ABSTRACT
Interleukin-6 (IL-6) is a highly pro-inflammatory cytokine involved in the etiopathology of several inflammatory diseases and cancer. As so, the inhibition of IL-6 signaling pathways has emerged as an attractive therapeutic avenue for the treatment of several chronic diseases. Since IL-6 trans-signaling was described as the pathological branch of IL-6, selective inhibitors were developed. Next-generation variants with increased trans-signaling specificity and potency emerged as great candidates for the treatment of several diseases, with reduced off-target effects. The highly time-consuming and costly processes involving recombinant protein production, however, have hampered the progress of anti-cytokine pharmaceuticals in clinic so far. Herein, we developed gene therapeutic modalities of IL-6-trans-signaling inhibitors as alternatives for sustained recombinant protein secretion. By using an IL-6-dependent lymphoma cell line and xenograft tumor model, we demonstrated the superior inhibitory potential of second-generation anti-IL-6 trans-signaling therapeutic. We compared the efficiency of distinct gene delivery modalities using a bioluminescent biomarker probe and observed consistent protein production via cell-based delivery. When delivered intratumorally, genetically engineered sgp130FlyRFc-secreting cells significantly reduced tumor burden and increased animal survival, representing a promising therapeutic avenue to be explored in clinically relevant gene delivery applications.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
