MONOCYTE-BASED GENE THERAPY FOR ACUTE NEUTROPHIL-MEDIATED TISSUE INJURY

This proposal is aimed at developing new treatments for lung diseases caused by excessive activation of cells used by the body to fight infection (neutrophils). In particular, a condition called adult respiratory distress syndrome is fatal in 40% of cases despite modern treatment, and survivors have considerable long-term problems..
The research involves manipulating another defence cell, the monocyte. Monocytes are recruited to inflamed sites alongside neutrophils and are amenable to therapeutic modification. I shall boost monocytes by providing extra copies of a protective gene called elafin. Elafin efficiently neutralises the main toxic product of neutrophils and makes monocytes switch off inflammation. I shall therefore insert elafin into human monocytes in order to reduce the toxic effects of ‘activated‘ neutrophils. I shall extend these findings to mice with the aim of reducing lung injury caused by neutrophils. These pre-clinical studies will lay the grounds for future clinical application in humans. Importantly, the principles of this proposal can be extended not only to other lung diseases associated with neutrophil malfunction (e.g. emphysema, bronchitis, cystic fibrosis) but to all causes of inflammation involving the neutrophil. The work therefore has potential implications for psoriasis, arthritis, peritonitis, and a host of other conditions

Neutrophil-mediated lung injury (NMLI) is central to the pathogenesis of conditions such as acute
lung injury and adult respiratory distress syndrome (ARDS). Mortality from these conditions
approaches 40%. Therefore new neutrophil-directed therapeutic strategies are required. Human
neutrophil elastase (HNE) is the key effector of NMLI. Because monocytes specifically access
inflamed alveoli, manipulation of circulating monocytes to express anti-elastases(such as secretory leukocyte protease inhibitor (SLPI) and elafin) has therapeutic potential. I propose that antielastase production by native monocytes is negligible, that monocytes mature into antielastase expressing macrophages in the inflamed lung,that this defence mechanism is overwhelmed in ARDS, and that genetic augmentation of antielastases in monocytes can obviate NMLI. In support of this I have demonstrated novel and consistent protective effects mediated by alveolar macrophages (AMs) but not monocytes against neutrophil-mediated epithelial injury in vitro. This protective effect by AMs is temporally associated with an increase in SLPI secretion. Against this background I aim to address
three key hypotheses:
1. Interaction with neutrophils specifically stimulates generation of protective levels of SLPI from
autologous AMs but not autologous blood monocytes in vitro.
2. Monocytes transfected with adenovirus encoding human elafin (Ad-elafin) consistently protects the lung against neutrophil-mediated injury in vitro and in vivo.
3. Adoptively transferred monocytes traffic rapidly and selectively to the inflamed lung where they differentiate to a macrophage phenotype capable of expressing SLPI. Using primary autologous cells isolated from healthy volunteers and patients with ARDS, I shall assess whether protective effects of AMs on NMLI are SLPI-dependent by transfecting AMs with SLPI siRNA. Human monocytes will be transfected with Ad-elafin to confer consistent protection in vitro.
Extending these studies to an in vivo setting, murine monocytes will be transfected with elafin and adoptively transferred to mice with established NMLI. In the same model, trafficking of adoptively administered monocytes will be assessed dynamically and non-invasively using ‘fluorine’ MRI. To determine the fate of the administered cells, I will administer monocytes isolated from transgenic GFP mice, then isolate these cells to determine phenotype.
This project offers unique training opportunities in cell and molecular biology, gene therapy, animal models of lung inflammation and ‘cutting edge’ imaging techniques. From a scientific perspective the proposal will dissect mechanisms by which monocytes and macrophages interact with neutrophils in vitro and in vivo. The development of a novel, targeted ‘monocyte-based gene therapy’ approach provides a potential strategy for the future treatment of NMLI.