Antisense Therapy – assisted suicide for HIV
Figure 1: Steps in the reverse transcription of retroviral RNA to produce double-stranded DNA, and the site of interference by oligodeoxynucleotides targeted to the polypurine tract.
Antisense oligonucleotides activate the self-destruction of retroviruses
Antisense technology sounds nonsencical to you? It is nothing of the sort (s. a.). After having made little headway as a therapeutic approach to retroviral infection, results reported by Moelling and colleagues1 might change this dramatically, if the prove robust and generalizable. The authors demonstrate the therapeutic efficacy of interfering viral reproduction (antisense-mediated silencing2) using a classic model: mice infected with the murine spleen focus-forming virus. As this method targets a common feature of the Retroviridae, the strategy should in theory be applicable to any retrovirus.The defining event of the retroviral replication cycle is the reverse transcription, that is the conversion of the RNA genome into double-stranded DNA3 (Fig. above). This process is followed by irreversible insertion of the newly synthesized DNA into the genome of the host cell. Like this a provirus is formed that directs the production of progeny virions.In previous studies using cultured cells, Moelling and her collaborators showed that antisense oligodeoxynucleotides against HIV-1 prevent viral replication, that the treatment causes a kind of suicide of the viral RNA genome, and that the effect is mediated entirely by the virus’ own enzyme equipment (reverse transcriptase5, 6, 7). The strategy works by essentially programming the virus to destroy itself. No other antiretroviral strategy works in this way; all drugs approved for use against HIV-1, for example, simply block a step in the viral replication cycle8, 9. The antisense treatment, in contrast, triggers the viral reverse transcriptase to destroy its own template, precluding reverse transcription and integration of the provirus.
In the new paper, the authors study the antisense treatment in living animals. Because there is no practical, small-animal model that supports the complete HIV-1 replication cycle, they used mice experimentally infected with the spleen focus-forming virus, that causes a severe leukemia in mice10. The logic was sound, as the approach should work essentially the same way for these viruses as for HIV-1. All that was required was to tinker with the hereditary information sequence. The authors found that pretreatment of virions with certain oligonucleotides prevents or reduces infection. Moreover, injecting mice with other oligonucleotides, during or soon after infection reduces infection and prolongs survival. Although the results might be attributed exclusively to the viral enzyme-activity, unrelated mechanisms cannot formally be excluded. For example, it is possible that cellular proteins (RNases) are included too.
Of course, the approach of Moelling and colleagues will need to clear many hurdles on the way to becoming a viable therapeutic option. Studies in nonhuman primates would be a valuable next step. Among other considerations, it will be important to assay the ability of target viruses to generate resistance mutations. There isn’t a single antiretroviral med that HIV-1 has not succeeded in evading.
Biodistribution is another potential concern. HIV-1 causes an infection of the whole body, primarily accumulating in lymphoid tissue and the circulatory and central nervous systems. In contrast, the injected oligonucleotides accumulate mainly in the liver. Even if tissue distribution is a concern, anti-HIV oligonucleotide treatment could still move forward as a candidate for preventing the infection (for example, as a component of a vaginal microbicide). Oligonucleotide treatment might also be appropriate for emergency post-exposure prophylaxis, or in situations where viral replication has already been substantially reduced by treatment with other inhibitors. As with all pharmacologic agents, issues of toxicity, pharmacokinetics, large scale production and patient adherence will also need to be addressed.
Beyond these challenges, there is certainly cause for optimism. Research on using antisense oligonucleotides as therapeutic agents already has a rich history in both the academic and commercial arenas, and much is known about oligonucleotide design, synthesis and biological activity2. Research and development in this area are likely to expand further. For example, previous research on antisense therapeutics for genetic diseases and tumors, which require repeated dosing over long periods of time, may be relevant to HIV-1, which may also require long-term treatment.
The antisense approach may be applicable to a broad range of retroviral diseases. Moelling and her colleagues have already demonstrated efficacy against two unrelated retroviruses—a primate lentivirus (HIV-1) and a gammaretrovirus of mice. Thus, the strategy should in principle be readily adapted to an array of retroviral pathogens of medical, environmental and agricultural importance, including bovine immunodeficiency virus, equine infectious anemia virus, feline immunodeficiency virus, feline leukemia virus, Koala retrovirus and the Jaagsiekte sheep retrovirus, to name just a few.
For those interested in details, here you go: during reverse transcription, the viral RNA serves as the template for polymerization of minus-strand DNA. RNase H–mediated cleavage of the viral RNA is necessary to free the minus strand for plus-strand DNA synthesis3. A cellular tRNA primes synthesis of minus-strand DNA (light blue). The RNase H domain of the retroviral reverse transcriptase cleaves the RNA strand of the resulting duplex into short fragments without cleaving within the polypurine tract. Instead, cleavage occurs precisely at the polypurine tract–U3 junction. The 3′ end of the polypurine tract RNA then serves as a primer for plus-strand synthesis (dark blue). A second cut at the polypurine tract–U3 junction facilitates removal of the primer. In both cases, cleavage must occur precisely at this junction because the first few bases of U3 are subsequently required for integration. As indicated in the shaded box, an antisense oligodeoxynucleotide complementary to the polypurine tract creates an RNA-DNA duplex that mimics the structure recognized by the reverse transcriptase, leading to premature cleavage of viral RNA at the polypurine tract–U3 junction before reverse transcription. The most effective oligodeoxynucleotide design includes an antisense strand that is perfectly complementary to the polypurine tract and a passenger strand that is partially complementary to the antisense strand. PPT, polypurine tract. ODN, oligodeoxynucleotide. Vertical arrows denote cleavage of the viral RNA by RNase H. Short arrows represent non-specific digestion of viral RNA during minus-strand synthesis, whereas long arrows indicate sites of highly specific cleavage defined by the structure of RNA/DNA duplex at the site of polypurine tract.
Only a short stretch of the viral RNA (typically 12–20 bases), known as the polypurine tract, is resistant to cleavage and remains firmly bound to its minus-strand DNA complement. The RNA-DNA duplex spanning the polypurine tract adopts an unusual local structure owing in part to atypical base pairing and the presence of unpaired bases. A specific interaction of the viral RNase H with this structure leads to precise cleavage at the 3′ end of the polypurine tract4, and the free 3′ end of this short, remaining stretch of hybridized RNA then initiates plus-strand DNA synthesis. After plus-strand synthesis is underway, RNase H must cleave once again, at precisely the same site, so that the RNA primer can be displaced. If all goes well—for the virus—the result is a complete, double-stranded DNA copy of the viral genome.
Matzen, K. et al. Nat. Biotechnol. 25, 669–674 (2007). | Article |
The original publication quoted above:
Nat Biotechnol. 2007 Jun;25(6):669-674. Epub 2007 Jun 3.
RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides.
 Institute of Medical Virology, University of Zurich, Gloriastrasse 30, 8006 Zurich, Switzerland.  Present addresses: Karlstrasse 41, 80333 Munich, Germany (K.M.) and Elswigstrasse 68, 23562 Lubeck, Germany (A.N.).  These authors contributed equally to this work.
The HIV-1 RNase H can be prematurely activated by oligodeoxynucleotides targeting the highly conserved polypurine tract required for second strand DNA synthesis. This inhibits retroviral replication in cell-free HIV particles and newly infected cells. Here we extend these studies to an in vivo model of retroviral replication. Mice that are chronically infected with the spleen focus-forming virus and treated with oligodeoxynucleotides that target the polypurine tract, exhibit either transient or long-term reductions in plasma virus titer, depending on the therapeutic regimen. Treatment prior to, during or shortly after infection can delay disease progression, increase survival rates and prevent viral infection. This strategy destroys viral RNA template in virus particles in serum as well as early retroviral replication intermediates in infected cells. As it targets events common to the replication cycle of all retroviruses, this approach may be broadly applicable to retroviruses of medical and agricultural importance.
PMID: 17546028 [PubMed – as supplied by publisher]
Nature Biotechnology 25, 669 – 674 (2007)
Published online: 3 June 2007 | doi:10.1038/nbt1311
RNase H-mediated retrovirus destruction in vivo triggered by oligodeoxynucleotides
The HIV-1 RNase H can be prematurely activated by oligodeoxynucleotides targeting the highly conserved polypurine tract required for second strand DNA synthesis1, 2, 3, 4, 5. This inhibits retroviral replication in cell-free HIV particles and newly infected cells1, 2, 3, 4. Here we extend these studies to an in vivo model of retroviral replication. Mice that are chronically infected with the spleen focus-forming virus and treated with oligodeoxynucleotides that target the polypurine tract, exhibit either transient or long-term reductions in plasma virus titer, depending on the therapeutic regimen. Treatment prior to, during or shortly after infection can delay disease progression, increase survival rates and prevent viral infection. This strategy destroys viral RNA template in virus particles in serum as well as early retroviral replication intermediates in infected cells. As it targets events common to the replication cycle of all retroviruses, this approach may be broadly applicable to retroviruses of medical and agricultural importance.
Institute of Medical Virology, University of Zurich, Gloriastrasse 30, 8006 Zurich, Switzerland.
Present addresses: Karlstrasse 41, 80333 Munich, Germany (K.M.) and Elswigstrasse 68, 23562 Lubeck, Germany (A.N.).
These authors contributed equally to this work.