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.

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.

Selecting a Cell Engineering Methodology During Cell Therapy Product Development.

When considering the development pathway for a genetically modified cell therapy product, it is critically important that the product is engineered consistent with its intended human use. For scientists looking to develop and commercialize a new technology, the decision to select a genetic modification method depends on several practical considerations. Whichever path is chosen, the developer must understand the key risks and potential mitigations of the cell engineering approach. The developer should also understand the clinical implications: permanent/memory establishment versus transient expression, and clinical manufacturing considerations when dealing with transplantation of genetically engineered cells. This review covers important topics for mapping out a strategy for developers of new cell-based therapeutics. Biological, technological, manufacturing, and clinical considerations are all presented to map out development lanes for the initiation and risk management of new gene-based cell therapeutic products for human use.

A continuous flow cell culture system for precision cell stimulation and time-resolved profiling of cell secretion.

Cells exchange substances with their surroundings during metabolism, signaling, and other functions. These fluxes are dynamic, changing in response to external cues and internal programs. Static cultures are inadequate for measuring these dynamics because the environments of the cells change as substances accumulate or deplete from medium, unintentionally affecting cell behavior. Static cultures offer limited time resolution due to the impracticality of frequent or prolonged manual sampling, and cannot expose cells to smooth, transient changes in stimulus concentrations. In contrast, perfusion cultures constantly maintain cellular environments and continuously sample the effluent stream. Existing perfusion culture systems are either microfluidic, which are difficult to make and use, or macrofluidic devices built from custom parts that neglect solute dispersion. In this study, a multiplexed macrofluidic perfusion culture platform was developed to measure secretion and absorption rates of substances by cells in a temporally controlled environment. The modular platform handles up to 31 streams with automated fraction collection. This paper presents the assembly of this dynamic bioreactor from commercially available parts, and a method for quantitatively handling the effects of dispersion using residence time distributions. The system is then applied to monitor the secretion of a circadian clock gene-driven reporter from engineered cells.

Tracking leukemic T-cell transcriptional dynamics in vivo with a blood-based reporter assay.

Transcriptional dynamics of cancer cells govern cell fate decisions and are therapeutically actionable drug targets. In this study, we engineered a circulating cancer cell line that secretes a luciferase reporter to capture constitutive and circadian clock-driven transcription dynamics over the course of a day. Engineered human leukemic T cells (Jurkat) were observed to rhythmically secrete luciferase in a continuous flow cell culture system. When transplanted in vivo, engineered leukemic cells caused circadian plasma luciferase activity and had expected pathological signs of leukemic disease. This technique is rapid and noninvasive, requiring only a few microliters of media or blood, and can aid in investigating relationships between in vivo cancer cell signaling and behavior, such as diet or sleep.

Kinetics of MSC-based enzyme therapy for immunoregulation.

BACKGROUND: Mesenchymal stromal cells (MSC) demonstrate innate and regulatory immunologic functions and have been widely explored for cell therapy applications. Mechanisms by which MSCs achieve therapeutic effects are theorized, though appropriate dosing and duration of these mechanisms in vivo warrant deeper investigation. One, rapid immunosuppressive function of MSCs is through ectoenzyme expression of CD73 and CD39 which cooperatively hydrolyze inflammatory, extracellular adenosine triphosphate (ATP) to anti-inflammatory adenosine. Extracellular ATP has a key role in autoimmune and inflammatory diseases, which administered MSCs have the potential to modulate in a timescale that is befitting of shorter acting therapeutic function. METHODS: In vitro experiments were performed to determine the hydrolysis rates of ATP by MSCs. Through kinetic modeling from experimental results, the rate at which a single cell can metabolize ATP was determined. Based on these rates, the ability of MSCs to downregulate inflammatory signaling pathways was prospectively validated using model system parameters with respect to two different mechanisms: extracellular ATP stimulates lymphocytes to suppress proliferation and induce apoptosis and with co-stimulation, it stimulates monocytes to release pro-inflammatory IL-1ß. MSCs were co-cultured with immune cells using transwell inserts and compared to immune cell only groups. RESULTS: Hydrolysis of ATP was efficiently modeled by first-order enzyme kinetics. For in vitro culture, the rate at which a single cell can hydrolyize ATP is 8.9 nmol/min. In the presence of extracellular ATP, cocultures of MSCs reduced cytotoxicity and allows for proliferation of lymphocytes while limiting IL-1ß secretion from monocytes. CONCLUSIONS: Such use of these models may allow for better dosing predictions for MSCs to be used therapeutically for chronic inflammatory diseases such as rheumatoid arthritis, diabetes, pancreatitis, and other systemic inflammatory response syndromes. For the first time, the effect of MSCs on ATP hydrolysis in immune cell response is quantitatively analyzed on a cell-molecule basis by modeling the hydrolysis as an enzyme-substrate reaction. The results also give insight into MSCs' dynamic response mechanisms to ameliorate effects of extracellular ATP whether it be through positive or negative regulation.