Intrabone Delivery of CD34+ Cells Using an Optimized Delivery System Does Not Enhance Engraftment in a Rhesus Macaque Model of Hematopoietic Stem Cell Transplantation
Authors: Stringaris Kate , Hoyt Robert F , Davidson Jan , Pantin Jeremy , Tisdale John F. , Uchida Naoya , Raines Lydia , Reger Robert , Sato Noriko , Dunbar Cynthia E. , Hunt Tim , Clevenger Randy , Krouse Allen , Metzger Mark , Bonifacino Aylin , Engels Theresa , Price Sandra , Choyke Peter , Donahue Robert E. , Childs Richard W. 
Published: October 2019

Abstract
Introduction: Umbilical cord blood (UCB) grafts are the only option for a significant minority of patients who require hematopoietic stem cell transplantation (HSCT) but lack a suitable related or unrelated donor. While UCB can serve as a suitable 'off the shelf' graft for many patients, units with the best HLA match often contain low and sometimes insufficient numbers of CD34+ cells for use in transplantation, particularly for adult patients. Furthermore, UCB grafts contain lower CD34+ cells number compared to BM or PBSC grafts, which leads to longer engraftment times and a higher risk of graft failure. BM and PBSC grafts are usually injected intravenously, homing to the bone marrow after several hours in the circulation. During this time, CD34+ cells are lost in the lungs, liver and spleen with typically < 20% making it to the bone marrow.1 Investigators have sought to overcome the limitation of low cell dose in UCB grafts and loss of CD34+ cells in the circulation by injecting CD34+ cells directly into the bone marrow space. However, we have recently shown that conventional intrabone delivery methods used in investigational trials to transplant UCB, result in low-level retention of hematopoietic progenitor cells in the intrabone space. Recently, we developed an optimized intrabone (OIB) transplant method using computer controlled low pressure and low volume injection (controlled infusion rate <0.2ml/min, total volume <5ml) under CT guidance. Utilizing porcine and rhesus macaque models, we have shown that OIB delivery of CD34+ cells improves intrabone retention, preventing circulation of CD34+ cells through the lungs, which is observed with conventional non-optimized intrabone methods.2 Here we conducted experiments using an autologous myeloablative transplant model in rhesus macaques comparing engraftment of gene-marked CD34+ cells transplanted intravenously, with cells transplanted using OIB delivery.
Methods: Rhesus macaques received GCSF and plerixafor mobilization prior to apheresis. Products were CD34+ selected using Miltenyi beads. CD34+ cells were split equally for transduction with lentiviral vectors encoding the reporters GFP and YFP. After myeloablative conditioning with 10Gy total body irradiation, half the graft was injected directly intra-bone using the OIB method, with the other half of the autograft simultaneously being injected intravenously via slow iv push. Peripheral blood samples were measured daily by flow cytometry to assess the proportion of engrafting cells deriving from each source. To address whether OIB transplantation could allow engraftment of low CD34+ cell numbers, as found in UCB units, the cell doses transplanted in one rhesus macaque recipient were reduced to only 0.5 x 106CD34+ cells/kg.
Results: CD34+ cells injected intrabone utilizing the OIB method engrafted in all 3 animals. However, flow cytometric analysis gating on GFP vs YFP positive neutrophils showed CD34+ cells injected utilizing OIB delivery did not engraft quicker than IV transplanted cells. Sequential monitoring of neutrophils over 30 days showed the contribution to hematopoiesis of OIB delivered cells was significantly lower than the cells injected IV (Table 1.)
Conclusions: We developed a novel intrabone delivery system that optimizes the retention of CD34+ cells into the bone marrow space. Although autologous CD34+ cells injected using this OIB transplant method were capable of engrafting in rhesus macaques that had undergone myeloablative conditioning, they did not engraft faster and contributed less to hematopoiesis than CD34+ cells simultaneously transplanted using conventional IV infusion. These data raise questions over whether intrabone delivery, even when using techniques to optimize intrabone retention, has utility in improving CD34+ cell engraftment.
References:
1. Van der Loo JC et al. Marrow and spleen seeding efficiencies of all murine hematopoietic stem cell subsets are de- creased by preincubation with hematopoietic growth factors. Blood. 1995; 85:2598-2606
2. Pantin et. al. Optimization of intrabone delivery of hematopoietic progenitor cells in a swine model using cell radiolabeling with 89zirconium. American J Transplant. 2015 Mar;15(3):606-17.

Disclosures
Davidson: Macrogenics: Employment. Pantin:NIH: Patents & Royalties: a patent application for an intrabone delivery device. Dunbar:National Institute of Health: Research Funding.