Method to bioengineer designer platelets using gene editing and stem cell methodologies
Methods of creating cells expressing specific platelet alloantigens by combining gene editing techniques and cell culture differentiation or expansion techniques employing pluripotent cells, including the steps of transfecting pluripotent cells with a plasmid encoding one or more guide RNAs targeting within a platelet alloantigen target locus and a nuclease in the presence of an HDR repair oligo and culturing the resulting cells to expand their numbers or to create a differentiated cell type of interest.
1. A method of creating a mammalian hematopoietic progenitor cell expressing a specific platelet alloantigen by combining gene editing techniques and cell culture differentiation or expansion techniques employing pluripotent cells, the method comprising the steps of:
a) providing one or more guide RNAs designed to target the ITGB3 gene around the Pl A locus;
b) ligating the guide RNA of step (a) into a plasmid which also expresses a Cas9 nuclease;
c) transfecting mammalian β 2 microglobulin-deficient induced pluripotent stem cells with the plasmid of step (b) in the presence of a homology-directed repair (HDR) template oligo encoding a single nucleotide substitution specific to alloantigen HPA-1;
d) cloning and selecting resulting clones that express the HPA-1 alloantigen; and
e) differentiating the selected clones into mammalian hematopoietic progenitor cells that express the HPA-1 alloantigen.
2. The method of claim 1 wherein the nuclease is Cas9n.
3. Mammalian hematopoietic progenitor cells created by the method of claim 1 .
4. The method of claim 1 , wherein the HDR template oligo additionally encodes a diagnostic restriction enzyme site at the target locus.
5. The method of claim 1 , wherein the plasmid additionally expresses a selectable marker.
6. The method of claim 1 , wherein the plasmid additionally expresses a reporter gene.
7. The method of claim 1 , wherein the mammalian β 2 microglobulin-deficient induced pluripotent stem cells are produced using one or more guide RNAs targeting the areas flanking the β 2 microglobulin gene of HLA and a Cas9n nuclease in mammalian induced pluripotent stem cells.
8. The method of claim 6 , wherein the reporter gene is GFP.
9. The method of claim 4 , wherein the diagnostic restriction enzyme site is a NciI restriction site.