AU2004202497A1 - Treatment for Renal Disease - Google Patents

Description

S&F Ref: 662765

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: The University of Sydney, a body corporate established pursuant to the University of Sydney Act 1989 (NSW), of Parramatta Road, Sydney, New South Wales, 2006, Australia Huiling Wu, Yiping Wang, Guoping Zheng, Stephen I.

Alexander, David C.H. Harris, Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Treatment for Renal Disease The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 1 Treatment for Renal Disease Technical Field The present invention relates to nucleic acid-based vaccines for the treatment or prevention of renal disease. The invention also relates to methods of treating or preventing, renal disease using nucleic acid-based vaccines and to methods of inducing protective immunity against renal disease using such vaccines.

Background of the Invention Renal diseases are a significant health issue across the world. In the United States alone it is estimated that approximately 20 million people suffer from chronic renal disease, most commonly diabetic renal disease and hypertensive renal disease. Many renal diseases share a common pathology of tubulointerstitial nephropathy or glomerulonephropathy and a common symptom of proteinuria. Such diseases account for a high percentage of all renal diseases and frequently develop into End-Stage Renal Disease (ESRD). Due to the lack of effective therapies, patients with ESRD typically require dialysis or kidney transplantation.

There is a clear need not only for effective therapies for ESRD but also for treatments of earlier stage renal diseases to prevent the development of ESRD.

Chemokines are pro-inflammatory glycoproteins that have the ability to attract and activate leukocytes. There is growing evidence that chemokines play a central role in inflammation and various diseases, including renal diseases. Increased expression of chemokines has been found in both human renal diseases and in animal models of acute glomerular or tubulointerstitial diseases (Sergerer, 2003; Anders et al, 2003). The selective role of chemokines in the trafficking of macrophages and T cells to sites of inflammation may be crucial in the evolution of renal injury as chemokine expression correlates with the local infiltration of effector cells and renal damage. In particular, in vivo studies in animals and humans suggest a pivotal role for the C-C chemokines monocycle chemoattractant protein 1 (MCP-1) and RANTES in producing renal inflammation (see, for example, Wang et al, 1997; Rangan et al, 2000). Studies in different immunologic models of renal disease suggest pathogenic roles for MCP-1 and RANTES in the recruitment of leukocytes into the interstitium and the resulting tissue damage in chronic proteinuric renal disease.

Accordingly, methods for the blockage or inhibition of chemokine activity or chemokinedependent pathways may offer therapeutic potential for the treatment of renal diseases. Blocking chemokine activity using neutralizing antibodies has been demonstrated to offer some protection in several models of renal injury. For example, treatment with MCP-1 antibodies reduced proteinuria and monocyte infiltration in rat nephrotoxic serum nephritis (Lloyd et al, 1997). However a major limitation in the treatment of chronic diseases with neutralizing antibodies is their immunogenicity, 2 that is the development of host antibodies to the therapeutic antibodies. Indeed the therapeutic application of anti-chemokine antibodies has proved to be ineffective or of limited effectiveness in renal disease treatment. The redundancy of chemokines and chemokine receptors may be a significant factor limiting the effectiveness of antibody therapy for renal disease.

There is a clear need for improved compositions and methods for inhibiting chemokine expression and treating renal diseases.

The present invention is predicated on the inventors findings that vaccination using naked DNA encoding MCP-1 and/or RANTES ameliorates the progression of renal disease in the ratadriamycin nephropathy model of chronic proteinuric renal disease.

Summary of the Invention According to a first aspect of the present invention there is provided a method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter and a polynucleotide encoding RANTES operably linked to a promoter.

1i The polynucleotide encoding MCP-1 may have the nucleotide sequence set forth in SEQ ID NO:1. The polynucleotide encoding RANTES may the nucleotide sequence set forth in SEQ ID NO:3.

MCP-1 may have the amino acid sequence as set forth in SEQ ID NO:2. RANTES may have the amino acid sequence as set forth in SEQ ID NO:4.

The subject may be human.

The renal disease may be a chronic proteinuric renal disease. The renal disease may be selected from the group consisting of: focal glomerulosclerosis, glomerulonephritis, diabetic renal disease, hypertensive renal disease, renal failure, end-stage renal disease, or a related condition.

The polynucleotide encoding MCP-1 and the polynucleotide encoding RANTES may be located in a single nucleic acid construct. The nucleic acid construct may be a DNA construct. The DNA construct may be a plasmid.

In an embodiment, the administration of the polynucleotides induces an immune response in the subject.

According to a second aspect of the present invention there is provided a method for inducing protective immunity against renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter and a polynucleotide encoding RANTES operably linked to a promoter.

According to a third aspect of the present invention there is provided an immunological composition comprising a polynucleotide encoding MCP-1 and a polynucleotide encoding 3 RANTES, wherein administration of the composition to a subject induces an immune response in the subject.

According to a fourth aspect of the present invention there is provided a composition for the treatment or prevention of renal disease comprising a polynucleotide encoding MCP-1 and a polynucleotide encoding RANTES, the polynucleotides being operably linked to a promoter.

According to a fifth aspect of the present invention there is provided a method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter.

According to a sixth aspect of the present invention there is provided a method for inducing protective immunity against renal disease in a subject, comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter.

According to a seventh aspect of the present invention there is provided an immunological composition comprising a polynucleotide encoding MCP-1, wherein administration of the composition to a subject induces an immune response in the subject.

According to an eighth aspect of the present invention there is provided a composition for the treatment or prevention of renal disease comprising a polynucleotide encoding MCP-1 operably linked to a promoter.

According to the methods and compositions of the fifth to the eighth aspects, the polynucleotide encoding MCP-1 may have the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5. MCP-1 may have the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:6.

Definitions As used herein, the term "protective immunity" refers to the ability of a molecule or composition administered to a subject to elicit an appropriate immune response in the subject and thereby provide protection to the subject from the development or progression of renal disease.

The term "polynucleotide" as used herein refers to a single- or double-stranded polymer of deoxyribonucleotide, ribonucleotide bases or known analogues of natural nucleotides, or mixtures thereof. The term "polypeptide" means a polymer made up of amino acids linked together by peptide bonds.

As used herein, the term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides.

As used herein the term "treatment", refers to any and all uses which remedy a disease state or symptoms, prevent the establishment of disease, or otherwise prevent, hinder, retard, or reverse the progression of disease or other undesirable symptoms in any way whatsoever.

As used herein the term "effective amount" includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic effect. The exact amount required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated, the particular agent being administered and the mode of administration and so forth. Thus, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.

Brief Description of the Drawings Embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings.

Figure 1. MCP-1 and RANTES mRNA expression in AN kidney measured by semiquantitative RT-PCR. Results are expressed as the mean ratio of chemokine gene densitometry score to 18s rRNA densitometry score SD. N 5 per group.

Figure 2. Proteinuria week 1 to week 4 post adriamycin administration following vaccination with a DNA vaccine encoding MCP-1 and RANTES. DV: DNA vaccination against MCP-1 and RANTES in AN DV control: DNA vaccination with empty pTarget vector as a control for vaccination in AN AN: injection with adriamyciniamycin alone PBS: injection with PBS only in normals as a control for adriamycin Mean SD. P<0.01).

Figure 3. Creatinine clearance (ml/min) following vaccination with a DNA vaccine encoding MCP-1 and RANTES. DV, DV control (ctrl) and AN as for Figure 2. Mean SD. <0.05).

Figure 4. Kidney sections at week 4 post adriamycin administration observed under the light microscope following vaccination with a DNA vaccine encoding MCP-1 and RANTES. Sections stained by periodic acid Schiff in DV group DV control AN and PBS (magnification 200x).

Figure 5. Renal interstitial infiltrates at week 4 post adriamycin administration following vaccination with a DNA vaccine encoding MCP-1 and RANTES. A. Macrophages, CD8+ and CD4+ T cells, and CD25+ infiltrates in renal interstitium in the DV, DV control, AN and PBS groups p <0.0001). Results are expressed as mean SD of cells per 400 x field. B. Representative immunoperoxidase-stained kidney sections from the AN and DV groups. (magnification 200x).

Figure 6. Antibody production (MCP-1 antibody and RANTES antibody) measured by ELISA in sera of AN rats following vaccination with a DNA vaccine encoding MCP-1 and RANTES. P <0.005).

Figure 7. Creatinine clearance (ipmol/L) week 1 to week 4 post adriamycin administration following vaccination with a DNA vaccine encoding wild-type MCP-1 or modified MCP-1 (A) empty vector control Mean SD. p <0.05).

Best Mode of Performing the Invention 1o Adriamycin nephropathy (AN) is an animal model of human renal disease. AN is induced in mice and rats by a single intravenous injection of adriamycin, which is directly toxic to the kidney, leading to chronic proteinuric renal disease similar to human focal segmental glomerulosclerosis (Rangan et a/,1999). As disclosed herein, the present inventors have shown that using a novel therapeutic approach, that of vaccination using DNA encoding both MCP-1 and RANTES significantly reduces proteinuria, interstitial infiltrates, specifically T cells and macrophages, and protects against renal injury in AN. Vaccination using DNA encoding a modified MCP-1 protein alone also provided enhanced protection over that achieved using DNA encoding wild-type MCP-1.

Further, vaccination using DNA encoding MCP-1 and RANTES, produces significantly higher levels of specific antibodies to MCP-1 and RANTES than in the absence of vaccination.

Accordingly, one aspect of the present invention relates to a method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter.

Another aspect of the present invention relates to a method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter and a polynucleotide encoding RANTES operably linked to a promoter.

The invention further relates to a method for inducing protective immunity against renal disease using the polynucleotides of these aspects and to compositions for inducing an immune response or for treating or preventing renal disease, the compositions comprising one or more of the polynucleotides of these aspects.

In particular embodiments, the polynucleotides are administered to subjects in a vector. The vector may be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of foreign sequences, their introduction into eukaryotic cells and the expression of the introduced sequences. Typically the vector is a eukaryotic expression vector and may include expression control and processing sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals and transcription termination sequences.

In embodiments in which both the MCP-1 and RANTES-encoding polynucleotides are administered, the polynucleotides may be located on separate nucleic acid constructs or on the S same construct. In embodiments in which the polynucleotides are located on the same construct, they may be operably linked to the same of different promoters.

A nucleic acid construct in accordance with an embodiment of the present invention may comprise a vaccine, in particular a DNA vaccine. Accordingly, the present invention also relates to methods of DNA vaccination of subjects for the treatment or prevention of renal disease or to induce protective immunity against renal disease. The DNA vaccine may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers.

MCP-1 and RANTES sequences Those skilled in the art will appreciate that the precise sequences of the MCP-1-encoding and RANTES-encoding polynucleotides used according to the methods and compositions of the present invention may vary depending on a number of factors, for example the species of animal to be treated such that the sequences of the MCP-1 and RANTES polynucleotides are selected so as to be derived from the species to be treated. For example, in the treatment of human renal diseases polynucleotides encoding the human MCP-1 and RANTES chemokines may be used.

In a particular embodiment, the nucleotide sequence of the polynucleotide encoding MCP-1 is as set forth in SEQ ID NO:1 or a fragment or variant thereof, or displays sufficient sequence identity thereto to hybridise to the sequence of SEQ ID NO:1. In alternative embodiments, the nucleotide sequence of the polynucleotide may share at least 30%, 40%, 50%, 60%, 70%, 90%, 96%, 97%, 98% or 99% identity with the sequence set forth in SEQ ID NO:1.

In a particular embodiment, the nucleotide sequence of the polynucleotide encoding RANTES is as set forth in SEQ ID NO:3 or a fragment or variant thereof, or displays or displays sufficient sequence identity thereto to hybridise to the sequence of SEQ ID NO:3. In alternative embodiments, the nucleotide sequence of the polynucleotide may share at least 30%, 40%, 70%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identity with the sequence set forth in SEQ ID NO:3.

The polynucleotide encoding MCP-1 may encode a polypeptide having the amino acid sequence as set forth in SEQ ID NO:2. The polynucleotide encoding RANTES may encode a polypeptide having the amino acid sequence as set forth in SEQ ID NO:4. Within the scope of the term "polypeptide" as used herein are fragments and variants thereof.

The term "fragment" refers to a nucleic acid or polypeptide sequence that encodes a constituent or is a constituent of the full-length MCP-1 or RANTES chemokines. In terms of the polypeptide the fragment may possesses qualitative biological activity in common with the fulllength chemokine, or may be an immunogenic portion thereof, capable of inducing protective immunity against renal disease in a subject.

The term "variant" as used herein refers to substantially similar sequences. Generally, nucleic acid sequence variants encode polypeptides which possess qualitative biological activity in common. Generally, polypeptide sequence variants also possess qualitative biological activity in common. Further, these polypeptide sequence variants may share at least 25%, 30%, 35%, 1o 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

Those skilled in the art will also readily appreciate that various modifications may be made to the sequences of the polynucleotides encoding MCP-1 and RANTES such that modified variants of the MCP-1 and RANTES polypeptides are generated in which one or more portions of the encoded polypeptide is replaced by one or more portions of another polypeptide. The portion(s) replaced may correspond to a particular structural or functional domain of the MCP-1 or RANTES polypeptide. Such modifications are also included within the scope of the term "variant". For example, modifications may be made so as to enhance the immuno-protective properties of the MCP-1 or RANTES polypeptide or to otherwise increase the effectiveness of the polypeptide to treat or prevent renal disease.

In one such example, the polynucleotide encoding MCP-1 may be modified by PCR or other suitable technique to replace a surface loop region of MCP-1 with a corresponding region of an alternative protein, such as the P30 tetanus toxoid helper epitope. In one particular embodiment the polynucleotide sequence encoding the modified MCP-1 may have the nucleotide sequence as set forth in SEQ ID NO:5 or a variant thereof. The modified MCP-1 polypeptide may have the amino acid sequence set forth in SEQ ID NO:6 or a variant thereof.

It will be appreciated by those skilled in the art that the RANTES polynucleotide and polypeptide may be similarly modified.

Further, a variant polypeptide may include analogues, wherein the term "analogue" means a polypeptide which is a derivative of MCP-1 or RANTES, which derivative comprises addition, deletion, substitution of one or more amino acids, such that the polypeptide retains substantially the same function as native MCP-1 or RANTES. The term "conservative amino acid substitution" refers to a substitution or replacement of one amino acid for another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). For example, the substitution of the 8 charged amino acid glutamic acid (Glu) for the similarly charged amino acid aspartic acid (Asp) would be a conservative amino acid substitution.

Vaccines Compositions comprising polynucleotides encoding MCP-1 and/or RANTES may be administered in the form of a nucleic-acid based vaccine, in particular a DNA vaccine. The DNA vaccine may comprise naked DNA comprising one or more of the polynucleotides as defined herein.

A major limitation in treatment of chronic diseases with neutralizing antibodies is their immunogenicity (that is, the development of host anti-antibodies against therapeutic antibodies).

DNA vaccination, inducing immunization with plasmid DNA encoding antigen, represents a novel means of expressing antigen in vivo for the generation of both cellular and humoral immune responses against products of a given construct. Naked DNA vaccines promote a highly efficient protective immunity not only against foreign antigens, such as microbes and tumours but also self antigens, such as TCR V genes. A distinct advantage of DNA vaccines is the induction of cellular ~s or humoral immune response to autologous antigen. In addition, when co-delivered with plasmid- DNA encoding other molecules, there is the possibility of enhancement or modulation of the subsequent response to the DNA encoded antigen.

A typical vaccination regime is to deliver the vaccine in multiple doses, generally one, two or more equal doses.

Vaccination using nucleic acid-based vaccines according to the invention may provide protective immunity against renal disease to the subject being vaccinated. That is, the polypeptide(s) encoded by the nucleic acid vaccine may elicit a protective immune response in the subject, for example by inducing the production of autoantibodies against the encoded polypeptide(s). In this regard, the person skilled in the art will readily appreciate that not only fulllength MCP-1 and RANTES polynucleotide sequences may be used in a vaccine, but also fragments or variants thereof, wherein the fragments or variants are capable of encoding immunogenic polypeptides to elicit an immune response and thereby provide protective immunity against renal disease.

The efficiency of nucleic acid uptake into cells can be increased by pre-treatment of cells with one or more enhancing agents capable of enhancing the cellular uptake of nucleic acids. Many suitable agents are known to those skilled in the art. One such agent is bupivacaine, commonly used to enhance the efficiency of transduction of naked DNA. The person skilled in the art will readily appreciate which nucleic acid uptake-enhancing agents may be suitable and the appropriate concentrations of these agents.

Compositions and routes of administration Polynucleotides encoding MCP-1 and/or RANTES may be administered in the form of a composition, together with one or more pharmaceutically acceptable carriers. Compositions may be administered either therapeutically or preventively. In a therapeutic application, compositions are administered to a patient already suffering from a renal disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. The composition should provide a quantity of the compound or agent sufficient to effectively treat the patient. Typically in therapeutic applications the treatment would be for the duration of the disease state.

The therapeutically effective dose level for any particular patient will depend upon a variety of factors including: the disorder being treated and the severity of the disorder; activity of the compound or agent employed; the composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of sequestration of the agent or compound; the duration of the treatment; drugs used in combination or coincidental with the treatment, together with other related factors well known in medicine.

One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of agent or compound which would be required to treat applicable diseases.

Generally, an effective dosage is expected to be in the range of about 0.0001mg to about 1000mg per kg body weight per 24 hours; typically, about 0.001mg to about 750mg per kg body weight per 24 hours; about 0.01mg to about 500mg per kg body weight per 24 hours; about 0.1mg to about 500mg per kg body weight per 24 hours; about 0.1mg to about 250mg per kg body weight per 24 hours; about 1.0mg to about 250mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.0mg to about 200mg per kg body weight per 24 hours; about 1.Omg to about 100mg per kg body weight per 24 hours; about 1.0mg to about 50mg per kg body weight per 24 hours; about 1.0mg to about 25mg per kg body weight per 24 hours; about 5.0mg to about 50mg per kg body weight per 24 hours; about 5.0mg to about per kg body weight per 24 hours; about 5.0mg to about 15mg per kg body weight per 24 hours.

Alternatively, an effective dosage may be up to about 500mg/m 2 Generally, an effective dosage is expected to be in the range of about 25 to about 500mg/m 2 preferably about 25 to about 350mg/m 2 more preferably about 25 to about 300mg/m 2 still more preferably about 25 to about 250mg/m 2 even more preferably about 50 to about 250mg/m 2 and still even more preferably about 75 to about 150mg/m 2 Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the disease state being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques.

It will also be apparent to one of ordinary skill in the art that the optimal course of treatment, such as, the number of doses of the composition given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.

In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and accordingly may include a pharmaceutically acceptable carrier, diluent and/or adjuvant. These compositions can be administered by standard routes. In general, the compositions may be administered by the parenteral intravenous, intraspinal, subcutaneous or intramuscular) or oral route. More preferably administration is by the parenteral route, in particular intramuscularly.

The carriers, diluents and adjuvants must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.

Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil, sesame oil, arachis oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysolpoxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrolidone; agar; gum tragacanth or gum acacia, and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.

For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol.

11 Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration.

Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.

Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol.

Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, arachis oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.

Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, poly-vinyl-pyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.

12 The emulsions for oral administration may further comprise one or more emulsifying agents.

Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.

Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein.

The vaccines and other compositions of the present invention may be administered in combination with other therapies for the treatment or prevention of renal disease. For example, a vaccine or composition of the invention may be administered in combination with other agents known to assist to in the reduction or prevention of proteinuria or interstitial infiltrates. For such combination therapies, each component of the combination therapy may be administered at the same time, or sequentially in any order, or at different times, so as to provide the desired effect. Alternatively, the components may be formulated together in a single dosage unit as a combination product. When administered separately, it may be preferred for the components to be administered by the same route of administration, although it is not necessary for this to be so. Similarly a vaccine or composition of the invention may be administered in combination with other renal disease treatment regimes such as dialysis.

Renal diseases The methods and compositions of the present invention are applicable to the treatment and prevention of various renal diseases and renal injury, both primary and secondary. Secondary renal diseases include those diseases resulting from a pre-existing condition such as diabetes or hypertension. The methods and compositions of the present invention find application particularly in chronic inflammatory and autoimmune diseases characterised by pathological changes of tubulointerstitial nephropathy or glomerulonephropathy and by proteinuria. Diseases include, but are not limited to, focal segmental glomerulosclerosis, glomerulonephritis, diabetic renal disease, hypertensive renal disease, renal failure, End Stage Renal Disease, and related conditions.

The present invention will now be described with reference to specific examples, which should not be construed as in any way limiting the scope of the invention.

Examples General methods Induction of adriamycin nephropathy (AN) Inbred male Wistar rats were obtained from the Animal Care Facility, Westmead Hospital, Sydney, Australia. Rats weighing 130-180g at the age of 5- 6 weeks were used in all experiments.

13 AN was induced as described previously (Rangan et al, 2001). Rats received a single intracardiac injection of adriamycin (6.9 mg/kg; David Bull Laboratories, Victoria, Australia). Rats were kept in individual cages and allowed free access to water and regular rat chow ad libitum. Rats were divided into five groups: group A, DV: DNA vaccination against MCP-1 and RANTES in AN (n group B, DV control: DNA vaccination with empty pTarget vector as a control for vaccination in AN (n group C, AN: injection with adriamycin alone (n group D, PBS: injection with PBS only in normals as a control for adriamycin (n group E, DV alone: DNA vaccination against MCP-1 and RANTES in normal rats (n 3).

Blood from each animal was collected weekly. At 4 weeks after adriamycin administration, rats were sacrificed and each kidney was divided into three portions: one portion was placed in formalin, one in embedding OCT compound to be snap frozen for histological examination and another portion was snap frozen and used for RT-PCR analysis. The blood and spleen from each animal were also harvested.

The protocol was approved by the conjoint Animal Ethics Committee of the Children's Medical Research Institute and the Children's Hospital at Westmead, Sydney, Australia.

DNA vaccination MCP-1 and RANTES vaccine RNA was isolated from kidneys as previously described (Walters et al, 2001) and reverse transcribed into cDNA. Oligonucleotide primers used for the MCP-1 and RANTES genes were those described as follows (Youssef et al, 1998): MCP-1: sense 5'-ATGCAGGTCTCTTGTCACGCTTCTGGGC-3 antisense 5'-CTAGTTCTCTGTCATACTGGTCAC-3' and

RANTES:

sense 5'-ATGAAGATCTCTGCAGCTGCATCC-3 antisense 5'-CTAGCTCATCTCCAAATAGTTG-3.

All forward primers were designed to include an in-frame ATG. The PCR cycle profile consisted of denaturation at 95 0 C for 1min, annealing at 55 0 C for 1 min and extension at 72 0 C for 1 min for 35 cycles in a DNA thermal cycler (Perkin Elmer). Amplified PCR products were cloned into the pTarget plasmid Vector (Promega, Madison. WI) according to the manufacturer's instructions. Plasmid DNA from colonies with an insert was sequenced to confirm the insertion of the correct gene with the ATG in-frame. Large-scale preparation of plasmid DNA was performed using the Mega prep kit (QIAGEN, Hilden Germany). For DNA vaccination, animals were pretreated with 0.75% bupivacaine (Ip.l/g bodyweight; Sigma) by injection into the tibialis anterior muscle 1wk before the first vaccination. This is known to enhance the efficiency of DNA 14 vaccination. 200(pg of DNA was injected into the same site four times at weekly intervals. One week after the fourth DNA vaccination, rats were treated with adriamycin. Rats received a booster injection of DNA vaccine one week after adriamycin administration.

(ii) modified MCP-1 The rat MCP-1 gene was modified by replacing a surface loop region +109 to +138 (37 to 46 aa) by tetanus toxoid helper epitope sequence TTCACCAACTTCACCGTCAGCTTCTGGCTG CGCGTGCCCAAGGTCAGCGCCAGCCACCTGGAG using primer extension PCR.

The primers used were as follows: CCACTATGCAGGTCTCTGTC MCP-1 5'primer designed for amplification of the first overlap fragment of modified MCP-1; ATCACATTCCAAATCACACTAG MCP-1 3' primer (reverse) designed for amplification of the second overlap fragment of modified MCP-1; GACCTTGGGCACGCGCAGCCAGAAGCTGACGGTGAAGTTGGTGAAgtagcagcaggtgagtgg overlap primer 1 (reverse) from overlap region of P30 to adjacent MCP-1 sequence (lower case) 1i (sequence on antisense chain); and TGGCTGCGCGTGCCCAAGGTCAGCGCCAGCCACCTGGAGatgagtcggctggagaacta overlap primer 2 (forward) from overlap region of P30 to adjacent MCP-1 sequence (lower case).

MCP-1 5' primer and overlap primer 1 were used to amplify a first fragment of modified MCP- 1 gene, while overlap primer 2 and MCP-1 3' primer were used to amplify a second fragment of modified MCP-1. The first fragment was annealed to the second fragment at the overlap region.

This was then further extended and amplified with 5' and 3' MCP-1 primers. The modified MCP-1 PCR product was cloned into pTarget vector to make the modified MCP-1 DNA vaccine. The modified MCP-1 vaccine sequence was confirmed by DNA sequencing.

Assessment of renal function Sixteen hour urine samples were collected in metabolic cages weekly after adriamycin administration. Urine protein concentrations were determined by colorimetric assay (Biorad, Oakland, CA, USA). Blood and urine creatinine level 5 were analyzed as previously described (Rangan et al, 1999). Creatinine clearance was calculated as creatinine excretion divided by serum creatinine concentration.

Histology and morphometric analysis Kidneys were immersion-fixed in 10% neutral-buffered formalin and embedded in paraffin.

Sections 4 tm thick were stained with periodic acid-Schiff (PAS). A computer assisted image analysis system was used to quantify glomerulosclerosis and tubular atrophy (tubular dilation and tubule cell height) in the renal cortex, as described previously (Rangan et al, 1999). Interstitial volume and interstitial infiltrates were determined by 2 blinded observers who assessed 20 high powered sections/animal. Interstitial volume was graded according to the extent of cortical involvement on a scale of 0 to 4.

Immunohistochemistry Frozen sections were cut at 5 tm, fixed with cold acetone at 10 min and blocked with 0.3% H202 for 10 min to eliminate endogenous peroxidase and 10% goat serum for 10 min to minimize nonspecific antibody binding. Sections then were incubated with monoclonal antibodies for minutes, followed by biotinylated goat anti-mouse immunoglobulin (Ig) polyclonal antibody (BD PharMingen, San Diego, CA USA) and avidin-biotin-horseradish peroxidase complex for minutes each. The slides were incubated with 3,3- diaminobenzidine tetrahydrochloride to produce o1 a dark brown-colored end product. The monoclonal antibodies used were W3/25 for CD4 OX-8 for CD8*, NDS-61 for CD25 cells and CD68 for macrophages (Serotec, Oxford, UK). Staining was quantitated by a blinded observer counting 20 consecutive high-power fields per animal and expressed as cells per 400 x field.

Evaluation of anti-chemokine antibody in sera of DNA-vaccinated rats Anti-MCP-1 or anti-RANTES antibody titres (total Ig) were determined by a direct ELISA assay as described (Youssef et al, 1998; Wu et al, 2001). Briefly, 96 wells of an Immulon 1 ELISA microtiter plate (Dynatch Laboratories, Alexandria, VA) were coated with each recombinant rat chemokine MCP-1 and RANTES (Chemicon) at a concentration of 25 ng/well in 100pl of coating buffer and reacted sequentially with test rat sera, alkaline phosphatase conjugated sheep anti-rat Ig Fab fragments (Boehringer Mannheim, GmbH, Mannheim, Germany) and substrate solution p-nitrophenyl phosphate (Sigma) in carbonate buffer, pH 9.6. Absorbance was read at 405nm on an ELISA reader (Dynatech Laboratories). Triplicate sample ODs were read at 405nm, corrected for a control sample of known strongly positive serum OD sample OD /control positive serum OD x 100). The anti-MCP-1 or anti-RANTES antibody titer of the control serum was 1: 200.

Statistical analysis The results are expressed as mean ±SD. Statistical significance of difference between and among the groups was made by one way analysis of variance (ANOVA). A value of P 0.05 was considered significant.

Example 1 High levels of MCP-1 and RANTES mRNA expression in AN kidney Adriamycin nephropathy an animal model of human renal disease displays pathologic features including severe nephrotic syndrome, focal glomerular sclerosis and tubular injury with massive mononuclear cell infiltrates composed largely of macrophages and T cells. Previous studies have demonstrated that in AN overt proteinuria appears shortly after adriamycin administration, glomerular vacuolation and focal glomerular sclerosis appear by week 4 and 16 extensive focal and global glomerular sclerosis by week 6. Tubulointerstitial infiltrates are dominated by macrophages at week 2, and later by accumulation of both CD+4 and CD+8 T cells (Rangan et al, 1999, 2001, Wang et al 2000).

As reported previously (Rangan et al, 2000), the AN rat model was characterised by massive proteinuria immediately after adriamycin injection and progressed to renal failure. In the adriamycin rat at 4 6 weeks, severe nephrotic syndrome, glomerular sclerosis, tubular atrophy and interstitial fibrosis with massive interstitial mononuclear cell infiltration were observed. Expression of MCP-1 and RANTES mRNA in AN kidney was significantly increased compared with the controls (P<0.001) (Figure 1).

Example 2 DNA vaccination using MCP-1 and RANTES is protective in AN Vaccination using naked DNA encoding MCP-1 and RANTES significantly reduced proteinuria from week 1 to week 4 post adriamycin administration (Figure 2) compared with AN and DV controls. There was no significant difference in proteinuria between AN and DV control groups.

Control rats injected with only PBS did not develop proteinuria. Chemokine DNA vaccination had no effect on proteinuria in the normal rats, as there was no significant difference in proteinuria between PBS and DV alone groups at each time point.

Chemokine DNA vaccination significantly ameliorated renal dysfunction in rat AN. Serum creatinine in the DV group was significantly lower than in the DV control group (47.6 15.1 vs 70.0 14.8 umol/L, P 0.05 at week 2; 49.2 9.7 vs 70.0 10.5 umol/L, P <0.05 at week 3) and the AN group (81.0 6.2 and 83.3 8.6 umol/L, p 0.05 at week 2 and 3 respectively). Creatinine clearance in DV group was significantly higher than in the DV control and AN groups, p 0.05 (Figure 3) from week 2 to week 4 post adriamycin administration. There was no significant difference in creatinine clearance between AN and DV control groups. Normal rats injected with only PBS did not develop renal failure. The DNA vaccine encoding MCP-1 and RANTES had no effect on serum creatinine and creatinine clearance in the normal rats.

Chemokine DNA vaccination significantly alleviated the histologic manifestations of AN at week 4 (Table Light microscopic examination revealed that glomeruli and tubules were only mildly damaged at week 4 in the DV group, as compared with the DV control and the AN groups (Figure Morphometric analysis showed much less glomerulosclerosis in the DV group than in the DV control and AN groups (p 0.05). Most tubules were intact in DV group rats and tubular atrophy was significantly alleviated by chemokine DNA vaccination, in comparison with rats in the AN group and DV control groups. Furthermore, vaccination with chemokine reduced interstitial infiltrates in DV group in comparison to AN group and DV control group (p 0.05). Morphological data are summarized in Table 1.

17 Examination of renal interstitial infiltrates in AN rats at 4 weeks demonstrated significant infiltrates of macrophages (CD68), CD8 and CD4+ T cells, and CD25 cells in renal interstitium as compared with PBS control rats (p 0.005), as shown in Figures 5A and B. Macrophages, CD8+ and CD4+ T cells, and CD25+ cells in renal interstitium in the DV group were significantly less than in the AN and DV control groups (p <0.001). There were no significant differences in staining for these cell infiltrates in the interstitium between the AN and DV control groups. These immunohistochemical data also are summarized in Table 1.

Table 1. Quantitation of morphology and immunohistochemistry DV DVctrl AN PBS DValone group A Proup B group C Group D group E Morpholog&y Interstitial volume 1.3 1.2 a 2.8 ±0.8 3.3 0.6' 0 0 Interstitial infiltrates 1.6 0.9' 3.2 0.8 3.6 0.6 0 0 Glomerular sclerosis 7.6 4.2a 16.4 ±6.1 19.3 1.2 0 0 Tubular diameter pm 34.6 1 1.6 31.5 2.1 31.5 ±0.6c 41.6 0.9 42.3 0.8 Tubular cell height pm 10.9 03 a 9.9 0.9 103 0.2 C 12.8 0.3 12.5 ±0.2 Inmunohistochenmistn' (cells /400 x field) CD4' cells 22.6 13 .3 88.8 18.2 84.9± 1 6 1.1 1.4 1.4 1.2 CD8+ cells 8.3 ±3.6 b 37.7 ±9.2 33.9 5.1 d 0.8 ±0.2 0.4 0.3 Macrophages (CD68') 8.9 3.9b 27.7 7.3 29.6 6 .8d 1.8 0.5 0.6 0.4 cells 0.

4 2 ±0.

3 b 4.5 ±0.6 6.4 2.2 0.2 ±0.1 0.1 ±0.1 Value are expressed as mean SD.

Sp 0.05 compared with group 2 and group 3.

b P< 0.001 compared with group 2 and group 3.

p 0.001 compared with group 4 and group Sp 0.005 compared with group 4 and group Example 3 0t Anti-chemokine antibodies produced by DNA vaccination in vivo To test whether vaccination using DNA encoding with MCP-1 and RANTES induced the production of autoantibodies against gene products of each vaccinated DNA, serum samples of all animals from week 2 to week 4 were analyzed for specific autoantibodies against MCP-1 and RANTES by ELISA. Rats vaccinated with chemokine DNA had significantly higher levels of MCP-1 and RANTES autoantibody titers than rats in the AN, DV control group and PBS groups (P 0.005), as shown in Figure 6.

Example 4 Vaccination using a modified MCP-1 construct The efficacy of a vaccine comprising a polynucleotide encoding wild-type rat MCP-1 in treatment of AN was compared to that of a vaccine comprising a polynucleotide encoding a 18 modified MCP-1 in which the surface loop region at bases +109 to +138 (37 to 46 aa) was replaced by a P30 tetanus toxoid helper epitope sequence.

Morphological changes in AN rats following vaccination was semi-quantitated using PASstained kidney sections and given a score of 0 to The observer was blinded to the treatment groups. This revealed significant reductions in glomerular sclerosis and interstitial infiltrates in rats vaccinated with the modified MCP-1 vaccine compared to those vaccinated with the MCP-1 wild type vaccine or vector only controls (Table 2).

Table 2- Morphological changes in AN rats following vaccination with wild-type or modified MCP-1 encoding polynucleotides Vector control MCP-1 vaccine mMCP-1 vaccine n=4 n=3 n=3 Overall morphology 3.0 0 2.7 0.6 1.8 0.9 Glomerular sclerosis (0 2.5 0.5 2.0 0 0.8 0.9 Tubular atrophy (0 2.7 0.6 2.5 0.5 2.0 0.4 Interstitial infiltrates (0 3.0 0 2.5 0.5 1.4 0.5 o p 0.05 compared with vector control group.

p 0.05 compared with vector control and MCP-1 vaccine groups.

Further, creatinine clearance (mean SEM) of rats vaccinated with modified MCP-1 vaccine was maintained at a steady level compared to that of controls treated with vector or unmodified MCP-1 vaccine (Figure 7).

Example Compositions for treatment In accordance with the best mode of performing the invention provided herein, specific preferred compositions are outlined below. The following are to be construed as merely illustrative examples of compositions and not as a limitation of the scope of the present invention in any way.

Example Composition for Parenteral Administration A composition for intramuscular injection could be prepared to contain 1 mL sterile buffered water, and 1 mg of a suitable agent or compound.

Similarly, a composition for intravenous infusion may comprise 250 ml of sterile Ringer's solution, and 5 mg of a suitable agent or compound.

Example Injectable Parenteral Composition A composition suitable for administration by injection may be prepared by mixing 1% by 19 weight of a suitable agent or compound in 10% by volume propylene glycol and water. The solution is sterilised by filtration.

Example 5(C) Capsule Composition A composition of a suitable agent or compound in the form of a capsule may be prepared by filling a standard two-piece hard gelatin capsule with 50 mg of the agent or compound, in powdered form, 100 mg of lactose, 35 mg of talc and 10 mg of magnesium stearate.

References Anders HJ, Vielhauer V, Schlondorff D: Chemokines and chemokine receptors are involved in the o1 resolution or progression of renal disease. Kidney Int. 63:401-415, 2003.

Lloyd CM, Minto AW, Dorf ME, Proudfoot A, Wells TN, Salant DJ, Gutierrez-Ramos JC: RANTES and monocyte chemoattractant protein-1 (MCP-1) play an important role in the inflammatory phase of crescentic nephritis, but only MCP-1 is involved in crescent formation and interstitial fibrosis.

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Rangan GK, Wang Y, Tay YC, Harris DC: Inhibition of nuclear factor-kappaB activation reduces cortical tubulointerstitial injury in proteinuric rats. Kidney Int. 56:118-134, 1999.

Rangan GK, Wang Y, Tay YC, Harris DC: Cytokine gene expression in Adriamycin nephropathy: effects of antioxidant nuclear factor kappaB inhibitors in established disease. Nephron 86:482-490, 2000.

Rangan GK, Wang Y, Harris DC: Induction of proteinuric chronic glomerular disease in the rat (Rattus norvegicus) by intracardiac injection of doxorubicin hydrochloride. Contemp.Top.Lab Anim Sci. 40:44-49, 2001.

Segerer S: The role of chemokines and chemokine receptors in progressive renal diseases.

Am.J.Kidney Dis. 41:S15-S18, 2003.

Segerer S, Nelson PJ, Schlondorff D: Chemokines, chemokine receptors, and renal disease: from basic science to pathophysiologic and therapeutic studies. J.Am.Soc.Nephrol. 11:152-176, 2000.

Walters G, Wu H, Knight JF: Glomerular T cells in Heymann nephritis. Clin Exp Immunol 2001 Nov;126(2):319-25.319-325, 2001.

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Claims (43)

1. A method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter and a polynucleotide encoding RANTES operably linked to a promoter.

2. The method of claim 1 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1.

3. The method of claim 1 wherein the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3. o1 4. The method of claim 1 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1 and the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3. The method of claim 1 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 and RANTES has the amino acid sequence as set forth in SEQ ID NO:4.

6. The method of claim 1 wherein the subject is human.

7. The method of claim 1 wherein the renal disease is a chronic proteinuric renal disease.

8. The method of claim 1 wherein the renal disease is selected from the group consisting of: focal glomerulosclerosis, glomerulonephritis, diabetic renal disease, hypertensive renal disease, renal failure, end-stage renal disease, or a related condition.

9. The method of claim 1 wherein the polynucleotide encoding MCP-1 and the polynucleotide encoding RANTES are located in a single nucleic acid construct. The method of claim 9 wherein the nucleic acid construct is a plasmid.

11. The method of claim 1 wherein administration of the polynucleotides induces an immune response in the subject.

12. A method for inducing protective immunity against renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter and a polynucleotide encoding RANTES operably linked to a promoter.

13. The method of claim 12 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1. 22

14. The method of claim 12 wherein the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3. The method of claim 12 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1 and the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3.

16. The method of claim 12 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 and RANTES has the amino acid sequence as set forth in SEQ ID NO:4.

17. The method of claim 12 wherein the subject is human.

18. The method of claim 12 wherein the renal disease is a chronic proteinuric renal disease.

19. The method of claim 12 wherein the renal disease is selected from the group consisting of: focal glomerulosclerosis, glomerulonephritis, diabetic renal disease, hypertensive renal disease, renal failure, end-stage renal disease, or a related condition. The method of claim 12 wherein the polynucleotide encoding MCP-1 and the polynucleotide encoding RANTES are located in a single nucleic acid construct.

21. The method of claim 20 wherein the nucleic acid construct is a plasmid.

22. An immunological composition comprising a polynucleotide encoding MCP-1 and a polynucleotide encoding RANTES, wherein administration of the composition to a subject induces an immune response in the subject.

23. The composition of claim 22 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1.

24. The composition of claim 22 wherein the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3. The composition of claim 22 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1 and the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3.

26. The composition of claim 22 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 and RANTES has the amino acid sequence as set forth in SEQ ID NO:4.

27. The composition of claim 22 wherein the subject is human.

28. The composition of claim 22 wherein the composition is a vaccine.

29. The composition of claim 28 wherein the vaccine is a DNA vaccine. 23 A composition for the treatment or prevention of renal disease comprising a polynucleotide encoding MCP-1 and a polynucleotide encoding RANTES, the polynucleotides being operably linked to a promoter.

31. The composition of claim 30 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1.

32. The composition of claim 30 wherein the polynucleotide encoding RANTES has the nucleotide sequence set forth in SEQ ID NO:3.

33. The composition of claim 30 wherein the polynucleotide encoding MCP-1 has the nucleotide sequence set forth in SEQ ID NO:1 and the polynucleotide encoding RANTES has the 1o nucleotide sequence set forth in SEQ ID NO:3.

34. The composition of claim 30 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 and RANTES has the amino acid sequence as set forth in SEQ ID NO:4. The composition of claim 30 wherein the renal disease is a chronic proteinuric renal disease.

36. The composition of claim 30 wherein the renal disease is selected from the group consisting of: focal glomerulosclerosis, glomerulonephritis, diabetic renal disease, hypertensive renal disease, renal failure, end-stage renal disease, or a related condition.

37. The composition of claim 30 wherein the polynucleotide encoding MCP-1 and the polynucleotide encoding RANTES are located in a single nucleic acid construct.

38. The composition of claim 37 wherein the nucleic acid construct is a plasmid.

39. The composition of claim 30 wherein administration of the composition to a subject induces an immune response in the subject. The composition of claim 30 wherein the composition is a vaccine.

41. The composition of claim 40 wherein the vaccine is a DNA vaccine.

42. A method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter.

43. The method of claim 42 wherein the polynucleotide encoding MCP-1 has the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID

44. The method of claim 42 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:6. 24 A method for inducing protective immunity against renal disease in a subject, the method comprising administering to the subject an effective amount of a polynucleotide encoding MCP-1 operably linked to a promoter.

46. The method of claim 45 wherein the polynucleotide encoding MCP-1 has the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID

47. The method of claim 45 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:6.

48. An immunological composition comprising a polynucleotide encoding MCP-1, wherein administration of the composition to a subject induces an immune response in the subject.

49. The composition of claim 48 wherein the polynucleotide encoding MCP-1 has the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID The composition of claim 48 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:6.

51. A composition for the treatment or prevention of renal disease comprising a polynucleotide encoding MCP-1 operably linked to a promoter.

52. The composition of claim 51 wherein the polynucleotide encoding MCP-1 has the nucleic acid sequence set forth in SEQ ID NO:1 or SEQ ID

53. The composition of claim 51 wherein MCP-1 has the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:6.

54. A method for the treatment or prevention of renal disease in a subject, the method comprising administering to the subject an effective amount of a composition according to claim or 51. Dated 7 June, 2004 The University of Sydney Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON