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. 2014 Nov 25;23(2):310–320. doi: 10.1038/mt.2014.205

Multiplexing Seven miRNA-Based shRNAs to Suppress HIV Replication

Jang-Gi Choi 1, Preeti Bharaj 1, Sojan Abraham 1, Hongming Ma 1, Guohua Yi 1, Chunting Ye 1, Ying Dang 1, N Manjunath 1, Haoquan Wu 1,*, Premlata Shankar 1,*
PMCID: PMC4445613  PMID: 25358251

Abstract

Multiplexed miRNA-based shRNAs (shRNA-miRs) could have wide potential to simultaneously suppress multiple genes. Here, we describe a simple strategy to express a large number of shRNA-miRs using minimal flanking sequences from multiple endogenous miRNAs. We found that a sequence of 30 nucleotides flanking the miRNA duplex was sufficient for efficient processing of shRNA-miRs. We inserted multiple shRNAs in tandem, each containing minimal flanking sequence from a different miRNA. Deep sequencing of transfected cells showed accurate processing of individual shRNA-miRs and that their expression did not decrease with the distance from the promoter. Moreover, each shRNA was as functionally competent as its singly expressed counterpart. We used this system to express one shRNA-miR targeting CCR5 and six shRNA-miRs targeting the HIV-1 genome. The lentiviral construct was pseudotyped with HIV-1 envelope to allow transduction of both resting and activated primary CD4 T cells. Unlike one shRNA-miR, the seven shRNA-miR transduced T cells nearly abrogated HIV-1 infection in vitro. Additionally, when PBMCs from HIV-1 seropositive individuals were transduced and transplanted into NOD/SCID/IL-2R γc−/− mice (Hu-PBL model) efficient suppression of endogenous HIV-1 replication with restoration of CD4 T cell counts was observed. Thus, our multiplexed shRNA appears to provide a promising gene therapeutic approach for HIV-1 infection.

Introduction

Following successful clinical trials, there has been a resurgence of interest in using RNA interference (RNAi) to treat many diseases including cancer and HIV-1 infection.1 It has been well documented that short hairpin RNAs (shRNA) can be used to inhibit HIV-1 replication.2,3,4 However, one potential problem of shRNAs expressed under the commonly used Pol III promoters is that the overexpressed shRNA can cause toxicities by competing with endogenous miRNAs for cytoplasmic transport.5,6,7 Moreover, shRNAs in the cytoplasm that are processed by Dicer can also bind all Ago proteins, further compromising endogenous miRNA function.8 Several studies suggest that expression of shRNAs in an endogenous miRNA backbone (shRNA-miRs) can avoid the toxicities caused by saturation of RNAi machinery.6,9 For this purpose, generally shRNAs containing pri-miRNA processing signals are expressed under Pol III or Pol II promoters, where the shRNA is processed like endogenous miRNA, avoiding potential toxicities.9,10 Even so, a major concern in using any shRNA, including shRNA-miR for HIV-1 therapy is the rapid emergence of escape mutations since HIV-1 evolves very quickly due to highly error-prone reverse transcription.11,12 This problem can be minimized by using multiple shRNAs targeting host factors and viral genes.13 Therefore, it is important to be able to express multiple shRNAs in a single vector. However, attempts to express multiple shRNAs using tandem repeats of the same promoter were unsuccessful because the cassettes were prone for deletion due to homologous recombination.14 Similarly, expression of multiple shRNA-miRs using the same miRNA backbone is also likely to lead to deletion by homologous recombination. Thus, multiple shRNA-miRs have been expressed using the naturally occurring polycistronic miRNA clusters.15,16,17 However, currently the maximum number of shRNA-miRs that can be expressed in a polycistronic miRNA backbone is four.15 Although mathematical modeling suggests that a combination of four shRNAs may be sufficient to overcome escape, this requires all four shRNAs to be matched to each of the circulating 100's of viral variants and the viral quasispecies present in patients.18 However, if seven shRNAs are simultaneously expressed, it could cover nearly all HIV-1 strains by ensuring that at least four shRNAs are active against any given viral strain.18

miRNAs are transcribed as long (up to several kb) primary miRNAs (Pri-miRNAs) containing the miRNA duplex with long 5′ and 3′ flanking sequences.19,20 Pri-miRNAs are processed in the nucleus by Drosha/DGCR8 complex to generate pre-miRNAs,21,22 which are then exported to the cytoplasm for further processing by Dicer to generate mature miRNAs.23,24 Generally, long stretches of flanking sequences are used to design shRNA-miRs,25 in the hope that it will lead to processing by Drosha/DGCR8, just like primary miRNAs. Multiplexing shRNAs with different miRNA backbones by this method is impractical. However, recent knowledge of pri-miRNA processing in vitro and in vivo suggests that Drosha recognizes only a short stretch of flanking sequences for processing.26,27 Whether including just this stretch of flanking sequences in a shRNA-miR construct is enough for effective Drosha processing is not known. If indeed this is true, multiple shRNA-miRs can be easily expressed in a single vector via tandem repeats of different miRNA-based shRNA-miR expression cassettes.

Delivery of shRNA to the relevant (HIV-1 susceptible) target cells in vivo remains a great challenge. It is a common strategy to deliver shRNA via a VSV-G pseudotyped lentiviral vector because the VSV receptor is ubiquitously expressed in most cell types. VSV-G pseudotyping allows transduction of many different cell types. However, CD4 T cells, the major targets of HIV-1, can be transduced by VSV-G pseudotyped lentivirus only after activation, which could impact their repertoire and long-term survival.28,29 On the other hand, pseudotyping the lentiviral vector with HIV-1 envelope rather than VSV-G permits effective transduction of resting CD4 T cells and also ensures preferential targeting of HIV-1 susceptible cells.28,29

In this study, we have determined the minimal flanking sequence necessary for processing of shRNA-miRs. Using this information, we designed a common platform to express seven shRNA-miRs targeting CCR5 and six regions in the viral genome. We used HIV-1 pseudotyped lentiviral vector to deliver multiplexed shRNA-miRs to both resting and activated CD4 T cells to confer HIV-1 resistance in vitro and in vivo in a Hu-PBL mouse model.

Results

Design of multiplexed shRNA-miRs

The essential difference between conventional shRNA and shRNA-miR is that while the former resembles pre-miRNA that is processed by Dicer in the cytoplasm, the latter resembles pri-miRNA that is first processed by Drosha/DGCR8 in the nucleus.22,23,24 Although based on in vitro processing of select miRNAs it has been suggested that Drosha cuts the pri-miRNA ~11 nt from the lower stem-single stranded RNA (ssRNA) junction, this may not always hold true for all miRNAs, particularly in vivo.27 Moreover, whether including just these flanking sequences will enable pri-miRNA processing is not known. Thus, we initially wished to define the minimal flanking sequences required for processing of different miRNAs. Starting with miR-30a backbone with 150-nt flanking sequences, we shortened the flanking sequence length to 30, 20, and 15 nt and tested the impact on the functionality of the miRNA. For this, we cotransfected plasmids expressing miRNA with different flank lengths along with psiCHECK vector containing the relevant target site in the 3′ UTR of Rluc and measured activity by dual luciferase assay 24 hours later. Figure 1a shows that the functionality of pri-miR-30a with 150-nt flanking sequence is similar to that with 30- or 20-nt flanking sequence, while a 15-nt flanking sequence slightly diminishes functionality. We also tested the functionality of pri-miR-150 with 60- or 30-, 20- or 15-nt flanking sequences and did not detect any difference between 60- and 30-nt flanks (Figure 1b), suggesting that a 30-nt flanking sequence is long enough to ensure proper Drosha processing to retain the full functionality of miRNA. Thus, we decided to use 30-nt flanks for all miRNA backbones.

Figure 1.

Figure 1

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Optimized design of multiplexed shRNA-miRs. Plasmids encoding (a) pri-miR-30 or (b) pri-miR-150 containing indicated lengths of flanking sequence (10, 50, and 100 ng) were cotransfected with psiCHECK-2 vector (100 ng) harboring the target sequence in the 3′ UTR of the Renilla luciferase gene and gene silencing was tested by dual luciferase assay 24 hours later. Bar graphs represent mean + SD of triplicates. (c) Schematic of single and multiplexed shRNA-miR designed to express indicated 1, 2, 4, and 7 shRNA-miRs. “X” represents restriction enzyme sites. (d) Schematic showing the position of shRNA-miR target sites in the HIV-1 genome.

Multiplexing seven shRNA-miRs into a single construct without decreasing functionality of individual shRNAs

Multiple shRNAs cannot be expressed using tandem repeats of the same miRNA backbone because homologous recombination at the flanking sequences is likely to eliminate the insert. Therefore, we designed an artificial miRNA cluster to multiplex shRNA-miRs under different miRNA backbones. First, we determined that tandem expression of two shRNA-miRs with or without a spacer sequence between them did not affect the shRNA functionality, indicating that two tandem miRNAs can be directly combined (data not shown). Thus, we expressed different numbers (one, two, four, and seven) of shRNA-miRs, each with a different miRNA backbone (without any spacer sequence between the individual miRNAs) under the control of EF-1 α promoter, as illustrated in Figure 1c. The shRNA sequences were picked from previous studies showing the efficacy of conventional shRNAs targeting the CCR5 gene and highly conserved regions in the HIV-1 genome.2,30,31,32,33

We first tested if the shRNA-miRs in the multiplex are correctly processed and whether their expression is affected by the position of each individual shRNA-miR with respect to the promoter. siRNAs produced from one, two, four, or seven shRNA-miRs were determined using deep sequencing. For this, 293 T cells were transfected with either of the one, two, four, or seven shRNA-miR expression constructs and small RNAs isolated 48 hours later were cloned and deep sequenced. siRNAs were detected for each unit in all the constructs. Moreover, analysis of the dominant reads show accurate and predicted processing of six/seven shRNAs (Supplementary Figure S1). Only in one (Env), there were two forms, one as predicted, but with one  bp shift. However, even with some of the endogenous miRNAs, alternative processing is known to occur. Thus, the siRNAs generated appears to be the right ones.

Analysis of the read number frequencies revealed that the expression of individual shRNA-miRs in the multiplex is not affected by the position of that shRNA-miR with respect to the promoter. Although the small RNA reads showed three- to eightfold lower from the seven shRNA-miR construct as compared to one shRNA-miR (Supplementary Figure S2 and Table S1), the reads of all siRNAs in the construct (CCR5, Gag, Env, Tat, Pol1, Pol2, and Vif) were significantly higher than one of the most abundant endogenous miRNA, miR-16. Thus, the level of each of the seven shRNAs appears to be adequate to suppress all targeted genes.

We next tested individual shRNA-miR functionality in the multiplex. For this, we cotransfected plasmids expressing single or multiple shRNA-miRs along with psiCHECK vector containing relevant shRNA-miR target sites in the 3′ UTR of Rluc and measured activity by dual luciferase assay 24 hours later. All individual shRNA-miRs within all multiplexed vectors (expressing two, four, or seven shRNA-miRs) showed similar functionality compared to plasmids expressing only the individual shRNA-miRs (Figure 2a). This was seen at all different concentration of plasmids used for transfection. We also evaluated the toxicity of single, dual, and multiple shRNA-miR constructs. Transfection of Jurkat cells with multiple shRNA-miR expression construct was not toxic to the cells as determined by annexin V staining (Figure 2b).

Figure 2.

Figure 2

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Each shRNA-miR expressed from the multiplexed shRNA-miRs construct is as functionally competent as singly expressed shRNA-miRs. (a) Plasmids encoding indicated individual or multiplexed shRNA-miRs (2 shRNA = CCR5 and Vif; 4 shRNAs = CCR5, Gag, Pol 1 and Vif; 7 shRNAs = CCR5, Gag, Env, Tat, Pol 1, Pol 2, and Vif) were cotransfected (10, 50, and 100 ng) with psiCHECK vector (100 ng) containing shRNA-miR target sequences in the 3′ UTR of R-luc into 293 FT cells. Gene silencing was determined by dual luciferase assay performed 24 hours after cotransfection. The ratio of renilla luciferase (Rluc, reporter) to firefly luciferase (Fluc, internal control), normalized to the negative control mCherry vector expressing no shRNA-miR transfection is shown. The experiments were performed in triplicate. (b) Jurkat cells were transfected with single, dual, or multiple shRNA-miR encoding constructs and assayed for apoptosis by Annexin V staining. Staurosporine was used as positive control. (c) Indicated shRNA-miR expression vector-transduced PBMCs were subjected to MTS assay 2 and 4 days after transduction. Bar graphs represent mean + SD of triplicates.

We next cloned the different shRNA-miR expression cassettes (expressing one, two, four, and seven shRNAs) in a lentiviral vector that also expresses mCherry or Zs Green as markers. The vectors were pseudotyped with HIV-1 envelope and the resultant lentivirus was used for transduction of primary CD4 T cells. The lentiviral titer with multiplexed shRNA-miRs was around tenfold lower than one shRNA-miRs (data not shown), but still sufficient to perform experiments after concentration by ultracentrifugation.

To monitor toxicity, we cultured the cells for two, four, and seven days after lentivirus transduction and followed the percentage of mCherry+ cells by flow cytometry. Although mCherry expression levels varied between different constructs, the expression levels did not decline with time for any of the constructs (Supplementary Figure S3). We also tested for cell viability of the transduced CD4 T cells by the MTS assay on days 2 and 4 after transduction. We did not observe a decrease in the viability of transduced cells (Figure 2c). Thus, these data indicate that single and multiple shRNA-miR constructs were stable and had no obvious adverse effects on cell viability in vitro.

Functionality of shRNA-miRs targeting CCR5 and multiple HIV-1 genes in cell lines

The ability of single, dual, and multiple shRNA-miR constructs to knockdown CCR5 was tested in the TZM-bl cells. Cells were transduced with lentiviruses encoding two, four, and seven shRNA-miRs and after 48 hours, CCR5 expression was tested by flow cytometry. As shown in Figure 3a, all three constructs could effectively silence CCR5 expression as compared to cells transduced with lentivirus expressing mCherry alone. To test the efficacy of antiviral shRNA-miRs, we cotransfected 293 T cells with X4 tropic HIV-1 molecular clone pNL4-3 plasmid along with two, four, or seven shRNA-miR encoding vectors. Culture supernatants obtained 48 hours later were tested for viral replication by infecting TZM-bl cells encoding Tat-dependent luciferase. While as expected, supernatants from CCR5 shRNA-miR transfected cells had no effect on viral levels compared to control, that from antiviral shRNA treated samples showed increasingly (seven shRNA-miR<four shRNA-miR<two shRNA-miR) lower levels of infectivity, suggesting antiviral shRNA-miRs were also functional (Figure 3b). To evaluate the antiviral effect of CCR5 and antiviral shRNA-miRs in the context of lentivirus transduction, TZM-bl cells were transduced with HIV-1-pseudotyped lentiviruses encoding one, two, four, or seven shRNA-miRs and 24 hours later infected with either X4-tropic NL4-3 or R5 tropic HIV-1Bal at an MOI of 0.01. p24 levels in culture supernatants were tested 9 days after infection. Again, CCR5 shRNA-miR transduction had no effect on X4 tropic virus, but inhibited R5 tropic virus by 50% and antiviral shRNA-miRs showed increasing levels of inhibition, with seven shRNA-miR virtually abrogating infection with both X4 tropic and R5 tropic viruses (Figure 3c). Thus, our artificial miRNA clusters appears to be capable of expressing multiple functional shRNA-miRs and therefore can serve as good candidates for anti-HIV-1 therapy.

Figure 3.

Figure 3

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Multiplexed shRNA-miR silences CCR5 and inhibits HIV replication in cell lines. (a) TZM-bl cells were transduced with lentivirus encoding CCR5 shRNA-miR only or multiplexed shRNA-miRs and CCR5 expression assayed 48 hours later by flow cytometry. (b) 293 T cells were cotransfected with pNL4-3 plasmid together with plasmids encoding no shRNA-miR (mock), CCR5 shRNA-miR only or multiplexed shRNA-miRs. Supernatants collected 48 hours later were used to infect TZM-bl cells and after another 48 hours, cell lysates were analyzed for luciferase activity. (c) TZM-bl cells were transduced with HIV-1 env pseudotyped lentivirus encoding indicated shRNA-miRs and infected with HIV-1NL4-3 (left) and HIV-1BaL (right) at an MOI of 0.01. p24 antigen level in culture supernatants obtained 9 days after infection was measured by ELISA.

Resting T cells can be effectively transduced with HIV-1-env pseudotyped shRNA-miR expressing lentivirus

Having established that multiplexed shRNA-miRs are functional, we next tested the efficacy of CCR5 only versus CCR5 + six antiviral shRNA-miRs to resist HIV-1 infection in activated and resting primary CD4 T cells. Our goal was to develop a method to confer HIV-1 resistance to all T cells, regardless of activation status. Thus, we first tested the transduction efficiency of VSV-G and HIV-1 env-pseudotyped lentiviruses expressing mCherry. Primary CD4 T cells were transduced either before or after activation with PHA for 2 days and examined for mCherry expression 2 days after transduction. Flow cytometric analysis revealed that while both VSV-G and HIV-1 envelope-pseudotyped viruses transduced activated T cells with equal efficiency, effective transduction of resting T cell could be achieved only with the latter (Supplementary Figure S4). Thus, we used the HIV-1 envelope pseudotyped virus for testing antiviral efficacy. To evaluate the shRNA-miRs, we transduced resting or activated CD4 T cells with HIV-1 pseudotyped lentiviruses expressing no shRNA-miR, CCR5 shRNA-miR alone, or CCR5 + six antiviral shRNA-miRs. In this experiment, GFP-expressing lentivirus was used to track the transduced cells. After 48 hours, the cultures were infected with HIV-1 strains, NL4-3 or Bal (transduced resting T cells were activated with PHA before infection). Culture supernatants harvested on days 0, 3, 9, and 15 after infection were tested for released virus levels by p24 ELISA. The transduction levels were comparable between the different lentiviral constructs in both activated and resting CD4 T cells (Figure 4a,b). Again, similar to results obtained for TZM-bl cells, while CCR5 shRNA-miR was effective only against R5 virus, the seven shRNA-miR expressing lentivirus was able to nearly abrogate infection of both X4 and R5 tropic viruses in both activated and resting T cells (Figure 4a,b). Moreover, GFP expressing cells increased over time in the protected CCR5 shRNA-miR and seven shRNA-miR transduced cultures. The high enrichment of viable GFP-positive cells and the well preserved cell viability at the end of the experiment on day 15 points to a clear survival advantage of the shRNA-miR transduced cells following HIV-1 infection (Figure 4, Supplementary Figure S5). We conclude that seven shRNA-miR expressing lentivirus, pseudotyped with HIV-1 envelope provides a means to confer HIV-1 resistance in primary CD4 T cells.

Figure 4.

Figure 4

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HIV-1 env pseudotyped lentivirus encoding multiplexed shRNA-miRs transduces and protects both resting and activated T cells against HIV infection. (a) Activated and (b) resting T cells were transduced with HIV-1 env-pseudotyped lentivirus encoding no shRNA-miR (mock), CCR5 only shRNA-miR or 7 shRNA-miRs (transduction efficiency in terms of GFP expression is shown below the schematic of treatment), infected with HIV-1NL4-3 (left panels) or HIV-1Bal (right panels) and p24 antigen levels in culture supernatants were measured by ELISA on days 0, 3, 9, and 15 after infection (top panels). Cells were analyzed by flow cytometry for GFP expression on days 0, 3, 9, and 15 after infection (bottom panels).

Inhibition of HIV-1 replication in HIV-seropositive donor PBMCs transduced with HIV-1 env-pseudotyped seven shRNA-miR encoding lentivirus

Our previous results showed the effectiveness of shRNA-miRs to prevent infection. Next, we tested if we can inhibit the replication of HIV-1 from already infected cells. For this, PBMC obtained from HIV-1 seropositive individuals, either treatment naïve or on ART were used. Since in this case, we are mainly relying on antiviral shRNA-miRs, shRNA-miR targeting Tat only or seven shRNA-miRs were used to test efficacy. First, we made sure that the transduction efficacy of all the vectors to be similar by determining mCherry expression by flow cytometry (data not shown). To test shRNA efficacy, PBMCs from HIV-1 seropositive donors were depleted of CD8 T cells, transduced with shRNA-miR encoding lentiviruses and activated the next day (Figure 5a). The cells were cultured for 35 days and p24 antigen levels determined on days 3, 6, 18, and 35. As shown in Figure 5b, in the control lentivirus (expressing no shRNA-miR) transduced cells, HIV-1 replication showed a peak on day 18 and declined by day 35, presumably due to depletion of CD4 T cells by infection. In contrast, in the seven shRNA-miR transduced cultures, HIV-1 replication by day 18 was dramatically reduced in all 4 donor PMBCs and by day 35, p24 was virtually undetectable, even though the cultures contained healthy looking cells. In the Tat shRNA-miR only transduced cells, at day 18, HIV-1 replication was reduced in all four donors PBMCs. However, at later time points, Tat shRNA-miR transduced cell cultures showed increased HIV-1 replication (Figure 5b), indicating either inadequacy of Tat shRNA-miR antiviral activity or emergence of escape mutants. We tested for the presence of HIV-1 escape mutants by sequencing Tat shRNA-miR target regions in the virus present in the supernatants. Of the four donors, cultures from two showed emergence of viral escape mutants. Donor 2 had a mutation at nucleotide position 18 and donor 4 had mutation at position 4 of the Tat-targeted sequence (Figure 5c). In the seven shRNA-miR treated cultures, we could not recover virus to perform similar studies.

Figure 5.

Figure 5

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Inhibition of HIV-1 replication in HIV-seropositive donor PBMCs transduced with HIV-1 env-pseudotyped lentiviral vector expressing multiplexed shRNA-miRs. (a) Time course of treatment and testing. (b) PBMCs from four different HIV-1 seropositive donors were transduced with HIV-1 env-pseudotyped lentivirus encoding no shRNA-miR (mock), Tat shRNA-miR or 7 shRNA-miRs and p24 antigen level in culture supernatants measured by ELISA on indicated days after infection. (c) Culture supernatants from Tat shRNA-miR transduced cells in b, obtained on day 35 were sequenced for Tat shRNA-miR target region in the HIV-1 genome.

Multiplexed miRNA-based seven shRNA-miRs prevent CD4 T cell loss and HIV-1 replication in Hu-PBL mice reconstituted with PBMCs from HIV-1+ patients

Transplantation of gene modified T cells is being tried as a potential treatment in HIV-1 infected individuals.34 Thus, we next tested if reinfusion of seven shRNA-miR treated cells provides a therapeutic possibility by preclinical testing in the Hu-PBL mouse model. For this, we selected two HIV-1 infected individuals, one on ART (donor #1, viral load <20 copies/ml; CD4/CD3 ratio = 0.35) and the other, treatment naïve (donor #4, viral load: 67,420 copies/ml; CD4/CD3 ratio = 0.13). PBMCs were transduced with HIV-1 env-pseudotyped lentivirus expressing either no shRNA-miR (mock), only Tat shRNA-miR or seven shRNA-miRs and injected into NOD/SCID/IL2-Rγc−/− mice to evaluate their ability to engraft, expand and resist HIV-1 replication. CD4 T cell counts were monitored on days 8, 29, and 43 and serum p24 levels measured on day 43 (Figure 6a). In mice reconstituted with lentivirus only (no shRNA-miR) transduced T cells, for both donors, few CD4 T cells were found at all-time points tested and the cell numbers progressively declined over time, consistent with HIV-1 mediated depletion of xenogenically activated CD4 T cells that became productively infected with endogenous virus. In the Tat shRNA-miR only transduced group, CD4 T cells expanded (particularly noticeable in donor #1 on ART) by day 29, but declined by day 43, reflecting the initial effectiveness of a single antiviral shRNA-miR that failed at later time points. In contrast in the seven shRNA-miR transduced group, for both donors, CD4 T cells continued to increase and by day 43, constituted 87.4% (donor #1, on ART) and 52.2% (donor #4, treatment naïve) of PBMCs. Correspondingly, viral load was highest in no shRNA-miR control, intermediate in Tat shRNA-miR only and lowest in the seven shRNA-miR group (Figure 6b,c). Taken together, our results show that multiplexed miRNA-based seven shRNA can control HIV-1 replication and reverse CD4 T cell loss, thus providing a potential clinically viable treatment strategy.

Figure 6.

Figure 6

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Prevention of CD4 T cell loss and endogenous HIV-1 replication in 7 shRNA-miR treated HIV seropositive PBMC transplanted Hu-PBL mice. (a) Time course of treatment and analysis. (b,c) NOD/SCID/IL2-Rγc−/− mice were transplanted with PBMCs form two seropositive donors (b on ART) and (c, treatment naive) that were transduced ex vivo with HIV-1 env-pseudotyped lentivirus encoding either no shRNA-miR (mock), only Tat shRNA-miR or 7 shRNA-miRs and monitored for CD4 T cell counts on days 8, 29, and 43 and serum p24 levels on day 43. A representative dot plots of CD4 and CD8 T cell reconstitution on CD3 gated cells (left) and cumulative data from four mice on CD4 T cell levels (middle) and plasma p24 levels (right) is shown. P values between groups for p24 levels are indicated.

Discussion

Here, we have developed a general platform to easily express large numbers of shRNA-miRs using minimal flanking sequences from different endogenous miRNAs to express individual shRNA-miRs. Using this system, we have been able to express seven shRNA-miRs targeting CCR5 and six regions in the HIV-1 genome and show that this affords better protection in vitro as well as in vivo in Hu-PBL mice.

Newer gene editing technologies such as ZFN, TALEN and CRISPR/Cas9 have received a lot of attention lately as potential tools for gene therapy.34,35,36,37 The major advantage of these systems compared to RNAi is that the gene knockout is complete and heritable, so that a short-term treatment will achieve permanent gene disruption. However, although they may be superior approaches for knocking out dispensable host factors like CCR5 to confer HIV-1 resistance, they cannot be used to target viral genes in noninfected cells to preempt infection. Such an endeavor requires the gene editing systems to be active permanently, which results in considerable off-target effects and toxicity. Thus, currently these systems cannot be used to silence host factors and HIV-1 genes simultaneously, which will be required to confer HIV-1 resistance in a therapeutic setting in any meaningful manner. RNAi therefore remains the only system that has been shown to be able to silence host factors and HIV-1 genes at the same time.30,34 However, because HIV-1 is known to escape shRNA-mediated inhibition by mutating the target sites,11,12 it is important to be able to target multiple, highly conserved viral regions. As alluded to earlier, if seven shRNAs are simultaneously expressed, it could cover nearly all HIV-1-strains by ensuring that at least four shRNAs are active against any given viral strain.18 In attempts to express seven conventional shRNAs using plasmids containing tandem repeat of H1 promoters, it was noted that the expression of downstream shRNAs progressively declined.18 Another constraint in multiplexed shRNA expression using the same promoter is the instability of such vectors caused by rapid deletion of cassettes due to homologous recombination.38 Moreover, as noted earlier, miRNA-based shRNAs (shRNA-miRs) will be required to avoid perturbation of endogenous miRNA function. However, the limitation here is that so far, expressing multiple shRNA-miRs has been difficult and is limited to a maximum of four, using a polycistronic miRNA background.15 Moreover, although shRNA-miR expression under polycistronic miRNA background was described in 2011, so far this has not been tested for in vivo efficacy in humanized mouse models.

Advances in understanding Drosha processing of pri-miRNA should allow for rational design of shRNA-miRs. Although Drosha cleavage was previously reported to occur ~11 nt from the lower stem-ssRNA junction,26 we have recently found that the microprocessor measures the distances from both the lower and upper stem-ssRNA junctions to determine the cleavage site in human cells, and optimal distances from both structures are critical to the precision of Drosha processing.27 However, the optimal flanking sequence required to ensure Drosha processing of shRNA-miRs was not known. Our results show that incorporation of ~30 nt of flanking sequences may be enough to ensure processing of different shRNA-miRs. It must be cautioned that we cannot generalize it to all possible miRNA flanks because we actually tested different lengths for only two miRNA flanks. However, efficacy was seen for all seven shRNA-miRs in the backbone of other miRNA flanks of ~30 nt. This finding allows easy multiplexing by expression of shRNA-miRs in different miRNA backbones in tandem. Here, each shRNA-miR will be an independent module that can be easily changed and manipulated. Importantly, the functionality of individual shRNA-miRs within the multiplexed constructs did not decrease compared with constructs containing only single, nonmultiplexed shRNA-miR. In addition, our multiplexed shRNA-miRs were stable and had no obvious adverse effects on cells. More importantly, the seven shRNA-miR transduced resting T cells from HIV-1 seropositive individuals, when reconstituted in Hu-PBL mice led to restoration of CD4 T cell decline, suggesting the feasibility of using such therapy in humans. Thus, our lentiviral platform to express seven shRNA-miRs appears to be a significant advancement towards using RNAi for HIV-1 gene therapy.

Conventionally, VSV-G is used for pseudotyping lentiviruses because of its broad tropism for many different cell types. Nonetheless, cells in the G0 stage of the cell cycle, such as resting CD4 T cells are highly recalcitrant to transduction with VSV-G pseudotyped lentivirus.28 On the other hand, unlike VSV-G, CXCR4-tropic HIV-1 envelope has been shown to mediate fusion of lentiviral particles in both unstimulated and stimulated T cells.28 Our study also shows that lentivirus packaged with X4-tropic envelope from LAI can efficiently deliver shRNAs into resting CD4 T cells. This is particularly important since in HIV-1 infection, resting memory CD4 T cells are the well-known reservoirs of latent HIV-1 infection and delivery of shRNAs might prevent viral reactivation in these cells. In addition to selective shRNA delivery to HIV-1 susceptible T cells, the approach has other advantages over adoptive cellular therapy with activated CD4 T cells that have been gene modified and expanded in vitro.34 Studies in humans and mice have shown that less differentiated T cells can persist longer after transfer because they have longer telomeres and are not prone to activation-induced cell death.39 Furthermore, in the absence of prolonged culture, perturbation of the T cell repertoire is also likely to be minimized. As ex vivo transduction is the only external manipulation required, the approach would allow immediate reinfusion of the gene modified cells into the patient, which simplifies the therapy for wider clinical application.

In summary, we provide a compact and flexible design to express multiple shRNAs without inducing toxic effects or compromising their expression and efficacy. We used this system to express seven shRNA-miRs targeting the CCR5 gene and six regions in the viral genome and showed its effectiveness in suppressing HIV-1 infection in vitro and in vivo in the Hu-PBL model. We propose that this strategy provides a clinically viable approach for gene therapy in HIV-1 infection.

Materials and Methods

Plasmids and constructs. psiCHECK2 vectors were modified to express shRNA target sites in the Renilla luciferase 3′ UTR. For this, synthetic oligonucleotides for the forward and reverse strands of the target sequences were annealed and cloned into psiCHECK2 at the XhoI and NotI site. The oligonucleotide target sequences are listed in Supplementary Table S2. To determine the minimal flanking sequence required for efficient processing, we cloned miR-150 and miR-30a with different length of flanking sequences into pLB vector (Addgene plasmid 11619) at the HpaI and XhoI in front of the U6 promoter. The sequences inserted into the vectors are shown in Supplementary Table S3. The shRNAs were inserted into pLVX vector (Clontech plasmid 631987(mCherry) and 631982 (ZsGreen/GFP)) at the EcoRI and BamHI site. Multiplexed shRNAs were synthesized as ultramers (IDT Technologies) and cloned into pLVX vectors (Supplementary Figure S6). The sequences inserted into the vectors are shown in Supplementary Table S3.

Cell culture. 293 FT/T and TZM-bl cells were cultured as described elsewhere.40 PBMCs were obtained from healthy and HIV infected adult volunteers under an IRB approved protocol. CD4 T cells were isolated from PBMCs using CD4 T cell enrichment kits (Stem cell Technologies, Vancouver, British Columbia, Canada). CD8 Dynabeads (Invitrogen) were used for depleting CD8 T cells from PBMCs. The CD8 depleted PBMCs and isolated CD4 T cells were stimulated with PHA after transduction with lentivirus and were cultured at 37 °C in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml of penicillin-streptomycin with recombinant IL-2 (20 U/ml).

DNA transfection and dual-luciferase reporter assay. 293 FT cells were cotransfected using 10, 50, and 100 ng shRNA vector and 100 ng of psiCHECK2 plasmid harboring the target regions using Lipofectamine 2000. Dual luciferase assay was performed 24 hours later as reported earlier.41

Generation of lentiviral vector and transduction. The lentiviral vector, pLVX-IRES-mCherry or ZsGreen/GFP were purchased from Clontech. Oligonucleotides targeting viral Gag, Env, Tat, Pol, Vif and cellular coreceptor CCR5 were cloned into the EF-1α promoter–expressing lentiviral vector pLVX-IRES-mCherry or ZsGreen/GFP where ever applicable. 293 T cells were plated to 70–80% confluence in 150 mm dishes one day before transfection. The lentiviral vector and the helper pHR8.9VPR and env plasmids pCMV-VSV-G or HIV LAI envelope (kindly donated by Dr Una O'Doherty at University of Pennsylvania) were cotransfected in 293 T cells using calcium phosphate precipitation (Promega). Medium was replaced after 4 hours and supernatants were harvested as described.2,28 TZM-bl cells, resting/activated CD4 T cells and CD8 depleted PBMC from healthy and HIV infected patients were transduced at a multiplicity of infection (MOI) of 5–50 as described earlier.2 After transduction, the cells were washed twice with PBS and cultured in media for 48 hours. Transduction efficiency was determined by examining for mCherry or GFP expression by flow cytometry.

Assay for HIV replication in TZM–bl reporter cells. 2 µg of shRNA expression vectors and 100 ng of HIV-1 NL4-3 plasmid (NIH AIDS Research and Reference Reagent Program) were co transfected into 293 T cells using Lipofectamine 2000 reagent. The supernatants harvested 2 days after transfection was used for infecting equivalent number of TZM-bl cells in presence of 10 μg/ml DEAE-D. Tat-induced luciferase activities were determined in cell lysates 48 hours after infections using the Luciferase assay system (Promega) as previously described.42,43

Assay for toxicity of micro-RNA based shRNAs. To determine the toxicity of shRNA constructs, Jurkat cells were transfected with single, dual, and multiple shRNA constructs by Neon transfection system (Life Technologies). Transfected cells were harvested after 48 hours, stained with anti-Annexin V FITC antibody analyzed by flow cytometry. To determine vector cytotoxicity in CD4 T cells, mCherry expression was followed in transduced cells over time by flow cytometry. Lentivirally transduced CD8 depleted PBMCs were stimulated with PHA and cultured in presence of IL-2 and were monitored on day 0, 4, 7, and 12 for mCherry expression by flow cytometry. Lentivirus transduced cells harvested 2 and 4 days after transduction were also subjected to MTS Assay (Promega) according to manufactures instructions.

HIV-1 challenge assays. Untransduced and lentiviral vector transduced TZM-bl cells were infected with R5-tropic BaL and X4-tropic NL4-3 strains of HIV-1 at an MOIs 0.01 for 4 hours at 37 °C. 2 × 105 resting CD4 T cells and CD8 depleted PBMCs from normal and HIV seropositive donors were activated with PHA (2 µg/ml) after transduction with corresponding lentiviruses and cultured in the presence of IL-2. Cells were infected 48 hours after activation with HIV BaL and NL4-3, at MOIs of 0.01 and 0.001 respectively. Supernatants from TZM-bl cells and T cells/PBMCs were collected and analyzed for HIV replication by p24 ELISA assay (Perkin Elmer) as described previously.30

Sequence analysis of the tat shRNA target region of HIV-1. Viral RNAs from four different HIV-seropositive donors were analyzed for shRNA-induced mutations in the Tat-shRNA target region on day 15 (mock) or 31(Tat/seven shRNA) after infection as previously described.44,45 Viral RNA was extracted using the QIAamp RNA Kit (Qiagen) and first strand cDNA was synthesized using Superscript III First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) as per the manufacturer's instructions. DNA sequences were PCR amplified using Tat targeted primers sense 5′-TGT TGC TTT CAT TGC CAA GT-3′ and antisense primer 5′-TGA TGA GTC TGA CTG CCT TGA-3′. PCR was performed using the following thermal program: 95 °C for 2 minutes and then 35 cycles at 95 °C for 30 seconds, 57.8 °C for 30 seconds and 72 °C for 30 seconds, followed by 72 °C for 5 minutes. The PCR products were gel purified and cloned into the pCR2.1 TOPO vector and subsequently sequenced with the M13R primers.44,45

NOD/SCID-Hu PBL mouse model. NOD/SCID IL2rγcnull mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in specific pathogen free conditions at the Paul L. Foster School of Medicine, TTUHSC animal facility. Hu-PBL mice were generated as described.30 In brief, mice were conditioned with sublethal (2 Gy) whole-body irradiation. Lentivirus transduced HIV-seropositive donor PBMCs (2 × 106) were intravenously injected via the tail vein (in 0.2 ml PBS) into 6- to 7-week-old mice. Cell engraftment was tested 8, 29, and 43 days after transplantation by staining mouse PBMCs with human CD3, CD4, and CD8 antibodies and analyzing CD3 gated population for CD4 and CD8 expression. All mouse experiments had been approved by the TTUHSC IACUC and animal infection experiments were performed in bio-safety level 2 animal facility at TTUHSC.

Flow cytometry. Flow cytometry was performed to determine cell surface antigen expression by 30-minute incubation on ice with pertinent antibodies. The following monoclonal antibodies were used: human-specific monoclonal antibodies used were anti-CCR5 conjugated with FITC or APC (2D7/CCR5; BD Pharmingen), anti-human CD3 (FITC), CD4 (PB), CD8 (APC) and corresponding isotype control mAbs (BD Pharmingen). Data were acquired by BD FACS Canto II and analyzed on BD FACS Diva software v3.0. Overlays were made using FlowJo software v3.0 where ever applicable.

Small RNA deep sequencing. Small RNA libraries were constructed and sequenced in a similar manner as described previously.27,32 Briefly, 48 hours after the constructs were transfected into 293 FT cells, the small RNAs were purified with the miRNeasy kit (Qiagen, Valencia, CA) as per the manufacturer's instructions. Small RNA (50 ng) was ligated with 3′ and 5′ linkers (with barcode) using an improved ligation method that was optimized comprehensively to minimize the ligation bias between different small RNAs. The ligated small RNAs were reverse transcribed and amplified with the KAPA library amplification kit (KAPA Biosystems, Woburn, MA) for 10 cycles, and the library sequenced using the Illumina MiSeq, Salt Lake City, UT. All reads that were sequenced only once were discarded to lower the noise level.

Statistical analysis. The data were analyzed using Graph-Pad Prism software 5 (GraphPad Software, San Diego, CA) or Microsoft Excel. Results are given as means with SD. Comparisons were made using the Pearson two-tailed test. All data with P < 0.05 were considered significant.

SUPPLEMENTARY MATERIAL Figure S1. Left panel shows the each shRNA-miR hairpin. Figure S2. Frequency of individual siRNA reads generated from 7 shRNA-miRs. Figure S3. Representative data showing mCherry (vector alone, single and multiplex shRNA-miRs) expression in vector transduced PBMCs over time. Figure S4. Activated ( A CD4) and resting CD4 T ( R CD4) cells were transduced with VSV-G or HIV env pseudotyped lentivirus and mCherry expression determined 48 hours later by flow cytometry. Figure S5. Activated and resting CD4 T cells transduced with HIV-1 env-pseudotyped lentivirus encoding 7 shRNA-miRs in Figure 4 were monitored for GFP expression on days 0, 3, 9, and 15 after infection to determine enrichment of HIV-resistant cells. Figure S6. Schematic of multiplexed shRNA-miRs. Table S1. Dominant small RNA reads in transfected cells. Table S2. Oligonucleotide sequences used to insert target sites in the 3′ UTR of R-luc in psiCheck2 vector. Table S3. Sequences of the oligonucleotides used in the generation shRNAs.

Acknowledgments

We greatly appreciate the blood donors who volunteered for the study. The plasmid pNL4-3 was obtained through the National Institutes of Health (NIH) AIDS Reagent Program. We also thank Una O'Doherty at the University of Pennsylvania for providing us the HIVLAI envelope plasmid for lentiviral pseudotyping. This work was supported by NIH/NIAID grant RO1 AI071882 to P.S. J.G.C. designed and performed research, analyzed data and wrote the paper, P.B., S.A., H.M., G.Y., C.Y., and Y.D. performed research. M.N. analyzed data and wrote the paper, H.W. designed research and analyzed data, and P.S. designed research, analyzed data and wrote the paper. The authors declare no conflict of interest.

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