Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.
BACKGROUND
The development of inhibitory antibodies, referred to as inhibitors, against exogenous factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies.


OBJECTIVE
To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors.


METHODS
Platelet FVIII expression in preimmunized FVIII(null) mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined with a chromogenic assay. The transgene copy number per cell was quantitated with real-time PCR. Inhibitor titer was measured with the Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytically induced venous injury model. Integration sites were analyzed with linear amplification-mediated PCR.


RESULTS
Therapeutic levels of platelet FVIII expression were sustained in the long term without evoking an anti-FVIII memory response in the transduced preimmunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet FVIII expression resulting in phenotypic correction in preimmunized secondary and tertiary recipients.


CONCLUSIONS
Lentivirus-mediated platelet-specific gene transfer improves hemostasis in mice with hemophilia A with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies.
Department of Pediatrics, Medical College of Wisconsin, Blood Research Institute, BloodCenter of Wisconsin, Children's Research Institute, Children's Hospital of Wisconsin, Milwaukee, WI 53226, USA.
E L
EL
Kuether
J A
JA
Schroeder
S A
SA
Fahs
B C
BC
Cooley
Y
Y
Chen
R R
RR
Montgomery
D A
DA
Wilcox
Q
Q
Shi
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D016026:Bone Marrow Transplantation; D004195:Disease Models, Animal; D005169:Factor VIII / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015316:Genetic Therapy; D005822:Genetic Vectors; D005838:Genotype; D018380:Hematopoietic Stem Cell Transplantation; D006467:Hemophilia A / Q000097:blood / Q000235:genetics / Q000276:immunology / Q000628:therapy*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016254:Mutagenesis, Insertional; D010641:Phenotype; D060888:Real-Time Polymerase Chain Reaction; D013997:Time Factors; D014161:Transduction, Genetic
D001323:Autoantibodies; C427184:recombinant factor VIII SQ; D005169:Factor VIII