Enhancing prime editing in hematopoietic stem and progenitor cells by modulating nucleotide metabolism

Data availability

All amplicon sequencing data are publicly accessible from the National Center for Biotechnology Information BioProject database under accession number PRJNA1090890.

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Acknowledgements

This work was supported by the Doris Duke Foundation, the St. Jude Children’s Research Hospital Collaborative Research Consortium, the Harvard Stem Cell Institute and the National Institutes of Health (R01HL150669 and R01HL170629). S.L. holds a Banting Postdoctoral Fellowship from the Canadian Institutes of Health Research. A.C. was partially supported by a fellowship from the American Italian Cancer Foundation. CD34+ cells were provided by the Fred Hutch Cooperative Center of Excellence in Hematology (U54 DK106829). We thank D. Klatt and C. Brendel for their support in producing in-house Vpx VLPs, G. Casirati for his support in mRNA IVT, P. Wang and N. Kanarek for performing metabolic studies, W. Mannherz and S. Agarwal for insightful discussions, J. Zeng and N.R. Neri for technical support, and the GPP of the Broad Institute of MIT and Harvard for providing VSV-G envelope and SIV Vpx vectors.

Author information

Authors and Affiliations

Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, USA

Sébastien Levesque, Andrea Cosentino, Archana Verma, Pietro Genovese & Daniel E. Bauer

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

Sébastien Levesque, Andrea Cosentino, Archana Verma, Pietro Genovese & Daniel E. Bauer

Harvard Stem Cell Institute, Cambridge, MA, USA

Sébastien Levesque, Archana Verma, Pietro Genovese & Daniel E. Bauer

Broad Institute, Cambridge, MA, USA

Sébastien Levesque, Archana Verma, Pietro Genovese & Daniel E. Bauer

Department of Pediatrics, Harvard Medical School, Boston, MA, USA

Sébastien Levesque, Andrea Cosentino, Archana Verma, Pietro Genovese & Daniel E. Bauer

Milano-Bicocca University, Milan, Italy

Andrea Cosentino

Contributions

S.L. and D.E.B. conceived the study; S.L. and D.E.B. devised the methods; S.L., A.C., A.V., P.G. and D.E.B. carried out the investigation; S.L. wrote the original draft; S.L. and D.E.B. wrote, reviewed and edited the manuscript; P.G. and D.E.B. acquired the funding; and P.G. and D.E.B. provided supervision.

Corresponding author

Correspondence to
Daniel E. Bauer.

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Competing interests

S.L. and D.E.B. have filed a provisional patent application related to this work. All other authors have no competing interests.

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Extended data

Extended Data Fig. 1 Vpx VLP and dN supplementation enhance prime editing in HSPCs with the first-generation PE2 prime editor.

(a) PE quantification as determined by BEAT analysis from Sanger sequencing. 5 ×105 HSPCs were electroporated with PE3 RNAs (PE2 mRNA + epegRNA + nicking sgRNA) targeting ATP1A1 and treated with 5X Vpx VLP (GPP) or vehicle control. Genomic DNA was harvested 3 days post-nucleofection. n = 1 experiment (donor 1) replicated with HSPCs from donor 2 with similar results (See Extended Data Fig. 1b). (b) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. HSPCs were thawed and cultured in the presence or absence of 100 µM dNs for 24 hours. Following dN treatment, 5 ×105 HSPCs were electroporated with PE3 RNAs targeting ATP1A1 and cultured in the presence or absence of 100 µM each dN and the indicated concentration of Vpx VLPs (GPP) or vehicle control for 72 hours. Genomic DNA was harvested 3 days post-nucleofection. n = 1 experiment. (c) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. HSPCs were thawed and cultured in the presence or absence of the indicated concentration of each dN for 24 hours (before electroporation). Following dN treatment, 1.25 ×105 HSPCs were electroporated with PE3 RNAs targeting ATP1A1 and cultured in the presence or absence of the indicated concentration of each dN for 72 hours (after electroporation). Genomic DNA was harvested 3 days post-nucleofection. n = 1 experiment. Donor 2, circle. Donor 3, diamond.

Extended Data Fig. 2 Modulation of nucleotide metabolism has minimal impact on prime editing in cancer cell lines.

(a) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. K562 cells were electroporated with PE3max vectors targeting ATP1A1 and the indicated Vpx or SAMHD1 vector and cultured with the indicated concentration of each dN for 72 hours. An empty pUC19 vector was used as a negative control to normalize the concentration of DNA in all nucleofections. Genomic DNA was harvested 3 days post-nucleofection. n = 2 independent biological replicates performed at different times. (b) Same as in (a) with Jurkat cells.

Extended Data Fig. 3 Staggered PE3 nicks generate tandem duplications at ATP1A1 in HSPCs.

(a) CRISPResso2 amplicon sequencing allele plots after PE3 at ATP1A1. 2.5 ×105 HSPCs were electroporated with PE3max RNAs targeting ATP1A1 (Q118R_v2, +3 TTG to CCT) and cultured in the presence or absence of 5X Vpx VLPs (GPP) and 50 µM each dN. Genomic DNA was harvested 3 days post-nucleofection. Representative allele plots are from one of three independent biological replicates performed with CD34+ HSPCs from three different donors with equivalent results. Insertions are illustrated in red squares, and they represent tandem duplications of the genomic sequence found between the two nicking sites.

Extended Data Fig. 4 Modulation of nucleotide metabolism modestly improves PE at HBB.

(a) PE and indels quantification as determined by CRISPResso2 analysis from amplicon sequencing. 2.5 ×105 HSPCs were electroporated with PE3/PE3bmax RNAs targeting HBB and cultured in the presence or absence of 5X Vpx VLPs (GPP) and 50 µM each dNs. Genomic DNA was harvested 3 days post-nucleofection. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from one donor homozygous for the rs713040 allele (donor 2). Each biological replicate is illustrated with the same shade of grey. (b) Same as in (a) but HSPCs were electroporated with PE2max or PE4max (PE2max mRNA + hMLH1dn mRNA) RNAs targeting HBB and cultured in the presence or absence of 50 µM each dNs. Where indicated, an equimolar ratio of Vpx mRNA was co-delivered during electroporation. The mRNA molarity was normalized between all conditions with EGFP mRNA. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from three different donors. Donor 2, circle. Donor 6, empty diamond. Donor 7, triangle (down). (c) Representative CRISPResso2 amplicon sequencing allele plots from the experiments shown in (a) and (b). Representative allele plots are from one of three independent biological replicates performed with CD34+ HSPCs from three different donors with equivalent results. Reads with ≥ 0.2% frequency are shown, and the frequency of indels is indicated for each sample.

Extended Data Fig. 5 Modulation of nucleotide metabolism positively interacts with MMR-evading epegRNAs in quiescent HSPCs.

(a) Schematic representation of the RNAs used to co-deliver Vpx with PE3max components. The timeline for prime editing in quiescent CD34+ HSPCs is illustrated on the right. (b) PE and indels quantification as determined by BEAT and TIDE analysis from Sanger sequencing. 5 ×105 quiescent HSPCs were electroporated directly after thawing with Vpx mRNA and PE3max RNAs targeting HBB or B2M and cultured in the presence or absence of 50 µM each dN. Genomic DNA was harvested 3 days post-nucleofection. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from three different donors. (c) Same as in (b) for prime editing at ATP1A1 with MMR-evading substitutions. Donor 2, circle. Donor 6, empty diamond. Donor 7, triangle (down).

Extended Data Fig. 6 Omitting the nicking sgRNA abrogates indels and tandem duplications at ATP1A1 in HSPCs.

(a) CRISPResso2 amplicon sequencing allele plots from Fig. 2a. 2.5 ×105 HSPCs were electroporated with PE2max RNAs targeting ATP1A1 (Q118R_v3, +3 TTGG to CCTA) and cultured in the presence or absence of 25X in-house Vpx VLPs and 50 µM each dN. Genomic DNA was harvested 3 days post-nucleofection. Representative allele plots are from one of three independent biological replicates performed with CD34+ HSPCs from three different donors with equivalent results. Reads with ≥ 0.2% frequency are shown, and the frequency of indels is indicated for each sample.

Extended Data Fig. 7 Comparison of quantification accuracy between Sanger and amplicon sequencing at ATP1A1, B2M, and HBB (related to Fig. 2 and Extended Data Fig. 4).

(a) PE and indel quantification as determined by BEAT and TIDE analysis from Sanger sequencing. (See Fig. 2a for comparison with amplicon sequencing.) The MMR evasion strategy based on the installation of additional silent mutations and the epegRNAs used to target the ATP1A1 locus are illustrated. Point mutations are illustrated in orange. As described in Figs. 2, 2.5 ×105 HSPCs were electroporated with PE2max RNAs targeting ATP1A1 and cultured in the presence or absence of 50 µM each dN and 25X in-house Vpx VLPs. Genomic DNA was harvested 3 days post-nucleofection. Data are from two out of three independent biological replicates performed with both sequencing methods. (b) Same as in (a), but HSPCs were electroporated with Vpx mRNA and PE2max RNAs targeting B2M (See Fig. 2b for comparison with amplicon sequencing). Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from three different donors. (c) PE and indel quantification as determined by BEAT and TIDE analysis from Sanger sequencing (See Extended Data Fig. 4a for comparison with amplicon sequencing). As described in Extended Data Fig. 4, 2.5 x 105 HSPCs were electroporated with PE3/PE3bmax RNAs targeting HBB and cultured in the presence or absence of 5X Vpx VLPs (GPP) and 50 µM each dNs. Genomic DNA was harvested 3 days post-nucleofection. Data are plotted as mean ± SEM from n = 3 independent biological replicates performed with CD34+ HSPCs from one donor homozygous for the rs713040 allele (donor 2). Each biological replicate is illustrated with the same shade of grey. (d) Correlation of Sanger and amplicon sequencing quantifications at ATP1A1 (PE2max), B2M (PE2max), and HBB (PE3max and PE3bmax) for the 96 samples shown in (a-c).

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Levesque, S., Cosentino, A., Verma, A. et al. Enhancing prime editing in hematopoietic stem and progenitor cells by modulating nucleotide metabolism.
Nat Biotechnol (2024). https://doi.org/10.1038/s41587-024-02266-4

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Received: 16 October 2023

Accepted: 26 April 2024

Published: 28 May 2024

DOI: https://doi.org/10.1038/s41587-024-02266-4

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