Delivery of cas9 via arrdc1-mediated microvesicles (armms)
AssigneesPresident and Fellows of Harvard College, The Board of Trustees of the Leland Stanford Junior University
InventorsQuan Lu, Qiyu Wang, Stanley N. Cohen
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Abstract
Methods, systems, compositions and strategies for the delivery of WW domain-containing fusion proteins into cells in vivo, ex vivo, or in vitro via ARMMs are provided. Methods, systems, compositions and strategies for the delivery of Cas9 proteins and/or Cas9 variants into cells in vivo, ex vivo, or in vitro via fusion to ARMM associated proteins (e.g., ARRDC1 or TSG101) are also provided.
Claims22
-89. (canceled)
A fusion protein comprising:
- (i) a cargo protein, wherein the cargo protein is a transcription factor, a tumor suppressor, a developmental regulator, a growth factor, a metastasis suppressor, a pro-apoptotic protein, a nuclease, a recombinase, or a reprogramming factor;
- and
- (ii) a WW domain or variant thereof.
An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising:
- (i) a lipid bilayer;
- (ii) an ARRDC1 protein or variant thereof;
- and
- (iii) a Cas9 protein, wherein the Cas9 protein is linked to the ARRDC1 protein or variant thereof.
An arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM), comprising:
- (i) a lipid bilayer;
- (ii) an ARRDC1 protein or variant thereof;
- (iii) a TSG101 protein or variant thereof;
- and
- (iv) a Cas9 protein, wherein the Cas9 protein is linked to the TSG101 protein or variant thereof.
A Cas9 fusion protein comprising:
- (i) a Cas9 protein, wherein the Cas9 protein comprises an amino acid sequence that is at least about 85% identical to any one of SEQ ID NOs:
- 2, 4, and 5;
- and
- (ii) an ARRDC1 protein or variant thereof.
A Cas9 fusion protein comprising:
- (i) a Cas9 protein, wherein the Cas9 protein comprises an amino acid sequence that is at least about 85% identical to any one of SEQ ID NOs:
- 2, 4, and 5;
- and
- (ii) an TSG101 protein or variant thereof.
A microvesicle-producing cell comprising:
- (i) an ARRDC1 protein or variant thereof;
- and
- (ii) a cargo protein, wherein the cargo protein is capable of associating with the ARRDC1 protein via one or more WW domain or variant thereof.
A method of delivering a cargo protein to a target cell, the method comprising contacting the target cell with an arrestin domain-containing protein 1 (ARRDC1)-mediated microvesicle (ARMM) comprising:
- (i) a lipid bilayer;
- (ii) an ARRDC1 protein or variant thereof, and
- (iii) a cargo protein, wherein the cargo protein is linked to the ARRDC1 protein or variant thereof.
Dependencies are inferred from the claim text.