Chan James, Clements Warren, Field Judith, Nasa Zeyad, Lock Peter, Yap Felicia, Toh Ban-Hock, Alderuccio Frank
Department of Immunology, Monash University, Commercial Road, Prahran, Victoria 3181, Australia.
J Gene Med. 2006 Nov;8(11):1281-90. doi: 10.1002/jgm.968.
Type 1 diabetes (T1D) is a T-cell-dependent autoimmune disease resulting from destructive inflammation (insulitis) of the insulin-producing pancreatic beta-cells. Transgenic expression of proinsulin II by a MHC class II promoter or transfer of bone marrow from these transgenic mice protects NOD mice from insulitis and diabetes. We assessed the feasibility of gene therapy in the NOD mouse as an approach to treat T1D by ex vivo genetic manipulation of normal hematopoietic stem cells (HSCs) with proinsulin II followed by transfer to recipient mice.
HSCs were isolated from 6-8-week-old NOD female mice and transduced in vitro with retrovirus encoding enhanced green fluorescent protein (EGFP) and either proinsulin II or control autoantigen. Additional control groups included mice transferred with non-manipulated bone marrow and mice which did not receive bone marrow transfer. EGFP-sorted or non-sorted HSCs were transferred into pre-conditioned 3-4-week-old female NOD mice and insulitis was assessed 8 weeks post-transfer.
Chimerism was established in all major lymphoid tissues, ranging from 5-15% in non-sorted bone marrow transplants to 20-45% in EGFP-sorted bone marrow transplants. The incidence and degree of insulitis was significantly reduced in mice receiving proinsulin II bone marrow compared to controls. However, the incidence of sialitis in mice receiving proinsulin II bone marrow and control mice was not altered, indicating protection from insulitis was antigen specific.
We show for the first time that ex vivo genetic manipulation of HSCs to express proinsulin II followed by transplantation to NOD mice can establish molecular chimerism and protect from destructive insulitis in an antigen-specific manner.
1型糖尿病(T1D)是一种T细胞依赖性自身免疫性疾病,由产生胰岛素的胰腺β细胞的破坏性炎症(胰岛炎)引起。通过MHC II类启动子进行胰岛素原II的转基因表达或从这些转基因小鼠转移骨髓可保护非肥胖糖尿病(NOD)小鼠免受胰岛炎和糖尿病的侵害。我们评估了在NOD小鼠中进行基因治疗的可行性,该方法是通过用胰岛素原II对正常造血干细胞(HSC)进行体外基因操作,然后转移到受体小鼠中来治疗T1D。
从6-8周龄的NOD雌性小鼠中分离出HSC,并在体外用编码增强型绿色荧光蛋白(EGFP)和胰岛素原II或对照自身抗原的逆转录病毒进行转导。其他对照组包括接受未处理骨髓转移的小鼠和未接受骨髓转移的小鼠。将EGFP分选或未分选的HSC转移到预先处理的3-4周龄雌性NOD小鼠中,并在转移后8周评估胰岛炎情况。
在所有主要淋巴组织中均建立了嵌合体,未分选骨髓移植中的嵌合体比例为5-15%,EGFP分选骨髓移植中的嵌合体比例为20-45%。与对照组相比,接受胰岛素原II骨髓移植的小鼠中胰岛炎的发生率和程度显著降低。然而,接受胰岛素原II骨髓移植的小鼠和对照小鼠中涎腺炎的发生率没有改变,这表明对胰岛炎的保护是抗原特异性的。
我们首次表明,对HSC进行体外基因操作以表达胰岛素原II,然后移植到NOD小鼠中,可以以抗原特异性方式建立分子嵌合体并保护免受破坏性胰岛炎的侵害。