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.