IL322105A - Animals containing a modified klhdc7b locus - Google Patents

Description

WO 2024/163650 PCT/US2024/013826 ANIMALS COMPRISING A MODIFIED KLHDC7B LOCUS CROSS-REFERENCE TO RELATED APPLICATIONS [0001]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 hereby incorporate by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS AN XML FILE [0002]The Sequence Listing written in file “11348WO01 Sequence Listing XML” is 1kilobytes, was created on January 31, 2024, and is hereby incorporated in its entirety by reference. TECHNICAL FIELD [0003]Described are non-human animals, cells, and tissues, and methods for making and using them, that comprise a modified K1hdc7b locus, which may comprise a deletion and/or a replacement of an endogenous Klhdc7b gene or portion thereof. The non-human animals described may comprise phenotypes consistent with hearing loss.

BACKGROUND OF THE INVENTION

[0004]Auditory dysfunction in humans is associated with social isolation and cognitive decline, and thus, is an ongoing problem in the medical fields of otology and audiology. About 1.5 billion people live with hearing loss and over 34 million children exhibit deafness or hearing loss. [0005]Currently, very few cases of hearing loss can be cured. Audiological devices such as hearing aids have limitations including the inability to improve speech intelligibility. Of those impacted by hearing impairments, less than 20 percent presently use hearing instruments. In cases of age-related, noise- or drug-induced auditory dysfunctions, often the only effective way to currently "treat" the disorder or reduce its severity is prevention, such as by avoiding excessive noise and using ear protectors, practicing a healthy lifestyle, and avoiding exposure to ototoxic drugs and substances if possible.

WO 2024/163650 PCT/US2024/013826

[0006]The prevalence of hearing loss after damage to the mammalian cochlea has been thought to be due to a lack of spontaneous regeneration of hair cells and/or neurons, the primary components to detect sound. Humans are born with about 15,000 inner ear hair cells and hair cells do not regenerate after birth. [0007]Thus, there remains a long felt need to determine the biology and mechanisms involved in protecting protect auditory hair cells before injury and/or in preserving/promoting the function of existing cells after injury.

SUMMARY OF THE INVENTION

[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 an endogenous Klhdc7b gene, or portion thereof. The deletion may comprise, consist essentially of, or consist of a deletion of an open reading frame (orf) of an endogenous Klhdc7b gene at the endogenous Klhdc7b locus of the non-human animal nucleic acids, non-human animal cells, and non-human animals, e.g., the deletion spans between, but does not include or extend beyond, an endogenous start codon of the endogenous Klhdc7b gene and an endogenous stop codon of the endogenous Klhdc7b gene. In some embodiments, the deletion may be the result of a replacement of the endogenous Klhdc7b gene, or a portion thereof (e.g., an orf portion thereof) with an insert nucleic acid. In some embodiments, the insert nucleic acid may comprise a reporter gene and/or a gene encoding a selectable marker, optionally wherein the reporter gene is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter) and/or the gene encoding a selectable marker is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter), and/or wherein the reporter gene (and optional promoter) is flanked by site-specific recombination sequences and/or the gene encoding a selectable marker (and optional promoter) is flanked by site-specific recombination sequences. In non-limiting embodiments, a modified endogenous Klhdc7b locus comprises: (i) a nucleic acid sequence set forth as SEQ ID NO:5 and/or (ii) a nucleic acid sequence set forth as SEQ ID NO:6 or a nucleic acid sequence set forth as SEQ ID NO:7, and/or (iii) a nucleic acid sequence set forth as SEQ ID NO:38 or a nucleic acid sequence set forth as SEQ ID NO:39, and/or (iv) an 2 WO 2024/163650 PCT/US2024/013826 endogenous 5’ Klhdc7b untranslated region, optionally an intact endogenous 5’ Klhdc7b untranslated region, and/or (v) an endogenous 3’ Klhdc7b translated region, optionally an intact endogenous 3’ Klhdc7b untranslated region. Generally, an endogenous 5’ Klhdc7b untranslated region, optionally an intact endogenous 5’ Klhdc7b untranslated region, as described herein may be upstream of a deletion of an Klhdc7b gene or portion thereof, e.g., upstream of and operably linked to an Klhdc7b start codon, and/or an endogenous 3’ Klhdc7b translated region, optionally an intact endogenous 3’ Klhdc7b untranslated region, may be downstream of a deletion of an Klhdc7b gene or portion thereof, e.g., downstream of and operably linked to the endogenous stop codon of the endogenous Klhdc7b gene. In some embodiments, a non-human animal as described herein, e.g., a mouse homozygous for the modified Klhdc7b locus, may act as a model for hearing loss.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]The patent or application file 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. [0010] Figure 1provides predicted long or short isoforms of mouse or human KLHDC7b transcripts (top panel) and transcript levels (delta CT; y-axis) of long (unfilled bars) or short (filled bars) KLHDC7b transcripts found in tissue samples (x-axis) from a commercial mouse cDNA panel (bottom left), freshly isolated tissue (bottom middle), or a commercial human cDNA panel (bottom right). Lower values indicate higher expression. Error bars are SEM with 3-4 technical replicates. All data are normalized to Drosha, a housekeeping gene. Figure 1 shows that KLHDC7b transcripts are found in cochlea and other organs, and that expression patterns differ slightly in the mouse and human. The mouse gene long isoform including UTRs is located at position: mmlO chrl5:89, 384,917-89,388,867 with a size of 3,951 nucleotides. The coding region is located at position: mmlO chr!5;89, 384,917-89,388,708 with a size of 3,7nucleotides, and one exon. The short isoform is located at position: mmlO chrl5:89,386,891- 89,388,708 with a size of 1,818 nucleotides, one exon, and no annotated UTRs. The long forward primer is located at chrl5; 89385390-89385412, the long reverse primer is located at chrl5: 89385458-89385478, and the probe is located at chrl5: 89385413-89385437. The 3 WO 2024/163650 PCT/US2024/013826 overlapping forward primer is chrl5: 89388123-89388141, the reverse primer is chrl5:89388182-89388200, and the probe is chrl5: 89388143-89388165. The human gene putative long isoform transcript (including UTRs) is located at position: hg38 chr22:50,545,899- 50,551,023, with a size of 5,125 nucleotides. There is one coding exon on the plus strand. The coding region is located at position: hg38 chr22: 50,546,244-50,549,951, with a size of 3,708 nucleotides. The putative short human isoform including UTRs is located at position: hgchr22:50, 548,033-50,551,022 with a size of 2,990 nucleotides. The coding region is located at position hg38 chr22;50, 548,167-50,549,951 with a size of 1,785 nucleotides. 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: 50549582-50549600 on the plus strand, the reverse primer is located at chr22: 50549649-50549668 on the minus strand, and the probe is located at chr22: 50549601-50549620. [0011] Figure 2provides transcript levels ( | CT; y-axis) of overlapping (left) and long (right) KLHDC7b transcripts found in liver (square), brain (circle), temporal bone including cochlea (triangle), or kidney (diamond) isolated from postnatal day 1 (pl) mice, postnatal day 7 (p7) mice, 11-28 week old (adult) mice, or 63-70 week old (aged mice). P1 and P7 time points consisted of 5 mice each. The mice were not sexed at this age. Adult and aged mice were heterozygous for the B6.C AST-Cdh2 3AW+Kjn corrected allele. Adult mice were 4 female mice at 11-14 weeks, and 1 male at 24 weeks. Aged mice are 4 females aged 63-73 weeks. Each data point is a biological replicate, tissue from one mouse, and was calculated from the mean of three technical replicates. All data are normalized to Drosha, a housekeeping gene. Analysis was performed via Two-way ANOVA, using Tukey’s test 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 lifespan of a mouse, and is consistently expressed at relatively high levels in cochlea. [0012] Figures 3A-3Dprovide images of histological sections of cochlea labeled with RNA probes that detect the overlapping portion of the long and short KLHDC7b isoforms (red) or only the long KLHDC7b isoform (green), and immunostained for My07a, a hair cell marker (white) from: (3A) adult wildtype mice, shown next to illustrative (not to scale) cartoons of the cochlea anatomy, (3B)adult wildtype mice, magnified to show the Organ or Corti, (3C)adult wildtype WO 2024/163650 PCT/US2024/013826 mice, magnified to show the vestibular system, or (3D)embryonic mice. These images show KLHDC7b is expressed exclusively in hair cells within the cochlea. Probe labeling appears as small punctae. Punctae are only visible in hair cells. In Figure 3B,the inset of the Organ of Corti (middle), shows staining with each probe (long and overlapping), MY07A, a hair cell marker, and DAPI. Each probe is presented with MY07A (right, top two images), and each probe is presented alone (right, below two images). Neither probe labels any area outside of the hair cells, indicating hair-cell specific expression. In Figure 3C,similarly, vestibular hair cells are shown with MY07A labeling and each probe (right, top two images), with each probe also presented alone (right, bottom two images) to show that the probes overlap only with hair cells. In Figure 3D,similarly, it is shown that both long and overlapping KLHDC7b+ punctae (right, bottom two images) colocalize with MY07A (right, top two images) in labelled, developing hair cells of embryonic mice. [0013] Figure 4Aprovides an illustration (not-to-scale) of a mouse Klhdc7b gene and the length of a deletion of its open reading frame (orf), e.g., the genomic sequence spanning, but not including, the “start” and “stop” codons of the mouse Klhdc7b gene. The 3,787 bp orf is represented by the filled rectangle. The 5’ untranslated region of the Klhdc7b gene is represented by the unfilled rectangle upstream of the start codon. The 3’ untranslated region of the Klhdc7b gene is represented by the unfilled rectangle downstream of the stop codon. The asterisks indicate the locations of the upstream (4929mTU) and downstream (4929mTD2) primers for a loss-of-allele assay. The general location of the sequence encoding the Kelch domain is also shown. [0014] Figure 4Bprovides an illustration (not-to-scale) of a large targeting vector (LTVEC) made after replacement of the open reading frame of a mouse Klhdc7b gene, found in BAC clone RP23-241G24, with an 8,802 bp insert nucleic acid (“LacZ, Neo-SDC") As shown, the LTVEC comprises (a) a 140.5 kb 5’ homology arm that comprises intact 5’ untranslated sequences of a mouse Klhdc7b gene and a mouse Klhdc7b start codon from BAC clone RP23-241G24, (b) an 8,802 bp insert nucleic acid comprising the Lacz gene (grey arrow) inserted in-frame with the mouse Klhdc7b start codon and self-deleting cassette (Neo-SDC; black arrow) comprising a Neomycin gene (“Neo”) flanked by LoxP site-specific recombination sequences, and (c) a 12.kb 3’ homology arm that comprises intact 3’ untranslated sequences of a mouse Klhdc7b gene WO 2024/163650 PCT/US2024/013826 from BAC clone RP23-241G24. “A” indicates the location of the 5’ mouse UTR // Start, Acc// 5’ LacZ junction (SEQ ID NO: 5) and “B” indicates the location of the 3’ Neo // (loxP) //Nhel // 3’ mouse UTR junction (SEQ ID NO: 6). Sequences of these junctions are also provided. [0015] Figure 4Cprovides an illustration (not-to-scale) of a modified Klhdc7b locus after targeted homologous recombination with the LTVEC of Figure 4B and deletion of the neomycin cassette. Untranslated regions are depicted as unfilled rectangles, and the lacZ gene is depicted with a filled arrow. Also shown are the locations of a 5’ homology arm that comprises 5’ untranslated sequences of a mouse Klhdc7b gene and a mouse Klhdc7b start codon, and a 3’ homology arm that comprises 3’ untranslated sequences of a mouse Klhdc7b gene, of which 12.kb is from RP23-241G24. “A” indicates the location of the 5’ mouse UTR // Start, Acc65 // 5’ LacZ junction (SEQ ID NO: 5) and “C” indicates the location of the 3 ’ LacZ // Stop // (LoxP) H Nhel // 3’ mouse UTR junction (SEQ ID NO:7). Sequences of these junctions are also provided. The LacZ protein expressed from the modified Khldc7b locus depicted in this figure comprises an amino acid sequence set forth as SEQ ID NO:4. [0016] Figure 4Dprovides microscopy images showing that LacZ expression is present in hair cells of heterozygous (HET) and knockout (KO) mice. Both mice were males at 10 weeks of age. LacZ staining is visible exclusively in hair cells, both inner and outer. The darker shading indicates LacZ expression. [0017] Figure 4Eprovides fluorescence microscopy images for wildtype and KLHDC7B-/- (KO) mouse cochlea. RNAscope was performed using KLHDC7B probes for the long and overlapping transcripts. Transcripts are not present in hair cells of knockout mouse cochlea. The top row of images are from a wildtype (WT) cochlea, while the bottom images are from a KO cochlea. The leftmost column shows a merged image at 40X magnification of all four fluorescence channels (My07a, DAPI, long and overlapping KLHDC7B probes) depicting one full turn of the cochlea. The second column from the left is a zoomed-in merged fluorescence image to show only hair cells. The third column shows a zoomed-in single-channel fluorescence image of punctae representing labeling with the overlapping probe that are only located within hair cells (visualized as a bright white color in the third column). The fourth column shows a zoomed-in single-channel fluorescence image of punctae representing labeling with the long probe that are only located within hair cells. The WT cochlea shows labeling, while the KO WO 2024/163650 PCT/US2024/013826 cochlea does not, indicating that the KO mouse is missing RNA transcripts for Klhdc7b, as expected. Outlines of hair cells drawn over the image indicate that punctae are only present in hair cells. [0018] Figure 5Aprovides immunostaining images of whole mounted cochlea of WT and KLHDC7B KO mice at postnatal day (p)6, pll,p21 and 8 weeks. Hair cells appear normal at pbut some outer hair cells are missing at pl 1 (solid circle in knockout tissue). At p21, many hair cells are missing, and significant supporting cell scarring is visible where hair cells were previously located. Scarring is visible as a lattice structure in white stained for F-actin at p21 and weeks (dashed circle in knockout tissue). A pattern of circular staining above the outer hair cells represents abnormal MY07A labeling, suggesting hair cell death and possible engulfment by supporting cells (dotted circle in knockout tissue). Damage is worse at 8 weeks (solid rectangle in knockout tissue). [0019] Figure 5Bprovides images of histological sections of cochlea that are from 8 week old wildtype mice or 8 week old KLHDC7b knockout mice and that are immunostained for My07a, a hair cell maker (green); Tuj 1, a neuron marker (red); DAPI (blue). KO=knockout, OHC = outer hair cells, IHC = inner hair cells. These images show that hair cells are abnormal in KLHDC7b knockout mice at 8 weeks. Hair cells (depicted as the brightest staining in greyscale) appear to have an abnormal morphology, with cells spaced further apart, in a less organized fashion. The rightmost image, at the base, shows a very abnormal staining pattern. Neuronal staining, depicted as darker, diffuse staining in greyscale located below mostly inner hair cells (as opposed to the DAPI-labelled, largely circular nuclei with well-defined boundaries), is still present in KO tissue. [0020] Figure 6provides images of histological stains of whole mounted cochlea that are from week old wildtype mice or 8 week old KLHDC7b knockout mice and that are immunostained for My07a, a hair cell maker (green); Tuj 1, a neuron marker (red); DAPI (blue), and F-actin (white). WT = wildtype, KO=knockout. These images show that hair cells are missing with visible scars in KLHDC7b knockout mice at 8 weeks. As in Figure 5A,there is a visible lattice structure that indicates the presence of supporting cell scars. Fewer hair cells are also present. This damage is present in a base-to-apex gradient, with the base being the worst. [0021] Figures 7A-7Cprovide histological images of (A, C)apex or (B, C)base whole mounted sections of cochlea that are from 3 day old wildtype mice or 3 day old KLHDC7b knockout mice WO 2024/163650 PCT/US2024/013826 and that are immunostained for My07a, a hair cell maker (green); Tuj 1, a neuron marker (red); DAPI, and actin. All portions of the Organ of Corti, apex, middle and base, appear normal, with well-formed stereocilia visible and no missing hair cells in KO tissue. Figure 7Dprovides histological images of a cochlea from a 6-day KLHDC7b knockout mouse and that is immunostained for My07a, DAPI (blue), actin (white), and ZO-1 (red). Hair cells are present and have normal morphology, stereocilia, and ZO-1 localization. Each fluorescence channel is broken out individually in the right four panels. Figure 7Eprovides histological images of a cochlea from a 6-day old wildtype mouse and that is immunostained for My07a (green), DAPI (blue), actin (white), and ZO-1 (red). This wildtype mouse looks very similar to the knockout mouse tissue at the same age. Figure 7Fprovides histological images of a cochlea from 11-day old wildtype mouse and that is immunostained for My07a (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 stereocilia and ZO-1 staining. Figures 7G-7Hprovide histological images of cochlea from different 11-day old KLHDC7b knockout mice and that are immunostained for My07a (green), DAPI (blue), actin (white), and ZO-1 (red). Cell bodies of some outer hair cells of knockout, but not wild-type (compared with Figure 7F),show abnormal cytoplasmic localization of ZO-1, at a time point when only a few hair cells are missing. Figure 71provides histological images of a cochlea from a 21-day KLHDC7b knockout mouse and that is immunostained for My07a (green), DAPI (blue), actin (white), and ZO-1 (red). Note that MY07A staining shows missing hair cells as well as abnormally rounded shapes that are outside the normal outer hair cell locations, which may indicate hair cell engulfment by supporting cells. Some remaining hair cells show cytoplasmic localization of ZO-1. Actin staining shows significant scarring as indicated by the sawtooth-shaped lattice structure. Inner hair cells appear normal, with normal stereocilia. Figure 7Jprovides histological images of a cochlea from a 21-day old wildtype mouse and that is immunostained for My07a (green), DAPI (blue), actin (white), and ZO-1 (red). Compared to Figure 71,hair cells appear organized, with ZO-1 staining around the border of the top of the cell rather than in the cytoplasm. It is unknown whether these examples of ZO-mislocalization are due to a general cell death phenotype or a more specific phenotype caused by knockout of Klhdc7b. WT = wildtype, KO= KLHDC7b knockouts. These images show hair WO 2024/163650 PCT/US2024/013826 cells and stereocilia appear normal in knockout cochleae 3 days and 6 days after birth, with early signs of degeneration 11 days after birth, progressing to further degeneration 21 days after birth. [0022] Figure 8provides schematics of two hearing assays to assess cochlear function; an Auditory Brainstem Response (ABR) that measures function of inner hair cells and neurons (left panel) and Distortion Product Otoacoustic Emission (DPOAE) that measures function of outer hair cells (right panel). [0023] Figures 9A-9Cdemonstrate that KLHDC7B knockout mice have profound and progressive hearing loss, while heterozygous mice do not. (A) auditory brainstem responses (ABRs) were recorded in separate cohorts of KLHDC7B knockout (KO), heterozygous (HET) and wild-type (WT) mice at each time point. Top, ABR thresholds. For each age range listed on the x-axis, the data points to the left are WT, the data points in the middle are HET, and the data points to the right are KO. KO mice show profound hearing loss at hearing onset (2-3 weeks) that progresses to complete loss by 11-15 weeks at three measured frequencies, 8 kHz, 16kHz, and 32 kHz. 100 dB denotes no response. Middle, Wave 1 amplitude of KO mice is significantly smaller at 2-3 weeks than WT, while HET mice are not significantly different from WT. Middle right, average waveforms from KO and WT mice at 2-3 weeks. Shaded area denotes SEM. Bottom, At 11-15 weeks, ABR is absent in nearly all KO mice, while HETs and KO are not significantly different. Bottom right shows average waveforms of WT and KO mice as in middle. (B) AB Rswere recorded longitudinally beginning at 11-13 weeks (same data as above).Thresholds increase at all frequencies at 55-57 weeks, with no significant difference between WT and HET. Bottom, Wave I amplitude is also not different between HET and WT at any dB SPL level, and waveforms (right) are indistinguishable. All data were analyzed separately at each frequency. (C) Non-limiting and exemplary auditory brainstem responses (ABRs) from wildtype (WT), heterozygous KLHDC7b knockout (Het), and homozygous KLHDC7b knockout (KO) mice at day 17 day postnatally and 16 kHz (top panel). Also shown is a graph providing the hearing threshold (dB; y-axis) of these animals at 8 kHz, 16 kHz and 32 kHz (frequency; x-axis) assayed by ABR. These animals are also included in Figure 9A.WT = 5 males, 4 females. Het = 7 males, 10 females, KO = 2 male, 3 females, ns = not significant. * indicates p< 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. This figure shows homozygous KLHDC7b knockout mice have significant hearing loss at hearing onset.

WO 2024/163650 PCT/US2024/013826

[0024] Figure 10provides non-limiting and exemplary auditory brainstem responses (ABRs) from wildtype (WT) and KLHDC7b knockout (KO) mice at 6-weeks, 8-weeks, and 12-weeks of age and 16 kHz. Also shown are graphs providing the hearing threshold (dB; y-axis) of these animals at 8 kHz, 16 kHz, and 32 kHz (frequency; x-axis) assayed by ABR (top graph) or Distortion Product Otoacoustic Emission (DPOAE) (bottom graph). WT = 7 males, 2 females. KO = 4 males, 7 females. * indicates p< 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. This figure shows KLHDC7b knockout mice have profound hearing loss at 5-7 weeks, which is worse than the hearing loss at hearing onset, and progresses to complete deafness by about 12 weeks. Further information on this progression, with a larger number of animals, can be found in Figure 9A. [0025] Figures 11A-11Cshow graphs providing the hearing threshold (dB; y-axis) of wildtype (WT), heterozygous KLHDC7b knockout (het), or homozygous KLHDC7b (KO) animals at kHz, 16 kHz and 32 kHz (frequency; x-axis) assayed by ABR (left graph) or Distortion Product Otoacoustic Emission (DPOAE) (right graph). In Figure 11A:WT = 3 males, 6 females. Het = males, 2 females. KO = 4 males, 3 females. * indicates p< 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001. This figure shows adult heterozygous KLHDC7b knockout mice are not deaf at 11-13 weeks. Figures 11Band 11Cprovide ABRs for the KO mice and Het mice, respectively, tested at 16kHz. This figure shows the time course of hearing loss, and that hearing loss increases between 17 days and 7-8 weeks in homozygous KLHDC7b mice, and that heterozygous mice do not have hearing loss as late as 32 weeks. [0026] Figures 12A-12Cprovide images of (A)cochlear explants of wildtype mouse incubated in gentamycin-Texas Red (GtTR), Texas red alone, or media alone to show hair cells in cochlear explant cultures take up gentamycin-Texas red (GtTR) through the mechanotransduction channel. (B) cochlear explants were immunostained for My07a (white); DAPI (blue), and actin (green). The top panels depict merged fluorescence images of all channels. The middle panels depict zoomed-in merged fluorescence images of the areas represented by the white boxes in the images above. The bottom panels depict the single-channel fluorescence of GtTR for the zoomed-in images. In greyscale, the bright staining in the merged fluorescence images indicates colocalization of actin and GtTR staining which is indicative of uptake of GtTR into hair cells. WT = wildtype, het = heterozygous KLHDC7b knockout mice, KO = homozygous KLHDC7b WO 2024/163650 PCT/US2024/013826 knockout mice. These images show hair cells in cochlear explant cultures take up gentamycin- Texas red (GtTR) through the mechanotransduction channel and the mechanotransduction complex appears functional in cochlear explant cultures of knockout mice since hair cells in explants from postnatal day 4-5 take up GTTR, gross morphology of hair cells appears normal, and hair cells are not missing in these mice. (C) cochlear explant cultures of wildtype (KLHDC7b+ +) and knockout (KLHDC7bAA) mice were treated with Gentamycin-Texas Red (GtTR) or Texas Red (TR) alone, then immunostained for MY07A and stained for F-actin. GtTR enters hair cells of both WT and KO mice, while Texas Red does not. [0027] Figure 13Aprovides fluorescence images of otic organoids differentiated from hiPSCs (human induced pluripotent stem cells). RNA scope probes against two otic markers, Sox2 (hair cells and supporting cells) and TUBBS (spiral ganglion neurons) show expression around a likely otic vesicle (left). The JK iPSC line is shown. qPCR for otic markers (right) shows an increase after differentiation in D70 IEO (day 70 inner ear organoids) versus iPSCs. Both the JK and GM lines show this increase. In Figure 13B,RNA scope probes were designed against the long (top panels) and overlapping (bottom panels) portions of the KLHDC7B human transcript, and RNA scope was performed in parallel with staining for My07a. The leftmost column depicts merged fluorescence images of otic organoids (My07a, DAPI and KLHDC7B probe). The second column depicts zoomed-in merged fluorescence images of areas with a high concentration of probe labelling. The third and fourth columns depict single-channel fluorescence images for the KLHDC7B probes and My07a, respectively. Both probes are shown to be located around a likely otic vesicle. Slices are from the same organoid. qPCR for KLHDC7B (right) shows an increase after differentiation. Analysis was performed via Two-way ANOVA, using Tukey’s test for post-hoc comparisons, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. [0028] Figure 14provides scanning electron microscopy images of cochlea isolated from wildtype (WT) or homozygous KLHDC7b (KO) animals at post-natal day 10 (pl0) or post-natal day 20 (p20). [0029] Figure 15provides histological images of fixed, paraffin embedded mouse cochlea stained with a custom-generated antibody against KLHDC7b. A 20x confocal images, top left, shows labeling exclusively in hair cells, and a 40x image, top right, shows that the labeling WO 2024/163650 PCT/US2024/013826 appears to be localized to the plasma membrane. Bottom left and right are no primary antibody controls showing no labeling. [0030] Figure 16provides histological images of cochlear samples isolated from wildtype (WT) or homozygous KLHDC7b (KO) animals that are labeled with a custom monoclonal anti-KLHDC7b antibody (white) and DAPI. [0031] Figure 17Aprovides histological images of HEK cell lines that are stably transfected with short (top row) or long (middle row) human Klhdc7b isoforms tagged with FLAG, or GFP tagged with FLAG (bottom row), and that are labeled with one of 10 different monoclonal anti- KLHDC7b antibodies (cyan; clone number depicted below), and stained for FLAG (red), GFP (green), and DAPI expression (dark blue). In greyscale, the brightest staining for the top and middle rows (short and long FLAG-tagged human Klhdc7b isoforms, respectively) indicates staining with the anti-KLHDC7b antibody. The brightest staining for the bottom row of control transfected cells indicates GFP staining, with no detectable anti-KLHDC7b labelling with all clones except clone 2, indicating some nonspecific labeling with this clone. [0032] Figure 17Bprovides histological images of the same HEK cell lines as described above labelled with two monoclonal anti-KLHDC7b antibodies designated as Antibody A (clone 1 in Figure 17A)and Antibody B(clone 3 in Figure 17A).The top and middle rows depict cells transfected with short and long FLAG-tagged human Klhdc7b isoforms, respectively. The bottom row depicts cells transfected with FLAG-tagged GFP. Columns 1 and 2 show all channels. Columns 3 and 4 show only FLAG staining and DAPI staining, Columns 5 and 6 show anti-KLHDC7B and DAPI staining, and columns 7 and 8 show GFP staining and DAPI staining. In greyscale, the brightest staining in the first six columns of the first two rows is indicative of colocalization of DAPI and FLAG, DAPI and anti-KLHDC7b, or all three, suggesting that the antibodies bind to cells expressing 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 labelling.

WO 2024/163650 PCT/US2024/013826 DESCRIPTION 1. Overview [0033]Kelch Domain Containing 7B (KLHDC7) is a protein member of the Kelch superfamily, which are proteins involved in cellular processes such as cytoskeletal rearrangement and protein degradation, and which proteins also have roles in extracellular communication, cell morphology, gene expression, and actin binding. Apart from its membership in the Kelch- domain containing protein superfamily, very little is known about this gene. Kelch domains are a set of repeating beta-sheet forming subunits that come together to form a tertiary structure known as a beta propeller. Kelch domain containing proteins have diverse subcellular locations and functions, so KLHDC7b’s membership in this family does not clarify its role within the cell (Adams et al., 2000). There are few publications specifically examining KLHDC7b. It has been shown to be upregulated, yet also hypermethylated, in breast cancer cells (Martin-Pardillos and Cajal, 2019). Alterations in the Kelch superfamily are associated with various types of cancer, including leukemia, lung, prostate, brain, and Hodgkin's disease. KLHDC7B was identified as being hypermethylated, yet upregulated, in breast cancer cells. KLHDC7b has two predicted isoforms (long and short). [0034]Moderate levels of KLHDC7B expression are observed in cochlea, e.g., the hair cells of the ear, while outer hair cells seem to show slightly higher expression (gEAR portal). Predicted loss-of functions variants in KLHDC7B are associated with an increased risk of developing hair loss in humans. For example, a genetic alteration that changes the guanine nucleotide of position 3,778 in the human KLHDC7B reference (see, NCBI Accession No: NP_612442.3) to adenine has been observed to indicate that the human having such an alteration may have an increased risk of developing hearing loss, such as conductive hearing loss, sensorineural hearing loss, or neural hearing loss. The International Mouse Phenotyping Consortium (IMPC) indicates that exon deletion of Klhdc7b exhibits abnormal auditory brainstem response, abnormal ear morphology, shortened QT interval, abnormal locomotor behavior, decreased/abnormal startle reflex, and decreased prepulse inhibition (www.mousephenotype.org/data/genes/MGI:3648212). [0035]Shown herein is evidence that non-human animals comprising a knockout mutation of an endogenous Klhdc7b gene progressively develop hearing loss. In comparison to wildtype control animals, Klhdc7b knockout mice demonstrate increased hearing loss, and have profound WO 2024/163650 PCT/US2024/013826 hearing loss at the onset of hearing and near complete hearing loss by 11-15 weeks. Hair cells in the cochlea are missing, e.g., at time points where the mice are profoundly deaf, although the mechanotransduction complex appears to be functional at earlier time points. For example, hair cells develop normally, showing functional mechanotransduction complexes in culture, but begin to die around postnatal day 11-12, with the characteristic morphology of a supporting cell scar that is known to form beneath a dead hair cell (Wagner and Shin, 2019), as indicated by histology and scanning electron microscopy. Scanning electron microscopy (SEM) shows no gross abnormalities in stereocilia morphology prior to the onset on hair cell death and confirms outer hair cell loss at three weeks of age. RNA scope results show that KLHDC7 is expressed specifically 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. Thus, the non-human animals disclosed herein may be useful in determining the key biological players and/or mechanisms in preventing hearing loss and/or preserving hearing function. [0036]Provided herein are nucleic acids (e.g., non-human animal nucleic acids isolated from non-human animals), non-human animal cells, and non-human animals comprising a modified endogenous Kelch domain containing 7B (Klhdc7b) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of an endogenous Klhdc7b gene, or portion thereof. The deletion may comprise, consist essentially of, or consist of a deletion of an open reading frame (orf) of an endogenous Klhdc7b gene at the endogenous Klhdc7b locus of the non-human animal nucleic acids, non-human animal cells, and non-human animals, e.g., the deletion spans between, but does not include or extend beyond, an endogenous start codon of the endogenous Klhdc7b gene and an endogenous stop codon of the endogenous Klhdc7b gene. In some embodiments, the deletion may be the result of a replacement of the endogenous Klhdc7b gene, or a portion thereof (e.g., an orf portion thereof) with an insert nucleic acid. In some embodiments, the insert nucleic acid may comprise a reporter gene and/or a gene encoding a selectable marker, optionally wherein the reporter gene is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter) and/or the gene encoding a selectable is operably linked to a promoter (e.g., an endogenous Klhdc7b promoter), and/or wherein the reporter gene (and optional promoter) is flanked by site-specific recombination sequences and/or the gene encoding a selectable marker WO 2024/163650 PCT/US2024/013826 (and optional promoter) is flanked by site-specific recombination sequences. In non-limiting embodiments, a modified endogenous Klhdc7b locus comprises: (i) a nucleic acid sequence set forth as SEQ ID NO:5 and/or (ii) a nucleic acid sequence set forth as SEQ ID NO:6 or a nucleic acid sequence set forth as SEQ ID NO:7, and/or (iii) a nucleic acid sequence set forth as SEQ ID NO:38 or a nucleic acid sequence set forth as SEQ ID NO:39, and/or (iv) an endogenous 5’ Klhdc7b untranslated region, optionally an intact endogenous 5’ Klhdc7b untranslated region, and/or (v) an endogenous 3’ Klhdc7b translated region, optionally an intact endogenous 3’ Klhdc7b untranslated region. Generally, an endogenous 5’ Klhdc7b untranslated region, optionally an intact endogenous 5’ Klhdc7b untranslated region, as described herein, may be upstream of a deletion of Klhdc7h gene or portion thereof, e.g., upstream of and operably linked to an Klhdc7b start codon, and/or an endogenous 3’ Klhdc7b translated region, optionally an intact endogenous 3’ Klhdc7b untranslated region may be downstream of a deletion of an Klhdc7b gene or portion thereof, e.g., downstream of and operably linked to the endogenous stop codon of the endogenous Klhdc7b gene. II. Non-Human Cells andNon-Human Animals Comprising a ModifiedKlhdc7b Locus [0037]Non-human animal cells and non-human animals comprising a modified Klhdc7b locus as described herein are provided. The cells or non-human animals can be heterozygous or homozygous for the modified Klhdc7b locus. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. In some embodiments, provided herein is a non-human animal cell comprising a genetically modified endogenous Klhdc7b locus as described herein. The non-human animal cell can be a cochlear cell (e.g., an inner hair cell or an outer hair cell), a pluripotent cell, an ES cell, or a germ cell. [0038]In some embodiments, the disclosure further provides methods for making any non- human animal, or reagents required for making the non-human animal as described herein. [0039]The non-human animal cells provided herein can be, for example, any non-human cell comprising a modified Klhdc7b locus as described herein. The cells can be eukaryotic cells, which include, for example, fungal cells (e.g., yeast), plant cells, animal cells, mammalian cells, non-human mammalian cells, and human cells. An animal can be, for example, a mammal, fish, WO 2024/163650 PCT/US2024/013826 or bird. A mammalian cell can be, for example, a non-human mammalian cell, a rodent cell, a rat cell, a mouse cell, or a hamster cell. Other non-human mammals include, for example, non- human primates, monkeys, apes, orangutans, cats, dogs, rabbits, horses, bulls, deer, bison, livestock (e.g., bovine species such as cows, steer, and so forth; ovine species such as sheep, goats, and so forth; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostrich, geese, ducks, and so forth. Domesticated animals and agricultural animals are also included. The term “non-human” excludes humans. [0040]The cells can also be any type of undifferentiated or differentiated state. For example, a cell can be a totipotent cell, a pluripotent cell (e.g., a human pluripotent cell or a non-human pluripotent cell such as a mouse embryonic stem (ES) cell or a rat ES cell), or a non-pluripotent cell. Totipotent cells include undifferentiated cells that can give rise to any cell type, and pluripotent cells include undifferentiated cells that possess the ability to develop into more than one differentiated cell types. Such pluripotent and/or totipotent cells can be, for example, ES cells or ES-like cells, such as an induced pluripotent stem (iPS) cells. ES cells include embryo- derived totipotent or pluripotent cells that can contribute to any tissue of the developing embryo upon introduction into an 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). [0041]The cells provided herein can also be germ cells (e.g., sperm or oocytes). The cells can be mitotically competent cells or mitotically-inactive cells, meiotically competent cells or meiotically-inactive cells. Similarly, the cells disclosed herein can also be primary somatic cells or cells that are not a primary somatic cell. Somatic cells include any cell that is not a gamete, germ cell, gametocyte, or undifferentiated stem cell. Suitable cells provided herein also include primary cells. Primary cells include cells or cultures of cells that have been isolated directly from an organism, organ, or tissue. Primary cells include cells that are neither transformed nor immortal. Primary cells include any cell obtained from an organism, organ, or tissue which was not previously passed in tissue culture or has been previously passed in tissue culture but is incapable of being indefinitely passed in tissue culture. Such cells can be isolated by conventional techniques.

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[0042]Suitable cells include cells of the ear, e.g., cells involved with 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) may be a suitable cell provided herein. [0043]Other suitable cells provided herein include immortalized cells. Immortalized cells include cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation or alteration, have evaded normal cellular senescence and instead can keep undergoing division. Such mutations or alterations can occur naturally or be intentionally induced. Examples of immortalized cell lines are myofiber cell lines. Immortalized or primary cells include cells that can be used for culturing or for expressing recombinant genes or proteins. [0044]The cells provided herein also include one-cell stage embryos (i.e., fertilized oocytes or zygotes). Such one-cell stage embryos can be from any genetic background (e.g., B6.Cast- Cdb23Ahl+ for mice), can be fresh or frozen, and can be derived from natural breeding or in vitro fertilization. [0045]The cells provided herein can be normal, healthy cells, or can be diseased or mutant- bearing cells. [0046]Tissues, e.g., cochlear explants, comprising the nucleic acids and/or cells, and/or isolated from the non-human animals, described herein are also provided. [0047]Non-human animals comprising a modified Klhdc7b locus as described herein can be made by the methods described elsewhere herein. An animal can be, for example, a mammal, fish, or bird. 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., bovine species such as cows and steer; ovine species such as sheep and goats; and porcine species such as pigs and boars). Birds include, for example, chickens, turkeys, ostrich, geese, and ducks. Domesticated animals 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. [0048]The non-human animals can be from any genetic background. For example, suitable mice can be from a B6.Cast-Cdh23A111+ strain, 129 strain, a C57BL/6 strain, a mix of 129 and C57BL/6, a BALB/c strain, or a Swiss Webster strain. Examples of 129 strains include 129P1, WO 2024/163650 PCT/US2024/013826 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129Sl/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, herein incorporated by reference in its entirety for all purposes. Examples of C57BL strains include C57BL/A, C57BL/An, C57BL/GrFa, C57BL/Kal_wN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. Suitable mice can also be from a mix of an aforementioned 1strain and an aforementioned C57BL/6 strain (e.g., 50% 129 and 50% C57BL/6). Likewise, suitable mice can be from a mix of aforementioned 129 strains or a mix of aforementioned BL/strains (e.g., the 129S6 (129/SvEvTac) strain). [0049]Similarly, rats can be from any rat strain, including, for example, an ACI rat strain, a Dark Agouti (DA) rat strain, a Wistar rat strain, a LEA rat strain, a Sprague Dawley (SD) rat strain, or a Fischer rat strain such as Fisher F344 or Fisher F6. Rats can also be obtained from a strain derived from a mix of two or more strains recited above. For example, a suitable rat can be from a DA strain or an ACI strain. The ACI rat strain is characterized as having black agouti, with white belly and feet and an RTlavl haplotype. Such strains are available from a variety of sources including Harlan Laboratories. The Dark Agouti (DA) rat strain is characterized as having an agouti coat and an RTlavl haplotype. Such rats are available from a variety of sources including Charles River and Harlan Laboratories. Some suitable rats can be from an inbred rat strain. See, e.g., US 2014/0235933, herein incorporated by reference in its entirety for all purposes. III. Methods of Making Non-Human Animals Comprising a Modified Klhdc 7b Locus [0050]Various methods are provided for making a non-human animal comprising a modified Klhdc7b locus as disclosed elsewhere herein. Any convenient method or protocol for producing a genetically modified organism is suitable for producing such a genetically modified non-human animal. See, e.g., 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 herein incorporated by reference in its entirety for all purposes. Such genetically modified non- human animals can be generated, for example, through gene knock-in at a targeted Klhdc7b locus.

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[0051]For example, the method of producing a non-human animal comprising a modified Klhdc7b locus can comprise: (1) modifying the genome of a pluripotent cell to comprise the modified A/72،7c7Z> locus; (2) identifying or selecting the genetically modified pluripotent cell comprising the modified Klhdc7b locus; (3) introducing the genetically modified pluripotent cell into a non-human animal host embryo cells in vitro, and (4) implanting and gestating the host embryo cells in a surrogate mother. Optionally, the host embryo comprising modified pluripotent cell (e.g., a non-human ES cell) can be incubated until the blastocyst stage before being implanted into and gestated in the surrogate mother to produce an FO non-human animal. The surrogate mother can then produce an FO generation non-human animal comprising the modified A/A،7c7Z> locus. [0052]The methods can further comprise identifying a cell or animal having a modified target genomic locus. Various methods can be used to identify cells and animals having a targeted genetic modification. [0053]The screening step can comprise, for example, a quantitative assay for assessing modification of allele (MOA) of a parental chromosome. For example, the quantitative assay can be carried out via a quantitative PCR, such as a 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 a non-targeted reference locus. The primer set can comprise a fluorescent probe that recognizes the amplified sequence. [0054]Other examples of suitable quantitative assays include fluorescence-mediated in situ hybridization (FISH), comparative genomic hybridization, isothermic DNA amplification, quantitative hybridization to an immobilized probe(s), INVADER® Probes, TAQMAN® Molecular Beacon probes, or ECLIPSE™ probe technology (see, e.g., US 2005/0144655, incorporated herein by reference in its entirety for all purposes). [0055]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, for example, through recombination by (a) introducing into the cell one or more targeting vectors comprising an insert nucleic acid flanked by 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites, wherein the insert nucleic acid comprises a modified Klhdc7b locus or portion thereof (e.g., a modified WO 2024/163650 PCT/US2024/013826 Klhdc7b gene comprising a deletion of its open reading frame); and (b) identifying at least one cell comprising in its genome the insert nucleic acid integrated at the target genomic locus. [0056]Accordingly, also provided herein is a method of making a genetically modified cell, e.g., an ES cell, comprising contacting the cell with one or more targeting vectors comprising an insert nucleic acid flanked by 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites, wherein the insert nucleic acid comprises a modified Klhdc7b locus or portion thereof (e.g., a modified Klhdc7b gene comprising a deletion of its open reading frame), such that upon homologous recombination between the 5’ and 3’ homology arms and the corresponding 5’ and 3’ target sites, 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. [0057]Alternatively, the modified pluripotent cell can be generated by (a) introducing into the cell: (i) a nuclease agent, wherein the nuclease agent induces a nick or double-strand break at a recognition site within the target genomic locus; and (ii) one or more targeting vectors comprising an insert nucleic acid flanked by 5’ and 3’ homology arms corresponding to 5’ and 3’ target sites located in sufficient proximity to the recognition site, wherein the insert nucleic acid comprises the modified Klhdc7b locus; and (c) identifying at least one cell comprising a modification (e.g., integration of the insert nucleic acid) at the target genomic locus. Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used. Examples of suitable nucleases include a Transcription Activator-Like Effector Nuclease (TALEN), a zinc-finger nuclease (ZFN), a meganuclease, and Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) systems or components of such systems (e.g., CRISPR/Cas9). See, e.g., US 2013/0309670 and US 2015/0159175, each of which is herein incorporated by reference in its entirety for all purposes. [0058]The donor cell can be introduced into a host embryo at any stage, such as the blastocyst stage or the pre-morula stage (i.e., the 4 cell stage or the 8 cell stage). Progeny that are capable of transmitting the genetic modification though the germline are generated. See, e.g., USPatent No. 7,294,754, herein incorporated by reference in its entirety for all purposes. [0059]Alternatively, the method of producing the non-human animals described elsewhere herein can comprise: (1) modifying the genome of a one-cell stage embryo to comprise the modified Klhdc7b locus using the methods described above for modifying pluripotent cells; (2) WO 2024/163650 PCT/US2024/013826 selecting the genetically modified embryo; and (3) implanting and gestating the genetically modified embryo into a surrogate mother. Progeny that are capable of transmitting the genetic modification though the germline are generated. [0060]Nuclear transfer techniques can also be used to generate the non-human mammalian animals. Briefly, methods for nuclear transfer can include the steps of: (1) enucleating an oocyte or providing an enucleated oocyte; (2) isolating or providing a donor cell or nucleus to be combined with the enucleated oocyte; (3) inserting the cell or nucleus into the enucleated oocyte to form a reconstituted cell; (4) implanting the reconstituted cell into the womb of an animal to form an embryo; and (5) allowing the embryo to develop. In such methods, oocytes are generally retrieved from deceased animals, although they may be isolated also from either oviducts and/or ovaries of live animals. Insertion of the donor cell or nucleus into the enucleated oocyte to form a reconstituted cell can be by microinjection of a donor cell under the zona pellucida prior to fusion. Fusion may be induced by application of a DC electrical pulse across the contact/fusion plane (electrofusion), by exposure of the cells to fusion-promoting chemicals, such as polyethylene glycol, or by way of an inactivated virus, such as the Sendai virus. A reconstituted cell can be activated by electrical and/or non-electrical means before, during, and/or after fusion of the nuclear donor and recipient oocyte. Activation methods include electric pulses, chemically induced shock, penetration by sperm, increasing levels of divalent cations in the oocyte, and reducing phosphorylation of cellular proteins (as by way of kinase inhibitors) in the oocyte. The activated reconstituted cells, or embryos, can be cultured in media and then transferred to the womb of an animal. See, e.g., US 2008/0092249, WO 1999/005266, US 2004/0177390, WO 2008/017234, and US Patent No. 7,612,250, each of which is herein incorporated by reference in its entirety for all purposes. [0061]The various methods provided herein allow for the generation of a genetically modified non-human FO animal wherein the cells of the genetically modified FO animal comprise the modified Klhdc7b locus. It is recognized that depending on the method used to generate the FO animal, the number of cells within the FO animal that have the modified Klhdc7b locus will vary. The introduction of the donor ES cells into a pre-morula stage embryo from a corresponding organism (e.g., an 8-cell stage mouse embryo) via for example, the VELOCIMOUSE® method allows for a greater percentage of the cell population of the FO animal to comprise cells having WO 2024/163650 PCT/US2024/013826 the nucleotide sequence of interest comprising 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 Fanimal can comprise a cell population having the targeted modification. [0062]The cells of the genetically modified F0 animal can be heterozygous for the modified Klhdc7b locus. In some embodiment heterozygous F0 mice may be bred to generate progeny that are homozygous for the modified Klhdc7b locus. [0063]In some embodiments, the disclosure provides a method of making a non-human animal, a non-human animal cell, or a non-human animal genome of described herein, comprising inserting a nucleic acid sequence comprising a modified Klhdc7b locus into the genome of the non-human animal, the genome of the non-human animal cell, or the non-human animal genome. IV. Nucleic Acids Comprising a Modified Kihdc 7b Locus [0064]As described elsewhere herein, a variety of nucleic acids (e.g., targeting vectors) can be specifically used for such purposes. In some embodiments, a non-human animal nucleic acid comprising a modified endogenous Kelch domain containing 7B (Klhdc7K) locus, wherein the modified endogenous Klhdc7b locus comprises a deletion of an endogenous Klhdc7b gene, or portion thereof, may be used. In some non-human animal nucleic acid embodiments, the deletion comprises, consists essentially or, or consists of a deletion of an open reading frame (orf) of the endogenous Klhdc7b gene. [0065] Insert nucleic acids [0066]In some non-human animal nucleic acid embodiments, the modified endogenous Klhdc7b locus further comprises an insert nucleic acid. The “insert nucleic acid ” or “insert polynucleotide ” comprises a segment of DNA that one desires to integrate at the target locus. In one embodiment, the insert nucleic acid comprises one or more polynucleotides of interest. In other embodiments, the insert nucleic acid can comprise one or more expression cassettes. A given expression cassette can comprise a polynucleotide of interest, a polynucleotide encoding a selection marker and/or a reporter gene along with the various regulatory components that influence expression. [0067]Any polynucleotide of interest may be contained in the various insert polynucleotides and thereby integrated at the target Klhdc7b locus. The methods disclosed herein, provide for at least WO 2024/163650 PCT/US2024/013826 1, 2, 3, 4, 5, 6 or more polynucleotides of interest to be integrated into the targeted Klhdc7b genomic locus of interest. [0068]In one embodiment, the polynucleotide of interest contained in the insert nucleic acid encodes a reporter. In another embodiment, the polynucleotide of interest encodes for a selectable marker. [0069]In one embodiment, the polynucleotide of interest can be flanked by site-specific recombination sequences. In a specific embodiment, the site-specific recombination sequences flank a segment encoding a reporter and/or a segment encoding a selectable marker. [0070]Non-limiting examples of polynucleotides of interest, including selection markers and reporter genes that can be included within the insert nucleic acid are discussed in detail elsewhere herein. [0071]The polynucleotide of interest within the insert polynucleotide when integrated at the target Klhdc7b locus can introduce one or more genetic modifications into the cell. The genetic modification can comprise a deletion of an endogenous nucleic acid sequence (e.g., a deletion of an open reading frame) and/or the addition of an exogenous or heterologous or orthologous polynucleotide into the target genomic locus. In one embodiment, the genetic modification comprises a replacement of an endogenous nucleic acid sequence with an exogenous polynucleotide of interest at the target genomic locus. Thus, methods provided herein allow for the generation of a genetic modification comprising a knockout, a deletion, a replacement (“knock-in ”), or a combination thereof in a target Klhdc7b locus. Such modifications may occur upon integration of the first, second, third, fourth, fifth, six, seventh, or any subsequent insert polynucleotides into the target genomic locus. [0072]The polynucleotide of interest within the insert polynucleotide and/or integrated at the target genomic locus can comprise a sequence that is native or homologous to the cell it is introduced into; the polynucleotide of interest can be heterologous to the cell it is introduced to; the polynucleotide of interest can be exogenous to the cell it is introduced into; the polynucleotide of interest can be orthologous to the cell it is introduced into; or the polynucleotide of interest can be from a different species than the cell it is introduced into. The term “homologous” in reference to a sequence is a sequence that is native to the cell. The term “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, WO 2024/163650 PCT/US2024/013826 if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. The term “exogenous” in reference to a sequence is a sequence that originates from a foreign species. The term "orthologous" is a polynucleotide 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 from any organism of interest including, but not limited to, a prokaryote, a eukaryote, a non-human, a rodent, a hamster, a mouse, a rat, a human, a monkey, an avian, an agricultural mammal or a non- agricultural mammal. The polynucleotide of interest can further comprise a coding region, a non-coding region, a regulatory region, or a genomic DNA. Thus, the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, and/or any of the subsequent insert polynucleotides can comprise such sequences. [0073]In one embodiment, the polynucleotide of interest can range from about 500 nucleotides to about 200kb as described above. The polynucleotide of interest can be from about 5nucleotides to about 5kb, from about 5kb to about 200kb, from about 5kb to about lOkb, from about lOkb to about 20kb, from about 20kb to about 30kb, from about 30kb to about 40kb, from about 40kb to about 50kb, from about 60kb to about 70kb, from about 80kb to about 90kb, from about 90kb to about lOOkb, from about lOOkb to about 1lOkb, from about 120kb to about 130kb, from about 130kb to about 140kb, from about 140kb to about 150kb, from about 150kb to about 160kb, from about 160kb to about 170kb, from about 170kb to about 180kb, from about 180kb to about 190kb, or from about 190kb to about 200kb. [0074]The polynucleotide of interest within the insert polynucleotide and/or inserted at the target genomic locus can encode a polypeptide, can encode an RNA, can encode an miRNA, or it can comprise any regulatory regions or non-coding regions of interest including, for example, a regulatory sequence, a promoter sequence, an enhancer sequence, a transcriptional repressor- binding sequence, a Kozak consensus segment, a start codon, or a deletion of a non-protein- coding sequence, but does not comprise a deletion of a protein-coding sequence. [0075]In one embodiment, the insert nucleic acid comprises a regulatory element, including for example, a promoter, an enhancer, or a transcriptional repressor-binding element. [0076]In further embodiments, the insert nucleic acid comprises a conditional allele. In one embodiment, the conditional allele is a multifunctional allele, as described in US 2011/0104799, which is incorporated by reference in its entirety. In specific embodiments, the conditional allele WO 2024/163650 PCT/US2024/013826 comprises: (a) an actuating sequence in sense orientation with respect to transcription of a target gene, and a drug selection cassette in sense or antisense orientation; (b) in antisense orientation a nucleotide sequence of interest (NSI) and a conditional by inversion module (COIN, which utilizes an exon-splitting intron and an invertible genetrap-like module; see, for example, US 2011/0104799, which is incorporated by reference in its entirety); and (c) recombinable units that recombine upon exposure to a first recombinase to form a conditional allele that (i) lacks the actuating sequence and the DSC, and (ii) contains the NSI in sense orientation and the COIN in antisense orientation. [0077]In one embodiment, the genetic modification comprises a deletion of ^Klhdc7b gene or a portion thereof, e.g., a deletion comprising, consisting essentially of, or consisting of an open reading frame of aKlhdc7b gene. In one embodiment, the nucleic acid sequence of the targeting vector can comprise a polynucleotide that when integrated into the genome will produce a genetic modification of a region of the mammalian, non-human animal, or a non-human mammalian Klhdc7b locus, wherein the genetic modification at the Klhdc7b locus results in a loss-of-function of the Klhdc7b. In one embodiment, a Klhdc7b knockout (“null allele) is generated. In another embodiment, a disruption in the Klhdc7b locus is generated. In further embodiments, the insert nucleic acid results in the replacement of a portion of the mammalian, non-human animal, or non-human mammalian Klhdc7b gene, with an insert nucleic acid sequence comprising a heterologous sequence. [0078]In some embodiments, the insert nucleic acid replaces the endogenous Klhdc7b gene, or portion thereof (e.g., the orf portion thereof), that is deleted. Thus, in some non-human animal nucleic acid embodiments, the genetic modification of the Klhdc7b locus can comprise a replacement of or an insertion/addition to the Klhdc7b locus or a portion thereof with an insert nucleic acid. [0079]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 selection marker nucleic acid sequence. [0080]In one embodiment, the promoter is constitutively active promoter.

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[0081]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 an 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 a tet-Off promoter. In one embodiment, the chemically- regulated promoter is a steroid regulated promoter. In one embodiment, the steroid regulated promoter is a promoter of a rat glucocorticoid receptor. In one embodiment, the steroid regulated promoter is a promoter of an estrogen receptor. In one embodiment, the steroid-regulated promoter is a promoter of an ecdysone receptor. In one embodiment, the chemically-regulated promoter is a metal-regulated promoter. In one embodiment, the metal-regulated promoter is a metalloprotein promoter. In one embodiment, the inducible promoter is a physically-regulated promoter. In one embodiment, the physically-regulated promoter is a temperature-regulated promoter. In one embodiment, the temperature-regulated promoter is a heat shock promoter. In one embodiment, the physically- regulated promoter is a light-regulated promoter. In one embodiment, the light-regulated promoter is a light-inducible promoter. In one embodiment, the light-regulated promoter is a light-repressible promoter. [0082]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 glia-specific promoter. In one embodiment, the promoter is a muscle cell-specific promoter. In one embodiment, the promoter is a heart 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 WO 2024/163650 PCT/US2024/013826 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. [0083]In one embodiment, the promoter is a developmentally-regulated promoter. In one embodiment, the developmentally-regulated promoter is active only during an embryonic stage of development. In one embodiment, the developmentally-regulated promoter is active only in an adult cell. [0084]In specific embodiments, the promoter may be selected based on the cell type. Thus, the various promoters find use in a eukaryotic cell, a mammalian cell, a non-human mammalian cell, a pluripotent cell, a non-human pluripotent cell, a human pluripotent cell, a human ES cell, a human adult stem cell, a developmentally-restricted human progenitor cell, a human iPS cell, a human cell, a rodent cell, a rat cell, a mouse cell, a hamster cell, a fibroblast or a CHO cell. [0085]In some embodiments, the insert nucleic acid comprises a nucleic acid flanked with site- specific recombination target sequences. It is recognized the while the entire insert nucleic acid can be flanked by such site-specific recombination target sequences, any region or individual polynucleotide of interest within the insert nucleic acid can also be flanked by such sites. The site-specific recombinase can be introduced into the cell by any means, including by introducing the recombinase polypeptide into the cell or by introducing a polynucleotide encoding the site- specific recombinase into the 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 that is endogenous to the cell, a promoter that is heterologous to the cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter. Site-specific recombination target sequences, which can flank the insert nucleic acid or any polynucleotide of interest in the insert nucleic acid can include, but are not limited to, loxP, 10x511,10x2272,10x66,10x71,10xM2,10x5171, FRT, FRT11, FRT71, attp, att, FRT, rox, or a combination thereof. [0086]In some embodiments, the site-specific recombination sites flank a polynucleotide encoding a selection marker and/or a reporter gene contained within the insert nucleic acid. In such instances following integration of the insert nucleic acid at the targeted locus the sequences between the site-specific recombination sites can be removed.

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[0087]In one embodiment, the insert nucleic acid comprises a polynucleotide encoding a selection marker. The selection marker can be contained in a selection cassette. Such selection markers include, but are not limited, to neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puro1), blasticidin S deaminase (bsr1), xanthine/guanine phosphoribosyl transferase (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 with site-specific recombination target sequences. [0088]The insert nucleic acid can further comprise a reporter gene operably linked to a promoter. Such reporter genes can be operably linked to a promoter active in the cell. Such promoters can be an inducible promoter, a promoter that is endogenous to the reporter gene or the cell, a promoter that is heterologous to the reporter gene or to the cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter. [0089]In some cases, the insert nucleic acid comprises a reporter gene. In one embodiment, the reporter gene is positioned in the Klhdc7b locus in operable linkage with the endogenous Klhdc7b promoter. Such a modification allows for the expression of the reporter gene driven by the endogenous Klhdc7b promoter. Alternatively, the reporter gene is not placed in operable linkage with the endogenous Klhdc7b promoter. [0090]In some cases, the insert nucleic acid comprises a reporter gene. In one embodiment, the reporter gene is positioned in the K1hdc7b locus in operable linkage with an endogenous Klhdc7b start codon. Such a modification may allow for the expression of the reporter gene driven by the endogenous X7A،7c7/> promoter. [0091]Any reporter (or detectable moiety) can be used in the methods and compositions provided herein. Non-liming examples of reporters include, for example, P־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.

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[0092]The following description is a non-limiting example utilizing a lacZ reporter gene that encodes for P־galactosidase. The methods and compositions described herein can be performed with any reporter gene. [0093]Also provided herein are polynucleotides or nucleic acid molecules comprising the various components employed in a targeted genomic integration system provided herein for targeting a A75tifc 75 locus (i.e., any one of or any combination of nuclease agents, recognition sites, insert nucleic acids, polynucleotides of interest, reporter sequences, targeting vectors, selection markers, and other components). [0094]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 may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. Polynucleotides can comprise deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues, and any combination these. The polynucleotides provided herein also encompass all forms of sequences including, but not limited to, single-stranded forms, double- stranded forms, hairpins, stem-and-loop structures, and the like. [0095]Further provided are recombinant polynucleotides comprising the various components of the targeted genomic integration system for targeting a Klhdc7b locus. The terms “recombinant polynucleotide ” and “recombinant DNA construct ” are used interchangeably herein. A recombinant construct comprises an artificial or heterologous combination of nucleic acid sequences, e.g., regulatory and coding sequences that are not found together in nature. In other embodiments, a recombinant construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector. If a vector is used, then the choice of vector is dependent upon the method that is used to transform the host cells as is well known to those skilled in the art. For example, a plasmid vector can be used. Genetic elements required to successfully transform, select, and propagate host cells comprising WO 2024/163650 PCT/US2024/013826 any of the isolated nucleic acid fragments provided herein are also provided. Screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, immunoblotting analysis of protein expression, or phenotypic analysis, among others. [0096]In specific embodiments, one or more of the components of the targeted genomic integration system for targeting aKlhdc7b locus described herein can be provided in an expression cassette for expression in a prokaryotic cell, a eukaryotic cell, a bacterial, a yeast cell, or a mammalian cell or other organism or cell type of interest. The cassette can include 5' and 3' regulatory sequences operably linked to a polynucleotide provided herein. “Operably linked” comprises a relationship wherein the components operably linked function in their intended manner. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may 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 instance, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. The cassette may additionally contain at least one additional polynucleotide of interest to be co-introduced into the organism. Alternatively, the additional polynucleotide of interest can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of a recombinant polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selection marker genes. [0097]The expression cassette can include in the 5-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a recombinant polynucleotide provided herein, and a transcriptional and translational termination region (i.e., termination region) functional in mammalian cell or a host cell of interest. The regulatory regions (i.e., promoters, transcriptional regulatory regions, Kozak sequence, and translational termination regions) and/or a polynucleotide provided herein may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or a polynucleotide provided herein may be heterologous to the host cell or to each other. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the WO 2024/163650 PCT/US2024/013826 polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. Alternatively, the regulatory regions and/or a recombinant polynucleotide provided herein may be entirely synthetic. [0098]The termination region may be native with the transcriptional initiation region, may be native with the operably linked recombinant polynucleotide, may be native with the host cell, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the recombinant polynucleotide, the host cell, or any combination thereof. [0099]In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. [0100]A number of promoters can be used in the expression cassettes provided herein. The promoters can be selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of different promoters in the expression cassettes to modulate the timing, location and/or level of expression of the polynucleotide of interest. Such expression constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a Kozak consensus sequence, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal. [0101]The expression cassette containing the polynucleotides provided herein can also comprise a selection marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. [0102]Where appropriate, the sequences employed in the methods and compositions (i.e., the polynucleotide of interest, the nuclease agent, etc.) may be optimized for increased expression in the cell. That is, the genes can be synthesized using codons preferred in a given cell of interest including, for example, mammalian-preferred codons, human-preferred codons, rodent-preferred WO 2024/163650 PCT/US2024/013826 codon, mouse-preferred codons, rat-preferred codons, hamster-preferred codons, etc. for improved expression. [0103]The various methods and compositions provided herein can employ selection markers. Various selection markers can be used in the methods and compositions disclosed herein. Such selection markers can, for example, impart resistance to an antibiotic such as G418, hygromycin, blastocidin, neomycin, or puromycin. Such selection markers include neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N- acetyltransferase (puror), and blasticidin S deaminase (bsrr). In still other embodiments, the selection marker is operably linked to an inducible promoter and the expression of the selection marker is toxic to the cell. Non-limiting examples of such selection markers include xanthine/guanine phosphoribosyl transferase (gpt), hahypoxanthine-guanine phosphoribosyltransferase (HGPRT) or herpes simplex virus thymidine kinase (HSV-TK). The polynucleotide encoding the selection markers are operably linked to a promoter active in the cell. [0104] Targeting Vectors [0105]Targeting vectors are employed to introduce the insert nucleic acid into the Khldc7b locus of interest of the eukaryotic, non-human, mammalian, non-human mammalian, human, rodent, mouse, rat or hamster nucleic acid. In some embodiments, a nucleic acid molecule (e.g., 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 3’ homology arm are configured to undergo homologous recombination with a non-human animal Klhd7bc locus of interest, and following homologous recombination with a non-human animal Klhdc7b locus of interest, the modified Klhdc7b gene replaces the non-human animal Klhdc7b gene at the non- human animal Klhdc7b locus of interest and is operably linked to an endogenous promoter that drives expression of the modified non-human animal Klhdc7b gene at the non-human animal Klhdc7b locus of interest. In some embodiments, the nucleic acid molecule (e.g., targeting vectors) comprises a nucleic acid sequence set forth as SEQ ID NO:5, a nucleic acid sequence set forth as SEQ ID NO:6, a nucleic acid sequence set forth as SEQ ID NO:7, a nucleic acid sequence set forth as SEQ ID NO:38, or a nucleic acid sequence set forth as SEQ ID NO:39.

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[0106]Also described are various methods of using the genetically modified non-human animals described herein.

BRIEF DESCRIPTION OF THE SEQUENCES [0107]The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5’ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3’ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus.

Table 1:Summary description of sequences SEQ ID NO Description Features 1 Mus Musculus Klhdc7b. mRNA, NM_001160178.2Length: 5,764 bp CDS: 859-4,650 Exons: 1-5,764; 2 Mus musculus Klhdc7b, protein,NP_001153650.2Length; 1,263 aa 3 LacZ replacing Klhdc7b, DNA Length; 3,078 bp 4 LacZ replacing Klhdc7b, protein Length: 1,023 aa 5 ’ mouse UTR // Start, Acc65 //5 ’ LacZ 5 ’ mouse UTR. 5 ׳ LacZ See Fig. 4B and 4C bottom panel, box A. 6 3’ Neo // (LoxP) // Nhel //3’ mouse UTR 5' mouse UTR, 5 ’ LacZ WO 2024/163650 PCT/US2024/013826 See Fig. 4B bottom panel, box B. 7 3’ LacZ //Stop //(LoxP) // Nhel //3’mouse UTR’ LacZ, 3 ’ mouse UTR See Fig. 4C bottom panel, box C. 8 4929mTU forward primer See, Table 3 9 4929mTU probe (BHQ) See, Table 3 4929mTU reverse primer See. Table 3 11 4929mTD forward primer See. Table 3 12 4929mTD probe (BHQ) See. Table 3 13 4929mTD reverse primer See, Table 3 14 LacZ forward primer See, Table 3 LacZ probe (BHQ) See, Table 3 16 LacZ reverse primer See, Table 3 17 Neo forward primer See, Table 3 18 Neo probe (BHQ) See. Table 3 19 Neo reverse primer See. Table 3 m_sKLHDC7b probe See, Table 2 21 m_sKLHDC7b Forward Primer See, Table 2 22 m_sKLHDC7b Reverse Primer See, Table 2 23 m_LKLDHC7b probe See, Table 2 24 m_LKLDHC7b Forward Primer See, Table 2 m_LKLDHC7b Reverse Primer See. Table 2 26 m both KLHDC7b probe See. Table 2 WO 2024/163650 PCT/US2024/013826 27 m_both_KLHDC7b Forward Primer See. Table 2 28 m_both_KLHDC7b Reverse Primer See, Table 2 29 hKLHDC7b_s_both probe See, Table 2 11KLHDC7b_s_both Forward Primer See, Table 2 31 11KLHDC7b_s_both Reverse Primer See, Table 2 32 hKLHDC7b_l_sUTR probe See. Table 2 33 hKLHDC7b_l_sUTR Forward Primer See. Table 2 34 hKLHDC7b_l_sUTR Reverse Primer See. Table 2 hKLHDC7b_l probe See, Table 2 36 hKLHDC7b_l Forward Primer See, Table 2 37 11KLHDC7b_l Reverse Primer See, Table 2 38 Klhdc7b KO allele w ith Neo cassette See, Fig 4B 39 Klhdc7b KO allele without Neo cassette See. Fig. 4C 40 Mouse KLHDC7b short isoform -FLAG See. Table 4 41 Mouse KLHDC7b long isoform -FLAG See. Table 4 42 AS-1303 : Human Short isoform -FLAG See, Table 5 43 AS-1304 :Human Long isoform -FLAG See, Table 5 44 AS-1305 :H2B-GFP-FLAG See, Table 5 45 Mus Musculus Klhdc7b short isoform, mRNASee, NCBI Accession Number:NM_001160178.1 46 Mus musculus Klhdc7b short isofonn. proteinSee, NCBI Accession Number:NP_001153650.1 WO 2024/163650 PCT/US2024/013826 47 Homo sapiens KLHDC7B. mRNA,NM_138433.5Length: 5,125 bp CDS: 346-4053 Exons: 1-5,125 48 Homo sapiens KLHDC7B, protein, NP_612442.3Length: 1235 aa 49 Homo sapiens KLHDC7B short isoform. mRNANucleotides 2269-4053 of SEQ ID NO: 47 Length: 1785 bp 50 Homo sapiens KLHDC7B short isoform, proteinAmino acids 642-1235 of SEQ ID NO: Length: 594 aa

[0108]Mouse Klhdc7b comprises the amino acid sequence set forth as SEQ ID NO:2, which may be encoded by a nucleic acid comprising the sequence set forth as SEQ ID NO: 1. A short isoform of mouse Klhdc7b may also be expressed, comprising the amino acid sequence set forth as SEQ ID NO: 46, encoded by the nucleotide sequence set forth as SEQ ID NO: 45. 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 may also be expressed, comprising amino acids 642-1235 of SEQ ID NO: 48 (set forth as SEQ ID NO: 50), encoded by nucleotides 2269-4053 of SEQ ID NO: (set forth as SEQ ID NO: 49). [0109]The terms “protein,” “polypeptide,” and “peptide,” are used interchangeably herein, and include polymeric forms of amino acids of any length, including coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The terms also include polymers that have been modified, such as polypeptides having modified peptide backbones. The term domain can refer to any part of a protein or polypeptide having a particular function or structure.

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[0110]Proteins are said to have an “N-terminus” and a “C-terminus.” The term “N-terminus” relates to the start of a protein or polypeptide, terminated by an amino acid with a free amine group (-NH2). The term “C-terminus” relates to the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). [0111]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 comprising purine bases, pyrimidine bases, or other natural, chemically modified, biochemically modified, non-natural, or derivatized nucleotide bases. [0112]Nucleic acids are said to have “5’ ends” and “3’ ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5’ phosphate of one mononucleotide pentose ring is attached to the 3’ oxygen of its neighbor in one direction via a phosphodiester linkage. An end of an oligonucleotide is referred to as the “5’ end” if its 5’ phosphate is not linked to the 3’ oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3’ end” if its 3’ oxygen is not linked to a 5’ phosphate of another mononucleotide pentose ring. A nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5’ and 3’ ends. In either a linear or circular DNA molecule, discrete elements are referred to as being “upstream” or 5’ of the “downstream” or 3’ elements. [0113]The term "genomically integrated” refers to a nucleic acid that has been introduced into a cell such that the nucleotide sequence integrates into the genome of the cell and is capable of being inherited by progeny thereof. Any protocol may be used for the stable incorporation of a nucleic acid into the genome of a cell. [0114]As used herein, "embryonic stem cell" or "ES cell" includes an embryo-derived totipotent or pluripotent cell that is capable of contributing to any tissue of the developing embryo upon introduction into an embryo. The term “pluripotent cell ” includes an undifferentiated cell that possesses the ability to develop into more than one differentiated cell types. [0115]As used herein, “targeting vector, ” “large targeting vector, ” or "LTVEC" includes targeting vectors for eukaryotic cells that are derived from fragments of cloned genomic DNA WO 2024/163650 PCT/US2024/013826 larger than those typically used by other approaches intended to perform homologous gene targeting in eukaryotic cells. Examples of LTVEC, include, but are not limited to, bacterial homologous chromosome (BAG) and yeast artificial chromosome (YAC). Generally, a targeting vector may comprise a recombinant nucleic acid that can be introduced by homologous recombination, non-homologous-end-joining-mediated ligation, or any other means of recombination to a target position in the genome of a cell. [0116]As used herein, “site-specific recombination sequence ” includes a nucleotide sequence that is recognized by a site-specific recombinase and that can serve as a substrate for a recombination event. [0117]As used herein “site-specific recombinase ” includes a group of enzymes that can facilitate recombination between "site-specific recombination sequences". Examples of "site- specific recombinase" include, but are not limited to, Cre, Flp, and Dre recombinases. [0118]As used herein “germline” in reference to a nucleic acid sequence includes a nucleic acid sequence that can be passed to progeny and, e.g., may be found in germ cells (e.g., oocytes and sperm) of the non-human animal. [0119]As used herein “operably linked” and the like refers to components that are linked to function together in their intended manner. In one instance, a nucleic acid sequence encoding a protein may be operably linked to regulatory sequences (e.g., promoter, enhancer, silencer sequence, etc.) so as to retain proper transcriptional regulation. Operable linkage can include such sequences being contiguous with each other or acting in trans (e.g., a regulatory sequence can act at a distance to control transcription of the coding sequence). Operable linkage may also refer to one or more polypeptide fused together, e.g., as a fusion protein, such that each of the individual polypeptides retains its individual biological activity. [0120]As used herein, “locus ” refers to a segment of DNA within a larger nucleic acid molecule that generally contains the non-coding and coding sequences of a gene. For example, a Klhdc7b locus generally contains the non-coding and/or coding sequences of aKlhdc7b gene, which encodes a Klhdc7b protein. [0121]The term “gene” refers to a DNA sequence in a nucleic acid that codes for a product (e.g., an RNA product and/or a polypeptide product) and includes the coding region interrupted with non-coding introns and untranslated regions (UTRs) located adjacent to the coding region on WO 2024/163650 PCT/US2024/013826 both the 5’ and 3’ ends such that the gene corresponds to the full-length mRNA (including the 5’ and 3’ untranslated sequences). The term “gene” may also include other non-coding sequences including regulatory sequences (e.g., promoters, enhancers, and transcription factor binding sites), polyadenylation signals, internal ribosome entry sites, silencers, insulating sequence, and matrix attachment regions. These sequences may be close to the coding region of the gene (e.g., within 10 kb) or at distant sites, and they influence the level or rate of transcription and translation of the gene. [0122]“Open reading frame,” “orf,” and the like as used herein encompasses a portion of a DNA molecule (e.g., gene) that, when translated into amino acids, contains no stop codons. Generally, an open reading frame spans a gene sequence between the start and stop codons of the 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 the messenger RNA (mRNA) transcript thereof to be translated by a ribosome. In eukaryotes, a start codon codes for methionine. A “stop codon ” is a trinucleotide sequence within the gene, or mRNA transcript thereof, that signals the stop of protein synthesis. [0123]The term “allele” refers to a variant form of a gene. Some genes have a variety of different forms, which are located at the same position, or genetic locus, on a chromosome. A diploid organism has two alleles at each genetic locus. Each pair of alleles represents the genotype of a specific genetic locus. Genotypes are described as homozygous if there are two identical alleles at a particular locus and as heterozygous if the two alleles differ. [0124]A “promoter” is a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular polynucleotide sequence. A promoter may additionally comprise other regions which influence the transcription initiation rate. The promoter sequences disclosed herein modulate transcription of an operably linked polynucleotide. A promoter can be active in one or more of the cell types disclosed herein (e.g., a eukaryotic cell, a non-human mammalian cell, a human cell, a rodent cell, a pluripotent cell, a one-cell stage embryo, a differentiated cell, or a combination thereof). A promoter can be, for example, a constitutively active promoter, a conditional promoter, an inducible promoter, a temporally restricted promoter (e.g., a developmentally regulated promoter), or a spatially restricted promoter (e.g., a cell-specific or WO 2024/163650 PCT/US2024/013826 tissue-specific promoter). Examples of promoters can be found, for example, in WO 2013/176772, herein incorporated by reference in its entirety for all purposes. [0125]Hearing loss may refer to one or more decreased responses to auditory stimuli by a non- human animal compared to that of a control (e.g., wildtype) non-human animal. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 5% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 10% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 15% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 20% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 25% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 50% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 55% compared to that of a control (e.g., wildtype) non-human animal of the same age. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein may be considered to have hearing loss when its responses to auditory stimuli is decreased by about 60% compared to that of a control (e.g., wildtype) non-human animal of the same age. [0126]Hearing loss may be determined as a decrease in responses to auditory stimuli (e.g., a decrease in the function of inner ear hair cells, outer ear hair cells, and/or neurons (e.g., spiral WO 2024/163650 PCT/US2024/013826 ganglion neurons)) as measured by an auditory brainstem response assay and/or distortion product otoacoustic emission assay, both assays of which are generally depicted in Figure 8. Other assays that may be used to determine a response to auditory stimuli (e.g., function of inner hair cells, outer hair cells, and/or neurons such as spiral ganglion neurons) include those well- known in the art, e.g., electrocochleography (e.g., with a needle electrode or a cotton-wick electrode), and compound action potentials. [0127]In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein exhibits hearing loss when the threshold decibel (dB) level for its hearing is greater than 1.5-fold to 2.0-fold the threshold dB level for a hearing by a control (e.g., wildtype) animal, e.g., at 8 kHz, 16, and/or 32 kHz as measured by auditory brainstem response assay. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein exhibits hearing loss when the threshold decibel (dB) level for its hearing, e.g., at 8 kHz, 16, and/or 32 kHz as measured by auditory brainstem response assay, is greater than or about dB. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein exhibits hearing loss when the threshold decibel (dB) level for its hearing, e.g., at 8 kHz, 16, and/or 32 kHz as measured by auditory brainstem response assay, is greater than or about 40 dB. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein exhibits hearing loss when the threshold decibel (dB) level for its hearing, e.g., at 8 kHz, 16, and/or 32 kHz as measured by auditory brainstem response assay, is greater than or about 60 dB. In some embodiments, an animal genetically modified to comprise a nucleic acid as described herein exhibits hearing loss when the threshold decibel (dB) level for its hearing, e.g., at 8 kHz, 16, and/or 32 kHz as measured by auditory brainstem response assay, is greater than or about 70 dB. Profound deafness and the like may be determined when the threshold decibel of an auditory stimulus that generates an auditory brainstem response in an auditory brainstem response assay is about or above 80-90 decibels. [0128]The term “viral vector ” refers to a recombinant nucleic acid that includes at least one element of viral origin and includes elements sufficient for or permissive of packaging into a viral vector particle. The vector and/or particle can be utilized for the purpose of transferring DNA, RNA, or other nucleic acids into cells either ex vivo or in vivo. Numerous forms of viral vectors are known.

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[0129]The term “wild type" includes entities having a structure and/or activity as found in a normal (as contrasted with mutant, diseased, altered, or so forth) state or context. Wild type genes and polypeptides often exist in multiple different forms (e.g., alleles). [0130]The expression “gross mutant phenotype” refers to a significant difference or variation in phenotype between an engineered non-human mouse of the disclosure and a “wild type.” [0131]The term “endogenous” refers to a nucleic acid sequence that occurs naturally within a nucleic acid, a cell or non-human animal. For example, an endogenous Klhdc7b sequence of a non-human animal refers to a native Klhdc7b sequence that naturally occurs at the endogenous Klhdc7b locus in the non-human animal. Similarly, an endogenous Klhdc7b sequence of a non- human animal nucleic acid or cell refers to a native Klhdc7b sequence that naturally occurs at the endogenous Klhdc7b locus in the non-human animal nucleic acid or cell. [0132]The term “variant” refers to a nucleotide sequence differing from the sequence most prevalent in a population (e.g., by one nucleotide) or a protein sequence different from the sequence most prevalent in a population (e.g., by one amino acid). [0133]The term “fragment” or “portion” when referring to a protein means a protein that is shorter or has fewer amino acids than the full-length protein. The term “fragment” or “portion” when referring to a nucleic acid means a nucleic acid that is shorter or has fewer nucleotides than the full-length nucleic acid. A fragment can be, for example, an N-terminal fragment (i.e., removal of a portion of the C-terminal end of the protein), a C-terminal fragment (i.e., removal of a portion of the N-terminal end of the protein), or an internal fragment. [0134]“Sequence identity” or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards 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 WO 2024/163650 PCT/US2024/013826 adjustment are well known. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California). [0135]“Percentage of sequence identity” includes the value determined by comparing two optimally aligned sequences (greatest number of perfectly matched residues) over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise specified (e.g., the shorter sequence includes a linked heterologous sequence), the comparison window is the full length of the shorter of the two sequences being compared. [0136]Unless otherwise stated, sequence identity/similarity values include the value obtained using GAP Version 10 using the following parameters; % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. “Equivalent program” includes any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10. [0137]The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, or leucine for another non-polar residue.

WO 2024/163650 PCT/US2024/013826 Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, or between glycine and serine. Additionally, 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 acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (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 a polar residue for a non-polar residue. Typical amino acid categorizations are summarized below.

WO 2024/163650 PCT/US2024/013826 Alanine Ala A Nonpolar Neutral 1.8 Arginine Arg R Polar Positive -4.5 Asparagine Asn N Polar Neutral -3.5Aspartic acid Asp D Polar Negative -3.5 Cysteine Cys C Nonpolar Neutral 2.5 Glutamic acid Glu E Polar Negative -3.5Glutamine Gin Q Polar Neutral -3.5 Glycine Gly G Nonpolar Neutral -0.4 Histidine His H Polar Positive -3.2Isoleucine He 1 Nonpolar Neutral 4.5 Leucine Leu L Nonpolar Neutral 3.8 Lysine Lys K Polar Positive -3.9 Methionine Met M Nonpolar Neutral 1.9 Phenylalanine Phe F Nonpolar Neutral 2.8 Proline Pro P Nonpolar Neutral -1.6Serine Ser s Polar Neutral -0.8 Threonine Thr T Polar Neutral -0.7 Tryptophan Trp W Nonpolar Neutral -0.9 Tyrosine Tyr Y Polar Neutral -1.3 Valine Val V Nonpolar Neutral 4.2

[0138]A “homologous” sequence (e.g., nucleic acid sequence) includes a sequence that is either identical or substantially similar to a known reference sequence, such that it is, for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the known reference sequence. Homologous sequences can include, for example, orthologous sequence and paralogous sequences. Homologous genes, for example, WO 2024/163650 PCT/US2024/013826 typically descend from a common ancestral DNA sequence, either through a speciation event (orthologous genes) or a genetic duplication event (paralogous genes). "Orthologous" genes include genes in different species that evolved from a common ancestral gene by speciation. Orthologs typically retain the same function in the course of evolution. “Paralogous” genes include genes related by duplication within a genome. Paralogs can evolve new functions in the course of evolution. [0139]“Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur and that the description includes instances in which the event or circumstance occurs and instances in which it does not. [0140]Designation of a range of values includes all integers within or defining the range, and all subranges defined by integers within the range. [0141]Unless otherwise apparent from the context, the term “about” encompasses values within a standard margin of error of measurement (e.g., SEM) of a stated value. [0142]The term “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). [0143]The term “or” refers to any one member of a particular list and also includes any combination of members of that list. [0144]The singular forms of the articles “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a protein” or “at least one protein” can include a plurality of proteins, including mixtures thereof. [0145]Statistically significant means p <0.05, p < 0.01, p < 0.001, or p < 0.0001.

[0146]While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details 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 act as limitations on the scope of the claims.

WO 2024/163650 PCT/US2024/013826 EXAMPLES [0147]The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Characterization of KLHDC7b Expression [0148]According to the University of California at Santa Cruz (UCSC) genome browser, KLHDC7b has two possible isoforms that differ only in which methionine is the predicted start site (see Figure 1).Since the short isoform is contained entirely within the long isoform, it is not possible to show that the short isoform is present on its own, but it is possible to show whether the long isoform is present. Thus, two sets of primers and probes were designed. One set detected the presence of transcripts only within the long portion of the transcript, and the other set detected transcripts within the overlapping portion (referred to interchangeably herein as “short” or “overlapping”). [0149]A mouse multiple tissue cDNA panel was used to show that KLHDC7b is expressed in multiple organs, including liver, brain and testes of mice (Figure 1,bottom left). The long probe and overlapping probe show similar levels of expression, perhaps indicating that the short form may rarely be expressed on its own in mice. Figure 1(bottom middle) also shows KLHDC7b expression in freshly collected mouse liver and kidney samples at levels comparable levels to those of the commercial cDNA panel. Additionally, Figure 1(bottom middle) provides data showing KLHDC7b is also expressed in freshly isolated cochlea. High-expressing and low- expressing tissue according to the cDNA panel (liver and kidney, respectively) was collected to confirm that the results of fresh tissue collection were comparable to the cDNA panel (Figure 1, bottom middle). [0150]Long and overlapping probes were also designed for the human form of the transcript, which also has two putative isoforms, and qPCR was performed with a human multiple tissue cDNA panel (Figure 1,bottom right). Both probes indicated expression in liver and testes of human, with a slightly different pattern of tissue expression compared with mouse, and notably less expression in brain. The overlapping portion was expressed at higher levels than the long form, perhaps indicating that the short form may be present on its own in humans more often than in mice.

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[0151]To determine whether KLHDC7b was expressed throughout the lifespan of mice, mouse tissue was collected at four time points: postnatal day 1 (pl), postnatal day 7 (p7), 11-28 weeks postnatal (adult), and 63-70 days postnatal (aged), and qPCR was performed using the overlapping (Figure 2,left) and long (Figure 2,right) probes and primers. At pl, KLHDC7b expression was highest in cochlea compared with all the other tissues, and it remained relatively high in adult and aged mice as well. In other tissues, expression was higher in adult than it was in neonates. In liver, the expression increased markedly in aged mice.

[0152]qPCR [0153]qPCR probes for the long and overlapping portions of the mouse (m) or human (h) KLHDC7b transcripts were designed using BioSearch RealTimeDesign qPCR Assay Design software (RealTimeDesign qPCR Assay Design Software | LGC Biosearch Technologies) and tested for specificity via the UCSC genome browser BLAT function. Table 2provides the probes used in this Example. Table 2 Probe name Probe Forward Primer Reverse Primer m_sKLHDC7b TTTATGCCATTGGTGGCGAGTGC(SEQID NO:20) GGTGGCCCTGGATGGAATG(SEQID NO:21) TCTGTGCGTGGGTCATAGC(SEQID NO:22) m_LKLDHC7b TCCCCATCATGATCCACTTTACCCC(SEQID NO:23) CCGCAAAGCAAAGGATATTCCTG(SEQID NO:24) ATGCCTCCAGCTGCATCTC TA(SEQID NO:25) m_both_KLHDC7b ACTATGGTTCTTAGAAGCCACCCCTTCC(SEQID NO:26) CCCAGAAAGACGAATCTCAAATAGC(SEQID NO:27) CTGCAGGACTCTCCGGATCTG(SEQID NO:28) hKLHDC7b_s_both AGTGCCAGCCACCGGCGTTC(SEQID NO:29) TTGGGACGA GTGCCCATAC(SEQID NO:30) AGCAGGTCGAAGCGGTACAG(SEQID NO:31) WO 2024/163650 PCT/US2024/013826 hKLHDC7b_l_sUTR AGAGTGTGGCTCTCCCCAG GC(SEQ ID NO:32) CCCAGCTGACGGGTCAAAG(SEQ. ID NO:33)TGGCTTCTAAGAACCATGGCAAT(SEQ ID NO:34) hKLHDC7b_l AGGCCAGGCACTGCCCTCCT(SEQ ID NO:35) GAGCGGAAGGAAAGGAGCCT(SEQ ID NO:36) CCGAGGAGTGAAGTGGATCAG(SEQ ID NO:37)

[0154]The m_sKLHDC7b probe was designed to a portion of the transcript that overlapped between the two putative long and short isoforms. The m_both_KLHDC7b was designed across the junction between the long and short isoforms, and the m_LKLDHC7b was designed against a portion of the transcript that is only present in the long isoform. Primers and probes were designed similarly for the human transcripts. [0155]KLHDC7b probes were ordered from IDT (Integrated DNA Technologies) as Primetime qPCR assays in the FAM/ZEN/IBFQ dye combination. For experiments using the mouse (Panel I cat # 636745, Panel III cat # 636757, Takara) and human (Panel I cat # 636742, Panel II cat # 636743, Takara) cDNA panels, cDNA was treated similarly to RNA and the protocol was followed, using Taqman Fast Advanced Master Mix (example catalog number, 4444557, Thermofisher scientific). Plates were run on the Viaa7 Thermocycler up to 45 cycles, using Drosha, a housekeeping gene, as a control for both human and mouse experiments, with the Drosha probe added to an equivalent amount of cDNA. Data was analyzed and plotted using GraphPad Prism. [0156]For the collection of fresh tissue, mice were euthanized under CO2or decapitated if they were under 7 days old. Organs were collected and placed in RNA later. Cochleae were snap frozen to maintain RNA integrity for later extractions. For RNA Extractions; Tissue/Cells were homogenized in TRIzol, and chloroform was used for phase separation. The aqueous phase, containing total RNA, was purified using MagMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies) according to manufacturer ’s specifications. Genomic DNA was removed using RNase-Free DNase Set (Qiagen). [0157]mRNA was reverse-transcribed into cDNA using SuperScript® VILOTM Master Mix (Invitrogen by Life Technologies). cDNA was amplified with the SensiFAST Probe Lo-ROX49 WO 2024/163650 PCT/US2024/013826 (Meridian) using the 12K Flex System (Applied Biosystems). An endogenous control gene was used to normalize any cDNA input differences. Data was reported as the comparative CT method using ACT compared with Drosha, a housekeeping gene.

Example 2: Detecting KLHDC7b Expression in the Cochlea [0158]After finding that KLHDC7b is expressed in cochlea, an RNA scope was performed to examine which cell types within the cochlea express KLHDC7b. Similar to the qPCR probes, two probes to the overlapping and long portions of the transcript were made. The probes were applied in conjunction with immunostaining for My07a, a hair cell marker. Both probes bound to transcripts exclusively within hair cells. Both probes were present in inner, outer, and vestibular hair cells (Figures 3B-3C).RNA scope was also performed on embryonic mice at approximately el6-17. Both probes were present in what appear to be developing hair cells, showing that KLHDC7b is expressed very early in development (Figure 3D).

[0159]RNA scope with or without antibody co-detection [0160]Animals 21 days and older were sacrificed by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Cochleae were dissected and placed 1-4 hours or overnight in 4% PF A on a shaker, followed by three washes in PBS and stored at 4 °C until processing. [0161]For RNA scope in paraffin embedded sections, slides were baked in the HybEZ Oven at degrees for thirty minutes. After baking, slides were deparaffinized with 2 washes of xylene for 5 minutes each, then 100% ethanol for 2 x 1 minute. Slides were allowed to air dry. [0162]Organoid slices were thawed at room temperature, then washed for 5 min in PBS with agitation to remove OCT. Sections were post-fixed with cold 10% neutral-buffered formalin (NBF) for 15 min at 4 degrees. They were then dehydrated for 5 minutes each in 50% ethanol, 70% ethanol, and 100% ethanol and allowed to dry at room temperature. [0163]For both paraffin embedded cochlear sections and explants, hydrogen peroxide from the RNA scope kit was added to each slide and incubated for 10 minutes at room temperature, then washed twice with distilled water. Co-detection target retrieval reagent (ACD Bio) was heated in a vegetable steamer (Oster), and slides were placed in hot co-detection target retrieval reagent for WO 2024/163650 PCT/US2024/013826 minutes for cochleae and 5 minutes for organoids, followed by two washes in distilled water. Slides were then washed in PBS plus 0.1% tween-20 (PBS-T). A hydrophobic barrier was drawn around the sections on the slides. Antibody (My07a, Proteus, 25-6790) was diluted at a concentration of 1:200 in Co-detection antibody diluent and incubated overnight at 4 degrees in a humidified chamber. [0164]After primary incubation, slides were washed in PBS-T 3 times for 2 minutes each. Then they were post-fixed with 10% neutral buffered formalin in a fume hood for 30 minutes. Afterward, they were washed again 4x 2 minutes with PBS-T. Slides were then treated with protease plus reagent (ACD Bio) at 40 degrees in the HybEZ oven for 30 minutes. [0165]Fluorescent in situ hybridization, or RNA scope, was then performed in accordance with the protocol from ACD Bio. Briefly, probes were heated at 40 degrees for 10 minutes, wash buffer was heated at 40 degrees for 20 minutes and then diluted to the correct concentration. Probes were applied to the slides and placed in a humidified chamber in the oven at 40 degrees for 2 hours. All incubation steps moving forward were performed in a humidified chamber at degrees, and all washes were done with wash buffer. After probe hybridization, slides were washed 2x2 minutes with wash buffer. Then, for the amplification steps, Amp 1 (ACD bio) was incubated for 30 minutes, 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. [0166]After amplification, depending on the number of channels used, slides were incubated in HRP Cl for 15 minutes, followed by 2 x 2 minute washes, then Opal 520 at 1:15000 for minutes followed by 2 x 2 minute washes. Slides were incubated with HRP blocker for minutes, followed by 2 x 2 minute washes. Then the slides were incubated with HRP C2 for minutes, followed by 2 x 2 minute washes, then Opal 570 at 1:1500 for 30 minutes followed by x 2 minute washes. Slides were then again incubated with HRP blocker for 15 minutes, followed by 2 x 2 minute washes. Opal 570 at 1:1500 for 30 minutes followed by 2 x 2 minute washes. Slides were then again incubated with HRP blocker for 15 minutes, followed by 2 x 2 minute washes, then 30 minutes in Opal 690 at 1:1500, 2x2 minute washes and 15 minutes of HRP block. If only one channel was used, it was Opal 570 and subsequent channel HRP steps were skipped.

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[0167]Finally, secondary antibody (donkey anti-rabbit Alexa 647) was diluted to 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. DAPI was applied to the slides for seconds, shaken off the slide, then Prolong Gold Antifade mountant was applied and a coverslip was placed over the sections and allowed to dry overnight at room temperature. [0168]For RNA scope without co-detection (for organoid markers), steps were similar, but after antigen retrieval slides were placed in 100% ethanol and allowed to dry overnight. The post- fixation after antibody incubation was skipped, and the secondary antibody incubation was skipped. [0169]Microscopy [0170]Images of RNA scope and immunostained samples were acquired on a confocal microscope (either Zeiss LSM 780 or Zeiss LSM 880), with 20x and lOOx objectives. Z-stacks were taken with the appropriate size for the objective and numerical aperture or tile scans were acquired as necessary. Post hoc image processing and analysis was performed with Fiji and Imaris (PLA quantification).

Example 3: Knock Out of endogenous mouse Klhdc7b gene [0171]A targeting vector for knocking out an endogenous Klhdc7b gene was constructed using bacterial artificial chromosome (BAC) clones and VELOCIGENE® technology (see, e.g., U.S. Patent No. 6,586,251 and Valenzuela et al. (2003) High-throughput engineering of the mouse genome coupled with high-resolution expression analysis, Nature Biotechnology 21 (6);652-659; incorporated herein by reference). [0172] BACclone RP23-241G24 containing a mouse Klhdc7b gene was used and modified as follows. Briefly, a DNA fragment was generated to include a mouse 5’ homology nucleotide sequence of 100 bp (mHU), a LacZ gene (3,075bp) downstream and in frame with the ATG starting site of the mouse Klhdc7b gene, followed by a self-deleting Neomycin cassette of 4,8bp, and a 3’ mouse homology sequence of 100 bp (mHD). This DNA fragment was used to modify BAC clone RP23-241G24 through homologous recombination in bacterial cells. As result, a full knockout (KO) of the region encoding mouse Klhdc7b genomic fragment of 3,7bp in the BAC clone was replaced by the LacZ gene and Neo-self-deleting cassette (SDC) of WO 2024/163650 PCT/US2024/013826 8,202 bp. Specifically, the entire mouse Klhdc7b orf (mm9, chrl5:89, 215,351-89,219,137) was replaced with the LacZ-Neo SDC insert nucleic acid leaving intact 5’ and 3’ untranslated regions (UTRs) (Figures 4A-4B).The resulting modified BAC clone included, from 5’ to 3’, (i) a 5’ mouse homology arm containing about 140.5 kb of mouse genomic DNA including a mouse Klhdc7b 5’ UTR and ATG; (ii) LacZ cDNA of 3,075 bp, (iii) a self-deleting Neomycin cassette of about 4,809 bp, followed by (iv) a 3’ mouse homology arm of 12.6 kb containing the mouse Klhdc7b 3’ UTR and the remaining mouse genomic DNA in the original BAC clone (Figures 4A-4B).The amino acid sequence of the protein encoded by the LacZ cDNA is set forth in SEQ ID NO:4. The LacZ cDNA sequence is set forth as SEQ ID NO:3. [0173]The modified BAC clone containing the KO of Klhdc7b gene, as described above, was used to electroporate mouse embryonic stem (ES) cells to create modified ES cells comprising a Klhdc7b KO gene. Positively targeted ES cells containing a.Klhdc7b KO gene were identified by an assay (Valenzuela et al., supra) that detected the presence of the LacZ and Neo sequences and confirmed the loss and/or retention of mouse Klhdc7b sequence by TaqMan assay (SEQ ID NOs:8-19, Table 3).Once a correctly targeted ES cell clone had been selected, it was electroporated into an early-stage blastocyst (8 cells Morula stage) to generate F0 mice. The Neomycin selection cassette was removed by crossing the progeny generated from the ES clone with a deletor rodent strain that expresses a Cre recombinase (Figure 4C). [0174]Selected ES cell clones (with or without the cassette) were used to implant female B6.Cast-Cdh23A111+ mice using the VELOCIMOUSE® method (see, e.g., U.S. Pat. No. 7,294,7and Poueymirou et al., 2007, Nature Biotech. 25(l):91-99) to generate a litter of pups containing a Klhdc7b KO allele in the genome. B6.Cast-Cdh23 Ahl+ mice were used since mice on this background comprise an allele-corrected form of Cdh23 to prevent known age-related hearing loss that occurs in C57BL6 mice. [0175]Mice bearing the Klhdc7b KO allele were confirmed and identified by genotyping of DNA isolated from tails snips using a modification of allele assay (Valenzuela et al., supra) that detects the presence/absence of the Klhdc7b gene sequence. Pups are genotyped and cohorts of animals heterozygous for the Klhdc7b locus were selected for characterization. Animals homozygous KO for the Klhdc7b locus were made by crossing heterozygous animals.

WO 2024/163650 PCT/US2024/013826 Table 3 NAME Primer Sequence (5’-3’) SEQ ID NO 4929mTU Forward TGGGAGGCGGTTGTACTC (SEQ ID NO:8) Probe (BHQ) ACCTTGCTGGCACTAGCGGTGGT (SEQ ID NO :9) Reverse GCAAAGCCAGTGCTAAGGT (SEQ ID NO: 10) 4929mTD Forward GGCTTCCTATACCGCTTTGAC (SEQ ID NO: 11) Probe (BHQ) TCCAGCTATCGCACTTATCTGA (SEQ ID NO: 12) Reverse CTCCAGGAGCCGGTCACT (SEQ ID NO: 13) LacZ Forward GGAGTGCGATCTTCCTGAGG (SEQ ID NO: 14) Probe (BHQ) CGATACTGTCGTCGTCCCCTCAAACTG (SEQ ID NO: 15) Reverse CGCATCGTAACCGTGCATC (SEQ ID NO: 16) Neo Forward GGTGGAGAGGCTATTCGGC (SEQ ID NO: 17) Probe (BHQ) TGGGCACAACAGACAATCGGCTG (SEQ ID NO: 18) Reverse GAACACGGCGGCATCAG (SEQ ID NO: 19)

[0176]The mice were born at normal Mendelian ratios and appeared phenotypically normal. LacZ staining was present in cochlea of KLHDC7B+/" (HET) and KLHDC7B ־־ (KO) mice, in what appear to be hair cells (Figure 4D),confirming replacement of the Klhdc7b gene with the LacZ cassette. No other cells in the cochlea, including vestibular hair cells, appear to show LacZ labeling. Moreover, RNAScope probes against the long and overlapping transcripts of KLHDC7B do not label hair cells of KLHDC7B" ־ (KO) mice (Figure 4E),confirming the absence of transcribed Klhdc7b mRNA.

[0177]LacZ staining of knockout mice [0178]Mice were anesthetized with Ketamine/xylazine and fixed via perfusion with 2% paraformaldehyde. After perfusion, tissues were dissected and sliced into 1-5 mm pieces, fixed min at room temperature, washed in PBS for 30 min and stained in beta galactosidase (lacZ) 54 WO 2024/163650 PCT/US2024/013826 staining solution overnight at 4 C. After staining tissues were washed in cold PBS for 15 min and postfixed in 4% formaldehyde at 4°C overnight with mixing. Tissues were cleared with glycerol by incubating in 50% glycerol for a day at RT and then 70% glycerol for a day. Tissues were photographed on a Zeiss dissection microscope and stored atRT in 70% glycerol. Tissues were subsequently decalcified overnight with immunocal and dissected, then photographed using an Axioscan slide scanner (Zeiss).

Example 4; Characterization of KLHDC7b Knockout Mice [0179]The phenotype of cochleae and the auditory responses of knockout animals as described in Example 3 are examined to determine the phenotypic effects of the KLHDC7b knockout. [0180]Histology [0181]At postnatal day (p) 6, the Organ of Corti of KO mice appears normal, with intact hair cells stained for MY07A and stereocilia staining for F-actin (Figure 5A).However, at pit, a few outer hair cells appear to be missing and are not labeled for MY07A or F-actin. By p21, many outer hair cells are missing and visible supporting cell scars, which are known to form when hair cells die (Wagner and Shin, 2019). Circular stained areas of MY07A are present and could be hair cells being engulfed by supporting cells or hair cells being extruded from the sensory epithelium. By 8 weeks of age, almost no outer hair cells are present and there is significant scarring (Figure 5A).At 8 weeks, hair cells in KLHDC7b knockout mice have abnormal morphology that is worse at the base than the apex (Figure 5B).In the whole mount, hair cells appear to be missing, again worse in the base than 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).At postnatal day 6, hair cells also appear normal. ZO-1, a tight junction protein, shows diffuse cytoplasmic staining in some hair cells only in knockout mice at pl 1 and p21, but not earlier, suggesting that this protein may be mislocalized in KO tissue around the time of cell death (Figures 7D-7J).

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[0182]ABR (Auditory brainstem response) - measuring function of inner hair cells and neurons, and DPOAE (Distortion product otoacoustic emissions) - measuring the function of outer hair cells. [0183]To determine the impact of KLHDC7b knockout on hearing in mice, ABR and DPOAE hearing assays were performed (Figure 8).KLHDC7B ־־ (KO), KLHDC7B+/" (Het), and KLHDC7B+ + (WT) mice underwent Auditory Brainstem Response (ABR) tests at different ages to assess their hearing (Figure 9).Mice begin hearing at two weeks of age. At postnatal day (pl7, 2-3 weeks), KO mice show profound hearing loss with significantly elevated hearing thresholds and a significantly diminished Wave I amplitude. These thresholds become progressively elevated with most mice showing no response at 11-15 weeks of age (Figure 9A). Heterozygous mice have normal ABR thresholds and Wave I amplitude compared with WT mice as late as 57 weeks (Figure 9B).At the onset of hearing (around day 17), KLHDC7b knockout mice exhibit hearing loss, with significantly elevated ABR thresholds compared to wild type (Figure 9C).The heterozygotes have no hearing loss (Figure 9C).Hearing loss appears progressive. At 8 weeks, knockout mice are profoundly deaf at all tested frequencies, with some responses maintained at the highest frequencies tested by both ABR and DPOAE (Figure 10). Heterozygous mice are not deaf as of 30 weeks (Figures 11A-11C).

[0184]Histology [0185]For immunostaining paraffin-embedded sliced cochlea on slides, slides were first baked at 60 degrees in the HybEZ oven for one hour to melt the wax, then washed in xylene twice for three minutes each. Slides were then rehydrated in 100% ethanol for 2 x three minutes, then 95% ethanol for 2 x 3 minutes, then 70% ethanol for 3 minutes, 50% ethanol for 3 minutes, distilled water for 5 minutes, and 3 x PBS for 2 minutes each. [0186]Blocking solution [2% weight per volume bovine serum albumin, 5% normal donkey serum, 0.01 % triton-x 100 in PBS] was made. A hydrophobic barrier was drawn around the sections on the slide, and slides were placed in a humidified chamber and covered with blocking solution, then incubated at room temperature for one hour. Primary antibodies were diluted in blocking solution, the block on the slides was removed, and primary antibody solution added to the slides, then placed at 4 °Cand incubated overnight. After primary incubation, slides were WO 2024/163650 PCT/US2024/013826 washed three times in PBS, then covered with secondary antibodies and cell stains diluted in blocking solution and incubated for 1 hour. Afterward, slides were washed 3 x in PBS, then mountant (Prolong Diamond or Prolong gold) was placed on the slide and covered with a coverslip, then allowed to dry overnight before imaging on a confocal microscope. [0187]For whole mount samples, the procedure was similar. Dissected sections were washed three times in PBS, incubated with blocking buffer for 1 hour with shaking at room temperature, incubated overnight with primary antibodies in blocking solution at 4 °C or room temperature, then washed 3 x with PBS. Secondary antibodies and cell stains were diluted in blocking buffer and samples were incubated for 1 hour at room temperature, rinsed 3x with PBS, then placed on slides and covered with mountant and a coverslip, then allowed to dry overnight before imaging on a confocal microscope. Phalloidin was only used for whole mounts, not slides.

[0188]Auditory brainstem response (ABR) [0189]Auditory brainstem response recordings were performed in a soundproof booth. Equipment was calibrated each day using a microphone to confirm that the sound level presented was as expected. Animals were anesthetized with an intraperitoneal injection of ketamine/xylazine (12 mg/kg, 0.5 mg/kg) and placed in a heated cage. Puralube ointment was placed on the eyes after several minutes once the animal was no longer responsive. [0190]Once the mouse no longer responded to toe pinch, the mouse was placed on a heating pad in the sound booth. Electrodes were plugged into the preamplifier. The needle end of the electrode was placed subcutaneously with the positive electrode at the cheek of the animal (near the cochlea), the negative electrode at the midline of the skull on top of the head, and the ground electrode in the contralateral cheek. The ear being recorded (right ear for these experiments) was positioned 7.5 mm away from the speaker, which was in an open field configuration, rather than closed field in a tube directly placed in the ear. The booth was closed and recordings began. [0191]Recordings were performed at three pure-tone frequencies (8 kHz, 16 kHz, and 32 kHz), with 512 presentations of each stimulus averaged at each sound pressure level presented. Each frequency was played at 90 decibels (dB), followed by 80, 70, 60, and down by 10 dB until 50. From 50 dB to 15 dB the dB level was dropped by 5 dB instead of 10. The waveform of the auditory brainstem response was recorded. When a waveform was no longer visible, the sound WO 2024/163650 PCT/US2024/013826 pressure level of the previous waveform was called as the threshold for that frequency. Once threshold was reached, stimuli at 1-2 sound pressure levels was recorded, but subsequent recordings were skipped. Thresholds were recorded for each animal and grouped by age or sex of the animal. Animals were then placed in a heated recovery cage and returned to the home cage once they were ambulatory. [0192]ABR analysis [0193]ABR recordings were processed in Matlab. Traces were first smoothed with a moving median filter with a kernel of 50 time points (each 10 ms recordings contained 244 time points). This was to remove slow wave noise occasionally observed in recordings and had a minimal effect on recordings that were not noisy. [0194]ABR thresholds were called manually by two experimenters on traces presented in a blinded, random order, and run through an algorithm adapted from the Liberman lab (Suthakar and Liberman, 2019). Briefly, the covariance between pairs of adjacent decibel traces was calculated and plotted. These points were fit to a curve using a sigmoid or logarithmic function, and the decibel level at which the function crossed below a set criterion level was recorded as the threshold of hearing. The threshold values called by the algorithm were compared to manually called thresholds. Traces with no discernible ABR response were called at 100 dB. If the difference between the manual and automated thresholds was greater than 15 dB, traces were examined and the manual threshold was used; otherwise, the automated threshold was used. [0195]For wave 1 amplitude and latency, peaks were detected using a semi-automated method where a peak and trough was estimated within a time window to encompass wave 1, then checked by a user and corrected if necessary. [0196]Distortion product otoacoustic emissions (DPOAE) [0197]For DPOAE, recordings, the speakers were also calibrated daily. DPOAE measurements were taken on their own or immediately after the ABR while the animal was still anesthetized. If the DPOAE was collected on its own, animals were anesthetized as for the ABR recordings. After the animals were no longer responsive to toe pinch, they were placed in the recording booth. [0198]Two speakers were used to present two frequencies equally spaced around the three frequencies measured for ABR (8 kHz, 16 kHz, and 32 kHz). The speakers were used in a WO 2024/163650 PCT/US2024/013826 closed-field configuration, with tubes connecting the speaker to a sensitive microphone with a cut pipette tip on the end, which was then placed inside the ear canal and angled toward the eardrum. Stimuli were presented with 100 averages. The distortion product was detected at the expected frequency, and called manually by the user as positive if the signal was above the noise.

Example 5 - Gentamycin-Texas Red Mechanotransduction Assay in Cochlear Explant Cultures [0199]Mechanotransduction defects can cause hair cell death, so mechanotransduction (MET) complexes were assessed before the onset of cell death. Mice do not begin hearing until two weeks of age, but the mechanotransduction channels can be assessed in cultured Organ of Corti using a gentamycin-Texas red assay (GtTR). Gentamycin is known to be taken up by the MET complex, and by conjugating it to a dye, Texas red, the presence of functional MET complexes can be ascertained. GtTR labeled hair cells similarly in cultured Organs of Corti of KO and WT mice, indicating that MET complexes are assembled and functional. Hair cells in cochlear explant cultures appear to take up similar amounts of GTTR (Figures 12A-12C),indicating that MET complexes are likely functional and KLHDC7b is not required for assembly of these complexes. [0200]Human inner ear RNA is extremely difficult to obtain due to the fragile nature of hair cells and the time required to remove the temporal bone. Therefore, to examine the expression of KLHDC7b in human otic tissue, otic organoids were generated using a protocol similar to Koehler et al., 2017. These organoids begin with human induce pluripotent stem cells (iPSCs) and are given small molecules to coax them to pass through multiple developmental stages to become mature inner ear organoids at around day 70, when they contain hair cells, supporting cells, and neurons. Expression of markers of inner ear cells in mature otic organoids, including neurons (TUBB3) and hair cells (OTOF, MY07A, MYO15A), increased markedly compared with expression levels in human iPSCs (Figure 13A,right). Importantly, KLHDC7B also increased its expression level in mature otic organoids, measured using the overlapping probe (Figure 13B,right). Since otic organoids have spatially distinct areas containing a greater concentration of inner ear cell types, RNA scope was performed to examine the expression pattern of otic markers and KLHDC7b. Otic markers, including SOX2 and TUBB3, were found WO 2024/163650 PCT/US2024/013826 to be concentrated to some spatial areas that resemble otic vesicles as in Koehler et al., 20 (Figure 13A,left). RNA scope against the long and overlapping transcripts of KLHDC7B was performed together with immunostaining against My07a, a hair cell marker. KLHDC7B probes are shown labeling slices of the same otic organoid (Figure 13B,left). While the labeling was not only within hair cells, it was concentrated to the area around what appear to be otic vesicles. The two slices labeled with long and overlapping probes were from nearby slices of the same otic organoid, and they were located in the same region of the organoid, near a circle of probable hair cells labeled with Myo7a that are visible in the image of the overlapping KLHDC7B probe.

[0201]Cochlear explant cultures [0202]Cochlear explant cultures were performed with mouse pups at postnatal days 0-5. The cell culture plates used were 35 mm dishes with a circular punchout and a glass coverslip glued to the bottom (Matsunami, #D35-14-0-U). Collagen bubbles were prepared by mixing 1.7 pl 1 N NaOH, 10 pl lOx PBS, 67 pl rat tail collagen (set pipet to 68 uL), 21.3 pl H2O. Ten pl of this solution was placed on the coverslip and cured at 37 °C in a humidified cell culture incubator with 5% CO2 for 30-40 minutes. Collagen bubbles were covered in PBS until use and stored at degrees for up to 2 months. Explant culture media was prepared as follows: 90 ml DMEM/Fwithout phenol red, 7 ml fetal bovine serum, 1 ml penicillin G, and 1 ml L-glutamine. [0203]The mice were decapitated, and the skull bisected. Cochlea was removed and placed in Leibovitz/L15 media. The bone was opened and Organ of Corti was removed, stria vascularis removed. PBS was removed from the culture plate and replaced with cochlear explant media. The Organ of Corti was placed on the collagen bubble and most of the media removed to allow the explant to adhere to the collagen. Plates were placed in a humidified cell culture incubator at degrees with 5% CO2. After dissections were finished, 200 pl of explant culture media was added to the dish and cultures were brought from the vivarium to the lab, and 600 pl of culture media was added. [0204]Gentamycin-texas red (GtTR) assay [0205]Explant cultures were kept in culture and treated 2-3 days later with gentamycin-Texas Red (GtTR) or Texas red (TR) alone. GtTR was 5 pg/ml, and TR 12.5 pg/ml. GtTR or TR were diluted in cell culture media. Existing media on the explant was removed, and 600 pL of GtTR or WO 2024/163650 PCT/US2024/013826 TR containing media was added and treated for 20 minutes. Explant cultures were rinsed with media, PBS, and then fixed for 15 minutes in 4% PF A and rinsed 3x with PBS and stored at degrees for several days, after which they underwent immunostaining with the same protocol that was used for whole mount cochlea as described herein. [0206]Otic Organoids [0207]Inner ear organoids were produced according to Zhang et al. (2021) "A simplified method for generating human inner ear organoids from pluripotent stem cells" PROTOCOL (Version 1) doi.org/10.21203/rs.3.pex-1708/vl, incorporated herein in its entirety by reference. Briefly, human iPSCs were aggregated to form embryoid bodies in a chemically defined medium to induce ectoderm placode formation. Upon day 8 of the induction, the Wnt signaling agonist CHIR99012 (Tocris Cat# 4423) was added to the media to stimulate otic vesicle formation. Subsequently, organoids were cultured in maturation medium for upwards of 100 days to allow for otic sensory epithelium to mature. For these experiments, organoids were harvested at day 70. [0208]RNA scope and Immunofluorescence of Otic Organoids [0209]10 pm cryosections of fixed-frozen fully differentiated otic organoids were used for RNA scope. Some slides were use for RNA scope combined with immunofluorescence, while others were only used for RNA scope as described below. For both conditions, RNA scope, slides were baked in the HybEZ Oven at 60 degrees for thirty minutes. After baking, slides post-fixed in cold 4% paraformaldehyde (PFA) in lx PBS for 15 min at 4 degrees. Slide were then dehydrated for minutes each at room temperature in 50% ethanol, 70% ethanol, and 100% ethanol. Slides were allowed to air dry at room temperature. [0210]Hydrogen peroxide from the RNA scope kit was added to each slide and incubated for minutes at room temperature, then washed twice with distilled water. Co-detection target retrieval reagent for co-treated slides or target retrieval reagent for RNA scope alone (ACD Bio), and a different container of distilled water was heated in a vegetable steamer (Oster), and slides were placed in hot distilled water for 10 seconds, then into co-detection target retrieval or target retrieval reagent for 5 minutes, followed by two washes in room temperature distilled water. [0211]Co-detection slides were then washed four times in PBS plus 0.1% tween-20 (PBS-T) for minutes. RNA scope alone slides were dehydrated in 100% ethanol for 3 minutes, then allowed WO 2024/163650 PCT/US2024/013826 to air dry. A hydrophobic barrier was drawn around the sections on the slides. RNA scope alone slides were stored at room temperature overnight. Co-detection slides were treated with primary antibody (My07a, Proteus, 25-6790) diluted at a concentration of 1 ;200 in Co-detection antibody diluent and incubated overnight at 4 degrees in a humidified chamber. [0212]For co-detection slides, after primary incubation, slides were washed in PBS-T 3 times for 2 minutes each. Then they were post-fixed with 10% neutral buffered formalin in a fume hood for 30 minutes. Afterward, they were washed again 4x 2 minutes with PBS-T. Both RNA scope alone and co-detection slides were then treated with protease III reagent (ACD Bio) at degrees in the HybEZ oven for 30 minutes. [0213]Fluorescent in situ hybridization, or RNA scope, was then performed in accordance with the protocol from ACD Bio. Briefly, probes were heated at 40 degrees for 10 minutes, wash buffer was heated at 40 degrees for 20 minutes and then diluted to the correct concentration. Probes were applied to the slides and placed in a humidified chamber in the oven at 40 degrees for 2 hours. All incubation steps moving forward were performed in a humidified chamber at degrees, and all washes were done with wash buffer. After probe hybridization, slides were washed 2x2 minutes with wash buffer. Then, for the amplification steps, Amp 1 (ACD bio) was incubated for 30 minutes, 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. [0214]After amplification, slides were incubated in HRP Cl for 15 minutes, followed by 2 x minute washes, then Opal 520 at 1:15000 for 30 minutes followed by 2 x 2 minute washes. Then with HRP blocker for 15 minutes, followed by 2 x 2 minute washes. Then the slides were incubated with HRP C2 for 15 minutes, followed by 2 x 2 minute washes, then Opal 570 at 1:15000 for 30 minutes followed by 2 x 2 minute washes. Then with HRP blocker for minutes, followed by 2 x 2 minute washes. [0215]RNA scope alone slides were treated with HRP C3 for 15 minutes, followed by 2 x minute washes, then Opal 690 at 1:15000 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. [0216]For co-detection slides, secondary antibody (donkey anti-rabbit Alexa 647) was diluted to ;500 in co-detection antibody diluent and incubated for 30 minutes at room temperature in a WO 2024/163650 PCT/US2024/013826 humidified chamber, followed by 2 x 2 minute washes in PBS-T. For both conditions, DAPI was applied to the slides for 30 seconds, shaken off the slide, then Prolong Gold Antifade mountant was applied and a coverslip was placed over the sections and allowed to dry overnight at room temperature. [0217]Microscopy [0218]Images of RNA scope and immunostained samples were acquired on a confocal microscope, with 20x and lOOx objectives. Z-stacks or tile scans were acquired as necessary.

Example 6 - Scanning Electron Microscopy [0219]While hair cells appear normal before they begin to die at pl l, their morphology and function before the onset of cell death was examined. Scanning electron microscopy (SEM) was performed just before the onset of cell death at plO, and later at p22 after there is significant death. Similar areas of the Organ of Corti are depicted in the SEM images of Figure 14.Both heterozygous mice, which have no hearing loss phenotype as late as 50 weeks, and KO mice have grossly normal stereocilia before the onset of cell death (Figure 14,left). Even at p22, after many outer hair cells are clearly missing, the stereocilia morphology on remaining outer hair cells appears normal (Figure 14,right).

[0220]Scanning Electron Microscopy [0221]The cochleae were extracted from the temporal bone. A tiny hand-drilled hole was made on the apex and the stapes was removed and oval window pierced. Then the samples were fixed overnight in a mixture of 3% Formaldehyde, 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 (Electron Microscopy Sciences, Hatfield, PA) supplemented with 2 mM of CaC12. The samples were then washed with 0.1 M sodium cacodylate buffer, decalcified for 24 hours in 10% formic acid (Immunocal), washed 3 times, and dissected to three flat turns. The bone layer, strial ligament and tectorial membrane were carefully removed from each turn and cochlear sections were placed into porous baskets. Afterwards, the samples were dehydrated through a graded series of ethanol (5%, 10%, 20%, 40%, 60%, 80% and 100%), critical-point dried using a manual CPD (BAL-TEC 030) in liquid C02, sputter coated with 5nm Platinum and imaged with a field-emission scanning electron microscope (Zeiss Sigma VP).

WO 2024/163650 PCT/US2024/013826 Example 7 - Evaluating localization of KLHDC7b by custom monoclonal anti-KLHDC7b antibody antibodies [0222]Several custom monoclonal antibodies against mouse KLHDC7b were generated 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 binding of the custom anti- KLHDC7b antibody phenotyping. The cell lines were validated by Genscript by western blot and immunohistochemistry for the FLAG tag. As shown in Figure 15,anti-KLHDC7b antibodies exclusively labeled both inner and outer hair cells wildtype mouse cochlea, appearing to be localized to the membrane. Furthermore, the anti-KLHDC7b antibodies stained the plasma membrane of hair cells in paraffin embedded cochlear slices isolated from wildtype mice, but not in knockout KLHDC7b mice (Figure 16).The anti-KLHDC7b antibodies were also capable of recognizing human KLHDC7b expressed in transiently transfected HEK cells (Figures 17A- 17B; Table 5).

[0223]Transient transfection of HEK cells with mouse KLHDC7B constructs for custom antibody generation [0224]HEK-293 cells were cultured in a T75 flask 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 hours, they were collected for western blot or immunostaining, for which they were lifted off the plate, fixed, and embedded and sliced, placed on slides for antibody testing.

Table 4:Vectors designed to transfect cell lines for expression of mouse Klhdc7b transcriptsName/pur poseSequence (5’-3’) Short FLAG KLHDC7B (SEQID NO: 40) atgcattagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgegttacataacttacggtaaatggccc gcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgac gtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatga cggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctatta ccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtca atgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgt gtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcgattacgccaagctcgaaattaaccctcact aaagggaacaaaagctggagctccaccgcggtggcggccgccaccatggattacaaggatgacgacgataaggactataaggacg64 WO 2024/163650 PCT/US2024/013826 atgatgacaaggactacaaagatgatgacgataaaggtggttctggtatgGTTCTTAGAAGCCACCCCTTCCCC AGGCAAGAAAAGGCCCAAGGTAGTGCCCCAAGGGCAGATCCGGAGAGTCCTGC AGGGCCCAGCCCATCCACATACTCTGAAGATATACAACCAGGGTCCTCCTCTGAA GGCGCCATCTTCAGCCTCCAGGACAAACACAGACCAGTCTTAGTCACAGCAGTA GGTTCAGAGAGCCTAGCTGAGGCTAGATGTGAAACACAACCAGTGAGTTTAGAG ACTAATGCATCTCTCACCCCGGACCCGAGAGGGGATGAAACCAAGCAGAAGAGT CTAGACTTGCTGCCCCCGGCTGCAGAGCCTGTGGCTTCCCCGCAGGTCCCTGTTT CGGCACAGCCCGGTCCTGTACCGCCTACTTCTATGAGGCAGGATGCTCAGCCCAC CCCTCAGCCTCGGCAACGGAGGAGGAAACGCAGCATAGCTCAGAGCTCCGAACA CATACTGATCCAGGAAGTACCCCAGCAGCCCCAGGGGCAGGTGCGAAAGGAGG GAGCCAGACCTACAGACTGCAGGGACATCCTCACAGAGAAGCAGAAAGAGACC CAAAAGCTTATGGCTTTTCTGCAGAAGCCTGGGAGTTGGGGAGTGGTGGAGGGG CCTCATAAGTACTGCTCACAGACCTCGCAGCCTAGCAGCCTAGTGGCGCCGTGGC CCCCGAAGCTAGATCTAGGGAGCTGCCTGGAGGTTTTGGCCTTCGCCCAGCAGCA CGGAGAACTGGGCTTAGCCCAGGAGACCTATGTCTTGATGAGCAACAACTTACT GCATGTTCTGGGAGACCCACACCTCTACCGACAGCTGAGTGGAGCAGACAGGGA GCGCATCCTGAACCTGCGGACGTGCCAGGGCCAGACAGTGCTGGGGGTCCTCGT GCTACCCAGCCTTTATCAGGTGAGCCGCTCGGGGCTCACGAGGGGCCCTCCCAGC GAGGAAGCTCCTGCAGCCAGGTCTGAGCACTTGCATCTTCACACATACCTCCATG TATTCAACCCACAAGAGAATGTGTGGTGGCCCCTGACTCAAGTACCAGAGGAGG TCCCCCTTCGGGGCTGTGGCCTCTGCACTATGCATAACTATCTGTTTCTGGCAGGC GGCATCAGGGGTTCTGGTGCCAAGGCTGTCTGCTCCAACAAGGTGTTCTGCTACA ACCCTCTGACCAATATTTGGAGCCAGATTCGGCCCATGCAGCAAGCCCGGGCCC AGCTCAAGCTGGTGGCCCTGGATGGAATGCTTTATGCCATTGGTGGCGAGTGCCT ATACAGCATGGAGCGCTATGACCCACGCACAGATACCTGGACCTTGAGAGCATC CCTCCCTGAGGGCACCTTTCCCGTGGCCCACGAGGCTGTGGTCTGCCGAGGAGAG ATCTACGTCACAGGGGGTCATCTCTTTTACCGCCTGCTCAGATACAGCCCCATGA AAGACTCATGGGATGAGTGTCCCTACAGCGCCAGCCACCGACGCTCCAGTGATA TGGTGGCGCTAGGGGGCTTCCTATACCGCTTTGACTTGTTGCGGGGTGTGGGTGC TGCGGTGATGCGCTACAACACAGTGACCGGCTCCTGGAGCCGGGCAGCCTCCCT GCCACTGTCTGACCCCGCCCCACTCCACTGCACGGTATTGGGCAACACCATTTAC TGTCTCAACCACCAGGTCACGGCTACCTTCACAGTTTCGGAGGGGACCGCCCTGT TTCAGGCCAAGGAGCTGCAGCCCTTCCCACTGGGAAGTAAGGGGGTCCTCTATCC GTTCACTCTGACTCTGCCCCCTAAGACCTGGTTGCAGACCACAATCTGAaagcttatcg ataccgtcgacctcgagggggggcccggtaccttaattaattaaggtaccaggtaagtgtacccaattcgccctatagtgagtcgtatta caattcactcgatcggctcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgac cctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtg gggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggaacgcgtaaattgtaagcgttaatatttt gttaaaattegegttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagacc gagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtcta tcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaag ggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgc tagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcaggt ggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgat aaatgcttcaataatattgaaaaaggaagaatcctgaggcggaaagaaccagctgtggaatgtgtgtcagttagggtgtggaaagtcc ccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagc aggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgccc agttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagta gtgaggaggcttttttggaggcctaggcttttgcaaagatcgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggat tgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgt gttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagc gcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattg ggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgc atacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgt65 WO 2024/163650 PCT/US2024/013826 cgatcaggatgatctggacgaagaacatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggc gaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccgg ctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagaacttggcggcgaatgggctgaccgcttcc tcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcg aaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgtt ttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccctagggggaggctaactgaaaca cggaaggagacaataccggaaggaacccgcgctatgacggcaataaaaagacagaataaaacgcacggtgttgggtcgtttgttcat aaacgeggggttcggtcccagggctggcactctgtcgataccccaccgagaccccattggggccaatacgccegegtttcttccttttc cccaccccaccccccaagttcgggtgaaggcccagggctcgcagccaacgtcggggcggcaggccctgccatagcctcaggttac tcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaac gtgagttttegttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgegtaatctgctgcttgc aaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga gcgcagataccaaataclgllcllclagtgtagccglagllaggccaccactlcaagaaclctgtagcaccgcctacatacclcgclclgc taatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcag cggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagct atgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacga gggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtca ggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctg cgttatcccctgattctgtggataaccgtattaccgcc Long FLAG KLHDC7B (SEQID NO: 41) atgcattagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggccc gcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgac gtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatga cggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctatta ccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtca atgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgt gtacggtgggaggtctatataagcagagctggtttagtgaaccgtcagatccgctagcgattacgccaagctcgaaattaaccctcact aaagggaacaaaagctggagctccaccgcggtggcggccgccaccatggattacaaggatgacgacgataaggactataaggacg atgatgacaaggactacaaagatgatgacgataaaggtggttctggtatgCTCCTGGGCACTCTGGAGGCTGGC GGGCCCCTCTGGGGCTGGGATGGTGATGACGGAGATGATGACTGGGAGGCGGTT GTACTCACCTTGCTGGCACTAGCGGTGGTAGCCACCTTAGCACTGGCTTTGCACT GGTTTGGCTCCAGGGAGGAGCAGGAGACAGCATCTGCAGCCCCAGGTGTGCAAC CTGCTCAGGCAGGAGGAGCCAGGCAGGCCCTGCACCCAAAGCCCAAGGACAGTG AGGGGCAGAGCAAGGGACAGCAGACACCAGAGGCTCCTGGAGATGTCCAGAGG AGGTCTGCTGCAGCTAGGACCCAGGACCCAGGGCCATCGATAGACAGAAGTATG GCTTCAGATGCCCGGCTGCTGCACAAGACAACTGGAGAGGTGACCACCAGAGGG GCCACCCTAGCAGCCCTGGGAAAGAAAAGAAAGGAGCCAGCCAAGCCAGAAAC TTTCTCCCCAGGACACCGCAAAGCAAAGGATATTCCTGTCCCCATCATGATCCAC TTTACCCCTCGAAGTCTCGACAGCAAAATAGAGATGCAGCTGGAGGCATCTGGA ACCTATGGTAAGGGGACAGCTGGGCTAGGCCCTATCCATACGGAGCAACAGGAC ACCACCAGCCCCTGGCACCTGCCTGTGGGGCCTCTTGGCCCTCTGGGGAGAGGCC TAGGCAGCTTGCGCCGGCGCCGGCGGCCTGGTGCCAGCTCAGGAGACAGACCCT GCCGTCCTCTAAAGCTGGATCCTCTCCGCCTGGGCACCGTGGTGAGTGTGTGGGA TGCTGTGGACATGGCCGGTTCCGCCGGCCAAGACAATCTCACCAGCAGTCTCTCT GCAGGCCCCTCGTTGCACCACGCAGACAAGGGGCTCAAGGCTCCTCAGGAGATG GGCCTAGCTGATGCCGTGGACAGCCGGCCTCAGGCAGACTCCTTCAAGGATGTG TGTGGGAGAAGCAGCCTCCGTCTCCCGCCCACCAGCACTGCAGGGTCAGGGGCT AAGGGTGCTGAGAGCAAGGAGACTGGATCCCCCTCCCCTAGGGAGACTCGGAGC ATCCTGGCTAGTAAGGAAGCCAGGTCCCGGGAGGGTGGGGCAGGGGTTGTCACC AGCAACGTCAGCCCCTCCCAAGGTGCCTTACCAGAAGAGCTGCACTCTGAGGCTT GTACAGTGGACCAAGTGGCTGGCCAGAGCAAGATGGAGAAACTAGGAGAGGGC CACCTCAATGGGAAAGGGAGAGGGTCGTGCCAAGGCAAGGAAAGTAAGGACGT AGCTGGCAGGCCAGCTGCAGAGCAGAGCTTTGCGCCTATCATGGATACAAGGAA GTCCACACACGGGCTCAGCAGCACCCCCTCTGACACCCTTCCAGCCTCTGCTCCC66 WO 2024/163650 PCT/US2024/013826 ACCCGGGACACTGAGCTGGTCCCTTCCTCCACCACAGCAGTGCATCCCAGCCCTG GGTTCCTTCACACAGTCTCTACCTCTGCTCCTTTAGACAATTTGCCCCTGGACGTT AGCCAGGGTCCTTCTGGTGGTGACAGTCTCAGGGTAGGGCCAGCCCCACTCCCA ACCCCAACTTTAACCTCAGCAGGAGCCTCAACTTCAGCTCCCACAGTGATCCTAG CATCTCTCCCAGTATCACCCCCAGCCTCAACTGCAGCCTCACCCTTAACCTCCCC AGCCCCAGGTACAGCAGCCCCAACCTCAACTTCAACAGCAACCCCATCTCTAAC ATTACCCCTCTGCCCAGCCCCGGTTACAGCCTTAACCACAACCTCGACACCAACC CCATCCCCAACTCCAGCCACAGCCTTAACCCTAATCTCATCAGCAAACCCGCCCC CAACATCACCCCCAGCTCTAGCCTCTGCCCCAGTTGCAGCCCAAGATCCTGTAGC AGACACTGAGCCAAGGCCTAAGAATACGGAATCTAATGTGGGCCTCTCCAAGAG CTCCCCAGAAAGACGAATCTCAAATAGCTGGGGCAATTTAATTACTATGGTTCTT AGAAGCCACCCCTTCCCCAGGCAAGAAAAGGCCCAAGGTAGTGCCCCAAGGGCA GATCCGGAGAGTCCTGCAGGGCCCAGCCCATCCACATACTCTGAAGATATACAA CCAGGGTCCTCCTCTGAAGGCGCCATCTTCAGCCTCCAGGACAAACACAGACCA GTCTTAGTCACAGCAGTAGGTTCAGAGAGCCTAGCTGAGGCTAGATGTGAAACA CAACCAGTGAGTTTAGAGACTAATGCATCTCTCACCCCGGACCCGAGAGGGGAT GAAACCAAGCAGAAGAGTCTAGACTTGCTGCCCCCGGCTGCAGAGCCTGTGGCT TCCCCGCAGGTCCCTGTTTCGGCACAGCCCGGTCCTGTACCGCCTACTTCTATGA GGCAGGATGCTCAGCCCACCCCTCAGCCTCGGCAACGGAGGAGGAAACGCAGCA TAGCTCAGAGCTCCGAACACATACTGATCCAGGAAGTACCCCAGCAGCCCCAGG GGCAGGTGCGAAAGGAGGGAGCCAGACCTACAGACTGCAGGGACATCCTCACA GAGAAGCAGAAAGAGACCCAAAAGCTTATGGCTTTTCTGCAGAAGCCTGGGAGT TGGGGAGTGGTGGAGGGGCCTCATAAGTACTGCTCACAGACCTCGCAGCCTAGC AGCCTAGTGGCGCCGTGGCCCCCGAAGCTAGATCTAGGGAGCTGCCTGGAGGTT TTGGCCTTCGCCCAGCAGCACGGAGAACTGGGCTTAGCCCAGGAGACCTATGTCT TGATGAGCAACAACTTACTGCATGTTCTGGGAGACCCACACCTCTACCGACAGCT GAGTGGAGCAGACAGGGAGCGCATCCTGAACCTGCGGACGTGCCAGGGCCAGA CAGTGCTGGGGGTCCTCGTGCTACCCAGCCTTTATCAGGTGAGCCGCTCGGGGCT CACGAGGGGCCCTCCCAGCGAGGAAGCTCCTGCAGCCAGGTCTGAGCACTTGCA TCTTCACACATACCTCCATGTATTCAACCCACAAGAGAATGTGTGGTGGCCCCTG ACTCAAGTACCAGAGGAGGTCCCCCTTCGGGGCTGTGGCCTCTGCACTATGCATA ACTATCTGTTTCTGGCAGGCGGCATCAGGGGTTCTGGTGCCAAGGCTGTCTGCTC CAACAAGGTGTTCTGCTACAACCCTCTGACCAATATTTGGAGCCAGATTCGGCCC ATGCAGCAAGCCCGGGCCCAGCTCAAGCTGGTGGCCCTGGATGGAATGCTTTAT GCCATTGGTGGCGAGTGCCTATACAGCATGGAGCGCTATGACCCACGCACAGAT ACCTGGACCTTGAGAGCATCCCTCCCTGAGGGCACCTTTCCCGTGGCCCACGAGG CTGTGGTCTGCCGAGGAGAGATCTACGTCACAGGGGGTCATCTCTTTTACCGCCT GCTCAGATACAGCCCCATGAAAGACTCATGGGATGAGTGTCCCTACAGCGCCAG CCACCGACGCTCCAGTGATATGGTGGCGCTAGGGGGCTTCCTATACCGCTTTGAC TTGTTGCGGGGTGTGGGTGCTGCGGTGATGCGCTACAACACAGTGACCGGCTCCT GGAGCCGGGCAGCCTCCCTGCCACTGTCTGACCCCGCCCCACTCCACTGCACGGT ATTGGGCAACACCATTTACTGTCTCAACCACCAGGTCACGGCTACCTTCACAGTT TCGGAGGGGACCGCCCTGTTTCAGGCCAAGGAGCTGCAGCCCTTCCCACTGGGA AGTAAGGGGGTCCTCTATCCGTTCACTCTGACTCTGCCCCCTAAGACCTGGTTGC AGACCACAATCTGAaagcttatcgataccgtcgacctcgagggggggcccggtaccttaattaattaaggtaccaggtaa gtgtacccaattcgccctatagtgagtcgtattacaattcactcgatcggctcgctgatcagcctcgactgtgccttctagttgccagccat ctgttgtttgeccctececegtgecttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcatt gtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgct ggggaacgcgtaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcg gcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtgg actccaacglcaaagggcgaaaaaccglclalcagggcgatggcccactacgtgaaccalcaccctaalcaagtllttlggggtcgag gtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaag gaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgc gcttaatgcgccgctacagggcgcgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaa67 WO 2024/163650 PCT/US2024/013826 atatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagaatcctgaggcggaaagaaccagctgt ggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaac caggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccct aactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgag gccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaagatcgatcaagagacaggat gaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggca caacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccg gtgccctgaatgaactgcaagacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttg tcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagt atccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgag cgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagaacatcaggggctcgcgccagccgaactgttc gccaggctcaaggcgagcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaat ggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctga agaacttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttctt gacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgc cgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagegeggggatctcatgctggagttctt cgcccaccctagggggaggctaactgaaacacggaaggagacaataccggaaggaacccgcgctatgacggcaataaaaagaca gaataaaacgcacggtgttgggtcgtttgttcataaacgcggggttcggtcccagggctggcactctgtcgataccccaccgagaccc cattggggccaatacgcccgcgtttcttccttttccccaccccaccccccaagttcgggtgaaggcccagggctcgcagccaacgtcg gggcggcaggccctgccatagcctcaggttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaaga tcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttctt gagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctacc aactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaag aactctgtagcaccgcctacatacctegctctgctaatectgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttgg actcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgac ctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaa gcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccac ctctgacttgagegtcgatttttgtgatgctcgtcaggggggeggagectatggaaaaacgccagcaacgeggcctttttacggttcctg gccttttgctggccttttgctcacatgttctttcctgegttatcccctgattctgtggataaccgtattaccgcc

[0225]Stably transfected Cell Lines [0226]Transfections were performed using Lipofectamine 3000 in accordance with the manufacturer instructions. Cell culture media was changed the day of transfection, and DNA was diluted in Opti-mem with P-3000. Lipofectamine was diluted in Opti-mem. Then, the DNA and lipofectamine solutions were mixed and incubated at room temperature for 10-15 minutes. This mixture was added to the cells and cultures were checked or collected 48-72 hours later. Cell lines were generated by Genscript using lentiviruses to stably express either the short or long isoforms of mouse KLHDC7b tagged with FLAG at the C-terminus. Cell lines were clonalized and validated using western blot against FLAG and immunohistochemistry against the FLAG tag.

WO 2024/163650 PCT/US2024/013826

[0227]Transient transfection of HEK cells with human KLHDC7B constructs for antibody testing [0228]HEK-293 cells were grown on a 96 well plate and transiently transfected using Lipofectamine 3000 according to the manufacturer ’s instructions with plasmids expressing FLAG tagged short KLHDC7B-3XFLAG, long KLHDC7B-3XFLAG, and H2B-GFP-3XFLAG (Table 5).After 48-72 hours, cells were rinsed with PBS, fixed with 4% PF A at room temperature for 20 minutes and rinsed with PBS 3x, then immunostained with an antibody against FLAG (Thermofisher, MAI-91878) at 1:200, and each of 10 reagent antibody clones at a concentration of 1:600, and GFP-transfected samples were stained with anti-GFP (abeam, abl3970) then imaged using an Opera Phenix automated confocal microscope.

Table 5:Vectors designed to transfect cell lines for expression of human Klhdc7b transcripts.Constructcode sequence GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA GTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTA AGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGC GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAA TCAATTACG G G GTCATTAGTTCATAG CCC ATATATG G AGTTCCG CGTTAC ATAACTTACG GTAAATG GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT AACG CCAATAG G G ACTTTCC ATTG ACGTCAATG G GTG G AGTATTTACG GTAAACTGCCCACTTG G C AGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGG CATTATG CCCAGTAC ATG ACCTTATGG G ACTTTCCTACTTG G CAGTACATCTACGTATTAGTCA AS-1303 Short-FLAG TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC G G G G ATTTCCAAGTCTCCACCCCATTG ACGTCAATG G G AGTTTGTTTTG G CACC AAAATC AACG G G ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGG AGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAA(SEQIDTACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGCCACCATGGTTCTTANO:42) GAAGCCACCCCTTCCCCAGGCAAGACAGGCCCCAAGGGAGTGTCCCGAGGGCGGTTCCCGGGAGCCCCGTGG GTCCCAG C ACTTCCACACACTCTG AGG ACAG ACACG G CCCCTCTTCTTC AGTG G G G ACAGTCATAGGGACAGGTACAGGGGGCCTGGTTGAGGCTGGAGGTCAGCCACAGCCAAGAAGCTCCG WO 2024/163650 PCT/US2024/013826 AGACCAACGGATCGCCCAGCCCAGACCCTCCCCCAGGCCTAAGAGGAGAGGGAACCAGGGAGAA AAGTCTAGACCCGCTGCCCCAAGCCGCGATGCCCAGGGGCCCCGCACAGCCCCCCGCGCAGAGGC CGCCTGGCCCCGCGGCCTCCTCCTCTGCGAGGCGCTCACAGCCGGTACCCCAGCTACGGAAACGCA GCAGGTGCGAAATCGCCCCGAGCTCGGAGCAGGAGGTCAGGCCGGCCGCCTCGGGGGACCCTCA AGGGGAGGCGCCGGGGGAGGGGGGCAGCCCTGCCGGCCGCAGCGGGGCGCTCACGGAAAAGCA GGAGGAGGCCCGGAAGCTCATGGTGTTTCTGCAGAGGCCCGGGGGTTGGGGGGTGGTGGAGGG GCCCCGGAAGCCCAGCTCCCGGGCCCTGGAGCCCGCCACGGCGGCAGCCCTGCGGCGGCGGCTG GACCTGGGCAGTTGCCTGGACGTGCTGGCCTTTGCCCAGCAGCACGGAGAGCCCGGCCTGGCGCA G G AG ACCTACG CG CTGATG AGCG ACAACCTG CTG CGAGTG CTG G G AG ACCCGTG CCTCTACCG CC GGCTGAGCGCGGCCGACCGCGAGCGCATCCTCAGCCTGCGGACCGGCCGGGGCCGGGCGGTGCT GGGCGTCCTCGTACTGCCCAGCCTCTACCAGGGGGGCCGCTCAGGGCTCCCCAGGGGCCCTCGTG GCGAGGAGCCTCCTGCGGCGGCCCCTGTGTCCCTGCCTCTACCTGCGCACCTGCATGTGTTCAACCC CCGGGAGAACACCTGGCGGCCCCTGACCCAGGTGCCCGAGGAGGCCCCGCTTCGGGGCTGCGGTC TCTGCACCATGCACAACTACCTGTTTCTGGCGGGGGGCATCCGTGGCTCCGGTGCCAAGGCCGTCT GCTCCAACGAGGTCTTCTGCTACAACCCTCTGACCAACATCTGGAGCCAGGTTCGGCCCATGCAGC AGGCCCGAG CCCAG CTCAAG CTG GTG G CCCTG G ACG G G CTGCTCTATG CCATCG GTGG CG AATG C CTGTACAGCATGGAGTGCTACGACCCGCGAACAGACGCCTGGACCCCACGCGCGCCACTCCCCGCA GGCACCTTCCCTGTGGCCCACGAGGCTGTGGCCTGCCGTGGGGACATCTACGTCACCGGGGGTCAC CTCTTCTACCGCCTGCTCAGGTACAGCCCCGTGAAGGATGCTTGGGACGAGTGCCCATACAGTGCC AGCCACCGGCGTTCCAGCGACATCGTGGCACTGGGGGGCTTCCTGTACCGCTTCGACCTGCTGCGG GGCGTGGGCGCCGCCGTGATGCGCTACAACACAGTGACCGGCTCCTGGAGCAGGGCTGCCTCCCT GCCCCTGCCCGCCCCCGCCCCACTGCACTGCACCACCCTGGGCAACACCATTTACTGCCTCAACCCC CAGGTCACTGCCACCTTCACGGTCTCTGGGGGGACTGCCCAGTTCCAGGCCAAGGAGCTGCAGCCC TTCCCCTTGGGGAGCACCGGGGTCCTCAGTCCATTCATCCTGACTCTGCCCCCTGAGGACCGGCTGC AGACCTCACTCGGTGGTGGTTCTGGTGATTACAAGGATGACGACGATAAGGACTATAAGGACGAT GATGACAAGGACTACAAAGATGATGACGATAAATGACTCGAGTCTAGAGGGCCCGTTTAAACCCG CTG ATCAG CCTCG ACTGTG CCTTCTAGTTG CC AG CCATCTGTTGTTTG CCCCTCCCCCGTG CCTTCCT TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCT GAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAA GACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTG GGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTA CG CG CAG CGTG ACCG CTACACTTG CCAG CG CCCTAG CG CCCG CTCCTTTCG CTTTCTTCCCTTCCTTT CTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTA GTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGC70 WO 2024/163650 PCT/US2024/013826 CCTGATAGACGG Hill CGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAA ACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGC CTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTC AGTTAGG GTGTGG AAAGTCCCCAG GCTCCCCAG CAGG CAG AAGTATGCAAAG CATG CATCTCAATT AGTCAG CAACCAG GTGTGG AAAGTCCCCAG G CTCCCCAG CAG G CAG AAGTATG CAAAGCATG CAT CTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTC CGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCT CTGAGCTATTCCAGAAGTAGTGAGGAGGCHI IIIGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGG GAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAAC AAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCA CAACAG ACAATCG G CTG CTCTG ATG CCG CCGTGTTCCG G CTGTCAG CG C AG G G G CG CCCG GTTCTT TTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTG GCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACT GGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAG TATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCA CCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATG ATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATG CCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAAT GGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCG TTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTAC GGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGG GACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCAC CGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCA GCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTAC AAATAAAG CAATAG CATCACAAATTTCACAAATAAAG CAI 1 1 1 1 1 ICACTG CATTCTAGTTGTG GTTT GTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAAT CATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGG AAG CATAAAGTGTAAAG CCTG G G GTG CCTAATG AGTG AG CTAACTCACATTAATTG CGTTG CG CTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGA TAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG TTGCTGGCGI 11 1 1CCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCT71 WO 2024/163650 PCT/US2024/013826 CTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGC ACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAG GCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTA TCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAA CCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTC AAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGA TTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAA TC AATCTAAAGTATATATG AGTAAACTTG GTCTG ACAGTTACCAATG CTTAATCAGTG AGG C ACCTA TCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATA CGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGC GCAACGTTGTTG CCATTG CTACAG G CATCGTG GTGTCACG CTCGTCGTTTG GTATG G CTTCATTCAG CTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTC CTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA GTCATTCTG AG AATAGTGTATG CG G CG ACCG AGTTG CTCTTG CCCG G CGTCAATACG G G ATAATAC CG CG CC ACATAG CAG AACTTTAAAAGTG CTCATCATTG G AAAACGTTCTTCG G G G CG AAAACTCTC AAG G ATCTTACCG CTGTTG AG ATCCAGTTCG ATGTAACCCACTCGTG CACCCAACTG ATCTTCAG CA TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGA ATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCI1 1 1 1CAATATTATTGAAGCATTTATCA GGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC GCG CACATTTCCCCG AAAAGTG CCACCTG ACGTC AS-1304 GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATA long-FLAG GTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGC(SEQIDGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAANO:43)TCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACG CCAATAG G G ACTTTCC ATTG ACGTCAATG G GTG G AGTATTTACG GTAAACTGCCCACTTG G CAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC WO 2024/163650 PCT/US2024/013826 CTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC GGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGG ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGG AGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAA TACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGCCACCATGATCCAG GGCACCTTGGAGCCAGATGGTCCCCTCTGGGGCTGGGACTGGGACAGTGACAATGACTGGGATAG TG CTGTG CTG G CCCTCCTG G CG CTGG CTGTG GTG G CTG CC ACAG CG CTG G CCTTACACTG GTTTG G CTCCG G G CACG ATCAAG AG G CG G CAG AACCG GTGTCCACAGCCCTCG G G G CTCAACCTCATCAG G CAGGAGGAGCTGAGCTGGCCCTGCAACCGAAGTCTAAGGTCAGTGATGGCAGCGAGGGGCAGAG CCCAGGGCAGGGGAAACCAGAGCCCCCAGGACGCGGCCAGCAGAGCCCTGTCCCTGCTGCAGCGC CGGGCGGGGGCCTGGCCGCCATGGCCCGGCTTCCACTCAAGACGGCTGTCGAGGAGGCCCGCAG AGAGGCATTAGGACAGCAACGGGGCAGTGCCACCCCCGCGGCCCCCCGAGCGGAAGGAAAGGAG CCTCCCAGGCCAGGCACTGCCCTCCTGGGCAGGAGCGAAGCAGGGGGGATGTCCGCCCCCCTCCT GATCCACTTCACTCCTCGGAGCCCTGGCAGCGAAGCGGAGGCGGAGACAGGTGGTGTCAGGGCGT CCTCTCGCCAGGCCGCAGGCCCCGCGGGGCAACAGGACACTGGCCCCTGGCAGGCGGGCGCGGG GCCCTCGGGCTCGATGGGGAGAGGCCGGGGCCGGCGGCGGCGGATGGACGCTGGCTCGGGAGA CAGAGCCCGCCGCCCCCGGAAACTGGACCCGCTCCGCCTGGGCGCCGCGGGGAGCGTGTGGGAC GCGGTGGACGGGGCCGCCGCCCTGGACGCCCACGCGCGCGGCCTCCCCACAGGACCCCCACTCGC CCAGGAGCCCGCACTCCCGGCGCTGCCCGCTCCCCGCGCCCTGCAGCCTGGGTCTCAGACGGAAG GCTCTGGGGCCAAGGGTGGCTGGAGCAGGGAGGCCTCGGGGGTCCCTGCCCCCGGAGGAGGCTG GCCCTGGGTCAGCAGGGAGGTCCCGGGCACCCGGAGCTTTGGCCCAGCCCCAGACTCCACGCGCC CCTGGCTAGAGAGTCCGCCTCAAGGTCGCCCACTCTCGTCCCAAGGGCCGGGTGCCACAGGGGCCT ACGATGCCGGCGAGGCCGGGGCTGACAGCTCCCGAGATAACAGTCCTGCCGCTGACCTGGGGCCC ACCCGGCCCCCGGAGCAAGCAAAGCCGGCTGCAGCCGGCCACAGCCGCGCGCCCTCCCGGAGCCG TGAGCCTCGCCCGCGCTCCGCCTCCCCGCCCGCAGCTCCCGGCCCGGGGTTCCCACCTGAAGCCCTGACTCTCCCCTCTCCTTCAGACI 1 1 1 1GCCCCTGGAGGTTACCCAGGATCCTTCCGTGGGCGAAAATC TCAGAGCGGCGCCAGCCCCAAGTTCAGCCTCAGCCCAAGTCTTAACTTCAGCTCCAGCCTCAGTCCT AGCCCCAGCCCTGGCTTCATCCCCCAGCTCAGCACCAACCTCAGCCACCACCTCAACCTCATCCCCCA CCTCAGCCCCAGCCCCAGCTCCAACCTCAGCTCCAACTTCAACCCCAGCCCCAGCCCCAAGTCCAGC TGCAGCCGCAACTCCAGCCCCAGCCCCAGTCCCAGTCCCAACCCTCACACCCCCATCCCCAGCCCTA ACCCCAGTCCCAACCCCAGCCCTAAGCCCAGCTCCAACTCCAGCCCTAACCCCAGCCGCATCCCCAG CCCTAACCCCAGTCCCAACCCCAGCCCTAAGCCCAGCTCCAACTCCAGCCCCAACCCCAGCCGCATC CCCTGCCCCAGCCCCCACCTCAGCCCCAACCCCAACCCCAGCCGCATCCCCTGCCCCAGCTGACGGG73 WO 2024/163650 PCT/US2024/013826 TCAAAGCCTCAGGAGAGTGTGGCTCTCCCCAGGCGCTACCAGGAGGGGCAGGTCTCAGCCAGCTG GGGAAACCTTATTGCCATGGTTCTTAGAAGCCACCCCTTCCCCAGGCAAGACAGGCCCCAAGGGAG TGTCCCGAGGGCGGTTCCCGGGAGCCCCGTGGGTCCCAGCACTTCCACACACTCTGAGGACAGACA CGGCCCCTCTTCTTCAGTGGGGACAGTCATAGGGACAGGTACAGGGGGCCTGGTTGAGGCTGGAGGTCAGCCACAGCCAAGAAGCTCCGAGACCAACGGATCGCCCAGCCCAGACCCTCCCCCAGGCCTAA GAGGAGAGGGAACCAGGGAGAAAAGTCTAGACCCGCTGCCCCAAGCCGCGATGCCCAGGGGCCC CGCACAGCCCCCCGCGCAGAGGCCGCCTGGCCCCGCGGCCTCCTCCTCTGCGAGGCGCTCACAGCCGGTACCCCAGCTACGGAAACGCAGCAGGTGCGAAATCGCCCCGAGCTCGGAGCAGGAGGTCAGG CCGGCCGCCTCGGGGGACCCTCAAGGGGAGGCGCCGGGGGAGGGGGGCAGCCCTGCCGGCCGC AGCGGGGCGCTCACGGAAAAGCAGGAGGAGGCCCGGAAGCTCATGGTGTTTCTGCAGAGGCCCG GGGGTTGGGGGGTGGTGGAGGGGCCCCGGAAGCCCAGCTCCCGGGCCCTGGAGCCCGCCACGGC GGCAGCCCTGCGGCGGCGGCTGGACCTGGGCAGTTGCCTGGACGTGCTGGCCTTTGCCCAGCAGCACGGAGAGCCCGGCCTGGCGCAGGAGACCTACGCGCTGATGAGCGACAACCTGCTGCGAGTGCTG GGAGACCCGTGCCTCTACCGCCGGCTGAGCGCGGCCGACCGCGAGCGCATCCTCAGCCTGCGGAC CGGCCGGGGCCGGGCGGTGCTGGGCGTCCTCGTACTGCCCAGCCTCTACCAGGGGGGCCGCTCAG GGCTCCCCAGGGGCCCTCGTGGCGAGGAGCCTCCTGCGGCGGCCCCTGTGTCCCTGCCTCTACCTG CGCACCTGCATGTGTTCAACCCCCGGGAGAACACCTGGCGGCCCCTGACCCAGGTGCCCGAGGAG GCCCCGCTTCGGGGCTGCGGTCTCTGCACCATGCACAACTACCTGTTTCTGGCGGGGGGCATCCGT GGCTCCGGTGCCAAGGCCGTCTGCTCCAACGAGGTCTTCTGCTACAACCCTCTGACCAACATCTGGA GCCAGGTTCGGCCCATGCAGCAGGCCCGAGCCCAGCTCAAGCTGGTGGCCCTGGACGGGCTGCTC TATGCCATCGGTGGCGAATGCCTGTACAGCATGGAGTGCTACGACCCGCGAACAGACGCCTGGAC CCCACGCGCGCCACTCCCCGCAGGCACCTTCCCTGTGGCCCACGAGGCTGTGGCCTGCCGTGGGGACATCTACGTCACCGGGGGTCACCTCTTCTACCGCCTGCTCAGGTACAGCCCCGTGAAGGATGCTTG GGACGAGTGCCCATACAGTGCCAGCCACCGGCGTTCCAGCGACATCGTGGCACTGGGGGGCTTCC TGTACCGCTTCGACCTGCTGCGGGGCGTGGGCGCCGCCGTGATGCGCTACAACACAGTGACCGGCTCCTGGAGCAGGGCTGCCTCCCTGCCCCTGCCCGCCCCCGCCCCACTGCACTGCACCACCCTGGGCA ACACCATTTACTG CCTCAACCCCCAG GTC ACTG CCACCTTCACG GTCTCTG G G G G G ACTG CCC AGTT CCAG G CC AAG G AG CTG C AG CCCTTCCCCTTGG G G AG C ACCG G G GTCCTCAGTCCATTC ATCCTG AC TCTGCCCCCTGAGGACCGGCTGCAGACCTCACTCGGTGGTGGTTCTGGTGATTACAAGGATGACGA CGATAAGGACTATAAGGACGATGATGACAAGGACTACAAAGATGATGACGATAAATGACTCGAGT CTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGT TTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATG AGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGAC AGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTT74 WO 2024/163650 PCT/US2024/013826 CTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTA AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGC TCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG G CTCCCTTTAG G GTTCCGATTTAGTG CTTTACG G CACCTCG ACCCCAAAAAACTTG ATTAG G GTG AT GGTTCACGTAGTGGGCCATCGCCCTGATAGACGG 1 1 1 1 1CGCCCTTTGACGTTGGAGTCCACGTTCT TTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTA TAAG G G ATTTTG CCG ATTTCG G CCTATTG GTTAAAA AATG AG CTG ATTTAACAAAAATTTAACG CG A ATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAG TATG CAAAGCATG CATCTCAATTAGTCAG C AACCAG GTGTG G AAAGTCCCCAG G CTCCCCAG C AG G CAG AAGTATG CAAAGCATG CATCTCAATTAGTCAG CAACCATAGTCCCG CCCCTAACTCCG CCCATC CCG CCCCTAACTCCG CCCAGTTCCG CCCATTCTCCG CCCCATG G CTG ACTAA1 1111111ATTTATG CA GAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTA GGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGA GGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAG GCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTC AGCGCAGGGGCGCCCGGTTCI 1 1 1 IGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGA CGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTG TCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCT CACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGAT CCG GCTACCTG CCCATTCG ACCACCAAGCG AAACATCG CATCG AG CG AG CACGTACTCGG ATG G AA GCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTT CG CCAG G CTCAAG G CG CGCATG CCCG ACG G CG AG G ATCTCGTCGTG ACCCATG G CG ATG CCTG CT TGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGG CGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGG GCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCT TCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTG CCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGG ACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTT TATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCAI II II II CACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACC TCTAG CT AG AG CTTG G CGTAATCATG GTCATAG CTGTTTCCTGTGTG AAATTGTTATCCG CTCACAA TTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAA CTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATT AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCA75 WO 2024/163650 PCT/US2024/013826 CTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAC GGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGI1 11 1CCATAGGCTCCGCCCCCCTGACGAGCATCA CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTC CCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGG G AAG CGTG G CG CTTTCTCATAG CTC ACG CTGTAGGTATCTC AGTTCG GTGTAG GTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG AAG AACAGTATTTG GTATCTG CG CTCTG CTG AAG CCAGTTACCTTCG GAAAAAGAGTTGU 1 AGU 1 C1 1 bA 1LLGGCAAALAAACCALCGL 111 כ 1 נ AGCGG 111 1 1 )נ 1 1 1 1 1 1 1 כ 1 נ GLAAGCAGCAGA TTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGT GGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCC TTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTG AG G CACCTATCTCAG CG ATCTGTCTATTTCGTTCATCCATAGTTG CCTG AC TCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATAC CGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAG CGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAG TAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACG CTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCC ATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCA GTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTT TTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCT TGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTG CACCCA ACTG ATCTTCAG CATCTTTTACTTTCACCAG CGTTTCTG G GTG AG CAAAAACAG G AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCMill CAA1 A1 1 A1 1 GAAGLA 1 1 1 A 1 CAbbb 1 1 A1 1 b 1 C 1 LA 1 GAGLGGA 1 ACAI Alli bAA 1 b 1 A1 1TAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC AS-1305 GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAA WO 2024/163650 PCT/US2024/013826 H2B-GFP-TCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGFLAG (SEQ.GCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGT ID NO: 44)AACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGC AGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGC CTGG CATTATG CCCAGTAC ATG ACCTTATGG G ACTTTCCTACTTG G CAGTACATCTACGTATTAGTCA TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCAC G G G G ATTTCC AAGTCTCCACCCCATTG ACGTC AATG G G AGTTTGTTTTG G CACCAAAATCAACG G G ACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGG AGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAA TACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTGCCACCATGGGACCA GAGCCAGCGAAGTCTGCTCCCGCCCCGAAAAAGGGCTCCAAGAAGGCGGTGACTAAGGCGCAGA AGAAAGGCGGCAAGAAGCGCAAGCGCAGCCGCAAGGAGAGCTATTCCATCTATGTGTACAAGGTT CTGAAGCAGGTCCACCCTGACACCGGCATTTCGTCCAAGGCCATGGGCATCATGAACTCGTTTGTG AACGACATTTTCGAGCGCATCGCAGGTGAGGCTTCCCGCCTGGCGCATTACAACAAGCGCTCGACC ATCACCTCCAGGGAGATCCAGACGGCCGTGCGCCTGCTGCTGCCTGGGGAGTTGGCCAAGCACGC CGTGTCCGAGGGTACTAAGGCCATCACCAAGTACACCAGCGCTAAGGATCCACCGGTCGCCACCAT GGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGAC GTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACC TACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC ATGCCCGAAGGCTACGTCCAGGAGCGCACCATC1 1C1 1CAAGGACGACGGCAACTACAAGACCCGC GCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCA TGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCC GACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGGGTGG TGGTTCTGGTGATTACAAGGATGACGACGATAAGGACTATAAGGACGATGATGACAAGGACTACA AAGATGATGACGATAAATGACTCGAGTCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTG TGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT TCTG G GG G GTG G G GTG G G G C AG G AC AG CAAG G G G G AG G ATTG G G AAG AC AATAG CAG G C ATG C TGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATC CCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCT77 WO 2024/163650 PCT/US2024/013826 ACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGG CTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTC GACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGG Hill CGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA ACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAAT GAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAA AGTCCCC AG G CTCCCC AG CAG G CAG AAGTATG CAAAG CATG CATCTCAATTAGTCAG CAACCAG GT GTG G AAAGTCCCCAG G CTCCCCAG CAG G CAG AAGTATG CAAAG CATG CATCTCAATTAGTCAG CAA CCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCC CATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGA AGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCA TTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACG CAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGC TGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCI 1111GTCAAGACCGAC CTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGG CGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGA AGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGAT GCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGC ATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCA TCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATC TCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATT CATCGACTGTG G CCG G CTG G GTGTG G CG G ACCG CTATCAG G AC ATAG CGTTG G CTACCCGTG ATAT TGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGA TTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAA TGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAG GTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCT GGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATC ACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGT ATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTC CTGTGTG AAATTGTTATCCG CTC ACAATTCCACAC AACATACG AG CCG G AAG CATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTC GGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGT ATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA78 WO 2024/163650 PCT/US2024/013826 TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGI 1 1 1 ICCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGAC AGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTG CCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATC GCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGT TCTTGAAGTG GTG G CCTAACTACG G CTACACTAG AAG AACAGTATTTG GTATCTG CG CTCTG CTG A AG CCAGTTACCTTCG G AAAAAG AGTTG GTAG CTCTTG ATCCG G CAAAC AAACCACCG CTG GTAG CG GTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT ATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATAT ATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCT ATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCA TCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATA AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCC ATTG CTACAG G CATCGTG GTGTCACG CTCGTCGTTTG GTATG G CTTCATTCAG CTCCG GTTCCCAAC GATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT CGTTGTCAG AAGTAAGTTG G CCG CAGTGTTATCACTCATG GTTATG G CAG CACTG CATAATTCTCTT ACTGTCATGCCATCCGTAAGATGCI 1 1 1CTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCT GTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCA GCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACG GAAATGTTGAATACTCATACTCTTCC1 1 1 1 ICAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCG AAAAGTG CCACCTG ACGTC Example 8 - Discussion [0229]KLHDC7b is expressed in cochlea and other organs. Within the cochlea, KLHDC7b is expressed exclusively in hair cells as measured by RNA scope and immunofluorescence with a WO 2024/163650 PCT/US2024/013826 custom-generated antibody. KLHDC7 is also expressed in human otic organoids, with both the long and overlapping portions of the transcript being detectable by RNA scope. [0230]KLHDC7b does not appear to be required for the development of the cochlea or hair cells of the inner ear, or for assembly of the mechanotranduction complex, but appears to be necessary for the maintenance of hearing. Hair cells are missing at time points where the mice are profoundly deaf as indicated by immunohistochemistry and scanning electron microscopy. [0231]KLHDC7b appears to be localized to the plasma membrane of hair cells as indicated by immunostaining with a custom anti-KLHDC7b antibody. KLHDC7b also appears mislocalized to the cytoplasm in some hair cells at time points when the hair cells begin to degenerate.

Claims (78)

WO 2024/163650 PCT/US2024/013826 CLAIMS What is claimed is:

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

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

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

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

7. The genetically modified non-human animal nucleic acid of claim 6, wherein the promoter is an endogenous Klhdc7b promoter, wherein the endogenous Klhdc7b promoter drives expression of the reporter gene. WO 2024/163650 PCT/US2024/013826

8. The genetically modified non-human animal nucleic acid of any one of claims 5-7, wherein the reporter gene encodes a reporter that is 3-galactosidase, Green Fluorescent Protein (GFP), enhanced Green Fluorescent Protein (eGFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, enhanced yellow fluorescent protein (EYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

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

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

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

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

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

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

15. The genetically modified non-human animal nucleic acid of claim 14, wherein the WO 2024/163650 PCT/US2024/013826 modified endogenous Klhdc7b locus comprises:a nucleic acid sequence set forth as SEQ ID NO:5 and/ora nucleic acid sequence set forth as SEQ ID NO:6 or a nucleic acid sequence set forth as SEQ ID NO :7, and/ora nucleic acid sequence set forth as SEQ ID NO:38 or a nucleic acid sequence set forth as SEQIDNO:39.

16. The genetically modified non-human animal nucleic acid of any one of claims 1-15, wherein the modified endogenous Klhdc7b locus comprises an endogenous 5’ Klhdc7b untranslated region and/or an endogenous 3’ Klhdc7b untranslated region, andwherein the endogenous 5’ Klhdc7b untranslated region is upstream of the deletion of the endogenous X7A،/c7/> gene, or portion thereof, andwherein the endogenous 3’ Klhdc7b untranslated region is downstream of the deletion of the endogenous Klhdc7b gene, or portion thereof.

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

18. A non-human animal genome comprising the non-human animal nucleic acid of any one of claims 1-17, wherein the modified endogenous Kelch domain containing 7B (Klhdc7b) locus of the non-human animal nucleic acid replaces &Klhdc7b locus of the non-human animal genome. WO 2024/163650 PCT/US2024/013826

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

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

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

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

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

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

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

26. The genetically modified non-human animal cell of claim 25 wherein the promoter is an endogenous promoter,wherein the endogenous Klhdc7b promoter drives expression of the reporter gene. WO 2024/163650 PCT/US2024/013826

27. The genetically modified non-human animal cell of any one of claims 24-26, wherein the reporter gene encodes a reporter that is -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.

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

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

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

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

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

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

34. The genetically modified non-human animal cell of claim 33, wherein the mouse cell is a B6.Cast-Cdh23Ahl+ mouse cell. WO 2024/163650 PCT/US2024/013826

35. The genetically modified non-human animal cell of claim 33 or claim 34, wherein the endogenous A7/z6/c7׳A locus comprises;a nucleic acid sequence set forth as SEQ ID NO:5 and/ora nucleic acid sequence set forth as SEQ ID NO:6 or a nucleic acid sequence set forth as SEQ ID NO :7, and/ora nucleic acid sequence set forth as SEQ ID NO:38 or a nucleic acid sequence set forth as SEQIDNO;39.

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

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

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

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

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

41. A genetically modified non-human animal comprising a modified endogenous Kelch domain containing 7B (Klhdc7b) locus,wherein the modified endogenous Klhdc7b locus comprises a deletion of an endogenous Klhdc7b gene, or a portion thereof. WO 2024/163650 PCT/US2024/013826

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

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

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

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

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

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

48. The genetically modified non-human animal of any one of claims 45-47, wherein the reporter gene encodes a reporter that is -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. WO 2024/163650 PCT/US2024/013826

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

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

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

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

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

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

55. The genetically modified non-human animal of claim 54, wherein the mouse is a B6.Cast-Cdh23Ahl+ mouse.

56. The genetically modified non-human animal of claim 54 or claim 55, wherein the endogenous A7/7،7c7/1׳ locus comprises:a nucleic acid sequence set forth as SEQ ID NO:5 and/ora nucleic acid sequence set forth as SEQ ID NO:6 or a nucleic acid sequence set forth as SEQ ID NO :7, and/ora nucleic acid sequence set forth as SEQ ID NO:38 or a nucleic acid sequence set forth as SEQIDNO:39. WO 2024/163650 PCT/US2024/013826

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

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

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 after birth.

60. The genetically modified non-human animal of any one of claims 57-59, wherein:the genetically modified non-human animal exhibits normal cochlea development compared to a wildtype control non-human animal;the genetically modified non-human animal exhibits hearing loss about 17 days after birth;the genetically modified non-human animal exhibits hearing loss at 17 days after birth and hearing loss increases throughout the life of the genetically modified non-human animal;the genetically modified non-human animal exhibits profound deafness at about 8 weeks 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 wildtype control non-human animal, optionally wherein hair cells appear normal at birth, further optionally wherein degeneration of hair cells is seen after 11 days postnatally; and/orthe genetically modified non-human animal exhibits normal mechanotransduction in the cochlea as measured by a Gentamycin-Texas Red Assay.

61. A method of making a Klhdc7b knockout non-human animal pluripotent cell comprising deleting an endogenous Klhdc7b gene, or a portion thereof, to form a modified endogenous Klhdc7b locus. WO 2024/163650 PCT/US2024/013826

62. The method of claim 61, wherein deleting the endogenous K111dc7b gene, or portion thereof, comprises deleting an orf of the endogenous Klhdc7b gene.

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

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

65. The method of claim 64, wherein the insert nucleic acid comprises:a reporter gene;a reporter gene operably linked to a promoter, wherein the promoter drives expression of the reporter gene;a reporter gene operably linked to an endogenous Klhdc7b promoter, wherein the endogenous Klhdc7b promoter drives expression of the reporter gene;a reporter gene, wherein the reporter gene encodes a reporter selected from the group consisting of oP־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/ora gene encoding a selectable marker flanked by site-specific recombination sequences.

66. The method of any one of claims 61-65, wherein deleting comprises contacting the non- human animal pluripotent cell with a targeting vector comprising the genetically modified non­ WO 2024/163650 PCT/US2024/013826 human animal nucleic acid of any one of claims 1-17,wherein the targeting vector comprises a 5’ homology arm upstream of the genetically modified non-human animal nucleic acid and a 3’ homology arm downstream of the genetically modified non-human animal nucleic acid, andwherein the 5’ homology arm and the 3’ homology arm target an endogenous Klhdc7b locus of the non-human animal pluripotent cell.

67. The method of any one of claims 61-66, wherein the non-human animal pluripotent cell is a rodent pluripotent cell.

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

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

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

71. A method of making a Klhdc7b knockout non-human animal comprising gestating the non-human animal ES cell of claim 70 in a surrogate mother,wherein the surrogate mother produces a progeny non-human animal comprising the modified endogenous Klhdc7b locus in its germline genome.

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

73. The method of claim 71 or 72, wherein the non-human animal, surrogate mother, and progeny are each a rodent. WO 2024/163650 PCT/US2024/013826

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

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

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

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

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