B-cell-specific transduction for anti-HIV antibody and B-cell receptor expression

项目总结 针对HIV-1的中和抗体在抑制HIV-1复制方面非常有效。然而，血清 抗体的浓度必须长时间保持在治疗水平才能消除 HIV-1感染细胞，因为持续感染的细胞驻留在深层组织中。因此，多个 注射抗体是必要的，这可能是昂贵和劳动密集型的。基因治疗 载体可用于产生治疗性抗体。然而，通过任何基因治疗的转基因表达 载体，包括慢病毒载体，可以诱导针对转基因的免疫反应，从而减少 转基因产物的治疗效果和转导细胞的清除。因此，重要的是要 在能够对转基因产品产生耐受性的细胞类型中表达转基因。生理上的B细胞 表达由基因重组和突变产生的各种有价值的抗体区域。B细胞 已知会导致对有价值地区的耐受性。B细胞的这种能力以前被用来诱导 自身抗原耐受治疗自身免疫性疾病和凝血因子IX治疗自身免疫性疾病 血友病。因此，B细胞的转导很可能产生长期的抗HIV-1抗体，而不是 诱导对抗体的免疫反应。我们已经开发出慢病毒载体，可以特异性地 全身给药后转导所需的靶细胞类型。该载体可选择性地传导脾 不结合任何B细胞靶向配体的B细胞。通过利用我们的B细胞特异性转导 慢病毒载体，我们将尝试在B细胞中特异性表达抗HIV-1蛋白，以培养对 转基因产品。我们将通过我们的靶向载体进一步提高转基因表达的特异性 将其与B细胞特异性启动子的转录B细胞靶向相结合。使用这种高度特异的B细胞 转基因表达系统，我们将表达eCD4-Ig，一种高效的抗HIV-1试剂，由IgG和 免疫功能正常小鼠体内的可溶性CD_4。我们将研究eCD4-Ig的表达，特别是在B细胞中的表达， 可通过诱导发育耐受而诱导eCD4-Ig的长期表达。接下来我们将对这两种情况进行快递 分泌和膜固定型eCD4-Ig在B细胞中产生抗HIV-1B细胞受体(BCR) 在B细胞上。我们将研究抗HIV-1 BCR与HIV-1包膜蛋白结合后是否会产生信号， 诱导转导细胞分化生长。转导的B细胞的生长将增加 血清eCD_4-Ig浓度。将转导细胞分化为记忆B细胞和浆细胞 通过延长转导B细胞的寿命来延长eCD4-Ig的表达周期。然后我们将评估 B细胞特异性表达eCD4-Ig对人源化BLT小鼠HIV-1感染的治疗作用这些 实验的目的是开发一种新的基因治疗性抗体，这种抗体不仅可以用于治疗 HIV-1感染，也有其他类型的传染病。

PROJECT SUMMARY Neutralizing antibodies against HIV-1 are very potent in suppressing HIV-1 replication. However, the serum concentrations of the antibodies must be kept at therapeutic levels for long periods of time to eliminate HIV-1-infected cells, because persistently infected cells reside in the deep tissue. Therefore, multiple administrations of the antibodies are required, which could be expensive and labor-intensive. Gene therapy vectors can be used to produce therapeutic antibodies. However, transgene expression by any gene therapy vectors, including lentiviral vectors, can induce immune reactions against the transgenes, thereby decreasing the therapeutic effects of transgene products and eliminating the transduced cells. Therefore, it is important to express transgenes in the cell types that can develop tolerance to transgene products. B-cells physiologically express wide varieties of valuable antibody regions generated by recombination and mutations in genes. B-cells are known to induce tolerance to the valuable regions. This ability of B-cells was previously used to induce tolerance to self-antigens for therapy of autoimmune diseases and coagulation factor IX for treatment of hemophilia. Therefore, transduction of B-cells is likely to generate long-term anti-HIV-1 antibodies without inducing immune reactions to the antibodies. We have developed lentiviral vectors that can specifically transduce desired target cell types after systemic administration. The vector can selectively transduces splenic B-cells without conjugation of any B-cell-targeting ligand. By exploiting this B-cell-specific transduction by our lentiviral vectors, we will attempt to express anti-HIV-1 proteins specifically in B-cells to develop tolerance to the transgene products. We will further increase specificity of transgene expression with our targeting vector by combining it with transcriptional B-cell targeting by a B-cell-specific promoter. Using this highly B-cell-specific transgene expression system, we will express eCD4-Ig, a highly potent anti-HIV-1 reagent consisting of IgG and soluble CD4, in immunocompetent mice. We will investigate whether eCD4-Ig expression, specifically in B-cells, can induce long-term expression of eCD4-Ig by inducing developing tolerance. We will next express both secretory and membrane-anchored forms of eCD4-Ig in B-cells to generate an anti-HIV-1 B-cell receptor (BCR) on B-cells. We will investigate whether anti-HIV-1 BCR elicits signals upon binding to the HIV-1 envelope protein, inducing differentiation and growth of the transduced cells. The growth of transduced B-cells will increase the serum concentrations of eCD4-Ig. Differentiation of transduced cells to memory B-cells and plasma cells will extend the period of eCD4-Ig expression by prolonging the life span of transduced B-cells. We will then evaluate the therapeutic effects of B-cell-specific expression of eCD4-Ig in HIV-1 infection of humanized BLT mice. These experiments are designed to develop a novel gene therapeutic antibody that can be applied not only to therapy of HIV-1 infection, but also other types of infectious diseases.