Preclinical development of a nuclear-targeting biologic that safely increases stem cell expansion in vivo to accelerate recovery from neutropenia after chemotherapy and bone marrow transplant

造血干细胞移植(HSCT)被认为是治疗慢性粒细胞白血病患者的一种潜在选择。 在美国，每年有超过20,000例血液和骨髓癌以及超过20,000例造血干细胞移植。 然而，造血干细胞移植的风险很高，因为在移植之前需要切除患病的骨髓。 移植。直到移植的细胞植入并增殖到足以重建患者的免疫系统 系统中，患者被认为是中性粒细胞减少症，在此期间他们极易受到感染。除了……之外 中性粒细胞减少症可能危及患者的生命，因此需要较长的住院时间。 在美国，CAN在一定程度上推动了HSCT的昂贵成本，最高可达40万美元。当前临床上的一种策略 试验的目的是移植更多的捐赠者HSCs，以缩短中性粒细胞减少期。然而，这些方法 所有这些都依赖于捐赠者细胞的体外培养，这些细胞依赖于不完善的人工培养系统，这可能会破坏 干细胞的正常功能，而且可能昂贵得令人望而却步。特修斯之船的创新解决方案是 一种在移植前用于短暂、一次性接触供体细胞的药物，可改善 体内扩张。我们的药物是核转录因子同源框蛋白B4的专利突变体 (HOXB4(M))，促进无分化的HSC扩张。HOXB4(M)改善了降解性 抗性，这使我们能够克服HOXB4的几个缺点(例如，蛋白质半衰期短和 与基因过度表达相关的骨髓增殖性疾病)。通过延长其细胞内的半衰期， HOXB4(M)可用于临床，同时潜在地避免由成分引起的不良反应 过度表达。早期临床前数据表明，用HOXB4(M)处理细胞可以扩大所有主要的 造血干细胞，同时保持多能性。然而，目前HOXB4(M)的递送受到其渗透能力的限制 细胞，并定位于其生物活性所在的细胞核。 R61/R33提案的目标是通过改进HOXB4(M)的核性能来提高其效率和安全性 送货。这将通过执行3个目标来实现。在目标1(R61)中，我们将执行库 HOXB4(M)细胞穿透肽和核靶向基序最佳组合的筛选 核本地化。在目标2(R33)中，我们将评估最佳候选者的体外效率以选择一条线索 候选人。在目标3(R33)中，我们将演示长期的造血重建，没有不良反应 在小鼠造血干细胞移植研究中，我们的主要候选者体内的GVHD。成功完成此R61/R33计划 将展示改进的HOXB4(M)候选药物的可行性，该候选药物在较低浓度下保持效力 以及更短的曝光期。该项目将提供必要的数据，以支持未来在体内的广泛应用 提交IND申请所需的有效性和安全性研究。使用HOXB4(M)，我们可以绕过原始的 HOXB4的障碍和通过缩短中性粒细胞减少期改善体内扩张使HSCT患者受益 以减少感染、住院时间和成本。

Hematopoietic stem cell (HSC) transplants (HSCTs) are considered a potentially curative option for patients with certain cancers of the blood and bone marrow and over 20,000 HSCTs are performed in the US each year. However, HSCTs are associated with high risk due to the required removal of diseased bone marrow before transplantation. Until the transplanted cells engraft and proliferate enough to reconstitute the patient’s immune system, the patient is considered neutropenic during which they are highly susceptible to infection. In addition to the potentially life-threatening physical risk to the patient, neutropenia necessitates long hospitalization time that can in part drives the expensive cost of HSCTs ranging up to $400,000 in the US. A current strategy in clinical trials is to transplant larger pools of donor HSCs to shorten the neutropenic phase. However, these approaches all rely on ex vivo culturing of donor cells that depend on imperfect man-made culture systems that may disrupt the proper function of the stem cells and can be prohibitively expensive. Ship of Theseus’ innovative solution is a drug that will be used for a brief, one-time exposure to donor cells prior to transplantation that improves expansion in vivo. Our drug is a patented mutant of nuclear transcription factor, Homeobox protein B4 (HOXB4(m)), which promotes HSC expansion without differentiation. HOXB4(m) has improved degradation resistance, which enables us to overcome several shortcomings of HOXB4 (e.g., short protein half-life and myeloproliferative disorders associated with genetic overexpression). By elongating its intracellular half-life, HOXB4(m) is practical for clinical use while potentially avoiding adverse effects induced by constitutive overexpression. Early preclinical data indicating treating cells with HOXB4(m) can expand all major lineages of HSCs while maintaining multipotency. However, current delivery of HOXB4(m) is limited by its ability to penetrate cells and localize in the nucleus, the site of its bioactivity. The goal of this R61/R33 proposal is to improve efficiency and safety of HOXB4(m) by improving its nuclear delivery. This will be accomplished through the execution of 3 aims. In Aim 1 (R61), we will perform a library screen to identify optimal combinations of cell penetrating peptide and nuclear targeting motifs for HOXB4(m) nuclear localization. In Aim 2 (R33), we will assess in vitro efficacy of the top candidates to select a lead candidate. In Aim 3 (R33), we will demonstrate long-term hematopoietic reconstitution without adverse effects or GVHD of our lead candidate in vivo in a mouse HSCT study. Successful completion of this R61/R33 program will demonstrate feasibility of an improved HOXB4(m) candidate that maintains efficacy at lower concentrations and briefer exposure periods. This project will provide the necessary data to support future extensive in vivo efficacy and safety studies necessary for an IND submission. With HOXB4(m), we can circumvent the original hurdles of HOXB4 and improve expansion in vivo to benefit HSCT patients by shortening the neutropenic phase to reduce infection, hospitalization time, and cost.