Engineered Neural Stem Cells for Targeted Glioblastoma Therapy: Strategies for Cell Recognition and Response and Cytotoxicity

Abstract

Glioblastoma (GBM) remains the most aggressive primary brain malignancy, with median survival of 12–15 months despite maximal treatment. Its resistance to conventional therapy is driven by intratumoral heterogeneity, diffuse invasion, an immunosuppressive tumor microenvironment, and the blood-brain barrier, which restricts delivery of systemic biologics. Engineered neural stem cells (NSCs) offer a promising solution to these challenges owing to their intrinsic tumor tropism, which enables localized and sustained therapeutic delivery directly within the GBM microenvironment. This study describes the engineering of peripheral blood-induced NSCs (PBiNSCs) derived from adult blood through OCT4-free epigenetic reprogramming as a dual-strategy therapeutic platform for GBM. In the first strategy, PBiNSCs were engineered with a SyNthetic Intramembrane Proteolysis Receptor (SNIPR) system to couple tumor antigen recognition to inducible expression of light-chain leader PTEN-Long (lclPTENL), a secretable isoform of the tumor suppressor PTEN. This represents the first demonstration of SNIPR functionality in PBiNSCs, establishing them as a new cellular vehicle for synthetic receptor platforms. Initial validation using an anti-CD19 SNIPR confirmed antigen-dependent, dose-responsive activation in both bead-based and cell-based co-culture systems. The SNIPR was subsequently retargeted toward two GBM-associated antigens: IL13Rα2, using a mutant IL13(E13Y) ligand domain, and EGFRvIII, using the tumor-specific MR1 single-chain variable fragment. The MR1-EGFRvIII SNIPR demonstrated highly selective activation, with 74–81% BFP-positive cells in EGFRvIII-expressing primary GBM co-cultures compared to near-background levels in wild-type controls, an effect maintained in a 3D onco-neurosphere model. By contrast, the IL13(E13Y)-based SNIPR exhibited high sensitivity activation, attributed to residual IL13Rα2 cross-reactivity and the ultra-high affinity of the protein-based binding domain. SNIPR-driven lclPTENL expression was confirmed in CD19 co-culture conditions, and conditioned media from PBiNSCs constitutively expressing lclPTENL induced a statistically significant increase in p16 expression in primary GBM cells, demonstrating downstream biological activity and validating the therapeutic rationale for PTEN restoration. In the second strategy, PBiNSCs were engineered to constitutively secrete a bispecific Fc fusion protein simultaneously targeting CD47 a ubiquitous "don't eat me" signal overexpressed on GBM cells and either IL13Rα2 or EGFRvIII. The construct employs knob-into-hole Fc engineering and a P2A/furin co-expression strategy to produce a stable heterodimeric bispecific from a single transcript. Both the IL13/SIRPα and MR1/SIRPα variants were successfully expressed and secreted by engineered PBiNSCs, and binding to primary GBM cell lines was confirmed by Western blot. A novel antigen-affinity purification strategy using biotinylated EGFRvIII as a capture ligand enabled selective isolation of functionally intact bispecific protein. Collectively, these findings establish a proof-of-concept for a modular, cell-based delivery platform that combines the tumor-tropic properties of PBiNSCs with SNIPR-controlled therapeutic gene expression and innate immune checkpoint blockade, addressing multiple mechanisms of GBM treatment resistance simultaneously.