The use of combinations of small molecule drugs in highly active antiretroviral therapy (HAART) to stop or thwart HIV propagation has had a major impact on delaying the progression from HIV-1 infection to the development of AIDS. Despite this progress, there are problems associated with lifelong use of antiviral drug therapy, including toxicity, the emergence of virus resistant to multiple drugs, and the cost of daily medication. Gene therapy of human T lymphocytes and/or hematopoietic progenitor cells can be considered as a potential replacement or supplement to the current anti-HIV-1 therapies. Similar to small molecule therapies in which combinations of drugs targeting different steps in the viral replication cycle have been most effective, we believe that therapeutic RNAs must also be used in combinations to block various stages of the viral replication cycle to mitigate viral escape. On the basis of findings that both HIV-1 viral RNA transcripts and proteins localize in the nucleolus, we have previously demonstrated that nucleolar-localizing small RNAs can be potent therapeutic agents. For example, we had previously succeeded in inhibiting HIV-1 replication by individually expressing snoRNA chimeras, including the U16TAR and U16RBE RNA decoys that sequester the HIV-1 Tat and Rev proteins in the nucleolus, respectively (Michienzi et al.
). In addition, we also demonstrated that a nucleolar-localizing ribozyme targeting a conserved U5 sequence present in all HIV-1 transcripts had excellent HIV-1 inhibitory function (Michienzi et al.
; Unwalla et al.
). As a combinatorial approach to incorporate anti-HIV small RNAs with different mechanisms of action and target specificity, we multiplexed the aforementioned snoRNA chimeras in addition to siRNAs that cleave tat
mRNAs with the goal to block all transcript production and efficiently achieve suppression of HIV-1 replication.
We chose to use a single promoter and an intron-based platform to express combinations of siRNAs and snoRNAs. Both miRNAs and snoRNAs are processed from introns, thereby providing a rationale for our approach (Hirose et al.
). We previously engineered and optimized the polycistronic miRNA cluster referred to as MCM7 to coexpress three anti-HIV siRNAs from a single Pol II human U1 promoter (Aagaard et al.
). We have now carefully examined several aspects of using this system in a lentiviral vector backbone platform. The current constructs were further optimized for packaging efficiency by cloning the MCM7 transgene in the forward direction with respect to the CMV promoter in the lentiviral pHIV7-EGFP vector with the U1-specific transcriptional termination sequence. We were surprised by the superior packaging efficiency of our constructs, especially those with the transgene in the forward orientation, compared with the empty pHIV7-EGFP vector. Expression of the anti-HIV RNAs might be expected to negatively impact transcription of the full-length viral RNA genome during packaging because our pHIV7-EGFP lentiviral vector is dependent on HIV-1 Rev for packaging. Because all of the constructs contain at least one small RNA against HIV-1 Rev, it was expected that the viral titer of the constructs might be lower or equivalent at best to that of the parental pHIV7-EGFP vector. We have previously overcome this challenge by increasing the amount of HIV Rev-expressing plasmid (Li et al.
) or by inclusion of a plasmid that expresses an Ago2-targeting shRNA (H. Soifer, unpublished data) to minimize the RNAi activity in cells during packaging. In the present case, we did not find an advantage to downregulating Ago2 (data not shown) because the siRNA expression levels are relatively low compared with Pol III-transcribed shRNAs, and during packaging these do not effectively downregulate the viral transcripts. We also postulate that insertion of the MCM7 cassette produces a larger viral transcript (5.4
kb) whose size is closer to that of the natural HIV-1 RNA genome (9
kb) and therefore more favorable for packaging compared with the parental empty vector (3.9
kb). Indeed, we found a 2.5-fold increase in viral titer when the parental MCM7 intron lacking anti-HIV RNAs was incorporated (data not shown), similar to our gene therapy constructs carrying anti-HIV RNAs. Second, we observed the effect of transgene directionality on packaging efficiency, with the forward orientation yielding greater than 100-fold higher production of virus. It is likely that the transgene RNA transcript, when expressed from the U1 promoter in the reverse orientation, could create an opposing transcript during packaging that negatively impacts on levels of expression from an antisense effect.
In this study we have demonstrated the versatility of the MCM7 platform for expressing multiple siRNAs as miRNA mimics as well as snoRNAs from the polycistronic transcript, and efficient processing into mature and functional small RNAs that are readily detectable through Northern blotting analysis. Long-term inhibition of viral replication was evaluated by challenging stably transduced CEM T lymphocytes with HIV-1 NL4-3. The results of these analyses demonstrated that the MCM7-S1/S2M/S3B, MCM7-S1/S2M/U16TAR, and MCM7-S1/U16U5RZ/U16TAR constructs conferred complete protection against viral replication and spread during the 1-month challenge. Interestingly, the MCM7-U16RBE/S2M/U16TAR and MCM7-U16RBE/U16U5RZ/U16TAR constructs did not significantly inhibit HIV replication despite the fact that the small RNAs were actively expressed and readily detectable by Northern blotting. The U16 chimeras in these constructs had demonstrated antiviral activity when individually expressed from the parental vector with the Pol III U6 promoter (Michienzi et al.
; Unwalla et al.
). This discrepancy is most likely related to the difference in expression levels of these RNAs in the context of the intronic MCM7 platform driven by the Pol II U1 promoter versus independently from the Pol III U6 promoter.
The importance of optimal levels of RNA expression for anti-HIV activity and cell viability is supported by the observation that there was selection for transduced CEM T lymphocytes with optimal anti-HIV RNA expression during HIV infection. This phenomenon has also been observed for transduced CEM T lymphocytes harboring a single copy of the transgene (data not shown), reflecting selection of cells with more transcriptionally active integration sites. We evaluated overall RNA expression by qRT-PCR and showed persistent expression during challenge, and therefore it is likely that there is selective pressure for cells with optimal expression to provide antiviral activity in the absence of cellular toxicity.
In addition to RNA expression levels as a determinant for the effectiveness of an RNA-based gene therapy, the nature of the small RNAs should also be considered and carefully balanced between toxicity and therapeutic efficacy. Because RNA decoys act as “sponges” and therefore function in a stoichiometric fashion, the expression level needs to be sufficiently high to achieve therapeutic efficacy, whereas siRNAs and ribozymes are capable of multiple turnover by cleaving their targets in a catalytic manner and should be functional with lower copies per cell. Constructs with the most potent antiviral activities in the context of the MCM7 intron platform tended to have higher RNA expression levels and usually contained more than two RNA agents that are catalytic in nature, such as an siRNA or ribozyme. It is interesting to note that all the constructs that exhibit antiviral activity in the viral challenge assay contain the S1 siRNA that targets both the HIV-1 tat
messages. Although our current data cannot demonstrate whether the other two small RNAs in the constructs have additive effects in antiviral activity, the principle of combinational therapy is to reduce viral escape in a long-term setting. We have previously demonstrated that the combination of three is superior to two and better than single small RNA agents in prolonging anti-HIV protection in long-term setting in a viral challenge assay (Li et al.
In summary, these studies represent the first example of incorporating combinations of snoRNA-based agents with siRNA-based agents within a single expression platform driven by a single Pol II promoter. We demonstrated the versatility of the MCM7 platform for expressing a variety of small antiviral RNAs in addition to miRNAs. We also demonstrated superior packaging of these constructs versus the parental empty pHIV7-EGFP lentiviral vector. The enhanced packaging efficiency was especially pronounced when the transgene was cloned in the forward orientation with respect to the packaging CMV promoter. Last, after HIV-1 challenges of CEM T lymphocytes transduced with the various RNA combinations, we found three small RNA combinations, MCM7-S1/S2M/S3B, MCM7-S1/S2M/U16TAR, and MCM7-S1/U16U5RZ/U16TAR, that strongly inhibited viral replication during the 1-month challenge. We also found that the pressure of HIV-1 infection resulted in selection of cells with optimal levels of anti-HIV gene expression. The two RNA combinations that contained two or more nucleolar RNAs did not significantly inhibit HIV replication, perhaps owing to the noncatalytic nature of RNA decoys versus the siRNAs and ribozyme. Our results suggest these factors should be carefully considered in designing an efficient RNA-based gene therapy.