RNA GUIDED ERADICATION OF HUMAN JC VIRUS AND OTHER POLYOMAVIRUSES
The present invention includes methods and compositions for elimination of polyoma viruses, such as John Cunningham Virus (JVC), from host cells, and the treatment of polyoma-virus related diseases, such as progressive multifocal leukoencephalopathy (PML). The compositions include isolated nucleic acid sequences comprising a CRISPR-associated endonuclease and a guide RNA, wherein the guide RNA is complementary to a target sequence in a polyoma virus.
1 . A composition for use in eliminating John Cunningham Virus (JCV) from a host cell infected with JCV, the composition comprising:
at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and
at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a JCV DNA.
2 . The composition according to claim 1 , wherein said at least one gRNA having a spacer sequence complementary to a target sequence in a JCV DNA is a further defined as at least one gRNA having a spacer sequence complimentary to a target sequence in the large T-antigen (T-Ag) encoding region of the JCV DNA.
3 . The composition according to claim 2 , wherein said at least one gRNA having a spacer sequence complimentary to a target sequence in the T-Ag encoding region of the JCV DNA includes a gRNA having a spacer sequence complementary to a target sequence in the TM1 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM2 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM3 region of the T-Ag encoding region, or any combination of said gRNAs.
4 . The composition according to claim 3 wherein said CRISPR-associated endonuclease is selected from a wild-type Cas9, a human-optimized Cas9, a nickase mutant Cas9, SpCas9(K855a), SpCas9(K810A/K1003A/r1060A), or SpCas9(K848A/K1003A/R1060A).
5 . The composition according to claim 4 , wherein said gRNA having a spacer sequence complementary to a target sequence in the TM1 region is gRNA m1, said gRNA having a spacer sequence complimentary to a target sequence in the TM2 region is gRNA m2, and said gRNA having a spacer sequence complimentary to a target sequence in the TM3 region is gRNA m3.
6 . The composition according to claim 5 , wherein said spacer sequence of said gRNA M1 is complementary to a target sequence including SEQ ID NOS: 1, 2, 3, or 4; said spacer sequence of said gRNA m2 is complementary to a target sequence including SEQ ID NOS: 5, 6, 7, or 8; and said spacer sequence of said gRNA m3 is complementary to a target sequence including SEQ ID NOS: 9, 10, 11, or 12.
7 . The composition according to claim 1 , wherein said CRISPR-associated endonuclease is Cpf1.
8 . A method of eliminating John Cunningham Virus (JCV) from a host cell infected with JCV, including the steps of:
treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence that is complementary to a target sequence in a JCV DNA; and
eliminating the JCV from the host cell.
9 . The method according to claim 8 , wherein the at least one gRNA having a spacer sequence complementary to a target sequence in a JCV DNA is a further defined as at least one gRNA having a spacer sequence complimentary to a target sequence in the large T-antigen (T-Ag) encoding region of the JCV DNA, and the method additionally includes, after the treating step, the step of deleting at least a segment of the JCV DNA situated in a coding region of T-Ag.
10 . The method according to claim 9 , wherein the at least one gRNA having a spacer sequence complimentary to a target sequence in the T-Ag encoding region of the JCV DNA includes a gRNA having a spacer sequence complementary to a target sequence in the TM1 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM2 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM3 region of the T-Ag encoding region, or any combination of said gRNAs.
11 . The method according to claim 10 , wherein the CRISPR-associated endonuclease is selected from a wild-type Cas9; a human-optimized Cas9; a nickase mutant Cas9; SpCas9(K855a); SpCas9(K810A/K1003A/r1060A); SpCas9(K848A/K1003A/R1060A); SpCas9 N497A, R661A, Q695A, Q926A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E; SpCas9 N497A, R661A, Q695A, Q926A L169A; SpCas9 N497A, R661A, Q695A, Q926A Y450A; SpCas9 N497A, R661A, Q695A, Q926A M495A; SpCas9 N497A, R661A, Q695A, Q926A M694A; SpCas9 N497A, R661A, Q695A, Q926A H698A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, L169A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, Y450A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M495A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M694A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M698A; SpCas9 R661A, Q695A, Q926A; SpCas9 R661A, Q695A, Q926A, D1135E; SpCas9 R661A, Q695A, Q926A, L169A; SpCas9 R661A, Q695A, Q926A Y450A; SpCas9 R661A, Q695A, Q926A M495A; SpCas9 R661A, Q695A, Q926A M694A; SpCas9 R661A, Q695A, Q926A H698A; SpCas9 R661A, Q695A, Q926A D1135E L169A; SpCas9 R661A, Q695A, Q926A D1135E Y450A; SpCas9 R661A, Q695A, Q926A D1135E M495A; or SpCas9 R661A, Q695A, Q926A, D1135E, M694A.
12 . The method according to claim 11 , wherein the gRNA having a spacer sequence complementary to a target sequence in the TM1 region is gRNA m1, the gRNA having a spacer sequence complimentary to a target sequence in the TM2 region is gRNA m2, and the gRNA having a spacer sequence complimentary to a target sequence in the TM3 region is gRNA m3.
13 . The method according to claim 12 , wherein the spacer sequence of gRNA m1 is complementary to a target sequence including SEQ ID NOS: 1, 2, 3, or 4; said spacer sequence of said gRNA m2 is complementary to a target sequence including SEQ ID NOS: 5, 6, 7, or 8; and said spacer sequence of said gRNA m3 is complementary to a target sequence including SEQ ID NOS: 9, 10, 11, or 12.
14 . The method according to claim 8 , wherein the CRISPR-associated endonuclease is Cpf1.
15 . A vector composition for use in eliminating John Cunningham Virus (JCV) from a host cell infected with JCV, including:
at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and
at least one guide RNA (gRNA) having a spacer sequence complementary to a target sequence in a JCV DNA,
said isolated nucleic acid sequences being included in at least one expression vector;
wherein said at least one expression vector induces the expression of said CRISPR-associated endonuclease and said at least one gRNA in a host cell.
16 . The vector composition according to claim 15 , wherein said at least one gRNA having a spacer sequence complementary to a target sequence in a JCV DNA is a further defined as at least one gRNA having a spacer sequence complimentary to a target sequence in the large T-antigen (T-Ag) encoding region of the JCV DNA.
17 . The vector composition according to claim 16 , wherein said at least one gRNA having a spacer sequence complimentary to a target sequence in the T-Ag encoding region of the JCV DNA includes a gRNA having a spacer sequence complementary to a target sequence in the TM1 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM2 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM3 region of the T-Ag encoding region, or any combination of said gRNAs.
18 . The vector composition according to claim 17 , wherein said CRISPR-associated endonuclease is selected from a wild-type Cas9; a human-optimized Cas9; a nickase mutant Cas9; SpCas9(K855a); SpCas9(K810A/K1003A/r1060A); SpCas9(K848A/K1003A/R1060A); SpCas9 N497A, R661A, Q695A, Q926A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E; SpCas9 N497A, R661A, Q695A, Q926A L169A; SpCas9 N497A, R661A, Q695A, Q926A Y450A; SpCas9 N497A, R661A, Q695A, Q926A M495A; SpCas9 N497A, R661A, Q695A, Q926A M694A; SpCas9 N497A, R661A, Q695A, Q926A H698A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, L169A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, Y450A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M495A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M694A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M698A; SpCas9 R661A, Q695A, Q926A; SpCas9 R661A, Q695A, Q926A, D1135E; SpCas9 R661A, Q695A, Q926A, L169A; SpCas9 R661A, Q695A, Q926A Y450A; SpCas9 R661A, Q695A, Q926A M495A; SpCas9 R661A, Q695A, Q926A M694A; SpCas9 R661A, Q695A, Q926A H698A; SpCas9 R661A, Q695A, Q926A D1135E L169A; SpCas9 R661A, Q695A, Q926A D1135E Y450A; SpCas9 R661A, Q695A, Q926A D1135E M495A; or SpCas9 R661A, Q695A, Q926A, D1135E, M694A.
19 . The vector composition according to claim 18 , wherein said gRNA having a spacer sequence complementary to a target sequence in the TM1 region is gRNA m1, said gRNA having a spacer sequence complimentary to a target sequence in the TM2 region is gRNA m2, and said gRNA having a spacer sequence complimentary to a target sequence in the TM3 region is gRNA m3.
20 . The composition according to claim 19 , wherein said spacer sequence of said gRNA m1 is complementary to a target sequence including SEQ ID NOS: 1, 2, 3, or 4; said spacer sequence of said gRNA m2 is complementary to a target sequence including SEQ ID NOS: 5, 6, 7, or 8; and said spacer sequence of said gRNA m3 is complementary to a target sequence including SEQ ID NOS: 9, 10, 11, or 12.
21 . The composition according to claim 15 , wherein said CRISPR-associated endonuclease Cas9 is Cpf1.
22 . The composition according to claim 15 , wherein said expression vector is selected from the group consisting of a lentiviral expression vector, a drug inducible lentiviral expression vector, an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a pox virus vector, and a plasmid vector.
23 . The expression vector composition according to claim 15 , wherein said CRISPR associated endonuclease and said at least one gRNA are incorporated into in a single expression vector.
24 . The expression vector composition according to claim 15 , wherein said CRISPR associated endonuclease and said at least one gRNA are incorporated into separate lentiviral expression vectors.
25 . A method of preventing John Cunningham Virus (JCV) infection of cells of a patient at risk of JCV infection, including the steps of:
determining that a patient is at risk of JCV infection;
exposing cells of the patient at risk of JCV infection to an effective amount of an expression vector composition including an isolated nucleic acid encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one isolated nucleic acid encoding at least one guide RNA (gRNA) including a spacer sequence complementary to a target sequence in a JCV DNA;
stably expressing the CRISPR-associated endonuclease and the at least one gRNA in the cells of the patient; and
preventing JCV infection of the cells of the patient.
26 . The method according to claim 25 , wherein the least one gRNA including a spacer sequence complementary to a target sequence in a JCV DNA is further defined as at least one gRNA including a spacer sequence complementary to a target sequence in the large T-antigen (T-Ag) encoding region of the JCV DNA.
27 . A pharmaceutical composition including:
at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease; and
at least one isolated nucleic acid sequence encoding at least one guide RNA (gRNA) having a spacer sequence that is complementary to a target sequence in a John Cunningham Virus (JCV) genome;
said isolated nucleic acid sequences being included in at least one expression vector.
28 . The pharmaceutical composition according to claim 27 , wherein said at least one gRNA having a spacer sequence complementary to a target sequence in a JCV DNA is a further defined as at least one gRNA having a spacer sequence complimentary to a target sequence in the large T-antigen (T-Ag) encoding region of the JCV DNA.
29 . The pharmaceutical composition according to claim 28 , wherein said at least one gRNA having a spacer sequence complimentary to a target sequence in the T-Ag encoding region of the JCV DNA includes a gRNA having a spacer sequence complementary to a target sequence in the TM1 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM2 region of the T-Ag encoding region, a gRNA having a spacer sequence complimentary to a target sequence in the TM3 region of the T-Ag encoding region, or any combination of said gRNAs.
30 . The pharmaceutical composition according to claim 29 , wherein said CRISPR-associated endonuclease is selected from a wild-type Cas9; a human-optimized Cas9; a nickase mutant Cas9; SpCas9(K855a); SpCas9(K810A/K1003A/r1060A); SpCas9(K848A/K1003A/R1060A); SpCas9 N497A, R661A, Q695A, Q926A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E; SpCas9 N497A, R661A, Q695A, Q926A L169A; SpCas9 N497A, R661A, Q695A, Q926A Y450A; SpCas9 N497A, R661A, Q695A, Q926A M495A; SpCas9 N497A, R661A, Q695A, Q926A M694A; SpCas9 N497A, R661A, Q695A, Q926A H698A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, L169A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, Y450A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M495A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M694A; SpCas9 N497A, R661A, Q695A, Q926A, D1135E, M698A; SpCas9 R661A, Q695A, Q926A; SpCas9 R661A, Q695A, Q926A, D1135E; SpCas9 R661A, Q695A, Q926A, L169A; SpCas9 R661A, Q695A, Q926A Y450A; SpCas9 R661A, Q695A, Q926A M495A; SpCas9 R661A, Q695A, Q926A M694A; SpCas9 R661A, Q695A, Q926A H698A; SpCas9 R661A, Q695A, Q926A D1135E L169A; SpCas9 R661A, Q695A, Q926A D1135E Y450A; SpCas9 R661A, Q695A, Q926A D1135E M495A; or SpCas9 R661A, Q695A, Q926A, D1135E, M694A.
31 . The pharmaceutical composition according to claim 30 , wherein said gRNA having a spacer sequence complementary to a target sequence in the TM1 region is gRNA m1, said gRNA having a spacer sequence complimentary to a target sequence in the TM2 region is gRNA m2, and said gRNA having a spacer sequence complimentary to a target sequence in the TM3 region is gRNA m3.
32 . The pharmaceutical composition according to claim 31 , wherein said spacer sequence of said gRNA M1 is complementary to a target sequence including SEQ ID NOS: 1, 2, 3, or 4; said spacer sequence of said gRNA m2 is complementary to a target sequence including SEQ ID NOS: 5, 6, 7, or 8; and said spacer sequence of said gRNA m3 is complementary to a target sequence including SEQ ID NOS: 9, 10, 11, or 12.
33 . The pharmaceutical composition according to claim 27 wherein said CRISPR-associated endonuclease Cas9 is Cpf1.
34 . The pharmaceutical composition according to claim 27 , wherein said expression vector is selected from the group consisting of a lentiviral expression vector, a drug inducible lentiviral expression vector, an adenovirus vector, an adeno-associated virus vector, a retroviral vector, a pox virus vector, and a plasmid vector.
35 . A method of treating a subject having a John Cunningham Virus (JCV) related disorder, including the step of administering to the subject an effective amount of a pharmaceutical composition according to claim 27 .
36 . The method according to claim 36 , wherein the JCV-related disorder is progressive multifocal leukoencephalopathy (PML).
37 . A kit for the treatment or prophylaxis of John Cunningham Virus (JCV) infection, including:
a measured amount of a composition comprising at least one isolated nucleic acid sequence encoding a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one nucleic acid sequence encoding one or more guide RNAs (gRNAs), wherein each of said one or more gRNAs includes a spacer sequence complementary to a target sequence in a JCV DNA; and
one or more items selected from the group consisting of packaging material, a package insert comprising instructions for use, a sterile fluid, a syringe and a sterile container.
38 . The kit according to claim 37 , wherein said expression vector is a lentiviral expression vector.
39 . A method of eliminating a polyoma virus from a host cell infected with a polyoma virus, including the steps of:
treating the host cell with a composition comprising a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, and at least one guide RNA (gRNA) having a spacer sequence that is complementary to a target sequence in a polyoma virus DNA and eliminating the polyoma virus from the host cell.
40 . The method according to claim 39 , wherein the at least one gRNA having a spacer sequence complementary to a target sequence in a polyoma virus DNA is a further defined as at least one gRNA having a spacer sequence complimentary to a target sequence in the large T-antigen (T-Ag) encoding region of the polyoma virus DNA.