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RNA INTERFERENCE:

THE NEXT REVOLUTION IN GENE THERAPY

by Emily Heikamp

RNA interference (RNAi) has revolutionized not only the basic field of molecular biology, but also its applications in pharmaceutical research, in which RNAi can be used both to identify drug targets and to treat diseases. While clinical trials are already underway for an RNAi-based treatment for macular degeneration, recent studies have shown promising results for HIV, cancer, and hypercholesterolemia. The strong appeal of this technique is its potency and specificity; its greatest challenge is the delivery of RNAi-based drugs, a process which is still in the nascent stages of research.

INTRODUCTION

What is RNA interference?

RNA interference (RNAi) is a powerful functional genomics tool by which double-stranded RNA (dsRNA) silences a gene by inhibiting expression of protein. This phenomenon was first observed when Fire (1998) injected dsRNA into the nematode Caenorhabditis elegans silenced genes whose sequence was complementary to the dsRNA. This technique was not immediately successful in mammalian systems because exogenous dsRNA triggered the cell’s antiviral interferon response instead of producing gene-specific silencing. The full potential of RNAi was not realized until Zamore (2000) discovered that synthetic 21-23 nucleotide small interfering RNA (siRNA) could produce sequence-specific gene silencing in mammalian cells. With this discovery, the components of the endogenous RNAi pathway began to unravel.

RNAi is mediated by dsRNA that is cleaved into 21-23 nucleotide siRNA by an endonuclease component of the ribonuclease-III type enzyme Dicer. The siRNA is incorporated into an enzyme complex called the RNA-induced silencing complex (RISC). The RISC complex guides the antisense strand of the siRNA to its complementary target sequence on messenger RNA (mRNA) as it exits the nucleus. Binding of antisense siRNA to the mRNA results in cleavage of the mRNA by a nuclease component of RISC called Slicer. Because the mRNA is degraded after cleavage, its protein product is not translated. Thus, RNAi is a specific, potent technique to effectively silence a gene by preventing the translation of its protein product.

With the discovery that RNAi could be used to silence genes in mammalian cells, scientists turned their attention to therapeutic applicatiosn of RNAi. Large-scale genetic screens with RNAi can be used to identify druggable targets, such as the protein product of the mutated gene or an upstream component in the pathway. However, some disease-causing genes cannot be targeted with traditional drug therapies, such as small molecules, proteins, or monoclonal antibodies. Could RNAi be used to silence the expression of aberrant genes in order to treat human diseases? Possible targets would be oncogenes, growth factors, viral replication genes, or single nucleotide polymorphisms in inherited genetic disorders. While the list of potential targets is endless, the development of RNAi-based drugs is hindered by the problem of drug delivery. By addressing the pharmacodynamic challenges of delivering RNAi-based drugs, scientists hope to harness the potential of this new method to effectively turn off bad genes.

The first RNAi success story: macular degeneration

Age-related macular degeneration is one of the most common causes of severe blindness, affecting more than 1.65 million people in the United States. This disease results in a loss of vision specifically in the macular region of the retina, which is used for high acuity vision. The wet form of macular degeneration is caused by the growth of blood vessels in the retina from the choroidal layer of the eye. The early stages of macular degeneration are characterized by the formation of drusen—small yellow deposits on the center of the retina. In wet macular degeneration, blood vessels may grow within the drusen material, resulting in hemorrhage or scar tissue on the retina. Approximately 1-2% of cases of age-related wet macular degeneration are caused by a missense mutation in a gene called Fibulin 5. The Fibulin 5 gene codes for a protein that belongs to a family of extracellular proteins that are expressed in the basement membranes of blood vessels.

Scientists at Acuity Pharmaceuticals are using RNAi to turn off the mutated Fibulin 5 gene in patients with wet macular generation. Acuity is the first company to apply to the Food and Drug Administration (FDA) for approval for a clinical trial using an RNAi-based drug. Short-interfereing RNA targeting the mutated Fibulin 5 gene can be directly injected into a patient’s eye. The success of this treatment is far from guaranteed, but this clinical trial is a landmark for the field of RNAi-based therapeutics. Other pharmaceutical companies such as Sirna and Alnylam have also applied for FDA approval for their own clinical trials to treat macular degeneration, but they contend that their technique will be different. Acuity’s method of injecting naked siRNA has downfalls—it is possible that not enough of the drug will reach its target mRNA because enzymes can degrade the siRNA. To make their siRNA resistant to enzyme destruction and to decrease the number of eye injections that a patient would need, Sirna and Alnylam are modifying the sugar-phosphate backbone of the siRNA. While these backbone modifications may stabilize and prolong the half-life of siRNA, achieving intracellular delivery at therapeutically effective doses is still a major challenge.

Cancer

Since the discovery of the first oncogenes, scientists have focused much effort on trying to switch off these mutated genes. Short interfering RNA targeting tumor-specific oncogenic fusion proteins could potentially kill cancerous cells while sparing normal cells. Perhaps the best cancer success story to date is that of imatinib—a small molecule tyrosine kinase inhibitor drug. Imatinib targets the Bcr-Abl oncoprotein that is characteristic of chronic myelogenous leukaemia (CML). Marketed as Gleevec, this drug has been successful because the Bcr-Abl fusion protein is exclusive to tumor cells. Despite the initial enthusiasm about Gleevec, a number of patients with CML have developed resistance to the drug (Cowan-Jacob 2004). RNA interference has been used both as an alternative to imatinib and in combination with imatinib in mouse models of CML. Wohlbold (2003) demonstrated that siRNA targeting the Bcr-Abl gene sensitized mice to treatment with imatinib mesylate. Others have shown that RNAi can be used to directly treat CML. Scherr (2003) showed that the expression of the Bcr-Abl oncoprotein could be prevented by siRNA targeting that gene. With these encouraging results, scientists are attempting to use RNAi to inhibit the expression of other oncoproteins with siRNA.

Although an RNAi-based cancer therapy seems promising, the efficient and stable delivery of siRNA to the site of the tumor will be particularly crucial to the treatment of metastatic cancer. Delivering siRNA to the tumor is much trickier than delivering siRNA to the retina in patients being treated for macular degeneration. In order to achieve systemic delivery of siRNA, some groups have made chemical modifications to the sugar phosphate backbone, thereby increasing the half-life of siRNA in the plasma by preventing degradation of siRNA by serum nucleases (Chiu 2003 & Czauderma 2003). However, enhancing siRNA stability is not enough to ensure that these drugs will penetrate cells and tissues in therapeutically effective concentrations. Flynn (2004) reported efficient delivery of liposome encapsulated siRNA in a mouse model, but this method has never reached clinical trials. To achieve systemic delivery of RNAi, scientists must determine which backbone modifications are the most useful for enhancing cellular and tissue uptake of siRNA, or they need to develop alternative carriers for siRNA.

Gene therapy offers one alternative but controversial method for delivering siRNA to silence mutant genes. Using retroviral vectors, scientists can deliver short hairpin RNA (shRNA) that become incorporated into the host genome. These shRNA are processed by the Dicer enzyme to yield therapeutically active siRNA; this method has several advantages over directly injecting naked siRNA. First, shRNA can provide a continuous, stable supply of gene-silencing siRNA if the shRNA gene is cloned downstream of a constitutively active promoter. Second, tissue-specific or inducible promoters can be used to control the location and timing of shRNA expression. However, using retroviruses to deliver shRNA raises many safety concerns, as similar attempts in the field of gene therapy have experienced varied success. Nevertheless, systemic delivery of retroviral vectors also poses problems that have yet to be addressed. While using RNAi for the treatment of cancer may seem very appealing, there is much left to be understood about the pharmacodynamic aspects of delivering siRNA drugs efficiently.

Human Immunodeficiency Virus (HIV)

Combination drug therapies have led to dramatic improvements in the quality of life for HIV-infected individuals. Yet despite the success of anti-retroviral drugs, new problems are emerging. Mutant variants of HIV have acquired drug resistance, while other problems are caused by certain combinations of drugs that are toxic to some patients. Where these approaches have failed, RNAi could be used to inhibit the expression of genes that are necessary for viral replication and infection. HIV was actually the first infectious disease targeted with RNAi, likely because much is known about the lifecycle and patterns of gene expression of this virus. Although there have been no clinical trials for RNAi-based, anti-retroviral HIV drugs, many in vitro studies report successful silencing of early and late HIV-encoded RNAs in both cell lines and haematopoietic stem cells. However, the high mutation rate of the HIV virus necessitates an alternative approach to targeting the virus itself. Instead, RNAi could be used to silence the expression of cellular cofactors that are required for HIV infection of T lymphocytes. Novina (2002) reported that siRNA targeting the CD4 receptor on T lymphocytes resulted in the inhibition of HIV in numerous human cell lines. Others have targeted the co-receptors CXCR4 and CCR5 with equal success (Qin 2003).

The progress of clinical trials for siRNA drugs targeting HIV has been hindered by inefficient delivery methods. Again, the delivery of RNAi-based, anti-retroviral drugs to infected T lymphocytes remains a challenge. Michienzi (2003) proposed that gene therapy using retroviral vectors to deliver shRNA to the patient’s haematopoietic stem cells would be the most directed approach (Figure 5). First, the patient would be given injections of a cytokine called granulocyte colony stimulating factor (GCSF) to mobilize the haematopoietic stem cells from the patient’s bone marrow. Then, haematopoietic stem cells would be collected from the periphery and the T lymphocyte population could be enriched ex vivo. Next, lentiviral vectors would be used to deliver the anti-HIV shRNA genes. These modified T cells would be re-infused into the patient and would presumably confer resistance to HIV infection.

Hypercholesterolemia

While exploring the potential of siRNA to silence genes involved in hypercholesterolemia, Soutschek (2004) found that cholesterol-conjugated siRNA have markedly improved pharmacological properties. Previously, siRNA was delivered systemically by high-pressure intravenous injection or by retroviral transduction. Using an alternative approach, Soutschek’s group found that conjugating a molecule of cholesterol to synthetic siRNAs enhanced both their stability in vivo and their resistance to serum nucleases. Using a mouse model for hypercholesterolemia, the researchers used siRNA targeting the apoB gene, which codes for a protein that is necessary for synthesizing low density lipoprotein (LDL). The apoB gene, which is expressed predominantly in the liver and jejunum, was effectively silenced in these mice. Their serum cholesterol levels were reduced by 44% due to decreased levels of LDL. Although this method seems like a fairly simple solution to the problem of systemic delivery of siRNA, Soutschek’s technique takes advantage of some basic pharmacologic principles. Because the siRNA were tagged with cholesterol, they were taken up by cells that expressed transmembrane cholesterol receptors. Furthermore, the siRNA reached their target organ—the liver—because all drugs that are injected intravenously are sent to the liver before they are distributed throughout the body. While this approach may be ideal for treating hypercholesterolemia, it is unlikely that this method can be applied to other siRNA therapeutics.

DISCUSSION

The key challenge in developing RNAi-based therapies for cancer, HIV, and many other diseases will be efficient, stable delivery of siRNA. Since the discovery that siRNA could silence genes in mammalian cells, RNAi has been applied to finding potential treatments for human disease. While the genetic targets for siRNA are already known and are numerous, the current obstacles are problems of pharmacologic importance. Ensuring that siRNA are not degraded by serum nucleases and that enough siRNA enters the cell to be pharmacologically effective are two problems that must be overcome before RNAi-based therapeutics become feasible.

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