Gene-drive in DNA viruses

Patent metadata

Field	Value
Publication number	US-12344837-B2
Application number	US-201917054760-A
Country	US
Kind code	B2
Filing date	May 28, 2019
Priority date	May 29, 2018
Publication date	Jul 1, 2025
Grant date	Jul 1, 2025

Abstract

In various embodiments methods of utilizing gene drive constructs in asexual organisms such as viruses are provided. In certain embodiments the methods involve transfecting or infecting a cell with a modified DNA virus containing a gene drive construct; and infecting the cell with the target virus where the genome of said target DNA virus is modified by insertion of the gene drive construct into the genome of the target DNA virus and a population of modified target viruses (containing the gene drive construct) is produced.

First claim

What is claimed is:

1. A method of modifying a target DNA virus, said method comprising:
transfecting or infecting a cell population with a modified DNA virus containing a gene drive construct; and
infecting said cell population with said target DNA virus
wherein the genome of said target DNA virus is modified by insertion of said gene drive construct into the genome of said target DNA virus thereby producing a modified target virus,
wherein said target DNA virus and said modified DNA virus are the same species of DNA virus.

2. The method of claim 1 , wherein:
said target DNA virus comprises a genome large enough to add a 6-7 kb gene drive sequence; and/or
said target DNA virus has a minimal viral genome size of about 50 kb; and/or
said target DNA virus has the capacity to undergo homologous recombination; and/or
said target DNA virus comprises a nuclear-replicating virus.

3. The method of claim 1 , wherein:
said target DNA virus and said modified DNA virus are from a viral family selected from the group consisting of Herpesviridae, Alloherpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae, Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae, Iridoviridae, Lavidaviridae, Marseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipoviridae, Tectiviridae, and Turriviridae; or
said target DNA virus and said modified DNA virus are from the Herpesviridae family; or
said target DNA virus and said modified DNA virus are selected from the group consisting of HHV-5 (cytomegalovirus), HHV-1, HHV-2, HHV-3 (varicella-zoster virus (VZV)), HHV-4 (Epstein-Barr virus (EBV)), HHV-6A and 6B, HHV-7, and HHV-8 (Kaposi's sarcoma-associated herpesvirus (KSHV)), C3Hv, CeHV-1, MuHV-4, SuHV1, BoHV-1, GaHV-1, and MDV; or
said target DNA virus and said modified DNA virus are selected from the group consisting of HHV-5 (cytomegalovirus), HHV-1, HHV-2, HHV-3 (varicella-zoster virus (VZV)), HHV-4 (Epstein-Barr virus (EBV)), HHV-6A and 6B, HHV-7, and HHV-8 (Kaposi's sarcoma-associated herpesvirus (KSHV)); or
said target DNA virus and said modified DNA virus are HHV-5; or
said target DNA virus and said modified DNA virus are adenovirus; or
said target DNA virus and said modified DNA virus are baculovirus.

4. The method of claim 1 , wherein:
said target DNA virus and said modified DNA virus are selected from the group consisting of Ascoviridae, Asfarviridae, Poxviridae, Iridoviridae, Marseilleviridae, Megaviridae, Pandoraviridae, Phycodnaviridae, and Pithoviridae; or
said target DNA virus and said modified DNA virus is a poxvirus or an African Swine fever virus.

5. The method of claim 1 , wherein said gene drive construct comprises a nucleic acid encoding a targeted endonuclease inserted into the genome of the modified DNA virus at a location corresponding to the location in the target DNA virus that is cleaved by said targeted endonuclease.

6. The method of claim 5 , wherein said gene drive construct comprises homology arms that permit insertion of said gene drive construct at a site cleaved by said endonuclease.

7. The method of claim 5 , wherein said targeted endonuclease comprises an endonuclease selected from the group consisting of a class 2 CRISPR/Cas endonuclease, a TALEN, and a zinc finger nuclease.

8. The method of claim 7 , wherein said targeted endonuclease comprises a class 2 CRISPR/Cas endonuclease and said gene drive construct further comprise a nucleic acid encoding a guide RNA.

9. The method of claim 8 , wherein the class 2 CRISPR/Cas endonuclease comprises a Cas9 protein.

10. The method of claim 8 , wherein said class 2 CRISPR/Cas endonuclease is a type V or type VI CRISPR/Cas endonuclease.

11. The method of claim 10 , wherein the class 2 CRISPR/Cas protein is selected from the group consisting of a Cpf1 polypeptide or a functional portion thereof, a C2c1 polypeptide or a functional portion thereof, a C2c3 polypeptide or a functional portion thereof, and a C2c2 polypeptide or a functional portion thereof.

12. The method of claim 8 , wherein said gene drive construct encodes at least one guide RNA, wherein said guide RNA directs said targeted endonuclease to a site in the genome of said target DNA virus where cleavage permits integration of said gene drive construct by homologous recombination.

13. The method of claim 1 , wherein said gene drive construct inserts into and disrupts a gene essential for viral infection and/or replication.

14. The method of claim 1 , wherein: said gene drive construct introduces a modification that inhibits replication and/or assembly of said target DNA virus and said modification is compensated for by expression of a gene by the unmodified target DNA virus in said cell to permit viral replication of the modified target DNA virus; or said gene drive construct introduces a modification that inhibits replication and/or assembly of said target DNA virus and said modification is compensated for by expression of a rescue gene within said gene drive construct allowing for replication and assembly of the modified target DNA virus.

15. The method of claim 1 , wherein said modified DNA virus and said target DNA virus are introduced into the cell population ex vivo.

16. The method of claim 1 , wherein said method comprises administering said modified DNA virus to an animal already infected with said target DNA virus whereby the modified DNA virus infects a cell population already infected with said target DNA virus.

17. The method of claim 1 , wherein said gene drive construct comprises a nucleic acid encoding a homing endonuclease inserted into the genome of the modified DNA virus at a location corresponding to the location in the target DNA virus that is cleaved by said homing endonuclease.

Assignees

• Buck Inst Res Aging

Inventors

• Walter Marius

Classifications

• C12N2710/16122

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2710/16121

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N2710/16022

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• C12N2710/16021

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title