Bacterial tRNase–Based Gene Therapy with Poly(β-Amino Ester) Nanoparticles for Suppressing Melanoma Tumor Growth and Relapse

Purpose: Current cancer treatments (chemotherapy, radiotherapy) suffer from a lack of selectivity for tumor cells, causing severe side effects and therapeutic resistance. While gene therapy is a promising alternative, viral vectors pose safety risks (immunogenicity, tumorigenicity), and nonviral vectors often suffer from low in vivo stability and transfection efficiency. There is a need for a safe, efficient nonviral delivery system paired with therapeutic genes that are highly specific to cancer cells.

Hypothesis: Bacterial tRNases (colicin D and VapC) will exhibit highly specific toxicity toward cancer cells (due to upregulated translation in cancer) while sparing normal cells. Biodegradable poly(β-amino ester) (PBAE) nanoparticles can safely and efficiently deliver these genes to cancer cells to induce apoptosis and inhibit tumor growth/relapse. Furthermore, PEGylation of PBAEs will improve nanoparticle stability in vivo and enhance therapeutic efficacy.

Aims: Synthesize and screen a small library of PBAEs to identify the optimal polymer structure for delivering plasmid DNA to melanoma cells with high transfection efficiency and low cytotoxicity. Evaluate the selective anticancer toxicity of the tRNase genes ColD595 and VapC delivered by the lead PBAE (B4S5E5) in various cancer and normal cell lines in vitro. Assess the antitumor effects of this tRNase gene therapy in mouse melanoma models, specifically regarding tumor growth suppression and delay of tumor relapse after surgical resection. Investigate whether PEGylation of the lead PBAE improves nanoparticle stability and enhances in vivo gene delivery and therapeutic outcomes.

Delivery system: Polymer: Poly(β-amino ester) (PBAE). The lead formulation identified was B4S5E5 (poly(1,4-butanediol diacrylate-co-5-amino-1-pentanol) end-capped with (PEO)4-bis-amine). A PEGylated version, B4S5-PEG, was also developed using 2 kDa methoxy PEG-thiol. Nanoparticle Type: Polyplex nanoparticles formed by self-assembly of cationic PBAE with negatively charged plasmid DNA. Payload: Plasmid DNA encoding bacterial tRNases: ColD595 (residues 595–697 of native colicin D) or VapC (virulence-associated protein C from Shigella flexneri 2a), under the control of a CMV promoter. EGFP and luciferase plasmids served as controls/reporters. Formulation: Polyplexes were prepared at a plasmid-to-polymer weight ratio of 1:30 or 1:60. The optimal ratio for B4S5E5 was 1:30. Size/Shape: Spherical nanoparticles ~200 nm in diameter (unmodified B4S5E5) and ~98-105 nm for PEGylated versions. Targeting: Passive targeting via direct intratumoral injection. No active targeting ligand was used.

Approach: In Vitro: Cancer cell lines (B16-F10 mouse melanoma, HeLa human cervical cancer, A375 human melanoma) and primary normal cells (MEFs, hADSCs) were transfected with EGFP, ColD595, or VapC plasmids. Doses were 1–4 µg per well. In Vivo: Female BALB/c-nude mice (~5–6 weeks old). Two subcutaneous B16-F10 melanoma models were established: 1. Tumor Regression Model: Intratumoral injections (25 µg plasmid per injection) when tumors reached ~6 mm/200 mm³. Four consecutive injections every 2 days. 2. Tumor Relapse Model: Surgical resection of tumors (~200 mm³), followed by four injections every 2 days at the resection site starting the day after surgery. Groups:* No treatment, pEGFP (control), pColD595, pVapC. For PEGylation studies: No treatment, pColD595/B4S5E5, pColD595/B4S5-PEG. n = 7–10 per group.

Key methods: Nanoparticle Characterization: ¹H NMR (polymer synthesis), TEM (morphology), DLS (size/stability), Gel retardation assay (DNase I protection). Transfection Efficiency: FACS (EGFP expression), Confocal microscopy, RT-PCR (mRNA levels of tRNases). Cytotoxicity/Apoptosis: MTT assay (cell viability), qPCR (caspase-3 expression), Annexin-V/PI staining + FACS (apoptosis quantification). In Vivo Efficacy: Tumor volume measurement (caliper), Survival rate. Biodistribution: IVIS imaging (luciferase activity in tumors and major organs). Histology/Immunohistochemistry: H&E staining (tissue degeneration), caspase-3 immunostaining (apoptosis marker), TUNEL assay (DNA fragmentation).

Key results: Transfection Efficiency: B4S5E5 (1:30 w/w) achieved 79 ± 2% transfection efficiency in B16-F10 cells with 83 ± 3% viability, outperforming Lipofectamine 2000 (72 ± 4% efficiency). Selective Toxicity: 2 µg of ColD595 and VapC reduced B16-F10 viability to 35 ± 1% and 31 ± 2%, and HeLa viability to 53 ± 2% and 54 ± 1%, respectively. No significant toxicity was observed in normal MEFs and hADSCs. Apoptosis Induction: Caspase-3 mRNA expression increased 2.0 ± 0.2-fold (ColD595) and 1.5 ± 0.3-fold (VapC) vs. controls. Apoptotic cell populations (Annexin-V+) were 31.9% (ColD595) and 41.7% (VapC) vs. 6.3% (NT). In Vivo Tumor Suppression: Tumor growth rate was significantly reduced, with tumor volume at day 12 reduced by more than half compared to controls. Caspase-3 positive cells were 54 ± 25% (ColD595) and 47 ± 20% (VapC) vs. 3 ± 2% (NT). TUNEL-positive cells were 29 ± 13% (ColD595) and 24 ± 16% (VapC) vs. 5 ± 6% (NT). PEGylation Enhancement: B4S5-PEG resisted aggregation in 10% FBS (maintaining ~105 nm size). In the relapse model, complete recovery (no visible tumor at day 50) was achieved in 55% (6/11) of pColD595/B4S5-PEG treated mice, compared to 21% (4/19) for unmodified B4S5E5 and 10% (2/20)* for no treatment.

Interpretation: The authors claim this is the first demonstration of a nonviral, biodegradable PBAE-based gene therapy utilizing bacterial tRNase genes (ColD595 and VapC) for anticancer treatment. They highlight that these tRNases induce highly specific toxicity and apoptosis in cancer cells by inhibiting translation, while sparing normal cells. The study demonstrates that PBAE-mediated delivery suppresses melanoma tumor growth and delays relapse, with PEGylation further enhancing stability, gene delivery efficiency, and therapeutic outcomes (complete recovery in over half the mice). They propose this as a promising, clinically translatable supplemental therapy following surgical resection.

Limitations: Model Scope: The study is limited to in vitro cell lines and a single subcutaneous mouse melanoma model (B16-F10). No large animal or human clinical data is provided. Administration Route: The therapy relies on direct intratumoral injection, which limits applicability to accessible tumors and may not address metastatic disease. Long-term Safety: While acute toxicity in normal cells was low, long-term immunogenicity, off-target effects, and chronic toxicity of the bacterial tRNases and PBAE nanoparticles were not extensively evaluated. Tumor Heterogeneity: The efficacy was only tested in one tumor type (melanoma); generalizability to other cancer types remains to be validated.