Cell permeable proteins for genome engineering
AssigneeAltius Institute for Biomedical Sciences
InventorsALEXANDER J. FEDERATION, JOHN A. STAMATOYANNOPOULOS
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Abstract
The present disclosure provides genome engineering proteins, e.g., nucleic acid binding domains and/or functional domains that have a net positive charge and are cell permeable and can be introduced into the cells without the use of a carrier such as micelles, vesicles, liposomes, and the like.
Claims21
-143. (canceled)
A polypeptide comprising a nucleic acid-binding domain comprising:
- at least three repeat units comprising a 33-36 amino acid long sequence having at least 80% sequence identity to the amino acid sequence:
- (SEQ ID NO:
- 1)
- LTPDQ VVAIA SX12X13GG KQALE TVQRL LPVLC QDHG,
- having the sequence of SEQ ID NO:1 with one or more conservative amino acid substitutions thereto;
- and
- comprising at least one of the following amino acid substitutions relative to SEQ ID NO:1:
- D4K/R/H;
- S11K/R/H;
- Q23K/R/H;
- C30K/R/H;
- and D32K/R/H,
- wherein X12X13 is HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN, NI, KI, RI, HI, SI, NG, HG, KG, RG, RD, SD, HD, ND, KD, YG, YK, NV, HN, H*, HA, KA, N*, NA, NC, NS, RA, CI, or S*, where (*) means X13 is absent,
- wherein when the repeat unit comprises the substitution D4K, X12X13 is not HN, YK or YG or wherein when the repeat unit comprises the substitution D4K, the repeat unit further comprises at least one of the following substitutions S11K/R/H;
- Q23K/R/H;
- C30K/R/H;
- and D32K/R/H,
- wherein when the repeat unit comprises the substitution S11K, X12X13 is not RG or NI, or wherein when the repeat unit comprises the substitution S11K, the repeat unit further comprises at least one of the following substitutions D4K/R/H;
- Q23K/R/H;
- C30K/R/H;
- and D32K/R/H,
- wherein when the repeat unit comprises the substitution Q23K, X12X13 is not SI, CI, or NN, wherein when the repeat unit comprises the substitution Q23R, X12X13 is not NG, or the repeat unit further comprises at least one of the following substitutions D4K/R/H;
- S11K/R/H;
- C30K/R/H;
- and D32K/R/H,
- wherein when the repeat unit comprises the substitution C30R, X12X13 is not NS, HD, NI, NN, NH or NK, or the repeat unit further comprises at least one of the following substitutions D4K/R/H;
- S11K/R/H;
- Q23K/R/H;
- and D32K/R/H,
- wherein when the repeat unit comprises the substitution D32H, X12X13 is not NG, or the repeat unit further comprises at least one of the following substitutions D4K/R/H;
- S11K/R/H;
- Q23K/R/H;
- and C30K/R/H, and
- wherein the repeat unit has a net charge of at least +2.
A first binding member of a heterodimer, wherein the first binding member comprises an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:2 and comprises at least one of the following substitutions relative to the amino acid sequence of SEQ ID NO:2:
- D3K/R/H;
- E4K/R/H;
- T11K/R/H;
- D24K/R/H;
- D32K/R/H;
- S35K/R/H;
- E39K/R/H;
- D40K/R/H;
- E41K/R/H;
- D45K/R/H;
- D48K/R/H;
- L49K/R/H;
- T59K/R/H;
- and D66K/R/H and wherein the first binding member binds to a second binding member of the heterodimer, wherein the second binding member comprises an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:3.
A second binding member of a heterodimer, wherein the second binding member comprises an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:3 and comprises at least one of the following substitutions relative to the amino acid sequence of SEQ ID NO:3:
- D2K/R/H;
- D3K/R/H;
- E5K/R/H;
- T12K/R/H;
- T19K/R/H;
- D26K/R/H;
- E38K/R/H;
- D41K/R/H;
- E46K/R/H;
- E56K/R/H;
- E61K/R/H;
- T68K/R/H;
- and E74K/R/H and wherein the second binding member binds to a first binding member of the heterodimer, wherein the first binding member comprises an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO:2.
Dependencies are inferred from the claim text.