Mutant viral capsid libraries and related systems and methods
Provided are mutant viral capsid cell libraries, individual cells of such libraries, systems, vectors, and methods for generating the cell libraries, and methods of use thereof to screen for mutant viral capsids with desired characteristics.
1. A library of cells, comprising a plurality of eukaryotic cells, each of the plurality of eukaryotic cells comprising a heterologous polynucleotide comprising a coding sequence that encodes a non-naturally occurring mutant adeno-associated virus (AAV) capsid,
wherein the coding sequence is operably linked to a promoter on the heterologous polynucleotide,
wherein the heterologous polynucleotide is integrated into the genome of the eukaryotic cell and is flanked by hybrid integrase-specific DNA attachment sites,
wherein at least 90% of the cells in the library each encode a single mutant AAV capsid, and
wherein the plurality of eukaryotic cells comprise heterologous polynucleotides encoding a plurality of non-naturally occurring mutant AAV capsids.
2. The library of claim 1 , wherein the eukaryotic cells are mammalian cells or insect cells.
3. The library of claim 2 , wherein the eukaryotic cells are mammalian cells, and the mammalian cells are HEK-293 cells, HEK-293T cells, or HeLa cells.
4. The library of claim 2 , wherein the eukaryotic cells are insect cells, and the insect cells SF9 cells, sf21 cells, S2 (Schneider 2) cells, BTI-TN-5B1-4 cells, or Tni cells.
5. The library of claim 1 , wherein the heterologous polynucleotide is integrated into the genome of the cell at an integrase-specific DNA attachment site in the cellular genome selected from a pseudo attP site, a pseudo Bxb1 attP site, or a pseudo φC31 attP site.
6. The library of claim 1 , wherein the plurality of eukaryotic cells further comprise:
a polynucleotide that encodes Rep2;
a helper vector or plasmid; and/or
a helper virus.
7. The library of claim 6 , wherein the plurality of eukaryotic cells further comprises an adenovirus helper virus or a herpes virus helper virus.
8. The library of claim 1 , wherein the heterologous polynucleotide further comprises a coding sequence that encodes a reporter protein operably linked to a promoter.
9. The library of claim 8 , wherein the reporter protein is green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (GFP), mCherry, mRaspberry, mPlum, mTomato, dsRed, or luciferase.
10. The library of claim 1 , wherein the heterologous polynucleotide further comprises a coding sequence that encodes a drug-resistance protein operably linked to a promoter.
11. The library of claim 10 , wherein the drug-resistance protein is pac (puromycin), bsd (blasticidin), neo (G418), hygB (hygromycin B), and Sh ble (zeocin), or Sh bla (gentamycin).
12. The library of claim 1 , wherein the heterologous polynucleotide is comprises inverted terminal repeats (ITRs).
13. The library of claim 9 , wherein the heterologous nucleotide is flanked by hybrid integrase-specific DNA attachment sites.
14. The library of claim 10 , wherein the hybrid integrase-specific DNA attachment sites are attR and attL.
15. The library of claim 1 , wherein substantially all cells in the library comprise no more than one heterologous polynucleotide comprising the coding sequence that encodes the non-naturally occurring mutant AAV capsid.
16. The library of claim 1 , comprising at least 1×10 6 cells, wherein the mutant AAV capsid of each cell is distinct from the mutant capsid of substantially all of the other cells of the library.