FSHD Pre-clinical Development

项目摘要 我们的Wellstone研究已经确定了两类用于临床前体内测试的FSHD疗法 发展项目3的目标是验证这些候选治疗剂和/或药物组合物的体内功效。 在未来五年内为IND研究提供相关化合物。研究将在 与在药物开发方面具有专业知识和成功记录的行业合作伙伴合作， 资源，以支持拟议的研究战略。 待研究的一类FSHD药物是基于DUX 4吗啉代，FM 10，Wellstone 先前的研究已经确定了靶向DUX 4 4 qA多聚腺苷酸化序列，并敲低DUX 4 在FSHD患者细胞和FSHD异种移植肌肉中的转录活性。RNA-Seq研究进一步 确定FM 10不具有显著的脱靶效应，但在敲低表达方面有效 DUX 4靶基因的完整序列特定目标1的研究集中在体内体细胞递送和 FM 10序列对FSHD异种移植肌肉中DUX 4敲低的治疗效力，使用独特的， 由萨雷普塔Therapeutics开发和验证的无毒细胞穿透肽。其他PPMO 靶向DUX 4将使用FSHD原代患者肌源性细胞在FM 10序列区中鉴定， 优化治疗反应和RNA-Seq，以评估其靶向和脱靶活性。 待研究的第二类FSHD药物是4-甲基伞形酮（4-MU）和其他抑制剂 透明质酸（HA）信号通路。Wellstone的研究表明，4-MU是一种有效的抑制剂， DUX 4毒性和其他DUX 4分子病理学，这表明HA信号传导途径起着重要作用。 在DUX 4依赖性病理学中起重要作用，可能是通过涉及C1 QBP的机制，C1 QBP是一种蛋白质， 与DUX 4和HA结合。这将HA途径确定为额外FSHD治疗的靶点。 发展，也表明，4-MU，已经在使用作为一种抗痉挛治疗和广泛 在小鼠中研究了其抗癌和抗炎特性，是一种有前途的FSHD药物，可用于体内测试。 具体目的1和2集中于研究C1 QBP在DUX 4病理学中的功能以及C1 QBP在DUX 4病理学中的作用。 鉴定和测试靶向该通路以阻断DUX 4毒性的信号通路抑制剂。4-MU 将在全球范围内进行检查，以确定其全方位的靶向和脱靶效应，HA途径将 使用特异性途径抑制剂进行研究，以鉴定HA信号传导的其他靶点， 调查HA通路抑制剂研究将与Genea Biocells合作进行，Genea Biocells将 提供临床验证的HS信号通路抑制剂以及药物行业的专业知识和经验， 发展具有抑制DUX 4毒性的高效力以及低毒性和脱靶破坏的药物， 将在他莫昔芬诱导的Cre（TIC）中鉴定单独和与4-MU联合的体内活性 DUX 4小鼠模型中，并且如果合适，在FSHD异种移植肌肉模型中。

PROJECT SUMMARY Our Wellstone research has identified two classes of FSHD therapeutics for pre-clinical in vivo testing development. The goal of Project 3 is to validate the in vivo efficacy of these candidate therapeutics and/or related compounds for IND enabling studies within the next five years. Studies will be conducted in collaboration with industry partners with expertise and successful track records in drug development and with resources to support the proposed research strategies. One class of FSHD drugs to be investigated is based on the DUX4 morpholino, FM10, which Wellstone research has previously established targets the DUX4 4qA polyadenylation sequence and knocks down DUX4 transcriptional activity in FSHD patient cells and in FSHD xenograft muscle. RNA-Seq studies further established that FM10 does not have significant off-target effects, but is effective at knocking down expression of the entire suite of DUX4 target genes. Studies in Specific Aim 1 focus on in vivo somatic delivery and therapeutic potency of the FM10 sequence for DUX4 knockdown in FSHD xenograft muscle, using unique, non-toxic cell-penetrating peptides developed and validated by Sarepta Therapeutics. Additional PPMOs targeting DUX4 will be identified in the FM10 sequence region using FSHD primary patient myogenic cells to optimize therapeutic response and RNA-Seq to evaluate their on- and off-target activities. A second class of FSHD drugs to be investigated are 4-methylumbelliferone (4-MU) and other inhibitors of the hyaluronic acid (HA) signaling pathway. Wellstone research has shown that 4-MU is a potent inhibitor of DUX4 toxicity and other DUX4 molecular pathologies, which suggests that the HA signaling pathway plays an essential role in DUX4-dependent pathology, likely through a mechanism involving C1QBP, a protein that binds to both DUX4 and HA. This identifies the HA pathway as a target for additional FSHD therapeutic development, and also suggests that 4-MU, already in use as an antispasmodic therapeutic and extensively studied in mice for its anti-cancer and anti-inflammation properties, is a promising FSHD drug for in vivo testing. Specific Aims 1 and 2 focus on investigations of the functions of C1QBP in DUX4 pathology and the identification and testing of signaling pathway inhibitors that target this pathway to block DUX4 toxicity. 4-MU will be examined on a global level to identify its full range of on- and off-target effects, and the HA pathway will be studied using specific pathway inhibitors to identify additional targets of HA signaling for therapeutic investigation. HA pathway inhibitor studies will be conducted in collaboration with Genea Biocells, who will provide clinically validated HS signaling pathway inhibitors as well as industry expertise and experience in drug development. Drugs with high potency for inhibiting DUX4 toxicity, and low toxicity and off target disruptions, will be identified for in vivo activity, alone and in combination with 4-MU, in the Tamoxifen-Inducible Cre (TIC) DUX4 mouse model and, if suitable, in the FSHD xenograft muscle model.