A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant

Brooks, Dominique L.; Whittaker, Madelynn N.; Said, Hooda; Dwivedi, Garima; Qu, Ping; Musunuru, Kiran; Ahrens-Nicklas, Rebecca C.; Alameh, Mohamad-Gabriel; Wang, Xiao

doi:10.1016/j.xhgg.2023.100253

Cited by 7 publications

(11 citation statements)

References 22 publications

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“…3c ). Notably, editing rates were similar when the scAAV2/9 dose was decreased from 2.5 x 10 13 vg/kg to 1 x 10 12 vg/kg (20% vs. 26%; Fig. 3d ), and when animals were analyzed 4 months after treatment instead of 1 week after treatment ( Fig.…”

Section: Resultsmentioning

confidence: 77%

“…However, due to the low homology-directed repair (HDR) frequency in the liver only 1% of hepatocytes were corrected, which proved insufficient to resolve hyperphenylalaninemia. Circumventing the need for HDR, we and others have previously employed base editing to repair pathogenic PKU mutations at rates that led to therapeutic reduction of Phe levels [9][10][11][12][13] . Nonetheless, even though base editing holds promise for clinical use in PKU patients, the technology is largely limited to the correction of transition point mutations, excluding patients with different types of mutations.…”

Section: Introductionmentioning

confidence: 99%

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Treatment of a genetic liver disease in mice through transient prime editor expression

Rothgangl,

Ioannidi,

Weber

et al. 2024

Preprint

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Prime editing is a versatile genome editing technology that does not rely on DNA double-strand break formation and homology-directed repair (HDR). This makes it a promising tool for correcting pathogenic mutations in tissues consisting predominantly of postmitotic cells, such as the liver. While recent studies have already demonstrated proof-of-concept for in vivo prime editing, the use of viral delivery vectors resulted in prolonged prime editor (PE) expression, posing challenges for clinical application. Here, we developed an in vivo prime editing approach where we delivered the pegRNA using self-complementary adeno-associated viral (scAAV) vectors and the prime editor using nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs). This methodology led to transient expression of the PE for 48h and 26% editing at the Dnmt1 locus using AAV doses of 2.5x1013 vector genomes (vg)/kg and a single dose of 3mg/kg mRNA-LNP. When targeting the pathogenic mutation in the Pahenu2 mouse model of phenylketonuria (PKU), we achieved 4.3% gene correction using an AAV dose of 2.5x1013 vg/kg and three doses of 2 mg/kg mRNA-LNP. Editing was specific to the liver and the intended locus, and was sufficient to reduce blood L-phenylalanine (Phe) levels from over 1500 umol/l to below the therapeutic threshold of 600 umol/l. Our study demonstrates the feasibility of in vivo gene correction in the liver with transient PE expression, bringing prime editing closer to clinical application.

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Section: Resultsmentioning

confidence: 77%

Section: Introductionmentioning

confidence: 99%

Treatment of a genetic liver disease in mice through transient prime editor expression

Rothgangl,

Ioannidi,

Weber

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…From a regulatory perspective, such a strategy could greatly streamline the development of editing therapies, which will be essential if the field is to move towards just-intime, bespoke treatments for individual patients with inborn errors of metabolism and other serious conditions with unmet medical need, particularly conditions in which there is limited time from diagnosis to progression to irreversible pathology causing significant morbidity and mortality. SpRY-ABE8.8 mRNA were produced via in vitro transcription (IVT) and purification as previously described 4,5 . In brief, a plasmid DNA template containing a codon-optimized ABE8.8 or SpRY-ABE8.8 coding sequence and a 3' polyadenylate sequence was linearized.…”

Section: Discussionmentioning

confidence: 99%

“…LNP formulation. LNPs were formulated as previously described 4,5,27 , with the lipid components (SM-102, 1,2-distearoyl-sn-glycero-3-phosphocholine, cholesterol, and PEG-2000 at molar ratios of 50:10:38.5:1.5) being rapidly mixed with an aqueous buffer solution containing ABE8.8 or SpRY-ABE8.8 mRNA and either PAH1 gRNA or PAH2 gRNA in a 1:1 ratio by weight in 25 mM sodium acetate (pH 4.0), with an N:P ratio of 5.6. The resulting LNP formulations were subsequently dialyzed against sucrose-containing buffer, concentrated using Amicon Ultra-15 mL Centrifugal Filter Units (Millipore Sigma), sterile-filtered using 0.2-µm filters, and frozen until use.…”

Section: Methodsmentioning

confidence: 99%

Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs

Whittaker,

Brooks,

Quigley

et al. 2024

Preprint

Self Cite

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Phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase (PAH) gene, fumarylacetoacetate hydrolase (FAH) gene, and alpha-L-iduronidase (IDUA) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in thePAHandIDUAgenes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase (HPD) gene, a modifier of HT1, via adenine base editing. Here we evaluated the off-target editing profiles of therapeutic lead guide RNAs (gRNAs) that, when combined with adenine base editors correct the recurrentPAHP281L variant,PAHR408W variant, orIDUAW402X variant or disrupt theHPDgene in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveal dramatically reduced off-target editing and reduced bystander editing. Lastly, in a humanizedPAHP281L mouse model, we showed that when formulated in lipid nanoparticles (LNPs) with adenine base editor mRNA, selected hybrid gRNAs revert the PKU phenotype, substantially enhance on-target editing, and reduce bystander editingin vivo. These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies.

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“…Brooks and colleagues entitled “A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant”. 1 …”

Section: Main Textmentioning

confidence: 99%

Letter to the editor

Harding,

Martinez

2024

Human Genetics and Genomics Advances

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A base editing strategy using mRNA-LNPs for in vivo correction of the most frequent phenylketonuria variant

Cited by 7 publications

References 22 publications

Treatment of a genetic liver disease in mice through transient prime editor expression

Treatment of a genetic liver disease in mice through transient prime editor expression

Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs

Letter to the editor

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