Delivering large genes using adeno-associated virus and the CRE-lox DNA recombination system.

Adeno-associated virus (AAV) is a safe and efficient gene delivery vehicle for gene therapies. However, its relatively small packaging capacity limits its use as a gene transfer vector. Here, we describe a strategy to deliver large genes that exceed the AAV's packaging capacity using up to four AAV vectors and the CRE-lox DNA recombination system. We devised novel lox sites by combining non-compatible and reaction equilibrium-modifying lox site variants. These lox sites facilitate sequence-specific and near-unidirectional recombination of AAV vector genomes, enabling efficient reconstitution of up to 16 kb of therapeutic genes in a pre-determined configuration. Using this strategy, we have developed AAV gene therapy vectors to deliver IFT140 , PCDH15 , CEP290 , and CDH23 and demonstrate efficient production of full-length proteins in cultured mammalian cells and mouse retinas. Notably, this approach significantly surpasses the trans-splicing and split-intein-based reconstitution methods in efficiency, requiring lower doses, minimizing or eliminating the production of truncated protein products, and offering flexibility in selecting splitting positions. The CRE-lox approach described here provides a simple and effective platform for producing AAV gene therapy vectors beyond AAV's packaging capacity.

Subretinal gene therapy delays vision loss in a Bardet-Biedl Syndrome type 10 mouse model.

Blindness in Bardet-Biedl syndrome (BBS) is caused by dysfunction and loss of photoreceptor cells in the retina. BBS10, mutations of which account for approximately 21% of all BBS cases, encodes a chaperonin protein indispensable for the assembly of the BBSome, a cargo adaptor important for ciliary trafficking. The loss of BBSome function in the eye causes a reduced light sensitivity of photoreceptor cells, photoreceptor ciliary malformation, dysfunctional ciliary trafficking, and photoreceptor cell death. Cone photoreceptors lacking BBS10 have congenitally low electrical function in electroretinography. In this study, we performed gene augmentation therapy by injecting a viral construct subretinally to deliver the coding sequence of the mouse Bbs10 gene to treat retinal degeneration in a BBS10 mouse model. Long-term efficacy was assessed by measuring the electrical functions of the retina over time, imaging of the treated regions to visualize cell survival, conducting visually guided swim assays to measure functional vision, and performing retinal histology. We show that subretinal gene therapy slowed photoreceptor cell death and preserved retinal function in treated eyes. Notably, cone photoreceptors regained their electrical function after gene augmentation. Measurement of functional vision showed that subretinal gene therapy provided a significant benefit in delaying vision loss.

Progressive retinal degeneration of rods and cones in a Bardet-Biedl syndrome type 10 mouse model.

Bardet-Biedl syndrome (BBS) is a multi-organ autosomal-recessive disorder caused by mutations in at least 22 different genes. A constant feature is early-onset retinal degeneration leading to blindness. Among the most common forms is BBS type 10 (BBS10), which is caused by mutations in a gene encoding a chaperonin-like protein. To aid in developing treatments, we phenotyped a Bbs10 knockout (Bbs10-/-) mouse model. Analysis by optical coherence tomography (OCT), electroretinography (ERG) and a visually guided swim assay (VGSA) revealed a progressive degeneration (from P19 to 8 months of age) of the outer nuclear layer that is visible by OCT and histology. Cone ERG was absent from at least P30, at which time rod ERG was reduced to 74.4% of control levels; at 8 months, rod ERG was 2.3% of that of controls. VGSA demonstrated loss of functional vision at 9 months. These phenotypes progressed more rapidly than retinal degeneration in the Bbs1M390R/M390R knock-in mouse. This study defines endpoints for preclinical trials that can be utilized to detect a treatment effect in the Bbs10-/- mouse and extrapolated to human clinical trials.

Differential requirement of NPHP1 for compartmentalized protein localization during photoreceptor outer segment development and maintenance.

Nephrocystin (NPHP1) is a ciliary transition zone protein and its ablation causes nephronophthisis (NPHP) with partially penetrant retinal dystrophy. However, the precise requirements of NPHP1 in photoreceptors are not well understood. Here, we characterize retinal degeneration in a mouse model of NPHP1 and show that NPHP1 is required to prevent infiltration of inner segment plasma membrane proteins into the outer segment during the photoreceptor maturation. We demonstrate that Nphp1 gene-trap mutant mice, which were previously described as null, are likely hypomorphs due to the production of a small quantity of functional mRNAs derived from nonsense-associated altered splicing and skipping of two exons including the one harboring the gene-trap. In homozygous mutant animals, inner segment plasma membrane proteins such as syntaxin-3 (STX3), synaptosomal-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2) accumulate in the outer segment when outer segments are actively elongating. This phenotype, however, is spontaneously ameliorated after the outer segment elongation is completed. Consistent with this, some photoreceptor cell loss (~30%) occurs during the photoreceptor maturation period but it stops afterward. We further show that Nphp1 genetically interacts with Cep290, another NPHP gene, and that a reduction of Cep290 gene dose results in retinal degeneration that continues until adulthood in Nphp1 mutant mice. These findings demonstrate that NPHP1 is required for the confinement of inner segment plasma membrane proteins during the outer segment development, but its requirement diminishes as photoreceptors mature. Our study also suggests that additional mutations in other NPHP genes may influence the penetrance of retinopathy in human NPHP1 patients.

Photoreceptor cilia, in contrast to primary cilia, grant entry to a partially assembled BBSome.

The BBSome is a protein complex consisting of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 that associates with intraflagellar transport complexes and specializes in ciliary trafficking. In primary cilia, ciliary entry requires the fully assembled BBSome as well as the small GTPase, ARL6 (BBS3). Retinal photoreceptors possess specialized cilia. In light of key structural and functional differences between primary and specialized cilia, we examined the principles of BBSome recruitment to photoreceptor cilia. We performed sucrose gradient fractionation using retinal lysates of Bbs2-/-, Bbs7-/-, Bbs8-/- and Bbs3-/- mice to determine the status of BBSome assembly, then determined localization of BBSome components using immunohistochemistry. Surprisingly, we found that a subcomplex of the BBSome containing at least BBS1, BBS5, BBS8 and BBS9 is recruited to cilia in the absence of BBS2 or BBS7. In contrast, a BBSome subcomplex consisting of BBS1, BBS2, BBS5, BBS7 and BBS9 is found in Bbs8-/- retinas and is denied ciliary entry in photoreceptor cells. In addition, the BBSome remains fully assembled in Bbs3-/- retinas and can be recruited to photoreceptor cilia in the absence of BBS3. We compared phenotypic severity of their retinal degeneration phenotypes. These findings demonstrate that unlike primary cilia, photoreceptor cilia admit a partially assembled BBSome meeting specific requirements. In addition, the recruitment of the BBSome to photoreceptor cilia does not require BBS3. These findings indicate that the ciliary entry of the BBSome is subjected to cell-specific regulation, particularly in cells with highly adapted forms of cilia such as photoreceptors.

Limited time window for retinal gene therapy in a preclinical model of ciliopathy.

Retinal degeneration is a common clinical feature of ciliopathies, a group of genetic diseases linked to ciliary dysfunction, and gene therapy is an attractive treatment option to prevent vision loss. Although the efficacy of retinal gene therapy is well established by multiple proof-of-concept preclinical studies, its long-term effect, particularly when treatments are given at advanced disease stages, is controversial. Incomplete treatment and intrinsic variability of gene delivery methods may contribute to the variable outcomes. Here, we used a genetic rescue approach to 'optimally' treat retinal degeneration at various disease stages and examined the long-term efficacy of gene therapy in a mouse model of ciliopathy. We used a Bardet-Biedl syndrome type 17 (BBS17) mouse model, in which the gene-trap that suppresses Bbs17 (also known as Lztfl1) expression can be removed by tamoxifen administration, restoring normal gene expression systemically. Our data indicate that therapeutic effects of retinal gene therapy decrease gradually as treatments are given at later stages. These results suggest the presence of limited time window for successful gene therapy in certain retinal degenerations. Our study also implies that the long-term efficacy of retinal gene therapy may depend on not only the timing of treatment but also other factors such as the function of mutated genes and residual activities of mutant alleles.

The absence of BBSome function decreases synaptogenesis and causes ectopic synapse formation in the retina.

Photoreceptors possess ribbon synapses distinct from the conventional synapses in the brain. Little is known about the function of the BBSome, a complex integral in ciliary and intracellular trafficking, in ribbon synaptic formation. We performed immunohistochemistry using retinas from Bardet-Biedl Syndrome (BBS) mouse models and found that BBS mutant animals have significantly fewer ribbon synapses in the outer plexiform layer and increased ectopic synapses in the outer nuclear layer compared to controls. Many ectopic synapses in BBS mutant retinas are associated with horizontal cell axonal processes that aberrantly intrude into the outer nuclear layer. To determine whether this horizontal cell phenotype is a consequence of retinal degeneration, we examined this phenotype in mice with photoreceptor-specific inactivation of the BBSome induced by Cre recombinase driven by the rhodopsin promoter. At three months of age, despite retinal degeneration, Bbs8floxed/floxed; Rho-Cre+ mice lack the aberrant intrusion of horizontal cell processes. At 6 months, some horizontal cell processes intrude into the outer nuclear layer in Bbs8floxed/floxed; Rho-Cre+ mice, but the phenotype does not recapitulate the phenotypic severity observed in young congenital BBS mutant mice. Therefore, the lack of BBSome function negatively impacts retinal synaptogenesis, and causes horizontal cell defects in a potentially cell-autonomous fashion.

The myosin-tail homology domain of centrosomal protein 290 is essential for protein confinement between the inner and outer segments in photoreceptors.

Mutations in the centrosomal protein 290 (CEP290) gene cause various ciliopathies involving retinal degeneration. CEP290 proteins localize to the ciliary transition zone and are thought to act as a gatekeeper that controls ciliary protein trafficking. However, precise roles of CEP290 in photoreceptors and pathomechanisms of retinal degeneration in CEP290-associated ciliopathies are not sufficiently understood. Using conditional Cep290 mutant mice, in which the C-terminal myosin-tail homology domain of CEP290 is disrupted after the connecting cilium is assembled, we show that this domain is essential for protein confinement between the inner and the outer segments. Upon disruption of the myosin-tail homology domain, inner segment plasma membrane proteins, including syntaxin 3 (STX3), synaptosome-associated protein 25 (SNAP25), and interphotoreceptor matrix proteoglycan 2 (IMPG2), rapidly accumulated in the outer segment. In contrast, localization of endomembrane proteins was not altered. Trafficking and confinement of most outer segment-resident proteins appeared to be unaffected or only minimally affected in Cep290 mutant mice. One notable exception was rhodopsin (RHO), which severely mislocalized to inner segments during the initial stage of degeneration. Similar mislocalization phenotypes were observed in Cep290rd16 mice. These results suggest that a failure of protein confinement at the connecting cilium and consequent accumulation of inner segment membrane proteins in the outer segment, along with insufficient RHO delivery, is part of the disease mechanisms that cause retinal degeneration in CEP290-associated ciliopathies. Our study provides insights into the pathomechanisms of retinal degenerations associated with compromised ciliary gates.

Genotypic and phenotypic characterization of the Sdccag8Tn(sb-Tyr)2161B.CA1C2Ove mouse model.

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of disorders that present with end-stage renal failure in childhood/adolescence, kidney cysts, retinal degeneration, and cerebellar hypoplasia. One disorder that shares clinical features with NPHP-RC is Bardet-Biedl Syndrome (BBS). Serologically defined colon cancer antigen 8 (SDCCAG8; also known as NPHP10 and BBS16) is an NPHP gene that is also associated with BBS. To better understand the patho-mechanisms of NPHP and BBS caused by loss of SDCCAG8 function, we characterized an SDCCAG8 mouse model (Sdccag8Tn(sb-Tyr)2161B.CA1C2Ove) generated by Sleeping Beauty Transposon (SBT)-mediated insertion mutagenesis. Consistent with the previously reported, independent SDCCAG8 mouse models, our mutant mice display pre-axial polydactyly in their hind limbs. In addition, we report patterning defects in the secondary palate, brain abnormalities, as well as neonatal lethality associated with developmental defects in the lung in our mouse model. The neonatal lethality phenotype is genetic background dependent and rescued by introducing 129S6/SvEvTac background. Genetic modifier(s) responsible for this effect were mapped to a region between SNPs rs3714172 and rs3141832 on chromosome 11. While determining the precise genetic lesion in our mouse model, we found that SBT insertion resulted in a deletion of multiple exons from both Sdccag8 and its neighboring gene Akt3. We ascribe the patterning defects in the limb and the secondary palate as well as lung abnormalities to loss of SDCCAG8, while the developmental defects in the brain are likely due to the loss of AKT3. This mouse model may be useful to study features not observed in other SDCCAG8 models but cautions are needed in interpreting data.

BBSome function is required for both the morphogenesis and maintenance of the photoreceptor outer segment.

Genetic mutations disrupting the structure and function of primary cilia cause various inherited retinal diseases in humans. Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, pleiotropic ciliopathy characterized by retinal degeneration, obesity, postaxial polydactyly, intellectual disability, and genital and renal abnormalities. To gain insight into the mechanisms of retinal degeneration in BBS, we developed a congenital knockout mouse of Bbs8, as well as conditional mouse models in which function of the BBSome (a protein complex that mediates ciliary trafficking) can be temporally inactivated or restored. We demonstrate that BBS mutant mice have defects in retinal outer segment morphogenesis. We further demonstrate that removal of Bbs8 in adult mice affects photoreceptor function and disrupts the structural integrity of the outer segment. Notably, using a mouse model in which a gene trap inhibiting Bbs8 gene expression can be removed by an inducible FLP recombinase, we show that when BBS8 is restored in immature retinas with malformed outer segments, outer segment extension can resume normally and malformed outer segment discs are displaced distally by normal outer segment structures. Over time, the retinas of the rescued mice become morphologically and functionally normal, indicating that there is a window of plasticity when initial retinal outer segment morphogenesis defects can be ameliorated.

Photoreceptor outer segment as a sink for membrane proteins: hypothesis and implications in retinal ciliopathies.

The photoreceptor outer segment (OS) is a unique modification of the primary cilium, specialized for light perception. Being homologous organelles, the primary cilium and the OS share common building blocks and molecular machinery to construct and maintain them. The OS, however, has several unique structural features that are not seen in primary cilia. Although these unique features of the OS have been well documented, their implications in protein localization have been under-appreciated. In this review, we compare the structural properties of the primary cilium and the OS, and propose a hypothesis that the OS can act as a sink for membrane proteins. We further discuss the implications of this hypothesis in polarized protein localization in photoreceptors and mechanisms of photoreceptor degeneration in retinal ciliopathies.

RPGR, a prenylated retinal ciliopathy protein, is targeted to cilia in a prenylation- and PDE6D-dependent manner.

RPGR (retinitis pigmentosa GTPase regulator) is a ciliary protein associated with several forms of inherited retinal degenerative diseases. PDE6D is a ubiquitously expressed prenyl-binding protein and involved in ciliary targeting of prenylated proteins. The current working model for the RPGR function depicts that RPGR acts as a scaffold protein to recruit cargo-loaded PDE6D to primary cilia. Here, we present evidence demonstrating an alternative relationship between RPGR and PDE6D, in which RPGR is a cargo of PDE6D for ciliary targeting. We found that the constitutive isoform of RPGR, which is prenylated, requires prenylation for its ciliary localization. We also found that there are at least two independent ciliary targeting signals in RPGR: one within the N-terminal region that contains the RCC1-like domain and the other near the prenylation site at the C-terminus. Ablation of PDE6D blocked ciliary targeting of RPGR. Our study indicates that prenylated RPGR is one of the cargos of PDE6D for ciliary trafficking and provides insight into the mechanisms by which RPGR is targeted to cilia.

Gadd45a Protein Promotes Skeletal Muscle Atrophy by Forming a Complex with the Protein Kinase MEKK4.

Skeletal muscle atrophy is a serious and highly prevalent condition that remains poorly understood at the molecular level. Previous work found that skeletal muscle atrophy involves an increase in skeletal muscle Gadd45a expression, which is necessary and sufficient for skeletal muscle fiber atrophy. However, the direct mechanism by which Gadd45a promotes skeletal muscle atrophy was unknown. To address this question, we biochemically isolated skeletal muscle proteins that associate with Gadd45a as it induces atrophy in mouse skeletal muscle fibers in vivo We found that Gadd45a interacts with multiple proteins in skeletal muscle fibers, including, most prominently, MEKK4, a mitogen-activated protein kinase kinase kinase that was not previously known to play a role in skeletal muscle atrophy. Furthermore, we found that, by forming a complex with MEKK4 in skeletal muscle fibers, Gadd45a increases MEKK4 protein kinase activity, which is both sufficient to induce skeletal muscle fiber atrophy and required for Gadd45a-mediated skeletal muscle fiber atrophy. Together, these results identify a direct biochemical mechanism by which Gadd45a induces skeletal muscle atrophy and provide new insight into the way that skeletal muscle atrophy occurs at the molecular level.

Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in Bardet-Biedl syndrome.

Compartmentalization and polarized protein trafficking are essential for many cellular functions. The photoreceptor outer segment (OS) is a sensory compartment specialized for phototransduction, and it shares many features with primary cilia. As expected, mutations disrupting protein trafficking to cilia often disrupt protein trafficking to the OS and cause photoreceptor degeneration. Bardet-Biedl syndrome (BBS) is one of the ciliopathies associated with defective ciliary trafficking and photoreceptor degeneration. However, precise roles of BBS proteins in photoreceptor cells and the underlying mechanisms of photoreceptor degeneration in BBS are not well understood. Here, we show that accumulation of non-OS proteins in the OS underlies photoreceptor degeneration in BBS. Using a newly developed BBS mouse model [Leucine zipper transcription factor-like 1 (Lztfl1)/Bbs17 mutant], isolated OSs, and quantitative proteomics, we determined 138 proteins that are enriched more than threefold in BBS mutant OS. In contrast, only eight proteins showed a more than threefold reduction. We found striking accumulation of Stx3 and Stxbp1/Munc18-1 and loss of polarized localization of Prom1 within the Lztfl1 and Bbs1 mutant OS. Ultrastructural analysis revealed that large vesicles are formed in the BBS OS, disrupting the lamellar structure of the OS. Our findings suggest that accumulation (and consequent sequestration) of non-OS proteins in the OS is likely the primary cause of photoreceptor degeneration in BBS. Our data also suggest that a major function of BBS proteins in photoreceptors is to transport proteins from the OS to the cell body or to prevent entry of non-OS proteins into the OS.

INPP5E interacts with AURKA, linking phosphoinositide signaling to primary cilium stability.

Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause the ciliopathies known as Joubert and MORM syndromes; however, the role of INPP5E in ciliary biology is not well understood. Here, we describe an interaction between INPP5E and AURKA, a centrosomal kinase that regulates mitosis and ciliary disassembly, and we show that this interaction is important for the stability of primary cilia. Furthermore, AURKA phosphorylates INPP5E and thereby increases its 5-phosphatase activity, which in turn promotes transcriptional downregulation of AURKA, partly through an AKT-dependent mechanism. These findings establish the first direct link between AURKA and phosphoinositide signaling and suggest that the function of INPP5E in cilia is at least partly mediated by its interactions with AURKA.

A mutation in the mouse ttc26 gene leads to impaired hedgehog signaling.

The phenotype of the spontaneous mutant mouse hop-sterile (hop) is characterized by a hopping gait, polydactyly, hydrocephalus, and male sterility. Previous analyses of the hop mouse revealed a deficiency of inner dynein arms in motile cilia and a lack of sperm flagella, potentially accounting for the hydrocephalus and male sterility. The etiology of the other phenotypes and the location of the hop mutation remained unexplored. Here we show that the hop mutation is located in the Ttc26 gene and impairs Hedgehog (Hh) signaling. Expression analysis showed that this mutation led to dramatically reduced levels of the Ttc26 protein, and protein-protein interaction assays demonstrated that wild-type Ttc26 binds directly to the Ift46 subunit of Intraflagellar Transport (IFT) complex B. Although IFT is required for ciliogenesis, the Ttc26 defect did not result in a decrease in the number or length of primary cilia. Nevertheless, Hh signaling was reduced in the hop mouse, as revealed by impaired activation of Gli transcription factors in embryonic fibroblasts and abnormal patterning of the neural tube. Unlike the previously characterized mutations that affect IFT complex B, hop did not interfere with Hh-induced accumulation of Gli at the tip of the primary cilium, but rather with the subsequent dissociation of Gli from its negative regulator, Sufu. Our analysis of the hop mouse line provides novel insights into Hh signaling, demonstrating that Ttc26 is necessary for efficient coupling between the accumulation of Gli at the ciliary tip and its dissociation from Sufu.

CAPN5 gene silencing by short hairpin RNA interference.

BACKGROUND: The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis. FINDINGS: Two of four shRNA plasmids reduced mutant CAPN5 RNA in a stable cell line. Similar knockdown was observed in SH-SY5Y cells that natively express CAPN5. Lactose dehydrogenase assays showed that down-regulation of CAPN5 was not cytotoxic. CONCLUSIONS: CAPN5 expression can be suppressed by shRNA-based RNA interference. Further testing in ADNIV models will determine the potential of gene silencing as a strategy to treat, delay, or prevent blindness in ADNIV patients.

Essential role of the chaperonin CCT in rod outer segment biogenesis.

PURPOSE: While some evidence suggests an essential role for the chaperonin containing t-complex protein 1 (CCT) in ciliogenesis, this function remains poorly understood mechanistically. We used transgenic mice, previously generated in our lab, and characterized by a genetically-induced suppression of CCT in rod photoreceptors as well as a malformation of the rod sensory cilia, the outer segments, to gain new insights into this underlying molecular mechanism. METHODS: The CCT activity in rod photoreceptors of mice was suppressed by overexpressing the chaperonin inhibitor, phosducin-like protein short, and the ensuing changes of cellular morphology were analyzed by light and electron microscopy. Protein expression levels were studied by fluorescent microscopy and Western blotting. RESULTS: Suppressing the chaperonin made the photoreceptors incompetent to build their outer segments. Specifically, the CCT-deficient rods appeared unable to expand the outer segment plasma membrane, and accommodate growth of this compartment. Seeking the molecular mechanisms underlying such a shortcoming, we found that the affected rods could not express normal levels of Bardet-Biedl Syndrome (BBS) proteins 2, 5, and 7 and, owing to that deficiency, were unable to assemble the BBSome, a multisubunit complex responsible for ciliary trafficking. A similar effect in response to the chaperonin suppression was also observed in cultured ciliated cells. CONCLUSIONS: Our data provide new evidence indicating the essential role of the chaperonin CCT in the biogenesis of vertebrate photoreceptor sensory cilia, and suggest that it may be due to the direct participation of the chaperonin in the posttranslational processing of selected BBS proteins and assembly of the BBSome.

The centriolar satellite protein AZI1 interacts with BBS4 and regulates ciliary trafficking of the BBSome.

Bardet-Biedl syndrome (BBS) is a well-known ciliopathy with mutations reported in 18 different genes. Most of the protein products of the BBS genes localize at or near the primary cilium and the centrosome. Near the centrosome, BBS proteins interact with centriolar satellite proteins, and the BBSome (a complex of seven BBS proteins) is believed to play a role in transporting ciliary membrane proteins. However, the precise mechanism by which BBSome ciliary trafficking activity is regulated is not fully understood. Here, we show that a centriolar satellite protein, AZI1 (also known as CEP131), interacts with the BBSome and regulates BBSome ciliary trafficking activity. Furthermore, we show that AZI1 interacts with the BBSome through BBS4. AZI1 is not involved in BBSome assembly, but accumulation of the BBSome in cilia is enhanced upon AZI1 depletion. Under conditions in which the BBSome does not normally enter cilia, such as in BBS3 or BBS5 depleted cells, knock down of AZI1 with siRNA restores BBSome trafficking to cilia. Finally, we show that azi1 knockdown in zebrafish embryos results in typical BBS phenotypes including Kupffer's vesicle abnormalities and melanosome transport delay. These findings associate AZI1 with the BBS pathway. Our findings provide further insight into the regulation of BBSome ciliary trafficking and identify AZI1 as a novel BBS candidate gene.