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 -…

This Project Grant from the National Heart, Lung and Blood Institute, part of the Department of Health and Human Services, provides $914,900 to Ship Of Theseus LLC under the Cardiovascular Diseases Research program (CFDA 93.837). The funding supports preclinical development of a nuclear-targeting biologic to safely increase stem cell expansion in vivo and accelerate recovery from neutropenia following chemotherapy and bone marrow transplant.

Specifically, the grant will advance a patented mutant of the nuclear transcription factor HOXB4 (HOXB4(M)) that promotes hematopoietic stem cell expansion without differentiation. The goals are to improve HOXB4(M)'s nuclear delivery efficiency and safety by identifying optimal combinations of cell penetrating peptides and nuclear localization motifs. In vitro and in vivo studies in mice will assess top candidates to select a lead biologic demonstrating long-term hematopoietic reconstitution without adverse effects. Successful completion will provide data to support future efficacy and safety studies necessary for an investigational new drug application. The technology aims to shorten the risky neutropenic phase after transplants and reduce costs by circumventing limitations of ex vivo culturing methods.

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 - 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.