US 11,702,644 B2
Methods and compositions for in vivo gene editing based cell-type-specific cellular engineering
Yuki Oka, Pasadena, CA (US); and Allan-Hermann Pool, Pasadena, CA (US)
Assigned to California Institute of Technology, Pasadena, CA (US)
Filed by California Institute of Technology, Pasadena, CA (US)
Filed on Apr. 9, 2020, as Appl. No. 16/845,009.
Claims priority of provisional application 62/832,504, filed on Apr. 11, 2019.
Claims priority of provisional application 62/832,759, filed on Apr. 11, 2019.
Prior Publication US 2020/0347369 A1, Nov. 5, 2020
Int. Cl. C12N 9/22 (2006.01); A61K 9/00 (2006.01); A61K 35/12 (2015.01); A61K 48/00 (2006.01); C12N 15/11 (2006.01); C12N 15/86 (2006.01); C12N 15/90 (2006.01); A61K 38/00 (2006.01); A61P 25/04 (2006.01); A61P 25/28 (2006.01)
CPC C12N 9/22 (2013.01) [A61K 9/0043 (2013.01); A61K 35/12 (2013.01); A61K 48/005 (2013.01); C12N 15/11 (2013.01); C12N 15/86 (2013.01); C12N 15/90 (2013.01); A61K 38/00 (2013.01); C12N 2310/20 (2017.05); C12N 2750/14143 (2013.01); C12N 2800/80 (2013.01)] 19 Claims
 
1. A method of incorporating an effector gene into the genome of a cell, comprising:
introducing into a cell:
(i) a programmable nuclease or a nucleic acid encoding the programmable nuclease, wherein the programmable nuclease is Cas9;
(ii) a targeting molecule or a nucleic acid encoding the targeting molecule, wherein the targeting molecule is capable of associating with the programmable nuclease, optionally wherein the targeting molecule comprises single strand DNA or single strand RNA, further optionally wherein the targeting molecule comprises a single guide RNA (sgRNA), and
(iii) a donor nucleic acid or a nucleic acid encoding the donor nucleic acid, wherein the donor nucleic acid comprises the structure 5′-[recognition site]- [splice acceptor site]-[translation frame linker]-[self-cleaving peptide sequence]-[effector gene]-3′ and/or the structure 5′-[recognition site]-[splice acceptor site]-[target gene coding sequence downstream of the intron]-[self-cleaving peptide sequence]-[effector gene]-3′, and an optional transcript stabilization element,
wherein the cell comprises a target gene differentially expressed in a unique cell type and/or in a cell during a unique cell state, wherein the target gene comprises an intron comprising the recognition site, and
wherein the targeting molecule is complementary to the recognition site and the programmable nuclease is capable of cleaving the recognition site, whereby the donor nucleic acid is capable of being incorporated into the intron through non-homologous end joining (NHEJ)-dependent DNA repair.