Clinical Trial of MGMT(P140K) Gene Therapy in the Treatment of Pediatric Patients with Brain Tumors.

Gene transfer targeting hematopoietic stem cells (HSC) in children has shown sustained therapeutic benefit in the treatment of genetic diseases affecting the immune system, most notably in severe combined immunodeficiencies affecting T-cell function. The HSC compartment has also been successfully targeted using gene transfer in children with genetic diseases affecting the central nervous system, such as metachromatic leukodystrophy and adrenoleukodystrophy. HSCs are also a target for genetic modification in strategies aiming to confer drug resistance to chemotherapy agents so as to reduce off-target toxicity, and to allow for chemotherapy dose escalation with the possibility of enhanced therapeutic benefit. In a trial of this strategy in adult glioma patients, significant engraftment of gene-modified HSCs expressing a mutant of the DNA repair protein O6-methyl-guanine-methyl-transferase (MGMT(P140K)) showed potential in conferring drug resistance against the combined effect of O6-benzylguanine (O6BG)/temozolomide (TMZ) chemotherapy. The aim was to test the safety and feasibility of this approach in children with poor prognosis brain tumors. In this Phase I trial, seven patients received gene-modified HSC following myelo-suppressive conditioning, but with only transient low-level engraftment of MGMT(P140K) gene-modified cells detectable in four patients. All patients received O6BG/TMZ chemotherapy following infusion of gene-modified cells, with five patients eligible for chemotherapy dose escalation, though in the absence of demonstrable transgene-mediated chemoprotection. Since all gene-modified cell products met the criteria for release and assays for engraftment potential met expected outcome measures, inadequate cell dose, conditioning chemotherapy, and/or underlying bone-marrow function may have contributed to the lack of sustained engraftment of gene-modified cells. We were able to demonstrate safe conduct of a technically complex Phase I study encompassing manufacture of the gene therapy vector, genetically modified cells, and a drug product specifically for the trial in compliance with both local and national regulatory requirements.

Tropomyosin regulates cell migration during skin wound healing.

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.

NEDD9 stabilizes focal adhesions, increases binding to the extra-cellular matrix and differentially effects 2D versus 3D cell migration.

The speed of cell migration on 2-dimensional (2D) surfaces is determined by the rate of assembly and disassembly of clustered integrin receptors known as focal adhesions. Different modes of cell migration that have been described in 3D environments are distinguished by their dependence on integrin-mediated interactions with the extra-cellular matrix. In particular, the mesenchymal invasion mode is the most dependent on focal adhesion dynamics. The focal adhesion protein NEDD9 is a key signalling intermediary in mesenchymal cell migration, however whether NEDD9 plays a role in regulating focal adhesion dynamics has not previously been reported. As NEDD9 effects on 2D migration speed appear to depend on the cell type examined, in the present study we have used mouse embryo fibroblasts (MEFs) from mice in which the NEDD9 gene has been depleted (NEDD9 -/- MEFs). This allows comparison with effects of other focal adhesion proteins that have previously been demonstrated using MEFs. We show that focal adhesion disassembly rates are increased in the absence of NEDD9 expression and this is correlated with increased paxillin phosphorylation at focal adhesions. NEDD9-/- MEFs have increased rates of migration on 2D surfaces, but conversely, migration of these cells is significantly reduced in 3D collagen gels. Importantly we show that myosin light chain kinase is activated in 3D in the absence of NEDD9 and is conversely inhibited in 2D cultures. Measurement of adhesion strength reveals that NEDD9-/- MEFs have decreased adhesion to fibronectin, despite upregulated α5ß1 fibronectin receptor expression. We find that ß1 integrin activation is significantly suppressed in the NEDD9-/-, suggesting that in the absence of NEDD9 there is decreased integrin receptor activation. Collectively our data suggest that NEDD9 may promote 3D cell migration by slowing focal adhesion disassembly, promoting integrin receptor activation and increasing adhesion force to the ECM.