EP-4735615-A1 - METHOD FOR SITE-SPECIFIC INTRODUCTION OF GENETIC ELEMENTS IN ENGINEERED LOCI BY BIMODAL RECOMBINASE-MEDIATED CASSETTE EXCHANGE (BIRMCE)

Abstract

Described herein are systems and methods for genetic manipulation of mammalian cells. Also described are non-human animal models utilizing these systems, methods of generating these non-human animals, and methods of using these non-human animals.

Inventors

• BREUNIG, Joshua
• AYALA-SARMIENTO, Alberto

Assignees

• Cedars-Sinai Medical Center

Dates

Publication Date

20260506

Application Date

20240627

Claims (20)

1. 1. A system, comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a transgene or a nucleic acid encoding an RNA, (ii) the transgene or the nucleic acid encoding the RNA, and (iii) recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site; (b) two recombinases specific to the recombinase recognition sites.
2. 2. The system of claim 1, wherein the donor vector further comprises at least a third recombinase recognition site, and wherein the system further comprises at least a third recombinase specific to the at least third recombinase recognition site.
3. 3. The system of claim 1, further comprising a mammalian cell comprising a locus targeted by the donor vector and the two recombinases, and optionally the at least third recombinase.
4. 4. The system of any one of claims 1-3, wherein the two recombinases are provided by (i) one expression vector, comprising two genes encoding recombinases specific to their recognition sites, or (ii) two expression vectors, a first expression vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second expression vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or (iii) one mRNA encoding the two recombinases specific to their recognition sites, or (iv) two mRNA, a first mRNA encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and the second mRNA encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or (v) one viral vector comprising two genes encoding recombinases specific to their recognition sites, or (vi) two viral vectors, a first viral vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second viral vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or (vii) one recombinant protein comprising the unidirectional recombinase and the bidirectional recombinase, or (viii) two recombinant proteins, a first recombinase protein that is specific to the unidirectional recombinase recognition site, and a second recombinase protein that is specific to the bidirectional recombinase recognition site.
5. 5. The system of claim 4, wherein in (i) the one expression vector comprising two genes encoding recombinases specific to their recognition sites, the encoded recombinases are fused together; or in (iii) the one mRNA encoding the two recombinases specific to their recognition sites, the encoded two recombinases are fused together; or in (v) the one viral vector comprising two genes encoding recombinases specific to their recognition sites, the encoded recombinases are fused together; or in (viii) the two recombinant proteins are fused together.
6. 6. The system of claim 4, wherein any one of the recombinase is fused to one or more proteins other than the recombinase.
7. 7. The system of claim 5, wherein any one of the two fused recombinases are further fused to one or more proteins other than tire recombinase.
8. 8. The system of any one of claims 2-7, wherein the at least a third recombinases is provided by (iv) one expression vector, comprising a gene encoding the at least third recombinase specific to the third recombinase recognition site, or (x) one mRNA encoding tire at least third recombinase specific to the at least third recognition site, or (xi) one viral vector comprising a gene encoding the at least third recombinase specific to the at least third recombinase recognition site, or (xii) one recombinant protein comprising the at least third recombinase that is specific to the at least third recombinase recognition site.
9. 9. Tire system of claim 8, wherein in (iv) the one expression vector, comprising a gene encoding the at least third recombinase specific to the third recombinase recognition site, the expression vector further comprises a gene encoding one or more proteins than the third recombinase and the encoded third recombinase is fused to the encoded one or more proteins, or in (x) the one mRNA encoding the at least third recombinase specific to the at least third recognition site, the mRNA further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused to the encoded one or more proteins, or in (xi) the one viral vector comprising a gene encoding the at least third recombinase specific to the at least third recombinase recognition site, the one viral vector further encodes one or more proteins other than the third recombinase, and the encoded recombinase is further fused to the encoded one or more proteins, or in (xii) the one recombinant protein comprising the at least third recombinase that is specific to tire at least third recombinase recognition site is fused to one or more proteins other than the third recombinase.
10. 10. The system of any one of claims 1-9, wherein the unidirectional recombinase recognition site is upstream from the bidirectional recombinase recognition site.
11. 11. The system of any one of claims 1-9, wherein the unidirectional recombinase recognition site is downstream to a promoter.
12. 12. Tire system of any one of claims 1-11, wherein the donor vector further comprises an intron, part of an intron, or at least one splice acceptor site, and optionally, the unidirectional recombinase recognition site is embedded into an intron or part of the intron.
13. 13. The system of any one of claims 1-12, wherein the unidirectional recombinase is Bxbl.
14. 14. The system of any one of claims 1-12, wherein the unidirectional recombinase is selected from Bxb 1 , Phic31 , PhiBT 1 , PhiC 1 , MR11 , R4, TP901 - 1 , A 118, FC 1 , PhiRV, TG 1 , Phi370.1 , Wp, BL3 , SPBc, K38, and any mutants thereof.
15. 15. The system of any one of claims 1-12, wherein the bidirectional recombinase is Flp.
16. 16. The system of any one of claims 1-12. wherein the unidirectional recombinase is Bxbl and the bidirectional recombinase is selected from FLp. Cre, VCre. SCre. Nigri, Panto. Vika, or a mutant thereof.
17. 17. The system of any one of claims 2-12, wherein the third recombinase is selected from Bxb 1 , Phic31 , PhiBTl, PhiCl, MR11, R4, TP901-1, A118, FC1, PhiRV, TGI, Phi370.1, Wp, BL3, SPBc, K38, FLp, Cre, VCre. SCre, Nigri, Panto, Vika, or a mutant thereof.
18. 18. The system of any one of claims 1-17, wherein the unidirectional recombinase recognition site is attB.
19. 19. The system of any one of claims 1-17, wherein the unidirectional recombinase recognition site is attP.
20. 20. The system of any one of claims 1-17, wherein the bidirectional recombinase recognition site is flippase recognition target (FRT), loxP, VloxP, SloxP, nox, or pox.

Description

METHOD FOR SITE-SPECIFIC INTRODUCTION OF GENETIC ELEMENTS IN ENGINEERED LOCI BY BIMODAL RECOMBINASE-MEDIATED CASSETTE EXCHANGE (BIRMCE) CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application includes a claim of priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No. 63/523,550, filed June 27, 2023, the entirety of which is hereby incorporated by reference. FIELD OF INVENTION [0002] This invention relates to genetic manipulation: for example, in cells, organ models, and non-human animal models. BACKGROUND [0003] All publications herein are incorporated by reference to tire same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Tire following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. [0004] Stable integration and/or edition of genetic elements into specific loci is a very difficult task. Although there are different technologies trying to achieve this, it is still not possible to do it in a quick, clean, specific, cheap, and effortless way. MADR technology is a technology that can do it with the aforementioned features. One of the “Achilles heels” of MADR, and other similar technologies, is that it depends on the elimination of the exogenous genetic elements in order to close the system and to have the expression of only one exogenous genetic element. Tirus, the purpose of Bimodal Recombinase-Mediated Cassette Exchange (biRMCE) is that it can speed the stable expression of one exogenous genetic element even in the presence of different genetic elements. This principle enhances the use of genetic cassette exchanges for different applications where stable integration and/or edition of genetic elements into specific loci is desired. [0005] Genetic cassette exchanges through recombinases arc techniques used to integrate exogenous genetic elements into engineered loci in different types of cells. For example, the use of one recombinase targeting its heterotypic recognition sites, nevertheless, it has been found that there can be cross recombination between the heterotypic sites causing confounds. Other similar works use integrases targeting their respective recognition sites but the main issue with these systems is that they integrate not only the genetic cassettes elements but also the complete genetic vector which carries non-desired sequences. One existing solution is the sequential or simultaneous use of different types of recombinases, e.g., Flp & Cre, to avoid cross recombination between the recognition sites and to increase the efficiency of genetic cassette exchanges. However, recombinases perform reversible recombination reactions that make the system non-stable and not efficient when genetic cassettes earn ing different types of genetic elements are used. [0006] Accordingly, there remains a need in the art for robust and highly stable methods and systems for site-specific introduction of genetic elements in engineered loci. SUMMARY OF THE INVENTION [0007] Tire following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope. [0008] Various embodiments provide for a system, comprising: (a) a donor vector, comprising: (i) one or more polyadenylation signals or transcription stop element upstream from a transgene or a nucleic acid encoding an RNA, (ii) the transgene or the nucleic acid encoding the RNA, and (iii) recombinase recognition sites comprising at least one unidirectional recombinase recognition site and at least one bidirectional recombinase recognition site; and (b) two recombinases specific to the recombinase recognition sites. [0009] In various embodiments, the donor vector can further comprise at least a third recombinase recognition site, and the system can further comprise at least a third recombinase specific to the at least third recombinase recognition site. [0010] In various embodiments, the system can further comprise a mammalian cell comprising a locus targeted by the donor vector and the two recombinases, and optionally the at least third recombinase. [0011] In various embodiments, the two recombinases can be provided by (i) one expression vector, comprising two genes encoding recombinases specific to their recognition sites, or (ii) two expression vectors, a first expression vector comprising one gene encoding a first recombinase that is specific to the unidirectional recombinase recognition site, and a second expression vector comprising one gene encoding a second recombinase that is specific to the bidirectional recombinase recognition site, or (iii) one mRNA encoding the two recombinases