Animals containing an altered KLHDC7B locus

Abstract

Genetically modified non-human animals lacking Klhcd7b expression are described. Methods and compositions for disrupting, deleting, and / or replacing the Klhcd7b coding sequence are described. Genetically modified mice that can be used as models of deafness or severe hearing loss are also described. Also described are cells, tissues, and embryos genetically modified to contain a loss-of-function form of Klhcd7b. Described herein are nucleic acids (e.g., non-human animal nucleic acids isolated from non-human animals) comprising a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, non-human animal cells, and non-human animals, wherein the modified endogenous Klhdc7b locus comprises a deletion of the endogenous Klhdc7b gene or a portion thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 482,724, filed February 1, 2023, U.S. Provisional Application No. 63 / 484,087, filed February 9, 2023, and U.S. Provisional Application No. 63 / 585,784, filed September 27, 2023, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Reference to sequence listing submitted as an XML file The sequence listing set forth in file "11348WO01 Sequence Listing XML" is 119 kilobytes, was created on January 31, 2024, and is incorporated herein by reference in its entirety.

[0003] Non-human animals, cells, and tissues containing modified Klhdc7b loci, which may include deletions and / or substitutions of the endogenous Klhdc7b gene or portions thereof, and methods for making and using them are described. The described non-human animals may have a phenotype consistent with hearing loss. [Background technology]

[0004] Human hearing impairment is associated with social isolation and cognitive decline and is a continuing problem in the medical fields of otology and audiology. Approximately 1.5 billion people suffer from hearing loss, and over 34 million children exhibit hearing loss or hearing loss.

[0005] Currently, there are very few cases of hearing loss that can be cured. Hearing devices such as hearing aids have limitations, such as an inability to improve speech intelligibility. Fewer than 20% of people affected by hearing impairment currently use hearing aids. In the case of age-related or noise- or drug-induced hearing impairment, the only effective method currently available to "treat" the impairment or reduce its severity is often prevention, such as avoiding excessive noise and using ear protection, practicing a healthy lifestyle, and, where possible, avoiding exposure to ototoxic drugs and substances.

[0006] The prevalence of hearing loss after damage to the mammalian cochlea has been attributed to the failure of hair cells and / or neurons, the key components for sound detection, to spontaneously regenerate. Humans are born with approximately 15,000 inner ear hair cells, which do not regenerate after birth.

[0007] Thus, there remains a long-felt need to determine the biological effects and mechanisms involved in protecting auditory hair cells before injury and / or preserving / promoting the function of existing cells after injury. Summary of the Invention [Means for solving the problem]

[0008] 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 the endogenous Klhdc7b gene or a portion thereof. The deletion can comprise, consist essentially of, or consist of a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene in the endogenous Klhdc7b locus of the non-human animal nucleic acid, non-human animal cell, and non-human animal, for example, the deletion extends between, but does not include, or does not 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 be the result of replacing 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 can include a reporter gene and / or a gene encoding a selectable marker, optionally where the reporter gene is operably linked to a promoter (e.g., the endogenous Klhdc7b promoter), and / or the gene encoding the selectable marker is operably linked to a promoter (e.g., the endogenous Klhdc7b promoter), and / or 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 as SEQ ID NO: 5, and / or (ii) the nucleic acid sequence set forth as SEQ ID NO: 6 or the nucleic acid sequence set forth as SEQ ID NO: 7, and / or (iii) the nucleic acid sequence set forth as SEQ ID NO: 38 or the nucleic acid sequence set forth as SEQ ID NO: 39, and / or (iv) the endogenous 5'Klhdc7b untranslated region, optionally an intact endogenous 5'Klhdc7b untranslated region, and / or (v) the endogenous 3'Klhdc7b translated region, optionally an intact endogenous 3'Klhdc7b untranslated region.Generally, the endogenous 5'Klhdc7b untranslated region, optionally an intact endogenous 5'Klhdc7b untranslated region described herein, can be located upstream of the deletion of the Klhdc7b gene or a portion thereof, e.g., upstream of the Klhdc7b start codon, and / or the endogenous 3'Klhdc7b translated region, optionally an intact endogenous 3'Klhdc7b untranslated region, can be located downstream of the deletion of the Klhdc7b gene or a portion thereof, e.g., downstream of the endogenous stop codon of the endogenous Klhdc7b gene. In some embodiments, the non-human animals described herein, e.g., mice homozygous for an altered Klhdc7b locus, can serve as models of hearing loss.

[0009] This patent or application contains at least one drawing executed 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. [Brief explanation of the drawings]

[0010] [Figure 1]The predicted long or short isoforms of mouse or human KLHDC7b transcripts (top panel) are shown, along with the transcript levels (delta Ct; y-axis) of long (open bars) or short (filled bars) KLHDC7b transcripts found in tissue samples (x-axis) from a commercial mouse cDNA panel (bottom left), freshly isolated tissues (bottom center), or a commercial human cDNA panel (bottom right). Lower values ​​indicate higher expression. Error bars are SEM using 3–4 technical replicates. All data are normalized to the housekeeping gene, Drosha. Figure 1 shows that KLHDC7b transcripts are found in the cochlea and other organs, and that their expression patterns differ slightly between mice and humans. The mouse gene long isoform, including its UTR, is located at mm10 chr15:89,384,917–89,388,867 and is 3,951 nucleotides in size. The coding region is located at mm10 chr15:89,384,917-89,388,708, is 3,792 nucleotides in size, contains one exon, and is a short isoform located at mm10 chr15:89,386,891-89,388,708, is 1,818 nucleotides in size, contains one exon, and does not contain annotated UTRs. 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 (including UTRs) of the human gene is located at hg38 chr22:50,545,899-50,551,023 and is 5,125 nucleotides in size. There is one coding exon on the plus strand. The coding region is located at hg38 chr22:50,546,244-50,549,951 and is 3,708 nucleotides in size.The putative short human isoform (including UTRs) is located at hg38 chr22:50,548,033-50,551,022 and is 2,990 nucleotides in size. The coding region is located at hg38 chr22:50,548,167-50,549,951 and is 1,785 nucleotides in size. The long forward primer is located at chr22:50546689-50546708 on the plus strand, the reverse primer is located at chr22:50546769-50546789 on the minus strand, and the probe is located at chr22:50546712-50546731. The overlapping forward primer is located at chr22:50549582 to 50549600 on the plus strand, the reverse primer is located at chr22:50549649 to 50549668 on the minus strand, and the probe is located at chr22:50549601 to 50549620. [Figure 2-1] Transcription levels (ΔCT; y-axis) of the duplicated KLHDC7b transcripts found in liver (squares), brain (circles), temporal bones including the cochlea (triangles), or kidneys (diamonds) isolated from mice at postnatal day 1 (p1), postnatal day 7 (p7), 11–28 weeks of age (adult), or 63–70 weeks of age (aged). Each P1 and P7 time point consisted of five mice. Mice were not sexed at this age. Adult and aged mice were heterozygous for the B6.CAST-Cdh23Ahl+ / Kjn corrector allele. Adult mice were four females aged 11–14 weeks and one male aged 24 weeks. Aged mice were four females aged 63–73 weeks. Each data point represents a biological replicate of tissue from one mouse and is calculated from the average of three technical replicates. All data are normalized to the housekeeping gene, Drosha. Analysis was performed via two-way ANOVA with Tukey's test used for post-hoc comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 2 shows that KLHDC7b expression changes over the mouse lifespan and is consistently expressed at relatively high levels in the cochlea. [Figure 2-2]The transcript levels (ΔCT; y-axis) of the long KLHDC7b transcript are shown in the liver (squares), brain (circles), temporal bones including the cochlea (triangles), or kidneys (diamonds) isolated from mice at postnatal day 1 (p1), postnatal day 7 (p7), 11–28 weeks of age (adult), or 63–70 weeks of age (aged). Each P1 and P7 time point consisted of five mice. Mice were not sexed at this age. Adult and aged mice were heterozygous for the B6.CAST-Cdh23Ahl+ / Kjn corrector allele. Adult mice were four females aged 11–14 weeks and one male aged 24 weeks. Aged mice were four females aged 63–73 weeks. Each data point represents a biological replicate of tissue from one mouse and is calculated from the average of three technical replicates. All data are normalized to the housekeeping gene, Drosha. Analysis was performed via two-way ANOVA with Tukey's test used for post-hoc comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Figure 2 shows that KLHDC7b expression changes over the mouse lifespan and is consistently expressed at relatively high levels in the cochlea. [Figure 3A-1] Images of histological sections of the cochlea from adult wild-type mice, shown alongside a schematic diagram of cochlear anatomy (not to scale), are provided. These sections were labeled with RNA probes detecting overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling appears as small punctae, which are visible only within hair cells. [Figure 3A-2]Shown alongside a schematic diagram of cochlear anatomy (not to scale) are images of histological sections of the cochlea from adult wild-type mice labeled with RNA probes detecting overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling is represented as small dots, which are visible only within hair cells. [Figure 3A-3] Shown alongside a schematic diagram of cochlear anatomy (not to scale) are images of histological sections of the cochlea from adult wild-type mice labeled with RNA probes detecting overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling is represented as small dots, which are visible only within hair cells. [Figure 3A-4] Shown alongside a schematic diagram of cochlear anatomy (not to scale) are images of histological sections of the cochlea from adult wild-type mice labeled with RNA probes detecting overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling is represented as small dots, which are visible only within hair cells. [Figure 3B]These images show histological sections of the cochlea from an adult wild-type mouse, with the organ of Corti shown enlarged. The sections were labeled with RNA probes detecting either the overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling appears as small dots, which are visible only within hair cells. The inset of the organ of Corti (center) shows staining with each probe (long and overlapping), the hair cell marker MYO7A, and DAPI. Each probe is shown together with MYO7A (top two images on the right) and alone (bottom two images on the right). Neither probe labeled regions outside of hair cells, indicating hair cell-specific expression. [Figure 3C] These images show histological sections of the cochlea from an adult wild-type mouse, with the vestibular system magnified. These sections were labeled with RNA probes detecting either the overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling appears as small dots, which are visible only within hair cells. Similarly, in Figure 3C, vestibular hair cells are shown with MYO7A labeling and each probe (top two images on the right), and each probe is also presented alone (bottom two images on the right), demonstrating that the probes overlap only with hair cells. [Figure 3D]Images of histological sections of embryonic mouse cochleae are shown, labeled with RNA probes detecting either the overlapping long and short KLHDC7b isoforms (red) or the long KLHDC7b isoform alone (green), and immunostained for the hair cell marker Myo7a (white). These images demonstrate that KLHDC7b is expressed exclusively in hair cells within the cochlea. The probe labeling appears as small dots, which are visible only within hair cells. Figure 3D also shows that both long KLHDC7b and overlapping KLHDC7b dots (bottom two images on the right) colocalize with Myo7a (top two images on the right) in labeled developing hair cells from embryonic mice. [Figure 4A] A diagram (not to scale) of the deletion length of the mouse Klhdc7b gene and its open reading frame (orf) (e.g., the genomic sequence spanning but not including the "start" and "stop" codons of the mouse Klhdc7b gene) is provided. The 3,787 bp 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 location of the upstream primer (4929mTU) and downstream primer (4929mTD2) for the allelic loss assay. The general location of the sequence encoding the Kelch domain is also shown. [Figure 4B]FIG. 1 provides a diagram (not to scale) of the large targeting vector (LTVEC) generated after replacing the open reading frame of the mouse Klhdc7b gene found in BAC clone RP23-241G24 with an 8,802 bp insert nucleic acid ("LacZ, Neo-SDC"). As shown, the LTVEC contains (a) a 140.5-kb 5' homology arm containing the intact 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 nucleic acid containing a self-deleting cassette (Neo-SDC; black arrow) containing the LacZ gene inserted in frame with the mouse Klhdc7b start codon (gray arrow) and the neomycin gene (Neo) flanked by LoxP site-specific recombination sequences, and (c) a 12.6-kb 3' homology arm containing the intact 3' untranslated sequence of the mouse Klhdc7b gene from BAC clone RP23-241G24. "A" indicates the location of the 5' mouse UTR / / start, Acc65 / / 5' LacZ junction (SEQ ID NO: 5), and "B" indicates the location of the 3' Neo / / (loxP) / / NheI / / 3' mouse UTR junction (SEQ ID NO: 6). The sequences of these junctions are also provided. [Figure 4C] A diagram (not to scale) of the modified Klhdc7b locus after targeted homologous recombination with LTVEC and deletion of the neomycin cassette in Figure 4B is provided. The untranslated region is depicted as an open rectangle, and the lacZ gene is depicted with a filled arrow. The positions of the 5' homology arm containing the 5' untranslated sequence of the mouse Klhdc7b gene and the mouse Klhdc7b start codon, as well as the 3' homology arm (12.6 kb from RP23-241G24) containing the 3' untranslated sequence of the mouse Klhdc7b gene, are also shown. "A" indicates the position of the 5' mouse UTR / / start, Acc65 / / 5' LacZ junction (SEQ ID NO: 5), and "C" indicates the position of the 3' LacZ / / stop / / (LoxP) / / NheI / / 3' mouse UTR junction (SEQ ID NO: 7). The sequences of these junctions are also provided. The LacZ protein expressed from the modified Khldc7b locus depicted in this figure comprises the amino acid sequence shown as SEQ ID NO:4. [Figure 4D] Microscopic images are provided showing that LacZ expression is present in hair cells of heterozygous (HET) and knockout (KO) mice. Both mice were 10-week-old males. LacZ staining is visible only in both inner and outer hair cells. Darker shading indicates LacZ expression. [Figure 4E] Fluorescence microscopy images of wild-type and KLHDC7B- / - (KO) mouse cochleae are shown. RNA scoping was performed using a KLHDC7B probe for the long and overlapping transcripts. The transcripts are absent in the hair cells of the knockout mouse cochlea. The top image is from a wild-type (WT) cochlea, and the bottom image is from a KO cochlea. The leftmost column shows a 40x magnification image of all four fluorescent channels (Myo7a, DAPI, the long KLHDC7B probe, and the overlapping KLHDC7B probe) representing one entire turn of the cochlea. The second column from the left is a merged fluorescent image magnified to show only the hair cells. The third column shows a magnified single-channel fluorescent image of a small spot showing labeling by the overlapping probe, located exclusively within the hair cells (visualized as bright white in the third column). The fourth column shows magnified single-channel fluorescence images of small dots showing labeling by the long probe, located exclusively within hair cells. WT cochleae show labeling, but KO cochleae do not, indicating that, as expected, the KO mice lack Klhdc7b RNA transcripts. The outlines of hair cells on the images indicate that the small dots are exclusively present in hair cells. [Figure 5A]Immunostained images of whole-mount cochleae from WT and KLHDC7B KO mice at postnatal days (p) 6, p11, p21, and 8 weeks are shown. 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 scarring of supporting cells is visible where hair cells previously were located. Scarring is visible as a lattice structure stained white for F-actin at p21 and 8 weeks (dashed circle in knockout tissue). Circular staining patterns on outer hair cells represent aberrant labeling of MYO7A, suggesting possible hair cell death and phagocytosis by supporting cells (dotted circle in knockout tissue). Damage worsens at 8 weeks (solid rectangle in knockout tissue). [Figure 5B] Images of histological sections of the cochlea from 8-week-old wild-type or 8-week-old KLHDC7b knockout mice are shown, 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 that hair cells are abnormal in KLHDC7b knockout mice at 8 weeks. Hair cells (shown as the lightest staining in grayscale) appear to have abnormal morphology, with wider intercellular spacing and less order. The rightmost image shows a highly abnormal staining pattern in the base. Neuronal staining, represented as a darker, diffuse staining in grayscale (in contrast to the clearly outlined, roughly round nuclei labeled with DAPI) located primarily beneath the inner hair cells, is still present in the KO tissue. [Figure 6]Histological staining images of whole-mount cochleae from 8-week-old wild-type or 8-week-old KLHDC7b knockout mice are provided. They were immunostained for the hair cell marker Myo7a (green), the neuronal marker Tuj1 (red), DAPI (blue), and F-actin (white). WT = wild-type; KO = knockout. These images show hair cell loss and visible scarring in KLHDC7b knockout mice at 8 weeks. As in Figure 5A, there is a visible lattice structure indicating the presence of supporting cell scarring. There are also fewer hair cells. This damage is present as a gradient from base to apex, with the base being most severe. [Figure 7A] Histological images of (A, C) apical or (B, C) basal whole-mount sections of the cochlea from 3-day-old wild-type or 3-day-old KLHDC7b knockout mice are shown, immunostained for the hair cell marker Myo7a (green), the neuronal marker Tuj1 (red), DAPI (blue), and actin. All parts of the apical, middle, and basal regions of the organ of Corti appear normal, with well-formed stereocilia visible in the KO tissue and no hair cell defects. [Figure 7B] Histological images of (A, C) apical or (B, C) basal whole-mount sections of the cochlea from 3-day-old wild-type or 3-day-old KLHDC7b knockout mice are shown, immunostained for the hair cell marker Myo7a (green), the neuronal marker Tuj1 (red), DAPI (blue), and actin. All parts of the apical, middle, and basal regions of the organ of Corti appear normal, with well-formed stereocilia visible in the KO tissue and no hair cell defects. [Figure 7C]Histological images of (A, C) apical or (B, C) basal whole-mount sections of the cochlea from 3-day-old wild-type or 3-day-old KLHDC7b knockout mice are shown, immunostained for the hair cell marker Myo7a (green), the neuronal marker Tuj1 (red), DAPI (blue), and actin. All parts of the apical, middle, and basal regions of the organ of Corti appear normal, with well-formed stereocilia visible in the KO tissue and no hair cell defects. [Figure 7D] Histological images of the cochlea from a 6-day-old KLHDC7b knockout mouse are shown, immunostained for Myo7a, DAPI (blue), actin (white), and ZO-1 (red). Hair cells are present and have normal morphology, stereocilia, and ZO-1 localization. In the four panels on the right, each fluorescent channel is labeled separately. [Figure 7E] Histological images of the cochlea from a 6-day-old wild-type mouse are shown, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red), which closely resembles the tissue of a knockout mouse at the same age. [Figure 7F] Histological images of the cochlea from an 11-day-old wild-type mouse are provided, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red) from the apical turn of the cochlea. Hair cells are present and appear normal, as do the stereocilia and ZO-1 staining. [Figure 7G] Histological images of cochleae from two different 11-day-old KLHDC7b knockout mice are shown, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Unlike wild-type mice, some outer hair cell somas in the knockout mice show abnormal cytoplasmic localization of ZO-1 (compare Figure 7F), a time point when only a few hair cells are missing. [Figure 7H]Histological images of cochleae from two different 11-day-old KLHDC7b knockout mice are shown, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Unlike wild-type mice, some outer hair cell somas in the knockout mice show abnormal cytoplasmic localization of ZO-1 (compare Figure 7F), a time point when only a few hair cells are missing. [Figure 7I] Histological images of the cochlea from a 21-day-old KLHDC7b knockout mouse are shown, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Note that MYO7A staining reveals hair cell loss as well as abnormally rounded shapes outside the normal outer hair cell location, which may suggest engulfment of hair cells by supporting cells. Some remaining hair cells show cytoplasmic localization of ZO-1. Actin staining reveals significant scarring, as indicated by a sawtooth-shaped lattice structure. Inner hair cells appear normal, and stereocilia are normal. [Figure 7J] Histological images of cochleae from 21-day-old wild-type mice are shown, immunostained for Myo7a (green), DAPI (blue), actin (white), and ZO-1 (red). Compared to Figure 7I, hair cells appear more organized, and ZO-1 stains the upper border of the cells rather than within the cytoplasm. It is unclear whether these instances of ZO-1 mislocalization represent a general cell death phenotype or a more specific phenotype caused by the Klhdc7b knockout. WT = wild-type; KO = KLHDC7b knockout. These images demonstrate that in the knockout cochleae, hair cells and stereocilia appear normal at postnatal days 3 and 6, early signs of degeneration at postnatal day 11, and further progression of degeneration at postnatal day 21. [Figure 8] Schematic diagrams of two hearing assays for assessing cochlear function are provided: auditory brainstem response (ABR) (left panel), which measures inner hair cell and neuronal function, and distortion product otoacoustic emissions (DPOAE) (right panel), which measures outer hair cell function. [Figure 9A-1] This figure shows that KLHDC7B knockout mice have severe and progressive hearing loss, whereas heterozygous mice do not. Auditory brainstem responses (ABRs) were recorded at each time point in separate cohorts of KLHDC7B knockout (KO), heterozygous (HET), and wild-type (WT) mice. Top row: ABR thresholds. For each age range listed on the x-axis, the left data point is for WT, the middle data point is for HET, and the right data point is for KO. KO mice exhibit severe hearing loss during auditory development (2-3 weeks), which progresses to complete hearing loss at three measured frequencies (8 kHz, 16 kHz, and 32 kHz) by 11-15 weeks. 100 dB indicates no response. Middle row: Wave 1 amplitude in KO mice is significantly smaller than that in WT at 2-3 weeks, while HET mice are not significantly different from WT. Right of middle row: Average waveforms of KO and WT mice at 2-3 weeks. The shaded area represents the SEM. Bottom panel: At 11-15 weeks, ABRs are absent in almost all KO mice, but there is no significant difference between HET and KO. The right panel of the bottom panel shows the average waveforms of WT and KO mice, as in the middle panel. [Figure 9A-2]This figure shows that KLHDC7B knockout mice have severe and progressive hearing loss, whereas heterozygous mice do not. Auditory brainstem responses (ABRs) were recorded at each time point in separate cohorts of KLHDC7B knockout (KO), heterozygous (HET), and wild-type (WT) mice. Top row: ABR thresholds. For each age range listed on the x-axis, the left data point is for WT, the middle data point is for HET, and the right data point is for KO. KO mice exhibit severe hearing loss during auditory development (2-3 weeks), which progresses to complete hearing loss at three measured frequencies (8 kHz, 16 kHz, and 32 kHz) by 11-15 weeks. 100 dB indicates no response. Middle row: Wave 1 amplitude in KO mice is significantly smaller than that in WT at 2-3 weeks, while HET mice are not significantly different from WT. Right of middle row: Average waveforms of KO and WT mice at 2-3 weeks. The shaded area represents the SEM. Bottom panel: At 11-15 weeks, ABRs are absent in almost all KO mice, but there is no significant difference between HET and KO. The right panel of the bottom panel shows the average waveforms of WT and KO mice, as in the middle panel. [Figure 9A-3]This figure shows that KLHDC7B knockout mice have severe and progressive hearing loss, whereas heterozygous mice do not. Auditory brainstem responses (ABRs) were recorded at each time point in separate cohorts of KLHDC7B knockout (KO), heterozygous (HET), and wild-type (WT) mice. Top row: ABR thresholds. For each age range listed on the x-axis, the left data point is for WT, the middle data point is for HET, and the right data point is for KO. KO mice exhibit severe hearing loss during auditory development (2-3 weeks), which progresses to complete hearing loss at three measured frequencies (8 kHz, 16 kHz, and 32 kHz) by 11-15 weeks. 100 dB indicates no response. Middle row: Wave 1 amplitude in KO mice is significantly smaller than that in WT at 2-3 weeks, while HET mice are not significantly different from WT. Right of middle row: Average waveforms of KO and WT mice at 2-3 weeks. The shaded area represents the SEM. Bottom panel: At 11-15 weeks, ABRs are absent in almost all KO mice, but there is no significant difference between HET and KO. The right panel of the bottom panel shows the average waveforms of WT and KO mice, as in the middle panel. [Figure 9B-1] This shows that KLHDC7B knockout mice have severe and progressive hearing loss, whereas heterozygous mice do not. ABRs were recorded longitudinally starting at 11–13 weeks (same data as above). Thresholds increased at all frequencies between 55–57 weeks, with no significant differences between WT and HET. Bottom row: Wave 1 amplitudes were also indistinguishable between HET and WT at any dB SPL level, and the waveforms (right) were indistinguishable. All data were analyzed separately for each frequency. [Figure 9B-2] This shows that KLHDC7B knockout mice have severe and progressive hearing loss, whereas heterozygous mice do not. ABRs were recorded longitudinally starting at 11–13 weeks (same data as above). Thresholds increased at all frequencies between 55–57 weeks, with no significant differences between WT and HET. Bottom row: Wave 1 amplitudes were also indistinguishable between HET and WT at any dB SPL level, and the waveforms (right) were indistinguishable. All data were analyzed separately for each frequency. [Figure 9C]Figure 9A shows that KLHDC7B knockout mice have severe and progressive hearing loss, whereas heterozygous mice do not. Non-limiting, exemplary auditory brainstem responses (ABRs) at 16 kHz (top panel) from wild-type (WT), heterozygous KLHDC7b knockout (Het), and homozygous KLHDC7b knockout (KO) mice at postnatal day 17. Also shown is a graph providing the hearing thresholds (dB; y-axis) of these animals at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) as assayed 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, and **** indicates p<0.0001. This figure shows that homozygous KLHDC7b knockout mice have significant hearing loss during auditory development. [Figure 10-1] Non-limiting, exemplary auditory brainstem responses (ABRs) at 16 kHz from 6-, 8-, and 12-week-old wild-type (WT) and KLHDC7b knockout (KO) mice are shown. Also shown are graphs providing the hearing thresholds (dB; y-axis) of these animals at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) as assessed by ABR (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, and **** indicates p<0.0001. This figure shows that KLHDC7b knockout mice have severe hearing loss at 5–7 weeks, which is worse than the hearing loss observed during auditory development and progresses to complete hearing loss by approximately 12 weeks. Further information on this progression using a larger number of animals can be found in Figure 9A. [Figure 10-2]Non-limiting, exemplary auditory brainstem responses (ABRs) at 16 kHz from 6-, 8-, and 12-week-old wild-type (WT) and KLHDC7b knockout (KO) mice are shown. Also shown are graphs providing the hearing thresholds (dB; y-axis) of these animals at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) as assessed by ABR (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, and **** indicates p<0.0001. This figure shows that KLHDC7b knockout mice have severe hearing loss at 5–7 weeks, which is worse than the hearing loss observed during auditory development and progresses to complete hearing loss by approximately 12 weeks. Further information on this progression using a larger number of animals can be found in Figure 9A. [Figure 11A] Graphs are shown providing hearing thresholds (dB; y-axis) of wild-type (WT), heterozygous KLHDC7b knockout (het), or homozygous KLHDC7b (KO) animals at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) as assayed by ABR (left graph) or distortion product otoacoustic emissions (DPOAEs) (right graph). In Figure 11A, 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, and **** indicates p<0.0001. This figure demonstrates that adult heterozygous KLHDC7b knockout mice did not lose hearing at 11–13 weeks of age. [Figure 11B] ABRs for KO and Het mice tested at 16 kHz are shown. The figure shows the time course of hearing loss, which increases from day 17 to weeks 7-8 in homozygous KLHDC7b mice, while heterozygous mice have no hearing loss even at week 32. [Figure 11C]ABRs for KO and Het mice tested at 16 kHz are shown. The figure shows the time course of hearing loss, which increases from day 17 to weeks 7-8 in homozygous KLHDC7b mice, while heterozygous mice have no hearing loss even at week 32. [Figure 12A] We provide images of wild-type mouse cochlear explants incubated in gentamicin-Texas red (GtTR), Texas red alone, or medium alone, demonstrating that hair cells in cochlear explant cultures take up gentamicin-Texas red (GtTR) through mechanotransduction channels. [Figure 12B] Cochlear explants were immunostained for Myo7a (white), DAPI (blue), and actin (green). The top panel shows a merged fluorescence image of all channels. The middle panel shows a magnified merged fluorescence image of the area indicated by the white square in the top image. The bottom panel shows a single-channel fluorescence image of GtTR in the magnified image. In grayscale, bright staining in the merged fluorescence image indicates colocalization of actin and GtTR staining, indicating GtTR incorporation 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 channel. In cochlear explant cultures from knockout mice, hair cells in postnatal day 4-5 explants take up GTTR. The gross morphology of hair cells in these mice appears normal, and no hair cells are missing, suggesting that the mechanotransduction complex is functional. [Figure 12C]Cochlear explant cultures from wild-type (KLHDC7b+ / +) and knockout (KLHDC7bΔ / Δ) mice were treated with gentamicin-Texas Red (GtTR) or Texas Red (TR) alone, and then immunostained for Myo7A and stained for F-actin. GtTR, but not Texas Red, enters hair cells in both WT and KO mice. [Figure 13A] Fluorescence images of ear organoids differentiated from hiPSCs (human induced pluripotent stem cells) are provided. RNAscope probes for two ear markers, Sox2 (hair cells and supporting cells) and TUBB3 (spiral ganglion neurons), show expression around what is likely the otic vesicle (left). The JK iPSC line is shown. qPCR for ear markers (right) shows an increase after differentiation of D70 IEOs (inner ear organoids at day 70) compared to iPSCs. Both the JK and GM lines show this increase. [Figure 13B] RNAscope probes were designed for the long (top panel) and overlapping (bottom panel) portions of the KLHDC7B human transcript, and RNAscope was performed in parallel with staining for Myo7a. The leftmost column represents merged fluorescence images (Myo7a, DAPI, and KLHDC7B probes) of otic organoids. The second column represents a magnified merged fluorescence image of an area of ​​high probe labeling. The third and fourth columns represent single-channel fluorescence images of the KLHDC7B probe and Myo7a, respectively. Both probes are shown to be located around the likely otic vesicle. Sections are from the same organoid. qPCR for KLHDC7B (right) shows an increase after differentiation. Analysis was performed via two-way ANOVA with Tukey's test used for post-hoc comparisons. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 14] Scanning electron microscopy images of cochleae isolated from wild-type animals (WT) or homozygous KLHDC7b animals (KO) at postnatal day 10 (p10) or postnatal day 20 (p20) are provided. [Figure 15]These images show histological images of fixed, paraffin-embedded mouse cochleae stained with a custom-made antibody against KLHDC7b. The 20x confocal image on the top left shows labeling only in hair cells, while the 40x image on the top right shows that labeling appears to be localized to the cell membrane. The bottom left and bottom right are no-primary antibody controls, showing no labeling. [Figure 16] Histological images of cochlear samples isolated from wild-type animals (WT) or homozygous KLHDC7b animals (KO) labeled with a custom monoclonal anti-KLHDC7b antibody (white) and DAPI are provided. [Figure 17A] Histological images of HEK cell lines stably transfected with FLAG-tagged short (top) or long (middle) human Klhdc7b isoforms or FLAG-tagged GFP (bottom) are shown. They were labeled with one of 10 different monoclonal anti-KLHDC7b antibodies (cyan; clone numbers are indicated at the bottom) 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 the anti-KLHDC7b antibody. 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, suggesting some nonspecific labeling in this clone. [Figure 17B]Histological images of the same HEK cell line as above labeled with two monoclonal anti-KLHDC7b antibodies, designated Antibody A (Clone 1 in Figure 17A) and Antibody B (Clone 3 in Figure 17A), are provided. The top and middle rows represent cells transfected with the short and long FLAG-tagged human Klhdc7b isoforms, respectively. The bottom row represents cells transfected with FLAG-tagged GFP. Columns 1 and 2 show all channels. Columns 3 and 4 show FLAG and DAPI staining only, columns 5 and 6 show anti-KLHDC7B and DAPI staining, and columns 7 and 8 show GFP 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, suggesting that the antibodies bind to cells expressing the long and short isoforms of Klhdc7b. The brightest staining in the bottom row of control transfected cells indicates GFP staining, with no detectable anti-KLHDC7b labeling. DETAILED DESCRIPTION OF THE INVENTION

[0011] I. Overview Kelch domain-containing 7B (KLHDC7b) is a member of the Kelch superfamily of proteins, which are involved in cellular processes such as cytoskeletal reorganization and protein degradation, as well as in extracellular communication, cell morphology, gene expression, and actin binding. Little is known about this gene beyond its membership in the Kelch domain-containing protein superfamily. Kelch domains are sets of repeated beta-sheet-forming subunits that together form a tertiary structure known as a beta-propeller. Because Kelch domain-containing proteins vary in their cellular location and function, KLHDC7b's membership in this family does not clarify its role within the cell (Adams et al., 2000). Few publications have specifically investigated KLHDC7b. It has also been shown to be upregulated and hypermethylated in breast cancer cells (Martin-Pardillos and Cajal, 2019). Alterations in this Kelch superfamily protein have been associated with various types of cancer, including leukemia, lung cancer, prostate cancer, brain cancer, and Hodgkin's disease. KLHDC7B was found to be hypermethylated but upregulated in breast cancer cells. KLHDC7b has two predicted isoforms (long and short).

[0012] Moderate levels of KLHDC7B expression have been observed in the cochlea, e.g., in the hair cells of the ear, with slightly higher expression appearing in the outer hair cells (gEAR portal). Predicted loss-of-function variants in KLHDC7B are associated with an increased risk of developing hearing loss in humans. For example, a genetic change that changes the guanine nucleotide at position 3,778 of human KLHDC7B (see NCBI accession number NP_612442.3) to adenine has been found to indicate that individuals with such a change may be at increased risk of developing hearing loss, such as conductive hearing loss, sensorineural hearing loss, or nerve hearing loss. The International Mouse Phenotyping Consortium (IMPC) has shown that exon deletion of Klhdc7b results in abnormal auditory brainstem responses, abnormal ear morphology, shortened QT interval, abnormal locomotor behavior, suppressed / abnormal startle reflex, and reduced prepulse inhibition (www.mousephenotype.org / data / genes / MGI:3648212).

[0013] Herein, we present evidence that non-human animals containing a knockout mutation of the endogenous Klhdc7b gene progressively develop hearing loss. Compared to wild-type controls, Klhdc7b knockout mice exhibit increased hearing loss, with severe hearing loss during auditory development, reaching near-total deafness by 11–15 weeks of age. Cochlear hair cells, for example, are defective at the time the mice are severely hearing-deprived, although mechanotransduction complexes appear to be functional at earlier time points. For example, hair cells develop normally and exhibit functional mechanotransduction complexes in culture, but begin to die around postnatal days 11–12, with the characteristic morphology of supporting cell scars (Wagner and Shin, 2019) known to form beneath dying hair cells, as shown by histology and scanning electron microscopy. Scanning electron microscopy (SEM) reveals no gross abnormalities in stereocilia morphology before hair cell death begins, and outer hair cell loss is confirmed at 3 weeks of age. RNAscope results indicate that KLHDC7b is specifically expressed in hair cells. A custom anti-KLHDC7b antibody generated against KLHDC7B confirms hair cell specificity. These data suggest a role for KLHDC7B in the maintenance of cochlear hair cells. Therefore, the non-human animals disclosed herein may be useful for identifying important biological players and / or mechanisms in preventing hearing loss and / or maintaining hearing function.

[0014] 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 the endogenous Klhdc7b gene or a portion thereof. The deletion can comprise, consist essentially of, or consist of a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene in the endogenous Klhdc7b locus of the non-human animal nucleic acid, non-human animal cell, and non-human animal, for example, the deletion extends between, but does not include, or does not 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 be the result of replacing 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 can include a reporter gene and / or a gene encoding a selectable marker, optionally where the reporter gene is operably linked to a promoter (e.g., the endogenous Klhdc7b promoter), and / or the gene encoding the selectable marker is operably linked to a promoter (e.g., the endogenous Klhdc7b promoter), and / or 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 as SEQ ID NO: 5, and / or (ii) the nucleic acid sequence set forth as SEQ ID NO: 6 or the nucleic acid sequence set forth as SEQ ID NO: 7, and / or (iii) the nucleic acid sequence set forth as SEQ ID NO: 38 or the nucleic acid sequence set forth as SEQ ID NO: 39, and / or (iv) the endogenous 5'Klhdc7b untranslated region, optionally an intact endogenous 5'Klhdc7b untranslated region, and / or (v) the endogenous 3'Klhdc7b translated region, optionally an intact endogenous 3'Klhdc7b untranslated region.Generally, the endogenous 5'Klhdc7b untranslated region, optionally an intact endogenous 5'Klhdc7b untranslated region described herein, may be located upstream of the deletion of the Klhdc7b gene or a portion thereof, e.g., upstream of the Klhdc7b start codon, and / or the endogenous 3'Klhdc7b translated region, optionally an intact endogenous 3'Klhdc7b untranslated region, may be located downstream of the deletion of the Klhdc7b gene or a portion thereof, e.g., downstream of the endogenous stop codon of the endogenous Klhdc7b gene.

[0015] II. Non-human cells and animals containing modified Klhdc7b loci Non-human animal cells and animals are provided that contain the modified Klhdc7b locus described herein. The cells or non-human animals can be heterozygous or homozygous for the Klhdc7b locus. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous when two identical alleles are present at a particular locus, and as heterozygous when the two alleles are different. In some embodiments, provided herein are non-human animal cells that contain a genetically modified endogenous Klhdc7b locus described herein. The non-human animal cells can be cochlear cells (e.g., inner hair cells or outer hair cells), pluripotent cells, ES cells, or germ cells.

[0016] In some embodiments, the present disclosure further provides methods for producing any of the non-human animals or reagents necessary to produce the non-human animals described herein.

[0017] The non-human animal cells provided herein can be, for example, any non-human cell containing a modified Klhdc7b locus described herein. The cells can be eukaryotic cells, including, for example, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, non-human mammalian cells, and human cells. The animals can be, for example, mammals, fish, or birds. The mammalian cells can be, for example, non-human mammalian cells, rodent cells, rat cells, mouse cells, or hamster cells. Other non-human mammals include, for example, non-human primates, monkeys, apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, and livestock (e.g., cattle breeds such as cows and steers, ovine breeds such as sheep and goats, and porcine breeds such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, ducks, etc. Domestic and agricultural animals are also included. The term "non-human" excludes humans.

[0018] Cells can also be of any type, undifferentiated or differentiated. For example, cells can be totipotent cells, pluripotent cells (e.g., human pluripotent cells or non-human pluripotent cells such as mouse embryonic stem (ES) cells or rat ES cells), or non-pluripotent cells. Totipotent cells include undifferentiated cells that can give rise to any cell type, while pluripotent cells include undifferentiated cells that have the ability to develop into multiple differentiated cell types. Such pluripotent and / or totipotent cells can be, for example, ES cells or ES-like cells such as induced pluripotent stem (iPS) cells. ES cells include embryo-derived totipotent or pluripotent cells that, when introduced into an embryo, can contribute to any tissue of the developing embryo. ES cells can be derived from the inner cell mass of a blastocyst and can differentiate into cells of any of the three vertebrate germ layers (endoderm, ectoderm, and mesoderm).

[0019] The cells provided herein can also be germ cells (e.g., sperm or oocytes). The cells can be mitotically competent or mitotically inactive, meiotically competent or meiotically inactive. 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, gamete cells, or undifferentiated stem cells. Suitable cells provided herein also include primary cells. Primary cells include cells or cell cultures directly isolated from an organism, organ, or tissue. Primary cells include cells that are not transformed or immortal. Primary cells include any cells obtained from an organism, organ, or tissue that have not previously been passaged in tissue culture, or that have previously been passaged in tissue culture but cannot be passaged indefinitely in tissue culture. Such cells can be isolated by conventional techniques.

[0020] Suitable cells include cells of the ear, e.g., cells involved in hearing, e.g., neurons, hair cells, etc. Spiral ganglion neurons, and cochlear hair cells (e.g., inner hair cells, outer hair cells, or supporting cells of the cochlea), cells of the organ of Corti (e.g., Hensen's cells, Deiters' cells, pillar cells, inner phalangeal cells, and border cells) can be suitable cells provided herein.

[0021] Other suitable cells provided herein include immortalized cells. Immortalized cells include cells derived from multicellular organisms that do not normally proliferate indefinitely, but which, due to mutations or alterations, can avoid normal cellular aging and instead continue to divide. Such mutations or alterations can occur naturally or can be intentionally induced. An example of an immortalized cell line is a myofiber cell line. Immortalized or primary cells include cells that can be used to culture or express recombinant genes or proteins.

[0022] The cells provided herein also include one-cell embryos (i.e., fertilized oocytes or zygotes). Such one-cell embryos can be of any genetic background (e.g., B6.Cast-Cdh23 in the case of mice). Ahl+ ), which may be fresh or frozen and may be derived from natural mating or in vitro fertilization.

[0023] The cells provided herein can be normal, healthy cells, or cells that have a disease or mutation.

[0024] Also provided are tissues, eg, cochlear explants, comprising the nucleic acids and / or cells described herein and / or isolated from a non-human animal.

[0025] Non-human animals comprising the modified Klhdc7b locus described herein can be produced by methods described elsewhere herein. The animals can be, for example, mammals, fish, or birds. Non-human mammals include, for example, non-human primates, monkeys, apes, orangutans, cats, dogs, horses, bulls, deer, bison, sheep, rabbits, rodents (e.g., mice, rats, hamsters, and guinea pigs), and livestock (e.g., cattle breeds such as cows and steers, ovine breeds such as sheep and goats, and porcine breeds such as pigs and wild boars). Birds include, for example, chickens, turkeys, ostriches, geese, and ducks. Domestic and agricultural animals are also included. The term "non-human animal" excludes humans. Preferred non-human animals include, for example, rodents such as mice and rats.

[0026] The non-human animal may be of any genetic background. For example, a suitable mouse may be B6.Cast-Cdh23 Ahl+The 129 strain may be a 129 strain, a C57BL / 6 strain, a 129 and C57BL / 6 mix, a BALB / c strain, or a 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, e.g., Festing et al. (1999) Mammalian Genome 10:836, the entire contents of which are incorporated herein by reference 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 a mix of the aforementioned 129 strain and the aforementioned C57BL / 6 strain (e.g., 50% 129 and 50% C57BL / 6). Similarly, suitable mice can be a mix of the aforementioned 129 strains or a mix of the aforementioned BL / 6 strains (e.g., 129S6 (129 / SvEvTac) strain).

[0027] Similarly, rats can be of 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 the Fischer rat strain, such as Fisher F344 or Fisher F6. Rats can also be obtained from strains derived from a mix of two or more of the above strains. For example, suitable rats can be of the DA strain or the ACI strain. The ACI rat strain has a white abdomen and legs and RT1 av1 The dark agouti (DA) rat strain is characterized as having a black agouti haplotype and is available from a variety of sources, including Harlan Laboratories.RT1 The rats are characterized as having the av1 haplotype. Such rats are available from a variety of sources, including Charles River and Harlan Laboratories. Some suitable rats may be from inbred rat strains. See, e.g., US2014 / 0235933, the entire contents of which are incorporated herein by reference for all purposes.

[0028] III. Methods for producing non-human animals containing an altered Klhdc7b locus Various methods are provided for producing non-human animals comprising the modified Klhdc7b locus disclosed elsewhere herein. Any convenient method or protocol for producing genetically modified organisms is suitable for producing such genetically modified non-human animals. 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. Such genetically modified non-human animals can be generated, for example, through targeted gene knock-in at the Klhdc7b locus.

[0029] For example, a method for producing a non-human animal comprising a modified Klhdc7b locus can include (1) modifying the genome of a pluripotent cell to comprise a modified Klhdc7b locus, (2) identifying or selecting genetically modified pluripotent cells comprising the modified Klhdc7b locus, (3) introducing the genetically modified pluripotent cells into a non-human animal host embryonic cell in vitro, and (4) implanting the host embryonic cell into a surrogate mother for gestation. Optionally, the host embryo comprising the modified pluripotent cells (e.g., non-human ES cells) can be incubated to the blastocyst stage and then implanted into a surrogate mother for gestation to produce an F0 non-human animal. The surrogate mother can then produce an F0 generation non-human animal comprising the modified Klhdc7b locus.

[0030] The method can further include identifying cells or animals that have the modified target genomic locus. A variety of methods can be used to identify cells and animals that have the targeted genetic modification.

[0031] The screening step can include, for example, a quantitative assay for evaluating the allele modification (MOA) of parent chromosomes. For example, the quantitative assay can be carried out through quantitative PCR, such as real-time PCR (qPCR). The real-time PCR can utilize a first primer set that recognizes the target locus and a second primer set that recognizes the non-target reference locus. The primer set can include a fluorescent probe that recognizes the amplified sequence.

[0032] Other examples of suitable quantitative assays include fluorescence in situ hybridization (FISH), comparative genomic hybridization, isothermal DNA amplification, quantitative hybridization to immobilized probe(s), INVADER® probe, TAQMAN® molecular beacon probe, or ECLIPSE™ probe technology (see, e.g., US2005 / 0144655, the entire contents of which are incorporated herein by reference for all purposes).

[0033] An example of a suitable pluripotent cell is an embryonic stem (ES) cell (e.g., a mouse ES cell or a rat ES cell). The modified pluripotent cell can be generated by recombination, for example, by (a) introducing into a cell one or more targeting vectors containing an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites, the insert nucleic acid comprising a modified Klhdc7b locus or a portion thereof (e.g., a modified Klhdc7b gene comprising a deletion of the open reading frame), and (b) identifying at least one cell whose genome comprises the insert nucleic acid integrated at the target genomic locus.

[0034] Accordingly, also provided herein are methods for producing genetically modified cells (e.g., ES cells), the methods comprising contacting a cell with one or more targeting vectors comprising an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites (the insert nucleic acid comprising a modified Klhdc7b locus or a portion thereof (e.g., a modified Klhdc7b gene comprising a deletion of the open reading frame)), such that upon homologous recombination between the 5' and 3' target sites corresponding to the 5' and 3' homology arms, 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' and 3' target sites.

[0035] Alternatively, modified pluripotent cells can be generated by (a) introducing into a cell (i) a nuclease agent (a nuclease agent that introduces a nick or double-stranded break at a recognition site within a target genomic locus) and (ii) one or more targeting vectors containing an insert nucleic acid flanked by 5' and 3' homology arms corresponding to 5' and 3' target sites present in sufficient proximity to the recognition site (the insert nucleic acid comprises a modified Klhdc7b locus), and (c) identifying at least one cell that contains a modification at the target genomic locus (e.g., integration of the insert nucleic acid). Any nuclease agent that introduces a nick or double-stranded 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 CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) / CRISPR-associated (Cas) systems or components of such systems (e.g., CRISPR / Cas9).See, for example, US2013 / 0309670 and US2015 / 0159175, each of which is incorporated herein by reference in its entirety for all purposes.

[0036] Donor cells can be introduced into host embryos at any stage, such as blastocyst stage or pre-morula stage (i.e., 4-cell stage or 8-cell stage).Generate offspring that can transmit genetic modification through germline.See, for example, U.S. Patent No. 7,294,754.Its entirety is incorporated herein by reference for all purposes.

[0037] Alternatively, the methods for producing non-human animals described elsewhere herein may include (1) modifying the genome of a one-cell stage embryo to contain a modified Klhdc7b locus using the methods described above for modifying pluripotent cells, (2) selecting a genetically modified embryo, and (3) implanting the genetically modified embryo into a surrogate mother to gestate, producing offspring capable of transmitting the genetic modification through the germline.

[0038] Nuclear transfer techniques can also be used to generate non-human mammals. Briefly, methods for nuclear transfer can include the steps of: (1) enucleating an oocyte or preparing an enucleated oocyte; (2) isolating or preparing 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 reconstituted cell; (4) implanting the reconstituted cell into the uterus of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are typically retrieved from deceased animals, but may also be isolated from either the oviduct and / or ovary of a living animal. Insertion of the donor cell or nucleus into the enucleated oocyte to form the reconstituted cell can be accomplished by microinjecting the donor cell beneath the zona pellucida prior to fusion. Fusion can be induced by applying a DC electrical pulse across the contact / fusion surface (electrofusion), exposing the cells to a fusion-promoting chemical such as polyethylene glycol, or by an inactivated virus such as Sendai virus. The reconstituted cells can be activated by electrical and / or non-electrical means before, during, and / or after fusion of the nuclear donor with the recipient oocyte. Activation methods include electrical pulses, chemically induced shocks, sperm penetration, increasing the level of divalent cations in the oocyte, and reducing the phosphorylation of cellular proteins in the oocyte (via kinase inhibitors). The activated reconstituted cells or embryos can be cultured in a medium and then transferred to the uterus of an animal. See, e.g., US2008 / 0092249, WO1999 / 005266, US2004 / 0177390, WO2008 / 017234, and U.S. Patent No. 7,612,250, each of which is incorporated herein by reference in its entirety for all purposes.

[0039] 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. Naturally, the number of cells in the F0 animals that contain the modified Klhdc7b locus will vary depending on the method used to generate the F0 animals. Introducing donor ES cells into pre-morula stage embryos (e.g., 8-cell stage mouse embryos) from the corresponding organisms, for example, via the VELOCIMOUSE® method, can increase the percentage of the cell population of the F0 animals that contain cells with the desired nucleotide sequence containing the targeted genetic modification. 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 cellular contribution of the non-human F0 animal can comprise a cell population having the targeted modification.

[0040] The 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.

[0041] In some embodiments, the present disclosure provides a method of producing a non-human animal, non-human animal cell, or 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 genome of the non-human animal.

[0042] IV. Nucleic Acids Comprising Modified Klhdc7b Loci As described elsewhere herein, various nucleic acids (e.g., targeting vectors) can be specifically used for such purposes. In some embodiments, non-human animal nucleic acids can be used that contain a modified endogenous Kelch domain-containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus contains a deletion of the endogenous Klhdc7b gene or a portion thereof. In some non-human animal nucleic acid embodiments, the deletion comprises, essentially consists of, or consists of a deletion of the open reading frame (orf) of the endogenous Klhdc7b gene.

[0043] Inserted nucleic acid

[0044] In some non-human animal nucleic acid embodiments, the modified endogenous Klhdc7b locus further comprises an insert nucleic acid. An "insert nucleic acid" or "insertion polynucleotide" comprises a segment of DNA for which integration at the target locus is desired. 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, along with various regulatory components that affect expression.

[0045] Any polynucleotide of interest can be included within various insertion polynucleotides and thereby integrated into the target Klhdc7b locus. The methods disclosed herein provide for at least 1, 2, 3, 4, 5, 6, or more polynucleotides of interest to be integrated into the targeted Klhdc7b genomic locus of interest.

[0046] In one embodiment, the polynucleotide of interest contained in the insert nucleic acid encodes a reporter, hi another embodiment, the polynucleotide of interest encodes a selectable marker.

[0047] In one embodiment, the polynucleotide of interest may be flanked by site-specific recombination sequences, hi certain embodiments, the site-specific recombination sequences flank the segment encoding a reporter and / or the segment encoding a selectable marker.

[0048] Non-limiting examples of polynucleotides of interest, such as selectable markers and reporter genes, that may be included within the insert nucleic acid are discussed in detail elsewhere herein.

[0049] When integrated into the target Klhdc7b locus, the polynucleotide of interest within the insert polynucleotide can introduce one or more genetic modifications into the cell. 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, heterologous, or orthologous polynucleotide to the target genomic locus. In one embodiment, the genetic modification involves replacing an endogenous nucleic acid sequence with an exogenous polynucleotide of interest at the target genomic locus. Thus, the methods provided herein enable the generation of genetic modifications including knockouts, deletions, substitutions ("knock-ins"), or combinations thereof, at the target Klhdc7b locus. Such modifications can occur upon integration of the first, second, third, fourth, fifth, sixth, seventh, or any subsequent insert polynucleotide into the target genomic locus.

[0050] The polynucleotide of interest within the insert polynucleotide and / or the polynucleotide of interest integrated at the target genomic locus may comprise a sequence that is native 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 originate from a different species than the cell into which it is introduced. The term "homologous" with respect to a sequence refers to a sequence that is native to the cell. The term "heterologous" with respect to a sequence refers to a sequence that originates from a foreign species, or, even if originating from the same species, a sequence that has been substantially altered in composition and / or genomic locus from its native form by deliberate human intervention. The term "exogenous" with respect to a sequence refers to a sequence that originates from a foreign species. The term "ortholog" refers to a polynucleotide derived from one species that is functionally equivalent to a known reference sequence in another species (i.e., a species variant). The polynucleotide of interest can be derived 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 can further comprise coding regions, non-coding regions, regulatory regions, or genomic DNA. Thus, the first, second, third, fourth, fifth, sixth, seventh, and / or any subsequent inserted polynucleotides can comprise such sequences.

[0051] In one embodiment, the polynucleotide of interest can range from about 500 nucleotides to about 200 kb, as described above. The polynucleotide of interest can range 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, or from about 90 kb to about 100 kb. kb, about 100 kb to about 110 kb, about 120 kb to about 130 kb, about 130 kb to about 140 kb, about 140 kb to about 150 kb, about 150 kb to about 160 kb, about 160 kb to about 170 kb, about 170 kb to about 180 kb, about 180 kb to about 190 kb, or about 190 kb to about 200 kb.

[0052] The polynucleotide of interest within the insert polynucleotide and / or the inserted polynucleotide of interest at the target genomic locus can encode a polypeptide, can encode an RNA, can encode an miRNA, or the polynucleotide of interest can include any regulatory or non-coding region of interest, such as a regulatory sequence, a promoter sequence, an enhancer sequence, a transcriptional repressor binding sequence, a Kozak consensus segment, a start codon, or can include a deletion of a non-protein-coding sequence, but not a deletion of a protein-coding sequence.

[0053] In one embodiment, the insert nucleic acid comprises a regulatory element, such as, for example, a promoter, enhancer, or transcriptional repressor binding element.

[0054] In a further embodiment, the insert nucleic acid comprises a conditional allele. In one embodiment, the conditional allele is a multifunctional allele as described in US2011 / 0104799, the entirety of which is incorporated by reference. In certain embodiments, the conditional allele comprises: (a) an operating sequence in the sense orientation relative to transcription of the target gene, and a drug selection cassette (DSC) in the sense or antisense orientation; (b) a nucleotide sequence of interest (NSI) and a conditional inversion module (COIN, a conditional by inversion module utilizing an exon-splitting intron and an invertible gene trap-like module, see, e.g., US2011 / 0104799, the entirety of which is incorporated by reference) in the antisense orientation; and (c) a recombinable unit that recombines upon exposure to a first recombinase to form a conditional allele that (i) lacks the operating sequence and the DSC, and (ii) comprises the NSI in the sense orientation and the COIN in the antisense orientation.

[0055] In one embodiment, the genetic modification comprises a deletion of the Klhdc7b gene or a portion thereof, for example, 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 can comprise a polynucleotide that, when integrated into the genome, results in a genetic modification of a region of the Klhdc7b locus of a mammal, non-human animal, or non-human mammal, wherein the genetic modification at the Klhdc7b locus results in a loss-of-function form of Klhdc7b. In one embodiment, a Klhdc7b knockout ("null allele") is generated. In another embodiment, the Klhdc7b locus is disrupted. In a further embodiment, the inserted nucleic acid replaces a portion of the Klhdc7b gene of a mammal, non-human animal, or non-human mammal with an inserted nucleic acid sequence comprising a heterologous sequence.

[0056] In some embodiments, the insert nucleic acid replaces the endogenous Klhdc7b gene or a portion thereof (e.g., its orf portion), and the gene or a portion thereof is deleted. Thus, in some non-human animal nucleic acid embodiments, the genetic modification of the Klhdc7b locus can include replacing the Klhdc7b locus or a portion thereof with the insert nucleic acid, or inserting / adding the insert nucleic acid into the Klhdc7b locus or a portion thereof.

[0057] 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 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 selectable marker nucleic acid sequence.

[0058] In one embodiment, the promoter is a constitutively active promoter.

[0059] 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 rat glucocorticoid receptor promoter. In one embodiment, the steroid-regulated promoter is the estrogen receptor promoter. In one embodiment, the steroid-regulated promoter is the ecdysone receptor promoter. 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 light-repressible promoter.

[0060] 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-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.

[0061] In one embodiment, the promoter is a developmentally regulated promoter. In one embodiment, the developmentally regulated promoter is active only during embryonic development. In one embodiment, the developmentally regulated promoter is active only in adult cells.

[0062] In certain embodiments, promoters can be selected based on cell type.Therefore, various promoters are utilized 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.

[0063] In some embodiments, the insert nucleic acid comprises a nucleic acid flanked by site-specific recombination target sequences. Of course, the entire insert nucleic acid can be flanked by such site-specific recombination target sequences, but any region within the insert nucleic acid or individual polynucleotides of interest can also be flanked by such sites. The site-specific recombinase can be introduced into a cell by any means, including introducing a recombinase polypeptide into the cell or introducing a polynucleotide encoding the site-specific recombinase into a host cell. The polynucleotide encoding the site-specific recombinase can be located within the insert nucleic acid or within a separate polynucleotide. The site-specific recombinase can be operably linked to a promoter active in the cell, including, for example, an inducible promoter, a promoter endogenous to the cell, 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 can flank the insert nucleic acid or any polynucleotide of interest within the insert nucleic acid include, but are not limited to, loxP, lox511, lox2272, lox66, lox71, loxM2, lox5171, FRT, FRT11, FRT71, attp, att, FRT, rox, or combinations thereof.

[0064] In some embodiments, the site-specific recombination sites flank polynucleotides encoding selectable markers and / or reporter genes contained within the insert nucleic acid, in such cases, the sequences between the site-specific recombination sites can be removed after integration of the insert nucleic acid at the targeted locus.

[0065] In some embodiments, the insert nucleic acid comprises a polynucleotide encoding a selectable marker. The selectable marker may be included in a selection cassette. Such a selectable marker may include neomycin phosphotransferase (neomycin phosphotransferase). r ), hygromycin B phosphotransferase (hyg r ), puromycin-N-acetyltransferase (puro 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 site-specific recombination target sequences.

[0066] 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 cell, a promoter heterologous to the reporter gene or cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter.

[0067] In some cases, the inserted nucleic acid comprises a reporter gene. In one embodiment, the reporter gene is placed in the Klhdc7b locus in operable linkage with endogenous Klhdc7b promoter. Such modification allows the reporter gene to be expressed by the endogenous Klhdc7b promoter. Alternatively, the reporter gene is not placed in operable linkage with endogenous Klhdc7b promoter.

[0068] In some cases, the inserted nucleic acid comprises a reporter gene. In one embodiment, the reporter gene is placed in the Klhdc7b locus in operable linkage with the endogenous Klhdc7b start codon. Such modification can allow the reporter gene to be expressed by the endogenous Klhdc7b promoter.

[0069] Any reporter (or detectable moiety) can be used in the methods and compositions provided herein. Non-limiting examples of reporters include, for example, β-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.

[0070] The following description is a non-limiting example utilizing the lacZ reporter gene encoding β-galactosidase. The methods and compositions described herein can be performed with any reporter gene.

[0071] Also provided herein are polynucleotides or nucleic acid molecules that include various components (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) used in the targeted genome integration systems provided herein to target the Klhdc7b locus.

[0072] The terms "polynucleotide," "polynucleotide sequence," "nucleic acid sequence," and "nucleic acid fragment" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide can be a polymer of RNA or DNA that is single- or double-stranded and optionally contains 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 mixtures thereof. A polynucleotide can contain deoxyribonucleotides, and ribonucleotides include both naturally occurring molecules and synthetic analogs, as well as any combination thereof. The polynucleotides provided herein also encompass sequences in any form, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0073] Additionally, recombinant polynucleotides containing various components of a targeted genome integration system for targeting the Klhdc7b locus are provided. The terms "recombinant polynucleotide" and "recombinant DNA construct" are used interchangeably herein. A recombinant construct includes an artificial or heterologous combination of nucleic acid sequences, e.g., regulatory and coding sequences not found together in nature. In other embodiments, a recombinant construct may include regulatory and coding sequences from different sources, or regulatory and coding sequences 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. When a vector is used, the choice of vector will depend on the method used to transform host cells, as is well known to those skilled in the art. For example, a plasmid vector can be used. Also provided are the genetic elements necessary for successful transformation, selection, and propagation of host cells containing any of the isolated nucleic acid fragments provided herein. Screening can be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, etc.

[0074] In certain embodiments, one or more components of the targeted genome integration system for targeting the Klhdc7b locus described herein can be provided in an expression cassette for expression in a prokaryotic, eukaryotic, bacterial, yeast, or mammalian cell, or other organism or cell type of interest. The cassette can include 5' and 3' regulatory sequences operably linked to the polynucleotide provided herein. "Operably linked" includes a relationship in which the operably linked components function in their intended manner. For example, an operably linked linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional linkage that allows expression of the polynucleotide of interest. Operably linked elements can be contiguous or non-contiguous. 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 example, 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 control. The cassette can further contain at least one additional polynucleotide of interest to be co-introduced into the organism. Alternatively, the additional polynucleotides of interest may be provided on multiple expression cassettes comprising multiple restriction and / or recombination sites for insertion of recombinant polynucleotides under the transcriptional control of regulatory regions. The expression cassettes may further contain a selectable marker gene.

[0075] An expression cassette can include, in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., promoter), a recombinant polynucleotide provided herein, and a transcriptional and translational termination region (i.e., termination region) functional in a mammalian cell or host cell of interest. The regulatory region (i.e., promoter, transcriptional regulatory region, Kozak sequence, and translational termination region) and / or the polynucleotide provided herein can be native / analogous to the host cell or to each other. Alternatively, the regulatory region and / or polynucleotide provided herein can be heterologous to the host cell or to each other. For example, a promoter operably linked to a heterologous polynucleotide can be from a species different from that from which the polynucleotide is derived, or, even if from the same / analogous species, one or both have been substantially altered from their original form and / or genomic locus or are not the native promoter of the polynucleotide to which it is operably linked. Alternatively, the regulatory region and / or recombinant polynucleotide provided herein can be entirely synthetic.

[0076] The termination region may be native to the transcription initiation region, may be native to the operably linked recombinant polynucleotide, may be native to the host cell, or may originate from a source separate from (i.e., foreign or heterologous) the promoter, recombinant polynucleotide, host cell, or any combination thereof.

[0077] In preparing expression cassettes, various DNA fragments may be manipulated to provide DNA sequences in the proper orientation. To this end, adapters or linkers may be used to join DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0078] Several promoters can be used in the expression cassettes provided herein. The promoter can be selected based on the desired result. Of course, different promoters can be used in the expression cassette to enhance various applications by adjusting the timing, location, and / or level of expression of the polynucleotide of interest. Such expression constructs can also optionally include a promoter regulatory region (e.g., one that confers inducible, constitutive, environmentally or developmentally regulated, or cell- or tissue-specific / selective expression), a transcription initiation site, a Kozak consensus sequence, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0079] Expression cassettes containing the polynucleotides provided herein may also contain a selectable marker gene for the selection of transformed cells. The selectable marker gene is used to select transformed cells or tissues.

[0080] Optionally, the sequences used in the methods and compositions (i.e., polynucleotides of interest, nuclease agents, etc.) can be optimized for increased expression in cells, i.e., genes can be synthesized using codons that are preferred in a given cell of interest, e.g., mammalian-preferred codons, human-preferred codons, rodent-preferred codons, mouse-preferred codons, rat-preferred codons, hamster-preferred codons, etc., for improved expression.

[0081] The various methods and compositions provided herein may employ selectable markers. A variety of selectable markers may be used in the methods and compositions disclosed herein. Such selectable markers may confer resistance to antibiotics, such as G418, hygromycin, blasticidin, neomycin, or puromycin. Examples of such selectable markers include neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror), and blasticidin S deaminase (bsrr). In yet other embodiments, the selectable marker is operably linked to an inducible promoter, and expression of the selectable marker is toxic to the cell. Non-limiting examples of such selectable markers include xanthine / guanine phosphoribosyltransferase (gpt), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), or herpes simplex virus thymidine kinase (HSV-TK). The polynucleotide encoding the selectable marker is operably linked to a promoter active in the cell.

[0082] Targeting Vector

[0083] The targeting vector is used to introduce an insert nucleic acid into a desired Khldc7b locus of a eukaryotic, non-human, mammalian, non-human mammalian, human, rodent, mouse, rat, or hamster nucleic acid. In some embodiments, the nucleic acid molecule (e.g., the targeting vector) described herein 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 a Klhd7bc locus of interest in the non-human animal, and after homologous recombination with a Klhdc7b locus of interest in the non-human animal, the modified Klhdc7b gene replaces the non-human animal Klhdc7b gene at the Klhdc7b locus of interest in the non-human animal and is operably linked to an endogenous promoter that drives expression of the modified non-human animal Klhdc7b gene at the Klhdc7b locus of interest in the non-human animal. In some embodiments, the nucleic acid molecule (e.g., a targeting vector) comprises the nucleic acid sequence set forth as SEQ ID NO:5, the nucleic acid sequence set forth as SEQ ID NO:6, the nucleic acid sequence set forth as SEQ ID NO:7, the nucleic acid sequence set forth as SEQ ID NO:38, or the nucleic acid sequence set forth as SEQ ID NO:39.

[0084] Also described are various methods of using the genetically modified non-human animals described herein.

[0085] A brief description of arrays The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases and three-letter codes for amino acids. The nucleotide sequences follow the standard convention of starting at the 5'-terminus of the sequence and proceeding toward the 3'-terminus (i.e., from left to right in each column). Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the shown strand. The amino acid sequences follow the standard convention of starting at the amino-terminus of the sequence and proceeding toward the carboxy-terminus (i.e., from left to right in each column). [Table 1-1] [Table 1-2] [Table 1-3]

[0086] Mouse Klhdc7b comprises the amino acid sequence set forth as SEQ ID NO:2, which can be encoded by a nucleic acid comprising the sequence set forth as SEQ ID NO:1. A short isoform of mouse Klhdc7b comprising the amino acid sequence set forth as SEQ ID NO:46, encoded by the nucleotide sequence set forth as SEQ ID NO:45, can also be expressed. The human KLHDC7B amino acid and coding sequences are set forth as NCBI accession numbers NP_612442.3 (SEQ ID NO:48) and NM_138433.5 (SEQ ID NO:46), respectively. A short isoform of human KLHDC7B comprising amino acids 642 to 1235 of SEQ ID NO:48 (set forth as SEQ ID NO:50), encoded by nucleotides 2269 to 4053 of SEQ ID NO:47 (set forth as SEQ ID NO:49), can also be expressed.

[0087] 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 amino acids that are chemically or biochemically modified or derivatized. The terms also include modified polymers, 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.

[0088] 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, which ends with the amino acid having a free amine group (-NH2). The term "C-terminus" refers to the end of an amino acid chain (protein or polypeptide), which ends with a free carboxyl group (-COOH).

[0089] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, include polymeric forms of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof. Nucleic acids and polynucleotides can include single-, double-, and multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, and polymers that contain purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases.

[0090] Nucleic acids are said to have a "5' end" and a "3' end." This is because mononucleotides react to form oligonucleotides in such a way that the 5' phosphate of one mononucleotide pentose ring is unidirectionally linked to the 3' oxygen of the next via a phosphodiester bond. An end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of the mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to the 5' phosphate of another mononucleotide pentose ring. A nucleic acid sequence can also be said to have a 5' end and a 3' end, even if it is internal to a larger oligonucleotide. In either a linear or circular DNA molecule, individual elements are referred to as being "upstream" or 5' of the "downstream" or 3' element.

[0091] The term "genomically integrated" refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence can be incorporated into the genome of the cell and passed on to its progeny. Any protocol can be used for stable integration of a nucleic acid into the genome of a cell.

[0092] As used herein, "embryonic stem cells" or "ES cells" include totipotent or pluripotent cells derived from an embryo that, when introduced into an embryo, can contribute to any tissue of the developing embryo. The term "pluripotent cells" includes undifferentiated cells that have the capacity to develop into multiple differentiated cell types.

[0093] As used herein, "targeting vector," "large targeting vector," or "LTVEC" includes targeting vectors for eukaryotic cells that are derived from cloned fragments of genomic DNA that are larger than those typically used in other approaches aimed at achieving homologous gene targeting in eukaryotic cells. Examples of LTVEC include, but are not limited to, bacterial homologous chromosomes (BACs) and yeast artificial chromosomes (YACs). Generally, targeting vectors can contain recombinant nucleic acids that can be introduced into a target location within a cell's genome by homologous recombination, ligation by non-homologous end joining, or any other recombination means.

[0094] As used herein, a "site-specific recombination sequence" includes a nucleotide sequence that can be recognized by a site-specific recombinase and serve as a substrate for a recombination event.

[0095] As used herein, "site-specific recombinase" includes a group of enzymes that can promote recombination between "site-specific recombination sequences." Examples of "site-specific recombinases" include, but are not limited to, Cre recombinase, Flp recombinase, and Dre recombinase.

[0096] As used herein, "germline" with respect to a nucleic acid sequence includes nucleic acid sequences that can be passed on to offspring and that can be found, for example, in the germ cells (e.g., oocytes and sperm) of a non-human animal.

[0097] As used herein, "operably linked" and the like refer to components linked to function together in the intended manner. In one example, 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 appropriate transcriptional control. Operable linkage can include sequences where such sequences are adjacent to each other or act in trans (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 fused together, for example, as a fusion protein, such that each individual polypeptide retains its individual biological activity.

[0098] As used herein, a "locus" refers to a segment of DNA within a larger nucleic acid molecule that generally includes non-coding and coding sequences of a gene. For example, the Klhdc7b locus generally includes the non-coding and / or coding sequences of the Klhdc7b gene that encodes the Klhdc7b protein.

[0099] The term "gene" refers to a DNA sequence within a nucleic acid that encodes a product (e.g., an RNA product and / or a polypeptide product), including the coding region interrupted by non-coding introns and untranslated regions (UTRs) adjacent to the coding region at both the 5' and 3' ends, such that the gene corresponds to the full-length mRNA (including 5' and 3' untranslated sequences). The term "gene" also includes 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 located near (e.g., within 10 kb) or at distant sites within the coding region of a gene and affect the level or rate of transcription and translation of the gene.

[0100] As used herein, "open reading frame," "orf," and the like encompass a portion of a DNA molecule (e.g., a gene) that does not contain a stop codon when translated into amino acids. Generally, an open reading frame spans the genetic sequence between the start and stop codons of a gene and may or may not include the start and / or stop codons, but generally does not extend beyond the start or stop codons. A "start codon" is the first codon of a gene or its messenger RNA (mRNA) transcript that is translated by ribosomes. In eukaryotes, the start codon encodes methionine. A "stop codon" is a trinucleotide sequence within a gene or its mRNA transcript that directs the end of protein synthesis.

[0101] The term "allele" refers to a variant form of a gene. Some genes have different forms that exist at the same location, or locus, on a chromosome. Diploid organisms have two alleles at each locus. Each pair of alleles represents a genotype at a particular locus. A genotype is described as homozygous if two identical alleles are present at a particular locus, and heterozygous if the two alleles are different.

[0102] A "promoter" is a regulatory region of DNA that typically contains a TATA box that can direct RNA polymerase II to begin RNA synthesis at the appropriate transcription start site of a particular polynucleotide sequence. Promoters may further contain other regions that affect the rate of transcription initiation. The promoter sequences disclosed herein modulate the transcription of an operably linked polynucleotide. The promoters may be active in one or more of the cell types disclosed herein (e.g., eukaryotic cells, non-human mammalian cells, human cells, rodent cells, pluripotent cells, one-cell stage embryos, differentiated cells, or combinations thereof). The promoters may be, for example, constitutively active, conditional, inducible, temporally restricted (e.g., developmentally regulated), or spatially restricted (e.g., cell-specific or tissue-specific). Examples of promoters can be found, for example, in WO2013 / 176772, the entire contents of which are incorporated herein by reference for all purposes.

[0103] Hearing loss may refer to a decrease in one or more responses to auditory stimuli by a non-human animal compared to that of a control (e.g., wild-type) non-human animal. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response to an auditory stimulus is reduced by about 5% compared to that of a control (e.g., wild-type) non-human animal of the same age. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response to an auditory stimulus is reduced by about 10% compared to that of a control (e.g., wild-type) non-human animal of the same age. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response to an auditory stimulus is reduced by about 15% compared to that of a control (e.g., wild-type) non-human animal of the same age. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response to an auditory stimulus is reduced by about 20% compared to that of a control (e.g., wild-type) non-human animal of the same age. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response 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. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response 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. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response 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. In some embodiments, an animal genetically modified to contain a nucleic acid described herein may be considered to have hearing loss if its response to an auditory stimulus is reduced by about 60% compared to the response of a control (e.g., wild-type) non-human animal of the same age.

[0104] Hearing loss may be determined as a decreased response to auditory stimuli (e.g., decreased function of inner hair cells, outer hair cells, and / or neurons (e.g., spiral ganglion neurons)), measured by an auditory brainstem response assay and / or a distortion product otoacoustic emissions assay, both of which are generally illustrated in Figure 8. Other assays that may be used to determine responses to auditory stimuli (e.g., function of inner hair cells, outer hair cells, and / or neurons such as spiral ganglion neurons) are well known in the art, such as electrocochleography (e.g., using needle or cotton-core electrodes) and compound action potentials.

[0105] In some embodiments, an animal genetically modified to comprise a nucleic acid described herein exhibits hearing loss when its threshold decibel (dB) level of hearing, e.g., at 8 kHz, 16 kHz, and / or 32 kHz, as measured by an auditory brainstem response assay, is 1.5-2.0 times greater than the threshold decibel (dB) level of hearing of a control (e.g., wild-type) animal. In some embodiments, an animal genetically modified to comprise a nucleic acid described herein exhibits hearing loss when its threshold decibel (dB) level of hearing, e.g., at 8 kHz, 16 kHz, and / or 32 kHz, as measured by an auditory brainstem response assay, is about 50 dB or greater. In some embodiments, an animal genetically modified to comprise a nucleic acid described herein exhibits hearing loss when its threshold decibel (dB) level of hearing, e.g., at 8 kHz, 16 kHz, and / or 32 kHz, as measured by an auditory brainstem response assay, is about 40 dB or greater. In some embodiments, an animal genetically modified to contain a nucleic acid described herein exhibits hearing loss when its threshold decibel (dB) level of hearing is about 60 dB or greater, e.g., at 8 kHz, 16 kHz, and / or 32 kHz, as measured by an auditory brainstem response assay. In some embodiments, an animal genetically modified to contain a nucleic acid described herein exhibits hearing loss when its threshold decibel (dB) level of hearing is about 70 dB or greater, e.g., at 8 kHz, 16 kHz, and / or 32 kHz, as measured by an auditory brainstem response assay. Severe hearing loss, etc., can be determined when the threshold decibel of an auditory stimulus that elicits an auditory brainstem response in an auditory brainstem response assay is about 80-90 decibels or greater.

[0106] The term "viral vector" refers to a recombinant nucleic acid that contains at least one element of viral origin and contains elements sufficient for or that allow packaging into a viral vector particle. The vector and / or particle can be used to transfer DNA, RNA, or other nucleic acids into cells, either ex vivo or in vivo. Many forms of viral vectors are known.

[0107] The term "wild-type" includes entities having a structure and / or activity found in a normal state or context (as opposed to a mutant, diseased, altered, etc.). Wild-type genes and polypeptides often exist in multiple alternative forms (e.g., alleles).

[0108] The phrase "gross mutant phenotype" refers to a significant difference or variation in phenotype between an engineered non-human mouse of the present disclosure and a "wild-type" mouse.

[0109] The term "endogenous" refers to a nucleic acid sequence that naturally occurs in a nucleic acid, cell, or non-human animal.For example, the endogenous Klhdc7b sequence of a non-human animal refers to the natural Klhdc7b sequence that naturally occurs in the endogenous Klhdc7b locus of a non-human animal.Similarly, the endogenous Klhdc7b sequence of a non-human animal nucleic acid or cell refers to the natural Klhdc7b sequence that naturally occurs in the endogenous Klhdc7b locus of a non-human animal nucleic acid or cell.

[0110] The term "variant" refers to a nucleotide sequence that differs (e.g., by one nucleotide) from the sequence most prevalent in a population, or a protein sequence that differs (e.g., by one amino acid) from the sequence most prevalent in a population.

[0111] The term "fragment" or "portion" with respect to a protein refers to a protein that is shorter or has fewer amino acids than the full-length protein. The term "fragment" or "portion" with respect to a nucleic acid refers to 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., removal of a portion of the C-terminus of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminus of the protein), or an internal fragment.

[0112] "Sequence identity" or "identity" in the context of two polynucleotide or polypeptide sequences refers to residues that are the same in two sequences when aligned to maximize correspondence over a defined comparison window. When percentage sequence identity is used in the context of proteins, non-identical residue positions often differ by conservative amino acid substitutions. In conservative amino acid substitutions, an amino acid residue is replaced with another amino acid residue that has similar chemical properties (e.g., charge or hydrophobicity) and thus does not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity." Means for making this adjustment are well known. This typically involves scoring conservative substitutions as partial rather than full mismatches, thereby increasing the percentage of sequence identity. Thus, for example, if identical amino acids are given a score of 1 and non-conservative substitutions are given a score of 0, conservative substitutions would be given a score of 0-1. Scoring of conservative substitutions is calculated, for example, as implemented in the program PC / GENE (Intelligenetics, Mountain View, California).

[0113] "Percentage of sequence identity" includes values ​​determined by comparing two optimally aligned sequences in a comparison window (the maximum number of perfectly matched residues), and the portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences. This percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to determine the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to determine the percentage of sequence identity. Unless otherwise specified (e.g., including heterologous sequences to which the shorter sequence is linked), the comparison window is the full length of the shorter of the two sequences being compared.

[0114] Unless otherwise specified, sequence identity / similarity values ​​include those obtained using GAP version 10 with the following parameters: for nucleotide sequence % identity and % similarity, a GAP weight of 50 and a length weight of 3, and the nwsgapdna.cmp scoring matrix; for amino acid sequence % identity and % similarity, a GAP weight of 8 and a length weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. "Equivalent program" includes any sequence comparison program that produces alignments with identical nucleotide or amino acid residue matches and identical percent sequence identity for two sequences, when compared to corresponding alignments produced by GAP version 10.

[0115] The term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a nonpolar (hydrophobic) residue, such as isoleucine, valine, or leucine, for another nonpolar residue. Similarly, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as arginine for lysine, glutamine for asparagine, or glycine for serine. Furthermore, the substitution of a basic residue, such as lysine, arginine, or histidine, for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid, for another, are further examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a nonpolar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, for a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a nonpolar residue. Typical amino acid classifications are summarized below. [Table 2]

[0116] A "homologous" sequence (e.g., a nucleic acid sequence) includes a sequence that is identical or substantially similar to a known reference sequence, e.g., 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 the known reference sequence. Homologous sequences can include, for example, orthologous and paralogous sequences. For example, homologous genes typically derive from a common ancestral DNA sequence through either a speciation event (orthologous genes) or a gene duplication event (paralogous genes). "Orthologous" genes include genes from different species that evolved from a common ancestral gene through speciation. 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.

[0117] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and both the occurrence and non-occurrence of the event or circumstance are included in the description.

[0118] Designation of a range of values ​​includes all integers within or defining the range, and all subranges defined by integers within the range.

[0119] Unless otherwise clear from the context, the term "about" encompasses values ​​within the standard error of measurement (eg, SEM) of the stated value.

[0120] The term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted as alternatives ("or").

[0121] The term "or" refers to any one member of a particular list and also includes any combination of members of that list.

[0122] 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" can include a plurality of proteins (including mixtures thereof).

[0123] Statistically significant means p≦0.05, p<0.01, p<0.001, or p<0.0001.

[0124] While the present invention has been particularly shown and described with reference to numerous embodiments, it will be understood by those skilled in the art that changes in form and detail may be made to 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 serve as limitations on the scope of the claims. [Example]

[0125] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0126] Example 1: Characterization of KLHDC7b expression According to the University of California, Santa Cruz (UCSC) genome browser, KLHDC7b has two possible isoforms that differ only in the predicted methionine at the start site (see Figure 1). Because the short isoform is completely contained within the long isoform, it is not possible to demonstrate the existence of the short isoform alone, but it is possible to demonstrate the presence or absence of the long isoform. Therefore, two sets of primers and probes were designed: one set to detect the presence of the transcript only within the long portion of the transcript, and the other set to detect the transcript within the overlapping portion (interchangeably referred to herein as the "short" or "overlap").

[0127] Using a mouse multi-tissue cDNA panel, we demonstrated that KLHDC7b is expressed in multiple organs, including mouse liver, brain, and testis (Figure 1, bottom left). The long and overlapping probes showed similar expression levels, suggesting that the short form is likely not expressed solely in mice. Figure 1 (bottom center) also shows KLHDC7b expression in freshly collected mouse liver and kidney samples at levels comparable to those of a commercially available cDNA panel. Furthermore, Figure 1 (bottom center) provides data showing that KLHDC7b is also expressed in freshly isolated cochleae. High- and low-expressing tissues (liver and kidney, respectively) were collected by the cDNA panel, confirming that the fresh tissue results were comparable to those of the cDNA panel (Figure 1, bottom center).

[0128] We also designed long and overlapping probes for the human transcript with two putative isoforms and performed qPCR using a human multi-tissue cDNA panel (Figure 1, bottom right). Both probes showed expression in human liver and testis, with slightly different tissue expression patterns compared to mouse, particularly lower expression in the brain. The overlapping portion was expressed at higher levels than the long form, possibly indicating that the short form may exist independently in humans at a higher frequency than in mice.

[0129] To determine whether KLHDC7b was 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 (aged). qPCR was performed using overlapping probes (Figure 2, left) and long probes (Figure 2, right) and primers. At p1, KLHDC7b expression was highest in the cochlea compared with all other tissues and remained relatively high in adult and aged mice. In other tissues, expression was higher in adults than in neonates. In the liver, expression significantly increased in aged mice.

[0130] qPCR

[0131] qPCR probes for the long and overlapping portions of the mouse (m) or human (h) KLHDC7b transcript were designed using BioSearch's RealTimeDesign qPCR Assay Design Software (RealTimeDesign qPCR Assay Design Software | LGC Biosearch Technologies) and tested for specificity using the BLAT function of the UCSC Genome Browser. Table 2 lists the probes used in this example. [Table 3]

[0132] The m_sKLHDC7b probe was designed to the portion of the transcript that overlaps between the two putative long and short isoforms. m_both_KLHDC7b was designed across the junction between the long and short isoforms, and m_LKLDHC7b was designed to the portion of the transcript that is only present in the long isoform. Primers and probes were designed similarly for the human transcript.

[0133] The KLHDC7b probe was ordered from IDT (Integrated DNA Technologies) as a Primetime qPCR assay using the FAM / ZEN / IBFQ dye combination. For experiments using the mouse cDNA panel (Panel I Catalog No. 636745, Panel III Catalog No. 636757, Takara) and the human cDNA panel (Panel I Catalog No. 636742, Panel II Catalog No. 636743, Takara), cDNA was treated similarly to RNA and used Taqman Fast Advanced Master Mix (e.g., Catalog No. 4444557, Thermofisher Scientific) according to the protocol. Plates were run in a Viaa7 Thermocycler with equal amounts of cDNA plus the Drosha probe, using the housekeeping gene Drosha as a control for both human and mouse experiments. Data were analyzed and plotted using GraphPad Prism.

[0134] For fresh tissue collection, mice were euthanized under CO2 or decapitated if under 7 days of age. Organs were collected and placed in RNA-Later. Cochleae were flash-frozen to preserve RNA integrity for later extraction. For RNA extraction, tissues / cells were homogenized in TRIzol and phase separation was performed using chloroform. The aqueous phase containing total RNA was purified using the MagMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies) according to the manufacturer's specifications. Genomic DNA was removed using an RNase-Free DNase Set (Qiagen).

[0135] mRNA was reverse transcribed into cDNA using SuperScript® VILO™ Master Mix (Invitrogen by Life Technologies). cDNA was amplified with SensiFAST Probe Lo-ROX (Meridian) using a 12K Flex System (Applied Biosystems). An endogenous control gene was used to normalize for differences in cDNA input. Data are reported as the comparative Ct method using ΔCt relative to the housekeeping gene, Drosha.

[0136] Example 2: Detection of KLHDC7b expression in the cochlea After finding that KLHDC7b is expressed in the cochlea, we performed RNA scoping to determine which cell types in the cochlea express KLHDC7b. Similar to the qPCR probes, we generated two probes for overlapping and long portions of the transcript. These probes were applied in combination with immunostaining for the hair cell marker Myo7a. Both probes bound exclusively to the transcript in hair cells. Both probes were present in inner hair cells, outer hair cells, and vestibular hair cells (Figures 3B-3C). RNA scoping was also performed on embryonic mice at approximately e16-17. Both probes were present in what appeared to be developing hair cells, indicating that KLHDC7b is expressed very early in development (Figure 3D).

[0137] RNAScope with or without antibody co-detection

[0138] Animals aged 21 days or older were sacrificed by transcardial perfusion with phosphate-buffered saline (PBS) followed by 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 in PBS and stored at 4°C until processing.

[0139] For RNAscope on paraffin-embedded sections, slides were baked in a HybEZ oven at 60°C for 30 minutes. After baking, slides were deparaffinized by washing twice in xylene for 5 minutes each, followed by 2 x 1 minute washes in 100% ethanol. Slides were allowed to air dry.

[0140] Organoid sections were thawed at room temperature and then washed in PBS for 5 minutes with agitation to remove OCT. Sections were post-fixed in cold 10% neutral buffered formalin (NBF) for 15 minutes at 4°C. They were then dehydrated in 50% ethanol, 70% ethanol, and 100% ethanol for 5 minutes each and allowed to dry at room temperature.

[0141] For both paraffin-embedded cochlear sections and explants, hydrogen peroxide from the RNAScope kit was added to each slide, incubated at room temperature for 10 minutes, and then washed twice with distilled water. Co-detection target retrieval reagent (ACD Bio) was heated in a vegetable steamer (Oster). Slides were placed in the hot co-detection target retrieval reagent for 15 minutes for cochleae and 5 minutes for organoids, followed by two washes in distilled water. Slides were then washed with PBS + 0.1% Tween®-20 (PBS-T). A hydrophobic barrier was drawn around the sections on the slides. Antibody (Myo7a, Proteus, 25-6790) was diluted 1:200 in co-detection antibody diluent and incubated overnight at 4°C in a humidified chamber.

[0142] After the primary incubation, the slides were washed three times with PBS-T for 2 minutes each. They were then post-fixed in 10% neutral buffered formalin for 30 minutes in a fume hood. After that, they were washed again with PBS-T four times 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.

[0143] Fluorescence in situ hybridization or RNAscope was then performed according to ACD Bio's protocol. Briefly, the probe was heated at 40°C for 10 minutes, and the wash buffer was heated at 40°C for 20 minutes before dilution to the appropriate concentration. The probe was applied to the slide and placed in a humidified oven chamber at 40°C for 2 hours. All subsequent incubation steps were performed in a humidified oven chamber at 40°C, and all washes were performed with wash buffer. After probe hybridization, the slide was washed 2 x 2 minutes with wash buffer. Next, the amplification step involved a 30-minute incubation with Amp 1 (ACD Bio) followed by 2 x 2-minute washes. Amp 2 was incubated for 30 minutes followed by 2 x 2-minute washes. Amp 3 was incubated for 15 minutes followed by 2 x 2-minute washes.

[0144] After amplification, depending on the number of channels used, slides were incubated in HRP C1 for 15 minutes followed by 2 x 2 minute washes, then 1:15000 Opal 520 for 30 minutes followed by 2 x 2 minute washes. Slides were incubated in HRP blocker for 15 minutes followed by 2 x 2 minute washes. Slides were then incubated in HRP C2 for 15 minutes followed by 2 x 2 minute washes, then 1:1500 Opal 570 for 30 minutes followed by 2 x 2 minute washes. Slides were then again incubated in HRP blocker for 15 minutes followed by 2 x 2 minute washes. Slides were then incubated in 1:1500 Opal 570 for 30 minutes followed by 2 x 2 minute washes. Slides were then again incubated in HRP blocker for 15 minutes, followed by 2 x 2 minute washes, then incubated in 1:1500 Opal 690 for 30 minutes, 2 x 2 minute washes, and a 15 minute HRP block. If only one channel was used, Oval 570 was used and the HRP step for the subsequent channel was omitted.

[0145] Finally, the secondary antibody (donkey anti-rabbit Alexa 647) was diluted 1:500 in co-detection antibody diluent and incubated for 30 min at room temperature in a humidified chamber, followed by 2 × 2 min washes in PBS-T. DAPI was applied to the slide for 30 s and shaken off the slide, after which Prolong Gold Antifade mounting medium was applied and a coverslip was placed on the section and allowed to dry overnight at room temperature.

[0146] For RNAscope (for organoid markers) without co-detection, the steps were similar, except that after antigen retrieval, slides were placed in 100% ethanol and allowed to dry overnight, post-fixation after antibody incubation was omitted, and secondary antibody incubation was omitted.

[0147] Microscopy

[0148] Images of RNAscope and immunostained samples were acquired with a confocal microscope (either a Zeiss LSM 780 or Zeiss LSM 880) using 20x and 100x objectives. Z-stacks were acquired at a size appropriate for the objective and numerical aperture, or tile scans were acquired as needed. Post-hoc image processing and analysis were performed in Fiji and Imaris (PLA quantification).

[0149] Example 3: Knockout of the endogenous mouse Klhdc7b gene A targeting vector for knocking out the endogenous Klhdc7b gene was constructed using bacterial artificial chromosome (BAC) cloning and VELOCIGENE® technology (see, e.g., U.S. Pat. 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, which are incorporated herein by reference).

[0150] The BAC clone RP23-241G24 containing the mouse Klhdc7b gene was modified as follows. Briefly, a DNA fragment was generated containing a 100-bp mouse 5' homologous nucleotide sequence (mHU), a LacZ gene (3,075 bp) located in frame downstream of the ATG start site of the mouse Klhdc7b gene, followed by a 4,809-bp self-deleting neomycin cassette, and a 100-bp 3' mouse homologous sequence (mHD). This DNA fragment was used to modify the BAC clone RP23-241G24 through homologous recombination in bacterial cells. As a result, a complete knockout (KO) of the region encoding the 3,787-bp mouse Klhdc7b genomic fragment in the BAC clone was replaced with an 8,202-bp 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 the 5' and 3' untranslated regions (UTRs) intact (Figures 4A–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) a 3,075-bp LacZ cDNA, (iii) a 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 from the original BAC clone (Figures 4A–4B). The amino acid sequence of the protein encoded by the LacZ cDNA is shown in SEQ ID NO: 4. The LacZ cDNA sequence is shown as SEQ ID NO: 3.

[0151] The modified BAC clone containing the Klhdc7b gene KO described above was used to electroporate mouse embryonic stem (ES) cells to generate modified ES cells containing the Klhdc7b KO gene. Appropriately 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 loss and / or retention of the mouse Klhdc7b sequence was confirmed by TaqMan assay (SEQ ID NOS: 8-19, Table 3). Once correctly targeted ES cell clones were selected, they were electroporated into early-stage blastocysts (8-cell morula stage) to generate F0 mice. The neomycin selection cassette was removed by crossing offspring generated from the ES clones with a deleter rodent strain expressing Cre recombinase (Figure 4C).

[0152] Selected ES cell clones (with or without the cassette) were used to generate female B6.Cast-Cdh23 mice by the VELOCIMOUSE® method. Ahl+ The resulting allele was then transplanted into mice (see, e.g., U.S. Patent No. 7,294,754 and Poueymirou et al., 2007, Nature Biotech. 25(1):91-99) to generate a litter of mice containing the Klhdc7b KO allele in their genome. Ahl+ Mice were used because mice of this background contain an allele-correcting form of Cdh23 that prevents the known age-related hearing loss that occurs in C57BL6 mice.

[0153] Mice carrying the Klhdc7b KO allele were confirmed and identified by genotyping DNA isolated from tail snips using a modified version of the allele assay (Valenzuela et al., supra) that detects the presence / absence of Klhdc7b gene sequences. Pups were genotyped, and a cohort of animals heterozygous for the Klhdc7b locus was selected for characterization. Homozygous KO animals for the Klhdc7b locus were generated by mating with heterozygous animals. [Table 4]

[0154] Mice were born in normal Mendelian ratios and appeared phenotypically normal. LacZ staining revealed that KLHDC7B + / - (HET) and KLHDC7B - / - In the cochlea of ​​(KO) mice, the Klhdc7b gene was present in presumed hair cells (Figure 4D), confirming that the Klhdc7b gene had been replaced with the LacZ cassette. Other cells in the cochlea, including vestibular hair cells, did not appear to display LacZ labeling. Furthermore, RNAScope probes for the long and overlapping transcripts of KLHDC7B revealed that the Klhdc7b gene was present in presumed hair cells (Figure 4D). - / - (KO) mice hair cells were not labeled ( Fig. 4E ), confirming the absence of transcribed Klhdc7b mRNA.

[0155] LacZ staining of knockout mice

[0156] Mice were anesthetized with ketamine / xylazine and fixed by perfusion with 2% paraformaldehyde. After perfusion, tissues were dissected, sectioned into 1-5 mm pieces, fixed for 30 minutes at room temperature, washed for 30 minutes in PBS, and stained overnight at 4°C in beta-galactosidase (lacZ) staining solution. After staining, tissues were washed for 15 minutes in cold PBS and post-fixed overnight at 4°C in 4% formaldehyde with mixing. Tissues were cleared in glycerol by incubating in 50% glycerol at room temperature for 1 day, then in 70% glycerol for 1 day. Tissues were photographed under a Zeiss dissecting microscope and stored in 70% glycerol at room temperature. Tissues were then decalcified overnight using immunocal, dissected, and then photographed using an Axioscan slide scanner (Zeiss).

[0157] Example 4: Characterization of KLHDC7b knockout mice The cochlear phenotype and auditory responses of the knockout animals described in Example 3 will be examined to determine the phenotypic effects of KLHDC7b knockout.

[0158] Histological examination

[0159] At postnatal day (p) 6, the organ of Corti in KO mice appeared normal, with intact hair cells stained for Myo7A and intact stereocilia stained for F-actin (Figure 5A). However, at p11, some outer hair cells appeared missing and did not label for Myo7A or F-actin. By p21, many outer hair cells were missing, and supporting cell scars, known to form when hair cells die, were visible (Wagner and Shin, 2019). Circular areas of MYO7A staining were present, which may represent hair cells being engulfed by supporting cells or hair cells being extruded from the sensory epithelium. By 8 weeks of age, outer hair cells were largely absent, and significant scarring was present (Figure 5A). At 8 weeks, hair cells in KLHDC7b knockout mice had morphological abnormalities, worse at the base than at the apex (Figure 5B). In whole mounts, hair cells appear to be missing, more severely at the base than at the apex (Figure 6). At postnatal day 3 (p3) in whole mount samples, hair cells do not appear to be missing, and stereocilia appear normal (Figures 7A-7C). Even at postnatal day 6, hair cells appear normal. The tight junction protein ZO-1 showed diffuse cytoplasmic staining in some hair cells only in knockout mice at p11 and p21, but not at earlier time points, suggesting that this protein may be mislocalized in the KO tissue before or after cell death (Figures 7D-7J).

[0160] 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.

[0161] To determine the effect of KLHDC7b knockout on mouse hearing, ABR and DPOAE hearing assays were performed (Figure 8). - / - (KO), KLHDC7B + / -(Het), and KLHDC7B + / + Auditory brainstem response (ABR) testing was performed on WT mice at different ages to assess hearing (Figure 9). Mice develop hearing at 2 weeks of age. At postnatal day 17 (p17, 2-3 weeks), KO mice exhibit severe hearing loss, with significantly elevated hearing thresholds and significantly reduced I-wave amplitude. These thresholds gradually increase, and most mice show no response by 11-15 weeks of age (Figure 9A). Heterozygous mice have normal ABR thresholds and I-wave amplitudes even at 57 weeks compared to WT mice (Figure 9B). At the time of hearing development (approximately day 17), KLHDC7b knockout mice exhibit hearing loss, with significantly elevated ABR thresholds compared to wild-type mice (Figure 9C). Heterozygotes do not exhibit hearing loss (Figure 9C). Hearing loss appears to be progressive. At 8 weeks, knockout mice were profoundly deaf at all frequencies tested, with some retained responses at the highest frequency tested in both ABR and DPOAE (Fig. 10). Heterozygous mice were not deaf at 30 weeks (Figs. 11A-11C).

[0162] Histological examination

[0163] To immunostain paraffin-embedded sectioned cochleae on slides, slides were first baked in a HybEZ oven at 60°C for 1 hour to melt the wax, then washed twice in xylene for 3 minutes each. Slides were then rehydrated in 100% ethanol for 2 × 3 minutes, then 95% ethanol for 2 × 3 minutes, then 70% ethanol for 3 minutes, 50% ethanol for 3 minutes, distilled water for 5 minutes, and PBS for 3 × 2 minutes each.

[0164] A blocking solution (2% w / v bovine serum albumin, 5% normal donkey serum, 0.01% Triton-x100 in PBS) was prepared. A hydrophobic barrier was drawn around the sections on the slides, and the slides were placed in a humidified chamber. After covering with the blocking solution, the slides were incubated at room temperature for 1 hour. The primary antibody was diluted in the blocking solution, the blocking solution was removed from the slides, and the primary antibody solution was added to the slides. The slides were then placed at 4°C and incubated overnight. After the primary incubation, the slides were washed three times in PBS, then covered with secondary antibody and cell stain diluted in blocking solution and incubated for 1 hour. The slides were then washed three times in PBS, and mounting medium (Prolong Diamond or Prolong Gold) was placed on the slides, covered with a coverslip, and allowed to dry overnight before being imaged using a confocal microscope.

[0165] A similar procedure was performed for whole-mount samples. Dissected sections were washed three times in PBS, incubated in blocking buffer for 1 hour with shaking at room temperature, and then incubated overnight with primary antibodies in blocking solution at 4°C or room temperature, followed by three washes with PBS. Secondary antibodies and cell stains were diluted in blocking buffer, and the samples were incubated for 1 hour at room temperature. After rinsing three times with PBS, they were placed on slides, covered with mounting medium and coverslips, and allowed to dry overnight before being imaged by confocal microscopy. Phalloidin was used only for whole-mount samples, not for slides.

[0166] Auditory brainstem response (ABR)

[0167] Auditory brainstem response recordings were performed in a soundproof room. The device was calibrated daily using a microphone to ensure 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 heated cages. After the animals became unresponsive for several minutes, Pularube ointment was applied to the eyes.

[0168] Once the mouse no longer responded to a toe pinch, it was placed on a heating pad inside a soundproof chamber. Electrodes were inserted into a preamplifier. The needle tips of the electrodes were placed subcutaneously, with the anode on the animal's cheek (near the cochlea), the cathode on the midline of the skull at the top of the head, and the ground electrode on the opposite cheek. The recording ear (the right ear in these experiments) was positioned 7.5 mm from the speaker, which was in an open-field configuration rather than a closed-field configuration with a tube inserted directly into the ear. The soundproof chamber was closed, and recording began.

[0169] Recordings were performed at three pure-tone frequencies (8 kHz, 16 kHz, and 32 kHz), averaging 512 presentations of each stimulus at each presented sound pressure level. Each frequency was played at 90 decibels (dB), followed by 80, 70, and 60, decreasing in 10 dB increments until 50. From 50 dB to 15 dB, the dB level was decreased in 5 dB increments rather than 10 dB increments. Auditory brainstem response waveforms were recorded. When a waveform became invisible, the sound pressure level of the previous waveform was determined as the threshold for that frequency. Once threshold was reached, stimuli at one or two sound pressure levels were recorded, but further recordings were skipped. Thresholds were recorded for each animal, and the animals were grouped by age or sex. Animals were then housed in a heated recovery cage and returned to their home cages when ambulatory.

[0170] ABR analysis

[0171] ABR recordings were processed in Matlab®. Traces were first smoothed with a moving median filter using 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 effect on clean recordings.

[0172] ABR thresholds were determined manually by two experimenters for traces presented in a blinded, random order and subjected to 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 sigmoidal or logarithmic function, and the decibel level below which the function established a reference level was recorded as the hearing threshold. The threshold determined by the algorithm was compared to the manually determined threshold. Traces without a clear ABR response were obtained at 100 dB. If the difference between the manual and automatic thresholds exceeded 15 dB, the trace was inspected and the manual threshold was used; otherwise, the automatic threshold was used.

[0173] For wave 1 amplitude and latency, peaks were detected using a semi-automated method in which peaks and troughs were estimated within a time window including wave 1, which was then reviewed by the user and corrected if necessary.

[0174] Distortion Product Otoacoustic Emissions (DPOAEs)

[0175] For DPOAE recording, the loudspeaker was also calibrated daily. DPOAE measurements were performed either alone or immediately after ABR while the animals were still anesthetized. For DPOAEs alone, animals were anesthetized as for ABR recording. After the animals no longer responded to toe pinching, they were placed in the recording chamber.

[0176] Two speakers were used to present two equally spaced frequencies centered around the three frequencies measured for ABR (8 kHz, 16 kHz, and 32 kHz). The speakers were used in a closed-field configuration. The speakers were connected by tubing to a highly sensitive microphone with a cut pipette tip, which was then inserted into the ear canal and tilted toward the eardrum. Stimuli were presented 100 times, averaged. Distortion components were detected at the expected frequencies, and if the signal exceeded the noise, the user manually marked it as positive.

[0177] Example 5 - Gentamicin-Texas Red mechanical signaling assay in cochlear explant cultures Because defects in mechanical signaling can lead to hair cell death, we assessed the mechanotransduction (MET) complex before cell death. Although hearing does not develop in mice until 2 weeks of age, mechanotransduction channels can be assessed in cultured organs of Corti using the gentamicin-Texas Red assay (GtTR). Gentamicin is known to be taken up by the MET complex, and conjugating it to the dye Texas Red can confirm the presence of a functional MET complex. GtTR similarly labeled hair cells in cultured organs of Corti from KO and WT mice, indicating that the MET complex was assembled and functional. Hair cells in cochlear explant cultures appeared to take up similar amounts of GTTR (Figures 12A-12C), indicating that the MET complex was likely functional and that KLHDC7b was not required for the assembly of these complexes.

[0178] Human inner ear RNA is extremely difficult to obtain due to the fragile nature of hair cells and the time required to extract the temporal bone. Therefore, to examine KLHDC7b expression in human ear tissue, we generated ear organoids using a protocol similar to that described by Koehler et al., 2017. These organoids begin with human induced pluripotent stem cells (iPSCs) and are induced to undergo multiple developmental stages by the administration of small molecules to become mature inner ear organoids at approximately day 70. At this time, mature inner ear organoids contain hair cells, supporting cells, and neurons. Expression of inner ear cell markers, including neuron (TUBB3) and hair cell (OTOF, MYO7A, MYO15A) markers, in mature ear organoids was significantly increased compared to expression levels in human iPSCs (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 where inner ear cell types are highly concentrated, we performed RNA scoping to examine the expression patterns of otic markers and KLHDC7b. Otic markers, including SOX2 and TUBB3, were found to be concentrated in several spatial regions resembling the otic vesicle, as shown by Koehler et al., 2017 (Figure 13A, left). RNA scoping for KLHDC7B long and overlapping transcripts was performed along with immunostaining for the hair cell marker Myo7a. The KLHDC7B probe has been shown to label sections of the same otic organoid (Figure 13B, left). Labeling was not exclusively within hair cells but was concentrated in the area surrounding the presumed otic vesicle. The two sections labeled with the long and overlapping probes were from nearby sections of the same otic organoid, and they were located in the same region of the organoid, near the circle of putative hair cells labeled with Myo7a, visible in the image of the overlapping KLHDC7B probe.

[0179] Cochlear explant culture

[0180] Cochlear explant culture was performed using postnatal day 0–5 mouse pups. The cell culture plate used was a 35 mm dish with a circular opening and a glass cover glass attached to the bottom (Matsunami, #D35-14-0-U). A collagen bubble was prepared by mixing 1.7 μl of 1N NaOH, 10 μl of 10x PBS, 67 μl of rat tail collagen (pipette setting 68 μL), and 21.3 μl of HO. 10 μl of this solution was placed on the cover glass and allowed to harden for 30–40 minutes at 37°C in a humidified cell culture incubator with 5% CO2. The collagen bubble was covered with PBS and stored at 4°C for up to 2 months until use. Explant culture medium was prepared as follows: 90 ml of DMEM / F12 without phenol red, 7 ml of fetal bovine serum, 1 ml of penicillin G, and 1 ml of L-glutamine.

[0181] The mouse was decapitated and the skull was bisected. The cochlea was removed and placed in Leibovitz / L15 medium. The bone was opened, the organ of Corti was removed, and the stria vascularis was dissected. PBS was removed from the culture plate and replaced with cochlear explant medium. The organ of Corti was placed on a 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°C with 5% CO2. After dissection, 200 μl of explant culture medium was added to the dish. The culture was then transferred from the breeding room to the laboratory, and 600 μl of culture medium was added.

[0182] Gentamicin-Texas Red (GtTR) assay

[0183] Explant cultures were maintained in culture for 2–3 days and then treated with gentamicin-Texas Red (GtTR) or Texas Red (TR) alone. GtTR was 5 μg / ml, and TR was 12.5 μg / ml. GtTR or TR was diluted in cell culture medium. The existing medium on the explants was removed, and 600 μL of GtTR or TR-containing medium was added and treated for 20 minutes. Explant cultures were rinsed with medium and PBS, then fixed in 4% PFA for 15 minutes, rinsed three times with PBS, and stored at 4°C for several days. Afterwards, they were immunostained using the same protocol used for whole-mount cochleae described herein.

[0184] Ear organoids

[0185] Inner ear organoids were generated according to PROTOCOL (Version 1) of Zhang et al. (2021) "A simplified method for generating human inner ear organoids from pluripotent stem cells" (doi.org / 10.21203 / rs.3.pex-1708 / v1, incorporated herein by reference in its entirety). Briefly, human iPSCs were aggregated in a chemically defined medium to form embryoid bodies, and ectodermal placode formation was induced. On day 8 of induction, the Wnt signaling agonist CHIR99012 (Tocris Catalog No. 4423) was added to the medium to stimulate otic vesicle formation. Organoids were then cultured in maturation medium for over 100 days to allow the otic sensory epithelium to mature. For these experiments, organoids were harvested on day 70.

[0186] RNAscope and immunofluorescence of ear organoids

[0187] Fixed, frozen, fully differentiated ear organoids were used for RNAscope (10 μm cryosections). Some slides were used for RNAscope in combination with immunofluorescence, while others were used for RNAscope alone, as described below. For RNAscope, slides were baked in a HybEZ oven at 60°C for 30 minutes. After baking, slides were post-fixed in cold 4% paraformaldehyde (PFA) in 1x PBS at 4°C for 15 minutes. Slides were then dehydrated in 50% ethanol, 70% ethanol, and 100% ethanol for 5 minutes each at room temperature. Slides were then air-dried at room temperature.

[0188] Hydrogen peroxide from the RNAScope kit was added to each slide, incubated for 10 minutes at room temperature, and then washed twice with distilled water. Co-detection target retrieval reagent for co-treated slides or target retrieval reagent for RNAScope alone (ACD Bio) and a separate container of distilled water were heated in a vegetable steamer (Oster). Slides were placed in the hot distilled water for 10 seconds, then in the co-detection target retrieval reagent or target retrieval reagent for 5 minutes, followed by two washes in distilled water at room temperature.

[0189] The co-detection slides were then washed four times for 2 minutes each in PBS + 0.1% Tween®-20 (PBS-T). The RNAScope 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 RNAScope slides were stored overnight at room temperature. The co-detection slides were treated with primary antibody (Myo7a, Proteus, 25-6790) diluted 1:200 in the co-detection antibody diluent and incubated overnight at 4°C in a humidified chamber.

[0190] For the co-detection slides, after the primary incubation, the slides were washed three times with PBS-T for 2 minutes each. They were then post-fixed in 10% neutral buffered formalin for 30 minutes in a fume hood. After that, they were washed again four times with PBS-T for 2 minutes each. Both the RNAScope-only slides and the co-detection slides were then treated with Protease III reagent (ACD Bio) in a HybEZ oven at 40°C for 30 minutes.

[0191] Fluorescence in situ hybridization or RNAscope was then performed according to ACD Bio's protocol. Briefly, the probe was heated at 40°C for 10 minutes, and the wash buffer was heated at 40°C for 20 minutes before dilution to the appropriate concentration. The probe was applied to the slide and placed in a humidified oven chamber at 40°C for 2 hours. All subsequent incubation steps were performed in a humidified oven chamber at 40°C, and all washes were performed with wash buffer. After probe hybridization, the slide was washed 2 x 2 minutes with wash buffer. Next, the amplification step involved a 30-minute incubation with Amp 1 (ACD Bio) followed by 2 x 2-minute washes. Amp 2 was incubated for 30 minutes followed by 2 x 2-minute washes. Amp 3 was incubated for 15 minutes followed by 2 x 2-minute washes.

[0192] After amplification, slides were incubated in HRP C1 for 15 minutes followed by 2 x 2 minute washes, then 1:15000 Opal 520 for 30 minutes followed by 2 x 2 minute washes. Next, a 15 minute incubation in HRP blocker followed by 2 x 2 minute washes. Slides were then incubated in HRP C2 for 15 minutes followed by 2 x 2 minute washes, then 1:15000 Opal 570 for 30 minutes followed by 2 x 2 minute washes. Next, a 15 minute incubation in HRP blocker followed by 2 x 2 minute washes.

[0193] RNAscope alone slides were treated with HRP C3 for 15 minutes followed by 2 x 2 minute washes, then incubated with 1:15000 Opal 690 for 30 minutes followed by 2 x 2 minute washes, then treated with HRP blocker for 15 minutes followed by 2 x 2 minute washes.

[0194] For co-detection slides, the secondary antibody (donkey anti-rabbit Alexa 647) was diluted 1:500 in co-detection antibody diluent and incubated for 30 minutes at room temperature in a humidified chamber, followed by 2 x 2 minute washes in PBS-T. In both conditions, DAPI was applied to the slide for 30 seconds and shaken off, after which Prolong Gold Antifade mounting medium was applied, a coverslip was placed on the section, and the section was allowed to dry overnight at room temperature.

[0195] Microscopy

[0196] Images of RNAscope and immunostained samples were acquired with a confocal microscope using 20x and 100x objectives. Z-stacks or tile scans were acquired as needed.

[0197] Example 6 - Scanning Electron Microscopy Although hair cells appear normal until they begin to die at p11, we examined their morphology and function before the onset of cell death. Scanning electron microscopy (SEM) was performed at p10, just before the onset of cell death, and then at p22, after significant death had occurred. A similar region of the organ of Corti is illustrated in the SEM images in Figure 14. Both heterozygous and KO mice, which do not have a hearing loss phenotype at 50 weeks, have grossly normal stereocilia before the onset of cell death (Figure 14, left). Even at p22, many outer hair cells are clearly missing, but the morphology of the stereocilia on the remaining outer hair cells appears normal (Figure 14, right).

[0198] Scanning electron microscopy

[0199] The cochlea was removed from the temporal bone. A very small hole was drilled at the apex with a hand drill, the stapes was removed, and the oval window was drilled. The specimen was then fixed overnight in a mixture of 3% formaldehyde and 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences, Hatfield, PA) supplemented with 2 mM CaCl2. The specimen was then washed in 0.1 M sodium cacodylate buffer, decalcified in 10% formic acid (Immunocal) for 24 hours, washed three times, and dissected into three flat turns. The bone layer, spiral ligament, and tectorial membrane were carefully removed from each turn, and the cochlear sections were placed in porous baskets. The samples were then dehydrated through a graded ethanol series (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 of platinum, and imaged with a field emission scanning electron microscope (Zeiss Sigma VP).

[0200] Example 7 - Assessment of KLHDC7b localization with custom monoclonal anti-KLHDC7b antibody(ies) We generated several custom monoclonal antibodies against mouse KLHDC7b using transiently transfected HEK cells (Table 4). Cell lines stably expressing the mouse long and short isoforms of KLHDC7b were generated and used to test the phenotypic binding of our custom anti-KLHDC7b antibodies. These cell lines were validated by Western blot and immunohistochemistry for the FLAG tag using Genscript. As shown in Figure 15, the anti-KLHDC7b antibody labeled only both inner and outer hair cells in wild-type mouse cochleae and appeared to be localized to the membrane. Furthermore, the anti-KLHDC7b antibody stained the plasma membrane of hair cells in paraffin-embedded cochlear sections isolated from wild-type mice but not from knockout KLHDC7b mice (Figure 16). The anti-KLHDC7b antibody was also able to recognize human KLHDC7b expressed in transiently transfected HEK cells (FIGS. 17A-17B, Table 5).

[0201] Transient transfection of HEK cells with mouse KLHDC7B constructs for custom antibody generation

[0202] 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 were used as a negative control. After 48–72 h, they were harvested for Western blot or immunostaining, detached from the plate, fixed, embedded, sectioned, and placed on slides for antibody testing. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0203] Stably transfected cell lines

[0204] Transfections were performed using Lipofectamine 3000 according to the manufacturer's instructions. Cell culture medium was replaced on the day of transfection, and DNA was diluted in Opti-Mem with P-3000. Lipofectamine was diluted in Opti-Mem. The DNA and Lipofectamine solutions were then mixed and incubated at room temperature for 10–15 minutes. This mixture was added to the cells, and the cultures were checked or harvested 48–72 hours later. Cell lines were generated by Genscript using lentivirus to stably express either the short or long isoform of mouse KLHDC7b tagged with FLAG at the C-terminus. Cell lines were cloned and verified using Western blot for FLAG and immunohistochemistry for the FLAG tag.

[0205] Transient transfection of HEK cells with human KLHDC7B constructs for antibody testing

[0206] HEK-293 cells were grown in 96-well plates and transiently transfected with plasmids expressing FLAG-tagged short-chain KLHDC7B-3XFLAG, long-chain KLHDC7B-3XFLAG, and H2B-GFP-3XFLAG using Lipofectamine 3000 according to the manufacturer's instructions (Table 5). After 48–72 h, cells were rinsed with PBS, fixed with 4% PFA for 20 min at room temperature, rinsed three times with PBS, and immunostained with an antibody against FLAG (Thermofisher, MA1-91878) at a concentration of 1:200 and 10 different reagent antibody clones at a concentration of 1:600 ​​each. GFP-transfected samples were stained with anti-GFP (Abcam, ab13970) and subsequently imaged using an Opera Phoenix automated confocal microscope. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10] [Table 6-11]

[0207] Example 8 - Discussion KLHDC7b is expressed in the cochlea and other organs. Within the cochlea, KLHDC7b is expressed exclusively in hair cells, as determined by RNAscope and immunofluorescence using custom-made antibodies. KLHDC7b is also expressed in human ear organoids, where both the long and overlapping portions of the transcript are detectable by RNAscope.

[0208] KLHDC7b does not appear to be required for the development of cochlear or inner ear hair cells or for the assembly of mechanotransduction complexes, but it appears to be required for the maintenance of hearing: hair cells are lost at time points when mice are severely deaf, as shown by immunohistochemistry and scanning electron microscopy.

[0209] As shown by immunostaining with a custom anti-KLHDC7b antibody, KLHDC7b appears to be localized to the plasma membrane of hair cells. KLHDC7b also appears to be mislocalized to the cytoplasm of some hair cells at the time they begin to degenerate.

Claims

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

2. 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. 2. 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 modified endogenous Klhdc7b locus further comprises an insert nucleic acid; The genetically modified non-human animal nucleic acid according to any one of claims 1 to 3, wherein the insert 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 reporter gene is operably linked to a promoter; The genetically modified non-human animal nucleic acid of claim 5 , wherein the promoter drives expression of the reporter gene.

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

8. The genetically modified non-human animal nucleic acid according to any one of claims 5 to 7, wherein the reporter gene encodes a reporter 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, and combinations thereof.

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

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

11. 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. 10. 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 modified endogenous Klhdc7b locus is the nucleic acid sequence shown as SEQ ID NO: 5, and / or the nucleic acid sequence set forth as SEQ ID NO: 6 or the nucleic acid sequence set forth as SEQ ID NO: 7, and / or The nucleic acid sequence shown as SEQ ID NO: 38 or the nucleic acid sequence shown as SEQ ID NO: 39 The genetically modified non-human animal nucleic acid of claim 14, comprising:

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

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

18. 18. A non-human animal genome comprising the non-human animal nucleic acid of any one of claims 1 to 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 according to any one of claims 1 to 17, or the non-human animal genome according to 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. 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. 22. A genetically modified non-human animal cell as described in claim 20 or claim 21, wherein the deletion extends 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 modified endogenous Klhdc7b locus comprises an inserted nucleic acid; 23. The genetically modified non-human animal cell according to any one of claims 20 to 22, wherein the insert nucleic acid replaces the deleted endogenous Klhdc7b gene or a portion thereof.

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

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

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

27. The genetically modified non-human animal cell according to any one of claims 24 to 26, wherein the reporter gene encodes a reporter 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. 28. The genetically modified non-human animal cell of any one of claims 23 to 27, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the reporter gene.

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

30. 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 according to any one of claims 19 to 30, wherein the non-human animal cell is a rodent cell.

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

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

34. The mouse cells were B6.Cast-Cdh23 Ａｈｌ＋ 34. The genetically modified non-human animal cell of claim 33, which is a mouse cell.

35. the endogenous Klhdc7b locus the nucleic acid sequence shown as SEQ ID NO: 5, and / or the nucleic acid sequence set forth as SEQ ID NO: 6 or the nucleic acid sequence set forth as SEQ ID NO: 7, and / or The nucleic acid sequence shown as SEQ ID NO: 38 or the nucleic acid sequence shown as SEQ ID NO: 39 35. The genetically modified non-human animal cell of claim 33 or claim 34, comprising:

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

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

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

39. The genetically modified non-human animal cell according to any one of claims 19 to 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 according to any one of claims 1 to 17, the non-human animal genome according to claim 18, or the non-human animal cell according to any one of claims 19 to 39.

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

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

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

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

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

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

48. 48. The genetically modified non-human animal of any one of claims 45 to 47, wherein the reporter gene encodes a reporter that is 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. 49. The genetically modified non-human animal of any one of claims 45 to 48, wherein the insert nucleic acid comprises site-specific recombination sequences flanking the reporter gene.

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

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

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

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

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

55. The mice were B6.Cast-Cdh23 Ａｈｌ＋ 55. The genetically modified non-human animal of claim 54, which is a mouse.

56. the endogenous Klhdc7b locus the nucleic acid sequence shown as SEQ ID NO: 5, and / or the nucleic acid sequence set forth as SEQ ID NO: 6 or the nucleic acid sequence set forth as SEQ ID NO: 7, and / or The nucleic acid sequence shown as SEQ ID NO: 38 or the nucleic acid sequence shown as SEQ ID NO: 39 56. The genetically modified non-human animal of claim 54 or claim 55, comprising:

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

58. 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. 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 at about 17 days of age.

60. 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 at about 17 days of age; the genetically modified non-human animal exhibits hearing loss at about 17 days of age, and the hearing loss progresses throughout the life of the genetically modified non-human animal; the genetically modified non-human animal exhibits severe hearing loss at 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, and further optionally, wherein degeneration of the hair cells is observed after 11 days postnatal; and / or the genetically modified non-human animal exhibits normal mechanical signaling in the cochlea as measured by a gentamicin-Texas Red assay; 60. The genetically modified non-human animal according to any one of claims 57 to 59.

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

62. 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. 62. 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. 64. The method of any one of claims 61 to 63, wherein deleting the endogenous Klhdc7b gene or a portion thereof comprises replacing the endogenous Klhdc7b gene or a portion thereof with an inserted nucleic acid.

65. The insert nucleic acid is reporter genes, 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 encoding a reporter 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; 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 65. The method of claim 64, comprising:

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

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

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

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

70. The method according to any one of claims 61 to 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 gestating the non-human animal ES cell of claim 70 in a surrogate mother; The method, wherein the surrogate mother produces a progeny non-human animal, the progeny non-human animal comprising the modified endogenous Klhdc7b locus in its germline genome.

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

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

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

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

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

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

78. 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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