Targeted, programmable, and precise tandem duplication in the mammalian genome.

Tandem duplications are frequent structural variations of the genome and play important roles in genetic disease and cancer. However, interpreting the phenotypic consequences of tandem duplications remains challenging, in part owing to the lack of genetic tools to model such variations. Here, we developed a strategy, tandem duplication via prime editing (TD-PE), to create targeted, programmable, and precise tandem duplication in the mammalian genome. In this strategy, we design a pair of in trans prime editing guide RNAs (pegRNAs) for each targeted tandem duplication, which encode the same edits but prime the single-stranded DNA (ssDNA) extension in opposite directions. The reverse transcriptase (RT) template of each extension is designed homologous to the target region of the other single guide RNA (sgRNA) to promote the reannealing of the edited DNA strands and the duplication of the fragment in between. We showed that TD-PE produced robust and precise in situ tandem duplications of genomic fragments ranging from ∼50 bp to ∼10 kb, with a maximal efficiency up to 28.33%. By fine-tuning the pegRNAs, we achieved simultaneous targeted duplication and fragment insertion. Finally, we successfully produced multiple disease-relevant tandem duplications, showing the general utility of TD-PE in genetic research.

Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells.

Prime editors consisting of Cas9-nickase and reverse transcriptase enable targeted precise editing of small DNA pieces, including all 12 kinds of base substitutions, insertions and deletions, while without requiring double-strand breaks or donor templates. Current optimized prime editing strategy (PE3) uses two guide RNAs to guide the performance of prime editor. One guide RNA carrying both spacer and templating sequences (pegRNA) guides prime editor to produce ssDNA break and subsequent extension, and the other one produces a nick in the complementary strand. Here, we demonstrated that positioning the nick sgRNA nearby the templating sequences of the pegRNA facilitated targeted large fragment deletion and that engineering both guide RNAs to be pegRNAs to achieve bi-direction prime editing (Bi-PE) further increase the efficiency by up to 16 times and improved the accuracy of editing products by 60 times. In addition, we showed that Bi-PE strategy also increased the efficiency of simultaneous conversion of multiple bases but not single base conversion over PE3. In conclusion, Bi-PE strategy expanded the editing scope and improved the efficiency and the accuracy of prime editing system, which might have a wide range of potential applications.

Discovery of a potent inhibitor targeting the cap-binding domain of the PB2 subunit of influenza RNA-dependent RNA polymerase.

Influenza pandemics have emerged as a significant global public health and security concern. PB2, a crucial subunit of the influenza RNA-dependent RNA polymerase (RdRP), has been identified as a promising target for influenza treatment. We herein report the discovery of a potent novel PB2 inhibitor, 7-51A, with a KD value of 1.64 nM as determined by ITC. The high activity of 7-51A was elucidated by the co-crystal structure of the PB2-7-51A complex, and comparative analysis revealed unique interactions that had never been observed before. The preliminary pharmacological evaluation indicated that 7-51A exhibited commendable cellular safety, hepatic microsomal metabolic safety and stability. Collectively, 7-51A was found to be an effective PB2 inhibitor and could be used as a lead compound for further studies.

Mechanisms of spacer acquisition by sequential assembly of the adaptation module in Synechocystis.

CRISPR-Cas immune systems process and integrate short fragments of DNA from new invaders as spacers into the host CRISPR locus to establish molecular memory of prior infection, which is also known as adaptation in the field. Some CRISPR-Cas systems rely on Cas1 and Cas2 to complete the adaptation process, which has been characterized in a few systems. In contrast, many other CRISPR-Cas systems require an additional factor of Cas4 for efficient adaptation, the mechanism of which remains less understood. Here we present biochemical reconstitution of the Synechocystis sp. PCC6803 type I-D adaptation system, X-ray crystal structures of Cas1-Cas2-prespacer complexes, and negative stained electron microscopy structure of the Cas4-Cas1 complex. Cas4 and Cas2 compete with each other to interact with Cas1. In the absence of prespacer, Cas4 but not Cas2 assembles with Cas1 into a very stable complex for processing the prespacer. Strikingly, the Cas1-prespacer complex develops a higher binding affinity toward Cas2 to form the Cas1-Cas2-prespacer ternary complex for integration. Together, we show a two-step sequential assembly mechanism for the type I-D adaptation module of Synechocystis, in which Cas4-Cas1 and Cas1-Cas2 function as two exclusive complexes for prespacer processing, capture, and integration.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.

Cancer Targeted Gene Therapy for Inhibition of Melanoma Lung Metastasis with eIF3i shRNA Loaded Liposomes.

Eukaryotic translation initiation factors 3i (eIF3i) is a proto-oncogene that is overexpressed in various tumors, reducing its expression by eIF3i shRNA is a promising strategy to inhibit tumor growth or metastasis. Tumor cell is the target of eIF3i shRNA so that tumor-site accumulation could be important for fulfilling its therapeutic effect. Thus, the iRGD modified liposome (R-LP) was rationally synthesized to enhance the antitumor effect by active targeted delivery of eIF3i shRNA to B16F10 melanoma cells. R-LP encapsulating eIF3i shRNA gene (R-LP/sheIF3i) were prepared by a film dispersion method. The transfection experiment proves that R-LP could effectively transfect B16F10 cells. R-LP/sheIF3i notably restrained the migration, invasion, and adhesion of melanoma cells in vitro. In a mouse model of lung metastasis, R-LP/sheIF3i administered by intravenous injection suppressed pulmonary metastasis of melanoma by dramatically downregulated eIF3i expression and subsequently inhibiting tumor neovascularization and tumor cells proliferation in vivo. Our results provide a basis for tumor cells targeting strategies to reduce the expression of eIF3i by RNAi in the treatment of tumor metastasis.

A simple method based on Sanger sequencing and MS Word wildcard searching to identify Cas9-induced frameshift mutations.

Recent advances in targeted genome editing have enabled sequence-specific modifications in eukaryotic genomes. As it can be easily reprogrammed, the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 nuclease system has been studied extensively and is now a widely used genome editing tool. Generally, Cas9 nucleases are designed to target the coding regions in exons of protein-coding genes, which are expected to cause frameshift indel mutations and interrupt protein expression. In such cases, it is often necessary to separate single clones that harbor double frameshift mutant alleles from clones that harbor the wild-type allele or an in-frame mutant allele. We developed a simple and efficient method to identify frameshift mutations in diploid genomes based on Sanger sequencing and MS Word wildcard searching (SWS). As indel mutations induced by Cas9 are varied, Sanger sequencing of PCR products from a single mutant genome will generate double peaks that begin at the indel sites. By positioning the putative sequences deduced from the double peak regions in the sequencing graph onto the wild-type sequence by MS Word wildcard searching, it is possible to predict exactly how many nucleotides were deleted or inserted in each allele of the genome. The SWS strategy greatly facilitates the process of identifying single clones with biallelic frameshift mutations from pooled cells or model organisms.

Polymeric Nanomedicine for Combined Gene/Chemotherapy Elicits Enhanced Tumor Suppression.

Combination treatment through simultaneous delivery of DNA and anticancer drugs with nanoparticles has been demonstrated to be an elegant and efficient approach for cancer therapy. Herein, we employed a combination therapy for eliminating both the tumor cells and intratumoral neovascular network based on the nanoplatform we designed. Pigment epithelium-derived factor (PEDF) gene, a powerful antiangiogenic agent, and the clinically widely used chemotherapy agent paclitaxel (PTX) were simultaneously encapsulated in the same nanoparticle by a modified double-emulsion solvent evaporation method. The dual-drug-loaded nanoparticles (D/P-NPs) exhibited a uniform spherical morphology and released PTX and PEDF gene in a sustained manner. D/P-NPs showed an enhanced antitumor effect on C26 and A549 cells and a stronger inhibitory activity on proliferation of HUVECs. Moreover, D/P-NPs could dramatically elevate the PEDF expression levels in both C26 and A549 cells in comparison with PEDF gene loaded nanoparticles and significantly promote the cellular uptake of PTX. Additionally, microtubules were stabilized and G2/M phase arrest along with a higher subG1 cell population was induced by D/P-NPs in contrast to PTX or PTX loaded nanoparticles. Besides, D/P-NPs showed sustained release of PTX and PEDF gene in tumors as well as long-term gene expression. A significantly improved anticancer effect was also demonstrated in a C26 subcutaneous tumor model using this combinational therapy. D/P-NPs could sharply reduce the microvessel density and significantly promoted tumor cell apoptosis in vivo. More importantly, the in vivo distribution, serological and biochemical analysis, and H&E staining revealed that D/P-NPs had no obvious toxicity. Our study suggested that this novel polymeric nanomedicine had great potential for improving the therapeutic efficacy of combined gene/chemotherapy of cancer.

Transdifferentiation of fast skeletal muscle into functional endothelium in vivo by transcription factor Etv2.

Etsrp/Etv2 (Etv2) is an evolutionarily conserved master regulator of vascular development in vertebrates. Etv2 deficiency prevents the proper specification of the endothelial cell lineage, while its overexpression causes expansion of the endothelial cell lineage in the early embryo or in embryonic stem cells. We hypothesized that Etv2 alone is capable of transdifferentiating later somatic cells into endothelial cells. Using heat shock inducible Etv2 transgenic zebrafish, we demonstrate that Etv2 expression alone is sufficient to transdifferentiate fast skeletal muscle cells into functional blood vessels. Following heat treatment, fast skeletal muscle cells turn on vascular genes and repress muscle genes. Time-lapse imaging clearly shows that muscle cells turn on vascular gene expression, undergo dramatic morphological changes, and integrate into the existing vascular network. Lineage tracing and immunostaining confirm that fast skeletal muscle cells are the source of these newly generated vessels. Microangiography and observed blood flow demonstrated that this new vasculature is capable of supporting circulation. Using pharmacological, transgenic, and morpholino approaches, we further establish that the canonical Wnt pathway is important for induction of the transdifferentiation process, whereas the VEGF pathway provides a maturation signal for the endothelial fate. Additionally, overexpression of Etv2 in mammalian myoblast cells, but not in other cell types examined, induced expression of vascular genes. We have demonstrated in zebrafish that expression of Etv2 alone is sufficient to transdifferentiate fast skeletal muscle into functional endothelial cells in vivo. Given the evolutionarily conserved function of this transcription factor and the responsiveness of mammalian myoblasts to Etv2, it is likely that mammalian muscle cells will respond similarly.

Ammonia drives dendritic cells into dysfunction.

Ammonia levels are often elevated in patients with cirrhosis or tumors. Patients with these diseases are immunocompromised. In this study, we investigated the effects of ammonia on a member of the immune cell family, the dendritic cells (DCs). Our results demonstrated that ammonia diminished cell count, phagocytosis, and lymphocyte stimulation of DCs. Ammonia also induced DC swelling, excessive reactive oxygen species production, and mitochondrial damage, which may constitute the underlying mechanism of ammonia-induced DC dysfunction. In ammonium chloride (NH4Cl)-loaded mice, DCs exhibited lowered phagocytosis and a weakened immune response to the chicken OVA vaccine. DCs from patients with cirrhosis or ammonia-treated healthy human blood both exhibited diminished phagocytosis. Moreover, tumor cell conditioned medium drove DCs into dysfunction, which could be reversed by ammonia elimination. In a murine colon carcinoma model, we found that ammonia could regulate tumor growth involving DCs and their related immune response. These findings reveal that ammonia could drive DCs into dysfunction, which contributes to the immunocompromised state of patients with cirrhosis or tumors.

Liposomes as a Novel Ocular Delivery System for Brinzolamide: In Vitro and In Vivo Studies.

The objective of this study was to investigate the potential of liposomes as an ophthalmic delivery system for brinzolamide (Brz) to enhance the local glaucomatous therapeutic effect. The liposomes of Brz (Brz-LPs) were produced by the thin-film dispersion method with a particle size of 84.33 ± 2.02 nm and an entrapment efficiency of 98.32 ± 1.61%. Differential scanning calorimetry (DSC) and X-ray powder diffraction (XRD) analysis proved that Brz was successfully entrapped into Brz-LPs. Brz-LPs displayed a biphasic release pattern in vitro with burst release initially and sustained release afterwards. The corneal permeability was measured using modified Franz-type diffusion cells, and Brz-LPs showed 6.2-fold increase in the apparent permeability coefficient when compared with the commercial available formulation (B rz-Sus). Moreover, Brz-LPs (1 mg/mL Brz) showed a more sustained and effective intraocular pressure reduction (5-10 mmHg) than Brz-Sus (10 mg/mL Brz) in white New Zealand rabbits. Therefore, Brz-LPs were a hopeful formulation of Brz for glaucoma treatment and worthy of further investigation.

The translation initiation factor eIF3i up-regulates vascular endothelial growth factor A, accelerates cell proliferation, and promotes angiogenesis in embryonic development and tumorigenesis.

Vascular endothelial growth factor A (VEGFA) is a critical proangiogenic factor that is activated by hypoxia at both the transcriptional and post-transcriptional levels. In hypoxia conditions, stabilized hypoxia-inducible factor 1α (HIF1A) is the key regulator for transcriptional activation of VEGFA. However, the post-transcriptional control of VEGFA expression remains poorly understood. Here, we report that the eukaryotic translation initiation factor 3i (eIF3i) is required for VEGFA protein expression in both normal embryonic and tumorigenic angiogenesis. eIF3i is dynamically expressed in the early stages of zebrafish embryogenesis and in human hepatocellular carcinoma tissues. eIF3i homozygous mutant zebrafish embryos show severe angiogenesis defects and human hepatocellular cancer cells with depletion of eIF3i to induce less angiogenesis in tumor models. Under hypoxia, the HIF1A protein can interact with its binding sequence in the eIF3i promoter and activate eIF3i transcription. The expression of VEGFA, which should rise in hypoxia, is significantly inhibited by eIF3i siRNA treatment. Moreover, eIF3i knockdown did not cause a general translation repression but specifically reduced the translation efficiency of the VEGFA mRNAs. Taken together, our results suggest that eIF3i is induced by HIF1A under hypoxia and controls normal and tumorigenic angiogenesis through regulating VEGFA protein translation.

A Primary Primitive Neuroectodermal Tumor Arising from Left Subclavian Vein and Extending along Left Brachiocephalic Vein and Superior Vena Cava into Right Atrium.

Primitive neuroectodermal tumor (PNET) is an extremely rare malignancy thought to be derived from fetal neuroectodermal precursor cells. It usually occurs in central and peripheral nervous system or soft tissue and bone, while intravenous or intracavitary PNET is considered as an extremely rare tumor. We reported a case of a 44-year-old woman who presented with the left unilateral facial and neck swelling. Magnetic resonance imaging revealed a tape-shaped solid mass within left subclavian vein, left brachiocephalic vein, superior vena cava, and right atrium; the proximal end proportion occupied almost the entire right atrium with a pedicle flip protruded into the right ventricle. Ultrasonography revealed an irregular hypoechnoic mass arising from the left subclavian vein, which extended along the left brachiocephalic vein and superior vena cava into the right atrium and up to the right ventricle. Positron emission tomography-computed tomography revealed several hypermetabolic thyroid nodules with no evidence of intravenous hyperactive lesion. The patient underwent tumor resection under cardiopulmonary bypass. At 15 days postoperatively, total thyroidectomy and resection of the left subclavian vein were simultaneously performed. The patient received chemotherapy and radiotherapy later. Histologically, the neoplasm displayed small, round, blue cells with hyperchromatic nuclei and scant cytoplasm. The neoplastic cells showed a strong immunopositivity for CD99, synaptophysin, CD56, CD57, and friend leukemia integration 1, thus confirming a diagnosis of the PNET. Histopathological examination of the thyroid showed papillary carcinoma. Thus, this PNET had no definitive organ or tissue of origin, which primarily originated from the left subclavian vein with tumor extension along the superior vena cava to the right ventricle.

ENC1-like integrates the retinoic acid/FGF signaling pathways to modulate ciliogenesis of Kupffer's Vesicle during zebrafish embryonic development.

The left-right asymmetry is an essential feature shared by vertebrates. Cilia-driven counterclockwise flow in the mammalian node structure leads to the left-right asymmetric distribution of signals and subsequent asymmetric patterning. Although several signaling pathways have been identified in the specification of node ciliated cells, little is known about the direct downstream effectors of these signaling pathways. Here, we showed that zebrafish Ectoderm-Neural Cortex1-like (enc1l) is expressed in the Kupffer's Vesicle (KV), an equivalent structure of the mammalian node in zebrafish, and is necessary for KV ciliogenesis. Loss-of-function of enc1l increased the number and decreased the length of KV cilia. The enc1l expression in the KV region was specifically regulated by retinoic acid (RA), FGF, and Wnt signaling pathways. In addition, knocking down enc1l or ectopic enc1l expression was able to rescue the KV cilium defects caused by alteration of RA and FGF signaling, but not Wnt signaling. Taken together, these data indicate thatEnc1l is a direct downstream effector of RA and FGF signaling pathways and modulates KV ciliogenesis in the zebrafish embryo.

Nuclear factor kappa B signaling initiates early differentiation of neural stem cells.

Inflammatory mediators, many of which activate the signaling of nuclear factor kappa B (NFκB), have received increasing attention in the field of neurogenesis. NFκB signaling regulates neurite outgrowth and neural plasticity as well as the proliferation/apoptosis and terminal differentiation of neural stem cells (NSCs). Early neurogenesis from NSCs produces identical progeny through symmetric division and committed daughter cells through asymmetric division. Here, we show that NFκB signaling is required for NSC initial differentiation. The canonical IKKß/IκBα/p65 pathway is activated during the initial stages of neural differentiation induced by treatment with TNFα or withdrawal of epidermal growth factor/basic fibroblast growth factor. NSC-specific inhibition of NFκB in transgenic mice causes an accumulation of Nestin(+) /Sox2(+) /glial fibrillary acidic protein(+) NSCs. Inhibition of NFκB signaling in vitro blocks differentiation and asymmetric division and maintains NSCs in an undifferentiated state. The induction of initial differentiation and asymmetry by NFκB signaling occurs through the inhibition of C/EBPß expression. Our data reveal a novel function of NFκB signaling in early neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases.

Recent advances in therapeutic CRISPR-Cas9 genome editing: mechanisms and applications.

Recently, clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 derived editing tools had significantly improved our ability to make desired changes in the genome. Wild-type Cas9 protein recognizes the target genomic loci and induced local double strand breaks (DSBs) in the guidance of small RNA molecule. In mammalian cells, the DSBs are mainly repaired by endogenous non-homologous end joining (NHEJ) pathway, which is error prone and results in the formation of indels. The indels can be harnessed to interrupt gene coding sequences or regulation elements. The DSBs can also be fixed by homology directed repair (HDR) pathway to introduce desired changes, such as base substitution and fragment insertion, when proper donor templates are provided, albeit in a less efficient manner. Besides making DSBs, Cas9 protein can be mutated to serve as a DNA binding platform to recruit functional modulators to the target loci, performing local transcriptional regulation, epigenetic remolding, base editing or prime editing. These Cas9 derived editing tools, especially base editors and prime editors, can introduce precise changes into the target loci at a single-base resolution and in an efficient and irreversible manner. Such features make these editing tools very promising for therapeutic applications. This review focuses on the evolution and mechanisms of CRISPR-Cas9 derived editing tools and their applications in the field of gene therapy.

Case report: A case of delayed cutaneous metastases from signet-ring cell mixed-type gastric cancer.

Background: Signet-ring cell gastric carcinoma is a highly malignant tumor, with the characteristics of strong invasiveness, rapid progression, a high degree of malignancy, and generally poor prognosis. The most common site of metastases is the abdominal organs, especially the liver, while delayed cutaneous metastases are rare. Case presentation: We report a case of cutaneous metastases on the head, groin, and thigh, which recurred 7 years after signet-ring cell gastric carcinoma surgery. The patient was diagnosed with a 2.0×1.5×1.0cm tumor at the angle of stomach, and treated with Billroth II distal gastrectomy accompanied with D2 lymph node dissection. According the pathology, the stage was pT1N3M0. Then the patient received two cycles of oxaliplatin and tegafur chemotherapy, which was discontinued due to the inability to tolerate the side effects of chemotherapy. Seven years after the surgery, the patient initially presented with a fleshy mass on the head and beaded nodules in the groin; then, the mass gradually became larger, along with the thighs turning red, swollen, and crusty. Firstly, the patient was diagnosed with "lower extremity lymphangitis" and treated mostly with anti-inflammatory, promote lymphatic return, detumescence and elastic force cannula in vascular surgery department. However, the symptoms relieved insufficient. Finally, the skin biopsy indicates a signet-ring cell gastric carcinoma cutaneous metastasis. The whole-body PET-CT examination showed multiple nodules with increased metabolism. Then the patient was transferred to The Department of Oncology for further chemotherapy. Conclusion: Our case highlights that gastric tumor recurrence and metastasis should be highly suspected when skin lesions appear in patients with signet-ring cell gastric carcinoma. At the same time, multidisciplinary consultation and close cooperation between surgeons, oncologists, and dermatologists are of great significance to the diagnosis and treatment of this disease.

DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.

In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.

Dual-AAV split prime editor corrects the mutation and phenotype in mice with inherited retinal degeneration.

The prime editor (PE) can edit genomes with almost any intended changes, including all 12 possible types of base substitutions, small insertions and deletions, and their combinations, without the requirement for double strand breaks or exogenous donor templates. PE demonstrates the possibility of correcting a variety of disease-causing mutations and might expand the therapeutic application of gene editing. In this study, PE was optimized based on a dual-adeno-associated virus (AAV) split-intein system in vitro by screening different split sites and split inteins. We found that splitting PE before amino acid 1105(Ser) of SpCas9 with Rma intein resulted in the highest on-target editing. The orientations of pegRNA and nicking sgRNA in the AAV vector were further optimized. To test the in vivo performance of the optimized dual-AAV split-PE3, it was delivered by subretinal injection in rd12 mice with inherited retinal disease Leber congenital amaurosis. The prime editors corrected the pathogenic mutation with up to 16% efficiency in a precise way, with no detectable off-target edits, restored RPE65 expression, rescued retinal and visual function, and preserved photoceptors. Our findings establish a framework for the preclinical development of PE and motivate further testing of PE for the treatment of inherited retinal diseases caused by various mutations.

ApoA-II directs morphogenetic movements of zebrafish embryo by preventing chromosome fusion during nuclear division in yolk syncytial layer.

The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation.