In preclinical studies, neural stem cell (NSC)-based delivery of oncolytic virus has shown great promise in the treatment of malignant glioma. Ensuring the success of this therapy will require critical evaluation of the spatial distribution of virus after NSC transplantation. In this study, the patient derived GBM43 human glioma line was established in the brain of athymic nude mice, followed by administration NSCs loaded with conditionally replicating oncolytic adenovirus (NSC-CRAd-S-pk7). We determined the tumor coverage potential of oncolytic adenovirus by examining NSC distribution using magnetic resonance (MR) imaging and by three-dimensional reconstruction from ex vivo tissue specimens. We demonstrate that unmodified NSCs and NSC-CRAd-S-pk7 exhibit a similar distribution pattern with most prominent localization occurring at the tumor margins. We were further able to visualize the accumulation of these cells at tumor sites via T2-weighted MR imaging as well as the spread of viral particles using immunofluorescence. Our analyses reveal that a single administration of oncolytic virus-loaded NSCs allows for up to 31% coverage of intracranial tumors. Such results provide valuable insights into the therapeutic potential of this novel viral delivery platform.
SummaryPre-clinical studies indicate that neural stem cells (NSCs) can limit or reverse CNS damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-MYC (LM-NSC008) that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes, and astrocytes. These LM-NSC008 cells were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, LM-NSC008 distributed specifically to sites of traumatic brain injury (TBI). These data support the therapeutic development of immortalized LM-NSC008 cells for allogeneic use in TBI and other CNS diseases.
Cell-based therapies hold great promise for a myriad of clinical applications. However, as these therapies move from phase I to phase II and III trials, there is a need to improve scale-up of adherent cells for the production of larger good manufacturing practice (GMP) cell banks. As we advanced our neural stem cell (NSC)-mediated gene therapy trials for glioma to include dose escalation and multiple treatment cycles, GMP production using cell factories (CellStacks) generated insufficient neural stem cell (NSC) yields. To increase yield, we developed an expansion method using the hollow fiber quantum cell expansion (QCE) system. Seeding of 5.2 × 107 NSCs in a single unit yielded up to 3 × 109 cells within 10 days. These QCE NSCs showed genetic and functional stability equivalent to those expanded by conventional flask-based methods. We then expanded the NSCs in 7 units simultaneously to generate a pooled GMP-grade NSC clinical lot of more than 1.5 × 1010 cells in only 9 days versus 8 × 109 over 6 weeks in CellStacks. We also adenovirally transduced our NSCs within the QCE. We found the QCE system enabled rapid cell expansion and increased yield while maintaining cell properties and reducing process time, labor, and costs with improved efficiency and reproducibility.
Several preclinical studies indicate that neural stem cells can limit or reverse CNS damage by cell replacement, regeneration or delivery of various factors and therapeutic agents to sites of degeneration or tumor. Allogeneic NSC lines have several advantages over autologous ones, including "off the shelf" usage, greater availability to patients, and cost effectiveness. Genetic modification of NSCs for immortalization and retention of stem cell properties with c-myc or v-myc gene has been shown to be clinically useful in trials for stroke (ReNeuron), and brain tumors (Aboody et al). Here, we propose to express L-myc variants in human fetal brain derived NSCs, to extend NSC lifespan and stem cell characteristics. We have isolated and grown NSCs from freshly dispersed 10 week old human fetal brain tissue and propagated in culture. We then transduced these cells with retroviral vectors containing the L-myc gene. NSCs stably expressing L-myc gene (hNSC.Lmyc) were isolated for further characterization. hNSC.Lmyc cells propagated through passage 15, demonstrating a normal karyotype, expression of stem cell markers (Sox-2, A2B5 and nestin) and low expression of differentiation markers (Olig 2 and vimentin) in vitro. We have also demonstrated that hNSC.Lmyc cells retain their tumor-specific migratory abilities in vitro and in vivo for longer periods of time as compared to untransduced primary NSC pools. We further characterized these hNSC.Lmyc cells for gene expression profiling and genome wide DNA sequencing analysis. Somatic mutation analysis comparing early and late passages of hNSC.Lmyc cells showed no change in the genomic sequences. hNSC.Lmyc cells were able to self renew and differentiate into neurons, oligodendrocytes and astrocytes in vitro. In this study we generated and characterized hNSC.Lmyc line that has potential therapeutic use in numerous central nervous system (CNS) diseases.
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