October 23, 2019  |  

TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity.

Designer nucleases have been successfully employed to modify the genomes of various model organisms and human cell types. While the specificity of zinc-finger nucleases (ZFNs) and RNA-guided endonucleases has been assessed to some extent, little data are available for transcription activator-like effector-based nucleases (TALENs). Here, we have engineered TALEN pairs targeting three human loci (CCR5, AAVS1 and IL2RG) and performed a detailed analysis of their activity, toxicity and specificity. The TALENs showed comparable activity to benchmark ZFNs, with allelic gene disruption frequencies of 15-30% in human cells. Notably, TALEN expression was overall marked by a low cytotoxicity and the absence of cell cycle aberrations. Bioinformatics-based analysis of designer nuclease specificity confirmed partly substantial off-target activity of ZFNs targeting CCR5 and AAVS1 at six known and five novel sites, respectively. In contrast, only marginal off-target cleavage activity was detected at four out of 49 predicted off-target sites for CCR5- and AAVS1-specific TALENs. The rational design of a CCR5-specific TALEN pair decreased off-target activity at the closely related CCR2 locus considerably, consistent with fewer genomic rearrangements between the two loci. In conclusion, our results link nuclease-associated toxicity to off-target cleavage activity and corroborate TALENs as a highly specific platform for future clinical translation. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.


October 23, 2019  |  

Vector design Tour de Force: integrating combinatorial and rational approaches to derive novel adeno-associated virus variants.

Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8?×?10(5) complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.


October 23, 2019  |  

Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery.

The simplicity of site-specific genome targeting by type II clustered, regularly interspaced, short palindromic repeat (CRISPR)-Cas9 nucleases, along with their robust activity profile, has changed the landscape of genome editing. These favorable properties have made the CRISPR-Cas9 system the technology of choice for sequence-specific modifications in vertebrate systems. For many applications, whether the focus is on basic science investigations or therapeutic efficacy, activity and precision are important considerations when one is choosing a nuclease platform, target site and delivery method. Here we review recent methods for increasing the activity and accuracy of Cas9 and assessing the extent of off-target cleavage events.


October 23, 2019  |  

Bioengineered viral platform for intramuscular passive vaccine delivery to human skeletal muscle.

Skeletal muscle is ideal for passive vaccine administration as it is easily accessible by intramuscular injection. Recombinant adeno-associated virus (rAAV) vectors are in consideration for passive vaccination clinical trials for HIV and influenza. However, greater human skeletal muscle transduction is needed for therapeutic efficacy than is possible with existing serotypes. To bioengineer capsids with therapeutic levels of transduction, we utilized a directed evolution approach to screen libraries of shuffled AAV capsids in pools of surgically resected human skeletal muscle cells from five patients. Six rounds of evolution were performed in various muscle cell types, and evolved variants were validated against existing muscle-tropic serotypes rAAV1, 6, and 8. We found that evolved variants NP22 and NP66 had significantly increased primary human and rhesus skeletal muscle fiber transduction from surgical explants ex vivo and in various primary and immortalized myogenic lines in vitro. Importantly, we demonstrated reduced seroreactivity compared to existing serotypes against normal human serum from 50 adult donors. These capsids represent powerful tools for human skeletal muscle expression and secretion of antibodies from passive vaccines.


October 23, 2019  |  

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3′-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.


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.


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