RNAi vs. HIV - Difficulties faced by RNA Interference against HIV


[HOME] BACKGROUND [RNAi] [Difficulties] [Terms] FEATURED EXPERIMENTS [RNAi vs HIV mRNA] [RNAi Combination Therapy] [RNAi Against Receptors] [RNAi by shRNA] [Computer Simulations] [SUMMARY] RESOURCES [Papers] [Websites] [Companies]	Difficulties faced by RNA Interference against HIV RNAi is the third RNA based approach that has been developed in recent times. Antisense RNA and Ribozymes (the first two approaches) have shown little promise in a real clinical setting. Many of the experiments carried out by RNAi are "proof of principle" studies that show the effectiveness of RNAi in vitro. At this stage in its development RNAi faces many problems, especially against a highly adaptive enemy like HIV. This page will focus on general problems faced by RNAi, as well as problems that are specific to RNAi as a potential therapy against HIV. 1. Specificity Too High 2. Interferon Response 3. Unwanted Genes Silenced 4. Delivery Problems 1. Specificity Too High One of the central features of RNAi is that highly specific siRNAs are used to target mRNA of the gene to be silenced. This high level of specificity could be a disadvantage when it comes to fighting a rapidly evolving virus like HIV. As one can see from the graphic to the right, the phylogenetic tree of HIV has many branches. HIV-1 itself is divided into three groups--M, N, and O. According to Wikipedia, the M group is the most common. Most North Americans are infected with subtype B (Group M). Subtypes A and D are found in Africa, while subtype C is found in Africa and Asia. The subtypes of group M are based upon differences within the entire genome. When HIV penetrates a cell, reverse transcriptase immediately begins to reverse transcribe the viral RNA into DNA. Evolution has favoured an HIV version of reverse transcriptase that is unreliable and highly error prone. This initial reverse transcription event results in mutations that give the virus resistance to anti-HIV therapies. In an RNAi situation, where siRNA molecules base pair with HIV mRNA in a very specific manner, a single mutation in the base pairing sequence can compromise binding. Mutation is not the only source of the HIV virus' resistance to RNAi. If a person is infected with more than one type of HIV, an RNA strand from each type can be incorporated into the same virus. When working on the viral RNA, reverse transcriptase jumps between the two RNA strands. The crossing over effect creates a recombinant provirus that could be resistant to anti-HIV therapies. Gao et al. (1996) found that the HIV-1 epidemic in Thailand is caused by a recombinant version of the virus that has integrated subtypes A and E. The full length article is available from PubMed Central (Click on PDF to the top left after page loads). In 2003, Boden et al. found that a simple point mutation in the area of HIV mRNA targeted by siRNAs can neutralize the effects of RNAi. The article is titled "Human Immunodeficiency Virus Type 1 Escape from RNA Interference," and is available on the Journal of Virology website. In 2004, Westerhout et al. found that insertion or deletion mutations in the HIV-1 genome can cause alterations in the secondary structure of HIV mRNA. The alterations take away siRNA binding ability. This experiment is titled "HIV-1 can escape from RNA Interference by evolving an alternative structure in its RNA genome" (Nucleic Acids Research Journal). 2. Interferon Response The introduction of high levels of foreign RNA to the human body can result in the immune system beginning the interferon response. Interferons are proteins produced by cells in the immune system such as T-cells, B-cells, macrophages and fibroblasts [Wikipedia]. The response is undesirable for several reasons. During a study testing the effectiveness of RNAi against HIV, the interferon response could strengthen the fight against the HIV virus. Any positive effects observed might be attributed to RNAi, when indeed they were caused by the interferon response. During the early days of the Antisense RNA approach, the positive results that were thought to be a result of the therapy were actually caused by the interferon response. Today, interferon response detection packages are available for use during RNAi experiments. A strong immune response such as this can also put a tremendous amount of strain on the body. If the patient undergoing the therapy is weak, a strong immune response could cause severe adverse effects or even death. In addition, if RNAi is attempting to silence a gene within the human genome (such as the gene for the CCR5 receptor that HIV needs to get into some CD+ cells), a strong immune response will achieve nothing. The interferon response can be caused by long double stranded RNA even when these RNAs are present in low concentrations. To circumvent this problem, RNAi researchers have introduced siRNAs directly, instead of relying on the enzyme DICER to cleave long dsRNA into siRNAs. However, siRNAs have a limited lifetime. The mRNA to be silenced is constantly being produced, and a constant supply of siRNA is required for stable RNAi. This problem has been overcome by using constructs encoding shRNA to produce a constant supply of siRNA (see navigation bar to the left for experiment involving shRNA). It was thought that shRNAs were too small to give rise to the interferon response. On the contrary, in 2003 Bridge et al. published an article in Nature Genetics showing that in high concentrations, shRNA can activate the interferon response. The full text article titled "Induction of an interferon response by RNAi vectors in mammalian cells" can be accessed in PDF format by clicking on the link. 3. Unwanted Genes Silenced In 2004, Scacheri et al. attempted to use RNA Interference to silence the human MEN1 gene. In addition to neutralizing MEN1 RNA, the siRNAs used in this experiment also inhibited the TP53 and CDKN1A genes--the genes responsible for proteins p53 and p21 respectively. p53, a cell cycle regulating protein, is shown bound to DNA in the graphic to the right. The researchers theorized that p53 and p21 inhibition could have occurred because of partial sequence matching between the siRNAs used and the protein mRNAs. The complete paper titled "Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells" can be found at PubMed Central. 4. Delivery Problems One of the toughest obstacles faced by RNAi research involves the delivery of siRNA into the cell. siRNA injected directly into the bloodstream has a short half-life--it is excreted by the kidneys very fast. In addition to this, the phospholipid bilayer is not designed to allow for the uptake of nucleic acids. There are two approaches currently taken to overcome the problem of getting past the bilayer. One involves modification of the charged RNA backbone, and the other involves complexing RNA with a lipid molecule. Both approaches have shown some success. In a "proof of principle" study, where RNAi is simply applied to a culture of cells in media, delivery is not a problem. However, in real-life trials, delivery is a major issue. In a recent study, Judy Lieberman from Harvard Medical School used high-pressure injection to deliver interfering RNA molecules to mice. The targeted gene was effectively silenced, but several mice later died of heart failure. This was a result of the injection volume making up about 20% of the mouse's total blood volume. The article explaining the delivery problems outlined here can be found at the PLOS-Biology website. Lentiviruses have been used in several "proof of principle" studies to deliver siRNAs to cells. However, it remains to be seen how well they will do in clinical trials. In a clinical trial, viral vectors have the added responsibility of avoiding the host's immune system. This is only a general list of the difficulties faced by RNAi research in relation to HIV. Each study incorporating RNAi will additionally face its own specific set of difficulties. These might range from ineffective siRNAs, to cell specific resistance to vectors.

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Difficulties faced by RNA Interference against HIV

RNAi is the third RNA based approach that has been developed in recent times. Antisense RNA and Ribozymes (the first two approaches) have shown little promise in a real clinical setting. Many of the experiments carried out by RNAi are "proof of principle" studies that show the effectiveness of RNAi in vitro. At this stage in its development RNAi faces many problems, especially against a highly adaptive enemy like HIV. This page will focus on general problems faced by RNAi, as well as problems that are specific to RNAi as a potential therapy against HIV.

1. Specificity Too High

2. Interferon Response

3. Unwanted Genes Silenced

4. Delivery Problems

1. Specificity Too High

One of the central features of RNAi is that highly specific siRNAs are used to target mRNA of the gene to be silenced. This high level of specificity could be a disadvantage when it comes to fighting a rapidly evolving virus like HIV.

As one can see from the graphic to the right, the phylogenetic tree of HIV has many branches. HIV-1 itself is divided into three groups--M, N, and O. According to Wikipedia, the M group is the most common. Most North Americans are infected with subtype B (Group M). Subtypes A and D are found in Africa, while subtype C is found in Africa and Asia.

The subtypes of group M are based upon differences within the entire genome. When HIV penetrates a cell, reverse transcriptase immediately begins to reverse transcribe the viral RNA into DNA. Evolution has favoured an HIV version of reverse transcriptase that is unreliable and highly error prone. This initial reverse transcription event results in mutations that give the virus resistance to anti-HIV therapies. In an RNAi situation, where siRNA molecules base pair with HIV mRNA in a very specific manner, a single mutation in the base pairing sequence can compromise binding.

Mutation is not the only source of the HIV virus' resistance to RNAi. If a person is infected with more than one type of HIV, an RNA strand from each type can be incorporated into the same virus. When working on the viral RNA, reverse transcriptase jumps between the two RNA strands. The crossing over effect creates a recombinant provirus that could be resistant to anti-HIV therapies. Gao et al. (1996) found that the HIV-1 epidemic in Thailand is caused by a recombinant version of the virus that has integrated subtypes A and E. The full length article is available from PubMed Central (Click on PDF to the top left after page loads).

In 2003, Boden et al. found that a simple point mutation in the area of HIV mRNA targeted by siRNAs can neutralize the effects of RNAi. The article is titled "Human Immunodeficiency Virus Type 1 Escape from RNA Interference," and is available on the Journal of Virology website.

In 2004, Westerhout et al. found that insertion or deletion mutations in the HIV-1 genome can cause alterations in the secondary structure of HIV mRNA. The alterations take away siRNA binding ability. This experiment is titled "HIV-1 can escape from RNA Interference by evolving an alternative structure in its RNA genome" (Nucleic Acids Research Journal).

2. Interferon Response

The introduction of high levels of foreign RNA to the human body can result in the immune system beginning the interferon response. Interferons are proteins produced by cells in the immune system such as T-cells, B-cells, macrophages and fibroblasts [Wikipedia]. The response is undesirable for several reasons.

During a study testing the effectiveness of RNAi against HIV, the interferon response could strengthen the fight against the HIV virus. Any positive effects observed might be attributed to RNAi, when indeed they were caused by the interferon response. During the early days of the Antisense RNA approach, the positive results that were thought to be a result of the therapy were actually caused by the interferon response. Today, interferon response detection packages are available for use during RNAi experiments.

A strong immune response such as this can also put a tremendous amount of strain on the body. If the patient undergoing the therapy is weak, a strong immune response could cause severe adverse effects or even death. In addition, if RNAi is attempting to silence a gene within the human genome (such as the gene for the CCR5 receptor that HIV needs to get into some CD+ cells), a strong immune response will achieve nothing.

The interferon response can be caused by long double stranded RNA even when these RNAs are present in low concentrations. To circumvent this problem, RNAi researchers have introduced siRNAs directly, instead of relying on the enzyme DICER to cleave long dsRNA into siRNAs. However, siRNAs have a limited lifetime. The mRNA to be silenced is constantly being produced, and a constant supply of siRNA is required for stable RNAi. This problem has been overcome by using constructs encoding shRNA to produce a constant supply of siRNA (see navigation bar to the left for experiment involving shRNA). It was thought that shRNAs were too small to give rise to the interferon response. On the contrary, in 2003 Bridge et al. published an article in Nature Genetics showing that in high concentrations, shRNA can activate the interferon response. The full text article titled "Induction of an interferon response by RNAi vectors in mammalian cells" can be accessed in PDF format by clicking on the link.

3. Unwanted Genes Silenced

In 2004, Scacheri et al. attempted to use RNA Interference to silence the human MEN1 gene. In addition to neutralizing MEN1 RNA, the siRNAs used in this experiment also inhibited the TP53 and CDKN1A genes--the genes responsible for proteins p53 and p21 respectively. p53, a cell cycle regulating protein, is shown bound to DNA in the graphic to the right.

The researchers theorized that p53 and p21 inhibition could have occurred because of partial sequence matching between the siRNAs used and the protein mRNAs. The complete paper titled "Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells" can be found at PubMed Central.

4. Delivery Problems

One of the toughest obstacles faced by RNAi research involves the delivery of siRNA into the cell. siRNA injected directly into the bloodstream has a short half-life--it is excreted by the kidneys very fast. In addition to this, the phospholipid bilayer is not designed to allow for the uptake of nucleic acids. There are two approaches currently taken to overcome the problem of getting past the bilayer. One involves modification of the charged RNA backbone, and the other involves complexing RNA with a lipid molecule. Both approaches have shown some success.

In a "proof of principle" study, where RNAi is simply applied to a culture of cells in media, delivery is not a problem. However, in real-life trials, delivery is a major issue. In a recent study, Judy Lieberman from Harvard Medical School used high-pressure injection to deliver interfering RNA molecules to mice. The targeted gene was effectively silenced, but several mice later died of heart failure. This was a result of the injection volume making up about 20% of the mouse's total blood volume. The article explaining the delivery problems outlined here can be found at the PLOS-Biology website.

Lentiviruses have been used in several "proof of principle" studies to deliver siRNAs to cells. However, it remains to be seen how well they will do in clinical trials. In a clinical trial, viral vectors have the added responsibility of avoiding the host's immune system.

This is only a general list of the difficulties faced by RNAi research in relation to HIV. Each study incorporating RNAi will additionally face its own specific set of difficulties. These might range from ineffective siRNAs, to cell specific resistance to vectors.