Abstract
Gene therapy for sickle cell disease will require efficient delivery of a tightly regulated and stably expressed gene product to provide an effective therapy. In this study we utilized the non-viral Sleeping Beauty (SB) transposon system using the SB100X hyperactive transposase to transduce human cord blood CD34+ cells with DsRed and a hybrid IHK–β-globin transgene. IHK transduced cells were successfully differentiated into multiple lineages which all showed transgene integration. The mature erythroid cells had an increased β-globin to γ-globin ratio from 0.66±0.08 to 1.05±0.12 (p = 0.05), indicating expression of β-globin from the integrated SB transgene. IHK–β-globin mRNA was found in non-erythroid cell types, similar to native β-globin mRNA that was also expressed at low levels. Additional studies in the hematopoietic K562 cell line confirmed the ability of cHS4 insulator elements to protect DsRed and IHK–β-globin transgenes from silencing in long-term culture studies. Insulated transgenes had statistically significant improvement in the maintenance of long term expression, while preserving transgene regulation. These results support the use of Sleeping Beauty vectors in carrying an insulated IHK–β-globin transgene for gene therapy of sickle cell disease.
Highlights
Hematopoietic stem cells (HSCs) are an attractive target for genetic modification to treat diseases such as sickle cell anemia, bthalassemia, and severe combined immunodeficiency (SCID), among others [1,2]
The development of leukemia in 4 out of 20 patients enrolled in a trial of MLV-based therapy for X-SCID continues to be a cautionary example to the field of gene therapy [9,10]
In this study we have shown that primary human CD34+ cells can be transduced with the Sleeping Beauty (SB) construct, pKT2/meIF-SB100XIHK-b-globin, can express hemoglobin in mature erythroid progeny, while maintaining tight, erythroid-specific expression
Summary
Hematopoietic stem cells (HSCs) are an attractive target for genetic modification to treat diseases such as sickle cell anemia, bthalassemia, and severe combined immunodeficiency (SCID), among others [1,2]. The ability of HSCs to be harvested with relative ease, withstand ex vivo manipulation and their long established clinical use in transplants allows for numerous potential gene therapy strategies. This is supported by a recent report of successful treatment of a b-thalassemia patient using a recombinant hemoglobin-expressing lentivirus to transduce autologous CD34+ cells [3]. The current methods for gene transfer in clinical trials rely principally on the use of modified retroviruses such as Moloney murine leukemia virus (MMLV) and lentivirus, which have been known to insert into actively transcribed genes and potentially promote oncogenesis [6,7,8]. The development of leukemia in 4 out of 20 patients enrolled in a trial of MLV-based therapy for X-SCID continues to be a cautionary example to the field of gene therapy [9,10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
