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  • br further experiments using patient derived primary

    2020-08-28


    further experiments using patient-derived primary human GBM cell line GBM1A. GBM1A, which is capable of forming measurable mass-effect tumors when injected orthotopically, mimics what is seen in the clinic and is complimentary to GBM line 612. GBM1A SCH58261 were transfected with a mixture of five siRNA oligos targeting Robo1, YAP1, NKCC1, survivin, and EGFR. Western blotting showed simultaneous knockdown of all five genes in transfected cells compared to cells treated with the scRNA control (Fig. 4A). In addition, transfection with each siRNA se-quence separately as well as the mixture of all five siRNA sequences caused a decrease in the viability of GBM1A cells compared to the scRNA-treated controls (Fig. 4B). It should be noted that the cells transfected with all five siRNAs received a 120 nM total siRNA dose, or 24 nM of each siRNA, and the cells transfected with only a single siRNA sequence received the relatively higher dose of 120 nM of only that single siRNA. The effect of the combination therapy was even more pronounced when comparing 24 nM dosage of each individual siRNA to 24 nM of all siRNAs in combination (Fig. 4C).
    The viability of the cell line tested here, GBM1A, was sensitive to the knockdown of certain genes, particularly EGFR and survivin, while knockdown of some, like YAP1, had only a modest effect on GBM1A viability; interestingly, however, all five sequences in combination (24 nM each) had a statistically similar (p > 0.05) effect on GBM1A cells to that of a 5-fold higher dose (120 nM) of EGFR or survivin knockdown alone. Similarly, certain genes, particularly NKCC1 and EGFR, played in important role in GBM1A migration while others, like Robo1 and survivin, had little or no effect. Transfection with a mixture of all five genes resulted in decreased migration statistically similar to that seen with NKCC1, the siRNA that had the greatest effect on mi-gration individually (Fig. 4D). This highlights the benefit of our com-bination delivery system, in that we are able to target multiple 
    Fig. 5. Simultaneous knockdown of three genes following in vivo transfection of primary human GBM in a subcutaneous tumor model with a single formulation of bioreducible PBAE nanoparticles.
    pathways of GBM malignancy simultaneously and without a loss in efficacy of each individual siRNA. The independence of each siRNA's efficacy could be useful in tailoring siRNA combinations to target spe-cific tumor subtypes with different behaviors or gene expression pro-files.
    2.4. In vivo efficacy of siRNA delivered in combination via PBAEs
    In addition to verifying that in vitro delivery of siRNA combinations could cause human glioma cell death, it was important to evaluate if this strategy could also be effective in vivo. We first used a flank tumor model for ease of tumor access for protein analysis. As shown in Fig. 5, co-delivery of siRNAs against survivin, EGFR, and NKCC1 in combi-nation resulted in simultaneous in vivo knockdown of all three proteins, measured by Western blotting, demonstrating that these nanoparticles can successfully transfect tumor cells in vivo.
    In addition to the subcutaneous model, we also established a more clinically relevant human orthotopic GBM1A tumor model in mice and intratumorally injected nanoparticles to co-deliver all five functional siRNA sequences (siRobo, siYAP1, siNKCC1, siEGFR, and siSurvivin) simultaneously (Fig. 6). In previous work, we have demonstrated that intratumoral injection of R646 RNA nanoparticles into orthotopic brain tumors results in nanoparticle penetration throughout the tumor tissue [42]. In this work, our goal was to change the malignant, proliferative phenotype of these cells to lead to either tumor death or slowed tumor growth. Using an In Vivo Imaging System (IVIS) and systemically ad-ministered luciferin, we tracked the size of the luciferase-positive tumor over time and also performed histology on excised brains 14 days after the start of treatment to measure differences in the tumor burden among the groups. Tumors treated with the five siRNA sequences were less luminescent than those treated with control scRNA (7 ± 3 × 107 photons/sec flux vs. 17 ± 5 × 107 photons/sec, respectively, Fig. 6C). Image analysis of histological sections also showed significantly (p < 0.05) lower tumor burden in combination siRNA-treated mice (3.5 ± 0.6 mm2) than in scRNA-treated mice (6.9 ± 0.4 mm2) (Fig. 6A–B, D).
    3. Discussion r> This study demonstrates the ability of bioreducible PBAE-based nanoparticles to deliver functional siRNA to GBM cells both in vitro and in vivo, resulting in tumor cell death or slowed growth while avoiding major effects on healthy brain cells. The bioreducible, disulfide-con-taining PBAE R646 is of particular interest as an siRNA delivery agent, as it is achieves siRNA release specifically in the cytosol of cells, which is roughly three orders of magnitude more reducing than the extra-cellular space [43]. This targeted release is thought to improve siRNA delivery efficacy while also preventing any unwanted toxicity from the polymer by promoting its quick degradation inside the cell.