Title:  Microencapsulated DNA for vaccination and gene therapy

United States Patent:  6,667,294

Issued:  December 23, 2003

Inventors:  Jones; David Hugh (Devizes, GB); Farrar; Graham Henry (Salisbury, GB); Clegg; James Christopher Stephen (Salisbury, GB)

Assignee:  Microbiological Research Authority (Salisbury, GB)

Appl. No.:  745515

Filed:  November 12, 1996

Abstract

A microparticle contains DNA coding for a polypeptide and 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.

SUMMARY OF THE INVENTION

In the present invention, it is desired to deliver, in vivo, DNA encoding proteins with immunogenic, enzymatic or other useful biological activity, usually under the control of an active eukaryotic promoter. Objects of the invention include improvement on vaccination therapies known in the art and improvement upon prior art gene therapy methods.

Improvement of or alternatives to existing compositions and methods are desirable as these existing methods are known to contain a number of drawbacks.

WO-A-95/05853 describes administration of naked polynucleotides which code for desired gene products. However, the compositions and methods in this publication are suitable only for injection, requiring sterile procedures, being in itself an unpleasant and awkward route of administration.

WO-A-94/23738 purports to provide a process in which encapsulated DNA is released from the particles in the body of the recipient and then taken up by cells, although no accomplished in vivo examples are presented.

The invention seeks to provide novel compositions and methods of administration thereof that improve upon existing vaccination and gene therapy techniques and are effective in vivo, or at least overcome some of the problems or disadvantages identified in known compositions and methods.

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.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, a first aspect of the invention provides a pharmaceutical composition comprising DNA encapsulated in a polymer, said DNA comprising a sequence coding for a polypeptide and wherein the composition is adapted to induce expression in a recipient of the coding sequence. Preferably, the coding sequence is accompanied by a promoter promoting expression of the sequence. Where the pharmaceutical composition is 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, a tissue--or cell type--specific promoter is used.

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

A composition of the invention suitable for vaccination 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.

A specific embodiment of the invention, described in an example below, contains a DNA sequence coding for a protein. It has been administered in vivo and has been found to induce expression in a mammal of that protein. The expression was detected by measurement of production of antibodies specific for that protein. The composition of the specific embodiment comprises microparticles in the size range up to about 10 .mu.m.

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 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.

An alternative composition, according to the invention, contains a sequence coding for a non-immunogenic product. Such a composition is particularly useful for gene therapy, wherein it is desired to express in a recipient a gene product that is non-expressed, or expressed at a level that it is desired to increase. In this case, a composition of the invention comprises a DNA sequence coding for a desired product, and wherein following administration of the composition expression of the coding sequence results in an increased level of the desired product, giving the gene therapy effect.

It is again a feature of the invention that microparticles for gene therapy applications have sizes in the range 0.01 .mu.m to 30 .mu.m, preferably 1 .mu.m to 10 .mu.m, to target their uptake by phagocytosis.

Specific embodiments of the invention, for use in vaccination, comprise a DNA sequence coding for a protein or an immunogenic component thereof, or an immunogenic fragment or variant thereof, of a virus, bacterium or other pathogenic microorganism. The protein is, for example, an HIV protein, an influenza virus protein, a measles virus protein, a hepatitis virus protein (such as hepatitis surface antigen) or a pertussis protein. 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.

Specific embodiments of the invention, suitable for gene therapy, comprise a DNA sequence coding for an enzyme or another protein needed for the treatment of genetic disease. For example, a DNA sequence coding for glucocerebrosidase is suitable for the treatment of Gaucher's disease.

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 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. 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 on animals, in particular animals. 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 oral use and a person of skill in the art will be able to adapt polymers of the art into the microparticles of the present invention without difficulty. In this connection, the disclosures of EP-A-0451390, WO-A-95/31184 and WO-A-95/31187 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 a transcription termination and polyadenylation downstream of the coding sequence.

It is particularly preferred that the DNA be double stranded, circular and super coiled. It has been observed that during manufacture of particles the DNA is subjected to severe shear forces. Using particular mild 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 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 over as long a time frame as possible. 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.

In use of a specific embodiment of the invention, described in an example below, a plasmid encoding luciferase is under control of the human cytomegalovirus immediate early promoter and is encapsulated within PLG in particles around two .mu.m in size. This encapsulated DNA was administered to mice and elicited anti-luciferase antibodies that were detected over a period of several weeks. The production of antibodies in response to encapsulated DNA according to the invention was compared with antibody production in response to administration of naked DNA intraperitoneally and orally. In both cases, encapsulated DNA elicited equivalent or significantly higher amounts of IgG and IgM, and also evoked a significant IgA response.

A second aspect of the invention provides a method of encapsulating DNA in a polymer such that biological activity of the DNA is retained to a significant extent. 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 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.

It is essential that DNA is incorporated into the microparticles, and DNA incorporation is increased by preparing a 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%. 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.

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 speed 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 speeds are below 8000 rpm, preferably below 6000 rpm, and in a specific embodiment described below the speed is about 3000 rpm.

The method may be performed at ambient temperature, which is convenient for laboratory and industrial purposes, and may also be performed at below ambient temperature improves the stability of the plasmid DNA during the encapsulation procedures. The temperature of the method may be reduced to below 20^oC., below 10^oC. or even below 5^oC. In an embodiment of the invention, the method is carried out at below ambient temperature using a reduced amount of microparticle precursor compared to the amount used at ambient temperature.

The parameters of the method are thus chosen to promote formation of microparticles of 10 .mu.m diameter or less and to promote incorporation of DNA into microparticles, and to avoid damage to the DNA such that the DNA can not be expressed in the recipient.

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.

In a further embodiment of the invention there is provided a method of making a pharmaceutical composition comprising preparing a DNA construct for expression of a coding sequence within the construct, and forming around the construct a polymer particle of size between 0.01 .mu.m and 30 .mu.m, wherein the construct remains capable of inducing expression of the coding sequence. In use, when the construct is separated from the particle, it induces expression of the coding sequence. The particle is preferably formed by emulsifying a solution of a polymer plus DNA plus alcohol.

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 demonstrated by the composition.

In a specific embodiment of the invention, a composition is made by preparing a solution of a plasmid of double stranded, supercoiled DNA comprising a coding sequence and a eukaryotic promoter. Separately, a 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. After centrifugation and resuspension of particles the DNA within retains 25% of its activity.

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

Claim 1 of 28 Claims

What is claimed is:

1. A synthetic composition comprising a polymer microcapsule and DNA, wherein the DNA (a) is inside the microcapsule, (b) comprises a promoter linked to a sequence coding for an immunogen, and (c) exhibits at least 25% of its pre-encapsulation activity, as assayed by transformation of competent bacteria; and wherein the microcapsule is 10 .mu.m or less in diameter.

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