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A systematic knowledge of the roles of DNA repair genes at the level of the organism has been limited due to the lack of appropriate experimental approaches using animal model systems. Zebrafish has become a powerful vertebrate genetic model system with availability due to the ease of genome editing and large-scale phenotype screening. Here, we generated zebrafish mutants for 32 DNA repair and replication genes through multiplexed CRISPR/Cas9-mediated mutagenesis. Large-scale phenotypic characterization of our mutant collection revealed that three genes (atad5a, ddb1, pcna) are essential for proper embryonic development and hematopoiesis; seven genes (apex1, atrip, ino80, mre11a, shfm1, telo2, wrn) are required for growth and development during juvenile stage and six genes (blm, brca2, fanci, rad51, rad54l, rtel1) play critical roles in sex development. Furthermore, mutation in six genes (atad5a, brca2, polk, rad51, shfm1, xrcc1) displayed hypersensitivity to DNA damage agents. Our zebrafish mutant collection provides a unique resource for understanding of the roles of DNA repair genes at the organismal level.

G(M2) gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in β-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb(-/-)) of the G(M2) gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or -LacZ and monitored for serum β-hexosaminidase activity, motor function, and survival. Brain G(M2) ganglioside, β-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB-treated SD mice survived until 43 weeks (P0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB-treated mice had a significant increase in brain β-hexosaminidase activity, and a reduction in G(M2) ganglioside storage and neuroinflammation compared to adult SD-HexB- and SD-LacZ-treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other G(M2) gangliosidoses through early rAAV9 based systemic gene therapy.

Collective cell migration is critical for normal development, tissue repair and cancer metastasis. Migration of the posterior lateral line primordium (pLLP) generates the zebrafish sensory organs (neuromasts, NMs). This migration is promoted by the leader cells at the leading edge of the pLLP, which express the G protein-coupled chemokine receptor Cxcr4b and respond to the chemokine Cxcl12a. However, the mechanism by which Cxc112a/Cxcr4b signaling regulates pLLP migration remains unclear. Here we report that signal transduction by the heterotrimeric G protein subunit Gβ1 is essential for proper pLLP migration. Although both Gβ1 and Gβ4 are expressed in the pLLP and NMs, depletion of Gβ1 but not Gβ4 resulted in an arrest of pLLP migration. In embryos deficient for Gβ1, the pLLP cells migrated in an uncoordinated fashion and were unable to extend protrusions at the leading front, phenocopying those in embryos deficient for Cxcl12a or Cxcr4b. A transplantation assay showed that, like Cxcr4b, Gβ1 is required only in the leader cells of the pLLP. Analysis of F-actin dynamics in the pLLP revealed that whereas wild-type leader cells display extensive actin polymerization in the direction of pLLP migration, counterparts defective for Gβ1, Cxcr4b or Cxcl12a do not. Finally, synergy experiments revealed that Gβ1 and Cxcr4b interact genetically in regulating pLLP migration. Collectively, our data indicate that Gβ1 controls migration of the pLLP, likely by acting downstream of the Cxcl12a/Cxcr4b signaling. This study also provides compelling evidence for functional specificity among Gβ isoforms in vivo.

Zebrafish inner ear development is characterized by the crystallization of otoliths onto immotile kinocilia that protrude from sensory “hair” cells. The stereotypical formation of these sensory structures is dependent on the expression of key patterning genes and on Ca2+ signals. One potential target of Ca2+ signaling in the inner ear is the type II Ca2+/calmodulin-dependent protein kinase (CaMK-II), which is preferentially activated in hair cells, with intense activation at the base of kinocilia. In zebrafish, CaMK-II is encoded by seven genes; the expression of one of these genes (camk2g1) is enriched in hair cells. The suppression of camk2g1 expression by antisense morpholino oligonucleotides or inhibition of CaMK-II activation by the pharmacological antagonist, KN-93, results in aberrant otolith formation without preventing cilia formation. In fact, CaMK-II suppression results in additional ciliated hair cells and altered levels of Delta–Notch signaling members. DeltaA and deltaD transcripts are increased and DeltaD protein accumulates in hair cells of CaMK-II morphants, indicative of defective recycling and/or exocytosis. Our findings indicate that CaMK-II plays a critical role in the developing ear, influencing cell differentiation through extranuclear effects on Delta–Notch signaling. Continued expression and activation of CaMK-II in maculae and cristae in older embryos suggests continued roles in auditory sensory maturation and transduction.

The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Mis-regulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants have been identified affecting different components of the Hh/Gli signaling system. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified several known (e.g. ptc1 and nkx2.2a) as well as a large number of novel Hh regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of mis-regulation during tumorogenesis.

We have identified a mutation in the zebrafish gene claudinj generated by retroviral integration. Mutant embryos display otoliths severely reduced in size, no response to tapping stimulus, and an inability to balance properly suggesting vestibular and hearing dysfunction. Antisense in situ hybridization to the cldnj gene showed expression first in the otic placode and later asymmetric expression in the otic vesicle. Morpholino inhibition of claudinj expression showed similar defects in otolith formation. Phylogenetic analysis of claudin sequences from multiple species demonstrates that claudinj was part of a gene expansion that began in the common ancestor of fish and humans, but additional fish specific gene duplications must have also occurred.

Top of pageAbstract Although patients with X-linked severe combined immunodeficiency (X-SCID) were cured with hematopoietic stem cell (HSC) gene therapy, three of the treated X-SCID patients developed leukemia. In these clinic trials, moloney murine leukemia virus (MLV) was used as the vector. Since recent studies suggest lentiviral vector may be the better choice, it was used in our research projects of HSC gene therapy in mice. These mice served as a model to investigate whether lentivectors are associated with cancer development as MLV. We examined (1) the distribution of lentiviral vector insertions in the mouse genome and (2) tumor development. Genomic DNA was isolated from peripheral blood of three recipient mice two months after transplantation of bone marrow stem cells transduced ex vivo with lentiviral vectors. The DNA was pooled and subjected to a high-throughput method to rapidly clone the genomic regions adjacent to proviral integrations. A total of 1152 clones were sequenced. 1038 sequences had high quality insert, many of which were duplicates. Among the 246 unique integration sites, 115 integration sites (47%) were located in genes identified in RefSeq, while only 9 (3.0%) lentiviral integrations were located |[plusmn]| 1kb relative to the transcription start sites. These results indicate that compared with MLV, lentivectors do not preferentially integrate around transcription start sites and the reduced propensity of lentiviral vectors to incorporate into promoter-proximal regions may contribute to their safety in gene therapy. This claim is supported by our observation that there was no rapid weight loss, no enlarged thymus, lymph nodes, liver, or spleen, and no other tumor appearance among these bone marrow reconstituted mice even 18 months post transplant.

The recent demonstration that HIV-1, MLV, and AAV all strongly favor integration into active genes has raised significant concerns about the future of gene insertion-based therapies. The Sleeping Beauty (SB) transposon has shown tremendous success in preclinical gene therapy models and is a promising alternative to viral-based in vivo delivery methods, and herein, we investigated its target site preferences. Using a plasmid rescue strategy, we isolated 524 unique, unselected transposition events from adult mouse liver and compared the chromosomal features associated with these coordinates with 10,000 computer-simulated random integrations. SB insertions were distributed evenly on each chromosome, but were highly favored in 2-6 bp simple sequence repeats (p < 0.0001) and were strongly disfavored in genomic LINEs (p < 0.0001). Also, SB showed a marked preference for integration into localized AT-rich palindromes, especially the consensus sequence GNATATATANC (p < 0.01). These attributes are consistent with a bendable target DNA structure and suggest a two-fold dyad symmetry at the insertion site. We also mapped SB insertions with respect to the RefSeq genes defined by the UCSC mouse genome database. Results showed that ~20% (103/524) of transposon insertions mapped within at least one RefSeq gene, which was not statistically different from the control set (p=0.3). These intragenic insertions were distributed evenly along the transcripts without orientational preference, but were almost exclusively within introns. Interestingly, all five of the exon insertions we isolated were in the reverse orientation of transcription and all mapped near the 3′ end of genes, very close to the translational stop site. Moreover, even though SB does not appear to actively target intragenic regions, it showed a weak but significant tendency to insert upstream of genes (p=0.009), especially ± 5 kb of the transcriptional start site (p < 0.005) and within known CpG islands (p=0.0001). We therefore analyzed an Affymetrix chip dataset available on the web to test whether preferences for promoter regions might be due in part to transcriptional activity. However, results showed no statistical difference between the 44 SB-targeted and 70 randomly-targeted RefSeq genes represented on the array (p=0.25), suggesting that chromatin accessibility, rather than active tethering, may be involved in SB's regional insertion site preferences. Currently, we are analyzing a large number of insertions from mouse and human cell lines in order to test the generality of these preferences in different tissues and/or species, and are determining whether or not genetic selection introduces any bias during SB insertion site selection. Collectively, these data demonstrate a nonrandom distribution for SB insertions in mammals, and suggest that SB integration may be significantly safer for stable in vivo gene delivery than many widely used viral-based vectors.

Recent reports on severe adverse effects induced by retroviral integration into the LMO2 proto-oncogene in humans and the demonstration of preferential AAV serotype 2 (rAAV2) vector integration into genes in our previous study with a limited number of integration sites in mice1, have raised a possible concern about rAAV-mediated insertional mutagenesis. In order to further address this issue, we have expanded our previous study and performed a high-throughput analysis of rAAV2 integration sites isolated from in vivo selected hereditary tyrosinemia type I (HTI) mouse hepatocytes transduced with a human fumaryl acetoacetate hydrolase (FAH)-expressing rAAV2 shuttle vector, AAV-EF1α-hFAH.AOS. Briefly, total liver DNA was isolated from 4 HTI mice that underwent a 7-month in vivo selection of transplanted hepatocytes isolated from the donor HTI mice having received 3.0×1011 vector genomes of AAV-EF1α-hFAH.AOS via the portal vein and undergone an 8-week in vivo selection. The whole proviral rAAV2 vector genome and flanking genomic DNA sequences were isolated as a plasmid from the liver DNA by a plasmid rescue technique as previously described1. All four mice had a different donor HTI mouse, therefore, any integrations found in different mice should be considered as independent integration events. To date, we have characterized 307 independent integration events and found: 1) 191 of 307 (62%) of integrations occurred in genes; 2) there was a strong bias toward integrating into host genomes around transcription start sites, as has been observed in murine leukemia virus integrations (approximately a quarter of the total rAAV2 integrations occurred within ±1 kb from transcription start sites); 3) rAAV integration involved large genomic deletions of over 1 kb in 15% of the cases, suggesting that such large deletions may not be rare events associated with rAAV2 integration. The most striking finding was that among the 307 independent integration events, at least 4 integrations from 3 different mice (1.5 % of total integrations and 2.1 % of the genes targeted by rAAV) were found within a 30-kb region in the mouse ubiquitin C gene, and at least 4 integrations from 4 different mice fell on another sequence stretch of approximately 2.5 kb. Our preliminary analysis by restriction enzyme mapping and sequencing over a thousand base pairs has indicated that the hot spot of 2.5 kb in length likely resides in a proto-oncogene, the Evi-1 gene. It should be noted that murine leukemia found in a retroviral gene marking study was induced by insertional mutagenesis at this locus. Although the oncogenic potential of this gene in the liver is not known, overexpression of this gene can transform not only hematopoietic cells but also other cell types. Thus, the preliminary results in our high-throughput rAAV2 integration site analysis further emphasize the importance of pursuing the study on the elucidation of the mechanisms of rAAV vector integration and its consequences in the host.