title: Functional aspects of factor VIII expression after transplantation of genetically-modified hematopoietic stem cells for hemophilia A.
abstract: BACKGROUND
  Major complications with respect to the development of gene therapy treatments for hemophilia A include low factor VIII (fVIII) expression and humoral immune responses resulting in inhibitory anti-fVIII antibodies. We previously achieved sustained curative fVIII activity levels in hemophilia A mice after nonmyeloablative transplantation of genetically-modified hematopoietic stem cells (HSCs) encoding a B-domain deleted porcine fVIII (BDDpfVIII) transgene with no evidence of an immune response.
  
  
  METHODS
  Mouse HSCs were transduced using MSCV-based recombinant virus encoding BDDpfVIII and transplanted into hemophilia A mice. Transplanted mice were followed for donor cell engraftment, fVIII expression and activity, and generation of anti-fVIII immune response.
  
  
  RESULTS
  We now show that: (i) the protein expressed by hematopoietic cells has a specific activity similar to that of purified protein; (ii) BDDpfVIII expressed from hematopoietic cells effectively induces thrombus formation, which is shown using a new method of in vivo analysis of fVIII function; (iii) naïve and pre-immunized mice receiving HSC gene therapy are nonresponsive to challenges with recombinant human fVIII; (iv) nonresponsiveness is not broken after stringent challenges with BDDpfVIII; and (v) T cells from these mice are unresponsive to BDDpfVIII presentation. Furthermore, stem cells isolated from donors with high titer anti-human fVIII antibodies show no defects in donor cell engraftment or the ability to express BDDpfVIII.
  
  
  CONCLUSIONS
  These results demonstrate that HSC gene therapy can be an effective alternative treatment for individuals with hemophilia A and may benefit patients by inducing immunological nonresponsiveness to fVIII replacement products.
authors.affiliation: Department of Pediatrics, Aflac Cancer Center and Blood Disorders Service, Emory University and Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
authors.forename: Lucienne M
authors.initials: LM
authors.lastname: Ide
authors.forename: Neal N
authors.initials: NN
authors.lastname: Iwakoshi
authors.forename: Bagirath
authors.initials: B
authors.lastname: Gangadharan
authors.forename: Shawn
authors.initials: S
authors.lastname: Jobe
authors.forename: Robert
authors.initials: R
authors.lastname: Moot
authors.forename: David
authors.initials: D
authors.lastname: McCarty
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Doering
authors.forename: H Trent
authors.initials: HT
authors.lastname: Spencer
mesh_terms: D000818:Animals; D005169:Factor VIII / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006467:Hemophilia A / Q000628:therapy*; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: C078147:F8 protein, human; D005169:Factor VIII