A Modified ANK-1 Promoter Directs Uniform, Copy Number Dependent Gamma Globin Gene Expression at Therapeutic Levels of a in a Lentivirus Vector.

Abstract 3565 Poster Board III-502 Successful gene therapy for hemoglobin disorders beta-thalassemia and sickle cell disease (SCD) has been hindered by inefficient expression of beta-like globin genes from viral vectors. It is estimated that transduction of ∼25% of hematopoietic stem cells (HSC) to express a beta-like globin gene at ∼20% the level of endogenous alpha-globin would be therapeutic. The expression of the beta-like globin genes in their native locus is a precisely regulated process that relies on the coordinated activities of several cis-acting enhancer elements that make up the beta-globin locus control region (LCR). Expression of therapeutic levels of beta-like globin in mouse models and patient cells requires inclusion of LCR elements. These LCR elements are susceptible to silencing, reduce virus titer and can potentially activate oncogenes at the site of proviral insertion. To minimize these risks we have developed a novel approach using non-globin erythroid promoters to drive high levels of globin gene expression independent of enhancers. Previously, we showed that a minimal 276-bp promoter of the human ankyrin gene (ANK-1) does not contain an enhancer element, but does contain a barrier element that allows erythroid-specific, uniform, position independent and copy-number dependent expression of a linked gamma-globin gene. However, the level of gamma-globin expression from the ANK-1 promoter (4% of mouse alpha-globin per copy) was sub-therapeutic. TFIID, a component of the transcription initiation complex, binds a 9-bp sequence (TGCGGTGAG) within the wild type ANK-1 promoter. A dinucleotide deletion within this sequence that disrupts the binding of TFIID and results in ankyrin deficient Hereditary Spherocytosis (Gallagher et al. Hum Mol Genet.14: 2501-9. 2005). We hypothesized that sequence modifications of the ANK1 promoter in this region would increase the affinity for TFIID and increase expression. Mutational analysis of the wildtype TFIID site identified a number of sequence variants that increase expression. An ANK-1 promoter sequence variant that differs from the wildtype TFIID sequence in the first three nucleotides (GCGGGTGAG; GC-ANK-1), results in a 7-fold increase in transcriptional activity in K562 cells. We developed five independent transgenic mouse lines that carry this GC-ANK-1 promoter linked to a human gamma globin gene. Expression in all five lines is erythroid-specific, uniform, position independent and copy-number dependent. Moreover, the average level of gamma-globin mRNA is 34 ± 14 % of mouse alpha-globin mRNA per transgene copy, a 8.5-fold increase in gamma-globin expression over mice harboring the wildtype ANK-1 promoter. We developed a simplified lentiviral vector that contains the CG-ANK-1/gamma-globin gene. This vector was produced at high titer and transduced 57% (23/40) of mouse colony forming unit Spleen (CFU-S) cells. In CFU-S containing single copies of the provirus, the level of gamma-globin mRNA was 6.8 ± 2.9 % of mouse alpha-globin per copy. In CFU-S with two and three copies of the GC-ANK-1 gamma-globin provirus, expression of gamma-globin mRNA averaged 20% ± 1.6% and 30% of mouse alpha-globin per copy respectively, demonstrating copy-number dependent of expression. Overall, an analysis of 16 CFU-S foci demonstrated that the CG-ANK-1/gamma-globin vector expressed gamma globin mRNA at levels that average 8.5 ± 2.0% of mouse alpha-globin. Our data demonstrate that just two copies of the simplified CG-ANK-1/gamma-globin lentiviral vector could potentially produce the levels of gamma-globin necessary for effective treatment of the hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.