Upskilling the cell therapy manufacturing workforce: design, implementation, and evaluation of a massive open online course.

Cell therapies have gained prominence as a promising therapeutic modality for treating a range of diseases. Despite the recent clinical successes of cell therapy products, very few formal training programs exist for cell therapy manufacturing. To meet the demand for a well-trained workforce, we assembled a team of university researchers and industry professionals to develop an online course on the principles and practice of cell therapy manufacturing. The course covers the basic cell and systems physiology underlying cell therapy products, in addition to explaining end-to-end manufacturing from cell acquisition through to patient treatment, industrialization, and regulatory processes. So far, over 10,000 learners have enrolled in the course, and over 90% of respondents to the course exit survey indicated that they were 'very likely' or 'likely' to recommend the course to a peer. In this paper, we discuss our experience in the collaborative design and implementation of the online course, as well as lessons learned from quantitative and qualitative student feedback. We believe that this course can serve as a model for how academia and industry can collaborate to create innovative, scalable training programs to meet the demands of the modern biotechnology workforce.

Mechanistic modeling explains the production dynamics of recombinant adeno-associated virus with the baculovirus expression vector system.

Current manufacturing processes for recombinant adeno-associated viruses (rAAVs) have less-than-desired yields and produce significant amounts of empty capsids. The increasing demand and the high cost of goods for rAAV-based gene therapies motivate development of more efficient manufacturing processes. Recently, the US Food and Drug Administration (FDA) approved the first rAAV-based gene therapy product manufactured in the baculovirus expression vector system (BEVS), a technology that demonstrated production of high titers of full capsids. This work presents a first mechanistic model describing the key extracellular and intracellular phenomena occurring during baculovirus infection and rAAV maturation in the BEVS. The model predictions are successfully validated for in-house and literature experimental measurements of the vector genome and of structural and non-structural proteins collected during rAAV manufacturing in the BEVS with the TwoBac and ThreeBac constructs. A model-based analysis of the process is carried out to identify the bottlenecks that limit full capsid formation. Vector genome amplification is found to be the limiting step for rAAV production in Sf9 cells using either the TwoBac or ThreeBac system. In turn, vector genome amplification is hindered by limiting Rep78 levels. Transgene and non-essential baculovirus protein expression in the insect cell during rAAV manufacturing also negatively influences the rAAV production yields.

Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS).

The Consortium on Adventitious Agent Contamination in Biomanufacturing (CAACB) collected historical data from 20 biopharmaceutical industry members on their experience with the in vivo adventitious virus test, the in vitro virus test, and the use of next generation sequencing (NGS) for viral safety. Over the past 20 years, only three positive in vivo adventitious virus test results were reported, and all were also detected in another concurrent assay. In more than three cases, data collected as a part of this study also found that the in vivo adventitious virus test had given a negative result for a sample that was later found to contain virus. Additionally, the in vivo adventitious virus test had experienced at least 21 false positives and had to be repeated an additional 21 times all while using more than 84,000 animals. These data support the consideration and need for alternative broad spectrum viral detection tests that are faster, more sensitive, more accurate, more specific, and more humane. NGS is one technology that may meet this need. Eighty one percent of survey respondents are either already actively using or exploring the use of NGS for viral safety. The risks and challenges of replacing in vivo adventitious virus testing with NGS are discussed. It is proposed to update the overall virus safety program for new biopharmaceutical products by replacing in vivo adventitious virus testing approaches with modern methodologies, such as NGS, that maintain or even improve the final safety of the product.

Mechanistic model for production of recombinant adeno-associated virus via triple transfection of HEK293 cells.

Manufacturing of recombinant adeno-associated virus (rAAV) viral vectors remains challenging, with low yields and low full:empty capsid ratios in the harvest. To elucidate the dynamics of recombinant viral production, we develop a mechanistic model for the synthesis of rAAV viral vectors by triple plasmid transfection based on the underlying biological processes derived from wild-type AAV. The model covers major steps starting from exogenous DNA delivery to the reaction cascade that forms viral proteins and DNA, which subsequently result in filled capsids, and the complex functions of the Rep protein as a regulator of the packaging plasmid gene expression and a catalyst for viral DNA packaging. We estimate kinetic parameters using dynamic data from literature and in-house triple transient transfection experiments. Model predictions of productivity changes as a result of the varied input plasmid ratio are benchmarked against transfection data from the literature. Sensitivity analysis suggests that (1) the poorly coordinated timeline of capsid synthesis and viral DNA replication results in a low ratio of full virions in harvest, and (2) repressive function of the Rep protein could be impeding capsid production at a later phase. The analyses from the mathematical model provide testable hypotheses for evaluation and reveal potential process bottlenecks that can be investigated.

Cellular pathways of recombinant adeno-associated virus production for gene therapy.

Recombinant adeno-associated viruses (rAAVs) are among the most important vectors for in vivo gene therapies. With the rapid development of gene therapy, current rAAV manufacturing capacity faces a challenge to meet the emerging demand for these therapies in the future. To examine the bottlenecks in rAAV production during cell culture, we focus here on an analysis of cellular pathways of rAAV production, based on an overview of assembly mechanisms first in the wild-type (wt) AAV replication and then in the common methods of rAAV production. The differences analyzed between the wild-type and recombinant systems provide insights into the mechanistic differences that may correlate with viral productivity. Based on these analyses, we identify potential barriers to high productivity of rAAV and discuss future directions for improvement to meet the emerging needs set by the growth of rAAV-based therapy and the needs of patients.

Chiral Structure Formation during Casting of Cellulose Nanocrystalline Films.

A Landau-de Gennes formulation coupled with a mass-transfer equation was used to track the evaporation front and the development of chiral microstructures during the casting of sulfuric acid-hydrolyzed cellulose nanocrystal (CNC) films. These simulations are compared to thin-film casting experiments that used analogous processing parameters and environments. The results show that the initial concentration, chiral strength, surface anchoring, speed of drying, and the influence of initial shear alignment all affect the uniformity of the microstructure and the orientation of the chiral director. In this report, we aim to show that under optimal casting conditions, the lateral size of planar microstructural domains that exhibit uniform selective reflection can be achieved on the order of millimeters.

Hyaluronic acid modification of RNase A and its intracellular delivery using lipid-like nanoparticles.

Developing safe and effective nanosystems to deliver active and therapeutic proteins to targeted cells and organs is an important tool for many biomedical applications. We present here a simple and efficient strategy for this purpose: delivering hyaluronic acid (HA)-modified RNase A (RNase A-HA) in nanocomplex with cationic lipid-like molecules (lipidoids) to cancer cells, resulting in targeted inhibition of cancer proliferation. The chemical conjugation of RNase A with HA both increased the supramolecular interaction with carrier lipidoids, promoting protein encapsulation efficacy, and facilitated cancer cell targeting via interaction with overexpressed CD44. Through confocal laser scanning microscopy and flow cytometry analysis, we demonstrated that protein/lipidoid nanoparticles could facilely enter cells with high CD44 expression, and inhibit cell proliferation in a dose-dependent manner.

In Vivo Peripheral Nerve Repair Using Tendon-Derived Nerve Guidance Conduits.

There is an urgent need for a peripheral nerve repair product that can match or exceed the abilities of the current "gold-standard", nerve autografts. Using a sectioning-based fabrication technique, decellularized tendon sections formed into tubular conduits that maintain the native structure of the collagen. Our previous studies have demonstrated that these collagen structures provide nanotopographical growth guidance cues for regenerating neurons and support glia. Here, the regenerative abilities of the tendon-derived nerve guidance conduits to repair a critically sized defect (15 mm) are evaluated in a rat sciatic nerve model. Using the conduits, functional recovery occurs at a similar rate to isografts, when evaluated with a sciatic function index test. However, muscular recovery, as measured by gastrocnemius weight, was not as great in the conduit-treated group. Both conduit and isograft repairs are histologically evaluated using Masson's trichrome stain and immunofluorescent staining for neurofilament-160 and S100 (markers for neurons and Schwann cells, respectively). This evaluation shows that by week 14, conduits promote regrowth of both neuronal tissue and some physiological support structures, such as blood vessels and epi/perineurium-like structures. Lastly, positive staining for these two markers at week 14 is calculated as a quantitative means of assessment, and shows greater total content of neurofilament-160 and S100 in conduits than in isografts, but a smaller percent area, which may be a result of the greater cross-sectional area of the conduit.

Reactive oxygen species-responsive protein modification and its intracellular delivery for targeted cancer therapy.

Herein we report a convenient chemical approach to reversibly modulate protein (RNase A) function and develop a protein that is responsive to reactive oxygen species (ROS) for targeted cancer therapy. The conjugation of RNase A with 4-nitrophenyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzyl carbonate (NBC) blocks protein lysine and temporarily deactivates the protein. However, the treatment of RNase A-NBC with hydrogen peroxide (one major intracellular ROS) efficiently cleaves the NBC conjugation and restores the RNase A activity. Thus, RNase A-NBC can be reactivated inside tumor cells by high levels of intracellular ROS, thereby restoring the cytotoxicity of RNase A for cancer therapy. Due to higher ROS levels inside tumor cells compared to healthy cells, and the resulting different levels of RNase A-NBC reactivation, RNase A-NBC shows a significant specific cytotoxicity against tumor cells.

Dendritic cell-based immunotherapy in prevention and treatment of renal cell carcinoma: efficacy, safety, and activity of Ad-GM·CAIX in immunocompetent mouse models.

The dendritic cell vaccine DC-Ad-GM·CAIX is an active, specific immunotherapy with the potential of providing a safe and effective therapy against renal cell carcinoma (RCC). Using immunocompetent Balb/c mouse models we tested the efficacy and mechanism of the vaccine to prevent and treat the growth of a syngeneic RCC (RENCA) engineered to overexpress the human TAA carbonic anhydrase IX (NPR-IX). In a prevention model, NPR-IX tumor development was specifically and significantly delayed by 13 days in DC-Ad-GM·CAIX-treated mice (P < 0.001), tumor volumes were 79% smaller (day 24, P < 0.007), and body weight was maintained at study termination compared with the controls. Six of these mice remained tumor-free for > 1 year. In a treatment model, NPR-IX tumors remained smaller in DC-Ad-GM·CAIX-treated mice for 8 days (P < 0.002), achieving a 60% growth inhibition at termination. No vaccine-related organ toxicity was observed in either model. The critical mechanistic parameter separating responsive from nonresponsive tumors was hCAIX protein expression, demonstrated by aggressive growth of tumors that did not express hCAIX protein and in sham-treated mice (DC-Ad-Null). No murine serum anti-hCAIX antibodies were detected. Moreover, altered mechanisms of immunoediting as a means for immune evasion were suggested by differential gene expression (Ccl1, Hmgb1, Fgl2, Cd209a, and Klra2) and therapy evasion miRNAs (miR-1186, miR-98, miR-5097, miR-1942, and miR-708) in tumors that evaded DC-Ad-GM·CAIX immunotherapy. This is the first study in immunocompetent mice that provides a proof of concept for the specificity, efficacy, safety, and activity of the DC-Ad-GM·CAIX immunotherapy, forming the basis for a first-in-human phase I trial in RCC patients.