Title:  Method of making microencapsulated DNA for vaccination and gene therapy

United States Patent:  6,270,795

Assignee:  Microbiological Research Authority (GB)

Filed:  May 15, 1998

Foreign Application Priority Data:  Nov 09, 1995[GB] (9523019); Jan 31, 1996[GB] (9601929); Nov 11, 1996[WO] (PCT/GB96/02770); May 15, 1997[GB] (9709900)

A method of making a microparticle that contains DNA coding for a polypeptide is described in which a solvent extraction method is used and solvent extraction takes place at elevated temperature. Oral administration of the microparticle leads to its expression. DNA coding for an immunogen is for stimulating antibody formation in a recipient and DNA coding for a non-immunogenic polypeptide is for gene therapy applications. DNA is incorporated into the microparticle without destruction of its function.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is known that DNA is readily damaged so that it is no longer capable of inducing expression of a gene product. Surprisingly, the inventors have succeeded in devising a technique for encapsulation of DNA within polymer particles, such that the DNA retains sufficient integrity to induce expression of a gene product coded thereby. The inventors have also succeeded in devising a DNA-containing microparticle suitable for mammalian vaccination or for gene therapy.

Accordingly, a first aspect of the invention provides a composition for expression of a DNA coding sequence in a recipient, the composition comprising a polymer microparticle and DNA, wherein the DNA is in aqueous solution, is inside the microparticle and comprises said coding sequence, and wherein the microparticle is 10 .mu.m or less in diameter and induces expression of said coding sequence following oral administration to a recipient.

Preferably, the polymer is soluble in organic solvent and thereby suitable for formation of microparticles by solvent extraction.

A second aspect of the invention provides a method of encapsulating DNA in a polymer microparticle, comprising providing a (water-in-oil)-in-water emulsion, adding this emulsion to excess of a further aqueous phase to extract the oil phase and thereby form microparticles, wherein the further aqueous phase is added at elevated temperature.

The method enables useful biological activity, ie transducing activity, of the DNA to be retained in the microparticle which is suitable for vaccination and/or gene therapy depending upon its coding sequence. Thus, according to the method of the invention, an aqueous solution of DNA is prepared and added to an oil phase with agitation to form a water-in-oil emulsion. The water droplets in this emulsion contribute the aqueous DNA in the final microparticle. This emulsion is added to a second aqueous phase, forming the double (water-in-oil)-in-water emulsion, and extraction of solvent at elevated temperature leads to improved incorporation of DNA into the microparticle.

In an embodiment of the second aspect, a method for encapsulating DNA within a polymer particle, said DNA being capable of inducing expression of a coding sequence within said DNA, comprises preparing a (water-in-oil)-in-water emulsion to form microparticles and separating subsequently produced DNA-containing microparticles by centrifugation. Resultant microparticles preferably have sizes in the range 0.01 .mu.m to 30 .mu.m, more preferably 1 .mu.m to 10 .mu.m.

The method of the invention is carried out under conditions that ensure at least a minimum portion of the DNA is not damaged during manufacture of the particles and thereby retains its ability to induce expression of its gene coding sequence.

A typical method of the invention comprises:

(a) preparing an aqueous solution of DNA, said DNA comprising a sequence coding for a polypeptide in operative combination with at least a promoter sequence and optionally other sequences regulating or otherwise directing transcription of the DNA, said DNA being adapted to express the polypeptide in a mammalian recipient;

(b) preparing a solution of polymer in an organic solvent;

(c) forming an emulsion of the aqueous DNA solution in the organic polymer solution;

(d) preparing an aqueous surfactant solution;

(e) forming a double emulsion of (I) the emulsion from (c) in (II) the aqueous surfactant solution;

(f) at elevated temperature, dispersing or otherwise removing the organic solvent so as to form microparticles of polymer having sizes up to 10 .mu.m in diameter and which contain said DNA; and

(g) recovering the microparticles.

Recovery of microparticles from the solution is optionally by centrifuging the solution to form a pellet of microparticles, separating the pellet from the supernatant solution and resuspending the pellet in a desired solution, typically water. This step is conveniently repeated several times to prepare a purified preparation.

It is essential that step (f) is carried out so as to disperse or otherwise remove the organic solvent and form microparticles of the desired size without melting of the polymer into a congealed mass. According to the invention, this step is carried out at elevated temperature, that is to say (i) a temperature higher than that for steps (a)-(e), or (ii) a temperature above ambient temperature. The organic solvent used for the oil phase is generally more volatile than water, and it has surprisingly been found that carrying out the dispersal step at elevated temperature promotes uptake of DNA into the microparticle. Suitable above-ambient elevated temperatures are at least 25^oC., and preferably at least 30^oC. Beyond about 60^oC. there is the risk of the organic solvent boiling off, which must of course be avoided. However, with most solvents particularly good results are obtainable at 35^oC. and above.

In embodiments of the invention, forming of the double emulsion takes place below ambient temperature, and in these embodiments it is preferred that step (f) be carried out at a temperature at least 5^oC. higher, and more preferably at least 10^oC. higher.

The elevated temperature suitable to disperse the organic solvent may vary according to the choice of organic solvent. Solvent is removed under conditions that set the polymer into microparticles that contain DNA and avoiding melting of the polymer. In specific embodiments of the invention the organic solvent is dichloromethane and this is efficiently dispersed at a temperature of about 35^oC.

It is essential that DNA is incorporated into the microparticles, and DNA incorporation is optionally increased by preparing an aqueous solution of DNA plus an alcohol, adding microparticle polymer and forming microparticles therefrom. The alcohol content of the solution suitably varies between 1% and 60% and preferably between 5% and 40%. The role of the alcohol is to alter the chemistry of the transition from the aqueous to the oil phases, and the advantageous result of using alcohol in this way is to provide for improvements in DNA uptake into the microparticle. In specific embodiments of the invention the alcohol content is around 15-35%, more particularly 20-30% for microparticles made from PLG, producing DNA incorporation of 25% and above, up to 50-60%. Ethanol is particularly suitable; methanol and propanol and other alcohols that do not denature DNA are also suitable, and the alcohol is preferably a straight chain or branched C₂ -C₁₀ alcohol. However, a further feature of using elevated temperature to obtain microparticles from the double emulsion of step (e) is that when an elevated temperature is used for this purpose then it is further an option for the aqueous solution of DNA to contain a lesser amount of alcohol, such as 5% or less, and even to contain no alcohol.

A thus further aspect of the invention resides in a method of encapsulating an aqueous solution of DNA in a polymer microparticle, comprising

providing a (water-in-oil)-in-water emulsion containing the DNA solution; and

adding this emulsion to excess of a further aqueous phase to extract the oil phase and thereby form microparticles,

wherein the aqueous solution of DNA comprises alcohol.

It is also preferred that the emulsification step or steps of the method be carried out under conditions of reduced shear stress, and this is optionally achieved by use of an emulsifying energy, such as speed in the case of an emulsifying mixer, that is sufficient to obtain an emulsion and to form microparticles in the desired size range but not so high that all DNA is damaged by excessive shear. In an embodiment of the invention described below the emulsifying mixer speed is modified so that at least 25% DNA activity (assayed by transformation of competent bacteria or transfection of cultured cells) is retained in the resultant microparticles that contain DNA. Suitable mixer speeds are below 8000 rpm, preferably below 6000 rpm, and in specific embodiments described below the speeds are about 3000 rpm or about 2000 rpm.

A range of surfactants are suitable for use in the method of the invention, and the present invention is not limited to the particular surfactant used in the examples, polyvinylalcohol. Other acceptable surfactants are known in the art. The surfactant has the role of stabilising the double emulsion of aqueous DNA solution in polymer plus organic solvent in surfactant. Choice of aqueous surfactant is a matter for the skilled person and this choice may be made with regard to the choice of polymer and polymer solvent. Likewise, choice of polymer solvent is not limited to that used in the examples, dichloromethane, but encompasses any suitable organic solvent for formation of the double emulsion and subsequent formation of microparticles therefrom.

The steps preliminary to and during formation of microparticles are thus adapted to input sufficient energy so as to form microparticles in the desired size range, which is typically 0.01-10 microns, but not so much energy that DNA is damaged during the process to such an extent that the resultant microparticles are not capable of inducing expression of the polypeptide coded by the DNA. There is a balance required as more vigourous agitation such as through higher mixer speeds typically results in smaller microparticle sizes, and desired sizes are fairly small. But, DNA may be damaged by excessive agitation. On the other hand, reducing the energy input during emulsion formation may have the effect that no emulsion is formed and no microparticles can be obtained. The invention enables a balance of these competing factors, to provide for formation of microparticles retaining an acceptable degree of transducing activity in their encapsulated DNA.

Method steps (a)-(e) may be performed at ambient temperature, which is convenient for laboratory and industrial purposes, and may also be performed at below ambient temperature as this may improve the stability of the plasmid DNA during the encapsulation procedures. The temperature of these steps may optionally be reduced to below 20^oC., below 10^oC. or even below 5^oC. In an embodiment of the invention, steps (a)-(e) of the method is carried out at below ambient temperature using a reduced amount of microparticle precursor compared to the amount used at ambient temperature and then step (f) is carried out at ambient or elevated temperature.

The parameters of the method are thus chosen to promote formation of microparticles of 10 .mu.m diameter or less, to promote incorporation of DNA into microparticles, and to avoid damage to the DNA such that the resultant microparticles contain functional DNA that can be expressed in the recipient following oral administration.

For any particular choice of polymer and DNA variations in the method may be necessary to obtain best results. The efficiency of a method can be assessed by transformation or transfection assays. In the transformation assay used by the inventors, DNA is recovered from microparticles by dissolution with organic solvent, quantitated and used to transform bacteria--ampicillin selection determines successful transformants. In the transfection assay, recovered DNA is used to transfect eukaryotic cells in culture, which culture is then assayed for presence of the antigen or gene therapy product. These assays have demonstrated that DNA recovered from microparticles produced by the method of the invention can retain 50-60% and up to 80% of the activity of the original DNA, indicating high efficiency of incorporation of functional DNA into microparticles.

The method of the invention is adapted to produce pharmaceutical compositions of the first aspect of the invention. The steps of the method are adapted so that, in a resultant composition which contains many DNA containing polymer particles, a useful proportion of particles contain active DNA, i.e. DNA that has not been damaged by the method such that its ability to induce expression of its coding sequence is lost. DNA activity is measured as a percentage of activity prior to the particle forming step.

An acceptable level of DNA biological activity is at least 10% and preferably at least 25%, though for particularly fragile DNA a lower percentage may be acceptable so long as, in use, a therapeutic effect is obtained using the composition.

In a specific embodiment of the invention, a composition is made by preparing an aqueous solution of a plasmid of double stranded, supercoiled DNA comprising a coding sequence and a eukaryotic promoter. Separately, an organic polymer solution is prepared. The two solutions are mixed together and emulsified at a speed between 1000 and 4000 rpm. A solution of a stabilizing agent is then added and the new mixture emulsified at a speed between 1000 and 4000 rpm. Subsequently, the organic solvent is dispersed or otherwise removed so as to set the polymer into microparticles containing the plasmid DNA, and this step is carried out at 30^oC. or higher. After centrifugation and resuspension of particles the DNA within retains 25% or more of its activity.

Accordingly, a second aspect of the invention provides a pharmaceutical composition comprising a plurality of microparticles in a pharmaceutically acceptable carrier, wherein said microparticles are composed of or comprise polymer and contain an aqueous solution of DNA, which DNA comprises a sequence coding for a polypeptide, wherein the composition is adapted to induce expression in a recipient of the polypeptide and wherein the polypeptide is selected from:

(a) the antigens FHA, PT, 68 kd-Pertactin, tetanus toxin, gp48, NS1, Capsid, gp350, NS3, SA, I, NP E, M, gp340, F, H, HN, 35 kd protein, BP1, E1, E2, C, M, E and MSHA; and

(b) immunogenic fragments, variants and derivatives of the polypeptides of (a).

Preferably, the coding sequence is accompanied by a promoter sequence promoting expression of the coding sequence. In embodiments of the pharmaceutical composition for use on mammals, it is convenient to use a eukaryotic promoter and especially a promoter that operates in a wide variety of tissue types. In particular embodiments of the invention, the DNA comprises a tissue--or cell type--specific promoter.

In use, the pharmaceutical composition is orally administered, and the coding sequence is expressed leading to desired therapeutic effects.

A composition of the invention is suitable for vaccination and contains a sequence coding for an immunogen. Following administration of the composition, expressed immunogen elicits production of antibodies within the recipient, thereby contributing to vaccination of the recipient.

Generally, the microparticles of the invention are intended to enter cells of the recipient by phagocytosis, for example phagocytosis by macrophages or other antigen presenting cells. Subsequently, the body of the microparticle breaks down in the intracellular space and the DNA is released. It is preferred that the microparticles of the invention are in the size range 0.01 .mu.m to 30 .mu.m, with 0.1 .mu.m to 10 .mu.m being a more preferred range. These sizes have been found to be suitable for reliably achieving in vivo expression of the DNA. It is also to be noted that agents promoting uptake of the DNA are not needed in microparticles of the invention--as the microparticle size determines its uptake.

Further, where the composition is for oral use, it can conveniently also contain a taste-enhancing agent. The term "taste-enhancing agent" is intended to encompass sweeteners, flavourings and agents that mask any unpleasant taste from other components of the composition. It can conveniently be enterically coated or co-administered with an appropriate antacid formulation.

In a specific embodiment described below, a preparation of microparticles contains a DNA sequence coding for a measles protein. Oral administration of the microparticles elicited an increase in antibodies specific for that protein. Likewise, another microparticle preparation contains a DNA sequence coding for a rotavirus protein. Oral administration of these microparticles preparation elicited anti-rotavirus protein antibodies and a protective effect against challenge by the virus.

The inventors have thus provided DNA encapsulated within a polymer such that the ability of DNA to code for a desired gene product is substantially not affected by the encapsulation process. It is known that DNA can readily be damaged by emulsifying and other steps necessary for production of polymer particles. The inventors have provided for encapsulation of DNA such that sufficient operative DNA is encapsulated for a biological effect to be obtainable upon oral administration of the encapsulated DNA.

The invention offers advantages, in that encapsulated DNA is suitable for oral administration, avoiding the unpleasant and awkward aspects associated with having to inject DNA preparations described in the prior art. Specific embodiments in examples described below have been successful in inducing immunogen-specific antibodies in response to oral administration of a composition of the invention. In addition, the encapsulated DNA formulation is suitable for drying, e.g. freeze drying, into a form that is stable over long periods and is suitable for storage. Further, for many vaccine applications it would be advantageous if, as well as a systemic humoral and cell-mediated immune response, immunity at mucosal surfaces could also be evoked. Specific embodiments of the invention, described below, have been demonstrated to elicit significant increases in specific IgA antibodies, following oral administration. The invention thus provides a pharmaceutical composition comprising DNA within a polymer particle, the DNA encoding a polypeptide, and the composition being adapted to induce mucosal polypeptide specific IgA antibodies in a recipient.

The polymer of the microparticle of the invention preferably is both biodegradable and non-toxic. More preferably, the polymer is suitable for formation of microparticles by a solvent extraction method--for which the polymer should be soluble in an organic solvent so as to form a solution of polymer which will form an water-in-oil emulsion under the conditions described and further solidify around the internal water droplet when the solvent is extracted from the (water-in-oil)-in-water double emulsion.

Suitable polymers include lactide containing polymers and glycolide containing polymers and copolymers of 0-100:100-0 lactide:glycolide. In a specific embodiment of the invention, the polymer comprises poly (DL-lactide-co-glycolide), otherwise referred to as PLG, chosen as it has been approved for human and veterinary use.

The products of the invention are typically for in vivo use vaccination of animals, in particular humans. The polymer of the microparticle should therefore be non toxic in vivo and suitable for pharmaceutical use. The polymer should further be biodegradable--either by consisting of or comprising biodegradable polymer--so that it releases its DNA in the recipient. There exists in the art an extensive literature on polymers suitable for human and animal use. In this connection, the disclosures of EP-A-0451390, WO-A-95/31184 and WO-A-95/31 187 are incorporated herein by reference.

The DNA contained within the particle will typically comprise double stranded DNA. The construction of a suitable DNA sequence for use in the invention will be appreciated by persons of skill in the art. It is preferred that the sequence comprises both a transcriptional promoter and a gene coding sequence. It is further preferred that the DNA sequence provides for transcription termination and polyadenylation downstream of the coding sequence.

It is particularly preferred that the DNA be double stranded, circular and super coiled or coiled to some extent. It has been observed that during manufacture of microparticles the DNA is subjected to severe shear forces. Using particular particle manufacturing conditions, the inventors have managed to retain functional DNA, though have observed that previously supercoiled DNA may become partly converted to the open circular form in the process.

Plasmid DNA or DNA derived therefrom by conventional manipulations is particularly suitable and is used in the specific embodiments of the invention described below. As there is extensive literature relating to plasmid manufacture a person of skill in the art will readily be able to prepared a plasmid suitable for the microparticle of the invention. In general, plasmids incorporating any eukaryotic promoter sequence are suitable.

A further optional feature of the invention is that DNA-containing polymer particles can be manufactured so as to have different half-lives in vivo. When administering an antigen during vaccination, it may be advantageous for the antigen to be delivered in two distinct time frames, such as an initial short term dose followed by a slower, long term dose over a long time frame. A particular embodiment of the invention provides a vaccine comprising first and second vaccine components, the first vaccine component comprising polymer-encapsulated DNA wherein the DNA includes a sequence coding for an immunogen and wherein the polymer has a first half life in vivo, and a second vaccine component comprising polymer-encapsulated DNA, wherein the DNA contains a sequence coding for an immunogen and wherein the polymer has a second half-life in vivo. The respective half-lives could be up to 5 days and more than 5 days. In one example, the immunogen of the first and second vaccine components are the same. Alternatively, the respective vaccine components can contain DNA sequences coding for different immunogens.

In an embodiment of the invention, the half-lives of the respective first and second vaccine components are up to two days, and more than two weeks. In a further embodiment, the first and second half-lives differ by at least an order of magnitude.

A third aspect of the invention provides a pharmaceutical composition comprising polymer-encapsulated DNA and having a reduced water content, such as less than 5% by weight. This composition is suitable for long term storage while retaining the ability of the DNA, upon administration to a recipient, to induce expression of a coding sequence within said DNA.

A method of preparing a pharmaceutical composition for storage, is to dry, such as by freeze drying, a pharmaceutical composition according to the first aspect of the invention. It is preferred that the dried composition has a water content of less than 5%, though the precise water content will be determined by the period of drying used.

A fourth aspect of the invention provides a method of vaccination comprising administering a vaccine according to the first aspect of the invention. Vaccination can thus be obtained by eliciting antibodies to the immunogen expressed from the gene coding sequence. As will be appreciated, the immunogen can be a component of a virus or bacterium or other pathogenic microorganism, or can be an analogue of said immunogen such that antibodies against the analogue are effective against the pathogen itself.

According to a fifth aspect of the invention there is provided use of a microparticle according to the first aspect of the invention in manufacture of a medicament for inducing production of IgA antibodies.

In a specific embodiment of the invention described in an example below, the particle material is PLG. The size of particles produced by the method of the invention are generally in the range of 0.01-30 .mu.m, preferably 1-10 .mu.m. Other suitable polymer formulations for DNA-containing particles according to the present invention include poly-lactic acid, poly-hydroxybutyrate, poly hydroxyvalerate, poly (hydroxybutrate/valerate), ethyl cellulose, dextran, polysaccharides, polyalkylcyanoacrylate, poly-methyl-methacrylate, poly(e-caprolactone) and mixtures of all of these components.

As will be appreciated by a person of skill in the art, a wide range of DNA sequences and constructs are suitable for use in this invention. In particular, the invention can be practised incorporating a wide range of plasmid vectors already well known and characterised in the art. Typically, a plasmid vector used in this invention will include a cDNA that codes for the desired gene product. The selection of additional components for the DNA sequence, such as promoters, reporter genes and transcription termination sequences can be made by a person of skill in the art according to common general knowledge concerning construction of known plasmid vectors.

The preferred administration route for compositions of the invention is the oral route, meaning that compositions of the invention should preferably be designed to avoid significant degradation while passing through the stomach with its high acid levels. It is known that uptake of microparticles of less than 10 .mu.m in size occurs, inter alia, in the M cells of the intestine, and thus inclusion of DNA containing particles in this size range can be advantageous in promoting uptake at this intestinal location. Other modifications to the nature and character and components of the polymer can be made within the concept of the invention.

Claim 1 of 6 Claims

What is claimed is:

1. A method of encapsulating an aqueous solution of DNA in a polymer microparticle, the method comprising:

(a) providing an aqueous solution of DNA;

(b) providing a solution of polymer in an organic solvent;

(c) forming a water-in-oil emulsion containing the aqueous DNA solution in the solution of polymer in organic solvent;

(d) combining the water-in-oil emulsion with a second aqueous solution to form a (water-in-oil)-in-water emulsion; and

(e) adding the (water-in-oil)-in-water emulsion to excess of a further aqueous phase to extract the oil phase and thereby form a plurality of polymer microparticles;

wherein the aqueous solution of DNA has an alcohol content of 1 to 40% and the further aqueous phase is at least 5^oC. higher in temperature than the (water-in-oil)-in-water emulsion.

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