Synthetic introns for selective targeting of RNA splicing factor-mutant leukemia

总结 许多癌症携带影响RNA剪接因子的复发性、功能改变突变， RNA剪接中的序列特异性变化，促进疾病的发生和进展。这些 “剪接体突变”是骨髓增生异常综合征（MDS）中最常见的一类突变， 相关的血液疾病，其中有几个有效的，FDA批准的治疗。尽管频率很高 剪接体突变和相应的对新疗法的需求，目前还不存在 特异性和选择性地靶向这些病变。 在这里，我们建议通过创造新的精确疗法来解决这种临床需求， 杀死剪接体突变的细胞。我们的跨学科团队由一位具有专业知识的医生和科学家组成 在癌症生物学和病人护理（阿卜杜勒-瓦哈卜），一个基础科学家与RNA剪接的专业知识， 功能基因组学（布拉德利）和具有药物输送专业知识的生物工程师（海勒）。初步 在实验中，我们开发了“合成内含子”技术来利用改变的RNA剪接活性， 由剪接体突变引起的癌症特异性基因表达，表明合成 内含子允许在癌细胞中高度选择性地表达治疗有效载荷， 并使用该系统抑制体内多种癌症类型的生长（North et 等人，Nature Biotechnology，2022）。我们还证明了合成内含子能够同时和 选择性递送多种治疗有效载荷，并允许顺式和 反式作用序列元件和剪接因子，其控制由以下引起的促肿瘤发生错误剪接 复发性剪接体突变。 我们现在将在这些初步研究的基础上开发基于内含子的合成疗法， 骨髓肿瘤，包括MDS、急性髓性白血病（AML）和慢性粒单核细胞白血病 （CMML），并另外利用合成内含子来理解在细胞中异常剪接的机制基础。 目的：1、探讨常见疾病的分子机制 由于不同的SF 3B 1突变诱导的等位基因特异性剪接变化;目标2，开发合成内含子， 使每种常见突变的RNA剪接因子能够选择性地表达治疗性蛋白质， 白血病;目的3，优化体内递送并严格测试用于治疗白血病的免疫刺激疗法 SF 3B 1突变型造血系统恶性肿瘤 这些研究的意义在于，它们将开发一种新的 这项技术能够对癌症相关剪接体突变进行机制研究， 用于治疗性靶向这些突变的特定手段。健康相关性是，拟议的工作 将创造特定的治疗产品，用于治疗目前几乎没有有效的癌症类型，FDA- 批准的治疗。

SUMMARY Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors, resulting in sequence-specific changes in RNA splicing that promote disease initiation and progression. These “spliceosomal mutations” are the most common class of mutations in myelodysplastic syndromes (MDS) and related hematologic disorders, which have few effective, FDA-approved treatments. Despite the high frequency of spliceosomal mutations and corresponding need for new therapeutics, there currently exist no therapies that specifically and selectively target these lesions. Here, we propose to address this clinical need by creating new precision therapeutics that selectively kill cells with spliceosomal mutations. Our interdisciplinary team consists of a physician-scientist with expertise in cancer biology and patient care (Abdel-Wahab), a basic scientist with expertise in RNA splicing and functional genomics (Bradley), and a bioengineer with expertise in drug delivery (Heller). In preliminary experiments, we developed the “synthetic intron” technology to harness altered RNA splicing activity caused by spliceosomal mutations to drive cancer-specific gene expression, showed that synthetic introns permit highly selective expression of therapeutic payloads in cancer cells while leaving healthy cells unharmed, and used this system to suppress the growth of diverse cancer types in vivo (North et al, Nature Biotechnology, 2022). We additionally demonstrated that synthetic introns enable simultaneous and selective delivery of multiple therapeutic payloads and allow for detailed mechanistic dissection of the cis- and trans-acting sequence elements and splicing factors that govern pro-tumorigenic mis-splicing caused by recurrent spliceosomal mutations. We will now build on these preliminary studies to develop synthetic intron-based therapeutics for myeloid neoplasms, including MDS, acute myeloid leukemia (AML), and chronic myelomonocytic leukemia (CMML), and additionally utilize synthetic introns to understand the mechanistic basis for aberrant splicing in these diseases as follows: Aim 1, Dissect and exploit the molecular mechanisms underlying common as well as allele-specific splicing changes induced by different SF3B1 mutations; Aim 2, Develop synthetic introns that enable selective therapeutic protein expression for each of the commonly mutated RNA splicing factors in leukemia; Aim 3, Optimize in vivo delivery and rigorously test an immunostimulatory therapy for treating SF3B1-mutant hematopoietic malignancies. The significance of these studies is that they will develop a new technology that enables mechanistic studies of cancer-associated spliceosomal mutations and also provides a specific means for therapeutically targeting these mutations. The health relatedness is that the proposed work will create specific therapeutic products for treating cancer types that currently have few effective, FDA- approved treatments.