Titers of HIV-based Vectors Encoding shRNAs are Reduced by a Dicer-dependent Mechanism
Authors: Ananthalakshmi Poluri, Richard E. Sutton
Journal: Molecular Therapy (2007)

Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs. Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs. IntroductionViral vectors are widely used in studies of gene function and for gene transfer purposes. These vectors are also used for downregulating gene expression at a post-transcriptional level.1Olivier A Jeanson-Leh L Bouma G Compagno D Blondeau J Seye K et al.A partial down-regulation of WASP is sufficient to inhibit podosome formation in dendritic cells.Mol Ther. 2006; 13: 729-737Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar,2Szulc J Wiznerowicz M Sauvain MO Trono D Aebischer P A versatile tool for conditional gene expression and knockdown.Nat Methods. 2006; 3: 109-116Crossref PubMed Scopus (337) Google Scholar Vectors encoding short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) achieve post-transcriptional gene silencing through a Dicer-dependent pathway. When complexed with transactivation response RNA binding protein, Dicer recruits components of the RNA-induced silencing complex for assembling. Dicer then directs RNA-induced silencing complex to the mRNA complementary to the si/shRNA sequence, where the endonuclease Argonaute 2 cleaves the target mRNA.3Hammond SM Dicing and slicing—The core machinery of the RNA interference pathway.FEBS Lett. 2005; 579: 5822-5829Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar Induction of this pathway by transfected siRNAs typically remains effective for only a few cell divisions. Stably integrated vectors, including those based upon retroviral and lentiviral vectors, can constitutively express the shRNA and result in long-term silencing.HIV-based vectors are capable of transducing non-dividing cells, including hematopoietic stem cells, at high efficiency.4Miyoshi H Blomer U Takahashi M Gage FH Verma IM Development of a self-inactivating lentivirus vector.J Virol. 1998; 72: 8150-8157Crossref PubMed Google Scholar Because they are of high titer and now have an improved safety profile, these vectors are being increasingly considered for human gene therapy. Phase I clinical trials utilizing retroviral vectors targeting viral gene products have been conducted in the past,5Ranga U Woffendin C Verma S Xu L June CH Bishop DK et al.Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.Proc Natl Acad Sci USA. 1998; 95: 1201-1206Crossref PubMed Scopus (121) Google Scholar,6Woffendin C Ranga U Yang Z Xu L Nabel GJ Expression of a protective gene-prolongs survival of T cells in human immunodeficiency virus-infected patients.Proc Natl Acad Sci USA. 1996; 93: 2889-2894Crossref PubMed Scopus (172) Google Scholar,7Kohn DB Bauer G Rice CR Rothschild JC Carbonaro DA Valdez P et al.A clinical trial of retroviral-mediated transfer of a rev-responsive element decoy gene into CD34+ cells from the bone marrow of human immunodeficiency virus-1-infected children.Blood. 1999; 94: 368-371PubMed Google Scholar and vectors coding for a variety of anti-HIV genes are still being evaluated as potential therapeutic agents.8Levine BL Humeau LM Boyer J MacGregor RR Rebello T Lu XB et al.Gene transfer in humans using a conditionally replicating lentiviral vector.Proc Natl Acad Sci USA. 2006; 103: 17372-17377Crossref PubMed Scopus (412) Google ScholarMany of the anti-HIV gene products under consideration are RNA-based, such as anti-sense RNA targeting HIV envelope and the packaging signal,9Veres G Escaich S Baker J Barske C Kalfoglou C Ilves H et al.Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4+ lymphocytes.J Virol. 1996; 70: 8792-8800PubMed Google Scholar trans-activation response decoys,10Fraisier C Irvine A Wrighton C Craig R Dzierzak E High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.Gene Ther. 1998; 5: 1665-1676Crossref PubMed Scopus (29) Google Scholar ribozymes targeting the U5 region of incoming proviral RNA to prevent integration,11Sun LQ Ely JA Gerlach W Symonds G Anti-HIV ribozymes.Mol Biotechnol. 1997; 7: 241-251Crossref PubMed Scopus (20) Google Scholar and siRNAs and shRNAs targeting essential viral genes.12Lee NS Dohjima T Bauer G Li H Li MJ Ehsani A et al.Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells.Nat Biotechnol. 2002; 20: 500-505Crossref PubMed Scopus (137) Google Scholar,13Banerjea A Li MJ Leila R Lee NS Rossi J Akkina R Efficacy of lentiviral vector transduced anti-HIV-1 siRNAs in progenitor cell derived T cells and macrophages.Mol Ther. 2003; 7: S263Google Scholar A disadvantage of all RNA-based strategies (with the possible exception of anti-sense RNAs) is that small changes in the viral RNA target sequence may abolish the antiviral effects.14Das AT Brummelkamp TR Westerhout EM Vink M Madiredjo M Bernards R et al.Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition.J Virol. 2004; 78: 2601-2605Crossref PubMed Scopus (394) Google Scholar,15Boden D Pusch O Lee F Tucker L Ramratnam B Human immunodeficiency virus type 1 escape from RNA interference.J Virol. 2003; 77: 11531-11535Crossref PubMed Scopus (370) Google Scholar,16Sabariegos R Gimenez-Barcons M Tapia N Clotet B Martinez MA Sequence homology required by human immunodeficiency virus type 1 to escape from short interfering RNAs.J Virol. 2006; 80: 571-577Crossref PubMed Scopus (66) Google Scholar In order to counter this, cellular genes have also been targeted. Although the approach that involves downregulating cellular genes such as CD4 and nuclear factor-κ B has been successful, the encoded proteins are vital for normal cellular physiology, and the functional disruption of these proteins may have untoward effects on both the cell and the organism.17Surabhi RM Gaynor RB RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication.J Virol. 2002; 76: 12963-12973Crossref PubMed Scopus (185) Google Scholar,18Novina CD Murray MF Dykxhoorn DM Beresford PJ Riess J Lee SK et al.siRNA-directed inhibition of HIV-1 infection.Nat Med. 2002; 8: 681-686Crossref PubMed Scopus (735) Google ScholarAlternatively, non-essential cellular proteins may be targeted. For example, chemokine (C-C motif) receptor 5 (CCR5) is a coreceptor required for M-tropic HIV entry. The downregulation of this cell surface receptor has not been shown to have detrimental effects in macrophages in vitro. 19Bai J Gorantla S Banda N Cagnon L Rossi J Akkina R Characterization of anti-CCR5 ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse model in vivo.Mol Ther. 2000; 1: 244-254Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar,20Bai J Rossi J Akkina R Multivalent anti-CCR ribozymes for stem cell-based HIV type 1 gene therapy.AIDS Res Hum Retroviruses. 2001; 17: 385-399Crossref PubMed Scopus (39) Google Scholar Individuals who have a 32-bp homozygous deletion21Liu R Paxton WA Choe S Ceradini D Martin SR Horuk R et al.Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.Cell. 1996; 86: 367-377Abstract Full Text Full Text PDF PubMed Scopus (2549) Google Scholar in the coding sequence of CCR5 have a normal immune response and are relatively resistant to HIV infection and show slower disease progression.22Huang Y Paxton WA Wolinsky SM Neumann AU Zhang L He T et al.The role of a mutant CCR5 allele in HIV-1 transmission and disease progression.Nat Med. 1996; 2: 1240-1243Crossref PubMed Scopus (1197) Google Scholar,23Quillent C Oberlin E Braun J Rousset D Gonzalez-Canali G Metais P et al.HIV-1-resistance phenotype conferred by combination of two separate inherited mutations of CCR5 gene.Lancet. 1998; 351: 14-18Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar HIV-based vectors encoding shRNA against the viral coreceptor CCR5 have been shown to downregulate surface expression of CCR5, prevent viral entry, and reduce viral replication.24Qin XF An DS Chen IS Baltimore D Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5.Proc Natl Acad Sci USA. 2003; 100: 183-188Crossref PubMed Scopus (637) Google ScholarIn fact, retroviral and lentiviral vectors encoding both shRNAs and miRNAs have been widely used for modulating gene expression levels in order to inhibit an impressive variety of cellular and viral functions. To date, careful analyses have not been performed to determine whether the presence of the encoded shRNA affects vector production or titers. Here we demonstrate that HIV-based vectors encoding shRNAs have titer values that are 30-fold lower than those of non-shRNA encoding vectors. Co-transfection of a plasmid expressing the shRNA's cognate mRNA largely reversed this effect, as did a number of inhibitors of Dicer. Similar effects were observed using a conventional retroviral vector. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) demonstrated that, in comparison with empty vector, shRNA vectors had significantly lower vector RNA levels in the producers. Taken together, these data suggest that the shRNA acts on the RNA vector itself, but the effects can be reversed by inhibiting Dicer function. These results may have implications for the optimization of large-scale production and clinical use of RNA vectors encoding shRNAs.ResultsHIV-based vectors encoding shRNAs have lower titersWe obtained from Si-Yi Chen three shRNAs directed against CCR5 and one control against humanized red shifted green fluorescent protein (hrGFP) in the vector pSUPER25Brummelkamp TR Bernards R Agami R A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3946) Google Scholar (Figure 1a). In transient transfection assays of 293T cells, all three reduced surface expression of a co-transfected cytomegalovirus (CMV)-driven CCR5 cDNA plasmid, as judged by both transfection efficiency and mean fluorescence intensity of CCR5+ cells (Figure 1b). These shRNAs were cloned into an HIV-based vector having both long-terminal repeats (LTRs) intact, all cis -acting sequences including the central polypurine tract and, in place of nef, a bicistronic cassette encoding both enhanced green fluorescent protein (eGFP) and blasticidin S deaminase (bsd) driven by the mouse phosphoglycerate kinase promoter (Figure 1c). shRNAs in the HIV vector were as functional and specific as those in the parental pSUPER plasmid (Figure 1d).Vesicular stomatitis virus (VSV) G-pseudotyped vectors were produced by transient co-transfection of 293T cells, and the end-point was titered using eGFP epifluorescence on naive human targets. It was surprising that the titer of any vector encoding a functional shRNA was 30-fold lower than that of the empty vector control (3.0–6.0 × 103 IU/ml versus 1.0–2.1 × 105 IU/ml) (Figure 2a). Co-transfection of the producers with either CMV-driven or murine leukemia virus (MLV) LTR-driven CCR5 expression plasmid (Figure 2b) along with the shRNA vectors resulted in an increase in the shRNA-encoding vector titers tenfold. This effect was specific to the anti-CCR5 vectors, and there was no increase in the anti-hrGFP vector titer (Figure 2a). In order to verify that the increase in titer was caused by the presence of the cognate mRNA, CXCR4 expression plasmid (Figure 2b) was co-transfected into the producer cells. The resultant titers were found to be unchanged, again 30-fold lower than those of the empty vector (Figure 2a). The observed low titer of hrGFP shRNA vector in the presence of CCR5 mRNA, and also that of CCR5 shRNA vector in the presence of CXCR4 mRNA, suggests that the increase in titer depends upon the presence of cognate mRNA.Figure 2Vectors encoding short hairpin RNAs (shRNAs) have lower titers than the empty vector. (a, c) vesicular stomatitis virus (VSV) G-pseudotyped particles were produced by transient co-transfection of 293T producer cells with indicated vectors and plasmids and titered on HOS TK- cells. Enhanced green fluorescent protein (eGFP+) cells (a) or blasticidin-resistant colonies (c) were enumerated as described in the text. For both a and c, P < 0.05 when compared with the empty vector (*) and with both empty vector and shRNA vector (**). Error bars in this and subsequent figures indicate standard deviation. (b) Co-receptor expression constructs pcDNA3-CCR5, pBABE-Puro-CCR5, pBABE-PuroΔLTR-CCR5, pBABE-PuroΔ32-CCR5, and pBABE-Puro-CXCR4. Striped rectangles indicate long-terminal repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to confirm vector titer measurements and the absence of pseudotransduction, target cells exposed to the vector were also passaged into blasticidin S-containing medium. The number of blasticidin-resistant colonies correlated very well with the quantity of eGFP positive cells, indicating that either was a reliable measure of vector titer (Figure 2c).Reduced titers are correlated with a decrease in unspliced vector RNA levelsSeveral control experiments were performed in order to determine the cause of the decrease in the shRNA-encoding vector titer values. The target sequence in the anti-CCR5 shRNA-2 vector is entirely included in the deletion present in the Δ32CCR5 allele. Co-transfection of MLV vector plasmid encoding the Δ32CCR5 variant (Figure 2b) did not increase shRNA vector titers (Figure 2a). An MLV vector plasmid encoding full-length CCR5, but with the LTR deleted, failed to drive CCR5 expression (Figure 2b). Also, co-transfection of the ΔLTR-CCR5 plasmid did not increase the shRNA vector titer (Figure 2a). Flow cytometry was performed on the producer cells to determine both the percentage of transfection efficiency and the mean fluorescence intensity of eGFP+ cells after each vector transfection. The cells were equally transfected in all cases, with a transfection efficiency of >90% and approximately equivalent mean fluorescence intensities of the eGFP+ cells (Figure 3a).Figure 3Short hairpin RNA (shRNA) vectors reduce the level of vector RNA. (a) Flow-cytometric analysis of transfected cells indicating the percentage of enhanced green fluorescent protein (eGFP+) producer 293T cells. The mean fluorescence intensity (MFI) of eGFP+ cells is indicated at the top of each bar. (b) Transfected cells were lysed, and the detergent lysates were immunoblotted using either anti-HIV or anti-actin serum; the different HIV Gag proteins and actin are indicated by arrows. Protein (10, 20, and 30 μg) from cell lysates (wedge) were used for detecting HIV structural proteins and actin, and supernatant (2.5, 5, and 7.5 μl) (wedge) was used for detecting CA. Boxed areas indicate that the autoradiograph is a composite image from several different gels. (c) Total RNA from transfected cells was used for reverse transcriptase–polymerase chain reaction (RT-PCR). Size-separated DNA products were Southern-blotted using radioactive probes specific for, bsd and β-actin. Boxed areas indicate that the autoradiograph is a composite image, with some intervening lanes removed. (d) Quantitative RT-PCR for vector transcripts was performed using SYBR-green and the primers indicated in Figure 2a, as described in the text. The decrease in HIV-based shRNA vector transcripts relative to the empty vector is indicated as a multiple. P < 0.05 when compared with empty vector (*) and with both empty vector and shRNA vector (**).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The levels of HIV structural proteins expressed from the packaging vector were determined using Western blot analysis of the transfected cell lysates, with actin as a loading control. Similar amounts of Gag p55 and Capsid p24 were detected in all cases, independent of the transfer vector, indicating that the lower titers of the shRNA vectors were unlikely to be caused by decreased levels of the structural proteins required for pseudotyped particle formation. Analysis of CA in vector supernatants by immunoblot also revealed roughly equivalent amounts, further confirming that the reduction in observed titers was not a result of decreased levels of structural proteins (Figure 3b).In order to determine RNA levels in the producers RT-PCR, followed by either ethidium bromide staining or Southern blotting, demonstrated that levels of cellular β-actin and the vector's internal promoter-driven bsd transcripts were unaffected by the presence of the shRNA vector (Figure 3c). Reverse transcription followed by real-time PCR was used for determining whether there were lower levels of unspliced vector RNA in the presence of vector-encoded shRNA. With β-actin used as an internal control, real-time quantitation assays showed that unspliced viral RNA levels closely mirrored the titer reduction seen for shRNA vectors relative to non-shRNA vectors (Figure 3d). Empty vector RNA levels were ∼18-fold higher than those of the anti-CCR5 shRNA vector. In the presence of CCR5 expression plasmid, the shRNA vector RNA levels increased significantly and were only approximately threefold lower than those of empty vector. This shows that co-transfection of CCR5 expression plasmid caused a sixfold increase in the anti-CCR5 shRNA vector RNA levels.Decrease in titer is independent of shRNA target or position in the vectorIn order to address the question of whether the low titers of shRNA vectors were limited to a particular shRNA, an HIV vector encoding shRNA targeting human cyclin E1 (hCycE) was constructed (Figure 4a). Pseudotyped particles were produced by co-transfecting producer cells with or without hCycE expression construct. Anti-hCycE shRNA vector had a lower titer than empty vector, but this was partly reversed by co-transfecting with hCycE expression plasmid (Figure 4b). This suggests that functional shRNA-encoding HIV vector titers are reduced, irrespective of the target. The titers of HIV vectors encoding shRNA against cyclin T1 (cycT1), however, were only twofold lower than those of the empty vector (data not shown). It is likely that the more moderate reduction observed in this case was caused by high levels of expression of endogenous cycT1 in the producer 293T cells.Figure 4The decrease in titer is independent of short hairpin RNA (shRNA) target, position, and orientation but dependent on vector context. (a) Vector pHIV-H1-Cyclin-E-shRNA-PIGIB with cassette consisting of H1-promoter driving expression of anti-Cyclin E shRNA inserted into empty vector at the Hpa I site. (b) eGFP+ titers of pHIV-H1-Cyclin-E-shRNA-PIGIB on HOS TK- cells. P < 0.05 when compared with empty vector (*) and with both empty vector and shRNA vector (**). Error bars indicate SD. (c) pSUPER plasmids encoding anti-CCR5 shRNAs were individually co-transfected along with the empty vector into 293T producer cells, and the resultant pseudotyped particles were titered on target cells using eGFP expression. (d) Structures of pHIV-CCR5-shRNA-3-H1-PIGIB, pHIV-5′H1-CCR5-shRNA-3-PIGIB, pHIV-5′CCR5-shRNA-3-H1-PIGIB, pHIV-hrGFP-shRNA-H1-PIGIB, pHIV-5′H1-hrGFP-shRNA-PIGIB, and pHIV-5′hrGFP-shRNA-H1-PIGIB. End-point eGFP titers were determined on HOS TK- cells using epifluoresence microscopy. P < 0.05 when compared with shRNA vector (**). (e) Structure of pHIV-CCR5shRNA-PIGIB and end-point eGFP titers, as determined on HOS-TK- cells by fluorescence-activated cell sorting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to confirm that the reduction in titer is dependent upon the shRNA being encoded within the vector and not due to stimulation of the interferon pathway, we co-transfected empty vector in trans with various shRNAs encoded in the pSUPER plasmid. These included the three different shRNAs targeting CCR5 and one targeting hrGFP. Co-transfection of the pSUPER plasmids had no effect on vector titers (Figure 4c), thereby indicating that the shRNA had to be encoded within the vector proper in order to reduce titers.Each of the HIV-based vectors has a total of three promoters: the HIV LTR, the phosphoglycerate kinase promoter, and the H1 promoter (Figure 2a). The vector-encoded shRNAs are under H1-promoter regulation, and therefore RNA polymerase-III-dependent. There was the remaining possibility that the combination, orientation, or placement of promoters within the vector might be affecting shRNA expression or titer. The titers were therefore analyzed after varying the H1 promoter position and orientation within HIV vectors encoding shRNA against CCR5 or hrGFP (Figure 4d). Regardless of shRNA cassette position or orientation, titers of shRNA vectors were consistently 30-fold lower than those of empty vector (Figure 4d), and anti-CCR5 shRNA vector titers continued to increase tenfold when co-transfected with pCMV-CCR5.Removal of the H1 promoter should arrest the production of shRNAs, thus resulting in a higher vector titer. In order to test this, an HIV vector was constructed consisting of only anti-CCR5 shRNA in the absence of H1 promoter, and the titers were determined by end-point dilution (Figure 4e). The titer values of empty vectors (1.8 × 105 IU/ml) were less than twofold greater than promoter-less CCR5 shRNA vector (1.1 × 105 IU/ml), as measured by eGFP fluorescence. These results provide further evidence that pol III-transcribed shRNAs encoded by HIV vectors are capable of decreasing vector titers.Low titer of HIV vectors encoding shRNAs is Dicer-dependentWe anticipated that inhibition of Dicer function would increase vector RNA levels and titers. Three inhibitors of Dicer were therefore tested: Nodamura-virus B2 protein (NoV-B2),26Sullivan CS Ganem D A virus-encoded inhibitor that blocks RNA interference in mammalian cells.J Virol. 2005; 79: 7371-7379Crossref PubMed Scopus (138) Google Scholar Adenovirus VA.1 RNA,27Lu SH Cullen BR Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and microRNA biogenesis.J Virol. 2004; 78: 12868-12876Crossref PubMed Scopus (299) Google Scholar,29Andersson MG Haasnoot PCJ Xu N Berenjian S Berkhout B Akusjarvi G Suppression of RNA interference by adenovirus virus-associated RNA.J Virol. 2005; 79: 9556-9565Crossref PubMed Scopus (275) Google Scholar and siRNA directed against Dicer itself.30Li HW Li WX Ding SW Induction and suppression of RNA silencing by an animal virus.Science. 2002; 296: 1319-1321Crossref PubMed Scopus (698) Google Scholar When NoV-B2 expression plasmid was co-transfected into producer cells along with HIV vectors encoding shRNA against either CCR5 or hrGFP, vector titers increased tenfold, whereas the titer value of the empty vector remained unaffected (Figure 5a). VA.1 RNA also inhibits Dicer by blocking its ability to process shRNAs and pre-miRNAs.28Sano M Kato Y Taira K Sequence-specific interference by small RNAs derived from adenovirus VAI RNA.FEBS Lett. 2006; 580: 1553-1564Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Co-transfection with a plasmid encoding Adenovirus VA.1 RNA (but not control plasmid) improved the titers of vectors encoding either anti-CCR5 or hrGFP shRNA, again without affecting the titer of the empty vector (Figure 5a).Figure 5Low titers of HIV short hairpin RNA (shRNA) vectors are partially reversed by Dicer inhibitors. (a) Plasmids encoding NoV-B2 protein, Adenovirus VA.1 RNA, or control plasmids were co-transfected as indicated, and the resultant pseudotyped particles were titered by end-point dilution on HOS TK- cells. (b) Anti-Dicer or scrambled small interfering RNA (siRNA) and HIV-based shRNA vectors were co-transfected into 293T cells. (c) Dicer inhibitors were transfected in combination as indicated. (d) Quantitative RT-PCR for vector transcripts was performed using SYBR-green and the primers indicated in Figures 5a and 5c, as described in text. The decrease in HIV-based shRNA vector transcripts relative to the empty vector is indicated as a multiple. For (a–d) P < 0.05 when compared with empty vector (*) and with both empty vector and shRNA vector (**).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to directly inhibit Dicer 293T producers were transfected with anti-Dicer siRNA. This increased the shRNA-encoding vector titers 20-fold, whereas there was no discernible effect of the scrambled siRNA (Figure 5b). The greater increase in titer observed after the use of anti-Dicer siRNA relative to either NoV-B2 or VA.1 RNA DNA plasmid co-transfection may be a result of either a more immediate or direct action of the siRNA or a higher co-transfection efficiency. Cognate mRNA expression plasmid, NoV-B2, VA.1 RNA, and Dicer siRNA were transfected in combination together with shRNA-encoding vector into the 293T cells. This resulted in shRNA vector titers that were approximately twofold lower than those of empty vectors (9.8–11 × 104 IU/ml versus 2.2 × 105 IU/ml (Figure 5c). Quantitative real-time RT-PCR analysis showed that in the presence of NoVB2 protein and VA.1 RNA the CCR5 shRNA vector RNA level increased sixfold (Figure 5d). In the presence of anti-Dicer siRNAs the shRNA vector RNA level was decreased only twofold compared to that of the empty vector (Figure 5d).Retroviral shRNA vectors also have reduced titersAn MLV-based vector pBABE-Bleo-H1-CCR5-shRNA-1 encoding anti-CCR5 shRNA-1 under the regulation of the pol III promoter was constructed (Figure 6a). The titer of VSV G-pseudotyped empty MLV vector pBABE-Bleo was ∼2.0 × 105 IU/ml. The titer of the MLV vector encoding anti-CCR5 shRNA was ∼20-fold lower, ∼1.1 × 104 IU/ml. Co-transfection of the 293T producers with CMV promoter-regulated CCR5 expression plasmid increased the shRNA vector titer 15-fold, to ∼1.6 × 105 IU/ml (Figure 6b), which was statistically significant.Figure 6Retroviral short hairpin RNA (shRNA) vector has low titers. (a) Schematic of murine leukemia virus (MLV)-based vectors encoding bleomycin resistance (pBABE-Bleo, top) and H1 promoter regulating chemokine (C-C motif) receptor 5 (CCR5) shRNA (pBABE-Bleo-H1-CCR5 shRNA-1, bottom). Striped rectangles represent the MLV LTRs. (b) VSV G-pseudotyped vectors were produced in the presence or absence of C