Ben Towne Center for Childhood Cancer Research, Seattle Children's Research Institute, Seattle, Washington, USA.
Mozart Therapeutics, Seattle, Washington, USA.
J Immunother Cancer. 2022 Jun;10(6). doi: 10.1136/jitc-2021-003770.
Human immune cells, including monocyte-derived macrophages, can be engineered to deliver proinflammatory cytokines, bispecific antibodies, and chimeric antigen receptors to support immune responses in different disease settings. When gene expression is regulated by constitutively active promoters, lentiviral payload gene expression is unregulated, and can result in potentially toxic quantities of proteins. Regulated delivery of lentivirally encoded proteins may allow localized or conditional therapeutic protein expression to support safe delivery of adoptively transferred, genetically modified cells with reduced capacity for systemic toxicities.
In this study, we engineered human macrophages to express genes regulated by hypoxia responsive elements included in the lentiviral promoter region to drive conditional lentiviral gene expression only under hypoxic conditions. We tested transduced macrophages cultured in hypoxic conditions for the transient induced expression of reporter genes and the secreted cytokine, interleukin-12. Expression of hypoxia-regulated genes was investigated both transcriptionally and translationally, and in the presence of human tumor cells in a slice culture system. Finally, hypoxia-regulated gene expression was evaluated in a subcutaneous humanized-mouse cancer model.
Engineered macrophages were shown to conditionally and tranisently express lentivirally encoded gene protein products, including IL-12 in hypoxic conditions in vitro. On return to normoxic conditions, lentiviral payload expression returned to basal levels. Reporter genes under the control of hypoxia response elements were upregulated under hypoxic conditions in the presence of human colorectal carcinoma cells and in the hypoxic xenograft model of glioblastoma, suggesting utility for systemic engineered cell delivery capable of localized gene delivery in cancer.
Macrophages engineered to express hypoxia-regulated payloads have the potential to be administered systemically and conditionally express proteins in tissues with hypoxic conditions. In contrast to immune cells that function or survive poorly in hypoxic conditions, macrophages maintain a proinflammatory phenotype that may support continued gene and protein expression when regulated by conditional hypoxia responsive elements and naturally traffic to hypoxic microenvironments, making them ideal vehicles for therapeutic payloads to hypoxic tissues, such as solid tumors. With the ability to fine-tune delivery of potent proteins in response to endogenous microenvironments, macrophage-based cellular therapies may therefore be designed for different disease settings.
人类免疫细胞，包括单核细胞衍生的巨噬细胞，可以被工程改造以递呈促炎细胞因子、双特异性抗体和嵌合抗原受体，以支持不同疾病情况下的免疫反应。当基因表达受组成性激活启动子调控时，慢病毒载体的有效基因表达不受调控，可能导致潜在的毒性蛋白数量增加。慢病毒编码蛋白的调节性递呈可能允许局部或条件性治疗性蛋白表达，从而支持过继转移的、经基因修饰的细胞的安全递呈，降低全身毒性的能力。
在这项研究中，我们通过将慢病毒启动子区域中的缺氧反应元件包含在内，对人巨噬细胞进行工程改造，以驱动仅在缺氧条件下的条件性慢病毒基因表达。我们测试了在缺氧条件下培养的转导巨噬细胞中瞬时诱导表达报告基因和分泌细胞因子白细胞介素-12 的情况。在切片培养系统中存在人类肿瘤细胞的情况下，我们从转录和翻译水平研究了缺氧调节基因的表达，并在皮下人源化小鼠癌症模型中评估了缺氧调节基因的表达。
研究表明，工程化的巨噬细胞能够在体外缺氧条件下条件性和瞬时表达慢病毒编码的基因蛋白产物，包括白细胞介素-12。当回到常氧条件下时，慢病毒有效负载的表达会回到基础水平。在存在人结直肠癌细胞的情况下，缺氧反应元件控制的报告基因在缺氧条件下上调，并且在胶质母细胞瘤的缺氧异种移植模型中也上调，这表明系统工程细胞递呈具有局部基因递呈能力，有可能用于癌症。
表达缺氧调节有效负载的巨噬细胞有可能被全身给药，并在缺氧条件下的组织中条件性表达蛋白。与在缺氧条件下功能或存活不良的免疫细胞不同，巨噬细胞保持促炎表型，当受条件性缺氧反应元件调节并自然流向缺氧微环境时，可能支持持续的基因和蛋白表达，使它们成为缺氧组织（如实体瘤）治疗性有效负载的理想载体。通过精细调节对内源性微环境的有效蛋白递呈，基于巨噬细胞的细胞疗法可因此用于不同的疾病情况。
