CA2190121A1 - Polymeric gene delivery system - Google Patents
Polymeric gene delivery systemInfo
- Publication number
- CA2190121A1 CA2190121A1 CA002190121A CA2190121A CA2190121A1 CA 2190121 A1 CA2190121 A1 CA 2190121A1 CA 002190121 A CA002190121 A CA 002190121A CA 2190121 A CA2190121 A CA 2190121A CA 2190121 A1 CA2190121 A1 CA 2190121A1
- Authority
- CA
- Canada
- Prior art keywords
- gene
- polymer
- cells
- polymeric
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
- A61K9/1605—Excipients; Inactive ingredients
- A61K9/1629—Organic macromolecular compounds
- A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
- A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
- A61K9/2004—Excipients; Inactive ingredients
- A61K9/2022—Organic macromolecular compounds
- A61K9/2027—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
Abstract
A means for obtaining efficient introduction of exogenous genes into a patient, with long term expression of the gene, is disclosed. The gene, under control of an appropriate promoter for expression in a particular cell type, is encapsulated or dispersed with a biocompatible, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time, for example a period of three to twelve months or longer. The matrix is preferably in the form of a microparticle such as a microsphere (where the gene is dispersed throughout a solid polymeric matrix) or microcapsule (gene is stored in the core of a polymeric shell), a film, an implant, or a coating on a device such as a stent. The size and composition of the polymeric device is selected to result in favorable release kinetics in tissue. The size is also selected according to the method of delivery which is to be used, typically injection or administration of a suspension by aerosol into the nasal and or pulmonary areas. The matrix composition car be selected to not only have favorable degradation rates, but to be formed of a material which is bioadhesive, to further increase the effectiveness of transfer when administered to a mucosal surface.
Description
Wo 95/24929 ' ; 2 1 9 0 1 2 1 F~
p~lr.YM~. TC GENE DELIVERY SYSTE~S
Background of The Invention The present invention i8 generally in the area of drug delivery devices and i5 specifically in the 5 area of polymeric drug delivery devices.
"Gene Therapy" is general1y defined as the introduction and expression of an ~uyt:~uus gene in an animal to supplement or replace a defective or missing gene. Examples that have received a great 10 deal of recent atti~nt;nn include the genes missing in cystic fibrosis and severe, ' in~d n ~1 _ f; r i ~ n ~y . Al though t . A p rogre 8 8 has been made in the area of gene therapy, obtaining high level and long term expression of the desired 15 proteins remains elusive.
In the ma~ority of cases, a retroviral vector is used to introduce the gene to be expressed into appropriate cells . Gene transf er is most commonly achieved through a cell-r- ~ '; A~ c ViVC~ therapy in 20 which cells from the blood or tissue are - genetically modif ied in the laboratory and subsequently returned to the patient. The clinical studies by Steven Rns~nh~rg, et al ., " Immunotherapy of patients with metastatic mAlAn( using tumor-25 infiltrating lymphocytes and IL-2", Preliminary report, New England J. ~ed., 319 (1988) 1676-1680, using in vitro-activated I~K and TIL for tumor destruction illustrates this approach. In other cases, the vector carrying the gene to be expressed 30 is introduced into the patient, for example, by i nhA~ n into the lungs in the case of cystic f ibrosis . Transf ected cells have also been implanted, alone or ~n~AArAA~ ed within a protective membrane that protects the cells from 35 the ;nfl. ory response of the body while at the same time allowing the gene product to diffuse out of the - A~ There have also been reports of WOgS/24929 ~ , ?~q~121 the direct inj ection of an C:~-Jyt~ U8 gene in combination with an d~L.,~, iate promoter, into tissue, with some transient expression being notèd.
Viral vectors have been widely used in gene 5 trans~er, due to the relatively high efficiency of transfection and potential long term effect through the actual integration into the host ' s genome .
However, there are still ~^.^,n~^.^rnC about the risks involved in the use of viruses. Activation of 10 proto-oncogenes and reversion to wild-type viruses from replication incompetent viruses are some important potential hazards of viral delivery of genes .
Since the discovery that naked DNA is taken up 15 by muscle cells and transiently expressed in vivo, and subsec,uent reports, by Wolff, Jon Aal, et, "Direct gene transfer into mouse muscle in vivo, "
Science, 247, 1465-1468, 1990; and Wolff, Jon A, "Human dystrop~in expression in mdx mice after 20 intramuscular injection of DNA constructs, " Nature, 352, 815-818, 1991, there has been increasing interest in using non-viral vehicles for in vivo transf ections .
Plasmid DNA, which can function episomally, 25 has been used with 1 ;ros encapsulation, CaP0~
precipitation and electroporation as an alternative to viral transfections. Recent clinical trials with liposome encapsulated DNA in treating ~ n~
illustrates this approach to gene therapy, as 30 reported by Nabel, J.G., et al., ~Direct gene transfer with DNA-liposome, 1 ~Y^^ in r^~ Ar Expression, biological activity and lack of toxicity in humans", Proc. Nat. Acad. sci. U.S.A., 90 (1993) 11307-11311. A foreign gene coding for 35 HLA-B was introduced into sllhrlltAn^^~llc sites of 1 ;~n~ tumors. Expression of the new gene and the absence of an anti-DNA host response was ~ WO95/24929 ~ 2 1 90 1 2 1 1~
confirmed. Wolff, Jon A, "Persistence of plasmid DNA and expression in rat brain cells in vivo, "
~xperimental Neurology, 115, 400-413, 1992, also reported expression of plasmid DNA. Thus, direct gene transfer offers the potential to introduce DNA
encoding proteins to treat human diseases.
The - -h~n; r~-- for cellular uptake of exogenous DNA and subsequent expression are not clear but gene transf er with naked DNA is associated with several characteristics. Unlike in the case of oligonucleotides, which are typically a maximum of twenty to thirty nucleotides in length, genes encoding most molecules of therapeutic interest are quite large, and therefore considerably more dif f icult to introduce into cells other than through retroviral vector, or in vitro, by chemical manipulation, 80 that the efficiency of transfer is low. In most reported cases to date, only transient high level expression of up to a few weeks or months has been observed. Although low level e~pression and short term expression are two important drawbacks with direct DNA transfer, transfections with naked DNA have several advantages over viral transfers. Most importantly, concerns related to the; t~gpn; city and transforming capability of viruses are avoided. In addition, naked DNA is easy to produce in large quantities, is ;nP~nqive, and can be injected at high c -ncPntration into localized tissue sites 3 0 allowing gene expression in si tu without extensive ex vi vo therapy .
The following additional articles review the current state of gene therapy and the problems associated therewith: Blau, Helen M, "Muscling in on gene therapy," Nature, 364, 673-675, 1993;
Cohen, Jon, "Naked DNA points way to vaccines, ~
Science, 259, 1691-1692, 1993; Dagani, Ron, "Gene .. . . . ... . . ... .. . . . .. . . . . _ _ WOgs/24929 ~-.r~ 2~'~0121 r_l,. / ~
therapy advance, anti-XIV antibodies work inside cells, ~ C&EN, 3-4, 1993; Felgner, Philip L, "Lipofectamine reagent: A new, higher ~ff;~;~n~-y polycationic liposome transfection reagent, "
FocuEI/Gibco, 15, 73-78, 1993; Liu, Margaret A et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein, "
Science, 259, 1745-1749, 1993; Marx, Jean, "A first step toward gene therapy for hemophilia B, "
Science, 262, 29-30, 1993; Mulligan, Richard C, "The basic science of gene therapy, " Science, 260, 926--931, 1993; Nicolau, ~--1 A~l~Al, et, In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I, ~ Proc. Natl . Acad. Sci. rJSA, 80, 1068-1072, 1983; Partridge, Terence A, "Muscle transfection made easy, " Nature, 352, 757-758, 1991; Wilson, James M, "Vehicles for gene therapy, "
Nature, 365, 691-692, 1993; Wivel, et aI., "Germ-2~ line gene modification and disease prevention: Some medical and ethical perspectives, " Science, 262, 533-538, 1993; and Woo, Savio L Cal, et, "In vivo gene therapy of hemophilia B: sustained partial correction in Factor IX-deficient dogs, ~ Science, 262, 117-119, 1993.
Gene therapy is one of the most promising areas of research today. It would therefore be extremely useful if one had an efficient way to introduce genes into cells which yielded long term 30 expression.
It is therefore an object of the present invention to provide a means f or ef f icient transf er of exogenous genes to cells in a patient.
It is a further object of the present 35 invention to provide a meanR for long term expression of exogenoug geneg in p~t jent~, ~ W0 95/24929 ; ;~ 9 0 ~ 2 1 r~ 7 It is a further object of the present invention to provide a means for increasing or decreasing the ;nfl; tnry response to implanted polymeric devices.
It is a still further object of the present invention to provide a method for; ; 7Ation of individuals over a more prolonged period of time than is achieved by a single or multiple ; 7Ation protocol .
It is another object of the present i~vention to provide a method for targeting of gene delivery either to tissue cellg or to ; n f 1 tory type cells .
8umnary of the Invention A means for obtaining efficient introduction of exogenouæ genes into a patient, with long term expression of the gene, is disclosed. The gene, under control of an appropriate promoter f or expression in a particular cell type, is encapsulated or dispersed with a biocompatible, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time, for example, a period of three to twelve months or longer. The matrix is preferably in the form of a microparticle such as a microsphere (where the gene is dispersed throughout a solid polymerlc matrix) or microcapsule (gene is stored in the core of a polymeric shell), although other forms including film8, ~At;n~s, gels, 3 0 implants, and stents can also be used . The gene, or other DNA or RNA to be delivered, can be incorporated directly into the polymer, or first inc~,L~"~ted into another material ~nhAn~ ;n~
penetration of the DNA through the cell wall, such as liposomes or surfAc~An~. The size and composition of the polymeric device is selected to wo g~l24929 ~ , J ~ P~~ 07 result in favorable release kinetics in tiEsue.
The size i8 alBo selected according to the method of delivery which is to be used, typically inj ection into a tissue or administration of a 5 suspension by aerosol into the nasal and/or p~ ry areas. The matrix composition can be selected to not only have favorable degradation rates, but to be formed of a material which is bioadhesive, to further increase the effectiveness 10 of transfer when administered to a mucosal surface, or selected not to degrade but to release by diffusion over an extended period.
Examples demonstrate the effectiveness of the system in animals.
Detailed Description of the Invention Gene transfer iE achieved using a polymeric delivery system which releases entrapped genes, usually in ~ ; nAt j on with an appropriate promoter for expression of the gene, into ~uLLuullding 20 tissue. Efficacy of transfer is achieved by: a) releasing the ~ene for prolonged period of time, b) m;n'm;~;ng diffusion of the gene out of the delivery system (due to its size) so that release is predominantly degradation ~r~nA~nt, and c) 25 improving the~transient time of expression and the low inf ection seen by direct gene therapy . In case of non-erodible polymers, the device is fUL~ lt BO that the gene is released via dif fusion . This is achieved by creating porous systems or adding 3 0 soluble bulking agents that create pores as they leach out of the system.
The Poly3neric Matrice~
Selection of Polvmer Both non-biodegradable and biodegradable 35 matrices can be used for delivery of genes, although biodegradable matrices are preferred.
~ Wo9~/24929 ~ 4 2 1 9~ 1 2 1 P~ v.,.07 These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release prof iles . The polymer is selected based on 5 the period over which releage is desired, generally in the range of at least three months to twelve months, although longer periods may be desirable.
In some cases linear release may be most useful, although in others a pulse release or "bulk 10 release" may provided more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90~ by weight of water), and can optionally be crosslinked with multivalent ion~ or polymers.
High molecular weight genes can be delivered partially by diffusion but mainly by degradation of the polymeric system. In this case, biodegradable polymers, bioerodible hydrogels, and protein delivery systems are particularly preferred.
20 Representative synthetic polymers are: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, 25 polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyuret_anes and co-polymers thereof, alkyl cellulose, llydL~ycLlkyl celluloses, cellulose ethers, celluloGe esters, nitro n~ llo~e5, polymers of acrylic and methacrylic 30 ester~, methyl cellulose, ethyl CPl~ se, 11YdL~Y~L~Y1 cellulose, hydroxy-propyl methyl ~I~1 1111n~Ie~ 11YdL~J~Y~ULY1 methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate rh~h~ e, 35 carboxylethyl cellulose, c~11ll1n~e triacetate, C~llll1nqe sulphate sodium salt, poly(methyl methacrylate), poly (ethyl methacrylate), wo gs/24929 . ~ } 2 1 9 0 1 2 1 P-"~ i~Q7 ~
poly (butylmethacrylate ), poly ( isobutyl methacrylate), poly (hexyl th~~rylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), 5 poly (methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glyco1), poly(ethylene oxide), poly(ethylene tererhth~l~te), poly(vinyl alcohols), poly(vinyl acetate, poly 10 vinyl chloride, polystyrene and polyvinylpyrrolidone .
Examples of non-biodegradable polymers include ethylene vinyl acetate, poly (meth) acrylic acid, polyamides, copolymers and mixtures thereof.
Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly (valeric acid), and poly (lactide-co-20 caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, 25 hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these 3 0 materials degraae either by enzymatic hydrolysis or ~O~u~e to water in v~vo, by surface or bulk ero8ion .
R;~:~rlh~R;ve polymers of particular interest include bioerodible hydrogels described by H.S.
35 Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the tf~at-h;n~s of which are incorporated herein, polyhyaluronic ~ W095/24929 ~ " ~ 21 9 ~1 21 r~ vv vlQ3~07 acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates ), poly ( ethyl methacrylates ), po ly ( butyl m~ t h A rrylate ), po ly ( i 8 0butyl 5 methacrylate), poly (hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl 10 acrylate).
Selection of Matrix Form and Si~e In the preferred embodiment, the polymeric matrix is a microparticle between nanometers and one millimeter in t~ trr~ more preferably between =~
0.5 and 100 microns for administration via injection or inhalation (aerosol).
The microparticles can be microspheres, where gene is dispersed within a solid polymeric matrix, or microcapsules, where the core is of a different material than the polymeric shell, and the gene is dispersed or suspended in the core, which may be liquid or solid in nature . Unless specif ically defined herein, microparticles, microspheres, and microcapsules are used interchangeably.
Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard terhn;q~ p~ or even a gel such as a hydrogel. The polymer can also be in the form of a coating or part of a stent or catheter, vascular graf t, or other prosthetic device .
Methods ~or Makinq the Matrix The matrices can be f ormed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.
W093l24929 ,' ~ . 2 1 9 (~ 1 2 I P~ 3; , ~
Micro~phere Preparation Bioerodible microspheres can be ~L~dL~:d using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987); Mathiowitz, et al., Reactive Polvrners 6, 275-283 (1987); and Mathiowitz, et al., J. A~l. Polvmer Sci. 35, 755-774 (1988), the t~rh;n~ of which are incorporated herein. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz, et al., Srilnn;nr Microsoo~y 4,329-340 (1990); Mathiowitz, et al., J.
A~l. PolYmer Sci. 45, 125-134 (1992); and Benita, et al., J. ph~rm~ Sci. 73, 1721-1724 (1984), the t~Arh;n~R of which are lnc:u.~uurc.ted herein.
In solvent evaporation, described for example, in Mathiowitz, et al., (1990), Benita, and U.S.
Patent No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent. The DNA, either i~ soluble form or dispersed as ~ine particles, is added to the polymer solution, and the mixture is sl~p~n~l~d in an aSIueous phase that rrnt~inR a surface active agent such as poly(vinyl alcohol). The resulti~lg emulsion is stirred until most of the organic solvent evaporates, leaYing solid microspheres.
In general, the polymer can be dissolved in methylene rhlrr~G Several different polymer rr,nr~ntrations can be used, for example, between o . 05 and 0 . 20 g/ml . After loading the solution with DNA, the solution is sll~p~n~ l in 200 ml of vigorously stirring distilled water cr,ntA;ninS 19~
(w/v) poly (vinyl alcohol) (Sigma Chemical Co ., St .
Louis, M0). After four hours of stirring, the organic solvent will have evaporated from the W095/24929 ' .""~`f~ 2~90~2 1 P~ 07 polymer, and the resulting microspheres will be washed with water and dried overnight in a lyo~ll; 1 i 71:.r, Microspheres with different sizes (1-1000 5 microns) and morphologies can be obtained by this method which is useful for relatively stable polymers such as polyesters and polystyrene.
However, labile polymers such as polyanhydrides may degrade due to exposure to water. For these 10 polymers, hot melt encapsulation and solvent removal may be pref erred .
In hot melt encapsulation, the polymer i8 f irst melted and then mixed with the solid particles of DNA, preferably sieved to less than 50 15 ,~lm. The mixture is s--~rf~n~ d in a non-miscible solvent such as silicon oil and, with c~nt; nl7~
stirring, heated to 5C above the melting point of the polymer. Once the ~mlll ~ n is st~hl 1; 71~1, it is cooled until the polymer particles solidify.
20 The resulting microspheres are washed by decantation with petroleum ether to give a free-f lowing powder. Microspheres with diameters between one and 1000 microns can be obtained with this method. The external surface of spheres 25 prepared with this techni~ue are usually smooth and dense. This procedure is useful with water labile polymers, but is limited to use with polymers with molecular weights between 1000 and 50000.
Solvent removal was primarily designed for use 30 with polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then gll~p~n~
in oil, such as silicon oil, by stirring, to form 35 an ~ n. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike solvent Wo95l24929 ~ 901 21 r~
evaporation, this method can be used to make microspheres from polymers with high melting point~
and a wide range of molecular weights.
Microspheres having a ~; -tPr between one and 300 microns can be obtained with this procedure. The psrtPrniql morphology of the spheres is highly dependent on the type of polymer used.
In ~pray drying, the polymer is dissolved in methylene chloride (0 . 04 g/ml) . A known amount of 0 active drug is suspended (if insoluble) or co-dissolved (if soluble) in the polymer solution.
The solution or the dispersion is then spray-dried.
Typical process parameters for a mini-spray drier are as f ollows: polymer concentration = O . 04 g/ml, inlet temperature = 24C, outlet temperature = 13 to 15C, aspirator setting = 15, pump setting = 10 ml/min, spray flow = 600 N~h-l, and nozzle .1; -tPr = 0 . 5 mm. Microspheres ranging in ~; i t~r between one and ten microns can be ~lhtAinP~l with a morphology which depends on the selection of polymer .
Double walled microspheres can be prepared according to U.S. Patent No. 4,861,627 to Mathiowitz .
Hydrogel microspheres made of gel-type polymers such as alginate or polyphosphi~7~nPq or other dicarboxylic polymer3 can be prepared by dissolving the polymer in an aqueous solution, suspending the r~tPr;i~l to be incorporated into the mixture, and extruding the polymer mixture through a microdroplet forming device, equipped with a nitrogen gas iet. The resulting microspheres fal into a slowly stirring, ionic hardening bath, as described, for example, by Salib, et al., Pharmazeutische Industrie 40-llA, 1230 (1978), the tPi~rh; n~f: of which are incorporated herein. The advantage of this system is the ability to further ~ WogSl24929 ~ 2 ~ 9012 r ~
modify the 8urface of the microspheres by coating them with polycationic polymers such as polylysine, after fabrication, for example, a8 described by 3Jim, et al., J. Pharm. Sci. 70, 351-354 (1981).
5 For example, in the case of alginate, a hydrogel can be formed by ionically crosslinking the alginate with calcium ions, then crosslinking the outer surface of the microparticle with a polycation such as polylysine, after fabrication.
10 The microsphere particle size will be controlled using various size extruders, polymer flow rate8 and gas f low rates .
Chitosan microsphere8 can be prepared by dissolving the polymer in acidic solution and 15 crosslinking with tripolyphosphate. For example, ~ LJ~yl ~thylcellulose (CMC) microsphere are prepared by dissolving the polymer in an acid solution and precipitating the microspheres with lead ions. Alginate/polyethylene imide (Pl~I) can 20 be prepared to reduce the amount of carboxyl groups on the alginate microcapsules. Table 1 summarizes various hydrogels, rrnr,~ntrations, ionic baths, and stirring rates used to manufacture them.
T~blu 1: E. "~ ^n o:E Hydrog~l M~trie~
25 llydrog-l Hydrog~l di~olviny bath ionie b~th ~tirrir~g eone~n. ~H TemDC eonC~m. (w/v) rat~(rD~n) chitosan 1. 0% 5 . 0 23 3t tripolJ- 170 phoQph~te alginate 2.09~ 7.4 50 1.3% cale:.um 160 3 0 chlori~e algi~ate/ 2.0~/ 7.4 50 1.3~ calc_um 160 PEI 6 . 0% chloride Carboxy 2.0t 7.4 50 10.0~ lead 100 methyl nitrate 35 cellulose Other device forms Other delivery systems including f ilms, coatings, pellet8, slabs, and devices can be fabricated using solvent or melt casting, and 40 extrusion, as well as standard methods for making composites. The polymer can be produced by first _ .. _ . .. , ., .. ,, . ., ., . , , _ _ _ _ _ _ _ _ _ _ _ _ . _ _ wogs/24929 ; '~ 9 0 1 ~ 0 mixing l u and DNA as described by Sawhney, et al., and polymerizing the monomers with W light.
The polymerization can be carried out in vitro as well as in vivo. Thus, any biocompatible glue 5 could be also used to incorporate the DNA.
Loading o~ Gene The range of loading of the gene to be delivered i9 typically between about 0 . 01% and 9096, r~n~1;n~ on the form and size of the gene to be delivered and the target tissue.
Selection of Gene~ to be Inco ~o qted Any genes that would be useful in r-~rl~r; n~ or supple t; ng a desired function, or achieving a desired ef ~ect- such as the inhibition o~ tumor growth, could be introduced using the matrices described herein. A_ used herein, a "gene~ i9 an isolated nucleic acid molecule of greater than thirty nucleotides, preferably one hundred nucleotides or more, in length.
r ~ A of genes which replace or supplement function include the genes encoding missing enzymes such as ~r~Pnns;n~ m;nAqe ~ADA) which has been uqed in clinical trials to treat ADA def iciency and rnf~tnrs such as insulin and coagulation factor VIII.
Genes which effect regulation can also be administered, alone or in rr-binAt;nn with a gene suppl: ;n~ or rf-pl~,-;ns a specific function.
~or example, a gene encoding a protein which 3 0 suppresses expression of a particular protein-Pnro~l;n~ gene, or vice versa, which induces expresses of a protein-~n-o~;n~ gene, can be administered in the matrix.
Examples of genes which are useful in stimulation of the immune response include viral antigens and tumor antigens, as well as cytokines W095/24929 i ~ 21 9012 ~ r~
(tumor necroeis factor) and inducer9 of cytokines (endotoxin), and various pharmacological agents.
The chronic immune responEe to the polymeric matrix is mediated by the action of a variety of growth factors including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth _actors (FGFs), tran8forming growth factors (TGF-~ and TGF-$, interleukin-1 (II,-1), and tumor necrosis factor (TNF). Inhibitors of these infl~r~~tory mediators in combination with a gene to be delivered other than the immune inhibitor would be effective in decreasing the normal ;nfl tory response directed toward the polymeric matrix. By inhibiting the amount of encapsulation of the matrix, the effective release would be further ~ n~d. r ~ of materials which could inhibit encapsulation include antisense mRNA to suppress fibrin or collagen fo~r-~inn, inhibitors of EGF, PDGF, FGFs, TGF-~Y, TGF-i~, IL-1 and TNF and anti-;nfl: tory agents such as corticosteroids and cyclosporin.
Genes can be obtained using literature references or from commercial suppliers. They can be synthesized using solid phase synthesis if relatively small, or obtained in expression vectors, for example, as deposited with the American Type Culture Collection, Rockville, MD.
Selection of vector~ to be introduced in - ' in~ with the gene.
A8 used herein, vectors are agents that transport the gene into the cell without - degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Promoters can be general promoters, yielding expression in a variety of 11 ~n cells, or cell specific, or even nuclear versus cytoplasmic ~re~; f; ~. These are known to those skilled in the art and can be constructed using woss/24929 ` ~ i 2 ~ 901~1 r~".J~
standard molecular biology protocols. Although as demonstrated by the examples, the genes will diffuse out of the polymeric matrix into the surrounding cells where they are expressed, in a 5 preferred ~mh~ t, the q,enes are delivered in combination with a vector to further enhance uptake and expression. Vectors are divided into two classes:
a) Biological agents derived from viral, 10 hact~r; ~1 or other sources .
b) Chemical/ physical methods that increase the potential for gene uptake, directly introduce the gene into=the nucleus or target the gene to a cell receptor ~ =
Bioloqical Vectors Viral vectors have higher transaction (ability to introduce genes) abilities than do most chemical or physical methods to introduce genes into cells.
Retroviral vectors are the vectors most 2C commonly used in clinical trials, since they carry a larger genetic payload than other viral vectors.
However, they are not useful in non-proliferating cells .
Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation. However, many people may have pre-existing ~nt;hn~lies negating effectiveness ahd they are difficult to produce in quantity .
3 0 Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. However, they cannot be tr~n~m; tt~i from host to host and there are some safety issues since they can enter other cells.
Plasmids are not integrated into the genome and the vast maj ority of them are present only f rom ~ W0 9~/24929 ' ; ` ' ' ` 2 19 0 12 1 r~ . 7 a f ew weeks to several month9, 80 they are typically very safe. Xowever, they have lower expression levels than retroviruses and since cells have the ability to identify and eventually shut 5 down foreign gene expregsion, the t~r~n~;n~
release of DNA f rom the polymer to the target cells substantially increases the duration of functional expression while ~ ;nt~;n;n~ the benefit of the - ---safety as80ciated with non-viral transfeetions.
Chf~m; cal/phvsieal veetors Other methods to direetly introduee geneæ into cells or exploit receptors on the surf ace of eells include the use of liposomes and lipid8, ligands f or specif ic eell surf aee receptors, cell 15 receptors, and caleium phosphate and other chemieal mediators, microinjections directly to single cells, electroporation and homologous reeombination. The ehemieal/physical methods have a number of problems, however, and will typieally not 20 be used with the polymeric matrices described herein. For example, ehemicals mediators are impractical for in vivo use: when ealeium phosphate is used there appears to be very low transduetion rate, when sodium butyrate is used the inserted 25 gene is highly unstable and when glyeerol is used inserted gene is rapidly lost.
As demonstrated by the examples, it ha8 been diseovered that it is possible to incorporate nucleic acid molecules into liposomes or complexed 3 0 to liposomes which are t~en entrapped or otherwise incorporated into the polymeric matrix f or delivery to cells. The ratio of liposome to polymer 801ution is important in ~ t~orm;n;n~ whether the liposomes will remain as separate entities during 35 the process for incorporation into the polymeric matrix. If the ratio of solvent is too high, the rhn~Ph~l;rid Will dissolve into the polymer W09s/24929 ! ~ , 2 1 9~ ~ 21 r~
solvent, rather than rPm~;nlns as part of the liposome bilayer. This is a function of the liposome composition, polymer cnnopntration~ and solvent composition. The liposomes increase the efficiency of the transfer of the DNA into the cells. Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINf~ and ~IPOFECTACEf~, which are f ormed of cationic lipids such as N- [1- (2,3 dioleyloxy) -pro?yl] -n,n,n-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDA~3). Numerous methods are also rllhl; F~hP~l for making liposomes, known to those skilled in the art.
As demonstrated by the following example, any nucleic acid molecule, including genes as def ined above, antisense, ribozymes and ribozyme targeting molecules, and probes, def ined as an oli~-nl-~ 1P~ tide of at least fourteen to seventeen nucleotides in length, can be mixed with, or incorporated into, the liposomes. The liposome-nucleic acid molecule mixture is then mixed with a polymer solution. A8 noted above, the polymer solution can be formed by melting of a polymer, llltinn of a polymer in a solvent, or selection of a polymer t~Lat is a liquid under certain conditions, such as an acidic pH or room temperature, which is then sol; ~; f j ed by a change in conditions.
rltical Co~poE~ition~
When the matrix is in the form of microparticles, the microparticles can be sll~pon~Pd in any appropriate phar--ceutl~l carrier, such as saline, for administration to a patient. In the most preferred f ' orl; t, the microparticles will be stored in dry or lyrrh; 1; ~od form until tol y before administration . They will then ~ w09i5/24929 ' ~ ~ i `;` 2 1 r~ 3~!~
be suspended in sufficient solution for admini stration .
In some cases, it may be desirable to administer the microparticles in combination with an adjuvant to enhance the ;nfl. tory response against the polymer and thereby increase the l; k.-l; hnod of phagocytosis by macrophages and other hematopoietic cells, with subsequent expression of the gene specifically within these cells, or, in the case where the microparticles contain an anti-cancer agent, to enhance the ;nfli tnry reaction against the tumor cells in combination with the effect of the anti-cancer agent.
The polymeric microparticles can be administered by injection, infusion, implantation, orally (not preferred), or administration to a mucosal surface, for example, the nasal-pharyngeal region and/or lungs using an aerosol, or in a cream, ointment, spray, or other topical carrier, for example, to rectal or vaginal areas.
Other ~ ; r-nt- S ~ such as slabs, coatings, rods, and other devices are preferably administered by impl _nt_l-i nr in the area where release is desired .
The materials can also be incorporated into an appropriate vehicle for transdermal delivery as well as stents. Appropriate vehicles include o; ~-, lotions, patches, and other standard delivery means.
Targeti~g of cell populatio~ through polymer material characteri3tic~.
- Studies with plaismid release using PI.A/PC~
biodegradable polymers indicate that the majority of transfected cells, assessed with the $--Ji~lA.-tvsidase reporter gene, are ;nfl: tnry cells involved in the "foreign bodyD response. In general, degrading non-degrading polymers evoke a stronger infli tnry response when compared to .
095/24929 ~ 9 ~ _l/L~ 'tO7 non-biodegrading polymers. A strong foreign body response results in a thick layer of macrophages, f ibroblasts, and lymphocytes around the implant .
Because the polymer release device relies on diffusion for n.~v~ t of its particles, a strong inflammatory response will limit the effective distance of diffusion. Accordingly, biodegrading polymers can be uged to target i nl~l i tory cells due to the inability o~ the plasmid DNA (pDNA) to migrate across the reactive tissue layer to the site specific tissue_ A more biocc _-t;hle material which induces a weaker response from the host will result in a thinner layer of infl i tnry cells, enabling the released pDNA to migrate across the ;nfli tory cells to the indigenous cells to be transfected.
Protection of Pli~smid DNA from [mmediate Degradation Tris-EDTA (TE) is the buffer of choice for DNA
due primarily to it' 5 inhibitory effect on nucleases. It is known that storage of DNA in other buffers or plain water results in degradation of the DNA even at 4 o C (Wolf f, et al ., Biotechniques 11(4), 474-485 (1991) ) . The polymer release system can be loaded to deliver plain DNA
or DNA with buf fer salts to be used as carriers or protecting agents depending on the choice of buffer used .
Incorporation of ~ntiinfll tories and Immune ~nhi/nt ~8; Treatment of Canc~rs In recent years, rnn~;~iprable attention has been f ocused on the use of gene therapy to treat various diseases including cancer. Generally, gene therapy f or cancer therapeutics either targets the cells of the immune system to enhance their ability to kill malignant cells or directly targets the cancer cells to regulate their proliferation or .
Wo gsl24929 l ; 2 1 9 0 1 2 1 l ~ ,,J/t enhance some f~ r function which will result in a stronger activation of the immune response.
Most types of cancer are characterized by frequent relapses during the course of treatment 5 and the cnnt;n~ non-specific and/or specific activation of the immune system resulting from gene therapy is crucial. Second, cell targeting is a major limitation of current vectors and implantation of a controlled release device 10 directly inside a tumor where the DNA is released locally is one alternative to ex vivo therapy or the development of effective ligand specific vectors. As indicated by the prevalence of ex vi~o therapy, targeting hematopoietic cells is 15 especially difficult. The histological results from the implant site in the studies described in the examples below, reveal a substantial ;nfli tory response :juLLuu~ding the intramuscular implant. The well known "foreign body" host 2 0 response can be used to an advantage as this migration of lymphocytes and antigen presenting cells raises the pnsR;h;1;ty of directing the transfection to these specific cell pOp~ ;
Tumors elicit both the humoral and cell-25 mediated immune response, and lymphocytes,particularly cytotoxic T cells and NK cells, as well as macrophages, are known to play a crucial role in tumor elimination. Gene therapy for cancer treatment either targets these cells or the 30 malignant cells themselves. An implant releasing naked DNA for long term functional gene transfer which can target ;nfl: tory cells and/or tumor cells could signif icantly improve cancer therapy.
The approaches used include upregulation of 35 class I MHC expression, transduction of antige~
presenting cells with tumor-specific antigens, W095/24929 - 2 1 qO ~ ~1 r~l,.,.. /
cytokine immunotherapy, transfection of tumor cells with tumor auppressor genes and anti-sense therapy.
The malignant transformation of cells is often characterized by a reduction of class I M~C
5 expression leading to a severe depression of the CT~-r^~ t~-l immune response. An increase in class I ME~C expression on tumor cells could facilitate the activation of the immune system against these altered self-cells. Transfection of genes for 10 cytokines such as tumor necrosis factor (TNF) into tumor cells or tumor suppressor genes such as p53 can be used to limit the ability of tumor ells to multiply . Anti - sense therapy targets cell prolif eration or the production of necessary 15 proteins such as tumor angiogenesis factor (TAF) by complf~m~n~ry RNA hybri~;7~t~nn to block transcription of specific genes.
The immune system can be activated and induced to attack speci~ic cells using cytokines such as 20 Proleukin or monoclonal antibodies. For example, cancer cells proliferate in part due to a decreased immune response against the transformed cells. The matrices described herein provide a means to allow recognition and provocation of a response to cancer 25 cells. For example, genes - coding ~or antigens, such as the aberrant epithelial mucin of breast cancer, and monoclonal ~nt;horl;f-~ directed against tumor antigens have been shown to have potential in st~ ;n~ immune destruction of malignant cells.
3 0 These genes, alone or in combination with monoclonal zlnt;hn~l;es~ can be delivered to the tumor sites in the polymeric matrices to achieve inhibition of the tumor cells.
Cancer cells can also be treated by 35 introducing chemotherapy drug resistant genes into healthy cells to protect them against the toxicity of drug therapy, or by the insertion of appropriate 95/24929 ~ ir ,~ ~; 2 ~ 9 û 1 2 ~ u..,.,lQ~07 vector8 cnntAinin~ cytotoxic genes or blocking genes into a tumor mass to ~1 ;rG;nAte cancer cells.
In a preferred embodiment, the immune system is specif ically stimulated against antigens or proteins on the surf ace of the cancer cells .
These approaches can be used in vi tro and in vivo. Tn vitro, the cells can be removed from a patient, the gene inserted into the cell and the cells reintroduced into the patient. In vivo, the gene can be directly introduced into the body either systematically or in localized sites.
Another approach is to use suicide gene8 that cau8e cell death when they are activated or when their product is ~o~l nt~cl with a pharmaceutical .
The primary limitation of the method is the fact that the gene 8hould be targeted to the cancer cell and not to normal cell. Current approach to ~v~ the problem is direct injection of the vectors into a localized area where normal cells do not proliferate. This would be greatly facilitated using the polymeric devices described herein. The advantages of polymeric devices in this setting include r~nt;nllmlR and protracted release of the incorporated pharmaceutical. This increases the l;kf~lih~od that the int~n~ purposes, ~or example, treatment of cancerous cells, will be achieved.
r The method' and materials of the present invention will be further understood by reference to the following non-limiting ~
3xample 1: Expression of linear and 8~ oiled plasmid DNA ~n~rs~ t~d in polymeric; _ls-nt~ in vuscle tissue of rats.
The study described in this example confirms the feasibility of in vivo transfections using biodegradable polyester blends to release linear or supercoiled plasmid DNA. Although only short term _ _ _ _ _ _, _ _ _ _ .. .. . . . _ _ _ _ . _ .
WogS/24929 ~ 9~ 1 2 1 P~ J~07 ~
expression was studied in this study, polymer devices releasing drugs offer the potential for sustained long term delivery of naked DNA.
Marker aenes are used to study the r v I of 5 engineered cells rf~nt:~in;n~ exogenous genes, as well as the vectors and genes introduced with the vectors, to inbure that the genes remain where they are introduced. Almost all of the initial research into gene therapy is with marker genes. Preferred 10 marker genes are those whose product is innocuous and which can be readily detected by simple laboratory tools. An appropriate marker gene is ~-galactosidase (~-gal), since expression is readily detected by addition of X-gal, a substrate which 15 yields a blue color when the active enzyme is present .
r~nr~ ulation of linear and s~ coiled ~-aal codinq DNA in a PLA blend 1 g PI.A (300K) and 2 g PI~ (2K) was dissolved 20 in lO ml of methylene chloride and 5 drops of Span 85. The mixture was divided into two aliauots of 5 ml and 100 ul of either circular or linear DNA
(between 1 and 2 mg/ml diluted 1:5 in buffer) was introduced into the aliauots. Each mixture was 25 mixed well and ali~uoted into glass vials (1 ml/vial). Between 20 ~g and 40 ILg of ~-gal plasmid DNA was encapsulated in each glass vial. The glass vials were left in the refrigerator for four days to evaporate the methylene chloride and then 30 lyop~; l; 7~
T laI~tation of DNA/PIA ~ellets Each sample was first sterilized with ethanol for 5 min and then washed with PBS-penicillin/streptomycin for 5 min. Surgery was 35 done on Sprague Dawley rats. Linear DNA was implanted into the lef t leg and supercoiled DNA
implanted into the right. T, l;lnt.c were inserted into incised muscle - either in the vastus or the _ _ . _ _ _ _ _ _ _ _ _ ' ' ~ ~ `t ~ t.
~ WO 9St24929 2 1 9 0 1 2 1 hamstring. The muscle was sutured back together and then the skin was sutured closed. Rats were sacrificed for analysis at two weeks.
Result 5 Rats were perfused with Pho5phate Buffered Saline (PBS) with 2500 units of heparin followed by 3% paraformaldehyde and 0 . 296 glutaraldehyde in PBS .
The tissue was post-fixed with 396 paraformaldehyde followed by 1596 sucrose/PBS. 33xcised muscles were cut with a cryostat and stained with X-gal.
Xistology of the implant sites revealed a substantial infl: ~ory response around the film at two weeks and two months. The bulk of the ,(~-gal positive staining was localized to this area with few muscle cells exhibiting positive staining. The cells present around the implant probably consists of phagocytic cells, lymphocytes and fibroblasts.
As expected, transfection was more ef~icient with supercoiled DNA.
2 0 Example 2: In v~ tro transf ection with pRSV ,~-g~l .
NIH3T3 f ibroblasts were plated onto a 6 well tissue culture dish with 1 ml of D-MEM (1096 Fetal calf serum with penicillin/streptomycin). 24 hours after plating, the cells were transfected with pRSV
,B-gal control plasmids as per Promega Profection Transfection system.
Plate 1: 10 t~l pRSV-Z (3.4 t~g) Calcium Phosphate Precipitated Plate 2: 30 tll pRSV-Z (10.2 llg) Calcium Phosphate Precipitated Plate 3: 10 t~l pRSV-Z (3.4 ~g) Naked DNA
Plate 4: 30 /Ll pRSV-Z (10.2 ~g) Naked DNA
Plate 5: DNA/PLA
3 5 Plate 6: Control Plate 5 with the PLA pellet was placed into the well with 4 ml of media to counter the effect of the decrease in pH. After 24 hours, the DNA/PLA
pellet was removed and the media left unchanged.
:
WO9~/24929 ~ q Q ~ 2 ~ p ", ~ Q7 At 48 hours, the cells were fixed and stained with X-Gal (1 ml/plate) overnight.
Result 8 The efficiency of transfection was very low.
5 All plates except the control well had a handful of blue staining cells. There was no observable dif f erences in the number of blue cells among the 5 plates. It was interesting to note that the plate with the DNA/P~A had similar levels of staining as 10 the other plates even after the fact that half the cells had died and ~ ti~rh~rl due to the PLA
degradation .
Example 3: Duratio~ of Expreasion with pSV ,B-gal DNA Encapsulated I~to PLA
Blends.
Tn Vl tro r-elea8e of ~lasmid DNA
pSV ~-gal was amplified i~ H3101 and purified with Qiagen' 8 Mega Prep . 500 ~l of plasmid in Tris-EDTA buffer (67 . 5 ~g) was lyorh; 1 i 7~-1 and 20 resuspended into 100 ~Ll of sterile dEI2O and incorporated into P~A. O . 05 g PLA (2K) and O . 05 g (300K) was dissolved in 1 ml of methylene chloride and 1 drop of Span~K 85. After the polymer was in solution, 100 1ll of plasmid (67 . 5 ~g) was added to 25 the mixture and vortexed for 15 sec. The resulting f ilm was lef t in a ref rigerator overnight and subsequently lyophilized overnight.
This f ilm was incubated with 1. 0 ml of TE
buffer at 37C and sample sUprrn~t~ntq tested at 24 3 0 hours and at 4 days f or the presence of released DNA. DNA was assayed by agarose gel electrophoresis on the ~lrrrn :t~nt~.
The results based on the gel of the gUp~rn~t~nt after 24 hours of inrllh~t;r,n show that 35 a substantial amount of plasmid was released.
Af ter 4 days, the results indicate that there was a first phase of release due to the diffusion of plasmid molecules which are close to the surface of ~ WO 9SI24929 ~ ~ 2 1 9 0 1 2 1 P~
the device followed by a slower release at 4 days due to the low degradation rate of the polymer which was too low to be measured.
In vivo transfection levels 3 mg P~A (2K) and 1 mg PLA (lOOK) were dissolved in methylene chloride (0.25 ml). 1 drop of Span~ 85 and 20 ~1 of plasmid (20 ,llg) was added to the solution and homogenized for 1 minute. This solution was air dried in a glass vial for 3 hours in a sterile hood. The brittle film was ground into fine granules and pressed into a pellet form.
Three of these DNA rnn~;l;nln~ pellets were made as well as three control pellets without DNA. All pellets were lyo~h;l 7ed overnight to extract residual solvents.
Three rats received DNA/P~A in their left hamstring and control/PLA in their right hamstring.
Pellets were inserted into incised hamstrings and the muscles closed with 6- 0 Vicryl . Three rats received an injection of pSV ~-gal plasmids (20 ILg in 100 ~1 of TE buffer) over a minute long period ~:
in their left leg and 100 ~11 of plain TE bufier in their right leg as controls . The site of inj ection was marked with suture.
25 Rat ID Le~t Riqht T 1 ~n~ l~uration R112 DNA/PLA Control/DNA 1 week RllO DNA/PLA Control/DNA ~ weeks Rlll DNA/PLA Control/DNA 10 weeks RllS DNA/bu~fer Control/bu~fer l week 30 ~1~ DNA/bu~fer Control/buf~er 5 weeks Rats were perfused with PBS/heparin, followed by 4~ paraformaldehyde, and post-fixed in 49c paraformaldehyde followed by 1596 and 259~
sucrose/P~3S. Excised muscles were cut with a 35 cryostat and 6tained with X-Gal.
Results Tn vI tro release studies indicate that plasmid DNA can be incorporated into polymers without .. , . . . , , _ _ _ _ _ _ Wo 95/24929 ~ . " ~ P~ 07 degradation through manuf acturing processes and released in functional form for possible uptake by surrounding cells.
In vivo etudies reveal that with a 20 ~g loading of DNA into the polymer, there is substantial transfection of ;nfl; tn~y cells at 1 and 5 weeks aa confirmed by X-gal staining and immunoblotting. At 10 weeks, there was no difference in ~It;~in;n~ intensity between the control PLA and DNA/PLA. This is believed to be due to the resu=lt of the low loading (20 llg) of the polymer such that af ter one week the release rate was below half maximal levels. Investigators using direct injection use doses in the 100 ,Lg range to see their effects. A higher initial loading, which will lead to Cnnt; nl~etl release of higher amounts of DNA from polymers, should prolong transfection durations. Rats injected with 20 ILg of DNA in solution showed no transf ection at 1 and 5 weeks .
Example 4: Compari~on of Pla2~mid DNA Relen~e From ~o~_ ' hle ~d Non~ s A~lins Polymer~ .
Release of plasmids from biodegradable and non-degradable polymer was compared to test the 2~ feasibility of targeting either inflammatory cells or tissue speci_ic cells by selection of polymer material. Plasmid DNA was incorporated into a non-degradable elastomer, ethylene vinyl acetate copolymer (EVAc~ and implanted into the same site in different animals as PI~/PC~ implants. EVAc is a very biocompatible polymer which can be --nllf~c~ ed into a mi.L.,~-J~ .,us structure through which DNA can diffuse into the surrounding tissue.
En~-~nsulation of ~RSV ~-qal into Polvmers.
pRSV ,~-gal in H~3101 was purchased from the ATCC (American Type Culture ~'nllert;nn, Rockville, MD). The pl~r~ were grown and purified with Promega' 8 Maxi Prep . 1 ml of a 0 .19~ solution of ~ Wogs/2~929 ~ 2 t 90 ~ 2 ~ u~ 7 ELVAX40 (Dupont) in methylene chloride was vortexed with 645.2 ,ul of pRSV ,~-gal (200 ~lg), frozen in liquid nitrogen and lyoph; l; 7ed. The resulting mixture was extruded at 55C into a rod shaped 5 form.
PLA (2K) and PCL (112K) were dissolved in methylene chloride in a 3 :1 ratio and 80 mg of the polymer vortexed with 322 . 6 /11 of pRSV ~-gal (100 llg). The mixture wa8 left in the refrigerator for 10 2 days and lyorh; 1; 7P~I .
Im~:1lantation of the PolYmers.
The EVAc/DNA and PLA/DNA were implanted into rat ham8trings along with their control on opposite sides and 6acrificed at 2 weekE.
Results.
E~istological staining with X-gal reveals positive staining of muscle cells as well as ; nf 1 i tory cells in close proximity to the EVAc polymeric implant at two weeks post-impli~ntati,-n.
In comparison, the PLA/PCL implant reveals positive staining of mostly ; nf 1 tory cells only, in accordance with the earlier data regarding biodegradable polymers.
Thus the selection of a biodegradable or non-degradable polymer implant can be used to target delivery to {nfl tnry cells or tissue cells (for example, muscle). Comparison of PLA/PCL and the EVAc implants illustrates the different transfected cell populations. Specifically, the PLA/PCL
implant results in almost exclusive transfection of ; nf 1 i tnry cells while the EVAc implant results in a large number of transfected muscle cells.
Example 5: Stability of pl~ a during manu~i~cturing p~ e ~F .
Pla8mid DNA is known to be sensitive to high temperatures, physical manipulations and other such factors. Because fabrication techniques require different manipulation of plasmids, methods must be WO9~/24929 ~ ?`~ 9r:J 1 2 1 P~
selected to avoid substantial degradation of plasmids. The following studies were conducted to determine the ef f ect of exposure of plasmids to heating, sonication and solvents such as methylene chloride, as analyzed by agarose gel electrophoresis for changes in topology and/or degradation .
Effect of heatinq I,yorh i 1; 7~orl plasmids were added to dry polymer (PC~) and heated up to 850C ~or 5 minutes and swirled into a 850C corn oil bath as per hot melt microsphere preparation protocols Electrophoresis of extracted plasmids indicate that the majority of the loaded pl A~rmi lc had lost their supercoiled topology and degraded into a linear f orm .
Effect o~ 80nication A mixture of polymer (PCI-) and plasmid solution was sonicated f or 5 seconds .
Electrophoresis of the ~'YtrA~'t~d plasmids show that 2 0 there is some degradation f rom sonicatioll . Thus, sonication of plasmid/polymer solutions ior dispersion in the polymer matrix can result in substantial des~radation of plasmids.
Ef f ect of methYlene chloride eY~osure J~yorh; 1; 7ed plasmids and E~l Arm; iC in solution were eYposed to methylene chloride and vortexed together for 30 seconds. Agarose gel electrophoresis of the extracted ~1 Arm; ~lc show that there are no detectAhl ~ degradation due to methylene t~hlnritl~ exposure.
Results These experiments illustrate that plasmids are sensitive to some of the physical m~n;r~llAtions required by various fabrication techniques and can result in the incorporation of linear, nicked or degraded plasmids In vi tro and ill vivo transfection e~ficiency is highest with supercoiled WO s i/24929 , 2 ~ 9 0 1 2 1 ~ 07 DNA . Thus, manuf acturing methods should be selected or optimized to preserve the supercoiled topology of the DNA within the polymer matrix.
Example 6: In vi tro release assay.
In vitro evaluation of plasmid releasing polymer systems was perf ormed by incubating the devices in buffers such as Tris-EDTA and periodically replacing the supernatant with fresh buffer. The ;n~1lh~tirm buffer was analyzed for plasmids with agarose gel electrophoresis to assess plasmid topology. In addition, the plasmid~3 present in the release buffers were precipitated with the erOmegarM Profection Calcium Phosphate n Trangfection System and transfected into C2C12 murine muscle cell~ to as~ay for bioactivity.
Mate r; ~ l ~ tested 1) Ethylene vinyl acetate (EVAc) ELVAX'1940 2) Polylactic acid tPLA) Mw 2k /
polycaprolactone (PCL) Mc 112k-3 :1 blend 3 ) Polylactic acid (PLA) Mw 2k /
polycaprolactone (PCL) Mw 75k - 2 :1 blend 4) Polycaprolactone (PCL) Mw 75k 5) Polylactic acid (PLA) Mw 2K / polylactic acid (PLA) Mw 300k - 2:1 blend 6) Polylactic acid (PLA) Mw 2k / polylactic acid (PLA) Mw lOOk - 2 :1 blend 7) Fatty acid dimer (FAD) / sebacic acid (SA:FA, 50 :50) The fabrication met~ods, materials, and reRults are 3~ ~ummari~ed in ~able 1.
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The plasmids were evaluated by agarose gel electrophoresis of the plasmids in the release buf f er . The results ehowed degraded and intact supercoiled plasmids in the samples of pRSV/i3gal S loaded PLA/PCL (112k), pRSV/i3gal loaded PI,A/PC~
(112k), and pRSV/~gal loaded P~A/PCI, (112k).
Agarose gel electrophoresis show undegraded plasmids in the release buffer, as reflected by clean bands, in pRSV/$gal loaded EVAc, pRSV/~gal 10 loaded EVAc. and pRSV/i3gal loaded EVAc.
This provides further evidence that the effect of processing conditions on the plasmids can be controlled by selection of the polymer and processing conditions.
The plasmids were also evaluated for biological activity. Sllr~rnAt~nt~ containing the released plasmids were precipitated with calcium phosphate and transfected into C2C12 muscle cells according to Promega'M Profection protocols. Cells 20 were assayed for ,B-galactosidase activity 48 hours post-transfection with X-gal histochemistry. The results obtained with positive control stock pSV/,~-gal (10 ~g) CaP, transfection of C2C12 cells were compared with the results obtained with CaP~
25 transfection with the DNA released over a period of four weeks into the supernatant from EVAc-pSV/,l3gal rods .
The in vitro transfection ~ff;r;~n~y, as measured by the number of ~-gal positive cells, of 3 0 released plasmids were lower than that seen with stock plasmid DNA . One explanation f or this difference could be due to the fact that stock rlAPm; tl~ are 9596 monomeric supercoiled plasmids while the plAf'm;~ relea~ed from the polymers have 35 open circular or mullimeric supercoiled topology.
WO 95/24929 ; F ~
However, as demonstrated above, the release occurs over a longer period of time than can be obtained with stock plasmid DNA.
Example 7: Mi~ t.h~l~ delivery i~ r~t aorta/adventitial layer ~or pla~mid release targeting vascular smooth muscle and endothelium.
The following study was conducted to demonstrate that the microspheres Pn~rsul~ting plasmids could be used to target smooth muscle and endothelium for release of plasmids directly to these cells.
Methods ~nr~ MAterials:
A segment of the aorta/vena cava approximately 2 centimeters in length was isolated. A small hole in the adventitia was cut for insertion of a 27 gauge catheter. A blunt 25 gauge needle was used to separate the adventitia from the medial layer.
A 27 gauge catheter was inserted into the space and the microspheres were injected.
10 mg of 100 ~m sieved PLGA/PCL microspheres suspended in a sodium hyaluronate solution were inj ected into the aorta .
Cryosections were then made of the microspheres in the rat aorta. The results demonstrate the fo~;h;l;ty of the method and that the volume should be minimized to the extent possible, preferably fifty microliters.
Ex~ple 8: Release from pSV/,~Fgal PI.A:PCL(2:1) 3 0 microspheres .
The following study was to demonstrate that release rate is a function of degradation rate of the polymer used for release.
Microspheres were made by solvent removal f rom polylactic acid (PI~) IMw 2k) and polycaprolactone (PCL) (Mw 75k) (2:1) . Samples contain 19~ loading of pSV/F~gal dispersed dry or wet.
The results show that release was l-n~l-ote~ t~hle after one week, probably due to the 810w ~ WO 95/24929 ; , `3 ~ ` s 2 1 9 0 1 2 1 }~l/.J~ `,1 , degradation of the polymer. Faster release can be obtained using a f aster degrading polymer such as poly(FA:SA) fumaric co-sebacic acid. Another explanation could be due to the fact that with one 5 of the polymer blends used, the microspheres formed were double walled. Thus the outer layer could be slowing the release of the DNA contAl nPrl in the inner core.
Example 9: Relea~e of L ~ -/DNA Complexe~
with E:thylene Vinyl Aaetate polymer23 .
Liposome/DNA complexes have been used for both in vitro and in vivo transfection of llAn cells. For in vf tro applications, various formulations such as LIPOFECTIN~M (GIBCO BRL) and TRANSFECTAMTM (Promega) have been used to enhance cellular uptake of plasmid DNA. Although liposome/DNA complexes have high transfection efficiencies when used in tissue culture, direct application of liposome/DNA for in vivo applications is 8, ' -t limited. Limitations on the use of liposome/DNA complexes in vivo stem from their fast degradation, toxicity at high doses and inability to transfect certain cell types. In the case of skeletal muscle, direct injection of plasmid DNA is much more efficient than liposome delivery. Nonetheless, in certain tisaues such as the brain, liposome/DNA results in much higher expression levels compared to direct injection.
This study was done to assess the feasibility of releasing liposome/DNA complexes f rom polymer systems. In light of the fact that lipids are soluble in organic solvents, preserving the liposome/DNA complex in intact f orm within the polymer matrix was the prirnary goal.
`i 0121 wog5/24929 ! ~ '9 r Materials:
200 1ll of pSV/Bgal (1 ,ug/,ul) 2 0 o ,ul Lipof ectinTM
100 111 dEI20 1 ml EVAc/Methylene Chloride (5 solution) ~hQ~:
The plasmid DNA and lipid solutions were vortexed briei~ly and allowed to form complexes for 10 30 min. This solution was vortexed for 10 seconds with the polymer ~nl~t;nn/ frozen in liquid nitrogen and lyophilized overnight. The resulting matrix was either used with or without heat extrusion at 500C.
To assay the bioactivity of the liposome/DNA
nm~l F~ , the extruded rod and unextruded matrix (10 mg each) was incubated with C2C12 myoblasts for 48 hours. The cells were subsequently fixed and stained for ,B~ ctnsi~l~ce activity by X-gal 20 histQrh~m; ~try.
The results show that incubation with the unextruded matrix resulted in positive transfection while incubation with the extruded matrix resulted in no transfection. Sl~hs~ Pnt experiments with 25 the unextruded matrix show that liposome/DNA
complexes are released for up to one week ill vitro.
Modifications and variations of the method and compositions of the present invention will be obvious to thoae skilled in the art from the 30 foregoing detailed description. Such modif;~ t;nn~
and variations are ; nt~n~ to come within the scope of the following claims.
-
p~lr.YM~. TC GENE DELIVERY SYSTE~S
Background of The Invention The present invention i8 generally in the area of drug delivery devices and i5 specifically in the 5 area of polymeric drug delivery devices.
"Gene Therapy" is general1y defined as the introduction and expression of an ~uyt:~uus gene in an animal to supplement or replace a defective or missing gene. Examples that have received a great 10 deal of recent atti~nt;nn include the genes missing in cystic fibrosis and severe, ' in~d n ~1 _ f; r i ~ n ~y . Al though t . A p rogre 8 8 has been made in the area of gene therapy, obtaining high level and long term expression of the desired 15 proteins remains elusive.
In the ma~ority of cases, a retroviral vector is used to introduce the gene to be expressed into appropriate cells . Gene transf er is most commonly achieved through a cell-r- ~ '; A~ c ViVC~ therapy in 20 which cells from the blood or tissue are - genetically modif ied in the laboratory and subsequently returned to the patient. The clinical studies by Steven Rns~nh~rg, et al ., " Immunotherapy of patients with metastatic mAlAn( using tumor-25 infiltrating lymphocytes and IL-2", Preliminary report, New England J. ~ed., 319 (1988) 1676-1680, using in vitro-activated I~K and TIL for tumor destruction illustrates this approach. In other cases, the vector carrying the gene to be expressed 30 is introduced into the patient, for example, by i nhA~ n into the lungs in the case of cystic f ibrosis . Transf ected cells have also been implanted, alone or ~n~AArAA~ ed within a protective membrane that protects the cells from 35 the ;nfl. ory response of the body while at the same time allowing the gene product to diffuse out of the - A~ There have also been reports of WOgS/24929 ~ , ?~q~121 the direct inj ection of an C:~-Jyt~ U8 gene in combination with an d~L.,~, iate promoter, into tissue, with some transient expression being notèd.
Viral vectors have been widely used in gene 5 trans~er, due to the relatively high efficiency of transfection and potential long term effect through the actual integration into the host ' s genome .
However, there are still ~^.^,n~^.^rnC about the risks involved in the use of viruses. Activation of 10 proto-oncogenes and reversion to wild-type viruses from replication incompetent viruses are some important potential hazards of viral delivery of genes .
Since the discovery that naked DNA is taken up 15 by muscle cells and transiently expressed in vivo, and subsec,uent reports, by Wolff, Jon Aal, et, "Direct gene transfer into mouse muscle in vivo, "
Science, 247, 1465-1468, 1990; and Wolff, Jon A, "Human dystrop~in expression in mdx mice after 20 intramuscular injection of DNA constructs, " Nature, 352, 815-818, 1991, there has been increasing interest in using non-viral vehicles for in vivo transf ections .
Plasmid DNA, which can function episomally, 25 has been used with 1 ;ros encapsulation, CaP0~
precipitation and electroporation as an alternative to viral transfections. Recent clinical trials with liposome encapsulated DNA in treating ~ n~
illustrates this approach to gene therapy, as 30 reported by Nabel, J.G., et al., ~Direct gene transfer with DNA-liposome, 1 ~Y^^ in r^~ Ar Expression, biological activity and lack of toxicity in humans", Proc. Nat. Acad. sci. U.S.A., 90 (1993) 11307-11311. A foreign gene coding for 35 HLA-B was introduced into sllhrlltAn^^~llc sites of 1 ;~n~ tumors. Expression of the new gene and the absence of an anti-DNA host response was ~ WO95/24929 ~ 2 1 90 1 2 1 1~
confirmed. Wolff, Jon A, "Persistence of plasmid DNA and expression in rat brain cells in vivo, "
~xperimental Neurology, 115, 400-413, 1992, also reported expression of plasmid DNA. Thus, direct gene transfer offers the potential to introduce DNA
encoding proteins to treat human diseases.
The - -h~n; r~-- for cellular uptake of exogenous DNA and subsequent expression are not clear but gene transf er with naked DNA is associated with several characteristics. Unlike in the case of oligonucleotides, which are typically a maximum of twenty to thirty nucleotides in length, genes encoding most molecules of therapeutic interest are quite large, and therefore considerably more dif f icult to introduce into cells other than through retroviral vector, or in vitro, by chemical manipulation, 80 that the efficiency of transfer is low. In most reported cases to date, only transient high level expression of up to a few weeks or months has been observed. Although low level e~pression and short term expression are two important drawbacks with direct DNA transfer, transfections with naked DNA have several advantages over viral transfers. Most importantly, concerns related to the; t~gpn; city and transforming capability of viruses are avoided. In addition, naked DNA is easy to produce in large quantities, is ;nP~nqive, and can be injected at high c -ncPntration into localized tissue sites 3 0 allowing gene expression in si tu without extensive ex vi vo therapy .
The following additional articles review the current state of gene therapy and the problems associated therewith: Blau, Helen M, "Muscling in on gene therapy," Nature, 364, 673-675, 1993;
Cohen, Jon, "Naked DNA points way to vaccines, ~
Science, 259, 1691-1692, 1993; Dagani, Ron, "Gene .. . . . ... . . ... .. . . . .. . . . . _ _ WOgs/24929 ~-.r~ 2~'~0121 r_l,. / ~
therapy advance, anti-XIV antibodies work inside cells, ~ C&EN, 3-4, 1993; Felgner, Philip L, "Lipofectamine reagent: A new, higher ~ff;~;~n~-y polycationic liposome transfection reagent, "
FocuEI/Gibco, 15, 73-78, 1993; Liu, Margaret A et al., "Heterologous protection against influenza by injection of DNA encoding a viral protein, "
Science, 259, 1745-1749, 1993; Marx, Jean, "A first step toward gene therapy for hemophilia B, "
Science, 262, 29-30, 1993; Mulligan, Richard C, "The basic science of gene therapy, " Science, 260, 926--931, 1993; Nicolau, ~--1 A~l~Al, et, In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I, ~ Proc. Natl . Acad. Sci. rJSA, 80, 1068-1072, 1983; Partridge, Terence A, "Muscle transfection made easy, " Nature, 352, 757-758, 1991; Wilson, James M, "Vehicles for gene therapy, "
Nature, 365, 691-692, 1993; Wivel, et aI., "Germ-2~ line gene modification and disease prevention: Some medical and ethical perspectives, " Science, 262, 533-538, 1993; and Woo, Savio L Cal, et, "In vivo gene therapy of hemophilia B: sustained partial correction in Factor IX-deficient dogs, ~ Science, 262, 117-119, 1993.
Gene therapy is one of the most promising areas of research today. It would therefore be extremely useful if one had an efficient way to introduce genes into cells which yielded long term 30 expression.
It is therefore an object of the present invention to provide a means f or ef f icient transf er of exogenous genes to cells in a patient.
It is a further object of the present 35 invention to provide a meanR for long term expression of exogenoug geneg in p~t jent~, ~ W0 95/24929 ; ;~ 9 0 ~ 2 1 r~ 7 It is a further object of the present invention to provide a means for increasing or decreasing the ;nfl; tnry response to implanted polymeric devices.
It is a still further object of the present invention to provide a method for; ; 7Ation of individuals over a more prolonged period of time than is achieved by a single or multiple ; 7Ation protocol .
It is another object of the present i~vention to provide a method for targeting of gene delivery either to tissue cellg or to ; n f 1 tory type cells .
8umnary of the Invention A means for obtaining efficient introduction of exogenouæ genes into a patient, with long term expression of the gene, is disclosed. The gene, under control of an appropriate promoter f or expression in a particular cell type, is encapsulated or dispersed with a biocompatible, preferably biodegradable polymeric matrix, where the gene is able to diffuse out of the matrix over an extended period of time, for example, a period of three to twelve months or longer. The matrix is preferably in the form of a microparticle such as a microsphere (where the gene is dispersed throughout a solid polymerlc matrix) or microcapsule (gene is stored in the core of a polymeric shell), although other forms including film8, ~At;n~s, gels, 3 0 implants, and stents can also be used . The gene, or other DNA or RNA to be delivered, can be incorporated directly into the polymer, or first inc~,L~"~ted into another material ~nhAn~ ;n~
penetration of the DNA through the cell wall, such as liposomes or surfAc~An~. The size and composition of the polymeric device is selected to wo g~l24929 ~ , J ~ P~~ 07 result in favorable release kinetics in tiEsue.
The size i8 alBo selected according to the method of delivery which is to be used, typically inj ection into a tissue or administration of a 5 suspension by aerosol into the nasal and/or p~ ry areas. The matrix composition can be selected to not only have favorable degradation rates, but to be formed of a material which is bioadhesive, to further increase the effectiveness 10 of transfer when administered to a mucosal surface, or selected not to degrade but to release by diffusion over an extended period.
Examples demonstrate the effectiveness of the system in animals.
Detailed Description of the Invention Gene transfer iE achieved using a polymeric delivery system which releases entrapped genes, usually in ~ ; nAt j on with an appropriate promoter for expression of the gene, into ~uLLuullding 20 tissue. Efficacy of transfer is achieved by: a) releasing the ~ene for prolonged period of time, b) m;n'm;~;ng diffusion of the gene out of the delivery system (due to its size) so that release is predominantly degradation ~r~nA~nt, and c) 25 improving the~transient time of expression and the low inf ection seen by direct gene therapy . In case of non-erodible polymers, the device is fUL~ lt BO that the gene is released via dif fusion . This is achieved by creating porous systems or adding 3 0 soluble bulking agents that create pores as they leach out of the system.
The Poly3neric Matrice~
Selection of Polvmer Both non-biodegradable and biodegradable 35 matrices can be used for delivery of genes, although biodegradable matrices are preferred.
~ Wo9~/24929 ~ 4 2 1 9~ 1 2 1 P~ v.,.07 These may be natural or synthetic polymers, although synthetic polymers are preferred due to the better characterization of degradation and release prof iles . The polymer is selected based on 5 the period over which releage is desired, generally in the range of at least three months to twelve months, although longer periods may be desirable.
In some cases linear release may be most useful, although in others a pulse release or "bulk 10 release" may provided more effective results. The polymer may be in the form of a hydrogel (typically in absorbing up to about 90~ by weight of water), and can optionally be crosslinked with multivalent ion~ or polymers.
High molecular weight genes can be delivered partially by diffusion but mainly by degradation of the polymeric system. In this case, biodegradable polymers, bioerodible hydrogels, and protein delivery systems are particularly preferred.
20 Representative synthetic polymers are: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides, 25 polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyuret_anes and co-polymers thereof, alkyl cellulose, llydL~ycLlkyl celluloses, cellulose ethers, celluloGe esters, nitro n~ llo~e5, polymers of acrylic and methacrylic 30 ester~, methyl cellulose, ethyl CPl~ se, 11YdL~Y~L~Y1 cellulose, hydroxy-propyl methyl ~I~1 1111n~Ie~ 11YdL~J~Y~ULY1 methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate rh~h~ e, 35 carboxylethyl cellulose, c~11ll1n~e triacetate, C~llll1nqe sulphate sodium salt, poly(methyl methacrylate), poly (ethyl methacrylate), wo gs/24929 . ~ } 2 1 9 0 1 2 1 P-"~ i~Q7 ~
poly (butylmethacrylate ), poly ( isobutyl methacrylate), poly (hexyl th~~rylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), 5 poly (methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly (ethylene glyco1), poly(ethylene oxide), poly(ethylene tererhth~l~te), poly(vinyl alcohols), poly(vinyl acetate, poly 10 vinyl chloride, polystyrene and polyvinylpyrrolidone .
Examples of non-biodegradable polymers include ethylene vinyl acetate, poly (meth) acrylic acid, polyamides, copolymers and mixtures thereof.
Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly (valeric acid), and poly (lactide-co-20 caprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, 25 hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these 3 0 materials degraae either by enzymatic hydrolysis or ~O~u~e to water in v~vo, by surface or bulk ero8ion .
R;~:~rlh~R;ve polymers of particular interest include bioerodible hydrogels described by H.S.
35 Sawhney, C.P. Pathak and J.A. Hubell in Macromolecules, 1993, 26, 581-587, the tf~at-h;n~s of which are incorporated herein, polyhyaluronic ~ W095/24929 ~ " ~ 21 9 ~1 21 r~ vv vlQ3~07 acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates ), poly ( ethyl methacrylates ), po ly ( butyl m~ t h A rrylate ), po ly ( i 8 0butyl 5 methacrylate), poly (hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl 10 acrylate).
Selection of Matrix Form and Si~e In the preferred embodiment, the polymeric matrix is a microparticle between nanometers and one millimeter in t~ trr~ more preferably between =~
0.5 and 100 microns for administration via injection or inhalation (aerosol).
The microparticles can be microspheres, where gene is dispersed within a solid polymeric matrix, or microcapsules, where the core is of a different material than the polymeric shell, and the gene is dispersed or suspended in the core, which may be liquid or solid in nature . Unless specif ically defined herein, microparticles, microspheres, and microcapsules are used interchangeably.
Alternatively, the polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard terhn;q~ p~ or even a gel such as a hydrogel. The polymer can also be in the form of a coating or part of a stent or catheter, vascular graf t, or other prosthetic device .
Methods ~or Makinq the Matrix The matrices can be f ormed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art.
W093l24929 ,' ~ . 2 1 9 (~ 1 2 I P~ 3; , ~
Micro~phere Preparation Bioerodible microspheres can be ~L~dL~:d using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release 5,13-22 (1987); Mathiowitz, et al., Reactive Polvrners 6, 275-283 (1987); and Mathiowitz, et al., J. A~l. Polvmer Sci. 35, 755-774 (1988), the t~rh;n~ of which are incorporated herein. The selection of the method depends on the polymer selection, the size, external morphology, and crystallinity that is desired, as described, for example, by Mathiowitz, et al., Srilnn;nr Microsoo~y 4,329-340 (1990); Mathiowitz, et al., J.
A~l. PolYmer Sci. 45, 125-134 (1992); and Benita, et al., J. ph~rm~ Sci. 73, 1721-1724 (1984), the t~Arh;n~R of which are lnc:u.~uurc.ted herein.
In solvent evaporation, described for example, in Mathiowitz, et al., (1990), Benita, and U.S.
Patent No. 4,272,398 to Jaffe, the polymer is dissolved in a volatile organic solvent. The DNA, either i~ soluble form or dispersed as ~ine particles, is added to the polymer solution, and the mixture is sl~p~n~l~d in an aSIueous phase that rrnt~inR a surface active agent such as poly(vinyl alcohol). The resulti~lg emulsion is stirred until most of the organic solvent evaporates, leaYing solid microspheres.
In general, the polymer can be dissolved in methylene rhlrr~G Several different polymer rr,nr~ntrations can be used, for example, between o . 05 and 0 . 20 g/ml . After loading the solution with DNA, the solution is sll~p~n~ l in 200 ml of vigorously stirring distilled water cr,ntA;ninS 19~
(w/v) poly (vinyl alcohol) (Sigma Chemical Co ., St .
Louis, M0). After four hours of stirring, the organic solvent will have evaporated from the W095/24929 ' .""~`f~ 2~90~2 1 P~ 07 polymer, and the resulting microspheres will be washed with water and dried overnight in a lyo~ll; 1 i 71:.r, Microspheres with different sizes (1-1000 5 microns) and morphologies can be obtained by this method which is useful for relatively stable polymers such as polyesters and polystyrene.
However, labile polymers such as polyanhydrides may degrade due to exposure to water. For these 10 polymers, hot melt encapsulation and solvent removal may be pref erred .
In hot melt encapsulation, the polymer i8 f irst melted and then mixed with the solid particles of DNA, preferably sieved to less than 50 15 ,~lm. The mixture is s--~rf~n~ d in a non-miscible solvent such as silicon oil and, with c~nt; nl7~
stirring, heated to 5C above the melting point of the polymer. Once the ~mlll ~ n is st~hl 1; 71~1, it is cooled until the polymer particles solidify.
20 The resulting microspheres are washed by decantation with petroleum ether to give a free-f lowing powder. Microspheres with diameters between one and 1000 microns can be obtained with this method. The external surface of spheres 25 prepared with this techni~ue are usually smooth and dense. This procedure is useful with water labile polymers, but is limited to use with polymers with molecular weights between 1000 and 50000.
Solvent removal was primarily designed for use 30 with polyanhydrides. In this method, the drug is dispersed or dissolved in a solution of a selected polymer in a volatile organic solvent like methylene chloride. The mixture is then gll~p~n~
in oil, such as silicon oil, by stirring, to form 35 an ~ n. Within 24 hours, the solvent diffuses into the oil phase and the emulsion droplets harden into solid polymer microspheres. Unlike solvent Wo95l24929 ~ 901 21 r~
evaporation, this method can be used to make microspheres from polymers with high melting point~
and a wide range of molecular weights.
Microspheres having a ~; -tPr between one and 300 microns can be obtained with this procedure. The psrtPrniql morphology of the spheres is highly dependent on the type of polymer used.
In ~pray drying, the polymer is dissolved in methylene chloride (0 . 04 g/ml) . A known amount of 0 active drug is suspended (if insoluble) or co-dissolved (if soluble) in the polymer solution.
The solution or the dispersion is then spray-dried.
Typical process parameters for a mini-spray drier are as f ollows: polymer concentration = O . 04 g/ml, inlet temperature = 24C, outlet temperature = 13 to 15C, aspirator setting = 15, pump setting = 10 ml/min, spray flow = 600 N~h-l, and nozzle .1; -tPr = 0 . 5 mm. Microspheres ranging in ~; i t~r between one and ten microns can be ~lhtAinP~l with a morphology which depends on the selection of polymer .
Double walled microspheres can be prepared according to U.S. Patent No. 4,861,627 to Mathiowitz .
Hydrogel microspheres made of gel-type polymers such as alginate or polyphosphi~7~nPq or other dicarboxylic polymer3 can be prepared by dissolving the polymer in an aqueous solution, suspending the r~tPr;i~l to be incorporated into the mixture, and extruding the polymer mixture through a microdroplet forming device, equipped with a nitrogen gas iet. The resulting microspheres fal into a slowly stirring, ionic hardening bath, as described, for example, by Salib, et al., Pharmazeutische Industrie 40-llA, 1230 (1978), the tPi~rh; n~f: of which are incorporated herein. The advantage of this system is the ability to further ~ WogSl24929 ~ 2 ~ 9012 r ~
modify the 8urface of the microspheres by coating them with polycationic polymers such as polylysine, after fabrication, for example, a8 described by 3Jim, et al., J. Pharm. Sci. 70, 351-354 (1981).
5 For example, in the case of alginate, a hydrogel can be formed by ionically crosslinking the alginate with calcium ions, then crosslinking the outer surface of the microparticle with a polycation such as polylysine, after fabrication.
10 The microsphere particle size will be controlled using various size extruders, polymer flow rate8 and gas f low rates .
Chitosan microsphere8 can be prepared by dissolving the polymer in acidic solution and 15 crosslinking with tripolyphosphate. For example, ~ LJ~yl ~thylcellulose (CMC) microsphere are prepared by dissolving the polymer in an acid solution and precipitating the microspheres with lead ions. Alginate/polyethylene imide (Pl~I) can 20 be prepared to reduce the amount of carboxyl groups on the alginate microcapsules. Table 1 summarizes various hydrogels, rrnr,~ntrations, ionic baths, and stirring rates used to manufacture them.
T~blu 1: E. "~ ^n o:E Hydrog~l M~trie~
25 llydrog-l Hydrog~l di~olviny bath ionie b~th ~tirrir~g eone~n. ~H TemDC eonC~m. (w/v) rat~(rD~n) chitosan 1. 0% 5 . 0 23 3t tripolJ- 170 phoQph~te alginate 2.09~ 7.4 50 1.3% cale:.um 160 3 0 chlori~e algi~ate/ 2.0~/ 7.4 50 1.3~ calc_um 160 PEI 6 . 0% chloride Carboxy 2.0t 7.4 50 10.0~ lead 100 methyl nitrate 35 cellulose Other device forms Other delivery systems including f ilms, coatings, pellet8, slabs, and devices can be fabricated using solvent or melt casting, and 40 extrusion, as well as standard methods for making composites. The polymer can be produced by first _ .. _ . .. , ., .. ,, . ., ., . , , _ _ _ _ _ _ _ _ _ _ _ _ . _ _ wogs/24929 ; '~ 9 0 1 ~ 0 mixing l u and DNA as described by Sawhney, et al., and polymerizing the monomers with W light.
The polymerization can be carried out in vitro as well as in vivo. Thus, any biocompatible glue 5 could be also used to incorporate the DNA.
Loading o~ Gene The range of loading of the gene to be delivered i9 typically between about 0 . 01% and 9096, r~n~1;n~ on the form and size of the gene to be delivered and the target tissue.
Selection of Gene~ to be Inco ~o qted Any genes that would be useful in r-~rl~r; n~ or supple t; ng a desired function, or achieving a desired ef ~ect- such as the inhibition o~ tumor growth, could be introduced using the matrices described herein. A_ used herein, a "gene~ i9 an isolated nucleic acid molecule of greater than thirty nucleotides, preferably one hundred nucleotides or more, in length.
r ~ A of genes which replace or supplement function include the genes encoding missing enzymes such as ~r~Pnns;n~ m;nAqe ~ADA) which has been uqed in clinical trials to treat ADA def iciency and rnf~tnrs such as insulin and coagulation factor VIII.
Genes which effect regulation can also be administered, alone or in rr-binAt;nn with a gene suppl: ;n~ or rf-pl~,-;ns a specific function.
~or example, a gene encoding a protein which 3 0 suppresses expression of a particular protein-Pnro~l;n~ gene, or vice versa, which induces expresses of a protein-~n-o~;n~ gene, can be administered in the matrix.
Examples of genes which are useful in stimulation of the immune response include viral antigens and tumor antigens, as well as cytokines W095/24929 i ~ 21 9012 ~ r~
(tumor necroeis factor) and inducer9 of cytokines (endotoxin), and various pharmacological agents.
The chronic immune responEe to the polymeric matrix is mediated by the action of a variety of growth factors including epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth _actors (FGFs), tran8forming growth factors (TGF-~ and TGF-$, interleukin-1 (II,-1), and tumor necrosis factor (TNF). Inhibitors of these infl~r~~tory mediators in combination with a gene to be delivered other than the immune inhibitor would be effective in decreasing the normal ;nfl tory response directed toward the polymeric matrix. By inhibiting the amount of encapsulation of the matrix, the effective release would be further ~ n~d. r ~ of materials which could inhibit encapsulation include antisense mRNA to suppress fibrin or collagen fo~r-~inn, inhibitors of EGF, PDGF, FGFs, TGF-~Y, TGF-i~, IL-1 and TNF and anti-;nfl: tory agents such as corticosteroids and cyclosporin.
Genes can be obtained using literature references or from commercial suppliers. They can be synthesized using solid phase synthesis if relatively small, or obtained in expression vectors, for example, as deposited with the American Type Culture Collection, Rockville, MD.
Selection of vector~ to be introduced in - ' in~ with the gene.
A8 used herein, vectors are agents that transport the gene into the cell without - degradation and include a promoter yielding expression of the gene in the cells into which it is delivered. Promoters can be general promoters, yielding expression in a variety of 11 ~n cells, or cell specific, or even nuclear versus cytoplasmic ~re~; f; ~. These are known to those skilled in the art and can be constructed using woss/24929 ` ~ i 2 ~ 901~1 r~".J~
standard molecular biology protocols. Although as demonstrated by the examples, the genes will diffuse out of the polymeric matrix into the surrounding cells where they are expressed, in a 5 preferred ~mh~ t, the q,enes are delivered in combination with a vector to further enhance uptake and expression. Vectors are divided into two classes:
a) Biological agents derived from viral, 10 hact~r; ~1 or other sources .
b) Chemical/ physical methods that increase the potential for gene uptake, directly introduce the gene into=the nucleus or target the gene to a cell receptor ~ =
Bioloqical Vectors Viral vectors have higher transaction (ability to introduce genes) abilities than do most chemical or physical methods to introduce genes into cells.
Retroviral vectors are the vectors most 2C commonly used in clinical trials, since they carry a larger genetic payload than other viral vectors.
However, they are not useful in non-proliferating cells .
Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation. However, many people may have pre-existing ~nt;hn~lies negating effectiveness ahd they are difficult to produce in quantity .
3 0 Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature. However, they cannot be tr~n~m; tt~i from host to host and there are some safety issues since they can enter other cells.
Plasmids are not integrated into the genome and the vast maj ority of them are present only f rom ~ W0 9~/24929 ' ; ` ' ' ` 2 19 0 12 1 r~ . 7 a f ew weeks to several month9, 80 they are typically very safe. Xowever, they have lower expression levels than retroviruses and since cells have the ability to identify and eventually shut 5 down foreign gene expregsion, the t~r~n~;n~
release of DNA f rom the polymer to the target cells substantially increases the duration of functional expression while ~ ;nt~;n;n~ the benefit of the - ---safety as80ciated with non-viral transfeetions.
Chf~m; cal/phvsieal veetors Other methods to direetly introduee geneæ into cells or exploit receptors on the surf ace of eells include the use of liposomes and lipid8, ligands f or specif ic eell surf aee receptors, cell 15 receptors, and caleium phosphate and other chemieal mediators, microinjections directly to single cells, electroporation and homologous reeombination. The ehemieal/physical methods have a number of problems, however, and will typieally not 20 be used with the polymeric matrices described herein. For example, ehemicals mediators are impractical for in vivo use: when ealeium phosphate is used there appears to be very low transduetion rate, when sodium butyrate is used the inserted 25 gene is highly unstable and when glyeerol is used inserted gene is rapidly lost.
As demonstrated by the examples, it ha8 been diseovered that it is possible to incorporate nucleic acid molecules into liposomes or complexed 3 0 to liposomes which are t~en entrapped or otherwise incorporated into the polymeric matrix f or delivery to cells. The ratio of liposome to polymer 801ution is important in ~ t~orm;n;n~ whether the liposomes will remain as separate entities during 35 the process for incorporation into the polymeric matrix. If the ratio of solvent is too high, the rhn~Ph~l;rid Will dissolve into the polymer W09s/24929 ! ~ , 2 1 9~ ~ 21 r~
solvent, rather than rPm~;nlns as part of the liposome bilayer. This is a function of the liposome composition, polymer cnnopntration~ and solvent composition. The liposomes increase the efficiency of the transfer of the DNA into the cells. Liposomes are commercially available from Gibco BRL, for example, as LIPOFECTINf~ and ~IPOFECTACEf~, which are f ormed of cationic lipids such as N- [1- (2,3 dioleyloxy) -pro?yl] -n,n,n-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammonium bromide (DDA~3). Numerous methods are also rllhl; F~hP~l for making liposomes, known to those skilled in the art.
As demonstrated by the following example, any nucleic acid molecule, including genes as def ined above, antisense, ribozymes and ribozyme targeting molecules, and probes, def ined as an oli~-nl-~ 1P~ tide of at least fourteen to seventeen nucleotides in length, can be mixed with, or incorporated into, the liposomes. The liposome-nucleic acid molecule mixture is then mixed with a polymer solution. A8 noted above, the polymer solution can be formed by melting of a polymer, llltinn of a polymer in a solvent, or selection of a polymer t~Lat is a liquid under certain conditions, such as an acidic pH or room temperature, which is then sol; ~; f j ed by a change in conditions.
rltical Co~poE~ition~
When the matrix is in the form of microparticles, the microparticles can be sll~pon~Pd in any appropriate phar--ceutl~l carrier, such as saline, for administration to a patient. In the most preferred f ' orl; t, the microparticles will be stored in dry or lyrrh; 1; ~od form until tol y before administration . They will then ~ w09i5/24929 ' ~ ~ i `;` 2 1 r~ 3~!~
be suspended in sufficient solution for admini stration .
In some cases, it may be desirable to administer the microparticles in combination with an adjuvant to enhance the ;nfl. tory response against the polymer and thereby increase the l; k.-l; hnod of phagocytosis by macrophages and other hematopoietic cells, with subsequent expression of the gene specifically within these cells, or, in the case where the microparticles contain an anti-cancer agent, to enhance the ;nfli tnry reaction against the tumor cells in combination with the effect of the anti-cancer agent.
The polymeric microparticles can be administered by injection, infusion, implantation, orally (not preferred), or administration to a mucosal surface, for example, the nasal-pharyngeal region and/or lungs using an aerosol, or in a cream, ointment, spray, or other topical carrier, for example, to rectal or vaginal areas.
Other ~ ; r-nt- S ~ such as slabs, coatings, rods, and other devices are preferably administered by impl _nt_l-i nr in the area where release is desired .
The materials can also be incorporated into an appropriate vehicle for transdermal delivery as well as stents. Appropriate vehicles include o; ~-, lotions, patches, and other standard delivery means.
Targeti~g of cell populatio~ through polymer material characteri3tic~.
- Studies with plaismid release using PI.A/PC~
biodegradable polymers indicate that the majority of transfected cells, assessed with the $--Ji~lA.-tvsidase reporter gene, are ;nfl: tnry cells involved in the "foreign bodyD response. In general, degrading non-degrading polymers evoke a stronger infli tnry response when compared to .
095/24929 ~ 9 ~ _l/L~ 'tO7 non-biodegrading polymers. A strong foreign body response results in a thick layer of macrophages, f ibroblasts, and lymphocytes around the implant .
Because the polymer release device relies on diffusion for n.~v~ t of its particles, a strong inflammatory response will limit the effective distance of diffusion. Accordingly, biodegrading polymers can be uged to target i nl~l i tory cells due to the inability o~ the plasmid DNA (pDNA) to migrate across the reactive tissue layer to the site specific tissue_ A more biocc _-t;hle material which induces a weaker response from the host will result in a thinner layer of infl i tnry cells, enabling the released pDNA to migrate across the ;nfli tory cells to the indigenous cells to be transfected.
Protection of Pli~smid DNA from [mmediate Degradation Tris-EDTA (TE) is the buffer of choice for DNA
due primarily to it' 5 inhibitory effect on nucleases. It is known that storage of DNA in other buffers or plain water results in degradation of the DNA even at 4 o C (Wolf f, et al ., Biotechniques 11(4), 474-485 (1991) ) . The polymer release system can be loaded to deliver plain DNA
or DNA with buf fer salts to be used as carriers or protecting agents depending on the choice of buffer used .
Incorporation of ~ntiinfll tories and Immune ~nhi/nt ~8; Treatment of Canc~rs In recent years, rnn~;~iprable attention has been f ocused on the use of gene therapy to treat various diseases including cancer. Generally, gene therapy f or cancer therapeutics either targets the cells of the immune system to enhance their ability to kill malignant cells or directly targets the cancer cells to regulate their proliferation or .
Wo gsl24929 l ; 2 1 9 0 1 2 1 l ~ ,,J/t enhance some f~ r function which will result in a stronger activation of the immune response.
Most types of cancer are characterized by frequent relapses during the course of treatment 5 and the cnnt;n~ non-specific and/or specific activation of the immune system resulting from gene therapy is crucial. Second, cell targeting is a major limitation of current vectors and implantation of a controlled release device 10 directly inside a tumor where the DNA is released locally is one alternative to ex vivo therapy or the development of effective ligand specific vectors. As indicated by the prevalence of ex vi~o therapy, targeting hematopoietic cells is 15 especially difficult. The histological results from the implant site in the studies described in the examples below, reveal a substantial ;nfli tory response :juLLuu~ding the intramuscular implant. The well known "foreign body" host 2 0 response can be used to an advantage as this migration of lymphocytes and antigen presenting cells raises the pnsR;h;1;ty of directing the transfection to these specific cell pOp~ ;
Tumors elicit both the humoral and cell-25 mediated immune response, and lymphocytes,particularly cytotoxic T cells and NK cells, as well as macrophages, are known to play a crucial role in tumor elimination. Gene therapy for cancer treatment either targets these cells or the 30 malignant cells themselves. An implant releasing naked DNA for long term functional gene transfer which can target ;nfl: tory cells and/or tumor cells could signif icantly improve cancer therapy.
The approaches used include upregulation of 35 class I MHC expression, transduction of antige~
presenting cells with tumor-specific antigens, W095/24929 - 2 1 qO ~ ~1 r~l,.,.. /
cytokine immunotherapy, transfection of tumor cells with tumor auppressor genes and anti-sense therapy.
The malignant transformation of cells is often characterized by a reduction of class I M~C
5 expression leading to a severe depression of the CT~-r^~ t~-l immune response. An increase in class I ME~C expression on tumor cells could facilitate the activation of the immune system against these altered self-cells. Transfection of genes for 10 cytokines such as tumor necrosis factor (TNF) into tumor cells or tumor suppressor genes such as p53 can be used to limit the ability of tumor ells to multiply . Anti - sense therapy targets cell prolif eration or the production of necessary 15 proteins such as tumor angiogenesis factor (TAF) by complf~m~n~ry RNA hybri~;7~t~nn to block transcription of specific genes.
The immune system can be activated and induced to attack speci~ic cells using cytokines such as 20 Proleukin or monoclonal antibodies. For example, cancer cells proliferate in part due to a decreased immune response against the transformed cells. The matrices described herein provide a means to allow recognition and provocation of a response to cancer 25 cells. For example, genes - coding ~or antigens, such as the aberrant epithelial mucin of breast cancer, and monoclonal ~nt;horl;f-~ directed against tumor antigens have been shown to have potential in st~ ;n~ immune destruction of malignant cells.
3 0 These genes, alone or in combination with monoclonal zlnt;hn~l;es~ can be delivered to the tumor sites in the polymeric matrices to achieve inhibition of the tumor cells.
Cancer cells can also be treated by 35 introducing chemotherapy drug resistant genes into healthy cells to protect them against the toxicity of drug therapy, or by the insertion of appropriate 95/24929 ~ ir ,~ ~; 2 ~ 9 û 1 2 ~ u..,.,lQ~07 vector8 cnntAinin~ cytotoxic genes or blocking genes into a tumor mass to ~1 ;rG;nAte cancer cells.
In a preferred embodiment, the immune system is specif ically stimulated against antigens or proteins on the surf ace of the cancer cells .
These approaches can be used in vi tro and in vivo. Tn vitro, the cells can be removed from a patient, the gene inserted into the cell and the cells reintroduced into the patient. In vivo, the gene can be directly introduced into the body either systematically or in localized sites.
Another approach is to use suicide gene8 that cau8e cell death when they are activated or when their product is ~o~l nt~cl with a pharmaceutical .
The primary limitation of the method is the fact that the gene 8hould be targeted to the cancer cell and not to normal cell. Current approach to ~v~ the problem is direct injection of the vectors into a localized area where normal cells do not proliferate. This would be greatly facilitated using the polymeric devices described herein. The advantages of polymeric devices in this setting include r~nt;nllmlR and protracted release of the incorporated pharmaceutical. This increases the l;kf~lih~od that the int~n~ purposes, ~or example, treatment of cancerous cells, will be achieved.
r The method' and materials of the present invention will be further understood by reference to the following non-limiting ~
3xample 1: Expression of linear and 8~ oiled plasmid DNA ~n~rs~ t~d in polymeric; _ls-nt~ in vuscle tissue of rats.
The study described in this example confirms the feasibility of in vivo transfections using biodegradable polyester blends to release linear or supercoiled plasmid DNA. Although only short term _ _ _ _ _ _, _ _ _ _ .. .. . . . _ _ _ _ . _ .
WogS/24929 ~ 9~ 1 2 1 P~ J~07 ~
expression was studied in this study, polymer devices releasing drugs offer the potential for sustained long term delivery of naked DNA.
Marker aenes are used to study the r v I of 5 engineered cells rf~nt:~in;n~ exogenous genes, as well as the vectors and genes introduced with the vectors, to inbure that the genes remain where they are introduced. Almost all of the initial research into gene therapy is with marker genes. Preferred 10 marker genes are those whose product is innocuous and which can be readily detected by simple laboratory tools. An appropriate marker gene is ~-galactosidase (~-gal), since expression is readily detected by addition of X-gal, a substrate which 15 yields a blue color when the active enzyme is present .
r~nr~ ulation of linear and s~ coiled ~-aal codinq DNA in a PLA blend 1 g PI.A (300K) and 2 g PI~ (2K) was dissolved 20 in lO ml of methylene chloride and 5 drops of Span 85. The mixture was divided into two aliauots of 5 ml and 100 ul of either circular or linear DNA
(between 1 and 2 mg/ml diluted 1:5 in buffer) was introduced into the aliauots. Each mixture was 25 mixed well and ali~uoted into glass vials (1 ml/vial). Between 20 ~g and 40 ILg of ~-gal plasmid DNA was encapsulated in each glass vial. The glass vials were left in the refrigerator for four days to evaporate the methylene chloride and then 30 lyop~; l; 7~
T laI~tation of DNA/PIA ~ellets Each sample was first sterilized with ethanol for 5 min and then washed with PBS-penicillin/streptomycin for 5 min. Surgery was 35 done on Sprague Dawley rats. Linear DNA was implanted into the lef t leg and supercoiled DNA
implanted into the right. T, l;lnt.c were inserted into incised muscle - either in the vastus or the _ _ . _ _ _ _ _ _ _ _ _ ' ' ~ ~ `t ~ t.
~ WO 9St24929 2 1 9 0 1 2 1 hamstring. The muscle was sutured back together and then the skin was sutured closed. Rats were sacrificed for analysis at two weeks.
Result 5 Rats were perfused with Pho5phate Buffered Saline (PBS) with 2500 units of heparin followed by 3% paraformaldehyde and 0 . 296 glutaraldehyde in PBS .
The tissue was post-fixed with 396 paraformaldehyde followed by 1596 sucrose/PBS. 33xcised muscles were cut with a cryostat and stained with X-gal.
Xistology of the implant sites revealed a substantial infl: ~ory response around the film at two weeks and two months. The bulk of the ,(~-gal positive staining was localized to this area with few muscle cells exhibiting positive staining. The cells present around the implant probably consists of phagocytic cells, lymphocytes and fibroblasts.
As expected, transfection was more ef~icient with supercoiled DNA.
2 0 Example 2: In v~ tro transf ection with pRSV ,~-g~l .
NIH3T3 f ibroblasts were plated onto a 6 well tissue culture dish with 1 ml of D-MEM (1096 Fetal calf serum with penicillin/streptomycin). 24 hours after plating, the cells were transfected with pRSV
,B-gal control plasmids as per Promega Profection Transfection system.
Plate 1: 10 t~l pRSV-Z (3.4 t~g) Calcium Phosphate Precipitated Plate 2: 30 tll pRSV-Z (10.2 llg) Calcium Phosphate Precipitated Plate 3: 10 t~l pRSV-Z (3.4 ~g) Naked DNA
Plate 4: 30 /Ll pRSV-Z (10.2 ~g) Naked DNA
Plate 5: DNA/PLA
3 5 Plate 6: Control Plate 5 with the PLA pellet was placed into the well with 4 ml of media to counter the effect of the decrease in pH. After 24 hours, the DNA/PLA
pellet was removed and the media left unchanged.
:
WO9~/24929 ~ q Q ~ 2 ~ p ", ~ Q7 At 48 hours, the cells were fixed and stained with X-Gal (1 ml/plate) overnight.
Result 8 The efficiency of transfection was very low.
5 All plates except the control well had a handful of blue staining cells. There was no observable dif f erences in the number of blue cells among the 5 plates. It was interesting to note that the plate with the DNA/P~A had similar levels of staining as 10 the other plates even after the fact that half the cells had died and ~ ti~rh~rl due to the PLA
degradation .
Example 3: Duratio~ of Expreasion with pSV ,B-gal DNA Encapsulated I~to PLA
Blends.
Tn Vl tro r-elea8e of ~lasmid DNA
pSV ~-gal was amplified i~ H3101 and purified with Qiagen' 8 Mega Prep . 500 ~l of plasmid in Tris-EDTA buffer (67 . 5 ~g) was lyorh; 1 i 7~-1 and 20 resuspended into 100 ~Ll of sterile dEI2O and incorporated into P~A. O . 05 g PLA (2K) and O . 05 g (300K) was dissolved in 1 ml of methylene chloride and 1 drop of Span~K 85. After the polymer was in solution, 100 1ll of plasmid (67 . 5 ~g) was added to 25 the mixture and vortexed for 15 sec. The resulting f ilm was lef t in a ref rigerator overnight and subsequently lyophilized overnight.
This f ilm was incubated with 1. 0 ml of TE
buffer at 37C and sample sUprrn~t~ntq tested at 24 3 0 hours and at 4 days f or the presence of released DNA. DNA was assayed by agarose gel electrophoresis on the ~lrrrn :t~nt~.
The results based on the gel of the gUp~rn~t~nt after 24 hours of inrllh~t;r,n show that 35 a substantial amount of plasmid was released.
Af ter 4 days, the results indicate that there was a first phase of release due to the diffusion of plasmid molecules which are close to the surface of ~ WO 9SI24929 ~ ~ 2 1 9 0 1 2 1 P~
the device followed by a slower release at 4 days due to the low degradation rate of the polymer which was too low to be measured.
In vivo transfection levels 3 mg P~A (2K) and 1 mg PLA (lOOK) were dissolved in methylene chloride (0.25 ml). 1 drop of Span~ 85 and 20 ~1 of plasmid (20 ,llg) was added to the solution and homogenized for 1 minute. This solution was air dried in a glass vial for 3 hours in a sterile hood. The brittle film was ground into fine granules and pressed into a pellet form.
Three of these DNA rnn~;l;nln~ pellets were made as well as three control pellets without DNA. All pellets were lyo~h;l 7ed overnight to extract residual solvents.
Three rats received DNA/P~A in their left hamstring and control/PLA in their right hamstring.
Pellets were inserted into incised hamstrings and the muscles closed with 6- 0 Vicryl . Three rats received an injection of pSV ~-gal plasmids (20 ILg in 100 ~1 of TE buffer) over a minute long period ~:
in their left leg and 100 ~11 of plain TE bufier in their right leg as controls . The site of inj ection was marked with suture.
25 Rat ID Le~t Riqht T 1 ~n~ l~uration R112 DNA/PLA Control/DNA 1 week RllO DNA/PLA Control/DNA ~ weeks Rlll DNA/PLA Control/DNA 10 weeks RllS DNA/bu~fer Control/bu~fer l week 30 ~1~ DNA/bu~fer Control/buf~er 5 weeks Rats were perfused with PBS/heparin, followed by 4~ paraformaldehyde, and post-fixed in 49c paraformaldehyde followed by 1596 and 259~
sucrose/P~3S. Excised muscles were cut with a 35 cryostat and 6tained with X-Gal.
Results Tn vI tro release studies indicate that plasmid DNA can be incorporated into polymers without .. , . . . , , _ _ _ _ _ _ Wo 95/24929 ~ . " ~ P~ 07 degradation through manuf acturing processes and released in functional form for possible uptake by surrounding cells.
In vivo etudies reveal that with a 20 ~g loading of DNA into the polymer, there is substantial transfection of ;nfl; tn~y cells at 1 and 5 weeks aa confirmed by X-gal staining and immunoblotting. At 10 weeks, there was no difference in ~It;~in;n~ intensity between the control PLA and DNA/PLA. This is believed to be due to the resu=lt of the low loading (20 llg) of the polymer such that af ter one week the release rate was below half maximal levels. Investigators using direct injection use doses in the 100 ,Lg range to see their effects. A higher initial loading, which will lead to Cnnt; nl~etl release of higher amounts of DNA from polymers, should prolong transfection durations. Rats injected with 20 ILg of DNA in solution showed no transf ection at 1 and 5 weeks .
Example 4: Compari~on of Pla2~mid DNA Relen~e From ~o~_ ' hle ~d Non~ s A~lins Polymer~ .
Release of plasmids from biodegradable and non-degradable polymer was compared to test the 2~ feasibility of targeting either inflammatory cells or tissue speci_ic cells by selection of polymer material. Plasmid DNA was incorporated into a non-degradable elastomer, ethylene vinyl acetate copolymer (EVAc~ and implanted into the same site in different animals as PI~/PC~ implants. EVAc is a very biocompatible polymer which can be --nllf~c~ ed into a mi.L.,~-J~ .,us structure through which DNA can diffuse into the surrounding tissue.
En~-~nsulation of ~RSV ~-qal into Polvmers.
pRSV ,~-gal in H~3101 was purchased from the ATCC (American Type Culture ~'nllert;nn, Rockville, MD). The pl~r~ were grown and purified with Promega' 8 Maxi Prep . 1 ml of a 0 .19~ solution of ~ Wogs/2~929 ~ 2 t 90 ~ 2 ~ u~ 7 ELVAX40 (Dupont) in methylene chloride was vortexed with 645.2 ,ul of pRSV ,~-gal (200 ~lg), frozen in liquid nitrogen and lyoph; l; 7ed. The resulting mixture was extruded at 55C into a rod shaped 5 form.
PLA (2K) and PCL (112K) were dissolved in methylene chloride in a 3 :1 ratio and 80 mg of the polymer vortexed with 322 . 6 /11 of pRSV ~-gal (100 llg). The mixture wa8 left in the refrigerator for 10 2 days and lyorh; 1; 7P~I .
Im~:1lantation of the PolYmers.
The EVAc/DNA and PLA/DNA were implanted into rat ham8trings along with their control on opposite sides and 6acrificed at 2 weekE.
Results.
E~istological staining with X-gal reveals positive staining of muscle cells as well as ; nf 1 i tory cells in close proximity to the EVAc polymeric implant at two weeks post-impli~ntati,-n.
In comparison, the PLA/PCL implant reveals positive staining of mostly ; nf 1 tory cells only, in accordance with the earlier data regarding biodegradable polymers.
Thus the selection of a biodegradable or non-degradable polymer implant can be used to target delivery to {nfl tnry cells or tissue cells (for example, muscle). Comparison of PLA/PCL and the EVAc implants illustrates the different transfected cell populations. Specifically, the PLA/PCL
implant results in almost exclusive transfection of ; nf 1 i tnry cells while the EVAc implant results in a large number of transfected muscle cells.
Example 5: Stability of pl~ a during manu~i~cturing p~ e ~F .
Pla8mid DNA is known to be sensitive to high temperatures, physical manipulations and other such factors. Because fabrication techniques require different manipulation of plasmids, methods must be WO9~/24929 ~ ?`~ 9r:J 1 2 1 P~
selected to avoid substantial degradation of plasmids. The following studies were conducted to determine the ef f ect of exposure of plasmids to heating, sonication and solvents such as methylene chloride, as analyzed by agarose gel electrophoresis for changes in topology and/or degradation .
Effect of heatinq I,yorh i 1; 7~orl plasmids were added to dry polymer (PC~) and heated up to 850C ~or 5 minutes and swirled into a 850C corn oil bath as per hot melt microsphere preparation protocols Electrophoresis of extracted plasmids indicate that the majority of the loaded pl A~rmi lc had lost their supercoiled topology and degraded into a linear f orm .
Effect o~ 80nication A mixture of polymer (PCI-) and plasmid solution was sonicated f or 5 seconds .
Electrophoresis of the ~'YtrA~'t~d plasmids show that 2 0 there is some degradation f rom sonicatioll . Thus, sonication of plasmid/polymer solutions ior dispersion in the polymer matrix can result in substantial des~radation of plasmids.
Ef f ect of methYlene chloride eY~osure J~yorh; 1; 7ed plasmids and E~l Arm; iC in solution were eYposed to methylene chloride and vortexed together for 30 seconds. Agarose gel electrophoresis of the extracted ~1 Arm; ~lc show that there are no detectAhl ~ degradation due to methylene t~hlnritl~ exposure.
Results These experiments illustrate that plasmids are sensitive to some of the physical m~n;r~llAtions required by various fabrication techniques and can result in the incorporation of linear, nicked or degraded plasmids In vi tro and ill vivo transfection e~ficiency is highest with supercoiled WO s i/24929 , 2 ~ 9 0 1 2 1 ~ 07 DNA . Thus, manuf acturing methods should be selected or optimized to preserve the supercoiled topology of the DNA within the polymer matrix.
Example 6: In vi tro release assay.
In vitro evaluation of plasmid releasing polymer systems was perf ormed by incubating the devices in buffers such as Tris-EDTA and periodically replacing the supernatant with fresh buffer. The ;n~1lh~tirm buffer was analyzed for plasmids with agarose gel electrophoresis to assess plasmid topology. In addition, the plasmid~3 present in the release buffers were precipitated with the erOmegarM Profection Calcium Phosphate n Trangfection System and transfected into C2C12 murine muscle cell~ to as~ay for bioactivity.
Mate r; ~ l ~ tested 1) Ethylene vinyl acetate (EVAc) ELVAX'1940 2) Polylactic acid tPLA) Mw 2k /
polycaprolactone (PCL) Mc 112k-3 :1 blend 3 ) Polylactic acid (PLA) Mw 2k /
polycaprolactone (PCL) Mw 75k - 2 :1 blend 4) Polycaprolactone (PCL) Mw 75k 5) Polylactic acid (PLA) Mw 2K / polylactic acid (PLA) Mw 300k - 2:1 blend 6) Polylactic acid (PLA) Mw 2k / polylactic acid (PLA) Mw lOOk - 2 :1 blend 7) Fatty acid dimer (FAD) / sebacic acid (SA:FA, 50 :50) The fabrication met~ods, materials, and reRults are 3~ ~ummari~ed in ~able 1.
W095124929 ,. !; ~ 2~ 9~121 r~
., ~
_ -- V r~
) ~ ( ~ r ~ 1 t~ 1 1 L~ ' 1 r ._ rt S ; t t U _ ~,~ V ~
V ~ ~ I ; ~ 5 ; r rd ; o ' V S
.. _ tl ~E m a ' E r E C
'' S~
r~ N .~ .Y ~ r:' O
, ~ In In c~
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~r W0 95/24929 . ~ ~,? ~t, ~,~ 2 t 9 0 1 2 1 r~l,~J~ ~; /
The plasmids were evaluated by agarose gel electrophoresis of the plasmids in the release buf f er . The results ehowed degraded and intact supercoiled plasmids in the samples of pRSV/i3gal S loaded PLA/PCL (112k), pRSV/i3gal loaded PI,A/PC~
(112k), and pRSV/~gal loaded P~A/PCI, (112k).
Agarose gel electrophoresis show undegraded plasmids in the release buffer, as reflected by clean bands, in pRSV/$gal loaded EVAc, pRSV/~gal 10 loaded EVAc. and pRSV/i3gal loaded EVAc.
This provides further evidence that the effect of processing conditions on the plasmids can be controlled by selection of the polymer and processing conditions.
The plasmids were also evaluated for biological activity. Sllr~rnAt~nt~ containing the released plasmids were precipitated with calcium phosphate and transfected into C2C12 muscle cells according to Promega'M Profection protocols. Cells 20 were assayed for ,B-galactosidase activity 48 hours post-transfection with X-gal histochemistry. The results obtained with positive control stock pSV/,~-gal (10 ~g) CaP, transfection of C2C12 cells were compared with the results obtained with CaP~
25 transfection with the DNA released over a period of four weeks into the supernatant from EVAc-pSV/,l3gal rods .
The in vitro transfection ~ff;r;~n~y, as measured by the number of ~-gal positive cells, of 3 0 released plasmids were lower than that seen with stock plasmid DNA . One explanation f or this difference could be due to the fact that stock rlAPm; tl~ are 9596 monomeric supercoiled plasmids while the plAf'm;~ relea~ed from the polymers have 35 open circular or mullimeric supercoiled topology.
WO 95/24929 ; F ~
However, as demonstrated above, the release occurs over a longer period of time than can be obtained with stock plasmid DNA.
Example 7: Mi~ t.h~l~ delivery i~ r~t aorta/adventitial layer ~or pla~mid release targeting vascular smooth muscle and endothelium.
The following study was conducted to demonstrate that the microspheres Pn~rsul~ting plasmids could be used to target smooth muscle and endothelium for release of plasmids directly to these cells.
Methods ~nr~ MAterials:
A segment of the aorta/vena cava approximately 2 centimeters in length was isolated. A small hole in the adventitia was cut for insertion of a 27 gauge catheter. A blunt 25 gauge needle was used to separate the adventitia from the medial layer.
A 27 gauge catheter was inserted into the space and the microspheres were injected.
10 mg of 100 ~m sieved PLGA/PCL microspheres suspended in a sodium hyaluronate solution were inj ected into the aorta .
Cryosections were then made of the microspheres in the rat aorta. The results demonstrate the fo~;h;l;ty of the method and that the volume should be minimized to the extent possible, preferably fifty microliters.
Ex~ple 8: Release from pSV/,~Fgal PI.A:PCL(2:1) 3 0 microspheres .
The following study was to demonstrate that release rate is a function of degradation rate of the polymer used for release.
Microspheres were made by solvent removal f rom polylactic acid (PI~) IMw 2k) and polycaprolactone (PCL) (Mw 75k) (2:1) . Samples contain 19~ loading of pSV/F~gal dispersed dry or wet.
The results show that release was l-n~l-ote~ t~hle after one week, probably due to the 810w ~ WO 95/24929 ; , `3 ~ ` s 2 1 9 0 1 2 1 }~l/.J~ `,1 , degradation of the polymer. Faster release can be obtained using a f aster degrading polymer such as poly(FA:SA) fumaric co-sebacic acid. Another explanation could be due to the fact that with one 5 of the polymer blends used, the microspheres formed were double walled. Thus the outer layer could be slowing the release of the DNA contAl nPrl in the inner core.
Example 9: Relea~e of L ~ -/DNA Complexe~
with E:thylene Vinyl Aaetate polymer23 .
Liposome/DNA complexes have been used for both in vitro and in vivo transfection of llAn cells. For in vf tro applications, various formulations such as LIPOFECTIN~M (GIBCO BRL) and TRANSFECTAMTM (Promega) have been used to enhance cellular uptake of plasmid DNA. Although liposome/DNA complexes have high transfection efficiencies when used in tissue culture, direct application of liposome/DNA for in vivo applications is 8, ' -t limited. Limitations on the use of liposome/DNA complexes in vivo stem from their fast degradation, toxicity at high doses and inability to transfect certain cell types. In the case of skeletal muscle, direct injection of plasmid DNA is much more efficient than liposome delivery. Nonetheless, in certain tisaues such as the brain, liposome/DNA results in much higher expression levels compared to direct injection.
This study was done to assess the feasibility of releasing liposome/DNA complexes f rom polymer systems. In light of the fact that lipids are soluble in organic solvents, preserving the liposome/DNA complex in intact f orm within the polymer matrix was the prirnary goal.
`i 0121 wog5/24929 ! ~ '9 r Materials:
200 1ll of pSV/Bgal (1 ,ug/,ul) 2 0 o ,ul Lipof ectinTM
100 111 dEI20 1 ml EVAc/Methylene Chloride (5 solution) ~hQ~:
The plasmid DNA and lipid solutions were vortexed briei~ly and allowed to form complexes for 10 30 min. This solution was vortexed for 10 seconds with the polymer ~nl~t;nn/ frozen in liquid nitrogen and lyophilized overnight. The resulting matrix was either used with or without heat extrusion at 500C.
To assay the bioactivity of the liposome/DNA
nm~l F~ , the extruded rod and unextruded matrix (10 mg each) was incubated with C2C12 myoblasts for 48 hours. The cells were subsequently fixed and stained for ,B~ ctnsi~l~ce activity by X-gal 20 histQrh~m; ~try.
The results show that incubation with the unextruded matrix resulted in positive transfection while incubation with the extruded matrix resulted in no transfection. Sl~hs~ Pnt experiments with 25 the unextruded matrix show that liposome/DNA
complexes are released for up to one week ill vitro.
Modifications and variations of the method and compositions of the present invention will be obvious to thoae skilled in the art from the 30 foregoing detailed description. Such modif;~ t;nn~
and variations are ; nt~n~ to come within the scope of the following claims.
-
Claims (29)
1. A delivery system for a gene into a cell comprising a biocompatible polymeric device and a gene under the control of a promoter which is capable of being expressed in a mammalian cell.
wherein the gene is entrapped in the polymer and released over time into the surrounding cells when the polymeric device is implanted into tissue.
wherein the gene is entrapped in the polymer and released over time into the surrounding cells when the polymeric device is implanted into tissue.
2. The system of claim 1 wherein the polymeric device is selected from the groupconsisting of microparticles, microspheres, microcapsules, stents, coatings, implants, slabs, films, and gels.
3. The system of claim 1 wherein the polymeric device is formed of a biodegradable polymer.
4. The system of claim 3 wherein the polymer is selected from the group consisting of synthetic polymers, polysaccharides, proteins, and combinations thereof.
5. The system of claim 1 further comprising a component selected from the group consisting of compounds inhibiting inflammation due to the polymer device and compounds increasing inflammation due to the polymeric device.
6. The system of claim 1 wherein the gene includes a promoter and a transcription termination signal.
7. The system of claim 1 further comprising compounds selected from the group consisting of antiinflammatories, inhibitors of capsule formation, and inhibitors of cytokines.
8. The system of claim 1 wherein the polymer is bioadhesive.
9. The system of claim 1 comprising between 0.01 and 90% by weight gene.
10. A method for delivery of a gene into a cell in vivo comprising entrapping a gene under the control of a promoter which is capable of being expressed in a mammalian cell in a biocompatible polymeric device which releases the gene over a period of time following implantation of the polymer, and implanting the polymeric device into tissue where the gene is released into the surrounding tissue and expressed by the cells in the tissue.
11. The method of claim 10 wherein the polymeric device is selected from the group consisting of microparticles, microspheres, microcapsules, stents, coatings, implants, slabs, films, and gels.
12. The method of claim 10 wherein the polymeric device is formed of a biodegradable polymer.
13. The method of claim 12 wherein the polymer is selected from the group consisting of synthetic polymers, polysaccharides, proteins, and combinations thereof.
14. The method of claim 10 further comprising a component selected from the group consisting of compounds inhibiting inflammation due to the polymer device and compounds increasing inflammation due to the polymeric device.
15. The method of claim 10 wherein the gene includes a promoter, a transcriptiontermination signal, and the promoter is tissue specific.
16. The method of claim 10 further comprising administering with the device antiinflammatories, inhibitors of capsule formation, and inhibitors of cytokines.
17. The method of claim 10 for targeting release of the gene into inflammatory cells comprising selecting a biodegradable polymer as the polymer forming the device.
18. The method of claim 10 for targeting release of the gene into inflammatory cells comprising selecting a polymer which elicits an inflammatory reaction as the polymer forming the device.
19. The method of claim 10 for targeting release of the gene into tissue specific cells comprising selecting a non-biodegradable polymer as the polymer forming the device.
20. The method of claim 10 for targeting release of the gene into tissue specific cells comprising selecting a biocompatible polymer as the polymer forming the device.
21. The method of claim 10 further comprising selecting a bioadhesive polymer as the polymer forming the implant.
22. The method of claim 10 further comprising loading between 0.01 and 90% by weight gene into the polymer.
23. A method for delivery of nucleic acid molecules comprising incorporating thenucleic acid molecules in combination with liposomes into a matrix formed of a biocompatible polymer which releases the nucleic acid molecules and liposomes over a period of time following implantation of the polymeric matrix.
24. The method of claim 23 wherein the polymeric device is selected from the group consisting of microparticles, microspheres, microcapsules, stents, coatings, implants, slabs, films, and gels.
25. The method of claim 23 wherein the polymeric device is formed of a biodegradable polymer and the liposomes are formed of cationic lipids.
26. The method of claim 23 wherein the nucleic acid molecules are selected from the group consisting of genes under the control of a promoter, antisense, ribozymes and ribozyme targeting molecules, and probes.
27. A composition for delivery of nucleic acid molecules comprising nucleic acidmolecules in combination with liposomes incorporated into a matrix formed of a biocompatible polymer which releases the nucleic acid molecules and liposomes following implantation of the polymeric matrix.
28. The composition of claim 27 wherein the polymeric device is selected from the group consisting of microparticles, microspheres, microcapsules, stents, coatings, implants, slabs, films, and gels.
29. The composition of claim 27 wherein the polymeric device is formed of a biodegradable polymer and the liposomes are formed of cationic lipids.
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US21366894A | 1994-03-15 | 1994-03-15 | |
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CA2190121A1 true CA2190121A1 (en) | 1995-09-21 |
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CA002190121A Abandoned CA2190121A1 (en) | 1994-03-15 | 1995-03-15 | Polymeric gene delivery system |
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US (4) | US6262034B1 (en) |
EP (1) | EP0804249A2 (en) |
AU (1) | AU710504B2 (en) |
CA (1) | CA2190121A1 (en) |
WO (1) | WO1995024929A2 (en) |
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US5763416A (en) * | 1994-02-18 | 1998-06-09 | The Regent Of The University Of Michigan | Gene transfer into bone cells and tissues |
US5783567A (en) * | 1997-01-22 | 1998-07-21 | Pangaea Pharmaceuticals, Inc. | Microparticles for delivery of nucleic acid |
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1995
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- 1995-03-15 EP EP95914073A patent/EP0804249A2/en not_active Withdrawn
- 1995-03-15 AU AU21214/95A patent/AU710504B2/en not_active Ceased
- 1995-03-15 CA CA002190121A patent/CA2190121A1/en not_active Abandoned
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1997
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2001
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2003
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AU710504B2 (en) | 1999-09-23 |
WO1995024929A2 (en) | 1995-09-21 |
EP0804249A2 (en) | 1997-11-05 |
WO1995024929A3 (en) | 1995-12-28 |
US6475779B2 (en) | 2002-11-05 |
AU2121495A (en) | 1995-10-03 |
US20010051608A1 (en) | 2001-12-13 |
US20040126884A1 (en) | 2004-07-01 |
US20010020011A1 (en) | 2001-09-06 |
US6262034B1 (en) | 2001-07-17 |
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