Method to bioengineer designer platelets using gene editing and stem cell methodologies
Inventors
Newman, Peter Jay • Rao, Sridhar • Zhang, Nanyan • Zhi, Huiying
Assignees
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Publication Number
US-10725041-B2
Publication Date
2020-07-28
Expiration Date
Abstract
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.
Core Innovation
The invention describes a platform for creating designer platelets and designer red blood cells by gene editing of pluripotent cells, followed by cell culture differentiation or expansion to generate desired hematopoietic progenitors and platelet-lineage cells. A platelet alloantigen locus within the ITGB3 gene around the PlA locus is targeted using one or more guide RNAs combined with Cas9 nuclease activity. Editing is performed using an HDR repair template oligo that encodes a single nucleotide substitution specific to an alloantigen HPA-1, so that selected clones express the HPA-1 alloantigen.
Pluripotent cells that are deficient in β2 microglobulin are used, and optional engineering elements are applied to support downstream selection and confirmation. The plasmid-based delivery includes a plasmid that expresses a Cas9 nuclease, and the transfection is carried out in the presence of an HDR template oligo. Resulting edited clones are cloned and selected based on expression of the HPA-1 alloantigen and then differentiated into mammalian hematopoietic progenitor cells that express the HPA-1 alloantigen.
A further described aspect is optional co-editing related to HLA class I background by deleting β2 microglobulin (β2M), which is presented in the context of improving compatibility and reducing HLA-driven background in antibody detection. The platform is supported with proof-of-concept conversion of PlA1 to PlA2 in DAMI cells and iPSCs, with confirmation by diagnostic restriction site genotyping and additional detection approaches.
Claims Coverage
The independent claim is directed to a method for creating a mammalian hematopoietic progenitor cell expressing a specific platelet alloantigen from pluripotent cells using gene editing and differentiation. The coverage centers on inventive features combining guide RNAs targeting the ITGB3 gene near the PlA locus, Cas9 nuclease plasmid delivery, HDR using an oligo encoding an HPA-1 single nucleotide substitution, clone selection for alloantigen expression, and differentiation into hematopoietic progenitor cells expressing the alloantigen.
Guide RNAs targeting ITGB3 near PlA locus and Cas9 plasmid delivery
Providing one or more guide RNAs designed to target the ITGB3 gene around the PlA locus, and ligating the guide RNA of step (a) into a plasmid which also expresses a Cas9 nuclease.
HDR template oligo encoding HPA-1 single nucleotide substitution in β2 microglobulin-deficient iPSCs
Transfecting β2 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.
Cloning and selecting clones expressing the HPA-1 alloantigen and differentiating into hematopoietic progenitors
Cloning and selecting resulting clones that express the HPA-1 alloantigen, and differentiating the selected clones into mammalian hematopoietic progenitor cells that express the HPA-1 alloantigen.
HDR template includes a diagnostic restriction enzyme site
Providing the HDR template oligo encoding a diagnostic restriction enzyme site at the target locus.
Plasmid expresses a selectable marker
Using a plasmid that additionally expresses a selectable marker.
Reporter gene is GFP
Characterized in that the reporter gene is GFP.
β2 microglobulin-deficient iPSCs generated using guide RNAs flanking the β2 microglobulin gene and Cas9n
Producing β2 microglobulin-deficient induced pluripotent stem cells by using one or more guide RNAs targeting sequences flanking the β2 microglobulin gene of HLA and a Cas9n nuclease in mammalian induced pluripotent stem cells.
Diagnostic restriction enzyme site is an NciI restriction site
Specifying the diagnostic restriction enzyme site as an NciI restriction site.
Across the independent claim and dependent refinements provided, the inventive coverage is centered on editing the ITGB3 locus around PlA using guide RNAs with Cas9 nuclease plasmid delivery, executing HDR with an oligo encoding an HPA-1 single nucleotide substitution, and selecting and differentiating edited pluripotent clones into hematopoietic progenitors expressing the platelet alloantigen; dependent claim elements further specify diagnostic restriction-site incorporation, selectable marker and GFP reporter, and β2 microglobulin-deficient iPSC generation details, including Cas9n and NciI.
Stated Advantages
Optional co-editing to delete β2 microglobulin is described as reducing HLA-driven background in antibody detection and improving compatibility.
Documented Applications
Designer platelet creation by gene editing of pluripotent cells to generate hematopoietic progenitors/megakaryocytes/platelets expressing an alloantigen HPA-1 (e.g., PlA1 to PlA2 conversion).
Designer red cell applications are described as part of the platform for creating designer red blood cells using the gene-editing approach.
Antibody detection context is described where deletion of β2 microglobulin reduces HLA-driven background.
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