Editors' ChoiceGene Therapy

Let’s switch on AAV!

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Science Translational Medicine  05 Feb 2020:
Vol. 12, Issue 529, eaba9016
DOI: 10.1126/scitranslmed.aba9016


RNA switches expand the reach of AAV vectors.

One limitation to the use of adeno-associated virus (AAV) vectors for transient or intermittent gene transfer is the lack of genetic switches adapted to clinical translation. In particular, the introduction of off switches in AAV vectors allows inhibition of transgene expression in case something goes wrong with gene transfer. Current approaches are based on protein switches, potentially recognized as nonself by the immune system and with a size that further reduces the already limited space available in AAV vectors.

Zhong and colleagues proposed an alternative approach. They used ribozymes, small ribonucleic acids that act as very specialized enzymes, to control expression in an AAV gene transfer setting; specifically, they engineered a type III hammerhead ribozyme to improve its enzymatic activity. Ribozymes of this class, when included in a ribonucleic acid chain, specifically cleave that chain. When this cleavage occurs in the 3′ untranslated region of an mRNA,, it leads to mRNA degradation and reduced protein synthesis.

Zhong and colleagues took advantage of this interesting property to develop an off switch adapted to AAV gene transfer. They engineered the original hammerhead ribozyme into a version 60- to 80-fold more active in reducing the expression of the transgene compared with other ribozymes (switch-on). Then they developed an approach to interfere with this switch and induce transgene expression (switch-off). They used phosphorodiamidate morpholino oligomers, a class of oligonucleotides already approved for use in neuromuscular diseases. After testing different morpholinos, they identified a variant that was able to increase gene expression in cells by 300-fold in culture. Importantly, when they tested the ribozyme in combination with this morpholino, they increased transgene expression by a factor of 10 to 100 in mouse gastrocnemius muscle.

This study reports important progress, as it provides new molecular tools to control transgene expression in a gene transfer setting. One possible limitation of the approach is the poor delivery of morpholinos to muscle when injected intravenously. However, the use of different morpholino chemistries could represent a potential solution to this limitation. Although further testing is needed, this approach greatly enhances the dynamic range of hammerhead ribozymes that—combined with morpholino treatment—represent a unique tool to switch on transgene expression.

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