Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need

Brooks, Dominique L.; Whittaker, Madelynn N.; Qu, Ping; Musunuru, Kiran; Ahrens-Nicklas, Rebecca C.; Wang, Xiao

doi:10.1016/j.ajhg.2023.10.005

Cited by 10 publications

(12 citation statements)

References 21 publications

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“…The most frequently occurring pathogenic PAH variant worldwide is the c.1222C>T (p.Arg408Trp) variant (RefSeq: NM_000277.3), particularly prevalent in European countries and the United States. 3 We have found in a parallel study 4 being simultaneously published in The American Journal of Human Genetics that most individuals with PAH c.1222C>T variants experience chronic, severe Phe elevations, reflecting in part the limitations of the existing treatment options. 5 , 6 Genome editing offers the potential of a one-time curative therapy to permanently normalize blood Phe levels.…”

Section: Main Textmentioning

confidence: 99%

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

Brooks,

Whittaker,

Said

et al. 2024

Human Genetics and Genomics Advances

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Section: Main Textmentioning

confidence: 99%

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

Brooks,

Whittaker,

Said

et al. 2024

Human Genetics and Genomics Advances

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“…PAH variant c.1066-11G>A, a splicing defect resulting in an in-frame insertion of 3 amino acids, is the second most common pathogenic variant causing PKU worldwide, prevalent in Southern Europe and Latin America. Innovative RNA-based therapeutics such as splice switching antisense oligonucleotides (24) or HULC lncRNA mimics (8), gene editing approaches (9,10,25,26) or use of specific pharmacological chaperones (27,28), are theoretically applicable to target either the mutant pre-mRNA or the resulting protein, with the aim of correcting the pathological phenotype of this frequent variant. Due to the limitation in access to patients' tissues where the PAH gene is expressed (liver and to a lesser extent, kidney) the splicing defect was studied using minigenes and in a gene edited HepG2 cell line homozygous for the c.1066-11G>A variant, in which almost no detectable PAH protein and activity were detected.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, gene editing approaches, which have shown to be effective in correcting PKU variants in different mouse models (9,10,25,26), could provide a novel therapeutic approach for PKU patients with this specific variant. In this sense, the created avatar PKU mouse model with the humanized mutant (c.1066-11A) intron 10 fragment of the endogenous Pah gene is a suitable preclinical model for assessing the efficacy of adenine base editing as a therapeutic intervention, which could be applicable to a high number of PKU patients worldwide.…”

Section: Discussionmentioning

confidence: 99%

“…However, the murine mutations present in these models do not correspond to human variants, and, with the advent of gene editing technologies, mouse "avatar" models carrying variants identified in human patients (p.R261Q, p.R408Wy, p.P281L) have now been generated (7)(8)(9)(10). To date, no mouse models with PAH splicing variants have been characterised, despite their frequency in the PKU mutational spectrum.…”

Section: Introductionmentioning

confidence: 99%

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PAH DEFICIENT PATHOLOGY IN HUMANIZED c.1066-11G>A PHENYLKETONURIA MICE

Martínez-Pizarro,

Picó,

López-Márquez

et al. 2023

Preprint

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We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalentPAHvariant c.1066-11G>A. This variant creates an alternative 3’ splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. HomozygousPahc.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

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“…Previous studies established proof-of-concept for in vivo prime editing in the liver [15][16][17][18][19][20] .…”

Section: Introductionmentioning

confidence: 99%

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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Efficient in vivo prime editing corrects the most frequent phenylketonuria variant, associated with high unmet medical need

Cited by 10 publications

References 21 publications

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

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

PAH DEFICIENT PATHOLOGY IN HUMANIZED c.1066-11G>A PHENYLKETONURIA MICE

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

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