rna interference as an anticancer therapy: a patent perspective

Review

10.1517/13543770902838008 © 2009 Informa UK Ltd ISSN 1354-3776 475All rights reserved: reproduction in whole or in part not permitted

RNAinterferenceasananticancertherapy:apatentperspectiveDerek M DykxhoornThe University of Miami Miller School of Medicine, Miami Institute for Human Genomics, The John T Macdonald Foundation Department of Human Genetics and the Department of Microbiology and Immunology, Miami, FL 33136, USA

Background: The development of RNA interference (RNAi)-based gene silencing approaches has revolutionized biomedical research. These technologies have been applied for functional genomic studies in a variety of areas, including cancer research, by facilitating a better understanding of the mechanisms that underlie tumorigenicity and the identification of novel factors that either promote or inhibit oncogenic transformation. Objective: These approaches have laid the groundwork for the development of a novel class of genetic therapies. Preclinical results have exposed both the unique therapeutic opportunities and challenges that are encountered in adapting these technologies for clinical applications. These themes are reflected in the patent literature that has mirrored the rise in complexity and sophistication of RNAi-based therapeutic approaches. This review focuses on the identification of potential anticancer therapeutic targets and the development of clinically relevant delivery approaches. Conclusions: Thus far, the patent landscape in the RNAi field has been dominated by a handful of key patents that describe the original identification and charac-terization of inhibitory double-stranded RNA molecules. Only time will tell how these original patents will hold up in the face of the development of new approaches and reagents as RNAi-based therapeutics approach transition from the bench to the clinic.

Keywords: cancer, drug discovery, genetic therapy, RNA interference

Expert Opin. Ther. Patents (2009) 19(4):475-491

1. Introduction

RNA interference (RNAi) refers to a family of related gene silencing pathways that use small RNA molecules to guide the inhibition of gene expression in a sequence-specific manner. The term RNA interference was first coined by Fire et al. to describe the phenomenon in which the introduction of long double-stranded (ds)RNAs into Caenorhabditis elegans led to the silencing of the cognate target genes [1]. Biochemical and computational approaches have been used to profile the small RNA content of cells from diverse organisms and have identified a variety of small regulatory RNAs, including microRNAs (miRNAs), piwi-inter-acting RNAs and small interfering RNAs (siRNAs) [2-4]. Although similar in many ways, these small RNA molecules differ with respect to their length, precur-sor structure and the member of the argonaute (Ago)/piwi family of molecules with which they interact. These molecules regulate gene expression through a variety of transcriptional and post-transcriptional mechanisms and play important roles in the regulation of diverse cellular processes. Harnessing these endogenous pathways has allowed researchers to perform loss-of-function analyses in organ-isms that are intractable to conventional genetic approaches setting the stage for therapeutic gene silencing against a variety of pathogens and pathogenic states. This review focuses on the application of RNAi-based gene silencing approaches

1. Introduction

2. RNAi-based therapeutic targets

3. RNAi screens for novel anticancer

therapeutic targets

4. siRNA delivery

5. Conclusions and potential

challenges

6. Expert opinion

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476 ExpertOpin.Ther.Patents(2009) 19(4)

as a potential anticancer therapy with an emphasis on the current state of the patent literature.

1.1 miRNApathwayOf the small regulatory RNAs described until now, miRNAs are the most abundant and have the greatest sequence diversity [5,6]. Most miRNAs are encoded from non-protein coding regions of cellular genomes as RNA polymerase II generated, highly structured, primary miRNA transcripts (pri-miRNAs) (Figure 1A) [6,7]. Often, several miRNAs are expressed from the same transcript (polycistronic). The pri-miRNAs are sequentially processed by RNase III family members in part-nership with dsRNA-binding proteins. In the nucleus, the pri-miRNA is recognized and cleaved by the endoribonu-clease Drosha, associated with the dsRNA-binding protein DiGeorge syndrome critical region gene 8 (DGCR8), pro-ducing an ∼ 70 nt miRNA stem loop intermediate, the precursor miRNA (pre-miRNA) [8-10]. The pre-miRNA is translocated to the cytoplasm by the karyopherin exportin 5 in conjunction with ras-related nuclear protein GTPase [11]. Once in the cytoplasm, the pre-miRNA is cleaved by Dicer, with the help of the TAR RNA-binding protein, to release the transiently double-stranded mature miRNA [12,13]. This double-stranded miRNA is incorporated into the Ago-containing effector complex, often termed the miRNA-con-taining RNA-induced silencing complex or miRISC [14]. The passenger strand is dissociated leaving the active miRNA to regulate expression of the target gene. In animals, most miRNAs bind to partially complementary sites in the 3′ UTR of their targets [15-17]. Although this binding typically induces the repression of translation, at least a modest level of target mRNA degradation has been observed in many instances [18-20]. The 5′ region of the miRNA (nucleotides 2 – 7), referred to as the ‘seed region’, plays a critical role in determining the specificity of target site binding [21,22]. As a consequence, many miRNAs are capable of regulating a large number, potentially hundreds, of target mRNAs [19].

miRNAs play a central role in the regulation of diverse biological processes. Therefore, it is not surprising that the dysregulation of miRNA expression levels is associated with various pathogenic conditions, including tumorigenesis and metastatic development [23]. Depending on their targets, miRNAs can function as oncogenes (e.g., the miR-19 – 92 cluster [24]) or tumor suppressors (e.g., miR-15a and -16-1 [25]) and can lead to oncogenic transformation by either a loss or gain in their expression. The key role that miRNAs play in cancer development is supported by the overlap between genomic regions from which miRNAs are encoded and regions of the genome associated with cancer development, including gene amplifications, common breakpoint sites, fragile sites and regions associated with loss of heterozygos-ity [26]. Although this field is still in its infancy, miRNAs that regulate key aspects of tumor development including the control of cellular proliferation, cell signaling, angiogenesis, protein degradation, transcriptional regulation, apoptosis

and the immune response have been found. Beyond helping to understand important aspects of tumor biology, miRNAs can also be viewed as potential therapeutic targets, which can manipulated to impair the development of tumors, as well as serving as important diagnostic and prognostic markers. Doubtlessly, this will be an area of intense investigation in the future.

1.2 endo-siRNApathwayUntil recently, siRNAs in animal cells were thought to arise from exogenous sources such as the experimental introduction of dsRNA molecules or as by-products of viral replication. However, studies in Drosophila and mice have identified siRNAs, termed endogenous siRNAs (endo-siRNAs), which are produced from either inverted repeat sequences (such as hairpin RNAs) or bidirectional transcription of specific genetic loci (Figure 1B) [27-31]. Endo-siRNAs require Dicer and Ago2 for their biogenesis but lack any requirement for the microprocessor (Drosha and DGCR8), a necessary com-ponent of the miRNA pathway. These endo-siRNAs are transcribed in the nucleus as long dsRNA precursor mole-cules that are translocated to the cytoplasm. It is unclear at present if this translocation requires exportin 5. Once in the cytoplasm, the long dsRNA precursors are processively cleaved by the RNase III-type enzyme Dicer producing a pool of endo-siRNAs. Because these endo-siRNAs have a requirement for Ago2, the key catalytic component of the RNA-induced silencing complex (RISC), it is likely that they associate with Ago2 leading to the cleavage and release of the passenger strand. The association of the guide strand of the siRNA with RISC provides the specificity determi-nant that allows RISC to associate with and cleave the target mRNA [30,31]. The target mRNA is cleaved at the center of the siRNA:mRNA duplex, 10 nt from the 5′ end of the siRNA [32].

Long before the identification of endo-siRNAs, it was shown that RNAi could be induced in mammalian cells by the introduction of chemically synthesized siRNAs or the expression of short hairpin RNAs (shRNAs) from DNA vectors (Figure 1C) [33]. Similar to the endo-siRNAs and miRNA precursors, shRNAs are expressed in the nucleus, translo-cated to the cytoplasm and cleaved by Dicer to produce active siRNAs. Some researchers have reported increased efficiency of silencing when using slightly longer siRNAs (∼ 25 – 27 nt), which require Dicer processing, aptly called Dicer substrate siRNAs [34]. However, the conventional 21-mers described by Tuschl et al. (2001) that directly enter the RNAi pathway without Dicer processing remain the most widely used silencing agents [35].

2. RNAi-basedtherapeutictargets

Several genetic alterations, both somatic mutations in tumor cells and inherited germ-line mutations, contribute to the transformation of normal cells into cancerous cells. Although

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Figure1.RNAinterferencepathways. RNA interference pathways use short RNA molecules to guide the sequence-specific regulation of gene expression by inducing mRNA cleavage or the inhibition of translation and impairment of transcription. The targeted silencing of gene expression has principally been directed at the post-transcriptional mechanisms and only these will be considered here. A. miRNA pathway: miRNAs are encoded from highly structured primary transcripts (pri-miRNAs). These pri-miRNAs are processed in the nucleus by the RNase III-type enzyme Drosha aided by the dsRNA-binding protein DiGeorge syndrome critical region gene 8 into the precursor (pre-miRNA), which is transported to the cytoplasm by exportin 5. In the cytoplasm, the pre-miRNA is cleaved by the Dicer-TAR RNA-binding protein complex, producing the mature miRNA that enters the miRISC. The sense strand is released and the single-stranded antisense RNA molecule guides miRISC to partially complementary sites on the target mRNA leading to the inhibition of gene expression by inhibiting translation and/or cleaving the target mRNA. B. endo-siRNA pathway: long dsRNA molecules, either long hairpin RNAs or long dsRNAs produced from opposing promoters, are transported from the nucleus to the cytoplasm and processively cleaved by Dicer into endo-siRNAs, which associate with an Ago2-containing complex, presumably RISC, where one of the strands is lost. The single-stranded endo-siRNA guides the effector complex to the target mRNA leading to the silencing of gene expression. C. siRNA pathway: exogenously introduced siRNAs, either from DNA-based vectors expressing shRNAs or synthetic siRNAs transduced into cells, can be harnessed to experimentally silence target gene expression. shRNAs are exported to the cytoplasm using exportin 5 and cleaved by Dicer producing an active siRNA. Both the vector expressed siRNAs and the synthetic siRNAs are taken up by RISC where the sense strand is cleaved by argonaute 2 and released, leaving the single-stranded guide RNA associated with RISC. This activated RISC binds to the target mRNA, which is cleaved and degraded.dsRNA: Double-stranded RNA; endo-siRNA: Endogenous siRNA; miRISC: miRNA-containing RNA-induced silencing complex; miRNA: MicroRNA;

pre-miRNA: Precursor miRNA; pri-miRNA: Primary miRNA transcript; RISC: RNA-induced silencing complex; shRNA: Short hairpin RNA; siRNA: Small interfering RNA.



the alteration of many genes can contribute to the cancer phenotype, these cancer-promoting genes fall into one of three classes: oncogenes, tumor suppressor genes and stabil-ity genes [36]. The mutation of oncogenes or tumor suppres-sor genes that promote cancer development lead to the same physiological outcome, the increase in cell number resulting from the activation of cell division, the provision of nutri-ents through enhanced angiogenesis, or the inhibition of cell death (apoptosis) or cell cycle arrest [36,37]. Cancer-promoting mutations in oncogenes alter the activity of the gene product by rendering the gene either constitutively active or active under conditions in which the normal gene would be inac-tive. These mutations can take the form of gene amplifica-tions, translocations or the acquisition of point mutations, insertions or deletions that dysregulate the normal activity of the gene product [38]. On the other hand, cancer-promoting mutations in tumor suppressor genes lead to the inactivation of the gene product by the incorporation of deletions or insertions into the gene, the acquisition of nonsense muta-tions that lead to the premature termination of translation and the production of truncated proteins, the accumulation of inactivating missense mutations that impair the functionality of the gene product or impaired expression through epigenetic silencing [39].

Because oncogenes exert their tumorigenic activity by increasing their activity or their expression level, most anticancer RNAi-based silencing approaches have targeted this class of genes. Consequentially, many of these oncogenes are the basis for patent applications proposing the use of RNAi-based approaches as potential anticancer therapies. These oncogene targets include receptor tyrosine kinases and their ligands, signal transduction pathway components, GTPases, transcription factors, anti-apoptotic factors and pro-angiogenic factors [36,37].

2.1 SignaltransductionpathwaysThe receptor tyrosine kinases are a large and diverse family of cell surface molecules that recognize specific ligands and transmit that signal into the cell by initiating a phosphorylation cascade. This usually results in the alteration of gene expression patterns that promote such activities as cellular proliferation, cellular migration, cell differentiation and apoptosis [40,41]. Potential ligands include cytokines, survival factors, chemokines, hormones, transmitters, growth factors, extracellular matrix components and death factors [42]. These receptors are often mutated in human malignancies resulting in the overexpression of the receptor and/or the alteration of the ligand-binding domain or the kinase domain leading to their constitutive activation [43,44]. In addition, aberrant expression of the ligand(s) can promote tumor formation. Signals originating at the receptor can activate several signal transduction pathways (e.g., Janus kinase/STAT pathway [45], MAPK pathway [46] and the phosphoinositide 3-kinase (PI3K) pathway [47]) resulting in a signaling cascade [48]. Because these signal transduction pathways operate in a

similar manner, we examine the MAPK pathway in more detail to identify oncogenic alterations in the pathway components and potential anticancer targets (Figure 2). The activation of the EGFR on binding of either EGF or TGF-α leads to the autophosphorylation of the cytoplasmic tail of EGFR by its intracellular tyrosine kinase domain [49]. This autophospho-rylation activates several downstream proteins that associate with the phosphorylated tyrosines through their own phos-photyrosine-binding SH2 domains [43]. In turn, these down-stream signaling proteins initiate several signal transduction pathways, including the MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation. Mutations to the downstream components of these signal transduction pathways can bypass the need for receptor engagement [46,48,50]. Therefore, many of these downstream kinases are proto-oncogenes, normal genes that can become oncogenes when mutated or overexpressed. This group of genes includes the small-GTPase RAS and the MAPK kinase kinase RAF [50]. Given the important role these factors play in oncogenic transformation, it is not surprising that these molecules have been identified as potential targets for RNAi-based therapies. On engagement of the RTK (e.g., EGFR), RAS is converted from the inactive GDP-containing form to the active GTP-containing form by the guanine nucleotide exchange factor son of sevenless. The mutation of key residues within RAS produces constitutively active forms of RAS (e.g., RASV12), which have been found in a variety of tumor types including colorectal, pancreatic, non-small cell lung, thyroid, bladder, liver, kidney cancers and acute myelogenous leukemia [51]. RNAi-based silencing has been shown to have a high degree of specificity including the ability to recognize alleles of a gene that differ by a single nucleotide polymorphism. Brummelkamp et al. showed that siRNAs designed against the oncogenic form of RASV12 could be specifically targeted without altering expression of the wild-type allele [52]. This provides a unique mechanism to discriminate pathogenic and normal alleles of the same gene.

On binding with activated RAS, the serine/threonine-specific kinase RAF is competent to perpetuate the signaling cascade by engaging the MAPK kinase, MEK [46]. Similar to RAS, many human malignancies including melanomas, colon, thyroid, papillary and ovarian tumors contain mutation in RAF that render it constitutively active [53,54]. Until now, no oncogenic alleles of the MAPK kinase kinase and MAPK (or extracellular signal-regulated kinases) family of proteins have been identified. There are a variety of downstream targets of the MAPK pathways including cytoskeletal proteins, transcription factors, signaling molecules and apoptotic factors [42]. This diversity of targets produces a complex array of responses to these signaling pathways, such as activation of cellular proliferation, cell adhesion, cell migration, differentiation and angiogenesis. The transcription factor MYC is one of the downstream targets of the MAPK pathway, as well as a number of other mitogen-activated signaling pathways including the sonic hedgehog and WNT pathways [55-57]. MYC plays a central role in the

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regulation of cell proliferation by regulating the transcription of key cell cycle genes. Mutations that dysregulate MYC expression, including a chromosomal translocation of MYC associated with Burkitt’s lymphoma, promote tumorigenesis by altering the transcriptional profile of tumor cells [58,59].

In a manner similar to the MAPK pathway, components of other signal transduction pathways are also oncogenic when mutated. These include Janus kinase, Akt and PI3K (described below), which have all been identified as potential anticancer targets [60-62].

2.2 AngiogenesisSustained tumor growth requires the production of new blood vessels through angiogenesis. This allows the tumor to obtain a sufficient supply of oxygen and other nutrients while clearing toxic cellular waste products [63]. Under normal conditions, angiogenesis is strictly controlled and limited to several biological processes, including embryological develop-ment, wound healing and menstruation [64]. In addition to facilitating tumor growth, uncontrolled angiogenesis has been linked to the loss of vision associated with certain

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Figure2.OverviewoftheMAPKpathwayanditsroleinoncogenesis. Oncogenic transformation involves the dysregulation of cell growth. In many cases, this involves the alteration of signal transduction pathways that respond to a variety of extracellular signals. Mutations that alter the expression or activity of the components of these pathways permit the unfettered growth of cells and many of these proteins have been proposed as potential anticancer therapeutic targets. The MAPK pathway is used as an example of this process. Under normal circumstances, a ligand (e.g., TGF-α) binds to a receptor tyrosine kinase (e.g., EGFR) inducing the phosphorylation of tyrosine residues within the cytoplasmic tail of the receptor. Phosphotyrosine-binding proteins (e.g., Grb2) interact with the engaged receptor recruiting the GTP exchange factors son of sevenless, which facilitate the activation of Ras by replacing GDP with GTP. GTP-Ras associates and activates the MAPKKK Raf. This induces a phosphorylation cascade through the MAPKK, MEK, and the ERK. Phosphorylated ERK in turn can phosphorylate and activate a variety of substrates including transcription factors, apoptotic factors, structural proteins and more kinases (e.g.,Rrbosomal s6 kinase). Factors linked to oncogenic transformation have been highlighted in bold.ERK: Extracellular regulated kinase; MAPKK: MAP kinase kinase; MAPKKK: MAP kinase kinase kinase; MEK: Mitogen associated/extracellular regulated kinase.



forms of age-related macular degeneration (AMD). A variety of factors have been shown to have proangiogenic activity. One of the most well studied of these factors is the VEGF and its receptor, VEGF receptor (VEGFR or fms-related tyrosine kinase 1; FLT1) [65-70]. The association of VEGFs and the VEGFRs induce endothelial cell growth and cell migration leading to blood vessel formation during development. The ability to regulate the growth of new blood vessels have made VEGF and VEGFR attractive targets for RNAi-mediated silencing as a potential treatment for solid tumors and AMD [71-73]. In fact, the localized delivery of siRNAs targeting VEGFs and the VEGFRs has been shown to effectively inhibit neovascular-ization in AMD and was the first RNAi-based therapeutic approach to enter clinical trials [74].

Further proangiogenic factors have been identified as potential anticancer targets, including hepatocyte growth factor receptor (HGFR), angiopoietin and platelet derived growth factor (PDGFR) [64]. The dysregulation of HGFR (alternatively, mesenchymal–epithelial transition factor; MET) in cancers of the kidney, liver, stomach, breast and brain has been shown to promote cellular proliferation and angiogenesis [75-77]. Several patent applications have pro-posed the targeting of the proangiogenic factors angiopoietin 1 and angiopoietin 2 and their receptor, tyrosine kinase with immunoglobulin and EGF homology domains 2 [78]. Unlike VEGF, which initiates new blood vessel formation, angio-poietin is associated with the maturation and reorganization of blood vessels [79]. A similar proangiogenic effect has been found for PDGFR, which has been linked to several cancers including renal cell carcinomas, glioblastoma multiforme and gastrointestinal sarcomas [80-82].

2.3 RNAi,chemotherapyandradiationtherapyA number of recent papers have shown the benefit of combining chemo- or radiation therapy with RNAi treatment against genes involved in DNA repair and chromosomal stability, components of the pathways responsible for metabolizing potential drugs or efflux pathways responsible for drug resis-tance. This is exemplified by the effectiveness of combining chemotherapy with the silencing of the ATP-binding cas-sette subfamily B members (ABCB family; multidrug resistance (MDR)) that serve as ATP-dependent efflux pumps responsible for transporting a variety of substrates across the cell membrane including chemotherapeutic agents (e.g., doxorubicin) [83-87]. An adenoviral (AV) vector delivered shRNAs targeting hypoxia-inducible factor 1a, a factor that upregulates the expression of pro-angiogenic factors (e.g., VEGF and PDGF) under conditions of low oxygen, leads to an impairment in the growth of subcutaneously implanted tumor cells. This antitumor effect was enhanced when the mice were treated with radiation [88-90].

2.4 RNAiandstabilitygenesIn addition to oncogenes and tumor suppressor genes, the mutation of genes involved in maintaining genomic stability

has been linked to tumor development. This family of genes includes factors that are involved in DNA damage repair responses, such as nucleotide excision repair, base excision repair and mismatch repair proteins, as well as genes that are responsible for the maintenance of genome integrity [36]. Similar to tumor suppressor genes, the loss or inactivation of these genes promotes tumor development by increasing the overall mutation rate. This would limit their application as potential RNAi targets. However, Deweese et al. (2005) have proposed an approach that uses a replication defective AV vector (see section 4 below) to deliver siRNAs targeting DNA repair proteins (e.g., ATM, ATR and/or DNA PKCs) to hypersensitize tumor cells for treatment with a DNA damaging agent such as radiation therapy or chemotherapy (e.g., cisplatin) [91]. This approach has been validated by silenc-ing the expression of excision repair cross-complementing 1, a component of the nucleotide excision repair pathway, which sensitized tumor cells to cisplatin [92]. Chen et al. (2004) found that the RNAi-mediated silencing of the TEL-PDGFβR oncogene, produced by a common translocation found in chronic myelomonocytic leukemia, synergized with the tyrosine kinase inhibitor, imatinib (commonly known as Gleevec), increasing disease latency and the survival of treated mice [93,94].

2.5 RNAiandapoptosisGenes that suppress apoptosis are often mutated in cancers disrupting the intricate balance between cell growth and cell death. The overexpression of members of the Bcl-2 (B-cell lymphoma 2) family of anti-apoptotic factors, including Bcl-2, Bcl-XL and Bcl-w, protects tumor cells from normal cell death signals by promoting cellular proliferation in a variety of tumors (e.g., B-cell lymphomas, melanomas and cancers of the breast and lung) [38,95-97]. Conversely, the targeted silencing of Bcl-2 induced the regression of subcu-taneously implanted tumors in mice [98]. A number of signal transduction pathways promote or inhibit apoptosis. The serine/threonine protein kinase Akt1 promotes cell survival as part of the PI3K pathway that responds to extracellular survival factors, such as IGF1 [48]. Both PI3K and IGF1 can act as oncogenes and are themselves potential anticancer RNAi targets [60-62,99,100]. Akt1 exerts its survival effects by inhibiting the pro-apoptotic activity of the Bcl-2 family member Bad (Bcl-2-associated death promoter protein).

3. RNAiscreensfornovelanticancertherapeutictargets

The cost, time and difficulty associated with the identification of novel therapeutic targets have hindered the pace of anticancer drug development. However, recent technological advances have made it possible to perform large-scale functional genomic screens using RNAi-based gene silencing libraries to identify novel therapeutic targets in a rapid and cost effective manner. Using a limited library targeting 510 genes

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including most known and putative kinases, Cooke et al. (2003) performed an siRNA-based forward genetic screen to identify factors that modified cellular sensitivity to TRAIL-induced apoptosis including important roles for MYC and the WNT signaling pathways in mediating TRAIL sensitiv-ity [101]. An alternative to the transient transfection of chem-ically synthesized siRNAs for gene silencing is the expression of shRNAs from DNA-based vector systems. These shRNAs are processed by Dicer into active siRNAs [102]. Both plas-mid and viral vectors, in particular onco-retroviral- and len-tiviral-based systems, have been used to deliver siRNAs. This approach has been used extensively for gene function analysis as it allows for the development of cell lines that are stably silenced for a particular gene. These assays are partic-ularly adept at the identification of tumor suppressor genes as the silencing of such would lead to an increase in prolif-eration and the acquisition of anchorage-independent growth. For example, Brummelkamp et al. used on arrayed library that targeted 50 deubiquitinating enzymes to identify the tumor suppressor, cylindromatosis, as an important regulator of NF-κB activation by TNF-α [103].

During malignant transformation, cells undergo a series of genetic changes converting a normal cell into a cancerous lesion. These changes can be modeled by the ectopic expres-sion of combinations of oncogenes [104]. A conditionally transformed human BJ-fibroblast cell line expressing the catalytic subunit of telomerase (hTERT) and a temperature sensitive allele of the viral-derived oncogene SV40 large T antigen (active at 32°C but not 39°C) was used by Berns et al. (2004) to identify factors that would bypass p53-dependent proliferation arrest [105]. By screening a retroviral-based shRNA expression library targeting 7914 human genes, they found several factors that were able to bypass the p53-dependent proliferation arrest induced when these cells were grown at 39oC. These genes include the histone acetyl-transferase Tip60 and the histone deacetylase HDAC4. Sev-eral histone deacetylase genes, including HDAC4, have been proposed as potential anticancer targets for RNAi-based therapies [106]. In a similar set of experiments, human mam-mary epithelial cells expressing hTERT and a constitutively active SV40 large T antigen were screened for factors that permitted anchorage-independent growth after infection with an shRNA-expressing retroviral library. A barcoding system (a unique DNA sequence identifier specific for each shRNA construct) coupled with microarray deconvolution and DNA sequencing were used to identify the tumor suppressor genes from the isolated colonies. These genes included the previously characterized TGF-β receptor 2 (a potential anticancer target of several patents [107,108]) and phosphatase and tensin homologs, as well as the novel tumor suppressor RE1-silencing transcription factor/neuron-restrictive silencing factor, which functions by interfering with PI3K-dependent signaling [109]. Using a similar experimental approach, Kolfschoten et al. (2005) ascribed tumor suppressor function to the homeodomain pituitary transcription factor

PITX1 [110]. The silencing of PITX1 expression led to the upregulation of the RAS–GTPase activating protein RASAL1 and, consequentially, the activation of the RAS pathway.

Although screening with barcodes is not necessary for the identification of tumor suppressor genes as their silencing would permit the positive selection of silenced cells, they are necessary for the identification of factors that would impair or eliminate cell growth such as the screening for novel oncogenes or genes involved in drug sensitivity. shRNAs that are lost from the pool of total shRNAs due to the induction of cell death or an inhibition of proliferation can be identified using competitive hybridization of the barcode sequences. These ‘dropout’ screens for oncogenes rely on the ability to perform microarray analysis for identifying bar-code sequences that are lost from population of shRNA silenced cells. Staudt and colleagues (2006) used such an approach to identify factors that were necessary for the pro-liferation of diffuse large cell B-lymphoma cells in tissue culture. This led to the identification of components of the NF-κB pathway, which were necessary for the proliferation of active-type but not germinal center-type diffuse large cell B-lymphoma cells [111]. Recently, two papers described a technique for the screening of large pools of shRNAs that have a growth inhibitory effect in human cancer cell lines including the colon cancer lines DLD-1 and HCT116, the breast cancer cell lines HCC1954, MCF-10A, MDA-MB-435, MDA-MB-231, ZR-75.1 and T47D, as well as normal human mammary epithelial cells using novel half-hairpin barcodes for microarray deconvolution. Although many of the identified genes had generalized effects on all the cell lines examined in each study, there were subsets of genes that were selectively required for growth and proliferation of the different tumor cell lines, which may represent attractive, anticancer therapeutic targets [112,113].

4. siRNAdelivery

The biggest challenge for the therapeutic application of RNAi-based gene silencing approaches is the delivery of the siRNAs to the appropriate cell type. The route of siRNA administration will affect a number of parameters including the type of delivery vehicle used, tissue distribution, poten-tial off-target effects and the dose of siRNAs needed to achieve effective silencing [102,114,115]. The most successful applications of RNAi-based silencing have used the localized delivery of siRNAs to easily accessible organs and tissues. This has included the treatment of AMD through intraocular injection of siRNAs and the treatment of respiratory syncytial virus infection by intranasal or intratracheal instillation [71-73,116]. Another potential route of localized administration is through transepithelial absorption through the skin or mucosal surfaces such as oral, rectal or vaginal delivery. These approaches may be harnessed for the delivery of siRNAs to superficial tumors of the eye (retinoblastomas), nose, throat and neck (nasopharyngeal cancers and lung squamous cell



carcinomas), skin (melanomas) and mucosal surfaces (cervical and colorectal cancers). Although localized delivery may work for a handful of tumors, treatment of most tumor types requires a suitable systemic delivery approach. To deliver sufficient siRNAs to elicit a potent silencing response in the target cell type or tissue, systemic delivery approaches need to avoid the rapid clearance of the siRNAs from the circulatory system by renal filtration, they need to protect the siRNAs from serum nucleases and they need to achieve effective biodistribution. Two principle methods have been used for the delivery of siRNAs into target cells: i) the deliv-ery of chemically synthesized siRNAs (‘small molecule approach’) or ii) the expression of siRNA precursor mole-cules, shRNAs, which are processed by Dicer into the active siRNA from DNA-based vectors (‘gene therapy approach’).

4.1 DeliveryofsiRNAsasasmallmoleculedrugMost cells do not passively take up siRNAs from their environment with the possible exception of mucosal sur-faces [116-118]. Therefore, most applications require some agent that facilitates delivery of siRNAs to the cytoplasm where they associate with RISC and guide the sequence-specific silencing of gene expression. A variety of lipid-based trans-fection reagents are commonly used for the in vitro delivery of siRNAs into cultured cells [119]. Most of these systems use cationic lipids that help to neutralize the net negative charge of the siRNAs and permit transport across the cell membrane. However, many of these reagents are not amenable for in vivo use owing to their toxicity [120,121]. By altering the chemical composition of these lipids or using combinations of different lipids, it is possible to produce formulations that can effectively deliver siRNAs without overt toxicity. Using a synthetic cardiolipin analogue (CCLA), Chien et al. were able to deliver anti-raf siRNAs to subcutaneously implanted breast cancer tumor cells following tail vein injection in mice [122,123]. The formulation of liposomes that contain several lipid-species can facilitate efficient delivery and uptake into cells while stabilizing the liposome during for-mulation. Morrissey et al. (2005) used a combination of cationic and fusogenic lipids and coated the particles with a PEG-lipid complex that neutralizes the charged lipid surface, stabilizing the complex and creating a stable nucleic acid–lipid particle [124-126]. These stable nucleic acid–lipid particles trafficked to the liver and efficiently inhibited hepatitis B virus gene expression. In addition to serving as an effective antiviral agent, this approach could also be used as an anticancer agent for hepatocellular carcinomas resulting from chronic hepatitis B virus or hepatitis C virus infection. A similar strategy was used to deliver anti-ApoB siRNAs to the liver of non-human primates leading to a reduction of ApoB protein expression and a concomitant decrease in the levels of serum cholesterol and low-density lipoproteins [127]. The ease with which siRNAs can be delivered to the liver has made this organ an attractive target for RNAi-based gen silencing approaches.

In addition to cationic lipid reagents and liposomes, positively charged polymers such as polyethylenimine (PEI) have been shown to efficiently deliver siRNAs into the cyto-plasm of mammalian cells. Although there have been reports of PEI-mediated in vivo delivery of siRNAs (e.g., Urban-Klein et al. 2005 [128]), cytotoxicity issues have limited the in vivo utility of PEI [129]. This toxicity increases with the molecular mass of the polymer and the degree of branching. The use of shorter polymer lengths has decreased the toxicity of PEI but has compromised the efficiency of delivery [130]. However, PEG or cyclodextrin substitutions can reduce the toxicity associated with unmodified PEI, although maintaining effective delivery [131-134].

Naturally occurring polymers have been investigated as an alternative to synthetic polymers with the hope that these compounds would be more biocompatible and less toxic. Both chitosan and atelocollagen can associate with nucleic acids (DNA and RNA) and facilitate their delivery into cells. Chitosan is a linear polysaccharide produced by the deacetylation of chitin, a component of the exoskeleton of shellfish [135]. siRNAs can interact electrostatically with the polycation producing chitosan–siRNA nanoparticles or poly-plexes. This approach has been used for the delivery of anticancer siRNAs (e.g., an siRNA targeting the fusion protein BCR/ABL) into NIH 3T3 cells in vitro and for the systemic application of siRNAs to peritoneal macrophage cells by either intranasal instillation [136] or intraperitoneal injec-tion [137]. Importantly, no overt toxicity was seen in any of these experiments.

Atelocollagen, produced by the removal of the immunogenic C-terminal and N-terminal telopeptides from collagen by pepsin treatment, has also been shown to facilitate siRNA uptake by cells. siRNAs can bind noncovalently to the positive charged surface of atelocollagen. These atelocollagen–siRNA complexes protect the siRNA from degradation by serum nucleases and have been shown to effectively deliver siRNAs to prostate cancer (PC-3) and germ cell (NEC8) tumors on intratumoral or intravenous injection [138-140].

Systemic delivery approaches require delivery vehicles that will direct the siRNAs to the relevant cells to reduce potential toxicity to bystander cells and tissues, as well as lowering the therapeutic dose needed for effective silencing. Recently, several cell type specific delivery approaches have been developed to address this need. Targeted siRNA delivery to specific cell populations can be achieved by taking advantage of the specificity of different types of molecules for cell surface antigens. Several different types of molecules have been used to identify specific cell surface antigens including antibodies, ligands and aptamers.

Antibodies can recognize specific antigens in a highly selective manner. However, they have no means of binding and delivering nucleic acids. To facilitate siRNA delivery to the cytoplasm of target cells, the antigen-binding domain from a Fab or a single chain antibody can be fused to nucleic acid-binding motifs, either from the nucleic acid-binding

Dykxhoorn


domain of protamine or a polyarginine motif [141-144]. In each case, the siRNA binds noncovalently to the bifunctional protein molecule. Using a fragment antibody that specifically recognizes the HIV-1 glycoprotein (gp160), siRNAs could be targeted to cells that were infected with HIV-1 in vitro, as well as tumor cells, which transgenically express gp160 leading to a suppression of tumor growth [141,145]. A similar effect was seen using a single chain (Ab) to ErbB2. This approach allows exquisite specificity as demonstrated by the use of an antibody, which specifically recognizes a conforma-tionally distinct, activated form of the lymphocyte function-associated antigen-1 that delivered siRNAs only to cells that bore the activated but not the latent form of the receptor [142,146]. In an alternative approach, Peer et al. delivered siRNAs to gut mononuclear leukocytes expressing the β7 integrin mol-ecule on their surface [147]. Instead of conjugating the β7 integrin-specific Ab to protamine, they developed liposome-based stabilized nanoparticles with the anti-integrin β7 Ab covalently associated with the outer face of the nanoparticle. In this way, they successfully targeted cyclin D1 expression in an experimental model of dextran sodium sulfate-induced colitis. Cyclin D1 has been the subject of an anticancer RNAi-based therapeutic patent [148]. Although a number of studies have demonstrated delivery of siRNAs to solid tissues and tumors, a more difficult system would be to deliver to dif-fuse cell types such as lymphocytes that are dispersed through-out an organism. By fusing a single chain antibody specific for the pan T-lymphocyte marker CD7 to a polyarginine tract, Shankar and colleagues were able to deliver siRNAs into T cells in a humanized mouse model of HIV-1 [144,149]. Importantly, these approaches have all shown effective siRNA delivery without apparent toxicity to the animals.

Alternatively, cell type specific delivery can be achieved by using ligands (peptides, hormones, chemokines, etc.) that recognize specific cell surface molecules. One of the early experiments used a cholesterol molecule directly conjugated to the 3′ end of the passenger strand of an siRNA. This approach was shown to efficiently deliver siRNA targeting ApoB in cells of the liver and jejunum that bear lipoprotein receptors [150]. This led to a decrease in ApoB mRNA and protein levels and a concomitant decrease in cholesterol levels in the treated mice. Although effective at targeting siRNAs to the liver and jejunum, many more cell types bear lipoprotein receptors limiting the specificity and increasing the amount of material needed to get a therapeutic response. Most viruses take advantage of endogenous receptors present on the cell surface to gain access to specific cell types. A fragment of the rabies virus glycoprotein was shown to bind the nicotine acetylcholine receptor present on neuronal cells. The fusion of a polyarginine motif (nona-arginine peptide) capable of binding siRNAs to the peptide fragment of the rabies virus glycoprotein delivered siRNAs specifically to neuronal cells. Using such a mechanism, Kumar et al. were able to deliver siRNAs targeting Japanese encephalitis virus protecting mice from a lethal challenge with Japanese encephalitis virus [143,151].

Further potential ligands that have been suggested for the delivery of siRNAs are the human placental lactogen that binds to the prolactin receptor upregulated in many ovarian and breast cancers [152] or the anthrax toxin protective antigen that binds to the broadly expressed tumor endothelial marker 8 gene and the capillary morphogenesis protein 2 facilitating cellular uptake [153]. One limitation of the use of ligands is the effect that they will have on cellular physiology as the binding of a ligand to a cell surface receptor usually induces a signal transduction cascade that alters gene expression patterns.

Aptamers are structured nucleic acid molecules (RNA or DNA) that have been in vitro selected (systematic evolution of ligands by exponential enrichment) to bind with high affinity to specific target molecules. Although ligands and antibodies are purified from cultured mammalian cells, insect cells or bacteria, and require extensive purification before they are amenable for clinical applications, RNA aptamers can be produced by in vitro transcription and easily purified in high yield. In addition, RNA aptamers can be lyophilized and stored at ambient temperatures without loss of activity. RNA aptamers can be directly linked to the passenger strand of an siRNA, annealed to a chemically synthesized guide strand and deliver the siRNA into the appropriate cell type. An aptamer specific for the prostate-specific membrane antigen (PSMA) has been shown to effectively deliver anti-tumor siRNAs into prostate tumor cells in vitro and in vivo resulting in the inhibition of tumor growth [154,155]. In another approach, an aptamer specific for the HIV-1 glycoprotein was shown to target HIV-1 infected cells and led to the suppression of viral infection [126].

4.2 StabledeliveryofsiRNAsusingDNA-basedvectorsThe second widely used approach for the RNAi-mediated silencing of gene expression is through the expression of siRNA precursors from DNA-based vector [33,102,156-158]. Most of these approaches have taken advantage of viral vector systems, in particular AV, adeno-associated viral (AAV), oncoretroviral and lentiviral-based systems [158]. Viral-based systems are attractive due to the ease with which they can transduce cells relative to transfection-based approaches, they have specific tropisms (i.e., they infect specific cell type) and, in many cases, they can maintain expression of the siRNA for extended periods. In addition, by changing the viral surface proteins necessary for recognition of different cell types, a process called pseudotyping, the efficiency and specificity of the viral vectors can be expanded [159]. Although the altering of the viral surface proteins can alter the cell type(s) that can be infected, if the tumor cell lacks the appropriate surface antigen it will not be targeted by the viral vectors. This imposes severe restrictions on the types of tumors that are amenable to viral vector-based silencing approaches.

Adenovirus is a nonenveloped double-stranded DNA virus that replicates in the host cell nucleus as an episome (extrachromosomal DNA) reducing potential insertional mutagenesis. It can infect a variety of both dividing and



non-dividing cells. The current generation of AV vectors lacks the necessary structural and enzymatic proteins to produce replication-competent AV [160]. Therefore, recombinant AV production requires that the necessary viral factors be provided in trans from helper plasmids to get viral particle production. Recent papers have shown the benefit of delivering siRNAs encoded from an AV vector to target genes involved in DNA repair or components of the metabolic pathways or efflux pathways responsible for drug resistance to increase the potency of chemo- or radiation therapy [88,161]. In this way, therapeutic benefit was seen when IGF1 receptor, hypoxia-inducible factor 1α, ATM, ATR or PKCs were tar-geted in combination with traditional therapeutic approaches (see above) [91]. The expression of an shRNA targeting c-MET (mesenchymal-epithelial transition factor; HGFR) from a replication defective AV was able to suppress tumor growth of subcutaneously implanted mouse mammary tumor cells (DA3) after intratumoral injection [162]. Although most of the AV vectors remain latent, an oncolytic AV vector system expressing shRNAs against VEGF resulted in the effective targeting of tumor cells and an inhibition of angio-genesis [163]. Similar results have been achieved using oncolytic AV expressing siRNAs targeting hTert (the catalytic subunit of telomerase) and Ki-67 [164,165]. There are a number of features of AV vectors that may limit their applicability as a delivery system. One potential complication of the AV delivery system is pre-existing immunity, in particular, neutralizing antibodies, to adenovirus strains present in many individuals that may impair the effective transduction of target cells [158]. In addition, the broad host cell tropism means that many off-target cells will be infected [159,160]. Although earlier gen-eration of vectors were unable to maintain persistent transgene expression due in part to the stimulation of immune cells leading to the killing of the transduced cells and the loss of episomal DNA as the target cells replicate, longer-term expression can be achieved using the current generation of AV vectors.

AAV is a non-pathogenic single-stranded DNA virus that relies on the presence of another virus, either coinfection with an adenovirus or a herpesvirus, for completion of its life cycle [166]. In the absence of a helper virus, AAV can remain latent in cells replicating as an episome, only occa-sionally integrating into the host genome at a specific location on chromosome 19. AAV-based delivery approaches can be used to transduce a wide variety of dividing and non-dividing cells [159]. Although transgene size restrictions limit the potential utility of AAV-based transgene approaches, the small size of the siRNA expression cassette can be easily accommodated in these vectors. Similar to adenovirus vectors, preexisting host immunity may reduce the effectiveness of AAV-based delivery. Because there are a large number of strains of AAV (> 100 natural variants) that show different cell tropisms, AAV virus can be targeted to a wide variety of tissues by pseudotyping with the appropriate viral capsid [159]. Hildinger and colleagues have patented the general technique

of using AAV for the delivery of siRNAs without identifying potential targets [167]. AAV-mediated targeting of the tumor suppressor p53 or Caspase 8 was shown to effectively induce gene silencing in vitro [168].

Oncoretroviral and lentiviral vectors have been used extensively for the delivery of siRNAs to cells in vitro and in vivo. The ability to integrate into the host genome has been both a blessing and a curse for the therapeutic application of retroviral-based delivery strategies because it facilitates stable and long-term expression, but it also runs the risk of toxicity associated with potential insertional mutagenesis [169]. The range of cells that can be transduced by retroviral and lentiviral vectors can be broadened by the incorporation of various envelope glycoproteins into the viral particle in place of the endogenous glycoproteins [159]. Broad specificity glycoproteins, such as from the vesicular stomatitis virus and the lymphocytic choriomeningitis virus, allow for the trans-duction of a wide array of cell types, although the use of glycoproteins with a more narrow tropism, such as the rabies virus glycoprotein, can limit the infection to specific cell populations. Lentiviral and oncoretroviral vectors have been used for the delivery of siRNAs targeting a variety of cancer promoting genes, including Bcl-2, BRAF, SKP-2, VEGF, MDR1, BCR-ABL, TEL-PDGFβR and RasV12 [52,93,170-173].

The overexpression of shRNAs has, under some conditions, led to cytotoxic effects by overwhelming the RNAi machinery and impairing proper miRNA processing and function [174,175]. This principally arises in shRNA expression systems that use constitutively active, high expressing RNA pol III promoters (e.g., the U6 promoter) and can be mitigated by the use of weaker promoters that limit shRNA production [176,177]. In addition, tissue-specific and inducible promoters have been used that permit expression in specific cell types or under specific circumstances. The incorporation of the shRNA into a miRNA context that relies on processing through the entire miRNA biogenesis pathway has been found to lead to efficient silencing without overt toxicity [178].

5. Conclusionsandpotentialchallenges

Despite the great promise and wealth of preclinical data attesting to the utility of RNAi as a treatment for a variety of pathogenic states, there are concerns regarding the speci-ficity of the silencing response. Early studies suggested that siRNAs were remarkably specific with the ability to distin-guish between genes that differ by as little as a single nucle-otide polymorphism (e.g., the constitutively active RasV12 and the sickle allele of β-globin, the causative mutation responsi-ble for sickle cell anemia) [52,179]. This specificity has been called into question with the demonstration that the treatment of cells with siRNAs results in reduced mRNA levels for dozens perhaps even hundreds of genes. Typically these off-target effects are modest (< fourfold) but they can impact the observed phenotype [180,181]. Sequence analysis of the off-target mRNAs suggests that most have only low levels of

Dykxhoorn


sequence complementarity to the specific siRNA sequence used [182,183]. In fact, the critical parameter that determined the off-target mRNAs was not the presence of extensive complementarity between the miRNA and the target mRNA but rather the presence of short regions of complementarity involving the 5′ end of the siRNA strands, particularly nucleotides 2 – 7, known as the ‘seed region’ in miRNAs. These findings imply that many of the off-target effects are mediated through an miRNA-type silencing mechanism and may reflect an intrinsic feature of the silencing mechanisms mediated by the RNAi machinery [182,183]. It is a reasonable task to identify and select against siRNA sequences that have extensive complementarity with potential off-target mRNAs, but this becomes a nearly impossible task when dealing with short hexameric sequences. Because these off-target effects are sequence-specific, it is a good idea to test more than one siRNA sequence to ensure that the pheno-type being observed is a result of the correct targeting. This situation is more difficult when developing siRNAs as a potential therapeutic agent. In this instance, it is necessary to thoroughly test the siRNAs for potential toxicities that may result from off-target effects both in vitro, but more importantly in animal models and clinical safety trials.

Long dsRNAs, usually produced as a by-product of viral replication, can trigger a type 1 IFN response in mammalian cells. Because siRNAs fall below the length threshold needed to effectively activate an IFN response, it was believed that they would not induce the innate immune system. Early work in the field had assumed that no adverse immunologi-cal responses are seen on the treatment of cells with siRNAs. However, more recent studies have shown that in fact nonspecific responses to the siRNA can be generated. Although siRNAs cannot efficiently engage the type 1 IFN response mediated through the dsRNA-binding proteins (2′,5’-oligoadenylate synthetase, dsRNA-dependent protein kinase and the retinoic acid-inducible gene 1 [184]), there are specific siRNA sequence motifs, particularly GU-rich sequences such as UGUGU and GUCCUUCAA, that are recognized by toll like receptor (TLR) 7 and TLR8 [185-187]. TLRs respond to specific sets of pathogen-associated molecules (RNAs, lipid polysaccharides, etc.) [188]. This engagement of the TLRs stimulates the secretion of a variety of cytokines that can trigger inflammatory responses [184]. TLR7 and 8 are located in the endosomes of many leukocytes including myeloid dendritic cells, plasmacytoid dendritic cells, B cells and monocytes, and trafficking of the siRNAs through this compartment is necessary for activation of these responses [189]. As the list of sequence motifs that engage these receptors grows, it will be possible to screen out such sequences using siRNA prediction algorithms. In addition, it has been shown that the incorporation of chemically modified RNAs (e.g., 2′ O-methyl bases) can prevent recog-nition by TLRs [187]. In fact, substitution of as few as two 2′ O-methyl bases was sufficient to block TLR activation without altering the efficacy of the siRNA. With these new

insights in hand, it may be necessary to go back and examine more closely the role that these nonspecific responses have played in some of the key early experiments in the development of RNAi-based gene silencing approaches targeting various pathogenic states [190].

The therapeutic application of RNAi-based silencing approaches requires an investigation of the potential toxicities associated with the delivery vehicle. These toxicities will be dependent on the type of delivery system utilized. As already discussed, many of the lipid-based transfection systems used for the transduction of cultured cells have levels of toxicity, which preclude their use in therapeutic settings. Therefore, there is a need for the identification and development of novel lipid formulations with enhanced pharmacological properties and minimal cytotoxicity. Recently, Akinc et al. screened a combinatorial library of lipid-like materials, termed lipidoids, that can be easily synthesized and effec-tively deliver siRNAs into cells in vitro and in vivo with minimal to no cytotoxicity. They were able to identify sev-eral lipid formulations that effectively deliver siRNAs to the liver, lung and peritoneal macrophages from mice, rat and cynomolgus monkeys [191,192].

More concerns have been raised about the safety of viral vector-based shRNA delivery for the long-term silencing of gene expression. One of the major concerns with viral-based shRNA expression systems is the risk associated with inser-tional mutagenesis. This is especially true for retroviral vectors, which can integrate semi-randomly into the host genome and have been linked to the development of hematological malignancies in patients treated with retroviral gene therapy vectors for the correction of severe combined immunodefi-ciency disease [193,194]. In addition, the overexpression of shRNAs from lentiviral vectors and AAV vectors have been linked with cytotoxicity associated with the saturation of the RNAi machinery and the impairment of miRNA biogenesis. These effects can be offset by decreasing the amount of virus used to treat the cells and optimizing siRNA design [176,177]. More data from animal studies and safety trials in humans, particularly long-term analysis, will help to reveal any further challenges that the therapeutic application of RNAi-based silencing technologies face.

Despite these challenges, the therapeutic application of RNAi-based silencing approaches has a tremendous amount of potential for the treatment of a variety of intractable diseases, including cancer. The advent of several delivery platforms will permit the tailoring of the type of approach that will be most effective for a given disease, whether sustained silencing using a gene therapy approach or the application of synthetic siRNAs producing a transient silencing phenotype.

6. Expertopinion

RNAi has proven itself to be a powerful method for the targeted silencing of gene expression including the inhibition of disease-causing genes. The development of RNAi-based



silencing approaches as potential therapeutic modality has progressed at an extraordinary pace and has quickly surpassed other gene silencing approaches (e.g., antisense oligonucleotides and ribozymes). The use of RNAi-based silencing techniques has already become an indispensable tool for gene function analysis and there is little doubt that it will have an equally significant impact as a genetic therapy for a wide range of diseases, including anticancer applications.

Most patents filed proposing the use of RNAi as a potential therapeutic approach against cancer have focused on specific target genes that are important for tumor development. This includes oncogenes, genes involved in cell cycle regulation, DNA repair and genes that alter the tumor microenvironment (e.g., promoting angiogenesis). Because most of these patents have identified targets whose involvement in oncogenic transformation are already well characterized, they represent the low hanging fruit in the process of developing effective anticancer gene silencing approaches. As our understanding of the basic biology underlying the RNAi silencing pathways increases, so does the ease with which potent siRNAs can be identified. In fact, the current generation of siRNA design algorithms can predict effective siRNAs with a high degree of accuracy. The plethora of reagents that facilitate the efficient transduction of cultured cells has made testing the efficacy of specific siRNAs a reasonable prospect even in high-throughput applications. Taken together, it is clear that the identification of potential anticancer targets is not the limit-ing factor in the process of developing clinically feasible, RNAi-based anticancer approaches. This does not mean that there are no issues that need to be addressed regarding the design and testing of potential siRNA sequences for therapeutic applications, but merely that these challenges can be and are being overcome.

The primary challenge in translating RNAi-based mechanisms into clinical practice lies in the development of effective delivery approaches. Although the localized delivery of siRNAs to accessible sites has met with the most success, progress is being made in the delivery of siRNAs following systemic administration. Recent successes in the targeted delivery of siRNAs into specific cell types using antibody-, aptamer- and ligand-mediated approaches have begun to break down some of the barriers that have stood in the way of the thera-peutic application of siRNA-based gene silencing methods. In addition, the development of novel lipid formulations with enhanced pharmacological properties and improvements in DNA-based vector design have increased the effectiveness of these delivery approaches.

Even as these technologies continue the push forward towards the clinical application of siRNA-mediated gene silencing, they have uncovered further challenges. This has proven to be the case for the localized delivery of siRNAs to treat AMD. Animal studies demonstrated a reduction in angiogenesis after the intraocular injection of siRNAs targeting

VEGF or the VEGFR prompting the move into clinical trials. However, a more extensive examination in mice showed that angiogenesis could be broadly suppressed by the treatment with siRNAs in a sequence- and target- independent manner. This effect was mediated through the engagement of TLR3 receptors on the surface of endothelial cells inducing the release of IFN-γ and IL-12 [195]. These results point to the need for more stringent preclini-cal testing and the use of more controls to ensure the speci-ficity of the silencing response and the accuracy of the phenotype. Before RNAi-based silencing strategies can be brought into the clinic, it is essential to understand the possible difficulties that may impede progress. This can best be accomplished by extensive and comprehensive testing in model organisms.

The development of RNAi-based gene silencing approaches has occurred at a dizzying pace. In its first decade, RNAi has gone from a poorly understood silencing phenomenon in C.elegans to an integral research tool for gene function analysis. Not only have these tools and technologies been applied for high-throughput discovery and validation of drug targets, but as the extensive body of preclinical data show, harnessing RNAi holds a tremendous amount of therapeutic potential. Many of these developments have been the subject of patent applications. Despite the large numbers of patents filed regarding RNAi, only a small number of patents have been issued [196,197]. These have been dominated by several key patents that describe the identification and application of small double-stranded RNA molecules as mediators of gene silencing including the Kreutzer-Limmer patent (EP 1,144,623), the Fire and Mello patent (US 6,506,559), the Crooke patents (US 6,107,094 and US 5,898,221), and the Tuschl family of patents (Tuschl I (US 108,923) and Tuschl II (US 7,056,704 and US 7,078,196)). These groups of patents effectively cover the use of a broad range of small dsRNA molecules, both chemically modified and unmodified, for the inhibition of gene expression in a variety of organisms. Because the rate at which patents are issued lags behind the research being conducted, it is unclear how these ‘blanket patents’ will impact specific patents claiming specific delivery approaches and targets. This will become an increasingly important question as more and more RNAi-based approaches are being moved from the bench into the clinic. As the RNAi field continues to evolve and the ideas and technologies become more sophisticated, so will the patent landscape. More emphasis must be placed on the continued development of novel delivery methodologies for the promise of RNAi-based therapies to become a reality.

Declarationofinterest

The author states no conflict of interest and has received no payment in preparation of this manuscript.

Dykxhoorn


Bibliography1. Fire A, Xu S, Montgomery MK, et al.

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806-11

2. Farazi TA, Juranek SA, Tuschl T. The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members. Development 2008;135:1201-14

3. Matranga C, Zamore PD. Small silencing RNAs. Curr Biol 2007;17:R789-93

4. Hannon GJ, Rivas FV, Murchison EP, Steitz JA. The expanding universe of noncoding RNAs. Cold Spring Harb Symp Quant Biol 2006;71:551-64

5. Landgraf P, Rusu M, Sheridan R, et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 2007;129:1401-14

6. Kim VN, Nam JW. Genomics of microRNA. Trends Genet 2006;22:165-73

7. Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II. Embo J 2004;23:4051-60

8. Lee Y, Ahn C, Han J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature 2003;425:415-9

9. Denli AM, Tops BB, Plasterk RH, et al. Processing of primary microRNAs by the Microprocessor complex. Nature 2004;432:231-5

10. Landthaler M, Yalcin A, Tuschl T. The human DiGeorge syndrome critical region gene 8 and Its D. melanogaster homolog are required for miRNA biogenesis. Curr Biol 2004;14:2162-7

11. Lund E, Guttinger S, Calado A, et al. Nuclear export of microRNA precursors. Science 2004;303:95-8

12. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409:363-6

13. Chendrimada TP, Gregory RI, Kumaraswamy E, et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 2005;436:740-4

14. Tang G. siRNA and miRNA: an insight into RISCs. Trends Biochem Sci 2005;30:106-14

15. Sen GL, Blau HM. Argonaute 2/RISC resides in sites of mammalian mRNA decay known as cytoplasmic bodies. Nat Cell Biol 2005;7:633-6

16. Doench JG, Sharp PA. Specificity of microRNA target selection in translational repression. Genes Dev 2004;18:504-11

17. Doench JG, Petersen CP, Sharp PA. siRNAs can function as miRNAs. Genes Dev 2003;17:438-42

18. Bagga S, Bracht J, Hunter S, et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation. Cell 2005;122:553-63

19. Lim LP, Lau NC, Garrett-Engele P, et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005;433:769-73

20. Kutter C, Svoboda P. miRNA, siRNA, piRNA: knowns of the unknown. RNA Biol 2008;5:181-8

21. Lewis BP, Shih IH, Jones-Rhoades MW, et al. Prediction of mammalian microRNA targets. Cell 2003;115:787-98

22. Farh KK, Grimson A, Jan C, et al. The widespread impact of mammalian MicroRNAs on mRNA repression and evolution. Science 2005;310:1817-21

23. Dykxhoorn DM, Chowdhury D, Lieberman J. RNA interference and cancer: endogenous pathways and therapeutic approaches. Adv Exp Med Biol 2008;615:299-329

24. He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828-33

25. Cimmino A, Calin GA, Fabbri M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA 2005;102:13944-9

26. Calin GA, Sevignani C, Dumitru CD, et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 2004;101:2999-3004

27. Okamura K, Chung WJ, Lai EC. The long and short of inverted repeat genes in animals: microRNAs, mirtrons and hairpin RNAs. Cell Cycle 2008;7:2840-5

28. Okamura K, Balla S, Martin R, et al. Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster. Nat Struct Mol Biol 2008;15:998

29. Okamura K, Chung WJ, Ruby JG, et al. The Drosophila hairpin RNA pathway generates endogenous short interfering RNAs. Nature 2008;453:803-6

30. Golden DE, Gerbasi VR, Sontheimer EJ. An inside job for siRNAs. Mol Cell 2008;31:309-12

31. Okamura K, Lai EC. Endogenous small interfering RNAs in animals. Nat Rev Mol Cell Biol 2008;9:673-8

32. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 2001;15:188-200

33. Dykxhoorn DM, Novina CD, Sharp PA. Killing the messenger: short RNAs that silence gene expression. Nat Rev Mol Cell Biol 2003;4:457-67

34. Amarzguioui M, Lundberg P, Cantin E, et al. Rational design and in vitro and in vivo delivery of Dicer substrate siRNA. Nat Protoc 2006;1:508-17

35. Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494-8

36. Vogelstein B, Kinzler KW. Cancer genes and the pathways they control. Nat Med 2004;10:789-99

37. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57-70

38. Croce CM. Oncogenes and cancer. N Engl J Med 2008;358:502-11

39. Sherr CJ. Principles of tumor suppression. Cell 2004;116:235-46

40. Hubbard SR, Miller WT. Receptor tyrosine kinases: mechanisms of activation and signaling. Curr Opin Cell Biol 2007;19:117-23

41. Robinson DR, Wu YM, Lin SF. The protein tyrosine kinase family of the human genome. Oncogene 2000;19:5548-57

42. Yoon S, Seger R. The extracellular signal-regulated kinase: multiple substrates regulate diverse cellular functions. Growth Factors 2006;24:21-44

43. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001;411:355-65

44. Arteaga CL. Epidermal growth factor receptor dependence in human tumors: more than just expression? Oncologist 2002;7(Suppl 4):31-9

45. O’Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/Stat pathway. Cell 2002;109(Suppl):S121-31

46. Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein



kinase cascade for the treatment of cancer. Oncogene 2007;26:3291-310

47. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene 2008;27:5497-510

48. Steelman LS, Abrams SL, Whelan J, et al. Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia. Leukemia 2008;22:686-707

49. Hynes NE, Lane HA. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat Rev Cancer 2005;5:341-54

50. Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene 2007;26:3279-90

51. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003;3:459-65

52. Brummelkamp TR, Bernards R, Agami R. Stable suppression of tumorigenicity by virus-mediated RNA interference. Cancer Cell 2002;2:243-7

53. Rajagopalan H, Bardelli A, Lengauer C, et al. Tumorigenesis: RAF/RAS oncogenes and mismatch-repair status. Nature 2002;418:934

54. Davies H, Bignell GR, Cox C, et al. Mutations of the BRAF gene in human cancer. Nature 2002;417:949-54

55. McSwiggen J, Beigelman L. RNA interference meditaed inhibition of Myc and Myb gene expression or expression of genes involved in the myc and myb pathways using short interfering nucleic acid (siNA). WO2003US05326; 2003

56. Fujita DJ, Bjorge JD. Methods and reagents for inhibiting cell proliferation US20060453099; 2006

57. Khvorova A, Reynolds A, Leake D, et al. siRNA targeting v-myc myelocytomatosis viral oncogene homolog (MYC). US20070818547 2005

58. Frohling S, Dohner H. Chromosomal abnormalities in cancer. N Engl J Med 2008;359:722-34

59. Nilsson JA, Cleveland JL. Myc pathways provoking cell suicide and cancer. Oncogene 2003;22:9007-21

60. Macauley VM, Sohail M. Molecular targeting of the IGF-1 receptor. US20040996951; 2005

61. Khvorova A, Reynolds A, Leake D, et al. siRNA targeting insulin-like growth factor 1 receptor (IGF-1R). US20070732457; 2007

62. Evers BM, Rychahou P. SiRNA targeting PI3K signal transduction pathway and siRNA-based therapy. US20050085962; 2005

63. Bergers G, Benjamin LE. Tumorigenesis and the angiogenic switch. Nat Rev Cancer 2003;3:401-10

64. Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov 2007;6:273-86

65. Quay SC, McSwiggen J, Vaish NK, Ahmadian M. Nucleic Acid compounds for inhibiting VEGF gene expression and uses thereof. US2008003965020080228; 2008

66. Vargeese C, Jadhav V, Morrissey D. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA). US2005029939120051209; 2006

67. Kossen K, Jadhav V, Mcswiggen J, et al. RNA interference mediated inhibition of vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA). CA2004253708520040916; 2005

68. Liu Y, Lu PY, Woodle MC, Xie FY. Composition and methods of RNAi therapeutics for treatment of cancer and other neovascularization diseases. US2007082442620070629; 2008

69. Fougerollles AD, Frank-Kamenetsky M, Manoharan M, et al. IRNA agents targeting VEGF. US2006034008020060125; 2006

70. Trask DK, Bock J. Methods of inhibiting VEGF-C. US2006058558720061024; 2007

71. Shen J, Samul R, Silva RL, et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther 2006;13:225-34

72. Reich SJ, Fosnot J, Kuroki A, et al. Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol Vis 2003;9:210-6

73. Lu PY, Xie FY, Woodle MC. Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. Trends Mol Med 2005;11:104-13

74. Campochiaro PA. Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders. Gene Ther 2006;13:559-62

75. Gentile A, Trusolino L, Comoglio PM. The Met tyrosine kinase receptor in

development and cancer. Cancer Metastasis Rev 2008;27:85-94

76. Shinomiya N, Woude GFV. C-Met Sirna adenovirus vectors inhibit cancer cell growth, invasion and tumorigenicity. US20050599327; 2007

77. Khvorova A, Reynolds A, Leake D, et al. siRNA Targeting proto-oncogene MET. US20070980263; 2008

78. Reich SJ, Tolentino MJ. Compositions and methods for siRNA inhibition of angiopoietin 1and 2 and their receptor Tie2. US20040827759; 2004

79. Thurston G. Role of Angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis. Cell Tissue Res 2003;314:61-8

80. Kraus MH, Pierce JH, Fleming TP, et al. Mechanisms by which genes encoding growth factors and growth factor receptors contribute to malignant transformation. Ann NY Acad Sci 1988;551:320-35; discussion 36

81. Khvorova A, Reynolds A, Leake D, et al. siRNA targeting platelet-derived growth factor receptor beta polypeptide (PDGFR). US20070731890; 2008

82. McSwiggen J, Beigelman L, Chowrira BM. RNA interference mediated inhibition of platelet derived growth factor (PDGF) and platelet derived growth factor receptor (PDGFR) gene expression using short interfering nucleic acid (siNA). US20040923270; 2005

83. Wu H, Hait WN, Yang JM. Small interfering RNA-induced suppression of MDR1 (P-glycoprotein) restores sensitivity to multidrug-resistant cancer cells. Cancer Res 2003;63:1515-9

84. Stege A, Priebsch A, Nieth C, Lage H. Stable and complete overcoming of MDR1/P-glycoprotein-mediated multidrug resistance in human gastric carcinoma cells by RNA interference. Cancer Gene Ther 2004;11:699-706

85. Nieth C, Priebsch A, Stege A, Lage H. Modulation of the classical multidrug resistance (MDR) phenotype by RNA interference (RNAi). FEBS Lett 2003;545:144-50

86. Duan Z, Brakora KA, Seiden MV. Inhibition of ABCB1 (MDR1) and ABCB4 (MDR3) expression by small interfering RNA and reversal of paclitaxel resistance in human ovarian cancer cells. Mol Cancer Ther 2004;3:833-8

Dykxhoorn


87. McSwiggen J, Beigelman L, Thompson J. RNA interference mediated inhibition of MDR P-glycoprotein gene expression using short interfering nucleic acid (siNA). US20040918969; 2005

88. Zhang X, Kon T, Wang H, et al. Enhancement of hypoxia-induced tumor cell death in vitro and radiation therapy in vivo by use of small interfering RNA targeted to hypoxia-inducible factor-1alpha. Cancer Res 2004;64:8139-42

89. Akinc A, De Fougerolles A, Vornlocher HP, et al. RNAI modulation of HIF-1 and therapeutic uses thereof. EP20060785707; 2008

90. Usman N, McSwiggen J. RNA interference mediated inhibition of hypoxia inducible factor 1 (HIF1) gene expression using short interfering nucleic acid (siNA). WO2004US27294; 2005

91. Deweese TL, Collins SJ, Nelson WG. Engineered rnai adenovirus silencing expression (erase) of dna repair proteins. US20050534010; 2005

92. Chang IY, Kim MH, Kim HB, et al. Small interfering RNA-induced suppression of ERCC1 enhances sensitivity of human cancer cells to cisplatin. Biochem Biophys Res Commun 2005;327:225-33

93. Chen J, Wall NR, Kocher K, et al. Stable expression of small interfering RNA sensitizes TEL-PDGFbetaR to inhibition with imatinib or rapamycin. J Clin Invest 2004;113:1784-91

94. McSwiggen J, Beigelman L, Chowrira BM. RNA interference mediated inhibition of chromosome translocation gene expression using short interfering nucleic acid (siNA). US20040923522; 2005

95. Oltersdorf T, Elmore SW, Shoemaker AR, et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005;435:677-81

96. Khvorova A, Reynolds A, Leake D, et al. Functional and hyperfunctional siRNA directed against Bcl-2. US20070974878; 2008

97. McSwiggen J, Beigelman L. RNA interference mediated inhibition of B-cell CLL/Lymphoma-2 (BCL-2) gene expression using short interfering nucleic acid (siNA). US20040923516; 2005

98. Sonoke S, Ueda T, Fujiwara K, et al. Tumor regression in mice by delivery of Bcl-2 small interfering RNA with pegylated

cationic liposomes. Cancer Res 2008;68:8843-51

99. Sun H, Ghosh P, Kim Y. Methods of treating cancer by interfering with IGF-1 receptor signaling. WO2008US03393; 2008

100. McSwiggen J, Beigelman L, Chowrira B. RNA interference mediated inhibition of type 1 insulin-like receptor (IGF-1R) gene expression using short interfering nucleic acid (siNA). WO2003US05044; 2003

101. Aza-Blanc P, Cooper CL, Wagner K, et al. Identification of modulators of TRAIL-induced apoptosis via RNAi-based phenotypic screening. Mol Cell 2003;12:627-37

102. Dykxhoorn DM, Lieberman J. The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med 2005;56:401-23

103. Brummelkamp TR, Nijman SM, Dirac AM, Bernards R. Loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating NF-kappaB. Nature 2003;424:797-801

104. Hahn WC, Weinberg RA. Modelling the molecular circuitry of cancer. Nat Rev Cancer 2002;2:331-41

105. Berns K, Hijmans EM, Mullenders J, et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 2004;428:431-7

106. Jadhav V, Carrol JM, Terpstra AJ. RNA interference mediated inhibition of histone deacetylase (HDAC) gene expression using short interfering nucleic acid (siNA). AU20060261653; 2008

107. Vornlocher HP, Tan P, Constein R. RNAi modulation of TGF-beta and therapeutic uses thereof. EP20070753200; 2008

108. Guerciolini R, Robin H, McSwiggen J. RNA interference mediated inhibition of TGF-beta and TGF-beta receptor gene expression using short interfering nucleic acid (siNA). US20050054047; 2005

109. Westbrook TF, Martin ES, Schlabach MR, et al. A genetic screen for candidate tumor suppressors identifies REST. Cell 2005;121:837-48

110. Kolfschoten IG, van Leeuwen B, Berns K, et al. A genetic screen identifies PITX1 as a suppressor of RAS activity and tumorigenicity. Cell 2005;121:849-58

111. Ngo VN, Davis RE, Lamy L, et al. A loss-of-function RNA interference screen

for molecular targets in cancer. Nature 2006;441:106-10

112. Silva JM, Marran K, Parker JS, et al. Profiling essential genes in human mammary cells by multiplex RNAi screening. Science 2008;319:617-20

113. Schlabach MR, Luo J, Solimini NL, et al. Cancer proliferation gene discovery through functional genomics. Science 2008;319:620-4

114. Dykxhoorn DM, Palliser D, Lieberman J. The silent treatment: siRNAs as small molecule drugs. Gene Ther 2006;13:541-52

115. Dykxhoorn DM, Lieberman J. Knocking down disease with siRNAs. Cell 2006;126:231-5

116. Bitko V, Musiyenko A, Shulyayeva O, Barik S. Inhibition of respiratory viruses by nasally administered siRNA. Nat Med 2005;11:50-5

117. Palliser D, Chowdhury D, Wang QY, et al. An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection. Nature 2006;439:89-94

118. Li BJ, Tang Q, Cheng D, et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat Med 2005;11:944-51

119. Behlke MA. Progress towards in vivo use of siRNAs. Mol Ther 2006;13:644-70

120. Tousignant JD, Gates AL, Ingram LA, et al. Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice. Hum Gene Ther 2000;11:2493-513

121. Dass CR. Cytotoxicity issues pertinent to lipoplex-mediated gene therapy in-vivo. J Pharm Pharmacol 2002;54:593-601

122. Chien PY, Wang J, Carbonaro D, et al. Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo. Cancer Gene Ther 2005;12:321-8

123. Ahmad I, Zhang ZY, Zhang JA, et al. Lipid compositions and use thereof. CA20052559352; 2005

124. Morrissey DV, Lockridge JA, Shaw L, et al. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat Biotechnol 2005;23:1002-7

125. McSwiggen J, Morrissey D, Guerciolini R, et al. RNA interference mediated inhibition of hepatitis C virus (HCV) gene expression



using short interfering nucleic acid (siNA). WO2006US62252; 2007

126. Czech B, Malone CD, Zhou R, et al. An endogenous small interfering RNA pathway in Drosophila. Nature 2008;453:798-802

127. Zimmermann TS, Lee AC, Akinc A, et al. RNAi-mediated gene silencing in non-human primates. Nature 2006;441:111-4

128. Urban-Klein B, Werth S, Abuharbeid S, et al. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo. Gene Ther 2005;12:461-6

129. Leong KW, Mao HQ, Truong-Le VL, et al. DNA-polycation nanospheres as non-viral gene delivery vehicles. J Control Release 1998;53:183-93

130. Gary DJ, Puri N, Won YY. Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. J Control Release 2007;121:64-73

131. Schiffelers RM, Ansari A, Xu J, et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res 2004;32:e149

132. Pun SH, Bellocq NC, Liu A, et al. Cyclodextrin-modified polyethylenimine polymers for gene delivery. Bioconjug Chem 2004;15:831-40

133. Pun SH, Tack F, Bellocq NC, et al. Targeted delivery of RNA-cleaving DNA enzyme (DNAzyme) to tumor tissue by transferrin-modified, cyclodextrin-based particles. Cancer Biol Ther 2004;3:641-50

134. Davis ME, Pun SH, Bellocq NC, et al. Self-assembling nucleic acid delivery vehicles via linear, water-soluble, cyclodextrin-containing polymers. Curr Med Chem 2004;11:179-97

135. Borchard G, Junginger HE. Modern drug delivery applications of chitosan. Adv Drug Deliv Rev 2001;52:103

136. Howard KA, Rahbek UL, Liu X, et al. RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system. Mol Ther 2006;14:476-84

137. Howard KA, Paludan SR, Behlke MA, et al. Chitosan/siRNA nanoparticle-mediated TNF-alpha knockdown in peritoneal macrophages for anti-inflammatory treatment in a murine arthritis model. Mol Ther 2009;17:162-8

138. Hanai K, Takeshita F, Honma K, et al. Atelocollagen-mediated systemic DDS for nucleic acid medicines. Ann NY Acad Sci 2006;1082:9-17

139. Takeshita F, Minakuchi Y, Nagahara S, et al. Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo. Proc Natl Acad Sci USA 2005;102:12177-82

140. Minakuchi Y, Takeshita F, Kosaka N, et al. Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo. Nucleic Acids Res 2004;32:e109

141. Song E, Zhu P, Lee SK, et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol 2005;23:709-17

142. Peer D, Zhu P, Carman CV, et al. Selective gene silencing in activated leukocytes by targeting siRNAs to the integrin lymphocyte function-associated antigen-1. Proc Natl Acad Sci USA 2007;104:4095-100

143. Kumar P, Wu H, McBride JL, et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 2007;448:39-43

144. Kumar P, Ban HS, Kim SS, et al. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 2008;134:577-86

145. Lieberman J, Song E. Method of Delivering Rna Interference and Uses Thereof. US20050659386; 2008

146. Peer D, Shimaoka M, Lieberman J. Targeted delivery to leukocytes using protein carriers. WO2007US09975; 2007

147. Peer D, Park EJ, Morishita Y, et al. Systemic leukocyte-directed siRNA delivery revealing cyclin D1 as an anti-inflammatory target. Science 2008;319:627-30

148. Thompson J, McSwiggen J, Beigelman L. RNA interference mediated inhibition of cyclin D1 gene expression using short interfering nucleic acid (siNA). WO2003US03662; 2003

149. Shankar P, Lee SK, Swamy M, et al. Targeted delivery of siRNA. WO2008US52054; 2008

150. Soutschek J, Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 2004;432:173-8

151. Narasimhaswamy M, Shankar P, Kumar P. Method for delivery across the blood brain barrier. WO2007US12152; 2008

152. Lengyel E, Kossiakoff A, Piccirilli J. Receptor-mediated delivery: compositions and methods. WO2008014404; 2008

153. Wright DG, Murphy JR, Zhang Y. Cellular delivery of reagens that inhibit gene expression utilizing the anthrax toxin protective antigen (PA). WO2006091233; 2006

154. McNamara JO 2nd, Andrechek ER, Wang Y, et al. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras. Nat Biotechnol 2006;24:1005-15

155. Sullenger BA. Delivery method. WO2007US12927; 2007

156. Kim DH, Rossi JJ. Strategies for silencing human disease using RNA interference. Nat Rev Genet 2007;8:173-84

157. Grimm D, Kay MA. Therapeutic application of RNAi: is mRNA targeting finally ready for prime time? J Clin Invest 2007;117:3633-41

158. Grimm D, Kay MA. RNAi and gene therapy: a mutual attraction. Hematology Am Soc Hematol Educ Prog 2007;2007:473-81

159. Schaffer DV, Koerber JT, Lim KI. Molecular engineering of viral gene delivery vehicles. Annu Rev Biomed Eng 2008;10:169-94

160. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol 1999;10:440-7

161. Lee YJ, Imsumran A, Park MY, et al. Adenovirus expressing shRNA to IGF-1R enhances the chemosensitivity of lung cancer cell lines by blocking IGF-1 pathway. Lung Cancer 2007;55:279-86

162. Shinomiya N, Gao CF, Xie Q, et al. RNA interference reveals that ligand-independent met activity is required for tumor cell signaling and survival. Cancer Res 2004;64:7962-70

163. Yoo JY, Kim JH, Kwon YG, et al. VEGF-specific short hairpin RNA-expressing oncolytic adenovirus elicits potent inhibition of angiogenesis and tumor growth. Mol Ther 2007;15:295-302

164. Zheng JN, Pei DS, Sun FH, et al. Inhibition of renal cancer cell growth by oncolytic adenovirus armed short hairpin RNA targeting hTERT gene. Cancer Biol Ther 2009;8

165. Zheng JN, Pei DS, Mao LJ, et al. Inhibition of renal cancer cell growth in vitro and in vivo with oncolytic adenovirus armed short hairpin RNA

Dykxhoorn


targeting Ki-67 encoding mRNA. Cancer Gene Ther 2009;16:20-32

166. Daya S, Berns KI. Gene therapy using adeno-associated virus vectors. Clin Microbiol Rev 2008;21:583-93

167. Hildinger M, Auricchio A. Decreasing gene expression in a mammalian subject in vivo via AAV-mediated RNAi expression cassette transfer. US2005019927; 2005:80-7

168. Tomar RS, Matta H, Chaudhary PM. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene 2003;22:5712-5

169. Noguchi P. Risks and benefits of gene therapy. N Engl J Med 2003;348:193-4

170. Sumimoto H, Yamagata S, Shimizu A, et al. Gene therapy for human small-cell lung carcinoma by inactivation of Skp-2 with virally mediated RNA interference. Gene Ther 2005;12:95-100

171. Sumimoto H, Hirata K, Yamagata S, et al. Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi. Int J Cancer 2006;118:472-6

172. Sumimoto H, Miyagishi M, Miyoshi H, et al. Inhibition of growth and invasive ability of melanoma by inactivation of mutated BRAF with lentivirus-mediated RNA interference. Oncogene 2004;23:6031-9

173. Pichler A, Zelcer N, Prior JL, et al. In vivo RNA interference-mediated ablation of MDR1 P-glycoprotein. Clin Cancer Res 2005;11:4487-94

174. Grimm D, Streetz KL, Jopling CL, et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006;441:537-41

175. An DS, Qin FX, Auyeung VC, et al. Optimization and functional effects of stable short hairpin RNA expression in primary human lymphocytes via lentiviral vectors. Mol Ther 2006;14:494-504

176. An DS, Donahue RE, Kamata M, et al. Stable reduction of CCR5 by RNAi through hematopoietic stem cell transplant in non-human primates. Proc Natl Acad Sci USA 2007;104:13110-5

177. Giering JC, Grimm D, Storm TA, Kay MA. Expression of shRNA from a tissue-specific pol II promoter is an effective and safe RNAi therapeutic. Mol Ther 2008;16:1630-6

178. Dickins R, Hannon GJ, Lowe SW. Methods for producing miRNAs. EP1896587; 2008

179. Dykxhoorn DM, Schlehuber LD, London IM, Lieberman J. Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism. Proc Natl Acad Sci USA 2006;103:5953-8

180. Jackson AL, Bartz SR, Schelter J, et al. Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003;21:635-7

181. Lin X, Ruan X, Anderson MG, et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 2005;33:4527-35

182. Anderson EM, Birmingham A, Baskerville S, et al. Experimental validation of the importance of seed complement frequency to siRNA specificity. RNA 2008;14:853-61

183. Jackson AL, Burchard J, Schelter J, et al. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity. RNA 2006;12:1179-87

184. Sioud M. RNA interference and innate immunity. Adv Drug Deliv Rev 2007;59:153-63

185. Heil F, Hemmi H, Hochrein H, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004;303:1526-9

186. Judge AD, Sood V, Shaw JR, et al. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol 2005;23:457-62

187. Sioud M. Single-stranded small interfering RNA are more immunostimulatory than their double-stranded counterparts: a central role for 2’-hydroxyl uridines in immune responses. Eur J Immunol 2006;36:1222-30

188. Akira S, Takeda K. Toll-like receptor signalling. Nat Rev Immunol 2004;4:499-511

189. Sioud M. Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol 2005;348:1079-90

190. Robbins M, Judge A, Ambegia E, et al. Misinterpreting the therapeutic effects of siRNA caused by immune stimulation. Hum Gene Ther 2008

191. Akinc A, Zumbuehl A, Goldberg M, et al. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat Biotechnol 2008;26:561-9

192. Manoharan M, Rajeev KG, Akinc A, et al. Lipid containing formulations. WO2007US80331; 2008

193. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003;302:415-9

194. Hacein-Bey-Abina S, von Kalle C, Schmidt M, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348:255-6

195. Kleinman ME, Yamada K, Takeda A, et al. Sequence- and target-independent angiogenesis suppression by siRNA via TLR3. Nature 2008;452:591-7

196. Schmidt C. Negotiating the RNAi patent thicket. Nat Biotechnol 2007;25:273-5

197. Haussecker D. The business of RNAi therapeutics. Hum Gene Ther 2008;19:451-62

AffiliationDerek M DykxhoornThe University of Miami Miller School of Medicine, Miami Institute for Human Genomics, The John T Macdonald Foundation, Department of Human Genetics and the Department of Microbiology and Immunology, Miami, FL 33136, USA Tel: +1 305 243 7596; Fax: +1 305 243 2396; E-mail: [email protected]

rna interference as an anticancer therapy: a patent perspective

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