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

Abstract

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 Tmc1^Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1^Bth allele into the cochlea of neonatal Tmc1^Bth/+ 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 Tmc1^Bth/+ 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.

Extended Data Figure 1

Allele-selective editing of wild-type or Bth mutant Tmc1 in cleavage assays in vitro and in lipid-mediated delivery into primary fibroblasts. (a) In vitro Cas9:sgRNA-mediated Tmc1 DNA cleavage. 100 nM of a 995-bp DNA fragment containing wild-type Tmc1 (lanes 1-5) or Bth mutant Tmc1 (lanes 6-10) was incubated with 300 nM of each of the four Cas9:sgRNAs shown for 15 min at 37 °C. Expected cleavage products are 774-778 bp and 217-221 bp. M = 100-bp ladder; the lower two heavy bands are 500 and 1,000 bp. (b) Quantification of DNA cleavage in (a) by densitometry using imageJ. (c) Transfection efficiency comparison of HEK293T cells and wild-type primary fibroblasts. 50 ng GFP plasmid, 10 nM Cas9:FitC-Tmc1-mut3 sgRNA RNP, or 10 nM Cas9:CrRNA-Tmc1-mut3:atto-550-TracrRNA RNP were delivered into HEK293T cells or wild-type primary fibroblasts using 3 μL Lipofectamine 2000. For samples with GFP plasmid, the fraction of GFP-positive cells was measured by flow cytometry 24 h after delivery. For samples with Cas9:FitC-Tmc1-mut3 RNP, or Cas9:CrRNA-Tmc1-mut3:atto-550-TracrRNA RNP, media was removed 6 h after delivery. The cells were trypsinized, washed three times with 500 μL PBS containing 20 U/mL heparin, and subjected to flow cytometry. (d) Wild-type or Bth mutant Tmc1 allele editing in primary fibroblasts derived from wild-type or Bth/Bth mice as a function of the dose of Cas9:Tmc1-mut3:lipid complex. 12.5, 25, 50, 100, 200, or 400 nM of Cas9:Tmc1-mut3 were delivered into the primary fibroblasts using Lipofectamine 2000 in DMEM-FBS. (e) Lipid-mediated delivery of Cas9:sgRNA complexes into primary fibroblasts derived from wild-type or Bth/Bth mice. 100 nM of purified Cas9 protein and each wild-type Tmc1-targeting sgRNA (Tmc1-wt1, Tmc1-wt2, or Tmc1-wt3) or Bth mutant-targeting sgRNA (Tmc1-mut1, Tmc1-mut2, or Tmc1-mut3) were delivered into wild-type fibroblasts (red) and Bth/Bth fibroblasts (blue) using Lipofectamine 2000 in DMEM-FBS. Primary fibroblast cells were harvested 96 h after treatment. Genomic DNA was extracted and indels were detected by HTS. Values and error bars reflect the mean ± standard deviation of three or more biological replicates.

Extended Data Figure 2

Delivery of Cas9:Tmc1-mut3 sgRNA complexes into primary fibroblasts derived from wild-type or homozygous Bth/Bth mice using (a) seven commercially available lipids. LPF2000 = Lipofectamine 2000; RNAiMAX = Lipofectamine® RNAiMAX; LPF3000 = Lipofectamine 3000; CRISPRMAX = Lipofectamine CRISPRMAX; LTX = Lipofectamine LTX, or (b) ten biodegradable, bioreducible lipids. Lipid 1 = 75-O14B; Lipid 2 = 76-O14B; Lipid 3 = 80-O18B; Lipid 4 = 87-O16B; Lipid 5 = 113-O18B; Lipid 6 = 306-O12B; Lipid 7 = 306-O16B; Lipid 8 = 306-O18B; Lipid 9 = 400-O12B; Lipid 10 = 400-O16B. 100 nM purified Cas9:Tmc1-mut3 RNP was delivered using 3 μL of the cationic lipid shown in DMEM-FBS. Fibroblast cells were harvested 96 h after treatment, genomic DNA was extracted, and indels were detected by HTS. (c) Synthetic route and chemical structure of lipids. (d) Commercially available amine head groups used in lipid synthesis. Lipids were synthesized as previously described. Values and error bars reflect the mean ± standard deviation of three or more biological replicates.

Extended Data Figure 3

Off-target sites identified by GUIDE-seq after nucleofection of DNA plasmids encoding Cas9 and Tmc1-mut3 sgRNA into primary fibroblasts from Bth/+ mice. (a) 1,000 ng Cas9 plasmid, 300 ng Tmc1-mut3 sgRNA plasmid, 400 ng pmaxGFP plasmid, and 50 pmol double-stranded oligodeoxynucleotides (dsODN) were nucleofected into Bth/+ fibroblasts using a LONZA 4D-Nucleofector. Genomic DNA was extracted 96 h after nucleofection and subjected to GUIDE-seq as previously described. Off-T1 to off-T10 are ten off-target sites detected by GUIDE-seq. Mismatches compared to the on-target site are shown and highlighted in color. The Bth allele targeted by sgRNA Tmc1-mut3 is shown in the top row. (b) Indel frequency at the Tmc1 locus and at each of the off-target loci in Cas9:Tmc1-mut3 treated Bth/Bth primary fibroblasts following plasmid DNA nucleofection or following RNP delivery. For RNP delivery, 100 nM Cas9:Tmc1-mut3 RNP was delivered to the Bth/Bth fibroblasts using 3 μL Lipofectamine 2000. Indels were detected by HTS at the Tmc1 on-target site and at each off-target site. Red: samples nucleofected with DNA plasmids encoding Cas9 and Tmc1-mut3 sgRNA; blue: samples treated with Cas9:Tmc1-mut3 RNPs; grey: control samples nucleofected with unrelated dsDNA only.

Extended Data Figure 4

Cas9:Tmc1-mut3:lipid injection reduce hearing loss, improve acoustic startle response, and preserve stereocilia in Bth/+ mice. (a) Phalloidin labeling showed the preservation of stereocilia of IHCs in an ear 8 weeks after injection with Cas9:Tmc1-mut3 sgRNA at three frequency locations indicated, whereas the uninjected contralateral inner ear of the same mouse showed severe degeneration of stereocilia at locations corresponding to 16 and 32 kHz. The boxes indicate the stereocilia, which are shown at the bottom of each image at higher magnification. Scale bars: 10 μm. Similar results were observed in other injected ears that were immunolabeled (n = 5). (b) Representative ABR waveforms showing reduced threshold (red traces) at 16 kHz in a Cas9:Tmc1-mut3:lipid-injected Bth/+ ear (left) compared to an uninjected contralateral ear (right) after 4 weeks. (c) 8 weeks after Cas9:Tmc1-mut3 injection into Bth/+ ears (blue), mean ABR thresholds were significantly reduced at three frequencies. Uninjected Bth/+ ears (red) showed ABR thresholds > 85 dB at all frequencies after 8 weeks. ABR thresholds from wild-type C3H are shown in green. (d) ABR Wave 1 amplitudes following 90 dB SPL at 16 kHz were greater in injected Bth/+ ears than in uninjected ears 8 weeks after treatment. The horizontal bars represent mean values. (e) Startle responses at 16 kHz in individual Cas9:Tmc1-mut3 sgRNA-injected mice (blue) were significantly stronger (p < 0.001) than in uninjected mice (red) 8 weeks after treatment. Among the different frequencies assayed, the number of ears tested (n) varies within the range shown (see Supplementary Table 2). Statistical analyses of ABR thresholds, amplitudes, and startle responses were performed by two-way ANOVA with Bonferroni correction for multiple comparisons: *p < 0.05, **p < 0.01, and ****p < 0.0001. Values and error bars reflect mean ± SEM.

Extended Data Figure 5

Effect of in vivo injection of Cas9:sgRNA:lipid complexes on DPOAE thresholds. DPOAE thresholds 4 weeks after injection were elevated compared with uninjected ears at three frequencies following treatment with Cas9:Tmc1-mut3 sgRNA (a), and were elevated at two frequencies following treatment with Cas9:Tmc1-wt3 sgRNA, (b) Cas9:GFP sgRNA (c), or dCas9:Tmc1-mut1 sgRNA (d). (e) 8 weeks after Cas9:Tmc1-mut3 sgRNA injection, DPOAE thresholds were elevated at three frequencies in the injected group. Mean DPOAE thresholds of untreated wildtype (WT) C3H mice at 4 weeks (a) or 8 weeks (e) weeks of age are also shown in purple. Statistical analysis of DPOAE thresholds was performed by two-way ANOVA with Bonferroni correction for multiple comparisons: **p < 0.01, ***p < 0.001, and ****p < 0.0001. Values and error bars reflect mean ± SEM. Among the different frequencies assayed, the number of ears tested (n) varies within the range shown (see Supplementary Table 2). The elevation of DPOAE thresholds despite enhanced hair cell survival (Fig. 2d and 2g) suggests that the surviving OHCs may not be fully functional. IHCs can respond to sound and excite auditory nerve fibers in the absence of OHC amplification, although at higher SPLs. Thus, an improvement in ABR thresholds and suprathreshold amplitudes can occur without concomitant DPOAE enhancement if the functional improvements are restricted to the surviving IHCs.

Extended Data Figure 6

Hearing rescue is dependent on the Bth target specificity of the sgRNA, Cas9 nuclease activity, the presence of the Bth mutation, and the presence of the sgRNA. (a) In Bth/+ ears injected with Cas9:Tmc1-wt3:lipid, which targets the wild-type Tmc1 allele instead of the mutant Bth allele, ABR thresholds (blue) were comparable to or higher than those of uninjected controls (red) after 4 weeks. (b) Bth/+ ears injected with Cas9:GFP sgRNA:lipid (blue) did not show improved ABR thresholds 4 weeks after treatment. (c) Bth/+ ears injected with catalytically inactive dCas9:Tmc1-mut1:lipid did not show improved ABR thresholds 4 weeks after treatment. (d) ABR thresholds of wild-type C3H mice injected with Cas9:Tmc1-mut3:lipid showed similar patterns as the uninjected control inner ears at 4 weeks, except at 5.66 and 45.24 kHz where ABR thresholds were elevated. (e) Elevated DPOAE thresholds at three frequencies were observed after the treatment in (d). (f) Injection of Cas9:Lipofectamine 2000 (LPF2000) without sgRNA in Bth/+ mice did not improve ABR thresholds after 4 weeks. (g) Elevated DPOAE thresholds at 11 and 16 kHz were observed after the treatment in (f). Statistical analysis of ABR and DPOAE thresholds was performed by two-way ANOVA with Bonferroni correction for multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Values and error bars reflect mean ± SEM. Among the different frequencies assayed, the number of ears tested (n) varies within the range shown (see Supplementary Table 2).

Extended Data Figure 7

Hearing preservation following treatment with additional Tmc1-mut sgRNAs other than Tmc1-mut3. (a) Mean ABR thresholds were significantly reduced at three frequencies in ears injected with Cas9:Tmc1-mut1:lipid compared to uninjected Bth/+ ears after 4 weeks. (b) DPOAE thresholds were elevated in the same group of inner ears after Cas9:Tmc1-mut1 injection as in (a) after 4 weeks. (c) Mean ABR thresholds were significantly reduced at five frequencies in ears injected with Cas9:Tmc1-mut2:lipid compared to uninjected Bth/+ ears after 4 weeks. (d) DPOAE thresholds were elevated in the same group of inner ears after Cas9:Tmc1-mut2 injection as in (c) after 4 weeks. (e) Mean ABR thresholds were significantly reduced at three frequencies in ears injected with Cas9:Tmc1-mut4:lipid compared to uninjected Bth/+ ears after 4 weeks. (f) DPOAE thresholds were elevated in the same group of inner ears after Cas9:Tmc1-mut4:lipid injection as in (e) after 4 weeks. (g) Significantly stronger Wave 1 amplitudes were detected in ears injected with each of the Cas9:Tmc1-mut:lipid complexes shown at 16 kHz (80 and 90 dB SPL). (h) 8 weeks after Cas9:Tmc1-mut1:lipid injection into Bth/+ ears, mean ABR thresholds were significantly reduced at five frequencies compared to the uninjected Bth/+ ears, which showed ABR thresholds > 80 dB at all frequencies after 8 weeks. Mean ABR thresholds of untreated wildtype (WT) C3H mice of 8 weeks of age are shown in purple. Red arrows indicate no ABR response at the highest SPL level of 90 dB. (i) DPOAE thresholds were modestly elevated in the same group of inner ears after Cas9:Tmc1-mut1 injection as in (h) after 8 weeks. Mean DPOAE thresholds of untreated wildtype (WT) C3H mice of 8 weeks of age are shown in purple. Statistical analysis of ABR and DPOAE thresholds and Wave 1 amplitudes was performed by two-way ANOVA with Bonferroni correction for multiple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Values and error bars reflect mean ± SEM. Among the different frequencies assayed, the number of ears tested (n) varies within the range shown (see Supplementary Table 2).

Extended Data Figure 8

RNP delivery of Cas9:sgRNA:lipid complexes results in genome editing in adult hair cells. 6-week-old adult Atoh1-GFP cochlea were injected with 1 μL 25 μM Cas9:GFP sgRNA:lipid complex by canalostomy, with the cochlea harvested two weeks after injection. (a) Genome editing was detected by the loss of GFP (green, with GFP absence noted using cyan shapes) in inner hair cells (IHCs) and outer hair cells (OHCs) (b) Hair cells were labeled with a hair cell marker MYO7A (red) in the apex turn of cochlea. (c, d) In uninjected contralateral Atoh1-GFP cochlea, all hair cells were GFP-positive. Scale bars = 10 μm. Similar results were observed in other injected ears that were immunolabeled (n = 3).

Extended Data Figure 9

In vivo editing of the Tmc1 locus from Bth/+ ears injected with Cas9:Tmc1-mut3 sgRNA. A representation of the organ of Corti harvested at P5 for high-throughput DNA sequencing: (a) A confocal z-stack image showing the surface view of a dissected and labeled organ of Corti used for HTS. (b) A cross-sectional view of the organ of Corti (along the white line in (a)) showing the positions of hair cells (MYO7A), supporting cells (SOX2) and the cells from other cochlear regions that were used for quantification. LER: lesser epithelial ridge; GER: greater epithelial ridge; SE: sensory epithelium; Lib: The limbus region. DAPI-labeled nuclei are shown in blue. Quantification showed hair cells represented 1.45% ± 0.05% (mean ± SEM, n = 4) of all the cells in the dissected cochlea. Scale bars = 10 μm. (c) On-target and off-target in vivo editing of the Tmc1 locus in organ of Corti samples. No indels were observed at frequencies substantially above that of an untreated control sample at any of the ten off-target sites identified by GUIDE-seq (Off-T1 to Off-T10). Indels were detected by HTS at the Tmc1 on-target site and each off-target site from in vivo tissue samples dissected from the inner ear of neonatal mouse 5 days after Cas9:Tmc1-mut3 RNP injection (blue), or from untreated control samples (red).

Figure 1. Design of a genome-editing strategy to disrupt the Bth mutant allele

(a) SpCas9 sgRNAs were designed to target the mutant Tmc1 Bth allele, in which T1235 is changed to A (red). The protospacer (blue arrows) of each Bth-targeting sgRNA contains a complementary T (red) that pairs with the T1235A mutation in the Bth allele, but that forms a mismatch with wild-type Tmc1 allele. (b) Lipid-mediated delivery of Cas9:sgRNA complexes into primary fibroblasts derived from wild-type or homozygous Bth/Bth mice. 100 nM of purified Cas9 protein and 100 nM of each sgRNA shown were delivered using Lipofectamine 2000. Indels were quantitated by HTS. Values and error bars represent the mean ± SD of three or more independent biological replicates.

Figure 2. Effects of Cas9:Tmc1-mut3 sgRNA:lipid injection on hair-cell function and hair-cell survival in mice

(a) Representative transduction currents from inner hair cells (IHCs) of P0-P1 wild-type or Tmc1 Bth/Δ;Tmc2Δ/Δ mice that were uninjected, or injected with Cas9:Tmc1-mut3:lipid complex. (b) Maximal transduction current amplitudes for 135 IHCs from P1 wild-type C57B/L6 and Tmc1 Bth/Δ;Tmc2Δ/Δ mice. (i): uninjected wild-type C57B/L6 mice; (ii): wild-type C57B/L6 mice injected with Cas9:Tmc1-wt3:lipid; (iii): uninjected Tmc1 Bth/Δ;Tmc2Δ/Δ mice; (iv): Tmc1 Bth/Δ;Tmc2Δ/Δ mice injected with Cas9:GFP-targeting sgRNA:lipid; (v): Tmc1 Bth/Δ;Tmc2Δ/Δ mice injected with Cas9:Tmc1-mut3:lipid. Horizontal lines and error bars reflect mean ± SD. (c–e) Representative confocal microscopy images from (c) an uninjected Bth/+ cochlea; (d) the contralateral cochlea of the mouse in (c) injected with Cas9:Tmc1-mut3:lipid complex at P1; (e) an untreated wild-type C3H cochlea. Numbers in pink indicate approximate frequencies (in kHz) sensed by each region. Scale bars = 50 μm. (f, g) Quantification of IHC (f) and OHC (g) survival in Bth/+ mice relative to wild-type C3H mice 8 weeks after Cas9:Tmc1-mut3:lipid injection (blue) compared to uninjected (red) contralateral ears. Values and error bars reflect the means and SEMs of five biological replicates. Statistical tests in (b) are two-population T-tests, and in (f) and (g) are two-way ANOVA with Bonferroni correction: **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 3. Cas9:Tmc1-mut3 sgRNA:lipid injections reduce hearing loss in Bth/+ mice

(a) ABR thresholds in Bth/+ ears injected with Cas9:Tmc1-mut3:lipid (blue), uninjected Bth/+ ears (red), and wild-type C3H ears (green) after 4 weeks. (b) Amplitudes of ABR Wave 1 at 16 kHz in Cas9:Tmc1-mut3:lipid-injected ears (blue) compared with uninjected ears (red) after 4 weeks. Horizontal bars are mean values. (c) Mean ABR waveforms in Cas9:Tmc1-mut3:lipid-injected ears (blue) and uninjected ears (red). (d) Startle responses in Cas9:Tmc1-mut3:lipid-injected mice (blue) and in uninjected mice (red) 8 weeks after treatment. Red arrows in (a) indicate no ABR response at the highest stimulus level tested (90 dB). See also Supplementary Table 2. Statistical tests were two-way ANOVA with Bonferroni correction for multiple comparisons: **p < 0.01, ***p < 0.001, and ****p < 0.0001. Values and error bars reflect mean ± SEM.

Figure 4. Genome modification at Tmc1 induced by lipid-mediated delivery of Cas9:Tmc1-mut3 RNP into Bth/+ mice

(a) Tmc1 indel frequencies from tissue samples 5 days after injection of Cas9:Tmc1-mut3:lipid (blue) or from uninjected mice (red). Horizontal lines and error bars reflect mean ± SEM. Note that Bth allele indel frequencies in these heterozygous mice are approximately double the observed indel frequencies. (b) Analysis of indel-containing Tmc1 sequencing reads from four injected organ of Corti samples in (a). (c) The most abundant 16 Tmc1 sequences, grouped by similarity, from organ of Corti samples in (b). The T1235A Bth mutation is red.