Chinese Hamster Ovary (CHO) cell monoclonal antibody (mAb) production in continuous perfusion has witnessed a renewed interest within the biopharmaceutical industry. Widespread implementation of perfusion biomanufacturing, however, remains hindered by long process development timelines and high costs. Use of predictive scale-down platforms to generate large informative metabolic datasets and guide process development decisions is critical to decreasing a molecule's time to market. While scale-down platforms based on the pseudo perfusion concept have been previously reported, they have not been rigorously validated. They are often limited by oxygen transport or insufficient metabolic characterization, reducing their role to a preliminary screening tool. Here, we report the design and validation of a pseudo perfusion platform based on a phenotype-driven approach to ascertain that the process emulates continuous perfusion characteristics and is not oxygen limited. Beyond metabolic and cell size steady state, we show that our pseudo perfusion design enables cell cycle subpopulation and intracellular antibody expression steady state. We also demonstrate that pseudo perfusion robustly predicts amino acid demands in continuous perfusion bioreactors with exceptional linear correlation across a broad range of cell-specific perfusion rates. When coupling the pseudo perfusion platform developed here with a workflow for metabolic characterization, we significantly augment the dimensionality and reliability of data which can be generated at this scale to gain actionable insights towards perfusion process design, ultimately reducing process development timelines and the associated costs.

Synthetic microbial cocultures, which combine the designed capabilities of multiple microbes into one process, have significant potential for sustainable production of fuels and chemicals. Most studies of defined cocultures have tested relatively low cell densities in batch cultures, not the high cell density fed-batch or continuous processes with cell retention typically required to achieve industrially relevant volumetric productivities. Here, we explore the impact of increased cell density on isopropanol production from the syntrophic coculture of Clostridium ljungdahlii with a genetically modified Clostridium acetobutylicum (CACas9 Δhbd-p95ace02_atoB), which lacks 4-C metabolism while overexpressing an acetone-formation pathway on a plasmid. CACas9 Δhbd-p95ace02_atoB produces acetone without 4-C metabolites and C. ljungdahlii converts the acetone to isopropanol. To explore the potential of this coculture system to achieve supratheoretical isopropanol yields, we identify NADH-driven hydrogen conversion in CACas9 Δhbd-p95ace02_atoB as the thermodynamically limiting step for acetone production in monocultures and thus isopropanol production in cocultures. We then demonstrate the ability of C. ljungdahlii to mitigate this issue by eliminating detectable hydrogen accumulation in the coculture. Pseudo-perfusion cocultures showed that high cell densities combined with a high population fraction of C. ljungdahlii enable dramatic increases in isopropanol yields beyond the thermodynamic limitation imposed in CACas9 Δhbd-p95ace02_atoB monocultures. Finally, we demonstrate a continuous carbon-negative fermentation of glucose to isopropanol as the sole alcohol produced from glucose in a 3.6-L scale perfusion bioreactor.

Microbial conversion of renewable carbon sources into valuable chemicals can significantly reduce our reliance on fossil resources and decrease the carbon footprint of chemical manufacturing. Of emerging interest is anaerobic, nonphotosynthetic mixotrophy (ANP mixotrophy) that simultaneously converts renewable carbohydrates and C1 gases (CO2 and CO) into value-added chemical products in carbon-neutral and even carbon-negative fermentations. Despite significant advances in ANP mixotrophy over the past decade, several challenges remain. We discuss key challenges for carbon-neutral/negative ANP fermentations, notably the necessity of supplying additional electrons for reduced metabolite production, the slow adoption of genetic tools, uncertainties about carbon catabolite repression, and gas transfer limitations.

The core advantage of acetogens lies in their superior carbon management, achieved by engaging the Wood-Ljungdahl pathway to sequester CO₂ in situ and utilize exogenous CO₂. This advantage can be further exploited by coupling acetogens with other organisms in co-culture, leading to the increasingly explored concept of acetogenic co-cultures. We review and dissect schemes involving the co-fermentation of carbohydrates and exogenous gases, which present unique challenges and opportunities due to the nonlinearity of co-culture metabolic networks and complex, often unanticipated, interspecies interactions. The latter suggests that most, if not all, such co-cultures are mutualistic rather than commensalistic, contrary to previous assumptions. We discuss both fundamental and applied concepts, including co-culture stability and methods for quantitatively capturing population dynamics and interspecies interactions.

Syntrophic cocultures (hitherto assumed to be commensalistic) of Clostridium acetobutylicum and Clostridium ljungdahlii, whereby CO2 and H2 produced by the former feed the latter, result in interspecies cell fusion involving large-scale exchange of protein, RNA, and DNA between the two organisms. Although mammalian cell fusion is mechanistically dissected, the mechanism for such microbial-cell fusions is unknown. To start exploring this mechanism, we used RNA sequencing to identify genes differentially expressed in this coculture using two types of comparisons. One type compared coculture to the two monocultures, capturing the combined impact of interactions through soluble signals in the medium and through direct cell-to-cell interactions. The second type compared membrane-separated versus -unseparated cocultures, isolating the impact of interspecies physical contact. While we could not firmly identify specific genes that might drive cell fusion, consistent with our hypothesized model for this interspecies microbial cell fusion, we observed differential regulation of genes involved in C. ljungdahlii's autotrophic Wood-Ljungdahl pathway metabolism and genes of the motility machinery. Unexpectedly, we also identified differential regulation of biosynthetic genes of several amino acids, and notably of arginine and histidine. We verified that they are produced by C. acetobutylicum and are metabolized by C. ljungdahlii to its growth advantage. These and other findings, and notably upregulation of C. acetobutylicum ribosomal-protein genes, paint a more complex syntrophic picture and suggest a mutualistic relationship, whereby beyond CO2 and H2, C. acetobutylicum feeds C. ljungdahlii with growth-boosting amino acids, while benefiting from the H2 utilization by C. ljungdahlii.IMPORTANCEThe construction and study of synthetic microbial cocultures is a growing research area due to the untapped potential of defined multi-species industrial bioprocesses and the utility of defined cocultures for generating insight into complex, undefined, natural microbial consortia. Our previous work showed that coculturing C. acetobutylicum and C. ljungdahlii leads to a unique metabolic phenotype (production of isopropanol) and heterologous cell fusion events. Here, we used RNAseq to explore genes involved in and impacted by these fusions. First, we compared gene expression in coculture to each monoculture. Second, we utilized a transwell system to compare gene expression in mixed cocultures to cocultures with both species physically separated by a permeable membrane, isolating the impact of interspecies "touching" on the transcriptome. This study deepens our mechanistic understanding of the C. acetobutylicum-C. ljungdahlii coculture phenotype, laying the groundwork for reverse genetic studies of heterologous cell fusion in Clostridium cocultures.

There is a need for efficient and sustainable production of essential chemicals such as isopropanol and butanol from renewable sugar feedstocks. Microbial fermentations use glycolysis, and as result, a third of the sugar carbon is lost to CO2 through pyruvate decarboxylation to acetyl-CoA, the starting intermediate for the biosynthesis of most microbial metabolites. In nature, microbes exist in syntrophic consortia, allowing for mutually-beneficial interactions, the production of novel products, and the realization of novel benefits—including better carbon conservation—not seen in monocultures. We examined the impact of starting coculture cell densities, the gas atmosphere (N2, H2, or H2/CO2) and coculture species ratios (using a recently developed RNA-FISH flow cytometric assays) on metabolite production, yields and sugar-carbon utilization in serum bottles and bioreactors. Metabolic flux analysis identified the complex patterns by which the two species alter each other’s metabolism in a cell-density and gas-atmosphere dependent manner. For increased acetone production, we transformed Clostridium acetobutylicum with a plasmid (p95ace02a) expressing a synthetic acetone pathway comprising four native genes. This engineered C. acetobutylicum was cocultured with Clostridium ljungdahlii to capture the waste CO2 and H2 generated due to glucose catabolism by C. acetobutylicum, and to convert acetone into isopropanol. C. ljungdahlii activated the dormant acetate uptake in C. acetobutylicum, while coculture density dramatically impacted species ratios, electron management, and the H2 utilization of C. ljungdahlii. We achieved exceptionally-high concentrations of our desired products—246 mM isopropanol and 148 mM butanol—in 64 h, with about 85% of the production occurring before 32 h. We reached maximum productivities of 13.9 mM isopropanol/h and 10.4 mM butanol/h with 0.9 mol alcohol produced per mol of sugar consumed. Total product yields reached 84.7% on a C-mol basis, versus 65.6% that can be reached in a C. acetobutylicum monoculture. Engineered syntrophic cocultures can efficiently and tunably produce target chemicals including isopropanol and butanol for a renewable economy.

We have previously shown that mixotrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii – using sugars and H2 as substrates – increase sugar-substrate carbon and electron conversion via CO2 and H2 capture and synthesize valuable products, such as isopropanol and 2,3-butanediol, that neither species can make independently. In this pairing, growth of C. ljungdahlii is constrained by C. acetobutylicum , since C. ljungdahlii relies on C. acetobutylicum to convert glucose into CO2, which C. ljungdahlii can use as a carbon and electron sources. However, this dependence is unilateral; C. acetobutylicum’s growth is not constrained by C. ljungdahlii . Consequently, population ratios between the two species can vary substantially throughout the course of fermentation and in different fermentation setups, typically with the faster growing C. acetobutylicum outcompeting C. ljungdahlii . Population ratio is an important variable because it influences metabolite yields and productivity and likely also impacts the initiation and frequency of the heterologous cell fusion events we have documented between C. acetobutylicum and C. ljungdahlii . Thus, developing methods to rationally control and maintain the population ratio are important for both biotechnological applications and fundamental study of this coculture pairing. In this study we show that the different nitrogen utilization capabilities of these two organisms enable engineering of a mutualistic mixotrophic syntrophy in which C. ljungdahlii relies on C. acetobutylicum for carbon and electrons and C. acetobutylicum relies on C. ljungdahlii for nitrogen. First, we confirm that C. ljungdahlii , but not C. acetobutylicum , can convert nitrate into biologically useful ammonium, enabling the design of a culture medium in which C. acetobutylicum can only grow in the presence of C. ljungdahlii . Second, we test different ratios of nitrate to ammonium in batch cocultures and demonstrate that rapid nitrate utilization by C. ljungdahlii prevents C. acetobutylicum from becoming nitrogen-limited at any point in batch fermentation. Finally, we show that feeding different rates of nitrate to cocultures in fed-batch mode enables control of the coculture growth rate, maintenance of stable population ratios, and higher isopropanol and butanol yields in cocultures between C. acetobutylicum and C. ljungdahlii . ### Competing Interest Statement The authors have declared no competing interest. Advanced Research Projects Agency - Energy, AR0001505

Due to their ability to convert CO2, a greenhouse gas, into useful products, certain acetogenic bacterial species, such as Clostridium ljungdahlii , have been proposed as promising platform strains for renewable, carbon-negative chemical production. C. ljungdahlii , and similar acetogens, grows slowly and produce primarily acetate when grown on CO2 with H2 as the electron donor, but it grows quickly and can produce ethanol when grown on higher energy substrates, notably CO or fructose. Here, by utilizing different mixing strategies (and notably the first time use of roller bottles) to modulate the volumetric gas interfacial mass transfer coefficient (kLa), we show that, under both mixotrophic (sugar and gas utilization) and autotrophic conditions, C. ljungdahlii growth and CO2 fixation are primarily electron-limited due to the low solubility of H2 relative to CO and CO2. We demonstrate that, with sufficiently high H2 mass transfer, C. ljungdahlii can grow at similar high rates using CO2 as its sole carbon source compared to CO or fructose, a finding with significant implications for the use of acetogens in CO2-negative biomanufacturing, especially because at least 50% of CO used is oxidized and released as CO2. We also show that accumulation of fructose inhibits CO2 utilization by C. ljungdahlii under mixotrophic growth conditions, suggesting that a non-classical “catabolite repression” by fructose inhibits CO2 utilization. ### Competing Interest Statement The authors have declared no competing interest. Advanced Research Projects Agency - Energy, https://ror.org/03q1rgc19, AR0001505

Culture conditions have a profound impact on therapeutic protein production and glycosylation, a critical therapeutic-quality attribute, especially for monoclonal antibodies (mAbs). While the critical culture parameter of pH has been known since the early 1990s to affect protein glycosylation and production, detailed glycan and metabolic characterization and mechanistic understanding are critically lacking. Here, Chinese Hamster Ovary (CHO) cells were grown in bioreactors at pH 6.75, 7, and 7.25 (+/- 0.03) to examine how pH affects cell metabolism and site-specific N-linked glycosylation of the produced broadly neutralizing anti-HIV IgG1 mAb. VRC01 has N-linked glycosylation sites in both the Fc region and the Fab region, a situation not previously examined with respect to mAb glycosylation as affected by culture conditions. Using parsimonious Flux Balance Analysis (pFBA) and Flux Variability Analysis (FVA), we dissect and quantitate the impact of pH on cell growth, glucose/lactate metabolism, accumulation of the toxic metabolite ammonia, IgG production rates, and nonessential amino acid metabolism. pFBA revealed that beyond the established mechanism of glutamine conversion to glutamate, ammonia is also produced by the reaction converting serine to pyruvate, especially in the later phases of culture. pFBA also provided insights into the switch from ammonia production to consumption, notably due to depletion of glutamine, and consumption of glutamate and aspartate. We document that culture duration and pH alter the complex bimodal patterns (production/uptake) of several essential and non-essential amino acids. Site-specific N-linked glycan analysis using glycopeptide mapping demonstrated that pH significantly affects the glycosylation profiles of the two IgG1 sites. Fc region glycans were completely fucosylated but did not contain any sialylation. The Fab region glycans were not completely fucosylated but contained sialylated glycans. Bioreactor pH affected both the fucosylation and sialylation indexes in the Fab region and the galactosylation index of the Fc region. However, fucosylation in the Fc region was unaffected thus demonstrating that the effect of pH on site-specific N-linked glycosylation is complex.

Due to their ability to convert CO 2 , a greenhouse gas, into useful products, certain acetogenic bacterial species, such as Clostridium ljungdahlii , have been proposed as promising platform strains for renewable, carbon-negative chemical production. C. ljungdahlii , and similar acetogens, grows slowly and produce primarily acetate when grown on CO 2 with H 2 as the electron donor, but it grows quickly and can produce ethanol when grown on higher energy substrates, notably CO or fructose. Here, by utilizing different mixing strategies (and notably the first time use of roller bottles) to modulate the volumetric gas interfacial mass transfer coefficient (k L a), we show that, under both mixotrophic (sugar and gas utilization) and autotrophic conditions, C. ljungdahlii growth and CO 2 fixation are primarily electron-limited due to the low solubility of H 2 relative to CO and CO 2 . We demonstrate that, with sufficiently high H 2 mass transfer, C. ljungdahlii can grow at similar high rates using CO 2 as its sole carbon source compared to CO or fructose, a finding with significant implications for the use of acetogens in CO 2 -negative biomanufacturing, especially because at least 50% of CO used is oxidized and released as CO 2 . We also show that accumulation of fructose inhibits CO 2 utilization by C. ljungdahlii under mixotrophic growth conditions, suggesting that a non-classical “catabolite repression” by fructose inhibits CO 2 utilization.

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution. IMPORTANCE Investigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. While high butyrate levels induce toxic stress, effects of non-toxic levels on cell growth, health, metabolism, and sporulation remain unclear. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome to eliminate the butyrate synthetic pathway and thus butyrate formation. A xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. Non-toxic butyrate levels significantly affected growth, health, and sporulation of C. acetobutylicum. After deleting spo0A, the gene encoding the master regulator of sporulation, Spo0A, and conducting butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A-dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of crotonate, the precursor of butyrate biosynthesis, to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and potentially evolutionarily related Clostridium organisms.

The CHO VRC01 cell line produces an anti-HIV IgG1 monoclonal antibody containing N-linked glycans on both the Fab (variable) and Fc (constant) regions. Site-specific glycan analysis was used to measure the complex effects of cell culture process conditions on Fab and Fc glycosylation. Experimental data revealed major differences in glycan fractions across the two sites. Bioreactor pH was found to influence fucosylation, galactosylation, and sialylation in the Fab region and galactosylation in the Fc region. To understand the complex effects of process conditions on site-specific N-linked glycosylation, a kinetic model of site-specific N-linked glycosylation was developed. The model parameters provided mechanistic insights into the differences in glycan fractions observed in the Fc and Fab regions. Enzyme activities calculated from the model provided insights into the effect of bioreactor pH on site-specific N-linked glycosylation. Model predictions were experimentally tested by measuring glycosyltransferase-enzyme mRNA-levels and intracellular nucleotide sugar concentrations. The model was used to demonstrate the effect of increasing galactosyltransferase activity on site-specific N-linked glycan fractions. Experiments involving galactose and MnCl2 supplementation were used to test model predictions. The model is capable of providing insights into experimentally measured data and also of making predictions that can be used to design media supplementation strategies.

Native and engineered extracellular vesicles generated from human megakaryocytes (huMkEVs) or from the human megakaryocytic cell line CHRF (CHEVs) interact with tropism delivering their cargo to both human and murine hematopoietic stem and progenitor cells (HSPCs). To develop non-viral delivery vectors to HSPCs based on MkEVs, we first confirmed, using NOD-scid IL2Rγnull (NSG™) mice, the targeting potential of the large EVs, enriched in microparticles (huMkMPs), chosen for their large cargo capacity. 24 h post intravenous infusion into NSG mice, huMkEVs induced a nearly 50% increase in murine platelet counts. PKH26-labeled huMkEVs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs, thus confirming their receptor-mediated tropism for NSG HSPCs, and their potential to treat thromobocytopenias. We explored this tropism to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to NSG HSPCs both in vitro and in vivo. We loaded huMkEVs with plasmid DNA either through electroporation or by generating hybrid particles with preloaded liposomes. Both methods facilitated successful functional targeted delivery of pDNA, as tissue weight-normalized fluorescence intensity of the expressed fluorescent reporter was significantly higher in bone marrow than other tissues. Furthermore, the fraction of fluorescent CD117+ HSPCs was nearly 19-fold higher than other cell types within the bone marrow 72-h following administration of the hybrid particles, further supporting that HSPC tropism is retained when using hybrid particles. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases.

A new area of focus in Chinese hamster ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs or miRNA) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in miR-92a and miR-23a under normal culture conditions, in contrast to the high enrichment in let-7 family miRs (let-7c, let-7b, and let-7a) under both stress conditions. The latter suggests a preserved stress-responsive function of the let-7 miR family, one of the most highly preserved miR families across species, where among other functions, let-7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected-based on their profound enrichment-important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.

Chinese hamster ovary (CHO) cells release and exchange large quantities of extracellular vesicles (EVs). EVs are highly enriched in microRNAs (miRs, or miRNAs), which are responsible for most of their biological effects. We have recently shown that the miR content of CHO EVs varies significantly under culture stress conditions. Here, we provide a novel stoichiometric ("per-EV") quantification of miR and protein levels in large CHO EVs produced under ammonia, lactate, osmotic, and age-related stress. Each stress resulted in distinct EV miR levels, with selective miR loading by parent cells. Our data provide a proof of concept for the use of CHO EV cargo as a diagnostic tool for identifying culture stress. We also tested the impact of three select miRs (let-7a, miR-21, and miR-92a) on CHO cell growth and viability. Let-7a-abundant in CHO EVs from stressed cultures-reduced CHO cell viability, while miR-92a-abundant in CHO EVs from unstressed cultures-promoted cell survival. Overexpression of miR-21 had a slight detrimental impact on CHO cell growth and viability during late exponential-phase culture, an unexpected result based on the reported antiapoptotic role of miR-21 in other mammalian cell lines. These findings provide novel relationships between CHO EV cargo and cell phenotype, suggesting that CHO EVs may exert both pro- and antiapoptotic effects on target cells, depending on the conditions under which they were produced.

Gene therapy lacks tools to deliver in a safe and tissue-targeted manner. Limitations include toxicity, off-target effects due to targeting, and inability to repeat dose. We have developed a novel megakaryocyte-derived extracellular vesicle (MkEV)-based non-viral gene therapy delivery platform that preferentially targets bone marrow in vivo. Here we present work showing that STRM.BIO MkEVs: 1) pass through the liver and spleen to preferentially target bone marrow in mice and non-human primates (NHPs); 2) can selectively deliver pDNA cargo to long term HSCs to drive reporter protein expression exclusively in bone marrow following intravenous delivery in mice; and 3) present a platform to develop and deliver targeted gene therapies in vivo that are safe for repeat dosing. Our vision is to open the door to the future of medicine for patients living with rare diseases worldwide and bring gene therapy to life. Primary human CD34+ hematopoietic stem and progenitor cells (hHSPCs) were differentiated into megakaryocytes in vitro. MkEVs were harvested and loaded with pDNA via electroporation, followed by DNase treatment to remove free cargo. For in vivo biodistribution and cargo-mediated protein expression studies, STRM.BIO MkEVs were exogenously labeled with fluorescent dye (PKH26, Cell Tracker Deep Red, and/or DiD), loaded with cargo, and IV-injected into mouse tail veins (wild type or NSG) or NHPs (cynomolgus monkeys). Tissues were collected 16-48h after IV injection, with MkEV tissue biodistribution quantified by dye fluorescence (Mean Fluorescence Intensity (MFI), plate reader) normalized to tissue weight or genomic DNA input, and pDNA cargo quantified by qPCR. Additionally, quantification of MkEVs+ cells was performed by flow cytometry analysis of murine bone marrow derived sub-populations (lineage negative, c-Kit +, Sca-1 + (LSK); lineage positive (Lin +); and, long-term HSCs (LT-HSCs; Lin -/CD150 +/CD201 +)) cells. Protein expression from MkEV-loaded pDNA cargo was similarly determined by plate reader fluorescence data and confocal microscopy. In mouse biodistribution studies, DiD-labeled MkEVs were predominantly detected in bone marrow, followed by liver and spleen 16h-post tail vein injection (plate reader analyses: Bone Marrow - 1.1e 4 ± 0.2 SD; Liver - 9.6e 3 ± 1.0 SD; Spleen - 1.8e 4 ± 0.2 SD. MFI/g tissue; n=3mice/group). Flow cytometry analyses show MkEVs preferentially targeted the hematopoietic compartment (of the MkEV+ cells, 94 ± 1% CD45+ vs 6 ± 1% CD45-, respectively), specifically, the HSPCs (68%, LSK) and LT-HSCs (100%) 48h post intravenous delivery via tail vein injection. Pharmacokinetic studies show that while MkEVs can circulate through highly vascularized tissues like the liver and spleen, they accumulate in the bone marrow. Strikingly, miRFP encoded by the pDNA was exclusively detected in bone marrow (p<0.0001 vs. all other tissues; Fig. 1). In NHPs, dye-labeled MkEVSs were loaded with a MkEV-driven eGFP reporter plasmid. Studies showed preferential biodistribution to bone marrow, and preferential qPCR amplification of the pDNA cargo in bone marrow (both MFI/ ACTB and GFP/ ACTB were significantly higher vs. all other tissues: range p<0.05 through p<0.0001), Fig. 2. A tolerability and repeat dosing study was performed in NHPs. Monkeys were injected with 4 weekly doses of STRM.BIO MkEVs and serial bloodwork was obtained 6h post dose for cytokine evaluation and 5 days post dose for end organ damage assessment (n=4). The study showed no evidence of kidney, hematologic, or liver damage as evidenced by normal creatinine and electrolytes, no significant changes in complete blood counts, coagulation parameters, or liver function tests. There was no evidence of significant cytokine release or inflammatory changes on tissue histology. These data confirm that MkEVs are non-toxic and the platform is amenable to repeat dosing. Taken together, these data establish an advanced gene therapy platform that can target bone marrow specifically following in vivo administration in both mice and NHPs, potentially eliminating the need for current ex vivo approaches to treat rare blood disorders, and providing the critical ability to repeat dose as required in clinic. STRM.BIO is leveraging our platform to develop gene therapies for rare blood diseases in people.

Extracellular vesicles (EVs) are cornerstones of intercellular communication with exciting fundamental, clinical, and more broadly biotechnological applications. However, variability in EV composition, which results from the culture conditions used to generate the EVs, poses significant fundamental and applied challenges and a hurdle for scalable bioprocessing. Thus, an understanding of the relationship between EV production (and for clinical applications, manufacturing) and EV composition is increasingly recognized as important and necessary. While chemical stimulation and culture conditions such as cell density are known to influence EV biology, the impact of biomechanical forces on the generation, properties, and biological activity of EVs remains poorly understood. Given the omnipresence of these forces in EV preparation and in biomanufacturing, expanding the understanding of their impact on EV composition-and thus, activity-is vital. Although several publications have examined EV preparation and bioprocessing and briefly discussed biomechanical stresses as variables of interest, this review represents the first comprehensive evaluation of the impact of such stresses on EV production, composition and biological activity. We review how EV biogenesis, cargo, efficacy, and uptake are uniquely affected by various types, magnitudes, and durations of biomechanical forces, identifying trends that emerge both generically and for individual cell types. We also describe implications for scalable bioprocessing, evaluating processes inherent in common EV production and isolation methods, and propose a path forward for rigorous EV quality control.

Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR-486-5p and miR-22-3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude-dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR-486-5p and miR-22-3p cargo. However, while the shake flask-derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump-derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by "older" Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late-differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.