Novel treatments of ciliopathies

Abstract

This disclosure relates to compositions and methods for treating ciliophathies.

Description

MBHB Ref. No.23-1150-WO NOVEL TREATMENTS OF CILIOPATHIES STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under grant numbers NIH / NEI K08-EY022058, R01-EY025295, R01-EY-023295, R01-EY024932. The government has certain rights in the invention. BACKGROUND OF THE DISCLOSURE Field of Invention

[0002] This disclosure relates to compositions and methods for treating ciliopathies. SEQUENCE LISTING

[0003] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on 31 August 2023, having the file name “23-1150-US-PRO.xml” and is 16.5kb in size. Background

[0004] Mitochondria-related neurodegenerative diseases have been postulated to involve primary cilia function. The primary cilium is a solitary, immotile microtubule-based structure present on apical surface of many cell types, especially in sensory organs such as the eye and nose. Non-motile sensory cilia consist of an array of bundled microtubules (axoneme) that are covered by a plasma membrane. Axonemal microtubules arise from a specialized microtubule-nucleating center which is the basal body. Defects in cilium function have been associated with numerous human diseases including developmental delay, anosmia, renal cysts, and photoreceptor degeneration. A rare form of syndromic ciliopathy, Joubert Syndrome (JBS) is caused by mutations in genes that form the transition zone; one of the JBS genes ARMC9 is located at centriole basal body and interacts with CCDC66 and TOGARAM1 to stabilize cilia membrane. However, the precise mechanisms that link genetic mutations in Joubert to systemic diseases are unknown.

[0005] Prior electron microscopy studies of primary cilia show mitochondria to frequently localize near the bases of cilia in motile ciliated cells as well as sensory photoreceptors. Human fibroblasts also showed similar patterns of mitochondria distributionMBHB Ref. No.23-1150-WO at ciliary base based on cryo-electron tomography. It has been observed that microtubules project from the mother centriole at the cilium base toward mitochondria, suggesting a communication between the two organelles. However, how mitochondria involved in primary cilia formation are still unclear.

[0006] Thus there remains a need in the art for compositions and methods to address cilia- related diseases particularly ciliopathies. SUMMARY OF THE DISCLOSURE

[0007] This disclosure describes compositions and methods for treating ciliopathies.

[0008] In a first aspect, this disclosure provides a method for restoring cilia function in a cell that expresses a defective variant of a first gene needed for normal cilia function by expressing or overexpressing a second gene in the cell that when expressed therein restores cilia function in the cell, wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0009] In one embodiment of the first aspect disclosed herein, the second gene is NDUFAF2 (SEQ ID NOS.11 and 12).

[0010] In a second aspect, this disclosure provides a method for restoring cilia function in a cell that under-expresses or does not express a first gene needed for normal cilia function, comprising introducing into the cell a second gene that when expressed in the cell restores cilia function, wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0011] In one embodiment of the second aspect disclosed herein, the second gene is NDUFAF2 (SEQ ID NOS.11 and 12).

[0012] In a third aspect, this disclosure provides a method of treating or ameliorating ciliopathies in a subject that expresses a defective variant of a first gene needed for normal cilia function, the method comprising the steps of introducing into cells in the subject having the defective variant of the first gene a recombinant expression construct, wherein the construct comprises a second gene that when expressed is capable of correcting the ciliopathy in the subject, wherein the construct is delivered into an epithelial cell in the subject, and wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0013] In one embodiment of the third aspect disclosed herein, the recombinant expression construct comprises an adeno-associated virus (AAV).MBHB Ref. No.23-1150-WO

[0014] In one embodiment of the third aspect disclosed herein, the ciliopathy causes a kidney defect.

[0015] In one embodiment of the third aspec disclosed herein, the epithelial cell is a renal tubular epithelial cell.

[0016] In one embodiment of the third aspect disclosed herein, the AAV construct is introduced into the subject through the subject’s renal collecting duct.

[0017] In one embodiment of the third aspect disclosed herein, the treating results in reduction of ciliary defect symptoms.

[0018] In one embodiment of the third aspect disclosed herein, the second gene is NDUFAF2 (SEQ ID NOS.11 and 12).

[0019] In one embodiment of the third aspect disclosed herein, the steps further comprise administration of a therapeutically effective amount of a pharmaceutical formulation of a nicotinamide compound.

[0020] In a fourth aspect, this disclosure provides a method of treating a subject with a ciliopathy comprising: (a) obtaining a biological sample from the subject, (b) detecting DNA from the sample encoding a gene comprising at least one mutation or variant ssociated with cilia dysfunction, and (c) administering to the subject with the DNA mutation or variant associated with cilia dysfunction a nicotinamide pharmaceutical composition in an amount effective to maintain or improve cilia function.

[0021] In one embodiment of the fourth aspect disclosed herein, the the nicotinamide pharmaceutical composition comprises an active pharmaceutical ingredient that is NAD+.

[0022] In one embodiment of the fourth aspect disclosed herein, the composition further comprises a pharmaceutically acceptable carrier, a solvent, an adjuvant, a diluent, or a combination thereof.

[0023] In one embodiment of the fourth aspect disclosed herein, the composition is delivered systematically.

[0024] In one embodiment of the fourth aspect disclosed herein, the composition is administered by oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracranial, transdermal, intrathecal, intracranial, intravenous, or intracerebral administration.MBHB Ref. No.23-1150-WO

[0025] In one embodiment of the fourth aspect disclosed herein, the composition is administered orally.

[0026] In one embodiment of the fourth aspect disclosed herein, the subject has a ciliopathy selected from the group consisting of Joubert syndrome, developmental delay, anosmia, renal cysts, and photoreceptor degeneration.

[0027] In one embodiment of the fourth aspect disclosed herein, the ciliopathy is Joubert syndrome.

[0028] In a fifth aspect, this disclosure provides a pharmaceutical composition comprising a recombinant expression construct encoding a gene for treating a cilia related disorder, wherein the gene is ARMC9 and / or NDUFAF2.

[0029] In one embodiment of the fifth aspect disclosed herein, the recombinant expression construct comprises an adeno-associated virus (AAV).

[0030] In one embodiment of the fifth aspect disclosed herein, the composition further comprises an NAD+ supplement.

[0031] In one embodiment of the fifth aspect disclosed herein, the NAD+ supplement is administered to a subject at a therapeutically effective dose.

[0032] In one embodiment of the fifth aspect disclosed herein, the NAD+ supplement, is administered as a tablet or liquid formulation.

[0033] These and other features and advantages of this disclosure will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0035] Figures 1A-1J show loss of NDUFAF2 in retinal pigment epithelium (RPE) cells affected cilia formation. Fig.1A shows that NDUFAF2WT and NDUFAF2G258A wereMBHB Ref. No.23-1150-WO stably expressed in NDUFAF2- / - cells. Western blot analysis was performed with antibodies against NDUFAF2 and GAPDH and the results shown. Fig.1B shows cells that were stained with TOM20 (red) and NDUFAF2 (green) antibodies. DNA was stained with DAPI (blue). Scale bars as indicated. Fig.1C shows a positive signal of NDUFAF2 at mitochondria in the wild-type, NDUFAF2- / -, and NDUFAF2WT and NDUFAF2G258A -re-expressing RPE1 cells. Fig.1D shows cells that were serum starved for 2 days and the percentage of ciliated cells was quantified. More than 150 cells were analyzed for each independent experiment. Fig.1E shows cells that were serum starved for 2 days and analyzed by immunofluorescence with indicated antibodies. DNA (DAPI, blue). Scale bars as indicated. Fig.1F is a graph showing the percentage of cells with two CP110 dots at the centrioles in serum-starved cells. More than 150 cells were analyzed for each independent experiment. Fig.1G shows serum starvation of cells for 6 hours and measurement of ciliary vesicle and pre-ciliary vesicles. The percentage of cells with ciliary vesicles was measured with the antibody against myosin-Va. Fig.1H shows the distribution of ciliary vesicles and preciliary vesicles in NDUFAF2- / - cells expressing WT or G258A mutation, with > 150 cells that were analyzed for each independent experiment. Fig.1I shows cells that were serum-starved for 2 days and processed for immunostaining with antibodies as indicated. Fig.1J shows the intensity of NPHP1 signals at the centrioles that was quantified. More than 100 cells were analyzed for each independent experiment. The bars in graph represent mean ± SD, N=3. n.s., not significant. P<0.001(***).

[0036] Figures 2A-2I show Zebrafish Ndufaf2 morpholino-injected mutants that exhibit a ciliopathy phenotype. Fig.2A shows the protein level of control MO, Ndufaf2 morpholino- injected embryos, NDUFAF2WT and NDUFAF2G258A re-expressed in Ndufaf2 morpholino-injected embryos. Western blot analysis was performed with antibodies against NDUFAF2 and GAPDH. Fig.2B shows transmitted light images of body shape in control MO, Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re-expressed in Ndufaf2 morpholino-injected zebrafish larvae at 5 days post fertilization (dpf). Fig.2C shows quantification of body curve in control MO, Ndufaf2 morpholino- injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re-expressed in Ndufaf2 morpholino-injected zebrafish larvae at 5 dpf. More than 30 larvae were analyzed for each independent experiment. Fig.2D shows transmitted light images of eyes in control MO,MBHB Ref. No.23-1150-WO Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re- expressed in Ndufaf2 morpholino-injected zebrafish larvae at 3 dpf. Arrows point at the margins of the optic fissure. Fig.2E shows quantification of optic fissure closure in control MO, Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re-expressed in Ndufaf2 morpholino-injected zebrafish larvae at 3 dpf. More than 30 larvae were analyzed for each independent experiment. Fig.2F shows transmitted light images of heart in control MO, Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re-expressed in Ndufaf2 morpholino-injected zebrafish larvae at 5 dpf. Arrows point at the heart. Fig.2G shows quantification of heart failure in control MO, Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re- expressed in Ndufaf2 morpholino-injected zebrafish larvae at 5 dpf. More than 30 larvae were analyzed for each independent experiment. Fig.2H are confocal images of KV cells labeled with cilia marker, Arl13b (green) and Polyglutamylated Tubulin (red) in control MO, Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re- expressed in Ndufaf2 morpholino-injected zebrafish larvae at 8 somite-stage (ss). Fig.2I shows quantification of KV cilia number in control MO, Ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re-expressed in Ndufaf2 morpholino- injected zebrafish larvae. More than 30 larvae were analyzed for each independent experiment. The bars in graph represent mean ± SD, n.s., not significant. P<0.001(***).

[0037] Figures 3A-3J show ARMC9 interacts with NDUFAF2 via its N-terminal region and it requires the recruitment of NDUFAF2 to centriole. Fig.3A shows cells that were incubated with rat anti-ARMC9 and rabbit anti-NDUFAF2 antibodies then performed using anti-rabbit PLUS and anti-mouse MINUS PLA probes. Fig.3B shows 293T cells transfected with various expression constructs were analyzed by immunoprecipitation followed by western blots with indicated antibodies. Fig.3C is a diagram showing various ARMC9 mutants tagged with Flag. The ability of each construct in interacting with NDUFAF2 was also indicated. LiSH mean predicted lissencephaly type-1 like homology motif. CC mean coiled-coil domain. Fig.3D shows ARMC9 knockout in NDUFAF2 - / - cells. Western blot analysis was performed with antibodies against NDUFAF2, ARMC9 and Į-tubulin. Fig.3E shows cells that were serum starved for 2 days and the percentage of ciliated cells wasMBHB Ref. No.23-1150-WO quantified. More than 150 cells were analyzed for each independent experiment. Fig.3F shows cells that were serum starved for 2 days and analyzed by immunofluorescence with indicated antibodies. DNA (DAPI, blue). Scale bars as indicated. Fig.3G is a graph showing the percentage of cells with two CP110 dots at the centrioles in serum-starved cells. More than 150 cells were analyzed for each independent experiment. Fig.3H shows cells that were serum-starved for 2 days and processed for immunostaining with antibodies as indicated. Fig. 3I shows intensity of NPHP1 signals at the centrioles that was quantified.3J shows the quantification for 3I. More than 100 cells were analyzed for each independent experiment. The bars in the graph represent mean ± SD, n.s., not significant. P<0.001(***); P<0.01(**); P<0.05(*).

[0038] Figures 4A-4I show that expression of active NDUFAF2 rescued the mitochondrial defect in Joubert Syndrome patient, which is mutated in ARMC9 gene. Fig.4A shows a MRI T1 sequence of a 3-year-old patient demonstrating molar tooth sign (MTS) in midbrain. Fig.4B shows that NDUFAF2 was stably expressed in human conjunctiva fibroblasts (HConF) and Joubert Syndrome patient’s cells (JBTS), which is mutated in ARMC9 gene. Western blot analysis was performed with antibodies against NDUFAF2, ARMC9 and GAPDH. Fig.4C shows the oxygen consumption rate of HConF cells and JBTS cells were measured by Seahorse Analyzer. Fig.4D shows HConF cells and JBTS cells wherein NDUFAF2 was overexpressed in both cell lines that were used to analyze mitochondrial complex I activity. Fig.4E shows HConF cells and JBTS cells that were used to analyze the NAD+ / NADH ratio. Fig.4F are transmission electron microscope (TEM) images of HConF cells, JBTS cells and overexpressed NDUFAF2 in JBTS cells. There are 11 JBTS cells displayed tube-like cristae in stack and onion shapes. The total number of mitochondria in HConF cells, JBTS cells and overexpressed NDUFAF2 in JBTS cells were 12, 14 and 17, respectively. Scale bars is 200 nm. Fig.4G shows cells that were serum- starved for 2 days and processed for immunostaining with antibodies as indicated. Fig.4H shows the intensity of NPHP1 signals at the centrioles that was quantified. Fig 4I shows the quantification for 4H. More than 100 cells were analyzed for each independent experiment. The bars in the graph represent mean ± SD, n.s., not significant. P<0.001(***); P<0.01(**); P<0.05(*).MBHB Ref. No.23-1150-WO

[0039] Figures 5A-5F show that NAD+ supplement rescued the defection of ciliogenesis in JBTS, which is mutated in ARMC9 gene. Fig.5A shows the oxygen consumption rate of HConF cells, JBTS cells and treatment of nicotinamide in both cells that were measured by Seahorse Analyzer. Fig.5B shows HConF cells, JBTS cells and overexpressed NDUFAF2 in both cell lines that were used to analyze mitochondrial complex I activity. Fig.5C shows HConF cells and JBTS cells that were used to analyze the NAD+ / NADH ratio. Fig.5D shows cells that were serum-starved for 2 days and processed for immunostaining with antibodies as indicated. Fig.5E shows quantification of cilia length in HConF cells, JBTS cells and treatment of nicotinamide in both cells. More than 150 cells were analyzed for each independent experiment. Fig.5F shows intensity of NPHP1 signals at the centrioles that was quantified. More than 100 cells were analyzed for each independent experiment. Scale bar was 10 μm. The bars in graph represent mean ± SD, n.s., not significant. P<0.001(***); P<0.01(**); P<0.05(*).

[0040] Figures 6A-6B show an improvement in saccadic amplitude in JB patient upon nicotinamide supplementation. Fig.6A shows a schematic of eye tracking design. (6B) Joubert syndrome patient undergoing examination. Fig.6C shows a representative image of target, fixation point, and eye positions for eye tracking exam. Fig.6D shows a saccadic amplitude and SE for the last two sessions (101 trials) of with pre- and post- nicotinamide (oral 250 mg daily) (8707 saccades vs 3359 saccades in pre- and post- group respectively *, p<0.039).

[0041] Figures 7A-7C show NDUFAF2 mutation in a patient with bilateral retinal degeneration and optic nerve atrophy. Fig.7A shows A 28-yo woman presented with a novel NDUFAF2 missense mutation c.258 G>A. Fig.7B shows color fundus photography of both eyes showing pale optic nerve, attenuated blood vessels, and pigmentary retinopathy. Figure 7C shows red-free fundus imaging showing bilateral diffuse pigmentary degeneration with residual bullseye lesion in central macula.

[0042] Figures 8A-8D show NDUFAF2G258Aaffects mitochondrial activity. Figure 8A shows the editing sequencing outcomes in NDUFAF2- / -cells. The upstream was the sequence of RPE1 cells; the downstream were the sequence of two different NDUFAF2- / - cells. Fig. 8B shows mitochondrial activity in wild-type, NDUFAF2- / -, and NDUFAF2WTandMBHB Ref. No.23-1150-WO NDUFAF2G258A-re-expressing RPE1 cells. The oxygen consumption rate was measured by Seahorse Analyzer. Fig.8C shows the catalytic enzyme activity of complex I in purified mitochondria of wild-type, NDUFAF2- / -, and NDUFAF2WTand NDUFAF2G258A-re- expressing RPE1 cells. Fig.8D shows the NAD+ / NADH ratio in wild-type, NDUFAF2- / -, and NDUFAF2WTand NDUFAF2G258A-re-expressing RPE1 cells. The bars in each graph represent mean ± SD. Exact P values are indicated. The bars in each graph represent mean ± SD. Exact P values are indicated.

[0043] Figures 9A-9D show NDUFAF2 affect optic fissure closure but does not affect the number of otic vesicles. Fig.9A shows transmitted light images of eyes in control MO, ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WTand NDUFAF2G258Are- expressing in ndufaf2 morpholino-injected zebrafish larvae at 3 dpf. Arrows indicate the margins of the optic fissure. Fig.9B shows quantification of optic fissure closure in control MO, ndufaf2 morpholino-injected zebrafish larvae, NDUFAF2WT and NDUFAF2G258A re- expressing in ndufaf2 morpholino-injected zebrafish larvae at 3 dpf. > 30 larvae analyzed for each independent experiment. Fig.9C shows transmitted light images of eyes in control MO, ndufaf2 morpholino-injected zebrafish larvae, and ndufaf2 morpholino-injected zebrafish larvae re-expressing NDUFAF2WTand NDUFAF2G258Aat 2 dpf. Arrows point to otic vesicle margins. Fig.9D shows quantification of optic fissure closure in control MO, ndufaf2 morpholino-injected zebrafish larvae, and ndufaf2 morpholino-injected zebrafish larvae re- expressing NDUFAF2WTand NDUFAF2G258Aat 2 dpf. > 30 larvae were analyzed for each independent experiment.

[0044] Figures 10A-10F show photoreceptor dysfunction and degeneration in zebrafish ndufaf2 morpholino-injected mutants. Fig.10A shows a schematic diagram showing photoreceptor structure in zebrafish. Fig.10B shows light microscopic images showing retinal ganglion cells (RGC) in zebrafish larvae at 5 dpf. Zebrafish larvae are stained with Zn5 (green), DNA is stained with DAPI (blue). Scale bars as indicated. Fig.10C shows quantification of RGC signals in the GCL region of zebrafish larvae at 5 dpf.10 larvae analyzed for each independent experiment. Fig.10D shows light microscopic images showing photoreceptors in zebrafish larvae at 5 dpf. Zebrafish larvae are stained with rhodopsin (green) and recoverin (red), DNA is stained with DAPI (blue). The scale bars asMBHB Ref. No.23-1150-WO indicated. Fig.10E is the quantitative real-time PCR (RT-PCR) validation of rhodopsin in zebrafish larvae at 5 dpf. RT-PCR was repeated three times with different batches. Gene expression values are normalized to ȕ-actin. Fig.10F is the quantitative real-time PCR (RT- PCR) validation of transducin (gnat1 and gnat2) in zebrafish larvae at 5 dpf. RT-PCR was repeated three times with different batches. Gene expression values are normalized to ȕ-actin. Bars in the graph represent mean ± SD. Exact P values are indicated.

[0045] Figures 11A-11E show ARMC9 mutation-associated midbrain and retinal defects. Fig.11A shows anterior segment photos showing corneal exposure keratitis in right eye and inferior corneal transplant in left eye. Figure 11B shows retinal photograph of left eye showing early pigmentary retinal changes. Fig.11C and Fig.11D show Ocular coherence tomography of right eye showing attenuated blood vessels. Fig.11E shows optic nerve head photographs showing cupping of optic nerve in both eyes and myelinated optic nerve in left eye..

[0046] Fig.12A-12I show ARMC9 interacts with NDUFAF2 via its C-terminal region and it is required for cilia formation. Fig.12A and Fig.12B show 293T cells transfected with various expression constructs analyzed by immunoprecipitation followed by western blots with indicated antibodies. Fig.12C show the analysis of 293T cells transfected with various expression constructs by immunoprecipitation followed by western blots. Fig.12D depicts a schematic diagram showing various ARMC9 mutants tagged with Flag. The ability of each construct to interact with NDUFAF2 is also indicated. LiSH, mean predicted lissencephaly type-1 like homology motif. CC, mean coiled-coil domain. Fig.12E shows double knockout ARMC9- / -NDUFAF2- / -cells. Western blot analysis performed with antibodies against NDUFAF2, ARMC9, and Į-tubulin. Fig.12F shows percentage of ciliated cells after serum starving for 2 days. > 150 cells analyzed for each independent experiment. Fig.12G shows the analysis by immunofluorescence of cells serum starved for 2 days. DNA (DAPI, blue). Scale bars as indicated. Fig.12H depicts a graph showing the percentage of serum starved cells with two CP110 dots at the centrioles. > 150 cells analyzed for each independent experiment. Fig.12I shows immunostaining of cells serum starved for 2 days. The bars in each graph represent mean ± SD. Exact P values are indicated.MBHB Ref. No.23-1150-WO

[0047] Figures 13A-13E show NDUFAF2 rescued the defection of ciliogenesis in JBTS patient-derived cells, which is mutated in ARMC9 gene. Fig.13A shows representative cells stained by immunofluorescence for Mitotracker-red (red). Nuclei visualized with DAPI (blue). Scale bars 10 μm. Fig.13B shows mitochondrial length measured by Mytoe of HConF cells, JBTS cells, and both cell lines overexpressing NDUFAF2. > 100 cells measured. The bar line represents mean with standard deviation (mean ± SD). Fig.13C shows representative cells stained by immunofluorescence for 8-oxo-dg (red). Nuclei visualized with DAPI (blue). Scale bars 10 μm. Fig.13D shows quantitation of 8-oxo-dg signal in individual cells. Fig.13E shows immunostaining for polyglutamylated tubulin of cells serum starved for 2 days. The percentage of ciliated cells is quantified. > 150 cells analyzed for each independent experiment. The exact P values are indicated.

[0048] Figures 14A-14C show NAD+ supplement rescued defective zebrafish photoreceptor in vivo. Fig.14A shows the protein level of control MO, armc9 morpholino- injected embryos, Ndufaf2 mRNA and nicotinamide treatment in armc9 morpholino-injected embryos Western blot analysis was performed with antibodies against ARMC9, NDUFAF2 and GAPDH. Fig.14B shows transmitted light images of body shape in control MO and armc9 morpholino-injected embryos. Fig.14C shows images of photoreceptors in zebrafish larvae at 5 dpf. Zebrafish larvae stained for rhodopsin (green) and recoverin (red), DNA stained with DAPI (blue). The scale bars as indicated. Scale bar^is^50 μm. Bars in each graph represent mean ± SD. DETAILED DESCRIPTION

[0049] It is to be understood that the particular aspects of the specification are described herein are not limited to specific embodiments presented, and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

[0050] Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,”MBHB Ref. No.23-1150-WO “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0051] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indictates otherwise.

[0052] In some embodiments, percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated disclosure.

[0053] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0054] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. For example, “about 5%” means “about 5%” and also “5%.” The term “about” can also refer to ± 10% of a given value or range of values. Therefore, about 5% also means 4.5% - 5.5%, for example.

[0055] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0056] “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio or which have otherwise been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.MBHB Ref. No.23-1150-WO

[0057] As used herein, the term “pharmaceutically acceptable excipient” encompasses any of the standard pharmaceutical excipients, including carriers such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. Pharmaceutical compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Remington’s Pharmaceutical Sciences (20th ed., Mack Publishing Co.2000).

[0058] As used herein, the term “pharmaceutically acceptable salt” means a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction(s) with any other component of a pharmaceutical composition in which it may be contained.

[0059] As used herein, “ciliopathy”, “ciliopathies”, or “cilia disease” refers to a disease characterized by cilial dysfunction. “Cilial dysfunction” means the abnormal formation and / or function of cilia, including abnormal cilial location. Cilial dysfunction may affect the extracellular and / or intracellular portions of the cilia and it may be characterized by structural and / or functional irregularities.

[0060] Disease and disorders such as ciliopathies are often associated with one or more genetic mutations. In some embodiments of this disclosure, a subject or subject is diagnosed with having a particular disease or disorder and is also diagnosed to have a particular genetic mutation, for example, one that is known to be a cause of the disease or disorder in question, although it often cannot be proven that a particular patient’s disease or disorder is caused by the particular mutation that a person has been diagnosed with having. As used in this manner, the term “diagnosed to have a particular genetic mutation” means that a subject or patient has been tested, e.g., by DNA or RNA sequencing, protein profiling, or other suitable means, and found to have the mutation in question. However, many genetic diseases and disorders can have multiple genetic causes (e.g., mutations), and patients may have multiple mutations each of which may, under some circumstances, be sufficient to cause the disease or disorder, without it being subject to proof that a particular mutation causes a particular disease or disorder in a particular patient. Ciliopathies include, but are not limited to Joubert syndrome, Senior-Ldken syndrome, developmental delay, anosmia, renal cysts, and photoreceptorMBHB Ref. No.23-1150-WO degeneration. For instance, Ciliopathies such as Joubert syndrome cause multiple organ system disease and shortens lifeexpectancy. Joubert Syndrome is a rare form of ciliopathy characterized by midbrain defects, ocular motor apraxia, and developmental delays. Thirty- five identified genes encode proteins that play critical roles in the transition. ARMC9, one of the known Joubert genes, is located at basal body and interacts with CCDC66 and TOGARAM1 to stabilize cilia membrane. The renal diseases in ciliopathies can lead to failure to thrive, delayed growth, and renal failure requiring dialysis and renal transplant. The ocular phenotypes can lead to progressive blindness.

[0061] “Patient” refers to a warm blooded animal such as a mammal, preferably a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and disorders described herein.

[0062] As used herein, the term “subject” refers to both human and nonhuman animals. The term “nonhuman animals” of the disclosure includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, cat, horse, cow, chickens, amphibians, reptiles, and the like. The human subject can be of any age (e.g., an infant, child, or adult).

[0063] As used herein, “treatment” or “treating” refers to the clinical intervention made in response to a disease, disorder, or physiological condition of the subject or to which a subject can be susceptible. The aim of treatment includes the alleviation, ameliorating, or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder, or condition.

[0064] The term “administering” is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being inside the subject’s body. Such an administration can be by any route including, without limitation, oral, transdermal (for example, vagina, rectum, oral mucosa), by injection (for example, subcutaneous, intravenous, parenterally, intraperitoneally), or by inhalation (for example, oral or nasal). Pharmaceutical preparations are given by forms suitable for each administration route.MBHB Ref. No.23-1150-WO

[0065] As used herein, the term “preventing” or “prevention” of a disease includes causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease.

[0066] The terms “effective amount” or “therapeutically effective amount” refer to an amount sufficient to effect beneficial or desirable biological and / or clinical results. In other words, a “therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject.

[0067] The terms “express” or “expression” refer to transcription and translation of a nucleic acid coding sequence resulting in production of the encoded polypeptide. “Express” or “expression” also refers to antigens that are expressed on cell surfaces.

[0068] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this disclosure belongs.

[0069] In view of this disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need.

[0070] Disclosed herein are disclosed improved compositions and methods for treating or ameliorating ciliopathies, such as Joubert syndrome and others that rely on relationships mitochondrial function and primary cilia signaling.

[0071] The primary cilium is a solitary, immotile microtubule-based structure present on apical surface of many cell types, especially in sensory organs such as the eye and nose. Non- motile sensory cilia consist of an array of bundled microtubules (axoneme) that are covered by a plasma membrane. Axonemal microtubules arise from a specialized microtubule- nucleating center which is the basal body. Defects in cilium function have been associated with numerous human diseases including developmental delay, anosmia, renal cysts, and photoreceptor degeneration. A rare form of syndromic ciliopathy, Joubert Syndrome (JBS) is caused by mutations in genes that form the transition zone; one of the JBS genes termed ARMC9 is located at centriole basal body and interacts with CCDC66 and TOGARAM1 to stabilize cilia membrane. However, the precise mechanisms that link genetic mutations in Joubert to systemic diseases are unknown.MBHB Ref. No.23-1150-WO

[0072] Prior electron microscopy studies of primary cilia show mitochondria to frequently localize near the bases of cilia in motile ciliated cells as well as sensory photoreceptors. Human fibroblasts also showed similar patterns of mitochondria distribution at ciliary base based on cryo-electron tomography. It has been observed that microtubules project from the mother centriole at the cilium base toward mitochondria, suggesting a communication between the two organelles. However, how mitochondria are involved in primary cilia formation remains undetermined.

[0073] This disclosure relies upon the relationship illustrated herein between mitochondrial function and primary cilia signaling, with human patient-based evidence for therapeutic development for neurodegenerative diseases.

[0074] Mitochondria and cilia signaling have been previously proposed. Mitochondria- related neurodegenerative diseases have been postulated to involve primary cilia function. A relationship between mitochondria metabolism and primary cilia signaling illustrated herein involves an intrinsic mitochondrial complex 1 component, NDUFAF2, as a binding partner for ARMC9. Mutations in ARMC9 causes Joubert syndrome, a syndromic ciliopathy that presents with defects in the brain, kidney and the retina. Mutations in NDUFAF2 results in Leigh syndrome with severe inherited form of mitochondriopathy.

[0075] A novel mutation in NDUFAF2 (c.258G>A) found in a JBS patient was found to exhibit both mitochondrial and ciliary defects in vitro and in vivo. Using a rare disease patient-derived fibroblasts, inventors demonstrated a mechanistic link between an intrinsic mitochondrial protein NDUFAF2 and centriole protein ARMC9 during ciliogenesis. Furthermore, this invention discloses the mechanistic basis of NDUFAF2 function in ciliogenesis, which is dependent on tightly regulated NAD+ / NADH ratio. Specifically, it was found that that the mitochondrial protein could be involved in membrane vesicle docking, CP110 removal, and transition zone stabilization in cilia formation. Importantly, it is shown that a Joubert syndrome patient with ARMC9 mutation had decreased protein level of NDUFAF2, mitochondrial activity deficiency, and increased oxidative stress. Overexpression of NDUFAFA2 rescued the mitochondrial defect and cilia formation in JBTS fibroblast cells and armc9 deficient zebrafish larva.MBHB Ref. No.23-1150-WO

[0076] Intrinsically disordered protein regions (IDR) are important players in a variety of cellular processes such as transcription, translation, cell signaling, protein interaction, gene regulation networks, and also are linked to several human diseases. Centriole protein ARMC9 and mitochondrial protein NDUFAF2 were compared by sequence prediction (Predictor of Natural Disordered Regions, PONDR). Both of these contain IDR at C-terminal region. This finding raised the idea of the involvement of this property in primary cilia assembly. Taken together, the discoveries disclosed herein establish a relationship between mitochondrial signaling and primary cilia, supported by human evidence, thereby offering a new therapeutic perspective for patients with ciliopathies.

[0077] In some embodiments, this disclosure contemplates treating ciliopathy by the methods and compositions disclosed herein and substantiated by the examples below.

[0078] In one embodiment the term “first gene” comprises a gene that is mutated, defective, under-expressed, or does not express in a cell for normal cilia function. The mutated gene can be a corresponding mutated gene of a second gene. For example, in one embodiment of this invention, the cilia function can be restored in a cell that expresses a defective variant of a first gene that is needed for normal cilia function by expressing or overexpressing a second gene in the cell. The first gene is restored by expressing or overexpressing a second gene in the cell. The first gene can be ARMC9 and / or NDUFAF2. The second gene is NDUFAF2 (SEQ ID NOS.11 and 12). In another embodiment of this invention, the cilia function can be restored in a cell that under-expresses or does not express a first gene needed for normal function by introducing into the cell a second gene that when expressed in the cell restores cilia function. The first gene can be ARMC9 and / or NDUFAF2. The second gene is NDUFAF2 (SEQ ID NOS.11 and 12).

[0079] In some embodiments, this disclosure is directed to a method for restoring cilia function in a cell that expresses a defective variant of a first gene needed for normal cilia function by expressing or overexpressing a second gene in the cell that when expressed therein restores cilia function in the cell, wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0080] In some embodiments, the second gene is NDUFAF2 (SEQ ID NOS.11 and 12).MBHB Ref. No.23-1150-WO

[0081] In some embodiments, this disclosure is directed to a method for restoring cilia function in a cell that under-expresses or does not express a first gene needed for normal cilia function, comprising introducing into the cell a second gene that when expressed in the cell restores cilia function, wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0082] In some embodiments, the second gene is NDUFAF2 (SEQ ID NOS.11 and 12).

[0083] In some embodiments, this disclosure is directed to a method of treating ciliopathies in a subject that expresses a defective variant of a first gene needed for normal cilia function, the method comprising the steps of introducing into cells in the subject having the defective variant of the first gene a recombinant expression construct, wherein the construct comprises a second gene that when expressed is capable of correcting the ciliopathy in the subject, wherein the construct is delivered into an epithelial cell in the subject, and wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0084] In another embodiment, this disclosure is directed to a method of ameliorating ciliopathies in a subject that expresses a defective variant of a first gene needed for normal cilia function, the method comprising the steps of introducing into cells in the subject having the defective variant of the first gene a recombinant expression construct, wherein the construct comprises a second gene that when expressed is capable of correcting the ciliopathy in the subject, wherein the construct is delivered into an epithelial cell in the subject, and wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0085] Adeno-associated virus (AAV) is the smallest of known human viruses. It incorporates into the host cell's genome, but there is no evidence that it can cause malignant transformation. Because of these features it presents an attractive subject for creating vectors for gene therapy. AAV advantages for gene therapy include the lack of pathogenicity, the ability to infect non-dividing cells and the ability to stably integrate into the host cell genome at a specific site in the human 19th chromosome. The last feature makes it superior to retroviruses, which present threat of a random insertion and of mutagenesis, which is sometimes followed by development of a cancer. The AAV genome integrates most frequently into the site mentioned, while random incorporations into the genome take place with a negligible frequency. AAVs also present very low immunogenicity, restricted only to generation of neutralizing antibodies, while they induce no cytotoxic response. In someMBHB Ref. No.23-1150-WO embodiments, the recombinant expression construct comprises an adeno-associated virus (AAV).

[0086] In another embodiment, the methods of this invention provide gene therapy. In another embodiment, the methods of this invention provide the introduction of a functional gene into a target ciliated cell to restore proper protein production that is absent or deficient due to a genetic disorder or cilia dysfunction. In another embodiment, the methods of this invention provide the introduction of a functional gene into a target ciliated cell to induce protein production that is necessary for applications such as but not limited to a ciliopathy. In another embodiment, the methods of this invention provide the introduction of a functional gene into a target ciliated cell to induce protein production that is necessary for applications such as but not limited to kidney transplantation.

[0087] In another embodiment, the methods of this invention provide viral delivery systems that are based on viruses that have the ability to deliver genetic information into the host ciliated cell. In another embodiment, the methods of this invention provide that the coding regions of the virus are replaced by the genetic information of a therapeutic gene.

[0088] In another embodiment, the methods of this invention provide that the viral vector is introduced into producer cells providing the structural viral proteins in trans, production of nonreplicating virus particles containing the genetic information of a therapeutic gene is established. In some embodiments the therapeutic gene is the second gene. In another embodiment, the methods of this invention provide the ability to generate replication- defective viral vectors for virus-based gene delivery vehicles.

[0089] In another embodiment, the methods of this invention provide integrating vectors. In another embodiment, the methods of this invention provide non-integrating vectors. In another embodiment, the methods of this invention provide vectors based on adeno- associated virus and retroviruses (including lentivirus and foamy virus) that have the ability to integrate their viral genome into the chromosomal DNA of the host cell. In another embodiment, the methods of this invention provide a lifelong gene expression.

[0090] In another embodiment, the methods of this invention provide an AAV serotype 2 (AAV-2) vectors. In another embodiment, the methods of this invention provide that AAV-2 is efficient in transducing the ciliated cells of the invention. In another embodiment, theMBHB Ref. No.23-1150-WO methods of this invention provide that AAV-2 is efficient in engineering the ciliated cells of the invention. In another embodiment, the methods of this invention provide AAV-1 vectors. In another embodiment, the methods of this invention provide AAV-3 vectors. In another embodiment, the methods of this invention provide AAV-4 vectors. In another embodiment, the methods of this invention provide AAV-5 vectors. In another embodiment, the methods of this invention provide that AAV-5 is efficient in transducing epithelia cells. In another embodiment, the methods of this invention provide that AAV-5 is efficient in engineering airway epithelia cells. In another embodiment, the methods of this invention provide that the use of different AAV serotypes may allow targeting of the vector for tissue-specific transduction. In another embodiment, the methods of this invention provide altering the tropism of AAV vectors by chemical cross-linking of bispecific antibodies to the viral capsid and by the insertion of receptor specific epitopes in the cap proteins.

[0091] In another embodiment, the methods of this invention provide that the AAV vectors of the invention comprise cap proteins from different serotypes that overcome the problems of neutralizing antibodies.

[0092] In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 1. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 2. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 3. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 4. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 5. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 6. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 7. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 8. In another embodiment, this invention provides that the AAV used by the methods of this invention is an AAV serotype 9. In another embodiment, this invention provides that the AAV of this invention comprises AAV serotype 2 inverted terminal repeats (ITRs) and packaged into capsids from AAV serotype 1, 2, 5, 8, or 9.MBHB Ref. No.23-1150-WO

[0093] In another embodiment, this invention provides that the AAV vector comprises a reporter gene. In another embodiment, the reporter gene is a fluorescent protein. In another embodiment, the reporter gene comprises green fluorescent proteins such as but not limited to GFP, Emerald, Azami Green, or ZsGreenl. In another embodiment, the reporter gene comprises blue fluorescent proteins such as but not limited to EBFP or Sapphire. In another embodiment, the reporter gene comprises cyan fluorescent proteins such as but not limited to Cerulean, ECFP, AmCyanl or Midoriishi-Cyan. In another embodiment, the reporter gene comprises yellow fluorescent proteins such as but not limited to ZsYellowl, PhiYFP, Citrine, or Venus. In another embodiment, the reporter gene comprises orange fluorescent proteins such as but not limited to Kusabira-Orange or mOrange. In another embodiment, the reporter gene comprises red fluorescent proteins such as but not limited to: DsRed, HcRed, mPlum, mRaspberry, mTomato, mStrawberry. In another embodiment, the reporter gene comprises a green-to-red fluorescent Dendra.

[0094] In some embodiments, this disclosure is directed to a method of treating ciliopathies in a subject that expresses a defective variant of a first gene needed for normal cilia function, the method comprising the steps of introducing into cells in the subject having the defective variant of the first gene a recombinant expression construct, wherein the construct comprises a second gene that when expressed is capable of correcting the ciliopathy in the subject, wherein the construct is delivered into an epithelial cell in the subject, and wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0095] In another embodiment, this disclosure is directed to a method of ameliorating ciliopathies in a subject that expresses a defective variant of a first gene needed for normal cilia function, the method comprising the steps of introducing into cells in the subject having the defective variant of the first gene a recombinant expression construct, wherein the construct comprises a second gene that when expressed is capable of correcting the ciliopathy in the subject, wherein the construct is delivered into an epithelial cell in the subject, and wherein the defective first gene can be ARMC9 and / or NDUFAF2.

[0096] In some embodiments, the ciliopathy causes a kidney defect.

[0097] In some embodiments, the epithelial cell is a renal tubular epithelial cell.MBHB Ref. No.23-1150-WO

[0098] In some embodiments, the AAV construct is introduced into the subject through the subject’s renal collecting duct.

[0099] In some embodiments, the treating results in reduction of ciliary defect symptoms. [000100] In another embodiment, the term "second gene" comprises a wild-type gene that corrects the corresponding mutated gene or defective first gene. [000101] In another embodiment, the term " second gene" comprises growth factors that treat or reverse a pathology characterized by being responsive to growth factors as known to one of skill in the art. In another embodiment, the term "second gene" comprises anti- apoptotic genes that treat, ameliorate or reverse a pathology characterized by elevated, undesired apoptotic rate as known to one of skill in the art. [000102] In another embodiment, the term " second gene" comprises pro-apoptotic and / or anti-cancer genes that treat, ameliorate or reverse a pathology such as but not limited to a disease or uncontrolled tissue growth as known to one of skill in the art. In another embodiment, the term "second gene" comprises trophic genes encoding trophic proteins such as growth factors or immune system modulators which provide trophic support and are administered for example to a kidney cell. In some embodiments, the defective first gene can be ARMC9 and / or NDUFAF2. In some embodiments, the second gene is NDUFAF2 (SEQ ID NOS.11 and 12). [000103] In some embodiments, the steps further comprise administration of a therapeutically effective amount of a pharmaceutical formulation of a nicotinamide compound. [000104] In some embodiments, this disclosure is directed to a method of treating a subject with a ciliopathy comprising: (a) obtaining a biological sample from the subject, (b) detecting DNA from the sample encoding a gene comprising at least one mutation or variant ssociated with cilia dysfunction, and (c) administering to the subject with the DNA mutation or variant associated with cilia dysfunction a nicotinamide pharmaceutical composition in an amount effective to maintain or improve cilia function. [000105] In another embodiment, this disclosure is directed to a method of ameliorating a subject with a ciliopathy comprising: (a) obtaining a biological sample from the subject, (b) detecting DNA from the sample encoding a gene comprising at least one mutation or variantMBHB Ref. No.23-1150-WO associated with cilia dysfunction, and (c) administering to the subject with the DNA mutation or variant associated with cilia dysfunction a nicotinamide pharmaceutical composition in an amount effective to maintain or improve cilia function. [000106] “Nicotinamide” is one of the two principal forms of the B-complex vitamin niacin. The other principal form of niacin is nicotinic acid; nicotinamide, rather than nicotinic acid, however, is the major substrate for nicotinamide adenine dinucleotide (NAD+) biosynthesis in mammals, as discussed in detail herein. Nicotinamide, in addition to being known as niacinamide, is also known as 3-pyridinecarboxamide, pyridine-3-carboxamide, nicotinic acid amide, vitamin B3, and vitamin PP. This invention discloses nicotinamide or nicotinamide derivative supplements that may be utilized to treat a variety of ciliopathies. In some embodiments, the the nicotinamide pharmaceutical composition comprises an active pharmaceutical ingredient that is NAD+. [000107] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, a solvent, an adjuvant, a diluent, or a combination thereof. [000108] In some embodiments, the composition is delivered systematically. [000109] In some embodiments, the composition is administered by oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracranial, transdermal, intrathecal, intracranial, intravenous, or intracerebral administration. [000110] In some embodiments, the composition is administered orally. [000111] In some embodiments, the subject has a ciliopathy selected from the group consisting of Joubert syndrome, developmental delay, anosmia, renal cysts, and photoreceptor degeneration. [000112] In some embodiments, the ciliopathy is Joubert syndrome. [000113] In some embodiments, this disclosure is directed to a pharmaceutical composition comprising a recombinant expression construct encoding a gene for treating a cilia related disorder, wherein the gene is ARMC9 and / or NDUFAF2. [000114] In some embodiments, the recombinant expression construct comprises an adeno- associated virus (AAV). [000115] In some embodiments, the composition further comprises an NAD+ supplement.MBHB Ref. No.23-1150-WO [000116] In one embodiment of the fifth aspect disclosed herein, the NAD+ supplement is administered to a subject at a therapeutically effective dose. [000117] In some embodiments, the NAD+ supplement, is administered as a tablet or liquid formulation. [000118] Other diseases and disorders are contemplated for treatment herein. EXAMPLES [000119] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way. Example 1: Mitochondrial gene NDUFAF2 is involved in cilia formation at the initial steps Overview [000120] Mitochondria have been detected near the base of cilia in motile cilia cells and photoreceptors (Burkhalter, et al., J Clin Invest.2019). Human fibroblasts reveal similar pattern of mitochondrial at ciliary base based on cryo-electron tomography; microtubules have been observed to project from the mother centriole at the cilium base towards the mitochondria, suggesting a communication between the two organelles. However, how mitochondria involved in primary cilia formation are still unclear. [000121] A family with retinal and optic nerve degeneration was identified with a previously unreported NDUFAF2 mutation; a 28-year-old female presented in retinal degeneration and optic nerve atrophy with the c.258G>A missense mutation in NDUFAF2 gene (Figs.7A-7C). Mutations in NDUFAF2 previously have been reported in Leigh syndrome, a rare and progressive mitochondrial disorder that affects the central nervous system, specifically in the midbrain and basal ganglia. NDUFAF2 deficiency primary cells from NDUFAF2 knock-out mice reduce Complex-1 activity, increase oxidative stress and mitochondrial DNA deletion. This novel missense mutation was characterized and determined that a truncated and unstable form of NDUFAF2 was generated. [000122] To determine how Complex 1 of mitochondria can affect cilia structure and function, NDUFAF2 knockout cell line in RPE cells was generated, which confirmed theMBHB Ref. No.23-1150-WO mitochondrial defect by SeaHorse assay. Surprisingly, these deficient cells also exhibited primary cilia defects. Wild-type (NDUFAF2WT) was re-expressed and point mutation of NDUFAF2 (NDUFAF2G258A) into NDUFAF2- / - knockout cells. These stably expressed NDUFAF2 knockout cells were derived using single cell cloning (Fig.1A). Genotyping showed a clone of multiple base deletions in both alleles, predicting to produce severely truncated products consisting of only 38 amino acids in both of knockout clone cells (Figs. 8A). In addition, the oxygen consumption rate (OCR), complex I activity and NAD+ / NADH ratio were decreased in NDUFAF2- / -and NDUFAF2G258Acells (Figs.8B-8D). It was then determined whether the point mutation of NDUFAF2 affected its localization at mitochondria. RPE deficient NDUFAF2 clonal cells were generated (NDUFAF2- / -); NDUFAF2WTor disease-causing NDUFAF2G258Awere re-expressed and clonal cells were generated and confirmed by Sanger sequencing. These cells were stained with NDUFAF2 and TOM20 antibodies and confirmed that NDUFAF2G258Asignals could be detected at mitochondrial membrane (Figs.1B and 1C). Furthermore, it was observed that NDUFAF2- / -and NDUFAF2G258Aare decreased cilia formation (Fig.1D). [000123] To determine how NDUFAF2 contributed to ciliogenesis, steps during ciliogenesis were analyzed. First, it was determined whether NDUFAF2 affected CP110 removal. Most of NDUFAF2- / -and NDUFAF2G258Aexpressing clonal cells still showed two CP110 foci at the centrioles, indicating that the removal of CP110 was regulated by NDUFAF2 (Figs.1E and 1F). Next, it was determined whether NDUFAF2 mediated docking of membrane vesicles to centrioles. NDUFAF2- / -and NDUFAF2G258Aexpressing cells contained less ciliary vesicles when compared to the RPE and NDUFAF2WTexpressing cells, indicating that NDUFAF2 affected ciliary vesicle docking (Figs.1G and H). Removal of CP110 from the mother centrioles and the docking of membrane vesicles to the mother centrioles promoted transition zone assembly. The results also showed that NDUFAF2- / -and NDUFAF2G258A-expressing cells affected the recruitment of the transition zone protein NPHP1, thus affecting the establishment of the transition zone (Figs.1I and 1J). Taken together, these results demonstrated that mitochondrial gene NDUFAF2 is involved in initial steps of cilia formation, including membrane vesicles docking, CP110 removal and transition zone establishment.MBHB Ref. No.23-1150-WO Example 2: NDUFAF2 mutants exhibit a ciliopathy phenotype in vivo [000124] Ciliary genes are highly conserved between zebrafish and humans; zebrafish model system are frequently used to investigate human ciliopathies. Here, ndufaf2 in the zebrafish were knocked down by using morpholinos (MOs), then rescuing these morphants by co-injection of ndufaf2 mRNA (Fig.2A). Knocked down ndufaf2 and co-injection of ndufaf2G258AmRNA display multiple ciliopathies phonotype were observed, including severe body curvature, optic fissure closure and pericardial edema (Fig.2B-2G), but not optic vesicle defects. (Figs.9A-9D). Furthermore, the numbers of cilia in Kupffer’s vesicle (KV) are decreased in embryos with ndufaf2 knocked down, which can be rescued by co-injection of wildtype but not ndufaf2G258AmRNA (Figs.2H and 2I). Thus, NDUFAF2 is both necessary and sufficient for cilia formation. Furthermore, NDUFAF2 mutants cause photoreceptor dysfunction and degeneration in vivo, including shorter photoreceptor outer segment and reduced retinal ganglion cells (Figs.10A-10F), which phenocopies the human patient and confirms NDUFAF2 to play an essential role in both photoreceptor as well as optic nerve degeneration. Example 3: NDUFAF2 interacted with centriole protein ARMC9 and was recruited to the centriole by ARMC9 [000125] During cilia formation, mitochondria accumulated at the centriole base (Example 1; Burkhalter, et al., J Clin Invest.2019). It was determined whether centriole proteins at the basal body might interact with NDUFAF2 at the onset of ciliogenesis. In a pilot screening, NDUFAF2 was found to interact with centriole protein armadillo repeat motif–containing 9 (ARMC9). To determine whether centriole proteins at the basal body might interact with NDUFAF2 at the onset of ciliogenesis, CRISPR-Cas was used and stable cell lines was generated of NDUFAF2- / -; separately, wildtype gene was reintroduced to make stable NDUFAF2WTexpressing cells (Fig.3A). The interaction between NDUFAF2 and ARMC9 was characterized by performing proximity ligation assays (PLA) followed by immunostaining in RPE, NDUFAF2- / - and NDUFAF2WTexpressing cells (Fig.3A). The interaction between NDUFAF2 and ARMC9 was also characterized by performing immunoprecipitation followedMBHB Ref. No.23-1150-WO by western blot in 293T cells (Fig.3B and Fig.12A). Their interaction was not affected by the point mutation, as expression of NDUFAF2WTand NDUFAF2G258Ashowed no difference Fig.12B). [000126] ARMC9 deletion mutants were then generated to map its interacting region with NDUFAF2 (Fig.3C). A C-terminus deletion of ARMC9 (ARMC91-150) was found to abrogate its interaction with NDUFAF2, whereas the truncated form of ARMC9 carrying the C-terminal region (ARMC9151-665) was able to interact with NDUFAF2 (Fig.3B). In the detail, both of the deletion of ARMC9 C-terminus (ARMC91-350) and the deletion of ARMC9 N-terminus (ARMC9351-665) were able to interact with NDUFAF2, indicating that ARMC9 interacted with NDUFAF2 via its coiled-coil region and armadillo repeat (Fig.12C). [000127] Inventors determined whether NDUFAF2 was triggered by ARMC9 to the centriole and then involved in ciliogenesis. Then, the ARMC9 was knocked out in NDUFAF2 knockout cell line. The Western blotting showed that ARMC9 did not express in NDUFAF2 and ARMC9 double knockout cells (Fig.3D). The effects of NDUFAF2 and ARMC9 double knockout cells in promoting ciliogenesis were then confirmed. It was found that the percentage of ciliated cells dramatically reduced in NDUFAF2 / ARMC9 double knockout cells compared to NDUFAF2 knockout alone, further supporting that NDUFAF2 was triggered by ARMC9 and promoted ciliogenesis (Fig.3E). Here, inventors also found the percentage of CP110 removal and intensity of transition zone were reduced in NDUFAF2 and ARMC9 double knockout cells compared to NDUFAF2 knockout cell line and RPE cells (Figs.3F-3I). Thus, the data demonstrated a new model where the recruitment of NDUFAF2 to centriole by ARMC9 plays critical roles for ciliogenesis that promotes CP110 removal from the mother centrioles and transition zone stabilization. [000128] Next, whether known cilia-deficient cells exhibit mitochondria abnormalities and whether there was mitochondrial defecation in the patient cell line was determined. Here, it was found that the oxygen consumption rate (OCR), complex I activity and NAD+ / NADH ratio were reduced in JBTS cells compared to HconF cells. Metabolic assays using SeaHorse demonstrated a defect in mitochondrial respiration functions, confirming the hypothesis. To determine the possible mechanism, a targeted screen and identified a novel binding protein NDUFAF2 was performed. NDUFAF2, an essential assembly factor of Complex I withinMBHB Ref. No.23-1150-WO mitochondria, is one of the known genes to cause Leigh syndrome, rare and progressive neurological disorder that affects the central nervous system with early demise. Leigh syndrome is caused by mutations in mitochondrial genes; deficiency in mitochondrial Complex I has been found in this condition. Ndufaf2 knock-out mouse fibroblasts exhibited reduced Complex-1 activity and increased oxidative stress and mitochondrial DNA deletion. It was found that NDUFAF2 interacted with ARMC9. The interaction between NDUFAF2 and ARMC9 was characterized by performing proximity ligation assays (PLA). Example 4: NDUFAF2 rescues the mitochondrial defection and cilia formation in Joubert Syndrome patient [000129] Based on the observation that NDUFAF2 interacted with ARMC9 and deficiency to NDUFAF2 resulted in ciliary dysfunction, it was determined whether increased NDUFAF2 levels were able to rescue loss of ARMC9. Serendipitously, a Joubert syndrome family was recruited with a known mutation within ARMC9 that leads to the early termination of translation (Figs 11A-11E). The proband was a 9-year old patient demonstrating molar tooth sign (MTS) in midbrain and developed optic nerve colobomas, aptosis, and retinal degeneration (Figs 11A-11E). A patient-derived fibroblast line was generated and a previously noted ciliary defect was verified (Fig.4A and Fig.11A-11E). [000130] Because of this patient’s muscular hypotonia and ptosis, the existence of a mitochondria-defective phenotype in the patient’s fibroblasts was evaluated. It was found that protein levels of NDUFAF2 were reduced in these JBTS cells compared to wild-type conjunctiva fibroblast cells (HconF) (Fig.4B). NDUFAF2 was then overexpressed in JBTS cells and mitochondrial function was observed to be dramatically improved compared to untreated JBTS cells (Figs.4C-4E). Electron microscopy of mitochondrial cristae was then used to measure both treated and untreated JBTS cells and the structure of mitochondrial cristae in JBTS cells was compared to control (HconF) cells and NDUFAF2-overexpressing JBTS cells. It was found from these experiments that there were more onion-shaped crista and less crista in JBTS patient-derived cells compared to the other two cell lines (Fig.4F). Moreover, the JBTS patient-derived cells showed more filamentous types of mitochondria and higher oxidative stress levels as compared to other cell lines (Figs.13A-7D). Thus, theseMBHB Ref. No.23-1150-WO results demonstrated that ARMC9-deficient Joubert patient-derived cells exhibited mitochondrial defects, which can be rescued by NDUFAF2 expression. [000131] It was then evaluated whether the ciliary defects in JB patient-derived cells can be affected by NDUFAF2 expression. Here, the percentage of ciliated cell was not significantly different in JBTS cells compared than control (HconF) cells, but they displayed shorter cilia in JBTS patient-derived cells (Fig.13E). It is well-known that ARMC9 regulates the ciliary transition zone in JBTS, which prompted a determination of whether the stability of NPHP1 was affected in JBTS patient-derived cells. It was found that the intensity of NPHP1 was reduced in JBTS cells as compared to control cells. In NDUFAF2-overexpressing JBTS cells, NPHP1 remained localized at transition zone (Figs.4G and 4H). These results established that ARMC9 localized at the centriole basal body and interacted with NDUFAF2 to stabilize the ciliary transition zone. Example 5: NAD+ supplement rescued the mitochondrial defection and cilia deficiency in vivo and JBTS cells [000132] Mitochondrial complex I plays an important role in releasing reactive oxygen species (ROS) and oxidizing NADH. Experiments were performed to determine whether metabolic supplementation with nicotinamide compounds increased Complex 1 that exerted a similar effect as NDUFAF2 gene expression. In these experiments, nicotinamide (a form of vitamin B3 that utilitizes NAD+ as a substrate of complex I) was provided to cells and whether NAD+ supplementation could rescue mitochondrial and ciliary defects in JBTS cells was determined. Upon treatment with nicotinamide, elevated levels of oxygen consumption rate (OCR), increased complex I activity, and reduced NAD+ / NADH ratio was detected in JBTS cells as compared to untreated JBTS cells (Figs.5A-5C). Consistent with these findings, nicotinamide also corrected the cilia length deficiency and normalized NPHP1 in JBTS cells as compared to untreated JBTS cells (Figs.5D-5E). [000133] Furthermore, ARMC9 deletion displayed a thinner layer of photoreceptor in zebrafish, but did not affect body curve in larva stage (Latour, et. al., Clin Invest.2020and Fig.14B). Mopholino-mediated knockdown of armc9 was generated and defective rod outer segment observed in armc9 deficient larva; such defect can be rescued by co-injection ofMBHB Ref. No.23-1150-WO ndufaf2 mRNA (Figs.14B-14C). Separately, zebrafish armc9 larva treated solubilized nicotinamide restored these rod outers segment defects. Thus, NAD+ supplementation successfully remedied the mitochondrial defects in both cells and zebrafish larva lacking ARMC9. [000134] Taken together, these results showed that the mitochondrial complex I was involved in ciliogenesis by providing NAD+ supplement, then stabilizing transition zone formation. Accordingly, NAD+ supplementation can reverse mitochondrial and ciliary defects in JBTS cells, thus providing a new candidate therapy for ciliopathies patients. Example 6: Eye tracking results for Joubert patient [000135] To evaluate whether eye movement is affected in patients, a Tobii Pro Spectrum eye-tracking device was utilized for this experiment. This device has high precision and accuracy in capturing eye movements. The participant's eye movements were recorded using infrared oculography. Infrared light tracked the position and movements of the participant's eyes accurately and the device captured the participant's eye movements, including fixations, saccades, and other relevant eye-tracking metrics. The data collected provided insights into the participant's visual attention and gaze behavior. [000136] Based on the in vitro and in vivo evidence supporting nicotinamide in ARMC9 deficiency, low dose nicotinamide was administered to the JBS patient. For clinical endpoint measurements, ocular saccadic movements and sleep study were determined as endpoints. Patients with Joubert Syndrome develop ocular motor apraxia with difficulty initiating ocular saccades and central apneic suppression which results in shortened life expectancy, an average of 7 years. Ocular saccades are measured by eye tracking and central apnea was determined by sleep study. Daily oral supplementation was with 250 mg of nicotinamide for 2 months (PMID: 27654882). Pre- and post- NAD+ supplementation demonstrated marked improvement in patient’s motor skills per parents and objective eye movement studies (Fig. 6D).MBHB Ref. No.23-1150-WO Conclusion [000137] The novel mutation in NDUFAF2 (c.258G>A) found in a JBS patient was found to exhibit both mitochondrial and ciliary defects in vitro and in vivo. NDUFAF2 expression from heterologous introduction of this gene into was found to rescue human patient-derived cells with known ARMC9 deficiency. Further, NAD+ supplementation restored mitochondrial dysfunction in ARMC9 deficient cells, zebrafish, and mouse retinal degeneration. Finally, NAD+ supplementation ameliorated ocular motility and motor symptoms of the ARMC9 deficient patient (Figs.6A-6D). Taken together, the findings provided a basis for therapeutic intervention in JBS and other ciliopathies involving inactivating mutations in ARMC9 by introducing NDUFAF2 into the cells of such patients. These therapeutic interventions are useful for addressing an ameliorating neurodegenerative diseases associated or caused by pathological ciliopathies. Materials and Methods Cell culture and reagent [000138] 293T and 293FT were cultured in Dulbecco’s modified Eagle’s (DME) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The human telomerase-immortalized retinal pigment epithelial cells (hTERT-RPE1 or RPE1) were cultured in DME / F-12 (1:1) medium supplemented with 10% FBS and 1% penicillin- streptomycin. The human conjunctival fibroblasts (HConF, ScienCell Research Laboratories, #6570) from healthy human conjunctiva and conjunctiva cells from JBTS were cultured in DMEM / F-12 (1:1) medium supplemented with 10% FBS, Fibroblast Growth Supplement (FGS, ScienCell Research Laboratories, #2352), and 1% penicillin-streptomycin. Plasmids [000139] The human NDUFAF2 and ARMC9 cDNA were obtained from Origene (RC207387 and RC209499). To generate the epitope-tagged ARMC9, fragments were amplified by PCR and cloned into pcDNA3-FH. The pcDNA3-FH vector was derived from pcDNA3 (Thermo Fisher Scientific) but contained sequences for Flag and HA epitope tag between the HindIII and BamHI cloning sites. Thus, the expressing proteins expressed FlagMBHB Ref. No.23-1150-WO and HA epitope-tag at the N-terminus. Various ARMC9 mutants were cloned to pcDNA3-FH vector and used to analyze ARMC9-NDUFAF2 interaction (Figs.4A, 4B, and Fig.9A-9D). The NDUFAF2 and ARMC9 fragments were also sub-cloned into pCS vector from Addgene (12158) so that proteins could be stably expressed in RPE1, human conjunctival fibroblasts cells and conjunctiva cells of JBTS. Primary antibodies [000140] Primary antibodies were obtained from the following sources and used according to the manufacturers’ instructions: rabbit anti-NDUFAF2 (WB 1: 500; HPA054776; Sigma- Aldrich), mouse IgG2a anti-CEP164 (IF 1:1000; sc-515403; Santa Cruz), rabbit anti-CP110 (IF 1:1000; 12780-1-AP; Proteintech), mouse IgG2b anti-FOP (IF 1:1000; H00011116-M01; Abnova), anti-polyglutamylated tubulin (pGlu-Tu) (IF 1:3000; 901501; AdipoGen), rabbit anti-myosin-Va (IF 1:200; NBP1-92156; Novus Biologicals), rabbit anti-GAPDH (WB 1:5000; 60004-1-Ig; Proteintech), mouse IgG2a anti-Arl13b (IF 1:500; 75-287; Antibodies Incorporated), rabbit anti-TOMM20 (IF 1:1000; ab56783; Abcam), rabbit anti-ARMC9 (WB 1:500 in 5%BSA, HPA026671, Sigma-Aldrich), rat anti-ARMC9 (IF 1:100, 90703, BiCell Scientific), mouse IgG2b anti-8-oxo-Dg (IF 1: 250; 4354-MC-050; R&D systems), mouse anti-Flag (WB 1:5,000; F3165; Sigma-Aldrich), rabbit anti-NPHP1 (IF 1:1000; SAB1401267; Sigma-Aldrich), rabbut anti-Myc (WB 1:1000; 2278; Cell Signaling Technology),mouse IgG1 anti-Į-tubulin (WB 1: 1000; T6199; Sigma-Aldrich), mouse IgG1 anti-ZN5(IF 1:1000; ZDB-ATB-081002-19; Zebrafish International Resource Center), mouse IgG1 anti-Rhodopsin (IF 1:500; ab5417; Abcam), rabbit anti-Recoverin (IF 1:500; AB5585-I; Millipore), rabbit anti-GAPDH (WB 1:1000; 2118S; Cell Signaling Technology), MitoTracker™ Red CMXRos (Thermo Fisher Scientific, M7512). Transient transfection [000141] Transient transfections were performed using TurboFect™ Transfection Reagent (Thermo Fisher Scientific, R0533).3x106of 293T cells was plated on 60-mm plate for overnight. Cells were transfected with 2.5 ^g of expression constructs according to the manufacturer's instructions. Cells were harvested 48 h after transfection.MBHB Ref. No.23-1150-WO Lentivirus production and infection [000142] 5x105of 293FT cells were plated on 60-mm dish using TurboFect™ Transfection Reagent with following plasmids: 1.5 ^g of V-SVG, 2.5 ^g pCMV-gag-pol and 3.5 ^g of the pCS based constructs. The supernatant containing viral particles was harvested 48 h after transfection. Virus containing media was passed through a 0.45^m filter (Fisher scientific, 13-100-105). For the infection of RPE1 and human conjunctiva fibroblasts and conjunctiva cells of JBTS, 5×105of cells were seeded onto a 60-mm plate the night prior to infection and incubated with 3 ml of viral stock. The medium was changed to fresh culture medium 24 h after infection. Two days after infection, cells were maintained in culture medium. Generation of NDUFAF2 and ARMC9 knock-out RPE1 cells [000143] The targeting sequence for NDUFAF2 and ARMC9 was at exon 1 of NDUFAF2 (5’- GCAGTACAAGAACTGGAGAG-3’ (SEQ ID NO.: 13)) and exon 3 of ARMC9 (5’- GTTTCAAGTTCCATCCGAGAT-3 (SEQ ID NO.: 14)’), respectively. The target sequences were cloned into the gRNA cloning vector (pSLQ1654) containing Cas9 via the restriction enzyme BbsI (Thermo Fisher Scientific, FD1014). Knockout cells were all obtained through clonal propagation from a single cell. For genotyping, the following PCR primers were used: 5’- TCCAGGATGGAGGCCGACCT-3’ (SEQ ID NO.: 15), 5’- TAGTAGGGACCGCGGACGCA-3’ (SEQ ID NO.: 16) for NDUFAF2 alleles, and 5’- ATGGGGGACATTCTGGCTCAT-3’ (SEQ ID NO.: 17), 5’- GAGGGGTGCACCATAGGGTTG-3’ for ARMC9 alleles. PCR products were cloned and sequenced. Immunostaining [000144] Cells were grown on 0.1 mg / ml of poly-L-lysine coated coverslips and fixed with methanol at í20^ for 15 min. Cells were then washed three times with PBS and incubated in the blocking buffer that contained 3% bovine serum albumin (w / v) and 0.1% Triton X-100 in PBS for 30 min at RT. Primary antibodies were all diluted in the blocking buffer and incubated for 2 h at RT. Alexa Fluor 488–, 594–, or 647-conjugated goat secondaryMBHB Ref. No.23-1150-WO antibodies were used at 1:500 dilution (Thermo Fisher Scientific) and incubated for 1 h at RT. DNA was visualized using 4ƍ,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Coverslips were mounted on the slides with mounting medium (ProLong™ Gold Antifade; Thermo Fisher Scientific). Fluorescent images were obtained using a confocal microscope (LSM880 Zeiss confocal microscope). Images were acquired and processed by ZEN software (Carl Zeiss) or imageJ software (National Institutes of Health). Immunoblotting [000145] Cells were washed with ice-cold PBS twice and lysed in ice-cold RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate and 0.1% SDS) that contained protease inhibitors cocktail (Thermo Fisher Scientific, PI78430). Cell debris was removed by centrifugation at 13,500 g for 15 min at 4°C. Protein concentrations were determined by the BCA Protein Assay (Thermo Fisher Scientific, 23227). Equal amount of proteins were mixed with SDS sample buffer, boiled at 95°C for 5 min and separated by SDS-PAGE. The resolved proteins were then transferred on 0.45μm PVDF membranes (Thermo Fisher Scientific, 88518). Blots were blocked for 1h at RT with 5% non-fat milk in TBS-T (20mM Tris, PH 7.6, 137mM NaCl, and 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies in blocking solution. Membranes were washed three times with TBS-T and incubated with HRP-conjugated anti-mouse or anti- rabbit secondary antibodies for 1h at RT (Invitrogen, 31430 and 31460). After washing three times with TBS-T, proteins were visualized with ECL Western blotting substrate (Thermo Fisher Scientific, 34095). SIM imaging [000146] The three-dimensional structured illumination microscopy (3D-SIM) superresolution images were acquired using a Zeiss ELYRA PS.1 LSM780 system equipped with a Plan Apochromat 63× / NA 1.4 oil immersion objective (Carl Zeiss). Z stacks with an interval of 110ௗnm were used to scan the whole cells. The raw images were reconstructed using ZEN software under the default parameters (Carl Zeiss).MBHB Ref. No.23-1150-WO Immunoprecipitation [000147] A 60-mm dish of transfected cells were lysed in the buffer that contain 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, and 0.1% SDS together with phosphatase and protease inhibitor.1mg of cell lysates were suspended in 1 ml lysis buffer and incubated with 5 μl of anti-FLAG M2 beads (Sigma-Aldrich) for overnight at 4°C under gentle rotation. Beads were then washed three times with the lysis buffer that contained protease inhibitors. The immunocomplex were eluted by SDS sample buffer and separated by gels for western blot analysis. Quantification of images from immunostaining [000148] For quantification of fluorescent intensity with proteins at the centrioles and transition zone and quantification of cilia length, all cells were treated the same during the process of immunostaining and image acquisition. The ZEN software (Carl Zeiss) was used to analyze the image intensity and length. The same setting was applied to all images. A circle was drawn surrounding the centrioles and transition zone that the total pixel value of the marked region was then measured. The signal ratio of the marked region over the proteins at the centrioles was then normalized to the control group. All the quantifications were obtained from at least three experiments. Morpholino antisense oligonucleotides for injection into Zebrafish Embryos [000149] Wild type adult zebrafish were subjected to breeding events. The embryo in single cell stage were washed and collected in E3 water (water containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) in a petri dish. The two morpholinos were purchased from Gene Tool Company: NDUFAF2-MO1, targeting the translation 5’-UTR of NDUFAF2 (5’- TTGTACTGCACATGCAAACACGTTC-3’ (SEQ ID NO.:9)), NDUFAF2- MO2, targeting the translation start codon site of NDUFAF2 (5’- CAATGCGGCTCATCTCTGTGATTTA-3’(SEQ ID NO.:10)), and 0.4 mM MOs and 100 ng / uL RNA with a final concentration of 0.1% phenol red (Sigma-Aldrich, P0290) wereMBHB Ref. No.23-1150-WO injected into 1 cell stage embryos at nominated concentrations by using a PLI-100A microinjector (Harvard Medical Apparatus). After microinjection, embryos were placed into a Petri dish with E3 medium and incubated them at 28°C for development. Microinjection of in vitro transcribed mRNA into zebrafish embryos [000150] The cDNA of NDUFAF2 clone into a pcDNA3.1 vector and perform an in vitro synthesis reaction of large amounts of capped RNA using the mMESSAGE mMACHINE™ T7 Transcription Kit (Invitrogen™ Ambion, AM1344M). Then, RNA sample are purified by running it through the RNeasy Mini kit (Qiagen, 74104). On the day of the injection, 100 ng / uL RNA sample were thawed, then vortexed lightly and spinned down briefly with a final concentration of 0.1% phenol red (Sigma-Aldrich, P0290). TEM imaging [000151] CJ and JB cells were grown on Aclar film (Electron Microscopy Sciences, Hatfield, PA, USA)-based coverslips and fixed in 4% paraformaldehyde (15710, Electron Microscopy Sciences) and 2.5% glutaraldehyde (G5882, Sigma-Aldrich, St. Louis, MO, USA) in PBS buffer at 37°C for 30 min. Cells were then further postfixed in 1% Osmium Tetroxide (OsO4) in PBS buffer for 30 min on ice. After dehydrating in a graded series of ethanol, the cells were then infiltrated and embedded in EPON812 resin (Catalog #14120, Electron Microcopy Sciences) to generate a resin sample. A microtome (Ultracut UC6; Leica, Wetzlar, Germany) was used to cut the sample into serial sections (~90 nm thickness), which were then stained with 1% uranyl acetate and 1% lead citrate. Samples were imaged using the JEOL, JEM1400 transmission electron microscope with a LaB6 emitter. RT-PCR [000152] Real-time PCR was carried out in an Applied Biosystems Inc.(ABI) StepOne™ Real-Time PCR System using the Maxima SYBR Green / ROX qPCR Master Mix (2X) (Thermo Fisher Scientific, K0221). Primers for one rhodopsin marker, two transducin markers (gnat1 and gnat2), SHH signal markers (shh, Gli1 and PTCH1) and two nuclear DNA markers (ȕ-actin and GAPDH) were used. Amplifications were performed in 20 ^lMBHB Ref. No.23-1150-WO reactions comprising of 100 ng total DNA, 1X SYBR-Green PCR Master Mix with 0.5 ^M each primer. Triplicate reactions were performed for each marker in a 96-well plate using a three-step amplification program of initial denaturation at 95^ for 5 min, followed by 40 cycles of 95^ for 15 sec, 60^ for 30 sec and 72^ for 30 sec. The comparative cycle threshold (Ct) method was used to analyze the data by generating relative values of the amount of rhodopsin and transducin. Relative content was calculated after determination of the difference between Ct of the given rhodopsin marker and two transducin markers and that of the calibrator nuclear marker GAPDH. Each measurement was repeated in triplicate and a non-template control was included in each experiment. The sequences of each gene were: Rhodopsin (zebrafish), forward: 5’-AGTCCTGCCCAGACATCTAG-3’ (SEQ ID NO.:1); Rhodopsin, reverse: 5’-GTACTGTGGGTATTCGTATGGG-3’ (SEQ ID NO.:2); gnat1 (zebrafish), forward: 5’-CGTCAAGTTTGTGTTCGATGC-3’ (SEQ ID NO.:3); gnat1 (zebrafish), reverse: 5’-GAGGAAACGAGCTACAAGGAG-3’ (SEQ ID NO.:4); gnat2 (zebrafish), forward: 5’-CAAACCTGACTACCTTCCCAC-3’ (SEQ ID NO.:5); gnat2 (zebrafish), reverse: 5’-TCTTCCTCTCGGACCTCTG-3’ (SEQ ID NO.:6); ȕ-actin (zebrafish), forward: 5’-TGCTGTTTTCCCCTCCATTG-3’ (SEQ ID NO.:7); ȕ-actin (zebrafish), reverse: 5’-GTCCCATGCCAACCATCACT-3’ (SEQ ID NO.:8). Oxygen consumption rate (OCR) measurements [000153] OCR was measured using a Seahorse Biosciences XFe96 extracellular flux analyzer.1.25x105cells per well were seeded in XFe96 cell culture plates. Attachment of the cells was monitored after 24h, and cells were incubated overnight at 37°C with 5% CO2. Before the assay, cells were changed by Seahorse XF DMEM medium with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose and equilibrated for 1 h at 37^ without CO2. The OCR was measured afterwards using the following inhibitors: 2.5 ^M oligomycin, 2 ^M carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and 0.5 ^M rotenone with 0.5 ^M antimycin A (Agilent Technologies, 103015-100). For each condition, cycles were performed in triplicate with 3 min mixing followed by 3 min measurement. After completionMBHB Ref. No.23-1150-WO of the assay, the cell number per well was determined using the Cytation 5. The OCR was normalized to the cell number for each well. Determining catalytic activity of Complex I [000154] The fibroblast lysate were suspended in in 1 ml of 10 mM ice-cold hypotonic Tris buffer (pH 7.6). Then, using 26G needle took up and expelled the suspension for several times until it had a homogeneous solution. After that, 200 μl of 1.5 M sucrose solution added to the homogeneous solution and centrifuged at 600g for 10 min at 4 °C. Collecting the supernatant was centrifuged at 14,000g for 10 min at 4 °C. Then, the mitochondrial pellet suspended in 0.5 ml of 10 mM ice-cold hypotonic Tris buffer (pH 7.6). The homogeneous solution was frozen and thawed in liquid nitrogen and at 37 °C for 3 times. Enriched mitochondrial fractions were purified from fibroblasts lysate. The analysis of complex I activity in cells by performing rotenone-sensitive activities followed by detecting 340nm wavelength. Methods for assessing mitochondrial respiratory chain enzymatic activities on tissues and cultured cells NAD+ / NADH ratio [000155] The protocol was followed by abcam NAD / NADH Assay Kit (ab65348).2x106of cells were washed in 1x PBS and lysed in 500 ^l buffer (0.25M sucrose, 10mM Tris-HCl, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1mM EDTA, 1mM dithiothreitol and protease inhibitor). Cells were on ice for 15 mins and to shear cells by using 26G needle. And then, homogenates were centrifuged at 700g for 5 min at 4°C, and supernatants were collected and centrifuged at 10000g for 30 min at 4°C. The pellets were the mitochondrial fraction. The pellets were resuspended in 400^l NADH / NAD extraction buffer and extracted cells by two freeze / thaw cycles (20 mins on dry ice followed by 10 mins at RT). Then, extractions were vortexed for 10 sec. Extraction centrifuged for 5 minutes at 4°C at top speed in a cold microcentrifuge to remove any insoluble material. The supernatant was extracted, and enzyme was removed by passing through a 10 kD Spin Column (ab93349) before performing the assay. For preparing NADH sample, Aliquoting half of extraction and heating sample at 60°C for 30 mins. TheseMBHB Ref. No.23-1150-WO extractions were NAD decomposed samples. Preparing the standard curve and loading the sample into 96 wells followed by abcam NAD / NADH Assay Kit (ab65348). Proximity ligation assays (PLA) [000156] The protocol was followed by published paper (ref) (Duolink® In Situ Red Starter Kit Mouse / Rabbit; Milipore sigma; DUO92101). The 5x105of RPE, NDUFAF2- / -, NDUFAF2WT and NDUFAF2G258A cell were seed on the coverslides. The slides were fixed with 4% PFA in PBS for 20 minutes at room temperature (RT). For ARMC9 antibody, it was followed by fixing in ice-cold 100% methanol for 15 minutes at -20°C. Before the assay, the slides were washed twice with PBS then blocked cells with Duolink® block solution for 1 hr at RT. During PLA assays, the primary antibodies were diluted in Duolink® antibody dilution buffer and stained overnight at 4°C. Next day, the slides were washed with large volumn of 5% BSA in PBS for 10 minutes. Before the PLA assay, the 20 ^l total volume per reaction of secondary antibodies were prepared with 4 ^l PLUS antibody and 4 ^l MINUS antibody + 12 ^l Duolink® ab dilution buffer for 20 min at RT. After that, the secondary antibodies were added into slides and incubated at 37°C for 1 hr. Before ligation process of PLA assay, the slides were washed by 1x of buffer A twice for 5 minutes. During ligation process, the 20 ^l total volume per reaction of ligation mix were prepared added into slides with 4 ^l ligation stock,15.5^l ddH2O and 0.5 ^l ligase for 30 min at 37°C. Before amplification process of PLA assay, the slides were washed by 1x of buffer A twice for 5 minutes. During amplification process, the 20 ^l total volume per reaction of amplification mix were prepared and added into slides with 4 ^l amplification stock,15.75^l ddH2O and 0.25 ^l polymerase for 100 min at 37°C. After that, the slides were washed twice with 1x of buffer B each for 10 min. Then, the slides were washed once with 0.01x of buffer B for 1 min. The final step was to aspirate buffer B and then mounted Prolong Gold mounting medium with DAPI. Curr Protoc Immunol.2018 Nov; 123(1): e58. Published online 2018 Sep 20. doi: 10.1002 / cpim.58 Image analysis of mitochondrial lengthMBHB Ref. No.23-1150-WO [000157] Analyzing mitochondrial dynamics from fluorescence microscope images was followed by Mytoe. It was a software from Dr. Andre Ribeiro’s Lab (Lihavainen, et al. Bioinformatics, 2012). Eye-tracking study protocol [000158] Device used was the Tobii Pro Spectrum eye-tracking device was utilized for the study. This device is known for its high precision and accuracy in capturing eye movements. Tobii Pro Lab software was employed to control and analyze the eye-tracking data. This software provides comprehensive tools for data collection and analysis. The participant was positioned at an approximate distance of 61cm from the screen. This distance was chosen to ensure optimal tracking accuracy and participant comfort. A custom-designed fixation object was presented on the screen as the visual stimulus for the participant. The choice of a cartoon aimed to engage the participant and maintain their attention during the eye-tracking session. The participant's eye movements were recorded using infrared oculography. This technology utilizes infrared light to track the position and movements of the participant's eyes accurately. Throughout the session, the Tobii Pro Spectrum device captured the participant's eye movements, including fixations, saccades, and other relevant eye-tracking metrics. The data collected provided insights into the participant's visual attention and gaze behavior. Following the eye-tracking session, the collected data was analyzed using the Tobii Pro Lab software. Various eye-tracking metrics, such as gaze duration, fixation counts, and scan patterns, were examined to gain deeper insights into the participant's visual processing and attentional patterns. Statistical data analysis [000159] All data are representing as mean with standard deviation (SD) from at least 3 independent experiments. Experiment samples and numbers for statistical testing have been reported in the corresponding figure legends. All p-values are from the Student’s t-tests for two-group comparison (GraphPad Prism 8). n.s., not significant; P<0.001(***); P<0.01(**); P<0.05(*).MBHB Ref. No.23-1150-WO [000160] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments claimed. Thus, it should be understood that although the present description has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of these embodiments as defined by the description and the appended claims. Although some aspects of this disclosure can be identified herein as particularly advantageous, it is contemplated that this disclosure is not limited to these particular aspects of the disclosure. [000161] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. [000162] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. [000163] It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is / are referred to as comprising particular elements and / or features, certainMBHB Ref. No.23-1150-WO embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. SEQUENCESNDUFAF2 Full-length (SEQ ID NO.:11) 1 acccctactg cgggtcccgc tgctggcagc gctggaaact gggtggacgg catgggttgg 61 tctcaggatt tgttccgcgc cttgtggaga tcgctgtcaa gggaagtgaa ggagcacgtg 121 ggcacggacc aattcgggaa caaatactac tacatcccgc agtacaagaa ctggagagga 181 caaactattc gagagaaaag aattgtagaa gcagcaaata aaaaagaagt agactatgaa 241 gcaggggata ttccaacaga atgggaagct tggattagaa gaacaagaaa gactccacct 301 actatggagg aaatactaaa gaatgaaaaa cacagagaag aaatcaaaat aaaaagccaa 361 gatttttatg aaaaagaaaa actccttagt aaagagacca gtgaggaact cctgcctccaMBHB Ref. No.23-1150-WO 421 ccagttcaaa ctcaaattaa aggccatgcc tctgctccat actttggaaa ggaagaaccc 481 tcagtggctc ccagcagcac tggtaaaacc tttcagccag gatcctggat gccacgagat 541 ggcaagagcc acaatcaatg aatgcattat ggtcaaatct tttcatgtat atggatgtga 601 ctattttaac aaataaaaga agtgaaaagt ta NDUFAF2 Full-length (SEQ ID NO.:12) MGWSQDLFRA LWRSLSREVK EHVGTDQFGN KYYYIPQYKN WRGQTIREKR IVEAANKKEV DYEAGDIPTE WEAWIRRTRK TPPTMEEILK NEKHREEIKI KSQDFYEKEK LLSKETSEEL LPPPVQTQIK GHASAPYFGK EEPSVAPSST GKTFQPGSWM PRDGKSHNQMBHB Ref. No.23-1150-WO References 1. Burkhalter MD, Sridhar A, Sampaio P, Jacinto R, Burczyk MS, Donow C, Angenendt M; Competence Network for Congenital Heart Defects Investigators; Hempel M, Walther P, Pennekamp P, Omran H, Lopes SS, Ware SM, Philipp M. Imbalanced mitochondrial function provokes heterotaxy via aberrant ciliogenesis. J Clin Invest. 2019 May 16;129(7):2841-2855. doi: 10.1172 / JCI98890. PMID: 31094706; PMCID: PMC6597216. 2. Gonçalves J, Pelletier L. The Ciliary Transition Zone: Finding the Pieces and Assembling the Gate. Mol Cells.2017 Apr;40(4):243-253. doi: 10.14348 / molcells.2017.0054. Epub 2017 Apr 12. PMID: 28401750; PMCID: PMC5424270. 3. Latour BL, Van De Weghe JC, Rusterholz TD, Letteboer SJ, Gomez A, Shaheen R, Gesemann M, Karamzade A, Asadollahi M, Barroso-Gil M, Chitre M, Grout ME, van Reeuwijk J, van Beersum SE, Miller CV, Dempsey JC, Morsy H; University of Washington Center for Mendelian Genomics; Bamshad MJ; Genomics England Research Consortium; Nickerson DA, Neuhauss SC, Boldt K, Ueffing M, Keramatipour M, Sayer JA, Alkuraya FS, Bachmann-Gagescu R, Roepman R, Doherty D. Dysfunction of the ciliary ARMC9 / TOGARAM1 protein module causes Joubert syndrome. J Clin Invest.2020 Aug 3;130(8):4423-4439. doi: 10.1172 / JCI131656. PMID: 32453716; PMCID: PMC7410078. 4. Lihavainen E, Mäkelä J, Spelbrink JN, Ribeiro AS. Mytoe: automatic analysis of mitochondrial dynamics. Bioinformatics.2012 Apr 1;28(7):1050-1. doi: 10.1093 / bioinformatics / bts073. Epub 2012 Feb 9. PMID: 22321700.

Claims

MBHB Ref. No.23-1150-WO What is claimed is:

1. A method for restoring cilia function in a cell that expresses a defective variant of a first gene needed for normal cilia function by expressing or overexpressing a second gene in the cell that when expressed therein restores cilia function in the cell, wherein the defective first gene can be ARMC9 and / or NDUFAF2.

2. The method of claim 1, wherein the second gene is NDUFAF2.

3. A method for restoring cilia function in a cell that under-expresses or does not express a first gene needed for normal cilia function, comprising introducing into the cell a second gene that when expressed in the cell restores cilia function, wherein the defective first gene can be ARMC9 and / or NDUFAF2.

4. The method of claim 3, wherein the second gene is NDUFAF2.

5. A method of treating or ameliorating ciliopathies in a subject that expresses a defective variant of a first gene needed for normal cilia function, the method comprising the steps of introducing into cells in the subject having the defective variant of the first gene a recombinant expression construct, wherein the construct comprises a second gene that when expressed is capable of correcting the ciliopathy in the subject, wherein the construct is delivered into an epithelial cell in the subject, and wherein the defective first gene can be ARMC9 and / or NDUFAF2.

6. The method of claim 5, wherein the recombinant expression construct comprises an adeno- associated virus (AAV).

7. The method of claim 5, wherein the ciliopathy causes a kidney defect.

8. The method of claim 5, wherein the epithelial cell is a renal tubular epithelial cell.MBHB Ref. No.23-1150-WO 9. The method of claims 5 or 6, wherein the AAV construct is introduced into the subject through the subject’s renal collecting duct.

10. The method of any of claims 5-9, wherein the treating results in reduction of ciliary defect symptoms.

11. The method of claim 5, wherein the second gene is NDUFAF2.

12. The method of claim 5, further comprising administration of a therapeutically effective amount of a pharmaceutical formulation of a nicotinamide compound.

13. A method of treating a subject with a ciliopathy comprising: (a) obtaining a biological sample from the subject, (b) detecting DNA from the sample encoding a gene comprising at least one mutation or variant associated with cilia dysfunction, and (c) administering to the subject with the DNA mutation or variant associated with cilia dysfunction a nicotinamide pharmaceutical composition in an amount effective to maintain or improve cilia function.

14. The method of claim 13, wherein the nicotinamide pharmaceutical composition comprises an active pharmaceutical ingredient that is NAD+.

15. The method of any of claims 14, wherein the composition further comprises a pharmaceutically acceptable carrier, a solvent, an adjuvant, a diluent, or a combination thereof.

16. The method of any of claims 13, wherein the composition is delivered systematically.MBHB Ref. No.23-1150-WO 17. The method of any of claim 16, wherein the composition is administered by oral, buccal, rectal, parenteral, intraperitoneal, intradermal, intracranial, transdermal, intrathecal, intracranial, intravenous, or intracerebral administration.

18. The method of claim 17, wherein the composition is administered orally.

19. The method of any of claims 13-17, wherein the subject has a ciliopathy selected from the group consisting of Joubert syndrome, developmental delay, anosmia, renal cysts, and photoreceptor degeneration.

20. The method of claim 19, wherein the ciliopathy is Joubert syndrome.

21. A pharmaceutical composition comprising: a recombinant expression construct encoding a gene for treating a cilia related disorder, wherein the gene is ARMC9 and / or NDUFAF2.

22. The pharmaceutical composition of claim 21, wherein the recombinant expression construct comprises an adeno-associated virus (AAV).

23. The pharmaceutical composition of claim 22, wherein the composition further comprises an NAD+ supplement 24. The pharmaceutical composition of claim 23, is administered to a subject at a therapeutically effective dose.

25. The pharmaceutical composition of claim 24, wherein the NAD+ supplement is administered as a tablet or liquid formulation.

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