Benner S.,Firebird Biomolecular Sciences, LLC
Accounts of Chemical Research | Year: 2017
Life and the Earth have coevolved over the past four billion years to deliver a rich diversity of biological structure, from biomolecules to macrophysiology. One grand challenge seeks to interconnect these structures, in ways acceptable to both natural historians and physical scientists, to give an interconnecting web of models and experiments to create a planetary understanding of the phenomenon that we call "life". The molecular scientist wants experiments; the natural historian wants reference to Darwinian fitness. Paleogenetics offers both. © 2017 American Chemical Society.
Agency: Department of Defense | Branch: Army | Program: STTR | Phase: Phase II | Award Amount: 750.00K | Year: 2012
Because of chemical behaviors of real DNA molecules, the nanotechnology envisioned by the Defense Department in this Solicitation will be possible only if design automation software creates nanostructures that exploit artificially expanded genetic information systems (AEGIS). AEGIS DNA molecules have more than the four nucleotides (GACT) found in natural DNA. In Phase 1, we delivered a demonstration project showing that adding nucleotides to standard DNA supports three-dimensional self-assembling nanoarchitectures at the frontier of the field. We also improved several of the AEGIS components. This progress will allow Phase 2 to deliver kits for specialists, non-specialists, and students that combine AEGIS DNA with non-DNA linkers to give a nano AEGIS"Lego"kit. We will also deliver a nanostructure as a signaling element to detect DNA from insect-borne pathogens that cause rickettsial diseases (e.g. typhus). This project overlaps Firebird's business, which supports human diagnostics based on reagent innovations like AEGIS. Adding nanotechnology to its existing business makes commercial sense, and will help"dual uses"emerge from this project.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 169.58K | Year: 2015
DESCRIPTION provided by applicant Only a few inexpensive drugs can be used in the developing world for the treatment of AIDS Several of these target the HIV reverse transcriptase RT Accordingly patient therapy with these drugs often fails when the gene encoding RT undergoes mutation Thus the WHO recently reported that after months of treatment with anti RT drugs patients most often relapsed when the following mutations arose in RT a for nevirapine K N and Y C b for tenofovir and d T K R c for TC and FTC M V and d for thymidine analogs D N Therefore both for surveillance and for immediate patient care the NIAID and the CDC issued an SBIR solicitation for commercial technology transfer to develop an assay that could quickly and inexpensively detect these five mutations in patient samples in a resource limited environment The solicitation sought as a benchmark specification a level of detection LOD of molecules mL The work proposed here will deliver this assay with a much better LOD molecules mL exploiting technology developed under an NIAID R to the FfAME that ends in Thus an STTR grant format has been chosen to deliver an assay with the following features that make it easy and inexpensive The assay will do multiplexed amplification for regions of the RT gene that contain resistance conferring mutations in one assay avoiding the cost of five separate assays for each of the alleles This performance specification is possible because of FfAME Firebird self avoiding molecular recognition systems SAMRS The assay will exploit the isothermal helicase dependent amplification HDA not standard PCR This avoids both the cost of a PCR instrument and the power demands of thermal cycling Multiplexed HDA relies on technology recently developed in the Benner laboratory under the NIAID R grant including SAMRS reverse transcriptase HDA which has a level of detection LOD of molecules To ensure high coverage multiple primers covering of the sequence diversity surrounding the target site will be used SAMRS by preventing primer primer interactions makes this multiplicity of primers possible and allows them to be expanded without needing to redesign the multiplex The assay will amplify target xNA before SNP detection exploiting the very low noise of nested PCR using the FfAME Firebird technology known as andquot artificially expanded genetic information systemsandquot AEGIS The assay will detect amplicons using andquot orthogonal beaconsandquot also developed here These also rely on AEGIS technology to suppress background noise allowing detection by eye of as few as amplicons Readout will use immobilized beacons with fluorescence generated by a hand held battery operated LED with diagnosis made on the spot or if captured by a cell phone camera at a remote evaluation center Should cross reactivity be observed in Phase it will be reduced using aminoxy reversible terminators with engineered polymerases another innovation coming from the Benner laboratory PUBLIC HEALTH RELEVANCE Five mutations in the gene encoding HIV reverse transcriptase RT are commonly found when therapy with inexpensive anti RT drugs fails Recently the NIAID issued a call for innovative technologies that could inexpensively detect these mutations near points of care as assays based on these technologies could help optimize second line therapeutic regimens in the developing world as well as assist in surveillance around the world The work proposed here will deliver these assays by combining five separate chemical innovations exclusive to the applicant organizations FfAME and Firebird
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 150.00K | Year: 2015
DESCRIPTION provided by applicant Technology to deliver molecules andquot on demandandquot that bind to targets or catalyze reactions of a technologistandapos s choosing would have enormous commercial value in research manufacturing and medicine Thus this has been a andquot holy grailandquot of molecular science for a half century Even today chemical theory is inadequate to support de novo design of receptors ligands or catalyst having useful affinities or catalytic power Thus many have sought to bring Darwinism into the laboratory Here the hope is to re create under control of a technologist the evolutionary processes that create the powerful receptors ligands and catalysts found in Nature One approach to create laboratory Darwinism was begun by Gold Szostak and others to exploit the intrinsic ability of natural DNA and RNA collectively xNA molecules to direct their own replication These and others developed the technology that we andquot laboratory in vitro evolutionandquot LIVE which places libraries of xNA molecules under laboratory selection pressure to extract those that bind to targets Unfortunately because natural xNA molecules have only four building blocks with few functional groups they have low information density ambiguous folding and little intrinsic functional power and therefore have seen little commercial use Using synthetic biology and a second generation artificially expanded genetic information system AEGIS FfAME and Firebird scientists have created a fundamental breakthrough in LIVE technology This involves adding initially two but potentially up to eight additional nucleotides to standard xNA These can carry not only functionality found in natural amino acids but functionality in addition to that seen in natural proteins including antibodies which provide current state of the art technology for creating binding molecules To lay the grounds for this commercialization proposal we used AEGIS LIVE to create AEGIS aptamers for customers who were seeking molecules that bind specifically to breast and liver cancer cells This grant will fund the next logical step commercializing AEGIS LIVE via two overlapping business models a A custom research collaboration model where Firebird staff provide an AEGIS LIVE service at a fee to generate receptors ligands and catalysts that are chosen by customers and clients b A product generation model where Firebird uses AEGIS LIVE to generate receptors ligands and catalysts of its own choosing as commercial products in their own right Phase will perform AEGIS LIVE that for the first time targets a specific defined molecule glypican a protein that is key to the lethality of many cancers AEGIS LIVE will be compared with a standard LIVE without AEGIS as well as antibodies for the same protein target AEGIS aptamers will be delivered to our customer Dr Chen Liu at University of Florida to support his liver cancer research As well as generating AEGIS aptamers having value in their own right this work will demonstrate the flexibility of AEGIS LIVE develop supporting infrastructure and launch commercialization of technology that NIH referees note is andquot a game changerandquot PUBLIC HEALTH RELEVANCE Technology that delivers receptors and ligands with rapid turn around will ultimately capture markets worth tens of billions of dollars for therapeutics diagnostics and environmental surveillance among others Using synthetic biology and artificially expanded genetic systems researchers in the two collaborating organizations have created a fundamental breakthrough in this technology by combining chemical andquot coarse designandquot with laboratory evolution andquot fine tuningandquot This breakthrough has been proven through published work that provides detailed recipes to generate receptors that bind specifically to breast and liver cancer cells This project will now commercialize this technology Its proof of concept Phase will for the first time generate receptors that bind to defined molecular targets involve in cancer treatment These are commercial products in their own right but also will serve as demonstrations of the power of this technology that will attract contract research
Agency: Department of Defense | Branch: Army | Program: SBIR | Phase: Phase I | Award Amount: 100.00K | Year: 2014
Firebird will deliver a hand-held CLIA-waivable battery-powered device that will allow point-of-need detection of norovirus RNA, with a fluorescence readout excited by a battery-powered LED. The readout can be interpreted at the point-of-need, or transmitted by cell phone to a distant site. This devise is possible only through the application of five technologies to which Firebird has exclusive access: (a) Artificially expanded genetic information systems (AEGIS), which allow low-noise amplification of DNA and RNA (collectively xNA) from a sample; (b) self-avoiding molecular recognition systems (SAMRS), which allow introduction of multiple primers and probes to give highly multiplexed and highly flexible xNA-targeted assays; (c) isothermal xNA amplification architectures using helicase-dependent amplification (HDA), which allows xNA to be targeted without the thermocycling needed for PCR (PCR will not be forward-deployed by the Army); (d) AEGIS beacons, which allow signal readout without interference from competing natural xNA, allowing ?soup-to-nuts assays? without sample maniputations any more complicated than mixing; (e) The StrainTargeter bioinformatics platform, which supports the design of xNA-targeted assays for viruses (such as the norovirus) that are rapidly adapting. In Phase 1, the reagent support will be developed, with the Option period devoted to testing on Trisol inactivated norovirus samples.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 149.69K | Year: 2015
DESCRIPTION provided by applicant The public health challenges from RNA viruses are at a tipping point with dengue moving up Florida human transmission of chickungunya now established in Florida and transmitted by different mechanism Ebola While these have long been a challenge their incidence has been predominantly overseas In addition the health hazards of other RNA viruses better known in the United States remains unabated Eastern equine encephalitis is again emerging in New England with its mortality rate West Nile virus continues as an important health hazard Further given the speed of international travel and the ease with which the sequences of RNA viruses mutate a public health official can easily have a mosquito that carries an RNA virus from one of these classes that is nevertheless not detected by PCR with standard primers This creates a need for exactly the product that Firebirdandapos s technologies enable These technologies are Self avoiding molecular recognition systems SAMRS SAMRS primers do not interact with each other This allows unlimited multiplexing of PCR Further SAMRS allows additional targets to be added to an assay without the multiplex collapsing SAMRS also suppresses primer dimers in isothermal amplification e g RPA HDA making possible in Phase point of sampling kits SAMRS therefore supports a highly adaptable molecular diagnostics able to detect dozens of RNA species for essentially the cost of detecting one Artificially expanded genetic information systems AEGIS AEGIS adds nucleotides to the four in standard DNA and RNA collectively xNA Thus AEGIS generates primers that cannot complement any natural DNA no matter how complex the sample supporting assays with very low noise and very few false positives Universal base technology Biversals RNA viruses easily mutate exemplifying the consequences to public health recent surveillance of HIV infected patients with the goal of measuring incidence found that the viruses in of the isolates were not PCR amplified by standard primers Firebirdandapos s evolutionary analyses show that mutations often occur in silent sites in coding regions Thus Firebird scientist invented andquot biversalandquot nucleobases that prime on either A or G the Y biversal or on either T or C the R biversal Phase will generate a kit to detect common mosquito borne RNA viruses allowing public health staff to screen on a Luminex platform for all of these for less than $ less than the cost of detecting any two RNA viruses separately In Phase these will be moved to simpler platforms Firebirdandapos s pipeline investments allow us to prepare AEGIS and SAMRS building blocks in multi gram amounts and AEGIS and SAMRS oligonucleotides on demand Because surveillance assays are not heavily regulated and since its customers for those kits public health staff are well trained this product fits well within Firebirdandapos s current product line However strong performance of a low cost highly multiplexed easily adaptable and low false positive surveillance product will undoubtedly help Firebird raise capital for patient targeted applications of these technologies PUBLIC HEALTH RELEVANCE The public health challenges presented by RNA viruses are this year reaching a tipping point with dengue moving its way up the Florida peninsula human to human transmission of chickungunya now established in the United States which until this year was always imported from an exotic tropical locale Eastern equine encephalitis now being detected in New England with a mortality rate and of course operating by an entirely different mechanism of transmission Ebola which is uncontrolled at this moment in Western Africa This creates a need for exactly the product that Firebirdandapos s technologies enable a highly adaptable molecular diagnostics assay that is able to detect dozens of RNA species for essentially the same cost as detecting a single RNA virus design to manage the rapid divergence of these species and capable of extraordinarily few false positives This project will develop kits that allow a single public health official in a single step to test real biological samples for example mosquitoes caught in a single trap for all RNA viruses at the same time and with the same total cost as a test for any one of them separately Since such surveillance kits have few regulatory barriers Firebird will be able to bring these kits to market on its own before the end of the Phase project period
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 150.00K | Year: 2014
Firebird will deliver multiplexed assays that inexpensively detect and distinguish coccidioidomycosis, blastomycosis, and histoplasmosis by targeting the genomic DNA of their causative agents. This assay uses six Firebird technologies: 1. Artificially expanded genetic information systems (AEGIS), which support ultra-clean DNA amplification with ultra-low noise despite the complexity of realbiological samples. 2. Self-avoiding molecular recognition systems (SAMRS), which permit unlimited multiplexing of xNA targeted assays, and (consequently) low cost and extreme adaptability in such assays. 3. Conversion technologies that create AEGIS-containing amplicons easily detected by Luminex without interference from any naturally found xNA. 4. Evolution-based software to select genomic targets for genus and species discrimination. 5. Novel reagent-enzyme combinations that make fungal xNA accessible to amplification. 6. Novel isothermal amplification methods to allow the assays to work near points-of-care. The initial product will address the interest of the NCEZID in analyzing environmental samples, to avoid costly FDA regulation. Firebird's investment in pipelines allows us to prepare AEGIS and SAMRS building blocks in multi-gram amounts, and their oligonucleotides on demand. Thus, this product fits well within Firebird's current product line. Specific diagnosis of fungal infections will enhance public health by providing a mechanism to save lives and prevent death serious disease.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 150.00K | Year: 2016
DESCRIPTION provided by applicant To many notably Craig Venter andquot synthetic biologyandquot means simply andquot synthesizing large amounts of DNAandquot This type of synthetic biology actually began in the andapos s when Caruthers introduced a phosphoramidite based solid phase DNA synthesis architecture This allowed ICI and the Benner group to synthesize complete genes encoding biomedically useful proteins and enzymes for the first time The second example showed how biotechnological goals could better be met if the synthetic sequences were different from the sequences presented by Nature through codon optimization and watermarking inter alia Subsequent developments now allow semi routine synthesis of genes these are used in biotechnology gene therapy RNA therapy and elsewhere Extrapolation suggests that routine synthesis of whole genomes will soon be possible hopefully for less than the ca $ million spent to synthesize one in the Venter laboratory Unfortunately DNA has a rich biophysical chemistry that defeats any architecture that relies on autonomous assembly to make large DNA L DNA constructs by simply mixing synthetic fragments even within recombinogenic organisms However two innovations from the FfAME provide a new approach to creating L DNA constructs The first is an andquot artificially expanded genetic information systemandquot AEGIS AEGIS is a DNA like molecule that adds eight additional nucleobases that form four additional pairs the Z P S B K X V J pairs to the four natural nucleotides which form G C and A T pairs By increasing information density of DNA and avoiding non canonical interactions AEGIS allows autonomous self assembly of dozens of fragments to generate L DNA The second innovation is andquot transliterationandquot technology Transliteration allows rule based replacement of AEGIS nucleotides by standard nucleotides after a L DNA assembly is complete By converting S B Z and P to T A C and G respectively the AEGIS components can be replaced after they have served their role to guide autonomous self assembly converting GACTSBZP L DNA to entirely standard GACTTACG DNA To persuade commercial partners to engage with this technology FfAME scientists demonstrated this strategy using the simpler GACTSB six nucleotide AEGIS DNA to give in one assembly step an active full length and sequence correct gene encoding kanamycin resistance Parallel attempts with standard base DNA failed Phase project will transfer these innovations to Firebird which will deliver L DNA and whole plasmids by custom synthesis using an eight letter GACTSBZP alphabet This will require a adapting OligArch software to support design with this strategy b creating a pipeline to synthesize DNA nucleotide fragments using this alphabet and c providing demonstration products plasmids coding multiple enzymes for complete metabolic pathways to natural products assembled in a single step In Phase this technology will be merged with Firebirdandapos s E coli SEGUE strain Second Example of Genetics Undergoing Evolution that manages expanded DNA alphabets in cloning vehicles with one week andquot order to cloneandquot times for plasmids and viruses PUBLIC HEALTH RELEVANCE Whole gene synthesis today has a major commercial market in biomedical research and clinical practice with for example genes that include an RNA polymerase promoter being used to generate whole messenger RNA transcripts to be used in RNA based gene therapy The expensive part of whole gene construction is not the synthesis of the primary DNA oligonucleotide gene fragments which have now become quite inexpensive Rather the cost is the assembly of those fragments to give the full gene a process that requires considerable human involvement and risk of failure Recently scientists at the Foundation for Applied Molecular Evolution reported a technology that allows autonomous self assembly of dozens of the gene fragments largely without human attention Merritt et al Once transferred to Firebird this technology will support large DNA construction with applications ranging from biomanufacturing to diagnostics to therapy
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase I | Award Amount: 136.01K | Year: 2016
Commercial Polymerases to Create Receptors Ligands and Catalysts on Demand Firebird Biomolecular Sciences LLC University of Texas Steven A Benner Andrew D Ellington ABSTRACT Innovators at Firebird Biomolecular Sciences LLC have created several generations of an artificially ex panded genetic information system AEGIS that adds eight nucleobases that form four additional pairs Z P S B K X V J to the four natural nucleotides that form the standard G C and A T pairs AEGIS components carry functional groups not found in natural DNA and RNA collectively xNA NH SH COOH as well as some not found in any natural biopolymer e g B OH and ONH Thus AEGIS is a new kind of polymer replicable evolvable and adaptable like DNA but also having much of the functional potential of proteins In DNA AEGIS has been remarkably successful Six letter GACTSB AEGIS DNA enables two FDA approved products that measure hepatitis and HIV viral loads here the ability of AEGIS DNA to pair only with AEGIS DNA and not to background xNA orthogonality allowed these products to earn ca $ million each year in their ten year life The orthogonality of GACTZP AEGIS DNA as well as its ability to be replicated with high fidelity by DNA polymerases are today exploited in diagnostics and surveillance for respiratory disease viruses common and exotic arboviruses in mosquitoes noroviruses and coronaviruses e g SARS MERS Firebird now sees the opportunity to expand its product portfolio from AEGIS DNA to include AEGIS RNA Its scientists recently reported large scale syntheses of AEGIS RNA triphosphates and phosphoramidites these are now sold in its catalog Further Firebird scientists have shown that T RNA polymerase makes AEGIS RNA from AEGIS DNA although inefficiently and found reverse transcriptases that make AEGIS DNA from AEGIS RNA in both six and eight letter versions e g GACTSBZP These are leads for the proposed research RNA is well known to have a richer set of folds and more intrinsic potential to serve as receptors and ligands aptamers a term and technology invented by the subcontractor Functionalized AEGIS RNA will thus likely be better than AEGIS DNA in these biomolecules Functionalized AEGIS RNA will also have enhanced value in laboratory in vitro evolution LIVE experiments to create receptors ligands and catalysts on demand Should this vision be realized it will create a revolutionary transformation of diagnostics and therapeutics This vision requires better RNA polymerases and reverse transcriptases While Firebird could undertake their development in house advances in the Ellington laboratory at the University of Texas suggests that collabora tion would achieve this goal faster Ellington has already created directed evolution platforms to give T RNA polymerases and thermostable reverse transcriptases that accept unnatural but not AEGIS nucleotides This collaboration will apply those platforms to create enzymes that interconvert AEGIS DNA and AEGIS RNA The enzymes themselves will be commercial products they will also however support LIVE with this evolvable functionalized AEGIS biopolymer Phase will show the feasibility of using Ellington s platforms to create these enzymes Given feasibility the platforms will be applied in Phase to complete letter AEGIS alphabets Commercial Polymerases to Create Receptors Ligands and Catalysts on Demand Firebird Biomolecular Sciences LLC University of Texas Steven A Benner Andrew D Ellington NARRATIVE This project will generate for sale as well as for internal use at Firebird and the University of Texas RNA polymerases and reverse transcriptases that interconvert DNA and RNA built from a new type of biopolymer one that adds eight nucleobases that form four additional pairs Z P S B K X V J to the four natural nucleotides which form two natural pairs in the double helix G C and A T These polymerases acting with this artificially expanded genetic information system AEGIS have biomedical value as a result of their ability to create by laboratory in vitro evolution AEGIS RNA products that serve as receptors ligands and catalysts on demand AEGIS DNA and its distinct properties already have supported hundreds of millions of dollars worth of diagnostics products The RNA products that this research enableswill be used not only in diagnostics and therapy but also as research tools to help understand the newly discovered roles for RNA in natural and diseased biology
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 147.12K | Year: 2016
Eliminating Malaria from Haiti Reinventing DNA to Eradicate Endemic Parasites Firebird Biomolecular Sciences LLC University of Florida Steven A Benner John B Dame Abstract Eradication of Plasmodium falciparum the parasite that causes the most malarial deaths is the only way to finally treat the disease in any specific geographical region Haiti is one such region where a small population million geographical isolation and special ecology make eradication a real possibility Thus the Gates Foundation has just committed $ MM to the CDC Foundation to attempt this eradication Key to eradication is an assay that identifies human carriers asymptomatic individuals with active parasite infections That assay must a Require only a small sample of blood perhaps L b Detect very small numbers of living parasites perhaps as few as parasites per sample c Be as simple to operate as a conventional malarial rapid diagnostics test RDT and d cost less than $ to run These specifications are virtually impossible with classical molecular diagnostics However Firebird has developed many innovations over the past three years that make such specs possible including a sample prep processes that remove biohazard b whole nucleic acid capture without centrifugation c isothermal nucleic acid amplification using self avoiding molecular recognition systems SAMRS and artifi cially expanded genetic information systems AEGIS and d AEGIS molecular beacons Firebird has shown that these support assays that for example detect mosquito borne RNA viruses in one mosquito carcass The work is guided by work in the Dame lab showing that P falciparum ribosomal RNA present at copies per parasite can be detected in malaria infected blood using RT PCR The PCR assay does not meet the cost specs necessary for a LRE and is not as easy to run as an RDT but it shows the sensitivity of an assay directed at falciparum rRNA where detection is robust if a sample of blood contains just one organism This project will combine these innovations to develop an assay that detects falciparum rRNA as easily as an RDT but which much higher sensitivity One strength of the approach is its use of realistic samples of live falciparum in real blood to do benchmarking Costing less than $ the assay will generate fluorescence if falciparum is present that can be read directly or transmitted by cell phone camera for remote confirmation We will benchmark i a sample preparation work flow that releases P falciparum rRNA that is ii captured on a solid support where iii captured rRNA is amplified isothermally using primers with SAMRS and AEGIS nucleotides with iv amplicons detected by AEGIS beacons Results will be compared to samples analyzed by RT PCR Metrics for success include an ability to detect parasites in a sample In Phase the efficacy of the test will be validated in a small field trial with blood from individuals from in a malaria endemic area of Haiti again comparing with RT PCR This will as well support the epidemiological science behind carrier identifica tion and eradication as we still do not know the lowest level of parasitemia that can remain stably in an individual The product should gain WHO and Gates Foundation support and yield LRE tests for other agents Eliminating Malaria from Haiti Reinventing DNA to Eradicate Endemic Parasites Firebird Biomolecular Sciences LLC University of Florida Steven A Benner John B Dame NARRATIVE With a population of less than million the absence of non human hosts and geographical isolation Haiti on the island of Hispaniola presents an ideal opportunity to eradicate Plasmodium falciparum the parasite that causes malaria The success of this program and such programs in other jurisdictions depends on the availability of an inexpensive assay to detect the parasite in human carriers individuals who are infected but display no symptoms This work will deliver such an assay one that targets falcimarum ribosomal RNA but is as simple to run as a rapid diagnostics test RDT This will benefit not only the Haitian population but also will help in the eradication of malaria elsewhere protect the US from malaria tourism and support work developing the science behind the epidemiology of human carrier reservoirs all while offering a paradigm for diagnostics in low resource environments