678. Production of a Novel Recombinant AAV Vector Containing beta-Globin Gene and Essential Segments of LCR and Transfer of the Construct into the CD133+, Mesenchymal Stem Cell and Unrestricted Somatic Stem Cell (USSCs)

Abstract

Thalassemia is the world's most common hereditary disease and is a paradigm of monogenic genetic diseases. It is estimated that there are 80,000,000 carriers worldwide, thus beta thalassemia is a major genetic cause of morbidity and mortality. The only currently available curative treatment is replacement of the bone marrow with an allogeneic transplant. The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, AAV is currently being developed as a vector for human gene therapy. Our goal is to develop an AAV vector for treatment of hemoglobinopathies. We constructed a recombinant adeno-associated virus 2 (AAV) virions containing upstream erythroid cell-specific enhancers (HS2, a 501- bp fragment, HS3, a 307-bp fragment, HS4, a 343-bp fragment) from the locus control region of the human beta-globin gene cluster inserted upstream of a 3086-bp DNA fragment from a genomic clone of the human beta-globin gene. The total size of the recombinant vector was 4.56 kb. These recombinant virions were used to infect populations of CD133+ cells isolated from beta thalassemic human bone marrow. We obtained permanent and high-efficiency expression of a human beta globin gene in primary human hematopoietic stem cells. Three months post infection samples were analyzed to document the presence of the transduced human beta-globin gene sequences in the mentioned cells and good expression. We also infected mesenchymal stem cell and unrestricted somatic stem cells (USSCs) by this recombinant virus. We can detect our construct 3 months after transfection. We suggest that with further optimization of the system, this vector can be used for human gene therapy of thalassemia and sickle cell anemia. Thalassemia is the world's most common hereditary disease and is a paradigm of monogenic genetic diseases. It is estimated that there are 80,000,000 carriers worldwide, thus beta thalassemia is a major genetic cause of morbidity and mortality. The only currently available curative treatment is replacement of the bone marrow with an allogeneic transplant. The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, AAV is currently being developed as a vector for human gene therapy. Our goal is to develop an AAV vector for treatment of hemoglobinopathies. We constructed a recombinant adeno-associated virus 2 (AAV) virions containing upstream erythroid cell-specific enhancers (HS2, a 501- bp fragment, HS3, a 307-bp fragment, HS4, a 343-bp fragment) from the locus control region of the human beta-globin gene cluster inserted upstream of a 3086-bp DNA fragment from a genomic clone of the human beta-globin gene. The total size of the recombinant vector was 4.56 kb. These recombinant virions were used to infect populations of CD133+ cells isolated from beta thalassemic human bone marrow. We obtained permanent and high-efficiency expression of a human beta globin gene in primary human hematopoietic stem cells. Three months post infection samples were analyzed to document the presence of the transduced human beta-globin gene sequences in the mentioned cells and good expression. We also infected mesenchymal stem cell and unrestricted somatic stem cells (USSCs) by this recombinant virus. We can detect our construct 3 months after transfection. We suggest that with further optimization of the system, this vector can be used for human gene therapy of thalassemia and sickle cell anemia.