CN1187118A - Oral delivery of gene constructs - Google Patents

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

The present invention relates to the gene therapy vector of oral delivery.Or rather, it relate to oral delivery of gene constructs to the gastrointestinal tract target site to realize mucosal immunity or general immunity or to express topical application or treatment protein that whole body is used.

People's suggestion is treated multiple heredopathia and acquired disease with gene therapy.Somatic gene therapy concentrated on the bone marrow stem cell and from the lymphocyte of loop organization and solid tissue of transduceing in the past.The existing multiple delivering method of describing the related gene treatment comprises per nasal, through lung and method intramuscular.It should be noted that GI system (gastronintestinal system) can be used as the useful target of gene therapy, because it is easy to accept endoscope, the CFU-GM that in its glandular tube, contains immortality, and it can continue propagation (F.Ledley in vivo, J.Pediatric Gastroen-terology and Nutrition, 14:328-337 (1992)).

Gene therapy can two kinds of basic modes be carried out: by in vivo with by sending, then cell is returned to patient (M.Morsy et al.JAMA, 270:10:2338-2345 (November 17,1993) in vitro.Authorize the US Patent No.5 of French et al., described the antigenic specificity lymphocyte that gene is handled in 399,346 and be used for the delivery of gene product to target site or as whole body therapeutic.Sending lymphocyte is undertaken by central venous catheter.Also have and propose that making alternative with liposome and DNA-lipid complex passes through viral vector delivery of gene product in vivo.These complexs can external adding cell, non-intestinal injection or for the aerosolization of sending through lung to reach in vivo transfectional cell and tissue (D.Lasic et al, Science, 267:1275-1276 (March 3,1995)).

Through the intestinal delivery vector description is arranged also.H-Soriano-Br ü cher, AGA Abstracts, Gastroenterology 100:5,2 (May 1991) think, carry out in the intestinal somatic gene therapy may because of its accessibility organize than other more easy.Authors have described application non-replicating retroviral vector reporter gene have been delivered in the rat ileum.PCT/US 92/07029 has described adenovirus mediated gastrointestinal tract that therapeutic gene is transferred to and has used and/or the local treatment protein of using to produce whole body.By the capsular mode oral delivery of casing viral vector.PCT/US 94/04589 has described and has been used to transmit gene to cell with DNA is conjugated protein.Lactotransferrin is a kind of iron-binding protein, and it is simultaneously in conjunction with specific receptor on DNA and the target cell.Lactotransferrin-dna complex is bonded in small intestinal on the lactotransferrin receptor, and enters cell by receptor-mediated endocytosis.This lactotransferrin-dna complex can be used for oral delivery of gene, modulates with the butterfat micelle herein.

Existing people has studied the difficult point of gene transfer by enteric epithelium.Sweeter et al, ProcNatl Acad Sci, USA 85:9611-9615 (December 1988) is described as continuous proliferating cells material to enteric epithelium, and report, to in transgenic mice, expressing the analysis showed that of hGH reporter gene, in the gene expression of epithelium, cause area differentiation by cell-specific sex factor and Location factor (geographic factor).Jones et al, J Biol Chem 265:24,14684-14690 (1990) point out, the directed gene therapy of intestinal not only can have been realized but also catered to the need, because it is easily accepted by small intestinal, and it is to the viral infection sensitivity.Sandberg et al.Human Gene Therapy 5:323-329 (1994) also mentions especially with enteric epithelium this interesting practice of site as somatic cell gene therapy.Document enlightenment, the intestinal stem cell in the enteraden is the desirable target of gene therapy; And, intestinal is expanded help, thereby reduce fine hair, enlarge intervillous gap and remove intravital protectiveness rete malpighii near these cells.

Though it is the preferred sites of somatic cell gene therapy that these documents are all recognized intestinal, also need be at program time and enteral site delivery vector.

The permeability distributor that is used for the delivery treatments activating agent is that the present technique people know.This device is to send medicament to applied environment with expansion apparatus during a few hours, a couple of days or several months.This expansion apparatus absorbs liquid, expansion, emits useful medicament temper in check, normally constant mode from installing inside then.This permeability expansion apparatus is used in a period of time, controllably send medicament usually more slowly.Therefore, this class device is not used in this occasion usually: the initial release that promptly postpones medicament, then be rapid release, perhaps roughly introduce all medicaments or disposable all dosage forms introducing applied environments of medicament that will contain simultaneously (for example referring to US Patent Nos.3,845,770 and 3,916,899, it is for referencial use that they incorporate this paper into).Incorporate this paper US Patent No.5 for referencial use into, 342,624 have disclosed bipartite oral delivery capsule, and a part wherein is the bolt by the water-sensitive material.When bolt absorbs water, the delay of medicament initial release can take place, cause bolt swelling or expansion and discharge the medicament that is contained in wherein from the capsule body separation at last.Regrettably, bolt swelling and the last volume that causes in the capsule that discharges expand and make formation vacuum capsule in, make fluid in the fluid environment enter capsule and contact with activating agent.If the activating agent convection cell is responsive as viral vector that is of value to gene therapy and non-virus carrier, then this contact can cause the cohesion or the inactivation of medicament.In addition, the fluid that is absorbed by swollen water-sensitive bolt also can contact with the responsive activating agent of fluid.

The present invention relates to provide the method for people's gene treatment.This method comprises oral gene constructs or the carrier that is delivered to target site in the gastrointestinal tract as pill.

The invention still further relates to be used to gene therapy and delivery of gene constructs to gastrointestinal fluid permeability distributor.This device comprises the shell with two removable parts, and first removable part limits the water impermeability shell that contains gene constructed compositions.Extender in the device cause shell two parts with applied environment contact after separate, cause gene constructs to be delivered to target site in the gastrointestinal tract.

Accompanying drawing is not to draw in proportion, but in order to set forth each embodiment of the present invention.The structure that identical numeral is identical.

Fig. 1 represents the profile of an embodiment of apparatus of the present invention, and this device was in sealing or preparation form before putting into applied environment.

The device of Fig. 2 presentation graphs 1 is put into the residing duty in applied environment activation back, device is shown opens the release gene constructs to environment.

Fig. 3 represents the profile of another embodiment of apparatus of the present invention, and this device was in sealing or preparation form before putting into applied environment.

The device of Fig. 4 presentation graphs 3 is put into the residing duty in applied environment activation back, device is shown opens the release gene constructs to environment.

The present invention relates to delivery of gene constructs target site to stomach and intestine (GI) road method and Device. Sending of gene constructs temper Once you begin just finished rapidly. Delivery apparatus Be such design, this delivery apparatus enters after the intestines and stomach with pill all bases Import simultaneously target site in the intestines and stomach because making up temper.

Term " gene therapy " expression delivery of gene constructs or carrier to target site glues realizing Film immunity or general immunity or be that topical application or whole body are used expression treatment protein.

Term " pill " be illustrated in one hour or shorter and usually in 45 minutes, preferably in less than about 30 minutes time, discharging basically from device, full gene makes up temper." whole basically " gene constructed temper represent to be contained in the device more than about 85%, preferred about 95% or more and common more than 98% gene constructed temper.

Amount or application rate as the term " treatment effective dose " used in the literary composition or " treatment effective speed " expression gene make expression produce required therapeutic outcome, be generally useful result.

" treatment protein " expression provides certain pharmacological effect, the normally protein or the polypeptide of beneficial effect.This effect may concentrate on gastrointestinal tract or may be general.This class medicament example of being used for systemic application is including, but not limited to antitrypsin α ₁, blood coagulation factor VIII, plasma thromboplastin component and other coagulation factors, growth hormone, insulin and other peptide hormone, thyroliberin, thyrotropin and other pituitary hormone, interferon-ALPHA, β and δ, erythropoietin, somatomedin, tissue plasminogen activator, CD4, interleukin-1 receptor antagonist, Tumor Necrosis Factor Receptors and recombinant antibodies and antibody fragment or the like.Provide the treatment protein example of the local gene therapy of gastrointestinal tract to include but not limited to: pancreatin, Lactose enzyme, cytokine, interleukin-1 receptor antagonist, Tumor Necrosis Factor Receptors, recombinant antibodies and antibody fragment, tumor suppressor protein, cytotoxic protein matter or the like.These protein are of value to the multiple disease of treatment, including, but not limited to hemophilia and other hematopathy, retardation of growth, diabetes, leukemia, hepatitis, renal failure, HIV infects, heredopathia such as cerebrosidase defective and ADA Adenosine deaminase defective, hypertension, septic shock, autoimmune disease such as multiple sclerosis, Graves disease, systemic lupus erythematosus and rheumatoid arthritis, shock and Slim, cystic fibrosis, lactase deficiency, Crohn disease, inflammatory bowel, and human primary gastrointestinal cancers and other cancer.Further beneficial effect is promptly induced the oral patience (oral tolerance) to autoimmune disease, anaphylactoid treatment and prevention contact hypersensitivity, can realize by the genes produce of recombinant peptide; And peroral immunity method (oral immunization) can realize by recombinant virus, parasitic, antibacterial or other antigenic genes produce.

The comparable device structure, specific part convection cell of " impermeable " indication device and the component that is contained in device and the intestinal environment are impervious." semi permeable " expression shell convection cell is infiltrative, but to be contained in the device or the intestinal environment in other component be impervious.

It is interchangeable and represent genic combination that term " carrier " and " gene constructs " are used for literary composition, and its is with the factor coding treatment protein of regulating product expression." viral vector " is illustrated in virus or the relevant particle inner packing recombination of virus.So the effect of virus is to infect and gene integration is gone into target cell, gene will expression treatment protein like this.Useful viral vector includes but not limited to retrovirus, adenovirus, adeno associated virus, cowpox and simple skin ulcer exanthema virus in apparatus of the present invention.Otherwise " non-virus carrier " expresses possibility or recombination that can not be relevant with chemical compound or biologic artifact, but described two compounds stabilisations or promote cellular uptake DNA.In a this example, the recombinant DNA molecules that has a promoter and polyadenylation signal is impregnated in the DNA skeleton.Other example comprise use with the compound cation lipid of dna molecular and neutral lipid and interpolation make protein with receptor-mediated picked-up DNA performance or chimeric protein (for example referring to, MAMorsy et al, JAMA, 270:19, (November 17 for 2338-2345,1993) and FD Ledley, J Pediatric Gastroenterology and Nutrition 14:328-337 (1992), it is for referencial use all to incorporate this paper into).

Optional viral vector or the non-virus carrier that makes up with pharmaceutically acceptable carrier and other inert substance of term " gene constructed temper " or " carrier temper " expression.Should understand, can be with the gene constructed temper of mixing more than a kind of viral vector or non-virus carrier in apparatus of the present invention, and used term " carrier " or " gene constructs " are got rid of the application of two or more these class medicaments anything but.

The amount that is applied to viral vector in the delivery apparatus or non-virus carrier should be to cause treating the treatment protein expression of effective dose to reaching the required amount of expected results.In fact, its variable range is wide, depends on concrete medicament, sends the site, the curative effect of the order of severity of disease and expection.So,, be unpractical if regulation is mixed the amount ranges of the gene constructs or the carrier of device.

The pharmaceutically acceptable carrier that is suitable in the literary composition can comprise more than a kind of composition, for example: known those those class additives that maybe can impel gene constructs to disengage from delivery apparatus rapidly that help target cell picked-up medicament in buffer agent, viscosity adjustment carrier, surfactant, dyestuff, penetration enhancers, protease inhibitor, mucolytic agent or other temper composition and additive such as the present technique.Carrier can contain more than a kind of gene constructs.

The present invention includes delivery of gene constructs to the directed district of gastrointestinal.May distinguishing that gastrointestinal tract can be directed includes but not limited to distal small intestine, M cell, peyer's patches and last colon.This orientation can be impelled and sending site transduction gastrointestinal epithelial cell or M cell.For reaching targeted delivery, following apparatus can be oral.These devices will keep gene constructed temper so that send into stomach and prevent to pass intestinal into top, and be to send pill at target site.

Fig. 1 draws an embodiment profile of the delivery apparatus of delivery of gene constructs of the present invention.This device was sealing or preparation form before putting into applied environment.Distributor 1 comprises by first wall section 14 and second shell 12 that wall section 16 constitutes.First wall section 14 and second wall section 16 can mutual reverse slip suits.Shell 12 surrounds and limits inner chamber 18.First wall section 14 surrounds the part that contains gene constructed temper 20 in the inner chamber 18, and second wall section 16 surrounds and contain extender 22 in the inner chamber 18 to expand and to occupy spatial part in the chamber 18.Second wall section 16 also contains piston 24, and it is between medicament temper 20 and extender 22.Piston 24 can comprise the compositions that can not infiltrate the fluid passage substantially, so it will exist fluidic passage to be limited in the zone that contains gene constructed temper in chamber 18 in the expansion apparatus.Its work is to keep the integrity of gene constructed temper and extender substantially.In addition, importantly piston 24 can guarantee that the expansion drive power that extender 22 produces is applied directly to first wall section 14 to separate two wall sections.Therefore, piston 24 must have enough intensity, thickness and rigidity could shift driving force to first wall section 14.

Thereby first wall section 14 and second wall section 16 are in almost equal the two the formation frictional fit herein of its opening size.The friction that produces was enough to two wall sections are maintained together before the activate inflation device, but in case can not slip away to two wall sections of prevention greatly when applying expansion drive power.Other if desired friction then can be introduced projection or take other measure at the one side or the another side of first and second wall section contact surface.First wall section 14 can overlap closely with second wall section 16 fully, successive peripheral state.The opening of first wall section 14 is suitable for being assemblied in second wall section 16.

During work, along with extender 22 absorb and infiltration from the flow through fluid of second wall section 16 of applied environment, it just expands and promotes piston 24, and piston is slided in chamber 18.Along with extender 22 continues to expand, piston 24 promotes first wall sections 14, causes first wall section second wall section 16 that slip away.Two wall sections are separated, cause gene constructed temper 20 to be exposed in the applied environment, shown in the arrow 28 of Fig. 2.This class device is called as Chron-set ^(ALZA Corporation, Palo Alto CA) and at for example US PatentNo.5, further describe in 223,265 device, and it is for referencial use that the document is incorporated this paper into.

The distributor 1 that Fig. 2 has illustrated Fig. 1 is put into the duty after applied environment activates.Fig. 2 illustrates device 1 unlatching and discharges all gene constructed tempers 20 to environment simultaneously substantially.Extender 22 produces expansion drive power first wall section 14 is separated with second wall section 16 owing to causing cubical expansion from the environment absorption fluids.Arrow 28 indicators among Fig. 2 make up temper 20 and overflow by the opening of first wall section 14 from inner chamber 18, first wall section 14 now with environment UNICOM.

First wall section 14 can comprise the semipermeability compositions, i.e. fluid porous but be contained in gene constructs in the distributor 1 and other composition porous not; Perhaps it can comprise for the exchange between fluid, medicament and other composition and be impermeable compositions.If gene constructed temper is to the fluid sensitivity from the external fluid that exists in the applied environment; then first wall section 14 preferably is impermeability substantially for fluidic the entering in the external world, discharges and sends into applied environment from shell 12 until it so gene constructed temper 20 roughly shielded.

Because extender 22 is just worked after sucking extraneous fluid, so at least a portion in second wall section 16 should be semi-permeable with the part of extender 22 adjacency promptly; That is to say that the passing through of its convection cell is infiltrative, but to be contained in other composition in the distributor 1 to pass through be impervious basically.

Wall section 14 and the 16 optional for example flexibilizer of other composition that comprise.Being applicable to the impermeability and the semipermeability compositions of wall section 14 or 16, and suitable additive, is known in the present technique, and their example is disclosed in U.S.Pat.4, and in 874,388, it is for referencial use that the document is incorporated this paper into.

The shell 12 that comprises wall section 14 and 16 is nontoxic, biologically inert, and bodily tissue is nonallergic and non-irritating, and it can keep its physics and chemical integrity; That is to say that shell 12 during distribution can etch or degraded in applied environment.Following situation within the scope of the invention, promptly shell is insoluble during the application of being estimated just, but can be dissolved in the applied environment subsequently.So, imagine a kind of like this allotter, it is not subjected to the influence of its environment, dissolved state in the application scenario, perhaps it is microsolubility during the application of estimating, in case after its inclusions was removed like this, it just dissolves or etch is fallen, and does not stay adverse residue thing or ghost in the application scenario.

Extender or expandable driving element 22 be nontoxic, be nonallergic and biologically inert, it can be operated and separate first and second wall Duan Ercong distributor of the present invention discharges gene constructed temper 20.In one embodiment, extender 22 comprises osmopolymer (osmopolymer).Swelling or be expanded to equilibrium state after osmopolymer and water and the aqueous biological fluids effect.Osmopolymer has swollen ability in fluid, and in polymer architecture, keep quite a few suction with the fluid that absorbs.In another embodiment, extender 22 comprises penetrating agent (osmagent).Known penetrating agent is also as chemosmotic solute and chemical compound are arranged.The penetrating agent that can be used for the object of the invention comprises inorganic and organic compound, and they have through semipermeability is the osmotic pressure gradient of fluid permeability wall.In another embodiment, extender 22 comprises the penetrating agent that is scattered in the osmopolymer.Extender 22 can comprise a slice or one deck, or comprises a lot of sheets or layer, perhaps can be pressed into second wall section 16.Penetrating agent or osmopolymer are pressed into lamella and can are any suitable form such as particle, crystal, ball, granule or the like when being pressed into wall section 16.Penetrating agent and osmopolymer all are known and be described in for example U.S.Pat.Nos.3,865,108 in the present technique; 4,002,173; 4,207,893; In 4,327,725 and 4,612,008.

Have in some embodiments of the present invention, be in the piston 24 between activating agent temper and the extender, the power that extender is produced passes to first wall section 14 and keeps gene constructed temper and extender characteristic separately, and fluidic circulation between repressor gene structure temper and the extender substantially.It is known in the present technique being suitable for the representative raw material make piston 24, U.S.Pat.No.4 for example, report in 874,388.

Fig. 3 has set forth another embodiment of the distributor that delivery of gene constructs of the present invention uses.As shown in FIG., the shell 32 of distributor 30 has opening 34 and bolt 36.Shell 32 comprises permeability or semipermeability part 38 that contains extender 40 and the impermeability part 44 that contains gene constructed temper 20.Impermeability piston 42 is isolated extender 40 homogenic structure tempers 20.As shown in Figure 4, extender 40 will begin to expand in fluid environment, and cylindrical bolt 36 will separate with shell 32, so gene constructed temper 20 just is delivered to the target site in the gastrointestinal tract.This class device is called as Pulsincap ^TM(R.P.Scherer Co., Swindon England) and in EPO 0384642 further describe device, and it is for referencial use that the document is incorporated this paper into.

Separating before distributor and delivery of gene make up temper, be total time delay in about 1～24 hour scope, preferably between about 2～15 hours, and usually in 4～8 hours, and can control by certain methods.For example, the speed of fluid suction extender can be controlled by specifically selecting semi-permeable film for use.The expansion rate of extender can be controlled by the selection that extender is formed.The eclipsed length of the open end portion of first and second wall section can determine these two sections to separate the required time.Can be in conjunction with these methods of utilization.This class control method is known in present technique, and does not need too much experiment just can determine.

Though the concrete device of delivery of gene constructs specific part to the gastrointestinal tract has been described, also can be with other device known in the present technique, that pill can be delivered to target site in the gastrointestinal tract.An example of this device is Pulsincap ^TM, it comprises impermeable capsules and hydrogel suppository.This bolt is designed to swellable after the regular hour and from device, deviates from, so after predetermined delay, send the inclusions in the ball.For example referring to European Patent No.0384642, it is for referencial use that it is merged in this paper.Another example of sending the device of pill in the specific delays mode is Time-Clock ^, be described in U.S.Patent No.5, in 310,558, it is for referencial use to incorporate it into this paper.In this device, a core that contains activating agent is coated with hydrophobic layer; After the regular hour, this hydrophobic layer decomposes as scheduled, so release bioactive agent.Designed other device that discharges pill, for example, when activating by radiofrequency signal (for example referring to U.S.Patent No.3,894,538, it is for referencial use to incorporate this paper into) and cause (for example referring to U.S.Patent No.4 by ultrasound wave heating, 507,115, it is for referencial use to incorporate this paper into).The pill of describing in the application's book can be sent with the device with identical performance of known other any device or development from now on by above-mentioned device for administration of drugs.

If delivery of gene constructs rightly, then distributor must be delivered to the gastrointestinal specific part with gene constructed temper.Therefore, before delivery of gene constructs, need this device to pass through the certain position of gastrointestinal to another position.In some cases, can apply enteric coating in a part at least of distributor shell, this position promptly by semi-permeable film constitute, with the position of the activated dilatant adjacency of fluid.In a single day enteric coating will be kept perfectly under one's belt, will dissolve rapidly but arrive small intestinal, thereby allow fluid to be inhaled into to activate distributor.Enteric coating is known in present technique, and is for example coming into question in the following document: Remington ' s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA; And Polymers for Controlled Drug Delivery, Chapter 3, CRCPress, 1991.

As previously mentioned, gene constructs can prepare from viral vector or non-virus carrier.If the gene constructs preparation is from viral vector, then temper contains the specificity construction thing preparation of required genetic stew by generation.Virus genomic duplicate field lacks and is substituted by therapeutic gene, breeds replication-defective virus then, reclaims the purification reconcentration after lysis.

If the gene constructs preparation is from non-virus carrier, then temper can include only therapeutic gene, or this gene can be impregnated in for example liposome or cation lipid complex.

Can prepare the device shown in Fig. 1 and 2 with the preparation method of standard.The first segment shell 14 (storage) and second joint shell 16 (cap) mold or be extruded into required form respectively.Can be used for preparing second semipermeable materials that saves shell 16 and for example comprise the soft system of water enhanced (water flux enhanced) Hytrel ^Polyester elastomer (Du Pont), cellulose esters, the enhanced ethylene-vinyl acetate copolymer of the soft system of water, by known other semipermeable materials in the semi-permeable film of rigid polymer and soluble, low molecular weight compounds blending preparation and the present technique.The impermeability material that can be used for preparing first segment shell 14 for example comprises polyethylene, polystyrene, ethylene-vinyl acetate copolymer, Hytrel ^Known impermeability material in polyester elastomer (DuPont) and other present technique.

This device can be assembled according to following method: gene constructed temper 20 is placed first wall section 14 other end opposite with being initially opening." the double-layer osmotic bolt " that preparation is made of piston 24 and extender 22, its shape should make it to be inserted in second wall section 16.On rotary bi-layer tablet press with piston 24 and extender 22 tablet formings.This double-layer osmotic bolt is placed second wall section 16, and the gained assembly puts loads good first segment outer casing end, so piston 24 is adjacent with gene constructed temper 20.

If device shape of the present invention is with Fig. 3 and 4, also available standards preparation method.Shell 32 can be made into the hollow cylinder of end sealing, and this cylinder can be above-mentioned water permeability material, coats the impermeability coating in the part that contains gene constructed temper 20.Bolt 36 can be the material of water-swellable.Permeate bolt as the aforementioned by extender 40 and piston 42 preparations and can assemble this device.Bolt is put into the blind end of hollow cylinder, and extender 40 is adjacent with the blind end of cylinder.Then gene constructed temper is put into the shell 32 adjacent, again bolt 36 is inserted in the opening 34 of shell 32 with piston 42.

Provide as above description and only be easy to understand, should understand does not have unnecessary restriction to this, changes because those of skill in the art are very clear.

Following embodiment can not be considered to limit the scope of the invention as explanation of the present invention.According to accompanying drawing in this description, the literary composition and claim, those of skill in the art can understand variant and the equivalent example of these embodiment.

Embodiment 1

Make up the delivery apparatus of temper as following preparation delivery of gene of the present invention:

The infiltration actuating section of device is the double-layer tablet of compacting, and it comprises polymeric infiltration temper of 150mg (extender) and 50mg cerul flexible material (piston).

Polymeric infiltration temper consists of: 60wt% poly(ethylene oxide) (Polyox ^303, Union Carbide), 29wt% sodium chloride, 5wt% polyacrylic acid (Carbomer ^934P, B.F.Goodrich), 5wt% hydroxypropyl emthylcellulose E-5 and 1wt% ferrum oxide.During preparation, above-mentioned each component all sieves by 40 mesh sieves, and the component after the screening adds blender with suitable ratio.To do component fully mixed 10 minutes; Continuing slowly to add SDA 3A ethanol under the mixing then until forming wet stock.Again wet stock is sieved air-dry 18 hours of the particle that to wet by 20 mesh sieves.After the drying, another makes particle pass through 20 mesh sieves.

The wax flexible material is by 95wt% microwax (MF-2JH Durawax ^, Astor WaxCorp.) and 5wt% gelatin (Type A, 275-300 bloom) formation.During preparation, earlier each component is sieved by 40 mesh sieves, add blender with the delicate ratio again.Fully mix dry material and reach 10 minutes; Continuing in mixture, to add purified water lentamente under the stirring then.After forming wet stock, make mixture pass through 20 mesh sieves, dry particle down at 40 ℃ and reach 24 hours.After treating the particle drying, sieve by 20 mesh sieves again.

In hydraulic press or rotary tablet machine, will permeate temper and wax flexible material temper is pressed into the cylindrical shape double-layer tablet.The permeable face of this sheet is a convex surface, and meeting the shape of delivery apparatus, and the flexible material face of sheet is flat.Carry out tabletting and between bilayer, obtain well-balanced, unique contact surface.

For the storage part (first wall section) of preparation facilities, 70wt% cellulose acetate 320 and 30wt% polypropylene glycol are fully mixed, add the hopper of screw extruder then.When polymeric blends is extruded through the cartridge heater of extruder, under 127 ℃, be heated, enter the mould of system storage again.Inject the cooling of mould relief polymer composition, from the mould of opening, take out storage subsequently.

With the cap part (second wall section) of the method for making producing device of storage, cap consist of 70wt% cellulose acetate 320 and 30wt% polypropylene glycol.The thermopolymer compound is injected its cooling of cap mould relief, emit the cap of completion again.

Be the assembling delivery apparatus, should adopt craft or automatic charge machinery that required gene constructed temper is inserted the storage of making, to permeate the startup double-layer tablet again and put into the cap of making, make the blind end of convex surface permeable formation, and soft material layer reveal to the cap opening towards cap; Then the storage opening of filling in is inserted in the opening of cap, this two joint of jam-packed closely is nested together until cap, infiltration two synusia and storage.

Embodiment 2

Make delivery apparatus as embodiment 1, the device that will assemble is coated about 20mg methacrylic acid copolymer enteric coating (Eudragit then ^L100-55, Rohm Phar-ma).

Embodiment 3

The delivery apparatus of gland-containing virus carrier temper of the present invention is prepared as follows: remove the E1 district of adenoviral gene group, and with being described in Crystal, the α l-antitrypsin expression cassette among the PCT/US 92/07029 replaces, it is for referencial use that the document is incorporated this paper into.This replication-defective virus is breeding in 293 in human kidney cells, and trans E1 product is provided.Reclaim virus after the lysis, with concentrating behind the cesium chloride gradient purification.Then this adenoviral gene construction is mixed the delivery apparatus described in the embodiment 1.

Embodiment 4

The delivery apparatus that the present invention contains the non-virus carrier temper is prepared as follows: (Immunol.Lett.1989,20:237-240) dehydration of Miao Shuing-rehydration technology prepares liposome according to Gregori-adis and Panagiotidi.Distearoyl phosphatidylcholine, hydrogenated phospholipid acyl serine and cholesterol are dissolved in chloroform/methanol (9: 1 v/v) with 1: 1: 2 mol ratio.Behind the rotary evaporation, incorporate this paper Eaton et al. for referencial use into being described in, Biochemistry 25, and the aqueous solution of the gene constructs blood coagulation factor VIII expression plasmid among the 8343-8347 carries out rehydration to the dried lipid film, rotate 10 minutes down at 50-55 ℃.With this preparation lyophilizing.Part with 0.9%NaCl rehydration said preparation.This liposome/structure temper, a kind of with lyophilized form, another kind of with the rehydration form, be loaded into the delivery apparatus described in the embodiment 1 respectively.

Embodiment 5

Be similar to the release of 4 delivery apparatus (wherein respectively containing 500mg adenovirus vector (AV) temper) application USP paddling process (with 150rpm) test vector viral temper of embodiment 3.To put into the 500ml pH that contains 0.1% bovine albumin be 7 solution and stir with these devices.Observe the time of these devices, and whether test solution exists AV to determine release time and deenergized period to determine that cap and medicine storage are separated from each other.The plaque that can be used on standard in the responsive host cell system forms the existence of analyzing definite virus.Each device will be after 4～8 hours, less than all discharging its inclusions in during halfhour.

Embodiment 6

Test respectively contains 500mg adenovirus vector temper, is similar to the device of device among the embodiment 3 to determine whether to take place the transduction of gastrointestinal tract epithelial cell.The oral device of enteric coating as described in example 2 above that scribbles at the 0th day.When gene constructs is used for challenge, after 2 weeks that began to offer medicine, carry out booster shot.After booster shot, collect blood and saliva sample, and utilization elisa assay method is measured the IgA level of serum IgG and saliva.The obvious increase of IgG and/or IgA shows in the serum, has produced effective transfection and immunne response behind the dispensing gene constructs.Effective transfection and expression are represented in the remarkable increase of peptide of having expressed in the serum or protein level.

The present invention is made detailed description with reference to its some embodiment preferred, but should understand, can make various modifications and variations within the spirit and scope of the present invention.

Claims (15)

1. the device of people's gene treatment is provided, and this device comprises can be by the gene constructs of the oral form of patient, and this device can be treated protein to induce local the generation at target site for gene constructs to the gastrointestinal tract of targeted delivery pill.

2. the device of claim 1, treatment protein induce mucosal immunity wherein.

3. the device of claim 1, treatment protein induce general immunity wherein.

4. the device of claim 1, treatment protein wherein be local being suitable in gastrointestinal tract.

5. the device of claim 4, treatment protein wherein is applicable to the treatment enteropathy.

6. the device of claim 1, treatment protein wherein are secreted into the body circulation.

7. the device of claim 1, gene constructs wherein is a viral vector.

8. the device of claim 7, viral vector wherein is selected from: retrovirus, adenovirus, adeno associated virus, vaccinia virus and herpes simplex virus.

9. the device of claim 1, gene constructs wherein is a non-virus carrier.

10. the device of claim 9, non-virus carrier wherein is selected from: mix the promoter of DNA skeleton and polyadenylation signal, with the compound cation lipid of dna molecular, with the compound neutral lipid of dna molecular with make the protein with receptor-mediated picked-up DNA performance.

Make up temper to gastrointestinal fluid imbedibility distributor 11. be used for delivery of gene, it comprises separable two joints, first separable joint limits the water impermeability storage that contains gene constructed temper, wherein makes up temper with the target site delivery of gene of pill in gastrointestinal tract.

12. the device of claim 11, second separable joint limits the water permeability storage that contains the water-sensitive material, can apply malleation to first separable joint when the water-sensitive material soaks, and causes first to separate with second separable joint.

13. the device of claim 12, water-sensitive material wherein comprises water-soluble bloated material.

14. the device of claim 11, the separable joint of wherein first has an opening at least, and this opening is by second separable joint sealing.

15. the device of claim 14, second separable joint wherein comprises a water-soluble bloated bolt.

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