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1. WO2020109866 - ASSOCIATION ENTRE L'INTÉGRATION DE GÉNOMES VIRAUX DE VPH OU DE VIH ET LA GRAVITÉ ET/OU LE RÉSULTAT CLINIQUE DE TROUBLES TELS QUE LES LÉSIONS CERVICALES ASSOCIÉES AU VPH OU LES PATHOLOGIE DU SIDA

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CLAIMS

Claim 1. A method for detecting a level of integrated viral DNA or dormant DNA, such as proviral DNA, comprising:

removing episomal viral or vector nucleic acids from genomic DNA in a cell sample, and quantifying a number of integrations of viral DNA into the genomic DNA of the cells by a method of amplification of an integration region in the DNA sample;

thereby detecting a level of integrated viral DNA in the genomic DNA from the cell sample; and

optionally, performing a method of amplification on episomal nucleic acids removed from the sample.

Claim 2. A method according to claim 1 wherein the amplification method is quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH) or molecular combing.

Claim 3. The method of claim 1 or 2, wherein said step of removing comprises:

permeabilizing cell membranes in the cell sample by exposing the cells to an extracting salt solution for a time and under conditions sufficient to make the cellular membranes permeable to episomal viral or vector nucleic acids from the cells and for the episomal viral or vector nucleic acids to leak out of the cells into a medium,

separating the permeabilized cells from episomal viral or vector nucleic acids in the medium, and

performing qPCR on the separated permeabilized cells; and

optionally, isolating and performing qPCR on the episomal viral or vector nucleic acids in the medium.

Claim 4. The method of any one of claims 1 to 3, wherein said removing comprises: embedding cells into an agarose plug having an agarose concentration ranging from about 0.5 wt% to 1.5 wt%,

infusing the plugs containing the embedded cells with a proteolytic enzyme for a time and under conditions sufficient to substantially digest cellular proteins,

washing the plugs for a time and under conditions sufficient to remove episomal nucleic acids,

extracting cellular genomic DNA caught in the plugs and

performing qPCR on the genomic DNA extracted from the washed agarose plugs performing qPCR on the separated permeabilized cells; and

optionally, isolating and performing qPCR on the nucleic acids washed out of the agarose plugs.

Claim 5. The method of any one of claims 1 to 4, comprising partially depleting episomal HPV nucleic acids by biotinylating episomal nucleic acids and binding them (strept)avidin, thus removing them from a mixture of genomic and episomal nucleic acids.

Claim 6. The method of any one of claims 1 to 5, comprising:

isolating nucleic acids from the cell sample using a plasmid purification column, recovering genomic DNA from material eluting from the plasmid purification column, and

performing qPCR on the recovered genomic DNA; and, optionally,

isolating and performing qPCR on the material bound to the plasmid purification column.

Claim 7. The method of any one of claims 1 to 6, wherein the cell sample contains viral

DNA.

Claim 8. The method of any one of claims 1 to 7, wherein the cell sample contains HIV nucleic acids.

Claim 9. The method of any one of claims 1 to 7, wherein the cell sample contains human papilloma virus (HPV) nucleic acids.

Claim 10. The method of claim 9, further comprising detecting by PCR a ratio of DNA encoding HPV E2 ORF to DNA encoding HPV E6 ORF, especially by qPCR ; wherein the ratio of DNA encoding E2 ORF to DNA encoding E6 ORF represents an amount of an episomal form in relation to an integrated form.

Claim 1 1. The method of claim 10, wherein the ratio ofE2 ORF to E6 ORF is determined by real-time PCR using a set of primers and probes described by Table 1.

Claim 12. The method of any one of claims 9 to 11, further comprising determining HPV viral load comprising determining a ratio between E6/E7 and beta globin, MSH2 or at least one other human gene target.

Claim 13. The method of any one of claims 9 to 12, further comprising detecting at least one biomarker that covers a junction between a disrupted host gene and at least part of an integrated HPV DNA.

Claim 14. The method of claim 9, further comprising detecting at least one biomarker selected from the group consisting of MAPK10 (Gene ID: 5602), PTPN13 (Gene ID: 5783), NUDT15 (Gene ID: 55270), MED4 (Gene ID: 29079), ITM2B (Gene ID: 9445), RBI (Gene ID:

5925), LPAR6 (Gene ID: 10161), RAB1 1A (Gene ID: 8766), RPL13A (Gene ID: 23521), ZNF341 (Gene ID: 84905), OFD1 (Gene ID: 8481), DHRS3 (Gene ID: 9249), TBC1D22B (Gene ID: 55633), AFF3 (Gene ID: 3899), CXCL6 (Gene ID: 6372), PF4V1 (Gene ID: 5197), IMMP2L (Gene ID: 83943), MMP12 (Gene ID: 4321), WDR20 (Gene ID: 91833), ALDHA1A (Gene ID: 216), TPRG1 (Gene ID: 285386), TUBD1 (Gene ID: 51174), MAST4 (Gene ID: 375449), LOCI 00132167, NFIX (Gene ID: 4784), CCAT1 (Gene ID: 100507056), GPR137B (Gene ID: 7107), RAB22A (Gene ID: 57403), C9orf3, MACROD2 (Gene ID: 140733), DACH1 (Gene ID: 1602), ATP 10A (Gene ID: 57194), SPG11 (Gene ID: 80208), SORD (Gene ID:

6652), COL4A4 (Gene ID: 1286), GATSL1 (Gene ID: 729438), GATSL2 (Gene ID: 729438), MAP2 (Gene ID: 4133), EPN1 (Gene ID: 29924), ATXN3L (Gene ID: 92552), EGFL6 (Gene ID: 25975), and MAGI2 (Gene ID: 9863).

Claim 15. The method of claim 13, wherein forward and reverse oligonucleotide primers that specifically amplify an integration junction between host genomic DNA and integrated HPV DNA are used to produce the biomarker.

Claim 16. The method of claim 13, wherein the biomarker is human OFD1

(NG_008872.1) and is produced and the forward and reverse primers described by Table 2 are used to amplify an OFD1 gene/HPV16 and the HPV16/OFD1 gene junctions as biomarkers.

Claim 17. The method of claim 13, wherein at least one specific nucleic acids sequence complementary to an integration junction between host genomic DNA and integrated HPV DNA is used for the detection by hybridization of a biomarker.

Claim 18. The method of claim 13, wherein said HPV DNA is selected from a DNA from the group consisting of HPV strains 16, 18, 21 , 33, 45, 52 and 58.

Claim 19. A composition comprising one or more biomarkers as defined in any one of claims 15 or 16.

Claim 20. The composition of claim 19, wherein said one or more biomarkers is selected from the group consisting of MAPK10, PTPN13, NUDT15, MED4, ITM2B, RBI, LPAR6, RAB11A, RPL13A, ZNF341, OFD1, DHRS3, TBC1D22B, AFF3, CXCF6, PF4V1, IMMP2F, MMP12, WDR20, AFDHA1A, TPRG1 , TUBD1, MAST4, FOC100132167, NFIX, CCAT1 , GPR137B, RAB22A, C9orf3, MACROD2, DACH1 , ATP10A, SPG1 1, SORD, COF4A4, GATSF1, GATSF2, MAP2, EPN1, ATXN3F, EGFF6, and MAGI2.

Claim 21. A kit comprising standardized and purified biomarkers that hybridize with host cell DNA and with integrated viral DNA sequences, and optionally, control reagents, one or more other reagents, supplies and/or equipment useful for detecting viral integration, in particular a kit comprising one or more biomarkers of claim 20.

Claim 22. A method of preparation of genomic DNA containing suspected integrated viral DNA from the cell sample comprising:

embedding cells into an agarose plug having an agarose concentration ranging from about 0.5 wt% to 1.5 wt%,

infusing the plugs containing the embedded cells with a proteolytic enzyme for a time and under conditions sufficient to substantially digest cellular proteins,

washing the plugs for a time and under conditions sufficient to remove episomal nucleic acids,

extracting cellular genomic DNA caught in the plugs containing or not the integrated viral DNA.

Claim 23. _ A method for assessing a risk of having or developing a cervical cancer comprising: detecting or quantifying a number of integrations of HPV DNA into, or an integration pattern of HPV DNA in a sample of genomic DNA obtained from a patient or subject, thereby assessing the risk of having or developing cervical cancer, wherein said sample of genomic DNA is obtained by the method of claim 1.

Claim 24. The method of claim 23, wherein a greater number of instances, or a greater amount of, integrated HPV DNA is indicative of a high risk of having or developing cancer or is indicative of a more aggressive or higher grade cancer compared to a patient or subject having fewer instances or lesser amounts of integrated HPV DNA.

Claim 25. The method of claim 23, wherein a different pattern of HPV DNA integrations into genomic host DNA, compared to those in a control subject or patient, is indicative of a high risk of having or developing cancer or is indicative of a more aggressive or higher grade cancer.

Claim 26. The method of claim 23 that comprises assessing a risk of having a cervical cancer.

Claim 27. The method of claim 23 that comprises assessing the risk of developing a cervical cancer.

Claim 28 The method of claim 23, wherein said HPV DNA is from a high risk or pathogenic strain of HPV.

Claim 29. The method of claim 23, wherein said HPV DNA is from a high risk or pathogenic strain of HPV selected from the group consisting of HPV strains 16, 18, 21 , 33, 45, 52 and 58.

Claim 30. The method of claim 23, wherein said HPV is from a low risk or non-pathogenic strain of HPV.

Claim 31. The method of claim 23, wherein said HPV is from a low risk or non-pathogenic strain of HPV that is less pathogenic than any one of HPV strains 16, 18, 21, 33, 45, 52 and 58.

Claim 32. The method of any one of claims 23 to 28, further comprising detecting or quantifying a number of integrations of HPV DNA by comparison to a patient or subject not infected with HPV, or not infected with a pathogenic strain of HPV, having no lesions or other symptoms of HPV infection, or having substantially no antibody titer or cellular immunity to HPV or to a particular HPV strain.

Claim 33. The method of any one of claims 23 to 32, further comprising detecting or quantifying a number of integrations of HPV DNA by comparison to those in an earlier biological sample obtained from the same patient or subject.

Claim 34. The method of any one of claims 23 to 33, wherein said detecting or quantifying a number of integrations is performed using molecular combing of the genomic host DNA using probes that bind to HPV DNA sequences.

Claim 35. The method of any one of claims 23 to 34, wherein said detecting or quantifying a number of integrations is performed using molecular combing of the genomic host DNA using probes to HPV 16, 18, 31, 33, 45 35, 39, 51 , 52, 56, 58, 59, 66 and 68.

Claim 36. The method of claim 23, wherein said detecting or quantifying a number of integrations is performed using molecular combing of the genomic host DNA using probes that bind to or cover HPV DNA LI and L2, El and E2, and/or E6 and E7 sequences, wherein said probes may be labelled with the same different colored fluorescent tags.

Claim 37. The method of claim 23, wherein the patient or subject has a cervical dysplasia or has a positive PAP test.

Claim 38. The method of claim 23, wherein the patient of subject has been infected with human immunodeficiency virus (HIV), is immunosuppressed, has been exposed to diethylstilbestrol before birth, or is or has been treated for a precancerous cervical lesion or cervical cancer.

Claim 39. The method of claim 23, wherein the patient has or is at risk of having anal, vaginal, vulvar, penile or oropharyngeal cancer.

Claim 40. The method of claim 23, wherein the number or pattern of HPV integrations is quantified by, or correlated, with at least one of the following:

the number of HPV integration sites in host genomic DNA or the average number of such integrations,

the size in kb of HPV DNA integrations into host genomic DNA,

the number of HPV genomes integrated at each integration site,

the presence of absence of integrated HPV DNA,

the number of HPV integration sites per cellular genome,

the average number of HPV integration sites in host cells,

the mean number of HPV genomes integrated per integration site (or the mean size of integration sites),

maximum number of HPV genomes integrated per integration site (or the maximum size of integration sites),

minimum number of HPV genomes integrated per integration site (or minimum size of integration sites), or

number of HPV genomes integrated per cellular genome.

Claim 41. The method of claim 23, wherein the number or pattern of HPV integrations is correlated with at least one parameter of lesion status including:

normal histology (including all abnormalities without intraepithelial lesions or signs of viral infection such as metaplasia, cervicitis, decidual lesions or adenosis),

low grade (LG) lesion, corresponding to former CIN 1 ,

high grade (HG) lesion, corresponding to former CIN2, 3 and CIS (carcinoma in situ) or AIS (adenocarcinoma in situ);

normal cervix,

Grade 1 atypical transformation (AT),

Grade 2 atypical transformation (AT),

TAG2 a if there are no major signs,

TAG2 b if there are major signs,

TAG2 c when the appearance is suggestive of invasive cancer; and/or

atypical transformation (minor or major).

Claim 42. The method of claim 23, wherein the number or pattern of HPV integrations is correlated with at least one parameter of cytological classification including:

negative for intraepithelial lesion or malignancy,

abnormal squamous cells,

typical squamous cells (ASC),

of undetermined significance (ASC-US),

cannot exclude high-grade squamous intraepithelial lesion (ASC-H),

low-Grade Squamous Intraepithelial Lesion (LSIL),

high-Grade Squamous Intraepithelial Lesion (HSIL),

squamous cell carcinoma,

abnormal glandular cells,

atypical glandular cells (AGC): endocervical (not otherwise specified (NOS) or commented),

endometrial or not otherwise specified

atypical glandular cells, favor neoplastic: endocervical or not otherwise specified endocervical adenocarcinoma in situ (AIS), and/or

adenocarcinoma.