. 2024 Dec;8(12):1540-1552.

doi: 10.1038/s41551-024-01222-6. Epub 2024 Jun 17.

Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells

Sofia E Luna^#^{1

2}, Joab Camarena^#^{1

2}, Jessica P Hampton^{1

2}, Kiran R Majeti^{1

2}, Carsten T Charlesworth^{1

2}, Eric Soupene³, Sridhar Selvaraj^{1

2}, Kun Jia^{4

5

6}, Vivien A Sheehan⁷, M Kyle Cromer^{8

9

10}, Matthew H Porteus^{11

12}

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA, USA.
² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
³ Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA.
⁴ Department of Surgery, University of California San Francisco, San Francisco, CA, USA.
⁵ Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA.
⁶ Eli and Edythe Broad Center for Regeneration Medicine, University of California San Francisco, San Francisco, CA, USA.
⁷ Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
⁸ Department of Surgery, University of California San Francisco, San Francisco, CA, USA. kyle.cromer@ucsf.edu.
⁹ Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA. kyle.cromer@ucsf.edu.
¹⁰ Eli and Edythe Broad Center for Regeneration Medicine, University of California San Francisco, San Francisco, CA, USA. kyle.cromer@ucsf.edu.
¹¹ Department of Pediatrics, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.
¹² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.

^# Contributed equally.

PMID: 38886504
PMCID: PMC11668683
DOI: 10.1038/s41551-024-01222-6

Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells

Sofia E Luna et al. Nat Biomed Eng. 2024 Dec.

. 2024 Dec;8(12):1540-1552.

doi: 10.1038/s41551-024-01222-6. Epub 2024 Jun 17.

Authors

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA, USA.
² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.
³ Department of Pediatrics, University of California, San Francisco, Oakland, CA, USA.
⁴ Department of Surgery, University of California San Francisco, San Francisco, CA, USA.
⁵ Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA.
⁶ Eli and Edythe Broad Center for Regeneration Medicine, University of California San Francisco, San Francisco, CA, USA.
⁷ Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
⁸ Department of Surgery, University of California San Francisco, San Francisco, CA, USA. kyle.cromer@ucsf.edu.
⁹ Department of Bioengineering and Therapeutic Sciences, University of California San Francisco, San Francisco, CA, USA. kyle.cromer@ucsf.edu.
¹⁰ Eli and Edythe Broad Center for Regeneration Medicine, University of California San Francisco, San Francisco, CA, USA. kyle.cromer@ucsf.edu.
¹¹ Department of Pediatrics, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.
¹² Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA. mporteus@stanford.edu.

^# Contributed equally.

PMID: 38886504
PMCID: PMC11668683
DOI: 10.1038/s41551-024-01222-6

Abstract

Some gene polymorphisms can lead to monogenic diseases, whereas other polymorphisms may confer beneficial traits. A well-characterized example is congenital erythrocytosis-the non-pathogenic hyper-production of red blood cells-that is caused by a truncated erythropoietin receptor. Here we show that Cas9-mediated genome editing in CD34⁺ human haematopoietic stem and progenitor cells (HSPCs) can recreate the truncated form of the erythropoietin receptor, leading to substantial increases in erythropoietic output. We also show that combining the expression of the cDNA of a truncated erythropoietin receptor with a previously reported genome-editing strategy to fully replace the HBA1 gene with an HBB transgene in HSPCs (to restore normal haemoglobin production in cells with a β-thalassaemia phenotype) gives the edited HSPCs and the healthy red blood cell phenotype a proliferative advantage. Combining knowledge of human genetics with precise genome editing to insert natural human variants into therapeutic cells may facilitate safer and more effective genome-editing therapies for patients with genetic diseases.

PubMed Disclaimer

Conflict of interest statement

Competing interests: M.H.P. serves on the scientific advisory board of Allogene Tx and is an advisor to Versant Ventures. M.H.P., M.K.C. and J.C. have equity in Graphite Bio. M.H.P. has equity in CRISPR Tx and serves on the board of directors of Graphite Bio and Kamau Therapeutics. J.C., M.K.C. and M.H.P. have filed provisional patent number PCT/US2022/077951. All other authors declare no competing interests.

Figures

**Fig. 1. Cas9-guided *EPOR* truncation in HSPCs enhances erythroid proliferation.**
a, Schematic of HSPC editing and model of *tEPOR*’s effect. Representation of *EPOR* gene and location of the candidate sgRNA (*EPOR*-sg1) indicated by a line. Location of c.1316G>A mutation is denoted by the star. Created with BioRender.com. b, Frequency of indels created by *EPOR*-sg1 in primary human CD34⁺ HSPCs over the course of erythroid differentiation compared with control *HBB* sgRNA. Points represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 5 for *EPOR*-sg1 and n = 1 for control *HBB* sgRNA. *P = 0.0016 of day 0 versus day 14 by unpaired two-tailed t-test. c, Genome-editing strategy when using an AAV6 DNA repair template to introduce the *EPOR* c.1316G>A mutation followed by a *BGH*-poly(A) region and *UbC*-driven *GFP* reporter. d, Percentage of GPA⁺/CD71⁺ of live single cells on day 14 of differentiation. Bars represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 2–3 for HSPC and n = 3–4 for −EPO and +EPO conditions. *P = 0.0016 of −EPO versus HSPC conditions; **P = 0.003, ***P = 0.0001 of −EPO versus +EPO conditions by unpaired two-tailed t-test. e, Percentage of GFP⁺ cells of live single cells maintained in RBC media with or without EPO or HSPC media as determined by flow cytometry. Points represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 2 for HSPC condition and n = 3–4 for −EPO and +EPO conditions. *P = 0.04, **P = 0.0006 of day 0 versus day 14 by unpaired two-tailed t-test. f, Fold change in cell count throughout RBC differentiation (for example, if at day 0 starting cell numbers were 1 × 10⁵ cells total, then a fold count change of 1,000 would yield a total cell number of 1 × 10⁸ at day 14). Points represent mean ± s.e.m. Values represent biologically independent HSPC donors: n = 3 for mock and *EPOR*-sg1 + *BGH* and n = 2 for *EPOR*-sg1. Source data

**Fig. 2. Integration of *tEPOR* cDNA shows an erythroid-specific proliferative effect.**
a, Genome-editing strategy to introduce *tEPOR*-*T2A*-*YFP*-*BGH*-poly(A) cDNA at the *CCR5* locus with expression driven by a ubiquitous *UbC* promoter. b, Percentage of GPA⁺/CD71⁺ of live single cells on day 14 of differentiation following introduction of *tEPOR* at the *CCR5* locus. Bars represent median ± interquartile range. Values represent biologically independent HSPC donors: n = 2–3 for HSPC condition and n = 2–4 for −EPO and +EPO conditions. *P = 0.0018 for −EPO versus HSPC conditions, **P < 0.0001 for −EPO versus +EPO conditions by unpaired two-tailed t-test. c, Representative flow cytometry plots of one donor of *CCR5*-sg3 + *tEPOR*-edited HSPCs on day 14 of RBC differentiation in the +EPO condition. d, Percentage of YFP⁺ cells of live single cells as determined by flow cytometry. Points represent mean ± s.e.m. Values represent biologically independent HSPC donors: n = 2 for HSPC condition, n = 3 for −EPO condition and n = 3–4 for +EPO condition. *P = 0.0003 for day 0 versus day 14 by unpaired two-tailed t-test. e, Genome-editing strategy to introduce *tEPOR*-*T2A*-*YFP* cDNA at the *HBA1* locus by whole gene replacement to place integration cassette under regulation of the endogenous *HBA1* promoter. f, Percentage of GPA⁺/CD71⁺ of live single cells on day 14 of differentiation following introduction of *tEPOR* cDNA at the *HBA1* locus. Bars represent median ± 95% confidence interval. Values represent biologically independent HSPC donors: n = 2 for HSPC condition and n = 2–3 for −EPO and +EPO condition. *P = 0.0002 for −EPO to +EPO condition by unpaired two-tailed t-test. g, Representative flow cytometry plots of one donor of *HBA1*-sg4 + *tEPOR*-edited HSPCs on day 11 of RBC differentiation in the +EPO condition. h, Percentage of YFP⁺ cells of live single cells as determined by flow cytometry. Points represent mean ± s.e.m. Values represent biologically independent HSPC donors: n = 2 for HSPC condition and n = 3 for −EPO and +EPO condition. FSC-A, forward scatter area; FITC, fluorescein isothiocyanate. Source data

**Fig. 3. Therapeutic editing frequencies are achieved using bicistronic *HBB*-*tEPOR* cassette.**
a, Design of *HBB* (control) and *HBB*-*tEPOR* (bicistronic) AAV6 donor cassettes targeted to the *HBA1* locus by whole gene replacement. b, Percentage of GPA⁺/CD71⁺ of CD34⁻/CD45⁻ cells on day 14 as determined by flow cytometry. Points are shown as median ± 95% confidence interval. Values represent biologically independent HSPC donors: n = 2 for *HBB* and n = 3 for all other vectors. c, Percentage of edited alleles for control (*HBB*) and bicistronic *HBB*-*tEPOR* in cord-blood-derived CD34⁺ cells over the course of RBC differentiation. Points are shown as median ± 95% confidence interval. Values represent biologically independent HSPC donors: n = 2 for *HBB*, n = 3 for all other vectors. *P = 0.003 for *PGK*-*tEPOR*, P = 0.0055 for *tEPOR-T2A*, P = 0.0259 for *tEPOR-FuT2A*, P = 0.0003 for *IRES*-*tEPOR* (day 0 versus day 14) by unpaired two-tailed t-test. d, Fold change in edited alleles from the beginning (day 0) to end (day 14) of RBC differentiation.The dashed line represents no fold change. Bars represent median ± 95% confidence interval. e, Percentage of edited alleles for control (*HBB*) and bicistronic *HBB*-*tEPOR* vectors in cells from patients with SCD over the course of RBC differentiation. Points are shown as median ± 95% confidence interval. Values represent biologically independent HSPC donors: n = 3 for *PGK*-*tEPOR* and n = 4 for all other vectors. *P = 0.0061 for *PGK*-*tEPOR*, *P = 0.011 for *tEPOR-T2A*, *P = 0.0016 for *tEPOR-FuT2A*, *P = 0.0153 for *IRES*-*tEPOR* (day 0 versus day 14) by unpaired two-tailed t-test. f, Fold change in edited alleles from the beginning (day 0) to end (day 14) of RBC differentiation. The dashed line represents no fold change. Bars represent median ± 95% confidence interval. Source data

**Fig. 4. Multiplexed editing of *EPOR* and *HBA1* leads to robust increase in *HBB* mRNA within editing HSPCs.**
a, Schematic of multiplexed editing strategy with spike-in of unedited cells at the start of erythroid differentiation to model HSPC transplantation. b, Percentage of GPA⁺/CD71⁺ of CD34^–/CD45^– cells on day 14 of RBC differentiation as determined by flow cytometry. Points are shown as median ± interquartile range. n = 3 biologically independent HSPC donors. c, Percentage of edited alleles at *HBA1* in all multiplexed editing or spike-in conditions throughout RBC differentiation. Points are shown as median ± 95% confidence interval. n = 3 biologically independent HSPC donors. *P = 0.0332 for *HBB* + *tEPOR* 100%, *P = 0.0086 for *HBB* + *tEPOR* 30%, *P = 0.0122 for *HBB* + *tEPOR* 10% (day 0 versus day 14) by unpaired two-tailed t-test; **P = 0.0113 for *HBB* versus *HBB* + *tEPOR* 30% at day 14, **P = 0.008 for *HBB* versus *HBB* + *tEPOR* 10% at day 14 by unpaired two-tailed t-test. d, Fold increase in edited alleles on day 14 of differentiation of multiplexed conditions versus *HBB* only. The dashed line represents no fold change. Bars represent median ± 95% confidence interval. *P = 0.0315, **P = 0.0123 by unpaired two-tailed t-test. e, mRNA expression of integrated *HBB* at *HBA1* locus normalized to *HBB* expression from mock. *GPA* mRNA expression was used as a reference. n = 3 biologically independent HSPC donors. Bars represent median ± 95% confidence interval. Source data

**Extended Data Fig. 1. Indel analysis of HSPCs edited with most effective *EPOR* sgRNA (sg1) over course of erythroid differentiation.**
a, Frequency of five most common indels found in one HSPC donor targeted with *EPOR*-sg1 over the course of RBC differentiation. b, Fold enrichment of five most common indels over course of RBC differentiation in one HSPC donor targeted with *EPOR*-sg1. Source data

**Extended Data Fig. 2. Almost all cells edited at *EPOR* locus contain tEPOR at end of differentiation and show increased erythroid proliferation.**
a, Plot of percentage of GFP⁺ cells of live single cells maintained in RBC media +EPO (from data shown in Fig. 1e) overlaid with percentage of alleles containing indels in *EPOR* in same three biological donors. Points represent median ± 95% confidence interval. b, Cell count fold change in mock and edited cells maintained in RBC media +/- EPO or HSPC media. Points represent mean ± SEM. Values represent biologically independent HSPC donors N = 3 for Mock and *EPOR*-sg1+ BGH and N = 2 for sg1. Data for +EPO condition same as in Fig. 1f. Source data

**Extended Data Fig. 3. Enrichment of edited cells and RBC differentiation is only minorly affected by different concentration of EPO in media.**
a, Schematic depicting HSPC editing and subsequent RBC differentiation using different levels of EPO cytokine. b, Percentage of GFP⁺ cells of live single cells maintained in RBC media with 0 U/mL – 20 U/mL EPO from one biological HSPC donor. c, Fold increase of GFP⁺ cells at each concentration of EPO compared to 0 U/mL EPO at day 14 of RBC differentiation. Bars represent median ± 95% confidence interval. N = 2 biological HSPC donors. d, Percentage of GPA⁺/CD71⁺ of live single cells on day 14 of differentiation. Bars represent median ± 95% confidence interval. Values represent N = 2 biologically independent HSPC donors. Source data

**Extended Data Fig. 4. Off-target analysis of *EPOR*-sg1 by *in silico* prediction and targeted next-generation sequencing (NGS).**
a, Complete list of potential off-target sites predicted by COSMID with a score ≤5.5. b, Summary of off-target sites classified by region in genome. c, Detailed summary of 5 candidate off-target sites found in an exon or UTR of the genome. Sequence nucleotide mismatches highlighted in red, inserted nucleotides underlined, and missing nucleotides indicated by the ‘^’ symbol. PAM sequence highlighted in blue. d, Sequencing read depth of mock-edited and *EPOR*-sg1 edited sample sent for each off-target site. N = 1 biological HSPC donor. e, Frequency of indels detected in each sample at each off-target site. N = 1 biological HSPC donor. Source data

**Extended Data Fig. 5. Safe harbour integration of *tEPOR* leads to increased erythroid proliferation and editing frequencies with little impact at different concentrations of EPO.**
a, Fold change in edited alleles at *CCR5* over course of RBC differentiation +/- EPO or maintained in HSPC media measured by ddPCR. Points shown as median ± interquartile range. N = 3-4 biologically independent HSPC donors. b, Cell count fold change in mock and edited cells maintained in RBC media +/- EPO or HSPC media. Points represent mean ± SEM. Values represent biologically independent HSPC donors N = 3 for Mock and *CCR5*-sg3 + *tEPOR* and N = 2 for *CCR5*-sg3. c, Percentage of YFP⁺ cells of live single cells throughout RBC differentiation with 0 U/mL – 20 U/mL EPO from one biological HSPC donor. d, Fold increase of YFP⁺ cells at each concentration of EPO compared to 0 U/mL EPO at day 14 of differentiation. Bars represent median ± 95% confidence interval. N = 2 biological HSPC donors. e, Percentage of GPA⁺/CD71⁺ of live single cells on day 14 of differentiation. Bars represent median ± 95% confidence interval. Values represent N = 2 biologically independent HSPC donors. Source data

**Extended Data Fig. 6. Erythroid specific expression of *tEPOR* leads to increased editing frequencies and is not affected by EPO concentration.**
a, Fold change in edited alleles at *HBA1* over course of RBC differentiation +/- EPO measured by ddPCR. Points shown as median ± 95% CI. N = 3 biologically independent HSPC donors. b, Percentage of YFP⁺ cells of live single cells throughout RBC differentiation with 0 U/mL – 20 U/mL EPO in one biological HSPC donor. c, Fold increase of YFP⁺ cells at each EPO concentration compared to 0 U/mL EPO at day 14 of RBC differentiation. Bars represent median ± 95% confidence interval. N = 2 biological HSPC donors. d, Percentage of GPA⁺/CD71⁺ of live single cells on day 14 of differentiation. Bars represent median ± 95% confidence interval. Values represent N = 2 biologically independent HSPC donors. Source data

**Extended Data Fig. 7. Haemoglobin tetramer analysis of *tEPOR*-edited SCD patient HSPCs following RBC differentiation.**
a, Percentage of GPA⁺/CD71⁺ of CD34⁻/CD45⁻cells on day 14 of RBC differentiation as determined by flow cytometry. Points shown as median ± 95% confidence interval. N = 4 biologically independent HSPC donors. b, Ratio of adult haemoglobin to sickle haemoglobin from HPLC analysis of haemoglobin tetramers from differentiated SCD patient HSPCs. Bars shown as mean ± SD. N = 4 biologically independent HSPC donors. Source data

**Extended Data Fig. 8. Multiplexed editing shows maintenance of *EPOR* truncation at end of RBC differentiation and does not disrupt HSPC lineage formation.**
a, Percentage of GFP⁺ cells of live single cells on day 14 of RBC differentiation as determined by flow cytometry. Points represent median ± interquartile range. Values represent N = 3 biologically independent HSPC donors. b, CFU assay of mock, single edited, and multiplex edited HSPCs. Bars represent percent of total colonies: CFU-GEMM (multi-potential granulocyte, erythroid, macrophage, megakaryocyte progenitor cells), CFU-GM (colony forming unit-granulocytes and monocytes), and BFU-E (erythroid burst forming units). N = 1. c, Number of total colonies in CFU assay produced per 500 plated cells in each condition. Source data

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Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells

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Enhancement of erythropoietic output by Cas9-mediated insertion of a natural variant in haematopoietic stem and progenitor cells

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