In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA

Villiger, Lukas; Rothgangl, Tanja; Witzigmann, Dominik; Oka, Rurika; Lin, Paulo J.C.; Qi, Weihong; Janjuha, Sharan; Berk, Christian; Ringnalda, Femke; Beattie, Mitchell; Stoffel, Markus; Thöny, Beat; Hall, Jonathan; Rehrauer, Hubert; Boxtel, Ruben van; Tam, Ying K.; Schwank, Gerald

doi:10.1038/s41551-020-00671-z

Cited by 74 publications

(57 citation statements)

References 56 publications

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“…Whereas the limited cargo capacity of AAVs demands the use of a split protein delivered in two separate vectors, LNP technology allows editor mRNA and guide RNA to be formulated into a single particle. Both platform technologies have recently been used for in vivo genome base editing in mice to correct the metabolic liver disease phenylketonuria 94,127 and ex vivo modification of human hematopoietic stem cells 128 .…”

Section: Aav Vectorsmentioning

confidence: 99%

The current landscape of nucleic acid therapeutics

et al. 2021

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Section: Aav Vectorsmentioning

confidence: 99%

The current landscape of nucleic acid therapeutics

et al. 2021

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“…2c ). Previous studies suggested that unguided deamination by base editors is dose dependent 16 – 18 , providing a possible explanation for the low rates of off-target deamination in mRNA in vivo. Indeed, when we compared ABE expression levels in vitro and in vivo, we found that expression in transfected HEK293T cells was in the order of 3–4 magnitudes higher than in hepatocytes isolated from LNP- or AAV-treated mice (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For clinical application of base editing, the potential generation of off-target mutations represents a major concern. Off-target mutations could be single guide RNA (sgRNA) dependent 11 , 12 or sgRNA independent 13 – 15 and are influenced by the levels and duration of base editor expression 16 – 18 . Thus, the risks of generating off-target mutations in therapies are likely to depend on the delivery method and dose.…”

Section: Mainmentioning

confidence: 99%

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

et al. 2021

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Most known pathogenic point mutations in humans are C•G to T•A substitutions, which can be directly repaired by adenine base editors (ABEs). In this study, we investigated the efficacy and safety of ABEs in the livers of mice and cynomolgus macaques for the reduction of blood low-density lipoprotein (LDL) levels. Lipid nanoparticle–based delivery of mRNA encoding an ABE and a single-guide RNA targeting PCSK9, a negative regulator of LDL, induced up to 67% editing (on average, 61%) in mice and up to 34% editing (on average, 26%) in macaques. Plasma PCSK9 and LDL levels were stably reduced by 95% and 58% in mice and by 32% and 14% in macaques, respectively. ABE mRNA was cleared rapidly, and no off-target mutations in genomic DNA were found. Re-dosing in macaques did not increase editing, possibly owing to the detected humoral immune response to ABE upon treatment. These findings support further investigation of ABEs to treat patients with monogenic liver diseases.

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“…The most common and effective Cas systems to date (in editing efficiencies) are Streptococcus pyogenes Cas9 (SpCas), Staphylococcus aureus (SaCas), and Staphylococcus aureus -KKH (SaKKH). These three Cas species offer PAM versatility and have verified base editing potential ( Ran et al, 2015 ; Editas Medicine, 2021 ; Suh et al, 2021 ; Villiger et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

The Scope of Pathogenic ABCA4 Mutations Targetable by CRISPR DNA Base Editing Systems—A Systematic Review

2022

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Stargardt macular dystrophy (STGD1) is the most common form of inherited childhood blindness worldwide and for which no current treatments exist. It is an autosomal recessive disease caused by mutations in ABCA4. To date, a variety of gene supplementation approaches have been tested to create a therapy, with some reaching clinical trials. New technologies, such as CRISPR-Cas based editing systems, provide an exciting frontier for addressing genetic disease by allowing targeted DNA or RNA base editing of pathogenic mutations. ABCA4 has ∼1,200 known pathogenic mutations, of which ∼63% are transition mutations amenable to this editing technology. In this report, we screened the known “pathogenic” and “likely pathogenic” mutations in ABCA4 from available data in gnomAD, Leiden Open Variation Database (LOVD), and ClinVar for potential PAM sites of relevant base editors, including Streptococcus pyogenes Cas (SpCas), Staphylococcus aureus Cas (SaCas), and the KKH variant of SaCas (Sa-KKH). Overall, of the mutations screened, 53% (ClinVar), 71% (LOVD), and 71% (gnomAD), were editable, pathogenic transition mutations, of which 35–47% had “ideal” PAM sites. Of these mutations, 16–20% occur within a range of multiple PAM sites, enabling a variety of editing strategies. Further, in relevant patient data looking at three cohorts from Germany, Denmark, and China, we find that 44–76% of patients, depending on the presence of complex alleles, have at least one transition mutation with a nearby SaCas, SpCas, or Sa-KKH PAM site, which would allow for potential DNA base editing as a treatment strategy. Given the complexity of the genetic landscape of Stargardt, these findings provide a clearer understanding of the potential for DNA base editing approaches to be applied as ABCA4 gene therapy strategies.

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In vivo cytidine base editing of hepatocytes without detectable off-target mutations in RNA and DNA

Cited by 74 publications

References 56 publications

The current landscape of nucleic acid therapeutics

The current landscape of nucleic acid therapeutics

In vivo adenine base editing of PCSK9 in macaques reduces LDL cholesterol levels

The Scope of Pathogenic ABCA4 Mutations Targetable by CRISPR DNA Base Editing Systems—A Systematic Review

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