The successful engraftment of genetically modified hematopoietic stem cells (HSCs) without toxic conditioning is a desired goal for HSC gene therapy. To this end, we have examined the combination of intrabone marrow transplantation (iBMT) and in vivo expansion by a selective amplifier gene (SAG) in a nonhuman primate model. The SAG is a chimeric gene consisting of the erythropoietin (EPO) receptor gene (as a molecular switch) and c-Mpl gene (as a signal generator). Cynomolgus CD34+ cells were retrovirally transduced with or without SAG and returned into the femur and humerus following irrigation with saline without prior conditioning. After iBMT without SAG, 2-30% of colony-forming cells were gene marked over 1 year. The marking levels in the peripheral blood, however, remained low (<0.1%). These results indicate that transplanted cells can engraft without conditioning after iBMT, but in vivo expansion is limited. On the other hand, after iBMT with SAG, the peripheral marking levels increased more than 20-fold (up to 8-9%) in response to EPO even at 1 year posttransplant. The increase was EPO-dependent, multilineage, polyclonal, and repeatable. Our results suggest that the combination of iBMT and SAG allows efficient in vivo gene transduction without marrow conditioning.

Mutant calreticulin (CALR) has been shown to play a causal role in the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF) via activation of the thrombopoietin receptor MPL. The oncogenic property of mutant CALR originates from a +1 frameshift mutation in its carboxyl-terminal domain, which is found in approximately 30% of patients with ET and PMF. Because the domain is uniquely found in mutant CALR, it has been recognized as a neoantigen and can therefore be used to target CALR-mutant cells using immunotherapy. In the present study, we found that a large portion of the domain generated by the frameshift in the mutant CALR was cleaved by an endoprotease belonging to the subtilisin family in multiple cell lines and primary cells. The cleaved form of mutant CALR was detected in the cell lysate; however, it was more abundant in the culture supernatant, implying that the cleavage occurred on the cell surface and/or outside the cells. Using mass spectrometric analysis, we determined the cleavage site of mutant CALR. To examine whether the cleavage was required for the oncogenic properties of mutant CALR, we introduced point mutations at the cleavage site. The mutant CALR construct that was resistant to protease cleavage exhibited full oncogenic capacity when expressed in UT-7/TPO cells. Consistent with this observation, chemical inhibition of the protease, which blocked the cleavage of mutant CALR, did not interfere with mutant CALR-dependent cell growth in UT-7/TPO cells. Next, we generated B3, a rat monoclonal antibody that recognized the mutant-specific sequence, even after cleavage. B3 recognized both the uncleaved and cleaved forms of mutant CALR by immunoblot in cell lysates prepared from the platelets and peripheral blood cells of CALR-mutant ET and PMF patients. B3 also recognized mutant CALR expressed on the cell surfaces of monocytes and granulocytes from CALR-mutant ET and PMF patients. Based on these results, we developed B3-chimera, a mouse chimeric antibody of B3, and evaluated its therapeutic potential for ET in vivo. We used an ET mouse model created by transplantation of LSK (lin-sca1+c-kit+) cells transduced with CALR del52 into the bone marrow. Intravenous injection of B3-chimera markedly suppressed the thrombocytosis induced by CALR del52, which was associated with a significant reduction in the megakaryocyte count in the bone marrow (Figure 1). In conclusion, we demonstrated that targeting the cleaved form of mutant CALR on the cell surface was a promising strategy for the treatment of CALR-mutant myeloproliferative neoplasms. Disclosures Akazawa: ITOCHU CHEMICAL FRONTIER Corporation: Membership on an entity's Board of Directors or advisory committees. Komatsu:Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

No abstract

It has recently been reported that bone marrow cells can efficiently engraft without marrow conditioning when implanted directly into the bone marrow cavity (intra-bone marrow transplantation, iBMT) in mice. We have successfully examined the efficacy of autologous iBMT in a cynomolgus monkey model in conjuction with an in vivo expansion of transplanted cells by a selective amplifier transgene (Ueda et al., 2004) and provide here the detailed parameters of our iBMT method. We injected retrovirally-marked autologous CD34 + cells directly into the non-conditioned marrow cavity of the femur and humerus after gently irrigating the cavity with saline. This transplant procedure was safely performed without pulmonary embolism. Gene-marked cells were not detectable in the peripheral blood at one hour and one day after iBMT as assessed by sensitive PCR, indicating that iBMT hardly generated a systemic delivery of transplanted cells. On the other hand, 2 to 30% of clonogenic hematopoietic colonies produced from the implanted marrow were gene-marked at 6-12 months after iBMT. Our iBMT method for non-human primates is thus discussed in terms of longlived hematopoietic stem/progenitor cells, bone marrow niche and long-term engraftment after iBMT without myeloablative conditioning.