GENE THERAPY Subretinal Space with a HITI–AAV Construct Designed to Insert a Missing Exon from the Mertk Gene (The Causative Mutation in This Model)

RESEARCH HIGHLIGHTS

Nature Reviews Genetics | Published online 5 Dec 2016; doi:10.1038/nrg.2016.158

were recapitulated in the adult brain and other tissues in further experi- ments, demonstrating the versatility of HITI in vivo. The team then explored the potential of HITI for therapeutic gene replacement in an established rat model of retinitis pigmentosa. Adeno-associated virus (AAV) vectors, which have shown efficacy in humans, were used for HITI

Patrick26/iStockphoto/Thinkstock Images/Getty Patrick26/iStockphoto/Thinkstock delivery. Three-week-old rats (in which retinal degradation has already started to occur) were injected in the GENE THERAPY subretinal space with a HITI–AAV construct designed to insert a missing exon from the Mertk gene (the causative mutation in this model). In vivo gene editing in Analyses carried out 4 weeks after injection revealed increased levels of Mertk transcript and protein, and non-dividing cells preservation of the thickness of the photoreceptor outer nuclear layer compared with control-treated ani- Targeted gene integration in cassette, with the aim of subsequent mals. Moreover, electroretinography non-dividing cells presents a great NHEJ-mediated ligation of the revealed a partial rescue of visual challenge, as existing genome editing insertion cassette into the genomic function following delivery of HITI; techniques typically rely on the target site. cone response was fourfold higher homology-directed repair (HDR) The researchers first evaluated and overall photoreceptor activity pathway, which is most active during and optimized HITI in vitro by using was increased in treated compared DNA replication. In a new study the system to introduce fluorescent with untreated eyes. published in Nature, scientists have protein genes at specific sites in Finally, single-cell genotyping developed a technique that enables dividing cells (a human embryonic of mouse tissues demonstrated DNA knock-in in non-dividing cells kidney cell line) or non dividing cells 90–95% on-target specificity following in vivo and used it to partially restore (primary mouse neurons). In both systemic delivery of HITI and visual function in a rat model of cell types, targeted knock-in with biallelic integration of the transgene retinitis pigmentosa — a hereditary HITI was highly efficient compared in 30–50% of cells. Encouragingly, degenerative disease that causes with other methods, including HDR. sequence analysis of predicted blindness. Having established the in vitro off-target sites revealed minimal

cone response The new approach — called efficiency of HITI, the team then occurrence of insertions or deletions. homology independent targeted tested it in live animals. To enable the In enabling targeted transgene was fourfold integration (HITI) — combines targeting of non-dividing cells, they insertion into postmitotic cells higher and the CRISPR–Cas9 system with the designed HITI constructs in which in vivo, HITI should prove an inval- overall cell’s non-homologous end-joining Cas9 expression could be induced uable tool in neuroscience research. (NHEJ) pathway for double-strand using the drug tamoxifen. The Furthermore, it holds promise for a photoreceptor break (DSB) repair, which is active constructs were delivered to mouse range of therapeutic gene-replacement activity was in dividing and non-dividing cells. embryo brains by electroporation applications, subject to further increased Short sequences from the genomic and Cas9 expression was induced optimization. in treated target site are incorporated into a 10 days after birth (when most Denise Waldron donor construct, where they flank the nerve cells have ceased dividing). compared with ORIGINAL ARTICLE Suzuki, K. et al. In vivo insertion cassette. The Cas9 enzyme Immunofluorescence microscopy of genome editing via CRISPR/Cas9 mediated untreated eyes is then targeted, via a guide RNA, mouse brain tissue 21 days after homology-independent targeted integration. to create DSBs at these sequences in birth revealed efficient transgene Nature http://dx.doi.org/10.1038/nature20565 (2016) the genomic target site and donor insertion. Notably, these findings