Argonaute proteins are key determinants of RNAi efficacy,toxicity, and persistence in the adult mouse liver

shRNA overexpression from viral gene therapy vectors can trigger cytotoxicity leading to organ failure and lethality in mice and rats. This process likely involves saturation of endogenous cellular RNAi factors including exportin-5 (Xpo-5). Here, we have shown that Xpo-5 overexpression enhanced shRNA efficiency in the liver of adult mice but increased hepatotoxicity. We identified the 4 members of the human Argonaute (Ago) protein family as downstream factors involved in saturation of endogenous cellular RNAi, all of which were able to interact with shRNAs in cells and mice. In Ago/shRNA coexpression studies, Ago-2 (Slicer) was the primary rate-limiting determinant of both in vitro and in vivo RNAi efficacy, toxicity, and persistence. In adult mice, vector-based Ago-2/Xpo-5 coexpression enhanced U6-driven shRNA silencing of exogenous and endogenous hepatic targets, reduced hepatotoxicity, and extended RNAi stability by more than 3 months. Use of weaker RNA polymerase III promoters to minimize shRNA expression likewise alleviated in vivo toxicity and permitted greater than 95% persistent knockdown of hepatitis B virus and other transgenes in mouse liver for more than 1 year. Our studies substantiate that abundant small RNAs can overload the endogenous RNAi pathway and reveal possible strategies for reducing hepatotoxicity of short- and long-term clinical gene silencing in humans.     

Vectors for RNA interference

Introduction of double-stranded RNA into cells causes gene silencing in a sequence-specific manner, involving the coordinated activity of enzymes such as Dicer and RNA-induced silencing complex. Several groups have recently demonstrated that this phenomenon of RNA interference (RNAi) occurs in mammalian cells when small interfering (si)RNAs are used, and have developed vector-based siRNA expression systems that can induce RNAi in living cells. These vector systems use polymerase III promoters, such as U6 or H1, and are classified into two groups based on the form of expressed RNA, tandem or hairpin. This review describes the basis for, and methodology of siRNA expression vectors for mammalian cells.     

Protection from HIV-1 infection of primary CD4 T cells by CCR5 silencing is effective for the full spectrum of CCR5 expression

Stable gene silencing by RNA interference (RNAi) can be achieved by expression of small hairpin RNAs (shRNAs) from RNA polymerase III promoters. We have tested lentiviral vectors expressing shRNAs targetting CCR5 in primary CD4 T cells from donors representing various CCR5 and CCR2 genetic backgrounds covering the full spectrum of CCR5 expression levels and permissiveness for HIV-1 infection. A linear decrease in CCR5 expression resulted in a logarithmic decrease in cellular infection, giving up to three logs protection from HIV-1 infection in vitro. Protection was maintained at very high multiplicity of infection. This and other recent reports on RNAi should open a debate about the use of RNAi gene therapy for HIV infection.     

Short-hairpin RNA expressed from polymerase III promoters mediates RNA interference in mosquito cells

Putative U6snRNA polymerase III (PolIII) promoters were cloned from the Anopheles gambiae and Aedes aegypti genomes. The PolIII promoters were tested for their ability to express short-hairpin RNA (shRNA) targeted to firefly luciferase and to mediate RNA interference (RNAi) knockdown of a co-transfected luciferase reporter gene vector in AG-55 Anopheles gambiae and ATC-10 Aedes aegypti cells. Promoters capable of silencing expression of the co-transfected luciferase plasmid by up to 95% in AG-55 cells and up to 75% in ATC-10 cells were identified. RNase protection experiments allowed detection of the 19 nt luciferase short-interfering RNA (siRNA) in transfected cells. These findings indicate that mosquito U6snRNA gene promoters can be used for production of shRNA to induce the RNAi response in mosquito cells.     

Adenovirus vector-mediated doxycycline-inducible RNA interference

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.     

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.     

Artificial MicroRNAs as siRNA Shuttles: Improved Safety as Compared to shRNAs In vitro and In vivo

RNA interference (RNAi) provides a promising therapeutic approach to human diseases. However, data from recent reports demonstrate that short-hairpin RNAs (shRNAs) may cause cellular toxicity, and this warrants further investigation of the safety of using RNAi vectors. Earlier, in comparing hairpin-based RNAi vectors, we noted that shRNAs are highly expressed and yield an abundance of unprocessed precursors, whereas artificial microRNAs (miRNAs) are expressed at lower levels and are processed efficiently. We hypothesized that unprocessed shRNAs arise from the saturation of endogenous RNAi machinery, which poses likely a burden to cells. In this study, we tested that hypothesis by assessing the relative effects of shRNAs and artificial miRNAs on the processing and function of miRNAs. In competition assays, shRNAs disrupted miRNA biogenesis and function, whereas artificial miRNAs avoided this interference even when dosed to silence as effectively as shRNAs. We next compared the safety of these vectors in mouse cerebella, and found that shRNAs cause Purkinje cell neurotoxicity. By contrast, artificial miRNA expression was well tolerated, resulting in effective target gene silencing in Purkinje cells. These findings, together with data from earlier work in mouse striata, suggest that miRNA-based platforms are better suited for therapeutic silencing in the mammalian brain.     

RNA interference mediated in human primary cells via recombinant baculoviral vectors.

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.     

慢病毒介导环氧化酶-2的沉默对食管鳞癌细胞增殖的抑制效应

目的制备环氧化酶-2(COX-2)的RNA干扰(RNAi)重组慢病毒(lentivirus),观察COX-2表达沉默对人食管鳞癌(ESCC)细胞增殖的影响。方法将目的基因短发卡RNA(sh RNA)载体与包装载体共转染293T细胞,收集上清液后浓缩纯化、滴度测定、感染ESCC细胞;应用荧光显微镜观察包装效率及感染效率,实时荧光定量PCR及Western blot评价COX-2基因沉默效应,MTS法检测细胞的增殖。结果成功制备了沉默COX-2的重组慢病毒,该病毒感染ESCC细胞后,COX-2 m RNA和蛋白的表达量分别降低了80%和50%左右,细胞的增殖明显减慢。结论慢病毒介导的sh RNA干扰能有效沉默ESCC细胞COX-2基因,COX-2表达沉默抑制ESCC细胞增殖。     

Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors.

Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. We examined the effects of shRNA expression in primary human lymphocytes (PBLs) using lentiviral vectors bearing different RNA polymerase III promoters. We found that the U6 promoter is more efficient than the H1 promoter for shRNA expression and for reducing expression of CCR5 in PBLs. However, shRNA expression from the U6 promoter resulted in a gradual decline of the transduced cell populations. With one CCR5 shRNA this decline could be attributed to elevated apoptosis but another CCR5 shRNA that caused cytotoxicity did not show evidence of apoptosis, suggesting sequence-specific mechanisms for cytotoxicity. In contrast to the U6 promoter, PBLs transduced by vectors expressing shRNAs from the H1 promoter could be maintained without major cytotoxic effects. Since a lower level of shRNA expression appears to be advantageous to maintaining the shRNA-transduced population, lentiviral vectors bearing the H1 promoter are more suitable for stable transduction and expression of shRNA in primary human T lymphocytes. Our results suggest that functional shRNA screens should include tests for both potency and adverse metabolic effects upon primary cells.     

Bioreducible poly (β-amino esters)/shRNA complex nanoparticles for efficient RNA delivery

RNA interference (RNAi) mediating gene silencing is a promising approach in the area of gene therapy, but it still is a major challenge to find new non-viral vectors with high transfection efficiency and low toxicity until today. In this work, three novel bioreducible poly (β-amine esters) (PAEs) with different amino monomers in the main chain were designed and synthesized by Michael addition polymerization. All PAEs could condense shRNA into complex nanoparticles with particle size (60-200 nm) and positive surface charges (>+10 mV). The PAEs/shRNA complex nanoparticles (PAENs) were stable under the extracellular physiological condition, while it would degrade in the reductive environment due to the cleavage of the disulfide bonds in the PAEs main chain. PAENs could achieve efficient cellular uptake and EGFP silencing in HEK-293 cells and U-87 MG cells with low cytotoxicity. The high accumulation of PAENs in tumor and high silencing efficiency of intra-tumor EGFP expression occurred when PAENs were intravenously injected into BALB/c mice bearing U-87 MG-GFP tumor. The relationship between the polymer structure and RNAi efficiency and cytotoxicity showed that the density of nitrogen atoms in PAEs backbone and the existence of disulfide bonds demonstrated the remarkable influence on in vitro and in vivo gene silencing efficiency and cytotoxicity. These experimental results suggested that the PAENs could be a promising non-viral vector for efficient RNA delivery.     

Recombinant self-complementary adeno-associated virus serotype vector-mediated hematopoietic stem cell transduction and lineage-restricted, long-term transgene expression in a murine serial bone marrow transplantation model

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.     

Foamy virus vectors for gene transfer

Foamy virus (FV) vectors are efficient gene delivery vehicles that have shown great promise for gene therapy in preclinical animal models. FVs or spumaretroviruses are not endemic in humans, but are prevalent in nonhuman primates and in other mammals. They have evolved means for efficient horizontal transmission in their host species without pathology. FV vectors have several unique properties that make them well suited for therapeutic gene transfer including a desirable safety profile, a broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. They mediate efficient and stable gene transfer to hematopoietic stem cells (HSCs) in mouse models, and in the canine large animal model. Analysis of FV vector integration sites in vitro and in hematopoietic repopulating cells shows they have a unique integration profile, and suggests they may be safer than gammaretroviruses or lentiviral vectors. Here, properties of FVs relevant to the safety and efficacy of FV vectors are discussed. The development of FV vector systems is described, and studies evaluating their potential in vitro, and in small and large animal models, is reviewed.     

Evaluation of safety and efficacy of RNAi against HIV-1 in the human immune system (Rag-2(-/-)gammac(-/-)) mouse model

RNA interference (RNAi) gene therapy against HIV-1 by stable expression of antiviral short hairpin RNAs (shRNAs) can potently inhibit viral replication in T cells. Recently, a mouse model with a human immune system (HIS) was developed that can be productively infected with HIV-1. In this in vivo model, in which Rag-2(-/-)gamma(c)(-/-) mice are engrafted with human CD34(+)CD38(-) hematopoietic precursor cells, we evaluated an anti-HIV RNAi gene therapy. Human hematopoietic stem cells were transduced with a lentiviral vector expressing an shRNA against the HIV-1 nef gene (shNef) or the control vector. We observed normal development of the different cell subsets of the immune system. However, although initial transduction efficiencies were similar for both vectors, a reduced percentage of transduced human immune cells was observed for the shNef vector after establishment of the HIS in vivo. Further studies are required to fully evaluate the safety implications. When we infected the mature human CD4(+) T cells from the HIS mouse ex vivo with HIV-1, potent inhibition of viral replication was scored in shNef-expressing cells, confirming efficacy. When challenged with an shNef-resistant HIV-1 variant, equal replication was scored in control and shNef-expressing cells, confirming sequence-specificity of the RNAi therapy. We thus demonstrated that an antiviral RNAi-based gene therapy on blood stem cells leads to HIV-1-resistant T cells in vivo, an important proof of concept in the clinical development of RNAi against HIV-1.