title: In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA; Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI, USA; Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Schroeder
authors.forename: Y
authors.initials: Y
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Fang
authors.forename: D A
authors.initials: DA
authors.lastname: Wilcox
authors.forename: Q
authors.initials: Q
authors.lastname: Shi
mesh_terms: D000818:Animals; D001792:Blood Platelets / Q000276:immunology*; D016026:Bone Marrow Transplantation; D015254:DNA Modification Methylases / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005818:Genetic Engineering; D006467:Hemophilia A / Q000276:immunology*; D007108:Immune Tolerance / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D010641:Phenotype; D019076:Transgenes; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: D025521:Tumor Suppressor Proteins; D015254:DNA Modification Methylases; C528331:MGMT protein, mouse; D045643:DNA Repair Enzymes
keywords: blood platelet; factor VIII; genetic therapy; hemophilia A; immune tolerance