EP1224333A2

EP1224333A2 - Methods and compositions for correlating ccr5 expression with essential hypertension

Info

Publication number: EP1224333A2
Application number: EP00977297A
Authority: EP
Inventors: Thomas R. O'brien
Original assignee: US Department of Health and Human Services
Current assignee: US Department of Health and Human Services
Priority date: 1999-10-14
Filing date: 2000-10-02
Publication date: 2002-07-24
Also published as: AU1495201A; WO2001027334A2; WO2001027334A3; CA2387355A1

Abstract

The present invention provides a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising determining the presence in the subject of a genotype of the subject's CCR5 gene that correlates with an increased risk of developing essential hypertension, the presence of the genotype identifying the subject as having an increased risk of developing essential hypertension. Also provided is a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising: a) correlating the presence of a genotype of the subject's CCR5 gene with an increased risk of developing essential hypertension; and b) determining the genotype of the subject's CCR5 gene, whereby a subject having a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension is identified as having an increased risk of developing essential hypertension. In addition, the present invention provides a method of identifying a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension comprising: a) determining the nucleic acid sequence of the CCR5 gene from a subject; and b) correlating the presence of the nucleic acid sequence of step (a) with the presence of essential hypertension in the subject, whereby the nucleic acid sequence of the CCR5 gene identifies a genotype correlated with an increased risk of developing essential hypertension.

Description

METHODS AND COMPOSITIONS FOR CORRELATING CCR5 EXPRESSION

WITH ESSENTIAL HYPERTENSION

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention provides methods and compositions for identifying human subjects with an increased risk of having essential hypertension. In particular, this invention relates to the identification and characterization of mutations in the human CCR5 gene correlated with increased risk of developing essential hypertension.

BACKGROUND ART

Chemokines (chemoattractant cytokines), chemokine receptors, and host genetic factors are at the forefront of human immunodeficiency virus type 1 (HIV-1) research (1). The β- class (CC) chemokines play a critical role in the inflammatory process through recruitment or activation of lymphocytes, monocytes, mast cells, and eosinophil (2). By binding with CC-chemokine receptor 5 (CCR5), β- chemokines RANTES, MlP-lα and MlP-l β can also prevent in vitro infection of CD4+ cells (3). CCR5 is the major co-receptor for macrophage-tropic HIV-1 strains which predominate during early infection(4-8).

A 32 base-pair deletion (Δ32) of the CCR5 gene results in a truncated non-functional receptor for β- chemokines and HIV-1 (9-12). This allele has a frequency of -10% in Caucasians, but is more rare or absent in other populations (9-13). CCR5- Δ32/Δ32 homozygotes (about 1% of Caucasians) are highly resistant to infection by HIV-1 (9-12, 14), although this resistance is not absolute (15-17). HIV-1 -infected CCR5 heterozygotes (CCR5-+I Λ32) have delayed progression to AIDS (10, 12, 18, 19). Lymphocytes from CC7Ϊ5-Δ32/Δ32 homozygotes produce elevated levels of CCR5 ligand β- chemokines in vitro (8), and these proteins can bind chemokine receptors other than CCR5 (2). Studies in genetically altered mice indicate that the absence of CCR5 or the presence of higher levels of β- chemokines results in various phenotypic expressions. Mice lacking CCR5 have impaired macrophage function and an enhanced T cell dependent immune response (21). Higher levels of MlP-lα are associated with Coxsackievirus-induced myocarditis in mice (22).

Homozygosity for the CCR5-Δ32 allele may also have beneficial effects. The high allele frequency in Caucasians may reflect positive genetic selection, perhaps through protection against an infectious agent other than HIV-1 (23).

However, no previous studies have demonstrated a correlation between mutations in the CCR5 gene and blood pressure.

Essential hypertension is considered to be a quantitative disorder and is generally characterized in terms of marginal, moderate or severe hypertension. For example, marginal essential hypertension can be generally defined by a systolic blood pressure value greater than 120 mm Hg, up to about 140 mm Hg and diastolic blood pressure value greater than 90, up to about 100 mm Hg. Moderate essential hypertension can be generally defined by a systolic blood pressure greater than 140 mm Hg, up to about 180 mm Hg and a diastolic blood pressure value greater than 100 mm Hg, up to about 120 mm Hg. Severe essential hypertension can be generally defined by a systolic blood pressure value greater than 180 mm Hg and a diastolic blood pressure value greater than 120 mm Hg. These values do not define the absolute limits for the classification of essential hypertension as marginal, moderate or severe and can vary up to ± 5 mm Hg for each value. The present invention provides novel methods for identifying a CCR5 genotype correlated with an increased risk of developing essential hypertension as well as methods for identifying an individual having an increased risk of developing essential hypertension due to the presence in the individual of a CCR5 genotype correlated with such an increased risk.

SUMMARY OF THE INVENTION

The present invention provides a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising determining the presence in the subject of a genotype of the subject's CCR5 gene that correlates with an increased risk of developing essential hypertension, the presence of the genotype identifying the subject as having an increased risk of developing essential hypertension.

Further provided herein is a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising: a) correlating the presence of a genotype of the subject's CCR5 gene with an increased risk of developing essential hypertension; and b) determining the genotype of the subject's CCR5 gene, whereby a subject having a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension is identified as having an increased risk of developing essential hypertension.

A method of identifying a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension is also provided, comprising: a) determining the nucleic acid sequence (genotype) of the CCR5 gene from a subject; and b) correlating the presence of the nucleic acid sequence (genotype) of step (a) with the presence of essential hypertension in the subject, whereby the nucleic acid sequence (genotype) of the CCR5 gene identifies a genotype correlated with an increased risk of developing essential hypertension.

In a further embodiment, the present invention provides a method for identifying an agent that enhances the activity of CCR5, comprising: (a) contacting a cell that expresses CCR5 with the agent; (b) measuring the amount of CCR5 activity in the cell of step (a); and (c) comparing the amount of CCR5 activity measured in the cell of step (a) with the amount of CCR5 activity in control cells not contacted with the agent, whereby an amount of CCR5 activity in the cells of step (a) greater than the amount of CCR5 activity in the control cells identifies an agent that enhances the activity of CCR5.

Additionally provided is a method for identifying an agent that enhances the expression of CCR5, comprising: (a) contacting a cell that expresses CCR5 with the agent; (b) measuring the amount of CCR5 expression in the cell of step (a); and (c) comparing the amount of CCR5 expression measured in the cell of step (a) with the amount of CCR5 expression in control cells not contacted with the agent, whereby an amount of CCR5 expression in the cells of step (a) greater than the amount of CCR5 expression in the control cells identifies an agent that enhances the expression of CCR5.

Also provided in the present invention is a method of treating or preventing essential hypertension in a subject comprising administering to a subject an effective amount of CCR5, a CCR5 activity-enhancing agent and/or a CCR5 expression-enhancing agent, in a pharmaceutically acceptable carrier, thereby treating or preventing essential hypertension in the subject. Further provided is a method of treating or preventing essential hypertension in a subject comprising administering to a subject an effective amount of a nucleic acid encoding CCR5 under conditions whereby the nucleic acid is expressed in a cell in the subject, thereby treating or preventing hypertension.

Various other objectives and advantages of the present invention will become apparent from the following description.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, "a" can mean one or more than one. For example, "a cell" can mean a single cell or a multiple of cells.

The present invention provides the surprising discovery of a correlation between CCR5 genotype and blood pressure in a subject. Thus, the present invention provides a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising determining the presence in the subject of a genotype of the subject's CCR5 gene that correlates with an increased risk of developing essential hypertension, the presence of the genotype identifying the subject as having an increased risk of developing essential hypertension. For example, the genotype correlated with an increased risk of developing essential hypertension can comprise the CCR5-Δ32/Δ32 genotype, which is a 32 base pair deletion within the coding region of the CCR-5 chemokine receptor gene (39), as described by Samson et al. (38). As another example, the genotype can be a complete deletion of the CCR5 gene in a subject or the presence of a CCR5 allele (m303), having a single point mutation (T- A) at position 303 (40). It is also contemplated that other genotypes of the CCR5 gene correlated with hypertension can be identified by the methods described herein which can be used to identify a subject having an increased risk of developing essential hypertension. Also provided herein is a method for identifying a human subject as having an increased risk of developing essential hypertension, comprising: a) correlating the presence of a genotype of the subject's CCR5 gene with an increased risk of developing essential hypertension; and b) determining the genotype of the subject's CCR5 gene, whereby a subject having a genotype of the CCR5 gene conelated with an increased risk of developing essential hypertension is identified as having an increased risk of developing essential hypertension.

In addition, the present invention provides a method of identifying a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension comprising: a) determining the genotype of the CCR5 gene from a subject; and b) correlating the presence of the genotype of step (a) with the presence of essential hypertension in the subject, whereby the genotype of the CCR5 gene identifies a genotype correlated with an increased risk of developing essential hypertension.

The CCR5 genotype of a subject is identified by determining the nucleic acid sequence of the CCR5 genes of the subject according to standard molecular biology protocols well known in the art as described for example in Sambrook et al. (1989) and as set forth in the Examples provided herein.

A genotype of the CCR5 gene can be identified as correlated with an increased risk of developing essential hypertension on the basis of well-known statistical analyses of the incidence of a particular CCR5 genotype in individuals diagnosed with essential hypertension. Specifically, the CCR5 genotype of a subject is determined by nucleic acid sequencing or other assays well known in the art and the presence or absence of essential hypertension in the subject is also determined. These data are then statistically analyzed to establish a conelation between a specific CCR5 nucleic acid sequence (genotype) and the presence or absence of essential hypertension (phenotype), thereby establishing a correlation between a CCR5 genotype and an increased risk of developing essential hypertension.

The present invention further contemplates a method for identifying a human subject as having a decreased risk of developing essential hypertension, comprising determining the presence in the subject of a genotype of the subject's CCR5 gene that correlates with a decreased risk of developing essential hypertension, the presence of the genotype identifying the subject as having a decreased risk of developing essential hypertension. Genotypes of the CCR5 gene correlated with hypertension are identified by the methods described herein, which are then be used to identify a subject having a decreased risk of developing essential hypertension.

Also provided herein is a method for identifying a human subject as having a decreased risk of developing essential hypertension, comprising: a) correlating the presence of a genotype of the subject's CCR5 gene with a decreased risk of developing essential hypertension; and b) determining the genotype of the subject's CCR5 gene, whereby a subject having a genotype of the CCR5 gene correlated with a decreased risk of developing essential hypertension is identified as having a decreased risk of developing essential hypertension.

In addition, the present invention provides a method of identifying a genotype of the CCR5 gene conelated with a decreased risk of developing essential hypertension comprising: a) determining the genotype of the CCR5 gene from a subject; and b) correlating the presence of the genotype of step (a) with the absence of essential hypertension in the subject, whereby the genotype of the CCR5 gene identifies a genotype conelated with an increased risk of developing essential hypertension. The CCR5 genotype of a subject is identified by determining the nucleic acid sequence of the CCR5 genes of the subject according to standard molecular biology protocols well known in the art as described for example in Sambrook et al. (1989) and as set forth in the Examples provided herein.

A genotype of the CCR5 gene can be identified as conelated with a decreased risk of developing essential hypertension on the basis of well-known statistical analyses of the incidence of a particular CCR5 genotype in individuals with normal blood pressure. Specifically, the CCR5 genotype of a subject is determined by nucleic acid sequencing or other assays well known in the art and as described herein and the blood pressure of the subject is also determined. These data are then statistically analyzed to establish a conelation between a specific CCR5 nucleic acid sequence (genotype) and the absence of essential hypertension (phenotype), thereby establishing a conelation between a CCR5 genotype and a decreased risk of developing essential hypertension.

It is further contemplated herein that the discovery of a conelation between CCR5 genotype and an increased risk of developing essential hypertension can by used to develop a variety of therapeutic approaches to the treatment and/or prevention of essential hypertension in a subject.

Thus, further provided in the present invention is a method for identifying an agent that enhances the activity of CCR5, comprising: (a) contacting a cell that expresses CCR5 with the agent; (b) measuring the amount of CCR5 activity in the cell of step (a); and (c) comparing the amount of CCR5 activity measured in the cell of step (a) with the amount of CCR5 activity in control cells not contacted with the agent, whereby an amount of CCR5 activity in the cells of step (a) which is greater than the amount of CCR5 activity in the control cells identifies an agent that enhances the activity of CCR5. Additionally, the present invention provides method for identifying an agent that enhances the expression of CCR5, comprising: (a) contacting a cell that expresses CCR5 with the agent; (b) measuring the amount of CCR5 expression in the cell of step (a); and (c) comparing the amount of CCR5 expression measured in the cell of step (a) with the amount of CCR5 expression in control cells not contacted with the agent, whereby an amount of CCR5 expression in the cells of step (a) which is greater than the amount of CCR5 expression in the control cells identifies an agent that enhances the expression of CCR5.

As set forth herein, the amount of activity of CCR5 is determined according to methods well known in the art for quantitative measurement of the activity of a chemokine receptor. For example, a specific physiological activity of cells producing the chemokine receptor can be measured in response to contacting the cells with various chemokine ligands. For example, a ligand of the CCR5 receptor, (e.g., antibodies which bind CCR5, RANTES, MlP-lα, MlP-lβ, MCP-1, MCP-2, MCP-3, MCP-4 and/or eotaxin (41)) can be employed in a variety of well-known assays for measuring activity of CCR5 (see e.g., ref . 39, 60, 61) to provide a quantitative measurement of CCR5 activity.

As also set forth herein, the amount of expression of CCR5 is determined according to methods well known in the art for quantitative measurement of the expression of a chemokine receptor. For example, expression of the CCR5 receptor can be quantitatively measured by determining the amount of CCR5 receptor mRNA in a cell, the amount of CCR5 receptor protein in a cell and/or the number of CCR5 receptors present on the surface of a cell, etc. according to protocols well known in the art (see, e.g., ref. 39, 42).

The cell type used in the screening assays of this invention can be any cell type which produces either normally functioning CCR5 or CCR5 encoded by a defective CCR5 gene (e.g., having a genotype as described herein). The cells can produce normal CCR5 or defective CCR5 endogenously or as a result of having nucleic acid encoding normal CCR5 or defective CCR5 introduced into the cell via mechanisms well known in the art (e.g., transduction, transfection, transformation, etc.).

Any agent which has the potential of enhancing CCR5 activity and/or CCR5 expression can be used in the screening methods of this invention and identified as having said activity-enhancing or expression-enhancing activity according to the protocols provided herein. The agents identified by the screening methods described herein are then used in methods of treating and/or preventing hypertension in a subject, as set forth in detail herein below.

Thus, the present invention further provides compositions to be employed in the methods of this invention for the treatment and/or prevention of essential hypertension in a subject. In particular, the present invention provides a composition comprising 1) an isolated CCR5 protein in a pharmaceutically acceptable carrier, 2) an isolated nucleic acid encoding CCR5 protein in a pharmaceutically acceptable carrier, 3) a CCR5 activity- enhancing agent, and/or 4) a CCR5 expression-enhancing agent in a pharmaceutically acceptable carrier. These compositions can be present separately or in any combination.

The compositions of the present invention can also include other medicinal agents, pharmaceutical agents, caniers, diluents, immunostimulatory cytokines, etc. For example, the compositions of this invention can include agents that activate or stimulate T cells, including, but not limited to IL-2, IL-10, IL-12, IL-15, TNF-α, IFN-γ and HIV-1 Tat protein (43-46). Actual methods of preparing such compositions are known, or will be apparent, to those skilled in this art (Remington 's Pharmaceutical Sciences (Martin, E.W., ed., latest edition), Mack Publishing Co., Easton, PA.). By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected protein, nucleic acid or agent without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.

"Isolated CCR5 protein" as used herein means CCR5 receptor protein is sufficiently free of contaminants or cell components with which proteins normally occur and is present in such concentration as to be the only significant protein present in the sample. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the CCR5 protein in a form in which it can be used therapeutically.

It is understood that, where desired, modification and changes may be made in the structure of CCR5 having the amino acid sequence set forth in reference 39 herein and still obtain a protein having like or otherwise desirable characteristics. Such changes may occur in natural isolates or may be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art.

For example, certain amino acids may be substituted for other amino acids in a CCR5 protein without appreciable loss of functional activity of CCR5. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a CCR5 amino acid sequence (or, of course, the underlying nucleic acid sequence) and nevertheless obtain CCR5 protein with like properties. It is thus contemplated that various changes may be made in the sequence of the CCR5 amino acid sequence (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity.

It is also understood that the CCR5 protein of this invention may also contain conservative substitutions where a naturally occu ing amino acid is replaced by one having similar properties and which does not alter the function of the polypeptide. Such conservative substitutions are well known in the art. Thus, it is understood that, where desired, modifications and changes may be made in the nucleic acid and/or amino acid sequence of the CCR5 protein of the present invention and still obtain a CCR5 protein having like or otherwise desirable characteristics.

"Nucleic acid" as used herein refers to single- or double-stranded molecules which may be DNA, comprised of the nucleotide bases A, T, C and G, or RNA, comprised of the bases A, U (substitutes for T) , C, and G. The nucleic acid may represent a coding strand or its complement. Nucleic acids may be identical in sequence to the sequence which is naturally occurring or may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. Furthermore, nucleic acids may include codons which represent conservative substitutions of amino acids as are well known in the art.

As used herein, the term "isolated nucleic acid" means a nucleic acid separated or substantially free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids. The isolation of nucleic acids can therefore be accomplished by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids, as are well known in the art (e.g., see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). The nucleic acids of this invention can be isolated from cells according to methods well known in the art for isolating nucleic acids. Alternatively, the nucleic acids of the present invention can be synthesized according to standard protocols well described in the literature for synthesizing nucleic acids. Modifications to the nucleic acids of the invention are also contemplated, provided that the essential structure and function of the peptide or polypeptide encoded by the nucleic acid are maintained.

The nucleic acid encoding the CCR5 protein can be part of a recombinant nucleic acid construct comprising any combination of restriction sites and/or functional elements as are well known in the art which facilitate molecular cloning and other recombinant DNA manipulations. Thus, the present invention further provides a recombinant nucleic acid construct comprising a nucleic acid encoding CCR5 protein.

The present invention further provides a vector comprising a nucleic acid encoding CCR5 protein. The vector can be an expression vector which contains all of the genetic components required for expression of the nucleic acid in cells into which the vector has been introduced, as are well known in the art. The expression vector can be a commercial expression vector or it can be constructed in the laboratory according to standard molecular biology protocols. The expression vector can comprise viral nucleic acid including, but not limited to, alphavirus, vaccinia virus, adenovirus, retrovirus and/or adeno-associated virus nucleic acid. The nucleic acid or vector of this invention can also be in a liposome or a delivery vehicle which can be taken up by a cell via receptor-mediated or other type of endocytosis.

The nucleic acid of this invention can be in a cell, which can be a cell expressing the nucleic acid whereby CCR5 protein is produced in the cell. In addition, the vector of this invention can be in a cell, which can be a cell expressing the nucleic acid of the vector whereby CCR5 protein is produced in the cell. It is also contemplated that the nucleic acids and/or vectors of this invention can be present in a host animal (e.g., a transgenic animal) which expresses the nucleic acids of this invention and produces CCR5 protein.

The nucleic acid encoding the CCR5 protein of this invention can be any nucleic acid that functionally encodes the CCR5 protein of this invention. To functionally encode the CCR5 protein (i.e., allow the nucleic acids to be expressed), the nucleic acid of this invention can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences.

Preferred expression control sequences are promoters derived from metallofhionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc. A nucleic acid encoding a selected peptide or polypeptide can readily be determined based upon the genetic code for the amino acid sequence of the selected peptide or polypeptide and many nucleic acids will encode any selected peptide or polypeptide. Modifications in the nucleic acid sequence encoding the peptide or polypeptide are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression of the peptide or polypeptide to make production of the peptide or polypeptide inducible or repressible as controlled by the appropriate inducer or repressor. Such methods are standard in the art (see. e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989.). The nucleic acid of this invention can be generated by means standard in the art, such as by recombinant nucleic acid techniques and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.

It is contemplated that the above-described compositions of this invention can be administered to a subject or to a cell of a subject to impart a therapeutic benefit. Thus, the present invention further provides a method of treating or preventing essential hypertension in a subject, comprising administering to a subject an effective amount of CCR5 protein in a pharmaceutically acceptable carrier, thereby treating or preventing essential hypertension in the subject.

A method of treating or preventing essential hypertension in a subject is also provided, comprising administering to a subject an effective amount of a nucleic acid encoding CCR5 under conditions whereby the nucleic acid is expressed in a cell in the subject, thereby treating or preventing hypertension.

In addition, the present invention provides a method of treating or preventing essential hypertension in a subject, comprising administering to a subject an effective amount of a CCR5 expression enhancing agent and/or an effective amount of a CCR5 activity-enhancing agent in a pharmaceutically acceptable carrier, thereby treating or preventing essential hypertension in the subject.

The methods of the present invention further contemplate the administration of the proteins, nucleic acids and enhancing agents of this invention in any combination to treat and/or prevent essential hypertension in a subject.

The CCR5 protein and/or enhancing agents of this invention can be administered to a cell of a subject either in vivo or ex vivo. Administration to a cell of the subject in vivo, as well as administration to the subject, can be oral, parenteral (e.g., intravenous), by intramuscular injection, by intraperitoneal injection, by subcutaneous injection, transdermal, extracorporeal, topical, or the like. Also, the protein or enhancing agent of this invention may be pulsed onto dendritic cells which are isolated or grown from patient cells, according to methods well known in the art, or onto bulk PBMC or various cell subfractions thereof from a patient. The exact amount of the protein or enhancing agent required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular protein or enhancing agent used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every protein or enhancing agent. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein (Remington 's Pharmaceutical Sciences (Martin, E.W., ed., latest edition), Mack Publishing Co., Easton, PA.).

If ex vivo methods are employed, cells or tissues can be removed and maintained outside the subject's body according to standard protocols well known in the art. The proteins or enhancing agents of this invention can be introduced into the cells via known mechanisms for uptake of proteins into cells (e.g., phagocytosis, pulsing onto class I MHC- expressing cells, liposomes, etc.). The cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

The nucleic acid encoding CCR5 protein of this invention can also be administered to the cells of the subject either in vivo and/or ex vivo. If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The nucleic acids of this invention can be introduced into the cells via any gene transfer mechanism, such as, for example, virus-mediated gene delivery, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes. The transduced cells can then be infused (e.g., in a pharmaceutically acceptable ca ier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. For in vivo methods, the nucleic acid encoding CCR5 can be administered to the subject in a pharmaceutically acceptable carrier as further described below.

In the methods described above which include the administration and uptake of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the CCR5 protein. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTLN, LIPOFECTAMiNE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).

As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g.,47,48). The recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding TK and/or ANP. The exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (49), adeno-associated viral (AAV) vectors (50), lentiviral vectors (51), pseudotyped retroviral vectors (52). Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanisms (see, for example, 53). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.

The mode of administration of the nucleic acid or vector of the present invention can vary predictably according to vehicle for delivery as well as the tissue being targeted. For example, for administration of the nucleic acid or vector in a liposome, catheterization of an artery upstream from the target organ is a prefened mode of delivery, because it avoids significant clearance of the liposome by the lung and liver.

The nucleic acid or vector may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, although intravenous administration is typically preferred. The exact amount of the nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disease being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every nucleic acid or vector. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein (see, e.g., Remington's Pharmaceutical Sciences (Martin, E.W., ed., latest edition), Mack Publishing Co., Easton, PA).

As one example, if the nucleic acid of this invention is delivered to the cells of a subject in an adenovirus vector, the dosage for administration of adenovirus to humans can range from about 10⁷ to 10⁹ plaque forming unit (pfu) per injection but can be as high as 10¹² pfu per injection (54, 55). Ideally, a subject will receive a single injection. If additional injections are necessary, they can be repeated at six month intervals for an indefinite period and/or until the efficacy of the treatment has been established. Parenteral administration of the nucleic acid or vector of the present invention, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

In the methods of the present invention which describe the treatment and/or prevention of essential hypertension, the efficacy of the treatment can be monitored according to clinical protocols well known in the art for monitoring the treatment of hypertension or hypertension-related renal disorders. For example, such clinical parameters as blood pressure measurement, renal sodium excretion, urine volume, urinary sediment and/or urine creatinine can be monitored according to methods standard in the art. Ideally, these parameters would be measured at about ten days after gene delivery. A clinician would look for a reduction in systolic and/or diastolic blood pressure, or the absence of proteinuria, edema, hematuria, azotemia and casts as well as increased urine volume and sodium excretion as indicators of the efficacy of the treatment methods of this invention.

In a further embodiment, the present invention provides a method of treating and/or preventing essential hypertension in a subject, comprising administering to the subject an effective amount of an agent which reduces the effects of increased levels of the CCR5 ligands, MlP-lα, MlP-lβ and RANTES produced by CCR5Δ32 homozygotes (56, 57). Such an agent may, for example, downregulate production and/or expression of these ligands (58, 59) or act in a manner to reduce signaling of the major angiotensin receptor, AT-1 (32). The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

Subjects. Subjects were enrolled in the Multicenter Hemophilia Cohort Study (MHCS) (24) or the Washington and New York Men's Research Study (DCG). (25) MHCS is a prospective cohort study of 2,117 patients enrolled in the United States and Europe beginning in 1982. At 6-12 month intervals, clinical status information is recorded, a physical examination is performed, and blood is collected. DCG is a study of 245 homosexual men enrolled in 1982 at primary care physicians' offices. Patient blood was collected and interviews were conducted at enrollment and about every 12 months subsequently, but follow-up of most HIV-1 uninfected subjects was discontinued in 1991. Both studies were reviewed and approved by the relevant institutional review boards, and all subjects provided informed consent to participate. The present analysis is limited to HIV-1 -uninfected subjects.

Demographic and clinical (including hematologic) data from the studies were recorded on standardized forms and entered into a computer database. Clinical conditions that were assessed were primarily those associated with hemophilia, liver disease, HIV infection, or sexually transmitted infections. In addition, records of clinical care were available for 11 CCR5-Δ32/A 32 homozygotes; we systematically abstracted information from these records. To examine a possible association between CC ?5-Δ32/Δ32 homozygosity and hypertension, we also abstracted information on blood pressure and hypertension treatment from clinical records of CCR5-A32/A32 homozygotes and selected CCR5- +/+ comparison subjects. Laboratory Methods. Hemato logic measures were determined directly with an automated cell counter, or by an automated count of white blood cells and a manual differential count. The percentage of CD4+ lymphocytes was measured by flow cytometry using the whole blood lysate method (26) or methods appropriate for frozen lymphocyte specimens (27). Assays for antibodies to HIV-1, hepatitis B virus (HBV) and hepatitis C virus (HCV), as well as for HBV surface antigen and the HCV branched DNA (bDNA) were performed as previously described (28). For CCR5Δ32 homozygotes, we measured antibodies to ten common viruses (Mayo Medical Laboratories, Rochester, MN) if adequate volumes of archived serum or plasma were available. These antibodies were measured by immuno fluorescence assay or enzyme-linked immunoassay (cytomegalovirus and rubella). Measles antibody results were confirmed by a measles neutralization assay (28a).

CCR5 genotype was determined by polymerase chain reaction (PCR) amplification of DNA, followed by single-stranded conformational polymorphism analysis as previously described (10). Some subjects could not be genotyped because no DNA specimen was available or for technical reasons.

Data Analysis. As all of the HIV-1 uninfected CC ?5-Δ32/Δ32 subjects were white or white Hispanic U.S. males, we limited the comparison group to similar subjects. For repeatedly measured variables (e.g., lymphocyte count), we determined the mean value for each subject and then calculated the grand mean for each genotype. Measurements taken before the subject reached 18 years of age were excluded. For comparisons of hepatic enzyme levels among hemophilic subjects infected with HCV, measurements before the initial positive HCV antibody test were excluded, as were measurements taken when the hepatitis B surface antigen result was positive. Subject characteristics and results from laboratory assays were compared using the two-sided Mann- Whitney- Wilcoxon rank sum test for continuous measures (29) and the chi-square test for categorical data (30). We used Fisher's exact test (Epi-Info, version 6.04a) and the exact binomial test to compare serologic assay results for the CCR5Δ32 homozygotes with results expected in a normal healthy population; expected values were calculated using seroprevalence data obtained from the commercial laboratory which performed the assays (Wold AD. Mayo Clinic, Rochester, MN).

In a substudy of hypertension prevalence, we abstracted information from clinic records of CCi?5-Δ32/Δ32 homozygotes and selected CCR5-+/+ subjects (matched by age and site of enrollment) for the period January 1, 1985 through August 1, 1997. We considered a subject hypertensive if the mean of his last two systolic pressure readings was > 140 mm Hg, if the mean of his last two diastolic pressure readings was_> 90 mm Hg, or if he had ever been treated for hypertension. These data were analyzed as a series of strata each comprised of a CCR5 -A32IA32 homozygote and up to five CCR5-+I+ subjects. The common relative risk was determined by the Mantel-Haenszel method, the 95% confidence interval (95% CI) by test-based methods, and the p-value by the Cochran-Mantel-Haenszel statistic (30).

Among 219 HIV-1 uninfected white hemophiliacs genotyped for C 5-Δ32, there were 175 (79.9%) CCR5-+/+ subjects, 32 (14.6%) CCR5-+I 32 heterozygotes, and 12 (5.5%) CCR5-A32/A32 homozygotes. Among 97 white or white Hispanic homosexual men, there were 73 (75.3%) CCR5-+/+ subjects, 21 (21.6%) CCR5-+/ A32 heterozygotes, and 3 (3.1%) CCR5-A32/A32 homozygotes. The median age at study entry was 25 years for hemophiliacs and 33 years for homosexual men. Although most hemophiliacs, regardless of CCR5 genotype, had factor VIII deficiency (hemophilia A), CCR5-A32/A32 subjects had received greater amounts of blood products. For example, seven often CCi?5-Δ32/Δ32 hemophiliacs with available blood product exposure information had received >20,000 units of non-heat treated factor VIII concentrate between 1978 to 1985, compared to 8% of subjects with other genotypes (p=0.001). The elevated frequency of CCR5-A32IA32 homozygosity in this study population and the greater blood product exposure among such hemophiliacs reflect the resistance of CCΛ5-Δ32/Δ32 homozygotes to HIV-1 infection.

The small number of deaths precluded a meaningful analysis of genotype-specific mortality: five CCR5-+/+ individuals died during the follow-up period and one CCi?5-Δ32/Δ32 hemophiliac died as a result of trauma. To assess morbidity, we abstracted diagnostic information from the study records of the 15 HIV-1 uninfected CCR5-A32IA32 subjects and, for 11 subjects, from clinical records covering 10 to 26 years of follow-up (Table 1). As expected, many men with hemophilia had clinical evidence of arthropathies (10/12), bleeding complications (8/12), and hepatitis or hepatomegaly (5/12). Two of the three homosexual men had reported at least one sexually transmitted infection. Six of the 15 CCR5-A32/A32 subjects had lymphadenopathy (any enlarged lymph node) noted during at least one study visit, but lymphadenopathy was similarly common among subjects with other CCR5-A32 genotypes. Most other health conditions were limited to only one or two subjects, but the diagnosis of hypertension was found for six of 11 CC Ϊ5-Δ32/Δ32 homozygotes with available clinical records.

To examine the possible association between CCR5- A32I 32 homozygosity and hypertension, we abstracted data on anti-hypertensive medications and the two most recent blood pressure readings from clinic records of the 11 evaluable CC/Ϊ5-Δ32/Δ32 homozygotes and age-matched CG 5— ⁽7+ subjects from the same study site. This review confirmed the diagnosis of hypertension for six CO?5-Δ32/Δ32 homozygotes and revealed a seventh CCR5-A32/A32 homozygote who met our criteria. Two CCR5-A32/A32 homozygotes were hypertensive on the basis of treatment, two were hypertensive on the basis of blood pressure, and three were hypertensive on the basis of both treatment and blood pressure; no blood pressure was in the severe range (systolic_> 180 or diastolic > 110). No age- and site-matched subject was available for one of the 11 CCR5-A32IA32 homozygotes, a 64 year old on anti-hypertensive treatment. We compared the ten remaining CCR5- Δ32/Δ32 homozygotes (mean age, 37.1 years) to 31 matched CCR5- subjects (mean age, 37.9 years). Five of the CCR5-^Jrl+ subjects had evidence of hypertension, yielding a 2.8-fold higher prevalence of hypertension among CCΛ5-Δ32/Δ32 homozygotes (95% CI, 1.2-6.4; p = 0.01). Compared to matched CCR5-+/+ subjects, CO5-Δ32/Δ32 homozygotes had more clinic visits (mean, 11.0 and 15.6 visits, respectively), more often had hemophilia A (66.7%> and 88.9%, respectively), more often received >20,000 units of Factor VIII concentrate (37.5% and 88.9%, respectively), and less often received >20,000 units of Prothrombin Complex concentrate (40.0% and 0.0%, respectively). Greater Prothrombin Complex concentrate use among CCR5 — <-/+ subjects reflects patients with Factor VIII deficiency who had inhibitors to Factor VIII concentrate. Rather than receiving Factor VIII concentrate, such patients were treated with Prothrombin Complex concentrate which, for unknown reasons, carried a lower risk of HIV-1 infection.

We compared CCR5-A 2/Δ32 homozygotes to other subjects with regard to hematologic and lymphocyte subset measurements (Table 2). Values for hemoglobin, red blood cells, and platelets were similar across the genotypes. Total lymphocytes were higher for CCR5-A32IA 32 subjects than CCR5- +/+ subjects in each cohort (the difference was not statistically significant for homosexual men), but none of the 90 separate total lymphocyte count measurements among 13 C05-Δ32/Δ32 homozygotes exceeded the upper limit of normal (4,800 cells/mm³). When total lymphocyte counts among heterozygotes were compared to those for CCR5-+/+ subjects, a similar increase was noted among hemophiliacs (p=0.02), but not homosexual men. Compared to CCR5^-/+ subjects, total CD4+ lymphocytes were higher among hemophilic CCi?5-Δ32/Δ32 homozygotes, but not among homosexual men. There were no consistent differences between genotypes for other white blood cell types.

For 10 CCJ 5-Δ32/Δ32 homozygotes, blood chemistry results were found in the clinic record. Comparing the most recent result to the testing laboratory's reference range, hepatic enzymes and bilirubin were commonly elevated among the HCV-infected hemophiliacs, but abnormal results for other measures (BUN, creatinine, uric acid, CO2, calcium, phosphorus, albumin, electrolytes, lactate dehydrogenase, alkaline phosphatase, total protein) were observed in only one or two CCR5-A32I Δ32 subjects each. Hepatic enzyme results were available from study records for most hemophiliacs. Among HCV-infected hemophiliacs, mean AST and ALT levels were 67% and 117% higher among CCR5- A32IA32 homozygotes than CCR5-^I+ subjects (Table 2). Elevated hepatic enzymes among CCR5-Δ32/Δ32 hemophiliacs were not explained by higher viral burden, as serum HCV bDNA levels did not vary by genotype (Table 2).

Consistent with the high prevalence of blood borne infections among persons with hemophilia, antibody was detected in 11/11 CCR5- Δ32/Δ32 hemophiliacs tested for hepatitis B virus and 10/12 (83.3%) tested for hepatitis C virus. For 9 of 10 common viruses, antibody prevalence was similar in CC Ϊ5-Δ32/Δ32 subjects and the reference population (Table 3). Antibody to measles virus was, however, less common than expected among CCR5-A32/A32 subjects, especially in subjects born before 1957 (4/7 or 57%>) who would be expected to have high levels of immunity acquired through natural exposure to measles virus. In an attempt to verify this association in a general population, we determined CCR5-A32 genotype for a group of U.S. blood donors bom before 1957. All 14 CCΛ5-Δ32/Δ32 homozygotes in this group had detectable measles antibody levels. In addition, PBMCs from C05-Δ32/Δ32 or CCR5-+/+ individuals replicated Edmonston wild type measles virus or the Bilthoven strain of measles virus to similar levels, indicating that CCR5 was not required for infection with these strains.

Although our study population was relatively young (median age at study entry, 25 years for hemophiliacs and 33 years for homosexual men), seven of 11 CCR5-A32IA32 homozygotes with evaluable clinical records were hypertensive on the basis of anti-hypertensive therapy or blood pressure. Compared to matched CCR5^-/+ subjects, CCR5-A32/A32 homozygotes were 2.8-fold more likely to be hypertensive. Although a substantial proportion of blood pressure variation may be genetically determined, the few recognized Mendelian forms of hypertension result from rare alleles which cause severe hypertension by altering the renin-angiotensin system (31).

Although the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

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61. Lacy et al. Blood 1999; 94:23-32. Table 1. Clinical conditions, year of birth, and length of study observation and clinical review for 15 men with the CCR5-AZ21AZ2 genotype.

Length of Length of Year of Study Clinical Record I

Subject Birth Enrollment Review Reported or Observed Conditions *

Hemophil iacs

1 1974 8 years 16 years - 1, t, §. -splenomegaly,, migraine headaches

2 1954 14 years ^' 11 years t. ll

3 1951 6 years 16 years t, drverϋculrrjs

4 1960 8 years n/a t, colitis

5 1961 9 years 14 years t_> §_> H_> splenomegaly, cardiovascular abnormalities, nasai septal deviation, allergies

6 1932 8 years 12 years t_> ÷_> ii. H. gastrointestinal problems, respiratory dysfunction, other cardiovascular abnormalities, skin disorders, benign prostatic hypertrophy, kidney stone, depression

7 1966 12 years 25 years f, t, |], shingles, pneumonia, Mallory- Weiss tear, gastroenteritis

8 1954 13 years 24 years f, t, nevi, indirect inguinal hernia, possible cartilaginous neoplasm

9 1970 9 years 24 years t» t. §. II. chicken pox, rosacea, herpes labialis, peptic ulcer disease, epidydimitjs, learning disability

10 1942 δyears 19 years 1. 1, §. ||. celiulrrjs, testjcuiar pain/sweffing, kidney mass, depression, pseudotumor

11 1961 1 year** 10 years *. §

12 1957 3 years n/a t, skin ulcers; subject died at age 35 (secondary to trauma) TSBI-E 1 (cont.J

Homosexual Men

13 1958 1 year** n a

14 1947 9 years n/a §, , gonorrhea, body lice, non- gonorrheal penile discharge, non- herpes anal problem, herpes labialis, ski rash, muscle ache, difficulty with concentration/mood, respiratory infection dysfunction, anal fissure

15 1947 9 years 26 years ||, H, syphilis, -body Bee, non-herpes anal problem, colorectal polyps, obesity, diabetes, respiratory infection/ dysfunction, joint/muscle disorder, difficulty with concentration mood, childhood impetigo, eye disorder, neurologic disorder

* Common conditions included the following: t Arthropathy (10 hemophiliacs),

$ Bleeding complications (8 hemophiliacs)

§ Hepatitis orhepatomegaiy (5 hemophQiacs, 1 homosexual)

|| Hypertension (5 hemophiiiacs, 1 homosexual)

1T Lymphadenopathy (3 hemophiiiacs, 3 homosexuals) n/a - No clinical records were available for this subject ** Subject had a single study visit

Table 2. Mean values for hematologlc measures, lymphocyte subsets, hepatic enzymes, and HCV levels, by cohort and CCRS genotype.

Grand mean (Number ol f subjects) <p- /alue>

Male Hemophiliacs I Homosexual Mer 1

+/+ +/Δ32 Δ32/Δ32 +/+ +/Δ32 Δ32/Δ32

Hemoglobin (g/dL) 16.0 (^27) 18.4 (21) <0.09> 16.2 (11) <0.72> 16.6 (66) 16.0 (19) <0.02> 16.3 (2) <0.θθ>

RBC (10¹² cells/L) 4.97 (127) 4.9β (21) <0.82> β.09 (11) <0.33> -

Platelets (10³ cells/mm³) 246 (126) 2(54 (21) <0.8β> 272 (11) <0.58> 262 (6S) 267 (19) <0.72> 286 (2) <0.6β> BC (10³ cell8/mm⁸) β.47 (128) 6.94 (21) <0.24> 7.08 (11) <0.18> 6.80 (73) 6.66 (20) <0.69> 6.39 (3) <0.12>

Lymphocytes (cellβ/mm³ 1809 (127) 2132 (21) <0.02> 2194 (11) <0.01> 2397 (84) 2361 (19) <0.75> 3018 (2) <0.13>

Nβutrophlla (cells/mm⁸) 3892 (127) 3922 (21) <0.83> 4180 (11) <0.38> 3782 (64) 3769 (19) <0.82> 4218 (2) <0.68> σi

Eoβinophlls (cells/mm³) 164 (118) 168 (20) <0.Θ7> 128 (11) <0.25> 122 (63) 117 (19) <0.47> 294 (2) <0.01> onocytes (cells/mm³) 418 (119) 626 (20) <0.07> 444 (11) <0.34> 383 (63) 384 (19) <0.βθ 322 (2) <0.7β>

Baβophlls (cells/mm³) 43 (112) 87 (20) <0.67> 33 (10) <Q.32> 48 (63) .39 (19) <0.75> 69 (2) <0.4Θ>

Bands (cells/mm³) 28 (107) 48 (20) <0.39> 21 (10) <0.19> 106 (60) 60 (19) <0,9Θ> 74 (2) <0.Θ3>

CD4+ cells (cβllβ/mm³) 781 (119) U42 (20) <0.11> 916 (11) <0.04> 980 (73) 923 (21) <0.47> 893 (3) <0.73>

CD8+ cells (cellβ/mm³) 807 (119) (334 (20) <0.03> 889 (11) <0.19> 609 (73) 700 (21) <0.41> 761 (3) <0.17>

HCV (log₁₀ copleβ/mL) * S.69 (94) 4.S0 (17) <0.08> δ.67 (8) <0.37>

A8T (U/L) * 61 (90) 82 (1β) <0.51> 86 (8) <0.02>

ALT (U L) * 72 (94) 87 (16) <0.15> 186 (8) <0.01>

For white blood cell differential counts, the number of observations vary because results for eostnophilβ, monocytββ, baaophll^β, and bwnd^β were not entered for some subjects.

* HCV-infected hemophiliacs only.

Table 3. Number of CCR5Δ32 Homozyotes with Antibodies Against Selected Viral Infections

Cryopreserved Serum from 11 Male Hemophαlacs and 2 Homosexual Men

-

Proportion of

Number of Reference

Subjects with Population with

Virus Antibody Antibody p-vaiue t

Cytomegalovirus 5 47% 0.74

Epstein-Barr virus 13 85% 0.24

Varicella zoster virus 12 98% 0.46

Respiratory syncytial virus 13 95% 1.00

Herpes simplex virus 9 72% 1.00

Influenza A virus 13 93% 0.78

Influenza B virus 13 93% 0.78

Rubeola meastest 9 99/105 0.01 - ion bβfbra 1857 (h"7) 4 19/19 0.01 - tan in or after 1957 fø*β) 5 80/85 0.39

Mumps 12 93% 1.00

Rubella 11 93% 0.46

- Statistically significant, oe=0.05

• Data for reference population proviαeo by cαranerάal laboratory

(Wold AD. Mayo Cδriic, Rochester, MR ISA. Personal communication.) t Binomial test, except for rabeoia/measies (Fisher's exact test).

Avaflabϋfty of reference popuiarion data did not permit Fishery exact calculations for an viruses. X Subjects bom before 1957 were less ϋkeiy to have received measles vaccine (licensed in the

USA in 1963) and more Ukery to have bad natural exposure to measles (ACtP. MMWR 1989;

38CS-9)rl-18).

Claims

What is claimed is:

1. A method for identifying a human subject as having an increased risk of developing essential hypertension, comprising determining the presence in the subject of a genotype of the subject's CCR5 gene that correlates with an increased risk of developing essential hypertension, the presence of the genotype identifying the subject as having an increased risk of developing essential hypertension.

2. The method of claim 1, wherein the genotype comprises the CCR5- Δ32/Δ32 genotype.

3. A method for identifying a human subject as having an increased risk of developing essential hypertension, comprising: a) correlating the presence of a genotype of the subject's CCR5 gene with an increased risk of developing essential hypertension; and b) determining the genotype of the subject's CCR5 gene, whereby a subject having a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension is identified as having an increased risk of developing essential hypertension.

4. The method of claim 3, wherein the genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension comprises the CCR5-Δ32/Δ32 genotype.

5. A method of identifying a genotype of the CCR5 gene correlated with an increased risk of developing essential hypertension comprising: a) determining the nucleic acid sequence (genotype) of the CCR5 gene from a subject; and b) correlating the presence of the nucleic acid sequence (genotype) of step (a) with the presence of essential hypertension in the subject, whereby the nucleic acid sequence (genotype) of the CCR5 gene identifies a genotype correlated with an increased risk of developing essential hypertension.

6. A method for identifying an agent that enhances the activity of CCR5, comprising:

(a) contacting a cell that expresses CCR5 with the agent;

(b) measuring the amount of CCR5 activity in the cell of step (a); and (c) comparing the amount of CCR5 activity measured in the cell of step (a) with the amount of CCR5 activity in control cells not contacted with the agent, whereby an amount of CCR5 activity in the cells of step (a) greater than the amount of CCR5 activity in the control cells identifies an agent that enhances the activity of CCR5.

7. A method for identifying an agent that enhances the expression of CCR5, comprising:

(a) contacting a cell that expresses CCR5 with the agent;

(b) measuring the amount of CCR5 expression in the cell of step (a); and

(c) comparing the amount of CCR5 expression measured in the cell of step (a) with the amount of CCR5 expression in control cells not contacted with the agent, whereby an amount of CCR5 expression in the cells of step (a) greater than the amount of CCR5 expression in the control cells identifies an agent that enhances the expression of CCR5.

8. A method of treating or preventing essential hypertension in a subject comprising administering to a subject an effective amount of CCR5 in a pharmaceutically acceptable carrier, thereby treating or preventing essential hypertension in the subject.

9. A method of treating or preventing essential hypertension in a subject, comprising administering to a subject an effective amount of a CCR5 activity enhancing agent in a pharmaceutically acceptable carrier, thereby treating or preventing essential hypertension in the subject.

10. A method of treating or preventing essential hypertension in a subject, comprising administering to a subject an effective amount of a CCR5 expression enhancing agent in a pharmaceutically acceptable carrier, thereby treating or preventing essential hypertension in the subject.

11. A method of treating or preventing essential hypertension in a subject comprising administering to a subject an effective amount of a nucleic acid encoding CCR5 under conditions whereby the nucleic acid is expressed in a cell in the subject, thereby treating or preventing hypertension.