Cell Permeable Proteins for Genome Engineering
OwnerAlexander J. Federation, Kyle Siebenthall +3inventors
InventorsAlexander J. Federation, Kyle Siebenthall, Aidan Quigley, Alister PW Funnell, 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.
Claims90
A polypeptide comprising a nucleic acid-binding domain (NBD) comprising:
- at least three repeat units (RUs) comprising a 33-36 amino acid long sequence having at least 80% sequence identity to the amino acid sequence:
- LTPDQ VVAIA SX12X13GG KQALE TVQRL LPVLC QDHG (SEQ ID NO:1), or
- having the sequence of SEQ ID NO:1 with one or more conservative amino acid substitutions thereto;
- and comprising one or both of the following amino acid substitutions relative to SEQ ID NO:1:
- E20R/K/H and Q31K/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 RUs comprise the substitution Q31K/R/H,
- X12X13 is not NK, YK or HN,
- the amino acid at position 32 is not P,
- the RUs further comprise the substitution E20R/K/H, and/or
- the RUs are 33-34 amino acid long;
- wherein when the RUs comprise the substitution E20R/K/H,
- X12X13 is not HD, HN, KG, KI, or
- the amino acid at position 32 is not P,
- the RUs further comprise the substitution Q31K/R/H, and/or
- the RUs are 33-34 amino acid long.
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 and wherein the second binding member is fused to a nuclear localization sequence (NLS).
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 and wherein the first binding member is fused to a nuclear localization sequence (NLS).
The first binding member of 63, wherein the functional domain is fused to the C-terminus of the first binding member and the NLS is fused to the N-terminus of the first binding member.
Dependencies are inferred from the claim text.