Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45 000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Insights from CTTACC: immune system reset by cellular therapies for chronic illness after trauma, infection, and burn.

BACKGROUND AIMS: In this paper, we present a review of several selected talks presented at the CTTACC conference (Cellular Therapies in Trauma and Critical Care) held in Scottsdale, AZ in May 2023. This conference review highlights the potential for cellular therapies to "reset" the dysregulated immune response and restore physiologic functions to normal. Improvements in medical care systems and technology have increasingly saved lives after major traumatic events. However, many of these patients have complicated post-traumatic sequelae, ranging from short-term multi-organ failure to chronic critical illness. METHODS/RESULTS: Patients with chronic critical illness have been found to have dysregulated immune responses. These abnormal and harmful immune responses persist for years after the initial insult and can potentially be mitigated by treatment with cellular therapies. CONCLUSIONS: The sessions emphasized the need for more research and clinical trials with cellular therapies for the treatment of a multitude of chronic illnesses: post-trauma, radiation injury, COVID-19, burns, traumatic brain injury (TBI) and other chronic infections.

Investigating dynamics of lentiviral vector secretion from HEK293T producer cells using a fractionated perfusion system.

Mammalian cell culture is quickly becoming the go to engineering vehicle to mass produce viral vectors in a manner that is safe, convenient, reproducible, and cost and scale effective. Human embryonic kidney (HEK293) cells, in particular, have been utilized and customized (via differentiated transgene expression, modified culture parameters, addition of cytostatic culture agents) to increase vector yields. However, less attention has been made to understanding innate processes within the cells (such as, immune response, cell cycle, metabolism) themselves to better control or increase viral vector product yield. Accordingly, herein, the variation in viral production was studied from HEK cells over time using a one-way perfusion system and bioreactor to study the impact of external factors on secretion dynamics without retrotransduction. Specifically, the impact of cell density on viral titer, transduction efficiency, and LDH, was studied. Next, we look at the impact of using an inflammatory reporter cell line on viral output, and the secretion dynamics from HEK cells when we use sodium butyrate (cell cycle arrest agent). Lastly, we assess how downregulation of the PDK pathway increases viral titer. Altogether, we investigated the impact of various interventions to increase transient protein expression and viral output from HEK cells in a controlled and measurable environment to ultimately increase the efficiency of HEK cells for downstream clinical applications.

Simulating Interclonal Interactions in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is one of the most common types of cancers, accounting for 37% of B-cell tumor cases globally. DLBCL is known to be a heterogeneous disease, resulting in variable clinical presentations and the development of drug resistance. One underexplored aspect of drug resistance is the evolving dynamics between parental and drug-resistant clones within the same microenvironment. In this work, the effects of interclonal interactions between two cell populations-one sensitive to treatment and the other resistant to treatment-on tumor growth behaviors were explored through a mathematical model. In vitro cultures of mixed DLBCL populations demonstrated cooperative interactions and revealed the need for modifying the model to account for complex interactions. Multiple best-fit models derived from in vitro data indicated a difference in steady-state behaviors based on therapy administrations in simulations. The model and methods may serve as a tool for understanding the behaviors of heterogeneous tumors and identifying the optimal therapeutic regimen to eliminate cancer cell populations using computer-guided simulations.

Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries.

Sequence verification of plasmid DNA is critical for many cloning and molecular biology workflows. To leverage high-throughput sequencing, several methods have been developed that add a unique DNA barcode to individual samples prior to pooling and sequencing. However, these methods require an individual plasmid extraction and/or in vitro barcoding reaction for each sample processed, limiting throughput and adding cost. Here, we develop an arrayed in vivo plasmid barcoding platform that enables pooled plasmid extraction and library preparation for Oxford Nanopore sequencing. This method has a high accuracy and recovery rate, and greatly increases throughput and reduces cost relative to other plasmid barcoding methods or Sanger sequencing. We use in vivo barcoding to sequence verify >45,000 plasmids and show that the method can be used to transform error-containing dispersed plasmid pools into sequence-perfect arrays or well-balanced pools. In vivo barcoding does not require any specialized equipment beyond a low-overhead Oxford Nanopore sequencer, enabling most labs to flexibly process hundreds to thousands of plasmids in parallel.

Dynamics of Ex Vivo Mesenchymal Stromal Cell Potency under Continuous Perfusion.

Mesenchymal stromal cells (MSCs) are a candidate for cell immunotherapy due to potent immunomodulatory activity found in their secretome. Though studies on their secreted substances have been reported, the time dynamics of MSC potency remain unclear. Herein, we report on the dynamics of MSC secretome potency in an ex vivo hollow fiber bioreactor using a continuous perfusion cell culture system that fractionated MSC-secreted factors over time. Time-resolved fractions of MSC-conditioned media were evaluated for potency by incubation with activated immune cells. Three studies were designed to characterize MSC potency under: (1) basal conditions, (2) in situ activation, and (3) pre-licensing. Results indicate that the MSC secretome is most potent in suppressing lymphocyte proliferation during the first 24 h and is further stabilized when MSCs are prelicensed with a cocktail of pro-inflammatory cytokines, IFNγ, TNFα, and IL-1ß. The evaluation of temporal cell potency using this integrated bioreactor system can be useful in informing strategies to maximize MSC potency, minimize side effects, and allow greater control for the duration of ex vivo administration approaches.

A cell culture system to model pharmacokinetics using adjustable-volume perfused mixing chambers.

The pharmacokinetic (PK) profile of a drug is an essential factor in determining its efficacy, yet it is often neglected during in vitro cell culture experiments. Here, we present a system in which standard well plate cultures may be "plugged in" and perfused with PK drug profiles. Timed drug boluses or infusions are passed through a mixing chamber that simulates the PK volume of distribution specific to the desired drug. The user-specified PK drug profile generated by the mixing chamber passes through the incubated well plate culture, exposing cells to in vivo-like PK drug dynamics. The effluent stream from the culture may then optionally be fractionated and collected by a fraction collector. This low-cost system requires no custom parts and perfuses up to six cultures in parallel. This paper demonstrates a range of PK profiles the system can produce using a tracer dye, describes how to find the correct mixing chamber volumes to mimic PK profiles of drugs of interest, and presents a study exploring the effects of differing PK exposure on a model of lymphoma treatment with chemotherapy.

3D Microcapsules for Human Bone Marrow Derived Mesenchymal Stem Cell Biomanufacturing in a Vertical-Wheel Bioreactor.

Microencapsulation of human mesenchymal stromal cells (MSCs) via electrospraying has been well documented in tissue engineering and regenerative medicine. Herein, we report the use of microencapsulation, via electrospraying, for MSC expansion using a commercially available hydrogel that is durable, optimized to MSC culture, and enzymatically degradable for cell recovery. Critical parameters of the electrospraying encapsulation process such as seeding density, correlation of microcapsule output with hydrogel volume, and applied voltage were characterized to consistently fabricate cell-laden microcapsules of uniform size. Upon encapsulation, we then verified ~ 10x expansion of encapsulated MSCs within a vertical-wheel bioreactor and the preservation of critical quality attributes such as immunophenotype and multipotency after expansion and cell recovery. Finally, we highlight the genetic manipulation of encapsulated MSCs as an example of incorporating bioactive agents in the capsule material to create new compositions of MSCs with altered phenotypes.

Allometric-like scaling of AAV gene therapy for systemic protein delivery.

The use of adeno-associated virus (AAV) as a gene delivery vehicle for secreted peptide therapeutics can enable a new approach to durably manage chronic protein insufficiencies in patients. Yet, dosing of AAVs have been largely empirical to date. In this report, we explore the dose-response relationship of AAVs encoding a secreted luciferase reporter to establish a mathematical model that can be used to predict steady-state protein concentrations in mice based on steady-state secretion rates in vitro. Upon intravenous administration of AAV doses that scaled multiple logs, steady-state plasma concentrations of a secreted reporter protein were fit with a hyperbolic dose-response equation. Parameters for the hyperbolic model were extracted from the data and compared with create scaling factors that related in vitro protein secretion rates to in vivo steady-state plasma concentrations. Parathyroid hormone expressed by AAV was then used as a bioactive candidate and validated that the model, with scaling factors, could predict the plasma hormone concentrations in mice. In total, this model system confirmed that plasma steady-state concentrations of secreted proteins expressed by AAVs can be guided by in vitro kinetic secretion data laying groundwork for future customization and model-informed dose justification for AAV candidates.

An Analysis of Transcriptomic Burden Identifies Biological Progression Roadmaps for Hematological Malignancies and Solid Tumors.

Biological paths of tumor progression are difficult to predict without time-series data. Using median shift and abacus transformation in the analysis of RNA sequencing data sets, natural patient stratifications were found based on their transcriptomic burden (TcB). Using gene-behavior analysis, TcB groups were evaluated further to discover biological courses of tumor progression. We found that solid tumors and hematological malignancies (n = 4179) share conserved biological patterns, and biological network complexity decreases at increasing TcB levels. An analysis of gene expression datasets including pediatric leukemia patients revealed TcB patterns with biological directionality and survival implications. A prospective interventional study with PI3K targeted therapy in canine lymphomas proved that directional biological responses are dynamic. To conclude, TcB-enriched biological mechanisms detected the existence of biological trajectories within tumors. Using this prognostic informative novel informatics method, which can be applied to tumor transcriptomes and progressive diseases inspires the design of progression-specific therapeutic approaches.

Scaffold-mediated switching of lymphoma metabolism in culture.

BACKGROUND: Diffuse large B cell lymphoma (DLBCL) is an aggressive subtype of non-Hodgkin lymphoma (NHL) and accounts for about a third of all NHL cases. A significant proportion (~40%) of treated DLBCL patients develop refractory or relapsed disease due to drug resistance which can be attributed to metabolomic and genetic variations amongst diverse DLBCL subtypes. An assay platform that reproduces metabolic patterns of DLBCL in vivo could serve as a useful model for DLBCL. METHODS: This report investigated metabolic functions in 2D and 3D cell cultures using parental and drug-resistant DLBCL cell lines as compared to patient biopsy tissue. RESULTS: A 3D culture model controlled the proliferation of parental and drug-resistant DLBCL cell lines, SUDHL-10, SUDHL-10 RR (rituximab resistant), and SUDHL-10 OR (obinutuzumab resistant), as well as retained differential sensitivity to CHOP. The results from metabolic profiling and isotope tracer studies with D-glucose-13C6 indicated metabolic switching in 3D culture when compared with a 2D environment. Analysis of DLBCL patient tumor tissue revealed that the metabolic changes in 3D grown cells were shifted towards that of clinical specimens. CONCLUSION: 3D culture restrained DLBCL cell line growth and modulated metabolic pathways that trend towards the biological characteristics of patient tumors. Counter-intuitively, this research thereby contends that 3D matrices can be a tool to control tumor function towards a slower growing and metabolically dormant state that better reflects in vivo tumor physiology.

Unbiased discovery of autoantibodies associated with severe COVID-19 via genome-scale self-assembled DNA-barcoded protein libraries.

Pathogenic autoreactive antibodies that may be associated with life-threatening coronavirus disease 2019 (COVID-19) remain to be identified. Here, we show that self-assembled genome-scale libraries of full-length proteins covalently coupled to unique DNA barcodes for analysis by sequencing can be used for the unbiased identification of autoreactive antibodies in plasma samples. By screening 11,076 DNA-barcoded proteins expressed from a sequence-verified human ORFeome library, the method, which we named MIPSA (for Molecular Indexing of Proteins by Self-Assembly), allowed us to detect circulating neutralizing type-I and type-III interferon (IFN) autoantibodies in five plasma samples from 55 patients with life-threatening COVID-19. In addition to identifying neutralizing type-I IFN-α and IFN-ω autoantibodies and other previously known autoreactive antibodies in patient plasma, MIPSA enabled the detection of as yet unidentified neutralizing type-III anti-IFN-λ3 autoantibodies that were not seen in healthy plasma samples or in convalescent plasma from ten non-hospitalized individuals with COVID-19. The low cost and simple workflow of MIPSA will facilitate unbiased high-throughput analyses of protein-antibody, protein-protein and protein-small-molecule interactions.

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

Bioactive, full-length parathyroid hormone delivered using an adeno-associated viral vector.

Delivering the parathyroid hormone (PTH) gene has been attempted preclinically in a handful of studies, but delivering full-length PTH (1-84) using adeno-associated viral (AAV) vectors has not. Given the difficulty in achieving therapeutic levels of secreted proteins using gene therapy, this study seeks to determine the feasibility of doing so with PTH. An AAV vector was used to deliver human PTH driven by a strong promoter. We demonstrate the ability to secrete full-length PTH from various cell types in vitro. PTH secretion from hepatocytes was measured over time and a fluorescent marker was used to compare the secretion rate of PTH in various cell types. Potency was measured by the ability of PTH to act on the PTH receptors of osteosarcoma cells and induced proliferation. PTH showed potency in vitro by inducing proliferation in two osteosarcoma cell lines. In vivo, AAV was administered systemically in immunocompromised mice which received xenografts of osteosarcoma cells. Animals that received the highest dose of AAV-PTH had higher liver and plasma concentrations of PTH. All dosing groups achieved measurable plasma concentrations of human PTH that were above the normal range. The high-dose group also had significantly larger tumors compared to control groups on the final day of the study. The tumors also showed dose-dependent differences in morphology. When looking at endocrine signaling and endogenous bone turnover, we observed a significant difference in tibial growth plate width in animals that received the high-dose AAV as well as dose-dependent changes in blood biomarkers related to PTH. This proof-of-concept study shows promise for further exploration of an AAV gene therapy to deliver full-length PTH for hypoparathyroidism. Additional investigation will determine efficacy in a disease model, but data shown establish bioactivity in well-established models of osteosarcoma.

Proceedings of the ISCT scientific signature series symposium, "Advances in cell and gene therapies for lung diseases and critical illnesses": International Society for Cell & Gene Therapy, Burlington VT, US, July 16, 2021.

The ISCT Scientific Signature Series Symposium "Advances in Cell and Gene Therapies for Lung Diseases and Critical Illnesses" was held as an independent symposium in conjunction with the biennial meeting, "Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Diseases," which took place July 12-15, 2021, at the University of Vermont. This is the third Respiratory System-based Signature Series event; the first 2, "Tracheal Bioengineering, the Next Steps" and "Cellular Therapies for Pulmonary Diseases and Critical Illnesses: State of the Art of European Science," took place in 2014 and 2015, respectively. Cell- and gene-based therapies for respiratory diseases and critical illnesses continue to be a source of great promise and opportunity. This reflects ongoing advancements in understanding of the mechanisms by which cell-based therapies, particularly those using mesenchymal stromal cells (MSCs), can mitigate different lung injuries and the increasing sophistication with which preclinical data is translated into clinical investigations. This also reflects continuing evolution in gene transfer vectors, including those designed for in situ gene editing in parallel with those targeting gene or cell replacement. Therefore, this symposium convened global thought leaders in a forum designed to catalyze communication and collaboration to bring the greatest possible innovation and value of cell- and gene-based therapies for patients with respiratory diseases and critical illnesses.

Automated Assessment of Cancer Drug Efficacy On Breast Tumor Spheroids in Aggrewell™400 Plates Using Image Cytometry.

Tumor spheroid models have proven useful in the study of cancer cell responses to chemotherapeutic compounds by more closely mimicking the 3-dimensional nature of tumors in situ. Their advantages are often offset, however, by protocols that are long, complicated, and expensive. Efforts continue for the development of high-throughput assays that combine the advantages of 3D models with the convenience and simplicity of traditional 2D monolayer methods. Herein, we describe the development of a breast cancer spheroid image cytometry assay using T47D cells in Aggrewell™400 spheroid plates. Using the Celigo® automated imaging system, we developed a method to image and individually track thousands of spheroids within the Aggrewell™400 microwell plate over time. We demonstrate the use of calcein AM and propidium iodide staining to study the effects of known anti-cancer drugs Doxorubicin, Everolimus, Gemcitabine, Metformin, Paclitaxel and Tamoxifen. We use the image cytometry results to quantify the fluorescence of calcein AM and PI as well as spheroid size in a dose dependent manner for each of the drugs. We observe a dose-dependent reduction in spheroid size and find that it correlates well with the viability obtained from the CellTiter96® endpoint assay. The image cytometry method we demonstrate is a convenient and high-throughput drug-response assay for breast cancer spheroids under 400 µm in diameter, and may lay a foundation for investigating other three-dimensional spheroids, organoids, and tissue samples.

Massively parallel DNA target capture using long adapter single stranded oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology.

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the linker sequence for the mature LASSO probe through Cre-LoxP intramolecular recombination. This strategy generates high quality LASSO probes libraries (≈46% of correct probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 Escherichia coli ORFs spanning from 400- to 5000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, were able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

Assembly of Long-Adapter Single-Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets.

Genome DNA sequencing has become an affordable means to resolve questions about the genetic background of life. However, the biological functions of many DNA-encoded sequences are still relatively unknown. A highly scalable and cost-effective cloning method to select natural DNA targets from genomic templates is therefore urgently needed to enable rapid understanding of the biological products of genomes. One such method involves LASSO probes, which are long single-stranded DNA oligonucleotides designed with a universal adapter that is used to link two sequences that are complementary to a genomic target of interest. Through a pooled assembly method, LASSOs can be made for multiplex DNA capture. Herein, we describe a robust, efficient method to assemble LASSO probe libraries using a Cre-recombinase-mediated reaction and a protocol for multiplex genome target capture. The starting components are a pre-LASSO probe library comprising short DNA oligo pools designed in silico and an Escherichia coli plasmid (pLASSO) that incorporates the pre-LASSO library. Through internal recombination of pLASSO with its inserts, a mature LASSO library in final configuration can be made with high purity. Assembly of a LASSO probe library takes 4 days, and target capture can be performed in a single day. With an exponentially growing list of new genomes available for investigation, this method can enable the rapid production of ORFeome libraries for high-throughput screening to identify biological functions as a complementary approach to understand genome functional biology. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assembly of LASSO probes Support Protocol 1: Generation of pLASSO vectors Support Protocol 2: Preparation of pre-LASSOs Basic Protocol 2: Massively parallel capture of large DNAs using LASSO probes.

Pharmacological effects of ex vivo mesenchymal stem cell immunotherapy in patients with acute kidney injury and underlying systemic inflammation.

Mesenchymal stem cells (MSCs) have natural immunoregulatory functions that have been explored for medicinal use as a cell therapy with limited success. A phase Ib study was conducted to evaluate the safety and immunoregulatory mechanism of action of MSCs using a novel ex vivo product (SBI-101) to preserve cell activity in patients with severe acute kidney injury. Pharmacological data demonstrated MSC-secreted factor activity that was associated with anti-inflammatory signatures in the molecular and cellular profiling of patient blood. Systems biology analysis captured multicompartment effects consistent with immune reprogramming and kidney tissue repair. Although the study was not powered for clinical efficacy, these results are supportive of the therapeutic hypothesis, namely, that treatment with SBI-101 elicits an immunotherapeutic response that triggers an accelerated phenotypic switch from tissue injury to tissue repair. Ex vivo administration of MSCs, with increased power of testing, is a potential new biological delivery paradigm that assures sustained MSC activity and immunomodulation.

3D host cell and pathogen-based bioassay development for testing anti-tuberculosis (TB) drug response and modeling immunodeficiency.

Tuberculosis (TB) is a global health threat that affects 10 million people worldwide. Human Immunodeficiency Virus (HIV) remains one of the major contributors to the reactivation of asymptomatic latent tuberculosis (LTBI). Over the recent years, there has been a significant focus in developing in-vitro 3D models mimicking early events of Mycobacterium tuberculosis (Mtb) pathogenesis, especially formation of the granuloma. However, these models are low throughput and require extracellular matrix. In this article, we report the generation of a matrix-free 3D model, using THP-1 human monocyte/macrophage cells and mCherry-expressing Mycobacterium bovis BCG (Bacilli Camille Guérin), henceforth referred as 3D spheroids, to study the host cell-bacterial interactions. Using mCherry-intensity-based tracking, we monitored the kinetics of BCG growth in the 3D spheroids. We also demonstrate the application of the 3D spheroids for testing anti-TB compounds such as isoniazid (INH), rifampicin (RIF), as well as a host-directed drug, everolimus (EVR) as single and combinational treatments. We further established a dual infection 3D spheroid model by coinfecting THP-1 macrophages with BCG mCherry and pseudotype HIV. In this HIV-TB co-infection model, we found an increase in BCG mCherry growth within the 3D spheroids infected with HIV pseudotype. The degree of disruption of the granuloma was proportional to the virus titers used for co-infection. In summary, this 3D spheroid assay is an useful tool to screen anti-TB response of potential candidate drugs and can be adopted to model HIV-TB interactions.