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WO2009034661A1 - Procédé de diagnostic et induction d'une résistance à un virus - Google Patents

Procédé de diagnostic et induction d'une résistance à un virus Download PDF

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Publication number
WO2009034661A1
WO2009034661A1 PCT/JP2007/068591 JP2007068591W WO2009034661A1 WO 2009034661 A1 WO2009034661 A1 WO 2009034661A1 JP 2007068591 W JP2007068591 W JP 2007068591W WO 2009034661 A1 WO2009034661 A1 WO 2009034661A1
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Prior art keywords
virus
snp
hiv
subject
resistance
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PCT/JP2007/068591
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English (en)
Inventor
Yasuyoshi Kanari
Masaaki Miyazawa
Shinji Irie
Mario S. Clerici
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Toppan Printing Co., Ltd.
Immunoclin Ltd.
Osaka Industrial Promotion Organization
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Application filed by Toppan Printing Co., Ltd., Immunoclin Ltd., Osaka Industrial Promotion Organization filed Critical Toppan Printing Co., Ltd.
Priority to PCT/JP2007/068591 priority Critical patent/WO2009034661A1/fr
Priority to PCT/JP2008/067213 priority patent/WO2009035170A1/fr
Publication of WO2009034661A1 publication Critical patent/WO2009034661A1/fr

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    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention is related generally to the analytical testing of the samples obtained from a subject, and more particularly the method for detecting genetic predisposition of a subject to resistance to virus.
  • the invention also provides a chip and kit for detecting such genetic predisposition.
  • the invention further provides an application of identified genetic predisposition to induce resistance to virus infection.
  • biomarkers such as single nucleotide polymorphisms (SNPs), nucleotide insertion mutations, nucleotide deletion mutations, microsatellite markers or other DNA sequence variations.
  • SNPs single nucleotide polymorphisms
  • nucleotide insertion mutations nucleotide insertion mutations
  • nucleotide deletion mutations microsatellite markers or other DNA sequence variations.
  • inherited polymorphisms and somatic mutations in oncogenes or tumor suppressor genes are widely regarded as being indicative of a susceptibility to certain cancers, especially in view of the associations between mutated oncogenes or deleted tumor suppressor genes and certain cancers.
  • some individuals are highly susceptible or resistant to infection, especially viral infection.
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • HIV- 1 human immunodeficiency virus type 1
  • HIV-I seroconversion Reference 1, O'Brien et al., "Polygenic and multifactorial disease gene association in man", Annu. Rev. Genet., 2000, vol.34, pp.563-591; Reference 2, O'Brien et al., "Human genes that limit AIDS", Nat. Genet., 2004, vol.36, pp.565-574).
  • MHC major histocompatibility complex
  • AIDS restriction genes can explain only up to 15 % of the host determinants that control HIV- I/AIDS (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574; Reference 10, Fellay, J. et al., "A whole-genome association study of major determinants for host control of HIV-I", Science, Published online July 19 2007, 10.1126/science.1143767), and the presence of other resistance genes has been predicted (Reference 2, O'Brien et al., "Human genes that limit AIDS", 2004, Nat. Genet., vol.36, pp.565-574).
  • HIVl -exposed but uninfected or exposed seronegative (ESN) individuals who have evidence of multiple and repeated exposures to HIV-I through unprotected sexual contacts, but nevertheless possess no serum IgG reactive to the viral antigens (Reference 11, Beyrer, C. et al., "Epidemiologic and biologic characterization of a cohort of human immunodeficiency virus type 1 highly exposed, persistently seronegative female sex workers in northern Thailand", J.Infect.Dis., 1999, vol.79, pp.59-68; Reference 12, Liu H.
  • the inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 30, WO2004/035825, International publication of PCT application, 29 April 2004).
  • the inventors of the present invention genotyped 74 HIV-exposed but uninfected and 77 HIV-infected individuals enrolled from the same geographical region.
  • SNPs are significantly associated with ESN individuals under a dominant gene hypothesis at the CardlO, CDC42EP1 and GRAP2 ⁇ oci (Reference 31, WO2006/067506, International publication of PCT application, 29 June 2006).
  • exact molecular genetic mechanisms that explain the observed resistance of ESN individuals to HIV-I acquisition had not been provided.
  • Croker, B.A. et al. "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376-3386.
  • Biasin M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide -like 3G: a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964.
  • the inventors of the present invention performed expression analyses of all the genes and open reading frames located in the candidate segment of human chromosome 22 and extensive genome sequencing, and identified previously unforeseen association between certain sequence polymorphisms in the Rac2 gene locus and the ESN (HIV- 1 -exposed but uninfected or exposed seronegative) status.
  • the sequence polymorphism associated with ESN confers a higher enhancer activity, and is associated with poor replication of CCR5-tropic HIV in peripheral blood mononuclear cells.
  • SNPs and specific haplotypes of them are representative of the above sequence polymorphism and thus predictive of a resistance to HIV-I infection, and these are designated by the following SNP ID Nos: rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73
  • the present invention provides:
  • An ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprising : a) genotyping a sample of the subject at a site of at least one SNP selected from the group consisting of SNPs designated by following SNP ID Nos: (i) rs739041, (ii) rs739042, (iii) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570; and b) assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP is a resistance allele, wherein the resistance allele is as follows (SNP ID No.
  • An ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprising: a) genotyping a sample of the subject at sites of SNPs designated by following SNP ID Nos: (i) rs2284037, (ii) rs739042 and (iii) rs73904i; and b) assessing the subject as having genetic predisposition to resistance to virus if a haplotype is present in the genotyped sample, wherein the haplotype comprises (SNP ID No. : allele): (i) rs2284037 : T; (ii) rs739042 : T; (iii) rs739041 : T.
  • An ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprising: a) genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (i ⁇ ) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (v ⁇ ) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582
  • [20] (6) A method for inducing or enhancing resistance to virus in a subject, comprising enhancing the Rac2 gene expression in the subject.
  • nucleic acid exhibiting the Rac2 enhancer activity wherein the nucleic acid is selected from the following (A) and (B):
  • A a nucleic acid having the nucleotide sequence of SEQ ID No ⁇ 32; and (B) a nucleic acid having more than 80% identity with the nucleotide sequence of SEQ ID No:32.
  • a method for screening a candidate therapeutic agent against virus comprising: a) contacting an agent with a nucleic acid fragment having a partial or entire nucleotide sequence of the nucleic acid according to the above (10) or (ll); b) measuring an interaction between the agent and the nucleic acid fragment; and c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
  • a method for screening a candidate therapeutic agent against virus comprising: a) administering an agent to a biological material; b) measuring an expression level of the Rac2 gene in the biological material; and c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.
  • a method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus comprising: a) detecting genetic predisposition of subjects by the method according to any one of the above methods (l) - (5); b) stratifying the subject by the result of the detected genetic predisposition; c) giving a treatment to the stratified subject! and d) assessing the effect of the prevention, reduction, prophylaxis or treatment to the stratified subject.
  • each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO ' 6, 4, 3, 26, 11, 7 and 12.
  • a chip used in the method according to the above method (2) comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs2284037, (ii) rs739042 and (iii) rs739041, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO ⁇ 3, 4 and 6.
  • a chip used in the method according to the above method (3) comprising a base plate and probe sets detecting SNPs and nucleotide polymorphisms designated by the SNP ID Nos (i) ss73405466, ( ⁇ ) rs9798725, (iii) ss73405467, (iv) rs5995400, (v) rs6000619, (vi) rs5756570, (vii) rs36110509, (viii) rs2899284, Gx) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) ss73405477, (xviii) ss73405479, (xix
  • a chip used in the method according to the above method (4) comprising a base plate and probe sets detecting SNPs designated by the SNP ID Nos (i) rs9610683 and (ii) rs9610682, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO ⁇ l and 2.
  • a chip used in the method according to the above method (5) comprising a base plate and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos ⁇ (i) rs9610683, (ii) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (x ⁇ ) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii) s
  • kits used in the method according to the above method (l), comprising a reagent and at least one probe detecting SNP selected from the group consisting of SNPs designated by the SNP ID Nos (i) rs739041, (ii) rs739042, (in) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570, wherein each probe of the probe sets comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: 6, 4, 3, 26, 11, 7 and 12.
  • kits used in the method according to the above method (5) comprising a reagent and at least one probe detecting SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos: (i) rs9610683, (ii) rs9610682, (iii) rs2284036, (iv) rs9798725, (v) ss73405467, (vi) rs5995400, (vii) rs36110509, (viii) rs2899284, (ix) rs6000618, (x) rs6000617, (xi) rs9610677, (xii) rs6000616, (xiii) rs9610676, (xiv) rs9610675, (xv) rs9622582, (xvi) ss73405476, (xvii)
  • Figure 1 is an illustration of the distribution of SNPs and nucleotide polymorphisms in the Rac2 locus.
  • Figure 2 shows a result of DNA microarray analysis comparing the changes in gene expression levels during the HIV-I antigenic stimulation in peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • Figure 3 is a coefficient map of linkage disequilibrium (LD) between each pair of SNP alleles.
  • Figure 4 is a graph showing luciferase activities induced by the Rac2 promoter and the polymorphic region 1 or 2 indicated in Figure 1.
  • Figure 5 is a graph showing changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides.
  • Figure 6 is a graph showing HIV-I p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the Rac2 region 1 genotypes.
  • Figure 7 is an illustration of locations and spans of known genes and open reading frames in the candidate region in human chromosome 22.
  • Figure 8 is a table showing allele distribution at known SNP loci in chromosome 22 among the ESN and HIV-1-infected individuals.
  • Figure 9-Al, Figure 9-A2, Figure 9 ⁇ 3, Figure 9-A4, Figure 9-Bl, Figure 9-B2, Figure 9-B3, Figure 9-B4, Figure 9-Cl, Figure 9-C2, Figure 9-C3, Figure 9-C4, Figure 9"Dl, Figure 9-D2, Figure 9-D3 and Figure 9-D4 are tables showing genotypes at all detected polymorphic loci between the CardlO and U2rb loci and the results of case-control analyses between the ESN and HIV-1-infected individuals.
  • AIC Akaike Information Criterion.
  • the present invention provides an ex vivo method, a chip and a kit detecting genetic predisposition of a subject to resistance to virus. Further, the present invention provides a method, biopharmaceutical and nucleic acid for inducing or enhancing resistance to virus in a subject.
  • virus examples include, but are not limited to, Human immunodeficiency virus (HIV), Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV) and Influenza (Flu) virus.
  • HIV Human immunodeficiency virus
  • HPV Human papilloma virus
  • HCV Hepatatis C virus
  • HHV Human herpes virus
  • CMV Human herpes virus
  • CMV Cytomegalovirus
  • SRSV Small round structured virus
  • Influenza virus Influenza virus
  • the virus is HIV, HPV or HCV. More preferably, the virus is HIV. HIV includes HIV-I and HIV-2.
  • genetic predisposition refers to genetic factors which influence the phenotypes of an organism. However, the effect of the genetic predisposition can be modified by environmental conditions.
  • the term "subject”, as used herein, refers to an object for the method for detecting genetic predisposition.
  • the subject is a primate such as human, monkey and ape. More preferably, the subject is human. Most preferably, the human is Caucasian.
  • the term "resistance to virus”, as used herein, refers to an act or power to prevent virus infection or virus-induced disease progression.
  • the present invention is based on the research demonstrating that particular SNPs and haplotypes located in the Rac2 gene region are significantly associated with ESN (HIV- 1-exposed but uninfected or exposed seronegative) individuals. Summary of the research is described below.
  • the inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423.
  • the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Figure 8 for complete listing of the observed genotypes).
  • the present invention provides use of the above listed SNPs and nucleotide sequence polymorphisms for diagnosis of resistance to HIV infection.
  • rs739041 (i) rs739042, (iii) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570 are more significant for detecting genetic predisposition to resistance to virus.
  • the SNP ID Nos are identification numbers used in dbSNP which is world's largest database for nucleotide variations and is part of the US National Center for Biotechnology Information (NCBI).
  • the "rs” ID number is an identification tag assigned by NCBI to a group of SNPs that map to an identical location. The rs ID number is assigned after submission.
  • the "ss” ID number is simply a unique identifier assigned by NCBI when SNP is submitted to dbSNP.
  • the above described SNP genotypes are highly linked to each other within each of the above Rac2 regions, with the levels of LD being higher among the ESN than in HIV-infected individuals in the region 2 ( Figure 3).
  • the polymorphic loci within the regions 1 and 2 constitute small numbers of haplotypes.
  • FIG. 1 illustrates the observed haplotypes divided in three blocks.
  • Haplotypes of the left block consist of SNPs designated by SNP ID Nos. rs9610683 and rs9610682.
  • Haplotypes of the central block consist of SNPs designated by SNP ID Nos. rs2284037, rs739042 and rs739041.
  • Hapotypes of the right block consist of SNPs and nucleotide polymorphisms designated by SNP ID Nos.
  • ss73405466 rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss73405484 and ss73405485.
  • the haplotype CA in the left block, TTT in the central block, and the haplotype (GT+CAC— -TTTACATCTTTGCAAA") in the right block were significantly accumulated in ESN individuals.
  • the present invention provides use of the above three haplotypes for diagnosis of resistance to HIV infection.
  • the genomic fragments harbouring each of the polymorphic regions 1 and 2 isolated from representative healthy control individuals possessing homozygous R/R or S/S haplotype were cloned and inserted into the downstream of the luc gene along with the known Rac2 promoter in the upstream.
  • the genomic DNA fragment corresponding to the region 1 was placed downstream of the luc gene, the expression levels of the luciferase was higher than that induced with the promoter alone, indicating that the Rac2 region 1 is an enhancer of the gene expression.
  • the present invention provides novel enhancer having improved activity enhancing the Rac2 gene expression.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • p24 concentrations were significantly lower as compared to what observed in supernatants of homozygous S/S cells upon infection with either of the two CCR5-tropic HIV-I isolates ( Figure 6).
  • p24 concentrations in the supernatants of cells infected with CXCR ⁇ tropic HIV clone or isolate were not different between the S/S and R-harbouring cells.
  • the Rac2 region 1 haplotype R is functionally more active, and is associated with a restricted replication of CCR5"tropic HIV-I.
  • Rh2 is required for the migration of Bl lymphocytes, a specific subpopulation of B-lymphocytes, from the peritoneal cavity to gut-associated lymphoid tissues (Reference 27, Croker, B.A. et al., "The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-Ia and marginal zone B lymphocytes", J.Immunol., 2002, vol.168, pp.3376"3386), and Bl-derived plasma cells are the predominant sources of pathogen-reactive mucosal IgA (Reference 28, Fagarasan, S.
  • this virus resistance mechanism mediated by the Rac2 gene expression also has effect against all other viruses infected via mucosal route such as Human papilloma virus (HPV), Hepatatis C virus (HCV), Human herpes virus (HHV), Cytomegalovirus (CMV), Small round structured virus (SRSV), Influenza (Flu) virus and the like.
  • HPV Human papilloma virus
  • HCV Hepatatis C virus
  • HHV Human herpes virus
  • CMV Cytomegalovirus
  • SRSV Small round structured virus
  • Influenza Influenza virus and the like.
  • SNP associated with differences in the course of HCV infection is located in the region close to the Rac2 gene (Reference 32, Saito, T., et al., "Genetic variations in humans associated with differences in the course of hepatitis C", Biochemical and Biophysical Research Communication (BBRC), 2004, vol.317, pp.335-341).
  • the present invention provides an ex vivo method, a chip and a kit for detecting genetic predisposition of a subject to resistance to virus.
  • the present invention provides a method and a biopharmaceutical (Rac2 protein or the Rac2 gene inducer) for inducing or enhancing resistance to virus in a subject by utilizing virus resistance mechanism mediated by Rac2.
  • a biopharmaceutical Rost2 protein or the Rac2 gene inducer
  • the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing Rac2 or the Rac2 regulatory region as a target molecule. And the present invention provides use of the Rac2 enhancer region for a gene therapy to induce resistance to virus in a subject.
  • the ex vivo method for detecting genetic predisposition of a subject to resistance to virus comprises following steps (a) and (b): the step (a) is genotyping a sample of the subject at a site of at least one SNP or nucleotide polymorphism selected from the group consisting of SNPs and nucleotide polymorphisms designated by following SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73
  • step (b) is assessing the subject as having genetic predisposition to resistance to virus if an allele at the site of genotyped SNP or nucleotide polymorphism is a resistance allele, wherein the resistance allele is as follows (SNP ID No. : allele) : rs9010683 : C; rs9610682 : A; rs2284037 : T; rs739042 : T; rs2284036 : T; rs739041 : C; ss73405466 : G; rs9798725 : T; ss73405467 : GGCCTCATCCTTCCAAGTTC
  • AAGTTCAG AAGTTCAG; rs5995400 : C; rs6000619 : C; rs5756570 : G; rs36110509 : gap, ' rs2899284 : T; rs6000618 : T; rs6000617 : T; rs9610677 : A; rs6000616 : C; rs9610676 : A; rs9610675 : T; rs9622582 : C; ss73405476 : T; ss73405477 : T; ss73405479 : T; rs5756568 : G; rs933223 : T; rs933222 : A; rs933321. : A; ss73405482 : A; ss73405484 : gap; and ss73405485 : TCCCATAATCCAGGTGGGAGGAACC
  • rs739041 (i) rs739042, (iii) rs2284037, (iv) rs933223, (v) rs6000619, (vi) ss73405466 and (vii) rs5756570 are more significant for detecting genetic predisposition to resistance to virus.
  • the term "genotyping” refers to a process of determining the genetic constitutions such as nucleotide sequence of an individual with a biological assay.
  • sample refers to a biological sample or material obtained from a subject, for example a cell, tissue, organ, blood, hair or the like.
  • site refers to a position or location of specific nucleotide or polynucleotide in the genome.
  • nucleotide polymorphism refers to difference of nucleotide sequence between members of a species (or between paired chromosomes in an individual).
  • SNP single nucleotide polymorphism
  • allele refers to a particular form of a gene or DNA sequence at a specific chromosomal location (locus).
  • the ex vivo method of the present invention comprises (a) genotypying a sample of the subject at sites of SNPs and nucleotide polymorphism constituting a haplotype; and (b) assessing the subject as having genetic predisposition of resistance to virus if the haplotype which is significantly accumulated in ESN individuals is present.
  • haplotype refers to a set of closely linked genetic markers present on one chromosome which tends to be inherited together.
  • the haplotypes significantly accumulated in ESN individuals are following three haplotypes :
  • a haplotype comprising: (SNP ID No. : allele) (i) rs9610683 : C; and (ii) rs9610682 : A;
  • a haplotype comprising: (SNP ID No. : allele) (i) rs2284037 : T; (ii) rs739042 : T; and (iii) rs739041 : T; and
  • a haplotype comprising: (SNP ID No. : allele) (i) rs9010683 : C; (ii) rs9610682 : A; (iii) rs2284036 : T; (iv) rs9798725 : T; (v) ss73405467 : GGCCTCATCCTTCCAAGTTCAAGTTCAG; (vi) rs5995400 : C; (vii) rs36110509 : gap; (viii) rs2899284 : T; (ix) rs6000618 : T; (x) rs6000617 : T; (xi) rs9610677 : A; (xii) rs6000616 : C; (xiii) rs9610676 : A; (xiv) rs9610675 : T; (xv) rs9622582 : C; (xvi) ss734054
  • any techniques publicly known in the art can be used, including single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC), direct DNA sequencing, Invader assay, and TaqMan assay.
  • SSCP single-strand conformation polymorphism
  • DPLC denaturing high-performance liquid chromatography
  • direct DNA sequencing Invader assay
  • TaqMan assay TaqMan assay
  • SNP or nucleotide polymorphism can be detected by the single-strand conformation polymorphism (SSCP) analysis in which electrophoretic separation of single -stranded nucleic acids based on subtle differences in sequence (often a single base pair) results in a different secondary structure and a measurable difference in mobility through a gel (Reference 33, Orita, M. et al., "Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction", Genomics, 1989, vol.5(4), pp.874-879).
  • SSCP single-strand conformation polymorphism
  • nucleotide polymorphism can be detected by the heteroduplex analysis by denaturing high-performance liquid chromatography (DHPLC) that is based on DNA heteroduplex formation and separation of heteroduplex from homoduplex molecular species by means of ion-pair reverse phase HPLC (Reference 34, Oefner, P.J. et al., "DNA mutation detection using denaturing high performance liquid chromatography", Current protocols in human genetics, 1998, p.7 10:1-7 10 12).
  • SNP or nucleotide polymorphism can be detected by direct DNA sequencing, such as the chain termination method (Reference 35, Sanger, F. et al., "DNA sequencing with chain-terminating inhibitors", Proceedings of the National Academy of Sciences of the United States of America (PNAS), 1977, vol.74(l2), pp.5463-5467), which are well known in the art.
  • a subsequence of the Rac2 gene region encompassing the SNPs and nucleotide polymorphisms is amplified and either cloned into a suitable plasmid and then sequenced, or sequenced directly.
  • SNP can be detected by Invader assay.
  • Invader assay three single -stranded DNA chains form a ternary complex with one base-pair overlap.
  • This complex is composed of a DNA target oligonucleotide, which contains the SNP sequence of interest, and two other oligonucleotides.
  • the upstream oligonucleotide is designated as invader oligo and downstream oligonucleotide is designated as a probe.
  • These three oligonucleotide strands hybridize to one another, forming a one base-pair junction causing the 5' end of the probe to form a unhybridized 5' flap.
  • This 5' flap is then cleaved by a Cleavase enzyme, resulting in the release of the 5' flap of the probe.
  • FRET cassette is labeled with a fluorophore (F) and a quencher (Q) so cleavage between them generates a fluorescence signal.
  • FRET cassette is self hybridized at 5' portion, and 5' flap is hybridized with 3' portion of the FRET cassette so as to form a ternary complex with one base-pair overlap.
  • Cleavase enzyme cleavage of 5' end of FRET cassette by Cleavase enzyme causes fluorescence signal (Reference 36, U.S. Patent No. 5,846,717; Reference 39, Lyamichev, V.
  • SNP can be detected by TaqMan assay which uses two types (wild type and mutant type) of probes having fluorescence dye and quencher and utilizes 5 1 to 3' nuclease activity of Taq polymerase. DNA fragment harboring SNP site is amplified by PCR primer. On the process of the amplification, TaqMan probe anneals to the DNA.
  • the TaqMan probe is degraded by the 5' to 3' nuclease activity of the Taq polymerase. With the degradation of the TaqMan probe, fluorescence dye is separated by quencher and fluorescence is generated. Where there is a mismatch between the Taqman probe and DNA, the probe is not degraded, and fluorescence does not occur. SNP can be detected by measurement of this fluorescence (Reference 37, Holland, P.M.
  • the present invention provides a method for inducing or enhancing resistance to virus in a subject. This method comprises a step of enhancing the Rac2 gene expression in the subject.
  • the step of enhancing the Rac2 gene expression can be performed by any technique known in the art, for example, modulation of the Rac2 enhancer region by using a DNA-binding protein, peptide, oligonucleotide, nucleotide analogue or chemical compound. And RNAi technique may be used for inhibiting an expression of the Rac2 repressor in order to enhance the Rac2 gene expression.
  • the present invention provides use of Rac2 protein in the preparation of a medicament for a treatment or prophylaxis of virus-induced disease.
  • the present invention provides a method for treatment or prophylaxis of virus-induced disease comprising administering Rac2 protein to a subject.
  • Rh2 protein refers to a full length polypeptide of Rac2 protein, its homologue or functional fragment thereof.
  • the amino acid sequence and coding sequence of Rac2 is registered in Genbank under accession number NM_002872.
  • the present invention provides a nucleic acid exhibiting the Rac2 enhancer activity (the polymorphic region l) which is selected from the following nucleic acids ' • (A) a nucleic acid having the nucleotide sequence of SEQ ID No:32, ' and (B) a nucleic acid having more than 80%, 90% or 95% identity with the nucleotide sequence of SEQ ID No:32.
  • the present invention provides a nucleic acid exhibiting improved Rac2 enhancer activity, which has specific alleles at the sites of SNPs in the nucleotide sequence of SEQ ID No.32 as follows (base number in the SEQ ID No.32 : aUele): (i) 132 : C; (ii) l44 : A; (iii) 1276 : T; (iv) 1959 : T; (v) 2301 :T; and (vi) 2379 : T.
  • the term "identity”, as used herein, refers to a percentage of identical nucleic acid bases among two nucleotide sequences which are aligned in high order match. The percentage of the identity can be calculated by BLAST program under default setting (Reference 40, Altschul, S. F. et al, "Gapped BLAST and PSI-BLAST: A new generation of protein database search programs", Nucleic Acids Res., 1997, vol.25, pp.3389-3402).
  • the nucleic acid may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 38).
  • PNAs peptide nucleic acids
  • Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry.
  • the nucleic acid of the present invention can be used as a Rac2 enhancer and can be utilized for a gene therapy in order to enhance the Rac2 gene expression and induce resistance to virus in a subject.
  • the present invention provides a method for screening a candidate therapeutic agent against virus by utilizing the Rac2 enhancer as a target molecule. This method comprises following steps: (a) contacting an agent with a nucleic acid fragment having the partial or entire nucleotide of the Rac2 region 1 nucleic acid of the present invention; (b) measuring an interaction between the agent and the nucleic acid fragment! and (c) selecting the agent having the interaction with the nucleic acid fragment as a candidate therapeutic agent against virus.
  • a candidate therapeutic agent refers to a chemical compound, lead compound or biological molecule which has a potential activity to induce resistance to virus in a subject.
  • the present invention provides a method for screening a candidate therapeutic agent against virus. This method comprises the following steps: (a) administering an agent to a biological material; (b) measuring an expression level of the Rac2 gene in the biological material; and (c) selecting the agent enhancing expression level of the Rac2 gene in the biological material as a candidate therapeutic agent against virus.
  • biological material refers to the material which possesses a fragment of or the entire Rac2 gene or the Rac2 gene regulatory element, for example, DNA fragment, a cell, a tissue, an organ, an organism and individual.
  • the present invention provides a method for performing clinical trial for prevention, reduction, prophylaxis or treatment against virus. This method comprises following steps: (a) detecting genetic predisposition of subjects by the detecting method of the present invention,' (b) stratifying the subject by the result of the detected genetic predisposition;
  • phase I phase II
  • phase III phase III clinical trials
  • stratifying the subject refers to arrangement of each individual in a clinical trial population in a hierarchical order according to any status such as resistance level to virus infection or virus disease progression.
  • the present invention provides a chip or a kit for detecting genetic predisposition of a subject for resistance to virus.
  • the chip comprises base plate and at least one probe detecting SNP or nucleotide polymorphism.
  • the kit comprises reagent and at least one probe detecting SNP or nucleotide polymorphism.
  • the SNP or nucleotide polymorphism is selected from the group consisting of SNPs and nucleotide polymorphisms designated by the SNP ID Nos rs9610683, rs9610682, rs2284037, rs739042, rs2284036, rs739041, ss73405466, rs9798725, ss73405467, rs5995400, rs6000619, rs5756570, rs36110509, rs2899284, rs6000618, rs6000617, rs9610677, rs6000616, rs9610676, rs9610675, rs9622582, ss73405476, ss73405477, ss73405479, rs5756568, rs933223, rs933222, rs933321, ss73405482, ss
  • the probes detecting each SNP or nucleotide polymorphism designated by each SNP ID Nos comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID NO: l (for rs9610683), 2 (for rs9610682), 3 (for rs2284037), 4 (for rs739042), 5 (for rs2284036), 6 (for rs73904l), 7 (for ss73405466), 8 (for rs9798725), 9 (for ss73405467), 10 (for rs5995400), 11 (for rs6000619), 12 (for rs5756570), 13 (for rs36110509), 14 (for rs2899284), 15 (for rs6000618), 16 (for rs6000617), 17 (for rs9610677), 18 (for rs6000616), 19 (for rs9610676), 20 (for rs9610675), 21
  • sequences designated by SEQ ID Nos 1 - 31 represent flanking region sequences harboring each SNP or nucleotide polymorphism designated by each SNP ID Nos.
  • the term "chip” refers to a tool or instrument for biological analysis having a portable size. The scope of the "chip” includes a plate or array used in biological experiments.
  • base plate refers to a material holding a probe or probe sets.
  • reagent refers to any chemical compound, solution or protein for a reaction detecting SNP or nucleotide polymorphism.
  • examples of the reagent include, but are not limited to, polymerase, Clevase enzyme, restriction enzyme, ligase, fluorescent dye, quencher, reaction buffer, hybridization buffer or etc.
  • the probe sequence may comprise DNA, RNA or nucleic acid analogs such as uncharged nucleic acid analogs including, but not limited to, peptide nucleic acids (PNAs) which are disclosed in International publication WO 92/20702 (Reference 38).
  • PNAs peptide nucleic acids
  • Such sequences can routinely be synthesized using variety of techniques currently available. For example, a sequence of DNA can be synthesized using conventional nucleotide phosphoramidite chemistry. Once synthesized, oligonucleotide probes may be labeled by any well-known methods.
  • the probe comprises more than 7 bases of partial sequence or partial complementary sequence of SEQ ID Nos 1 - 31.
  • the probe has 7 to 100 bases, more preferably, 10 to 50 bases, and most preferably, 15 to 30 bases.
  • the probe can comprise any sequence other than partial sequence or partial complementary sequence of SEQ ID Nos 1- 31.
  • the probe may comprise linker sequence fixed to the base plate or 5' flap sequence for invader reaction.
  • the probe is used for detecting SNP or nucleotide polymorphism in any form.
  • the probe can be used as "invader oligo" or “probe” in the invader assay described above.
  • the probe can be used as "TaqMan probe”.
  • ESN ESN, their HIV-1-infected partner, and unexposed control individuals were enrolled with written informed consents as described previously (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024), and genetic analyses were performed with an approval from the ethical committee of the Kinki University School of Medicine. PBMC were stimulated with a mixture of HIV-I Gag and Env peptides as described (Reference 18, Clerici, M.
  • Genomic DNA was amplified with primers (enlisted in the SEQUENCE LISTING under SEQ ID Nos: 33 - 72) and sequenced. Case-control analyses of the observed genotypes, calculation of the coefficients of LD, and extraction of haplotype compositions were performed by using the SNPAlyze ver. 5.1 (DYNACOM Co., Ltd., Yokohama, Japan). Putative Rac2 regulatory regions were cloned into the pGL3 plasmid (Promega Corporation, Madison, U.S.A.), transfected into human Jurkat cells, and the Dual-Luciferase Reporter Assays (Promega) were performed. The in vitro infection of PBMC with HIV-I and measurements of p24 were performed as described elsewhere (Reference 29, Biasin, M. et al., J. Infec. Dis., 2007, vol.195, pp.960-964).
  • the inventors of the present invention previously mapped an ESN-associated gene locus in a segment of human chromosome 22 harbouring the microsatellite markers D22S277, D22S272, and D22S423 (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HlVl-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015-1024; Reference 30, WO2004/035825, International publication of PCT application, 29 April 2004).
  • the inventors of the present invention genotyped 74 ESN and 77 HIV-infected individuals enrolled from the same area of Italy at multiple loci of known SNP (see Figure 7 for their chromosomal locations and Figure 8 for complete listing of the observed genotypes).
  • PBMC peripheral blood mononuclear cells
  • Total RNA was prepared from antigen-stimulated PBMC as described (Reference 29, Biasin, M. et al., "Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide -like 3G : a possible role in the resistance to HIV of HIV-exposed seronegative individuals", J.Infec.Dis., 2007, vol.195, pp.960-964), and cDNA was produced in the presence of an RNase inhibitor by using the T7-oligo(dT)24 primer. The resultant cDNA was purified, and biotinylated cRNA was prepared by using Biotin-16UTP and MEGAscript transcription kit (Ambion, Inc., Austin, U.S.A.).
  • Two to 10 oligodeoxynucleoti.de probes were designed for each of the genes enlisted in Figure 7 by using the TargetSpecifier (CombiMatrix Corporation, Mukilteo, U.S.A.) and synthesized on microarray chips. After prehybridization, denatured biotin-conjugated cRNA samples were hybridized at 45 0 C, overnight. After washing and blocking, microarray chips were incubated with Cy3-conjugated streptavidin and washed vigorously. A fluorescence image of each microarray was scanned by a GenePix (Molecular Devices Corporation, Union City, U.S.A.), and analyzed by using the Microarray Imager (CombiMatrix Corporation).
  • TargetSpecifier CombiMatrix Corporation, Mukilteo, U.S.A.
  • LD between SNP was analyzed by likelihood ratio test as described (Reference 17, Kanari, Y. et al., "Genotypes at chromosome 22ql2-13 are associated with HIV-1-exposed but uninfected status in Italians", AIDS, 2005, vol.19, pp.1015- 1024), and exact P values were obtained.
  • the inventors of the present invention sequenced the chromosomal segments spanning the Myh9, I12rb, Rac2-Pscd4, CardlO, and Lgalsl loci (see Figure 7 for the location of the sequenced segments), and compared the frequencies of ESN and HIV-1-infected individuals possessing a particular allele at each of the observed polymorphic loci.
  • the inventors of the present invention detected no sequence polymorphisms with a significant case -control difference at the Myh9, I12rb, and Lgalsl loci.
  • the SNP genotypes were highly linked to each other within each of the above Rac2 regions, with the levels of LD being higher among the ESN than in HIV-infected individuals in the region 2 ( Figure 3).
  • the polymorphic loci within the regions 1 and 2 constitute small numbers of haplotypes.
  • Figure 3 shows distribution of observed SNP with significant case-control differences by adopting either a dominant (red lines) or a recessive (blue lines) hypothesis. P values shown are those calculated by ⁇ 2 (chi-square) test. See Figure 9 for odds ratios and l values calculated by Fisher's exact test. A representative value is shown for each cluster of SNP with significant case -control differences in the Rac2 locus, with the upper and lower brackets corresponding respectively to the regions 1 and 2 in Figure 1. Coefficients of LD between each pair of SNP alleles are shown here in /lvalues and colour-scaled by using SNPAlyze ver. 5.1.
  • Figure 1 shows distribution of sequence polymorphisms (arrows) in the Rac2 locus with those showing significant case -control differences between ESN and HIV-infected groups shown in red. Longer arrows indicate SNP with case-control differences of P ⁇ 0.006. Chromosomal regions showing >50 % sequence homologies between humans and mice are indicated with horizontal lines labelled HH for high homology. In the polymorphic region 2, there were also gaps (small red squares in the diagram) and base deletions (-)' ⁇ ⁇ l, a 28-bp deletion (-) relative to the database -reported genome sequence (+); ⁇ 2, an 82-bp insertion (+) relative to the reported genome sequence (-); and ⁇ 3, a 34"bp insertion (+).
  • Haplotypes in each of the three blocks are those identified by the Four Gamete method using SNPAlyze ver. 5.1, and all observed linkages between each haplotype across the blocks are shown. Exact frequencies of sequenced chromosomes with each observed linkage are shown only for the major ones indicated with thick lines. The numbers of individuals possessing a particular haplotype are compared between the two groups by Fisher's exact test.
  • the above polymorphic regions 1 and 2 colocalized with the regions that showed >50 % sequence homology between humans and mice ( Figure l), indicating that these polymorphic regions may contain functional regulatory elements.
  • the genomic fragments harbouring the polymorphic region 1 or 2 isolated from representative R/R and S/S healthy control individuals were cloned and inserted into the downstream of the luc gene along with the known Rac2 promoter (Reference 25, Ladd, PD. et al., "Identification of a genomic fragment that directs hematopoietic- specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333) in the upstream.
  • the Rac2 promoter is known to be strong and promiscuous (Reference 25, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333), the luc constructs harbouring the region 1 fragment induced significantly higher luciferase activities in transfected human Jurkat cells than that containing the promoter alone ( Figure 4), indicating the presence of an enhancer element within this genomic fragment. More importantly, the region 1 fragment of the R haplotype induced significantly higher luciferase activities than that of the S halotype, revealing a functional difference between the two haplotypes.
  • the region 2 fragments apparently reduced the expression of the luc gene regardless of the haplotype, in consistency with the previously indicated presence of a repressor in this region (Reference 25, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333).
  • the region 1 haplotypes were also associated with in vivo expression levels of the Rac2 in HIV-I antigen-stimulated PBMC.
  • levels of the Rac2 expression were examined quantitatively by real-time PCR in cultured PBMC, a 2.5-fold higher level of the Rac2 message was seen after 6 hours of stimulation with the HIV-I peptides in the individuals possessing the homozygous R/R haplotype in region 1.
  • levels of the Rac2 expression did not change after the antigenic stimulation in cells homozygous for the S haplotype in region 1 ( Figure 5).
  • haplotypic differences in the region 2 did not affect the Rac2 induction after antigenic stimulation, again indicating that the polymorphic region 1, but not the region 2, harbours a functional enhancer of the Rac2 expression.
  • the Rac2 region 1 haplotypes also affected HIVl replication in vitro.
  • PBMC from healthy control individuals possessing the homozygous S/S hap Io type or harbouring the R hap Io type in region 1 were infected in vitro with HIV-I, different behaviours were observed.
  • FIG. 5 shows changes in the expression levels of the Rac2 gene in PBMC stimulated with the HIV-I peptides.
  • Real-time PCR analyses were performed with samples obtained from 10 ESN and 6 HIV-I -infected individuals, which were prepared as described for Figure 2, and the results were re-grouped based on the separately determined region 1 and region 2 genotypes. Data are shown here as ratios of the Rac2 expression between 1 and 6 hours after the antigenic stimulation calculated as 2 MCT , and bars indicate S.E.M.
  • Statistical analyses were done by two-tailed Welch's ttest.
  • Figure 6 shows replication of HIV-I in cultured PBMC. Uninfected healthy control individuals were genotyped with written informed consent for the Rac2 region 1 and region 2 haplotypes, and their PBMC were infected with each of the 4 different HIV-I clones or isolates, or with the dual-tropic isolate 89.6. None of the individuals included here possessed the CCR5 ⁇ 32 allele. Data shown here are p24 concentrations measured at 3 and 5 days after virus inoculation grouped by the region 1 genotypes, with horizontal bars indicating median values.
  • the 2504-bp region 1 and 8904-bp region 2 genomic fragments were amplified by PCR using the following oligonucleotide primers and LA-Taq polymerase (TAKARA BIO, Inc., Ohtsu, Japan):
  • the cloned Rac2 genomic fragments were inserted to the SaR restriction site located downstream of the luc gene within the pGL3 plasmid (Promega Corporation, Madison, U.S.A.) harbouring the previously described Rac2core promoter (-260 to +130 bp) (Reference 25, Ladd, RD. et al., "Identification of a genomic fragment that directs hematopoietic-specific expression of Rac2 and analysis of the DNA methylation profile of the gene locus", Gene, 2004, vol.341, pp.323-333).
  • Each of the resultant reporter constructs along with the pRL-TK were cotransfected into 1.0 to 1.2 x 10 6 human Jurkat cells using an amaxa Nucleofector (amaxa AG, Cologne, Germany) with the solution V and condition C- 17 according to the manufacturer's recommendations. After 18, 21 and 24 hours, cells were lysed and expressed luciferase activities were measured using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's protocol. [112] Method of RNA Extraction and Real-Time PCR Analyses
  • PBMC peripheral blood mononuclear cells
  • HIV-I p24 Absolute levels of HIV-I p24 were measured using the Alliance HIV-I p24 ELISA kit (PerkinElmer Inc., Waltham, U.S.A.). HIV-I BaL and IIIB were provided by Drs. S. Gartner, M. Popovic, and R. Gallo (Courtesy of the NIH AIDS Research and Reference Reagent Program). HIV-I primary isolates were kind gifts from Prof. C. -F. Perno, University of Roma, Tor Vergata, Italy.

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Abstract

La présente invention concerne un procédé ex vivo, une puce et un kit détectant la prédisposition génétique d'un sujet à la résistance à un virus. Selon l'invention, le procédé, la puce et le kit utilisent des SNP et des polymorphismes nucléotidiques spécifiques accumulés dans des individus ESN ('Exposed SeroNegative' : exposés au VIH-1 mais non infectés) en tant que marqueurs de résistance au virus. L'invention concerne en outre des moyens préventifs et thérapeutiques de conférer une résistance à une infection virale.
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