Abstract 3754


An ultimate goal of gene therapy is the development of effective strategies to correct mutant genomic sequences in pathologic cells. To that end, studies have been undertaken to evaluate the therapeutic potential of an oligo/polynucleotide-based sequence-specific gene modification strategy, small fragment homologous replacement (SFHR) in the correction of the mutation giving rise to sickle cell anemia. Small DNA fragments (SDFs) comprising the sickle cell anemia mutation (an A>T transversion in codon 6) and flanking DNA sequences in the human b-globin gene were introduced into Hematopoietic Stem/Progenitor Cells (HSPCs). The studies presented indicated modification at the level of DNA, RNA, and protein when cells were differentiated into erythrocytes.


In this study, SFHR was used to convert A>T in codon 6 of the b-globin gene in CD34+/CD38-/Lin- HSPCs isolated from full term umbilical cord blood as a proof of principle. HSPCs were transfected with a defined number of a 559-bp SDF using the Amaxa electroporation (nucleofection) system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. Erythrocytes were also analyzed using antibodies that differentiate between wild-type hemoglobin A (HBA) and sickle cell hemoglobin S (HBS).


RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated conversion of bA- to bS-globin. Sequencing of the 430-bp amplicon showed the A > T conversion. Analysis of the transfected wild-type HSPC-derived erythrocytes with HBA and HBS specific antibodies demonstrated the presence of a subpopulation of cells expressing HBS. These data are consistent with previous studies showing both conversion of bS- to bA-globin in SC1 cells and bA- to bS-globin in HSPCs after electroporation and microinjection of SDF, respectively.


These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that they demonstrate long-term SFHR-mediated modification of b-globin following mass transfection by electroporation of HSPCs.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.