Delivery of Pesticides to Plant Parasitic Nematodes Using Tobacco Mild Green Mosaic Virus as a Nanocarrier

Plant parasitic nematodes are a major burden to the global agricultural industry, causing a $157 billion loss each year in crop production worldwide. Effective treatment requires large doses of nematicides to be applied, putting the environment and human health at risk. Challenges are to treat nematodes that are located deep within the soil, feeding on the roots of plants. To attack the problem at its roots, we propose the use of tobacco mild green mosaic virus (TMGMV), an EPA-approved herbicide as a carrier to deliver nematicides. TMGMV self-assembles into a 300 × 18 nm soft matter nanorod with a 4 nm-wide hollow channel. This plant virus is comprised of 2130 identical coat protein subunits, each of which displays solvent-exposed carboxylate groups from Glu/Asp as well as Tyr side chains, enabling the functionalization of the carrier with cargo. We report (1) the successful formulation and characterization of TMGMV loaded with ∼1500 copies of the anthelmintic drug crystal violet (CV), (2) the bioavailability and treatment efficacy of _CVTMGMV <i>vs</i> CV to nematodes in liquid cultures, and (3) the superior soil mobility of _CVTMGMV compared to free CV

Soil mobility of synthetic and virus-based model nanopesticides

Large doses of chemical pesticides are required to achieve effective concentrations in the rhizosphere, which results in the accumulation of harmful residues. Precision farming is needed to improve the efficacy of pesticides, but also to avoid environmental pollution, and slow-release formulations based on nanoparticles offer one solution. Here, we tested the mobility of synthetic and virus-based model nanopesticides by combining soil column experiments with computational modelling. We found that the tobacco mild green mosaic virus and cowpea mosaic virus penetrate soil to a depth of at least 30 cm, and could therefore deliver nematicides to the rhizosphere, whereas the Physalis mosaic virus remains in the first 4 cm of soil and would be more useful for the delivery of herbicides. Our experiments confirm that plant viruses are superior to synthetic mesoporous silica nanoparticles and poly(lactic-co-glycolic acid) for the delivery and controlled release of pesticides, and could be developed as the next generation of pesticide delivery systems

Generation of murine tumor models refractory to αPD-1/-L1 therapies due to defects in antigen processing/presentation or IFNγ signaling using CRISPR/Cas9.

Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications

Diffusion and Uptake of Tobacco Mosaic Virus as Therapeutic Carrier in Tumor Tissue: Effect of Nanoparticle Aspect Ratio

Nanoparticle-based technologies, including platforms derived from plant viruses, hold great promise for targeting and delivering cancer therapeutics to solid tumors by overcoming dose-limiting toxicities associated with chemotherapies. A growing body of data indicates advantageous margination and penetration properties of high aspect-ratio nanoparticles, which enhance payload delivery, resulting in increased efficacy. Our lab has demonstrated that elongated rod-shaped and filamentous macromolecular nucleoprotein assemblies from plant viruses have higher tissue diffusion rates than spherical particles. In this study, we developed a mathematical model to quantify diffusion and uptake of tobacco mosaic virus (TMV) in a spheroid system approximating a capillary-free segment of a solid tumor. Model simulations predict TMV concentration distribution with time in a tumor spheroid for different sizes and cell densities. From simulations of TMV concentration distribution, we can quantify the effect of TMV aspect ratio (e.g., nanorod length-to-width) with and without cellular uptake by modulated surface chemistry. This theoretical analysis can be applied to other viral or nonviral delivery systems to complement the experimental development of the next generation of nanotherapeutics

Detection and Imaging of Aggressive Cancer Cells Using an Epidermal Growth Factor Receptor (EGFR)-Targeted Filamentous Plant Virus-Based Nanoparticle

Molecular imaging approaches and targeted drug delivery hold promise for earlier detection of diseases and treatment with higher efficacy while reducing side effects, therefore increasing survival rates and quality of life. Virus-based nanoparticles are a promising platform because their scaffold can be manipulated both genetically and chemically to simultaneously display targeting ligands while carrying payloads for diagnosis or therapeutic intervention. Here, we displayed a 12-amino-acid peptide ligand, GE11 (YHWYGYTPQNVI), on nanoscale filaments formed by the plant virus potato virus X (PVX). Bioconjugation was used to produce fluorescently labeled PVX-GE11 filaments targeted toward the epidermal growth factor receptor (EGFR). Cell detection and imaging was demonstrated using human skin epidermoid carcinoma, colorectal adenocarcinoma, and triple negative breast cancer cell lines (A-431, HT-29, MDA-MB-231), all of which upregulate EGFR to various degrees. Nonspecific uptake in ductal breast carcinoma (BT-474) cells was not observed. Furthermore, co-culture experiments with EGFR⁺ cancer cells and macrophages indicate successful targeting and partitioning toward the cancer cells. This study lays a foundation for the development of EGFR-targeted filaments delivering contrast agents for imaging and diagnosis, and/or toxic payloads for targeted drug delivery

Effects of β2m, <i>Jak1</i>, and LMP2 single-gene deletion on the intra-tumoral immune cell landscape.

Quantification of macrophages, myeloid derived suppressor cells (MDSC) of mononuclear (M-MDSC) or polymorphonuclear origin (PMN-MDSC), dendritic cells (DC), CD4+ T-lymphocytes (CD4), regulatory CD4+ T-lymphocytes (Tregs), CD8+ T-lymphocytes (CD8), and natural killer cells (NK) per milligram of individual EMT6 wt and designated gene KO tumors, identified by the designated phenotype. KO cell lines: B2m KO clone #2 (442–6), Jak1 KO clone #1 (450–4), and Psmb9 KO clone #1 (454–5).Data are representative of 2 independent experiments with 4–7 mice per experiment. Box plots denote values from each tumor, with median±SD. Tumors were analyzed when their volume reached ~1000 mm3. Two-tailed t-test, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.</p

Validation of CRISPR gene knockout single-cell clones.

(A) Oncoplot of mutations in selected genes in the generated CRISPR clones. (B) Corresponding western blot quantification of designated proteins in the B2m, Jak1, and LMP2 gene knockout (KO) clones previously incubated for 24 h in the presence or absence of murine IFNγ. Beta-actin was used as loading control. Blue boxes denote protein expression of the KO gene in each corresponding clone. All three KOs were performed once, independently of each other.</p

Gene knockout tumor models are unresponsive to immune checkpoint inhibitor therapies.

Generic treatment schedule for all EMT6/EMT6 knockout (KO) (A) and MC38/MC38 B2m KO (C) tumor experiments. Arrows indicate days post tumor implant (day 0) when mice received αPD-1 or αPD-L1. Treatment was initiated when tumors reached an average volume of 50 mm3. (EMT6/EMT6 KO) or 60 mm3 (MC38/MC38 B2m KO). Graphs show (B) representative tumor growth curves (mean ± SEM) and (C) survival of EMT6 wt and gene KO tumors (n = 6-7/group) treated as depicted in the schematic in Fig 5A. Insets denote median overall survival (mOS) and number of mice without palpable tumors per treatment group at the time of study termination (day 50). (E) Graphs show tumor growth curves (mean ± SEM) of MC38 wt and B2m KO tumors (n = 5-7/group) treated as depicted in the schematic in Fig 5D. Inset denotes tumor growth in individual mice implanted with MC38 B2m KO cells and treated as depicted in Fig 5D. Black dashed lines depict days of αPD-1 or αPD-L1 doses. Tumor growth curves and corresponding survival of each KO tumor model were performed once independently of each other. The corresponding wt EMT6 data were performed three times independently of each other (data pooled). KO cell lines: EMT6 B2m KO clone #2 (442–6), EMT6 Jak1 KO clone #1 (450–4), EMT6 Psmb9 KO clone #1 (454–5), and MC38 B2m KO clone #3. Ordinary two-way ANOVA with Tukey’s multiple comparisons test (B), or two-tailed Log-rank (Mantel–Cox) (C). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.</p

S1 raw images.

Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.</div

Sequencing results.

Summary table of the Sanger sequencing, whole exome sequencing, and western blotting results in EMT6 KO clones. Transcript and protein change columns describe the position and change according to the Human Genome Variation Society (HGVS) nomenclature.</p