Knocking down schistosomes - promise for lentiviral transduction in parasites.

TREPAR-1371; No. of Pages 9

Review

Knocking down schistosomes – promise for lentiviral transduction in parasites Jana Hagen*, Jean-Pierre Y. Scheerlinck, and Robin B. Gasser Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Victoria 3010, Australia

Underpinned by major advances in our understanding of the genomes of schistosomes, progress in the development of functional genomic tools is providing unique prospects to gain insights into the intricacies of the biology of these blood flukes, their host relationships, and the diseases that they cause. This article reviews some key applications of double-stranded RNA interference (RNAi) in Schistosoma mansoni, appraises delivery systems for transgenesis and stable gene silencing, considers ways of increasing efficiency and specificity of gene silencing, and discusses the prospects of using a lentivirus delivery system for future functional genomic–phenomic explorations of schistosomes and other parasites. The ability to achieve effective and stable gene perturbation in parasites has major biological implications and could facilitate the development of new interventions. Schistosomes and the need for functional genomic– phenomic studies Schistosomiasis is a major neglected tropical disease, affects 300 million people globally, and is responsible for 300 000 deaths each year [1–3]. This debilitating disease is caused by a chronic infection with one or more schistosomes (blood flukes) including Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium. No vaccines are available, and treatment relies mainly on the use of a single drug, praziquantel, to which drug resistance appears to be emerging [4]. In a complex life cycle (Figure 1), S. mansoni is transmitted from an infected aquatic snail (Biomphalaria) to humans via skin penetration. Following schistosomule migration, adult worms develop and dwell in intestinal and hepatic blood vessels. Eggs released from female worms become embedded in the liver parenchyma and intestinal wall where they trigger immune-mediated granuloma formation [5]; some eggs pass through this wall into the gut lumen and are then excreted into the environment to complete the life cycle. Corresponding author: Hagen, J. ([email protected]). Keywords: helminth; Schistosoma; RNA interference; microRNA; short interfering RNA. * Current address: Department of Life Sciences, Imperial College, London SW7 2AZ, UK. 1471-4922/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2015.03.009

The granulomata that form in tissues are the principal cause of disease, ultimately leading to complications including fibrosis and periportal hypertension [6,7]. Although numerous studies (reviewed in [6]) have explored the pathogenesis

Glossary Dicer: RNase III enzyme that cleaves dsRNA to siRNA. Drosha: RNase III enzyme that initiates processing of miRNAs by cleaving primary miRNA transcripts to stem-loop structures (pre-mature miRNA). Genotoxicity: damage to the genomic information that can lead to mutagenesis and cancer. Lentivirus: a member of the Retroviridae that can infect dividing and resting cells. Long terminal repeat (LTR): identical sequences resulting from reverse transcription flanking proviral DNA that contain promoter/enhancer sequences and a termination signal. MicroRNA (miRNA): small non-coding RNA involved in post-transcriptional regulation of gene expression. miRNA-adapted short hairpin RNA (shRNAmir): synthetic short hairpin RNAs that contain the flanking regions of a natural miRNA such that they are expressed as primary miRNAs. Mutagenesis: introduction of stable, heritable changes to the genetic information. Omega-1: immunomodulatory protein that is secreted by S. mansoni eggs. Oncogene: a gene with the potential to cause cancer. Promoter: sequence in DNA that is recognized by RNA polymerase enzymes to initiate transcription of a gene. Pseudotyping: altering envelope proteins of a virus to expand the range of host cells that can be infected (tissue tropism) by combination of virus vectors with foreign virus envelope proteins. Retroviridae: a family of viruses with a single-stranded (ss) RNA genome that is replicated after reverse transcription to proviral DNA and subsequent integration into the genome of the host cell. g-Retrovirus: a member of the Retroviridae that can infect dividing cells. Reverse transcription: process in that the enzyme reverse transcriptase uses a single-stranded RNA template to synthesize a ssDNA molecule referred to as complementary DNA (cDNA). RNA interference: process of post-transcriptional regulation of gene expression that is induced by double-stranded (ds) RNA and leads to the degradation of mRNA or transcriptional regression. RNA trigger: dsRNA molecule that induces RNA interference. RNA-induced silencing complex (RISC): a multiprotein complex that incorporates mature miRNAs or one strand of siRNA and initiates cleavage of the mRNA at a sequence complementary to the miRNA/siRNA. Seed region: nucleotide hexamer or heptamer of the mature miRNA sequence at positions 2–7 or 2–8, respectively. Short hairpin RNA (shRNA): short RNA sequences with complementary sense and antisense regions that result in a stem-loop secondary structure resembling a hairpin. Small interfering RNA (siRNA): short dsRNA of 20–25 bp in length that can trigger RNAi. Transduction: utilizing a viral vector for the delivery of genetic information to a cell. Transgenesis: process of transferring a new gene into a cell with a view of producing a cell expressing this gene. Virion: a complete virus particle outside a host cell containing the virus genome and proteins protecting it (capsid) that can be surrounded by a lipid layer (envelope). Virus: infectious nucleic acid that replicates inside living cells.

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Cercariae enter skin

Schistosomula migrate to lungs, then to liver

Miracidia penetrate snail Snails shed cercariae

Adults mate in liver

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Adults migrate to mesenterics

Eggs deposited with feces in water

Eggs pass into small intesne

Adults live in mesenteric venule females lay eggs

Eggs pass into feces TRENDS in Parasitology

Figure 1. The life cycle of Schistosoma mansoni. Schistosome eggs are released in the feces from the human host. Upon contact with freshwater, miracidia hatch and penetrate the snail host (Biomphalaria spp.). Miracidia develop asexually into sporocysts in which further asexual propagation produces numerous cercariae. These motile stages actively penetrate the host skin, lose their tail, transform into schistosomula, and travel through the blood to the lungs and then the liver. After several days, schistosomula migrate to the portal venous system where they mature and unite. The adult worm pairs migrate to the mesenteric veins. Mature, gravid females release eggs, which pass into the lumen of excretory organs via induced inflammatory tissue-damaging processes. Modified from [94] with permission.

of the different forms of schistosomiasis, the molecular basis of the disease remains elusive. Knowledge gaps relate not only to the complexity of the biology of the parasites but also to technical obstacles. Major advances in our understanding of schistosome genomes [8–10], and gradual progress in the development of functional genomic techniques (reviewed in [11–14]), are providing new and exciting opportunities to study and understand the intricacies of the schistosome–host relationship and the pathogenesis of disease. Various tools, such as RNA interference (RNAi; see Glossary) using double-stranded RNA (dsRNA) or small interfering RNA (siRNA), have been utilized to study the functions of single genes [12]. However, some methods employed to date can have limitations, such as off-target (non-specific) perturbation effects [15,16] and inadequate stability of gene knockdown, which can interfere with subsequent phenotypic assessment in vitro or in vivo in host animals [17]. These issues are compounded by the challenge of consistently producing sufficient amounts of the various parasite stages in the laboratory for functional genomic analyses [18]. The present article reviews some key applications of RNAi in S. mansoni, appraises the delivery 2

systems for transgenesis and stable gene silencing, considers ways of increasing the specificity and efficiency of gene silencing, and discusses the prospects of using a lentivirus-based delivery system for future functional genomic–phenomic investigations of schistosomes and other parasites. Applications of RNAi to S. mansoni Following the first report of gene knockdown in schistosomes using synthetic long dsRNA [19], RNAi has been employed to study gene function in all life stages of S. mansoni (Figure 2). In conventional RNAi approaches, dsRNAs and siRNAs were usually delivered to the worms by soaking, electroporation, or particle bombardment. The processing of dsRNAs and longer hairpin RNAs (125– 500 bp) results in multiple siRNA sequences (21–25 nt) that can target different regions of the mRNA. Synthetic siRNAs can induce targeted gene knockdown at an efficiency comparable with dsRNAs [20]. However, the effectiveness of predicted siRNA requires experimental validation, which can be costly and time-consuming. The need to validate siRNAs before experimentation might be circumvented and the knockdown effect increased by using


Review


Delivery Soaking; Electroporaon Life stage Schistosomula Target gene Cathepsin B1.1 Luciferase

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Delivery Soaking; electroporaon; liposomes Target genes α-, β-integrin Annexin Asparaginyl endopepdase Calcineurin B Calcium ATPase 2 Calcium channel Calmodulin-1, -2 Calpain Calreculn Cathepsin B, C, D, L1 Cav2A DHHC domain

Life stage Adults; eggs; miracidia; schistosomula

Elongaon factor 1 α Fibrillarin Glucose transporter Glucogen synthase kinase-3 GST-26, -28 Gluthathione peroxidase Glycoprotein K5 Hexamer-binding protein Inhibin/acvin Lactate dehydrogenase Leucin aminopepdase

Myosin Methionine aminopepdase Neuroendocrine convertase N-myristol transferase Pepc kinase Protein kinase Cβ Protein kinase C receptor Protein phosphatase-2a Rho-1, -2 GTPase Ring box Scavenger receptor B Sm29

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Smeg Smad1, 2, 4 SPO1 Superoxide dismutase Syk kinase Tetraspanin-1, -2 Thioredoxin glutathione reductase TPx-1, -2 Zinc finger 1 14.3.3

Delivery Soaking, electroporaon, injecon, parcle bombardment Life stages Adults; schistosomula Target genes α-, β-integrin-1 Asparaginyl endopepdase Cathepsin B Glucose transporter-1, -2 Hypoxanthine guanine phosphoribosyl transferase Proteasome subunit SmRPN11/POH1 Serotonin transporter TGF-β receptor II TRENDS in Parasitology

Figure 2. RNA interference (RNAi) methods used in Schistosoma mansoni. Delivery of synthetic double-stranded (ds) or small interfering (si) RNA: upon uptake during soaking or facilitated by electroporation, injection, particle bombardment, or via liposomes, synthetic dsRNA or siRNA is recognized by Dicer and processed. The sense or antisense strand is incorporated into RISC, which can bind to the target mRNA sequence and initiate mRNA degradation. Delivery of short hairpin (sh)RNA: shRNA expression cassettes are delivered via Moloney murine leukemia virus (MMLV) or plasmid DNA ( piggyBac, pXL-II) by soaking or electroporation, respectively. Viral genomic RNA is released into the cytoplasm and transcribed reversely into proviral DNA. Proviral DNA can randomly integrate into the host genome in dividing cells and express shRNA. After export into the cytoplasm via exportin-5, shRNA is recognized and processed by Dicer, and enters the RNAi pathway to induce target mRNA degradation. For a full reference list of target genes see [12]. Abbreviations: LTR, long terminal repeat; Pol, RNA polymerase; RISC, RNA-induced silencing complex.

siRNA cocktails containing multiple target-specific siRNAs [21,22]. This approach has been utilized recently to achieve target gene knockdown in S. mansoni [23]. Although the use of different transfection reagents does not usually improve RNAi efficiency, electroporation can enhance knockdown effects compared with soaking, presumably because more dsRNA enters the cells [20,24,25]. However, it is not yet entirely understood whether electroporation is beneficial to knockdown efficiency [26]. To date, most studies employing RNAi have focused on investigating genes expressed by adult schistosomes, schistosomula, and miracidia (Figure 2). Targets (Figure 2) have included genes involved in schistosome development (e.g., CD36-like scavenger receptor B and glucose-transporter) [27,28], hemoglobin digestion (e.g., cathepsins B, C, D, and asparaginyl endopeptidase) [19,24–26,29–32], membrane proteins [23,33], and components of the transforming growth factor TGF-b and spleen tyrosine kinase (Syk) signaling or redox pathways [32,34–36]. In two studies [26,37], RNAi was also used as a screening tool to identify essential genes in S. mansoni; however, owing to a lack of discernible phenotypes, the authors (perhaps unjustifiably) concluded that the use of RNAi-based gene silencing for this purpose may be limited. There is only a small number of reports in which RNAi was used to study gene

function in S. mansoni eggs [38,39], although egg and cercarial stages of this species express high levels of Dicer [40] and are thus amenable to RNAi. For instance, Freitas et al. [38] showed that eggs laid ex vivo failed to fully mature after soaking in dsRNA targeting the inhibin/ activin (SmInAct) gene, encoding a member of the TGFb protein superfamily. In addition, Rinaldi et al. [39] demonstrated an 80% reduction in egg hatching upon knockdown of a leucine aminopeptidase gene. Thus, some studies conducted have shown clearly that RNAi can be used effectively for functional genomic investigations and to identify essential genes. The knockdown effect of synthetic siRNAs and dsRNAs in S. mansoni in vitro persists for several weeks [24,28]. However, a recent study [28] reported that longterm gene knockdown observed in vitro is not seen in vivo following the introduction of siRNA-treated S. mansoni into the definitive host. Therefore, effective studies of the biology of S. mansoni by loss of gene function require the stable production of interfering RNAs, which could be achieved by transgenesis. The complex life cycles and particular tissue/parenchymal structures of schistosomes, and the absence of immortalized cell lines, have somewhat impeded the development of a robust transgenesis system. However, there has been some promising progress using 3


Review delivery systems such as transposons and integrating retroviruses. Delivery systems for transgenesis and stable knockdown Various approaches have been assessed to achieve transgenesis of different life stages of S. mansoni; they include electroporation [29,41,42] and particle bombardment [43– 50] of plasmid DNA, and the introduction of transposons by electroporation [51] or retroviral transduction [11,52,53]. The utilization of transposons or retroviruses is of particular relevance because they enable random genomic integration, facilitating the establishment of transgenesis. Indeed, both of these delivery methods have been applied successfully to S. mansoni. First evidence of transgenesis in S. mansoni was reported by Morales et al. [51], who achieved genomic integration of a reporter gene after electroporation of schistosomula with the piggyBac transposon. However, non-viral systems are only able to transduce a small percentage of cells, as opposed to retroviral delivery systems which usually achieve a transduction efficiency of >90% [54]. Pseudotyping of retroviral envelopes with the fusogen vesicular stomatitis virus glycoprotein (VSV-G) allows the transduction of a broad range of mammalian and nonmammalian cells [54,55]. Upon fusion of the virus envelop with the host cell membrane, viral genomic RNA is released into the cytoplasm and is reverse-transcribed, producing the pre-integration complex (PIC), in conjunction with the enzyme integrase. The PIC can then integrate into various sites within the host genome as proviral DNA. The successful introduction of a reporter transgene into S. mansoni chromosomes by g-retroviral delivery has been achieved by employing replication-incompetent Moloney murine leukemia virus (MMLV) [53,56]. Although soaking appears to be sufficient for the transduction of eggs and other life cycle stages, Kines et al. [56,57] reported an increase in virus uptake and integration by square-wave electroporation. The transduction of eggs is attractive because they contain a high percentage of pluripotent cells derived from the germline [58]; hence, there is a prospect of using this developmental stage to produce transduced S. mansoni lines in which every cell is affected by the transgene delivered. Indeed, it has been shown that miracidia hatching from transduced S. mansoni eggs were able to continue the life cycle in the snail intermediate host [11]. However, higher transgene copy numbers correlated with a diminished infectivity of miracidia, and electroporation was not beneficial for the promotion of transgenic parasites in the snail host [11]. Recently, MMLV was applied to adult S. mansoni for RNAi (Figure 2) [31,59]. In a first study [31], the virus vector contained an expression cassette encoding a 250 bp hairpin structure, targeting the protease cathepsin B1, under the control of the SmACT1.1 promoter. Using this system, an 80% reduction of target gene expression was observed within 72 h. Following this proof-of-principle, MMLV [59] and pXL-BacII ( piggyBac) [59,60] were employed for the delivery of short hairpin (sh) RNA (21 bp) to S. mansoni, with expression cassettes under the control of schistosome or human Pol III U6 promoters, 4


respectively. The advantage of using a mammalian promoter, as opposed to a parasite-derived promoter, is that it allows validation of expression cassettes and of the efficacy of the RNAi trigger in mammalian cells [61]. Furthermore, the relatively short sequence length of Pol III promoters and the shRNA allow effective packaging of viral vectors into virions and the production of high functional virus titers [59]. However, despite the success of virus-delivered expression cassettes, improvements will be necessary to enhance the specificity and efficiency of the RNAi trigger, and to optimize the delivery and safety of the transducing virus. Exploiting microRNAs (miRNAs) for increased specificity and efficiency of knockdown A limitation of RNAi can be the induction of off-target effects (i.e., silencing of non-target RNAs). The processing of dsRNAs and longer hairpin RNAs (125–500 bp) results in multiple siRNA sequences (21–25 nt) that can target different regions of the mRNA. However, it is challenging to predict the siRNAs that will be processed, such that the use of long dsRNA triggers carries an intrinsic risk of offtarget effects. For instance, in a recent study [37], primary S. mansoni sporocysts were screened for emerging phenotypes after knockdown of 32 different genes by dsRNA treatment. Remarkably, while a similar phenotype of reduced body length was observed after knockdown of 11 different genes, body shortening could be linked to a significant and consistent knockdown of target gene transcripts for only six of these 11 genes. Therefore, it is possible that, at least in some cases, the observed phenotype might have resulted from off-target effects rather than from specific knockdown of the target gene. The sequence length of short hairpins (21 bp) might reduce the risk of off-target effects. shRNA expression cassettes, under the control of Pol III promoters, have been introduced into schistosomules of S. mansoni by electroporation of plasmid DNA or delivery via MMLV, and have resulted in efficient target gene knockdown [59,60,62]. However, the use of Pol III promoters can also lead to high, constitutive expression of the RNAi trigger. Importantly, abundance of the RNAi trigger can be cytotoxic as a result of saturation of the RNAi pathway [63] and might lead to a dysregulation of endogenous miRNAs, which can result in off-target effects [16]. Furthermore, one study [64] demonstrated that the sense strand was loaded predominantly into the RNA-induced silencing complex (RISC) when shRNA was used, further increasing the risk of off-target effects. Despite the unique structures of miRNAs (Box 1), sequence variation, particularly in the stem region, is usually tolerated without impairing their processing to mature and functional structures [65]. This characteristic was used to develop so-called second-generation shRNA structures which are adapted from the secondary structure of the primary human miR-30 [66,67]. In these miRNAadapted short hairpin RNAs (shRNAmirs), the mature miRNA-30 sequence encoded in the stem is replaced with an artificial mature miRNA sequence designed specifically to the target gene. Therefore, shRNAmirs possess the typical primary-miRNA secondary structure and contain


Review


Box 1. The RNAi pathway The miRNAs encoded in transcripts of endogenous genes are processed to pre-mature miRNAs by Drosha and are exported into the cytoplasm via exportin-5 (Figure I). Pre-miRNAs are recognized by Dicer and cleaved into small interfering RNAs (siRNA), and the antisense strand (mature miRNA) is incorporated into RISC. Binding of miRNAs with an imperfect match to the 30 -UTRs of the target mRNA results in translational repression of target gene expression. miRNAs perfectly matching mRNA coding regions induce target mRNA degradation.

DNA Exporn5 Dicer Drosha miRNA*/ miRNA Pri-miRNA

Pre-miRNA

RISC

ORF Translaonal repression

seed region (i.e., nucleotides 2–8 of the mature miRNA sequence) to the 30 -untranslated region (30 -UTR) of an unintended mRNA (seed complement) [15,75–77]. Because scrambled or irrelevant control RNAs have entirely different seed regions from the RNAi trigger, they could be considered as inappropriate controls for the exclusion of off-target effects induced by the relevant RNAi trigger. Therefore, one could argue that suitable control RNAs should contain a seed region that is identical to the target RNAi trigger. Importantly, the presence of miRNAs [78,79] and enzymes of the miRNA pathway (Drosha and Dicer) in S. mansoni [80] indicated that miRNA-adapted shRNA design could be applied to schistosomes. Therefore, exploiting the miRNA pathway of S. mansoni might lead to enhanced specificity, efficiency, and stability of the silencing effect as well as reduced ‘side effects’, such as cytotoxicity, as sometimes observed in mammalian cells [16,73].

3′-UTR

RISC

mRNA cleavage

TRENDS in Parasitology

Figure I. The RNAi pathway. Modified from [95].

Dicer and Drosha processing sites [67,68]. Owing to the distinct design of miRNAs, the antisense strand (miRNA) exhibits a thermodynamically unstable 50 -end, ensuring predominant incorporation into the RISC complex and reducing the risk of off-target effects induced by the sense strand sequence (miRNA*) [69]. To maximize the probability of specific target RNA degradation, new artificial miRNA sequences can be designed using siRNA prediction programs, such as designer for short interfering RNAs (DSIR) [70], to perfectly match coding regions of target mRNAs and induce mRNA cleavage. Remarkably, the expression of shRNAs as primary miRNAs can increase the efficiency of target gene knockdown by up to 10-fold [67]. Increased knockdown efficiencies might allow the use of a lower multiplicity of infection (MOI), which could decrease the risk of insertional mutagenesis due to virus integration [71,72]. Furthermore, relatively weak promoters could be employed to reduce both genotoxicity by transactivation and the abundance of the RNAi trigger, and this might prevent cytotoxicity caused by saturation of the RNAi pathway [16,73]. Because off-target effects of siRNAs can affect cell viability [63], it is particularly important to exclude off-target effects when a lethal phenotype is achieved. Furthermore, cytotoxicity in target cells has also been linked to overexpression of the RNAi trigger, leading to saturation of the RNAi machinery and dysregulation of endogenous miRNAs [16,73]. Controls employed for assessing off-target effects have commonly been RNAs with scrambled nucleotide sequences of the RNAi trigger [20,28,60] or an irrelevant, non-target sequence [37,39,59,74]. However, most off-target effects occur after binding of the miRNA

Advantages of lentiviral delivery systems The use of MMLV for the introduction of RNAi expression cassettes into S. mansoni provides proof-of-principle that this technology results in gene knockdown. However, all members of the g-retrovirus subfamily, including MMLV, lack nuclear transport signaling and can, therefore, only infect dividing cells. Furthermore, most MMLV-derived vectors contain active promoter/enhancer sequences in their 50 long terminal repeats (50 -LTR), which remain active upon integration of proviral DNA into the genome of the host cell, and can interact with and activate endogenous promoters, leading to uncontrolled expression of endogenous genes [72] (Figure 3A). In addition, g-retroviruses are prone to suboptimal termination because of weak polyadenylation signals encoded in the redundant region (R). Transcriptional read-through events into adjacent sequences in the host genome (Figure 3B) have been linked to insertional mutagenesis by transactivation of neighboring genes [71,72,81]. Moreover, transactivation of proto-oncogenes due to strong promoter enhancer sequences has been linked to the development of leukemia [71,82–84], highlighting possible adverse effects resulting from virus integration. Some of the limitations of MMLV delivery systems might be overcome through the use of lentiviruses. A major advantage of lentiviral vectors is the ability to infect both arrested and dividing cells, thus increasing transduction efficiency [85]. Usually HIV-derived lentiviral transduction systems have several safety features that minimize the risk of producing replication-competent lentiviruses [86]. In advanced packaging systems, genes required for the formation of infectious virions are split from the virus genome and are encoded in several helper plasmids that need to be co-transfected into the packaging cells together with the virus vector plasmid [86]. In addition, lentiviral vectors usually contain a self-inactivating 30 -LTR (SIN LTR) [87] which restricts virus replication to the packaging cell line and overcomes transactivation associated with promoter/enhancer sequences encoded in the proviral 50 LTR. Retroviral genomic RNAs are capped single-stranded RNAs (+ssRNA) with polyadenylated tails and contain unique, short regions at their 50 - and 30 -ends (U5 and U3, respectively) that are flanked by the R region. During 5


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Figure 3. Reduced genotoxicity and improved transgene expression upon delivery with lentiviral self-inactivating (SIN)-systems compared with g-retrovirus MMLV used for transduction of Schistosoma mansoni. During transduction, viral genomic RNA is released into the cytoplasm and is reverse-transcribed into proviral DNA. Proviral DNA is transported into the nucleus, where it randomly integrates into the genome of host cells. (A,B) MMLV. (A) Promoter/enhancer (P E) interaction of the 50 -long terminal repeat (LTR) with endogenous promoters can lead to transactivation of endogenous genes in transduced cells. (B) Weak termination signals lead to transcriptional read-through into adjacent sequences and partial expression of endogenous genes resulting in poor translatability of the transgene and potentially the activation of oncogenes. (C,D) HIVderived SIN-lentivirus (HIV-SIN). (C) Vectors with SIN LTRs have a deletion in the U3 region (dU3) which contains promoter/enhancer sequences required for virus replication. During reverse transcription, this deletion is transferred to the 50 -LTR such that transcription from proviral DNA is dependent on the internal promoter. (D) The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) increases transcript stability, nuclear export of transcripts, and their translatability in the target cell. c, packaging signal.

reverse transcription, the U3 and U5 regions are copied and transferred to either site, such that proviral DNA contains repeats of the U3-R-U5-region (Figure 3A), also referred to as LTRs. SIN LTRs have a deletion in the U3 region of the 30 -LTR, which contains the promoter/ enhancer sequences required for virus replication (Figure 3C). During reverse transcription, this deletion is copied to the 50 -LTR, such that virus replication is restricted to the packaging cell line (Figure 3C). Furthermore, the lack of 50 -promoter enhancer sequences greatly reduces the risk of transactivation of oncogenes. Finally, lentiviral vectors usually contain the woodchuck hepatitis virus post-transcriptional regulatory element (Figure 3D). This element increases transcript stability, nuclear export of transcripts, and their translatability, resulting in higher virus titers and enhanced transgene expression [88–90]. Latest generations of retroviral vectors can also contain a termination enhancing sequence, such as the polyadenylation enhancing element of the simian virus 40 (upstream sequence element, 2SV USE), which has been shown to almost completely prevent read-through transcription from neighboring genes [91]. Furthermore, the deletion from the U3 region provides space to add a chromatin insulator element flanking the provirus (Figure 3D). Insulation provides a barrier between the virus and endogenous genes and their promoters, and aids in the maintenance of transcriptionally 6

active chromatin, preventing epigenetic silencing of the viral expression cassette. The insulator element of the 50 hypersensitive site 4 (50 -HS4) of the chicken b-globulin gene (cHS4 insulator) has enhancer-blocking activity. This approach has been used successfully in MMLV and HIVderived systems [92,93] and was shown to lead to improved transgene expression and virus titers while minimizing transcriptional read-through responsible for the transactivation of endogenous genes. In summary, recent improvements to lentiviral delivery systems have greatly reduced the risks of adverse effects associated with integrating gene delivery systems and allow persistent transgene expression in the host cells. First application of the lentiviral transduction system to a schistosome Recently, Hagen et al. [61] successfully assessed a lentiviral transduction system for the delivery of a shRNAmir expression cassette to S. mansoni eggs under the control of the cytomegalovirus (CMV) promoter (Figure 4A). The use of this mammalian promoter allowed the verification of efficacy and efficiency of novel shRNAmirs in mammalian cell lines before their application to the parasite. Results showed knockdown of selected target genes by shRNAmirs, and that the miRNA pathway is available for RNAi studies in S. mansoni. Importantly, lentiviral transduction and shRNAmir-induced transcriptional gene knockdown had


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Figure 4. Knockdown (kd) of omega-1 transcripts in S. mansoni eggs upon lentiviral delivery of a microRNA-adapted short hairpin RNA (shRNAmir) expression cassette leads to decreased granuloma formation in BALB/c mice. (A) Lentiviral delivery of shRNAmir. During transduction, viral genomic RNA is released into the cytoplasm and reverse-transcribed into proviral DNA. Proviral DNA is transported into the nucleus where it randomly integrates into the genome of dividing and resting host cells. shRNAs are constitutively expressed as primary miRNAs (pri-miRNA), which are processed by Drosha to pre-mature miRNAs (pre-miRNA). After export into the cytoplasm via exportin-5, pre-miRNA are recognized and processed by Dicer and processed into miRNA*/miRNA duplexes. The antisense strand (mature miRNA) is incorporated into the RNA-induced silencing complex (RISC). miRNAs designed to perfectly match target mRNA-coding regions induce mRNA degradation. (B) Decrease in granuloma sizes in the lung tissue of mice 15 days after intravenous (i.v.) injection with wild type eggs or eggs transduced with virus particles encoding shRNAmirs targeting omega-1 transcripts (omega-1 knockdown, kd). Photomicrographs taken from lesions represent mean granuloma sizes (original magnification 200) [61]. Abbreviations: CMV, cytomegalovirus promoter; dU3, deletion in the U3 region; HIV-SIN, HIV-derived lentiviral vector with self-inactivating 30 -LTR; LTR, long terminal repeat; ORF, open reading frame; RISC, RNA-induced silencing complex; miRNA*, sense strand sequence.

no effect on the vitality or maturation of larval eggs stages, and this enabled, for the first time, immunomodulatory proteins involved in egg-induced pathogenesis to be studied in vivo [61]. Specifically, experimental studies in mice showed that downregulation of omega-1 transcripts, the major Th2-driving molecule in S. mansoni eggs, related to a decrease in granuloma size. The diminished granulomatous response to eggs related to decreased infiltration of

Box 2. Outstanding questions Investigation of integration events and transgene copy numbers. MMLV integrates predominantly close to promoter regions in mammalian cells [96]. However, Rinaldi et al. [11] have shown that integration is entirely random in schistosome genomes. HIVderived lentiviruses have been shown to predominantly integrate into transcriptionally active chromatin in mammalian cells, with some preferential integration sites related to the tethering factor (LEDGF/p75) of the integrase [55,97]. However, whether this is also true for S. mansoni remains an open question that needs to be addressed in future studies. Does lentiviral transduction lead to long-term stability of the expression cassette and/or is it subject to epigenetic control? Applicability of integration-deficient lentiviruses (IDLVs)? IDLVs have been demonstrated to be latent in mammalian cells [98]. Therefore, testing the applicability of integration defective viruses is warranted. Indeed, IDLVs might prevent negative effects associated with viral integration, in other words insertional mutagenesis. How specific and durable is shRNAmir-induced gene knockdown?

effector cells, such as CD4+ T cells and B cells, but also of macrophages and dendritic cells (DCs) (Figure 4) [61]. This first successful application of lentiviral transduction to a parasitic helminth, combined with the use of the RNAi pathway via the delivery of an artificial miRNA trigger, could provide a new avenue to explore parasite–host interactions in vivo. Furthermore, lentiviral transduction could lead to the production of a range of transgenic schistosome strains containing miRNA expression cassettes. The establishment of stable gene knockdown through multiple generations of S. mansoni would provide huge scope for studying aspects of schistosome biology and schistosomiasis. Outstanding questions and issues are listed in Box 2. Concluding remarks The evaluation of an alternative method of gene perturbation and its application to the dissection of the host response in mice against S. mansoni provide exciting opportunities for future investigations. Although lentiviral delivery of artificial miRNAs was established specifically for S. mansoni [61], similar approaches could be established to explore molecular functions in other parasites. Indeed, the use of the miRNA pathway to achieve stable gene knockdown provides prospects to gain a profound understanding of the different aspects of the molecular biology of various helminths and the diseases that they cause, and has potential to support the development of new intervention strategies against diverse parasites. 7


Review Acknowledgments Funding from the National Health and Medical Research Council (NHMRC) and the Australian Research Council (ARC) is gratefully acknowledged. This project was also partially supported by a Victorian Life Sciences Computation Initiative (VLSCI; grant VR0007) on its Peak Computing Facility at the University of Melbourne, an initiative of the Victorian Government. The authors thank colleagues who contributed to the research of J.H. [61]. J.H. was a grateful recipient of Melbourne International Research Scholarships (MIRS and MIFRS) from the University of Melbourne.

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9

Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells.

Knocking Down Snrnp200 Initiates Demorphogenesis of Rod Photoreceptors in Zebrafish.

Driving DNA transposition by lentiviral protein transduction.

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage.

Lentiviral transduction of mammary epithelial cells.

Knocking down disease: a progress report on siRNA therapeutics.

Knocking down the obstacles to functional genomics data sharing.

Kinetics of lentiviral vector transduction in human CD34(+) cells.

High-throughput screening identifies compounds that enhance lentiviral transduction.

Building up and knocking down: an emerging role for epigenetics and proteasomal degradation in systems consolidation.

Gene Transfer into Pluripotent Stem Cells via Lentiviral Transduction.

Knocking down CDK4 mediates the elevation of let-7c suppressing cell growth in nasopharyngeal carcinoma.

Lentiviral vector transduction of spermatozoa as a tool for the study of early development.

TRP channels in schistosomes.

Knocking-down Meloidogyne incognita proteases by plant-delivered dsRNA has negative pleiotropic effect on nematode vigor.

Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants.

Knocking down the expression of SYF2 inhibits the proliferation of glioma cells.

Role of the mammalian target of rapamycin pathway in lentiviral vector transduction of hematopoietic stem cells.

Effect of knocking down the insulin receptor on mouse rod responses.

Knocking down 10-Formyltetrahydrofolate dehydrogenase increased oxidative stress and impeded zebrafish embryogenesis by obstructing morphogenetic movement.

Chemical communication in adult schistosomes.

Anti-Apoptotic Effects of Lentiviral Vector Transduction Promote Increased Rituximab Tolerance in Cancerous B-Cells.

In vitro rescue of FGA deletion by lentiviral transduction of an afibrinogenemic patient's hepatocytes.

Simplified production and concentration of lentiviral vectors to achieve high transduction in primary human T cells.