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RNA interference (RNAi): a recently discovered part of the machinery of the human cell, and a novel possibility for future therapy
Written by David Margolis, MD, University of Texas, Southwestern Medical Center
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RNA (RiboNucleic Acid) is the message copied (transcribed) from our genes, which are made of DNA (DeoxyriboNucleic Acid). Message RNA is decoded by the cellular machinery, directing the proper assembly of amino acids into cellular proteins and enzymes. However, in recent years RNA has been found to be capable of a broader and broader array of functions. It is hypothesized that eons ago there was an "RNA world" in which very primitive organisms used only RNA and had no DNA.
The most recent newly understood function of RNA is known as RNA interference (RNAi). The appreciation of this phenomenon was so significant that it was named the discovery of the year by the prestigious journal Science. Although the application of this new understanding to the treatment of HIV is likely far off, despite statements by some that RNAi will soon be used to cure AIDS. However, RNAi is already in use as a research tool to understand how HIV works, and like other forms of gene therapy biotechnology companies are already exploring the possibility of clinical uses of RNAi. The organizers of the 10th Conference on Retroviruses and Opportunistic Infections (feb 10-14, 2003) devoted a symposia to explain RNAi and its possible future applications.
Phil Zamore (UMass, abstr. 49), one of the scientists involved in the initial description of RNAi, explained how RNAi works. Host message RNAs are single RNA strands. Some virus genomes, such as HIV, enter the cell as double RNA strands. A human cellular enzyme, appropriately named Dicer, exists that specifically recognizes double-stranded RNAs and chops them up into 21 to 25 nucleotide fragments. These fragments are known as silencing RNAs (siRNA). Double-stranded RNAs are not usually produced by human cells, and their presence often signifies that a virus has invaded.
These siRNA fragments are then picked up by another conglomeration of host cell enzymes called RISC (RNA-induced silencing complex). This complex unwinds a short double stranded siRNA into a short single strand. RISC then uses these single-strand siRNAs as bait, and hunts for any other RNA strands in the cell capable of binding the siRNA due to a complementary RNA sequence. When RISC finds an RNA that sticks to the fragment it is carrying, this tells RISC that it has found an RNA that must come from an invading virus. Another enzyme within RISC then chops up this RNA target.
As the host cell may have to cope with an invader generating multiple copies of foreign RNA, it needs to be able to generate multiple copies of siRNA to deal with the invaders. A family of RNA-dependent RNA polymerases, enzymes that use RNA templates to make copies of RNA molecules, can generate the numbers of siRNA molecules needed to silence and destroy incoming foreign RNA molecules.
John Rossi (City of Hope Med. Ctr, abstr. 50) then discussed one possible technology for the delivery of siRNA to target cells. Using the technology of retroviral gene delivery, in use in his lab and many others for gene therapy experiments, Rossi and coworkers delivered genes expressing siRNAs targeting message RNAs for several HIV-1 regulatory and structural genes. These siRNA-producing retroviral gene transfer vectors then were used to infect (transduce) primary T-lymphocytes as well as CD34+ hematopoietic progenitor cells. CD34+ stem cells into which anti-HIV-1 siRNA expression constructs were inserted were transplanted into mice. These mice (SCID-hu) are bred for the inability to reject human cell transplants, and human T cells that develop within these mice can later be harvested and challenged with HIV-1. siRNAs expressed in both primary T-lymphocytes and in CD34+ derived monocytes made these cells resistant to HIV-1 infection (see also Nat Biotechnol 2002; 20:500-5. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A, Salvaterra P, Rossi J).
Judy Lieberman (Harvard, abstr. 51) presented a more direct methods of siRNA therapy. siRNAs targeting host HIV receptors and viral genes were transfected individually or in combination into cell lines, as well as primary macrophages and T-cells. These cells were then challenged with HIV.
siRNAs that suppressed the host cell's production of the HIV surface co-receptor CCR5 or the viral capsid protein p24 both suppressed production of HIV in challenged cell lines and primary cells. The dual expression of both types of siRNA had synergistic antiviral effects.
As a proof of concept that siRNA can be used in a therapeutic fashion, Lieberman and colleagues demonstrated a direct therapeutic effect of siRNA in a mouse model of liver failure. In a dramatic experiment, the group induced liver failure by treating a mouse with a chemical toxin that induces spontaneous cell death (apoptosis) in the liver. Without therapy, liver failure leads to death in this mouse model system. siRNAs were injected intravenously into the mouse in massive quantities. These siRNAs were designed to block the production of a critical liver gene product that leads to apoptosis (cell death). Injected siRNAs were efficiently taken up by most of the mouse liver cells, suppressed apoptosis gene expression for 10 days, and prevented death of the animals.
Brian Cullen (Duke, abstr. 52) also presented studies from his laboratory using siRNAs targeted against the essential Tat and Rev regulatory proteins of HIV-1, or CCR5, the human cellular co-receptor for HIV-1. His laboratory showed that these specifically block the expression and function of these viral genes without which HIV cannot replicate. When expressed in human T-cell lines and primary lymphocytes, these siRNAs effectively block HIV replication.
The primary hurdle that must first be overcome before RNAi might become a useful therapy is the invention of clinically practical methods that can efficiently deliver siRNAs to the interior of target cell populations. This might be done using retroviral vectors, as in other forms of gene therapy, or with chemical modification of siRNA that prevent its degradation and turn these molecules into stable drugs. Once this substantial initial hurdle is surmounted, RNAi therapy might share obstacles familiar to patients and practioners: resistance and toxicity. As siRNA is targeted to a specific viral sequence, viral mutations within that sequence would enable escape from siRNA-mediated destruction. And as siRNAs are targeted to short sequences, it is likely that some siRNA molecules will share complete or substantial overlap with sequences found in normal human genes. siRNAs have not yet been tested in human systems in which the long-term side effects or toxicities of their expression can be measured. Interference with some normal human gene function over the long term seems possible, if not likely.
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