Improving Engraftment of Hematopoietic Stem Cell Gene Therapy

描述（由申请人提供）：造血干细胞（HSC）基因疗法（GT）有望治愈目前治疗不足或高毒性的疾病。开发有效的HSC-GT，实现更高水平的移植，减少调节方案的毒性和无免疫原性风险，仍然是一个未满足的需求。我们建议使用狗HSC-GT模型来研究这些关键问题，这些问题可以转化为血液和其他疾病治疗的重大进展。本研究旨在通过以下方法提高自体HSC-GT的疗效和安全性：(1)通过输注更多体外扩增基因修饰(GM)-HSC增加移植量，(2)通过消除或减少GM-HSC输注前的化疗/放疗条件降低毒性，(3)诱导对GM-HSC及其后代产生的治疗性蛋白/新抗原的免疫耐受。这些研究旨在取得将转化为改善人类HSC-GT的结果。目的1。增加自体GM-HSC的细胞剂量，体外扩增CD34+细胞2周。在扩增过程中，用非免疫原性分子“条形码”慢病毒载体转导CD34+细胞。采用高通量测序技术长期跟踪移植后GM-HSC后代的移植情况。比较14天扩增的GM-HSC和3天培养的GM-HSC在犬竞争性繁殖试验中的应用。目标2。减少GM-HSC长期移植所需的细胞毒性调理方案。为了增加体外扩增的GM-HSC的竞争再繁殖优势，在低剂量全身照射（TBI）之前使用CXCR4拮抗剂plerixafor （AMD3100）动员内源性HSC，然后输注GM-HSC。接下来，评估KIT （CD117）特异性酪氨酸激酶抑制剂（TKI）是否可以增加GM-HSC的植入。我们将检验gm -HSC比plerixafor动员或tki处理的内源性HSC具有竞争优势的假设。如果成功，我们将联合plerixafor + TKI来评估这种无细胞毒性方案是否可以在没有TBI的情况下实现GM-HSC植入。Plerixafor + TKI后GM-HSC输注可多次重复，进一步增加GM-HSC的植入。目标3。利用CTLA4-Ig共刺激阻断和药理学免疫抑制（环孢素和霉酚酸酯）建立对GM-HSC表达的新抗原的免疫耐受。最后，我们将测试最佳GM-HSC移植方案，以纠正具有r型PK基因的丙酮酸激酶（PK）缺乏症犬的红系疾病，从而实现功能性治愈贫血。在完成这三个目标后，我们将定义高度可翻译的方法来增加GM-HSC的植入，减少调节方案的毒性并诱导对GM-HSC的免疫耐受

DESCRIPTION (provided by applicant): Hematopoietic stem cell (HSC) gene therapy (GT) holds promise for curing diseases for which there is currently inadequate or highly toxic treatment. There remains an unmet need to develop effective HSC-GT that achieves a greater level of engraftment with reduced conditioning regimen toxicity and without risk of immunogenicity. We propose to use the dog model of HSC-GT to study these key problems that can translate into significant advances for treatment of hematological and other diseases. The aims of this proposal are designed to improve the efficacy and safety of autologous HSC-GT with (1) increased engraftment by infusion of greater numbers of ex vivo expanded gene modified (GM)-HSC, (2) reduced toxicity by eliminating or reducing chemo/radiotherapy conditioning prior to GM-HSC infusion, and (3) inducing immune tolerance to the therapeutic protein / neoantigen produced by GM-HSC and their progeny. The studies are designed to achieve results that will be translated into improving human HSC-GT. Aim 1. Increase the cell dose of autologous GM-HSC with a 2-week ex vivo expansion of CD34+ cells. During expansion, transduce the CD34+ cells with a non-immunogenic molecular "barcode" lentiviral vector. Use high throughput sequencing to track engraftment of GM-HSC progeny after transplantation long term. Compare 14-day expanded GM-HSC to 3-day cultured GM-HSC in dog competitive repopulation assays. Aim 2. Reduce the cytotoxic conditioning regimen needed for long term engraftment of GM-HSC. To increase the competitive repopulating advantage of ex vivo expanded GM-HSC, mobilize endogenous HSC with the CXCR4 antagonist plerixafor (AMD3100) just prior to low-dose total body irradiation (TBI) followed by infusion of GM-HSC. Next, assess if KIT (CD117)-specific tyrosine kinase inhibitor (TKI) can increase engraftment of GM-HSC. We will test the hypothesis that the GM-HSCs have a competitive advantage over the plerixafor mobilized, or TKI-treated endogenous HSC. If successful, we would combine plerixafor + TKI to assess if this non-cytotoxic regimen could achieve GM-HSC engraftment without TBI. Plerixafor + TKI followed by GM-HSC infusion may be repeated multiple times to further increase engraftment of GM-HSC. Aim 3. Establish immune tolerance to GM-HSC expressed neoantigens with CTLA4-Ig costimulatory blockade and pharmacologic immunosuppression (cyclosporine and mycophenolate mofetil). Finally, we will test the optimal GM-HSC transplant regimen to correct the erythroid disease in pyruvate kinase (PK) deficiency dogs with the R-type PK gene to achieve a functional cure of anemia. Upon completion of these three aims, we will have defined highly translatable approaches to increase engraftment of GM-HSC, reduce conditioning regimen toxicity and induce immune tolerance to GM-HSC