CRISPR system based antiviral therapy

Patent metadata

Field	Value
Publication number	US-12415000-B2
Application number	US-201816629310-A
Country	US
Kind code	B2
Filing date	Jul 6, 2018
Priority date	Jul 7, 2017
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

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 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 to 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.

First claim

The invention claimed is:

1. A Class 2, type VI CRISPR system effective to reduce a viral load in a eukaryotic subject and detect the presence of a virus, the system comprising:
(a) a Cas13 protein and/or a polynucleic acid encoding the Cas13 protein; and
(b) two or more guide RNAs and/or one or more polynucleic acids encoding said two or more guide RNAs that bind to one or more target viral, wherein the one or more target viral mRNAs comprise viral sequences that, when cleaved by the Cas13 protein, (i) results in the reduction in viral load for use in treating, suppressing, and/or alleviating viral pathogenesis, infection, propagation and/or replication in the subject, wherein the reduction in viral load is greater as compared to a Class 2, type VI CRISPR system having a single guide, and (ii) stimulates Cas13 protein collateral activity, and wherein the two or more guide RNAs are selected from a pool of tiled guide RNAs that are tiled across the coding strand of a viral genome starting every 50 nucleotides.

2. The CRISPR system according to claim 1 , wherein (a) said polynucleic acid encoding said polynucleic acid encoding the Cas13 protein comprises a regulatory element operably linked to a polynucleic acid encoding said Cas13 protein, (b) said polynucleic acid encoding said one or more guide RNAs comprises a regulatory element operably linked to a polynucleic acid encoding said one or more guide RNAs or both (a) and (b).

3. The CRISPR system according to claim 2 , wherein said regulatory element allows constitutive or inducible expression of said Cas13 protein and/or said one or more guide RNAs, optionally tissue specific expression.

4. The CRISPR system according to claim 1 , wherein said polynucleic acid encoding said one or more guide RNAs and/or said polynucleic acid encoding said Cas13 protein are comprised in one or more vectors, wherein the one or more vectors are one or more eukaryotic expression vectors.

5. The CRISPR system according to claim 4 , wherein said vector is a viral vector, or an adenoviral vector, or an AAV vector, or a retroviral vector.

6. The CRISPR system according to claim 1 , wherein the CRISPR system is effective to reduce viremia.

7. The CRISPR system according to claim 1 , wherein said subject is an animal subject, or a mammalian subject, or a human subject.

8. The CRISPR system according to claim 1 , wherein the one or more target RNAs comprise sequences of or that correspond to regions of a viral genome that are less likely to evolving resistance to the one or more guide RNAs.

9. The CRISPR system according to claim 1 , wherein (a) said Cas13 protein comprises one or more mutations, and wherein the one or more mutations affect catalytic activity and/or stability and/or specificity; (b) wherein said Cas13 protein is codon optimized; (c) wherein said Cas13 protein comprises a NLS or a NES; (d) wherein said Cas13 protein comprises a fusion protein; or (e) any combination of (a)-(d).

10. The CRISPR system according to claim 1 , wherein the system further comprises (c) a masking construct that produces a detectable signal or modifies a detectable signal in response to Cas13 protein collateral activity.

11. The CRISPR system according to claim 1 , wherein said Cas13 protein comprises one or more HEPN domains that comprise a RxxxxH motif sequence, wherein the RxxxxH motif sequence comprises a R{N/H/K]X1X2X3H sequence, and wherein X1 is R, S, D, E, Q, N, G, or Y, X2 is independently I, S, T, V, or L, and X3 is independently L, F, N, Y, V, I, S, D, E, or A.

12. The CRISPR system according to claim 1 , wherein the Cas13 protein is Cas13a, Cas13b, or Cas 13c.

13. The CRISPR system according to claim 12 , wherein the Cas13 protein is from an organism of a genus selected from the group consisting of: Leptotrichia, Listeria , Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma, Campylobacter , and Lachnospira;
or wherein the Cas13 protein is from an organism selected from the group consisting of: Leptotrichia shahii; Leptotrichia wadei (Lw2); Listeria seeligeri ; Lachnospiraceae bacterium MA2020; Lachnospiraceae bacterium NK4A179 ; Clostridium aminophilum DSM 10710 ; Carnobacterium gallinarum DSM 4847 ; Carnobacterium gallinarum DSM 4847 (second CRISPR Loci); Paludibacter propionicigenes WB4 ; Listeria weihenstephanensis FSL R9-0317; Listeriaceae bacterium FSL M6-0635 ; Leptotrichia wadei F0279 ; Rhodobacter capsulatus SB 1003 ; Rhodobacter capsulatus R121 ; Rhodobacter capsulatus DE442 ; Leptotrichia buccalis C-1013-b; Herbinix hemicellulosilytica; Eubacterium rectale ; Eubacteriaceae bacterium CHKCI004 ; Blautia sp. Marseille-P2398; and Leptotrichia sp. oral taxon 879 str. F0557, Lachnospiraceae bacterium NK4A144 ; Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp. TSL5-1 ; Pseudobutyrivibrio sp. OR37 ; Butyrivibrio sp. YAB3001 ; Blautia sp. Marseille-P2398 ; Leptotrichia sp. Marseille-P3007 ; Bacteroides ihuae ; Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia ; and Insolitispirillum peregrimim ; or
wherein the Cas13 protein is a L. wadei F0279 Cas13a protein.

14. The CRISPR system according to claim 1 , wherein the two or more guide RNAs designed to bind to the one or more target molecules comprise a synthetic mismatch, and wherein said mismatch is up- or downstream of a SNP or other single nucleotide variation in said target molecule.

15. The CRISPR system according to claim 1 , wherein the guide RNAs comprise a pan-viral guide RNA set that targets each virus and/or viral strain in a set of viruses.

16. The CRISPR system according to claim 1 , wherein the virus is a DNA virus, or a single stranded, or double stranded DNA virus, or a positive sense DNA virus, or a negative sense DNA virus, or an antisense DNA virus.

17. The CRISPR system according to claim 16 , wherein the virus is a Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae (including human herpes virus, and Varicella Zoster virus), Malocoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae, Baculoviridae, Cicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Maseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae, Turriviridae, Dinodnavirus, Salterprovirus, Rhizidovirus, or any combination thereof; or Simian virus 40, JC virus, or BK virus; or cowpox or smallpox; or African swine fever virus; or Human herpes virus or varicella Zoster virus.

18. The CRISPR system according to claim 1 , wherein the virus is a single-stranded, or double-stranded RNA virus, or a positive sense RNA virus, or a negative sense RNA virus, or an antisense RNA virus.

19. The CRISPR system according to claim 18 , wherein the virus is a Retroviridae virus, Lentiviridae virus, Coronaviridae virus, a Picornaviridae virus, a Caliciviridae virus, a Flaviviridae virus, a Togaviridae virus, a Bornaviridae, a Filoviridae, a Paramyxoviridae, a Pneumoviridae, a Rhabdoviridae, an Arenaviridae, a Bunyaviridae, an Orthomyxoviridae, or a Deltavirus; or
wherein the virus is Lymphocytic choriomeningitis virus, Coronavirus, HIV, SARS, Poliovirus, Rhinoviru

Assignees

• Broad Inst Inc
• Massachusetts Inst Technology
• Harvard College
• Massachuetts Institute Of Tech

Inventors

• Zhang Feng
• Gootenberg Jonathan S
• Abudayyeh Omar O
• Sabeti Pardis
• Myhrvold Cameron
• Freije Catherine Amanda

Classifications

• C12N2320/11

  for the determination of target sites, i.e. of active nucleic acids · CPC title

• C12N15/1131

  against viruses · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title