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

Abstract

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 10 9 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/L, well above the nadirs before lentivirus administration. After the injection of lentivirus, mean neutrophil counts ؎ SD were 12 460 ؎ 4240 cells/ L, significantly increased over both pretreatment values of 3040 ؎ 2540 cells/L (P < .0001) and neutrophil counts during G-CSF administration of 10 290 ؎ 4860 cells/L (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. IntroductionCyclic neutropenia is a rare disease that occurs both in man and gray collie dogs. In collie dogs, cyclic hematopoiesis is inherited as an autosomal recessive disease. 1,2 The dogs have 12-to 14-day cyclic variations in blood neutrophils, monocytes, lymphocytes, eosinophils, reticulocytes, and platelets due to periodic fluctuations in blood cell production by the bone marrow. 1,2 The recurrent severe neutropenia leads to bacterial infections and shortened life expectancy. The disorder can be cured by bone marrow transplantation from a normal dog to a gray collie as well as transferred from a gray collie to a normal littermate. 3,4 This transplantability strongly supports the concept that this is a disease of defective regulation of hematopoietic stem cells.Gray collie dogs with cyclic hematopoiesis have been successfully treated with granulocyte colony-stimulating factor (G-CSF). 5,6 The severe recurrent neutropenia in gray collie dogs was not abrogated by in vivo interleukin 3 (IL-3) or granulocytemacrophage colony-stimulating factor (GM-CSF) treatment. 5 In contrast, G-CSF prevented the recurrent neutropenia and obliterated periodic fluctuation of monocyte, eosinophil, reticulocyte, and platelet counts. 5,6 ...

In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer

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Robust, Reversible Gene Knockdown Using a Single Lentiviral Short Hairpin RNA Vector

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