Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents.

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.

Generating experimentally unrelated target molecule-binding highly functionalized nucleic-acid polymers using machine learning.

In vitro selection queries large combinatorial libraries for sequence-defined polymers with target binding and reaction catalysis activity. While the total sequence space of these libraries can extend beyond 1022 sequences, practical considerations limit starting sequences to ≤~1015 distinct molecules. Selection-induced sequence convergence and limited sequencing depth further constrain experimentally observable sequence space. To address these limitations, we integrate experimental and machine learning approaches to explore regions of sequence space unrelated to experimentally derived variants. We perform in vitro selections to discover highly side-chain-functionalized nucleic acid polymers (HFNAPs) with potent affinities for a target small molecule (daunomycin KD = 5-65 nM). We then use the selection data to train a conditional variational autoencoder (CVAE) machine learning model to generate diverse and unique HFNAP sequences with high daunomycin affinities (KD = 9-26 nM), even though they are unrelated in sequence to experimental polymers. Coupling in vitro selection with a machine learning model thus enables direct generation of active variants, demonstrating a new approach to the discovery of functional biopolymers.

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.

Altered expression of genes regulating inflammation and synaptogenesis during regrowth of afferent neurons to cochlear hair cells.

The spiral ganglion neurons constitute the primary connection between auditory hair cells and the brain. The spiral ganglion afferent fibers and their synapse with hair cells do not regenerate to any significant degree in adult mammalian ears after damage. We have investigated gene expression changes after kainate-induced disruption of the synapses in a neonatal cochlear explant model in which peripheral fibers and the afferent synapse do regenerate. We compared gene expression early after damage, during regeneration of the fibers and synapses, and after completion of in vitro regeneration. These analyses revealed a total of 2.5% differentially regulated transcripts (588 out of 24,000) based on a threshold of p<0.005. Inflammatory response genes as well as genes involved in regeneration of neural circuits were upregulated in the spiral ganglion neurons and organ of Corti, where the hair cells reside. Prominent genes upregulated at several time points included genes with roles in neurogenesis (Elavl4 and Sox21), neural outgrowth (Ntrk3 and Ppp1r1c), axonal guidance (Rgmb and Sema7a), synaptogenesis (Nlgn2 and Psd2), and synaptic vesicular function (Syt8 and Syn1). Immunohistochemical and in situ hybridization analysis of genes that had not previously been described in the cochlea confirmed their cochlear expression. The time course of expression of these genes suggests that kainate treatment resulted in a two-phase response in spiral ganglion neurons: an acute response consistent with inflammation, followed by an upregulation of neural regeneration genes. Identification of the genes activated during regeneration of these fibers suggests candidates that could be targeted to enhance regeneration in adult ears.

Publisher Correction: Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo base editing restores sensory transduction and transiently improves auditory function in a mouse model of recessive deafness.

Most genetic diseases arise from recessive point mutations that require correction, rather than disruption, of the pathogenic allele to benefit patients. Base editing has the potential to directly repair point mutations and provide therapeutic restoration of gene function. Mutations of transmembrane channel-like 1 gene (TMC1) can cause dominant or recessive deafness. We developed a base editing strategy to treat Baringo mice, which carry a recessive, loss-of-function point mutation (c.A545G; resulting in the substitution p.Y182C) in Tmc1 that causes deafness. Tmc1 encodes a protein that forms mechanosensitive ion channels in sensory hair cells of the inner ear and is required for normal auditory function. We found that sensory hair cells of Baringo mice have a complete loss of auditory sensory transduction. To repair the mutation, we tested several optimized cytosine base editors (CBEmax variants) and guide RNAs in Baringo mouse embryonic fibroblasts. We packaged the most promising CBE, derived from an activation-induced cytidine deaminase (AID), into dual adeno-associated viruses (AAVs) using a split-intein delivery system. The dual AID-CBEmax AAVs were injected into the inner ears of Baringo mice at postnatal day 1. Injected mice showed up to 51% reversion of the Tmc1 c.A545G point mutation to wild-type sequence (c.A545A) in Tmc1 transcripts. Repair of Tmc1 in vivo restored inner hair cell sensory transduction and hair cell morphology and transiently rescued low-frequency hearing 4 weeks after injection. These findings provide a foundation for a potential one-time treatment for recessive hearing loss and support further development of base editing to correct pathogenic point mutations.

Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses.

The success of base editors for the study and treatment of genetic diseases depends on the ability to deliver them in vivo to the relevant cell types. Delivery via adeno-associated viruses (AAVs) is limited by AAV packaging capacity, which precludes the use of full-length base editors. Here, we report the application of dual AAVs for the delivery of split cytosine and adenine base editors that are then reconstituted by trans-splicing inteins. Optimized dual AAVs enable in vivo base editing at therapeutically relevant efficiencies and dosages in the mouse brain (up to 59% of unsorted cortical tissue), liver (38%), retina (38%), heart (20%) and skeletal muscle (9%). We also show that base editing corrects, in mouse brain tissue, a mutation that causes Niemann-Pick disease type C (a neurodegenerative ataxia), slowing down neurodegeneration and increasing lifespan. The optimized delivery vectors should facilitate the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest.

Development of hRad51-Cas9 nickase fusions that mediate HDR without double-stranded breaks.

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.

Continuous evolution of base editors with expanded target compatibility and improved activity.

Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.

Publisher Correction: Continuous evolution of base editors with expanded target compatibility and improved activity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo base editing of post-mitotic sensory cells.

Programmable nucleases can introduce precise changes to genomic DNA through homology-directed repair (HDR). Unfortunately, HDR is largely restricted to mitotic cells, and is typically accompanied by an excess of stochastic insertions and deletions (indels). Here we present an in vivo base editing strategy that addresses these limitations. We use nuclease-free base editing to install a S33F mutation in ß-catenin that blocks ß-catenin phosphorylation, impedes ß-catenin degradation, and upregulates Wnt signaling. In vitro, base editing installs the S33F mutation with a 200-fold higher editing:indel ratio than HDR. In post-mitotic cells in mouse inner ear, injection of base editor protein:RNA:lipid installs this mutation, resulting in Wnt activation that induces mitosis of cochlear supporting cells and cellular reprogramming. In contrast, injection of HDR agents does not induce Wnt upregulation. These results establish a strategy for modifying posttranslational states in signaling pathways, and an approach to precision editing in post-mitotic tissues.

Improving the DNA specificity and applicability of base editing through protein engineering and protein delivery.

We recently developed base editing, a genome-editing approach that enables the programmable conversion of one base pair into another without double-stranded DNA cleavage, excess stochastic insertions and deletions, or dependence on homology-directed repair. The application of base editing is limited by off-target activity and reliance on intracellular DNA delivery. Here we describe two advances that address these limitations. First, we greatly reduce off-target base editing by installing mutations into our third-generation base editor (BE3) to generate a high-fidelity base editor (HF-BE3). Next, we purify and deliver BE3 and HF-BE3 as ribonucleoprotein (RNP) complexes into mammalian cells, establishing DNA-free base editing. RNP delivery of BE3 confers higher specificity even than plasmid transfection of HF-BE3, while maintaining comparable on-target editing levels. Finally, we apply these advances to deliver BE3 RNPs into both zebrafish embryos and the inner ear of live mice to achieve specific, DNA-free base editing in vivo.

Treatment of tumors with vitamin E suppresses myeloid derived suppressor cells and enhances CD8+ T cell-mediated antitumor effects.

Vitamin E has been shown to have strong anticarcinogenic properties, including antioxidant characteristics, making it an ideal candidate for use in combination with immunotherapies that modify the tumor microenvironment. The tumor microenvironment contains immunosuppressive components, which can be diminished, and immunogenic components, which can be augmented by immunotherapies in order to generate a productive immune response. In the current study, we employ the α-tocopherol succinate isomer of vitamin E to reduce immunosuppression by myeloid derived suppressor cells (MDSCs) as well as adoptive transfer of antigen-specific CD8+ T cells to generate potent antitumor effects against the HPV16 E7-expressing TC-1 tumor model. We show that vitamin E alone induces necrosis of TC-1 cells and elicits antitumor effects in TC-1 tumor-bearing mice. We further demonstrate that vitamin E reverses the suppression of T cell activation by MDSCs and that this effect is mediated in part by a nitric oxide-dependent mechanism. Additionally, treatment with vitamin E reduces the percentage of MDSCs in tumor loci, and induces a higher percentage of T cells, following T cell adoptive transfer. Finally, we demonstrate that treatment with vitamin E followed by E7-specific T cell adoptive transfer experience elicits potent antitumor effects in tumor-bearing mice. Our data provide additional evidence that vitamin E has anticancer properties and that it has promise for use as an adjuvant in combination with a variety of cancer therapies.