devices as described in Example 1.

EXAMPLE 5

Four delivery devices similar to the device of Example 3, each containing 500 mg of the adenovirus vector (AV) formulation are tested for release of the viral vector formulation using the USP paddle method (at 150 rpm). The devices are placed in 500 mL of pH 7 solution with 0.1% bovine albumin and stirred. The devices are observed to determine when the cap and drug vessel separate from each other, and the solution is tested for presence of AV to confirm the release time and the duration of release. The presence of virus can be determined by standard plaque forming assays in susceptible host cell lines. Each device will release its contents completely after 4 to 8 hours and over a short duration of time of less than half an hour.

EXAMPLE 6

Devices similar to the device of Example 3, each containing 500 mg of the adenovirus vector formulation are tested to determine if transduction of the Gl epithelial cells has occurred. The device, with enteric coating as described in Example 2, is orally administered on Day 0. A booster inoculation is administered 2 weeks following initial dosing when the gene construct is intended to elicit an immunological response. Blood and salivary samples are collected subsequent to booster inoculation and serum IgG and salivary IgA levels determined by ELISA assay. A significant rise in serum IgG and/or IgA is indicative of effective transfection and generation of immunological response by the administered gene construct. A significant

SUBSTITUTE SHEET rise in levels of expressed peptide or protein in the serum is indicative of efficacious transfection and expression.

This invention has been described in detail with particular reference to certain preferred embodiments thereof, but it will be understood that variations and modifications can be effected within the spirit and scope of the invention.

SUBSTITUTE SHEET

Claims

WHAT IS CLAIMED IS:

1. A device for providing human gene therapy, said device comprising a gene construct in a form that can be orally administered to a human patient, said device providing for targetted delivery of the gene construct as a bolus dose to the gastrointestinal tract to induce local production of a therapeutic protein at the target site.

2. The device of claim 1 wherein the therapeutic protein induces mucosal immunity.

3. The device of claim 1 wherein the therapeutic protein induces systemic immunity.

4. The device of claim 1 wherein the therapeutic protein is useful locally in the Gl tract.

5. The device of claim 4 wherein the therapeutic protein is useful for the treatment of bowel disease.

6. The device of claim 1 wherein the therapeutic protein is secreted into the systemic circulation.

7. The device of claim 1 wherein the gene construct is a viral vector.

8. The device of claim 7 wherein the viral vector is selected from the group consisting of retroviruses, adenoviruses, adeno-associated viruses, vaccinia and herpes simplex viruses.

BSTITUTE SHEET

9. The device of claim 1 wherein the gene construct is a nonviral vector.

10. The device of claim 9 wherein the nonviral vector is selected from the group consisting of a promoter and polyadenylation signal incorporated into a DNA backbone, a cationic lipid complexed with a DNA molecule, a neutral lipid complexed with a DNA molecule, and a protein that confers receptor-mediated uptake of DNA.

11. A fluid-imbibing dispensing device for the delivery of a gene construct formulation to the gastrointestinal tract, comprising two separable pieces, the first separable piece defining a water-impermeable container containing the gene construct formulation, wherein delivery of the gene construct formulation occurs in a bolus dose at a targeted site in the gastrointestinal tract.

12. The device of claim 11 , the second separable piece defining a water-permeable container containing a water-sensitive material which when wet will cause a positive pressure to be exerted on the first separable piece and result in the separation of the first and second separable pieces.

13. The device of claim 12 wherein the water-sensitive material comprises a water-swellable material.

14. The device of claim 1 1 wherein the first separable piece defines at least one orifice, said orifice being closed by a second separable piece.

15. The device of claim 14 wherein the second separable piece comprises a water-swellable plug.