Toxicity of repeated intravenous injection of gene therapeutics for X-CGD in mice

Abstract

We made gene therapeutics for X-chronic granulomatous disease (CGD) by transducing murine bone marrow-derived stem cells with MT-gp91 retrovirus and evaluated possible toxicity in mice as a prerequisite for human clinical trials. Male C57BL/6 mice were injected intravenously with gene therapeutics for X-CGD twice at an interval of two weeks at 5 × 10⁷ cells/kg and sacrificed 2 weeks after the last administration. Significant changes noted in gene therapeutics for X-CGD-treated animals were an increase in white blood cell counts and a slight decrease in albumin/globulin ratio. The red pulp hyperplasia in the spleen accompanied with an increase in organ weight was considered to result from the accumulation of gene therapeutics for X-CGD, bone marrow-derived stem cells, in the spleen. No anti-gp91 antibody was detected in the sera collected from the animals treated with gene therapeutics for X-CGD. No integration of gp91 DNA from retroviral vector was detected in chromosomal DNA of gonads in animals dosed with the test substance, indicating no potential of genomic integration. In conclusion, the repeated dose of gene therapeutics for X-CGD exerted no toxicity. The splenic red pulp hyperplasia and the increase observed in white blood cell counts and in spleen weights were considered as pharmacological changes induced by the treatment.

Keywords

chromosomal integration gene therapeutics immunogenicity toxicity

References

1 Dinauer MC Orkin SH . Molecular genetics of chronic granulomatous disease. Immunodefic Rev 1988; 1: 55–69.

2 Leusen JH de Klein A Hilarius PM Ahlin A Palmblad J Smith CI . Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. J Exp Med 1996; 184: 1243–1249.

3 Meischl C Roos D . The molecular basis of chronic granulomatous disease. Springer Semin Immunopathol 1998; 19: 417–434.

4 Calvino MC Maldonado MS Otheo E Munoz A Couselo JM Burgaleta C . Bone marrow transplantation in chronic granulomatous disease. Eur J Pediatr 1996; 155: 877–879.

5 Johnston RB Harbeck RJ Johnston RB . Recurrent severe infections in a girl with apparently variable expression of mosaicism for chronic granulomatous disease. J Pediatr 1985; 106: 50–55.

6 Saulnier SO Steinhoff D Dinauer MC Zufferey R Trono D Seger RA . Lentivirus-mediated gene transfer of gp91phox corrects chronic granulomatous disease (CGD) phenotype in human X-CGD cells. J Gene Med 2000; 2: 317–325.

7 Grez M Becker S Saulnier S Knoss H Ott MG Maurer A . Gene therapy of chronic granulomatous disease. Bone Marrow Transplant 2000; 25(Suppl. 2): S99–S104.

8 Dinauer MC Li LL Bjorgvinsdottir H Ding C Pech N . Long-term correction of phagocyte NADPH oxidase activity by retroviral-mediated gene transfer in murine X-linked chronic granulomatous disease. Blood 1999; 94: 914–922.

9 Till JE McCulloch EA Siminovitch L . A stochastic model of stem cell proliferation, based on the growth of spleen colony-forming cells. Proc Natl Acad Sci USA 1964; 51: 29–36.

10.

10 Austin TW Salimi S Veres G Morel F Ilves H Scollay R . Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells. Blood 2000; 95: 829–836.

11.

11 Forestell SP Dando JS Bohnlein E Rigg RJ . Improved detection of replication-competent retrovirus. J Virol Methods 1996; 60: 171–178.

12.

12 Tucker KA Lilly MB Heck L Rado TA . Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity. Blood 1987; 70: 372–378.

13.

13 Zhen L King AA Xiao Y Chanock SJ Orkin SH Dinauer MC . Gene targeting of X chromosome-linked chronic granulomatous disease locus in a human myeloid leukemia cell line and rescue by expression of recombinant gp91phox. Proc Natl Acad Sci USA 1993; 90: 9832–9836.

14.

14 Segal BH Leto TL Gallin JI Malech HL Holland SM . Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore) 2000; 79: 170–200.