Reduced and minimal manipulation manufacturing of genetically-modified cells
AssigneeFred Hutchinson Cancer Research Center
InventorsJennifer E. Adair, Reza Shahbazi
Life of this patent
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Abstract
Nanoparticles to genetically modify selected cell types within a biological sample that has been subjected to reduced or minimal manipulation are described. The nanoparticles deliver all components required for precise genome engineering and overcome numerous drawbacks associated with current clinical practices to genetically engineer cells for therapeutic purposes.
Claims83
A method of genetically modifying a hematopoietic stem and progenitor cell (HSPC) population in a biological sample comprising adding a gold nanoparticle (AuNP) to the biological sample, wherein the AuNP comprises
- a gold (Au) core that is less than 20 nm in diameter;
- a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) guide RNA (crRNA)-nuclease ribonucleoprotein (RNP) complex wherein the crRNA comprises a 3′ end and a 5′ end, wherein the 3′ end is conjugated to a spacer with a thiol modification, and the 5′ end is conjugated to the nuclease, and wherein the thiol modification is covalently linked to the surface of the Au core and wherein the crRNA has a sequence set forth in SEQ ID NO:
- 262;
- SEQ ID NO:
- 13;
- SEQ ID NO:
- 14;
- or SEQ ID NO:
- 241-261;
- a positively-charged polyethyleneimine polymer coating wherein the positively-charged polyethyleneimine polymer has a molecular weight of less than 2500 daltons, surrounds the RNP complex, and contacts the surface of the Au core;
- and
- a donor template comprising a homology-directed repair template (HDT) on the surface of the positively-charged polymer coating wherein the HDT template comprises a sequence set forth in SEQ ID NO:
- 48;
- SEQ ID NO:
- 4;
- SEQ ID NO:
- 15;
- SEQ ID NO:
- 33-41;
- SEQ ID NO:
- 44-47;
- or SEQ ID NO:
- 49-51;
- and
- a CD133 targeting ligand comprising a binding domain of antibody clone REA820, REA753, REA816, 293C3, AC141, AC133, or 7
- wherein the targeting ligand is linked to the nuclease through an amine-to-sulfhydryl crosslinker or a sulfhydryl-to-sulfhydryl crosslinker and
- wherein the HSPC population has not been exposed to electroporation, a viral vector encoding an HDT, or a magnetic cell separation process, and wherein the method results in no more than 30% HSPC cellular toxicity and provides a gene-editing efficiency within the HSPC population of at least 10%.
A method of genetically modifying a selected cell population in a biological sample comprising adding a gold nanoparticle (AuNP) to the biological sample, wherein the AuNP comprises
- a gold (Au) core that is less than 30 nm in diameter;
- a guide RNA (gRNA)-nuclease ribonucleoprotein (RNP) complex wherein the gRNA comprises a 3′ end and a 5′ end, wherein the 3′ end is conjugated to a spacer with a chemical modification, and the 5′ end is conjugated to the nuclease, and wherein the chemical modification is covalently linked to the surface of the Au core;
- a positively-charged polymer coating wherein the positively-charged polymer has a molecular weight of less than 2500 daltons, surrounds the RNP complex, and contacts the surface of the Au core;
- and
- a donor template comprising a homology-directed repair template (HDT) on the surface of the positively-charged polymer coating
- wherein the selected cell population has not been exposed to electroporation or a viral vector encoding an and wherein the method results in no more 30% cellular toxicity of the selected cell population and provides a gene-editing efficiency within the selected cell population of at least 10%.
A gold nanoparticle (AuNP) comprising
- a gold (Au) core that is less than 30 nm in diameter;
- a guide RNA-nuclease ribonucleoprotein (RNP) complex wherein the gRNA comprises a 3′ end and a 5′ end, wherein the 3′ end is conjugated to a spacer with a chemical modification, and the 5′ end is conjugated to the nuclease, and wherein the chemical modification is covalently linked to the surface of the Au core;
- a positively-charged polymer coating wherein the positively-charged polymer has a molecular weight of less than 2500 daltons, surrounds the RNP complex, and contacts the surface of the Au core;
- and
- a donor template comprising a homology-directed repair template (HDT) on the surface of the positively-charged polymer coating.
Dependencies are inferred from the claim text.