Selective recovery
Provided herein are methods of selective screening. In addition, various targeting proteins and sequences, as well as methods of their use, are also provided.
1. A method of generating an AAV with a desired tropism, the method comprising:
providing a population of rAAV genomes, wherein each of the rAAV genomes comprises:
an AAV capsid gene, and
two or more recombinase recognition sequences for a recombinase, wherein the recognition sequences are configured to allow generation of a recombinase-dependent change that is detectable;
transfecting a target cell expressing the recombinase with the population of rAAV genomes, whereby the recombinase induces a recombination event to generate a recombinase-dependent change in at least one of the rAAV genomes of the population, and wherein the recombinase-dependent change comprises an inversion of the sequences that are flanked by the recognition sequences;
detecting at least one recombinase-dependent change;
identifying an rAAV genome generated by the at least one recombinase-dependent change, wherein said identified rAAV genome comprises the inversion and encodes an AAV capsid with the desired tropism; and
obtaining sequence information of the AAV capsid gene or a portion thereof in the identified rAAV genome with the desired tropism.
2. The method of claim 1 , wherein the target cell is present in a tissue, an organ, an organism, or a combination thereof.
3. The method of claim 1 , wherein the capsid genes of the population of rAAV genomes encode mutant capsid proteins.
4. The method of claim 1 , wherein at least two of the two or more recombinase recognition sequences are in a head-to-head orientation.
5. The method of claim 4 , wherein the recombinase is a Cre recombinase.
6. The method of claim 5 , wherein the at least two of the two or more recombinase recognition sequences are two loxP sites or variants of loxP sites.
7. The method of claim 1 , wherein the target cell is a neuronal cell, a neural stem cell, an astrocytes, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium, a skeletal cell, or a cardiac muscle cell.
8. The method of claim 1 , wherein the target cell is located in a brain or spinal cord.
9. The method of claim 1 , further comprising recovering the rAAV that is identified to have a capsid with the desired tropism.
10. A method of generating an AAV with a desired tropism, the method comprising:
providing a population of rAAV genomes, wherein each of the rAAV genomes comprises:
an AAV capsid gene, and
one or more recognition sequences for a recombinase, wherein the one or more recognition sequences are configured to allow generation of a recombinase-dependent change that is detectable;
transfecting a target cell expressing the recombinase with the population of rAAV genomes, wherein the target cell is in a transgenic mammal comprising transgenic expression of the recombinase, and whereby the recombinase induces a recombination event to generate a recombinase-dependent change in at least one of the rAAV genomes of the population;
detecting at least one recombinase-dependent change;
identifying an rAAV genome generated by the at least one recombinase-dependent change wherein said identified rAAV genome encodes an AAV capsid with the desired tropism; and
obtaining sequence information of the AAV capsid gene or a portion thereof in the identified rAAV genome with the desired tropism.
11. The method of claim 10 , wherein the target cell is present in a tissue, an organ, an organism, or a combination thereof.
12. The method of claim 10 , wherein the capsid genes of the population of rAAV genomes encode mutant capsid proteins.
13. The method of claim 10 , wherein the recombinase is Cre recombinase.
14. The method of claim 13 , wherein the one or more recombinase recognition sequences comprise two or more recombinase-recognition sequences.
15. The method of claim 13 , wherein the recombinase-dependent change comprises an inversion of the sequences that are flanked by the recombinase-recognition sequences; and wherein the method comprises identifying the rAAV genomes that comprise the inversion.
16. The method of claim 14 , wherein at least two of the two or more recombinase-recognition sequences are in a head-to-head orientation.
17. The method of claim 16 , wherein the recombinase is a Cre recombinase.
18. The method of claim 17 , wherein the at least two of the two or more recombinase recognition sequences are two loxP sites or variants of loxP sites.
19. The method of claim 10 , wherein the target cell is a neuronal cell, a neural stem cell, an astrocytes, a tumor cell, a hematopoietic stem cell, an insulin producing beta cell, a lung epithelium, a skeletal cell, or a cardiac muscle cell.
20. The method of claim 10 , wherein the target cell is located in a brain or spinal cord.
21. The method of claim 10 , further comprising recovering the rAAV that is identified to have a capsid with the desired tropism.
22. The method of claim 10 , wherein the mammal is a mouse.