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Synthetic biology involves the engineering of proteins, signaling pathways and even whole organisms using modular designs and formats. A major tool of synthetic biology is artificial gene synthesis, which provides a direct means from a conceptualized DNA sequence to the corresponding physical DNA for the construction of a variety of biological components. To date, synthetic biology has often been used to answer fundamental questions in basic research, but now is poised to greatly enhance translational research. In this review, we discuss several translational applications of synthetic biology including the construction of novel diagnostics and vaccines, development of new synthetic pathways for drug screening and biosynthesis, and the creation of engineered viruses and microbes to fight human disease. Together these and other novel translational applications of artificial gene synthesis and synthetic biology have the opportunity to make major advances for improving human health.
Applied translational research directed at preventing, diagnosing and/or treating human disease still faces large gaps between basic scientific research and clinically applicable discoveries. Synthetic biology, involving the synthesis and engineering of biological molecules using modular designs and formats [1, 2], offers novel strategies to bridge the gap in translational research. An important feature of synthetic biology is the increasing application of artificial DNA synthesis to fabricate any DNA sequence, thereby providing a rapid means to create or eliminate restriction enzyme sites, generate single or multiple mutations, optimize codons, and generate new molecules, pathways and even whole genomes . Although the recent construction of the synthetic 1.1 MB genome of Mycoplasma mycoides highlights the extraordinary potential of artificial DNA synthesis technology , smaller scale projects are increasingly impacting many areas of biomedical research. While several excellent in-depth reviews are available on synthetic biology, this review focuses on highlighting current and future translational applications of synthetic biology.
The ability to synthesize and express the coding sequence for any open reading frame (ORF) through artificial gene synthesis may assist in developing diagnostic serologic immunoassays against a comprehensive panel of all known and potential pathogens. Analogous to nucleic acid pathogen arrays , a comprehensive human pathogen antigen panel would be a powerful resource for disease diagnostics and discovery. Unlike nucleic acid detection, which requires the presence of DNA/RNA from the pathogenic organism at the time of sampling, the detection of antibodies against a given pathogen by immunoassays offer the ability to document current and past exposure, which ultimately may be an important covariate for many human diseases.
One clear advantage of artificial gene synthesis for developing antigenic targets is that the corresponding natural DNA is not required, eliminating the time and effort needed to obtain the required template (Figure 1). The ability to define the particular codons in a synthetic gene is also extremely useful to achieve optimal levels of recombinant protein production in the designated bacterial, yeast, and/or mammalian cell expression system. Once the synthetic genes are constructed and cloned, recombinant protein can be produced and used in solid or liquid phase immunoassays. From our experience, artificial genes for antigenic targets in the size range of 300-500 bp are cost-effective and time -saving alternatives to using natural templates for a variety of filarial , helminthic , bacterial proteins  and viruses (Burbelo, unpublished). In these studies, luciferase-tagged antigens were engineered and used with the luciferase immunoprecipitation systems (LIPS) technology to yield diagnostic tests with high sensitivity, specificity, and a large dynamic range of antibody titer detection . These and other technologies employing artificial genes have the capacity to improve humoral response profiling against many important infectious agents by decreasing the time and effort required to generate recombinant proteins for testing.
The ability to detect patient antibodies using proteins expressed from synthetic genes could serve to accelerate human metagenomics towards translational research. Although the entire sequences of many novel genomes can often be rapidly obtained by nucleic acid amplification and deep sequencing methodologies , understanding their role in disease and determining the prevalence of infection in distinct human populations usually lags far beyond their discovery at the nucleic acid level. One important approach for providing evidence for in vivo expression of the pathogen and for performing epidemiological studies is the detection of host humoral responses to the infectious agent. Artificial gene synthesis offers a valuable short-cut from pathogen discovery to immunoassay development (Figure 1A). Now laboratories without physical access to the genomes of newly discovered potential pathogens can design synthetic proteins based on the ORFs of GenBank entries and use the corresponding recombinantly expressed protein to generate immunoassays to detect human antibodies. One example of the clinical application of artificial gene synthesis to immunodiagnostics is the recent discovery of Merkel cell polyomavirus (MCV) and its association with Merkel cell carcinoma, a rare human skin cancer . Two independent groups studied the immunoreactivity and prevalence of MCV infection in controls and Merkel cell carcinoma patients. In one study, Carter et al. obtained the natural VP1 MCV sequence by PCR cloning, performed site-directed mutagenesis to obtain a three amino substitution of VP1 needed to match the relevant MCV strain, and then used this DNA to generate re-combinant VP1 protein . Tolstov et al. employed a more direct route by synthetically cloning the MCV VP1 and VP2 genes using codons optimized for recombinant expression in human cells . From both studies, not only were high titer anti-MCV antibodies detected in over 90% of the patients with Merkel cell carcinoma, but also 50% of the control population had anti-MCV antibodies suggesting that MCV infection is widespread and not sufficient for cancer development, instead requiring additional immuno-suppression and other cofactors [12, 13]. These studies on MCV demonstrate the importance of developing immunoassays for novel agents and highlight the ability of synthetic genes to simplify and accelerate this area of human disease research.
In addition to single antigens, artificial gene synthesis allows the development of multi-epitope and chimeric antigens to achieve high levels of sensitivity and specificity in immunoassays to detect antibodies to infectious agents and even autoantibodies in autoimmune disease. The ability to design highly complex artificial antigens is often not possible using natural sequences. Artificial antigens may also provide higher diagnostic performance than the natural proteins because multiple immunodominant regions can be incorporated to increase the sensitivity of the test by the detection of low affinity antibodies. Specifically, artificial genes offer a fast and easy approach for generating a repeating peptide in a single protein by slightly altering the codons to prevent recombination (Figure 1B). For example, the synthetic protein VOVO combined four different immunodominant epitopes from Borrelia burgdorfi (Bb) including two repeated immunodominant peptides of VlsE and two of OspC . Use of the VOVO antigen in LIPS provided highly sensitive detection of Lyme disease, greater than any single natural Bb antigen and yielded a large dynamic range useful for potentially monitoring patient responses to treatment. Employing repeated peptides from a single recombinant synthetic protein in LIPS or other immunoassay formats may be a useful approach to develop antibody-based tests to other antigens, including those identified from phage display screening. It is also likely that artificial antigens based on bioinformatics, which incorporate consensus protein sequences derived from many different strains or different types of proteins may simplify the assay and provide better diagnostic performance than testing individual, natural strain-specific proteins. Chimeric antigens combining multiple proteins into one single antigen may also simplify testing and improve diagnostic performance (Figure 1C). In conclusion, artificial genes offer an important opportunity to increase the spectrum and quality of immunodiagnostics accelerating the agents relevant to human disease, improving diagnostics and gaining insight into disease pathogenesis.
The ability to synthesize the coding sequences for single proteins or even the entire genome of many viruses and microbes offers additional opportunities for translational research. The first human pathogen to be completely artificially synthesized was the 9 kb poliovirus in 2002 . One year later, the entire bacteriophage-X174 (5,386bp), non-pathogenic in humans, was assembled in only two weeks . Following the sequencing of DNA fragments of the 1918 Spanish influenza virus in 2007, this pandemic virus was resurrected by artificial DNA synthesis . Reconstruction of the genome of other viruses, including the bat severe acute respiratory syndrome (SARS)-like coronavirus, the likely original reservoir for human SARS, was used to study trans-species infection and zoonosis . In the recent outbreak of H1N1, the rapid determination of the exact genome of H1N1 from Mexico enabled many laboratories to immediately use synthetic genes to study the virus and develop vaccines. Moreover, even larger genomes have been synthetically engineered including 582,970 bp for Mycoplasma genitalium in 2008  and 1.1 MB for Mycoplasma mycoides in 2010 . In the case of the Mycoplasma mycoides, Gibson and colleagues were able to transfer this synthetic DNA into a recipient shell of another bacterium and demonstrated that this artificial genome was fully capable of growth and bacterial replication.
Just as they provide a more sensitive method of detecting patient antibodies in diagnostic immunoassays, novel multi-epitope and chimeric genes can be valuable vaccine targets. The design of highly complex artificial vaccine target proteins is often not possible using the natural sequences and in some cases, synthetic target antigens may have higher efficacy in inducing a vaccine response. For example, an artificial antigen was developed as a vaccine to Plasmodium vivax, a malarial species distinct from P. falciparum . This chimeric protein, VMP 001, encompassed two different variants of an immunodominant peptide of each strain, as well as other conserved regions from the circum-porozite protein. VMP 001 protein was also codon-optimized for expression in bacteria. ELISA studies with recombinant VMP 001 protein demonstrated immunoreactivity in 82% of patients acutely infected with P. falciparum . Moreover, immunization of mice with VMP 001 blocked sporozite infectivity suggesting that it might be an efficacious vaccine in humans. These results demonstrate the advantages of employing diverse epitopes in artificially designed proteins for maximizing the efficacy of a specific vaccine.
Bioinformatics can also be used to engineer artificial proteins to match the highly complex antigenic strain variations to induce greater immune response. In one study, extensive bioinformatics was used to design a consensus hemagglutinin surface protein based on 467 sequenced strains of H5N1 influenza virus . The sequence encoding this consensus hemag-glutinin protein, CHA5, was then codon-optimized for expression in mammalian cells. Studies in mice revealed that vaccination with CHA5 induced neutralizing responses to a variety of divergent H5N1 influenza viruses suggesting that this approach may prove useful to protect against many strains of H5N1 influenza viruses. Similarly, synthetic genes could significantly impact the study of immunoreactivity against the large diversity of HIV gp120 and HCV envelope proteins. Here a panel of diverse viral surface antigens could be easily generated and studied by immunoassays offering potential new insights for vaccine targets and development.
An extraordinarily powerful application of artificial whole viral genomes is a procedure called Synthetically Attenuated Virus Engineering (SAVE) . The SAVE approach is based on markedly weakening viruses and potentially other microbes by codon deoptimization (Figure 2A). Despite the fact that SAVE-modified viruses have proteomes identical to the virulent one, these less than optimal codons and codon pairs for the coding sequences render the pathogenic viruses unable to efficiently grow and replicate, thereby allowing the infected host an advantage in mounting a powerful immune response against the weakened virus. In an initial application of the technology, poliovirus expressing deoptimized coding sequences was made relatively innocuous in mice . SAVE has also been used to attenuate influenza viruses for vaccination and provided protection from subsequent challenge with wild type influenza virus . Since this SAVE approach can be performed very rapidly for the small influenza genomes, it may become the method of choice to make live attenuated viruses for vaccine production. In the future, scientists will likely efficiently redesign many other microbial genomes, including those from bacteria and other parasites, by the SAVE approach to generate non-effectively replicating organisms that might elicit a safer and more powerful immune response. Although SAVE does require further testing, this approach will likely provide a universal way to develop safe vaccine candidates for many pathogens because so many regions of the genome are deoptimized preventing reversion and/or recombination to a more virulent form. Many other human pathogenic viruses besides influenza, such as HBV, HCV, HPV, and HIV have relatively small genomes and could be studied further with the application of synthetic biology. In practice, the combination of large scale DNA sequencing, artificial gene synthesis and vaccine development using synthetically-produced antigens could allow for the rapid deployment of a new vaccine in response to a novel infection outbreak.
Due to the modular nature of protein structural and regulatory elements, it is possible to design and engineer new proteins by combining multiple sequence motifs from different proteins, create unique regulatory controls and design other novel pathways . One opportunity for the use of synthetic biology in translational research is to develop new drug screens, modify cells and engineer new synthetic pathways in mammalian cells to treat cancer, neurodegen-eration and infection. One such example is the approach taken by Weber et al. who used synthetic biology to screen for new anti-microbial compounds against Mycobacterium tuberculosis (TB) . A drug screen was developed based on the fact that the potent TB drug ethionamide is made cytotoxic in cells by the EthA enzyme of Mycobacterium tuberculosis. However, the expression of the EthA gene can also be inhibited by the EthA repressor (ETHR) thereby promoting resistance. To design a screen for drugs that would block repressor-mediated inhibition of EthA expression, a new circuit was synthesized. The EthA repressor with its DNA binding domain was fused to the generic transcriptional activator VP16 and then synthetic ETHR operator DNA sequences were coupled to the secreted alkaline phosphatase as the reporter for transcriptional activity . From screening candidate compounds, 2-phenylethyl-butyrate was found to potently inactivate the repressor and promote transcriptional activity. Unlike structure-based identification of potential compounds, this and other in vivo transcriptional screens have the added advantage of identifying compounds which are cell permeable. It seems highly likely that many other drug screens could be engineered for targeting other pathogen pathways. Furthermore, since alterations in transcription factors are a common feature in many cancers, additional drug screens employing synthetically engineered transcription pathways could potentially identify novel drugs useful for cancer therapy.
Synthetic biology also makes it practical and economically feasible to synthesize certain natural compounds, which can be used to treat a variety of human diseases, but are difficult to synthesize and/or are too costly to produce by standard chemical synthesis. In one study, ar-temisinin, an anti-malarial drug precursor normally isolated from the sweet wormwood (Artemisia annua) was synthesized using synthetic biology. Gene transfer and modification of several enzymes involved in this biosynthetic pathway were optimized for expression in yeast . Using this engineered pathway, industrial amounts of arteminsin were produced. The ability to reconstruct and optimize chemical biosynthesis in bacteria, yeast and other microorganisms may allow the large scale production of diverse compounds for drug therapy as an alternative to the cumbersome isolation of these products from their natural sources.
A number of highly useful drugs are derived from natural sources including the anti-cancer drug Taxol isolated from Pacific yew (Taxus brevifolia), the analgesic resiniferatoxin isolated from the resin spurge (Euphorbia resinifera) and many other drugs isolated from plants and microbes. The identification of new therapeutic natural products from bacteria and other natural resources could be greatly enhanced by synthetic biology. In particular, bacteria are a primary source of antibiotic, anti-cancer agents and other drugs. One approach for discovery of these compounds from bacteria involves using DNA from diverse bacterial species to biosynthesize these compounds in vitro . Since the genetic material needed to encode the biosynthesis of these compounds is often tightly clustered in the genome of bacteria, the entire natural product biosynthetic pathway for many of these novel compounds can be heterologously expressed as a single large genomic DNA insert in a recipient bacteria using DNA obtained from random environmental sampling (called eDNA) . Using this approach, antibacterial activity can be directly screened by identifying clones that produce zones of growth inhibition in the bacterial lawn. Once the compounds and pathways are identified, synthetic biology could again be employed to further optimize production of these compounds. Furthermore, if large scale cloning efforts were performed in eukaryotic cells, the resulting compounds could be directly screened for unique biological activities such as for inhibiting cell growth and for modulating signaling activity.
The ultimate use of synthetic biology is to engineer phage, viruses, bacteria and even mammalian cells for treating human disease. One such goal within reach involves destroying antibiotic-resistant bacteria using genetically-engineered bacteriophages. In one study, genetically altered bacteriophages were used to attack the bacterial biofilm, the extracellular matrix secreted by bacteria that plays a role in antibiotic resistance . Here T7 phages were engineered to express dispersin B, a bacterial biofilm degrading enzyme normally expressed in Actinobacillus actinomycetemcomitans. These modified phages were able to kill 99.97% of the biofilm producing cells through the combined activity of killing by phage infection along and the dispersin-based destruction of the polysaccharide biofilm . In another example, the bacterial transcriptional signaling involved in the induction of antibiotic resistance was targeted in E. coli . Here the lexA3 repressor, a protein normally found in bacteria that blocks a key stress response in E. coli, was engineered into the M13 phage and used to express this repressor in phage-infected cells. These engineered M13 phage were highly effective in increasing the susceptibility of the E. coli to be killed by the antibiotics in cell culture and in infected mice. Phage engineered in this manner may be useful treating patients with drug-resistant Mycobacterium tuberculosis. Although genetically-engineered phage are likely to meet regulatory resistance, in principle these engineered bacteriophages are safe for use in humans because they are specific for bacteria, not human cells and could be further engineered for human safety using additional synthetic biological approaches.
In addition to targeted therapy of bacterial infection, microbial genes can also be used to fight cancer. In one study, bacteria were synthetically engineered to invade cancer cells . Here E. coli were engineered to express the in-vasin gene from Yersinia psuedotuberculoisis, which causes mammalian cell membrane ruffling and bacterial cellular uptake into human epithelial cells through activation of the Rac GTPase. To specifically target cancer cells, the invasin gene was transcriptionally engineered under control of the hypoxia-activated transcriptional elements derived from different microbial genes (Figure 2B). E. coli cells expressing hypoxic-regulated invasin were able to invade cancer cells in a cell density dependent fashion related to hypoxia. Alternatively, the natural ability of some bacteria to target hypoxic regions within tumors has also been exploited. In one study, 26 species of anaerobic bacteria were compared for their ability to target colorectal cancer xenografts . The anaerobic bacterium Clostridium novyi looked promising for tumor colonization and oncolysis. Additional modification of Clostridium novyi by genetic deletion of its corresponding lethal toxin revealed that bacterial spores germinated exclusively within the hypoxic regions of cancers and had tumoricidal activity [29, 30]. It is possible that additional engineering of these bacteria with novel anticancer biosynthetic pathways might further increase the power of these bacteria to destroy tumors. These and other approaches may make it practical to use bacteria to specifically target tumor cells.
Although synthetic biology is still in its early phases, the general approach offers new tools to study proteins and genomes. As shown in Table 1, there are several areas that will make applied research proceed more rapidly and effectively with synthetic biology. Some of these advances could come from simply developing better tests for the diagnosis of infectious and autoimmune diseases, while others may come from modifying protein function to identifying new therapeutic compounds. Future studies modifying human cells including stem cells and combining synthetic biology with nanotechnology may potentially represent powerful new tools for treating certain human diseases. Since the field of translational synthetic biology is still expanding, it is likely that additional applications along with new approaches will further accelerate advances in human health.
This research was supported by the Intramural Research Program of the NIDCR, NIH.