Animals comprising modified KLHDC7B loci

Abstract

A genetically modified non-human animal lacking the expression of Klhcd7b is described. Methods and compositions for disrupting, deleting, and / or replacing the Klhcd7b coding sequence are described. Genetically modified mice useful as models of hearing loss or extremely severe deafness are also described. Loss of function cells, tissues, and embryos genetically modified to include Klhcd7b are also described.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 482,724, filed on February 1, 2023, U.S. Provisional Application No. 63 / 484,087, filed on February 9, 2023, and U.S. Provisional Application No. 63 / 585,784, filed on September 27, 2023, the disclosures of which are hereby incorporated by reference in their entirety.

[0003] Reference to a sequence listing submitted as an XML file

[0004] The sequence listing written in the file "11348WO01 Sequence Listing XML" is 119 kilobytes, was created on January 31, 2024, and is hereby incorporated by reference in its entirety. Technical Field

[0005] Non-human animals, cells, and tissues, as well as methods for making and using the same, are described, wherein the non-human animals, cells, and tissues comprise a modified Klhdc7b locus, which can comprise a deletion and / or replacement of an endogenous Klhdc7b gene or a portion thereof. The non-human animals described can comprise a phenotype consistent with hearing loss. Background Art

[0006] Human hearing impairment is associated with social isolation and cognitive decline and is therefore a persistent problem in the fields of otology and audiology. Approximately 1.5 billion people suffer from hearing loss, and more than 34 million children exhibit deafness or hearing loss.

[0007] Currently, few cases of hearing loss are curable. Hearing devices such as hearing aids have limitations, including an inability to improve speech intelligibility. Fewer than 20% of people affected by hearing impairment currently use hearing aids. In cases of age-related, noise- or medication-induced hearing dysfunction, the only effective way to "treat" the condition or reduce its severity is often prevention, such as by avoiding excessive noise and using ear protectors, adopting a healthy lifestyle, and minimizing exposure to ototoxic drugs and substances.

[0008] The prevalence of hearing loss after cochlear damage in mammals is believed to be due to a lack of spontaneous regeneration of hair cells and / or neurons, the main components that detect sound. Humans are born with approximately 15,000 inner ear hair cells, and hair cells do not regenerate after birth.

[0009] Therefore, there remains a long-term need to define the biology and mechanisms involved in protecting auditory hair cells before injury and / or maintaining / promoting the function of existing cells after injury. Summary of the Invention

[0010] Described herein are nucleic acids (e.g., non-human animal nucleic acids isolated from non-human animals), non-human animal cells, and non-human animals comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of an endogenous Klhdc7b gene or a portion thereof. The deletion can include, consist essentially of, or consist of a deletion of the open reading frame (ORF) of the endogenous Klhdc7b gene at the endogenous Klhdc7b locus of the non-human animal nucleic acid, non-human animal cell, and non-human animal, e.g., the deletion spans between, but does not include or extend beyond, the endogenous start codon of the endogenous Klhdc7b gene and the endogenous stop codon of the endogenous Klhdc7b gene. In some embodiments, the deletion can result from replacement of an endogenous Klhdc7b gene or a portion thereof (e.g., an ORF portion thereof) with an insert nucleic acid. In some embodiments, the insert nucleic acid may comprise a reporter gene and / or a gene encoding a selectable marker, optionally wherein the reporter gene is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter) and / or the gene encoding the selectable marker is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter), and / or wherein the reporter gene (and optional promoter) is flanked by site-specific recombination sequences and / or the gene encoding the selectable marker (and optional promoter) is flanked by site-specific recombination sequences. In non-limiting embodiments, the modified endogenous Klhdc7b locus comprises: (i) the nucleic acid sequence set forth in SEQ ID NO: 5 and / or (ii) the nucleic acid sequence set forth in SEQ ID NO: 6 or the nucleic acid sequence set forth in SEQ ID NO: 7, and / or (iii) the nucleic acid sequence set forth in SEQ ID NO: 38 or the nucleic acid sequence set forth in SEQ ID NO: 39, and / or (iv) the endogenous 5' Klhdc7b untranslated region, optionally the entire endogenous 5' Klhdc7b untranslated region, and / or (v) the endogenous 3' Klhdc7b translated region, optionally the entire endogenous 3' Klhdc7b untranslated region. In general, an endogenous 5'Klhdc7b untranslated region, optionally the entire endogenous 5'Klhdc7b untranslated region, as described herein, may be located upstream of a deletion of a Klhdc7b gene or a portion thereof, e.g., upstream of and operably linked to the Klhdc7b start codon, and / or an endogenous 3'Klhdc7b translated region, optionally the entire endogenous 3'Klhdc7b untranslated region, may be located downstream of a deletion of a Klhdc7b gene or a portion thereof, e.g., downstream of and operably linked to the endogenous stop codon of the endogenous Klhdc7b gene.In some embodiments, a non-human animal as described herein, such as a mouse homozygous for a modified Klhdc7b locus, can serve as a model for hearing loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The patent or application file contains at least one drawing printed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0012] Figure 1 The predicted long or short isoforms of mouse or human KLHDC7b transcripts (top panel) are presented, along with the transcript levels (ΔCT; y-axis) of the long (open bars) or short (filled bars) KLHDC7b transcripts found in tissue samples (x-axis) from commercial mouse cDNA panels (lower left), freshly isolated tissues (lower middle), or commercial human cDNA panels (lower right). Lower values ​​indicate higher expression. Error bars are SEM, with 3-4 technical replicates. All data are normalized to the housekeeping gene Drosha. Figure 1KLHDC7b transcripts are found in the cochlea and other organs, and their expression patterns differ slightly between mice and humans. The long isoform of the mouse gene, including the UTR, is located at mm10 chr15:89,384,917-89,388,867 and is 3,951 nucleotides long. The coding region is located at mm10 chr15:89,384,917-89,388,708 and is 3,792 nucleotides long with one exon. The short isoform is located at mm10 chr15:89,386,891-89,388,708 and is 1,818 nucleotides long with one exon and no annotated UTR. The long forward primer is located at chr15:89385390-89385412, the long reverse primer is located at chr15:89385458-89385478, and the probe is located at chr15:89385413-89385437. The overlapping forward primer is chr15:89388123-89388141, the reverse primer is chr15:89388182-89388200, and the probe is chr15:89388143-89388165. The putative long isoform transcript of the human gene (including the UTR) is located at the following positions: hg38 chr22:50,545,899-50,551,023 and is 5,125 nucleotides in size. There is one coding exon on the positive strand. The coding region is located at the following positions: hg38 chr22: 50,546,244-50,549,951, and is 3,708 nucleotides in size. The putative short human isoform, including the UTR, is located at the following positions: hg38 chr22: 50,548,033-50,551,022, and is 2,990 nucleotides in size. The coding region is located at the following positions: hg38 chr22: 50,548,167-50,549,951, and is 1,785 nucleotides in size. The long forward primer was located at chr22: 50546689-50546708 on the positive strand, the reverse primer was located at chr22: 50546769-50546789 on the negative strand, and the probe was located at chr22: 50546712-50546731. The overlapping forward primer was located at chr22: 50549582-50549600 on the positive strand, the reverse primer was located at chr22: 50549649-50549668 on the negative strand, and the probe was located at chr22: 50549601-50549620.

[0013] Figure 2Transcript levels (ΔCT; y-axis) of overlapping (left) and long (right) KLHDC7b transcripts found in liver (squares), brain (circles), temporal bone (including cochlea) (triangles), or kidney (diamonds) isolated from postnatal day 1 (p1) mice, postnatal day 7 (p7) mice, 11-28 weeks of age (adult) mice, or 63-70 weeks of age (old) mice are shown. P1 and P7 time points each consist of 5 mice. Mice at this age have not been sexed. Adult and old mice are sensitive to B6.CAST-Cdh23. Ahl+ / Kjn Correction allele heterozygosity. Adult mice were four female mice aged 11-14 weeks and one male mouse aged 24 weeks. Aged mice were four female mice aged 63-73 weeks. Each data point is a biological replicate, tissue from one mouse, and is calculated based on the mean of three technical replicates. All data were normalized to the housekeeping gene Drosha. Analysis was performed by two-way ANOVA with post hoc comparisons using the Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 2 It is shown that KLHDC7b expression changes with the lifespan of mice and is always expressed at a relatively high level in the cochlea.

[0014] Figures 3A to 3D Images of cochlear histological sections labeled with RNA probes detecting overlapping portions of the long and short KLHDC7b isoforms (red) or only the long KLHDC7b isoform (green) and immunostained for the hair cell marker Myo7a (white) from: (3A) an adult wild-type mouse, shown alongside an illustrative (not to scale) cartoon of the cochlear anatomy, (3B) an adult wild-type mouse, zoomed in on the organ or Corti, (3C) an adult wild-type mouse, zoomed in on the vestibular system, or (3D) an embryonic mouse. These images show that KLHDC7b is expressed exclusively in the hair cells within the cochlea. Probe markers appear as small dots. The dots are visible only in the hair cells. Figure 3B In the figure, the inset of the organ of Corti (center) shows staining with each probe (long and overlapping), MYO7A (a hair cell marker), and DAPI. Each probe is presented together with MYO7A (upper right two images) and each probe is presented alone (lower right two images). Neither probe labels any area outside of hair cells, indicating hair cell-specific expression. Similarly, in Figure 3C In the figure, vestibular hair cells are shown labeled with MYO7A and each probe (top two images on the right), and each probe is also presented alone (bottom two images on the right) to show that the probe overlaps only with the hair cells. Figure 3DMiddle, showing long and overlapping KLHDC7b+ dots (lower right two images) colocalizing with MYO7A (upper right two images) in labeled, developing hair cells of embryonic mice.

[0015] Figure 4A A graphic representation (not to scale) of the deletion length of the mouse Klhdc7b gene and its open reading frame (ORF) is provided, such as the genomic sequence spanning but excluding the "start" and "stop" codons of the mouse Klhdc7b gene. The 3,787b orf is represented by a solid rectangle. The 5' untranslated region of the Klhdc7b gene is represented by an open rectangle upstream of the start codon. The 3' untranslated region of the Klhdc7b gene is represented by an open rectangle downstream of the stop codon. Asterisks indicate the positions of the upstream (4929mTU) and downstream (4929mTD2) primers used for allelic loss determination. The approximate location of the sequence encoding the Kelch domain is also shown.

[0016] Figure 4B A schematic representation (not to scale) of a large targeting vector (LTVEC) generated by replacing the open reading frame of the mouse Klhdc7b gene found in the BAC clone RP23-241G24 with an 8,802 bp insert ("LacZ, Neo-SDC"). As shown, the LTVEC contains (a) a 140.5 kb 5' homology arm comprising the complete 5' untranslated sequence of the mouse Klhdc7b gene and the mouse Klhdc7b start codon from BAC clone RP23-241G24, (b) an 8,802 bp insert comprising the Lacz gene (grey arrow) inserted in-frame with the mouse Klhdc7b start codon and a self-deleting cassette (Neo-SDC; black arrow) comprising the neomycin gene ("Neo") flanked by LoxP site-specific recombination sequences, and (c) a 12.6 kb 3' homology arm comprising the complete 3' untranslated sequence of the mouse Klhdc7b gene from BAC clone RP23-241G24. "A" indicates the position of the 5' mouse UTR / / start, Acc65 / / 5' LacZ junction (SEQ ID NO: 5), and "B" indicates the position of the 3' Neo / / (loxP) / / NheI / / 3' mouse UTR junction (SEQ ID NO: 6). The sequences of these junctions are also provided.

[0017] Figure 4C Provided with Figure 4BSchematic representation of the Klhdc7b locus modified after targeted homologous recombination with the LTVEC of the mouse Klhdc7b gene and deletion of the neomycin cassette (not to scale). The untranslated region is depicted as a hollow rectangle, and the lacZ gene is depicted with a solid arrow. Also shown are the positions of the 5' homology arm comprising the 5' untranslated sequence of the mouse Klhdc7b gene and the mouse Klhdc7b start codon, and the 3' homology arm comprising the 3' untranslated sequence of the mouse Klhdc7b gene from RP23-241G24, 12.6 kb. "A" indicates the position of the 5' mouse UTR / / start, Acc65 / / 5' LacZ junction (SEQ ID NO: 5), and "C" indicates the 3' LacZ / / stop / / ( LoxP ) / / NheI / / 3' mouse UTR junction (SEQ ID NO: 7). The sequences of these junctions are also provided. The modified Khldc7b locus depicted in this figure expresses a LacZ protein comprising the amino acid sequence shown in SEQ ID NO: 4.

[0018] Figure 4D Provided are microscopic images showing LacZ expression in hair cells of heterozygous (HET) and knockout (KO) mice. Both mice were 10-week-old males. LacZ staining was only visible in hair cells (including inner and outer hair cells). Darker shadows indicate LacZ expression.

[0019] Figure 4E Fluorescence microscopy images of the cochlea of ​​wild-type and KLHDC7B- / - (KO) mice are provided. RNAscope was performed using the KLHDC7B probe for long and overlapping transcripts. Transcripts are absent in the hair cells of the knockout mouse cochlea. The top row of images is from the wild-type (WT) cochlea, while the bottom row of images is from the KO cochlea. The leftmost column shows a merged image of all four fluorescence channels (Myo7a, DAPI, long and overlapping KLHDC7B probes) at 40x magnification, depicting a complete convolution of the cochlea. The second column from the left is a magnified merged fluorescence image showing only the hair cells. The third column shows a magnified single-channel fluorescence image representing dots labeled with the overlapping probe, which are located only in the hair cells (visible as bright white in the third column). The fourth column shows a magnified single-channel fluorescence image representing dots labeled with the long probe, which are located only in the hair cells. WT cochlea shows labeling, while KO cochlea shows no labeling, indicating that KO mice lack RNA transcripts for Klhdc7b, as expected. The hair cell outlines drawn on the image indicate that the dots are present only in hair cells.

[0020] Figure 5AFigure 1 provides immunostaining images of whole mounted cochleae of WT and KLHDC7B KO mice at postnatal day (p) 6, p11, p21, and 8 weeks. Hair cells appear normal at p6, but some outer hair cells are missing at p11 (solid circle in knockout tissue). At p21, many hair cells are missing, and significant supporting cell scars are visible where the hair cells were previously located. At p21 and 8 weeks, scars are visible as a white lattice structure stained for F-actin (dashed circle in knockout tissue). The circular staining pattern above the outer hair cells represents abnormal MYO7A labeling, indicating that the hair cells die and may be engulfed by supporting cells (dashed circle in knockout tissue). The damage is more severe at 8 weeks (solid rectangle in knockout tissue).

[0021] Figure 5B Provided are images of cochlear histological sections from 8-week-old wild-type mice or 8-week-old KLHDC7b knockout mice and immunostained for the hair cell marker Myo7a (green); the neuronal marker Tuj1 (red); and DAPI (blue). KO = knockout, OHC = outer hair cell, IHC = inner hair cell. These images show hair cell abnormalities in KLHDC7b knockout mice at 8 weeks. The hair cells (depicted as the brightest stain in grayscale) appear to have abnormal morphology, with further increased intercellular spacing and poor organization. The bottom of the rightmost image shows a very abnormal staining pattern. Neuronal staining, depicted as a darker, grayscale diffuse staining located below the primary inner hair cells (rather than the DAPI-labeled, mostly round nuclei with clearly defined borders), is still present in the KO tissue.

[0022] Figure 6 Provided are images of whole-mount cochlear histology staining from 8-week-old wild-type mice or 8-week-old KLHDC7b knockout mice, immunostained for the hair cell marker Myo7a (green); neuronal marker Tuj1 (red); DAPI (blue) and F-actin (white). WT = wild type, KO = knockout. These images show the lack of visible scarring in the hair cells of KLHDC7b knockout mice at 8 weeks. As shown in Figure 5A In the image, a latticework pattern is visible, indicating scarring of supporting cells. Fewer hair cells are also present. This damage occurs gradually from the base to the apex, with the base being the most severely damaged.

[0023] 7A to 7CProvided are histological images of the (A, C) apex or (B, C) base of whole-mount cochlear sections from 3-day-old wild-type mice or 3-day-old KLHDC7b knockout mice and immunostained for the hair cell marker Myo7a (green); the neuronal marker Tuj1 (red); DAPI and actin. All parts of the organ of Corti (apex, middle and base) appear normal, with well-formed stereocilia and no missing hair cells in the KO tissue. Figure 7D Provided are histological images of the cochlea from 6-day KLHDC7b knockout mice immunostained for Myo7a, DAPI (blue), actin (white), and ZO-1 (red). Hair cells are present and have normal morphology, stereocilia, and ZO-1 localization. Each fluorescent channel is shown separately in the four images on the right. Figure 7E Provided are histological images of the cochlea from a 6-day-old wild-type mouse and immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Tissue from this wild-type mouse and knockout mouse of the same age looks very similar. Figure 7F Provided are histological images of the cochlea from an 11-day-old wild-type mouse immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red) from the apical gyrus of the cochlea. Hair cells are present and appear normal, as are stereocilia and ZO-1 staining. Figures 7G to 7H Provided are histological images of cochleae from two different 11-day-old KLHDC7b knockout mice immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Figure 7F The cell bodies of 100% (compared to 100% (100%)) showed abnormal cytoplasmic localization of ZO-1, while only a few hair cells were missing. Figure 7I Provided are histological images of the cochlea from a 21-day KLHDC7b knockout mouse, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Of note, MYO7A staining revealed a lack of hair cells and abnormal rounded shapes outside the normal outer hair cell location, which may indicate that the hair cells have been engulfed by supporting cells. Some of the remaining hair cells showed cytoplasmic localization of ZO-1. Actin staining revealed significant scarring, as indicated by a jagged lattice structure. Inner hair cells appeared normal, with normal stereocilia. Figure 7J Provided are histological images of cochleae from 21-day-old wild-type mice immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Figure 7IIn contrast, hair cells appear more organized, with ZO-1 staining localized around the apical border of the cells rather than in the cytoplasm. Whether these instances of ZO-1 mislocalization are due to a general cell death phenotype or a more specific phenotype caused by Klhdc7b knockout is unknown. WT = wild type, KO = KLHDC7b knockout. These images show that hair cells and stereocilia in knockout cochleae appear normal at 3 and 6 days after birth, show early signs of degeneration by 11 days after birth, and progress to further degeneration by 21 days after birth.

[0024] Figure 8 Schematic diagrams are provided of two audiometric tests used to assess cochlear function: the auditory brainstem response (ABR), which measures the function of inner hair cells and neurons (left panel), and the distortion product otoacoustic emissions (DPOAE), which measures the function of outer hair cells (right panel).

[0025] Figures 9A to 9C KLHDC7B knockout mice were shown to have profound and progressive hearing loss, while heterozygous mice did not. (A) Auditory brainstem responses (ABRs) were recorded in separate groups of KLHDC7B knockout (KO), heterozygous (HET), and wild-type (WT) mice at each time point. Top, ABR thresholds. For each age range listed on the x-axis, the data point on the left is WT, the data point in the middle is HET, and the data point on the right is KO. KO mice showed profound hearing loss at the onset of hearing loss (2-3 weeks), which progressed to complete loss at 11-15 weeks at three measured frequencies (8kHz, 16kHz, and 32kHz). 100dB represents no response. In the middle, the wave 1 amplitude of KO mice was significantly smaller than that of WT at 2-3 weeks, while HET mice did not differ significantly from WT. In the middle right, the average waveforms of KO and WT mice at 2-3 weeks. The shaded area represents the mean average (SEM). Bottom, at 11-15 weeks, ABRs are absent in almost all KO mice, while HET and KO are not significantly different. The lower right shows the average waveforms of WT and KO mice are the same as in the middle. (B) ABRs were recorded longitudinally starting from 11-13 weeks (data same as above). Thresholds increased at all frequencies at 55-57 weeks, with no significant differences between WT and HET. Bottom, Wave 1 amplitudes were also not different between HET and WT at any dB SPL level, and the waveforms (right) were indistinguishable. All data were analyzed separately at each frequency. (C) Non-limiting and exemplary auditory brainstem responses (ABRs) from wild-type (WT), heterozygous KLHDC7b knockout (Het), and homozygous KLHDC7b knockout (KO) mice at postnatal day 17 and 16 kHz (top panel). Also shown is a graph providing hearing thresholds (dB; y-axis) of these animals at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) as determined by ABR. These animals are also included in Figure 9A WT = 5 males, 4 females. Het = 7 males, 10 females, KO = 2 males, 3 females. ns = not significant. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. This figure shows that homozygous KLHDC7b knockout mice have significant hearing loss at the onset of hearing loss.

[0026] Figure 10 Non-limiting and exemplary auditory brainstem responses (ABRs) from wild-type (WT) and KLHDC7b knockout (KO) mice at 6, 8, and 12 weeks of age and 16 kHz are provided. Also shown are graphs providing hearing thresholds (dB; y-axis) at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) for these animals as measured by ABRs (top graph) or distortion product otoacoustic emissions (DPOAEs) (bottom graph). WT = 7 males, 2 females. KO = 4 males, 7 females. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. This figure shows that KLHDC7b knockout mice have profound hearing loss at 5-7 weeks of age that is more severe than the hearing loss at the onset of hearing loss and progresses to complete deafness by approximately 12 weeks of age. Further information on this progress and more animals can be found at Figure 9A Found in.

[0027] Figures 11A to 11C Graphs are shown providing hearing thresholds (dB; y-axis) at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) for wild-type (WT), heterozygous KLHDC7b knockout (het), or homozygous KLHDC7b (KO) animals as measured by ABR (left graph) or distortion product otoacoustic emissions (DPOAE) (right graph). Figure 11A Center: WT = 3 males, 6 females. Het = 6 males, 2 females. KO = 4 males, 3 females. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. This figure shows that adult heterozygous KLHDC7b knockout mice are not deafened at 11-13 weeks of age. Figure 11B and Figure 11C ABRs of KO and Het mice tested at 16 kHz are provided, respectively. This figure shows the time course of hearing loss, with homozygous KLHDC7b mice experiencing increasing hearing loss between 17 days and 7-8 weeks, and heterozygous mice not experiencing hearing loss until 32 weeks.

[0028] 12A to 12C(A) Images of cochlear explants from wild-type mice incubated in gentamycin-Texas Red (GtTR), Texas Red alone, or culture medium alone are provided to show that hair cells in cochlear explant culture take up gentamycin-Texas Red (GtTR) through mechanotransduction pathways. (B) Cochlear explants were immunostained for Myo7a (white); DAPI (blue); and actin (green). The top panel depicts a merged fluorescence image of all channels. The middle panel depicts a magnified merged fluorescence image of the area represented by the white box in the top panel. The bottom panel depicts a magnified single-channel fluorescence image of GtTR. In grayscale, bright staining in the merged fluorescence image indicates colocalization of actin and GtTR staining, indicating GtTR uptake into hair cells. WT = wild type, het = heterozygous KLHDC7b knockout mouse, KO = homozygous KLHDC7b knockout mouse. These images show that hair cells in cochlear explant cultures take up gentamicin-Texas Red (GtTR) through the mechanotransduction pathway, and that the mechanotransduction complex appears functional in cochlear explant cultures from knockout mice, as hair cells in postnatal day 4-5 explants take up GTTR, the gross morphology of hair cells appears normal, and there is no loss of hair cells in these mice. (C) Wild-type (KLHDC7b + / + ) and knockout (KLHDC7b Δ / Δ Cochlear explant cultures from WT and KO mice were treated with gentamicin-Texas Red (GtTR) or Texas Red (TR) alone, and then immunostained for MYO7A and stained for F-actin. GtTR, while Texas Red, did not, enter hair cells in both WT and KO mice.

[0029] Figure 13A Fluorescence images of ear organoids differentiated from hiPSCs (human induced pluripotent stem cells) are provided. RNA scope probes for two ear markers, Sox2 (hair cells and supporting cells) and TUBB3 (spiral ganglion neurons), show expression around the possible otic capsule (left). The JKiPSC line is shown. qPCR of ear markers (right) shows an increase after differentiation in D70IEO (70th day internal ear organoids) compared to iPSCs. Both the JK and GM lines show this increase. Figure 13BIn this study, RNA scope probes were designed targeting the long portion (top panel) and overlapping portion (bottom panel) of the KLHDC7B human transcript, and RNAscope was performed in parallel with staining for Myo7a. The leftmost column depicts a merged fluorescence image of an otic organoid (Myo7a, DAPI, and KLHDC7B probes). The second column depicts a magnified merged fluorescence image of an area with high probe labeling concentration. The third and fourth columns depict single-channel fluorescence images of the KLHDC7B probe and Myo7a, respectively. Both probes are shown localized around the probable otic capsule. Sections were obtained from the same organoid. qPCR of KLHDC7B (right) shows an increase after differentiation. Analysis was performed by two-way ANOVA, with post hoc comparisons performed using the Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0030] Figure 14 Presented are scanning electron microscopy images of isolated cochleae from wild-type (WT) or homozygous KLHDC7b (KO) animals at postnatal day 10 (p10) or postnatal day 20 (p20).

[0031] Figure 15 Provided are histological images of fixed, paraffin-embedded mouse cochleae stained with a custom-generated antibody against KLHDC7b. The 20x confocal image on the upper left shows labeling only in hair cells, and the 40x confocal image on the upper right shows labeling that appears to be localized to the plasma membrane. Unlabeled primary antibody controls are not shown on the lower left and right.

[0032] Figure 16 Provided are histological images of cochlear samples isolated from wild-type (WT) or homozygous KLHDC7b (KO) animals, labeled with a custom monoclonal anti-KLHDC7b antibody (white) and DAPI.

[0033] Figure 17A Provided are histological images of HEK cell lines stably transfected with FLAG-tagged short (top row) or long (middle row) human Klhdc7b isoforms or FLAG-tagged GFP (bottom row) and labeled with one of 10 different monoclonal anti-KLHDC7b antibodies (cyan; clone numbers are depicted below) and stained for FLAG (red), GFP (green), and DAPI expression (dark blue). In grayscale, the brightest staining in the top and middle rows (short and long FLAG-tagged human Klhdc7b isoforms, respectively) indicates staining with anti-KLHDC7b antibodies. The brightest staining in the bottom row of control-transfected cells indicates GFP staining. No anti-KLHDC7b labeling was detected in all clones except clone 2, indicating some nonspecific labeling for this clone.

[0034] Figure 17B Provided are histological images of the same HEK cell line as described above labeled with two monoclonal anti-KLHDC7b antibodies, designated Antibody A ( Figure 17A Clone 1) and antibody B ( Figure 17A 3). The top and middle rows depict cells transfected with short and long FLAG-tagged human Klhdc7b isoforms, respectively. The bottom row depicts cells transfected with FLAG-tagged GFP. Columns 1 and 2 show all channels. Columns 3 and 4 show FLAG staining and DAPI staining only, columns 5 and 6 show anti-KLHDC7B and DAPI staining, and columns 7 and 8 show GFP staining and DAPI staining. In grayscale, the brightest staining in the first six columns of the first two rows indicates colocalization of DAPI and FLAG, DAPI and anti-KLHDC7b, or all three, indicating that the antibody binds to cells expressing both the long and short isoforms of Klhdc7b. The brightest staining in the bottom row of control transfected cells indicates GFP staining, in which no anti-KLHDC7b labeling was detectable. DETAILED DESCRIPTION

[0035] I. Overview

[0036] Kelch domain-containing 7B (KLHDC7b) is a member of the Kelch superfamily of proteins involved in cellular processes such as cytoskeletal rearrangement and protein degradation. It also plays a role in extracellular communication, cell morphology, gene expression, and actin binding. Little is known about this gene, other than its membership in the Kelch domain-containing superfamily of proteins. The Kelch domain is a set of repeated β-sheet-forming subunits that bind together to form a tertiary structure called a β-propeller. Kelch domain-containing proteins have diverse subcellular locations and functions, so the fact that KLHDC7b belongs to this family does not clarify its role within the cell (Adams et al., 2000). Few publications specifically examine KLHDC7b. KLHDC7b has been shown to be upregulated in breast cancer cells, but is also hypermethylated (Martin-Pardillos and Cajal, 2019). Alterations in the Kelch superfamily have been associated with various types of cancer, including leukemia, lung cancer, prostate cancer, brain cancer, and Hodgkin's disease. KLHDC7B has been identified as hypermethylated but upregulated in breast cancer cells. KLHDC7b has two predicted isoforms (long and short).

[0037] Moderate levels of KLHDC7B expression are observed in the hair cells of the cochlea, such as the ear, while outer hair cells appear to show slightly higher expression (gEAR portal). Predicted loss-of-function variants in KLHDC7B are associated with an increased risk of developing hair loss in humans. For example, genetic alterations that change the guanine nucleotide at position 3,778 to an adenine in the human KLHDC7B reference (see, NCBI accession number: NP_612442.3) have been observed, indicating that people with such alterations may be at increased risk of developing hearing loss (such as conductive hearing loss, sensorineural hearing loss, or sensorineural hearing loss). The International Mouse Phenotyping Consortium (IMPC) indicates that exon deletions of Klhdc7b exhibit abnormal auditory brainstem responses, abnormal ear morphology, shortened QT intervals, abnormal motor behavior, reduced / abnormal startle reflexes, and reduced prepulse inhibition (www.mousephenotype.org / data / genes / MGI:3648212).

[0038] Evidence presented herein indicates that non-human animals harboring a knockout mutation of the endogenous Klhdc7b gene progressively develop hearing loss. Compared to wild-type control animals, Klhdc7b knockout mice exhibit increased hearing loss, with profound hearing loss at onset and near-complete hearing loss by 11-15 weeks of age. For example, hair cells in the cochlea are absent at the time point of profound deafness in mice, although mechanotransduction complexes appear functional at earlier time points. For example, hair cells develop normally and display functional mechanotransduction complexes in culture, but begin to die around postnatal days 11-12, with the characteristic morphology of supporting cell scars known to form beneath dying hair cells (Wagner and Shin, 2019), as indicated by histology and scanning electron microscopy. Scanning electron microscopy (SEM) revealed no overt abnormalities in stereocilia morphology before the onset of hair cell death and confirmed that outer hair cells are shed by three weeks of age. RNA scope results demonstrated that KLHDC7b is specifically expressed in hair cells. Custom anti-Klhdc7b antibodies generated against KLHDC7B demonstrated hair cell specificity. These data suggest that KLHDC7B plays a role in the maintenance of cochlear hair cells. Therefore, the non-human animals disclosed herein may help identify key biological factors and / or mechanisms for preventing hearing loss and / or preserving hearing function.

[0039] Provided herein are nucleic acids (e.g., non-human animal nucleic acids isolated from non-human animals), non-human animal cells, and non-human animals comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of an endogenous Klhdc7b gene or a portion thereof. The deletion can include, consist essentially of, or consist of a deletion of the open reading frame (ORF) of the endogenous Klhdc7b gene at the endogenous Klhdc7b locus of the non-human animal nucleic acid, non-human animal cell, and non-human animal, e.g., the deletion spans between, but does not include or extend beyond, the endogenous start codon of the endogenous Klhdc7b gene and the endogenous stop codon of the endogenous Klhdc7b gene. In some embodiments, the deletion can result from replacement of the endogenous Klhdc7b gene or a portion thereof (e.g., an ORF portion thereof) with an insert nucleic acid. In some embodiments, the insert nucleic acid may comprise a reporter gene and / or a gene encoding a selectable marker, optionally wherein the reporter gene is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter) and / or the gene encoding the selectable marker is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter), and / or wherein the reporter gene (and optional promoter) is flanked by site-specific recombination sequences and / or the gene encoding the selectable marker (and optional promoter) is flanked by site-specific recombination sequences. In non-limiting embodiments, the modified endogenous Klhdc7b locus comprises: (i) the nucleic acid sequence set forth in SEQ ID NO: 5 and / or (ii) the nucleic acid sequence set forth in SEQ ID NO: 6 or the nucleic acid sequence set forth in SEQ ID NO: 7, and / or (iii) the nucleic acid sequence set forth in SEQ ID NO: 38 or the nucleic acid sequence set forth in SEQ ID NO: 39, and / or (iv) the endogenous 5' Klhdc7b untranslated region, optionally the entire endogenous 5' Klhdc7b untranslated region, and / or (v) the endogenous 3' Klhdc7b translated region, optionally the entire endogenous 3' Klhdc7b untranslated region. In general, an endogenous 5'Klhdc7b untranslated region, optionally the entire endogenous 5'Klhdc7b untranslated region, as described herein, may be located upstream of a deletion of a Klhdc7b gene or a portion thereof, e.g., upstream of and operably linked to the Klhdc7b start codon, and / or an endogenous 3'Klhdc7b translated region, optionally the entire endogenous 3'Klhdc7b untranslated region, may be located downstream of a deletion of a Klhdc7b gene or a portion thereof, e.g., downstream of and operably linked to the endogenous stop codon of the endogenous Klhdc7b gene.

[0040] II. Non-human cells and non-human animals containing a modified Klhdc7b locus

[0041] Provided are non-human animal cells and non-human animals comprising a modified Klhdc7b locus as described herein. The cell or non-human animal can be heterozygous or homozygous for the modified Klhdc7b locus. Diploid organisms have two alleles at each genetic locus. Each pair of alleles represents the genotype of a particular genetic locus. If two identical alleles are present at a particular locus, the genotype is described as homozygous, and if the two alleles are different, the genotype is described as heterozygous. In some embodiments, provided herein is a non-human animal cell comprising an endogenous Klhdc7b locus genetically modified as described herein. The non-human animal cell can be a cochlear cell (e.g., an inner hair cell or an outer hair cell), a pluripotent cell, an ES cell, or a germ cell.

[0042] In some embodiments, the present disclosure further provides methods for making any non-human animal or reagents required to make a non-human animal as described herein.

[0043] The non-human animals provided herein can be, for example, any non-human cell comprising a modified Klhdc7b locus as described herein. The cell can be a eukaryotic cell, including, for example, a fungal cell (e.g., yeast), a plant cell, an animal cell, a mammalian cell, a non-human mammalian cell, and a human cell. The animal can be, for example, a mammal, a fish, or a bird. The mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non-human primates, monkeys, apes, gorillas, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., bovine species such as cows, steers, etc.; ovine species such as sheep, goats, etc.; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, etc. Domestic animals and agricultural animals are also included. The term "non-human" does not include humans.

[0044] Cell can also be any type of undifferentiated or differentiated state.For example, cell can be totipotent cell, pluripotent cell (for example people's pluripotent cell or non-human pluripotent cell, such as mouse embryonic stem (ES) cell or rat ES cell) or non-pluripotent cell.Totipotent cell includes the undifferentiated cell that can produce any cell type, and pluripotent cell includes the undifferentiated cell with the ability that develops into more than one differentiated cell type.Such multipotency and / or totipotent cell can be for example ES cell or class ES cell, such as induced pluripotent stem (iPS) cell.ES cell includes embryo-derived totipotent or pluripotent cell, and it can be conducive to any tissue of development embryo after being introduced in embryo.ES cell can be derived from the inner cell mass of blastocyst, and can be divided into the cell of any one in three vertebrate germ layers (endoderm, ectoderm and mesoderm).

[0045] The cells provided herein can also be germ cells (e.g., sperm or oocytes). The cells can be mitotically competent cells or mitotically inactive cells, meiotically competent cells or meiotically inactive cells. Similarly, the cells disclosed herein can also be primary somatic cells or cells that are not primary somatic cells. Somatic cells include any cells that are not gametes, germ cells, gametocytes or undifferentiated stem cells. Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures directly isolated from organisms, organs or tissues. Primary cells include cells that are neither transformed nor immortal. Primary cells include any cells obtained from organisms, organs or tissues that have not been passaged in tissue culture before or have been passaged in tissue culture before but cannot be passaged in tissue culture indefinitely. Such cells can be separated by conventional techniques.

[0046] Suitable cells include cells of the ear, such as cells related to hearing, such as neurons, hair cells, etc. Spiral ganglion neurons and cochlear hair cells (such as inner hair cells, outer hair cells or supporting cells of the cochlea), cells of the organ of Corti (such as Hensen's cells, Deiters' cells, pillar cells, inner phalangeal cells and border cells) can be suitable cells provided herein.

[0047] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells from multicellular organisms that usually do not proliferate indefinitely, but due to mutation or change, have avoided normal cell aging and can continue to divide instead. Such mutation or change can occur naturally or be induced intentionally. The example of an immortalized cell line is a myofibroblast line. Immortalized or primary cells include cells that can be used for cultivating or for expressing recombinant genes or proteins.

[0048] The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be from any genetic background (e.g., B6.Cast-Cdh23 for mice). Ahl+ ), can be fresh or frozen, and can be derived from natural breeding or in vitro fertilization.

[0049] The cells provided herein can be normal, healthy cells, or can be diseased or harbor a mutation.

[0050] Also provided are tissues comprising the nucleic acids and / or cells described herein and / or isolated from non-human animals, such as cochlear explants.

[0051] Non-human animals comprising a modified Klhdc7b locus as described herein can be produced by methods described elsewhere herein. The animal can be, for example, a mammal, fish, or bird. Non-human mammals include, for example, non-human primates, monkeys, apes, gorillas, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs), and livestock (e.g., bovine species such as cows and steers; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domestic animals and agricultural animals are also included. The term "non-human animal" does not include humans. Preferred non-human animals include, for example, rodents such as mice and rats.

[0052] The non-human animal may be from any genetic background. For example, a suitable mouse may be from B6.Cast-Cdh23 Ahl+ Strain, 129 strain, C57BL / 6 strain, mixture of 129 and C57BL / 6, BALB / c strain or Swiss Webster strain. Examples of 129 strains include 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1 / SV, 129S1 / Svlm), 129S2, 129S4, 129S5, 129S9 / SvEvH, 129S6 (129 / SvEvTac), 129S7, 129S8, 129T1 and 129T2. See, for example, Festing et al. (1999) Mammalian Genome 10:836, which is incorporated herein by reference in its entirety for all purposes. Examples of C57BL strains include C57BL / A, C57BL / An, C57BL / GrFa, C57BL / Kal_wN, C57BL / 6, C57BL / 6J, C57BL / 6ByJ, C57BL / 6NJ, C57BL / 10, C57BL / 10ScSn, C57BL / 10Cr, and C57BL / Ola. Suitable mice can also be derived from a mixture of the above-mentioned 129 strain and the above-mentioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be derived from a mixture of the above-mentioned 129 strain or a mixture of the above-mentioned BL / 6 strain (e.g., 129S6 (129 / SvEvTac) strain).

[0053] Similarly, rats can be from any rat strain, including, for example, the ACI rat strain, the Dark Agouti (DA) rat strain, the Wistar rat strain, the LEA rat strain, the Sprague Dawley (SD) rat strain, or a Fischer rat strain, such as Fisher F344 or Fisher F6. Rats can also be obtained from strains derived from a mixture of two or more of the above strains. For example, suitable rats can be from the DA strain or the ACI strain. The ACI rat strain is characterized by having a dark agouti gray color with a white abdomen and feet and RT1 av1 Haplotype. Such strains are available from a variety of sources, including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized by a wild gray coat and RT1 av1 Haplotype. Such rats are available from a variety of sources, including Charles River and Harlan Laboratories. Some suitable rats may be derived from inbred rat strains. See, for example, US 2014 / 0235933, which is incorporated herein by reference in its entirety for all purposes.

[0054] III. Methods for producing non-human animals comprising a modified Klhdc7b locus

[0055] Various methods for making non-human animals comprising a modified Klhdc7b locus as disclosed elsewhere herein are provided. Any convenient method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, for example, Cho et al. (2009) Current Protocols in Cell Biology 42:19.11:19.11.1–19.11.22 and Gama Sosa et al. (2010) Brain Struct. Funct. 214(2-3):91-109, each of which is incorporated herein by reference in its entirety for all purposes. For example, such genetically modified non-human animals can be produced by knocking in a gene at the targeted Klhdc7b locus.

[0056] For example, a method for producing a non-human animal comprising a modified Klhdc7b locus may comprise: (1) modifying the genome of a pluripotent cell to comprise a modified Klhdc7b locus; (2) identifying or selecting genetically modified pluripotent cells comprising a modified Klhdc7b locus; (3) introducing the genetically modified pluripotent cells into non-human animal host embryonic cells in vitro; and (4) implanting the host embryonic cells into a surrogate mother and gestating. Optionally, the host embryo comprising the modified pluripotent cells (e.g., non-human ES cells) may be incubated until the blastocyst stage and then implanted into a surrogate mother and gestated to produce an F0 non-human animal. The surrogate mother may then produce an F0 generation non-human animal comprising the modified Klhdc7b locus.

[0057] The method may further comprise identifying cells or animals having the modified target genomic locus.A variety of methods can be used to identify cells and animals having targeted genetic modifications.

[0058] The screening step can include, for example, a quantitative determination of the modification of alleles (MOA) for evaluating the parental chromosome. For example, quantitative determination can be performed via quantitative PCR such as real-time PCR (qPCR). Real-time PCR can utilize a first primer set that identifies the target locus and a second primer set that identifies a non-targeted reference locus. Primer sets can include a fluorescent probe that identifies an amplified sequence.

[0059] Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization with immobilized probes, Probe, Molecular beacon probe or ECLIPSE TM Probe technology (see, eg, US 2005 / 0144655, which is herein incorporated by reference in its entirety for all purposes).

[0060] Examples of suitable pluripotent cells are embryonic stem (ES) cells (e.g., mouse ES cells or rat ES cells). Modified pluripotent cells can be generated by recombination, for example, by: (a) introducing into a cell one or more targeting vectors comprising an insert nucleic acid flanked by 5' homology arms and 3' homology arms corresponding to the 5' target site and the 3' target site, wherein the insert nucleic acid comprises a modified Klhdc7b locus or a portion thereof (e.g., a modified Klhdc7b gene comprising a deletion of its open reading frame); and (b) identifying at least one cell comprising in its genome the insert nucleic acid integrated at the target genomic site.

[0061] Thus, also provided herein is a method for making a genetically modified cell (e.g., an ES cell), comprising contacting the cell with one or more targeting vectors comprising an insert nucleic acid flanked by 5' homology arms and 3' homology arms corresponding to a 5' target site and a 3' target site, wherein the insert nucleic acid comprises a modified Klhdc7b locus or a portion thereof (e.g., a modified Klhdc7b gene comprising a deletion of its open reading frame), such that upon homologous recombination between the 5' homology arms and the 3' homology arms and the corresponding 5' target site and the 3' target site, the insert nucleic acid is integrated into the genome of the cell at the target genomic locus, i.e., the genomic region between the 5' target site and the 3' target site.

[0062] Alternatively, modified pluripotent cells can be generated by: (a) introducing into a cell: (i) a nuclease agent, wherein the nuclease agent induces a nick or double-strand break at a recognition site within a target genomic locus; and (ii) one or more targeting vectors comprising an insert nucleic acid flanked by 5' and 3' homology arms corresponding to a 5' target site and a 3' target site located sufficiently close to the recognition site, wherein the insert nucleic acid comprises a modified Klhdc7b locus; and (c) identifying at least one cell comprising a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-strand break into the desired recognition site can be used. Examples of suitable nucleases include transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), meganucleases, and clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems or components of such systems (e.g., CRISPR / Cas9). See, e.g., US 2013 / 0309670 and US 2015 / 0159175, each of which is herein incorporated by reference in its entirety for all purposes.

[0063] Donor cells can be introduced into the host embryo at any stage, such as the blastocyst stage or the pre-morula stage (i.e., the 4-cell stage or the 8-cell stage). Produced offspring that can pass the genetic modification through the germline. See, for example, U.S. Patent No. 7,294,754, which is incorporated herein by reference in its entirety for all purposes.

[0064] Alternatively, a method of producing a non-human animal described elsewhere herein may comprise: (1) modifying the genome of a one-cell stage embryo to include a modified Klhdc7b locus using the methods described above for modifying pluripotent cells; (2) selecting the genetically modified embryo; and (3) implanting the genetically modified embryo into a surrogate mother and gestating the embryo. Progeny are generated that are capable of transmitting the genetic modification through the germline.

[0065] Nuclear transfer techniques can also be used to produce non-human mammals. Briefly, methods for nuclear transfer may include the following steps: (1) enucleating an oocyte or providing an enucleated oocyte; (2) isolating or providing a donor cell or nucleus to be combined with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstructed cell; (4) implanting the reconstructed cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are generally removed from dead animals, although they can also be isolated from the oviduct and / or ovary of living animals. Donor cells or nuclei can be inserted into enucleated oocytes to form reconstructed cells by microinjecting the donor cell under the zona pellucida before fusion. Fusion can be induced by applying a DC electric pulse (electrofusion) at the contact / fusion plane, by exposing the cells to a chemical that promotes fusion (such as polyethylene glycol), or by inactivating viruses (such as Sendai virus). Reconstruction cell can be activated by electrical means and / or non-electrical means before, during and / or after the fusion of nucleus donor and recipient oocyte.Activation method includes electric pulse, chemically induced shock, sperm penetration, increase the level of divalent cations in oocyte and reduce the phosphorylation (such as by kinase inhibitor) of cytoprotein in oocyte.Activated reconstruction cell or embryo can be cultivated in culture medium and then transferred to the uterus of animal.See, for example, US2008 / 0092249, WO 1999 / 005266, US 2004 / 0177390, WO 2008 / 017234 and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference in its entirety for all purposes.

[0066] The various methods provided herein enable the generation of genetically modified non-human F0 animals, wherein the cells of the genetically modified F0 animals contain a modified Klhdc7b locus. It will be appreciated that, depending on the method used to generate the F0 animal, the number of cells within the F0 animal that have the modified Klhdc7b locus will vary. By, for example, Method is introduced donor ES cell into the embryo (for example, 8 cell stage mouse embryo) before the morula of corresponding organism and can realize that the cell mass of F0 animal comprises the percentage ratio of the cell with the nucleotide sequence of interest that comprises targeted genetic modification is larger.For example, at least 50%, 60%, 65%, 70%, 75%, 85%, 86%, 87%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the cell contribution of non-human F0 animal can comprise the cell mass with targeted modification.

[0067] Cells of the genetically modified F0 animal can be heterozygous for the modified Klhdc7b locus. In some embodiments, heterozygous F0 mice can be bred to produce offspring that are homozygous for the modified Klhdc7b locus.

[0068] In some embodiments, the present disclosure provides a method of making a non-human animal, a non-human animal cell, or a non-human animal genome described herein, comprising inserting a nucleic acid sequence comprising a modified Klhdc7b locus into the genome of the non-human animal, the genome of the non-human animal cell, or the non-human animal genome.

[0069] IV. Nucleic Acids Comprising a Modified Klhdc7b Locus

[0070] As described elsewhere herein, a variety of nucleic acids (e.g., targeting vectors) are available specifically for such purposes. In some embodiments, non-human animal nucleic acids comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus can be used, wherein the modified endogenous Klhdc7b locus comprises a deletion of the endogenous Klhdc7b gene or a portion thereof. In some non-human animal nucleic acid embodiments, the deletion comprises, consists essentially of, or consists of a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene.

[0071] Insert nucleic acid

[0072] In some non-human animal nucleic acid embodiments, the modified endogenous Klhdc7b locus further comprises an insert nucleic acid. An "insert nucleic acid" or "insert polynucleotide" comprises a DNA segment that is desired to be integrated at the target locus. In one embodiment, the insert nucleic acid comprises one or more polynucleotides of interest. In other embodiments, the insert nucleic acid may comprise one or more expression cassettes. A given expression cassette may comprise a polynucleotide of interest, a polynucleotide encoding a selectable marker and / or a reporter gene, and various regulatory components that affect expression.

[0073] Any polynucleotide of interest can be included in various insert polynucleotides for integration at the target Klhdc7b locus. The methods disclosed herein provide at least 1, 2, 3, 4, 5, 6 or more polynucleotides of interest for integration at the targeted Klhdc7b genomic locus.

[0074] In one embodiment, the polynucleotide of interest contained in the insert nucleic acid encodes a reporter molecule. In another embodiment, the polynucleotide of interest encodes a selectable marker.

[0075] In one embodiment, the polynucleotide of interest may be flanked by site-specific recombination sequences. In a specific embodiment, the site-specific recombination sequences are flanked by segments encoding a reporter molecule and / or a segment encoding a selectable marker.

[0076] Non-limiting examples of polynucleotides of interest, including selectable markers and reporter genes that can be included within the insert nucleic acid, are discussed in detail elsewhere herein.

[0077] The polynucleotide of interest within the insertion polynucleotide can be introduced into the cell when it is integrated at the target Klhdc7b locus. The genetic modification can include deletion of an endogenous nucleic acid sequence (e.g., deletion of an open reading frame) and / or addition of an exogenous or heterologous or orthologous polynucleotide to the target genomic locus. In one embodiment, the genetic modification includes replacing an endogenous nucleic acid sequence with an exogenous polynucleotide of interest at the target genomic locus. Thus, the methods provided herein generate genetic modifications comprising knockout, deletion, replacement ("knock-in"), or a combination thereof, in the target Klhdc7b locus. Such modifications can occur after the first insertion polynucleotide, the second insertion polynucleotide, the third insertion polynucleotide, the fourth insertion polynucleotide, the fifth insertion polynucleotide, the sixth insertion polynucleotide, the seventh insertion polynucleotide, or any subsequent insertion polynucleotide is integrated into the target genomic locus.

[0078] The polynucleotide of interest inserted into the polynucleotide and / or integrated at the target genomic locus may comprise a sequence that is natural or homologous to the cell into which it is introduced; the polynucleotide of interest may be heterologous to the cell into which it is introduced; the polynucleotide of interest may be exogenous to the cell into which it is introduced; the polynucleotide of interest may be orthologous to the cell into which it is introduced; or the polynucleotide of interest may be from a species different from the cell into which it is introduced. The term "homologous" with respect to a sequence is a sequence that is natural to a cell. The term "heterologous" with respect to a sequence is a sequence derived from an alien species, or if derived from the same species, a sequence whose composition and / or genomic locus is substantially altered relative to its natural form by artificial deliberate intervention. The term "exogenous" with respect to a sequence is a sequence derived from an alien species. The term "orthologous" is a polynucleotide from a species that is functionally equivalent to a known reference sequence in another species (i.e., a species variant). The polynucleotide of interest may be from any organism of interest, including but not limited to prokaryotes, eukaryotes, non-humans, rodents, hamsters, mice, rats, humans, monkeys, birds, agricultural mammals, or non-agricultural mammals. The polynucleotide of interest may further comprise a coding region, a noncoding region, a regulatory region or a genomic DNA. Thus, any one of the first insertion polynucleotide, the second insertion polynucleotide, the third insertion polynucleotide, the fourth insertion polynucleotide, the fifth insertion polynucleotide, the sixth insertion polynucleotide, the seventh insertion polynucleotide and / or subsequent insertion polynucleotides may comprise such a sequence.

[0079] In one embodiment, as described above, the polynucleotide of interest can range from about 500 nucleotides to about 200 kb. The polynucleotide of interest can be from about 500 nucleotides to about 5 kb, from about 5 kb to about 200 kb, from about 5 kb to about 10 kb, from about 10 kb to about 20 kb, from about 20 kb to about 30 kb, from about 30 kb to about 40 kb, from about 40 kb to about 50 kb, from about 60 kb to about 70 kb, from about 80 kb to about 90 kb, from about 90 kb to about 100 kb, from about 100 kb to about 110 kb, from about 120 kb to about 130 kb, from about 130 kb to about 140 kb, from about 140 kb to about 150 kb, from about 150 kb to about 160 kb, from about 160 kb to about 170 kb, from about 170 kb to about 180 kb, from about 180 kb to about 190 kb, or from about 190 kb to about 200 kb.

[0080] The polynucleotide of interest inserted into the polynucleotide and / or inserted at the target genomic locus may encode a polypeptide, may encode an RNA, may encode a miRNA, or the polynucleotide of interest may comprise any regulatory or non-coding region of interest, including, for example, deletions of regulatory sequences, promoter sequences, enhancer sequences, transcriptional repressor binding sequences, Kozak consensus segments, start codons, or non-protein coding sequences, but not protein coding sequences.

[0081] In one embodiment, the insert nucleic acid comprises regulatory elements, including, for example, promoter, enhancer, or transcriptional repressor binding elements.

[0082] In a further embodiment, the insertion nucleic acid comprises conditional allele.In one embodiment, conditional allele is a multifunctional allele, as described in US2011 / 0104799, which is incorporated by reference as a whole. In a specific embodiment, conditional allele comprises: (a) a promoter sequence in a sense orientation relative to target gene transcription and a drug selection cassette in a sense or antisense orientation; (b) a nucleotide sequence of interest (NSI) and a conditional inversion module (COIN in an antisense orientation, which utilizes exon splitting introns and reversible gene trap-like modules; See, for example, US2011 / 0104799, which is incorporated by reference as a whole); and (c) a recombinant unit, which is recombined to form a conditional allele after being exposed to the first recombinase, wherein the conditional allele (i) lacks a promoter sequence and a DSC and (ii) contains an NSI in a sense orientation and a COIN in an antisense orientation.

[0083] In one embodiment, the genetic modification comprises a deletion of the Klhdc7b gene or a portion thereof, e.g., a deletion comprising, consisting essentially of, or consisting of the open reading frame of the Klhdc7b gene. In one embodiment, the nucleic acid sequence of the targeting vector may comprise a polynucleotide that, when integrated into the genome, results in genetic modification of a region of the Klhdc7b locus of the mammal, non-human animal, or non-human mammal, wherein the genetic modification at the Klhdc7b locus results in loss of function of Klhdc7b. In one embodiment, a Klhdc7b knockout ("null allele") is generated. In another embodiment, a disruption in the Klhdc7b locus is generated. In a further embodiment, the insertion of the nucleic acid results in replacement of a portion of the Klhdc7b gene of the mammal, non-human animal, or non-human mammal with an insert nucleic acid sequence comprising a heterologous sequence.

[0084] In some embodiments, the insert nucleic acid replaces a deleted endogenous Klhdc7b gene or a portion thereof (e.g., an orf portion thereof). Thus, in some non-human animal nucleic acid embodiments, genetic modification of the Klhdc7b locus can include replacing or inserting / adding the insert nucleic acid into the Klhdc7b locus or a portion thereof.

[0085] In some cases, the insert nucleic acid comprises a promoter. In one embodiment, the insert nucleic acid comprises a polynucleotide of interest operably linked to a promoter that drives the expression of the polynucleotide of interest. In one embodiment, the polynucleotide of interest comprises a reporter nucleic acid sequence. In another embodiment, the polynucleotide of interest comprises a selection marker nucleic acid sequence.

[0086] In one embodiment, the promoter is a constitutively active promoter.

[0087] In one embodiment, the promoter is an inducible promoter. In one embodiment, the inducible promoter is a chemically regulated promoter. In one embodiment, the chemically regulated promoter is an alcohol-regulated promoter. In one embodiment, the alcohol-regulated promoter is the alcohol dehydrogenase (alcA) gene promoter. In one embodiment, the chemically regulated promoter is a tetracycline-regulated promoter. In one embodiment, the tetracycline-regulated promoter is a tetracycline-responsive promoter. In one embodiment, the tetracycline-regulated promoter is a tetracycline operator sequence (tetO). In one embodiment, the tetracycline-regulated promoter is a tet-On promoter. In one embodiment, the tetracycline-regulated promoter is a tet-Off promoter. In one embodiment, the chemically regulated promoter is a steroid-regulated promoter. In one embodiment, the steroid-regulated promoter is the promoter for the rat glucocorticoid receptor. In one embodiment, the steroid-regulated promoter is the promoter for the estrogen receptor. In one embodiment, the steroid-regulated promoter is the promoter for the ecdysone receptor. In one embodiment, the chemically regulated promoter is a metal-regulated promoter. In one embodiment, the metal-regulated promoter is a metalloprotein promoter. In one embodiment, the inducible promoter is a physically regulated promoter. In one embodiment, the physically regulated promoter is a temperature regulated promoter. In one embodiment, the temperature regulated promoter is a heat shock promoter. In one embodiment, the physically regulated promoter is a light regulated promoter. In one embodiment, the light regulated promoter is a light inducible promoter. In one embodiment, the light regulated promoter is a photorepressible promoter.

[0088] In one embodiment, the promoter is a tissue-specific promoter. In one embodiment, the promoter is a neuron-specific promoter. In one embodiment, the promoter is a glial cell-specific promoter. In one embodiment, the promoter is a muscle cell-specific promoter. In one embodiment, the promoter is a cardiac cell-specific promoter. In one embodiment, the promoter is a kidney cell-specific promoter. In one embodiment, the promoter is a bone cell-specific promoter. In one embodiment, the promoter is an endothelial cell-specific promoter. In one embodiment, the promoter is an immune cell-specific promoter. In one embodiment, the immune cell promoter is a B cell promoter. In one embodiment, the immune cell promoter is a T cell promoter. In one embodiment, the promoter is a cochlear cell-specific promoter. In one embodiment, the cochlear cell-specific promoter is a hair cell-specific promoter. In one embodiment, the cochlear cell-specific promoter is a cochlear supporting cell-specific promoter.

[0089] In one embodiment, the promoter is a developmentally regulated promoter. In one embodiment, the developmentally regulated promoter is active only during the embryonic development stage. In one embodiment, the developmentally regulated promoter is active only in adult cells.

[0090] In a specific embodiment, the promoter can be selected based on the cell type. Thus, various promoters can be used in eukaryotic cells, mammalian cells, non-human mammalian cells, pluripotent cells, non-human pluripotent cells, human pluripotent cells, human ES cells, human adult stem cells, developmentally restricted human progenitor cells, human iPS cells, human cells, rodent cells, rat cells, mouse cells, hamster cells, fibroblasts, or CHO cells.

[0091] In some embodiments, the insertion nucleic acid comprises a nucleic acid that is flanked by a site-specific recombinant target sequence. Should be recognized that, although the whole insertion nucleic acid can be flanked by this type of site-specific recombinant target sequence, any region in the insertion nucleic acid or an independent polynucleotide of interest can also be flanked by this type of site. The site-specific recombinase can be introduced into the cell by any means, including by introducing the recombinase polypeptide into the cell or by introducing the polynucleotide of the coding site-specific recombinase into the host cell. The polynucleotide of the coding site-specific recombinase can be located in the insertion nucleic acid or in an independent polynucleotide. The site-specific recombinase can be operably connected to an active promoter in the cell, including for example an inducible promoter, to an endogenous promoter of the cell, to a promoter heterologous to the cell, a cell-specific promoter, a tissue-specific promoter or a developmental stage-specific promoter. Site-specific recombination target sequences that may flank the inserted nucleic acid or any polynucleotide of interest in the inserted nucleic acid may include, but are not limited to, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, or a combination thereof.

[0092] In some embodiments, the site-specific recombination sites flank the polynucleotide encoding a selectable marker and / or a reporter gene contained within the insert nucleic acid. In such cases, after the insert nucleic acid is integrated at the targeted locus, the sequence between the site-specific recombination sites can be removed.

[0093] In one embodiment, the insert nucleic acid comprises a polynucleotide encoding a selection marker. The selection marker may be contained in a selection cassette. Such selection markers include, but are not limited to, neomycin phosphotransferase (neo r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puromycin r ), blasticidin S deaminase (bsr r ), xanthine / guanine phosphoribosyltransferase (gpt) or herpes simplex virus thymidine kinase (HSV-k) or a combination thereof. In one embodiment, the polynucleotide encoding the selection marker is operably linked to a promoter active in the cell. In one embodiment, the polynucleotide encoding the selection marker is flanked by a site-specific recombination target sequence.

[0094] The insert nucleic acid may further comprise a reporter gene operably linked to a promoter. Such a reporter gene may be operably linked to a promoter active in the cell. Such a promoter may be an inducible promoter, a promoter endogenous to the reporter gene or the cell, a promoter heterologous to the reporter gene or the cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter.

[0095] In some cases, the insert nucleic acid comprises a reporter gene. In one embodiment, the reporter gene is located in the Klhdc7b locus and is operably linked to the endogenous Klhdc7b promoter. Such modification enables expression of the reporter gene driven by the endogenous Klhdc7b promoter. Alternatively, the reporter gene is not operably linked to the endogenous Klhdc7b promoter.

[0096] In some cases, the insert nucleic acid comprises a reporter gene. In one embodiment, the reporter gene is located in the Klhdc7b locus and is operably linked to the endogenous Klhdc7b start codon. Such modification may enable expression of the reporter gene driven by the endogenous Klhdc7b promoter.

[0097] Any reporter molecule (or detectable moiety) can be used in the methods and compositions provided herein. Non-limiting examples of reporter molecules include, for example, beta-galactosidase (encoded by the lacZ gene), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

[0098] The following description is a non-limiting example using the lacZ reporter gene encoding beta-galactosidase.The methods and compositions described herein can be performed using any reporter gene.

[0099] Also provided herein are polynucleotides or nucleic acid molecules comprising the various components employed in the targeted genomic integration system provided herein for targeting the Klhdc7b locus (i.e., any one or any combination of nuclease agents, recognition sites, insert nucleic acids, polynucleotides of interest, reporter sequences, targeting vectors, selectable markers, and other components).

[0100] The terms "polynucleotide," "polynucleotide sequence," "nucleic acid sequence," and "nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences, etc. A polynucleotide can be a polymer of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA can be composed of one or more segments of cDNA, genomic DNA, synthetic DNA, or a mixture thereof. A polynucleotide can include deoxyribonucleotides and ribonucleotides, including naturally occurring molecules and synthetic analogs, and any combination thereof. The polynucleotides provided herein also encompass all forms of sequence, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like.

[0101] Further provided are recombinant polynucleotides comprising the various components of a targeted genomic integration system for targeting the Klhdc7b locus. The terms "recombinant polynucleotide" and "recombinant DNA construct" are used interchangeably herein. Recombinant constructs comprise artificial or heterologous combinations of nucleic acid sequences, such as regulatory and coding sequences that do not occur together in nature. In other embodiments, recombinant constructs may comprise regulatory and coding sequences derived from different sources, or regulatory and coding sequences derived from the same source but arranged in a manner different from that found in nature. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector will depend on the method used to transform the host cell, as is well known to those skilled in the art. For example, plasmid vectors may be used. Also provided are the genetic elements required for successful transformation, selection, and propagation of host cells comprising any of the isolated nucleic acid fragments provided herein. Screening can be accomplished, inter alia, by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblot analysis of protein expression, or phenotypic analysis.

[0102] In specific embodiments, one or more components of the targeted genomic integration system for targeting the Klhdc7b locus described herein can be provided in an expression cassette for expression in prokaryotes, eukaryotes, bacteria, yeast cells, or mammalian cells, or other organisms or cell types of interest. The cassette can include a 5' regulatory sequence and a 3' regulatory sequence operably linked to a polynucleotide provided herein. "Operably linked" includes a relationship in which the operably linked components function in their intended manner. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional connection that enables expression of the polynucleotide of interest. Operably linked elements can be adjacent or non-adjacent. When used to refer to the joining of two protein coding regions, "operably linked" means that the coding regions are in the same reading frame. In another embodiment, a nucleic acid sequence encoding a protein can be operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.) to maintain proper transcriptional regulation. The cassette can additionally contain at least one additional polynucleotide of interest for co-introduction into an organism. Alternatively, the additional polynucleotides of interest can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for inserting a recombinant polynucleotide so that it is under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain a selectable marker gene.

[0103] The expression cassette can include a transcription and translation initiation region (i.e., a promoter), a recombinant polynucleotide as provided herein, and a transcription and translation termination region (i.e., a termination region) that is functional in a mammalian cell or host cell of interest in the 5'-3' transcriptional direction. The regulatory regions (i.e., promoters, transcriptional regulatory regions, Kozak sequences, and translational termination regions) and / or polynucleotides provided herein can be native / similar to the host cell or to each other. Alternatively, the regulatory regions and / or polynucleotides provided herein can be heterologous to the host cell or to each other. For example, the promoter operably linked to the heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or if from the same / similar species, one or both species are substantially modified relative to their original form and / or genomic locus, or the promoter is not the native promoter of the polynucleotide operably linked. Alternatively, the regulatory regions and / or recombinant polynucleotides provided herein can be completely synthetic.

[0104] The termination region may be naturally identical to the transcriptional initiation region, may be naturally identical to the operably linked recombinant polynucleotide, may be naturally identical to the host cell, or may be derived from another source (i.e., foreign or heterologous) of the promoter, the recombinant polynucleotide, the host cell, or any combination thereof.

[0105] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the correct orientation. To this end, adapters or linkers may be employed to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., transitions and transversions) may be involved.

[0106] A variety of promoters can be used for expression cassettes provided herein. Promoters can be selected based on the desired results. It should be recognized that different applications can be enhanced by using different promoters in expression cassettes to regulate the time, position and / or level of expression of the polynucleotide of interest. If desired, such expression constructs can also contain promoter regulatory regions (for example, a regulatory region that gives inducible, constitutive, environmental regulation or developmental regulation or cell-specific / selective or tissue-specific / selective expression), transcription initiation sites, Kozak consensus sequences, ribosome bind sites, RNA processing signals, transcription termination sites and / or polyadenylation signals.

[0107] The expression cassette containing the polynucleotides provided herein may also comprise a selectable marker gene for selecting transformed cells. The selectable marker gene can be used to select transformed cells or tissues.

[0108] Where appropriate, the sequences employed in the methods and compositions (i.e., polynucleotides of interest, nucleases, etc.) can be optimized to increase expression in cells. That is, genes can be synthesized using codons that are preferred in a given cell of interest, including, for example, mammalian preferred codons, human preferred codons, rodent preferred codons, mouse preferred codons, rat preferred codons, hamster preferred codons, etc., to improve expression.

[0109] The various methods and compositions provided herein can adopt selection markers. Various selection markers can be used for the methods and compositions disclosed herein. Such selection markers can, for example, confer resistance to antibiotics (such as G418, hygromycin, blasticidin, neomycin or puromycin). Such selection markers include neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror) and blasticidin S deaminase (bsrr). In other embodiments, the selection marker can be operably linked to an inducible promoter and the expression of the selection marker is toxic to the cell. Non-limiting examples of such selection markers include xanthine / guanine phosphoribosyltransferase (gpt), hypoxanthine-guanine phosphoribosyltransferase (HGPRT) or herpes simplex virus thymidine kinase (HSV-TK). The polynucleotide encoding the selection marker can be operably linked to an active promoter in the cell.

[0110] Targeting vector

[0111] A targeting vector is used to introduce an insert nucleic acid into the Khldc7b locus of interest in a eukaryotic, non-human, mammalian, non-human mammalian, human, rodent, mouse, rat, or hamster nucleic acid. In some embodiments, the nucleic acid molecule described herein (e.g., a targeting vector) comprises (i) a 5' homology arm upstream of the modified non-human animal Klhdc7b gene and (ii) a 3' homology arm downstream of the modified non-human animal Klhdc7b gene. In some embodiments, the 5' homology arm and the 3' homology arm are configured to undergo homologous recombination with the non-human animal Klhdc7b locus of interest, and upon homologous recombination with the non-human animal Klhdc7b locus of interest, the modified Klhdc7b gene replaces the non-human animal Klhdc7b gene at the non-human animal Klhdc7b locus of interest and is operably linked to an endogenous promoter that drives expression of the modified non-human animal Klhdc7b gene at the non-human animal Klhdc7b locus of interest. In some embodiments, the nucleic acid molecule (e.g., a targeting vector) comprises the nucleic acid sequence shown in SEQ ID NO:5, the nucleic acid sequence shown in SEQ ID NO:6, the nucleic acid sequence shown in SEQ ID NO:7, the nucleic acid sequence shown in SEQ ID NO:38, or the nucleic acid sequence shown in SEQ ID NO:39.

[0112] Various methods of using the genetically modified non-human animals described herein are also described.

[0113] Sequence Description

[0114] The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard alphabetical abbreviations for the nucleotide bases and the three-letter codes for the amino acids. The nucleotide sequences follow the standard convention of starting from the 5' end of the sequence and proceeding (i.e., from left to right in each row) to the 3' end. Only one strand of each nucleotide sequence is shown, but it is understood that the complementary strand is included by any reference to the displayed strand. The amino acid sequences follow the standard convention of starting from the amino terminus of the sequence and proceeding (i.e., from left to right in each row) to the carboxyl terminus.

[0115] Table 1: Summary description of sequences

[0116]

[0117]

[0118] Mouse Klhdc7b comprises the amino acid sequence set forth in SEQ ID NO:2 and is encoded by a nucleic acid comprising the sequence set forth in SEQ ID NO:1. A short isoform of mouse Klhdc7b can also be expressed, comprising the amino acid sequence set forth in SEQ ID NO:46 and encoded by the nucleotide sequence set forth in SEQ ID NO:45. The amino acid and coding sequences of human KLHDC7B are set forth in NCBI Accession Nos. NP_612442.3 (SEQ ID NO:48) and NM_138433.5 (SEQ ID NO:46), respectively. A short isoform of human KLHDC7B can also be expressed, comprising amino acids 642-1235 of SEQ ID NO:48 (set forth in SEQ ID NO:50) and encoded by nucleotides 2269-4053 of SEQ ID NO:47 (set forth in SEQ ID NO:49).

[0119] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides with modified peptide backbones. The term domain can refer to any portion of a protein or polypeptide that has a specific function or structure.

[0120] Proteins are said to have an "N-terminus" and a "C-terminus." The term "N-terminus" refers to the beginning of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH).

[0121] The terms "nucleic acid" and "polynucleotide," as used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modifications thereof. Nucleic acids and polynucleotides can include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers containing purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0122] Nucleic acids are considered to have a "5' end" and a "3' end" because mononucleotides react to make oligonucleotides in a way that the 5' phosphate of a mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. If the 5' phosphate of an oligonucleotide is not connected to the 3' oxygen of a mononucleotide pentose ring, the end of the oligonucleotide is referred to as the "5' end". If the 3' oxygen of an oligonucleotide is not connected to the 5' phosphate of another mononucleotide pentose ring, the end of the oligonucleotide is referred to as the "3' end". Even nucleic acid sequences located inside larger oligonucleotides are considered to have a 5' end and a 3' end. In linear or circular DNA molecules, discrete elements are referred to as being located "upstream" of the 5' element or "downstream" of the 3' element.

[0123] The term "genomic integration" refers to the introduction of a nucleic acid into a cell such that the nucleotide sequence is integrated into the cell's genome and can be inherited by its progeny. Any strategy can be used to stably incorporate a nucleic acid into the cell's genome.

[0124] As used herein, "embryonic stem cells" or "ES cells" include embryo-derived totipotent or pluripotent cells that are capable of contributing to the development of any tissue of the embryo after introduction into the embryo. The term "pluripotent cells" includes undifferentiated cells that have the ability to develop into more than one differentiated cell type.

[0125] As used herein, "targeting vector," "large targeting vector," or "LTVEC" includes targeting vectors for eukaryotic cells that are derived from fragments of cloned genomic DNA that are larger than fragments typically used by other methods for homologous gene targeting in eukaryotic cells. Examples of LTVECs include, but are not limited to, bacterial homologous chromosomes (BACs) and yeast artificial chromosomes (YACs). In general, a targeting vector can comprise a recombinant nucleic acid that can be introduced into a target location in the genome of a cell by homologous recombination, nonhomologous end joining-mediated ligation, or any other recombination means.

[0126] As used herein, a "site-specific recombination sequence" includes a nucleotide sequence that is recognized by a site-specific recombinase and can serve as a substrate for a recombination event.

[0127] As used herein, "site-specific recombinases" include a group of enzymes that can promote recombination between "site-specific recombining sequences." Examples of "site-specific recombinases" include, but are not limited to, Cre, Flp, and Dre recombinases.

[0128] As used herein, "germline" with reference to nucleic acid sequences includes nucleic acid sequences that are transmitted to progeny and, for example, are found in germ cells (eg, oocytes and sperm) of non-human animals.

[0129] As used herein, "operably linked" and the like refer to components that are linked together to function in their intended manner. In one instance, a nucleic acid sequence encoding a protein can be operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.) to retain appropriate transcriptional regulation. Operable linkage can include such sequences that are adjacent to or act in trans with each other (e.g., a regulatory sequence can act at a distance to control the transcription of a coding sequence). Operable linkage can also refer to one or more polypeptides being fused together, for example, as a fusion protein, such that each of the separate polypeptides retains its separate biological activity.

[0130] As used herein, "locus" refers to a DNA segment within a larger nucleic acid molecule that generally contains both non-coding and coding sequences of a gene. For example, the Klhdc7b locus generally contains non-coding and / or coding sequences of the Klhdc7b gene, which encodes the Klhdc7b protein.

[0131] The term "gene" refers to a DNA sequence encoding a product (e.g., an RNA product and / or a polypeptide product) in a nucleic acid and includes a coding region interrupted by non-coding introns and an untranslated region (UTR) near the coding region at the 5' end and the 3' end, such that the gene corresponds to a full-length mRNA (including a 5' untranslated sequence and a 3' untranslated sequence). The term "gene" may also include other non-coding sequences, including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequences, and matrix attachment regions. These sequences may be close to the coding region of a gene (e.g., within 10 kb) or at a distant site, and they affect the level or rate of transcription and translation of the gene.

[0132] As used herein, "open reading frame", "orf" and the like encompass a portion of a DNA molecule (e.g., a gene) that, when translated into amino acids, does not contain a stop codon. Generally speaking, an open reading frame spans the gene sequence between the start codon and the stop codon of a gene and may or may not include a start codon and / or a stop codon, but generally does not extend beyond a start codon or a stop codon. A "start codon" is the first codon that a gene or its messenger RNA (mRNA) transcript is translated by the ribosome. In eukaryotes, the start codon encodes methionine. A "stop codon" is a trinucleotide sequence within a gene or its mRNA transcript that indicates the termination of protein synthesis.

[0133] The term "allele" refers to a variant form of a gene. Some genes have multiple different forms that are located at the same position or genetic locus on a chromosome. Diploid organisms have two alleles at each genetic locus. Each pair of alleles represents the genotype of a particular genetic locus. If two identical alleles are present at a particular locus, the genotype is described as homozygous, and if the two alleles are different, the genotype is described as heterozygous.

[0134] " Promoter " is the regulatory region of DNA, which generally includes a TATA box that can guide RNA polymerase II to start RNA synthesis at the appropriate transcription start site of a specific polynucleotide sequence. The promoter may additionally include other regions that affect transcription initiation rate. Promoter sequences disclosed herein regulate the transcription of operably connected polynucleotides. Promoter can be active in one or more of the cell types disclosed herein (for example, eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, single cell stage embryos, differentiated cells or a combination thereof). Promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a time-limited promoter (for example, a developmentally regulated promoter) or a spatially restricted promoter (for example, a cell-specific or tissue-specific promoter). The example of a promoter can be found in, for example, WO2013 / 176772, which is incorporated herein by reference in its entirety for all purposes.

[0135] Hearing loss may refer to a decrease in one or more responses of a non-human animal to an auditory stimulus compared to one or more responses of a control (e.g., wild-type) non-human animal to an auditory stimulus. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 5% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 10% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 15% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 20% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 25% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 50% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, when the response of an animal genetically modified to comprise a nucleic acid as described herein to an auditory stimulus is reduced by about 55% compared to the response of a control (e.g., wild-type) non-human animal of the same age to an auditory stimulus, the animal may be considered to have hearing loss. In some embodiments, an animal genetically modified to contain a nucleic acid as described herein can be considered to have hearing loss when its response to auditory stimulation is reduced by about 60% compared to the response of a control (e.g., wild-type) non-human animal of the same age.

[0136] Hearing loss can be determined by a decrease in response to auditory stimulation (e.g., decreased function of inner ear hair cells, outer ear hair cells, and / or neurons (e.g., spiral ganglion neurons)) as measured by auditory brainstem response assays and / or distortion product otoacoustic emissions assays, both of which are generally described in Figure 8 Other assays that can be used to determine responses to auditory stimulation (e.g., function of inner hair cells, outer hair cells, and / or neurons such as spiral ganglion neurons) include assays well known in the art, such as electrocochleography (e.g., using needle electrodes or cotton wick electrodes) and compound action potentials.

[0137] In some embodiments, when the hearing threshold decibel (dB) level of an animal genetically modified to comprise a nucleic acid as described herein is greater than 1.5 to 2.0 times the hearing threshold dB level of a control (e.g., wild-type) animal, the animal exhibits hearing loss, e.g., at 8kHz, 16, and / or 32kHz, as measured by an auditory brainstem response assay. In some embodiments, when the hearing threshold decibel (dB) level of an animal genetically modified to comprise a nucleic acid as described herein is greater than or about 50dB, the animal exhibits hearing loss, e.g., at 8kHz, 16, and / or 32kHz, as measured by an auditory brainstem response assay. In some embodiments, when the hearing threshold decibel (dB) level of an animal genetically modified to comprise a nucleic acid as described herein is greater than or about 40dB, the animal exhibits hearing loss, e.g., at 8kHz, 16, and / or 32kHz, as measured by an auditory brainstem response assay. In some embodiments, when the hearing threshold decibel (dB) level of an animal genetically modified to comprise a nucleic acid as described herein is greater than or about 60 dB, the animal exhibits hearing loss, e.g., at 8 kHz, 16, and / or 32 kHz, as measured by an auditory brainstem response assay. In some embodiments, when the hearing threshold decibel (dB) level of an animal genetically modified to comprise a nucleic acid as described herein is greater than or about 70 dB, the animal exhibits hearing loss, e.g., at 8 kHz, 16, and / or 32 kHz, as measured by an auditory brainstem response assay. When the threshold decibel of an auditory stimulus that produces an auditory brainstem response in an auditory brainstem response assay is about or higher than 80-90 dB, it can be determined to be profound deafness, etc.

[0138] The term "viral vector" refers to a recombinant nucleic acid that contains at least one element of viral origin and includes elements sufficient or allowing for packaging into viral vector particles. The vector and / or particle can be used for the purpose of transferring DNA, RNA or other nucleic acids into cells in vitro or in vivo. Various forms of viral vectors are known.

[0139] The term "wild-type" includes an entity having structure and / or activity as found in a normal (as compared to mutated, diseased, altered, etc.) state or situation. Wild-type genes and polypeptides typically exist in multiple different forms (eg, alleles).

[0140] The expression "gross mutant phenotype" refers to a significant difference or change in phenotype between the engineered non-human mice of the present disclosure and the "wild type."

[0141] The term "endogenous" refers to a nucleic acid sequence that is naturally present in a nucleic acid, cell, or non-human animal. For example, an endogenous Klhdc7b sequence of a non-human animal refers to a native Klhdc7b sequence that is naturally present at the endogenous Klhdc7b locus of the non-human animal. Similarly, an endogenous Klhdc7b sequence of a non-human animal nucleic acid or cell refers to a native Klhdc7b sequence that is naturally present at the endogenous Klhdc7b locus of the non-human animal nucleic acid or cell.

[0142] The term "variant" refers to a nucleotide sequence that differs from the most prevalent sequence in a population (eg, by one nucleotide) or a protein sequence that differs from the most prevalent sequence in a population (eg, by one amino acid).

[0143] When referring to a protein, the term "fragment" or "portion" means a protein that is shorter or has fewer amino acids than the full-length protein. When referring to a nucleic acid, the term "fragment" or "portion" means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., a portion of the protein with the C-terminus removed), a C-terminal fragment (i.e., a portion of the protein with the N-terminus removed), or an internal fragment.

[0144] In the case of two polynucleotides or polypeptide sequences, "sequence identity" or "identity" refers to the identical residues in the two sequences when aligned over a specified comparison window to achieve maximum correspondence. When using percentage sequence identity for proteins, non-identical residue positions often differ due to conservative amino acid substitutions, in which amino acid residues are substituted by other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity), and therefore do not change the functional properties of the molecule. When the difference between the sequences is conservative substitutions, the percentage sequence identity can be adjusted upward to correct for the conservative nature of the substitutions. Sequences that differ due to such conservative substitutions are considered to have "sequence similarity" or "similarity." The means for making such adjustments are well-known. Typically, this involves counting conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Therefore, for example, where the score for identical amino acids is 1 and the score for non-conservative substitutions is zero, the score for conservative substitutions is between zero and 1. The score for conservative substitutions is, for example, calculated as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0145] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences (maximum number of perfectly matched residues) within a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (not containing additions or deletions) to optimally align the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Unless otherwise indicated (e.g., the shorter sequence includes linked heterologous sequences), the comparison window is the full length of the shorter of the two sequences being compared.

[0146] Unless otherwise indicated, sequence identity / similarity values ​​include values ​​obtained using GAP version 10 using the following parameters: % identity and % similarity for nucleotide sequences using a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent programs thereof. "Equivalent programs" include any sequence comparison program that, for any two sequences under consideration, produces an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP version 10.

[0147] The term "conservative amino acid substitution" refers to replacing an amino acid that is typically present in a sequence with a different amino acid having a similar size, charge or polarity. Examples of conservative substitutions include replacing another non-polar residue with a non-polar (hydrophobic) residue (such as isoleucine, valine or leucine). Similarly, examples of conservative substitutions include replacing another with a polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. In addition, replacing another basic residue with a basic residue such as lysine, arginine or histidine or replacing another acidic residue with an acidic residue such as aspartic acid or glutamic acid is another example of conservative substitution. Examples of non-conservative substitutions include replacing polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid or lysine with a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine or methionine, and / or replacing a non-polar residue with a polar residue. Typical amino acid classifications are summarized below.

[0148]

[0149]

[0150] "Homologous" sequences (e.g., nucleic acid sequences) include sequences that are identical or substantially similar to known reference sequences, such that they are, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to known reference sequences. Homologous sequences can include, for example, orthologous sequences and paralogous sequences. For example, homologous genes are typically derived from a common ancestral DNA sequence by speciation events (orthologous genes) or by genetic duplication events (paralogous genes). "Orthologous" genes include genes that evolved from a common ancestral gene by speciation in different species. Orthologs typically retain the same function during evolution. "Paralogous" genes include genes that are related by duplication within a genome. Paralogs can evolve new functions during evolution.

[0151] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when the event or circumstance occurs and instances when it does not.

[0152] A range of values ​​is specified to include all integers within or defining the range and all subranges defined by integers within the range.

[0153] Unless otherwise apparent from the context, the term "about" encompasses values ​​within the standard error of measurement (eg, SEM) of the stated value.

[0154] The term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0155] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0156] Unless the context clearly dictates otherwise, the singular articles "a," "an," and "the" include plural referents. For example, the term "a protein" or "at least one protein" may include a plurality of proteins, including mixtures thereof.

[0157] Statistically significant means p≤0.05, p<0.01, p<0.001, or p<0.0001.

[0158] While the invention has been particularly shown and described with reference to a number of embodiments, it will be understood by those skilled in the art that changes in form and details may be made in the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to limit the scope of the claims.

[0159] Example

[0160] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0161] Example 1: Characterization of KLHDC7b expression

[0162] According to the University of California at Santa Cruz (UCSC) genome browser, KLHDC7b has two possible isoforms that differ only in the presence of a methionine as the predicted start site (see Figure 1 Since the short isoform is completely contained within the long isoform, it is not possible to show the presence of the short isoform alone, but it is possible to show whether the long isoform is present. Therefore, two sets of primers and probes were designed. One set detects only the presence of transcripts within the long portion of the transcript, and the other set detects transcripts within the overlapping portion (interchangeably referred to herein as "short" or "overlap").

[0163] Using a mouse multi-tissue cDNA panel, we showed that KLHDC7b is expressed in multiple organs of mice, including liver, brain, and testis ( Figure 1 , lower left). The long probe and the overlapping probe showed similar expression levels, which may indicate that the short form may be rarely expressed alone in mice. Figure 1 (Middle bottom) also shows that the expression level of KLHDC7b in freshly collected mouse liver and kidney samples is comparable to that of the commercial cDNA kit. Figure 1 (Bottom middle) Data showing that KLHDC7b is also expressed in freshly isolated cochleae. High- and low-expressing tissues (liver and kidney, respectively) were collected according to the cDNA kit to confirm that the results of fresh tissue collection are comparable to those of the cDNA kit ( Figure 1 , middle and lower).

[0164] Long and overlapping probes were also designed for the human form of the transcript, which also has two putative isoforms, and were detected using a human multitissue cDNA set ( Figure 1qPCR was performed on the human (bottom right). Both probes indicated expression in human liver and testis, with slightly different tissue expression patterns compared to mice, and significantly less expression in the brain. The overlapping portion was expressed at higher levels than the long form, which may indicate that the short form can exist alone more frequently in humans than in mice.

[0165] To determine whether KLHDC7b is expressed throughout the mouse lifespan, mouse tissues were collected at four time points: postnatal day 1 (p1), postnatal day 7 (p7), postnatal weeks 11–28 (adult), and postnatal days 63–70 (old), and the expression of KLHDC7b was determined using overlapping ( Figure 2 , left) and long ( Figure 2 qPCR was performed using probes and primers (right). At p1, KLHDC7b expression was highest in the cochlea compared to all other tissues and remained relatively high in adult and aged mice. In other tissues, expression was higher in adults than in newborns. In the liver, expression was significantly increased in aged mice.

[0166] qPCR

[0167] qPCR probes against long and overlapping portions of the mouse (m) or human (h) KLHDC7b transcript were designed using BioSearch RealTimeDesign qPCR assay design software (RealTimeDesign qPCR assay design software | LGC Biosearch Technologies) and tested for specificity via the UCSC genome browser BLAT function. Table 2 provides the probes used in this example.

[0168] Table 2

[0169]

[0170]

[0171] The m_sKLHDC7b probe was designed for the portion of the transcript that overlaps between the two putative long and short isoforms. m_both_KLHDC7b was designed to span the junction between the long and short isoforms, and m_LKLDHC7b was designed for the portion of the transcript that is present only in the long isoform. For human transcripts, primer and probe designs were similar.

[0172] The KLHDC7b probe was ordered from IDT (Integrated DNA Technologies) as a Primetime qPCR assay in a FAM / ZEN / IBFQ dye combination. For experiments using mouse (Panel I, Catalog No. 636745, Panel III, Catalog No. 636757, Takara) and human (Panel I, Catalog No. 636742, Panel II, Catalog No. 636743, Takara) cDNA sets, cDNA was processed similarly to RNA and followed the protocol using Taqman Fast Advanced MasterMix (Example Catalog No. 4444557, Thermofisher Scientific). Plates were cycled up to 45 times on a Viaa7 thermal cycler. The housekeeping gene Drosha was used as a control for human and mouse experiments, and the Drosha probe was added to equal amounts of cDNA. Data were analyzed and plotted using GraphPad Prism.

[0173] To collect fresh tissue, mice were euthanized under CO2 or decapitated if they were under 7 days of age. Organs were collected and placed in RNA later. Cochleae were snap frozen to maintain RNA integrity for subsequent extraction. For RNA extraction: tissues / cells were homogenized in TRIzol and phase separated using chloroform. MagMAX was used according to the manufacturer's instructions. TM The aqueous phase containing total RNA was purified using the RT-PCR-96 Microarray Total RNA Isolation Kit (Ambion, Life Technologies), and genomic DNA was removed using the RNase-Free DNase Set (Qiagen).

[0174] use VILO TM mRNA was reverse transcribed into cDNA using Master Mix (Invitrogen, Life Technologies). cDNA was amplified using the SensiFAST Probe Lo-ROX (Meridian) using the 12K Flex System (Applied Biosystems). An endogenous control gene was used to normalize any cDNA input differences. Data reported are comparative CT methods using ΔCT compared to the housekeeping gene Drosha.

[0175] Example 2: Detection of KLHDC7b expression in the cochlea

[0176] After discovering that KLHDC7b is expressed in the cochlea, RNA scoping was performed to examine which cell types within the cochlea express KLHDC7b. Similar to the qPCR probes, two probes were generated for overlapping and long portions of the transcript. The probes were combined with immunostaining for the hair cell marker Myo7a. Both probes bound only to transcripts within hair cells. Both probes were present in inner hair cells, outer hair cells, and vestibular hair cells ( Figures 3B to 3C RNA scope was also performed on embryonic mice at approximately e16-17. Both probes were present in developing hair cells, indicating that KLHDC7b is expressed very early in development ( Figure 3D ).

[0177] RNA scope with or without antibody co-detection

[0178] Animals 21 days and older were sacrificed by transcardial perfusion with phosphate buffered saline (PBS) followed by transcardial perfusion with 4% paraformaldehyde (PFA) in PBS. Cochleae were dissected and placed in 4% PFA on a shaker for 1-4 hours or overnight, then washed three times with PBS and stored at 4°C until processed.

[0179] For RNA scoping in paraffin-embedded sections, bake the slides in a HybEZ oven at 60°C for 30 minutes. After baking, dewax the slides by washing them twice in xylene for 5 minutes each, followed by two washes in 100% ethanol for 1 minute each. Allow the slides to air dry.

[0180] Organoid slices were thawed at room temperature and then washed with agitation in PBS for 5 minutes to remove OCT. Slices were postfixed with cold 10% neutral buffered formalin (NBF) at 4°C for 15 minutes. They were then dehydrated in 50% ethanol, 70% ethanol, and 100% ethanol for 5 minutes each and allowed to dry at room temperature.

[0181] For paraffin-embedded cochlear sections and explants, hydrogen peroxide from the RNA scope kit was added to each slide and incubated at room temperature for 10 minutes, then washed twice with distilled water. The co-detection target repair reagent (ACD Bio) was heated in a vegetable steamer (Oster), and the slides were placed in the hot co-detection target repair reagent, the cochlea was placed for 15 minutes and the organoid was placed for 5 minutes, then washed twice with distilled water. The slides were then washed in PBS plus 0.1% Tween-20 (PBS-T). A hydrophobic barrier was drawn around the sections on the slides. Antibodies (Myo7a, Proteus, 25-6790) were diluted at a concentration of 1:200 in co-detection antibody diluent and incubated overnight at 4 degrees in a humidified chamber.

[0182] After the initial incubation, the slides were washed three times in PBS-T for 2 minutes each. They were then post-fixed in 10% neutral buffered formalin for 30 minutes in a fume hood. Afterwards, they were washed four more times in PBS-T for 2 minutes each. The slides were then treated with Protease Plus reagent (ACD Bio) in a HybEZ oven at 40°C for 30 minutes.

[0183] Fluorescence in situ hybridization or RNA scope was then performed according to the protocol from ACD Bio. In short, the probe was heated at 40 degrees for 10 minutes, the wash buffer was heated at 40 degrees for 20 minutes, and then diluted to the correct concentration. The probe was applied to the slide and placed in a humidified chamber of an oven for 2 hours at 40 degrees. All subsequent incubation steps were carried out in a humidified chamber at 40 degrees, and all washings were completed with wash buffer. After probe hybridization, the slide was washed 2 times with wash buffer for 2 minutes each. Then, for the amplification step, Amp 1 (ACD bio) was incubated for 30 minutes, followed by 2 washes of 2 minutes each. Amp 2 was incubated for 30 minutes, followed by 2 washes of 2 minutes each. Amp 3 was incubated for 15 minutes, followed by 2 washes of 2 minutes each.

[0184] After amplification, depending on the number of channels used, slides were incubated in HRP C1 for 15 minutes, followed by two 2-minute washes, and then incubated in Opal 520 at 1:15,000 for 30 minutes, followed by two 2-minute washes. Slides were incubated with HRP blocker for 15 minutes, followed by two 2-minute washes. Slides were then incubated with HRP C2 for 15 minutes, followed by two 2-minute washes, and then incubated with Opal 570 at 1:1500 for 30 minutes, followed by two 2-minute washes. Slides were then incubated again with HRP blocker for 15 minutes, followed by two 2-minute washes, and then in Opal 570 at 1:1500 for 30 minutes, followed by two 2-minute washes. The slides were then incubated again with HRP blocking reagent for 15 minutes, followed by two 2-minute washes, then incubated in 1:1500 Opal 690 for 30 minutes, two 2-minute washes, and a 15-minute HRP block. If only one channel was used, it was Opal 570, and the subsequent channel HRP step was skipped.

[0185] Finally, the secondary antibody (donkey anti-rabbit Alexa 647) was diluted to 1:500 in co-detection antibody diluent and incubated in a humidified chamber at room temperature for 30 minutes, followed by two 2-minute washes in PBS-T. DAPI was applied to the slides for 30 seconds, shaken off the slides, and then Prolong Gold Antifade mounting medium was applied and a coverslip was placed on the sections and allowed to dry overnight at room temperature.

[0186] For RNA scopes without co-detection (for organoid markers), the procedure is similar, but after antigen retrieval, slides are placed in 100% ethanol and allowed to dry overnight. Post-fixation after antibody incubation is skipped, and secondary antibody incubation is skipped.

[0187] microscope

[0188] Images of RNA scope and immunostained samples were acquired on a confocal microscope (Zeiss LSM 780 or Zeiss LSM 880) with 20x and 100x objectives. Z stacks were acquired depending on the size of the objective, and numerical aperture or tile scans were acquired as needed. Post-hoc image processing and analysis were performed using Fiji and Imaris (PLA quantification).

[0189] Example 3: Knockout of the endogenous mouse Klhdc7b gene

[0190] Using bacterial artificial chromosome (BAC) cloning and A targeting vector for knocking out the endogenous Klhdc7b gene was constructed using the technology (see, for example, U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotechnology 21(6):652-659; the patents and literature are incorporated herein by reference).

[0191] The BAC clone RP23-241G24 containing the mouse Klhdc7b gene was used and modified as follows. Briefly, a DNA fragment was generated containing a 100bp mouse 5' homologous nucleotide sequence (mHU), a LacZ gene (3,075bp) located downstream of the ATG start site of the mouse Klhdc7b gene and in frame with it, followed by a 4,809bp self-deleting neomycin cassette and a 100bp 3' mouse homologous sequence (mHD). This DNA fragment was used to modify the BAC clone RP23-241G24 by homologous recombination in bacterial cells. Thus, the complete knockout (KO) of the 3,787bp region encoding the mouse Klhdc7b genomic fragment in the BAC clone was replaced by an 8,202bp LacZ gene and a Neo-self-deleting cassette (SDC). Specifically, the entire mouse Klhdc7b orf (mm9, chr15:89,215,351-89,219,137) was replaced with the LacZ-Neo SDC insert, leaving intact the 5' untranslated region and the 3' untranslated region (UTR) ( Figures 4A to 4B The resulting modified BAC clone contained, from 5' to 3', (i) a 5' mouse homology arm containing approximately 140.5 kb of mouse genomic DNA including the mouse Klhdc7b 5'UTR and ATG; (ii) 3,075 bp of LacZ cDNA, (iii) an approximately 4,809 bp self-deleting neomycin cassette, followed by (iv) a 12.6 kb 3' mouse homology arm containing the mouse Klhdc7b 3'UTR and the remaining mouse genomic DNA in the original BAC clone ( Figures 4A to 4B The amino acid sequence of the protein encoded by LacZ cDNA is shown in SEQ ID NO: 4. The LacZ cDNA sequence is shown in SEQ ID NO: 3.

[0192] As described above, modified BAC clones containing the KO of the Klhdc7b gene were used to electroporate mouse embryonic stem (ES) cells to generate modified ES cells containing the Klhdc7b KO gene. Forward-targeted ES cells containing the Klhdc7b KO gene were identified by assays detecting the presence of LacZ and Neo sequences (Valenzuela et al., supra), and the loss and / or retention of the mouse Klhdc7b sequence was confirmed by TaqMan assays (SEQ ID NOs: 8-19, Table 3). Once the correctly targeted ES cell clones were selected, they were electroporated into early blastocysts (8-cell morula stage) to generate F0 mice. The neomycin selection cassette was removed by crossing the offspring generated by the ES clones with a deletion rodent strain expressing Cre recombinase. Figure 4C ).

[0193] use Methods (see, for example, US Pat. No. 7,294,754 and Poueymirou et al., 2007, Nature Biotech. 25(1):91-99) Selected ES cell clones (with or without the cassette) were implanted into female B6.Cast-Cdh23 Ahl+ mice to produce a litter containing the Klhdc7b KO allele. Ahl+ mice because mice on this background contain an allele-corrected form of Cdh23 that prevents the age-related hearing loss known to occur in C57BL6 mice.

[0194] Mice carrying the Klhdc7b KO allele were confirmed and identified by genotyping DNA isolated from tail snips using a modified allelic assay that detects the presence / absence of the Klhdc7b gene sequence (Valenzuela et al., supra). Pups were genotyped, and a cohort of animals heterozygous for the Klhdc7b locus was selected for characterization. Animals homozygous for the Klhdc7b locus were generated by crossing heterozygous animals.

[0195] Table 3

[0196]

[0197] Mice were born with normal Mendelian ratios and appeared phenotypically normal. LacZ staining was performed in KLHDC7B + / - (HET) and KLHDC7B - / - (KO) mice, where they appear to be hair cells ( Figure 4D ), confirming that the Klhdc7b gene was replaced with the LacZ cassette. Other cells in the cochlea, including vestibular hair cells, did not appear to show LacZ labeling. In addition, RNAScope probes against the long and overlapping transcripts of KLHDC7B did not label KLHDC7B. - / - Hair cells of (KO) mice ( Figure 4E ), thus confirming the absence of transcribed Klhdc7b mRNA.

[0198] LacZ staining of knockout mice

[0199] Mice were anesthetized with Ketamine / xylazine and fixed with 2% paraformaldehyde via perfusion. After perfusion, the tissue was dissected and cut into 1-5 mm fragments, fixed for 30 min at room temperature, washed for 30 min in PBS, and stained overnight in β-galactosidase (lacZ) staining solution at 4 ° C. After staining, the tissue was washed for 15 min in cold PBS and post-fixed overnight in 4% formaldehyde at 4 ° C. The tissue was made transparent by incubating in 50% glycerol at RT for one day and then incubating in 70% glycerol for one day. The tissue was photographed using a Zeiss dissecting microscope and stored in 70% glycerol at RT. Subsequently, the tissue was decalcified and dissected overnight with immunoglobulin and then photographed using an Axioscan slide scanner (Zeiss).

[0200] Example 4: Characterization of KLHDC7b knockout mice

[0201] The cochlear phenotype and auditory responses of the knockout animals as described in Example 3 were examined to determine the phenotypic effects of KLHDC7b knockout.

[0202] Histology

[0203] On postnatal day (p) 6, the organ of Corti of KO mice appeared normal, with intact hair cells stained for MYO7A and stereocilia stained for F-actin ( Figure 5A ). However, at p11, some outer hair cells appear absent and do not label for MYO7A or F-actin. By p21, many outer hair cells are absent and visible supporting cell scars are present, which are known to form when hair cells die (Wagner and Shin, 2019). Circular areas of MYO7A staining are present and may represent hair cells that have been engulfed by supporting cells or that have been extruded from the sensory epithelium. By 8 weeks of age, outer hair cells are almost absent and there is significant scarring ( Figure 5A At 8 weeks of age, hair cells in KLHDC7b knockout mice had abnormal morphology, with the base being worse than the apex ( Figure 5B In whole mount, hair cells appear absent, again more so at the base than at the apex ( Figure 6 In whole-mount samples taken at postnatal day 3 (p3), hair cells did not appear to be missing and stereocilia appeared normal ( 7A to 7C At postnatal day 6, hair cells also appeared normal. The tight junction protein ZO-1 showed diffuse cytoplasmic staining in some hair cells only at p11 and p21 in knockout mice, but not earlier, suggesting that this protein may be mislocalized in KO tissues around the time of cell death ( Figures 7D to 7J ).

[0204] ABR (auditory brainstem response) - measures the function of inner hair cells and neurons, and DPOAE (distortion product otoacoustic emissions) - measures the function of outer hair cells.

[0205] To determine the effect of KLHDC7b knockout on the hearing of mice, ABR and DPOAE audiometry were performed ( Figure 8 ). KLHDC7B - / - (KO), KLHDC7B + / - (Het) and KLHDC7B + / + (WT) mice underwent auditory brainstem response (ABR) testing at different ages to assess their hearing (Figure 9). Mice began to hear at two weeks of age. At postnatal day 17 (p17, 2-3 weeks), KO mice showed profound hearing loss, with significantly elevated hearing thresholds and significantly reduced wave I amplitudes. These thresholds progressively increased, with most mice showing no response at 11-15 weeks of age ( Figure 9A Compared with WT mice, heterozygous mice still had normal ABR thresholds and wave I amplitudes until 57 weeks ( Figure 9B At the onset of hearing loss (around day 17), KLHDC7b knockout mice showed hearing loss, and the ABR threshold was significantly elevated compared to wild-type mice ( Figure 9C Heterozygotes have no hearing loss ( Figure 9C Hearing loss appeared to be progressive. At 8 weeks, the knockout mice were profoundly deaf at all tested frequencies, but maintained some responses at the highest frequencies tested by ABR and DPOAE ( Figure 10 Heterozygous mice were no longer deaf at 30 weeks ( Figures 11A to 11C ).

[0206] Histology

[0207] For immunostaining of paraffin-embedded cochleae on slides, slides were first baked in a HybEZ oven at 60 degrees Celsius for one hour to melt the wax, followed by two three-minute washes in xylene. Slides were then rehydrated in 100% ethanol twice for three minutes each, then in 95% ethanol twice for three minutes each, then in 70% ethanol for three minutes, in 50% ethanol for three minutes, in distilled water for five minutes, and in 3xPBS for two minutes.

[0208] Make blocking solution [2% weight / volume bovine serum albumin, 5% normal donkey serum, 0.01% triton-x 100 in PBS]. Draw a hydrophobic barrier around the slice on the slide, and the slide is placed in a humidified chamber and covered with blocking solution, then incubated at room temperature for one hour. The primary antibody is diluted in blocking solution, the blocking agent on the slide is removed, and the primary antibody solution is added to the slide, then placed at 4 ° C and incubated overnight. After the initial incubation, the slide is washed three times with PBS, then covered with secondary antibody and cell stain diluted in blocking solution, and incubated for 1 hour. Afterwards, the slide is washed 3 times in PBS, then a mounting agent (Prolong Diamond or Prolong gold) is placed on the slide and covered with a cover slip, then allowed to dry overnight, and then imaged on a confocal microscope.

[0209] For whole-mount samples, the procedure is similar. Dissected sections are washed three times in PBS, incubated with blocking buffer for 1 hour at room temperature while shaking, incubated with primary antibodies in blocking solution overnight at 4°C or room temperature, and then washed three times with PBS. Secondary antibodies and cell stains are diluted in blocking buffer, and samples are incubated for 1 hour at room temperature, rinsed three times with PBS, then placed on slides and covered with mounting medium and coverslips, then allowed to dry overnight before imaging on a confocal microscope. Phalloidin is used only for whole mounts, not slides.

[0210] Auditory brainstem response (ABR)

[0211] Auditory brainstem response recordings were performed in a soundproof room. The equipment was calibrated daily using a microphone to confirm that the sound levels presented were as expected. Animals were anesthetized with an intraperitoneal injection of ketamine / xylazine (12 mg / kg, 0.5 mg / kg) and placed in a heated cage. After several minutes, once the animals were no longer responsive, Puralube ointment was placed on the eyes.

[0212] Once the mice no longer react to toe pinching, they are placed on a heating pad in a soundproof room. Electrodes are inserted into a preamplifier. The electrode pin end is placed subcutaneously, the positive electrode is placed at the animal cheek (near the cochlea), the negative electrode is placed at the midline of the skull, and the ground electrode is placed at the contralateral cheek. The recorded ear (right ear in these experiments) is located 7.5 mm from the speaker, in an open field configuration, rather than a closed field in a tube directly placed in the ear. The soundproof room is closed, and recording begins.

[0213] Recording is carried out at three pure tone frequencies (8kHz, 16kHz and 32kHz), and each stimulus is presented 512 times on average at each sound pressure level. Each frequency is played at 90 decibels (dB), followed by 80, 70, 60, and each time reduced by 10dB, until 50. From 50dB to 15dB, the dB level is reduced by 5dB each time, instead of 10. The waveform of the auditory brainstem response is recorded. When the waveform is no longer visible, the sound pressure level of the previous waveform is referred to as the threshold value of the frequency. Once the threshold value is reached, the stimulation of 1-2 sound pressure levels will be recorded, but subsequent recordings will be skipped. The threshold value of each animal is recorded and grouped by age or sex. The animal is then placed in a heated recovery cage and returned to the home cage once it can walk around.

[0214] ABR analysis

[0215] ABR recordings were processed in Matlab. Trajectories were first smoothed using a moving median filter with a kernel of 50 time points (each 10 ms recording contained 244 time points). This was done to remove slow wave noise occasionally observed in the recordings, with minimal impact on recordings without noise.

[0216] Two experimenters manually adjudicated ABR thresholds based on traces presented in a blinded, randomized order and run through an algorithm adapted from the Liberman lab (Suthakar and Liberman, 2019). Briefly, the covariance between pairs of adjacent decibel traces was calculated and plotted. These points were fitted to a curve using a sigmoid or logarithmic function, and the decibel level at which the function fell below a set standard level was recorded as the hearing threshold. The threshold called by the algorithm was compared with the manually adjudicated threshold. Traces with no discernible ABR response at 100 dB were adjudicated. If the difference between the manual and automatic thresholds was greater than 15 dB, the trace was checked and the manual threshold was used; otherwise, the automatic threshold was used.

[0217] For wave 1 amplitude and latency, peaks are detected using a semi-automatic method where estimates are made covering the peaks and valleys of wave 1 within a time window and then checked and corrected if necessary by the user.

[0218] Distortion product otoacoustic emissions (DPOAEs)

[0219] For DPOAE recordings, the speakers were also calibrated daily. DPOAE measurements were performed either alone while the animal was still anesthetized or immediately after the ABR. If collecting DPOAEs alone, the animal was anesthetized, just as for ABR recordings. After the animal no longer responded to the toe pinch, it was placed in the recording chamber.

[0220] Two loudspeakers were used to present two frequencies equidistant from the three frequencies measured around the ABR (8 kHz, 16 kHz, and 32 kHz). The loudspeakers were connected to a sensitive microphone using a closed-field configuration with a tube connected to a truncated pipette tip at the end, which was then placed in the ear canal and tilted toward the eardrum. The stimulus presented 100 averages. Distortion products were detected at the expected frequency, and if the signal was above the noise, the user manually judged it as positive.

[0221] Example 5 - Gentamycin-Texas Red Mechanotransduction Assay in Cochlear Explant Culture

[0222] Defects in mechanotransduction can lead to hair cell death, so the mechanotransduction (MET) complex is assessed before cell death begins. Mice do not begin to hear until two weeks of age, but mechanotransduction pathways can be assessed in cultured organs of Corti using the gentamicin-Texas Red assay (GtTR). Gentamycin is known to be taken up by the MET complex, and by combining it with the dye Texas Red, the presence of a functional MET complex can be determined. In cultured organs of Corti from KO and WT mice, GtTR labeled hair cells similarly, indicating that the MET complex is assembled and functional. Hair cells in cochlear explant cultures appear to take up similar amounts of GTTR ( 12A to 12C ), indicating that MET complexes may be functional and that KLHDC7b is not required for the assembly of these complexes.

[0223] Human inner ear RNA is extremely difficult to obtain due to the fragility of hair cells and the time required to remove the temporal bone. Therefore, to examine the expression of KLHDC7b in human ear tissue, ear organoids were generated using a protocol similar to Koehler et al., 2017. These organoids began with human induced pluripotent stem cells (iPSCs) and were given small molecules to coax them through multiple developmental stages to become mature inner ear organoids around day 70, at which point they contain hair cells, supporting cells, and neurons. Compared to the expression levels in human iPSCs, the expression of markers of inner ear cells, including neurons (TUBB3) and hair cells (OTOF, MYO7A, MYO15A) was significantly increased in mature ear organoids ( Figure 13A , right). Importantly, KLHDC7B expression levels were also increased in mature ear organoids, as measured using overlapping probes ( Figure 13B , right). Because otic organoids have spatially distinct regions with higher concentrations of inner ear cell types, RNA scoping was performed to examine the expression patterns of otic markers and KLHDC7b. We found that otic markers, including SOX2 and TUBB3, were concentrated in spatial regions of the otic capsule similar to those of Koehler et al., 2017 ( Figure 13A, left). RNAscope was performed for the long and overlapping transcripts of KLHDC7B and immunostained for the hair cell marker Myo7a. The KLHDC7B probe is shown to label sections of the same ear organoid ( Figure 13B (Image left). While labeling is not only present within hair cells, it is also concentrated in the surrounding area of ​​what appears to be the otic capsule. The two sections labeled with the long and overlapping probes are from nearby sections of the same otic organoid and are located in the same region of the organoid, near a circle of possible hair cells labeled with Myo7a, visible in the image with the overlapping KLHDC7B probe.

[0224] Cochlear explant culture

[0225] Cochlear explants were cultured from mouse pups aged 0-5 days after birth. The cell culture plates used were 35 mm culture dishes with round punched holes and glass coverslips glued to the bottom (Matsunami, No. D35-14-0-U). Collagen bubbles were prepared by mixing 1.7 μl 1N NaOH, 10 μl 10xPBS, 67 μl rat tail collagen (pipette set to 68 uL), and 21.3 μl H2O. Ten μl of this solution was placed on the coverslip and solidified in a humidified cell culture incubator at 37°C and 5% CO2 for 30-40 minutes. The collagen bubbles were covered with PBS before use and stored at 4 degrees for up to 2 months. Explant culture medium was prepared as follows: 90 ml DMEM / F12 without phenol red, 7 ml fetal bovine serum, 1 ml penicillin G, and 1 ml L-glutamine.

[0226] The mice were decapitated and the skulls were dissected. The cochlea was removed and placed in Leibovitz / L15 medium. The bones were opened and the organ of Corti was removed, removing the stria vascularis. The PBS was removed from the culture plate and replaced with cochlear explant medium. The organ of Corti was placed on the collagen bubble and most of the medium was removed to allow the explant to adhere to the collagen. The plate was placed in a humidified cell culture incubator at 37 degrees and 5% CO2. After the dissection was completed, 200 μl of explant medium was added to the culture dish and the culture was brought from the experimental animal room to the laboratory and 600 μl of medium was added.

[0227] Gentamicin-Texas Red (GtTR) assay

[0228] Explant cultures were maintained in culture medium and treated with gentamicin-Texas Red (GtTR) or Texas Red (TR) alone after 2-3 days. GtTR was 5 μg / ml and TR was 12.5 μg / ml. GtTR or TR was diluted in cell culture medium. The existing culture medium on the explants was removed, and 600 μL of culture medium containing GtTR or TR was added and treated for 20 minutes. The explant cultures were rinsed with culture medium, PBS, and then fixed in 4% PFA for 15 minutes and rinsed three times with PBS and stored at 4 degrees for several days before immunostaining using the same protocol as described for whole-mount cochleae.

[0229] ear organoids

[0230] Inner ear organoids were generated according to Zhang et al. (2021) "A simplified method for generating human inner ear organoids from pluripotent stem cells" PROTOCOL (Version 1) doi.org / 10.21203 / rs.3.pex-1708 / v1, which is incorporated herein by reference in its entirety. Briefly, human iPSCs were aggregated to form embryoid bodies in a chemically defined culture medium to induce ectodermal placode formation. After the 8th day of induction, the Wnt signaling agonist CHIR99012 (Tocris catalog number 4423) was added to the culture medium to stimulate otic capsule formation. Subsequently, the organoids were cultured in maturation medium for more than 100 days to allow the ear sensory epithelium to mature. For these experiments, organoids were harvested at day 70.

[0231] RNA scoping and immunofluorescence in ear organoids

[0232] 10 μm cryosections of fixed frozen fully differentiated ear organoids were used for RNA scope. Some slides were used for RNA scope in combination with immunofluorescence, while other slides were used only for RNA scope as described below. For the two conditions of RNAscope, the slides were baked in a HybEZ oven at 60 degrees for thirty minutes. After baking, the slides were post-fixed in cold 4% paraformaldehyde (PFA) in 1xPBS at 4 degrees for 15 minutes. The slides were then dehydrated at room temperature in 50% ethanol, 70% ethanol, and 100% ethanol for 5 minutes. The slides were allowed to air dry at room temperature.

[0233] Hydrogen peroxide from the RNA scope kit was added to each slide and incubated at room temperature for 10 minutes, followed by two washes with distilled water. Co-detection target repair reagent for co-processed slides or target repair reagent for RNA scope alone (ACD Bio) and separate containers of distilled water were heated in a vegetable steamer (Oster), and the slides were placed in hot distilled water for 10 seconds, then placed in co-detection target repair or target repair reagent for 5 minutes, followed by two washes in room temperature distilled water.

[0234] The co-detection slides were then washed four times for 2 minutes in PBS plus 0.1% Tween-20 (PBS-T). The individual RNA scope slides were dehydrated in 100% ethanol for 3 minutes and then air-dried. A hydrophobic barrier was drawn around the sections on the slides. The individual RNA scope slides were stored at room temperature overnight. The co-detection slides were treated with antibodies (Myo7a, Proteus, 25-6790) diluted 1:200 in co-detection antibody diluent and incubated overnight at 4 degrees in a humidified chamber.

[0235] For co-detection slides, after the initial incubation, the slides were washed three times in PBS-T for 2 minutes each. They were then post-fixed in 10% neutral buffered formalin for 30 minutes in a fume hood. Afterwards, they were washed again four times in PBS-T for 2 minutes each. The RNA scope and co-detection slides were then treated with Protease III reagent (ACD Bio) in a HybEZ oven at 40°C for 30 minutes.

[0236] Fluorescence in situ hybridization or RNA scope was then performed according to the protocol from ACD Bio. In short, the probe was heated at 40 degrees for 10 minutes, the wash buffer was heated at 40 degrees for 20 minutes, and then diluted to the correct concentration. The probe was applied to the slide and placed in a humidified chamber of an oven for 2 hours at 40 degrees. All subsequent incubation steps were carried out in a humidified chamber at 40 degrees, and all washings were completed with wash buffer. After probe hybridization, the slide was washed 2 times with wash buffer for 2 minutes each. Then, for the amplification step, Amp 1 (ACD bio) was incubated for 30 minutes, followed by 2 washes of 2 minutes each. Amp 2 was incubated for 30 minutes, followed by 2 washes of 2 minutes each. Amp 3 was incubated for 15 minutes, followed by 2 washes of 2 minutes each.

[0237] After amplification, slides were incubated in HRP C1 for 15 minutes, followed by two 2-minute washes, then incubated in 1:15,000 Opal 520 for 30 minutes, followed by two 2-minute washes. HRP blocking agent was then applied for 15 minutes, followed by two 2-minute washes. Slides were then incubated in HRP C2 for 15 minutes, followed by two 2-minute washes, then incubated in 1:15,000 Opal 570 for 30 minutes, followed by two 2-minute washes. HRP blocking agent was then applied for 15 minutes, followed by two 2-minute washes.

[0238] Separate RNA scope slides were treated with HRP C3 for 15 minutes, followed by two washes of 2 minutes each, and then treated with 1:15,000 Opal 690 for 30 minutes, followed by two washes of 2 minutes each. They were then treated with HRP blocking reagent for 15 minutes, followed by two washes of 2 minutes each.

[0239] For co-detection slides, the secondary antibody (donkey anti-rabbit Alexa 647) was diluted to 1:500 in co-detection antibody diluent and incubated in a humidified chamber at room temperature for 30 minutes, followed by two 2-minute washes in PBS-T. For both conditions, DAPI was applied to the slides for 30 seconds, shaken off the slides, and then Prolong Gold Antifade mounting medium was applied and a coverslip was placed on the sections and allowed to dry overnight at room temperature.

[0240] microscope

[0241] Acquire images of RNA scopes and immunostained samples on a confocal microscope using 20x and 100x objectives. Acquire Z-stacks or tile scans as needed.

[0242] Example 6 - Scanning Electron Microscopy

[0243] Although hair cells appear normal until they begin to die at p11, their morphology and function were examined before the onset of cell death. Scanning electron microscopy (SEM) examinations were performed before the onset of cell death (p10) and after significant cell death had occurred (p22). Similar regions of the organ of Corti are depicted in Figure 14 Both heterozygous mice (which do not develop a hearing loss phenotype until 50 weeks of age) and KO mice have largely normal stereocilia before cell death begins ( Figure 14 , left). Even after many outer hair cells are apparently absent at p22, the stereocilia on the remaining outer hair cells appear normal in morphology ( Figure 14 ,right).

[0244] Scanning electron microscopy

[0245] Cochlea is extracted from temporal bone.On apex, a small hole was drilled by hand, and stapes was taken out and pierced through oval window.Then sample was supplemented with 2mM CaCl 0.1M sodium cacodylate buffer (pH 7.4) (ElectronMicroscopy Sciences, Hatfield, PA) in 3% formaldehyde, 3% glutaraldehyde mixture in fixed overnight.Then sample was washed with 0.1M sodium cacodylate buffer, decalcified 24 hours in 10% formic acid (Immunocal), washed 3 times, and dissected into three tiling circles. Carefully from each circle, take out bone layer, striated ligament and tectorial membrane, and cochlear slices are placed in porous basket. Afterwards, the samples were dehydrated through a series of graded ethanol (5%, 10%, 20%, 40%, 60%, 80%, and 100%), critical point dried in liquid Co2 using a manual CPD (BAL-TEC 030), sputter-coated with 5 nm platinum, and imaged using a field emission scanning electron microscope (Zeiss Sigma VP).

[0246] Example 7 - Evaluation of Custom Monoclonal Anti-KLHDC7b Antibodies for Localization of KLHDC7b

[0247] Several custom monoclonal antibodies against mouse KLHDC7b were generated using transiently transfected HEK cells (Table 4). Cell lines stably expressing mouse long and short KLHDC7b isoforms were generated and used to test the binding of custom anti-KLHDC7b antibodies for phenotypic analysis. Genscript validated the cell lines for FLAG tagging by western blot and immunohistochemistry. Figure 15 As shown, the anti-KLHDC7b antibody only labeled the inner and outer hair cells of the wild-type mouse cochlea and appeared to be localized to the membrane. In addition, the anti-KLHDC7b antibody stained the plasma membrane of hair cells in paraffin-embedded cochlear sections isolated from wild-type mice, but not in KLHDC7b knockout mice ( Figure 16 The anti-KLHDC7b antibody also recognized human KLHDC7b expressed in transiently transfected HEK cells ( 17A to 17B ; Table 5).

[0248] Transient transfection of HEK cells with mouse KLHDC7B constructs to produce custom antibodies

[0249] HEK-293 cells were cultured in T75 flasks and transiently transfected with plasmids expressing FLAG-tagged short KLHDC7B (KLHDC7B-3XFLAG) and long KLHDC7B (KLHDC7B-3XFLAG). Untransfected cells served as negative controls. After 48-72 hours, cells were harvested for Western blotting or immunostaining, removed from the plate, fixed, embedded, and sectioned onto slides for antibody detection.

[0250] Table 4: Vectors designed for transfection of cell lines to express mouse Klhdc7b transcripts

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258] Stably transfected cell lines

[0259] Transfection was performed using Lipofectamine 3000 according to the manufacturer's instructions. On the day of transfection, the cell culture medium was changed, and the DNA was diluted in Opti-MEM containing P-3000. Lipofectamine was diluted in Opti-MEM. The DNA and liposome solution were then mixed and incubated at room temperature for 10-15 minutes. This mixture was added to the cells, and the cultures were examined or harvested after 48-72 hours. Cell lines were generated by Genscript using lentivirus to stably express the short or long isoforms of mouse KLHDC7b tagged at the C-terminus with a FLAG. The cell lines were cloned and verified using Western blotting for FLAG and immunohistochemistry for the FLAG tag.

[0260] Transient transfection of HEK cells with human KLHDC7B constructs for antibody testing

[0261] HEK-293 cells were grown in 96-well plates and transiently transfected with plasmids expressing FLAG-tagged short KL HDC7B-3XFLAG, long KL HDC7B-3XFLAG, and H2B-GFP-3XFLAG (Table 5) using Lipofectamine 3000 according to the manufacturer's instructions. After 48-72 hours, the cells were rinsed with PBS, fixed with 4% PFA for 20 minutes at room temperature, and rinsed three times with PBS. Immunostained with an antibody against FLAG (Thermofisher, MA1-91878) at a concentration of 1:200, and immunostained with each of the 10 reagent antibody clones at a concentration of 1:600. GFP-transfected samples were stained with anti-GFP (abcam, ab13970) and imaged using an Opera Phenix automated confocal microscope.

[0262] Table 5: Vectors designed for transfection of cell lines to express human Klhdc7b transcripts.

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] Example 8 - Discussion

[0278] KLHDC7b is expressed in the cochlea and other organs. Within the cochlea, KLHDC7b is expressed exclusively in hair cells, as measured by RNA scope and immunofluorescence using a custom-generated antibody. KLHDC7b is also expressed in human ear organoids, where both the long and overlapping portions of the transcript are detectable by RNA scope.

[0279] KLHDC7b does not appear to be required for the development of hair cells in the cochlea or inner ear, nor for the assembly of the mechanotransduction complex, but appears to be required for the maintenance of hearing. At the time point of profound deafness in mice, hair cells are absent, as indicated by immunohistochemistry and scanning electron microscopy.

[0280] KLHDC7b appears to localize to the plasma membrane of hair cells, as indicated by immunostaining with a custom anti-KLHDC7b antibody. KLHDC7b also appears to mislocalize to the cytoplasm in some hair cells as they begin to degenerate.

Claims

1. A genetically modified non-human animal nucleic acid comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of the endogenous Klhdc7b gene or a portion thereof.

2. The genetically modified non-human animal nucleic acid of claim 1, wherein the deletion comprises a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene.

3. The genetically modified non-human animal nucleic acid of claim 1 , wherein the deletion spans between, but does not include or extend beyond, the endogenous start codon of the endogenous Klhdc7b gene and the endogenous stop codon of the endogenous Klhdc7b gene.

4. The genetically modified non-human animal nucleic acid of any one of claims 1-3, wherein the modified endogenous Klhdc7b locus further comprises an insert nucleic acid, The inserted nucleic acid replaces the deleted endogenous Klhdc7b gene or a portion thereof.

5. The genetically modified non-human animal nucleic acid of claim 4, wherein the insert nucleic acid comprises a reporter gene.

6. The genetically modified non-human animal nucleic acid of claim 5, wherein the reporter gene is operably linked to a promoter, wherein the promoter drives the expression of the reporter gene.

7. The genetically modified non-human animal nucleic acid of claim 6, wherein the promoter is an endogenous Klhdc7b promoter, wherein the endogenous Klhdc7b promoter drives expression of the reporter gene.

8. The genetically modified non-human animal nucleic acid of any one of claims 5-7, wherein the reporter gene encodes a reporter molecule selected from the group consisting of β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

9. The genetically modified non-human animal nucleic acid of any one of claims 5-8, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the reporter gene.

10. The genetically modified non-human animal nucleic acid of any one of claims 4-9, wherein the insert nucleic acid comprises a gene encoding a selectable marker, and wherein the gene encoding the selectable marker is operably linked to a promoter.

11. The genetically modified non-human animal nucleic acid of claim 10, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the gene encoding the selectable marker.

12. The genetically modified non-human animal nucleic acid of any one of the preceding claims, wherein the non-human animal nucleic acid is a rodent nucleic acid.

13. The genetically modified non-human animal nucleic acid of claim 12, wherein the non-human animal nucleic acid is a rat nucleic acid.

14. The genetically modified non-human animal nucleic acid of claim 12, wherein the non-human animal nucleic acid is a mouse nucleic acid.

15. The genetically modified non-human animal nucleic acid of claim 14, wherein the modified endogenous Klhdc7b locus comprises: The nucleic acid sequence shown in SEQ ID NO: 5 and / or The nucleic acid sequence shown in SEQ ID NO: 6 or the nucleic acid sequence shown in SEQ ID NO: 7, and / or The nucleic acid sequence shown in SEQ ID NO: 38 or the nucleic acid sequence shown in SEQ ID NO:

39.

16. The genetically modified non-human animal nucleic acid of any one of claims 1 to 15, wherein the modified endogenous Klhdc7b locus comprises an endogenous 5' Klhdc7b untranslated region and / or an endogenous 3' Klhdc7b untranslated region, and wherein the endogenous 5' Klhdc7b untranslated region is located upstream of the deletion of the endogenous Klhdc7b gene or portion thereof, and wherein the endogenous 3' Klhdc7b untranslated region is located downstream of the deletion of the endogenous Klhdc7b gene or a portion thereof.

17. The genetically modified non-human animal nucleic acid of any one of claims 3 to 16, wherein the modified endogenous Klhdc7b locus comprises an endogenous 5' Klhdc7b untranslated region and / or an endogenous 3' Klhdc7b untranslated region, wherein the endogenous 5' Klhdc7b untranslated region is located upstream of the endogenous start codon of the endogenous Klhdc7b gene and is operably linked to the endogenous start codon of the endogenous Klhdc7b gene, and wherein the endogenous 3' Klhdc7b untranslated region is located downstream of the endogenous stop codon of the endogenous Klhdc7b gene and is operably linked to the endogenous stop codon of the endogenous Klhdc7b gene.

18. A non-human animal genome comprising the non-human animal nucleic acid of any one of claims 1-17, wherein the modified endogenous Kelch domain-containing 7B (Klhdc7b) locus of the non-human animal nucleic acid replaces the Klhdc7b locus of the non-human animal genome.

19. A genetically modified non-human animal cell comprising the genetically modified non-human animal nucleic acid of any one of claims 1-17 or the non-human animal genome of claim 18.

20. A genetically modified non-human animal cell comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of the endogenous Klhdc7b gene or a portion thereof.

21. The genetically modified non-human animal cell of claim 20, wherein the deletion comprises a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene.

22. The genetically modified non-human animal cell of claim 20 or claim 21, wherein the deletion spans between, but does not include or extend beyond, the start codon of the endogenous Klhdc7b gene and the stop codon of the endogenous Klhdc7b gene.

23. The genetically modified non-human animal cell of any one of claims 20-22, wherein the modified endogenous Klhdc7b locus comprises an insert nucleic acid, The inserted nucleic acid replaces the deleted endogenous Klhdc7b gene or a portion thereof.

24. The genetically modified non-human animal cell of any one of claims 20-23, wherein the insert nucleic acid comprises a reporter gene.

25. The genetically modified non-human animal cell of claim 24, wherein the reporter gene is operably linked to a promoter, wherein the promoter drives the expression of the reporter gene.

26. The genetically modified non-human animal cell of claim 25, wherein the promoter is an endogenous Klhdc7b promoter, The endogenous Klhdc7b promoter drives the expression of the reporter gene.

27. The genetically modified non-human animal cell of any one of claims 24-26, wherein the reporter gene encodes a reporter molecule selected from the group consisting of β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, and alkaline phosphatase.

28. The genetically modified non-human animal cell of any one of claims 23-27, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the reporter gene.

29. The genetically modified non-human animal cell of any one of claims 23-28, wherein the insert nucleic acid comprises a gene encoding a selectable marker, and wherein the gene encoding the selectable marker is operably linked to a promoter.

30. The genetically modified non-human animal cell of claim 29, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the gene encoding the selectable marker.

31. The genetically modified non-human animal cell of any one of claims 19-30, wherein the non-human animal cell is a rodent cell.

32. The genetically modified non-human animal cell of claim 31 , wherein the non-human animal cell is a rat cell.

33. The genetically modified non-human animal cell of claim 31 , wherein the non-human animal cell is a mouse cell.

34. The genetically modified non-human animal cell of claim 33, wherein the mouse cell is B6.Cast-Cdh23 Ahl+ Mouse cells.

35. The genetically modified non-human animal cell of claim 33 or claim 34, wherein the endogenous Klhdc7b locus comprises: The nucleic acid sequence shown in SEQ ID NO: 5 and / or The nucleic acid sequence shown in SEQ ID NO: 6 or the nucleic acid sequence shown in SEQ ID NO: 7, and / or The nucleic acid sequence shown in SEQ ID NO: 38 or the nucleic acid sequence shown in SEQ ID NO:

39.

36. The genetically modified non-human animal cell of any one of claims 19-35, wherein the non-human animal cell is a cochlear hair cell.

37. The genetically modified non-human animal cell of any one of claims 19-36, wherein the non-human animal cell is an embryonic stem (ES) cell or other pluripotent cell.

38. The genetically modified non-human animal cell of any one of claims 19-37, wherein the non-human animal cell is homozygous for the deletion.

39. The genetically modified non-human animal cell of any one of claims 19-38, wherein the non-human animal cell does not express a functional Klhdc7b protein.

40. A non-human animal comprising the genetically modified non-human animal nucleic acid of any one of claims 1-17, the non-human animal genome of claim 18, or the non-human animal cell of any one of claims 19-39.

41. A genetically modified non-human animal comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of the endogenous Klhdc7b gene or a portion thereof.

42. The genetically modified non-human animal of claim 41, wherein the deletion comprises a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene.

43. The genetically modified non-human animal of claim 41 or claim 42, wherein the deletion spans between, but not including or beyond, the start codon and the stop codon of the endogenous Klhdc7b gene.

44. The genetically modified non-human animal of any one of claims 41-43, wherein the modified endogenous Klhdc7b locus further comprises an insert nucleic acid, The inserted nucleic acid replaces the deleted endogenous Klhdc7b gene or a portion thereof.

45. The genetically modified non-human animal of claim 44, wherein the insert nucleic acid comprises a reporter gene.

46. ​​The genetically modified non-human animal of claim 45, wherein the reporter gene is operably linked to a promoter, wherein the promoter drives the expression of the reporter gene.

47. The genetically modified non-human animal of claim 46, wherein the promoter is an endogenous Klhdc7b promoter, wherein the endogenous Klhdc7b promoter drives expression of the reporter gene.

48. The genetically modified non-human animal of any one of claims 45-47, wherein the reporter gene encodes a reporter molecule selected from the group consisting of β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

49. The genetically modified non-human animal of any one of claims 45-48, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the reporter gene.

50. The genetically modified non-human animal of any one of claims 44-49, wherein the insert nucleic acid comprises a gene encoding a selectable marker, and wherein the gene encoding the selectable marker is operably linked to a promoter.

51. The genetically modified non-human animal of claim 50, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the gene encoding the selectable marker.

52. The genetically modified non-human animal of any one of claims 40-51, wherein the non-human animal is a rodent.

53. The genetically modified non-human animal of claim 52, wherein the non-human animal is a rat.

54. The genetically modified non-human animal of claim 52, wherein the non-human animal is a mouse.

55. The genetically modified non-human animal of claim 54, wherein the mouse is B6.Cast-Cdh23 Ahl+ mouse.

56. The genetically modified non-human animal of claim 54 or claim 55, wherein the endogenous Klhdc7b locus comprises: The nucleic acid sequence shown in SEQ ID NO: 5 and / or The nucleic acid sequence shown in SEQ ID NO: 6 or the nucleic acid sequence shown in SEQ ID NO: 7, and / or The nucleic acid sequence shown in SEQ ID NO: 38 or the nucleic acid sequence shown in SEQ ID NO:

39.

57. The genetically modified non-human animal of any one of claims 40-56, wherein the non-human animal is homozygous for the deletion.

58. The genetically modified non-human animal of claim 57, wherein the non-human animal lacks expression of functional Klhdc7b protein in cochlear hair cells.

59. The genetically modified non-human animal of claim 57 or claim 58, wherein the genetically modified non-human animal exhibits hearing loss and / or deafness approximately 17 days after birth.

60. The genetically modified non-human animal of any one of claims 57-59, wherein: The genetically modified non-human animal exhibits normal cochlear development compared to a wild-type control non-human animal; The genetically modified non-human animal exhibits hearing loss approximately 17 days after birth; The genetically modified non-human animal exhibits hearing loss about 17 days after birth and the hearing loss increases throughout the life of the genetically modified non-human animal; The genetically modified non-human animal exhibits profound deafness by about 8 weeks of age and / or complete hearing loss by about 11-15 weeks of age; The genetically modified non-human animal exhibits a loss of hair cells in the cochlea compared to a wild-type control non-human animal, optionally wherein the hair cells appear normal at birth, further optionally wherein degeneration of hair cells is observed after 11 days after birth; and / or The genetically modified non-human animals exhibit normal mechanotransduction in the cochlea as measured by a gentamicin-Texas Red assay.

61. A method for producing Klhdc7b knockout non-human animal pluripotent cells, comprising deleting an endogenous Klhdc7b gene or a portion thereof to form a modified endogenous Klhdc7b locus.

62. The method of claim 61, wherein deleting the endogenous Klhdc7b gene or a portion thereof comprises deleting an orf of the endogenous Klhdc7b gene.

63. The method of claim 61, wherein deleting the endogenous Klhdc7b gene or a portion thereof consists essentially of or consists of deleting an orf of the endogenous Klhdc7b gene.

64. The method of any one of claims 61-63, wherein deleting the endogenous Klhdc7b gene or a portion thereof comprises replacing the endogenous Klhdc7b gene or a portion thereof with an insert nucleic acid.

65. The method of claim 64, wherein the insert nucleic acid comprises: Reporter gene; a reporter gene operably linked to a promoter, wherein the promoter drives expression of the reporter gene; a reporter gene operably linked to an endogenous Klhdc7b promoter, wherein the endogenous Klhdc7b promoter drives expression of the reporter gene; a reporter gene, wherein the reporter gene encodes a reporter molecule selected from the group consisting of: oβ-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof; a reporter gene flanked by site-specific recombination sequences; a gene encoding a selectable marker; a gene encoding a selectable marker operably linked to a promoter; and / or A gene encoding a selectable marker flanked by site-specific recombination sequences.

66. The method of any one of claims 61-65, wherein deleting comprises contacting the non-human animal pluripotent cells with a targeting vector comprising a genetically modified non-human animal nucleic acid of any one of claims 1-17, wherein the targeting vector comprises a 5' homology arm upstream of the genetically modified non-human animal nucleic acid and a 3' homology arm downstream of the genetically modified non-human animal nucleic acid, and The 5' homology arm and the 3' homology arm target the endogenous Klhdc7b locus of the non-human animal pluripotent cells.

67. The method of any one of claims 61-66, wherein the non-human animal pluripotent cells are rodent pluripotent cells.

68. The method of any one of claims 61-67, wherein the non-human animal pluripotent cells are rat pluripotent cells.

69. The method of any one of claims 61-67, wherein the non-human animal is a mouse pluripotent cell.

70. The method of any one of claims 61-69, wherein the non-human animal pluripotent cells are non-human animal embryonic stem (ES) cells.

71. A method for producing a Klhdc7b knockout non-human animal, comprising incubating the non-human animal ES cell of claim 70 in a surrogate mother, wherein the surrogate mother produces an offspring non-human animal comprising the modified endogenous Klhdc7b locus in its germline genome.

72. The method of claim 71, further comprising breeding the progeny to produce offspring homozygous for the modified endogenous Klhdc7b locus.

73. The method of claim 71 or 72, wherein the non-human animal, surrogate mother, and offspring are each rodents.

74. The method of any one of claims 71-73, wherein the non-human animal, surrogate mother, and offspring are each rats.

75. The method of any one of claims 71-73, wherein the non-human animal, surrogate mother, and offspring are each a mouse.

76. A non-human animal tissue comprising the genetically modified non-human animal nucleic acid of any one of claims 1-17, the non-human animal genome of claim 18, or the non-human animal cell of any one of claims 19-39.

77. The non-human animal tissue of claim 76, wherein the non-human animal tissue is isolated from the non-human animal of any one of claims 40-60 or a non-human animal produced by the method of any one of claims 61-75.

78. The non-human animal tissue of claim 76 or claim 77, wherein the non-human animal tissue comprises cochlear explant cells.

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