Compositions and methods for targeted delivery of CRISPR-Cas effector polypeptides and transgenes
AssigneeThe Regents of the University of California
InventorsJennifer A. Doudna, Jennifer Hamilton, Connor Tsuchida
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Abstract
The present disclosure provides virus-like particles (VLPs) comprising: i) a CRISPR-Cas effector polypeptide; ii) a recombinant lentivirus comprising a nucleotide sequence encoding a therapeutic polypeptide having a length of from about 250 amino acids to about 3,000 amino acids, where the VLP comprises a pseudotyping viral glycoprotein and/or a polypeptide that provides for binding to a target cell. The present disclosure provides systems for producing a VLP. The present disclosure provides methods of delivering a therapeutic protein, using a VLP of the present disclosure.
Claims33
A virus-like particle (VLP) comprising:
- a) a Cas9 polypeptide, wherein the Cas9 polypeptide is present inside of the VLP as both:
- (i) a Gag-Cas9 fusion polypeptide, wherein the Cas9 polypeptide is linked to the C-terminus of a Gag polyprotein through a cleavable linker between the Gag polyprotein and the Cas9 polypeptide;
- and
- (ii) a proteolytically released form following proteolytic cleavage of the cleavable linker;
- b) a Pol polyprotein;
- c) lentiviral structural components comprising:
- (i) a matrix (MA) polypeptide;
- (ii) a capsid (CA) polypeptide;
- and
- (iii) a nucleocapsid (NC) polypeptide;
- d) a recombinant lentiviral vector comprising a nucleotide sequence encoding a therapeutic polypeptide selected from the group consisting of:
- a chimeric antigen receptor (CAR), a T-cell receptor, s synNotch polypeptide, a natural killer cell receptor, and an antibody;
- and
- e) a pseudotyping viral glycoprotein;
- and optionally
- f) a polypeptide that provides for binding to a target cell.
A VLP comprising:
- a) a Cas9 polypeptide, wherein the Cas9 polypeptide is present inside of the VLP as both:
- (i) a Gag-Cas9 fusion polypeptide, wherein the Cas9 polypeptide is linked to the C-terminus of an HIV-1 Gag polyprotein through an HIV-1 protease cleavage site between the HIV-1 Gag polyprotein and the Cas9 polypeptide;
- and
- (ii) a proteolytically released form following proteolytic cleavage of the HIV-1 protease cleavage site;
- b) an HIV-1 Pol polyprotein;
- c) HIV-1 lentiviral structural components comprising:
- (i) a MA polypeptide;
- (ii) a CA polypeptide;
- and
- (iii) a NC polypeptide;
- d) a recombinant lentiviral vector comprising a nucleotide sequence encoding a CAR;
- e) a pseudotyping viral glycoprotein;
- f) a polypeptide that provides for binding to a T cell;
- and
- g) one or more CRISPR/Cas guide RNAs comprising:
- (a) a CRISPR/Cas guide RNA targeting a B2M gene;
- or
- (b) a CRISPR/Cas guide RNA targeting a TRAC gene;
- or
- (c) both (a) and (b).
A VLP comprising:
- a) a Cas9 polypeptide, wherein the Cas9 polypeptide is present inside of the VLP as both:
- (i) a Gag-Cas9 fusion polypeptide, wherein the Cas9 polypeptide is linked to the C-terminus of an HIV-1 Gag polyprotein through an HIV-1 protease cleavage site between the HIV-1 Gag polyprotein and the Cas9 polypeptide;
- and
- (ii) a proteolytically released form following proteolytic cleavage of the HIV-1 protease cleavage site;
- b) an HIV-1 Pol polyprotein;
- c) HIV-1 lentiviral structural components comprising:
- (i) a MA polypeptide;
- (ii) a CA polypeptide;
- and
- (iii) a NC polypeptide;
- d) a CRISPR/Cas guide RNA targeting a genomic sequence of a TRAC gene;
- e) a recombinant lentiviral vector comprising a nucleotide sequence encoding a CAR, wherein the CAR comprises an antigen-binding domain specific for a cancer-associated antigen, wherein the nucleotide sequence encoding the CAR is flanked by homology regions corresponding to nucleotide sequences flanking a sequence of the TRAC gene targeted by the one or more CRISPR/Cas guide RNAs;
- f) a pseudotyping viral glycoprotein;
- and
- g) a polypeptide that provides for binding to a T cell, wherein the polypeptide that provides for binding to the T cell is an antibody.
Dependencies are inferred from the claim text.