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(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2019/010422 Al 10 January 2019 (10.01.2019) W ! P O PCT (51) International Patent Classification: JE, Catherine Amanda; c/o 17 Quincy Street, Cambridge, A61K 35/76 (2015.01) Massachusetts 02138 (US). (21) International Application Number: (74) Agent: NIX, F. Brent et al.; Johnson, Marcou & Isaacs, PCT/US20 18/04 1099 LLC, P.O. Box 691, Hoschton, Georgia 30548 (US). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 06 July 2018 (06.07.2018) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (25) Filing Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (26) Publication Langi English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, (30) Priority Data: KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, 62/530,029 07 July 2017 (07.07.2017) US MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (71) Applicants: THE BROAD INSTITUTE, INC. [US/US]; OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, 415 Main Street, Cambridge, Massachusetts 02142 (US). SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, MASSACHUSETTS INSTITUTE OF TECHNOLOGY TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. [US/US]; 77 Massachusetts Avenue, Cambridge, Massa (84) Designated States (unless otherwise indicated, for every chusetts 02139 (US). PRESIDENT AND FELLOWS OF kind of regional protection available): ARIPO (BW, GH, HARVARD COLLEGE [US/US]; 17 Quincy Street, Cam GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, bridge, Massachusetts 02138 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, (72) Inventors: ZHANG, Feng; c/o 415 Main Street, Cam TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, bridge, Massachusetts 02142 (US). GOOTENBERG, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, Jonathan S.; c/o 17 Quincy Street, Cambridge, Massachu MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, setts 02138 (US). ABUDAYYEH, Omar O.; c/o 77 Mass TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, achusetts Ave, Cambridge, Massachusetts 02139 (US). SA- KM, ML, MR, NE, SN, TD, TG). BETI, Pardis; c/o 17 Quincy Street, Cambridge, Massachu setts 02138 (US). MYHRVOLD, Cameron; c/o 17 Quin Published: cy Street, Cambridge, Massachusetts 02138 (US). FREI- — with international search report (Art. 21(3)) (54) Title: CRISPR SYSTEM BASED ANTIVIRAL THERAPY (57) Abstract: The present invention offers a new approach for highly multiplexed, programmable antiviral therapies that directly target viral RNA, and can be flexibly adapted to target novel viruses or emerging outbreak pathogens. Class 2, type VI CRISPR system-based therapies can be used in combination with existing antiviral compounds for virus es where such compounds exist, either by increasing their efficacy or by preventing the evolution of specific drug resistance mutations. Per haps most excitingly, if a virus evolves resistance to a specific guide Cells expressing Virus-specific RNA sequence, it is easy to switch to a different guide RNA sequence, Cas1 a guide or to design a new guide sequence to target the new mutation. Such ap proaches should prevent the widespread development of resistance to X X X Class 2, type VI CRISPR system-based therapies. ij Viral RNA genome Decreased viral replication o © Fig. 2A CRISPR SYSTEM BASED ANTIVIRAL THERAPY CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/530,029 filed July 7, 2017. The entire contents of the above-identified application are hereby fully incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under grant numbers MH1 007006 MH1 1049, and All 10818 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD [0003] The subject matter disclosed herein is generally directed to the use of CRISPR effector systems for use in treating, preventing, suppressing, and/or alleviating viral pathogenesis, infection, propagation, and/or replication in a subject. BACKGROUND [0004] The CRISPR-Cas systems of bacterial and archaeal adaptive immunity show extreme diversity of protein composition and genomic loci architecture. The CRISPR-Cas system loci has more than 50 gene families and there is no strictly universal genes indicating fast evolution and extreme diversity of loci architecture. So far, adopting a multi-pronged approach, there is comprehensive cas gene identification of about 395 profiles for 93 Cas proteins. Classification includes signature gene profiles plus signatures of locus architecture. A new classification of CRISPR-Cas systems is proposed in which these systems are broadly divided into two classes, Class 1 with multisubunit effector complexes and Class 2 with single-subunit effector modules exemplified by the Cas9 protein. Novel effector proteins associated with Class 2 CRISPR-Cas systems may be developed as powerful genome engineering tools and the prediction of putative novel effector proteins and their engineering and optimization is important. [0005] The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. Recently, the Class 2 type VI single-component CRISPR-Cas effector Casl3a, previously known as C2c2 (Shmakov et al. (2015) "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems"; Molecular Cell 60:1-13; doi: http://dx.doi.Org/10.1016/j.molcel.2015.10.008) was characterized as an RNA-guided Rnase (Abudayyeh et al. (2016), Science, [Epub ahead of print], June 2; "C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector"; doi: 10.1 126/science.aaf5573). It was demonstrated that C2c2 (e.g. from Leptotrichia shahii) provides robust interference against RNA phage infection. Through in vitro biochemical analysis and in vivo assays, it was shown that C2c2 can be programmed to cleave ssRNA targets carrying protospacers flanked by a 3' H (non- G) PAM. Cleavage is mediated by catalytic residues in the two conserved HEPN domains of C2c2, mutations in which generate a catalytically inactive RNA-binding protein. C2c2 is guided by a single crRNA and can be re-programmed to deplete specific mRNAs in vivo. It was shown that LshC2c2 can be targeted to a specific site of interest and can carry out non-specific RNase activity once primed with the cognate target RNA. These results broaden our understanding of CRISPR- Cas systems and demonstrate the possibility of harnessing Casl3, such as Casl3a, Casl3b, or Casl3c to develop a broad set of RNA-targeting tools. [0006] While interference with phage infection in prokaryotes has been demonstrated for LsCasl3a, it is currently unknown if Casl3 mediated antiviral therapy is feasible or even possible at all in a eukaryotic setting. Indeed, the extreme differences between prokaryotes and eukaryotes, further confounded by the very nature of prokaryotic versus eukaryotic viruses, including etiology and pathogenesis, makes extrapolation from prokaryotic immunity to eukaryotic immunity highly unpredictable. [0007] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY [0008] Antiviral drugs do not exist for most emerging viruses, and available direct-acting antivirals, which include small molecules, short interfering RNAs, and antibodies, typically target a small number of highly mutable viral proteins or RNAs. This is problematic because RNA viruses evolve rapidly and can easily acquire resistance to existing therapeutics. The present invention offers a new approach for highly multiplexed, programmable antiviral therapies that directly target viral RNA, and can be flexibly adapted to target novel viruses or emerging outbreak pathogens. Class 2, type VI CRISPR system-based therapies can be used in combination with existing antiviral compounds for viruses where such compounds exist, either by increasing their efficacy or by preventing the evolution of specific drug resistance mutations. Perhaps most excitingly, if a virus evolves resistance to a specific guide RNA sequence, it is easy to switch to a different guide RNA sequence, or design a new guide sequence to target the new mutation. Such approaches should prevent the widespread development of resistance to Class 2, type VI CRISPR system-based therapies. [0009] Current gold-standard pathogen diagnostics are often expensive, slow, and lack sufficient sensitivity to detect viral infections. Standard molecular amplification methods, such as RT-qPCR, typically require nucleic acid extraction and expensive thermocycling machinery. Immunoassays, such as ELISAs, can only detect single targets, cross-react to antigenically similar targets, and cannot be quickly developed or updated to deal with new or evolving threats. By means of example, and without limitation, CRISPR-based detection/diagnostic platforms, such as described in Grootenberg et al. (2017), "Nucleic acid detection with CRISPR-Casl3/C2c2", Science, 356(6336):438-442 can transform the diagnosis of viral diseases with single-molecule detection sensitivity and single nucleotide polymorphism specificity. [0010] The present inventors have surprisingly found that Class 2, type VI CRISPR systems are useful as an antiviral therapeutic or prophylactic. [0011] The present invention has, among others, the following objectives: (1) Dissecting viral targets and viral evolution in response to anti-viral therapy, including Class 2, type VI CRISPR system therapy, to enable robust targeting. Methods and guides are identified which effectively inhibit viral pathogenesis, such as but not limited to viral replication.
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  • Quantification and Epigenetic Evaluation of the Residual Pool Of

    Quantification and Epigenetic Evaluation of the Residual Pool Of

    www.nature.com/scientificreports OPEN Quantifcation and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long‑term nucleoside analogue‑treated patients Fanny Lebossé1,2,3, Aurore Inchauspé1,2, Maëlle Locatelli1,2, Clothilde Miaglia1,2,3, Audrey Diederichs1,2, Judith Fresquet1,2, Fleur Chapus1,2, Kamal Hamed4, Barbara Testoni1,2* & Fabien Zoulim1,2,3* Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long‑term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplifcation (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA‑associated histone tails post‑transcriptional modifcations (PTMs) by micro‑chromatin immunoprecipitation. Long‑term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all‑but‑one negative samples. Low preC/pgRNA level in telbivudine‑treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No diference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof‑of‑concept clinical trials aiming at a functional cure of chronic hepatitis B.