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. 2018 Nov 2;8(1):16304.
doi: 10.1038/s41598-018-34601-6.

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells

Jonathan Yen  1 , Michael Fiorino  2 , Yi Liu  1 , Steve Paula  1 , Scott Clarkson  1 , Lisa Quinn  3 , William R Tschantz  3 , Heath Klock  4 , Ning Guo  1 , Carsten Russ  1 , Vionnie W C Yu  1 , Craig Mickanin  1 , Susan C Stevenson  1 , Cameron Lee  5 , Yi Yang  6
Affiliations

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells

Jonathan Yen et al. Sci Rep. .

Abstract

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as β-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because β-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.

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Conflict of interest statement

At the time the research was performed, the authors were employees of Novartis. Several of the authors are inventors on patent applications owned by Novartis AG covering aspects of the work described in the manuscript.

Figures

Figure 1
Figure 1
Design and optimization of TRIAMF. (a) Schematic illustration of the single unit system. The device consists of a silicone washer (1), a stainless steel mesh (2), a hydrophilic track-etched polycarbonate filter membrane (3), and a PTFE washer (4). MD: membrane diameter; MT: membrane thickness; PD: pore diameter. Not drawn to scale. (b) Illustration of basic set up, including the pressure regulator and valve from a nitrogen source, connector, and the membrane holder. (c) Illustration of a 24-membranemanifold system. B2M knockout efficiency determined by FACS and cell recovery as a function of (d) membrane pore diameter and thickness; (e) applied pressure; (f) RNP concentration; (g) cell density (n = 6, 3 donors with duplicate of each donor). When not specified, the sample volume was kept at 50 μl, cell density at 8 × 107 cells/ml, nitrogen pressure at 5 PSI and 7 μm thick membranes with 8 μm pore diameter were used. (h) B2M knockout efficiency remained the same with further increased cell density from 108 to 2 × 108 cells/ml in 50 μl volume processed by TRIAMF using 25 μM of RNP, 7 μm thick membrane with 8 μm pore diameter under 5 PSI nitrogen pressure (n = 2). ***p < 0.001, **p < 0.002, one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. Bars represent standard deviation. Alan Abrams helped to produce the graphics in (a–c).
Figure 2
Figure 2
Genome editing of the HBG1 and HBG2 promoters using TRIAMF mediated delivery of RNPs. (a) HBG1 and HBG2 promoter and g1-RNP cutting sites. (b) The sequence of HBG1 and HBG2 target sites, the bar indicates targeting gRNA-1 with orange indicating the PAM site. The putative transcription repressor binding CCAAT box sequence is marked in red. Total indel percentages among total reads by NGS with top 5 frequent alleles. The 4.9 kb deletion between the two target sites as detected by qPCR. (n = 2, single donor, biological duplicate, indel patterns are mean of biological duplicates). (c) Representative flow cytometry plots showing HbF+ immunostaining of CD235a+/CD71−/low cells after erythroid differentiation for 3 weeks of untreated, mock treated (TRIAMF Mock), and g1-RNP edited (TRIAMF + RNP) HSPCs (see Fig. S5 for gating strategy). (d) Immunostaining of HSPCs for CD34 and CD90 markers after 7 day expansion post treatment. (e) Cell count of CD34+ and CD34+/CD90+ cells after 7-day expansion post treatment. (f) Colony forming unit assay. Numbers indicate mean +/− SD, n = 4 (single donor, biological duplicate with technical duplicate each).
Figure 3
Figure 3
Preservation of engraftment competency of HSPCs post TRIAMF treatment. 7 × 105 viable HSPCs (from the same donor as used in Fig. 2) 1-day post treatment were transplanted into each sub-lethally irradiated recipient mouse via tail vein injection. (a) Human CD45+ chimerism of untreated (n = 4 mice), mock treated (TRIAMF Mock, n = 5 mice), and g1-RNP edited (TRIAMF + RNP, n = 6 mice) in the peripheral blood 8, 12, 16, and 20 weeks post-transplantation. (b) Human CD45+ chimerism in mouse bone marrow 20 weeks post-transplantation. (c) Lineage distribution of human CD45+ cells isolated from mouse bone marrow 20 weeks post-transplantation. (d) Two aliquots of HSPCs from the same donor were processed by TRIAMF. One aliquot of cells was harvested 48 hours after treatment (Pre-Transplant, n = 1) and the other aliquot of cells were engrafted into NGS mice from which hCD45+ cells were harvested from mouse bone marrow 20 weeks after transplantation (Post-Transplant, n = 6 mice). The total mutation rate and top 5 most frequent indel alleles as a percentage of total NGS reads at HBG1 and HBG2 sites were shown. For the Post-Transplant group, indel patterns are the mean of all the mice. (e) Comparison of HbF+ immunostaining of CD235a+/CD71−/low cells after erythroid differentiation of HSPCs 48 hours post treatment (n = 2, technical duplicate) and hCD45+/CD34+ HSPCs harvested from mouse bone marrow 20 weeks post transplantation (n = 4–6 mice).
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