Compositions and methods for improving viral transduction

Abstract

Disclosed herein are fusion proteins comprising a CR3 domain of a low density lipoprotein (LDLR) and a polypeptide having phase behavior. Also provided herein are polypeptides having phase behavior. Further provided herein are methods of increasing or decreasing the efficiency of viral transduction comprising administering the fusion proteins or polypeptides having phase behavior described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 388,544, filed on July 12, 2022, the entire contents of which are incorporated herein by reference.

[0003] Reference Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (ISOL_010_01WO_SeqList_ST26.xml; size: 54,873 bytes; and creation date: July 11, 2023) are incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to compositions and methods for improving the efficiency of lentiviral transduction. The present disclosure also provides a fusion protein comprising a CR3 domain of LDLR and a polypeptide having a phase-like behavior, and a method of using the same to improve the efficiency of lentiviral transduction. The present disclosure also relates to using a polypeptide having a phase-like behavior to improve the efficiency of lentiviral transduction. Background Art

[0006] Lentiviral transduction is used for genetically engineered therapeutic cells. For example, lentiviral transduction is used to generate T cells, such as chimeric antigen receptor (CAR) T cells, which express chimeric antigen receptors targeting antigens on tumor cells on their surface, thereby causing tumor cell death. These therapies are life-saving. However, the transduction efficiency may be low, which leads to higher manufacturing costs. There is a need in the art for compositions and methods for improving the efficiency of lentiviral transduction to reduce the cost of CAR T treatment. The present disclosure addresses the needs of compositions and methods used to improve in the art. Summary of the invention

[0007] Provided herein are compositions comprising fusion proteins comprising the CR3 domain of the low density lipoprotein receptor (LDLR) and a polypeptide having a phase interaction. The compositions described herein unexpectedly enhance or block lentiviral transduction, depending on the identity of the polypeptide having a phase interaction.

[0008] Provided herein is a fusion protein comprising a CR3 domain of a low density lipoprotein (LDLR) that is at least 80% identical to a polypeptide of SEQ ID NO: 14 and a polypeptide that is at least 80% identical to a polypeptide of any one of SEQ ID NOs: 1, 2, or 11-13 and has a phase behavior. In embodiments, the CR3 domain of LDLR comprises a polypeptide that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide of SEQ ID NO: 14. In embodiments, the CR3 domain of LDLR comprises a polypeptide sequence of SEQ ID NO: 14. In embodiments, the polypeptide having phase behavior comprises a polypeptide that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NOs: 1, 2 or 11-13. In embodiments, the polypeptide having phase behavior comprises the polypeptide sequence of SEQ ID NO: 1. In embodiments, the polypeptide having phase behavior comprises the polypeptide sequence of SEQ ID NO: 2. In embodiments, the polypeptide having phase behavior comprises the polypeptide sequence of SEQ ID NO: 11. In embodiments, the polypeptide having phase behavior is located at the N-terminus of the CR3 domain of LDLR (e.g., the polypeptide having phase behavior is attached to the N-terminus of the CR3 domain of LDLR). In embodiments, the polypeptide having phase behavior is the C-terminus of the CR3 domain of LDLR (e.g., the polypeptide having phase behavior is attached to the C-terminus of the CR3 domain of LDLR). In an embodiment, the fusion protein includes a linker between the polypeptide having phase behavior and the CR3 domain of LDLR. In an embodiment, the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NOs: 21-38.

[0009] Provided herein is a method for increasing lentiviral transduction efficiency, comprising transducing a cell with a lentivirus and administering to the cell a fusion protein described herein. In embodiments, the lentiviral transduction efficiency is increased compared to the lentiviral transduction efficiency of a method that does not include administering to the cell transduced with the lentivirus a fusion protein described herein. In embodiments, the lentiviral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 321%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 421%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 5 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%. In embodiments, the method comprises administering a fusion protein comprising a polypeptide having similar behavior that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 11. In embodiments, the method comprises administering a fusion protein having the amino acid sequence of SEQ ID NO: 11. In embodiments, the method comprises administering a fusion protein having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. In embodiments, the method comprises administering a fusion protein having the amino acid sequence of SEQ ID NO: 5. In embodiments, the method increases lentiviral transduction efficiency by at least 120%.

[0010] In embodiments, a method of improving lentiviral transduction comprises transducing a cell with a lentivirus and administering to the cell a polypeptide having similar behavior; wherein the amino acid sequence of the polypeptide having similar behavior is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2. In embodiments, a method of improving lentiviral transduction comprises transducing a cell with a lentivirus and administering to the cell a polypeptide having similar behavior; wherein the polypeptide having similar behavior has an amino acid sequence of SEQ ID NO: 2. In embodiments, the method increases the efficiency of lentiviral transduction by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 321%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 421%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least In some embodiments, the method comprises increasing the lentiviral transduction efficiency by at least 90%. In some embodiments, the method comprises increasing the lentiviral transduction efficiency by at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%. In some embodiments, the method comprises increasing the lentiviral transduction efficiency by at least 90%. In some embodiments, the method comprises increasing the lentiviral transduction efficiency by at least 90% compared to the lentiviral transduction efficiency of a method that does not include administering a polypeptide having similar behavior to cells transduced with the lentivirus.

[0011] Provided herein are fusion proteins comprising a CR3 domain of a low density lipoprotein (LDLR) that is at least 80% identical to a polypeptide of SEQ ID NO: 14 and a polypeptide that is at least 80% identical to a polypeptide of any one of SEQ ID NOs: 1, 2, 11-13, and 41-42 and that has phase behavior. In embodiments, the CR3 domain of the LDLR of the fusion protein comprises a polypeptide that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide of SEQ ID NO: 14. In embodiments, the CR3 domain of the LDLR comprises the polypeptide sequence of SEQ ID NO: 14. In embodiments, the polypeptide of the fusion protein having phase behavior comprises a polypeptide that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NO: 1, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 42. In embodiments, the fusion protein comprises a linker between the polypeptide having phase behavior and the CR3 domain of LDLR. In embodiments, the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NOs: 21-38. In embodiments, the fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO:5.

[0012] Provided herein are methods for increasing lentiviral transduction efficiency, comprising transducing a cell with a lentivirus and administering a fusion protein as described herein, wherein lentiviral transduction of the cell administered with the lentivirus and the fusion protein is increased compared to a cell transduced with the lentivirus but without the fusion protein. In embodiments, the lentiviral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 321%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 421%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%. In embodiments, the lentiviral transduction efficiency is increased from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 20% to about 200%, from about 30% to about 200%, from about 40% to about 200%, from about 50% to about 200%, from about 60% to about 200%, from about 70% to about 200%, from about 80% to about 200%, from about 90% to about 200%. %, from about 100% to about 200%, from about 110% to about 200%, from about 120% to about 200%, from about 130% to about 200%, from about 140% to about 200%, from about 150% to about 200%, from about 160% to about 200%, from about 170% to about 200%, from about 180% to about 200%, from about 190% to about 200%, from about 20% to about 300%, from about 30% to about 300%, from about 40% to about 300%, from about 50% to about 300%, from about 60% to about 300%, from about 70% to about 300%, from about 80% to about 300%,from about 90% to about 300%, from about 100% to about 300%, from about 110% to about 300%, from about 120% to about 300%, from about 130% to about 300%, from about 140% to about 300%, from about 150% to about 300%, from about 160% to about 300%, from about 170% to about 300%, from about 180% to about 300%, from about 190% to about 300%, from about 200% to about 300%, from about 210% to about 300%, from about 220% to about 300%, from about 230% to about 300%, from about 240% to about 300%, from about 250% to about 300%, from about 260% to about 300%, from about 270% to about 300%, from about 280% to about 300%, from about 290% to about 300%, from about 310% to about 310%, from about 320% to about 310%, from about 330% to about 310%, from about 340% to about 310%, from about 350% to about 350%, from about 360% to about 370%, from about 380% to about 380%, from about 390% to about 390%, from about 400% to about 390%, from about 410% to about 390%, from about 420% to about 390%, from about 430% to about 390%, from about 440% to about 390%, from about 450% to about 390%, from about from about 20% to about 400%, from about 30% to about 400%, from about 40% to about 400%, from about 50% to about 400%, from about 60% to about 400%, from about 70% to about 400%, from about 80% to about 400%, from about 90% to about 400%, from about 100% to about 400%, from about 110% to about 400%, from about 120% to about 400%, from about 130% to about 400%, from about 140% to about 400%, from about 150% to about 400%, from about 160% to about 400%, from about 170% to about 400%, from about 180% to about 400%, from about 190% to about 400%, from about 200% to about 400%, from about 250% to about 250%, from about 260% to about 300%, from about 270% to about 300%, from about 280% to about 300%, from about 290% to about 300%, from about 300% to about 300%, from about 20% to about 400%, from about 30% to about 400%, from about 40% to about 400%, from about 50% to about 400%, from about 60% to about 400%, from about 70% to about 400%, from about 80% to about 400%, from about 90% to about 400%, from about 100% to about 400%, from about 110% to about 400%, from about 120% to about 400%, from about 130% to about from about 140% to about 400%, from about 150% to about 400%, from about 160% to about 400%, from about 170% to about 400%, from about 180% to about 400%, from about 190% to about 400%, from about 200% to about 400%, from about 210% to about 400%, from about 220% to about 400%, from about 230% to about 400%, from about 240% to about 400%, from about 250% to about 400%, from about 260% to about 400%, from about 270% to about 400%, from about 280% to about 400%, from about 290% to about 400%, from about 300% to about 400%, from about 310% to about 400%, from about 320% to about 400%, from about 330% to about 400%, from about 340% to about 400%, from about 350% to about 400%, from about 360% to about 400%, from about 370% to about 400%, from about 380% to about 400%, from about 390% to about 400%, from about 20% to about 500%, from about 30% to about 500%, from about 40% to about 500%, from about 50% to about 500%, from about 60% to about 500%, from about 70% to about 500%, from about 80% to about 500%,From about 90% to about 500%, from about 100% to about 500%, from about 110% to about 500%, from about 120% to about 500%, from about 130% to about 500%, from about 140% to about 500%, from about 150% to about 500%, from about 160% to about 500%, from about 170% to about 500%, from about 180% to about 500%, from about 190% to about 500%, from about 190% to about 500%, from about 110% to about 500%, from about 120% to about 500%, from about 130% to about 500%, from about 140% to about 500%, from about 150% to about 500%, from about 160% to about 500%, from about 170% to about 500%, from about 180% to about 500%, from about 190% to about 500%, from about From about 200% to about 500%, from about 210% to about 500%, from about 220% to about 500%, from about 230% to about 500%, from about 240% to about 500%, from about 250% to about 500%, from about 260% to about 500%, from about 270% to about 500%, from about 280% to about 500%, from about 290% to about 500%, from about 300% to about 500%, from about 500%, from about 310% to about 500%, from about 320% to about 500%, from about 330% to about 500%, from about 340% to about 500%, from about 350% to about 500%, from about 360% to about 500%, from about 370% to about 500%, from about 380% to about 500%, from about 390% to about 500%, from about 400% to about 500%, from about 410% to about 500%, from about 4 From about 20% to about 500%, from about 430% to about 500%, from about 440% to about 500%, from about 450% to about 500%, from about 460% to about 500%, from about 470% to about 500%, from about 480% to about 500%, or from about 490% to about 500%, wherein lentiviral transduction of cells administered with the lentivirus and the fusion protein is increased compared to cells transduced with the lentivirus but not administered with the fusion protein.

[0013] In embodiments, the method comprises administering a fusion protein comprising a polypeptide having similar behavior that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 42. In embodiments, the method comprises administering a fusion protein comprising a polypeptide having similar behavior that has an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 42.

[0014] In embodiments, the method comprises administering a fusion protein comprising a CR3 domain of LDLR that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of SEQ ID NO:14. In embodiments, the method comprises administering a fusion protein having an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. In embodiments, the lentiviral transduction efficiency is increased by at least 120%.

[0015] Provided herein are methods for increasing lentiviral transduction, comprising transducing a cell with a lentivirus and administering a polypeptide having similar behavior; wherein the amino acid sequence of the polypeptide having similar behavior is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12, wherein lentiviral transduction of the cell administered with the lentivirus and the polypeptide having similar behavior is increased compared to a cell administered with the lentivirus but without the polypeptide having similar behavior. In embodiments, the lentiviral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%, wherein lentiviral transduction of cells administered with the lentivirus and the polypeptide with similar behavior is increased compared to cells administered with the lentivirus but without the polypeptide with similar behavior. In embodiments, the lentiviral transduction efficiency is increased from about 10% to about 100%, from about 20% to about 100%, from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 20% to about 200%, from about 30% to about 200%, from about 40% to about 200%, from about 50% to about 200%, from about 60% to about 200%, from about 70% to about 200%, from about 80% to about 200%, from about 90% to about 200%, from about 100% to about 200%, from about 110% to about 200%,from about 120% to about 200%, from about 130% to about 200%, from about 140% to about 200%, from about 150% to about 200%, from about 160% to about 200%, from about 170% to about 200%, from about 180% to about 200%, from about 190% to about 200%, from about 20% to about 300%, from about 30% to about 300%, from about 40% to about 300%, from about 50% to about 300%, from about 60% to about 300%, from about 70% to about 300%, from about 80% to about 300%, from about 90% to about 300%, from about 100% to about 300%, From about 110% to about 300%, from about 120% to about 300%, from about 130% to about 300%, from about 140% to about 300%, from about 150% to about 300%, from about 160% to about 300%, from about 170% to about 300%, from about 180% to about 300%, from about 190% to about 300%, from about 200% to about 300%, from about 210% to about 300%, from about 220% to about 300%, from about 230% to about 300%, from about 240% to about 300%, from about 250% to about 300%, from about 260% to about 300%, from about 270% to about 300%, from about 280% to about 300%, from about 290% to about 300%, from about 310% to about 310%, from about 320% to about 310%, from about 330% to about 310%, from about 340% to about 310%, from about 350% to about 310%, from about 360% to about 310%, from about 370% to about 310%, from about 380% to about 380%, from about 390% to about 390%, from about 400% to about 390%, from about 410% to about 390%, from about 420% to about 390%, from about 430% to about 390%, from about 440% to about 390%, from about 450% to about 390%, from about 460% to about 390%, from about 470% to about 390%, from about 400%, from about 150% to about 400%, from about 160% to about 400%, from about 180% to about 400%, from about 200% to about 400%, from about 250% to about 400%, from about 200% to about 400%, from about 200% to about 400%, from about 250% to about 400%, from about 200% to about 400%, from about 200% to about 400%, from about 250% to about 400%, from about 200% to about 400%, from about 200% to about 400%, from about 250% to about 400%, from about 200% to about 400%, from about 250% to about 400%, from about 250% to about 400%, from about 250% to about 400%, from about 250% to about 400%, from about 250% to about 400%, from about 250% to about 400%, from about 250% to about 400%, from about 250% to about 400 400%, from about 250% to about 400%, from about 260% to about 400%, from about 270% to about 400%, from about 280% to about 400%, from about 290% to about 400%, from about 300% to about 400%, from about 310% to about 400%, from about 320% to about 400%, from about 330% to about 400%, from about 340% to about 400%, from about 350% to about 400%, from about 360% to about 400%, from about 370% to about 400%, from about 380% to about 400%, from about 390% to about 400%, from about 410% to about 410%, from about 420% to about 420%, from about 430% to about 430%, from about 440% to about 450%, from about 450% to about 450%, from about 460% to about 460%, from about 470% to about 470%, from about 480% to about 480%, from about 490% to about 490%, from about 500% to about 500%, from about 510% to about 400%,from about 320% to about 400%, from about 330% to about 400%, from about 340% to about 400%, from about 350% to about 400%, from about 360% to about 400%, from about 370% to about 400%, from about 380% to about 400%, from about 390% to about 400%, from about 20% to about 500%, from about 30% to about 500%, from about 40% to about 500%, from about 50% to about 500%, from about 60% to about 500%, from about 70% to about 500%, from about 80% to about 500%, from about 90% to about 90%, from about 100% to about 100%, from about 150% to about 150%, from about 100% to about 150%, from about 150% to about 150%, from about 150% to about 150%, from about 150% to about 150%, from about 150% to about 150%, from about 150% to about 150%, from about 150% to about 150%, from about 150% to about 150%, from about 500%, from about 100% to about 500%, from about 110% to about 500%, from about 120% to about 500%, from about 130% to about 500%, from about 140% to about 500%, from about 150% to about 500%, from about 160% to about 500%, from about 170% to about 500%, from about 180% to about 500%, from about 190% to about 500%, from about 200% to about 500%, from about 210% to about 500%, from about 220% to about 500%, from about 230% to about 500%, from about from about 240% to about 500%, from about 250% to about 500%, from about 260% to about 500%, from about 270% to about 500%, from about 280% to about 500%, from about 290% to about 500%, from about 300% to about 500%, from about 310% to about 500%, from about 320% to about 500%, from about 330% to about 500%, from about 340% to about 500%, from about 350% to about 500%, from about 360% to about 500%, from about 370% to about 500%, from about 380% to about 500% , from about 390% to about 500%, from about 400% to about 500%, from about 410% to about 500%, from about 420% to about 500%, from about 430% to about 500%, from about 440% to about 500%, from about 450% to about 500%, from about 460% to about 500%, from about 470% to about 500%, from about 480% to about 500%, or from about 490% to about 500%, wherein lentiviral transduction of cells administered with the lentivirus and the polypeptide having similar behavior is increased compared to cells administered with the lentivirus but without the polypeptide having similar behavior.

[0016] In embodiments, the lentiviral transduction efficiency is increased by at least 90%.In embodiments, the polypeptide having the same behavior has the amino acid sequence of SEQ ID NO:12.

[0017] These and other embodiments are further described in the following detailed description, examples, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 GFP expression (determined by flow cytometry) in HEK293T cells transduced with GFP-expressing lentivirus and incubated with reagents 1, 2, 3, 4, or 5 is shown. Fusion of the CR3 domain of LDLR to different polypeptides with similar behavior enhanced or blocked transduction of lentiviral particles. Reagents 1 and 4 had no effect on transduction efficiency, while reagents 2 and 5 significantly enhanced transduction efficiency and reagent 3 significantly blocked lentiviral transduction. Controls represent samples spiked with lentiviral particles but without the addition of reagents. ***p<0.001, ****p<0.0001, by one-way ANOVA with Dunnett post hoc test. Means + / - sem are presented. n = 6-9 experimental replicates per group.

[0019] Figure 2 The amount of lentivirus-associated p24 in the supernatant of HEK293T cells transduced with lentivirus expressing GFP and incubated with reagents 1, 2, 3, 4 or 5 is shown. The lentiviral particles in the supernatant of all samples had similar or higher levels compared to the control, indicating that the virus remained in the supernatant without causing damage. The control represents a sample spiked with lentiviral particles but without the addition of reagents. *p<0.05, according to one-way ANOVA with Dunnett's post hoc test. Expressed as mean + / - sem. n = 2-3 experimental replicates per group.

[0020] Figure 3A-3B GFP expression (measured by flow cytometry) in HEK293T cells transduced with lentivirus expressing GFP and co-incubated with reagent 4 or 5 after the mixture was incubated in 1.5 M NaCl for 0, 1, 2, 4, 6 or 12 hours. Figure 3A ) or in serum-free suspension medium ( Figure 3B ). Control represents samples spiked with lentiviral particles but without added reagents. *p<0.05, **p<0.01, ***p<0.001, by two-way ANOVA with Fisher's LSD post hoc test. Means + / - sem. n = 4 replicates per group.

[0021] Figure 4A-4B GFP expression (measured by flow cytometry) in HEK293T cells transduced with lentivirus expressing GFP and incubated with reagent 4 or 5 after the mixture was stored at 4-8°C for 0, 1, 2, 3, 4 or 5 weeks. Figure 4A) or in serum-free suspension medium ( Figure 4B ). Control represents samples spiked with lentiviral particles but without added reagents. *p<0.05, **p<0.01, ***p<0.001, by two-way ANOVA with Fisher's LSD post hoc test. Means + / - sem. n = 4 replicates per group.

[0022] Figure 5A-5B GFP expression (measured by flow cytometry) in HEK293T cells transduced with lentivirus expressing GFP and incubated with reagent 4 or 5 after the mixture was stored at 37°C for 0, 2, 4 or 6 hours. Figure 5A ) or in serum-free suspension medium ( Figure 5B ). Control represents samples spiked with lentiviral particles but without added reagents. *p<0.05, **p<0.01, ***p<0.001, by two-way ANOVA with Fisher's LSD post hoc test. Means + / - sem. n = 4 replicates per group.

[0023] Figure 6 GFP expression (determined by flow cytometry) in HEK293T cells transduced with lentivirus expressing GFP, co-incubated with reagent 4 or 5 after the mixture was stored at -80°C for 0, 1, 2, 3, 4 or 5 freeze-thaw cycles is shown. Lentivirus was produced in adherent medium containing serum. Control represents a sample spiked with lentiviral particles but without the addition of reagent. *p<0.05, **p<0.01, ***p<0.001, according to two-way ANOVA with Fisher's LSD post hoc test. Mean + / - sem is expressed. n = 4 replicates per group.

[0024] Figure 7A-7B Shown is GFP expression (determined by flow cytometry) in HEK293T cells transduced with adenovirus expressing GFP and incubated with Reagent 1 or 2. Control represents a sample spiked with adenoviral particles but without added reagent. n = 3 technical replicates per group. DETAILED DESCRIPTION

[0025] definition

[0026] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" may refer to one protein or a mixture of such proteins, and reference to "a method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0027] As used herein, the term "about" or "approximately" before a numerical value means a range of plus or minus 10% of the value. For example, "about 100" includes 90 and 110.

[0028] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative ("or").

[0029] It is specifically intended that the various features described herein can be used in any combination, unless the context dictates otherwise.

[0030] In addition, the present disclosure also contemplates that, in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To further illustrate, for example, if the specification indicates that a particular amino acid can be selected from A, G, I, L, and / or V, this expression also indicates that the amino acid can be selected from any subset of these one or more amino acids, such as A, G, I, or L; A, G, I, or V; A or G; only L; and so on, as if each such subcombination is expressly set forth herein. In addition, this expression also indicates that one or more particular amino acids can be abandoned. For example, in a particular embodiment, the amino acid is not A, G, or I; is not A; is not G or V; and so on, as if each such possible statement is expressly listed herein.

[0031] As used herein, the term "fragment" refers to a protein or polypeptide, including a truncated form of a protein or polypeptide. For example, a fragment of LDLR can include about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% or about 99% of the amino acid of the full-length LDLR. A fragment of the CR3 domain of LDLR can contain about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97% or about 99% of the amino acid of the full-length CR3 domain of LDLR. The full-length CR3 domain of LDLR has the amino acid sequence of SEQ ID NO: 14. In an embodiment, the fragment of the CR3 domain of LDLR includes at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, or at least 38 amino acids of the CR3 domain of LDLR.

[0032] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can comprise a protein sequence. The term "peptide" can refer to a short chain of amino acids, including, for example, natural peptides, recombinant peptides, synthetic peptides, or combinations thereof. Proteins and polypeptides can include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like.

[0033] When referring to a polypeptide, the term "modification" refers to a mutation, deletion or addition of an amino acid of the polypeptide. In an embodiment, the polypeptide is the CR3 domain of LDLR. The position of the modification within the CR3 domain of LDLR can be determined based on comparing the polypeptide sequence with SEQ ID NO: 14 (the amino acid sequence of the CR3 domain of LDLR).

[0034] A "polynucleotide" is a sequence of nucleotide bases and may be RNA, DNA, or a DNA-RNA hybrid sequence (including naturally occurring and non-naturally occurring nucleotides). In some embodiments, a polynucleotide is a single-stranded or double-stranded DNA sequence.

[0035] In the context of two or more nucleic acid or polypeptide sequences, the term "percent identity" refers to two or more sequences or subsequences that, when compared, have a specific percentage of nucleotides or amino acid residues that are identical. Unless otherwise indicated, the EMBL European Bioinformatics Institute (EMBL-EBI) tool EMBOSS needle, available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / , is used to determine the percent identity. The following default parameters can be used for EMBOSS needle pairwise alignments: matrix = BLOSUM62; gap opening = 10; gap extension = 0.5; terminal gap penalty = false; terminal gap opening = 10; terminal gap extension = 0.5. In an embodiment, the percent identity is calculated over the full length of the compared sequence. In some embodiments, the sequence identity is at each about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 35 amino acids, about 40 amino acids, about 45 amino acids, about 50 amino acids, about 55 amino acids, about 60 amino acids, about 65 amino acids, about 70 amino acids, about 75 amino acids, about 80 amino acids, about 85 amino acids, about 90 amino acids, about 95 amino acids, about 100 amino acids, about 105 amino acids, about 11 The comparison is calculated on a fragment of the compared sequence of about 10 amino acids, about 115 amino acids, about 120 amino acids, about 125 amino acids, about 130 amino acids, about 135 amino acids, about 140 amino acids, about 145 amino acids, about 150 amino acids, about 155 amino acids, about 160 amino acids, about 165 amino acids, about 170 amino acids, about 175 amino acids, about 180 amino acids, about 185 amino acids, about 190 amino acids, about 195 amino acids, or about 200 amino acids.

[0036] Compositions comprising a fusion protein comprising the CR3 domain of LDLR and a polypeptide having a phase-dependent manner

[0037] The present disclosure provides compositions comprising fusion proteins comprising a CR3 domain of LDLR and a polypeptide having phase behavior. In embodiments, the CR3 domain of LDLR is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 14. In an embodiment, the CR3 domain of LDLR comprises about 20 to about 39, about 21 to about 39, about 22 to about 39, about 23 to about 39, about 24 to about 39, about 25 to about 39, about 26 to about 39, about 27 to about 39, about 28 to about 39, about 29 to about 39, about 30 to about 39, about 31 to about 39, about 32 to about 39, about 33 to about 39, about 34 to about 39, about 35 to about 39, about 36 to about 39, about 37 to about 39, or about 38 to about 39 contiguous amino acids of SEQ ID NO:14. In an embodiment, the CR3 domain of LDLR comprises about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, or about 39 consecutive amino acids of SEQ ID NO: 14. In an embodiment, the CR3 domain of LDLR comprises 1 to 5 modifications, 1 to 4, 1 to 3, or 1 to 2 modifications compared to the polypeptide of SEQ ID NO: 14. In an embodiment, the CR3 domain of LDLR comprises 1, 2, 3, 4, or 5 modifications compared to the polypeptide of SEQ ID NO: 14.

[0038] In embodiments, the polypeptides having phase behavior include at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to the polypeptides of any one of SEQ ID NOs: 1, 2, 11-13, 39 and 41-42. In embodiments, the polypeptides having phase behavior have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications compared to the polypeptides of any one of SEQ ID NOs: 1, 2, 11-13, 39 and 41-42. In embodiments, the polypeptide having phase behavior comprises about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, 1 to about 9, or 1 to about 10 modifications compared to the polypeptide of any one of SEQ ID NOs: 1, 2, 11-13, 39, and 41-42.

[0039] In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:1. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:2. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:11. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:12. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:13. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:39. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:41. In an embodiment, the polypeptide having phase behavior comprises the amino acid sequence of SEQ ID NO:42.

[0040] In embodiments, the fusion protein is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NOs: 3-5 and 15-20. In embodiments, the fusion protein is a polypeptide having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications compared to the polypeptide of any one of SEQ ID NOs: 3-5 and 15-20. In embodiments, the fusion protein comprises about 1 to about 5, about 1 to about 6, about 1 to about 7, about 1 to about 8, 1 to about 9 or 1 to about 10 modifications compared to the polypeptide of any one of SEQ ID NOs: 3-5 and 15-20.

[0041] In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO: 3. In an embodiment, the fusion protein comprises the amino acid sequence of SEQ ID NO:5.

[0042] In an embodiment, the fusion protein comprises a polypeptide with phase behavior of SEQ ID NO: 1 and a CR3 domain of LDLR (SEQ ID NO: 14). In an embodiment, the fusion protein comprises a polypeptide with phase behavior of SEQ ID NO: 2 and a CR3 domain of LDLR (SEQ ID NO: 14). In an embodiment, the fusion protein comprises a polypeptide with phase behavior of SEQ ID NO: 11 and a CR3 domain of LDLR (SEQ ID NO: 14). In an embodiment, the fusion protein comprises a polypeptide with phase behavior of SEQ ID NO: 12 and a CR3 domain of LDLR (SEQ ID NO: 14). In an embodiment, the fusion protein comprises a polypeptide with phase behavior of SEQ ID NO: 13 and a CR3 domain of LDLR (SEQ ID NO: 14). In an embodiment, the fusion protein comprises a polypeptide with phase behavior of SEQ ID NO: 42 and a CR3 domain of LDLR (SEQ ID NO: 14).

[0043] In embodiments, provided herein are nucleic acids encoding fusion proteins as described herein. In embodiments, provided herein are nucleic acids encoding polypeptides having similar behavior as described herein. In embodiments, the polypeptides having similar behavior are encoded by a nucleic acid of any one of SEQ ID NOS: 6-8, or by a nucleic acid having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 6-8. In embodiments, the fusion protein is encoded by a nucleic acid of any one of SEQ ID NOS: 9-10, or by a nucleic acid having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOS: 9-10.

[0044] In an embodiment, the fusion protein comprises an N-terminal methionine. In an embodiment, the fusion protein lacks an N-terminal methionine.

[0045] In an embodiment, the fusion protein includes a linker between a polypeptide having phase behavior and the CR3 domain of LDLR. In an embodiment, the linker does not interfere with the function of the fusion protein described herein. In some embodiments, the linker can adopt various secondary structures, including but not limited to α-helix, β-strand and random coil. In some embodiments, the linker adopts α-helix and includes (EAAAK) n (SEQ ID NO: 21), wherein n is an integer from 1 to 20.

[0046] In some embodiments, the linker consists of (G4S) n (SEQ ID NO:22), wherein n can be an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In an embodiment, the polypeptide linker has repetitions of (SGGG)n (SEQ ID NO: 23), wherein n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In an embodiment, the polypeptide linker has (GGGS) n(SEQ ID NO:24), wherein n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

[0047] In some embodiments, the linker has an amino acid sequence of KESGSVSSEQLAQFRSLD (SEQ ID NO: 25). In some embodiments, the linker has an amino acid sequence of EGKSSGSGSESKST (SEQ ID NO: 26). In some embodiments, the linker comprises only glycine.

[0048] In some embodiments, the peptide linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a furin cleavage site.

[0049] In some aspects, the polypeptide linker is poly-(Gly) n A linker wherein n is an integer from 1 to 30 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (SEQ ID NO: 27). In some embodiments, the linker is selected from the group consisting of a dipeptide, a tripeptide, and a tetrapeptide. In an embodiment, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamate (LE), and serine-arginine (SR).

[0050] In some embodiments, the linker is selected from GKSSGSGSESKS (SEQ ID NO:28), GSTSGSGKSSEGKG (SEQ ID NO:29), GSTSGSGKSSEGSGSTKG (SEQ ID NO:30), GSTSGSGKPGSGEGSTKG (SEQ ID NO:31), EGKSSGSGSESKEF (SEQ ID NO:32), SRSSG (SEQ ID NO:33) and SGSSC (SEQ ID NO:34).

[0051] In some embodiments, the linker is a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a 2A peptide. 2A peptides are a class of 18-22 amino acid long peptides that induce ribosome skipping during protein translation in cells. In some embodiments, the 2A peptide is a T2A peptide having an amino acid sequence of EGRGSLLTCGDVEENPGP (SEQ ID NO: 35), a P2A peptide having an amino acid sequence of ATNFSLLKQAGDVEENPGP (SEQ ID NO: 36), an E2A peptide having an amino acid sequence of QCTNYALLKLAGDVESNPGP (SEQ ID NO: 37), or an F2A peptide having an amino acid sequence of VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 38). In some embodiments, the 2A peptide has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to any one of SEQ ID NO. 35-38.

[0052] In embodiments, the polypeptide having phase behavior is located at the N-terminus of the CR3 domain of LDLR. (e.g., the polypeptide having phase behavior is attached to the N-terminus of the CR3 domain of LDLR). In embodiments, the polypeptide having phase behavior is located at the C-terminus of the CR3 domain of LDLR (e.g., the polypeptide having phase behavior is attached to the C-terminus of the CR3 domain of LDLR).

[0053] Methods using a composition comprising a polypeptide or fusion protein having phase behavior, the fusion protein comprising the CR3 domain of LDLR and a polypeptide having phase behavior

[0054] In embodiments, methods for improving the efficiency of lentiviral transduction are provided herein. In embodiments, methods for improving the efficiency of lentiviral transduction include administering a fusion protein as described herein to cells. In embodiments, fusion proteins and lentiviruses are administered to cells simultaneously (i.e., within 15 minutes). In embodiments, the fusion protein is administered within 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours of lentivirus. In embodiments, before administering lentivirus to cells, fusion proteins are administered to cells. In embodiments, after administering lentivirus to cells, fusion proteins are administered to cells. In embodiments, the fusion protein has a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 3-5 or 15-20.

[0055] In an embodiment, the method for improving the transduction efficiency of a lentivirus comprises administering to a cell a polypeptide having a phase behavior as described herein. In an embodiment, a polypeptide having a phase behavior is administered simultaneously (i.e., within 15 minutes) with a lentivirus. In an embodiment, a polypeptide having a phase behavior is administered within 15 minutes, within 30 minutes, within 45 minutes, within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, within 8 hours, within 9 hours, within 10 hours, within 11 hours, within 12 hours, within 13 hours, within 14 hours, within 15 hours, within 16 hours, within 17 hours, within 18 hours, within 19 hours, within 20 hours, within 21 hours, within 22 hours, within 23 hours, or within 24 hours of the lentivirus. In an embodiment, a polypeptide having a phase behavior is administered to a cell before administering a lentivirus to the cell. In an embodiment, a polypeptide having a phase behavior is administered to a cell after administering a lentivirus to the cell. In embodiments, the polypeptide having phase behavior has a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1, 2, or 11-13.

[0056] In an embodiment, the lentiviral transduction efficiency (or the percentage of cells infected by the lentivirus) is assessed by flow cytometry. For example, in an embodiment, the lentivirus encodes a fluorescent protein, such as green fluorescent protein. The lentiviral transduction efficiency is the percentage of cells expressing the fluorescent protein. In other embodiments, the lentivirus encodes a cell surface protein. Cells transduced with a lentivirus encoding a cell surface protein can be incubated with a fluorescent antibody that binds to the cell surface protein. The lentiviral transduction efficiency is the percentage of cells that are bound to the fluorescent antibody.

[0057] In embodiments, the methods described herein provide improved lentiviral transduction efficiency compared to alternative methods. In embodiments, alternative methods include lentiviral transduction methods that do not administer a fusion protein or polypeptide having similar behavior as described herein. In embodiments, alternative methods are conventional methods for improving lentiviral transduction. Examples of conventional methods for improving lentiviral transduction include polybrene or the lentiviral enhancer TRANSDUX. TM Transduction in the presence of MAX.

[0058] In embodiments, the methods described herein increase lentiviral transduction efficiency by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 321%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%.

[0059] In embodiments, the method for increasing lentiviral transduction efficiency comprises administering a polypeptide having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2. In embodiments, administering a polypeptide having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 2 increases lentiviral transduction efficiency by at least 90% compared to alternative methods.

[0060] In an embodiment, the method for increasing the transduction efficiency of a lentivirus comprises administering a fusion protein having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5. In an embodiment, administering a fusion protein having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 5 increases the transduction efficiency of a lentivirus by at least 120%.

[0061] In embodiments, the methods described herein provide for reduced lentiviral transduction efficiency. In embodiments, the lentiviral transduction methods described herein comprising administering to cells a polypeptide or fusion protein having comparable behavior as described herein have a lentiviral transduction efficiency that is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 321%, at least 330%, at least 340%, at least 350%, at least 361%, at least 370%, at least 380%, at least 390%, at least 401%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 461%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 561%, at least 570%, at least 580%, at least 590%, 70%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%. In embodiments, the method of lentiviral transduction comprising administering a fusion protein having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3 reduces the lentiviral transduction efficiency by at least 80%, compared to a method that does not comprise administering a polypeptide having phase behavior.

[0062] In embodiments, the methods described herein provide for reduced adenoviral transduction efficiency. In embodiments, the adenoviral transduction efficiency of the adenoviral transduction methods described herein comprising administering a polypeptide having comparable behavior as described herein is reduced by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 321%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 401%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 530%, at least 540%, at least 550%, at least 560%, at least 570%, at least 580%, at least 590%, %, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%. In embodiments, the method comprises incubating the cells with an adenovirus and a polypeptide having a reciprocal effect. In embodiments, the polypeptide having phase behavior comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NOs: 1, 13, 39 and 41.

[0063] Methods for stabilizing lentivirus

[0064] In an embodiment, a method for stabilizing lentiviral particles is provided herein. As used herein, with respect to lentiviral particles, the term "stabilize / stabilizing" refers to the ability of a fusion protein to reduce the degradation or aggregation of a sample including multiple lentiviral particles, prevent lentiviral molecules from binding to other proteins, enhance the production of cells to synthesize lentiviral particles, or enhance the function of lentiviral particles. In an embodiment, under conditions such as salt, freeze-thaw cycles, at about 4-8°C or about 37°C, lentiviral particles incubated with the fusion protein described herein are stabilized compared to lentiviral particles not incubated with the fusion protein described herein.

[0065] In embodiments, the method of stabilizing a lentivirus comprises incubating the lentivirus with a fusion protein described herein. In embodiments, the fusion protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 4 and 5. In embodiments, the fusion protein comprises the CR3 domain of LDLR. In embodiments, the CR3 domain of the LDLR protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 14. In an embodiment, the fusion protein comprises a polypeptide having phase behavior, which comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO:42 and 2.

[0066] In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at room temperature in the presence of up to about 1.5 M NaCl for up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, or up to about 6 hours. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at room temperature in the presence of up to about 1.5 M NaCl for at least 6 hours. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable for 1 to about 6 hours, 1 to about 5 hours, 1 to about 4 hours, 1 to about 3 hours, or 1 to about 2 hours in the presence of up to about 1.5 M NaCl. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at room temperature in the presence of up to about 1.5 M NaCl for up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, or up to about 6 hours. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable in the presence of about 1.5 M NaCl at room temperature for at least about 6 hours. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable in the presence of about 1.5 M NaCl for 1 to about 6 hours, 1 to about 5 hours, 1 to about 4 hours, 1 to about 3 hours, or 1 to about 2 hours.

[0067] In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at about 4-8°C for up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, or up to about 6 weeks. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at about 4-8°C for at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 6 weeks. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at about 4-8°C for 1 to about 6 weeks, 1 to about 5 weeks, 1 to about 4 weeks, 1 to about 3 weeks, or 1 to about 2 weeks.

[0068] In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at about 37°C for up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, or up to about 6 hours. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at about 37°C for at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 6 hours. In embodiments, when the lentivirus is incubated with a fusion protein described herein, the lentivirus is stable at about 37°C for 1 to about 6 hours, 1 to about 5 hours, 1 to about 4 hours, 1 to about 3 hours, or 1 to about 2 hours.

[0069] In embodiments, when the lentivirus is incubated with a fusion protein as described herein, the lentivirus is stable in multiple freeze-thaw cycles. In embodiments, when the lentivirus is incubated with a fusion protein as described herein, the lentivirus is stable in at least 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 freeze-thaw cycles. In embodiments, when the lentivirus is incubated with a fusion protein as described herein, the lentivirus is stable in 1 to about 10, 1 to about 9, 1 to about 8, 1 to about 7, 1 to about 6, 1 to about 5, 1 to about 4, 1 to about 3, or 1 to about 2 freeze-thaw cycles.

[0070] sequence

[0071] In embodiments, provided herein are polypeptides or nucleic acids of Table A. In embodiments, provided herein are polypeptides or nucleic acids that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide or nucleic acid of Table A.

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] Example

[0081] Example 1. Polypeptides and / or fusion proteins described herein with phase behavior block or enhance lentiviral transduction

[0082] Objective: To evaluate the ability of polypeptides with phase interaction or fusion proteins comprising polypeptides with phase interaction and the CR3 domain of low-density lipoprotein receptor (LDLR) (agents 1-5 of Table B) to affect lentiviral transduction. Each fusion protein of SEQ ID NO: 3-5 comprises a different polypeptide with phase interaction.

[0083] Table B

[0084]

[0085] Methods – Cell culture: HEK293T cells were plated in 24-well plates. 500 μL of cells were suspended in complete culture medium (supplemented with 10% fetal bovine serum (FBS, GIBCO TM Catalog No. 10082147), 1x non-essential amino acids (NEAA, GIBCO TM Dulbecco's modified Eagle's medium (DMEM, GIBCO TM Catalog number A4192001)). Add 50,000 cells to each well. Incubate the plate overnight at 37°C, 5% CO2 in an incubator.

[0086] Methods – Lentiviral transduction: The next morning, replace the cell culture medium with 500 μL of fresh culture medium. Transduce 5 μL of lentivirus encoding green fluorescent protein (GFP) (4 x 10 6 vg / mL) was added to each well. Reagent 1, 2, 3, 4 or 5 was added to each well (1 μM). HEK293T cells transduced with lentivirus that were not incubated with reagent 1, 2, 3, 4 or 5 were used as controls. The lentivirus was incubated with the cells for 24 hours. p24 ELISA was evaluated to determine the viral load. Flow cytometry was performed to identify the amount of cells expressing GFP.

[0087] result: Figure 1 The percentage of cells expressing GFP compared to the control is shown. Reagents 1 and 4 had no effect on transduction efficiency. Reagents 2 and 5 significantly enhanced the efficiency of lentiviral transduction. In contrast, reagent 3 significantly blocked the efficiency of lentiviral transduction. It is noteworthy that reagents 3 and 5 contain different polypeptides with phase behavior. Reagent 3 includes a polypeptide with phase behavior of SEQ ID NO:1, and reagent 5 includes a polypeptide with phase behavior of SEQ ID NO:11. The data show that unexpectedly different polypeptides with phase behavior have different effects on lentiviral transduction. The viral load in the supernatant of each sample remained consistent ( Figure 2 ). This indicates that the virus remained in the supernatant without being damaged.

[0088] Example 2. The fusion proteins described herein stabilize lentivirus under various conditions

[0089] Purpose: To evaluate the ability of the polypeptides with phase behavior from Example 1 (agents 4 and 5 of Table B) to stabilize lentivirus under various conditions prior to transduction. Each protein of SEQ ID NO: 4 and 5 comprises a different polypeptide with phase behavior.

[0090] Methods – Cell culture: HEK293T cells were plated in 24-well plates. 500 μL of cells were suspended in complete culture medium (supplemented with 10% fetal bovine serum (FBS, GIBCO TM Catalog No. 10082147), 1x non-essential amino acids (NEAA, GIBCO TM Dulbecco's modified Eagle's medium (DMEM, GIBCO TM Catalog number A4192001)). Add 50,000 cells to each well. Incubate the plate overnight at 37°C, 5% CO2 in an incubator.

[0091] Methods – Lentivirus production: Lentiviruses were produced using either adherent cells in serum-containing medium or suspension cells in serum-free medium.

[0092] Methods – Lentiviral transduction: The next morning, replace the cell culture medium with 500 μL of fresh culture medium. Transduce 5 μL of lentivirus encoding green fluorescent protein (GFP) (4 x 10 6 vg / mL) was incubated with reagent 4 or 5 (10 μM). Subsequently, a composition containing lentivirus and reagent 4 or 5 was added to each well. HEK293T cells transduced with lentivirus that were not incubated with reagent 4 or 5 were used as controls. The lentivirus was incubated with the cells for 24 hours. Flow cytometry was performed to identify the amount of cells expressing GFP.

[0093] Methods – Stable at different temperatures: Aliquots containing reagent 4 or 5 mixed with lentivirus were stored at: 1) 4-8°C for 2 months, with additional samples taken weekly for testing; 2) 37°C for 6 hours, with additional samples taken every hour for testing; or 3) -80°C for 1 hour and then thawed on ice (one freeze-thaw cycle), and the freeze-thaw cycle was repeated for a total of 5 cycles, with additional samples taken after each freeze-thaw cycle for testing. Samples taken at different time points were transduced as described above.

[0094] Methods – Stable in high salt concentrations: Aliquots containing reagent 4 or 5 mixed with lentivirus were stored and 1.5 M NaCl was rotated for 12 h at room temperature, with additional samples taken every hour for testing. Samples taken at different time points were transduced with lentivirus as described above.

[0095] result: Figure 3A and 3B Shown is the stability of lentiviral particles measured by the percentage of GFP+ cells in 1.5 M NaCl from 0 to 12 hours. Figure 3A Shown is the stability of lentivirus produced in adherent cells in the presence of serum and Reagent 5 compared to controls. Figure 3B Shown is the stability of lentivirus produced in suspension cells compared to controls in the absence of serum but in the presence of Agents 4 or 5. The CR3 domain of LDLR provided enhanced, statistically significant stability for up to 6 hours compared to controls for both adherent and suspension cell produced lentivirus.

[0096] Figure 4A and 4B Shown is the stability of lentiviral particles measured by the percentage of GFP+ cells over 2 months at 4-8°C. Figure 4A Shown is the stability of lentivirus produced in adherent cells in the presence of serum and reagent 4 or 5 compared to controls. Figure 4B Shown is the stability of lentivirus produced in suspension cells in the absence of serum but in the presence of Agents 4 or 5 compared to controls. The CR3 domain of LDLR provided enhanced stability for up to 5 weeks compared to controls in both adherent and suspension conditions.

[0097] Figure 5A and 5B Shown is the stability of lentiviral particles measured by the percentage of GFP+ cells at 37°C for 6 hours. Figure 5A Shown is the stability of lentivirus produced in adherent cells in the presence of serum and Reagent 5 compared to controls. Figure 5BShown is the stability of lentivirus produced in suspension cells in the absence of serum but in the presence of Agents 4 or 5 compared to controls. The CR3 domain of LDLR provided enhanced, statistically significant stability for up to 6 hours compared to controls in both adherent and suspension conditions.

[0098] Figure 6 The stability of lentiviral particles measured by the percentage of GFP+ cells over 5 freeze-thaw cycles at -80°C is shown in serum-free suspension conditions, when lentivirus was produced with reagents 4 or 5 compared to controls. The CR3 domain of LDLR provided enhanced, statistically significant stability for up to 5 freeze-thaw cycles compared to controls.

[0099] These results indicate that the addition of agents 4 and / or 5 stabilized the lentivirus exposed to different temperatures (4-8°C, 37°C, and -80°C) and salt conditions (1.5 M NaCl), thereby maintaining the potency of the lentivirus.

[0100] Example 3. Peptides Described Herein with Phase Behavior Block Adenoviral Transduction

[0101] Purpose: To evaluate the ability of peptides with phase behavior (agents 1 and 2 of Table C) to affect adenoviral transduction.

[0102] Table C

[0103] Reagents describe 1 Polypeptide with phase behavior (SEQ ID NO: 1) 2 Polypeptide with phase behavior (SEQ ID NO: 39)

[0104] Methods – Cell culture: HEK293T cells were plated in 24-well plates. 500 μL of cells were suspended in complete culture medium (supplemented with 10% fetal bovine serum (FBS, GIBCO TM Catalog No. 10082147), 1x non-essential amino acids (NEAA, GIBCO TM Dulbecco's modified Eagle's medium (DMEM, GIBCO TM Catalog number A4192001)). 100,000 cells were added to each well. The plate was incubated overnight at 37°C, 5% CO2 in an incubator.

[0105] Methods – Adenoviral transduction: The next morning, replace the cell culture medium with 500 μL of fresh culture medium. Transduce 1 μL of adenovirus encoding green fluorescent protein (GFP) (1x 10 12vg / mL) was added to each well. Reagent 1 or 2 (0.1 μM, 1 μM, 10 μM, 100 μM) was added to each well. HEK293T cells transduced with adenovirus that were not incubated with reagent 1 or 2 were used as controls. Adenovirus was incubated with cells for 24 hours. INVITROGEN TM EVOS TM M5000 live cell imager was used to verify infectivity. Flow cytometry was performed to identify the amount of cells expressing GFP.

[0106] result: Fig. 7A and Figure 7B The percentage of cells expressing GFP compared to controls are shown for Agent 1 and Agent 2, respectively. Both Agents 1 and 2 blocked adenoviral transduction at concentrations greater than 1 μM (not tested at higher concentrations).

[0107] Implementation of the numbering of the present disclosure

[0108] Notwithstanding the appended claims, the present disclosure sets forth the following numbered embodiments:

[0109] 1. A fusion protein comprising a CR3 domain of a low density lipoprotein (LDLR) that is at least 80% identical to the polypeptide of SEQ ID NO: 14 and a polypeptide that is at least 80% identical to the polypeptide of any one of SEQ ID NOs: 1, 2 or 11-13 and has a phase-behavior.

[0110] 2. The fusion protein of embodiment 1, wherein the CR3 domain of the LDLR comprises a polypeptide that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of SEQ ID NO:14.

[0111] 3. The fusion protein according to embodiment 2, wherein the CR3 domain of the LDLR comprises the polypeptide sequence of SEQ ID NO:14.

[0112] 4. A fusion protein according to any one of embodiments 1-3, wherein the polypeptide having phase behavior includes a polypeptide that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NO: 1, 2 or 11-13.

[0113] 4. A fusion protein according to any one of embodiments 1-3, wherein the polypeptide having phase behavior comprises a polypeptide sequence of SEQ ID NO: 1.

[0114] 5. A fusion protein according to any one of embodiments 1-3, wherein the polypeptide having phase behavior comprises a polypeptide sequence of SEQ ID NO: 2.

[0115] 6. A fusion protein according to any one of embodiments 1-3, wherein the polypeptide having phase behavior comprises a polypeptide sequence of SEQ ID NO:11.

[0116] 7. A fusion protein according to any one of embodiments 1-6, wherein the polypeptide having phase behavior is the N-terminus of the CR3 domain of the LDLR.

[0117] 8. A fusion protein according to any one of embodiments 1-6, wherein the polypeptide having phase behavior is the C-terminus of the CR3 domain of the LDLR.

[0118] 9. A fusion protein according to any one of embodiments 1-6, comprising a linker between the polypeptide having phase behavior and the CR3 domain of the LDLR.

[0119] 10. A fusion protein according to embodiment 9, wherein the linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NOs: 21-38.

[0120] 11. A method for improving lentiviral transduction efficiency, comprising transducing cells with a lentivirus and administering the fusion protein according to any one of embodiments 1-10.

[0121] 12. The method of embodiment 11, wherein the lentiviral transduction efficiency is improved compared to the lentiviral transduction efficiency of the method that does not include administering the fusion protein of any one of embodiments 1-10.

[0122] 13. The method of embodiment 11, wherein the lentiviral transduction efficiency is improved compared to conventional methods for improving lentiviral transduction.

[0123] 14. The method of any one of embodiments 11-13, wherein the lentiviral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, At least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%.

[0124] 15. A method according to any one of embodiments 11-14, wherein the fusion protein comprises a polypeptide with phase behavior that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:11.

[0125] 16. A method according to embodiment 15, wherein the fusion protein comprises a polypeptide having a phase behavior and an amino acid sequence of SEQ ID NO: 11.

[0126] 17. A method according to any one of embodiments 11-16, wherein the fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:5.

[0127] 18. The method according to embodiment 17, wherein the fusion protein has an amino acid sequence of SEQ ID NO:5.

[0128] 19. The method of any one of embodiments 14-18, wherein the lentiviral transduction efficiency is increased by at least 120%.

[0129] 20. A method for improving lentiviral transduction, comprising transducing a cell with a lentivirus and administering a polypeptide having similar behavior; wherein the amino acid sequence of the polypeptide having similar behavior is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:2.

[0130] 21. A method according to embodiment 20, wherein the polypeptide having phase behavior has an amino acid sequence of SEQ ID NO: 2.

[0131] 22. The method of embodiment 20 or 21, wherein the viral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, at least 510%, at least 520%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%.

[0132] 23. The method of any one of embodiments 20-22, wherein the lentiviral transduction efficiency is increased by at least 90%.

[0133] 24. The method of any one of embodiments 20-23, wherein the lentiviral transduction efficiency is improved compared to the viral transduction efficiency of a method that does not include administering the polypeptide having phase behavior.

[0134] 25. The method of any one of embodiments 20-23, wherein the lentiviral transduction efficiency is improved compared to conventional methods for improving lentiviral transduction.

[0135] Join by reference

[0136] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. However, the reference to any reference, article, publication, patent, patent publication and patent application cited herein is not, and should not be taken as, an admission or any form of suggestion that it constitutes valid prior art or forms part of the common general knowledge in any country in the world. All of the following references are incorporated herein by reference in their entirety: U.S. Patent No. 11,015,174 and U.S. Application No. 2019 / 0055523.

Claims

1. A fusion protein comprising a CR3 domain of a low density lipoprotein (LDLR) that is at least 80% identical to the polypeptide of SEQ ID NO: 14 and a polypeptide that has a phase-matching behavior that is at least 80% identical to the polypeptide of any one of SEQ ID NOs: 1, 2, 11-13 or 41-42.

2. The fusion protein according to claim 1, wherein The CR3 domain of the LDLR comprises a polypeptide that is at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of SEQ ID NO:

14.

3. The fusion protein according to claim 2, wherein The CR3 domain of the LDLR includes the polypeptide sequence of SEQ ID NO:

14.

4. The fusion protein according to any one of claims 1 to 3, wherein The polypeptides having phase behavior include polypeptides that are at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of any one of SEQ ID NOs: 1, 2, 11-13 and 41-42.

5. The fusion protein according to any one of claims 1 to 4, wherein The polypeptide having phase behavior includes the polypeptide sequence of any one of SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:

42.

6. The fusion protein according to any one of claims 1 to 5, comprising a linker between the polypeptide having phase behavior and the CR3 domain of the LDLR.

7. The fusion protein according to claim 6, wherein The linker has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the polypeptide of any one of SEQ ID NOs: 21-38.

8. The fusion protein according to any one of claims 1 to 7, wherein The fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the polypeptide of SEQ ID NO:

5.

9. A method for improving lentiviral transduction efficiency, comprising transducing cells with a lentivirus and administering the fusion protein of any one of claims 1 to 8, wherein the lentiviral transduction of cells administered with the lentivirus and the fusion protein is improved compared to cells transduced with the lentivirus but not administered with the fusion protein.

10. The method according to claim 9, wherein: The lentiviral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, %, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%.

11. The method according to any one of claims 9 to 10, wherein: The fusion protein includes polypeptides with similar behavior that are at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 12 or SEQ ID NO:

42.

12. The method according to claim 11, wherein: The fusion protein includes a polypeptide having an amino acid sequence of SEQ ID NO: 12 or SEQ ID NO: 42 and having a phase-matching behavior.

13. The method according to any one of claims 9 to 12, wherein: The CR3 domain of the LDLR is a polypeptide having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:

14.

14. The method according to any one of claims 9 to 13, wherein: The CR3 domain of the LDLR is a polypeptide of SEQ ID NO:

14.

15. The method according to any one of claims 9 to 14, wherein: The fusion protein has an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

5.

16. The method according to any one of embodiments 9 to 15, wherein: The lentiviral transduction efficiency was increased by at least 120%.

17. A method for improving lentiviral transduction, comprising transducing cells with a lentivirus and administering a polypeptide having a similar behavior; wherein, The amino acid sequence of the polypeptide with similar behavior is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12, wherein lentiviral transduction of cells administered with the lentivirus and the polypeptide with similar behavior is increased compared to cells administered with the lentivirus but without the polypeptide with similar behavior.

18. The method according to claim 17, wherein: The lentiviral transduction efficiency is increased by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, at least 500%, %, at least 230%, at least 240%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 310%, at least 320%, at least 330%, at least 340%, at least 350%, at least 360%, at least 370%, at least 380%, at least 390%, at least 400%, at least 410%, at least 420%, at least 430%, at least 440%, at least 450%, at least 460%, at least 470%, at least 480%, at least 490%, or at least 500%.

19. The method according to any one of claims 17-18, wherein: The lentiviral transduction efficiency was increased by at least 90%.

20. The method according to any one of claims 17 to 19, wherein: The polypeptide having phase behavior has an amino acid sequence of SEQ ID NO:12.

Images