Faculty Collaboration Database

In vivo enrichment of genetically manipulated platelets for murine hemophilia B gene therapy. J Cell Physiol 2021 Jan;236(1):354-365

Date

06/09/2020

Pubmed ID

32510630

Pubmed Central ID

PMC7722216

DOI

10.1002/jcp.29861

Scopus ID

2-s2.0-85086088416 (requires institutional sign-in at Scopus site)   6 Citations

Abstract

Our previous studies have demonstrated that platelet-targeted factor IX (FIX) gene therapy can introduce sustained platelet-FIX expression in hemophilia B (FIX^null ) mice. In this study, we aimed to enhance platelet-FIX expression in FIX^null mice with O⁶ -methylguanine-DNA-methyltransferase (MGMT)-mediated in vivo drug selection of transduced cells under nonmyeloablative preconditioning. We constructed a novel lentiviral vector (2bF9/MGMT lentivirus vector), which harbors dual genes, the FIX gene driven by the αIIb promoter (2bF9) and the MGMT P140K gene under the murine stem cell virus promoter. Platelet-FIX expression in FIX^null mice was introduced by 2bF9/MGMT-mediated hematopoietic stem cell transduction and transplantation. The 2bF9/MGMT-transduced cells were effectively enriched after drug selection by O⁶ -benzylguanine/1,3-bis-2-chloroethyl-1-nitrosourea. There were a 2.9-fold higher FIX antigen and a 3.7-fold higher FIX activity in platelets, respectively, posttreatment compared with pretreatment. When a 6-hr tail bleeding test was used to grade the bleeding phenotype, the clotting time in treated animals was 2.6 ± 0.5 hr. In contrast, none of the FIX^null control mice were able to clot within 6 hr. Notably, none of the recipients developed anti-FIX antibodies after gene therapy. One of four recipients developed a low titer of inhibitors when challenged with rhF9 together with adjuvant. In contrast, all FIX^null controls developed inhibitors after the same challenge. Anti-FIX immunoglobulin G were barely detectable in recipients (1.08 ± 0.54 µg/ml), an 875-fold lower level than in the FIX^null controls. Our data demonstrate that using the MGMT-mediated drug selection system in 2bF9 gene therapy can significantly enhance therapeutic platelet-FIX expression, resulting in sustained phenotypic correction and immune tolerance in FIX^null mice.

Author List

Chen Y, Schroeder JA, Gao C, Li J, Hu J, Shi Q

Author

Qizhen Shi MD, PhD Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Blood Platelets
Female
Genetic Therapy
Genetic Vectors
Hematopoietic Stem Cells
Hemophilia B
Immune Tolerance
Lentivirus
Male
Mice
Mice, Inbred C57BL
O(6)-Methylguanine-DNA Methyltransferase
Phenotype
Promoter Regions, Genetic
Transduction, Genetic