Poster X1 - Engineering dual transgene plasmid tumor reprogramming immunological signals (4-1BBL and IL12) for effective clinical translation

Introduction: Prior work in the Green group has demonstrated that delivery of Signal 2 (4-1BBL) and Signal 3 (IL12) co-stimulatory molecules via Poly(beta-amino ester) nanoparticles (PBAE-NPs) has been successful in in orchestrating anti-tumor immune responses through T-cell activation . However, the current delivery approach involves multiple genetic constructs, posing challenges for clinical translation. As a result, we proposed several strategies to condense both Signal 2 and Signal 3 transgenes into one plasmid. In this project, we explore the effect of separating the two signals with a T2A self-cleaving domain versus a 25 amino acid linker comprised of Glycine and Serine motifs. This offers several advantages for downstream clinical translation: 1) it ensures both immunomodulatory signals are delivered into the same tumor cells, enhancing therapeutic efficacy, 2) it simplifies manufacturing processes, facilitating scalability, and 3) it streamlines regulatory approval pathways by minimizing the complexity of gene therapy formulations.

Materials and

Methods: Gene inserts were first ordered from Twist Bioscience, containing the reporter genes GFP and luciferase. Double digests were performed on these gene inserts to excise and ligate them into the pUNO plasmid backbone from Invivogen. These new constructs, named pUNO GFP T2A fluc (self cleaving) and pUNO GFP elasti fluc (elastin linker) were delivered into and IOMM-Lee meningioma cells and B16-F10 melanoma cells to evaluate expression levels of both reporter genes. GFP expression was evaluated via flow cytometry, and luciferase expression levels were evaluated using the Thermofisher Firefly Luc One-Step Glow assay kit.
Gene inserts for the therapeutic genes 4-1BBL and IL12 were also ordered from Twist Bioscience. The therapeutic genes were separated by either the T2A domain or the tether sequence, which consisted of a 25 amino acid repeat of Gly-Gly-Gly-Gly-Ser. We performed double digests on the T2A constructs with BamHI and NheI (New England Biolabs) restriction enzymes and were ligated into the pUNO backbone to create pUNO 2-T2A-3 and pUNO 3-T2A-2. Similarly, we performed double digests on the tethered insert with SalI and NheI (NEB) and ligated into the same pUNO backbone to form pUNO mTether.

Results, Conclusions, and Discussions: Results, Conclusions, & Discussion (350 words)
Delivery of PBAE nanoparticle carrying the dual transgene plasmids, pUNO GFP T2A fluc and pUNO GFP elasti fluc, were delivered into IOMM-Lee meningioma cells. Each plasmid was able to express both reporter genes, although expression levels were lower than in cells that received nanoparticles with only GFP or only luciferase.
After validating that delivery of two genes was viable using both a T2A domain and a linker sequence, we designed and created dual transgene plasmids containing both 4-1BBL and IL12. For the T2A constructs, we designed two plasmids, with 4-1BBL and IL12 flanking the T2A domain in both orientations. For the Gly4Ser construct, we designed the N terminus of the mouse IL12 protein to be tethered to the C terminus of the 4-1BBL protein, in order to ensure critical receptor binding domains were not occluded.
Delivery of pUNO 2-T2A-3 and pUNO mTether into B16F10 melanoma cells demonstrated expression of 41BBL, while delivery of pUNO 2-T2A-3 and pUNO 3-T2A-2 demonstrated expression of IL12, though expression of IL12 with the pUNO mTether plasmid may be difficult to quantify through ELISA assays due to the protein configuration. We plan to further co-culture B16F10 cells with splenocytes after dual transgene plasmid delivery to evaluate inflammatory response before testing these constructs for therapeutic efficacy in vivo.

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