Evaluation of lentiviral vector stability and development of ion exchange purification processes.

In this manuscript, we employ parallel batch stability and chromatographic screens in concert with linear and step gradient experiments to develop a high yield, HCP clearance anion exchange capture process for lentiviral vector (LVV) purification. An initial broad resin screen is carried out to determine anion exchange-based resins that exhibit high recovery of LVV. LVV stability is then evaluated and conditions are established where the vector exhibits good stability, namely phosphate buffer at pH 6.5-7.5, with low to moderate salt concentrations. A subsequent high-throughput batch screen is then carried out with a subset of resins selected from the first screen under stable conditions to identify optimal wash and elution steps to further improve product yield and protein clearance. Linear gradient experiments are also conducted in mini-column format to refine the operating conditions and final step gradient processes are established that exhibit greater than 70% yield of infectious LVV while also achieving up to 2.89 log reduction values (LRV) of HCPs during the process. The large set of stability and chromatographic data provided in this work represent an important contribution to knowledge in the field about the chromatographic efficacy of a wide range of resins for LVV bioprocessing under stable conditions.

Advancing a rapid, high throughput screening platform for optimization of lentivirus production.

BACKGROUND: Lentiviral vectors (LVVs) hold great promise as delivery tools for gene therapy and chimeric antigen receptor T cell (CAR-T) therapy. Their ability to target difficult to transfect cells and deliver genetic payloads that integrate into the host genome makes them ideal delivery candidates. However, several challenges remain to be addressed before LVVs are more widely used as therapeutics including low viral vector concentrations and the absence of suitable scale-up methods for large-scale production. To address these challenges, we have developed a high throughput microscale HEK293 suspension culture platform that enables rapid screening of conditions for improving LVV productivity. KEY RESULTS: High density culture (40 million cells mL-1 ) of HEK293 suspension cells in commercially available media was achieved in microscale 96-deep well plate platform at liquid volumes of 200 µL. Comparable transfection and LVV production efficiencies were observed at the microscale, in conventional shake flasks and a 1-L bioreactor, indicating that significant scale-down does not affect LVV concentrations and predictivity of scale-up. Optimization of production step allowed for final yields of LVVs to reach 1.5 × 107  TU mL-1 . CONCLUSIONS: The ability to test a large number of conditions simultaneously with minimal reagent use allows for the rapid optimization of LVV production in HEK293 suspension cells. Therefore, such a system may serve as a valuable tool in early stage process development and can be used as a screening tool to improve LVV concentrations for both batch and perfusion based systems.

3D tumor spheroid microarray for high-throughput, high-content natural killer cell-mediated cytotoxicity.

Immunotherapy has emerged as a promising approach to treating several forms of cancer. Use of immune cells, such as natural killer (NK) cells, along with small molecule drugs and antibodies through antibody dependent cell-mediated cytotoxicity (ADCC) has been investigated as a potential combination therapy for some difficult to treat solid tumors. Nevertheless, there remains a need to develop tools that support co-culture of target cancer cells and effector immune cells in a contextually relevant three-dimensional (3D) environment to provide a rapid means to screen for and optimize ADCC-drug combinations. To that end, here we have developed a high throughput 330 micropillar-microwell sandwich platform that enables 3D co-culture of NK92-CD16 cells with pancreatic (MiaPaCa-2) and breast cancer cell lines (MCF-7 and MDA-MB-231). The platform successfully mimicked hypoxic conditions found in a tumor microenvironment and was used to demonstrate NK-cell mediated cell cytotoxicity in combination with two monoclonal antibodies; Trastuzumab and Atezolizumab. The platform was also used to show dose response behavior of target cancer cells with reduced EC50 values for paclitaxel (an anti-cancer chemotherapeutic) when treated with both NK cells and antibody. Such a platform may be used to develop more personalized cancer therapies using patient-derived cancer cells.

Facile fabrication of antibacterial and antiviral perhydrolase-polydopamine composite coatings.

In situ generation of antibacterial and antiviral agents by harnessing the catalytic activity of enzymes on surfaces provides an effective eco-friendly approach for disinfection. The perhydrolase (AcT) from Mycobacterium smegmatis catalyzes the perhydrolysis of acetate esters to generate the potent disinfectant, peracetic acid (PAA). In the presence of AcT and its two substrates, propylene glycol diacetate and H2O2, sufficient and continuous PAA is generated over an extended time to kill a wide range of bacteria with the enzyme dissolved in aqueous buffer. For extended self-disinfection, however, active and stable AcT bound onto or incorporated into a surface coating is necessary. In the current study, an active, stable and reusable AcT-based coating was developed by incorporating AcT into a polydopamine (PDA) matrix in a single step, thereby forming a biocatalytic composite onto a variety of surfaces. The resulting AcT-PDA composite coatings on glass, metal and epoxy surfaces yielded up to 7-log reduction of Gram-positive and Gram-negative bacteria when in contact with the biocatalytic coating. This composite coating also possessed potent antiviral activity, and dramatically reduced the infectivity of a SARS-CoV-2 pseudovirus within minutes. The single-step approach enables rapid and facile fabrication of enzyme-based disinfectant composite coatings with high activity and stability, which enables reuse following surface washing. As a result, this enzyme-polymer composite technique may serve as a general strategy for preparing antibacterial and antiviral surfaces for applications in health care and common infrastructure safety, such as in schools, the workplace, transportation, etc.

Antimicrobial effects of positively charged, conductive electrospun polymer fibers.

In recent years, electrospun polymer fibers have gained attention for various antibacterial applications. In this work, the effect of positively charged polymer fiber mats as antibacterial gauze is studied using electrospun poly(caprolactone) and polyaniline nanofibers. Chloroxylenol, an established anti-microbial agent is used for the first time as a secondary dopant to polyaniline during the electrospinning process to make the surface of the polyaniline fiber positively charged. Both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli are used to investigate the antibacterial activity of the positively charged and uncharged polymer surfaces. The results surprisingly show that the polyaniline surface can inhibit the growth of both bacteria even when chloroxylenol is used below its minimum inhibitory concentration. This study provides new insights allowing the better understanding of dopant-based, intrinsically conducting polymer surfaces for use as antibacterial fiber mats.

Exploiting CRISPR Cas9 in Three-Dimensional Stem Cell Cultures to Model Disease.

Three-dimensional (3D) cell culture methods have been widely used on a range of cell types, including stem cells to modulate precisely the cellular biophysical and biochemical microenvironment and control various cell signaling cues. As a result, more in vivo-like microenvironments are recapitulated, particularly through the formation of multicellular spheroids and organoids, which may yield more valid mechanisms of disease. Recently, genome-engineering tools such as CRISPR Cas9 have expanded the repertoire of techniques to control gene expression, which complements external signaling cues with intracellular control elements. As a result, the combination of CRISPR Cas9 and 3D cell culture methods enhance our understanding of the molecular mechanisms underpinning several disease phenotypes and may lead to developing new therapeutics that may advance more quickly and effectively into clinical candidates. In addition, using CRISPR Cas9 tools to rescue genes brings us one step closer to its use as a gene therapy tool for various degenerative diseases. Herein, we provide an overview of bridging of CRISPR Cas9 genome editing with 3D spheroid and organoid cell culture to better understand disease progression in both patient and non-patient derived cells, and we address potential remaining gaps that must be overcome to gain widespread use.

Cis-regulatory analysis of Onecut1 expression in fate-restricted retinal progenitor cells.

BACKGROUND: The vertebrate retina consists of six major classes of neuronal cells. During development, these cells are generated from a pool of multipotent retinal progenitor cells (RPCs) that express the gene Vsx2. Fate-restricted RPCs have recently been identified, with limited mitotic potential and cell fate possibilities compared to multipotent RPCs. One population of fate-restricted RPCs, marked by activity of the regulatory element ThrbCRM1, gives rise to both cone photoreceptors and horizontal cells. These cells do not express Vsx2, but co-express the transcription factors (TFs) Onecut1 and Otx2, which bind to ThrbCRM1. The components of the gene regulatory networks that control the transition from multipotent to fate-restricted gene expression are not known. This work aims to identify and evaluate cis-regulatory elements proximal to Onecut1 to identify the gene regulatory networks involved in RPC fate-restriction. METHOD: We identified regulatory elements through ATAC-seq and conservation, followed by reporter assays to screen for activity based on temporal and spatial criteria. The regulatory elements of interest were subject to deletion and mutation analysis to identify functional sequences and evaluated by quantitative flow cytometry assays. Finally, we combined the enhancer::reporter assays with candidate TF overexpression to evaluate the relationship between the TFs, the enhancers, and early vertebrate retinal development. Statistical tests included ANOVA, Kruskal-Wallis, or unpaired t-tests. RESULTS: Two regulatory elements, ECR9 and ECR65, were identified to be active in ThrbCRM1(+) restricted RPCs. Candidate bHLH binding sites were identified as critical sequences in both elements. Overexpression of candidate bHLH TFs revealed specific enhancer-bHLH interactions. Nhlh1 overexpression expanded ECR65 activity into the Vsx2(+) RPC population, and overexpression of NeuroD1/NeuroG2/NeuroD4 had a similar effect on ECR9. Furthermore, bHLHs that were able to activate ectopic ECR9 reporter were able to induce endogenous Otx2 expression. CONCLUSIONS: This work reports a large-scale screen to identify spatiotemporally specific regulatory elements near the Onecut1 locus. These elements were used to identify distinct populations in the developing retina. In addition, fate-restricted regulatory elements responded differentially to bHLH factors, and suggest a role for retinal bHLHs upstream of the Otx2 and Onecut1 genes during the formation of restricted RPCs from multipotent RPCs.

High-throughput identification of factors promoting neuronal differentiation of human neural progenitor cells in microscale 3D cell culture.

Identification of conditions for guided and specific differentiation of human stem cell and progenitor cells is important for continued development and engineering of in vitro cell culture systems for use in regenerative medicine, drug discovery, and human toxicology. Three-dimensional (3D) and organotypic cell culture models have been used increasingly for in vitro cell culture because they may better model endogenous tissue environments. However, detailed studies of stem cell differentiation within 3D cultures remain limited, particularly with respect to high-throughput screening. Herein, we demonstrate the use of a microarray chip-based platform to screen, in high-throughput, individual and paired effects of 12 soluble factors on the neuronal differentiation of a human neural progenitor cell line (ReNcell VM) encapsulated in microscale 3D Matrigel cultures. Dose-response analysis of selected combinations from the initial combinatorial screen revealed that the combined treatment of all-trans retinoic acid (RA) with the glycogen synthase kinase 3 inhibitor CHIR-99021 (CHIR) enhances neurogenesis while simultaneously decreases astrocyte differentiation, whereas the combined treatment of brain-derived neurotrophic factor and the small azide neuropathiazol enhances the differentiation into neurons and astrocytes. Subtype specification analysis of RA- and CHIR-differentiated cultures revealed that enhanced neurogenesis was not biased toward a specific neuronal subtype. Together, these results demonstrate a high-throughput screening platform for rapid evaluation of differentiation conditions in a 3D environment, which will aid the development and application of 3D stem cell culture models.