- Short Communication
- Open Access
Gene correction of HBB mutations in CD34+ hematopoietic stem cells using Cas9 mRNA and ssODN donors
- Justin S. Antony†1, 2, 3,
- Ngadhnjim Latifi†1,
- A. K. M. Ashiqul Haque1,
- Andrés Lamsfus-Calle2,
- Alberto Daniel-Moreno2,
- Sebastian Graeter1,
- Praveen Baskaran4,
- Petra Weinmann1,
- Markus Mezger2,
- Rupert Handgretinger2 and
- Michael S. D. Kormann1Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 26 January 2018
- Accepted: 17 October 2018
- Published: 14 November 2018
β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control.
We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBBIVS1–110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34+ hematopoietic stem cells (HSCs).
Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBBIVS1–110 mutation showed indels in both K562 cells (up to 77%) and CD34+ hematopoietic stem cells—HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34+ HSCs.
Our approach provides guidance on non-viral gene correction in CD34+ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia.
- Gene correction
- Cas9 mRNA
β-Thalassemia (OMIM: 613985) is an inherited hematological disorder caused by mutations of the human hemoglobin beta (HBB) gene, leading to deficient β-globin expression and severe anemia . The current treatment options include allogeneic bone marrow transplantation and hematopoietic stem cell (HSC) transplantation but are limited to histo-compatible donors. However, gene therapy based on autologous transplantation of a lentiviral-transferred HBB gene to HSCs resulted in remarkable clinical benefit [2, 3]. Though the safety and efficacy of lentiviral-based gene therapy is positive in a treated patient, the transactivation of the proto-oncogene HMGA2 and more than 3500 unique integration sites in tested mouse model forewarns the possibility of insertional mutagenesis [2, 4]. Earlier retroviral gene therapy studies on other inherited diseases reported the treatment-related leukemogenesis [5–7], and lentiviral therapy resulted in T cell lymphoma in a mouse model of X-SCID due to random integration into oncogenes . Therefore, the ideal gene therapy with viral vectors must ensure targeted integration of a therapeutic HBB transgene in the endogenous locus. Otherwise, personalized gene-correction therapy with programmable nucleases and non-viral repair templates such as single-stranded oligonucleotides (ssODNs) must be employed as it is less likely to randomly integrate into the genome and result in a safe and precise gene editing . Though several studies targeted HBB gene with ZFNs, TALENs, and CRISPR/Cas9, no study has ever compared all the three gene-editing platforms simultaneously. Therefore, in the present study, we compared these three approaches for their target efficiency in parallel. Here, we tested the gene correction efficiency of strategically designed ssODNs as repair templates to target HBB gene. This is an important measure while editing the highly proliferating stem cell population to avoid clonal selection and thereby triggering oncogenesis.
To target the HBBIVS1–110 locus, we designed a sgRNA and several ssODNs (Fig. 1b; Additional file 1: Figure S1) with varying lengths of homology arms, symmetrical difference, and chemical modifications and complimentary to the strand targeted or not targeted by the guide-RNA . To evaluate the ability of CRISPR/Cas9 to correct the HBBIVS1–110 mutation by an exogenous DNA sequence, we introduced an NheI-tag (restriction site) into the ssODNs that can be stably integrated upon successful homology-directed repair (HDR) (Fig. 1b). We electroporated pX330.sg HBBIVS1–110 plasmid and ssODN donors harboring NheI-tag using a Neon system into K562 cells and evaluated the frequency of indels by T7EI assay and HDR-driven NheI integration by restriction fragment length polymorphism (RFLP) assay (Figs. 1c/d). Mean indel frequencies for the targeted loci were 44 ± 18%. We next determined whether any of the rationally designed ssODNs could stimulate gene targeting by HDR and found that most of the tested ssODNs resulted in similar HDR frequencies with the median of 20% (range 5–31%).
However, the absolute quantification of site-specific insertion events of the NheI tag at the HBBIVS1–110 locus by next-Generation sequencing showed 8% correction for modified ssODN (Fig. 2e; Additional file 1: Figure S5). All the analyses spotted improved gene correction for chemically modified ssODN in HSCs as reported earlier . No detectable off-target cleavage was found in six of the in silico predicted off-targets (Additional file 2: Table S4; Additional file 1: Figure S6) for the sgRNA-targeted HBBIVS1–110 locus. We found that CRISPR/Cas9 greatly facilitates targeted genome modification compared to TALENs and ZFNs, and introduction of new DNA sequences in HSCs using Cas9 mRNA and ssODN is feasible without viral vectors. However, correction of 8% in HSCs at ex vivo setting is sub-optimal. Therefore, further improvement on HDR efficacy and selection of corrected cells is needed to attain a meaningful gene correction of HBB mutations to treat β-thalassemia and other related genetic diseases.
Design of gene-editing tools
The targeting efficacy at the promoter of the HBB gene locus (200 bp upstream of the transcription start site) between three different gene-editing tools (CRSIPR/Cas9, TALENs, and ZFN) was evaluated (Additional file 1: Figure S1). The ZFN constructs targeting the promoter were ordered from Sigma-Aldrich (http://www.sigmaaldrich.com). The full amino acid sequences of the ZFN pair are shown in Additional file 2: Data S1. The targeting TALEN pair was designed with the help of the online tool ZiFiT Targeter Version 4.2 (http://zifit.partners.org) and assembled by fast ligation-based automatable solid-phase high-throughput (FLASH). Plasmids encoding TALE repeats harboring different repeat variable di-residues (RVDs) with their FLASH IDs are summarized in Additional file 2: Table S1. The sgRNAs for promoter (5′-AGCCAGTGCCAGAAGAGCCA-3′) and HBBIVS1–110 (5-GGGTGGGAAAATAGACCAAT-3′) were designed using CRISPR design tool. The respective oligo pairs encoding the 20-nt guide sequences are annealed and ligated into a pX330 vector consisting of a SpCas9 expression cassette and a U6 promoter driving the expression of chimeric sgRNA. The chemically modified sgRNA for HBBIVS1–110 mutation was synthesized by incorporating 2-O-methyl 3′phosphorothioate (MS) modification at three terminal nucleotides at both the 5′ and 3′ ends.
Cell culture and transfection
HEK-293 cells were cultured in DMEM (https://www.thermofisher.com) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. In HEK-293 cells, pDNA-encoded gene-editing tools (CRSIPR/Cas9, TALENs, and ZFN) were transfected using Lipofectamin 3000 at three different concentrations of 0.5, 1, and 1.5 μg. K562 cells were cultured in RPMI 1640 (https://www.thermofisher.com) supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO2. In K562 cells, 200 ng of pX330-Chimeric vector targeting HBBIVS1–110 loci was co-electroporated with 10 pmol of different ssODN using Neon Transfection System (https://www.thermofisher.com). Bone marrow-derived CD34+ HSCs from healthy donors were cultured in StemSpan™ serum-free medium II (SFEMII) containing StemSpan™ Cytokine Cocktail 110 (https://www.stemcell.com). 1 × 105 CD34+ HSCs were electroporated with 1.2 μg pX330 vector targeting HBBIVS1–110 mutation and 100 pmol of specified ssODN repair template using Lonza© 4D-Nucleofector™ (Program: EO 100). The similar amount of p.Max eGFP pDNA (1.2 μg) was electroporated as a transfection control. For Cas9 mRNA experiments, 1 × 105 CD34+ HSCs were co-electroporated with 5 μg chemically modified sgRNA, 10 μg Ca9 mRNA vector, and 100 pmol of specified ssODN repair template using Lonza© 4D-Nucleofector™ (Program: EO 100).
The ssODNs were synthesized by Metabion, Germany (www.metabion.com). The ssODNs were designed with an insertion site harboring an NheI recognition site (GCTAGC). The ssODNs were complimentary to either the strand targeted or not targeted by the gRNA, asymmetric or symmetric to the NheI tag. The chemically modified ssODN for HBBIVS1–110 mutation was synthesized by incorporating 2-O-methyl 3′phosphorothioate (MS) modification at three terminal nucleotides at both the 5′ and 3′ ends with 72 bp homology arms. The complete details can be found in Additional file 2: Table S2.
T7 endonuclease assay and RFLP assay
Five days post transfection, genomic DNA was isolated using Merchery-Nagel NucleoSpin Tissue Kit following the manufacturer’s instructions. The promoter region was amplified using the primer pair Prom-For_5′-GTAGACCACCAGCAGCCTAA-3′ and Prom-Rev_5′ TGGAGACGCAGGAAGAGATC-3′, and the region covering HBBIVS1–110 mutation was amplified using the primer pair IVS1–110-For_ 5′-GGGTTTGAAGTCCAACTCCTAA-3′ and 3′UTR-For_5′-AGAAAACATCAAGCGTCCCATA-3′. The target regions were amplified using the AmpliTag® Gold 360 MasterMix (https://www.thermofisher.com). The cycling parameters for both amplicons were as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1 min. PCR products were purified by ethanol precipitation, and 1 μg of PCR product was used for T7 endonuclease assay. Likewise, 1 μg of PCR product was used for the RFLP assay where amplicons bearing the NheI tag upon successful gene correction will result in a specific cleavage after the treatment with the NheI restriction enzyme. The readout of the T7 endonuclease assay and the RFLP assay were determined on a 1.5% agarose gel and on a Bioanalyzer chip using DNA1000 kit. The band intensities were quantified using ImageJ embedded in Fiji software (www.fiji.sc).
TIDE analysis and single-colony and next-generation sequencing
For analyzing allele modification frequencies with non-enzymatic assays, we used TIDE (Tracking of Indels by Decomposition) analysis by Sanger-sequencing the purified PCR products used for T7 assay and examined with the online TIDE software (http://tide.nki.nl.) with the respective control sample. To precisely measure the events of site-specific insertion of NheI tag at HBBIVS1–110 loci in CD34+ HSPCs, we performed two different approaches: (i) Sanger-based single-colony sequencing and (ii) next-generation sequencing. For single-colony sequencing, the HBBIVS1–110 region was PCR amplified from 5TS ssODN gene-corrected CD34+ HSCs and cloned into pJET1.2 vector using CloneJET PCR Cloning kit (https://www.thermofisher.com) and transformed into Top10 competent cells using standard cloning techniques. Closely, 192 (two 96-well plates) single colonies were processed for Sanger sequencing with pJET1.2 forward sequencing primer (5′-CGACTCACTATAGGGAGAGCGGC-3′) and analyzed for the presence for NheI recognition site (GCTAGC) using Geneious-R6 software. Three of 192 clones resulted in NheI insertion (1.5%). In case of next-generation sequencing (NGS), new primers were designed with the amplicon length of 150 bp to be sequenced with the Illumina platform (Forward 5′-AGAAACTGGGCATGTGGAGA-3′; Reverse 5′-CCATAACAGCATCAGGAGTGG-3′). Further, barcode-tagged PCR primers were used to multiplex samples (Additional file 2: Table S3), and are adapter ligated, size selected, and bridge amplified and proceeded with amplicon sequencing in HiSeq 4000 system (http://www.illumina.com). The standard R-Package was used for NGS analysis where the sequencing reads were pre-filtered for low-quality reads and mapped to the reference sequence using a BWA tool. Further, the number of indel-carrying sequence reads was calculated using SAM tools. The distribution plot was generated by calculating the size of the indels in sequence and calculating the median percentage for each indel class.
In vitro transcription (IVT) of Cas9 mRNA
The open reading frame of SpCas9 was PCR amplified from the pX330 vector and sub-cloned into the pVAX.120 vector consisting of a T7 promoter and 120 bp length of a poly-A tail using standard molecular biology techniques. The IVT reaction was performed in linearized plasmid using T7 RNA polymerase in MEGAscript T7 kit (https://www.thermofisher.com). All mRNAs were produced with an anti-reverse CAP analog (ARCA; [m7G(5′)G]) at the 5′ end (https://www.trilinkbiotech.com/). The IVT-mRNAs were made with following chemical modifications in the indicated ratios: 100% Pseudo-UTP and 25% s2-thio-UTP/5-methyl-CTP (https://www.trilinkbiotech.com/). All IVT mRNAs were purified using the MEGAclear kit (https://www.thermofisher.com) and quantified with nano-photometer and bioanalyzed for quality using the RNA6000 kit in Agilent 2100 Bioanalyzer (https://www.agilent.com).
Kruskal-Wallis or Wilcoxon-Mann-Whitney rank-sum tests were applied wherever appropriate to analyze the differences in indel induction among the gene-editing technologies and comparison between encoded pDNA Cas9 and Cas9 mRNA using Graphpad Prism v.6.0d (https://www.graphpad.com).
We thank Mr. Brian Weidensee for the help with the proofreading of the draft.
This work was supported by the Sanitätsrat Dr. Alexander Huebner und Gemahlin-Stiftung and by the UKT-fortüne grant (no. 2412-0-0). J.S.A. was financially supported by UKT-fortüne grant (to J.S.A., No. 2485-0-0) and Europe Research Council Starting Grant-ERC StG (to M.S.D.K., No. 637752). NL was supported by HMZ Private Foundation (to M.S.D.K). A.H. was supported by ERC StG (to M.S.D.K., No. 637752). A.L.C. was supported by Jürgen Man-chot-Stiftung. A.D.M. was supported by a UKT-fortüne grant (to M.M., No. 2412-0-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Availability of data and materials
All data generated or analyzed during this study are included in this published article (and its Additional files).
NL, JSA, RH, and MSDK conceived and designed the experiments. NL, JSA, AH, ALC, ADM, SG, and PW performed the experiments. JSA, NL, and PB analyzed the data. MSDK, MM, and RH contributed reagents/materials/analysis tools. JSA, NL, and MSDK wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Ethical approval for using CD34+ hematopoietic stem cells from healthy donors was obtained from Ethics commission, University Clinic of Tuebingen, Germany (829/2016BO2).
Consent for publication
M.S.D.K. is listed as main inventor on a patent application related to Nuclease encoding modified mRNA. M.S.D.K. is a main inventor on a patent licensed to the biopharmaceutical company, Ethris GmbH. M.S.D.K., A.D., A.H., and J.S.A. are inventors (with M.S.D.K. as main inventor) on a patent application related to modified mRNA encoding hCFTR.
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