Treatment of a metabolic liver disease by in vivo genome base editing in adult mice

Villiger, Lukas; Grisch-Chan, Hiu Man; Lindsay, Helen; Ringnalda, Femke; Pogliano, Chiara Balbo; Allegri, Gabriella; Fingerhut, Ralph; Häberle, Johannes; Matos, Joao; Robinson, Mark D.; Thöny, Beat; Schwank, Gerald

doi:10.1038/s41591-018-0209-1

Cited by 350 publications

(262 citation statements)

References 46 publications

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“…The large size of the ABE-NG and other base editors poses a major challenge for viral packaging and in vivo delivery. A dual trans-splicing adeno-associated virus (AAV) approach was previously used to deliver ABE 29 and a dual protein trans-splicing (PTS) approach using the split-intein moiety from Nostoc punctiforme (Npu) 46 was used to deliver CBE 47 . We attempted to adopt the PTS approach to deliver ABE.…”

Section: Resultsmentioning

confidence: 99%

Efficient precisein vivobase editing in adult dystrophic mice

Zhang

et al. 2020

Preprint

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†These authors contributed equally. Total word count: 9443ABSTRACT Background: Recent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice. Methods:We employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx 4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adenoassociated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx 4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively. Results:We showed efficient in vitro base correction of the dystrophin mutation carried in mdx 4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx 4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected. Conclusions:This study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.

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Section: Resultsmentioning

confidence: 99%

Efficient precisein vivobase editing in adult dystrophic mice

Zhang

et al. 2020

Preprint

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“…In transgenic animal models of CN1, repeated intravenous administration of UGT1A1 mRNA rescues a lethal phenotype and normalizes plasma B T , whereas a single injection of liver‐specific adeno‐associated virus (AAV) type 9 containing human UGT1A1 controls B T for life . However, normal liver growth (e.g., from about 120 g to 1,400 g in humans) dilutes out nonreplicating transgenes delivered by AAV; thus, genome editing might prove a more durable solution for very young patients.…”

Section: Discussionmentioning

confidence: 99%

Crigler‐Najjar Syndrome Type 1: Pathophysiology, Natural History, and Therapeutic Frontier

et al. 2020

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Background and Aims We describe the pathophysiology, treatment, and outcome of Crigler‐Najjar type 1 syndrome (CN1) in 28 UGT1A1 c.222C>A homozygotes followed for 520 aggregate patient‐years. Approach and Results Unbound (“free”) bilirubin (Bf) was measured in patient sera to characterize the binding of unconjugated bilirubin (BT) to albumin (A) and validate their molar concentration ratio (BT/A) as an index of neurological risk. Two custom phototherapy systems were constructed from affordable materials to provide high irradiance in the outpatient setting; light dose was titrated to keep BT/A at least 30% below intravascular BT binding capacity (i.e., BT/A = 1.0). Categorical clinical outcomes were ascertained by chart review, and a measure (Lf) was used to quantify liver fibrosis. Unbound bilirubin had a nonlinear relationship to BT (R2 = 0.71) and BT/A (R2 = 0.76), and Bf as a percentage of BT correlated inversely to the bilirubin–albumin equilibrium association binding constant (R2 = 0.69), which varied 10‐fold among individuals. In newborns with CN1, unconjugated bilirubin increased 4.3 ± 1.1 mg/dL per day. Four (14%) neonates developed kernicterus between days 14 and 45 postnatal days of life; peak BT ≥ 30 mg/dL and BT/A ≥ 1.0 mol:mol were equally predictive of perinatal brain injury (sensitivity 100%, specificity 93.3%, positive predictive value 88.0%), and starting phototherapy after age 13 days increased this risk 3.5‐fold. Consistent phototherapy with 33‐103 µW/cm2•nm for 9.2 ± 1.1 hours/day kept BT and BT/A within safe limits throughout childhood, but BT increased 0.46 mg/dL per year to reach dangerous concentrations by 18 years of age. Liver transplantation (n = 17) normalized BT and eliminated phototherapy dependence. Liver explants showed fibrosis ranging from mild to severe. Conclusion Seven decades after its discovery, CN1 remains a morbid and potentially fatal disorder.

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“…34 It may further expand the target compatibility to utilize different cytidine deaminases, such as CDA1, 35 APOBEC3A, 36,37 evolved APOBEC1 (eA1), and evolved CDA1 (eCDA1). 38,39 Additionally, finding or designing smaller variants of Cas9 and deaminase is also an effective approach in the future. However, NG-BEs cannot be packaged in a single adeno-associated virus (AAV) vector due to AAV packaging limit of ~4.7 kbp.…”

Section: Discussionmentioning

confidence: 99%

Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

et al. 2019

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Base editors, composed of a cytidine deaminase or an evolved adenine deaminase fused to Cas9 nickase, enable efficient C-to-T or A-to-G conversion in various organisms. However, the NGG protospacer adjacent motif (PAM) requirement of Streptococcus pyogenes Cas9 (SpCas9) substantially limits the target sites suitable for base editing. Quite recently, a new engineered SpCas9-NG variant, which can recognize minimal NG PAMs more efficiently than the present xCas9 variant. Here, we investigated the efficiency and PAM compatibility of SpCas9-NG-assisted cytidine base editors (CBEs) and adenine base editors (ABEs) in rabbits. In this study, we showed that NG-BE4max and NG-ABEmax systems can achieve a targeted mutation efficiency of 75%-100% and 80%-100% with excellent PAM compatibility of NGN PAMs in rabbit embryos, respectively. In addition, both base editors were successfully applied to create new rabbit models with precise point mutations, demonstrating their high efficiency and expanded genome-targeting scope in rabbits.Meanwhile, NG-ABEmax can be used to precisely mimic human Hoxc13 p.Q271R missense mutation in Founder (F0) rabbits, which is arduous for conventional ABEs to achieve due to a NGA PAM requirement. Collectively, NG-BE4max and NG-ABEmax systems provide promising tools to perform efficient base editing with expanded targeting scope in rabbits and enhances its capacity to model human diseases. K E Y W O R D Sbase editor, CRISPR/Cas9, NG PAMs, rabbit | 589 LIU et aL.

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Treatment of a metabolic liver disease by in vivo genome base editing in adult mice

Cited by 350 publications

References 46 publications

Efficient precisein vivobase editing in adult dystrophic mice

Efficient precisein vivobase editing in adult dystrophic mice

Crigler‐Najjar Syndrome Type 1: Pathophysiology, Natural History, and Therapeutic Frontier

Highly efficient base editing with expanded targeting scope using SpCas9‐NG in rabbits

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