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（HSC）移植（HSCT）被认为是一种潜在的治疗选择， 在美国每年进行超过20，000例造血干细胞移植。 然而，HSCT与高风险相关，因为在移植前需要去除患病的骨髓。 移植直到移植的细胞植入并增殖到足以重建患者的免疫力 系统中，患者被认为是贫血，在此期间，他们非常容易受到感染。除了 对患者潜在的危及生命的身体风险，中性粒细胞减少症需要长的住院时间， 在美国，HSCT的成本高达40万美元。临床上的一种现行策略 试验的目的是移植更大的供体造血干细胞库，以缩短造血干细胞减少期。然而，这些方法 都依赖于供体细胞的离体培养， 干细胞的正常功能，并且可能非常昂贵。忒修斯之船的创新解决方案是 一种在移植前短暂、一次性接触供体细胞的药物， 体内扩增我们的药物是核转录因子同源盒蛋白B4的专利突变体 （HOXB 4（m）），其促进HSC扩增而不分化。HOXB 4（m）具有改善的降解 抗性，这使我们能够克服HOXB 4的几个缺点（例如，蛋白质半衰期短， 与遗传过表达相关的骨髓增生性疾病）。通过延长其细胞内半衰期， HOXB 4（m）对于临床使用是实用的，同时潜在地避免了由组成型HOXB 4（m）引起的不良反应。 过度表达早期临床前数据表明，用HOXB 4（m）处理细胞可以扩增所有主要谱系， HSC同时保持多能性。然而，HOXB 4（m）的当前递送受到其渗透能力的限制。 细胞和定位在细胞核，其生物活性的网站。 该R61/R33提案的目标是通过改善HOXB 4（m）的核性能来提高其效率和安全性。 交付.这将通过实现三个目标来实现。在目标1（R61）中，我们将执行一个库 筛选以鉴定HOXB 4（m）的细胞穿透肽和核靶向基序的最佳组合 核定位在目标2（R33）中，我们将评估最佳候选药物的体外疗效，以选择电极导线 候选人在目标3（R33）中，我们将证明长期造血重建无不良反应 或我们的主要候选人在小鼠HSCT研究中的体内GVHD。成功完成R61/R33计划 将证明在较低浓度下维持疗效的改良HOXB 4（m）候选物的可行性 暴露时间更短该项目将提供必要的数据，以支持未来广泛的体内 IND申请所需的有效性和安全性研究。有了HOXB 4（m），我们可以绕过原来的 通过缩短血供减少期，改善体内扩增，使HSCT患者受益 以减少感染、住院时间和费用。

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.