Faculty Collaboration Database

In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection. J Thromb Haemost 2014 Aug;12(8):1283-93

Date

06/17/2014

Pubmed ID

24931217

Pubmed Central ID

PMC4127102

DOI

10.1111/jth.12633

Scopus ID

2-s2.0-84905667071 (requires institutional sign-in at Scopus site)   30 Citations

Abstract

BACKGROUND: Our previous studies have demonstrated that platelet-specific gene delivery to hematopoietic stem cells can induce sustained therapeutic levels of platelet factor VIII (FVIII) expression in mice with hemophilia A.

OBJECTIVE: In this study, we aimed to enhance platelet FVIII expression while minimizing potential toxicities.

METHODS: A novel lentiviral vector (LV), which harbors dual genes, the FVIII gene driven by the αIIb promoter (2bF8) and a drug-resistance gene, the MGMT(P140K) cassette, was constructed. Platelet FVIII expression in mice with hemophilia A was introduced by transduction of hematopoietic stem cells and transplantation. The recipients were treated with O(6)-benzylguanine followed by 1,3-bis-2 chloroethyl-1-nitrosourea monthly three or four times. Animals were analyzed by using polymerase chain reaction (PCR), quantitative PCR, FVIII:C assays, and inhibitor assays. Phenotypic correction was assessed by tail clipping tests and rotational thromboelastometry analysis.

RESULTS: Even using a low multiplicity of infection of 1 and a non-myeloablative conditioning regimen, after in vivo selection, the levels of platelet FVIII expression in recipients increased to 4.33 ± 5.48 mU per 10(8) platelets (n = 16), which were 19.7-fold higher than the levels obtained from the recipients before treatment. Quantitative PCR results confirmed that 2bF8/MGMT-LV-transduced cells were effectively enriched after drug-selective treatment. Fifteen of 16 treated animals survived tail clipping. Blood loss and whole blood clotting time were normalized in the treated recipients. Notably, no anti-FVIII antibodies were detected in the treated animals even after recombinant human B-domain deleted FVIII challenge.

CONCLUSION: we have established an effective in vivo selective system that allows us to enrich 2bF8LV-transduced cells, enhancing platelet FVIII expression while reducing the potential toxicities associated with platelet gene therapy.

Author List

Schroeder JA, Chen Y, Fang J, Wilcox DA, Shi Q

Authors

Qizhen Shi MD, PhD Professor in the Pediatrics department at Medical College of Wisconsin
David A. Wilcox 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
Bone Marrow Transplantation
DNA Modification Methylases
DNA Repair Enzymes
Disease Models, Animal
Enzyme-Linked Immunosorbent Assay
Genetic Engineering
Hemophilia A
Immune Tolerance
Mice
Mice, Inbred C57BL
Phenotype
Transgenes
Tumor Suppressor Proteins