microRNA-21 Blockade of Triple Negative Breast Cancer

三阴性乳腺癌(TNBC)是一种孤儿疾病，每年袭击4.6万名美国女性。TNBC 细胞缺乏人雌激素受体、孕激素受体和表皮生长因子受体2(Her2)， 现有药物的靶标。TNBC在标准护理化疗和放射治疗后复发，导致其死亡 受害者在4年内。有前景的新疗法，如仅使用聚腺苷二磷酸核糖聚合酶(PARP)抑制剂 使一小部分BRCA1/2突变患者受益。因此，TNBC显示出迫切需要 分子靶向治疗。大多数TNBC细胞和相关间质显示高microRNA21(miR-21)， 这会减少抑制细胞生长的肿瘤抑制蛋白。一种分子靶向治疗方法 在TNBC中阻断miR-21是我们的目标。前提：我们设计了一种强大的miR-21封闭剂，使用 氨甲基桥联核酸(BNA)，具有很强的碱基性，TM&GT；80°C，低毒，血清稳定， 通过胰岛素样生长因子1受体(IGF1R)连接到多肽配体以进行内吞作用。42%的 TNBC肿瘤显示结构性IGF1R信号。IGF1R的多肽配体提供了一种独特的策略 将miR-21阻滞剂特异性地输送到TNBC细胞。我们的代理与RNA中的miR-21配对- 诱导沉默复合体(RISC)，使靶mRNAs免于miR-21攻击。我们的miR-21BNA升高的肿瘤 抑制蛋白，受抑制的免疫检查点基因表达，增加细胞凋亡，减缓 在多个TNBC系中的扩散和迁移。无菌生理盐水中的BNA-肽可通过 输液。我们的策略优化了癌细胞的特异性传递，以阻止增殖和免疫检查点， 由Bound Treateutics LLC授权的PCT专利申请。假设：我们独一无二的 设计与受体配体连接的短microRNA阻断剂将引导TNBC细胞摄取并减缓 毒性最小的TNBC原位异种移植的生长。目标1：测量铅miR-21的影响 阻断剂对miR-21靶向mRNAs和Checkpoint增殖、凋亡、侵袭和细胞表达的影响 5种分子亚型TNBC细胞中的蛋白质。用铅测量肿瘤反应和免疫激活 MIR-21阻断剂在免疫活性同系小鼠原位移植TNBC 4T1-荧光素酶中的应用预测 结果：显著抑制肿瘤生长和免疫检查点，提高T细胞应答。目标 2：通过转录组分析检测铅miR-21阻滞剂对人肝细胞的毒性。量测 铅miR-21阻滞剂对小鼠肝肾血清标志物和体重的毒性。在目标上测量 和脱靶转录组效应、药代动力学和铅miR-21阻滞剂在体内的生物分布 老鼠模型。预测结果：对人肝细胞和小鼠宿主细胞的毒性最小。影响：我们 寻求概念验证，以降低BNA-肽TNBC治疗的商业化风险，使完全 IND和A期之前的效力、T细胞反应、毒理学和药代动力学的临床前研究 单剂安全性试验。我们预计，miRNA阻断将显著提高TNBC患者的存活率。

Triple negative breast cancer (TNBC) is an orphan disease that attacks 46,000 US women every year. TNBC cells lack human estrogen receptor, progesterone receptor, and epidermal growth factor receptor 2 (Her2), the targets of existing medicines. TNBC recurs after standard-of-care chemotherapy and radiation, killing its victims within 4 years. New promising therapies such as poly(ADP-ribose) polymerase (PARP) inhibitors only benefit a small subset of patients with BRCA1/2 mutations. Thus, TNBC shows a critical need for molecularly-targeted therapy. Most TNBC cells and associated stroma show high microRNA 21 (miR-21), which decreases tumor suppressor proteins that keep cell growth in check. A molecularly-targeted therapeutic to block miR-21 in TNBC is our objective. Premise: We have designed a strong miR-21 blocking agent using aminomethyl bridged nucleic acid (BNA), with strong basepairing, Tm >80°C, low toxicity, and serum stability, conjugated to a peptide ligand for endocytosis by the insulin-like growth factor 1 receptor (IGF1R). 42% of TNBC tumors show constitutive IGF1R signaling. The peptide ligand for IGF1R provides a unique strategy for delivering miR-21 blocker specifically into the TNBC cells. Our agent basepairs with miR-21 in the RNA- induced silencing complex (RISC), freeing target mRNAs from miR-21 attack. Our miR-21 BNA elevated tumor suppressor proteins, suppressed immune checkpoint gene expression, increased apoptosis, slowed proliferation and migration in multiple TNBC lines. BNA-peptide in sterile saline can be administered by infusion. Our strategy optimizes cancer cell-specific delivery to block proliferation and immune checkpoints, covered by a pending PCT patent application, licensed by Bound Therapeutics LLC. Hypothesis: Our unique design for short microRNA blockers conjugated to a receptor ligand will direct TNBC cell uptake and slow the growth of TNBC orthotopic xenografts with minimal toxicity. Aim 1: Measure the effects of the lead miR-21 blocker on proliferation, apoptosis, invasion, and cellular expression of miR-21 target mRNAs and checkpoint proteins in 5 molecular subtypes of TNBC cells. Measure tumor response and immune activation by the lead miR-21 blocker in TNBC 4T1-luciferase orthotopic xenografts in immunocompetent syngeneic mice. Predicted results: Significant inhibition of tumor growth and immune checkpoints, and elevation of T-cell response. Aim 2: Measure toxicity of the lead miR-21 blocker in human hepatocytes by transcriptome analysis. Measure toxicity of the lead miR-21 blocker in mice by liver and kidney serum markers and weight. Measure on-target and off-target transcriptome effects, pharmacokinetics, and biodistribution of the lead miR-21 blocker in the mouse model. Predicted results: Minimal toxicity to human hepatocytes and murine host cells. Impact: We seek proof-of-concept to derisk the commercialization of a BNA-peptide TNBC therapeutic, enabling a full preclinical study of potency, T-cell response, toxicology, and pharmacokinetics, prior to IND and a Phase I single agent safety trial. We expect that miRNA blockade will significantly increase TNBC patient survival.