Oligos Enable New Classes of Therapeutics

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Oligos Enable New Classes of Therapeutics
2006
Gail Dutton
GEN Updates in Biotechnology
Replicor is developing a broad-spectrum, oligonucleotide-based antiviral compound that has potential as a prophylactic and therapeutic agent in case of an influenza pandemic. It also has the potential to become a potent weapon against drug resistant viruses (like HIV) and for certain viral infections like hepatitis C, where there are large unmet medical needs.
This is just one of the new classes of oligonucleotide therapies that are drawing the interest of biotech firms. A number of research projects are focusing on the use of decoy oligos, aptamers, and strategies to inhibit fusion.
Replicor's emerging technology uses the sequence-independent activity of phosphorothioate oligos more than 30 bases long as amphipathic polymers that interact with viral fusion proteins and inhibit viral fusion and entry. One of the lead compounds, REP 9, "has shown antiviral activity against all 12 different families of enveloped virus," according to Andrew Vaillant, Ph.D., CSO.
Company reports indicate that REP 9 has the broadest spectrum of antiviral activity in vitro ever reported for a single chemical entity. Activity was demonstrated against all families of membrane-bound viruses. In the case of influenza A, aerosolized REP 9 was effective in vivo as a prophylactic and as a therapeutic treatment.
A 40mer fully degenerate phosphorothioate oligo, REP 9 targets the common chemical and structural properties of specific motifs found in most enveloped viruses that are required for the fusion of the viral membrane with the host cell membrane, thereby preventing entry of the virus into the cell.

Replicor's REP 9 targets the common chemical and structural properties of specific motifs found in most enveloped viruses that are required for the fusion of the viral membrane with the host cell membrane, thereby preventing entry of the virus into the cell.
"REP 9 targets a common protein motif essential for infection that is found in many enveloped viruses. This motif differs from the classical active site normally targeted by such small molecules as neuraminidase, reverse transcriptase, or protease inhibitors in that it is large and is defined not by amino acid sequence but structurally and chemically."
As Dr. Vaillant explains, the structure and chemistry of this motif are highly conserved in many enveloped viruses, but these motifs from specific viruses have no amino acid homology with each other. The implication, he continues, "is that simple point mutations will not alter the antiviral action of REP 9, which suggests that the development of resistance to REP 9 treatment will be unlikely."
Infection, the fusion of the viral and host cell membranes, "is catalyzed by a unique hinge." The ends of each leaf of that hinge contain an amphipathic alpha helix. "The association of these alpha helices with each other causes the hinge to fold, bringing both membranes into close proximity and inducing membrane fusion. By controlling the hydrophobicity and length of oligos, they can be designed to have similar amphipathic properties to those found in the alpha helices of viral fusion proteins, thus letting the DNA molecule bind tightly to these alpha helices, preventing the folding of the hinge and subsequent membrane fusion."
REP 9 is in preclinical development, with Phase I trials for the aerosol application against influenza and parenteral application against HCV expected to begin in 2007. So far, REP 9 has been validated in vivo against influenza A, respiratory syncytial virus, cytomegalovirus, herpes simplex 2, Ebola, and vaccinia. "Studies testing activity against SIV infection in macaques and HCV infection in vitro are under way," Dr. Vaillant says.
"REP 9 could be used as a prophylactic measure to prevent the spread of viral infection (in the event of a flu pandemic, for example) or to treat specific cases of viral infection," he says. "It has proven to be well-tolerated and effective against viral infection" using aerosol, intranasal, intraperitoneal, subcutaneous, and topical administration, says Dr. Vaillant.
Preliminary tests also indicate that it has potential as a virucidal cream for HSV-2 and HIV. It also could become a new therapy for HIV, hepatitis B and C, and other conditions in which individuals do not respond or poorly tolerate current antiviral therapies, he adds
Decoy ODNs
AnGes and Avontec are collaborating on a completely new class of therapeutics based on decoy oligodeoxynucleotides (decoy ODNs) that can be applied topically or locally via inhalation. According to Thomas Schulze, Ph.D., CEO, "the pilot data suggest that these therapeutics might be equivalent to steroids in terms of efficacy but might show a better safety profile."
These small, double-stranded DNA molecules down-regulate the expression of pro-inflammatory genes by binding to the transcription factors in a highly specific way that interferes with the way in which they bind to those gene's promoter regions. Therefore, the compound may attack multiple targets, as well as identical targets involved in different indications.
As Dr. Schulze elaborates, "The decoy ODN attenuates a pro-inflammatory transcription factor. Transcription factors play a pivotal role in regulating gene expression and many have been identified that are involved in the pathogenesis of inflammation by mediating the up-regulation of pro-inflammatory proteins like cytokins and cell surface receptors. Attenuation of pro-inflammatory transcription factor activity thus provides a new approach to the effective treatment of chronic inflammatory disease."
The decoys are taken up by the cell "without auxiliary means, making use of a membrane carrier system that is frequently expressed in mammalian cell types," Dr. Schulze says. "Inside the cell, they are protected against degradation by nucleases by the introduction of the phosphorothioate bonds within the DNA backbone."
Phase IIa trials have been completed for asthma and psoriasis. Broader studies are planned for 2006 and 2007.
Chemical Antibodies
Archemix is developing aptamers through the in vitro selection of nucleic acids. "We like to call them chemical antibodies," notes Markus Kurz, Ph.D., associate director of chemistry, because they offer the protein-to-protein blocking interactions of antibodies and the ease of chemical synthesis. "It's the best of both worlds," he says.

Mode of action of double-stranded DNA decoy oligonucleotides (decoy ODNs). The individual steps represent the following processes: 1. Ligand binding and receptor activation, 2. Transcription factor activation and dimerization, 3. Transcription factor translocation into the nucleus, 4. Initiation of gene transcription, 5. Cellular uptake of decoy ODNs, 6. Competitive biding of decoy ODN to transcription factor, 7. Inhibition of gene transcription.
Although Archemix focuses on therapeutics, aptamers technology also can be used for target validation and diagnostics. The company is developing cardiovascular applications, as well as oncology and inflammatory diseases. "We have Phase I data on anticoagulation (involving thrombin inhibition)," Dr. Kurz says. Because coagulation can be measured directly, Archemix gets data quickly. That, he says, is particularly important as "aptamers still are relatively new entities and aren't well-validated. We have a strong proof of principle."
The chemistry has few downsides and many advantages," Dr. Kurz says. Aptamers, he says, disrupt protein-to-protein interactions and work in the extracellular space. "By binding a protein agent, they can block binding with a receptor," he explains, unlike many oligo approaches, and offer little or no toxicity or immunogenicity.
Furthermore, because they are produced in vitro, therapeutic leads can be generated in relatively few weeks. The finished drugs can be administered intravenously and subcutaneously.
By choosing the right composition, the pharmacokinetic properties of aptamers are tunable. For example, the conjugation of PEG can confer longer circulating times in the bloodstream. Extrapolating data from primate studies, the circulating half life in humans is expected to be hundreds of hours. The other extreme is the thrombin inhibitor ARC183 that had a half life of only minutes.
The practical advantage here is that the anticoagulant is active during surgery and clears the body shortly afterward, eliminating the side effects triggered by current anticoagulants and their antidotes. Currently, Archemix is pursuing a second-generation molecule with increased potency.
The challenge is mainly marketing. "There's not much of an aptamers industry," Dr. Kurz says. Archemix was founded on aptamers technology and has more than 300 patents, effectively blocking any serious competition, although collaborations are ongoing and welcome.
So far, aptamers appear to offer ease of synthesis, low cost and improved safety features when compared to other therapeutic modalities, Dr. Kurz says. In terms of manufacturing, they take advantage of existing oligo infrastructure for manufacturing and supply chains.
RNA Targets
Topigen (TPI) uses its RNA-targeting technology to address respiratory disorders. Phase II trials are just beginning for the asthma compound TPI ASM8, following October's conclusion of Phase I trials in patients with allergic asthma.
According to Paolo Renzi, M.D., CSO and founder, TPI ASM8 has the potential to eliminate steroids as a treatment for some respiratory conditions. This investigational drug is delivered locally, using an inhaler to deposit it directly in the airways. Because of the turbulence, "the molecules deposit on the junction where the bronchi split up." Consequently, systemic side effects are avoided.

Multipathway target knockdown mechanism of action of Topigen's ASM8. By down-regulating the expression of CCR3 and the common beta chain for IL-3, IL-5