Selection of RNAi-based inhibitors for anti-HIV gene therapy

In the last decade, RNA interference (RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAi-based inhibitors against the chronic infection with human immunodeficiency virus type 1 (HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules (shRNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 shRNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three shRNAs. These shRNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome, exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial shRNA vector will soon move forward to the first clinical studies.     

A set cover approach to fast beam orientation optimization in intensity modulated radiation therapy for total marrow irradiation

The beam orientation optimization (BOO) problem in intensity modulated radiation therapy (IMRT) treatment planning is a nonlinear problem, and existing methods to obtain solutions to the BOO problem are time consuming due to the complex nature of the objective function and size of the solution space. These issues become even more difficult in total marrow irradiation (TMI), where many more beams must be used to cover a vastly larger treatment area than typical site-specific treatments (e.g., head-and-neck, prostate, etc). These complications result in excessively long computation times to develop IMRT treatment plans for TMI, so we attempt to develop methods that drastically reduce treatment planning time. We transform the BOO problem into the classical set cover problem (SCP) and use existing methods to solve SCP to obtain beam solutions. Although SCP is NP-Hard, our methods obtain beam solutions that result in quality treatments in minutes. We compare our approach to an integer programming solver for the SCP to illustrate the speed advantage of our approach.     

Dose optimization via index-dose gradient minimization

This paper presents an iterative optimization algorithm based on gradient minimization of index dose, defined as the product of physical dose and a numerical index. Acting as a template the index distribution is designed to represent the dosimetry that meets the dose volume histogram-based optimization objectives. The treatment dosimetry is optimized when the uniformity of the index-dose distribution is maximized. Prior to optimization the user can select all or only some of the beams to be intensity modulated. The remaining unmodulated beams can be either open or wedged photon beams, electron beams, or beams of previous treatments. The optimization result and treatment delivery efficiency can often be enhanced by including not only the IM photon beams but also all suitable fixed-beams available on the linac in the treatment plan. In addition, the doses from previous treatments can also be considered in the optimization of current treatment. Five clinical examples with different complexities in optimization objective are presented. The effects of two nonoptimization variables, beam setup and initial beam weights, on the quality of the dose optimization are also presented. The results are analyzed in terms of isodose distribution, dose volume histograms, and a dose optimization quality factor. The optimization algorithm, implemented in our in-house TPS PLanUNC, has been used in clinical application since 1996. The primary advantages of our optimization algorithm include computational efficiency, intensity modulation selection choice, and performance reliability for a wide range of clinical beam setups and optimization objectives.     

Targeted gene knockdown in zebrafish using negatively charged peptide nucleic acid mimics.

Negatively charged homo-oligomers of alternating trans-4-hydroxy-L-proline/phosphonate polyamides with DNA bases (HypNA-pPNA) display excellent hybridization properties toward DNA and RNA, while preserving the mismatch discrimination, nuclease resistance, and protease resistance of peptide nucleic acids (PNAs). Similar properties are associated with morpholino phosphorodiamidate (MO) DNA mimics, which have been used in the model vertebrate zebrafish (Danio rerio) for genome-wide, sequence-based, reverse genetic screens during embryonic development. We evaluated mixed sequence HypNA-pPNAs as an alternative to MOs, and found that even a single central DNA mismatch lowered the HypNA-pPNA melting temperature by 16 degrees C. We then observed that the melting temperatures of HypNA-pPNA 18-mers hybridized to RNA 25-mers were comparable to the melting temperatures of MO 25-mers, and that two HypNA-pPNA mismatches lowered the melting temperature with RNA by 18 degrees C. In zebrafish embryos we observed that HypNA-pPNA 18-mers displayed comparable potency to MO 25-mers as knockdown agents against chordin, notail, and uroD, with greater mismatch stringency. Finally we observed that a specific HypNA-pPNA 18-mer elicited the dharma (bozozok)(-/-) phenotype in zebrafish embryos, which MO 25-mers do not. HypNA-pPNAs designed to inhibit translation of specific zebrafish RNA targets thus demonstrated stringent hybridization properties, relative to DNA and MO oligomers, and present a valuable alternative for reverse genetic studies, enabling the targeting of previously inaccessible genes.     

Using siRNA technique to generate transgenic animals with spatiotemporal and conditional gene knockdown

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.     

PINK1 gene knockdown leads to increased binding of parkin with actin filament

Mutations in the PINK1 gene are known to cause early onset familial Parkinson's disease (PD). Genetic fruit fly model studies and rescue experiments with parkin overexpression suggest that PINK1 and parkin are associated via an unidentified mechanism. To gain additional insight into this interaction, we have investigated the impact of PINK1 deficiency on the biological function of parkin using actin filament dynamics. Actin is known to be associated with parkin and is a key regulator of eukaryotic cell death. PINK1 gene knockdown (KD) significantly increased actin aggregation and its binding with parkin. Known PD-related pathological conditions, such as oxidative stress and mitochondrial dysfunction, also increased actin aggregation and parkin binding. PINK1 KD resulted in the increased phosphorylation of cofilin, a protein important for the remodeling of actin filament and neurodegeneration. These results suggest that altered actin dynamics and increased association of parkin with actin filament might underlie the pathological conditions resulting from PINK1 deficiency.     

SSX基因家族研究进展

SSX基因家族由9个成员组成,其中SSX-1、SSX-2和SSX-4经常出现在滑膜肉瘤t(X18)染色体易位的SYT-SSX融合基因中.SSX基因家族可编码肿瘤/睾丸抗原,在睾丸以外的正常组织中,除甲状腺微弱表达外,没有发现它们的存在.HOM-MEL-40是最早用重组cDNA表达文库血清学分析技术确定的由SSX-2基因表达的抗原.近来的研究发现蛋白SSX-241-49是CD8+CTL免疫识别的优势表位,而SSX-237-58和SSX-219-34分别是CD4+T细胞识别相关的HLA-DR及HLA-DP限制表位.日本学者也发现SYT-SSX基因融合蛋白断裂点衍生肽是MHC Ⅰ分子HLA-A24和HLA-B7限制的CTL识别的表位.所有这些研究,证明SSX基因家族编码蛋白是肿瘤免疫治疗有前景的靶标之一.     

sox19b在斑马鱼胚胎眼睛发育中的作用

目的 通过抑制sox19b基因的表达,探讨sox19b基因在斑马鱼胚胎眼睛发育和形成中的作用。方法 通过显微注射sox19b 吗啉寡聚核苷酸（MO）抑制sox19b 基因的表达,观察胚胎发育过程中表型的变化;采用石蜡包埋组织切片及HE染色、RT-PCR和整封原位杂交等方法探讨敲除sox19b 后对斑马鱼胚胎眼睛发育的调控机制。 结果 敲除sox19b 基因后,斑马鱼胚胎眼睛发育受到影响,表现为眼睛变小或缺失,视网膜及晶状体结构发育异常(n =57/93);眼睛发育过程中重要调控基因 rx3、pax2a及 vsx2 等表达明显降低,进而影响眼睛的发育和形成。 结论 sox19b 基因作为转录调控因子,可以通过调节眼区转录因子的表达进而影响斑马鱼胚胎早期眼睛的发育和形成。     

Nucleofection mediates high-efficiency stable gene knockdown and transgene expression in human embryonic stem cells

High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.     

Effect of vegf gene knockdown on growth of the murine sarcoma cell line MS-K

The murine sarcoma cell line MS-K was previously established as a Ki-ras-positive cell line. Inoculation of this cell line under the flank of C3H/HeN mice results in the growth of large tumors with well-developed blood vessels within day 30 of transplantation without any metastasis because MS-K cells produce vascular endothelial growth factor (VEGF). To elucidate the role of VEGF in tumor formation in vivo, stable vegf-knockdown-MS-K clones were obtained using plasmid-based knockdown vectors. Interestingly, tumorigenesis was completely suppressed in a vegf-A-knockdown-MS-K clone [designated MS-K (A-KD)]. Proliferation and colony formation capacity of the MS-K (A-KD) cells in a semi-solid medium under low serum conditions was significantly lower than that of control MS-K (SCR) cells; however, the expression of vegf-receptor 1 (vegf-r-1) was not changed. Addition of the recombinant VEGF-A(165) partially restored the colony formation capacity of MS-K (A-KD) cells and caused the phosphorylation of VEGF-r-1 (Flt-1) in MS-K (Normal) cells. Furthermore, tumorigenicity of the vegf-r-1-knockdown-MS-K clone [designated MS-K (R1-KD)] had obviously delayed or strongly suppressed compared with the MS-K (Normal). These results indicate that Vascular endothelial growth factor-A, produced from MS-K, acts as a growth factor for MS-K cells itself and supports tumor formation in vivo by inducing the blood vessel formation.     

Functional delivery of DNAzyme with iron oxide nanoparticles for hepatitis C virus gene knockdown

DNAzyme is an attractive therapeutic oligonucleotide which enables cleavage of mRNA in a sequence-specific manner and thus, silencing target gene. A particularly important challenge in achieving the successful down-regulation of gene expression is to efficiently deliver DNAzymes to disease sites and cells. Here, we report the nanoparticle-assisted functional delivery of therapeutic DNAzyme for the treatment of hepatitis C by inducing knockdown of hepatitis C virus (HCV) gene, NS3. HCV NS3 gene encodes helicase and protease which are essential for the virus replication. The nanocomplex showed efficient NS3 knockdown while not evoking undesired immune responses or notable cytotoxicity. We also demonstrated the DNAzyme conjugated nanoparticle system could be applicable in vivo by showing the accumulation of the nanoparticles in liver, and more specifically, in hepatocytes. We believe that the present work is a successful demonstration of effective, functional, non-immunostimulatory DNAzyme delivery system based on inorganic nanoparticles with high potential for further therapeutic application of DNAzyme in the treatment of hepatitis C.     

Novel genes involved in Ciona intestinalis embryogenesis: characterization of gene knockdown embryos.

The sequenced genome of the urochordate ascidian Ciona intestinalis contains nearly 2,500 genes that have vertebrate homologues, but their functions are as yet unknown. To identify novel genes involved in early chordates embryogenesis, we previously screened 200 Ciona genes by knockdown experiments using specific morpholino oligonucleotides and found that suppression of the translation of 40 genes caused embryonic defects (Yamada et al. [2003] Development 130:6485-6495). We have since examined an additional 304 genes, that is, screening 504 genes overall, and a total of 111 genes showed morphological defects when gene function was suppressed. We further examined the role of these genes in the differentiation of six major tissues of the embryo: endoderm, muscle, epidermis, neural tissue, mesenchyme, and notochord. Based on the similarity of phenotypes of gene knockdown embryos, genes were categorized into several groups, with the suggestion that the genes within a given group are involved in similar developmental processes. For example, five were shown to be novel genes that are likely involved in beta-catenin-mediated endoderm formation. The type of large-scale screening used is, therefore, a powerful approach to identify novel genes with significant developmental functions, the details of which will be determined in future studies.     

GIPC gene family (Review)

GIPC1/GIPC/RGS19IP1, GIPC2, and GIPC3 genes constitute the human GIPC gene family. GIPC1 and GIPC2 show 62.0% total-amino-acid identity. GIPC1 and GIPC3 show 59.9% total-amino-acid identity. GIPC2 and GIPC3 show 55.3% total-amino-acid identity. GIPCs are proteins with central PDZ domain and GIPC homology (GH1 and GH2) domains. PDZ, GH1, and GH2 domains are conserved among human GIPCs, Xenopus GIPC/Kermit, and Drosophila GIPC/ LP09416. Bioinformatics revealed that GIPC genes are linked to prostanoid receptor genes and DNAJB genes in the human genome as follows: GIPC1 gene is linked to prostaglandin E receptor 1 (PTGER1) gene and DNAJB1 gene in human chromosome 19p13.2-p13.1 region; GIPC2 gene to prostaglandin F receptor (PTGFR) gene and DNAJB4 gene in human chromosome 1p31.1-p22.3 region; GIPC3 gene to thromboxane A2 receptor (TBXA2R) gene in human chromosome 19p13.3 region. GIPC1 and GIPC2 mRNAs are expressed together in OKAJIMA, TMK1, MKN45 and KATO-III cells derived from diffuse-type of gastric cancer, and are up-regulated in several cases of primary gastric cancer. PDZ domain of GIPC family proteins interact with Frizzled-3 (FZD3) class of WNT receptor, insulin-like growth factor-I (IGF1) receptor, receptor tyrosine kinase TrkA, TGF-beta type III receptor (TGF-beta RIII), integrin alpha6A subunit, transmembrane glycoprotein 5T4, and RGS19/RGS-GAIP. Because RGS19 is a member of the RGS family that regulate heterotrimeric G-protein signaling, GIPCs might be scaffold proteins linking heterotrimeric G-proteins to seven-transmembrane-type WNT receptor or to receptor tyrosine kinases. Therefore, GIPC1, GIPC2 and GIPC3 might play key roles in carcinogenesis and embryogenesis through modulation of growth factor signaling and cell adhesion.     

CARMA3基因敲减对结肠癌细胞HCT116生长和侵袭转移的抑制

目的:探讨CARMA3基因在人结肠癌细胞HCT116生长和侵袭转移中的作用及其机制。方法:选取高表达CARMA3的人结肠癌细胞株。应用慢病毒技术敲减CARMA3基因,puromycin筛选后构建稳定转染的HCT116-sh CARMA3细胞株。Real-time PCR和Western blot鉴定mRNA和蛋白表达的抑制情况。WST-1法和RTCA S16系统分析细胞增殖情况。集落形成实验观察集落形成。流式细胞术检测细胞周期。显微镜下观察上皮-间充质转化(EMT)形态变化。划痕实验和Transwell实验检测细胞迁移与侵袭能力的改变。Western blot分析相关分子变化,探讨可能机制。结果:4株人结肠癌细胞株中HCT116细胞的CARMA3 mRNA和蛋白表达量最高,构建稳定沉默CARMA3的HCT116-sh CARMA3细胞株,其中HCT116-sh CARMA3-93细胞中CARMA3的mRNA和蛋白受抑制最明显,将其作为细胞模型。相比于对照组,HCT116-sh CARMA3-93细胞形态发生EMT逆转,其增殖、集落形成、迁移和侵袭能力明显下降(P0.01)。HCT116-sh CARMA3-93的G_0/G_1细胞所占比例明显升高,S期细胞比例相应下降(P0.05)。信号通路分子Bcl10和NF-κB表达明显下调,MALT-1变化不明显;细胞周期相关蛋白cyclin D1显著下调,cyclin A表达略有下降;侵袭转移相关分子MMP-2和MMP-9的表达下调,MMP-7未见改变,TIMP-1和TIMP-2的表达上调;EMT相关分子E-cadherin的表达水平升高,N-cadherin、Snail、Slug和Twist的表达水平呈不同程度降低。结论:CARMA3可通过改变细胞周期和侵袭转移分子的表达、调控EMT来影响结肠癌细胞HCT116的生长和侵袭转移。这可能与NF-κB信号通路发生改变有关。     

Enhancement of antiviral capacity of transgenic silkworms against cytoplasmic polyhedrosis virus via knockdown of multiple viral genes

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a major pathogen of silkworms, causes serious economic losses in sericulture. The BmCPV genome contains 10 discrete dsRNA segments; among these, S1, S2, S3, S4, S6, and S7 encode virus structural proteins, whereas S5, S8, S9, and S10 encode nonstructural proteins. In an attempt to create an anti-BmCPV silkworm strain, we constructed transgenic RNAi vector pb-CNS for knockdown of S5, S8, S9, and S10, and pb-SNS targeting S1, S2, S4, S5, and S8. Transgenic silkworm line CNS and SNS were generated via microinjection of the practical diapause silkworm strain Furong. Following infection via the oral administration of a high dose of BmCPV, the mortality rates of the nontransgenic control, CNS, and SNS were 91%, 37%, and 41%, respectively. qPCR showed that the viral mRNA content in CNS and SNS was significantly lower than that in the nontransgenic line. The economic traits of CNS and SNS were not affected. These results suggest that the knockdown of multiple BmCPV genes significantly enhances the antiviral capacity of the silkworm.