Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells

Figures

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  Figure 1. Engraftment kinetics of CCR5-ZFN-edited and HBG CRISPR-edited NHP HSPCs in vivo (A) Average gene editing in peripheral blood. Statistical significance is denoted as ∗p < 0.05 and ∗∗p < 0.01. (B) Average CCR5 gene editing in bone marrow colony assays from pigtail macaques transplanted with ZFN-modified HSPCs. (C) Ratio of gene editing in HSPC infusion products ex vivo to post-transplantation peripheral blood or bone marrow total leukocytes. Data are derived from four SHIV-naive animals, 6–11 SHIV-infected animals (11 infusion products, of which in vivo samples were available from 6–11 over the course of up to 637 days' follow up), and three HBG CRISPR animals.

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  Figure 2. Improved engraftment of NHP HSPCs in humanized mice (A) ZFN editing efficiency in bulk NHP HSPCs and HSPC subsets from 4–7 donors cultured in FBS-containing medium (gray) or serum-free medium (black). Bulk: CD34+; Committed: CD34+CD90–CD45RA+; Early: CD34+CD90–CD45RA–; Primitive: CD34+CD90+CD45RA–. (B) Schematic of the workflow for studying engraftment of CCR5-edited NHP CD34+ cells in NSG mice. (C) CCR5 ZFN-edited or mock-edited NHP HSPCs were transplanted into NSG mice following culture in the indicated medium, followed by quantification of engraftment of NHP CD45+ cells. Time points with undetectable NHP cells are plotted as 0.01% for graphing purposes. (D) CCR5 MiSeq analysis of total cells from the infusion product, peripheral blood between 8 and 20 weeks post transplant, and necropsy. Because NHP HSPCs were obtained as surplus from autologous transplantation experiments, we were limited to 1 NHP donor per experimental arm in (C) and (D). Statistical significance is denoted as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.

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  Figure 3. RNase inhibitors rescue exogenous mRNA expression in NHP HSPCs (A) NHP HSPCs were electroporated with mRNA encoding GFP, with or without the indicated units of RNase inhibitor (RNasin) and assayed for GFP expression 24 h later by flow cytometry. (B) GFP expression with or without RNasin from three distinct NHP HSPC donors. Circles: bone marrow aspirate; squares: postmortem bone marrow pulp; triangles: mobilized apheresis. Open shapes: unmodified mRNA; filled shapes: mRNA modified with pseudo-uridine and 5-methylcytosine. (C) Median fluorescence intensity of data from (B). Statistical significance is denoted as ∗p < 0.05 and ∗∗p < 0.01.

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  Figure 4. RNase inhibitors enhance CCR5 ZFN editing in NHP HSPCs (A) NHP HSPCs (n = 3) derived from bone marrow aspirate (circles), postmortem bone marrow pulp (squares), and/or mobilized apheresis (triangles) were electroporated with unmodified mRNA encoding CCR5 ZFNs, with or without RNase inhibitor (RNasin), and assayed for CCR5 editing 5 days later. (B) Biallelic versus monoallelic editing in HSPC colonies derived from (A). (C) CCR5 editing in NHP HSPCs from 4–6 donors following electroporation with CCR5-targeted CRISPR ribonucleoprotein complexes (RNPs). Open circles: freshly isolated pigtail bone marrow cells; filled gray circle: cryopreserved rhesus bone marrow cells. (D) Biallelic versus monoallelic editing in HSPC colonies (n = 4) derived from sgRNA1-treated cells from (C). Statistical significance is denoted as ∗p < 0.05 and ∗∗p < 0.01.