AM Biotechnologies announced this month that it has received a $199,000 grant from the National Institute of General Medical Sciences to develop its core thioaptamer technology for synthesizing modified RNA oligonucleotides including siRNAs.
Thioaptamers are phosphorodithioate oligos that mimic DNA or RNA, and contain sulfur-modified phosphate ester backbones created using thiophosphoramidites and a sulfurization reagent, according to Xianbin Yang, the company’s director of research and development.
Designed to be nuclease-resistant with high affinity and specificity for protein targets, thioaptamers have been evaluated for their therapeutic potential in vitro by Yang and colleagues at the University of Texas Medical Branch, who reported in 2006 that one thioaptamer molecule, a double-stranded DNA oligo with a monothio-phosphate modified backbone, could be used to inhibit HIV infection.
About a year later, Yang and others from the school published in vivo data on the use of thioaptamers for treating infections caused by arenaviruses.
Currently, AM markets DNA thioaptamers and thiophosphoramidite reagents through partner Glen Research. However, the growing interest in RNAi has prompted AM to see if its technology can be used to create siRNAs with improved properties.
“This is a natural outgrowth of the work we’ve been doing on phosphorodithioates in DNA oligonucleotides,” Mark Shumbera, president of AM, told RNAi News this week. “We’ve found that appropriate modifications of selected positions in a DNA oligonucleotide can lead to aptamers with much higher binding affinities for target proteins and other target molecules,” as well as increased nuclease resistance.
These modifications also “make the molecule achiral, which eliminates any kind of stereoisomer problem that somebody might have with one of their oligonucleotides,” Ross Durland, AM’s director of product development, added.
“We’ve found that appropriate modifications of selected positions in a DNA oligonucleotide can lead to aptamers with much higher binding affinities for target proteins and other target molecules,” as well as increased nuclease resistance.
With the support of the NIGMS grant, AM is now developing “the RNA version of those thiophosphoramidites, which could be used to synthesize synthetic RNA molecules containing dithio modifications in the backbone,” Durland said. “We hope those will have significant applications in a lot of the areas of RNA biotechnology that are so hot these days [such as] RNAi and microRNAs.”
Shumbera compared the application of dithioate modifications to the 2’ O-methyl modifications commonly incorporated into siRNAs “using a special phosphoramidite that has the modification present. What we’re trying to do … is something analogous, except that the special phosphoramidite will be designed to create a dithioate modification instead,” he explained.
According to the abstract of AM’s phase I Small Business Innovation Research grant, the company plans to develop the chemistry and optimize the conditions required to produce four ribonucleoside thiophosphoramidites. It will then demonstrate high coupling yield in the synthesis of phosphorodithioate siRNAs, which will be evaluated for their gene-silencing properties in vitro.
Durland noted that while the grant project has an 18-month term, “we will probably wrap that up faster. We’ve already had significant progress on the grant [work], so I would say probably in 12 months we should [complete] our phase I” effort.
Once this work is completed, “the next step will be to determine [the phosphorodithioate siRNAs’] role and utility in the various RNAi fields,” Shumbera said. “Our intent is, after our phase I SBIR, to move to a phase II that would … determine [if] the dithioate modifications may lead to a more beneficial RNAi product.”
The phase I grant abstract said that phase II work is expected to include scaling up thiophosphoramidite reagent production to commercial-level quantities and quality; optimizing protocols for phosphorodithioate siRNA synthesis; and evaluating the positional effect of the dithioate modifications on siRNA activity in mammalian cells.
AM also plans to use phase II funding to examine the biodistribution and pharmacokinetics of the modified siRNAs with an eye toward marketing them for both research and therapeutic applications.
Shumbera said that AM sees near-term potential for the phosphorodithioate siRNAs as research tools, but that “there will be therapeutic applications if [the modifications] impart the types of characteristics we think they will.”
How the company will market the technology is not yet clear, however, but it is not likely to do so on its own.
“We are currently partnered with Glen Research … to market our DNA thiophosphoramidites, [but] … we haven’t come to any agreements for any of the RNA thiophosphoramidites yet with anyone,” Shumbera said.
Still, given AM’s limited resources as a small, five-employee firm, “we anticipate using a firm such as Glen to do our marketing and distribution [for RNA thiophosphoramidites], as well,” he added.