Development of Next-Generation Precision Medicine RNAi Therapeutics to Treat AML

摘要 急性髓系白血病（acute myeloid leukemia，AML）是最常见的恶性血液病之一。不幸的是， 由于化疗的高复发率，无病生存率（DFS）保持在40%。更大 超过80%的AML在其细胞表面上表达高水平的CD 33标记物。替代治疗 方法，包括抗体-药物偶联物（ADC），抗CD 33 Mylotarg（吉妥珠单抗）， 和放射性核素抗体偶联物（RAC）已经显示出一些有希望的结果。不过两 这些抗体治疗剂具有显著的副作用， 表达CD 33的正常细胞以及由于非特异性组织吸收而杀死正常细胞 和药物脱落。不幸的是，AML也不响应抗PD 1和PD-1 L检查点 抑制剂的由于染色体易位，MLL基因与许多其他基因融合，包括 AF 9基因，导致驱动AML生长和存活的ML融合癌基因。MLL的表达- AML中的AF 9驱动癌基因具有特别差的临床结果。因此，有一个伟大的 需要开发新的精确医学疗法，选择性地靶向并仅杀死AML肿瘤 细胞的致癌基因易位，同时保留正常的CD 33阳性细胞。RNA 干扰（RNAi）应答具有靶向MLL-AF 9和其它MLL易位的巨大潜力 融合基因不幸的是，尽管其有前途的治疗功能，由于其必要的负面影响， 由于siRNA具有带电荷的磷酸骨架，因此siRNA没有进入细胞的能力并且需要递送剂。到 为了解决RNAi传递问题，我们率先开发了下一代RNAi触发器，称为 核糖核中性（siRNN）前药。siRNN代表了一种“前药”方法， 电荷被生物可逆的磷酸三酯化学基团直接中和， 在细胞质中分裂，但不在细胞外。速率限制性RNAi递送步骤是从RNA中逃逸。 内体进入细胞质。为了增强内体逃逸，我们将合成下一代 内体逃逸结构域（EED）。我们将MLL-AF 9 siRNN RNAi触发物靶向递送至AML细胞 通过与抗CD 33抗体缀合，称为抗体-RNAi缀合物（ARC）。这个高的目标 风险/高收益技术开发建议是通过产生精确药物抗CD 33来治疗AML 选择性靶向MLL-AF 9融合癌基因并杀死AML的抗体-RNAi缀合物（ARC） 细胞基于其突变遗传学，但与ADC和RAC不同，对CD 33阳性细胞没有影响。 正常细胞

ABSTRACT Acute myeloid leukemia (AML) is one of the most prevalent hematological malignancies. Unfortunately, due to a high relapse rate from chemotherapy, disease-free survival (DFS) remains at 40%. Greater than 80% of AMLs express high levels of the CD33 marker on their cell surface. Alternative therapeutic approaches, including Antibody-Drug Conjugate (ADC), anti-CD33 Mylotarg (gemtuzumab ozogamicin), and Radionuclide Antibody-Conjugates (RACs), have shown some promising results. However, both of these antibody therapeutic agents have significant side effects and suffer from indiscriminant killing of normal cells expressing CD33 as well as from killing normal cells due to non-specific tissue absorption and drug shedding. Unfortunately, AML also does not respond to anti-PD1 and PD-1L check-point inhibitors. Due to chromosomal translocations, the MLL gene fuses to many other genes, including the AF9 gene, resulting in MLL-fusion oncogenes that drive AML growth and survival. Expression of MLL- AF9 driver oncogene in AML has a particularly poor clinical outcome. Consequently, there is a great need to develop novel precision medicine therapeutics that selectively targets and kills only AML tumor cells based on their oncogenic genetic translocations, while sparing normal CD33 positive cells. RNA Interference (RNAi) responses have great potential to target MLL-AF9 and other MLL translocation fusion genes. Unfortunately, despite its promising therapeutic features, due to their requisite negatively charged phosphate backbone, siRNAs have no ability to enter cells and require a delivery agent. To tackle the RNAi delivery problem, we pioneered development of a next-generation RNAi trigger, called RiboNucleic Neutral (siRNN) prodrugs. siRNNs represent a “Prodrug” approach where the negative charge is directly neutralized by a bioreversible phosphotriester chemical group that is selectively cleaved off in the cytoplasm, but not outside of cells. The rate-limiting RNAi delivery step is escape from the endosome into the cytoplasm. To enhance endosomal escape, we will synthesize a next-generation endosomal escape domain (EED). We will target delivery of MLL-AF9 siRNN RNAi triggers to AML cells by conjugation to anti-CD33 antibodies, called Antibody-RNAi Conjugates (ARCs). The goal of this high risk/high gain technology development proposal is treat AML by generating precision medicine anti-CD33 Antibody-RNAi Conjugates (ARCs) that selectively target the MLL-AF9 fusion oncogene and kill AML cells based on their mutant genetics, but unlike ADCs and RACs, have no effect on CD33-positive normal cells.