Ex Vivo Expansion of Human Hematopoietic Stem Cells Via Lipid Nanoparticle-Mediated HOXB4 mRNA Delivery
Authors: Perveen Sadia , Salisbury-Ruf Christi , Smith Richard H. , Araki Daisuke , Dagur Pradeep , Larochelle Andre 
Published: December 2024

Allogeneic transplantation and autologous ex vivo gene correction for blood disorders are often constrained by the limited availability of hematopoietic stem and progenitor cells (HSPCs). Furthermore, the inefficiency of current nuclease-based protocols for targeted integration or correction of therapeutic genes in human HSPCs results in a paucity of gene-edited cells suitable for transplantation. Ex vivo expansion of transplantable hematopoietic stem cells (HSCs) could effectively address these limitations. HOXB4, a member of the homeobox gene family, has emerged as a potent regulator of HSC self-renewal and expansion. Overexpression studies across various species have demonstrated its significant impact on HSC biology.

However, several challenges persist in current approaches for HOXB4 overexpression. Integrating retroviral vectors pose a risk of insertional mutagenesis, potentially leading to oncogenic transformation. The limited half-life of HOXB4 protein and the toxicity issues associated with electroporation in primary HSCs can constrain its long-term efficacy in HSC expansion. Additionally, achieving optimal levels of HOXB4 expression is critical, as both insufficient and excessive levels can adversely affect HSC function. Lipid nanoparticle (LNP)-mediated delivery of HOXB4 mRNA to human HSCs offers several potential advantages. This approach enables controlled, transient release and the potential for redosing, thereby prolonging the duration of HOXB4 overexpression in adult HSCs. Additionally, LNP-mediated delivery allows for transient rather than constitutive overexpression of HOXB4, mitigating the potential risks associated with prolonged overexpression.

In this study, we utilized a novel ionizable amino dendrimer lipid, 4A3-SSC-PH, known to promote efficient endosomal escape and rapid mRNA release. Utilizing the NanoAssemblr™ Ignite™ microfluidic system, we obtained HOXB4-encoding LNPS with an average diameter of 80-100 nm, a polydispersity index of less than 0.2, and an encapsulation efficiency between 95% and 98%. To assess transfection efficiency, LNPs encapsulating HOXB4 mRNA were applied to primary human mobilized peripheral blood CD34+ cells in vitro, and HOXB4 protein expression was subsequently analyzed via Western blot. The expression kinetics demonstrated peak HOXB4 protein levels at 12 hours post-transfection, with expression dissipating by 48 hours, at an LNP dose of 5 pg mRNA/cell. This method exhibited minimal cytotoxicity, with cell viabilities ranging from 90% to 95%. Based on these expression kinetics data, we cultured human HSC-enriched populations (characterized by CD34+CD38-CD45RA-CD90+ markers) for 10 days, supplementing with LNPs encapsulating HOXB4 mRNA every other day. Notably, the percentages and absolute numbers of phenotypic HSCs increased 2- and 3-fold, respectively, compared to untreated control cells. Xenotransplantation studies are planned to determine whether this approach facilitates the expansion of cells with long-term repopulation potential.

Overall, LNPs offer a novel, low-toxicity, and promising platform for transfecting human HSCs to overexpress proteins, thereby enhancing their therapeutic potential. Future research efforts are aimed at comprehensively understanding the impact of HOXB4 overexpression on HSC expansion and the maintenance of their stemness properties.