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
one or more recognition sequences for a Cre recombinase, wherein the one or more recognition sequences are configured to allow generation of a recombinase-dependent change that is detectable, and wherein the one or more recombinase recognition sequences comprises two Cre-recognition sites;
transfecting a target cell expressing the Cre recombinase with the population of rAAV genomes, whereby the Cre 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 Cre-recognition sites;
detecting at least one recombinase-dependent change; and
identifying an rAAV genome generated by the at least one recombinase-dependent change, wherein said identified rAAV genome comprises the inversion, and wherein said identified rAAV genome encodes an AAV capsid 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 the two Cre-recombinase recognition sites are two loxP or variants of loxP sites in a head-to-head orientation.
5. The method of claim 1 , wherein the target cell is a neuronal cell, a neural stem cell, an astrocytes, a tumor cell, a hematopoetic stem cell, an insulin producing beta cell, a lung epithelium, a skeletal cell, or a cardiac muscle cell.
6. The method of claim 1 , wherein the target cell is located in a brain or spinal cord.
7. The method of claim 1 , further comprising recovering the rAAV that is identified to have a capsid with the desired tropism.
8. 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 Cre-transgenic mouse, 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; and
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.
9. The method of claim 8 , wherein the target cell is present in a tissue, an organ, an organism, or a combination thereof.
10. The method of claim 8 , wherein the capsid genes of the population of rAAV genomes encode mutant capsid proteins.
11. The method of claim 8 , wherein the recombinase is Cre recombinase.
12. The method of claim 11 , wherein the one or more recombinase recognition sequences comprise two Cre-recognition sites.
13. The method of claim 11 , wherein the recombinase-dependent change comprises an inversion of the sequences that are flanked by the Cre-recognition sites; and wherein the method comprises identifying the rAAV genomes that comprise the inversion.
14. The method of claim 11 , wherein the two Cre-recombinase recognition sites are two loxP or variants of loxP sites in a head-to-head orientation.
15. The method of claim 8 , wherein the target cell is a neuronal cell, a neural stem cell, an astrocytes, a tumor cell, a hematopoetic stem cell, an insulin producing beta cell, a lung epithelium, a skeletal cell, or a cardiac muscle cell.
16. The method of claim 8 , wherein the target cell is located in a brain or spinal cord.
17. The method of claim 8 , further comprising recovering the rAAV that is identified to have a capsid with the desired tropism.