Degradable microparticles have broad utility as vehicles for drug delivery and form the basis of several FDA-approved therapies. Conventional emulsion-based methods of manufacturing produce particles with a wide range of diameters (and thus kinetics of release) in each batch. This paper describes the fabrication of monodisperse, drug-loaded microparticles from biodegradable polymers using the microfluidic flow-focusing (FF) devices and the drug delivery properties of those particles. Particles were engineered with defined sizes, ranging from 10 μm to 50 μm. These particles were nearly monodisperse (polydispersity index = 3.9 %). We incorporated a model amphiphilic drug (bupivacaine) within the biodegradable matrix of the particles. Kinetic analysis showed that the release of drug from these monodisperse particles was slower than that from conventional methods of the same average size but a broader distribution of sizes and, most importantly, exhibited a significantly lower initial burst than that observed with conventional particles. The difference in the initial kinetics of drug release was attributed to the uniform distribution of drug inside the particles generated using the microfluidic methods. These results demonstrated the utility of microfluidic FF for the generation of homogenous systems of particles for the delivery of drugs.

Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.

Natural infections expose the immune system to escalating antigen and inflammation over days to weeks, whereas nonlive vaccines are single bolus events. We explored whether the immune system responds optimally to antigen kinetics most similar to replicating infections, rather than a bolus dose. Using HIV antigens, we found that administering a given total dose of antigen and adjuvant over 1-2 wk through repeated injections or osmotic pumps enhanced humoral responses, with exponentially increasing (exp-inc) dosing profiles eliciting >10-fold increases in antibody production relative to bolus vaccination post prime. Computational modeling of the germinal center response suggested that antigen availability as higheraffinity antibodies evolve enhances antigen capture in lymph nodes. Consistent with these predictions, we found that exp-inc dosing led to prolonged antigen retention in lymph nodes and increased Tfh cell and germinal center B-cell numbers. Thus, regulating the antigen and adjuvant kinetics may enable increased vaccine potency.vaccination kinetics | antigen retention | humoral response | computational immunology | germinal center formation S ubunit vaccines based on recombinant protein antigens combined with adjuvants can safely elicit protective humoral immune responses in humans, and they have become a cornerstone of modern public health (1, 2). Recent advances in structure-based vaccine design (3, 4) and progress in the development of adjuvants that are safe and effective for prophylactic vaccines (5) have helped drive the field. However, several challenges remain: A number of protein vaccines, such as candidate vaccines against HIV and malaria, have tended to elicit short-lived immunity (6, 7). In HIV, broadly neutralizing antibodies (BNAbs) isolated from infected patients are generally characterized by high degrees of somatic hypermutation (SHM) (8), but methods to generate such highly mutated antibodies by vaccination remain unknown. SHM occurs in germinal centers (GCs) within lymphoid organs, and data from animal models demonstrate a critical role for follicular helper T cells in the induction of GCs and promotion of affinity maturation (9, 10). To date, methods to promote Tfh generation and long-lived germinal centers during vaccination remain unclear (11-15). Much attention has focused on the use of adjuvants to promote affinity maturation, but it remains unclear if adjuvants alone can provide the necessary immunological driving forces for promoting extensive affinity maturation (16).During acute infections, which often provoke robust germinal center responses and durable humoral immunity, microorganism replication typically occurs over the course of one to several weeks (17-19). During this time, recognition of molecular danger signals contained within the pathogen sustains stimulation of the innate immune system, and a continuous supply of antigen is provided to the adaptive immune system. In contrast to these patterns of antigen and inflammatory cues during infection, typical subunit vaccines...

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50 -70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia. plasma PCSK9 ͉ tissue LDLR levels P roprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the mammalian serine proprotein convertase family that typically functions in the proteolytic processing and maturation of secretory proteins (1, 2). PCSK9 was the first family member to be implicated in a dominantly inherited form of hypercholesterolemia (3). Mechanistic studies addressing the function of PCSK9 in mice and humans have demonstrated that overexpression or gain-of-function mutations in PCSK9 reduced low density lipoprotein receptor (LDLR) protein levels in liver, which significantly increased circulating plasma cholesterol both in mice and humans (4). Additional studies showed that the deletion of Pcsk9 in mice resulted in increased LDLR levels, accelerated the clearance of low density lipoprotein cholesterol (LDLc), and reduced circulating cholesterol levels (5). Recently, studies in mice have also shown that lowering PCSK9 transcript levels by antisense oligonucleotides resulted in reduced total cholesterol, LDLc, and HDL cholesterol (HDLc) in blood and increased LDLR levels in liver after 6 weeks of treatment (6). This effect was very similar to that observed in the Pcsk9 Ϫ/Ϫ mice (5). Collectively, these studies have clearly established a role for PCSK9 in cholesterol homeostasis.Validation of PCSK9 as an attractive therapeutic target for the treatment of hypercholesterolemia has come from genetic studies in humans. Cohen et al. (7) first identified loss-of-function mutations in PCSK9...

Double-stranded DNA break repair and homologous recombination in E. coli are initiated by the RecBCD enzyme, which unwinds and simultaneously degrades DNA from a double-stranded DNA end. This process is stimulated by cis-acting DNA elements, known as chi sites. Using both in vitro pairing and nuclease protection assays, we demonstrate that the translocating RecBCD enzyme, which has been activated by chi, coordinates the preferential loading of the homologous pairing protein, RecA, onto the resultant single-stranded DNA downstream of chi. This facilitated loading of RecA protein results in a substantial increase in both the efficiency and rate of in vitro recombination reactions and offers an explanation for stimulation of recombination and repair in vivo by chi.

Vaccines have had broad medical impact, but existing vaccine technologies and production methods are limited in their ability to respond rapidly to evolving and emerging pathogens, or sudden outbreaks. Here, we develop a rapid-response, fully synthetic, single-dose, adjuvant-free dendrimer nanoparticle vaccine platform wherein antigens are encoded by encapsulated mRNA replicons. To our knowledge, this system is the first capable of generating protective immunity against a broad spectrum of lethal pathogen challenges, including H1N1 influenza, Toxoplasma gondii, and Ebola virus. The vaccine can be formed with multiple antigen-expressing replicons, and is capable of eliciting both CD8+ T-cell and antibody responses. The ability to generate viable, contaminant-free vaccines within days, to single or multiple antigens, may have broad utility for a range of diseases.

Circular RNAs (circRNAs) are a class of singlestranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.

Homologous recombination in Escherichia coli is stimulated at DNA sequences known as X sites. Stimulation requires the multifunctional RecBCD enzyme, which is both a helicase and a 3' ~ 5' exonuclease. Upon recognition of a properly oriented X site, the 3' -9 5' exonuclease activity is attenuated. Here we show that in addition to attenuation of the 3' ~ 5' exonuclease activity, recognition of X by the RecBCD enzyme also up-regulates a nuclease activity of the opposite polarity, resulting in an enzyme that now preferentially degrades 5' ~ 3'. These results demonstrate that X is a unique regulatory element that converts the antirecombinogenic form of the RecBCD enzyme into a recombinogenic form by causing two distinct enzymatic changes: attenuation of the 3' ~ 5' nuclease activity, and up-regulation of the 5' ~ 3' nuclease activity. The consequence of X recognition is the production of a recombination intermediate possessing a 3'-ssDNA overhang terminating at the X sequence. This processing of a dsDNA end to a 3'-ssDNA overhang parallels that which occurs during the initation of homologous recombination in other pathways in E. coli, and in other organisms such as the yeast Saccharomyces cerevisiae.