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CRISPR-Cas9 base editors and their current role in human therapeutics

  • Walker S. Lahr
    Correspondence
    These authors contributed equally to this work.
    Affiliations
    Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA

    Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA

    Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA
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  • Christopher J. Sipe
    Correspondence
    These authors contributed equally to this work.Deceased.
    Affiliations
    Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA

    Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA

    Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA
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  • Joseph G. Skeate
    Affiliations
    Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA

    Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA

    Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA
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  • Beau R. Webber
    Correspondence
    Correspondence: Beau R. Webber, PhD, Masonic Cancer Center, University of Minnesota, 2231 6th St SE, Minneapolis, Minnesota, USA.
    Affiliations
    Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA

    Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA
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  • Branden S. Moriarity
    Correspondence
    Correspondence: Branden S. Moriarity, PhD, Masonic Cancer Center, University of Minnesota, 2231 6th St SE, Minneapolis, Minnesota, USA.
    Affiliations
    Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA

    Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA

    Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota, USA

    Stem Cell Institute, University of Minnesota, Minneapolis, Minnesota, USA

    Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, Minnesota, USA
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Open AccessPublished:January 10, 2023DOI:https://doi.org/10.1016/j.jcyt.2022.11.013

      Abstract

      Background

      Consistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification.

      Methods

      This review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes.

      Results

      CRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction.

      Conclusion

      As such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.

      Key Words

      Introduction

      Genetic mutations that cause genetic disorders or predispose individuals to an enhanced cancer risk are abundant. Nearly half of these pathogenic mutations are single nucleotide variants (SNVs) [
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      ], leading to a focus on understanding the occurrence and phenotypic consequences of particular SNVs. Parallel to this area of investigation, it is of high interest to correct or revert these SNVs to eliminate the problematic genotype completely [
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      ]. Historically, all therapeutically relevant targeted nuclease strategies induce double-strand breaks (DSBs) in genomic DNA to achieve gene editing. Transcription activator-like effector nucleases and zinc finger nucleases use a targeted DNA binding domain fused to an endonuclease domain of Fok1 [
      • Cermak T
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      ]. Meganucleases bind and create DSBs in palindromic, semi-palindromic (I-CreI) or non-palindromic (I-SceI) DNA sequences [
      • Silva G
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      Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy.
      ]. By contrast, the CRISPR-Cas9 platform uses a unique targeted single guide RNA (sgRNA), which complexes with the constant endonuclease Cas9 to induce targeted DSBs [
      • Jinek M
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      ]. These targeted nucleases can produce off-target effects—unwanted DSBs at nucleotide sequences in the genome that are highly similar to the desired DNA target site. Further, simultaneous DSBs within one genome, such as one at the on-target site or one or more at off-target sites, can result in chromosomal translocation events between these sites, which can severely alter gene regulation and function [
      • Frock RL
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      ]. DSBs have also been reported to induce cell cycle arrest [
      • Chapman JR
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      ]; p53 activation [
      • Haapaniemi E
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      CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response.
      ]; complex rearrangements of chromosomes, including large deletions of DNA [
      • Haapaniemi E
      • Botla S
      • Persson J
      • Schmierer B
      • Taipale J.
      CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response.
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      Large deletions induced by Cas9 cleavage.
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      CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.
      ,
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      UDiTaSTM, a genome editing detection method for indels and genome rearrangements.
      ,
      • Kosicki M
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      ]; alteration of expression in genes neighboring the DSB [
      • Oberdoerffer P
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      ATM-dependent chromatin changes silence transcription in cis to DNA double-strand breaks.
      ]; chromothripsis [
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      Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing.
      ]; and cancer [
      • Khanna KK
      • Jackson SP.
      DNA double-strand breaks: signaling, repair and the cancer connection.
      ]. Next-generation Cas9-derived “digital” genome editing tools—namely, Cas9 base editors (BEs) [
      • Komor AC
      • Kim YB
      • Packer MS
      • Zuris JA
      • Liu DR.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ,
      • Gaudelli NM
      • Komor AC
      • Rees HA
      • Packer MS
      • Badran AH
      • Bryson DI
      • et al.
      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
      ]—have the ability to perform single base conversion to correct a subset of SNVs, including C>T (G>A opposite strand) and A>G (T>C opposite strand). These tools provide a unique ability to correct pathogenic mutations and modify expression of genes (reactivate/suppress) without the need for creating a toxic DSB or DNA donor molecule for genetic correction, which greatly decreases the occurrence of unintended effects in the genome and enhances efficiency and efficacy of repair [
      • Fu Y
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      ].
      This review focuses on advances in BE technology, current applications to human therapeutics and the current and future landscape of in vivo delivery in clinical trials and beyond. A description of the mechanistic action of BEs will be followed by a summary with regard to broadening the editing toolbox by reducing the stringency of the Cas9 protospacer adjacent motif (PAM). Recent base editing strategies in the field of cancer immunotherapy as well as in genetic disorders that can be combated by editing single nucleotide polymorphisms will then be described. Therapeutic pre-clinical delivery of BEs, both ex vivo and in vivo, will be highlighted, with an emphasis on specific delivery methods: direct nucleic acid/protein delivery, lipid/polymer-based delivery and viral delivery. Finally, current and upcoming clinical trials involving in vivo delivery of BEs will be outlined, with an emphasis on and insight into the future applications and landscape of BE technology.

      Base Editors

      BEs are a fusion of a Cas9 nickase (nCas9) and a deaminase enzyme capable of performing base modifications through deamination, resulting in transition and transversion mutations. Similar to the CRISPR-Cas9 system, after nCas9 binds to its target DNA sequence, defined by a 20 base pair sgRNA, an “R-loop” is generated (Figure 1A) [
      • Nishimasu H
      • Ran FA
      • Hsu PD
      • Konermann S
      • Shehata SI
      • Dohmae N
      • et al.
      Crystal structure of Cas9 in complex with guide RNA and target DNA.
      ]. This R-loop results from localized denaturation upon Cas9 binding, exposing a short stretch of single-stranded DNA (ssDNA), which provides adequate space for the deaminase to act on the target base(s). Two main classes of BEs have emerged with high efficiency and product purity, cytosine BEs (CBEs) and adenosine BEs (ABEs), which convert a C:G base pair to a T:A base pair (G:C to A:T opposite strand) and an A:T base pair to a G:C base pair (T:A to C:G opposite strand), respectively [
      • Komor AC
      • Kim YB
      • Packer MS
      • Zuris JA
      • Liu DR.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ,
      • Gaudelli NM
      • Komor AC
      • Rees HA
      • Packer MS
      • Badran AH
      • Bryson DI
      • et al.
      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
      ].
      Fig 1
      Fig. 1Cytosine base editing. A nCas9, which nicks the opposite target strand, induces a DNA repair pathway response. The target C in the editing window is deaminated by a fused APOBEC1 protein to a U. A fused UGI prevents the cell from resolving this mismatch through the BER pathway. During DNA replication or repair, the U matches with an A, resulting in a C:G to T:A substitution. BER, base excision repair; gRNA, guide RNA; UGI, uracil glycosylase inhibitor.

      Cytosine BEs

      CBEs were the first class of BEs to be generated, CBEs first convert target cytosine bases to uracil then the edited strand is repaired to a thymine through DNA replication or repair (Figure 1B) [
      • Komor AC
      • Kim YB
      • Packer MS
      • Zuris JA
      • Liu DR.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ]. The first CBE, BE1, was a catalytically dead version of Streptococcus pyogenes Cas9 (dCas9) linked to an ssDNA targeting the cytidine deaminase enzyme from Rattus norvegicus (rAPOBEC1). BE1 induced low-level cytosine to uracil conversion efficiency in HEK293T cells, an outcome that was hypothesized to be due to high rates of intracellular uracil excision by the endogenous base excision repair pathway. Three improvements were then made to BE1 to generate the higher efficiency BE4. First, two phage-derived uracil glycosylase inhibitors were linked to the C-terminus of BE1 by a nine amino acid linker that functioned to inhibit uracil excision by uracil DNA glycosylase. The rAPOBEC1-dCas9 fusion was also optimized to 32 amino acids, and the dCas9 was converted to a D10A nCas9 to direct host repair to assume the nicked strand is the newly synthesized DNA strand, cloaking the strand deaminated by rAPOBEC1 (Figure 1A,B) [
      • Komor AC
      • Kim YB
      • Packer MS
      • Zuris JA
      • Liu DR.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ,
      • Komor AC
      • Zhao KT
      • Packer MS
      • Gaudelli NM
      • Waterbury AL
      • Koblan LW
      • et al.
      Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity.
      ]. Additional optimizations, such as limiting the use of rare codons and enhancing/optimizing online design tools and changes to the nuclear localization signal [
      • Koblan LW
      • Doman JL
      • Wilson C
      • Levy JM
      • Tay T
      • Newby GA
      • et al.
      Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.
      ], led to BE4max, which has highly efficient (>95%) cytosine deaminase editing at positions four through eight in the 5’ region of the protospacer (editing window) in human primary cells [
      • Webber BR
      • Lonetree CL
      • Kluesner MG
      • Johnson MJ
      • Pomeroy EJ
      • Diers MD
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      Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
      ,
      • Zeng J
      • Wu Y
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      • Shen AH
      • Shea D
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      Therapeutic base editing of human hematopoietic stem cells.
      ].

      Adenosine BEs

      ABEs were developed as a second class of BEs when it was recognized that adenosine deamination could generate an inosine intermediate [
      • Gaudelli NM
      • Komor AC
      • Rees HA
      • Packer MS
      • Badran AH
      • Bryson DI
      • et al.
      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
      ]. The Liu Lab (Cambridge, MA, USA) utilized directed evolution of the known bacterial double-stranded RNA adenosine deaminase enzyme ecTadA to perform deamination of ssDNA. This evolution was achieved through incorporation of 14 amino acid changes in TadA, which was then fused to an additional wtTadA and nCas9 D10A to generate ABE7.10. Because of the complete absence of inosine in the genome, no known endogenous mechanisms of inosine removal exist, and thus a glycosylase inhibitor was not necessary (Figure 2) [
      • Gaudelli NM
      • Komor AC
      • Rees HA
      • Packer MS
      • Badran AH
      • Bryson DI
      • et al.
      Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.
      ]. During DNA replication and cell division, this inosine will be read as a guanine and lead to permanent incorporation of a guanine. Some limitations of ABE7.10 are its efficiency of deamination and its generation of off-target edits within the editing window. Subsequently, phage-assisted evolution of the wtTaDA domain was performed to overcome the shortcomings of ABE7.10. This identified eight additional amino acid changes, resulting in a more processive and 590-fold more active form of ABE called ABE8e [
      • Richter MF
      • Zhao KT
      • Eton E
      • Lapinaite A
      • Newby GA
      • Thuronyi BW
      • et al.
      Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity.
      ]. ABE8e allows for efficient target adenosine base conversion to guanine at positions four through eight of the protospacer target site, and rescreening of the public archive of single nucleotide polymorphisms on ClinVar showed that this BE version could correct many of the known pathogenic transition mutations, which account for approximately 60% of all known pathogenic SNVs [
      • Landrum MJ
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      ].
      Fig 2
      Fig. 2Adenosine base editing. A nCas9, which nicks the opposite target strand, induces a DNA repair pathway response. The target A in the editing window is deaminated by a fused TadA protein to an I. During DNA replication or repair, the I is read as a G, resulting in an A:T to G:C substitution. gRNA, guide RNA.

      Expanding BE targeting range through removal of PAM restrictions

      Although BEs are exciting for the future treatment of disease, both CRISPR-Cas9 and BEs require a specific PAM sequence adjacent to the 20 base pair protospacer target sequence. The PAM sequence required for traditional Streptococcus pyogenes Cas9 is NGG (N = standard International Union of Pure and Applied Chemistry nucleotide code), which limits the number of SNVs that are targetable using BEs or traditional CRISPR-Cas9 methods. Because of this restriction, sgRNAs must place the target base within the editing window located 12–16 base pairs away from the PAM. In order to expand the utility of these enzymes and increase the amount of targetable SNVs, BEs can have the nCas9 domain altered to permit different PAM requirements. Many Cas9 PAM variants exist, including VQR-Cas9 (NGAN), EQR-Cas9 (NGAG), VRQR-Cas9 (NGCG), Cas9-NG [
      • Walton RT
      • Christie KA
      • Whittaker MN
      • Kleinstiver BP.
      Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.
      ,
      • Hirano S
      • Nishimasu H
      • Ishitani R
      • Nureki O.
      Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.
      ] and the “near-PAMless” (SpRY) Cas9 variant, which recognizes NRN (R = adenine or guanine) and, to a lesser extent, NYN (Y = cytosine or thymine) PAMs [
      • Walton RT
      • Christie KA
      • Whittaker MN
      • Kleinstiver BP.
      Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.
      ]. This increased flexibility combined with the development of software that predicts base editing outcomes, such as BE-HIVE and Honeycomb, has been and will continue to be critical to the expansion of base targeting with BEs [
      • Kluesner MG
      • Lahr WS
      • Lonetree CL
      • Smeester BA
      • Qiu X
      • Slipek NJ
      • et al.
      CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells.
      ,
      • Arbab M
      • Shen MW
      • Mok B
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      Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning.
      ].

      Clinical Applications

      Cancer immunotherapy

      One of the exciting applications of BEs for clinical therapy is in combination with ex vivo-manufactured allogeneic chimeric antigen receptor (CAR) or T-cell receptor-engineered lymphocytes. Autologous CAR T cells have been used with great success clinically to mount an immune response against leukemia and lymphoma [
      • June CH
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      CAR T cell immunotherapy for human cancer.
      ,
      • Sterner RC
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      ]. Allogeneic or “off-the-shelf” CAR T-cell therapies are of significant interest because of the cost-effective mass manufacturing and viability of lymphocytes from healthy donors. In theory, allogeneic T cells could be isolated from healthy donors, genetically engineered to express a CAR and then infused into unrelated cancer patients as a CAR T-cell therapy. Unfortunately, it has been well documented that without manipulation, a patient's immune cells can reject allogeneic T-cell products or the allogeneic T cells can attack the patient's healthy cells, a phenomenon known as graft-versus-host disease (GVHD) [
      • Perez C
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      ].
      Several methods have been employed in an attempt to circumvent the most harmful effects of allogeneic CAR T-cell therapy (i.e., GVHD). For instance, Cas9 nucleases have been deployed for gene knockout (KO) to generate allo-compatible CAR T cells by inactivating the endogenous T-cell receptor and/or removing beta-2 microglobulin, a structural component of the major histocompatibility complex, which has shown some effectiveness in evading GVHD in pre-clinical models [
      • Liu X
      • Zhang Y
      • Cheng C
      • Cheng AW
      • Zhang X
      • Li N
      • et al.
      CRISPR-Cas9-mediated multiplex gene editing in CAR-T cells.
      ]. These multiplex gene KO approaches using Cas9 nucleases can be highly efficient (>90% KO) but have the potential to induce translocations and other unwanted genomic insults. In light of this, researchers have begun to use BEs to achieve robust multiplex gene KO using premature stop codon induction [
      • Kuscu C
      • Parlak M
      • Tufan T
      • Yang J
      • Szlachta K
      • Wei X
      • et al.
      CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.
      ,
      • Dang L
      • Li G
      • Wang X
      • Huang S
      • Zhang Y
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      Comparison of gene disruption induced by cytosine base editing-mediated iSTOP with CRISPR/Cas9-mediated frameshift.
      ] and splice site disruption [
      • Kluesner MG
      • Lahr WS
      • Lonetree CL
      • Smeester BA
      • Qiu X
      • Slipek NJ
      • et al.
      CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells.
      ]. Beyond engineering for allogeneic compatibility, multiplex KO of checkpoint regulators such as CTLA4, PDCD1, SOCS3 and CISH has been used in T cells to enhance in vivo tumor clearance [
      • Webber BR
      • Lonetree CL
      • Kluesner MG
      • Johnson MJ
      • Pomeroy EJ
      • Diers MD
      • et al.
      Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
      ,
      • Zhu H
      • Blum RH
      • Bernareggi D
      • Ask EH
      • Wu Z
      • Hoel HJ
      • et al.
      Metabolic Reprograming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity.
      ]. This multiplex gene KO approach has demonstrated enhanced tumor clearance in comparison with CAR integration alone in multiple murine models of cancer [
      • Webber BR
      • Lonetree CL
      • Kluesner MG
      • Johnson MJ
      • Pomeroy EJ
      • Diers MD
      • et al.
      Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
      ,
      • Kluesner MG
      • Lahr WS
      • Lonetree CL
      • Smeester BA
      • Qiu X
      • Slipek NJ
      • et al.
      CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary and immortalized cells.
      ]. Utilization of BEs will most likely represent the future of multiplex gene KO, as BEs have demonstrated greatly reduced to undetectable levels of DSBs and translocations and thus may provide a way of tailoring specific gene edit combinations to target individual cancers as the next generation of precision medicine [
      • Haapaniemi E
      • Botla S
      • Persson J
      • Schmierer B
      • Taipale J.
      CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response.
      ,
      • Kosicki M
      • Tomberg K
      • Bradley A.
      Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.
      ,
      • Webber BR
      • Lonetree CL
      • Kluesner MG
      • Johnson MJ
      • Pomeroy EJ
      • Diers MD
      • et al.
      Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
      ,
      • Ihry RJ
      • Worringer KA
      • Salick MR
      • Frias E
      • Ho D
      • Theriault K
      • et al.
      p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.
      ,
      • Teboul L
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      ].

      Correction of SNVs in genetic disease

      Many rare autosomal diseases are caused by SNVs, which lead to non-functional or dysfunctional protein expression. Many SNVs that can be targeted with BEs have been defined for diseases such as severe combined immunodeficiency, Crigler–Najjar syndrome, sickle cell disease (SCD), beta thalassemia, cystic fibrosis and Fanconi anemia [
      • Liang P
      • Ding C
      • Sun H
      • Xie X
      • Xu Y
      • Zhang X
      • et al.
      Correction of β-thalassemia mutant by base editor in human embryos.
      ,
      • Geurts MH
      • de Poel E
      • Amatngalim GD
      • Oka R
      • Meijers FM
      • Kruisselbrink E
      • et al.
      CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank.
      ]. In the case of hematological diseases such as SCD (the most common inherited genetic disorder worldwide) and beta thalassemia, hematopoietic stem and progenitor cells (HSPCs) can be isolated from patients, base edited ex vivo and infused back into the individual after pre-conditioning [
      • Li C
      • Wang H
      • Georgakopoulou A
      • Gil S
      • Yannaki E
      • Lieber A.
      In Vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model.
      ]. Targeting HSPCs can also theoretically be done in vivo by first mobilizing the patient's HSPCs in the bloodstream and then delivering BE reagents intravenously, after which time the HSPCs will re-engraft in the bone marrow compartment [
      • Li C
      • Wang H
      • Georgakopoulou A
      • Gil S
      • Yannaki E
      • Lieber A.
      In Vivo HSC Gene Therapy Using a Bi-modular HDAd5/35++ Vector Cures Sickle Cell Disease in a Mouse Model.
      ,
      • Newby GA
      • Yen JS
      • Mayuranathan T
      • Lazzarotto CR
      • Li Y
      • Sheppard-Tillman H
      • et al.
      Base editing of haematopoietic stem cells rescues sickle cell disease in mice.
      ].
      As an example of BE phenotypic alleviation in SCD, the most common SCD SNV (A>T) in the hemoglobin subunit beta (HBB) gene causes a glutamine (GAG) to valine (GTG) substitution. This single amino acid change causes polymerization of hemoglobin proteins, which in turn causes red blood cells (RBCs), or erythrocytes, to form their pathogenic sickle shape. These mutant RBCs hemolyze frequently and block arteries, leading to pain crisis, organ failure and vaso-occlusion-related issues [
      • Salinas Cisneros G
      • Thein SL.
      Recent Advances in the Treatment of Sickle Cell Disease.
      ]. Because BE technology cannot convert T>A directly, several strategies have been employed to alleviate symptoms without directly correcting the pathogenic SNV back to wild-type. One strategy uses ABEs to convert the pathogenic valine (GTG) to alanine (GCG), which has been shown to be a benign mutation in individuals from the Makassar region of Indonesia. Given that this conversion could not be achieved with the current BEs, phage-assisted continuous evolution of the ABE (ABE-NRCH) was employed to deaminate the opposite strand A base to a G base, generating a C base through DNA replication, resulting in an alanine codon. This method produced high levels of targeted base editing in human SCD HSPC CD34+ cells and resulted in decreased levels of sickle-shaped RBCs [
      • Newby GA
      • Yen JS
      • Mayuranathan T
      • Lazzarotto CR
      • Li Y
      • Sheppard-Tillman H
      • et al.
      Base editing of haematopoietic stem cells rescues sickle cell disease in mice.
      ]. Corrected human HSPC CD34+ cells were then transplanted into irradiated mice, where edits persisted after 16 weeks and healthy HBB proteins were achieved, demonstrating pre-clinical therapeutic relevance.
      A separate strategy utilizes BEs to alleviate SCD symptoms by reactivating fetal hemoglobin (HbF), which has a much higher affinity for oxygen than adult hemoglobin [
      • Oski FA
      • Delivoria-Papadopoulos M.
      The shift to the left.
      ,
      • Pritišanac E
      • Urlesberger B
      • Schwaberger B
      • Pichler G.
      Fetal Hemoglobin and Tissue Oxygenation Measured With Near-Infrared Spectroscopy-A Systematic Qualitative Review.
      ]. In adults, circulating hemoglobin is composed of hemoglobin subunit alpha and HBB, whereas HbF is composed of hemoglobin subunit alpha and hemoglobin subunit gamma (HBG). Three months postnatally, HbF levels are reduced because of the silencing of HBG transcription by BCL11A, a transcriptional repressor that normally binds to the promoter region of HBG [
      • Shen Y
      • Li R
      • Teichert K
      • Montbleau KE
      • Verboon JM
      • Voit RA
      • et al.
      Pathogenic BCL11A variants provide insights into the mechanisms of human fetal hemoglobin silencing.
      ]. It was previously shown that creating small insertions or deletions with CRISPR-Cas9 in the erythroid-specific enhancer region of BCL11A halts the transcriptional repression by BCL11A in the erythroid compartment, leading to re-expression of HBG and, in turn, assembly of HbF [
      • Wu Y
      • Zeng J
      • Roscoe BP
      • Liu P
      • Yao Q
      • Lazzarotto CR
      • et al.
      Highly efficient therapeutic gene editing of human hematopoietic stem cells.
      ].
      Further examination of the genetic sequence of this enhancer revealed that several “hot spots” were the most relevant for disruption of subsequent BCL11A enhancement. Fortunately, some of these areas could be targeted with CBEs by making single C>T edits. Ex vivo genetic modification of SCD HSPC CD34+ cells with CBEs targeted to these erythroid-specific enhancers of BCL11A “hot spot” regions led to the reactivation of HBG transcription and thus HbF expression [
      • Wu Y
      • Zeng J
      • Roscoe BP
      • Liu P
      • Yao Q
      • Lazzarotto CR
      • et al.
      Highly efficient therapeutic gene editing of human hematopoietic stem cells.
      ]. Again, these corrected human HSPC CD34+ cells were transplanted into irradiated mice, where edits persisted after 16 weeks and HbF was highly expressed, outcompeting the assembly of adult hemoglobin with mutated HBB, leading to phenotypic improvement [
      • Zeng J
      • Wu Y
      • Ren C
      • Bonanno J
      • Shen AH
      • Shea D
      • et al.
      Therapeutic base editing of human hematopoietic stem cells.
      ].
      A second strategy for reactivation of HbF is targeting the binding of BCL11A and other transcriptional silencers on the promoter of HBG directly. In this in vivo mouse study [
      • Li C
      • Georgakopoulou A
      • Mishra A
      • Gil S
      • Hawkins RD
      • Yannaki E
      • et al.
      In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice.
      ], CBE and ABE constructs were packaged into CD46-targeting helper-dependent adenovirus (HDAd5/35++) and targeted single base conversion on the HBG promoter where lymphoma-related factor normally binds for transcriptional silencing. These “hot spot” sites are a form of naturally occurring mutation known as hereditary persistence of fetal hemoglobin, as these individuals have abnormally high levels of HbF [
      • Sharma DC
      • Singhal S
      • Woike P
      • Rai S
      • Yadav M
      • Gaur R.
      Hereditary persistence of fetal hemoglobin.
      ]. After injecting mice with virus, it was found that moderate levels of HSPC base editing (15–25% in both HBG1 and HBG2 promoter regions) equated to >40% HBG induction in peripheral blood erythrocytes [
      • Li C
      • Georgakopoulou A
      • Mishra A
      • Gil S
      • Hawkins RD
      • Yannaki E
      • et al.
      In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal γ-globin in β-YAC mice.
      ]. On the commercial side, Beam Therapeutics (Cambridge, MA, USA) is pursuing both the Makassar BE strategy (BEAM-102) and strategies to increase the expression of HbF (BEAM-101) as first-in-human clinical applications of BEs.

      Clinical Delivery

      Direct delivery of BE reagents

      Clinical delivery of CRISPR-Cas9 and BE reagents has commonly been through purified protein and chemically modified sgRNAs in the form of ribonucleoprotein (RNP) (NCT03745287), yet this approach has proven more difficult with BEs because of the size and folding properties of the BE protein [
      • Rees HA
      • Komor AC
      • Yeh WH
      • Caetano-Lopes J
      • Warman M
      • Edge ASB
      • et al.
      Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.
      ]. Thus, many groups are using messenger RNA (mRNA) encoding BEs in combination with chemically modified sgRNAs because these sgRNAs can be rapidly produced and do not require complex protein manufacturing [
      • Webber BR
      • Lonetree CL
      • Kluesner MG
      • Johnson MJ
      • Pomeroy EJ
      • Diers MD
      • et al.
      Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
      ,
      • Jiang T
      • Henderson JM
      • Coote K
      • Cheng Y
      • Valley HC
      • Zhang XO
      • et al.
      Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.
      ,
      • Gaudelli NM
      • Lam DK
      • Rees HA
      • Solá-Esteves NM
      • Barrera LA
      • Born DA
      • et al.
      Directed evolution of adenine base editors with increased activity and therapeutic application.
      ]. Direct ex vivo delivery of BE reagents is a process that involves isolation of target cells from the patient, delivery of BE reagents to the cells (typically via electroporation) and reinfusion of modified cells into the patient (Figure 3). Ex vivo delivery of BEs to human primary cells has become a common method for groups working with immune cells, stem cells, epithelial cells and retinal cells [
      • Webber BR
      • Lonetree CL
      • Kluesner MG
      • Johnson MJ
      • Pomeroy EJ
      • Diers MD
      • et al.
      Highly efficient multiplex human T cell engineering without double-strand breaks using Cas9 base editors.
      ,
      • Newby GA
      • Yen JS
      • Mayuranathan T
      • Lazzarotto CR
      • Li Y
      • Sheppard-Tillman H
      • et al.
      Base editing of haematopoietic stem cells rescues sickle cell disease in mice.
      ,
      • Levy JM
      • Yeh WH
      • Pendse N
      • Davis JR
      • Hennessey E
      • Butcher R
      • et al.
      Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.
      ]. This approach is attractive for two reasons: (i) a smaller starting population requires less cost and complexity of manufacture and (ii) after expansion and genetic modification ex vivo, the BE protein or mRNA will have dissipated by the time of infusion and gene editing, resulting in a modified cellular product that limits exposure of the host immune system to innocuous exogenous BEs, thus reducing the concern of immune activation against the foreign protein or mRNA [
      • Gough V
      • Gersbach CA.
      Immunity to Cas9 as an Obstacle to Persistent Genome Editing.
      ].
      Fig 3
      Fig. 3Ex vivo or in vivo base editing. Schematic representation of a clinical workflow for ex vivo manipulation of human leukocytes using electroporation and the BE system. Here, systemic in vivo delivery of AAV is represented by a split BE system because of cargo size limits of AAV. AAV, adeno-associated virus; PBMCs, peripheral blood mononuclear cells.

      Viral delivery

      Viruses such as adenovirus, recombinant adeno-associated virus (rAAV) and lentivirus can be used to deliver BE reagents both in vivo and ex vivo. rAAV has emerged as a popular candidate because of its low immunogenicity and largely transient, non-integrating nature [
      • Tong S
      • Moyo B
      • Lee CM
      • Leong K
      • Bao G.
      Engineered materials for in vivo delivery of genome-editing machinery.
      ]. Obtaining viruses with strict tropism can be challenging, and efforts to characterize rAAV serotypes to target various tissues are ongoing [
      • Srivastava A.
      In vivo tissue-tropism of adeno-associated viral vectors.
      ]. Another limitation is that viruses have packaging limits (approximately 4.7 kb with rAAV). To deliver cargo larger than this limit, multiple viruses need to be produced, increasing the overall cost of the therapy. Injections of rAAVs containing BEs that have been split into two viral preparations have been performed successfully. These separate rAAVs generate precursor proteins that are then spliced together after transduction using the intein system [
      • Levy JM
      • Yeh WH
      • Pendse N
      • Davis JR
      • Hennessey E
      • Butcher R
      • et al.
      Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.
      ]. Although these methods have proven to be effective in mice, these mice live in specific pathogen-free colonies and have little to no exposure to rAAV or Cas9. Groups have demonstrated that humans can produce antibodies against Streptococcus pyogenes Cas9 and various rAAV serotypes, which is a potential concern for translation [
      • Simhadri VL
      • McGill J
      • McMahon S
      • Wang J
      • Jiang H
      • Sauna ZE.
      Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population.
      ,
      • Boutin S
      • Monteilhet V
      • Veron P
      • Leborgne C
      • Benveniste O
      • Montus MF
      • et al.
      Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors.
      ]. Although viral delivery continues to be an efficient method of gene transfer in mice and template for homology-directed repair, much optimization is needed before the in vivo delivery method can become standard of care.

      Lipid/polymer-based delivery

      Lipid-like nanoparticles (LLNPs), virus-like particles and polymer-based nanoparticles overcome the limitations of in vivo viral delivery in part by improving in vivo tissue specificity and toxicity [
      • Zuris JA
      • Thompson DB
      • Shu Y
      • Guilinger JP
      • Bessen JL
      • Hu JH
      • et al.
      Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.
      ,
      • Banskota S
      • Raguram A
      • Suh S
      • Du SW
      • Davis JR
      • Choi EH
      • et al.
      Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.
      ]. Traditionally, this method relies on a negatively charged RNP packaged within an exosome, liposome or polymer for systemic infusion [
      • Banskota S
      • Raguram A
      • Suh S
      • Du SW
      • Davis JR
      • Choi EH
      • et al.
      Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.
      ,
      • Pack DW
      • Hoffman AS
      • Pun S
      • Stayton PS.
      Design and development of polymers for gene delivery.
      ]. LLNPs, virus-like particles and polymer-based nanoparticles have the ability to deliver large molecular payloads to tissues in the body and are traditionally less immunogenic than viruses [
      • Mintzer MA
      • Simanek EE.
      Nonviral vectors for gene delivery.
      ]. As this field has grown, charged liposomes that contain sgRNAs and protein have been used as a vehicle for intracellular delivery [
      • Zhao Y
      • Huang L.
      Lipid nanoparticles for gene delivery.
      ,
      • Rees HA
      • Komor AC
      • Yeh WH
      • Caetano-Lopes J
      • Warman M
      • Edge ASB
      • et al.
      Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.
      ,
      • Banskota S
      • Raguram A
      • Suh S
      • Du SW
      • Davis JR
      • Choi EH
      • et al.
      Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins.
      ,
      • Kay MA.
      State-of-the-art gene-based therapies: the road ahead.
      ]. With RNP delivery methods, the BE is able to penetrate and act on the target cell faster and is cleared rapidly from the host. As optimization of this delivery method continues, tissue specificity and delivery efficiency will also improve, and thus this method will likely emerge as the predominant form of delivery for in vivo studies and trials in years to come.

      The Future of BE Applications

      Current and upcoming clinical trials

      Several pre-clinical studies that utilize in vivo delivery of BEs to combat genetic disorders have been described in a recent publication [
      • Musunuru K
      • Chadwick AC
      • Mizoguchi T
      • Garcia SP
      • DeNizio JE
      • Reiss CW
      • et al.
      In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates.
      ], press releases and conference abstracts. The only in vivo trial thus far that has entered the clinic is the heart-1 trial (NCT05398029), which aims to treat heterozygous familial hypercholesterolemia, uncontrolled hypercholesterolemia and atherosclerotic cardiovascular disease. The novel gene therapy VERVE-101, which consists of ABE mRNA and sgRNA targeting the PCSK9 gene encapsulated in an LLNP, is used for complete silencing of protein expression in the liver. As of July 5, 2022, one patient has been infused, with an estimated 40 patients to be included over the next several years. The dosing of this first patient represents an important milestone for the future of BE-rooted therapies, which are trending in the direction of in vivo delivery.
      The remaining upcoming pre-clinical trials that have yet to move into phase 1b approval for human subject testing range in delivery method from LLNPs and rAAV to a novel subcutaneous injection of BE mRNA [
      • Kingwell K.
      Base editors hit the clinic.
      ]. Verve Therapeutics (Cambridge, MA, USA) has an additional pre-clinical trial in the pipeline that also utilizes LLNPs containing BEs to combat familial hypercholesterolemia, but by the silencing of the ANGPTL3 gene, which also regulates circulating cholesterol levels [
      • Wu L
      • Soundarapandian MM
      • Castoreno AB
      • Millar JS
      • Rader DJ.
      LDL-Cholesterol Reduction by ANGPTL3 Inhibition in Mice Is Dependent on Endothelial Lipase.
      ]. In addition to the aforementioned ex vivo BE investigational new drug approved and investigational new drug enabling studies to treat SCD and beta thalassemia, Beam Therapeutics is pursuing pre-clinical models of in vivo base editing. The first approach targets the R83C gene for glycogen storage disease 1a [
      • Arnaoutova I
      • Zhang L
      • Chen HD
      • Mansfield BC
      • Chou JY.
      Correction of metabolic abnormalities in a mouse model of glycogen storage disease type Ia by CRISPR/Cas9-based gene editing.
      ], the second targets the G1961E gene for Stargardt disease and the third targets SERPINA1 mRNA for alpha-1 antitrypsin deficiency [
      • Kingwell K.
      Base editors hit the clinic.
      ,
      • Packer MS
      • Chowdhary V
      • Lung G
      • Cheng LI
      • Aratyn-Schaus Y
      • Leboeuf D
      • et al.
      Evaluation of cytosine base editing and adenine base editing as a potential treatment for alpha-1 antitrypsin deficiency.
      ]. These pre-clinical studies that are being pushed into the clinic are exciting and provide evidence that the field of BE delivery is shifting into in vivo delivery methods.

      Broadening the BE toolbox

      As BEs have entered the clinic and their utility in both translation and basic science has been solidified, several groups have been working toward expanding their application by optimizing and creating new iterations as well as by designing creative strategies for mutation targeting. BEs have been continuously evolved to allow for varying PAM requirements based on the location of a specific disease mutation that was unreachable with the original enzyme. Theseare PAM variants of BE include in vivo murine delivery of an ABE-VRQR-requiring PAM to target the LMNA gene in the case of Hutchinson–Gilford progeria syndrome [
      • Koblan LW
      • Erdos MR
      • Wilson C
      • Cabral WA
      • Levy JM
      • Xiong ZM
      • et al.
      In vivo base editing rescues Hutchinson–Gilford progeria syndrome in mice.
      ] and ex vivo murine editing with an ABE-NRCH-requiring PAM in the aforementioned Makassar targeting strategy for SCD [
      • Newby GA
      • Yen JS
      • Mayuranathan T
      • Lazzarotto CR
      • Li Y
      • Sheppard-Tillman H
      • et al.
      Base editing of haematopoietic stem cells rescues sickle cell disease in mice.
      ]. In addition to evolving and creating new iterations of BEs, targeting strategies are being employed that do not correct disease mutations back to wild-type but instead target a base within the mutated codon to restore protein translation by removing a stop codon. As an example of this, the authors recently targeted the most common Fanconi anemia mutation worldwide—the Spanish founder mutation (FANCA c.295C>T). Here the authors used ABEs to convert the mutated stop codon to a different amino acid that was conserved in mammals, which restored the reading frame and led to phenotypic recovery in patient primary cells [
      • Sipe CJ
      • Kluesner MG
      • Bingea SP
      • Lahr WS
      • Andrew AA
      • Wang M
      • et al.
      Correction of Fanconi Anemia Mutations Using Digital Genome Engineering.
      ]. New enzyme creation for expanded mutational targeting and creative codon targeting strategies continue to further the possibilities of disease correction with BEs.

      Discussion

      Since the first BE was described in 2016, there has been an explosion of research aiming to bring this technology into the clinic for cancer immunotherapy and to address genetic disorders. Much of the attraction of BE technology in the clinical setting is based on circumventing the need to create a toxic DSB, removing the need for a DNA donor molecule and other concerns, such as high off-target editing and insertional mutagenesis. As improvements are concurrently being made in the space of in vivo delivery of nucleic acids and protein, in vivo BE delivery will dominate the field with regard to the treatment of genetic disorders and cancers once a robust delivery system is defined. Parallel to this, newer generations of Cas9 with loosened PAM requirements and reduced off-target effects will continue being created and will ultimately expand the list of targetable diseases that may undergo direct correction.

      Funding

      CJS was funded by the University of Minnesota's Stem Cell Institute INFUSE pre-doctoral award. JGS is supported by the T32HL007062-46 Hematology Research Training Program. BRW acknowledges funding from the National Institutes of Health (grant nos. R21CA237789, R21AI163731 and P01CA254849), Alex's Lemonade Stand Foundation, Children's Cancer Research Fund and Rein in Sarcoma. BSM acknowledges funding from the National Institutes of Health (grant nos. R01AI161017, R01AI161017, P01CA254849 and P50CA136393), Children's Cancer Research Fund and Fanconi Anemia Research Fund.

      Declaration of Competing Interest

      BRW and BSM have financial interests in Beam Therapeutics. WSL, BRW and BSM are inventors of the full patent “Lymphohematopoietic engineering using cas9 base editors” (WO2019178225A2). All authors’ interests were reviewed and are managed by the University of Minnesota in accordance with their conflict of interest policies.

      Author Contributions

      Conception and design of the study: WSL, CJS, BRW, BSM. Acquisition of data: WSL, CJS. Analysis and interpretation of data: WSL, CJS. Drafting or revising the manuscript: WSL, CJS, JGS, BRW, BSM. All authors have approved the final article.

      Acknowledgments

      During the revision of this manuscript, CJS unexpectedly passed away. The authors want to take this small space in his final article to acknowledge our colleague and friend. He lived life to the fullest and was a dedicated scientist and an astounding son, brother, friend, colleague and mentor to those who had the fortune of knowing him. Cheers mate.

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