RESUMO
Ex vivo gene therapy strategies avoid systemic delivery of viruses thereby mitigating the risk of vector-associated immunogenicity. Previously, we delivered autologous factor VIII (FVIII)-expressing blood outgrowth endothelial cells (BOECs) to hemophilia A mice and showed that these cells remained sequestered within the implanted matrix and provided therapeutic levels of FVIII. Prior to translating this strategy into the canine (c) model of hemophilia A, we increased cFVIII transgene expression by at least 100-fold with the use of the elongation factor 1 alpha (EF1α) promoter and a strong endothelial enhancer element. BOECs isolated from hemophilia A dogs transduced with this lentiviral vector express levels of cFVIII ranging between 1.0 and 1.5 U/mL per 10(6) cells over 24 hours. Autologous BOECs have been implanted into the omentum of 2 normal and 3 hemophilia A dogs. These implanted cells formed new vessels in the omentum. All 3 hemophilia A dogs treated with FVIII-expressing autologous BOECs developed anti-FVIII immunoglobulin G2 antibodies, but in only 2 of the dogs were these antibodies inhibitory. FVIII antigen levels >40% in the absence of FVIII coagulant function were detected in the circulation for up to a year after a single gene therapy treatment, indicating prolonged cellular viability and synthesis of FVIII.
Assuntos
Autoanticorpos , Inibidores dos Fatores de Coagulação Sanguínea , Células Endoteliais , Fator VIII , Expressão Gênica , Terapia Genética/métodos , Hemofilia A , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoenxertos , Inibidores dos Fatores de Coagulação Sanguínea/sangue , Inibidores dos Fatores de Coagulação Sanguínea/imunologia , Modelos Animais de Doenças , Cães , Células Endoteliais/metabolismo , Células Endoteliais/transplante , Fator VIII/biossíntese , Fator VIII/genética , Fator VIII/imunologia , Vetores Genéticos , Hemofilia A/sangue , Hemofilia A/genética , Hemofilia A/imunologia , Hemofilia A/terapia , Humanos , Lentivirus , Camundongos , Fator 1 de Elongação de Peptídeos/genética , Regiões Promotoras Genéticas , Transdução Genética , TransgenesRESUMO
Gene therapy for hemophilia A requires efficient delivery of the factor VIII gene and sustained protein expression at circulating levels of at least 1% to 2% of normal. Adeno-associated viral type 2 (AAV2) vectors have a number of advantages over other viral vectors, including an excellent safety profile and persistent gene expression. However, a major disadvantage is their small packaging capacity, which has hampered their use in treating diseases such as hemophilia A, cystic fibrosis, and muscular dystrophy, which are caused by mutations in large genes. Here we demonstrate that this can be overcome by using small regulatory elements to drive expression of a B-domain-deleted form of FVIII. The use of this vector for hepatic gene transfer in a canine model of hemophilia A resulted in the sustained (> 14 months) expression of biologically active FVIII. FVIII activity levels of 2% to 4% were achieved. These levels correlated with a partial correction in the whole-blood clotting time and cuticle bleeding time. In addition, immunoprecipitation analysis demonstrated the expression of canine FVIII of the predicted size in the plasma of injected animals. These data support the use of AAV2 vectors in human clinical trials to treat hemophilia A patients.