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WO1999023253A1 - Facteur-1 derivant de cellules stromales et son procede d'utilisation a des fins de diagnostique et de pronostique de pathogeneses du sida - Google Patents

Facteur-1 derivant de cellules stromales et son procede d'utilisation a des fins de diagnostique et de pronostique de pathogeneses du sida Download PDF

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WO1999023253A1
WO1999023253A1 PCT/US1998/022578 US9822578W WO9923253A1 WO 1999023253 A1 WO1999023253 A1 WO 1999023253A1 US 9822578 W US9822578 W US 9822578W WO 9923253 A1 WO9923253 A1 WO 9923253A1
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sdf
ofthe
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aids
hiv
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PCT/US1998/022578
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Cheryl Ann Winkler
Stephen J. O'brien
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Priority to AU11207/99A priority Critical patent/AU1120799A/en
Publication of WO1999023253A1 publication Critical patent/WO1999023253A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates generally to HIV infection and more specifically to a stromal cell derived factor (SDF-1) variant that is associated with resistance to or decreased susceptibility to HIV infection.
  • SDF-1 stromal cell derived factor
  • the AIDS epidemic is characterized by considerable variation in the incidence of HIV-1 infection, in the rate of progression to CD4-T lymphocyte depletion, and in the development of AIDS .
  • Part ofthe explanation for epidemiologic heterogeneity involves genetic variation in human loci that encode cellular factors which participate in HIV-1 infection and pathogenesis.
  • mutations in the CCR5 and CCR2 structural genes have been shown to be associated with delay in the median time required to develop AIDS, based on screens of mutant alleles in HIV-1 exposed cohorts (Samson, Nature 382:722, 1996; Dean et al, Science 273: 1856, 1996; Huang et al, Nature Med. 2:1240, 1996; Michael et al, Nature Med. 3:338, 1997; Zimmerman et al, Mol.
  • the CCR5- ⁇ 32 deletion mutation specifies a reading frame shift that truncates the CCR5 protein, an obligate co-receptor with CD4 for HIV-1 infection of macrophages and monocytes which are the principal cell reservoir for early HIV-1 infection (Liu et al., Cell 86:367, 1996; McNicholl et al, Emerg. Infect. Dis. 3:261, 1997; Alkhatib et al., Science 272:1955, 1996; Deng, et al., Nature 381:661, 1996; Dragic et al., Nature 381:667, 1996; Choe et al., Cell 85:1135, 1996; Doranz et al., Cell 85: 1149, 1996; J.
  • CCR5 deletion mutation homozygotes (CCR5- ⁇ 32/ ⁇ 32) are almost completely resistant to HIV-1 infections, even among individuals who are at high risk for infection.
  • the mechanism of CCR2 genetic restriction is less obvious, but likely relates to the ability of CCR2 also to serve as a receptor for HIV-1 infection of macrophages, monocytes and T-cells (Schuitamaker et al., J. Virol. 64:356, 1991; Schuitemaker et ⁇ /. J Virol 66:1354, 1992; Asjo, Lancet ⁇ :660, 1986; Connor et al, J. Virol. 67:1772, 1993; Roos et al, J. Infect. Dis. 165:427, 1992; Zhu et al, Science 261:1179, 1993).
  • HIV-1 strains isolated from recently infected individuals are predominantly M-tropic (macrophage or monocyte lineage tropic), NSI (non-syncytium inducing), and co-opt CC-chemokine receptor proteins as entry ports in combination with CD4 molecules. Later in the course of HIV- 1 infection, near the time at which AIDS symptoms are observed, a preponderance of T-tropic (T-lymphocyte cell line tropic) strains have been recovered . T-tropic strains induce the formation of syncytia in CD4-positive cell lines, infect PBMCs faster, and replicate more aggressively than the early M- tropic isolates.
  • T-tropic H ⁇ V-1 enters target cells using both CD4 and CXCR4 as obligate co- receptors.
  • CXCR4 is preferred, however, the terms fusin or HFAF have also been used to refer to the same molecule.
  • SDF-1 Stromal derived factor
  • pre-B-cell growth stimulating factor a powerful chemoattractant cytokine
  • the present invention is based on the discovery of a correlation between the presence of a mutation at nucleotide position 801 of SDF-1 and resistance to HrV infection. Based on this discovery, it is an object ofthe present invention to provide diagnostic and therapeutic approaches for identifying the mutation and down-regulating the CXCR4 receptor, respectively.
  • the invention provides an isolated polynucleotide encoding a stromal cell derived factor- 1 (SDF-1) variant having a nucleotide sequence set forth in SEQ ID NO: 1.
  • SEQ ID NO: 1 shows the nucleotide sequence of wild-type SDF-1 , with a G to A transition mutation at position 801.
  • the invention provides a method for determining the prognosis of a subject exposed to HIV-1.
  • the method is based on determining the presence of a SDF-1 variant nucleic acid in cells ofthe subject and correlating the presence ofthe variant on both alleles with prognosis of said subject.
  • the SDF-1 variant described in the present invention is a recessive mutation, thus both alleles must exhibit the mutation to affect HIV-1 susceptibility.
  • the cells are preferably PBLs from the subject.
  • the subject is a human.
  • the invention provides a method of determining susceptibility of a subject to HP/ infection by determining the SDF-1 allelic profile of a subject.
  • the method includes isolating the SDF-1 nucleic acid sequence and determining the presence or absence of a mutation in SDF-1 nucleic acid.
  • the invention also provides a method of inhibiting membrane fusion between HIV and a target cell that expresses CXCR4 or between an HIV -infected cell and a CD4 positive uninfected cell that expresses CXCR4, including contacting the target or CD4/CXCR4 positive cell with a CXCR4 down-regulating effective amount of a SDF-1 variant, thereby inhibiting membrane fusion.
  • the contacting may be by in vivo administration to a subject or by ex vivo administration to a cell, for example.
  • the invention provides a method of treating a subject having or at risk of having an HIV infection or disorder by administering to the subject, a therapeutically effective amount of an SDF-1 variant, such as SEQ ID NO:l.
  • the subject treated by the method ofthe invention may be suffering from AIDS or ARC.
  • the invention provides a method of treating a subject having a disorder associated with expression of CXCR4 including administering to the subject, an SDF-1 variant that suppresses CXCR4.
  • the subject ofthe invention is well suited for preparation of a kit for determining the SDF-1 allelic profile of a subject.
  • the kit includes amplification primers or hybridization probes which detect a transition mutation of G to A at nucleotide 801.
  • Figure 1 A-I are graphs showing Kaplan-Meier survival curves demonstrating the effect ofthe SDF1-3 ⁇ /3A genotype on progression to AIDS-1993, AIDS-1987, and death in: the MACS cohort (panels A-C) ; Caucasians in the ALIVE, MACS, MHCS, SFCC combined cohorts (panels D-F) ; and all ethnic groups in the four combined cohorts (panels G-I) .
  • Figure 2A-F are bar graphs which define disease category analysis of SDFl -3 A allele (panels A-C) and genotype (panels D-F) frequencies for each cohort and combined cohorts for the three endpoints, AIDS- 1993 (panels A and D), AIDS- 1987 (panels B and E), and death (panels C and F). Cutoffs, in years, were chosen as the time approximately half of all seroconverters had progressed to the outcomes. Times for the cutoffs were: 1) AIDS-1993; 7.5 year; 2) AIDS-1987, 8.5 years; 3) Death, 9.5 years.
  • Figure 3 A-I are graphs of Kaplan-Meier survival curves for the four protective genotypes for SDF 1 , CCR2, and CCR5 versus wild-type [+/+] at the three loci.
  • the protective genotypes are: SDF1-3A/3 ; CCR2-[+/64I], [641/641]; and CCR5-+/ ⁇ 32.
  • the four curves represent the following genotypes; 1) blue-+/+ at SDFl, CCR2 and CCR5; 2) green- CCR2/5 protein: one or more CCR2/5 protective genotypes and SDF-+/+ ; 3) orange- SDFl : SDF1-3 /3*A and CCR2/5-+/+; 4) pink-SDFl and CCR2/5: SDF1-3 ⁇ /3 and protection by one or more CCR2/5 protective genotype versus +/+.
  • Figure 4A-C shows the frequencies ofthe protective SDF 1-3 'A/3' A genotype alone (black) or in combination with at least one CCR2/5 protective genotype (CCR5- +/ ⁇ 32, CCR2-+/64I, and CCR2-64I/64I, cross hatch) in six intervals of increasing survivorship from midpoint (seroconverters) or imputed (seroprevalents) seroconversion dates in Caucasians.
  • Genotypic frequencies were determined separately for time to AIDS- 1993 (panel A), AIDS- 1987 (panel B), and to death (panel C) using seroconverters progressing to the outcome in less than 3.5 years, and including seroconverters and seroprevalents progressing to the outcomes in 3.5 ⁇ 7 years, ⁇ 10 years, 10 ⁇ 13 years, and 13 ⁇ 16 years, and ⁇ 16 years.
  • Figure 5 shows the nucleotide sequence of SDF 1 -3 'A (SEQ ID NO : 1 ), which is the SDF-1 variant ofthe invention.
  • the present invention is based on the identification of a variant of SDF-1 which appears to correlate in some subjects to resistance to HIV-1 infection. This enables therapeutic, prophylactic, prognostic and diagnostic approaches to AIDS. Further, the finding of this variant is useful for therapeutic approaches to inflammatory disorders associated with the expression of CXCR4, the receptor for the wild-type SDF-1 ligand. De ⁇ nitions
  • Allele A gene present in more than one form (different sequence) in a genome, is said to have multiple alleles.
  • wt - Wt stands for the wild type allele of SDF-1 , namely the gene without the G to A transition mutation at position 801.
  • SDF1-3 ⁇ stands for the mutant allele of SDF-1, found at a frequency of about 0.21 in the Caucasian population; 0.162 in the hispanic population; 0.057 in the African American population; and Asians, -0.257. It is also alternatively described as "SDF-1 variant”.
  • the SDF-1 gene (wt or variant) is present, alike most eukaryotic genes, as two copies/genome. If both copies are genetically alike, in regard to the absence or presence ofthe G to A mutation at position 801, the individual is homozygous, i.e., he is either wt/wt or SDF-3A/3A. Since the mutation is recessive, a homozygous mutation only will provide a meaningful protection.
  • Heterozygous If one copy each ofthe wt allele and the SDF1-3 ⁇ allele are present in one genome, the individual having such a genome is heterozygous. Since the mutation is recessive, a heterozygous mutation is not believed to afford the individual meaningful protection from HIV-1 infection.
  • Allelic profile A determination ofthe composition of an individuals genome in regard to the presence or absence, and the copy number, ofthe SDF1-3 ⁇ allele.
  • the invention provides an isolated polynucleotide encoding a stromal cell derived factor (SDF-1) variant (SDFl -3 A) having a nucleotide sequence which differs from wild-type SDF-1 by a single mutation.
  • SDF-1 stromal cell derived factor
  • the variant of the invention having a G to A transition at nucleotide 801 (counting from the ATG start codon) is set forth in Figure 5 and SEQ ID NO: 1.
  • a "variant" as used herein refers to a nucleotide sequence that is altered as compared to the wild-type sequence.
  • An exemplary variant ofthe invention differs from wild-type SDF-1 by only one nucleotide although other nucleotide changes are also included as long as the SDF-1 allele still correlates with a decreased progression to AIDS and preferably is still is able to down-regulate the CXCR4 receptor.
  • isolated includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • Polynucleotide sequences ofthe invention include DNA and RNA sequences which encode SDF-1 and which have a G to A transition mutation at nucleotide 801. It is understood that all polynucleotides encoding all or a portion of SDF1-3 ⁇ , but which include nucleotide 801 , are also included herein, as long as they encode a polypeptide with SDF-1 activity (e.g., bind to and down-regulate CXCR4).
  • Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides.
  • wild-type SDF-1 polynucleotide may be subjected to site-directed mutagenesis to produce a G to A substitution at position 801.
  • the polynucleotides ofthe invention include sequences that are degenerate as a result ofthe genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of SDF-1 polypeptide encoded by the nucleotide sequence is functionally unchanged (e.g., down-regulates CXCR4).
  • the polynucleotide encoding SDF 1-3 A includes the nucleotide sequence in FIGURE 5 (SEQ ID NO:l), as well as nucleic acid sequences complementary to that sequence.
  • a complementary sequence may include an antisense nucleotide.
  • the sequence is RNA
  • the deoxyribonucleotides A, G, C, and T of FIGURE 5 are replaced by ribo- nucleotides A, G, C, and U, respectively.
  • fragments (portions) ofthe above-described nucleic acid sequences that are at least 15 bases in length, which is sufficient to permit the fragment to selectively hybridize to DNA ofthe variant SDF 1-3 'A.
  • the fragments ofthe invention encompass position 801 (e.g., SEQ ID NO:l).
  • Selective hybridization refers to hybridization under moderately stringent or highly stringent physiological conditions (See, for example, the techniques described in Maniatis et al, 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporated herein by reference), which distinguishes related from unrelated nucleotide sequences.
  • nucleic acid hybridization reactions the conditions used to achieve a particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree of complementarity, nucleotide sequence composition (e.g., GC v. AT content), and nucleic acid type (e.g., RNA v. DNA) of the hybridizing regions ofthe nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.
  • An example of progressively higher stringency conditions is as follows: 2 x SSC/0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC/0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC/0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68 °C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, or each ofthe conditions can be used, e.g., for 10-15 minutes each, in the order listed above, repeating any or all ofthe steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • DNA sequences ofthe invention can be obtained by several methods.
  • the DNA can be isolated using hybridization or computer-based techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of interest; 4) computer searches of sequence databases for similar sequences; and 5) differential screening of a subtracted DNA library.
  • hybridization or computer-based techniques which are well known in the art. These include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) antibody screening of expression libraries to detect cloned DNA fragments with shared structural features; 3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the DNA sequence of
  • the SDF 1-3 A polynucleotide ofthe invention is derived from a mammalian organism. Screening procedures which rely on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the sequence encoding the protein in question, can be synthesized chemically. This requires that short, oligopeptide stretches of amino acid sequence must be known. The DNA sequence encoding the protein can be deduced from the genetic code, however, the degeneracy ofthe code must be taken into account. It is possible to perform a mixed addition reaction when the sequence is degenerate. This includes a heterogeneous mixture of denatured double-stranded DNA.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
  • Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization ofthe target DNA to that single probe in the mixture which is its complete complement (Wallace, et al, Nucl Acid Res., 9:879, 1981).
  • the production of labeled single or double- stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies ofthe cDNA which have been denatured into a single- stranded form (Jay, et al, Nucl Acid Res., 1 L2325, 1983).
  • SDF 1-3 'A nucleic acid include intragenic mutations (e.g., point mutation, nonsense (stop), missense, splice site and frameshift) and heterozygous or homozygous deletions. Detection of such alterations can be done by standard methods known to those of skill in the art including sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization. Such proteins can be analyzed by standard SDS-PAGE and/or immuno- precipitation analysis and/or Western blot analysis, for example.
  • intragenic mutations e.g., point mutation, nonsense (stop), missense, splice site and frameshift
  • Detection of such alterations can be done by standard methods known to those of skill in the art including sequence analysis, Southern blot analysis, PCR based analyses (e.g., multiplex PCR, sequence tagged sites (STSs)) and in situ hybridization.
  • STSs
  • DNA sequences encoding SDF 1-3 A can be expressed in vitro by DNA transfer into a suitable host cell.
  • "Host cells” are cells in which a vector can be propagated and its DNA expressed.
  • the term also includes any progeny ofthe subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term "host cell” is used. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
  • the SDFl -3 A polynucleotide sequences may be inserted into a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation ofthe SDF1-3 ⁇ genetic sequences.
  • Such expression vectors contain a promoter sequence which facilitates the efficient transcription ofthe inserted genetic sequence ofthe host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection ofthe transformed cells.
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al, Gene ,56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521. 1988) and baculovirus-derived vectors for expression in insect cells.
  • the DNA segment can be present in the vector operably linked to regulatory elements, for example, a promoter (e.g., T7, metallothionein I, or polyhedrin promoters).
  • Polynucleotide sequences encoding SDF 1-3 'A can be expressed in either prokaryotes or eukaryotes.
  • Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences ofthe invention.
  • a variety of host-expression vector systems may be utilized to express the SDF 1-3 'A coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the SDF 1-3 A coding sequence; yeast transformed with recombinant yeast expression vectors containing the SDF1-3 ⁇ coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the SDFl -3 A coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the SDF 1-3 coding sequence; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenovirus, vaccini
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see e.g., Bitter et al, 1987, Methods in Enzymology 153:516-544).
  • inducible promoters such as pL of bacteriophage ⁇ , plac, pt ⁇ , ptac (pt ⁇ -lac hybrid promoter) and the like may be used.
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription ofthe inserted SDFl -3 A. coding sequence.
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol. 2, 1988, Ed. Ausubel et al, Greens Publish. Assoc. & Wiley Interscience, Ch. 13; Grant et al, 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch.
  • yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol.l 1, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.).
  • vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
  • Eukaryotic systems and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur.
  • Eukaryotic cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, phosphorylation, and advantageously, plasma membrane insertion ofthe gene product may be used as host cells for the expression of SDFl -3 A.
  • Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered.
  • the SDFl -3 A coding sequence may be ligated to an adenovirus transcription translation control complex, e.g., the late promoter and tripartite leader sequence.
  • the vaccinia virus 7.5K promoter may be used. (e.g. , see, Mackett et al, 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al, 1984, J. Virol. 49: 857-864; Panicali et al, 1982, Proc. Natl. Acad. Sci.
  • vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al, 1981 , Mol. Cell. Biol. 1 : 486). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription ofthe inserted cDNA does not require integration ofthe plasmid into the host's chromosome, thereby yielding a high level of expression.
  • These vectors can be used for stable expression by including a selectable marker in the plasmid, such as, for example, the neo gene.
  • the retro viral genome can be modified for use as a vector capable of introducing and directing the expression ofthe SDF1-3 A gene in host cells (Cone & Mulligan, 1984, Proc. Natl. Acad. Sci. USA 81:6349-6353). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.
  • host cells can be transformed with the SDF 1-3 A cDNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler, et al, Cell H:223, 1977), hypoxanthine-guanine phosphoribosyl fransferase (Szybalska & Szybalski, Proc. Natl Acad. Sci.
  • adenine phosphoribosyltransferase genes can be employed in tk-, hgprt " or aprt " cells respectively.
  • antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al friendship Natl Acad. Sci. USA 77:3567, 1980; O'Hare, et al, Proc. Natl Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
  • ODC ornithine decarboxylase
  • Eukaryotic cells can also be cotransformed with DNA sequences encoding the SDF1-3 ⁇ ofthe invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the he ⁇ es simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein, (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • the invention includes a method for determining the prognosis of a patient exposed to HIV-1.
  • the patient may be asymptomatic or symptomatic for infection with HIV-1.
  • the prognosis ofthe patient is ascertained by determining the presence of a SDF-1 variant nucleic acid in cells ofthe subject and correlating the presence ofthe variant on both alleles with prognosis ofthe subject.
  • Appropriate cells include those susceptible to infection by HIV-l, such as peripheral blood leukocytes. Identification ofthe presence of a SDFl -3 A variant present on both alleles can be determined routinely by PCR amplification of SDF-1 as described herein followed by sequence analysis.
  • the allelic profile of a patient can be determined by employment of PCR technology.
  • the target nucleic acid to be amplified by PCR would be either the SDF-1 RNA (formation of a cDNA) or, in a preferred embodiment, the SDF-1 gene.
  • Primers would be designed on sequences flanking the putative SDF1-3A mutation site. By judiciously choosing the primers one can obtain a fragment whose size is indicative of the presence or absence ofthe deletion. For example, fragments whose size are between 75 to 450 nucleotides are preferred, though PCR products shorter and longer are acceptable.
  • the size consideration relates mostly to the ability to visualize the product after separation on an agarose gel. One skilled in the art would recognize many variations on this motif.
  • the PCR reaction may contain labeled oligonucleotides to facilitate subsequent detection ofthe PCR product.
  • the label can be, for example, radiolabeled nucleotides, or biotin inco ⁇ orating nucleotides.
  • Another variation ofthe technique would employ slurries other than agarose, or filters, for size separation ofthe PCR product. According to such a diagnostic procedure employing PCR, either wt or SDF 1-3 A homozygote will produce one product of a discrete, expected size, while the heterozygous individual will be identified by production of two differently sized products.
  • the prognosis ofthe patient improves by the detection of SDF 1-3 A on both alleles, since the mutation is recessive. Further, it may be desirable to analyze a mutation in the CCR2 receptor and/or analyze a mutation in the CCR5 receptor.
  • CCR5 and CCR2 Common alleles within the coding region for the chemokine and M-tropic HIV-1 co- receptor genes, CCR5 and CCR2, have been shown to delay the rate of progression to AIDS (4-9).
  • the mutant alleles CCR5- ⁇ 32 and CCR2-64I are dominant, genetically independent, and equally protective. An estimated 25-30% of long-term survivors who remain AIDS-free for > 16 years can be attributed to a protective genotype for either CCR5- ⁇ 32 or CCR2-64I (6,9).
  • a survival analysis ofthe relative contributions of CCR5- ⁇ 32, CCR2-64I, and SDF1-3 ⁇ genotypes (Fig. 3, Table 2) reaffirm the protective effects of CCR2, CCR5 and SDFl mutant genotypes on progression to AIDS when the influence ofthe other protective loci are considered as confounding variables (29,33).
  • the method for determining prognosis can be used to monitor subjects infected with HIV-1 and can be used to assess the results in clinical trials for pharmaceuticals, vaccines and other therapies as well.
  • the invention provides a method of determining susceptibility of a subject to HIV infection by determining the SDF-1 allelic profile of a subject.
  • the method includes isolating the SDF-1 nucleic acid sequence and determining the presence or absence of a mutation in SDF-1 nucleic acid similar to the method described above.
  • AIDS and infection by HIV has been recognized primarily in "at risk” groups, including homosexually active males, intravenous drug users, recipients of blood or blood products, and certain populations from Central Africa and the Caribbean.
  • the AIDS syndrome has also been recognized in heterosexual partners of individuals in all "at risk” groups and in infants of affected mothers. Thus, these groups of individual, in addition to other lower risk groups would be ideally suited for practicing the method ofthe invention to determine susceptibility.
  • the method ofthe invention is typically performed as described above, by PCR amplification of nucleic acid containing SDF-1 as described herein followed by sequence analysis. As in the method above, it may be desirable to determine the CCR2 and/or CCR5 allelic profile ofthe subject, in addition to the SDF-1 allelic profile. As described above and in the Examples below, it is believed that the SDF-1 variant and the CCR2 and CCR5 mutations act in a protective manner and the effect is additive.
  • Diagnostics are enabled by the present invention in that it is recognized that the infection is inhibited or reduced by individuals homozygous in regard to the SDFl -3'A allele and disease progression is reduced in individuals also having a CCR2 and/or CCR5 mutation.
  • the amplification of a nucleic acid can be accomplished by one of a number of methods known to one skilled in the art. By way of example, amplification by PCR is described below.
  • the invention also provides a method of inhibiting membrane fusion between HIV and a target cell that expresses CXCR4 or between an HIV-infected cell and a CD4 positive uninfected cell that expresses CXCR4, including contacting the target or CD4/CXCR4 positive cell with a CXCR4 down-regulating effective amount of a SDF-1 variant, thereby inhibiting membrane fusion.
  • the SDF-1 variant is SDF1-3 ⁇ (SEQ ID NO:l) as described herein.
  • the contacting may be by in vivo administration to a subject or by ex vivo administration to a cell, for example. While not wanting to be bound by a particular theory, it is believed that down-regulation by SDF1-3 ⁇ ofthe invention blocks the emergence and spread of T-tropic HTV-1, which requires CXCR4 as a co-receptor with CD4 to other cells.
  • the invention provides a method of treating a subject having a disorder associated with expression of CXCR4 including administering to the subject, an SDF-1 variant that suppresses CXCR4.
  • disorders include not only disorders associated with HIV-1 infection, but also inflammatory disorders.
  • an SDF1-3 ⁇ polynucleotide to a subject, either as a naked, synthetic polynucleotide or as part of an expression vector, can be effected via any common route (oral, nasal, buccal, rectal, vaginal, or topical), or by subcutaneous, intramuscular, intra-peritoneal, or intravenous injection.
  • Pharmaceutical compositions ofthe present invention are advantageously administered in the form of injectable compositions.
  • a typical composition for such pu ⁇ ose comprises a pharmaceutically acceptable solvent or diluent and other suitable, physiologic compounds.
  • the composition may contain polynucleotide and about 10 mg of human serum albumin per milliliter of a phosphate buffer containing NaCl.
  • SDF 1-3 'A polynucleotides expression vectors can be encapsulated within liposomes using standard techniques. Cationic liposomes would be preferred for delivery of nucleic acids. A variety of different liposome compositions and methods for synthesis are known to those of skill in the art. See, for example, U.S. Patent No. 4,844,904, No. 5,000,959, No. 4,863,740 and No. 4,975,282, the respective contents of which are hereby inco ⁇ orated by reference.
  • Liposomes are sometimes targeted to the cell type or tissue of interest (here PBLs or CD4+ cells) by the addition to the liposome preparation of a ligand, usually a polypeptide, for which a corresponding cellular receptor has been identified.
  • a ligand usually a polypeptide, for which a corresponding cellular receptor has been identified.
  • a likely such ligand would be gpl20 or gp21, or fragments thereof.
  • Examples of a cell receptors previously targeted include folate receptor which has recently been identified as a prominent tumor marker, especially in ovarian carcinomas. KB cells are known to vastly overexpress the folate receptor. See Campbell et al, Cancer Res. 51:6125 (1991).
  • liposome targeting including transferrin, protein A, ApoE, P-glycoprotein, ⁇ 2 -macroglobin, insulin, asiolofetuin, asialoorosomucoid, monoclonal antibodies with a variety of tissue specificity, biotin, galactose or lactose containing haptens (monovalent and tri- antennary), mannose, dinitrophenol, and vitamin B 12 .
  • the ligands are covalently conjugated to a lipid anchor in either pre-formed liposomes or are inco ⁇ orated during liposome preparation. See Lee & Low, J. Biol. Chem. 269:3198 (1994); Biochim. Biophys.
  • the association ofthe SDF 1-3 A polypeptide of the invention with an agent as described above includes association with additional targeting agents.
  • additional targeting agents For example, in order to gain access to the cytoplasm, a nucleic acid based therapeutic must overcome the plasma membrane barrier.
  • viral fusion peptides facilitate the delivery of viral DNA into the cytoplasm by promoting viral membrane fusion with the plasma membrane. For recent reviews on this subject, see Stegmann et al, Ann. Rev. Biophys. Chem. 18 . : 187 (1989).
  • the hemagglutinin (trimer) HA peptide N-terminal segment (a hydrophobic helical sequence) is exposed due to a conformational change induced by acidic pH in the endosome (pH 5-6), inserts into the target membrane, and mediates the fusion between the virus and the target endosomal membrane.
  • acidic pH in the endosome pH 5-6
  • amphipathic helix-forming oligopeptides have been designed to imitate the behavior ofthe viral fusion peptide. See, for example, Haensler & Szoka, Biocon. Chem. 4:372 (1993).
  • Nuclear localization signal peptides when attached covalently to a macromolecule such as a protein, have been shown to facilitate their translocation into the nucleus. See Goldfarb et al, Nature 322:641 (1986); Shreiber et al, Med. Sci. 8:134-39 (1992).
  • cellular targeting by the polypeptide ofthe invention and nucleus targeting yet other agents could be delivered in the nucleus of specific cells, for example DNA molecules.
  • treatment of prevention of HIV- 1 infection is achieved by introduction into a patient of CD4+ cells or bone marrow cells derived from donors.
  • such donors would be homozygous in respect to SDF 1-3' A.
  • the data suggests that about 21 in 100 Caucasian individuals would be homozygous for the deletion allele.
  • Donors will furthermore be HLA-matched individuals. They preferably would be blood relatives.
  • the blood typing of a potential donor in terms of compatibility is well known to one skilled in the art. See, for example, Beatty et al, Transplantation, 45: 714-8 (1988).
  • CD4+ cells from lymphocytes are also well known to one skilled in the art. Generally, such purification involves use of an antibody directed to the CD4 epitope.
  • umbilical cord blood stem cells are employed for transplantation.
  • CD34+ stem cells are isolated (usually by use of antibodies) and used for transplantation.
  • Cord blood stem cells and CD34+ cells are better tolerated, i.e. host rejection is limited, when compared with rejection of bone marrow or CD4+ cells.
  • CD34+ and cord cell transplantation can be used for adult recipients. Furthermore, they are a preferred source of transplantation tissue for infants.
  • bone marrow cells or CD4+ blood cells are isolated from the patient himself.
  • the isolated cells are transfected with a vector engineered to express an SDFl -3 A oligonucleotide, and transfected cells are selected.
  • Vectors for transfection of eukaryotic cells are well known in the art.
  • Such vectors have an origin of replication which allows replication and maintenance in the transfected cell.
  • the origin of replication may be a viral origin of replication.
  • One often used viral origin of replication is the SV40 replication region.
  • the vector, to be useful should contain a marker so transfected cells can be selected.
  • Such a marker often is a drug resistance gene.
  • the neo gene confirming the resistance to G418 is often used.
  • transplantation as described above would be accompanied by on-going antiviral treatments and more specifically anti-HIV- 1 treatments.
  • standard transplantation methodologies would generally be employed, which may contain such additional treatments as temporary immune suppression or lymphocyte growth and cytokine stimulation.
  • all method of treatments embodied by the present invention, the SDF 1-3 A approach as described above or the presently described transplantation or antibody treatment would each benefit and are compatible with standard anti-HIV- 1 treatments.
  • any treatment ofthe invention is augmented by known anti-HIV treatments.
  • anti-HIV- 1 treatments known to date include use of modified oligonucleotides, use of specific proteases, and specific anti-viral RNA nucleases.
  • administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
  • the pharmaceutical compositions are preferably prepared and administered in dose units.
  • Solid dose units are tablets, capsules and suppositories.
  • different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
  • the administration ofthe daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
  • compositions according to the invention are preferably administered intravenously.
  • other routes of administration is within the scope ofthe inventor.
  • the pharmaceutical compositions can be administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle.
  • Suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, Science, 249: 1527(1990), which is inco ⁇ orated herein by reference.
  • compositions according to the invention may be administered locally or systemically.
  • therapeutically effective dose is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms ofthe disease and its complications. Amounts effective for this use will, of course, depend on the severity ofthe disease and the weight and general state ofthe patient.
  • dosages used in vitro may provide useful guidance in the amounts useful for in situ administration ofthe pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders.
  • Various considerations are described, e.g., in Gilman et al.
  • the invention provides a method of treating a subject having or at risk of having an HIV infection or disorder by administering to the subject, a therapeutically effective amount of an SDF-1 variant, such as SEQ ID NO:l.
  • the subject treated by the method ofthe invention may be suffering from AIDS or ARC.
  • Administration ofthe variant may be by standard techniques as described above.
  • the SDF-1 variant is introduced into the cell using a carrier, such as a vector. Administration can be in vivo or ex vivo.
  • SDF1-3 ⁇ nucleic acids into cells affected by a SDF-1 disorder for the pu ⁇ ose of gene therapy, can be achieved using a recombinant expression vector, such as a chimeric virus or a colloidal dispersion system, such as a targeted liposome (see above discussion).
  • a recombinant expression vector such as a chimeric virus or a colloidal dispersion system, such as a targeted liposome (see above discussion).
  • a recombinant expression vector such as a chimeric virus or a colloidal dispersion system, such as a targeted liposome
  • the subject ofthe invention is well suited for preparation of a kit for determining the SDF-1 allelic profile of a subject.
  • the kit includes amplification primers or hybridization probes which detect a transition mutation of G to A at nucleotide 801.
  • Such primers for example, as described in detail in the Examples, can easily be designed based on the publicly available sequence for SDF-1 (GenBank L36033).
  • SDF1-3 ⁇ A role for SDF1-3 ⁇ in HIV-1 infection was investigated by genotyping 2419 HIV-1 infected patients and 435 HIV-1 exposed uninfected individuals. No significant differences in SDFl allele or genotype frequencies were observed in comparisons of exposed (or at risk) uninfected (HIV-1-) vs. infected (HIV-1+) individuals in any of the cohorts.
  • a collection of 138 extremely high risk, exposed-uninfected individuals (those with documented receipt of clotting factor prior to March 1984 when HIV-1 screening commenced or with frequent sexual encounters with high risk partners) also showed SDFl allele frequencies not significantly different from those of HIV- 1- infected individuals.
  • Example 1 Survival Analysis To assess the influence of SDFl genotype on progression of HIV- 1 infected patients to AIDS, a survival analyses was performed on a group of 867 seroconverter patients (those whose date of HIV-1 infection could be estimated precisely since they enrolled in the cohort before converting from HIV- 1 -antibody negative to HIV- 1 -antibody positive) from four cohorts by comparing the rate of progression to AIDS among different SDFl genotypes (+/+; +/3 and 3 A/3 A) using a Cox proportional hazards model. Three AIDS endpoints reflecting advancing morbidity were evaluated: 1) AIDS- 1993 definition as stipulated by the CDC (Center for Disease Control, Morb. Mort. Wkly. Rep. 36, suppl.
  • Figure 1 A-I are graphs showing Kaplan-Meier survival curves demonstrating the effect ofthe SDF1-3 ⁇ 3A genotype on progression to AIDS-1993, AIDS-1987, and death in: the MACS cohort (panels A-C) ; Caucasians in the ALIVE, MACS, MHCS, SFCC combined cohorts (panels D-F) ; and all ethnic groups in the four combined cohorts (panels G-I) .
  • the analysis was limited to seroconverters with an interval of ⁇ 3 years between last seronegative and first seropositive HIV-1 test. The midpoint between these two dates was used to estimate the seroconversion date (Cox, J.R. Stat Soc. B 34: 87, 1972).
  • SDF1-3A/3 ⁇ genotype survival was compared to that of SDF1-+/3 ⁇ and SDF1-+/+ genotype survival.
  • SDFl +/3 survival was compared to SDF1- +/+ survival.
  • the RH value for the combined Caucasian cohort sample was 0.65 for AIDS-1993, 0.36 for AIDS-1987, and 0.24 for death (lower values indicate increased protection, Table 1).
  • the tendency to display increased protection in later stages of HIV- 1 infection was also seen in MACS and SFCC cohorts indicating that the protection increases as some patients progress to an AIDS-1993 definition (including drop of CD4 T lymphocytes to ⁇ 200 cells/mm 3 ) the earliest step for most infected patients. This gradation was extended when time to CD4 ⁇ 200 cells mm 3 (alone without AIDS disease or death) was used as an endpoint, since in this case SDF 1-3 A/3 A protection is barely detectable and not statistically significant.
  • Protective genotypes include: CCR2-64I protection (CCR5-+/+, CCR2-+/64I or 641/641, and SDFl -+/+); CCR5- ⁇ 32 protection (CCR5-+/ ⁇ 32, CCR2-+/+, and SDFl -+/+); SDFl -3 'A/3 A plus at least one protective CCR allele (SDFl -3 'A/3 'A plus either CCR5-+/ ⁇ 32 or CCR2-+/64I). Further, protective genotypes at either CCR5 or CCR2 are referred to as "CCR protection". This gradation indicates that the SDF- 1-3 'A/3 'A protection is cummulative over the course of HIV- 1 infection, and is possibly related to interference with the appearance of T-cell tropic HIV-1 populations.
  • FIG. 2A-F are bar graphs which define disease category analysis of SDFl -3 'A allele (panels A-C) and genotype (panels D-F) frequencies for each cohort and combined cohorts for the three endpoints, AIDS- 1993 (panels A and D), AIDS-1987 (panels B and E), and death (panels cand F). Seroconverters who progressed to the designated outcomes before the cutoff time were compared to seroconverters plus seroprevalents who survived outcome-free for at least that long.
  • Imputed seroconversion dates for the seroprevalent subgroup for MHCS, HGDS, and ALIVE were provided by the cohort investigators (Hilgartner et al, Am. J. Pediatr. Hematol Oncol. 15_:208, 1993; Goedert et ⁇ /., N. Engl. J. Med. 321:1141, 1989; Lederman, et al, J. Infect. Dis. 172:228, 1995; Vlahov et al, NIDA Research Monograph Series 103 (Public Health Service, Alcohol and Drug Abuse
  • RR denotes relative risk of rapid progression for unprotected (SDF1-+/+ or SDF1-+/3 ) patients as compared to SDF1-3 ⁇ /3A patients, among Caucasians in the combined cohorts; relative risks are calculated as case/control odds ratios taking slow progressors as controls; i.e., the risk for each category is the ration ofthe number of rapid progressors to the number of slow progressors. 95% confidence intervals are in parentheses. P value is Fisher's exact test.
  • the relative risk for AIDS occurrence ranged from 3.0-9.1 for the three AIDS endpoints (Fig. 2A-F). Not one SDF 1-3 A/3 A homozygote was found among 63 patients from MHCS and SFCC that progressed to AIDS (by any definition) within 7.5 years compared to 4-5% frequency of SDF1-3A/3 ⁇ homozygotes in those who avoid AIDS for 9.5 years or longer.
  • the results of both the survival (Fig. 1 A-I, Table 1) and the defined disease category analyses (Fig. 2) reveal a strong recessive SDF1-3 association with protection against the clinical consequences of HIV-l infection.
  • the mutant alleles CCR5- ⁇ 32 and CCR2-64I are dominant, genetically independent, and equally protective. An estimated 25-30%) of long-term survivors who remain AIDS-free for > 16 years can be attributed to a protective genotype for either CCR5- ⁇ 32 or CCR2-64I.
  • a survival analysis ofthe relative contributions of CCR5- ⁇ 32, CCR2-64I, and SDFl- 3'A genotypes (Fig. 3 A-I, Table 2) reaffirm the protective effects of CCR2, CCR5 and SDFl mutant genotypes on progression to AIDS when the influence ofthe other protective loci are considered as confounding variables.
  • Figure 3 A-I are graphs of Kaplan-Meier survival curves for the four protective genotypes for SDFl, CCR2, and CCR5 versus wild-type [+/+] on progression to AIDS-1993, AIDS-1987, and death in: the MACS cohort (panels A-C) ; Caucasians in the ALIVE, MACS, MHCS, SFCC combined cohorts (panels D-F) ; and all ethnic groups in the four combined cohorts (panels G-I).
  • the protective genotypes are: SDF1-3A/3A; CCR2-[+/64I], [641/641]; and CCR5-+/ ⁇ 32.
  • the four curves represent the following genotypes; 1) blue-+/+ at SDFl , CCR2 and CCR5; 2) green- CCR2/5 protein: one or more CCR2/5 protective genotypes and SDF-+/+ ; 3) orange- SDF1 : SDF1-3A/3 ⁇ and CCR2/5-+/+; 4) pink-SDFl and CCR2/5: SDF1-3A/3A and protection by one or more CCR2/5 protective genotype versus +/+.
  • the proportion of patients who progress rapidly or delay AIDS onset as a consequence o ⁇ SDFl was estimated by computing the attributable risk of SDFl genotypes in extremely rapid ( ⁇ 3.5 yrs) and long term survivor (>16 yrs) disease categories.
  • the SDF-1 gene contains four exons over a 5.6 Kb region of chromosome 1 Oql 1.1 (Tashiro et al, Science, 261:600. 1993).
  • Two alternatively spliced transcripts which specify SDF-1 ⁇ and SDF-1 ⁇ are made from the gene and the isomers differ by the foreshortening of four carboxy terminal amino acids in SDF-1 (Tashiro et al, Science, 261 :600.
  • p 0.05 for AIDS- 1993; p ⁇ 0.01 for AIDS- 1987 and for death; Kaplan Meier log likelihood test.
  • FIG. 4A-C shows the frequencies ofthe protective SDFl -3 'A/3 A genotype alone (black) or in combination with at least one CCR2/5 protective genotype (CCR5-+/ ⁇ 32, CCR2-+/64I, and CCR2-64I/64I, cross hatch) in six intervals of increasing survivorship from midpoint (seroconverters) or imputed (seroprevalents) seroconversion dates in Caucasians.
  • Genotypic frequencies were determined separately for time to AIDS-1993 (panel A), AIDS-1987 (panel B), and to death (panel C) using seroconverters progressing to the outcome in less than 3.5 years, and including seroconverters and seroprevalents progressing to the outcomes in 3.5 ⁇ 7 years, ⁇ 10 years, 10 ⁇ 13 years, and 13 ⁇ 16 years, and ⁇ 16 years.
  • the number of individuals observed in each category is shown above the column.
  • the average frequency ofthe protective genotype for Caucasians is shown as an arrow.
  • SDFl -3 A/3 A homozygotes postpone AIDS onset raises several issues about the mechanism of viral restriction and AIDS pathogenesis.
  • the SDF 1 - 3'A mutation is located in the 3' untranslated region ofthe SDFl ⁇ gene transcript .
  • a screen of 8 homozygous SDF 1-3 A/3 A individuals for mutations in the four exons did not reveal any additional polymo ⁇ hisms. Absence of intragenic variants is not su ⁇ rising since the SDFl gene is highly conserved among mammals with only one amino acid difference observed between human and mouse homologues.
  • the 3 'UTR variant may influence mRNA transcript synthesis, persistence, transport, splice product abundance or response to transcription factors as have been reported for other 3'UTR systems (Mellors et al, Science 272:1167. 1996; O'Brien et al. . Am. Med. Assoc. 276:105, 1996).
  • the SDFl gene specifies alternatively spliced transcripts SDF-1 and SDF-l ⁇ that differ by the loss in SDF- l ⁇ of four carboxy terminal amino acids and in their 3 'UTR sequence. The development of specific reagents to assess these chemokine gene products and their transcripts would permit the investigation of SDFl genotype influence on SDF-1 protein availability and function.
  • SDF1-3 ⁇ /3A protection makes a prediction that the SDF 1 -3 A specific protein is more effective than the SDF- 1 + product in restricting late stage T-tropic viral load, a strong correlate and prognostic indicator of pathogenesis.
  • the SDFl alteration may impede AIDS onset simply by reducing availability of requisite CXCR4 co-receptors to HIV-l by up-regulation of SDFl , by increasing SDF-1 transcript stability, or by other interactions.
  • Such mechanisms would also account for the gradation in survival outcomes whereby SDF 1-3 'A 3 A effects are more pronounced in late stage outcomes (AIDS-1987 and death) than are evident with earlier stages of HIV-l infection (Fig. 1 A-I).
  • SDFl protection is recessive, making the prevalence of protected individuals below 5%>.
  • the combination of CCR2 or CCR5 plus SDFl protection is even more powerful (Fig. 4), but also very rare ( ⁇ 2% in non African ethnic groups and ⁇ 0.1 %> in
  • Table 1 shows a survival analysis for progression to 3 AIDS endpoints among HIV-l infected seroconverters for SDF 1-3 'A/3 'A versus SDF1-+/+ or SDF-+/3 ⁇ genotypes as in Figure 1 A-I.
  • Seroconverters for the ALIVE, MACS, SFCC, MHCS, and combined cohorts including only Caucasians, and for the combined cohorts with all ethnic groups included were analyzed using the Cox proportional hazards model.
  • the HGDS cohort was excluded since all the participants were HIV-l infected prior to study entry.
  • ALIVE is not analyzed as a separate cohort because the combination of the recency of cohort (1988) plus the low allele frequency among African Americans which comprise 94% ofthe cohort resulted in too few SDF 1-3 A/3 A AIDS outcomes to be statistically robust.
  • a log likelihood test (ldf) (LL), p value, and relative hazard (RH) were calculated for each variable in the analysis of AIDS outcomes. Time to AIDS-1993, AIDS-1987 and death were calculated from the midpoint ofthe last HIV- 1 negative test date and the first HIV-l positive test date. Seroconverters with an interval greater than 3 years between last negative and first positive were excluded from the analysis.
  • Table 2 shows a survival analysis of protection from progression to AIDS outcomes by SDF 1-3 'A/3 'A variant, CCR5 or CCR2 protective polymo ⁇ hisms, and a second analysis of any protection by any variant at CCR5, CCR2 and SDFl .
  • the analyses using the Cox proportional hazards model were performed as in Table 1.
  • Protective genotypes at SDFl were considered to be 3 ⁇ /3 ⁇ vs. 3A/+ or +/+; CCR2; 641/641 or 641/+ vs. +/+; CCR5- +/ ⁇ 32 or ⁇ 32/ ⁇ 32 VS. +/+.
  • the SDFl genotypes were analyzed three ways: 1) SDF1- 3'A/3'A versus SDF1-+/+ or SDF1-+/3 controlling for the protective genotypes of CCR2 and CCR5. 2) CCR2-641/641 or CCR2-+/641 or CCR5-+/ ⁇ 32 versus CCR5-+/+ and CCR2-+/+ (normal at tow loci) controlling for the protective genotype of SDFl . 3) SDF 1 -3 'A/3 A and/or one or more protective CCR2/5 genotypes versus +/+ at all three loci.

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Abstract

L'invention porte sur une séquence d'acide nucléique présentant une seule mutation de nucléotide dans la région 3' non traduite du transcript de l'ARNm du gène structurel pour le facteur dérivé du stroma (SDF1-3'A). Ladite mutation apparaît à une fréquence d'allèle de 6-26 % dans différents groupes raciaux. Le SDF-1, ligand principal du CXCR4, et récepteur de la transmembrane 7 à couplage G, qui, avec le CD4, constitue une voie d'entrée pour le VIH-1 T tropique, variété qui se développe fréquemment chez les patients atteints du SIDA juste avant la déplétion des lymphocytes CD4 T. L'invention porte également sur un procédé d'établissement de pronostiques pour un sujet exposé au VIH-1 et sur un procédé déterminant la susceptibilité d'un sujet à une infection par le VIH-1.
PCT/US1998/022578 1997-10-30 1998-10-23 Facteur-1 derivant de cellules stromales et son procede d'utilisation a des fins de diagnostique et de pronostique de pathogeneses du sida WO1999023253A1 (fr)

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Cited By (7)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6511826B2 (en) 1995-06-06 2003-01-28 Human Genome Sciences, Inc. Polynucleotides encoding human G-protein chemokine receptor (CCR5) HDGNR10
US6743594B1 (en) 1995-06-06 2004-06-01 Human Genome Sciences, Inc. Methods of screening using human G-protein chemokine receptor HDGNR10 (CCR5)
US6759519B2 (en) 1995-06-06 2004-07-06 Human Genome Sciences, Inc. Antibodies to human G-protein chemokine receptor HDGNR10 (CCR5receptor)
US6800729B2 (en) 1995-06-06 2004-10-05 Human Genome Sciences, Inc. Human G-Protein chemokine receptor HDGNR10 (CCR5 receptor)
US7160546B2 (en) 1995-06-06 2007-01-09 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
WO2001027330A3 (fr) * 1999-10-12 2001-12-27 Univ Texas Criblage de la susceptibilite d'une maladie par genotypage des genes ccr5 et ccr2
US7393634B1 (en) 1999-10-12 2008-07-01 United States Of America As Represented By The Secretary Of The Air Force Screening for disease susceptibility by genotyping the CCR5 and CCR2 genes
US7175988B2 (en) 2001-02-09 2007-02-13 Human Genome Sciences, Inc. Human G-protein Chemokine Receptor (CCR5) HDGNR10
US7393934B2 (en) 2001-12-21 2008-07-01 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10
US7501123B2 (en) 2004-03-12 2009-03-10 Human Genome Sciences, Inc. Human G-protein chemokine receptor (CCR5) HDGNR10

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