Through a mechanism known as RNA interference (RNAi), small interfering RNA (siRNA) molecules can target complementary mRNA strands for degradation, thus specifically inhibiting gene expression. The ability of siRNAs to inhibit gene expression offers a mechanism that can be exploited for novel therapeutics. Indeed, over the past decade, at least 21 siRNA therapeutics have been developed for more than a dozen diseases, including various cancers, viruses, and genetic disorders. Like other biological drugs, RNAi-based therapeutics often require a delivery vehicle to transport them to the targeted cells. Thus, the clinical advancement of numerous siRNA drugs has relied on the development of siRNA carriers, including biodegradable nanoparticles, lipids, bacteria, and attenuated viruses. Most therapies permit systemic delivery of the siRNA drug, while others use ex vivo delivery by autologous cell therapy. Advancements in bioengineering and nanotechnology have led to improved control of delivery and release of some siRNA therapeutics. Likewise, progress in molecular biology has allowed for improved design of the siRNA molecules. Here, we provide an overview of siRNA therapeutics in clinical trials, including their clinical progress, the challenges they have encountered, and the future they hold in the treatment of human diseases.
Since the first PEGylated product was approved by the Food and Drug Administration in 1990, PEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation, as the most established technology for extension of drug residence in the body, will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates.
Many types of mammalian cells can aggregate and differentiate into 3-D multicellular spheroids when cultured in suspension or a nonadhesive environment. Compared to conventional monolayer cultures, multicellular spheroids resemble real tissues better in terms of structural and functional properties. Multicellular spheroids formed by transformed cells are widely used as avascular tumor models for metastasis and invasion research and for therapeutic screening. Many primary or progenitor cells on the other hand, show significantly enhanced viability and functional performance when grown as spheroids. Multicellular spheroids in this aspect are ideal building units for tissue reconstruction. Here we review the current understanding of multicellular spheroid formation mechanisms, their biomedical applications, and recent advances in spheroid culture, manipulation, and analysis techniques.
Cancers are complex and heterogeneous pathological "organs" in a dynamic interplay with their host. Models of human cancer in vitro, used in cancer biology and drug discovery, are generally highly reductionist. These cancer models do not incorporate complexity or heterogeneity. This raises the question as to whether the cancer models' biochemical circuitry (not their genome) represents, with sufficient fidelity, a tumor in situ. Around 95% of new anticancer drugs eventually fail in clinical trial, despite robust indications of activity in existing in vitro pre-clinical models. Innovative models are required that better capture tumor biology. An important feature of all tissues, and tumors, is that cells grow in three dimensions. Advances in generating and characterizing simple and complex (with added stromal components) three-dimensional in vitro models (3D models) are reviewed in this article. The application of stirred bioreactors to permit both scale-up/scale-down of these cancer models and, importantly, methods to permit controlled changes in environment (pH, nutrients, and oxygen) are also described. The challenges of generating thin tumor slices, their utility, and potential advantages and disadvantages are discussed. These in vitro/ex vivo models represent a distinct move to capture the realities of tumor biology in situ, but significant characterization work still remains to be done in order to show that their biochemical circuitry accurately reflects that of a tumor.
Poly(butylene succinate) (PBS) and its copolymers are a family of biodegradable polymers with excellent biodegradability, thermoplastic processability and balanced mechanical properties. In this article, production of the monomers succinic acid and butanediol, synthesis, processing and properties of PBS and its copolymers are reviewed. The physical properties and biodegradation rate of PBS materials can be varied in a wide range through copolymerization with different types and various contents of monomers. PBS has a wide temperature window for thermoplastic processing, which makes the resin suitable for extrusion, injection molding, thermoforming and film blowing. Finally, we summarized industrialization and applications of PBS.
Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags.
Its remarkable ease and efficiency make the CRISPR (clustered regularly interspaced short palindromic repeats) DNA editing machinery highly attractive as a new tool for experimental gene annotation and therapeutic genome engineering in eukaryotes. Here, we report a versatile set of plasmids and vectors derived from adeno‐associated virus (AAV) that allow robust and specific delivery of the two essential CRISPR components – Cas9 and chimeric g(uide)RNA – either alone or in combination. All our constructs share a modular design that enables simple and stringent guide RNA (gRNA) cloning as well as rapid exchange of promoters driving Cas9 or gRNA. Packaging into potent synthetic AAV capsids permits CRISPR delivery even into hard‐to‐transfect targets, as shown for human T‐cells. Moreover, we demonstrate the feasibility to direct Cas9 expression to or away from hepatocytes, using a liver‐specific promoter or a hepatic miRNA binding site, respectively. We also report a streamlined and economical protocol for detection of CRISPR‐induced mutations in less than 3 h. Finally, we provide original evidence that AAV/CRISPR vectors can be exploited for gene engineering in vivo, as exemplified in the liver of adult mice. Our new tools and protocols should foster the broad application of CRISPR technology in eukaryotic cells and organisms, and accelerate its clinical translation into humans. The CRISPR/Cas9 system can be exploited for genome engineering in many species. The present study aims at helping to overcome one of the remaining hurdles, which is efficient delivery of the two essential components – Cas9 and gRNA – into cells in vitro or in vivo. Towards this aim, the authors created gene transfer vectors derived from non‐pathogenic Adeno‐associated viruses (AAV) whose parts – Cas9 promoter, Cas9 gene, polyA signal, gRNA promoter and gRNA – can be easily customized. The usefulness of the newly created vectors is shown in various cell types including liver cells of adult mice.
Despite the efforts that bioengineers have exerted in designing and constructing biological processes that function according to a predetermined set of rules, their operation remains fundamentally circumstantial. The contextual situation in which molecules and single-celled or multi-cellular organisms find themselves shapes the way they interact, respond to the environment and process external information. Since the birth of the field, synthetic biologists have had to grapple with contextual issues, particularly when the molecular and genetic devices inexplicably fail to function as designed when tested in vivo. In this review, we set out to identify and classify the sources of the unexpected divergences between design and actual function of synthetic systems and analyze possible methodologies aimed at controlling, if not preventing, unwanted contextual issues.
Biofuels are an attractive means to prevent a further increase of carbon dioxide emissions. Currently, gasoline is blended with ethanol at various percentages. However, butanol has several advantages over ethanol, such as higher energy content, lower water absorption, better blending ability, and use in conventional combustion engines without modification. Like ethanol, it can be produced fermentatively or petrochemically. Current crude oil prices render the biotechnological process economic again. The best-studied bacterium to perform a butanol fermentation is Clostridium acetobutylicum. Its genome has been sequenced, and the regulation of solvent formation is under intensive investigation. This opens the possibility to engineer recombinant strains with superior biobutanol-producing ability.
Industrial biotechnology and microbial metabolic engineering are poised to help meet the growing demand for sustainable, low‐cost commodity chemicals and natural products, yet the fraction of biochemicals amenable to commercial production remains limited. Common problems afflicting the current state‐of‐the‐art include low volumetric productivities, build‐up of toxic intermediates or products, and byproduct losses via competing pathways. To overcome these limitations, cell‐free metabolic engineering (CFME) is expanding the scope of the traditional bioengineering model by using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of target products. In recent years, the unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the development of engineering foundations for cell‐free systems. These efforts have led to activation of long enzymatic pathways (>8 enzymes), near theoretical conversion yields, productivities greater than 100 mg L –1 h –1 , reaction scales of >100 L, and new directions in protein purification, spatial organization, and enzyme stability. In the coming years, CFME will offer exciting opportunities to: (i) debug and optimize biosynthetic pathways; (ii) carry out design‐build‐test iterations without re‐engineering organisms; and (iii) perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility. Cell‐free metabolic engineering (CFME) is the practice of using in vitro ensembles of catalytic proteins prepared from purified enzymes or crude lysates of cells for the production of useful products. By avoiding constraints associated with product toxicity, focusing substrates towards the biosynthesis of a single product, and separating cellular growth objectives from engineering process objectives, CFME complements existing in vivo strategies and expands the scope of metabolic engineering. CFME offers exciting opportunities to debug and optimize enzymatic pathways and perform molecular transformations when bioconversion yields, productivities, or cellular toxicity limit commercial feasibility of whole‐cell fermentation.
Inkjet bioprinting is one of the most promising additive manufacturing approaches for tissue fabrication with the advantages of high speed, high resolution, and low cost. The limitation of this technology is the potential damage to the printed cells and frequent clogging of the printhead. Here we developed acrylated peptides and co‐printed with acrylated poly(ethylene glycol) (PEG) hydrogel with simultaneous photopolymerization. At the same time, the bone marrow‐derived human mesenchymal stem cells (hMSCs) were precisely printed during the scaffold fabrication process so the cells were delivered simultaneously with minimal UV exposure. The multiple steps of scaffold synthesis and cell encapsulation were successfully combined into one single step using bioprinting. The resulted peptide‐conjugated PEG scaffold demonstrated excellent biocompatibility, with a cell viability of 87.9 ± 5.3%. Nozzle clogging was minimized due to the low viscosity of the PEG polymer. With osteogenic and chondrogenic differentiation, the bioprinted bone and cartilage tissue demonstrated excellent mineral and cartilage matrix deposition, as well as significantly increased mechanical properties. Strikingly, the bioprinted PEG‐peptide scaffold dramatically inhibited hMSC hypertrophy during chondrogenic differentiation. Collectively, bioprinted PEG‐peptide scaffold and hMSCs significantly enhanced osteogenic and chondrogenic differentiation for robust bone and cartilage formation with minimal printhead clogging. Inkjet bioprinting is a promising approach for tissue fabrication, yet this technology is limited by potential damage to the printed cells and frequent clogging of the printhead. This study demonstrates the feasibility of using inexpensive and commercially available inkjet printers modified for advanced cartilage and bone tissue fabrication, with minimal printhead clogging. The resulting scaffold favors both osteogenic and chondrogenic differentiation of human stem cells, potentially providing engineered bone and cartilage tissue with enhanced biocompatibility and biodegradability.
Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Here, we review the advances in modeling and synthetic biology tools and how these tools can speed up the development of yeast cell factories. We also present an overview of metabolic engineering strategies for developing yeast strains for production of polymer monomers: lactic, succinic, and cis , cis ‐muconic acids. S. cerevisiae has already firmly established itself as a cell factory in industrial biotechnology and the advances in yeast strain engineering will stimulate development of novel yeast‐based processes for chemicals production. Industrial biotechnology enables production of chemicals from renewable feedstocks. Yeast Saccharomyces cerevisiae is an important industrial host for production of enzymes, pharmaceutical and nutraceutical ingredients and recently also commodity chemicals and biofuels. Metabolic modeling and synthetic biology tools can speed up the development of yeast cell factories.
Bioprinting based on thermal inkjet printing is a promising but unexplored approach in bone tissue engineering. Appropriate cell types and suitable biomaterial scaffolds are two critical factors to generate successful bioprinted tissue. This study was undertaken in order to evaluate bioactive ceramic nanoparticles in stimulating osteogenesis of printed bone marrow‐derived human mesenchymal stem cells (hMSCs) in poly(ethylene glycol)dimethacrylate (PEGDMA) scaffold. hMSCs suspended in PEGDMA were co‐printed with nanoparticles of bioactive glass (BG) and hydroxyapatite (HA) under simultaneous polymerization so the printed substrates were delivered with highly accurate placement in three‐dimensional (3D) locations. hMSCs interacted with HA showed the highest cell viability (86.62 ± 6.02%) and increased compressive modulus (358.91 ± 48.05 kPa) after 21 days in culture among all groups. Biochemical analysis showed the most collagen production and highest alkaline phosphatase activity in PEG‐HA group, which is consistent with gene expression determined by quantitative PCR. Masson's trichrome staining also showed the most collagen deposition in PEG‐HA scaffold. Therefore, HA is more effective comparing to BG for hMSCs osteogenesis in bioprinted bone constructs. Combining with our previous experience in vasculature, cartilage, and muscle bioprinting, this technology demonstrates the capacity for both soft and hard tissue engineering with biomimetic structures. Bioprinting based on thermal inkjet printing is a promising but unexplored approach in bone tissue engineering. The authors demonstrate the feasibility of using the inexpensive and commercially available inkjet printers modified for advanced bone tissue fabrication. For the first time, the ceramic nanoparticles and stem cells are successfully co‐printed for bone tissue construction in a layer by layer fashion. Bioprinting based on thermal inkjet printing technology demonstrates the capacity for both soft and hard tissue engineering with biomimetic structures.
Magnetic-nanoparticle-mediated intracellular hyperthermia has the potential to achieve localized tumor heating without any side effects. The technique consists of targeting magnetic nanoparticles to tumor tissue followed by application of an external alternating magnetic field that induces heat through Neel relaxation loss of the magnetic nanoparticles. The temperature in tumor tissue is increased to above 43 degrees C, which causes necrosis of cancer cells, but does not damage surrounding normal tissue. Among magnetic nanoparticles available, magnetite has been extensively studied. Recent years have seen remarkable advances in magnetite-nanoparticle-mediated hyperthermia; both functional magnetite nanoparticles and alternating-magnetic-field generators have been developed. In addition to the expected tumor cell death, hyperthermia treatment has also induced unexpected biological responses, such as tumor-specific immune responses as a result of heat-shock protein expression. These results suggest that hyperthermia is able to kill not only local tumors exposed to heat treatment, but also tumors at distant sites, including metastatic cancer cells. Currently, several research centers have begun clinical trials with promising results, suggesting that the time may have come for clinical applications. This review describes recent advances in magnetite nanoparticle-mediated hyperthermia.
From biomass to renewable chemicals: while industrial biotechnology offers a clear value proposition, a number of hurdles need to be addressed to fully realize the commercial potential of bio-based products and chemicals over the coming decade. A review of an early roadmap for biological production of chemicals from renewable sugars reveals a focus on those that would provide co-products for integrated biorefineries producing biofuels and bioenergy. A growing number of companies are now focusing on specialty chemicals as an entry point to build the bio-based economy.
Synthetic control of gene expression is critical for metabolic engineering efforts. Specifically, precise control of key pathway enzymes (heterologous or native) can help maximize product formation. The fundamental level of transcriptional control takes place at promoter elements that drive gene expression. Endogenous promoters are limited in that they do not fully sample the complete continuum of transcriptional control, and do not maximize the transcription levels achievable within an organism. To address this issue, several attempts at promoter engineering have shown great promise both in expanding the cell-wide transcriptional capacity of an organism and in enabling tunable levels of gene expression. Thus, this review highlights the recent advances and approaches for altering gene expression control at the promoter level. Furthermore, we propose that recent advances in the understanding of transcription factors and their DNA-binding sites will enable rational and predictive control of gene expression.
The cost-effective production of biofuels from renewable materials will begin to address energy security and climate change concerns. Ethanol, naturally produced by microorganisms, is currently the major biofuel in the transportation sector. However, its low energy content and incompatibility with existing fuel distribution and storage infrastructure limits its economic use in the future. Advanced biofuels, such as long chain alcohols and isoprenoid- and fatty acid-based biofuels, have physical properties that more closely resemble petroleum-derived fuels, and as such are an attractive alternative for the future supplementation or replacement of petroleum-derived fuels. Here, we review recent developments in the engineering of metabolic pathways for the production of known and potential advanced biofuels by microorganisms. We concentrate on the metabolic engineering of genetically tractable organisms such as Escherichia coli and Saccharomyces cerevisiae for the production of these advanced biofuels.
The technological utility of biomolecules (e.g. proteins, enzymes and DNA) can be significantly enhanced by combining them with ionic liquids (ILs) – potentially attractive ”green“ and ”designer“ solvents – rather than using in conventional organic solvents or water. In recent years, ILs have been used as solvents, cosolvents, and reagents for biocatalysis, biotransformation, protein preservation and stabilization, DNA solubilization and stabilization, and other biomolecule‐based applications. Using ILs can dramatically enhance the structural and chemical stability of proteins, DNA, and enzymes. This article reviews the recent technological developments of ILs in protein‐, enzyme‐, and DNA‐based applications. We discuss the different routes to increase biomolecule stability and activity in ILs, and the design of biomolecule‐friendly ILs that can dissolve biomolecules with minimum alteration to their structure. This information will be helpful to design IL‐based processes in biotechnology and the biological sciences that can serve as novel and selective processes for enzymatic reactions, protein and DNA stability, and other biomolecule‐based applications. The technological utility of biomolecules (e.g., proteins, enzymes and DNA) can be enhanced significantly by their employment in ionic liquids (ILs) – a potentially attractive ”green“ and ”designer“ solvent – rather than in conventional organics solvents or in aqueous media. The use of these biomolecules in ILs can dramatically increase the solubility, enhance activity and improve stability (both structural and chemical).
Mesenchymal stem cells (MSCs) respond to the elasticity of their environment, which varies between and within tissues. Stiffness gradients within tissues can result from pathological conditions, but also occur through normal variation, such as in muscle. MSC migration can be directed by shallow stiffness gradients before differentiating. Gradients with fine control over substrate compliance both in range and rate of change (strength) are needed to better understand mechanical regulation of MSC migration in normal and diseased states. We describe polyacrylamide stiffness gradient fabrication using three distinct systems, generating stiffness gradients of physiological (1 Pa/m), pathological (10 Pa/m), and step change ( 100Pa/m) strength. All gradients spanned a range of physiologically relevant elastic moduli for soft tissues (112 kPa). MSCs migrated to the stiffest region on each gradient. Time-lapse microscopy revealed that migration velocity correlated directly with gradient strength. Directed migration was reduced in the presence of the contractile agonist lysophosphatidic acid (LPA) and cytoskeleton-perturbing drugs nocodazole and cytochalasin. LPA- and nocodazole-treated cells remained spread and protrusive on the substrate, while cytochalasin-treated cells did not. Nocodazole-treated cells spread in a similar manner to untreated cells, but exhibited greatly diminished traction forces. These data suggest that a functional actin cytoskeleton is required for migration whereas microtubules are required for directed migration. The data also imply that, in vivo, MSCs may preferentially accumulate in regions of high elastic modulus and make a greater contribution to tissue repairs in these locations.
Recent advances in organ printing technology for applications relating to medical interventions and organ replacement are described. Organ printing refers to the placement of various cell types into a soft scaffold fabricated according to a computer-aided design template using a single device. Computer aided scaffold topology design has recently gained attention as a viable option to achieve function and mass transport requirements within tissue engineering scaffolds. An exciting advance pioneered in our laboratory is that of simultaneous printing of cells and biomaterials, which allows precise placement of cells and proteins within 3-D hydrogel structures. This advance raises the possibility of spatially controlling not only the scaffold structure, but also the type of tissue that can be grown within the scaffold and the thickness of the tissue as capillaries and vessels could be constructed within the scaffolds. Here we summarize recent advances in printing cells and materials using the same device.