Generation of New Cell Models to Screen Improved Vectors for Functional Hemoglobin Expression

Gene addition of a functional copy of β-globin and reactivation of fetal hemoglobin (HbF) are promising therapeutical approaches for β-globinopathies such as Sickle Cell Disease (SCD) and β-thalassemia (β-thal).

Results from the ongoing clinical trials for β-globinopathies, based on lentiviral delivery of the β-globin gene, indicate that a successful outcome is genotype-dependent. So far, the vectors used in the trials failed to produce curative Hb levels in most of the patients whose genotype leads to null adult hemoglobin (HbA) synthesis, like the β0/β0. Therefore, to achieve HbA synthesis at therapeutic levels in the most severe of genotypes, and with minimal vector copy number (VCN) per cell, more powerful and versatile vectors are required.

Our lab has engineered a new lentiviral vector, ALS10, that carries the β-globin gene, including the non-coding regions. In erythroblasts from patients with SCD and β-thal ALS10 induces HbA synthesis of 26.8% and 68.6%, respectively, with an average VCN=1.

In the present study we aim to streghten the efficacy of our gene addition approach. To do so, a shRNA^miR targeting the transcription factor BCL11A, a known repressor of γ□-globin, was incorporated into ALS10. The shRNA^miR sequences targeting BCL11A (Guda et. al. Molecular Therapy 2015) were flanked by an optimized backbone termed "miR-E" (Fellmann et. al. Cell Reports 2013) for the purpose of knocking down BCL11A and increasing HbF levels.

With this approach we expect to overcome some of the limitations of the vectors presently on clinical trials by simultaneous 1) production of transgenic HbA, 2) reactivation of endogenous HbF and 3) decreased production of endogenous mutant protein.

The development of gene addition and RNAi based therapies mainly relies on the isolation of cells from volunteer donors. Which is dependent on the establishment of ethical protocols and on the availability of donors. Moreover, the cells isolated by this procedure have a limited number of cycles before undergoing full differentiation into red blood cells and consequent death. The HUDEP-2 cell line (Kurita et. al. Plos One 2013) under the appropriate conditions, can be propagated indefinitely and, upon induction, differentiated into enucleated RBCs. Although it offers an efficient alternative system to overcome some of the limitations of using donor samples, HUDEP-2 are derived from healthy cells and produce almost exclusively HbA, which is a limitation in approaches based on the addition of a wt β-globin gene.

To overcome this limitation we used a CRISPR/Cas9 system to mutagenize the β-globin gene of the HUDEP-2 cell line. We have established 2 clones targeted in both alleles named M#9 and M#13. Clone M#9 is homozygous for the deletion of the triplet that encodes Glu6 and was predicted to translate into a shorter variant of the β-globin protein, made of 146 instead of 147aa. Clone M#13 presents the same deletion on 1 allele and a single nucleotide deletion on the second allele, which, by frameshift, causes a downstream premature stop codon that was predicted to encode a 19aa long protein. Chromatographic separation of the hemolysates of clones M#9 and M#13 confirmed the presence of a new hemoglobin variant that is eluted at a different time, distinguishable from both HbA and sickle hemoglobin (HbS). As expected, neither M#9 nor M#13 produce any HbA. From these properties, we predict that these 2 mutant HUDEP clones can be used as a a new cell model for the β-globinopathies, by providing a tool to measure the levels of HbA or F generated after lentiviral treatment.

We transduced M#9 and M#13 with a combined lentiviral vector with the miR-E-BCL11A sequence cloned in the 5' region of β-globin intron 2. Our preliminary results show that, upon integration of the dual lentiviral vector, both clones M#9 and M#13 reach levels of 41% HbF and 36% HbA with a simultaneous decrease in the endogenous mutant form to 17% of the total Hb produced. Western blot analyses confirmed a reduction of BCL11A protein levels and concurrent increase of γ-globin levels.

Our results show that M#9 and M#13 are a suitable platform to test both HbF and HbA induction therapies and confirm their versatility as a screening system for the various emerging therapeutic approaches for β-globinophaties. In addition, we show for the first time, that both HbA and F can be elevated using a single lentiviral construct, which could maximize the rate of Hb correction in patients with β-globinopathies.