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ABSTRACT: Prime editing_in vivo

PROVIDER: PRJNA667559 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12778000_1.fastq.gz	Fastqsanger.gz
	SRR12778000_2.fastq.gz	Fastqsanger.gz

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Project description:To investigate if the truncated PE can be dilivered by dual AAV8 vectors for in vivo prime editing. We injected the dual AAV8 into 10-week-old C57BL/6J mice . Livers were isolated 4 weeks after injection and next generation sequencing showed an average of 1.4% and 5.4% precise prime editing with the low and high AAV doses, respectively (Figure 4D ). This demonstrates that PECO-Mini can be efficiently delivered by dual AAVs for in vivo prime editing.

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Project description:Prime editing is a highly versatile CRISPR-based genome editing technology with the potential to correct the vast majority of genetic defects1. However, correction of a disease phenotype in vivo in somatic tissues has not been achieved yet. Here, we establish proof-of-concept for in vivo prime editing, that resulted in rescue of a metabolic liver disease. We first develop a size-reduced prime editor (PE) lacking the RNaseH domain of the reverse transcriptase (SpCas9-PERnH), and a linker- and NLS-optimized intein-split PE construct (SpCas9-PE p.1153) for delivery by adeno-associated viruses (AAV). Systemic dual AAV-mediated delivery of this variant in neonatal mice enables installation of a transversion mutation at the Dnmt1 locus with 15% efficiency on average. Next, we targeted the disease-causing mutation in the phenylalanine hydroxylase (Pah)enu2 mouse model for phenylketonuria (PKU). Correction rates of 1.5% using the dual AAV approach could be increased to up to 14% by delivery of full-length SpCas9-PE via adenoviral vector 5 (AdV5), leading to full restoration of physiological blood phenylalanine (L-Phe) levels below 120 µmol/L. Our study demonstrates in vivo prime editing in the liver at two independent loci, emphasizing the potential of PEs for future therapeutic applications.

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Project description:We addressed the question of primed CD8 T cell responsiveness to boost in a Balb/c mouse model of vaccination against gag of HIV-1, namely intramuscular (i.m.) prime with the Chimpanzee adenovector ChAd3-gag and i.m. boost with Modified Virus Ankara MVA-gag. In this setting, boost was more effective at day(d)100 than at d30 post-prime, as evaluated by multi-lymphoid organ assessment of gag-specific CD8 T cell frequency, CD62L-phenotype and in vivo killing activity at d45 post-boost. RNA-sequencing was used to compare memory signature of gag-specific spleen CD8 T cells at d100 post-prime with those at d30.

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2011-07-01 | E-MTAB-308 | biostudies-arrayexpress

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