genome editing Archives - Page 4 of 4

October 23, 2019 |

Adeno-associated virus genome population sequencing achieves full vector genome resolution and reveals human-vector chimeras

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.

October 23, 2019 |

Cas9-mediated allelic exchange repairs compound heterozygous recessive mutations in mice.

We report a genome-editing strategy to correct compound heterozygous mutations, a common genotype in patients with recessive genetic disorders. Adeno-associated viral vector delivery of Cas9 and guide RNA induces allelic exchange and rescues the disease phenotype in mouse models of hereditary tyrosinemia type I and mucopolysaccharidosis type I. This approach recombines non-mutated genetic information present in two heterozygous alleles into one functional allele without using donor DNA templates.

September 22, 2019 |

Autologous cell therapy approach for Duchenne muscular dystrophy using PiggyBac transposons and mesoangioblasts.

Duchenne muscular dystrophy (DMD) is a lethal muscle-wasting disease currently without cure. We investigated the use of the PiggyBac transposon for full-length dystrophin expression in murine mesoangioblast (MABs) progenitor cells. DMD murine MABs were transfected with transposable expression vectors for full-length dystrophin and transplanted intramuscularly or intra-arterially into mdx/SCID mice. Intra-arterial delivery indicated that the MABs could migrate to regenerating muscles to mediate dystrophin expression. Intramuscular transplantation yielded dystrophin expression in 11%-44% of myofibers in murine muscles, which remained stable for the assessed period of 5 months. The satellite cells isolated from transplanted muscles comprised a fraction of MAB-derived cells, indicating that the transfected MABs may colonize the satellite stem cell niche. Transposon integration site mapping by whole-genome sequencing indicated that 70% of the integrations were intergenic, while none was observed in an exon. Muscle resistance assessment by atomic force microscopy indicated that 80% of fibers showed elasticity properties restored to those of wild-type muscles. As measured in vivo, transplanted muscles became more resistant to fatigue. This study thus provides a proof-of-principle that PiggyBac transposon vectors may mediate full-length dystrophin expression as well as functional amelioration of the dystrophic muscles within a potential autologous cell-based therapeutic approach of DMD. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

September 21, 2019 |

Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.

CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.

July 19, 2019 |

Quantifying genome-editing outcomes at endogenous loci with SMRT sequencing.

Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a novel method for quantifying individual genome editing outcomes at any site of interest using single molecule real time (SMRT) DNA sequencing. We show that this approach can be applied at various loci, using multiple engineered nuclease platforms including TALENs, RNA guided endonucleases (CRISPR/Cas9), and ZFNs, and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach facilitates the evaluation of new gene editing technologies and permits sensitive quantification of editing outcomes in almost every experimental system used.

July 19, 2019 |

Editing out five Serpina1 paralogs to create a mouse model of genetic emphysema.

Chronic obstructive pulmonary disease affects 10% of the worldwide population, and the leading genetic cause is a-1 antitrypsin (AAT) deficiency. Due to the complexity of the murine locus, which includes up to six Serpina1 paralogs, no genetic animal model of the disease has been successfully generated until now. Here we create a quintuple Serpina1a-e knockout using CRISPR/Cas9-mediated genome editing. The phenotype recapitulates the human disease phenotype, i.e., absence of hepatic and circulating AAT translates functionally to a reduced capacity to inhibit neutrophil elastase. With age, Serpina1 null mice develop emphysema spontaneously, which can be induced in younger mice by a lipopolysaccharide challenge. This mouse models not only AAT deficiency but also emphysema and is a relevant genetic model and not one based on developmental impairment of alveolarization or elastase administration. We anticipate that this unique model will be highly relevant not only to the preclinical development of therapeutics for AAT deficiency, but also to emphysema and smoking research. Copyright © 2018 the Author(s). Published by PNAS.

July 7, 2019 |

Genome editing in human pluripotent stem cells: approaches, pitfalls, and solutions.

Human pluripotent stem cells (hPSCs) with knockout or mutant alleles can be generated using custom-engineered nucleases. Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases are the most commonly employed technologies for editing hPSC genomes. In this Protocol Review, we provide a brief overview of custom-engineered nucleases in the context of gene editing in hPSCs with a focus on the application of TALENs and CRISPR/Cas9. We will highlight the advantages and disadvantages of each method and discuss theoretical and technical considerations for experimental design. Copyright © 2016 Elsevier Inc. All rights reserved.

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