Persistent repression of tau in the brain using engineered zinc finger protein transcription factors.

Neuronal tau reduction confers resilience against ß-amyloid and tau-related neurotoxicity in vitro and in vivo. Here, we introduce a novel translational approach to lower expression of the tau gene MAPT at the transcriptional level using gene-silencing zinc finger protein transcription factors (ZFP-TFs). Following a single administration of adeno-associated virus (AAV), either locally into the hippocampus or intravenously to enable whole-brain transduction, we selectively reduced tau messenger RNA and protein by 50 to 80% out to 11 months, the longest time point studied. Sustained tau lowering was achieved without detectable off-target effects, overt histopathological changes, or molecular alterations. Tau reduction with AAV ZFP-TFs was able to rescue neuronal damage around amyloid plaques in a mouse model of Alzheimer's disease (APP/PS1 line). The highly specific, durable, and controlled knockdown of endogenous tau makes AAV-delivered ZFP-TFs a promising approach for the treatment of tau-related human brain diseases.

Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

Trait stacking via targeted genome editing.

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

Targeted genome editing across species using ZFNs and TALENs.

Evolutionary studies necessary to dissect diverse biological processes have been limited by the lack of reverse genetic approaches in most organisms with sequenced genomes. We established a broadly applicable strategy using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) for targeted disruption of endogenous genes and cis-acting regulatory elements in diverged nematode species.

Heritable targeted gene disruption in zebrafish using designed zinc-finger nucleases.

We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline disruption of genes in zebrafish (Danio rerio), in which targeted mutagenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of gene-disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.