686. Forced Fetal-Globin Reactivation Mediated by the TALE-ldb1 Fusion Protein

Abstract

Sickle cell disease (SCD) and thalassemia are very severe inherited disorders of the red cell and are amongst the world's most common genetic diseases. Thalassemia is caused by a serious lack of normal β-globin production in red cells whereas, sickle cell disease reflects a mutant hemoglobin that produces protein aggregates. The production of fetal-globin (HbF) may compensate for the reduction of β-globin and also it functions to inhibit sickling by participating in formation of mixed tetramers including both the γ and βS chain. Thus the induction of HbF holds tremendous promise to ameliorate the clinical symptoms of β -thalassemia and sickle cell disease (SCD). For this reason, the adult-to-fetal hemoglobin switch has been an area of long-standing interest among hematologists and basic scientists. The recent revolution in gene editing technology has provided new opportunities for high efficiency fetal-globin gene activation and provides strong potential for clinical benefit. Recently, it was reported that zinc finger artificial proteins that bind to the ? globin gene promoter could reactivate fetal-globin expression, via tethering the globin enhancer LCR to the ? globin gene promoter. We have designed Transcriptional Activator Like Effectors (TALE) targeting the ? globin gene promoters to reactivate ? globin gene expression. The EF1 short promoter was used initially to drive TALE gene expression. The TALEs were fused with a ldb1 dimerization domain, followed by a T2A GFP cassette. The various elements were then assembled into a lentiviral vector. To prevent deletions caused by repeats of TALEs during the lentivirus packing process, we changed the TALE's DNA coding sequence by using codon-optimized DNA for their expression. Both PCR and Southern blot analysis confirmed that TALEs remained as full-length intact DNA without deletion. We employed HUDEP-2 (human umbilical cord blood derived erythroid progenitor cells) to examine the function of TALEs with respect to fetal-globin gene reactivation. Quantitative RT-PCR was performed to determine the relative mRNA level of fetal-globin which was normalized to the alpha-globin mRNA content. Three of eight TALEs were found to activate fetal-globin expression in HUDEP-2 cells with increases in mRNA levels from several fold to more than 400-fold. Furthermore, we also observed a symmetrical reduction of β-globin mRNA by more than 50%. The fetal-globin level determined by HPLC was up to 40% of total globin, compared to 0.4% in control cells. To define the ability of the vector to transduce primitive CD34 cells, we performed methylcellulose culture after transduction of CD34 cells with TALE encoding lentiviral vectors at an MOI of 5. Individual red colonies were picked up to determine the vector copy number (VCN) by quantitative real time-PCR. Forty to fifty percent of the colonies were positive for vector insertion. In the future we are planning to do human CD34 cell erythroid differentiation to evaluate how the vector activates fetal-globin expression in red cells. To promote strong expression of TALE in the erythroid linage, we have replaced the EF1 short promoter with strong MND promoter or one erythroid specific ankrin promoter.