Randomized, Double-Blind, Placebo-Controlled Phase III Study of Paclitaxel ± Napabucasin in Pretreated Advanced Gastric or Gastroesophageal Junction Adenocarcinoma.

PURPOSE: To compare napabucasin (generator of reactive oxygen species) plus paclitaxel with paclitaxel only in patients with second-line advanced gastric or gastroesophageal junction (GEJ) adenocarcinoma. EXPERIMENTAL DESIGN: In the double-blind, phase III BRIGHTER study (NCT02178956), patients were randomized (1:1) to napabucasin (480 mg orally twice daily) plus paclitaxel (80 mg/m2 i.v. weekly for 3 of 4 weeks) or placebo plus paclitaxel. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), objective response rate (ORR), disease control rate (DCR), and safety. RESULTS: Overall, 714 patients were randomized (napabucasin plus paclitaxel, n = 357; placebo plus paclitaxel, n = 357). 72.1% were male, 74.6% had gastric adenocarcinoma, and 46.2% had peritoneal metastases. The study was unblinded following an interim analysis at 380 deaths. The final efficacy analysis was performed on 565 deaths (median follow-up, 6.8 months). No significant differences were observed between napabucasin plus paclitaxel and placebo plus paclitaxel for OS (6.93 vs. 7.36 months), PFS (3.55 vs. 3.68 months), ORR (16% vs. 18%), or DCR (55% vs. 58%). Grade ≥3 adverse events occurred in 69.5% and 59.7% of patients administered napabucasin plus paclitaxel and placebo plus paclitaxel, respectively, with grade ≥3 diarrhea reported in 16.2% and 1.4%, respectively. CONCLUSIONS: Adding napabucasin to paclitaxel did not improve survival in patients with pretreated advanced gastric or GEJ adenocarcinoma. Consistent with previous reports, the safety profile of napabucasin was driven by manageable gastrointestinal events; grade ≥3 diarrhea occurred at a higher frequency with napabucasin plus paclitaxel versus placebo plus paclitaxel.

Synthetic Biology Medicine and Bacteria-Based Cancer Therapeutics.

Spontaneous tumor regression following bacterial infection has been observed for hundreds of years. These observations along with anecdotal medical findings in 1890s led to the development of Coley's "toxins," consisting of killed Streptococcus pyogenes and Serratia marcescens bacteria, as the first cancer immunotherapy. The use of this approach, however, was not widely accepted at the time especially after the introduction of radiation therapy as a treatment for cancer in the early 1900s. Over the last 30-40 years there has been renewed interest in the use of bacteria to treat human solid tumors. This is based on the observation that various nonpathogenic anaerobic bacteria can infiltrate and replicate within solid tumors when given intravenously. Bacteria tested as potential anticancer agents include the Gram-positive obligate anaerobes Bifidobacterium and Clostridium, as well as the gram-negative facultative anaerobe Salmonella. Recent advances in synthetic biology and clinical success in cancer immunotherapy provide renewed momentum for developing bacteria-based cancer immunotherapy for cancer treatment and should allow greater potential for the development of novel therapeutic approaches for this devastating disease.

Two controls of cell proliferation underlie cancer relapse.

Much progress has been made in understanding the basis of cancer. Current therapies can effectively shrink tumors. But they frequently relapse, metastasize to other locations, and are lethal. Effective therapies are very much needed for preventing this relapse. Creation of a eukaryotic organism commences with one original stem cell, a fertilized egg, which multiplies and differentiates. Mutations of normal stem cells can produce cancer stem cells (CSC). These cells may resist chemotherapy, proliferate, and produce new tumors. Human chorionic gonadotrophin (hCG) is composed of two proteins (alpha and beta) that bind to the cell membrane and activate a number of intracellular pathways. hCG has been shown to activate the proliferation of cancer stem cells. Cyclin dependent regulation of the adult cells is created in normal differentiation and replaces the hCG regulation of stem cells. To selectively kill the cancer stem cells conventional cancer therapies could be followed with a therapy based on inactivating human chronic gonadotrophin (HCG). For example chemically modified prostaglandins like RU486 prevent binding of the unmodified steroid to hCG and inactivate hCG.

Napabucasin versus placebo in refractory advanced colorectal cancer: a randomised phase 3 trial.

BACKGROUND: Napabucasin is a first-in-class cancer stemness inhibitor that targets STAT3, which is a poor prognostic factor in colorectal cancer. This study aimed to test napabucasin in advanced colorectal cancer. METHODS: This study was a double-blind randomised phase 3 trial done at 68 centres in Canada, Australia, New Zealand, and Japan. Patients with advanced colorectal cancer with a good Eastern Cooperative Oncology Group (ECOG) performance status (0-1) for whom all available standard therapies had failed were eligible for the study. Patients were randomly assigned (1:1) to receive placebo or napabucasin through a web-based system with a permuted block method, after stratification by ECOG performance status, KRAS status, previous VEGF inhibitor treatment, and time from diagnosis of metastatic disease. Napabucasin 480 mg or matching placebo was taken orally every 12 h. All patients received best supportive care. The primary endpoint was overall survival assessed in an intention-to-treat analysis. This is the final analysis of this trial, which is registered at ClinicalTrials.gov, number NCT01830621. FINDINGS: Accrual began on April 15, 2013, and was stopped for futility on May 23, 2014, at which point 282 patients had undergone randomisation (138 assigned to the napabucasin group and 144 to the placebo group). Overall survival did not differ significantly between groups: median overall survival was 4·4 months (95% CI 3·7-4·9) in the napabucasin group and 4·8 months (4·0-5·3) in the placebo group (adjusted hazard ratio [HR] 1·13, 95% CI 0·88-1·46, p=0·34). The safety population included 136 patients in the napabucasin group and 144 patients in the placebo group. More patients who received napabucasin had any grade of treatment-related diarrhoea (108 [79%] of 136 patients), nausea (69 [51%]), and anorexia (52 [38%]) than did patients who received placebo (28 [19%] of 144 patients, 35 [24%], and 23 [16%], respectively). The most common severe (grade 3 or worse) treatment-related adverse events were abdominal pain (five [4%] patients receiving napabucasin vs five [3%] receiving placebo), diarrhoea (21 [15%] vs one [1%]), fatigue (14 [10%] vs eight [6%]), and dehydration (six [4%] vs one [1%]). 251 (89%) patients had data on pSTAT3 expression, of whom 55 (22%) had pSTAT3-positive tumours (29 in the napabucasin group, 26 in the placebo group). In a prespecified biomarker analysis of pSTAT3-positive patients, overall survival was longer in the napabucasin group than in the placebo group (median 5·1 months [95% CI 4·0-7·5] vs 3·0 months [1·7-4·1]; HR 0·41, 0·23-0·73, p=0·0025). INTERPRETATION: Although there was no difference in overall survival between groups in the overall unselected population, STAT3 might be an important target for the treatment of colorectal cancer with elevated pSTAT3 expression. Nevertheless, these results require validation. FUNDING: Canadian Cancer Society Research Institute and Boston Biomedical.

TLR7 tolerance is independent of the type I IFN pathway and leads to loss of anti-tumor efficacy in mice.

Systemic administration of small molecule toll-like receptor (TLR)-7 agonists leads to potent activation of innate immunity and to the generation of anti-tumor immune responses. However, activation of TLRs with small molecule agonists may lead to the induction of TLR tolerance, defined as a state of hyporesponsiveness to subsequent agonism, which may limit immune activation, the generation of anti-tumor responses and clinical response. Our data reveal that dose scheduling impacts on the efficacy of systemic therapy with the selective TLR7 agonist, 6-amino-2-(butylamino)-9-((6-(2-(dimethylamino)ethoxy)pyridin-3-yl)methyl)-7,9-dihydro-8H-purin-8-one (DSR-6434). In a preclinical model of renal cell cancer, systemic administration of DSR-6434 dosed once weekly resulted in a significant anti-tumor response. However, twice weekly dosing of DSR-6434 led to the induction of TLR tolerance, and no anti-tumor response was observed. We show that TLR7 tolerance was independent of type I interferon (IFN) negative feedback because induction of TLR7 tolerance was also observed in IFN-α/ß receptor knockout mice treated with DSR-6434. Moreover, our data demonstrate that treatment of bone marrow-derived plasmacytoid dendritic cells (BM-pDC) with DSR-6434 led to downregulation of TLR7 expression. From our data, dose scheduling of systemically administered TLR7 agonists can impact on anti-tumor activity through the induction of TLR tolerance. Furthermore, TLR7 expression on pDC may be a useful biomarker of TLR7 tolerance and aid in the optimization of dosing schedules involving systemically administered TLR7 agonists.

Suppression of cancer relapse and metastasis by inhibiting cancer stemness.

Partial or even complete cancer regression can be achieved in some patients with current cancer treatments. However, such initial responses are almost always followed by relapse, with the recurrent cancer being resistant to further treatments. The discovery of therapeutic approaches that counteract relapse is, therefore, essential for advancing cancer medicine. Cancer cells are extremely heterogeneous, even in each individual patient, in terms of their malignant potential, drug sensitivity, and their potential to metastasize and cause relapse. Indeed, hypermalignant cancer cells, termed cancer stem cells or stemness-high cancer cells, that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types. Moreover, such stemness-high cancer cells are resistant to conventional chemotherapy and radiation. Here we show that BBI608, a small molecule identified by its ability to inhibit gene transcription driven by Stat3 and cancer stemness properties, can inhibit stemness gene expression and block spherogenesis of or kill stemness-high cancer cells isolated from a variety of cancer types. Moreover, cancer relapse and metastasis were effectively blocked by BBI608 in mice. These data demonstrate targeting cancer stemness as a novel approach to develop the next generation of cancer therapeutics to suppress cancer relapse and metastasis.

Target gene abundance contributes to the efficiency of siRNA-mediated gene silencing.

The gene-silencing activity of a small interfering RNA (siRNA) is determined by various factors. Considering that RNA interference (RNAi) is an unparalleled technology in both basic research and therapeutic applications, thorough understanding of the factors determining RNAi activity is critical. This report presents observations that siRNAs targeting KRT7 show cell-line-dependent activity, which correlates with the expression level of KRT7 mRNA. By modulating the target mRNA level, it was confirmed that highly expressed genes are more susceptible to siRNA-mediated gene silencing. Finally, several genes that show different expression levels in a cell-line dependent manner were tested, which verified the expression-level-dependent siRNA activities. These results strongly suggest that the abundance of target mRNA is a critical factor that determines the efficiency of the siRNA-mediated gene silencing in a given cellular context. This report should provide practical guidelines for designing RNAi experiments and for selecting targetable genes in RNAi therapeutics studies.

RNA interference-mediated simultaneous silencing of four genes using cross-shaped RNA.

The structural flexibility of RNA interference (RNAi)-triggering nucleic acids suggests that the design of unconventional RNAi trigger structures with novel features is possible. Here, we report a cross-shaped RNA duplex structure, termed quadruple interfering RNA (qiRNA), with multiple target gene silencing activity. qiRNA triggers the simultaneous down-regulation of four cellular target genes via an RNAi mechanism. In addition, qiRNA shows enhanced intracellular delivery and target gene silencing over conventional siRNA when complexed with jetPEI, a linear polyethyleneimine (PEI). We also show that the long antisense strand of qiRNA is incorporated intact into an RNA-induced silencing complex (RISC). This novel RNA scaffold further expands the repertoire of RNAi-triggering molecular structures and could be used in the development of therapeutics for various diseases including viral infections and cancer.

Ionizing radiation induces stemness in cancer cells.

The cancer stem cell (CSC) model posits the presence of a small number of CSCs in the heterogeneous cancer cell population that are ultimately responsible for tumor initiation, as well as cancer recurrence and metastasis. CSCs have been isolated from a variety of human cancers and are able to generate a hierarchical and heterogeneous cancer cell population. CSCs are also resistant to conventional chemo- and radio-therapies. Here we report that ionizing radiation can induce stem cell-like properties in heterogeneous cancer cells. Exposure of non-stem cancer cells to ionizing radiation enhanced spherogenesis, and this was accompanied by upregulation of the pluripotency genes Sox2 and Oct3/4. Knockdown of Sox2 or Oct3/4 inhibited radiation-induced spherogenesis and increased cellular sensitivity to radiation. These data demonstrate that ionizing radiation can activate stemness pathways in heterogeneous cancer cells, resulting in the enrichment of a CSC subpopulation with higher resistance to radiotherapy.

Induction of cancer cell stemness by chemotherapy.

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.

Enhanced intracellular delivery and multi-target gene silencing triggered by tripodal RNA structures.

BACKGROUND: The development of gene interfering RNA (iRNA) molecules such as small interfering RNAs (siRNAs) and antagomirs provides promising therapeutic modalities for targeting specific mRNAs and microRNAs (miRNAs) involved in disease mechanisms. Therapeutic iRNA strategy against cancer or hypermutable viruses prefers targeting multiple genes simultaneously to achieve synergistic inhibition and to prevent resistance. METHODS: In the present study, we report chemically synthesized, multi-target gene interfering RNA structures based upon branched, tripodal interfering RNAs (termed T-tiRNAs). RESULTS: The T-tiRNAs could simultaneously inhibit up to three different mRNAs or miRNAs by harboring three siRNA or antagomir units. Moreover, when complexed with cationic delivery vehicles, T-tiRNAs showed enhanced gene interfering activity over conventional siRNAs or antagomirs as a result of increased intracellular delivery. CONCLUSIONS: The data obtained in the present study provide an example of synthetic multi-functional RNA structures that enable multiple gene interference in mammalian cells, which could become powerful tools for an efficient combinatorial iRNA strategy.

Structural diversity repertoire of gene silencing small interfering RNAs.

Since the discovery of double-stranded (ds) RNA-mediated RNA interference (RNAi) phenomenon in Caenorhabditis elegans, specific gene silencing based upon RNAi mechanism has become a novel biomedical tool that has extended our understanding of cell biology and opened the door to an innovative class of therapeutic agents. To silence genes in mammalian cells, short dsRNA referred to as small interfering RNA (siRNA) is used as an RNAi trigger to avoid nonspecific interferon responses induced by long dsRNAs. An early structure-activity relationship study performed in Drosophila melanogaster embryonic extract suggested the existence of strict siRNA structural design rules to achieve optimal gene silencing. These rules include the presence of a 3' overhang, a fixed duplex length, and structural symmetry, which defined the structure of a classical siRNA. However, several recent studies performed in mammalian cells have hinted that the gene silencing siRNA structure could be much more flexible than that originally proposed. Moreover, many of the nonclassical siRNA structural variants reported improved features over the classical siRNAs, including increased potency, reduced nonspecific responses, and enhanced cellular delivery. In this review, we summarize the recent progress in the development of gene silencing siRNA structural variants and discuss these in light of the flexibility of the RNAi machinery in mammalian cells.

Long double-stranded RNA-mediated RNA interference and immunostimulation: long interfering double-stranded RNA as a potent anticancer therapeutics.

In most applications, small interfering RNAs are designed to execute specific gene silencing via RNA interference (RNAi) without triggering nonspecific responses such as immunostimulation. However, in anticancer therapeutics, immunostimulation combined with specific oncogene silencing could be beneficial, resulting in the synergistic inhibition of cancer cell growth. In this study, we report an immunostimulatory long double-stranded RNA (dsRNA) structure with the ability to trigger RNAi-mediated specific target gene silencing, termed as long interfering dsRNA (liRNA). liRNA targeting Survivin mRNA not only efficiently and specifically triggered target gene silencing via RNAi, but also stimulated the protein kinase R pathway to induce the expression of interferon ß. As a result, the ability of Survivin-targeting liRNA to inhibit cancer cell growth was superior over conventional small interfering RNA or nontargeting dsRNA structures. Our results thus provide a simple yet efficient dual function immunostimulatory RNAi-triggering structure, which is potentially applicable for the development of anticancer therapeutics.

ARQ 197, a novel and selective inhibitor of the human c-Met receptor tyrosine kinase with antitumor activity.

The met proto-oncogene is functionally linked with tumorigenesis and metastatic progression. Validation of the receptor tyrosine kinase c-Met as a selective anticancer target has awaited the emergence of selective c-Met inhibitors. Herein, we report ARQ 197 as the first non-ATP-competitive small molecule that selectively targets the c-Met receptor tyrosine kinase. Exposure to ARQ 197 resulted in the inhibition of proliferation of c-Met-expressing cancer cell lines as well as the induction of caspase-dependent apoptosis in cell lines with constitutive c-Met activity. These cellular responses to ARQ 197 were phenocopied by RNAi-mediated c-Met depletion and further demonstrated by the growth inhibition of human tumors following oral administration of ARQ 197 in multiple mouse xenograft efficacy studies. Cumulatively, these data suggest that ARQ 197, currently in phase II clinical trials, is a promising agent for targeting cancers in which c-Met-driven signaling is important for their survival and proliferation.

Carvedilol in glioma treatment alone and with imatinib in vitro.

The purpose of the study was to investigate whether carvedilol has an antiproliferative effect alone and whether carvedilol provides an additive, synergistic or antagonistic effect on imatinib mesylate-induced cytotoxicity in both C6 glioma monolayer and spheroid culture. The C6 rat glioma chemoresistant experimental brain tumour cell line, that is notoriously difficult to treat with combination chemotherapy, was used both in monolayer and spheroid cultures. We treated C6 glioma cells with carvedilol alone and a combination of carvedilol and imatinib mesylate at a concentration of 10 microM. Following treatment, we evaluated cell proliferation index, bromodeoxyuridine labelling index (BrDU-LI), cell cycle distributions, apoptotic cell percentages, cAMP levels and three dimensional cell morphology at monolayer cultures. In addition BrDU-LI, volume and morphology of spheroids were also assessed. Carvedilol and imatinib mesylate alone reduced cell number, BrDU-LI, cAMP levels and spheroid volume. Carvedilol and imatinib mesylate arrested cells at G0/G1 phase in a time-dependent manner and time-independent manner, respectively. Carvedilol increased apoptosis rate only at the 24th h, but imatinib mesylate did for all time intervals. Interestingly carvedilol, drug with well-known protective effect on mitochondria, induced severe mitochondria damage, and imatinib mesylate induced autophagy confirmed only by transmission electron microscopy. These results suggest that carvedilol showed antitumour activity against rat C6 glioma cells and a combination of carvedilol with imatinib mesylate resulted in enhanced in vitro antitumour activity.

In vitro and in vivo gene silencing by TransKingdom RNAi (tkRNAi).

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.

Asymmetric shorter-duplex siRNA structures trigger efficient gene silencing with reduced nonspecific effects.

Small interfering RNAs (siRNAs) are short, double-stranded RNAs that mediate efficient gene silencing in a sequence-specific manner by utilizing the endogenous RNA interference (RNAi) pathway. The current standard synthetic siRNA structure harbors a 19-base-pair duplex region with 3' overhangs of 2 nucleotides (the so-called 19+2 form). However, the synthetic 19+2 siRNA structure exhibits several sequence-independent, nonspecific effects, which has posed challenges to the development of RNAi therapeutics and specific silencing of genes in research. In this study, we report on the identification of truncated siRNA backbone structures with duplex regions shorter than 19 bp (referred to as asymmetric shorter-duplex siRNAs or asiRNAs) that can efficiently trigger gene silencing in human cell lines. Importantly, this asiRNA structure significantly reduces nonspecific effects triggered by conventional 19+2 siRNA scaffold, such as sense-strand-mediated off-target gene silencing and saturation of the cellular RNAi machinery. Our results suggest that this asiRNA structure is an important alternative to conventional siRNAs for both functional genomics studies and therapeutic applications.

Asymmetric RNA duplexes mediate RNA interference in mammalian cells.

RNA interference (RNAi) has become an indispensable technology for biomedical research and has demonstrated the potential to become a new class of therapeutic. Current RNAi technology in mammalian cells relies on short interfering RNA (siRNA) consisting of symmetrical duplexes of 19-21 base pairs (bp) with 3' overhangs. Here we report that asymmetric RNA duplexes with 3' and 5' antisense overhangs silence mammalian genes effectively. An asymmetric interfering RNA (aiRNA) of 15 bp was incorporated into the RNA-induced silencing complex (RISC) and mediated sequence-specific cleavage of the target mRNA between base 10 and 11 relative to the 5' end of the antisense strand. The gene silencing mediated by aiRNA was efficacious, durable and correlated with reduced off-target silencing by the sense strand. These results establish aiRNA as a scaffold structure for designing RNA duplexes to induce RNAi in mammalian cells.

High-content fluorescent-based assay for screening activators of DNA damage checkpoint pathways.

Activation of DNA damage checkpoint pathways, including Chk2, serves as an anticancer barrier in precancerous lesions. In an effort to identify small-molecule activators of Chk2, the authors developed a quantitative cell-based assay using a high-content analysis (HCA) platform. Induction of phosphorylated Chk2 was evaluated using several different parameters, including fold induction, Kolmogorov-Smirnov score, and percentage of positively stained cells. These measurements were highly correlated and provided an accurate method for compound ranking/binning, structure-activity relationship studies, and lead identification. Screening for Chk2 activators was undertaken with a target-focused library and a diversified library from ArQule chemical space. Several compounds exhibited submicromolar EC( 50) values for phosphorylated Chk2 induction. These compounds were further analyzed for Chk2-dependent cytotoxicity, as assessed through a high-content cell death assay in combination with siRNA silencing of Chk2 expression. Several compounds were identified and showed specific inhibition or lethality in a target-dependent manner. Therefore, identification of DNA damage checkpoint pathway activators by HCA is an attractive approach for discovering the next generation of targeted cancer therapeutics.

TransKingdom RNA interference: a bacterial approach to challenges in RNAi therapy and delivery.

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with "undruggable" therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver silencing shRNA to target cells. This new approach, termed TransKingdom RNAi (tkRNAi), has several key advantages. First, tkRNAi may provide a viable means to accomplish therapeutic RNAi since non-pathogenic bacteria have a proven safety record in clinical applications. Second, tkRNAi eliminates the cost of siRNA manufacture since silencing shRNA are produced inside bacteria. Moreover, the intracellular mechanism of shRNA release inherent to tkRNAi may circumvent, or mitigate, the activation of host immune responses. Finally, tkRNAi may facilitate high-throughput in vivo functional genomics screening since bacteria-based RNAi libraries can be easily constructed, stored, reproduced and amplified, thereby allowing for the creation of a stable gene silencing system.