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1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 3E2, Canada
2 Department of Molecular Genetics, University of Toronto, Toronto, ON M5S 3E2, Canada
Correspondence
Sadhna Joshi
sadhna.joshi.sukhwal{at}utoronto.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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2 months). When the cells were precultured for 2–3 months prior to HIV-1 infection, inhibition was more prominent in cells transduced with MGIN-Rz1–7 than with HEG1-Rz1–7. Inhibition occurred at the level of viral entry, as no HIV-1 DNA could be detected. These results demonstrate that Rz1–7 confers excellent inhibition of R5-tropic HIV-1 replication at the level of entry. Therefore, we anticipate that this multimeric ribozyme will be beneficial for HIV-1 gene therapy. Two supplementary figures are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Strayer et al., 2005, Rossi et al., 2007). Strategies that inhibit viral entry are of particular interest, as they would prevent healthy cells from getting infected. HIV-1 entry is mediated by the specific interaction of the viral envelope glycoproteins with a cell-surface molecule, CD4, which serves as the primary receptor, and a chemokine receptor, either CCR5 or CXCR4, which serves as co-receptor (Berger et al., 1998). To inhibit viral entry, either the receptor or the co-receptor may be targeted. CD4 and CXCR4 cannot be downregulated because of their critical roles in immune function or cell maturation and homing (Kawabata et al., 1999; Onai et al., 2000; Zou et al., 1998). However, CCR5 is dispensable. The ccr5 gene is polymorphic and a mutant phenotype has been reported in the Caucasian population at a frequency of 1–2 % (Liu et al., 1996). The defective ccr5 gene (
32CCR5) in this population contains a 32 bp deletion. It gives rise to a truncated protein, 32CCR5, which is not expressed on the cell surface. Individuals homozygous for the defective ccr5 gene are resistant to HIV-1, although there are a few cases of X4- and R5x4-tropic HIV-1 infection in subjects with this genotype (Sheppard et al., 2002). Disease progression in heterozygotes is also slower than in individuals with normal ccr5 alleles (Dean et al., 1996; Huang et al., 1996; Michael et al., 1997). Resistance in 32CCR5 homozygotes implies that other co-receptors do not replace CCR5 for infection by R5-tropic HIV-1, which initiates transmission. Furthermore, CCR5 appears to be required for all routes of transmission, since 32CCR5 homozygous individuals among haemophiliacs and intravenous drug-users are also protected from HIV-1 transmission (Liu et al., 1996). Therefore, research has been focused on inhibiting CCR5 synthesis, cell-surface expression and function (Strayer et al., 2005; Rossi et al., 2007). The anti-CCR5 genes used to decrease co-receptor synthesis include antisense RNA, small interfering RNA (siRNA) and ribozymes.
A 653 nucleotide (nt)-long antisense RNA was designed against positions 187–839 within the CCR5 open reading frame (ORF) (Li et al., 2006). Transduced U937 cells expressing this antisense RNA showed 98 % reduction of surface CCR5 expression and conferred 55 % inhibition of R5-tropic HIV-1 (CN97001 strain; m.o.i. of 0.01) replication on day 12 post-infection (p.i.). However, expression of this antisense RNA may not be without side effects, as it possesses 87 % sequence similarity to the CCR2a and CCR2b mRNAs.
An siRNA targeting nt 554–572 within the CCR5 ORF conferred 48 % reduction of surface CCR5 expression in transfected U87 cells. It also displayed 79 % inhibition of R5-tropic HIV-1 (BaL strain; m.o.i. between 0.03 and 0.24) replication on day 2 p.i. (Martinez et al., 2002). CD4+ peripheral blood lymphocytes transduced with a lentiviral vector expressing an siRNA targeting nt 186–204 within the CCR5 ORF showed >90 % reduction of surface CCR5 expression and >95 % inhibition of R5-tropic HIV-1 replication on day 4 p.i. (Qin et al., 2003).
Ribozymes are catalytic RNAs that can be designed to recognize and cleave a specific RNA. The advantage of ribozymes over siRNA is that ribozymes do not require a cellular factor for their activity, have minimal cellular toxicity and do not induce an interferon response (Rossi, 1999; Shiota et al., 2004). Ribozyme-mediated cleavage in mammalian cells seems to occur quite efficiently (Jeang & Berkhout, 1992). A monomeric hammerhead ribozyme targeting position 23 within the CCR5 ORF is currently being evaluated as part of a triple combination gene therapy strategy in two clinical trials (Li et al., 2005; Rossi et al., 2007). Transduced PM1 cells expressing this ribozyme conferred 70 % inhibition of R5-tropic HIV-1 (BaL strain; m.o.i. of 0.02) replication on day 7 p.i. (Cagnon & Rossi, 2000). Macrophages derived from transduced CD34+ haematopoietic stem/progenitor cells showed 80 % inhibition of BaL virus (m.o.i. of 0.02) replication on day 17 p.i. (Bai et al., 2000). This ribozyme was also shown to confer a certain level of selective survival to transduced primary T cells and monocytes derived from the transduced CD34+ cells (Li et al., 2003).
Multimeric ribozymes have an increased probability of recognizing and cleaving at least one of the multiple target sites within the target mRNA. Therefore, a trimeric ribozyme was designed against nt 17, 153 and 249 within the CCR5 ORF (Bai et al., 2001). Transduced HOS.CD4/CCR5 cells expressing this trimeric ribozyme decreased CCR5 expression by 10–15 % and conferred 30 % inhibition of R5-tropic HIV-1 (BaL strain; m.o.i. of 0.001) replication on day 4 p.i. (Bai et al., 2001).
Since multimeric hammerhead ribozymes targeted against HIV-1 RNA inhibited virus replication better than monomeric ribozymes (Ramezani et al., 1997, 2002), we have developed and tested a multimeric hammerhead ribozyme that targets seven unique sites within the CCR5 mRNA. Gammaretroviral and lentiviral vectors were developed and used to express this multimeric ribozyme. Stably transduced PM1 cells were then tested for downregulation of CCR5 mRNA and surface CCR5 expression, for susceptibility to X4- and R5-tropic HIV-1 and for the absence of HIV-1 DNA.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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AAGTCCAA-3'; Rz2 target site, 5'-CGATAGGTA380CCTGGCTG-3'; Rz3 target site, 5'-CTGGCTGTC390GTCCATGC-3'; Rz4 target site, 5'-AAGAAGGTC520TTCATTAC-3'; Rz5 target site, 5'-CATACAGTC556AGTATCAA-3'; Rz6 target site, 5'-ATTGCAGTA811GCTCTAAC3'; and Rz7 target site, 5'-TAACAGGTT824GGACCAAG-3'. Cleavage sites are indicated by arrows (Fig. 1a).
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. Rz1F (containing a BamHI site), Rz2R and Rz3R (containing a ClaI site) were joined by overlapping PCR (Medina & Joshi, 1999) to yield a 189 bp product, Rz1–3. This PCR product was digested with BamHI and ClaI and cloned into the same sites in pGEM-7Zf(+) (Promega) to yield pGEM-Rz1–3. Similarly, Rz4F (containing a BamHI site), Rz5R, Rz6R and Rz7R (containing a ClaI site) were joined by overlapping PCR (Medina & Joshi, 1999) to yield a 230 bp product, Rz4–7. This PCR product was digested with BamHI and ClaI and cloned into the same sites in pGEM-7Zf(+) to yield pGEM-Rz4–7. Rz1–3 and Rz4–7 do not contain any nucleotides between individual ribozymes.
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In vitro cleavage activity of Rz1–3 and Rz4–7.
Rz1–3 and Rz4–7 RNAs were transcribed in vitro from pGEM-Rz1–3 and pGEM-Rz4–7, and the 32P-labelled target CCR5 RNA was transcribed in vitro from a plasmid designated pc.CCR5, as described by Ramezani & Joshi (1996). Cleavage reactions were performed by mixing the ribozymes and labelled CCR5 RNA at a 1 : 1 molar ratio followed by gel electrophoresis, as described by Ramezani & Joshi (1996). Briefly, Rz1–3 or Rz4–7 was mixed with the labelled CCR5 RNA in a buffer containing 40 mM Tris/HCl (pH 8.0) and 10 mM NaCl. After 5 min at 65 °C, the mixture was cooled stepwise to 37 °C; 13.3 mM MgCl2 was added and the incubation was continued for 30 min at 37 °C. The cleavage products were analysed on a 5 % polyacrylamide, 7 M urea gel.
Vector constructions.
The MGIN vector (Cheng et al., 1997) was previously modified in our laboratory to contain unique Csp45I and BglII sites downstream of the enhanced green fluorescence protein (egfp) gene (Ramezani et al., 2002). The Csp45I–BamHI fragment of pGEM-Rz1–7 was used to clone Rz1–7 into the modified MGIN vector at the Csp45I and BglII sites, to obtain MGIN-Rz1–7. The correct clone was identified and characterized by PCR and restriction enzyme analyses (Fig. 2).
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promoter; Ramezani et al., 2000]; the EcoRI–NotI fragment of MGIN (containing the egfp gene) or the same digestion fragment of MGIN-Rz1–7 (containing the egfp and Rz1–7 genes); and the NotI–SfiI fragment of the pHR'CMVLacZ vector (containing the HIV-1 3' LTR and the downstream sequences; Naldini et al., 1996). The correct clones were identified and characterized by PCR and restriction enzyme analyses (Fig. 2
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Transduction and selection of stable PM1 transductants.
PA317, a mouse packaging cell line (expressing the Moloney murine leukemia virus gag, pol and env genes; Miller & Buttimore, 1986) was transfected with the MGIN or MGIN-Rz1–7 vector, as described by Ramezani & Joshi (1996). The transfected cells were cultured for 3 weeks in medium containing 400 µg G418 ml–1 to select for stable transfectants. These cells were used to collect vector particles, which were filtered and stored at –70 °C until used.
To obtain lentiviral vector particles, a human embryonic kidney (293T) cell line was cotransfected with three plasmids: pCMV8.9 (expressing the HIV-1 Gag, Gag-Pol, Tat and Rev proteins; Ramezani & Joshi, 1996), pMD.G (expressing the VSV-G protein; Burns et al., 1993) and HEG1 or HEG1-Rz1–7, as described previously by Ramezani & Joshi (1996). The vector particles were collected on day 3 post-transfection, filtered and stored at –70 °C until used.
The MGIN, MGIN-Rz1–7, HEG1 and HEG1-Rz1–7 vector particles were used to transduce PM1 cells as described previously (Ramezani et al., 2002). The pools of green fluorescent stable PM1 transductants were sorted twice by a fluorescence activated cell sorter (FACS) and used in subsequent experiments.
PCR analysis of genomic DNA from stable PM1 transductants.
Genomic DNA was extracted from the individual pools of stable PM1 transductants, as described previously (Sambrook et al., 1989). Primers EGFP-F and MGIN-R were used to amplify the Rz1–7 gene from genomic DNA of the MGIN-Rz1–7-transduced cells (Table 2). Another primer pair, EGFP-F and HEG1-R, was used to amplify this gene from genomic DNA of the HEG1-Rz1–7-transduced cells. The endogenous β-actin gene was amplified as a control, using the β-actin-F and β-actin-R primer pair. PCRs were performed for 40 cycles (95 °C for 1 min, 56 °C for 1 min and 72 °C for 1.5 min), in a 5 µl reaction mixture containing 0.4 µM of each primer, 1xPCR buffer, 100 µM of each dNTP, 0.5 µg DNA and 2.5 Units Taq DNA polymerase. The PCR products were analysed by 2 % agarose gel electrophoresis.
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). The cDNAs were then PCR-amplified, as described above, using the primer pairs EGFP-F/MGIN-R, EGFP-F/HEG1-R, β-actin-F/β-actin-R or R5-F/R5-R. The RT-PCR products were analysed on a 2 % agarose gel.
Immunoflowcytometry analysis of stable PM1 transductants.
Transduced and untransduced PM1 cells (2x106) were washed twice with 10 ml PBS and resuspended in 250 µl PBS containing 2 % fetal calf serum (FCS) (Hyclone, USA). Anti-CCR5 mouse IgG2a
2D7 monoclonal antibody (mAb) (2.5 µl) (PharMingen) was added and the cells were incubated on ice in the dark for 30 min. The cells were then washed twice with PBS and incubated in a similar manner with 2.5 µl biotinylated goat anti-mouse IgG2a antibody (Southern Biotechnology Associates) and then with 2.5 µl allophycocyanin (APC)–streptavidin conjugate. Finally, the cells were washed twice with PBS, resuspended in 2 ml PBS containing 2 mM EDTA and 2 % FCS and analysed by flow cytometry.
HIV-1 susceptibility of stable PM1 transductants.
The pools of actively dividing stable PM1 transductants (6x105 cells in 1 ml) were each inoculated with the BaL (Gartner et al., 1986) or NL4-3 (Adachi et al., 1986) strain for 3 h at room temperature. The cells were then washed with PBS three times, suspended in 2 ml RPMI 1640 medium and cultured at 37 °C. One mililitre of each cell culture was collected every 3–4 days and replaced with 1 ml fresh medium. These aliquots were centrifuged at 500 g for 5 min; the cell pellets and supernatants were both stored at –70 °C. The supernatants of the frozen cultures were diluted as appropriate and the amount of HIV-1 p24 antigen was measured by ELISA (Beckman Coulter).
PCR analysis to detect HIV-1 DNA in HIV-infected PM1 transductants.
Genomic DNA was extracted from MGIN- and MGIN-Rz1–7-transduced frozen PM1 cell pellets from day 4 and 43 p.i. Genomic DNA was also extracted from HEG1- and HEG1-Rz1–7-transduced frozen PM1 cell pellets on day 4 and day 29 p.i. The primers T7-Tat-F and Tat-R (Table 2)were used to amplify by PCR a 424 bp region of the HIV-1 tat gene. The R5 F/R5-R primer pair was used to amplify by PCR a 465 bp region of the endogenous ccr5 gene. The PCRs were performed for 40 cycles (95 °C for 15 s, 53 °C for 1 min and 72 °C for 45 s) and the products analysed by 2 % agarose gel electrophoresis.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); these sites were not found elsewhere within the human genome. Starting from the 5' end, Rz1–7 was designed to contain Rz7, Rz6, Rz5, Rz4, Rz3, Rz2 and Rz1. Thus, individual ribozymes within this multimeric ribozyme should be able to recognize their respective target sites within the CCR5 mRNA in a collinear fashion. Each of these ribozymes was designed to contain 8 nt-long 5' and 3' flanking sequences, except for the 5' flanking sequences of Rz2, Rz4 and Rz6, and the 3' flanking sequences of Rz3, Rz5 and Rz7, which ranged between 10 and 16. Rz2 and Rz3, Rz4 and Rz5, and Rz6 and Rz7 have overlapping flanking sequences.
Rz1–3 and Rz4–7 were constructed by overlapping PCRs. In vitro cleavage reactions were performed to determine the activity of the cloned ribozymes. The expected lengths of the products resulting from cleavage of a 989 nt 32P-labelled CCR5 target RNA by Rz1–3 or Rz4–7 are shown in Fig. 1(b). The in vitro cleavage products are shown in Fig. 1(c). Rz1–3 cleaved the CCR5 RNA at three sites. Cleavage by Rz1 gave rise to 61 and 928 nt products, while cleavage by Rz2 or Rz3 resulted in 424 and 565 or 434 and 555 nt products, respectively. Cleavage by Rz1 and Rz2 or Rz3 produced 363 and 565, or 373 and 555 nt products, respectively, from the 928 nt fragment. Since Rz2 and Rz3 target sites are only 10 nt apart, their cleavage products could not be resolved from each other. Rz4–7 cleaved the CCR5 target RNA at four sites. Cleavage by Rz4 should have given rise to 564 and 425 nt products; however, these were not clearly detectable. Cleavage by Rz5 gave rise to 580 and 409 nt products. Cleavage of the 409 nt product by Rz6 or Rz7 gave rise to 275 and 134 or to 288 and 121 nt products, respectively. Cleavage by Rz6 or Rz7 yielded products that could not be resolved as they were very similar in size. These results show that Rz1–3 and Rz4–7 are active and can cleave the target RNA more than once. These ribozymes were then combined to yield Rz1–7. Rz1–7 contains the seven ribozymes with no intercalated nucleotides, except for 8 nt between Rz3 and Rz4.
Gammaretroviral and lentiviral vectors expressing Rz1–7
The mouse stem cell virus (MSCV)-based gammaretroviral vector MGIN (Cheng et al., 1997) contains the egfp gene, an internal ribosome entry site (IRES) and the neomycin phosphotransferase (neo) gene (Fig. 2). MGIN-Rz1–7 was engineered to express the Rz1–7 gene, which was cloned between the egfp gene and the IRES element. In the MGIN and MGIN-Rz1–7 vectors, the 5' LTR promoter allows constitutive expression of a bicistronic vector RNA, which permits translation of the two ORFs, EFGP and NEO.
An HIV-1-based lentiviral vector, HEG1, was designed to express the egfp gene under the control of an internal human elongation factor-1 (EF-1) promoter. The Rz1–7 gene was cloned downstream of the egfp gene in the HEG1 vector to yield HEG1-Rz1–7 (Fig. 2). In the HEG1 and HEG1-Rz1–7 vectors, the EF-1 promoter allows constitutive expression of EGFP mRNA, whereas the 5' LTR promoter allows inducible expression of vector RNA, which can also be spliced. Rz1–7 is present on all the transcripts.
Development of pools of stable PM1 transductants expressing Rz1–7
PM1 is a human CD4+ T lymphocyte-derived suspension cell line. PM1 cells were transduced with amphotropic MGIN, MGIN-Rz1–7, HEG1 and HEG1-Rz1–7 vector particles. Pools of stable PM1 transductants were sorted twice by FACS and used in subsequent experiments. The growth rates of transduced PM1 cells were comparable to those of untransduced PM1 cells.
The presence of the Rz1–7 gene was confirmed by PCR analysis of genomic DNAs isolated from various PM1 transductants using the EGFP-F/MGIN-R and EGFP-F/HEG1-R primer pairs (see Supplementary Fig. S1, available in JGV Online). These forward and reverse primers were designed to hybridize to sequences upstream and downstream of the ribozyme-cloning sites, respectively. No PCR product was detected in the untransduced samples (Supplementary Fig. S1a and b, lane 1). As expected, using the EGFP-F/MGIN-R primer pair, a 771 bp product was detected by PCR in the MGIN-transduced sample, whereas a 1083 bp product was obtained from the MGIN-Rz1–7-transduced sample when using the same primer pair (Supplementary Fig. S1a, lanes 2 and 3). Likewise, 834 and 1099 bp products were detected in the HEG1- and HEG1-Rz1–7-transduced cells, respectively, when the EGFP-F/HEG1-R primer pair was used (Supplementary Fig. S1b, lanes 2 and 3). Genomic DNAs from the untransduced and transduced cells were also analysed using the β-actin-F/β-actin-R primer pair to amplify a 474 bp region of the cellular β-actin gene (Supplementary Fig. S1a and b, lower panels).
Rz1–7 production was confirmed by RT-PCR analysis of total cellular RNA from various PM1 transductants. No RT-PCR product was obtained from untransduced cells (see Supplementary Fig, S2a and b, lane 1). RT-PCR using the EGFP-F/MGIN-R primer pair resulted in amplification of a 771 bp product from the MGIN-transduced cells and a 1083 bp product from the MGIN-Rz1–7-transduced cells (Supplementary Fig. S2a, lanes 2 and 3). RT-PCR using the EGFP-F/HEG1-R primer pair resulted in the amplification of an 834 bp product from the HEG1-transduced cells and a 1099 bp product from the HEG1-Rz1–7-transduced cells (Supplementary Fig. S2b, lanes 2 and 3). RT-PCR analysis using the β-actin-F/β-actin-R primer pair (as control) yielded the expected 353 bp product corresponding to spliced β-actin mRNA in all untransduced and transduced samples (Supplementary Fig. S2a and b, lower panels).
Rz1–7-mediated downregulation of CCR5 mRNA
Downregulation of CCR5 mRNA in MGIN-Rz1–7- and HEG1-Rz1–7-transduced cells was assessed by RT-PCR analysis. A 456 bp product, resulting from RT-PCR amplification of CCR5 mRNA, was detected in MGIN- and HEG1-transduced cells (Fig. 3a and b, lane 1). The absence of this band in MGIN-Rz1–7- and in HEG1-Rz1–7-transduced cells (Fig. 3a and b, lane 2) reveals that the CCR5 mRNA was cleaved by the multimeric ribozyme. Similar amounts of RNA were present in all samples, as shown by RT-PCR amplification of endogenous β-actin mRNA (Fig. 3, lower panels).
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2D7 mAb, followed by a biotinylated goat anti-mouse IgG2a antibody and an APC–streptavidin conjugate. The immunoflowcytometry results showed 90 and 99.6 % downregulation of surface CCR5 expression on PM1 cells expressing MGIN-Rz1–7 and HEG1-Rz1–7, respectively (Fig. 4).
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). Complete (100 %) inhibition of progeny virus production was observed up to day 87 (Fig. 5a) or 66 (Fig. 5b) p.i. in two independent experiments performed with the MGIN-Rz1–7-transduced cells challenged by the BaL strain at an m.o.i. of 0.225; the cells used in the experiment represented by Fig. 5(a) were cultured for 2–3 months prior to infection. Nearly total (99 %) inhibition of progeny virus production was observed up to day 66 p.i. when the MGIN-Rz1–7-transduced cells were challenged by the BaL strain at higher m.o.i. (0.675 and 2.025) (Fig. 5b). When HEG1-Rz1–7-transduced PM1 cells were cultured for 2–3 months and then challenged with the BaL strain at an m.o.i. of 0.225, progeny virus production was delayed (up to day 29 p.i.) and strongly diminished (80 % inhibition) up to day 60 p.i. (Fig. 5c). However, nearly total (99 %) inhibition of progeny virus production was observed up to day 66 p.i. when a fresh batch of HEG1-Rz1–7-transduced cells was challenged by the BaL strain at three different m.o.i. (0.225, 0.675 and 2.025) (Fig. 5d).
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, lanes 1, 3 and 5), as well as the untransduced and the MGIN- or HEG1-transduced PM1 cells infected with the BaL strain (Fig. 6a and b, lanes 2 and 6). No HIV-1 DNA could be detected at the two time points tested (day 4 and 43 p.i.) in the MGIN-Rz1–7-transduced PM1 cells (Fig. 6a, lane 4), whereas a faint band corresponding to this DNA was detectable by day 29 p.i. in the HEG1-Rz1–7-transduced cells (Fig. 6b, lane 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Furthermore, both of these multimeric ribozymes were shown to cleave the target RNA at more than one site. We have previously designed two other multimeric ribozymes, Rz10–14 and Rz1–14. The in vitro cleavage reactions performed with these multimeric ribozymes demonstrated clearly that Rz10, Rz11, Rz12, Rz13 and Rz14 within either Rz10–14 or Rz1–14 could cleave the target RNA equally well (Ramezani et al., 2002). Therefore, we believe that the in vitro cleavage results obtained for Rz1–3 and Rz4–7 are indicative of Rz1–7 activity. The choice of ribozyme target sites was limited by the availability of sequences that are unique to CCR5 mRNA. Some of the target sites were close to each other. As a result, Rz2 and Rz3, Rz4 and Rz5, as well as Rz6 and Rz7 contained overlapping flanking sequences, which may prevent the two ribozymes from acting simultaneously. However, this should not be a concern because a single cleavage at any of the seven ribozyme target sites is all that is required to inactivate the CCR5 mRNA.
We have used an MSCV-based gammaretroviral (MGIN) vector and an HIV-1-based lentiviral (HEG1) vector for delivery and expression of the multimeric ribozyme Rz1–7. RT-PCR analyses of total RNA extracted from PM1 cells transduced with MGIN-Rz1–7 or HEG1-Rz1–7 showed that both vectors were able to express Rz1–7 (see Supplementary Fig. S2). The Rz1–7 expressed in these cells was shown to be active, since the CCR5 mRNA (Fig. 3) and surface CCR5 co-receptor (Fig. 4) levels decreased.
As expected, high levels of progeny virus were produced when the untransduced cells, as well as MGIN-, MGIN-Rz1–7-, HEG1- or HEG1-Rz1–7-transduced PM1 cells, were challenged with the NL4-3 strain, indicating that the cells were permissive to X4-tropic HIV-1 replication. Control untransduced PM1 cells and MGIN- or HEG1-transduced cells could also be infected by the R5-tropic HIV-1 BaL strain. When the MGIN-Rz1–7- or HEG1-Rz1–7-transduced cells were challenged with the BaL strain (m.o.i. of 0.225, 0.675 and 2.025), 99–100 % inhibition of progeny virus production was observed for the duration of the experiment (2 months p.i.) (Fig. 5b, d). However, when the cells were cultured for 2–3 months prior to HIV-1 infection, inhibition of replication of the BaL strain (m.o.i. of 0.225) was more prominent when the multimeric ribozyme was expressed from the MGIN-Rz1–7 vector than from the HEG1-Rz1–7 vector. When the MGIN-Rz1–7-transduced cells were challenged with the BaL strain, 99 % inhibition of virus replication was observed compared with 80 % with the HEG1-Rz1–7-transduced cells (Fig. 5a, c). These results suggest Rz1–7 gene silencing from the HEG1 vector, but not the MGIN vector (Pannell & Ellis, 2001; Mok et al., 2007; Hawley, 1994).
PCR analyses at different time intervals confirmed that the inhibition of BaL virus replication in MGIN-Rz1–7- and HEG1-Rz1–7-transduced cells is at the level of entry, as no or very little HIV-1 DNA was detected by PCR (Fig. 6).
PM1 cells contain CXCR4, CCR1, CCR3, CCR4 and CCR5 receptors (De Clercq, 2000). In the absence of CCR5, if viral escape mutants capable of utilizing CXCR4, CCR1, CCR3 or CCR4 as co-receptor were generated, their replication should have led to progeny virus production, which was not the case. Therefore, significant inhibition of HIV-1 BaL virus replication in MGIN-Rz1–7 and HEG1-Rz1–7-transduced cells suggests that the progeny viruses produced from these cells do not correspond to escape viruses with altered tropism for CXCR4, CCR1, CCR3 or CCR4.
Based on the results obtained from in vitro cleavage activity (Fig. 1b, c), downregulation of CCR5 mRNA (Fig. 3), surface CCR5 co-receptor (Fig. 4) and inhibition of R5-tropic (but not X4-tropic) HIV-1 replication (Fig. 5) at the level of entry (Fig. 6) in PM1 cells transduced with vectors expressing Rz1–7, we feel strongly that the inhibition of virus replication observed in our experiments is due to a block at a step that is unique to entry of R5-tropic virus, namely the CCR5 co-receptor. Furthermore, the inhibition of R5-tropic HIV-1 replication observed with Rz1–7 is significantly better than a 653 nt antisense RNA targeting the CCR5 mRNA (Li et al., 2006). Therefore, we believe that the antiviral effect observed in our experiments is due to the activity of the ribozyme and not an antisense effect.
In conclusion, this study shows that Rz1–7-mediated cleavage of CCR5 mRNA results in almost complete inhibition of R5-tropic HIV-1 replication at the level of entry. Vectors expressing Rz1–7 will now be tested for inhibition of HIV-1 replication in transduced peripheral blood mononuclear cells and in the progeny of transduced CD34+ stem cells. We anticipate that this multimeric ribozyme will be beneficial for HIV-1 gene therapy.
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ACKNOWLEDGEMENTS |
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8.9 and pMD.G vectors. We are grateful to Dr A. L. Haenni for excellent scientific discussions and for critical reading of this manuscript. We also thank Dr A. Arora for the HEG1 vector construction and Dr M. Ameli for technical assistance. The following reagents were obtained through the AIDS Research and Reference Reagent Program, NIAID: HIV-1 NL4-3 and BaL strains, PM1 cell line and pc.CCR5. |
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Received 8 February 2008;
accepted 13 May 2008.
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) or with MGIN-Rz1–7 (
); the transduced cells were cultured for 2–3 months prior to HIV-1 infection. (b) Progeny virus production following HIV-1 infection of PM1 cells transduced with MGIN (open symbols) or MGIN-Rz1–7 (closed symbols) at an m.o.i. of 0.225 (circles), 0.675 (triangles) or 2.025 (squares). (c) Progeny virus production following HIV-1 infection at an m.o.i. of 0.225 of untransduced PM1 cells (x), PM1 cells transduced with HEG1 (