(M1230-03-18) Multimodal Immunogene Therapy via Nanoparticle Delivery System

Purpose: Designing a potency assay is a critical part of biologic drug development.  Dependent upon the mechanism of action of the drug, the potency assay can encompass multiple activities. In one innovative application, a potency assay is conceived for a nanoparticle delivery system that is employed to intravenously administer anti-cancer genes expressing DNA plasmids. This system utilizes positively charged nanoparticles that selectively bind to negatively charged cancer cells and enter them through endocytosis (Figure 1). The nanoparticle is designed to deliver the functioning Gene X to cancer cells while minimizing their uptake by normal tissue and elicit a multimodal mechanism of action including interruption of cell signaling pathways, re-establishment of pathways for apoptosis in cancer cells, and modulates the immune response against cancer cells.

Methods: The impact of the nanoparticles on caspase-mediated apoptosis was conducted through the optimization and evaluation of a potency assay.The experimental setup involved H1299 cells cultured at various cell densities ranging from 0.5X10⁵ cells/mL to 2X10⁵ cells/mL. The incubation period for drug-cell interaction was approximately 24 hours. To ensure a robust linear curve fit with adequate data points, the dose concentrations ranged from 7 µg/mL to 1.4 µg/mL. Apoptosis was quantified by measuring the activity of caspase released into the supernatant, an indicator of apoptotic processes. Quantification was achieved by employing the Promega Caspase Glo 3/7 Assay, where luminescence served as a proportional measure of the amount of caspase activity present. The acquired data was further processed using a linear regression fit model with calculations for mean, R², % CV, and relative potency carried out using Gen5 Secure, version 3.02 (Agilent Technologies).

Results: Results indicated discernible trends in caspase activity across different cell densities, delineating specific conditions that induced heightened apoptotic responses in H1299 cells. This approach, employing luminescence as a dependable indicator, offers valuable insights into the optimal conditions conducive to initiating programmed cell death concerning the studied drug product and H1299 cells. Representative linear curves for the potency assay featuring test samples at 70%, 100%, and 130% NDC are depicted in Figure 2, while Figure 3 illustrates the test sample linearity during assay development. This robust methodology provided a comprehensive understanding of the impact of the nanoparticles on caspase-mediated apoptosis under varied experimental conditions.

Conclusion: In conclusion, a cell-based potency method was developed for the quantitative determination of the multiple modes of action of a lipid nanoparticle containing Gene X. The assay was optimized and made ready for Phase II application. The assay was successfully applied to the evaluation of the LNP regarding its activity and functionality. This is a demonstration of the creation of a robust and high-functioning assay for the evaluation of a complex therapeutic in the gene therapy space. The evaluation of assay performance clearly demonstrates that it is ready for the control and characterization of the product through later phases of development.

References: 21 CFR Part 610 and ICH Q2 (R1) Guidelines
Bioanalytical method validation Guidance for Industry; FDA guidelines 2018


Non-Viral Delivery of Tumor Suppressor Gene


Dose Response Curves at 70%, 100% and 130% NDC


Sample Linearity for potency assay