Gene transfer technologies and new gene therapy strategies
6. Tie2-expressing monocytes: from contribution to angiogenesis and tissue regeneration to exploitation for targeting gene therapy to tumors
Luigi Naldini (Head of Unit), Unit of Angiogenesis and Tumor Targeting (Division of Regenerative Medicine, Stem Cells and Gene Therapy)
Bernhard Gentner (Project Leader), Giulia Escobar (PhD student), Silvia Nucera (MD, Post-doc), Anna Ranghetti (Technician), Lucia Sergi Sergi (Lab Technician)
Among tumor-infiltrating hematopoietic cells, we have previously characterized, both in mice and humans, a subset of monocytes that express the Angiopoietin receptor TIE2 (Tie2-expressing monocytes, TEMs) and are specifically recruited to tumors. Selective elimination of TEMs inhibits angiogenesis and tumor growth in several mouse tumor models without affecting the recruitment of other tumor-associated macrophages (TAMs), thus showing a non-redundant role of these cells in tumor angiogenesis. By performing gene profiling studies, we have shown that tumor infiltrating TEMs display a gene signature distinct from that of TAMs and consistent with enhanced pro-angiogenic and tissue remodeling functions and lower pro-inflammatory activity as compared to TAMs. Moreover, we have recently unveiled the requirement of the ANG2-TIE2 signaling axis for TEMs to exert pro-tumoral functions. In fact, selective knockdown of the TIE2 receptor in TEMs or blockade of the TIE2 receptor ligand ANG2 was sufficient to inhibit tumor angiogenesis and prevent the typical close association of TEMs with endothelial cells engaged in angiogenesis within the tumor. Our current model for promotion of vascular morphogenesis by TEMs is shown in the figure (taken from Mazzieri et al., Cancer Cell 2011, 19:512-26). Circulating TEMs express low-level TIE2 (A), but the receptor is upregulated (B) upon their extravasation in the tumor microenvironment and exposure to ANG2. Once in the tumor stroma, TEMs adhere to sprouting blood vessels which secrete high-level ANG2 in hypoxic tumor regions (B). In the presence of ANG2, TEMs promote vascular growth (C) and facilitate vascular anastomosis (D). We suggest that the ANG2-TIE2 signaling axis mediates iuxtacrine interactions between TEMs and endothelial cells that are required for sprouting angiogenesis.
The ability of both murine and human TEMs to home to tumors, and the selective expression of the TIE2 gene in TEMs among the differentiated progeny of hematopoietic stem cells, offers the possibility to exploit TEMs as cellular vehicle for the tumor-targeted delivery of bio-therapeutics. We designed LV with TEM-specific expression by virtue of the Tie2 enhancer/promoter sequence and exploited them for HSC gene transfer. Upon transplant of these engineered HSC into tumor-bearing mice we showed recruitment of their TEM progeny and selective transgene expression in primary tumors and metastases. Using this approach we provided proof-of-principle of a new gene and cell-based strategy to target a therapeutic IFNa transgene to tumors and demonstrated its therapeutic efficacy in mice. We showed that the local release of IFNa at the tumor site induced powerful activation of tumor-infiltrating macrophages, NK and T-cells, leading to effective anti-tumor responses and counteracting the pro-angiogenic and immunosuppressive influence of the tumor microenvironment. Importantly, we proved that the targeted delivery of IFNa was able to overcome the dose-limiting toxicity associated with conventional systemic administration of this cytokine. Furthermore, because TIE2 is also expressed by HSC, we more recently developed miRNA-regulated vectors to de-target transgene expression selectively from HSC, thus protecting these cells from any potential residual myelo-toxicity of this strategy. Moreover, we have generated a humanized version of this new platform and developed human hematochimeric mouse models to study anti-tumor immune responses upon IFNa delivery by human myeloid cells. Our current work aims to:
6.1 Improving safety and efficacy of TIE2-IFN gene delivery strategy
We will stringently assess the safety of our strategy by performing long-term follow up of hematopoietic reconstitution of mice transplanted with HSC engineered with the miRNA-regulated Tie2-Ifn vector and performing competitive serial transplantation. We will rule out adverse impact on protective immunity and potential development of autoimmune manifestations. As our initial dose-response studies indicated that safety is improved and efficacy is maintained when only a low fraction of HSC is gene modified, we will establish the threshold of safety and efficacy in mice to direct the development of a clinically compatible protocol, which would only modify a fraction of the infused HSCs.
6.2 Performing preclinical studies of the efficacy and safety of TIE2-IFN gene delivery strategy in human hematochimeric tumor bearing mice.
We will perform safety studies to include long-term follow up of multilineage hematopoietic reconstitution, bio-distribution and secondary transplantation to evaluate the preservation of stem cell properties of human HSCs modified by the miRNA-regulated TIE2-IFN vector. We will investigate the efficacy of our strategy with relevant human tumors that may best support the feasibility of a clinical translation of this strategy. Furthermore, we will investigate combining TEM-mediated IFNa gene delivery strategy with other immune therapy approaches for potential synergy in eliciting effective immune responses against established malignancies.