SYSTEMS AND METHODS FOR IN VIVO DUAL RECOMBINASE-MEDIATED CASSETTE EXCHANGE (dRMCE) AND DISEASE MODELS THEREOF

Abstract

Described herein are donor vectors and systems for use in in vivo dual recombinase-mediated cassette exchange. Also described are animal models for consistent, rigorous, and facile investigation of transgene expression. Further described are methods of screening for therapeutic drugs using these animal models, and methods of treatment.

1 . A system, comprising:
 a promoter-less donor vector, comprising
 a polyadenylation signal or transcription stop element upstream from a transgene or RNA, 
 the transgene or RNA, and 
 paired recombinase recognition sites; and 
   one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or   two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites.   
     
     
         2 . The system of  claim 1 , wherein the promoter-less donor vector further comprises a post-transcriptional regulatory element. 
     
     
         3 . The system of  claim 1 , wherein the promoter-less donor vector further comprises a polyadenylation signal downstream from the transgene or RNA. 
     
     
         4 . The system of  claim 1 , wherein the promoter-less donor vector comprises:
 PGK polyadenylation signal (pA);   trimerized SV40pA;   the transgene or RNA;   loxP and flippase recognition target (FRT);   a rabbit beta-globin pA; and   a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).   
     
     
         5 . The system of  claim 1 , wherein the paired recombinase recognition sites are loxP and flippase recognition target (FRT), and the recombinases are cre and flp. 
     
     
         6 . The system of  claim 1 , wherein the RNA is siRNA. 
     
     
         7 . The system of  claim 1 , wherein the RNA is shRNA. 
     
     
         8 . The system of  claim 1 , wherein the RNA is sgRNA. 
     
     
         9 . The system of  claim 1 , wherein the transgene or the RNA comprises disease associated mutations. 
     
     
         10 . A promoter-less donor vector, comprising:
 a polyadenylation signal or transcription stop element upstream from a transgene or RNA;   the transgene or RNA; and   paired recombinase recognition sites.   
     
     
         11 . The promoter-less donor vector of  claim 10 , further comprising a post-transcriptional regulatory element. 
     
     
         12 . The promoter-less donor vector of  claim 10 , further comprising a polyadenylation signal downstream from the transgene or RNA. 
     
     
         13 . The promoter-less donor vector of  claim 10 , comprising:
 PGK polyadenylation signal (pA);   trimerized SV40pA;   a transgene or RNA;   loxP and flippase recognition target (FRT);   a rabbit beta-globin pA; and   a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).   
     
     
         14 . A non-human animal model comprising the system of  claim 1 . 
     
     
         15 . The non-human animal model of  claim 14 , wherein dual Recombinase-Mediated Cassette Exchange has occurred. 
     
     
         16 . The non-human animal model of  claim 14 , wherein the animal is a mouse. 
     
     
         17 . A method of producing the non-human animal model of  claim 14 , comprising:
 providing a system, comprising:
 a promoter-less donor vector, comprising
 a polyadenylation signal or transcription stop element upstream from a transgene or RNA, 
 the transgene or RNA, and 
 paired recombinase recognition sites; and 
 
 one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or 
 two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites; 
   administering the system to the non-human animal; and   subjecting the non-human animal to electroporation.   
     
     
         18 . A method of screening a drug candidate, comprising:
 providing the non-human animal model of  claim 14 ;   administering the drug candidate; and   assessing the effects of the drug candidate on the non-human animal model.   
     
     
         19 . A method of treating a disease or condition in a subject, comprising:
 providing a cell comprising a system, comprising:
 a promoter-less donor vector, comprising
 a polyadenylation signal or transcription stop element upstream from a transgene or RNA, 
 the transgene or RNA, and 
 paired recombinase recognition sites; and 
 
 one expression vector, comprising two genes encoding recombinases specific to the paired recombinase recognition sites, or 
 two expression vectors, the first expression vector comprising one gene encoding a first recombinase that is specific to one of the paired recombinase recognition sites, and the second expression vector comprising one gene encoding a second recombinase that is specific to the other of the paired recombinase recognition sites; and 
   administering the cell to the subject.   
     
     
         20 . The method of  claim 19 , wherein the cell is a stem cell. 
     
     
         21 . (canceled)