STEM CELL PRESERVATION WITH NONTOXIC SUGARS

描述（由申请人提供）：可以用冷冻保存的造血干细胞移植治疗的骨髓疾病包括白血病、淋巴瘤和再生障碍性贫血。在冷冻保存领域，目前的观点是在移植前应去除冷冻保护剂。在二甲基亚砜（DM 80）的情况下，最流行的冷冻保护剂，有许多记录的影响，在细胞水平沿着频繁的临床报告的有害副作用的患者。使用非细胞毒性糖的能力应能够避免在目前采用的冷冻保存程序中使用DMSO。因此，在本提案中，我们将使用人类干/祖细胞开发利用新的细胞膜透化技术结合通常不可渗透的糖的保存方法。预计通过该技术冷冻保存的细胞将直接输注到患者体内。该技术还可以用于提供各种细胞的稳定长期储存，包括成熟血细胞，间充质干细胞，基于细胞的生物传感器和一些涉及基因治疗的医学疗法。 在本I期研究中将讨论以下具体目标： #1 H5孔和二价金属离子（用于开孔和投配的锌）浓度的优化; #2透化后孔隙去除的优化; #3在用海藻糖或蔗糖透化后以四种冷却速率进行受控速率冷冻保存的比较;以及 #4评估残留的H5细胞毒性和经处理的细胞的长期造血潜能。 这些目标将通过使用人CD 34+脐带血细胞和一组体外试验以及通过体内移植到NOD/SCID小鼠中来实现。该提案中的技术创新是基于细胞膜可逆透化的概念，使用工程孔在冷冻前用二糖装载细胞。透化技术采用基因工程造孔蛋白。二价金属离子调节孔的开口和定量。初步的可行性数据表明，用海藻糖冷冻保存穿孔的人造血祖细胞系。如果用穿孔的人细胞处理的小鼠表现出良好的存活和造血重建，则将提出II期SBIR研究以优化造血干细胞和祖细胞保存。

DESCRIPTION (provided by applicant): Diseases of the bone marrow that may be treated with cryopreserved hemopoletic stem cell transplantation indude the leukemias, lymphomas and aplastic anemia. In the field of cryopreservation, current opinion is that cryoprotective agents should be removed prior to transplantation. In the case of dimethylsulfoxide (DM80), the most popular cryoprotectant, there are many documented effects at the cellular level along with frequent clinical reports of detrimental side effects in patients. The ability to use non-cytotoxic sugars should enable the avoidance of DMSO in currently employed cryopreservation procedures. Therefore, in this proposal we will use human stem/progenitor cells to develop preservation methods utilizing a new cell membrane permeabilization technology in combination with sugars that are normally impermeable. It is anticipated that cells cryopreserved by this technology will be directly infusible into patients. This technology may also be leveraged to provide stable long-term storage of a variety of cells, including mature blood cells, mesenchymal stem cells, cell-based biosensors and some medical therapies involving gene therapy. The following specific aims will be addressed in this Phase I research study: #1 Optimization of H5 pore and divalent metal ion (zinc for pore opening and dosing) concentrations; #2 Optimization of postpermeabilization pore removal; #3 Comparison of controlled rate cryopreservation at four cooling rates following permeabilization with trehalose or sucrose; and #4 Evaluation of residual H5 cytotoxicity and the long-term hematopoietic potential of treated cells. These aims will be accomplished using human CD34+ cord blood cells and a panel of in vitro assays, and by transplantation in vivo into NOD/SCID mice. The technical innovations in this proposal are based upon the concept of reversible permeabilization of cell membranes using engineered pores to load the cells with disaccharides prior to freezing. The permeabilization technology employs a genetically engineered pore forming protein. The opening and dosing of the pore is regulated by divalent metal ions. Preliminary feasibility data is submitted demonstrating cryopreservation of a porated human hemopoietic progenitor cell line with trehalose. A Phase-Il SBIR study wilt be proposed to optimize hemopoletic stem and progenitor cell preservation if mice treated with porated human cells demonstrate good survival and hematopoietic reconstitution.