Cloning and sequencing of the canine neutrophil elastase cDNA.

Human cyclic neutropenia, also referred to as cyclic hematopoiesis, is a rare disease characterized by periodic fluctuations in blood cell production by the bone marrow and a corresponding recurrent severe neutropenia every 19-21 days. This results in bacterial infections and shortened life expectancy. Platelets, monocytes, lymphocytes, and reticulocytes cycle with the same periodicity. It has been determined that the neutrophil elastase (NE) gene is mutated in all cases of human cyclic hematopoiesis. Currently, the only animal model for this disease is the grey collie dog, in which there is a strikingly similar periodic neutropenia every 12-14 days. Towards the validation of this animal model, we have cloned and sequenced the canine NE cDNA from a normal dog.

Treatment of canine cyclic neutropenia by lentivirus-mediated G-CSF delivery.

Cyclic neutropenia is a rare disease that occurs both in humans and gray collie dogs and is characterized by recurrent severe neutropenia leading to bacterial infections and shortened life expectancy. Daily injections of recombinant granulocyte colony-stimulating factor (rG-CSF) are effective in shortening the period of severe neutropenia and reducing infections. After demonstrating that rG-CSF induced elevated neutrophil production in an affected dog, cytokine administration was stopped and 109 infectious units (IUs) of a lentivirus pseudotyped with vesicular stomatitis virus G protein (VSV-G) encoding canine G-CSF cDNA was administered intramuscularly. Serial blood cell counts showed elevated neutrophil production for longer than 17 months. Although neutrophil counts continued to cycle, the range at nadirs was from 3710 to 5300 cells/microL, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts +/- SD were 12 460 +/- 4240 cells/microL, significantly increased over both pretreatment values of 3040 +/- 2540 cells/microL(P <.0001) and neutrophil counts during G-CSF administration of 10 290 +/- 4860 cells/microL(P <.007). The changes in blood counts from lentivirus injection were associated with absence of clinical signs of infection and fever. The gray collie continued to gain weight and was no longer housed in a pathogen-free environment. Genomic DNA from muscle at injection sites was positive for provirus, whereas gonad, lung, spleen, heart, liver, kidney, leukocytes, and noninjected muscle samples were all negative for provirus. Thus, intramuscular administration of lentivirus encoding G-CSF provided sustained therapeutic levels of neutrophils, suggesting this approach may be applied for long-term treatment of patients with cyclic and other neutropenias.