The β-Hemoglobinopathies as a Model for the Development of Nonviral, Episomal Vectors for Gene Therapy.

The study of β-hemoglobinopathies and associated β-globin genes has revealed that genetic elements, such as the Locus Control Region (LCR) or the replication Initiation Region (IR) of the β-globin gene locus, are essential for the regulation of β-globin genes replication and expression. The LCR at 5' of the β-globin genes plays major role in the intricate regulation of transcription of the "β-like globin genes" expression in situ and in gene therapy protocols by viral gene transfer, ensuring globin gene expression independent from integration site and exerting a critical role in chromatin organization and boundary formation. The IR element, located at the 5' site of the HBB gene promoter, functions as the initiation point for physiological, bidirectional DNA replication, both in situ and within an episomal vector, and induces replication in positions that do not possess such capacity. It enhances plasmid replication, establishment, and transgene expression in the descendants of transfected human CD34+ cells during colony-forming cell assays. A third required genetic element is the promoter of the transgene(s). This is either the HBB gene native promoter or the CD34+ cell-functional ubiquitous promoter spleen focus-forming virus. Both promoters, in in vitro studies, can direct accurate, efficient transcription from episomal, S/MAR-based vectors. Mutations in the HBB gene native promoter as well as in LCR and IR lead to β-thalassemia. Another genetic element, the S/MAR, deriving from the 5' of the human β-interferon gene, ensures plasmid nonintegration and long-term nuclear retention in the prototype episomal vector pEPI-1 and derivative episomal vectors. Such S/MAR-based episomal vectors form the basis from which the genetic elements collectively- HBB gene promoter, LCR, and IR-represent a comprehensive model for the design of efficient episomal vectors with efficient transcription, replication, and long-term nuclear retention of vector for gene therapy applications for the β-hemoglobinopathies within the context of gene addition strategy.