Publications by authors named "Shaohua Yao"

44 Publications

A distinct structure of Cas1-Cas2 complex provides insights into the mechanism for the longer spacer acquisition in Pyrococcus furiosus.

Int J Biol Macromol 2021 Apr 14;183:379-386. Epub 2021 Apr 14.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, PR China. Electronic address:

In the adaptation stage of CRISPR-Cas systems, the Cas1-Cas2 integrase captures and integrates new invader-derived spacers into the CRISPR locus, serving as a molecular memory of prior infection. As of yet, the structural information of Cas1-Cas2 complex is available only for two species. Here we present the crystal structure of Cas1-Cas2 complex of Pyrococcus furiosus, which showed a distinct architecture from the known Cas1-Cas2 complexes. The shorter C-terminal tail of Pfu Cas2 directs the Cas1 dimers go in the opposite direction, resulting in a different prespacer binding mode. Based on our structural and mutagenesis results, we modeled a prespacer with a shorter duplex and longer 3' overhangs to bind Pfu Cas1-Cas2 complex. The prespacer preference was confirmed by EMSA, fluorescence polarization, and in vitro integration assays. This model provides a potential explanation for the longer spacer acquisition observed in P. furiosus when deleting both cas4 genes. Our study highlights the diversity of the CRISPR adaptation module.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.04.074DOI Listing
April 2021

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

Nucleic Acids Res 2021 03;49(5):2973-2984

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu 610041, P.R. China.

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.
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http://dx.doi.org/10.1093/nar/gkab105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7969031PMC
March 2021

Publisher Correction: A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Nature 2021 Feb;590(7844):E23

Key Laboratory of Human Disease Comparative Medicine, Chinese Ministry of Health, Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China.

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http://dx.doi.org/10.1038/s41586-020-03108-4DOI Listing
February 2021

Long-Term Metabolic Correction of Phenylketonuria by AAV-Delivered Phenylalanine Amino Lyase.

Mol Ther Methods Clin Dev 2020 Dec 13;19:507-517. Epub 2020 Jan 13.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.

Phenylketonuria (PKU) is an inherited metabolic disorder caused by mutation within phenylalanine hydroxylase (PAH) gene. Loss-of-function of PAH leads to accumulation of phenylalanine in the blood/body of an untreated patient, which damages the developing brain, causing severe mental retardation. Current gene therapy strategies based on adeno-associated vector (AAV) delivery of PAH gene were effective in male animals but had little long-term effects on blood hyperphenylalaninemia in females. Here, we designed a gene therapy strategy using AAV to deliver a human codon-optimized phenylalanine amino lyase in a liver-specific manner. It was shown that PAL was active in lysing phenylalanine when it was expressed in mammalian cells. We produced a recombinant adeno-associated vector serotype 8 (AAV8) viral vector expressing the humanized PAL under the control of human antitrypsin (hAAT) promoter (AAV8-PAL). A single intravenous administration of AAV8-PAL caused long-term correction of hyperphenylalaninemia in both male and female PKU mice (strain Pah). Besides, no obvious liver injury was observed throughout the treatment process. Thus, our results established that AAV-mediated liver delivery of PAL gene is a promising strategy in the treatment of PKU.
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http://dx.doi.org/10.1016/j.omtm.2019.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733040PMC
December 2020

sgBE: a structure-guided design of sgRNA architecture specifies base editing window and enables simultaneous conversion of cytosine and adenosine.

Genome Biol 2020 08 28;21(1):222. Epub 2020 Aug 28.

Laboratory of Biotherapy, National Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, Renmin Nanlu 17, Chengdu, 610041, Sichuan, China.

We present a base editing system, in which base editors are attached to different sites of sgRNA scaffold (sgBE). Each independent sgBE has its own specific editing pattern for a given target site. Among tested sgBEs, sgBE-SL4, in which deaminase is attached to the last stem-loop of sgRNA, yields the highest editing efficiency in the window several nucleotides next to the one edited by BE3. sgBE enables the simultaneous editing of adenine and cytosine. Finally, in order to facilitate in vivo base editing, we extend our sgBE system to an AAV-compatible Cas9, SaCas9 (Staphylococcus aureus), and observe robust base editing.
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http://dx.doi.org/10.1186/s13059-020-02137-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453718PMC
August 2020

A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.

Nature 2020 10 29;586(7830):572-577. Epub 2020 Jul 29.

Key Laboratory of Human Disease Comparative Medicine, Chinese Ministry of Health, Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.
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http://dx.doi.org/10.1038/s41586-020-2599-8DOI Listing
October 2020

Correction: Loss of augments the regeneration of nervous lateral line system through negative regulation of transcription.

Oncotarget 2019 Dec 24;10(67):7179-7180. Epub 2019 Dec 24.

Division of Cancer Biotherapy, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, P.R. China.

[This corrects the article DOI: 10.18632/oncotarget.11895.].
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http://dx.doi.org/10.18632/oncotarget.27337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935254PMC
December 2019

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

Mol Pharm 2020 01 10;17(1):229-238. Epub 2019 Dec 10.

State Key Laboratory of Biotherapy/Geriatrics and Cancer Center, West China Hospital and Collaborative Innovation Center for Biotherapy , Sichuan University , Chengdu , 610041 , China.

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 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 . Our results provide a basis for tumor cells targeting strategies to reduce the expression of eIF3i by RNAi in the treatment of tumor metastasis.
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http://dx.doi.org/10.1021/acs.molpharmaceut.9b00943DOI Listing
January 2020

Long-Term Correction of Copper Metabolism in Wilson's Disease Mice with AAV8 Vector Delivering Truncated ATP7B.

Hum Gene Ther 2019 12;30(12):1494-1504

National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, China.

Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by mutations in the gene encoding a liver active copper transport enzyme. Gene therapy with adeno-associated virus (AAV) carrying full-length ATP7B, which is about 4.4 kb, was shown to rescue copper metabolism disorder in WD mouse model. However, due to its relatively large size, the AAV vector containing full-length ATP7B could be oversized for its packaging capacity, which could lead to inefficient packaging. To this purpose, we engineered a truncated ATP7B mutant (tATP7B) that is about 3.3 kb in length and used for AAV gene therapy for WD mice. test showed that the excretion of copper outside the cells could be achieved with tATP7B as efficient as the full-length ATP7B. delivery of tATP7B to WD mice by AAV8 vectors corrected their copper metabolisms and significantly rescued copper accumulation-related syndromes, including reduced urinary copper excretion, increased serum ceruloplasmin, and improved liver damages. Thus, our study demonstrated that AAV gene therapy based on truncated ATP7B is a promising strategy in the treatment of WD.
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http://dx.doi.org/10.1089/hum.2019.148DOI Listing
December 2019

BE-PIGS: a base-editing tool with deaminases inlaid into Cas9 PI domain significantly expanded the editing scope.

Signal Transduct Target Ther 2019;4:36. Epub 2019 Sep 20.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, 610041 Chengdu, People's Republic of China.

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http://dx.doi.org/10.1038/s41392-019-0072-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6799832PMC
January 2021

Thermosensitive Micelles Encapsulating Phenylalanine Ammonia Lyase Act as a Sustained and Efficacious Therapy Against Colorectal Cancer.

J Biomed Nanotechnol 2019 Apr;15(4):717-727

Colorectal cancer has become the third most commonly diagnosed cancer worldwide, which has posed challenges to current conventional therapies. Hence, we propose an alternative approach to the existing therapeutics for colorectal cancer treatment. In this work, we prepared thermosensitive micelles based on poly(ethylene glycol)-poly(caprolactone)poly(ethylene glycol) (PEG-PCL-PEG, PECE) copolymers (PAL-micelles) that were used to encapsulate phenylalanine ammonia lyase (PAL) as an sustained drug delivery system. experiments suggested that PAL could inhibit the proliferation and induce apoptosis of tumor cells because the depletion of local phenylalanine blocked the synthesis of corresponding proteins. In addition, the release behavior and showed that PAL could be released from PAL-micelles in a controlled manner. Moreover, a significant inhibition of tumor growth was observed in the PAL-micellestreated xenograft mouse model compared to the control groups , but the systemic toxicity was not noteworthy. The antitumor efficacy was further confirmed by histological analysis of the tumor tissues with hematoxylin and eosin (H&E) and Ki-67 staining. The above results demonstrated that the PAL-micelles system could be considered an alternative strategy for colorectal cancer treatment.
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http://dx.doi.org/10.1166/jbn.2019.2734DOI Listing
April 2019

Structural and functional insights into the regulation of the lysis-lysogeny decision in viral communities.

Nat Microbiol 2018 11 15;3(11):1285-1294. Epub 2018 Oct 15.

Division of Respiratory and Critical Care Medicine, State Key Laboratory of Biotherapy, West China Hospital of Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu, China.

Communication is vital for all organisms including microorganisms, which is clearly demonstrated by the bacterial quorum-sensing system. However, the molecular mechanisms underlying communication among viruses (phages) via the quorum-sensing-like 'arbitrium' system remain unclear. Viral or host densities are known to be related to an increased prevalence of lysogeny; however, how the switch from the lytic to the lysogenic pathway occurs is unknown. Thus, we sought to reveal mechanisms of communication among viruses and determine the lysogenic dynamics involved. Structural and functional analyses of the phage-derived SAIRGA and GMPRGA peptides and their corresponding receptors, phAimR and spAimR, indicated that SAIRGA directs the lysis-lysogeny decision of phi3T by modulating conformational changes in phAimR, whereas GMPRGA regulates the lysis-lysogeny pathway by stabilizing spAimR in the dimeric state. Although temperate viruses are thought to share a similar lytic-lysogenic cycle switch model, our study suggests the existence of alternative strain-specific mechanisms that regulate the lysis-lysogeny decision. Collectively, these findings provide insights into the molecular mechanisms underlying communication among viruses, offering theoretical applications for the treatment of infectious viral diseases.
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http://dx.doi.org/10.1038/s41564-018-0259-7DOI Listing
November 2018

The RNA binding protein SORBS2 suppresses metastatic colonization of ovarian cancer by stabilizing tumor-suppressive immunomodulatory transcripts.

Genome Biol 2018 03 16;19(1):35. Epub 2018 Mar 16.

Department of Obstetrics and Gynecology, Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE and State Key Laboratory of Biotherapy, West China Second University Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, 610041, People's Republic of China.

Background: Ovarian cancer constitutes one of the most lethal gynecologic malignancies for females. Currently, early detection strategies and therapeutic options for ovarian cancer are far from satisfactory, leading to high diagnosis rates at late stages and disease relapses. New avenues of therapy are needed that target key processes in ovarian cancer progression. While a variety of non-coding RNAs have been proven to regulate ovarian cancer metastatic progression, the functional roles of RNA-binding proteins (RBPs) in this process are less well defined.

Results: In this study, we identify that the RBP sorbin and SH3 domain containing 2 (SORBS2) is a potent suppressor of ovarian cancer metastatic colonization. Mechanistic studies show that SORBS2 binds the 3' untranslated regions (UTRs) of WFDC1 (WAP four-disulfide core domain 1) and IL-17D (Interleukin-17D), two secreted molecules that are shown to act as metastasis suppressors. Enhanced expression of either WFDC1 or IL-17D potently represses SORBS2 depletion-mediated cancer metastasis promotion. By enhancing the stability of these gene transcripts, SORBS2 suppresses ovarian cancer invasiveness and affects monocyte to myeloid-derived suppressor cell and M2-like macrophage polarization, eliciting a tumor-suppressive immune microenvironment.

Conclusions: Our data illustrate a novel post-transcriptional network that links cancer progression and immunomodulation within the tumor microenvironment through SORBS2-mediated transcript stabilization.
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http://dx.doi.org/10.1186/s13059-018-1412-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5857099PMC
March 2018

Identify a Blood-Brain Barrier Penetrating Drug-TNB using Zebrafish Orthotopic Glioblastoma Xenograft Model.

Sci Rep 2017 10 30;7(1):14372. Epub 2017 Oct 30.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Sichuan, P.R. China.

The blood-brain barrier (BBB) is necessary for maintaining brain homeostasis, but it also represents a major challenge for drug delivery to the brain tumors. A suitable in vivo Glioblastoma Multiforme (GBM) model is needed for efficient testing of BBB crossable pharmaceuticals. In this study, we firstly confirmed the BBB functionality in 3dpf zebrafish embryos by Lucifer Yellow, Evans Blue and DAPI microinjection. We then transplanted human GBM tumor cells into the zebrafish brain, in which implanted GBM cells (U87 and U251) were highly mitotic and invasive, mimicking their malignancy features in rodents' brain. Interestingly, we found that, although extensive endothelial proliferation and vessel dilation were observed in GBM xenografts, the BBB was still not disturbed. Next, using the zebrafish orthotopic GBM xenograft model as an in vivo visual readout, we successfully identified a promising small compound named TNB, which could efficiently cross the zebrafish BBB and inhibit the progression of orthotopic GBM xenografts. These results indicate that TNB is a promising BBB crossable GBM drug worth to be further characterized in human BBB setting, also suggest the zebrafish orthotopic GBM model as an efficient visual readout for the BBB penetrating anti-GBM drugs.
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http://dx.doi.org/10.1038/s41598-017-14766-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662771PMC
October 2017

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

Lab Invest 2017 12 21;97(12):1500-1507. Epub 2017 Aug 21.

State Key Laboratory of Biotherapy, Cancer Center, West China Hospital, Sichuan University, 17# 3rd Section of South Renmin Road, Chengdu 610041, Sichuan, China.

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.
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http://dx.doi.org/10.1038/labinvest.2017.83DOI Listing
December 2017

Wolf-Hirschhorn Syndrome Candidate 1 (whsc1) Functions as a Tumor Suppressor by Governing Cell Differentiation.

Neoplasia 2017 Aug 24;19(8):606-616. Epub 2017 Jun 24.

State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, College of Life Science, Sichuan University, Chengdu, 610041, People's Republic of China. Electronic address:

Wolf-Hirschhorn syndrome candidate 1 (WHSC1) is a histone 3 lysine 36 (H3K36) specific methyltransferase that is frequently deleted in Wolf-Hirschhorn syndrome (WHS). Whsc1 is also found mutated in a subgroup of B-cell derived malignant diseases by genomic translocation or point mutation, both of which resulted in hyperactivity of WHSC1 mediated H3K36 methylation and uncontrolled cell proliferation, suggesting that whsc1 functions as an oncogene. However, here we provided evidences to show that whsc1 also has tumor suppressor functions. We used zebrafish as an in vivo model and generated homozygous whsc1 mutant lines via clustered regularly interspaced short palindromic repeats-associated protein Cas9 (CRISPR/Cas9) technology. Then western-blot (WB) and immunofluorescence (IF) were performed to analysis the expression level of H3K36Me2 and H3K36Me3, and we identified the diseased tissue via hematoxylin-eosin (HE) staining, IF staining or immunohistochemistry (IHC). Whsc1 lose-of-function led to significant decrease in di- and tri-methylation of H3K36. A series of WHS related phenotypes were found in whsc1 zebrafish, including growth retardation, neural development defects and heart failure. In addition, loss of function of whsc1 led to defects in the development of swim bladder, possibly through the dis-regulation of key genes in swim bladder organogenesis and inhibition of progenitor cell differentiation, which was correlated with its expression in this organ during embryonic development. At later stage, these whsc1 zebrafishes are inclined to grow tumors in the swim bladder. Our work suggested that whsc1 may function as a tumor suppressor by governing progenitor cell differentiation.
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http://dx.doi.org/10.1016/j.neo.2017.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487304PMC
August 2017

CRISPR/Cas9-mediated correction of human genetic disease.

Sci China Life Sci 2017 May 3;60(5):447-457. Epub 2017 May 3.

State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 system (CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs, this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity, and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining (NHEJ) and homology directed repair (HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.
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http://dx.doi.org/10.1007/s11427-017-9032-4DOI Listing
May 2017

Gene Therapy of Adult Neuronal Ceroid Lipofuscinoses with CRISPR/Cas9 in Zebrafish.

Hum Gene Ther 2017 07 5;28(7):588-597. Epub 2017 May 5.

1 State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, People's Republic of China.

Adult-onset neuronal ceroid lipofuscinosis (ANCL), one of the neuronal ceroid lipofuscinosis (NCLs), is an inherited neurodegenerative disorder with progressive neuronal dysfunction. Recently, mutations in the DNAJC5 gene that encodes cysteine-string protein alpha (CSPα) have been reported to be associated with familial autosomal-dominant ANCL (AD-ANCL). This study constructed an ANCL transgenic zebrafish model expressing the human mutant DNAJC5 (mDNAJC5) gene under the control of a zebrafish neuron-specific promoter. To investigate whether gene therapy based on genome-editing technology could treat ANCL, a panel of TALEN and Cas9 nucleases was designed to disrupt the mDNAJC5 gene in this transgenic animal model. By screening these nucleases, it was found that one nuclease that targeted the 5' coding region efficiently alleviated mDNAJC5 protein aggregates in the affected neurons. Therefore, this study provides a gene therapy strategy via the use of the CRISPR/Cas9 system to treat neural genetic diseases.
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http://dx.doi.org/10.1089/hum.2016.190DOI Listing
July 2017

eIF3i activity is critical for endothelial cells in tumor induced angiogenesis through regulating VEGFR and ERK translation.

Oncotarget 2017 Mar;8(12):19968-19979

State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.

Translational control is a critical step in the regulation of gene expression. Accumulating evidence shows that translational control of a subgroup of mRNAs tends to be selective. However, our understanding of the function of selective translational control in endothelial cells is still incomplete. We found that a key translational regulator, eIF3i, is highly expressed in endothelial cells during embryonic and tumor angiogenesis. Knockdown of eIF3i restrained cell proliferation and migration in endothelial cells. In zebrafish angiogenesis model, eIF3i mutant endothelial cells could not respond to induction signals from tumor mass. Mechanistically, we showed that eIF3i knockdown reduced VEGFR/ERK signaling by down-regulating VEGFR2 and ERK protein expression. Gene therapy model suggested that the growth and metastasis of cancer cells were suppressed by eIF3i shRNA. Therefore, our work established a selective translational regulatory mechanism during tumor induced angiogenesis and suggested that targeting eIF3i may be applicable for anticancer therapy.
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http://dx.doi.org/10.18632/oncotarget.15274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386737PMC
March 2017

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

ACS Nano 2017 01 17;11(1):95-111. Epub 2016 Nov 17.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and Collaborative Innovation Center for Biotherapy, Sichuan University , Chengdu 610041, P. R. China.

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.
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http://dx.doi.org/10.1021/acsnano.6b04261DOI Listing
January 2017

Long Noncoding RNA LINC00092 Acts in Cancer-Associated Fibroblasts to Drive Glycolysis and Progression of Ovarian Cancer.

Cancer Res 2017 03 13;77(6):1369-1382. Epub 2017 Jan 13.

Department of Obstetrics and Gynecology, Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE and State Key Laboratory of Biotherapy, West China Second Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, P.R. China.

The majority of patients with epithelial ovarian cancer are diagnosed at a late stage when the peritoneal metastases exist; however, there is little knowledge of the metastatic process in this disease setting. In this study, we report the identification of the long noncoding RNA LINC00092 as a nodal driver of metastatic progression mediated by cancer-associated fibroblasts (CAF). Prometastatic properties of CAFs and were found to associate with elevated expression of the chemokine CXCL14. In clinical specimens, elevated levels of CXCL14 in CAFs also correlated with poor prognosis. Notably, CXCL14-high CAFs mediated upregulation of LINC00092 in ovarian cancer cells, the levels of which also correlated with poor prognosis in patients. Mechanistic studies showed that LINC00092 bound a glycolytic enzyme, the fructose-2,6-biphosphatase PFKFB2, thereby promoting metastasis by altering glycolysis and sustaining the local supportive function of CAFs. Overall, our study uncovered a positive feedback loop in the metabolism of CXCL14-positive CAFs and ovarian cancer cells that is critical for metastatic progression. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-1615DOI Listing
March 2017

Erratum to: A zebrafish mosaic assay to study mammalian stem cells in real time in vivo.

J Mol Histol 2017 04;48(2):157

College of Life Science and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.

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http://dx.doi.org/10.1007/s10735-016-9704-1DOI Listing
April 2017

Loss of ZNF32 augments the regeneration of nervous lateral line system through negative regulation of SOX2 transcription.

Oncotarget 2016 Oct;7(43):70420-70436

Division of Cancer Biotherapy, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, P.R. China.

Human zinc finger protein 32 (ZNF32) is a Cys2-His2 zinc-finger transcription factor that plays an important role in cell fate, yet much of its function remains unknown. Here, we reveal that the zebrafish ZNF32 homologue zfZNF32 is expressed in the nervous system, particularly in the lateral line system. ZfZNF32 knock-out zebrafish (zfZNF-/-) were generated using the CRISPR-associated protein 9 system. We found that the regenerative capacity of the lateral line system was increased in zfZNF-/- upon hair cell damage compared with the wild type. Moreover, SOX2 was essential for the zfZNF32-dependent modulation of lateral line system regeneration. Mechanistic studies showed that ZNF32 suppressed SOX2 transcription by directly binding to a consensus sequence (5'-gcattt-3') in the SOX2 promoter. In addition, ZNF32 localizes to the nucleus, and we have identified that amino acids 1-169 (Aa 1-169) and each of three independent nuclear localization signals (NLSs) in ZNF32 are indispensable for ZNF32 nuclear trafficking. Mutating the NLSs disrupted the inhibitory effect of ZNF32 in SOX2 expression, highlighting the critical role of the NLSs in ZNF32 function. Our findings reveal a pivotal role for ZNF32 function in SOX2 expression and regeneration regulation.
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http://dx.doi.org/10.18632/oncotarget.11895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342562PMC
October 2016

A zebrafish mosaic assay to study mammalian stem cells in real time in vivo.

J Mol Histol 2016 Oct 23;47(5):437-44. Epub 2016 Aug 23.

College of life Science and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China.

The differentiation potentials of stem cells have been evaluated by various in vivo and in vitro assays. However, these assays have different limitations hindering efficient study of mammalian stem cells. Here we describe a rapid and powerful mosaic assay to study the differentiation potentials of stem cells in real time in vivo by using zebrafish embryo. We transplanted mouse neural stem cells into zebrafish embryos at different developmental stages and found that they mainly formed neural tissues while occasionally trans-differentiated into mesoderm- and endoderm-derived tissues. Because zebrafish embryo is transparent, the behaviors of transplanted mouse stem cells can be easily tracked in a real-time manner and at single-cell resolution. We expect that this assay may be widely applied to explore the in vivo behaviors of any stem cells available.
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http://dx.doi.org/10.1007/s10735-016-9688-xDOI Listing
October 2016

Pigment epithelial-derived factor gene loaded novel COOH-PEG-PLGA-COOH nanoparticles promoted tumor suppression by systemic administration.

Int J Nanomedicine 2016 25;11:743-59. Epub 2016 Feb 25.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, People's Republic of China.

Anti-angiogenesis has been proposed as an effective therapeutic strategy for cancer treatment. Pigment epithelium-derived factor (PEDF) is one of the most powerful endogenous anti-angiogenic reagents discovered to date and PEDF gene therapy has been recognized as a promising treatment option for various tumors. There is an urgent need to develop a safe and valid vector for its systemic delivery. Herein, a novel gene delivery system based on the newly synthesized copolymer COOH-PEG-PLGA-COOH (CPPC) was developed in this study, which was probably capable of overcoming the disadvantages of viral vectors and cationic lipids/polymers-based nonviral carriers. PEDF gene loaded CPPC nanoparticles (D-NPs) were fabricated by a modified double-emulsion water-in-oil-in-water (W/O/W) solvent evaporation method. D-NPs with uniform spherical shape had relatively high drug loading (~1.6%), probably because the introduced carboxyl group in poly (D,L-lactide-co-glycolide) terminal enhanced the interaction of copolymer with the PEDF gene complexes. An excellent in vitro antitumor effect was found in both C26 and A549 cells treated by D-NPs, in which PEDF levels were dramatically elevated due to the successful transfection of PEDF gene. D-NPs also showed a strong inhibitory effect on proliferation of human umbilical vein endothelial cells in vitro and inhibited the tumor-induced angiogenesis in vivo by an alginate-encapsulated tumor cell assay. Further in vivo antitumor investigation, carried out in a C26 subcutaneous tumor model by intravenous injection, demonstrated that D-NPs could achieve a significant antitumor activity with sharply reduced microvessel density and significantly promoted tumor cell apoptosis. Additionally, the in vitro hemolysis analysis and in vivo serological and biochemical analysis revealed that D-NPs had no obvious toxicity. All the data indicated that the novel CPPC nanoparticles were ideal vectors for the systemic delivery of PEDF gene and might be widely used as systemic gene vectors.
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http://dx.doi.org/10.2147/IJN.S97223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772918PMC
October 2016

Inducing Specific Immune Tolerance to Prevent Type 1 Diabetes in NOD Mice.

Pancreas 2016 07;45(6):882-8

From the *Department of Radiology, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province, China; †Donnellyl Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada; ‡Department of Cardiology, The Affiliated Hospital of Hangzhou Normal University, Institute of Ageing Research, School of Medicine, Hangzhou Normal University, Hangzhou; and §Department of Vascular Surgery, Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei Province, China.

Objectives: Proinsulin is the first autoantigen in type 1 diabetes (T1D). We reasoned that coupling hematopoietic stem cells (HSCs) transplantation with ex vivo transduction of syngeneic HSCs with lentiviral vectors to express proinsulin II could prevent T1D in nonobese diabetic (NOD) mice.

Methods: Hematopoietic stem cells were isolated from 6- to 8-week-old NOD female mice and transduced in vitro with lentiviral vectors encoding proinsulin II. Preconditioned 3- to 4-week-old female NOD mice were transplanted with transduced or nontransduced HSCs and compared with age-matched unmanipulated control. The insulitis, T1D development, and immune reconstitution were assessed.

Results: The mean (SD) insulitis score was significantly reduced (1.156 [0.575] vs 2.156 [0.892] or 3.043 [0.728], P = 0.009 or <0.001), and diabetes was nearly completely prevented (1/13 vs 5/12 or 4/9, P = 0.031 or 0.013) in recipients of transduced HSCs expressing proinsulin II as compared with recipients of nontransduced HSCs or unmanipulated control. Sialitis, reconstitution of peripheral blood leukocytes, and in vitro recall responses to ovalbumin were not different between 3 groups of mice.

Conclusions: Syngeneic transplantation of HSCs transduced ex vivo with lentiviral vectors to encode proinsulin II is a novel strategy to prevent T1D.
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http://dx.doi.org/10.1097/MPA.0000000000000603DOI Listing
July 2016

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

Mol Pharm 2016 Feb 6;13(2):663-76. Epub 2016 Jan 6.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy , Chengdu, Sichuan 610041, China.

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.
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http://dx.doi.org/10.1021/acs.molpharmaceut.5b00922DOI Listing
February 2016

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

AAPS PharmSciTech 2016 Jun 3;17(3):710-7. Epub 2015 Sep 3.

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan, 610041, China.

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.
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http://dx.doi.org/10.1208/s12249-015-0382-1DOI Listing
June 2016

CRISPR/Cas9-Mediated Genome Editing of Epigenetic Factors for Cancer Therapy.

Hum Gene Ther 2015 Jul;26(7):463-71

State Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy, West China Hospital, Sichuan University , Chengdu, China.

Advances in engineered recombinant nuclease have provided facile and reliable methods for genome editing. Especially with the development of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) system, the discovery of various versions of Cas9 proteins and delivery carriers, it is now practicable to introduce desired mutations into the genome, to correct disease-related mutations, and to activate or suppress genes of interest. Epigenetic regulators are often disturbed in cancer cells and are essential for the transformation of normal to cancerous cells. Tumor-related epigenetic alterations or epigenetic factor mutations play a major part during the various steps of carcinogenesis and affect a variety of cancer-related genes and a wide range of cancerous phenotypes. Therefore, epigenetic regulatory enzymes might be candidate targets for cancer therapy. In this review, we discuss prospects of CRISPR/Cas9-based genome editing in targeting epigenetics for cancer gene therapy.
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http://dx.doi.org/10.1089/hum.2015.067DOI Listing
July 2015