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Letters to the Editor
Figure 1. Initial constructs design, Hudep9 (M#9) line development and characterization of constructs efficiency. (A) Initial constructs used in the study: ALS10- T87Q, ATM1.1, ATM2.1, ATM2.2. The miR-E-BCL11A5 sequences were synthesized by Genscript USA (NJ) by combining the passenger and guide sequences of short hairpin RNA (shRNAmiR5) with the flanking miR-E backbone sequences.3,4 The combined miR-E-BCL11A5 sequences were cloned in intron 1 position c.79+36 of ALS10-T87Q (ATM1) and intron 2 positions c.303-163 and c.303-172 (ATM2.1 and ATM2.2, respectively) of the b-globin transgene. Viral production and titration were performed according to methods previously reported.15 All vectors include the human b-globin gene and regulatory elements (β-globin promot- er (bp) and portions of the locus control region (LCR) as in our previously described vector AnkT9).8 Some of these vectors present a miRNA as described in the main manuscript. Erythroid specific expression of the b-globin gene and shRNAmiR is achieved by using the b-globin promoter and its locus control region (LCR), in a pol II-promoter driven system. Transgene expression under these regulatory elements will be limited to the latest stages of erythroid development,8 prevent- ing unwanted effects as previously seen in B lymphocytes and hematopoetic stem cellks.10 (B) Representative chromatographic separation (high-performance liquid chromatography [HPLC])2 of hemolysates from the HUDEP-2 (top) and M#9 (middle) erythroblasts (developed using methodology previously described),15 showing fetal hemoglobin (HbF), adult hemoglobin (HbA), HbA2 and mutant HbA (MutHbA) peaks at day 7 of differentiation as described.1,15 At the bottom, a rep- resentative chromatographic separation15 of the M#9 cell line after transduction with one of the vectors that simultaneously produced HbAT87Q and induced HbF expression. (C) Relative proportion of HbF, HbA, HbA2 and MutHb in HUDEP-2 and M#9 quantified by HPLC at day 7 of differentiation. (D) Representative western blot showing HBA, HBB, HBG and BCL11A proteins in HUDEP-2 and M#9 at day 7 of differentiation. The evaluation was performed using rabbit anti- HBB (Proteintech, Chicago, Il), rabbit anti-HBA (Santa Cruz Biotechnology), mouse anti-BCL11A (Abcam, Cambridge, MA, USA) rabbit anti-HBG (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-GAPDH (Proteintech) primary antibodies. Secondary, anti-mouse and anti-rabbit IgG HRP-linked antibodies (Cell Signaling) were used for detection with the Amersham enhanced chemiluminescence (ECL) western blotting analysis system (GE Healthcare, Malborough, MA, USA). (E) Representative fluorescence-activated cell sorting (FACS) plots of HUDEP-2 and M#9 stained for GPA, CD71, c-KIT and CD36 markers adapted from and analyzed as previously described.8 (F) Percentage of total curative hemoglobins (HbAT87Q+HbF) over a range of viral integrations after transducing M#9 cells. (G) Proportion of the different hemoglobins produced in M#9 cells transduced with ALS10-T87Q with vector copy number (VCN)= 2.
Using a previous vector developed by our group indi- cated as AnkT9W, we generated ALS10-T87Q (Figure 1A), a vector that produces a functional b-globin gene that carries a modification (indicated as T87Q) with improved anti-sickling activity.7-8 Additionally, ALS10- T87Q vector includes the full sequence of the intron 2 of the b-globin gene (Figure 1A). We incorporated into the b-globin coding sequence a short hairpin RNA (shRNAmiR) targeting the transcription factor BCL11A, a known repressor of γ-globin.
The shRNAmiR sequences targeting BCL11A9-10 were flanked by an optimized backbone termed “miR-E” (11) to increase HbF levels through downregulation of BCL11A (Figure 1A). Our goal is to overcome some of the limitations of the vectors presently in clinical trials by simultaneous i) production of transgenic HbA; ii) reacti- vation of endogenous HbF; and iii) decrease in produc- tion of endogenous mutant b-globin mRNA and/or pro- tein.
The erythroid HUDEP-2 immortalized human cord blood cell line was generated by inducible expression of the encoding genes HPV16-E6/E7 derived from the human Papilloma virus 16.12 These cells can be used as a semi-primary adult hematopoietic cell model that can be propagated indefinitely in the presence of tetracycline or differentiated into red cells under appropriate culture conditions. In order to assess the level of HbAT87Q and HbF produced by the new vectors, we mutagenized the first exon of the human b-globin gene (HBB) in erythroid HUDEP-2 cells using the CRISPR/Cas9 system. We estab- lished a clonal cell line, named M#9, carrying a homozy- gous deletion of codon 6, thus generating a mutant HBB gene (Hb-mutant). This line produced a predominant mutant adult HbA (Hb-mutant) distinguishable from the endogenous wild-type (WT) HbA by high-performance liquid chromatography (HPLC) (Figure 1A and B, top and middle panel, and Figure 1C), which was also visible by western blot analysis (Figure 1D). The change did not alter erythroid maturation (Figure 1E). The HbAT87Q gen- erated by ALS10-T87Q and HbF induced by the shRNAmir were also both distinguishable from the Hb-mutant pro- duced by M#9 via chromatographic separation (Figure 1B, bottom panel). We were able to assess the relative production of functional HbAT87Q +HbF after gene trans- fer and correlate these values to VCN in a dose/effect relationship. Upon transduction in the M#9 cell line, ALS10-T87Q generated 18%, 23% and 44% of HbAT87Q with VCN=0.6, 1.0 and 2.0, respectively (Figure 1F and G). We cloned the miR-E-BCL11A sequence either in the
b-globin intron 1 (ATM1), or in two different regions of intron 2 (ATM2.1 and ATM2.2) (Figure 1A). Upon trans- duction of M#9, all ATM vectors reduced BCL11A level and induced HbF production by western blot (Online Supplementary Figure S1A), confirming functional repres- sion from the miR-E-BCL11A sequence. ATM1, the most effective among the ATM vectors, showed the highest HbF induction (Online Supplementary Figure S1B) and an HbA production equivalent to ~90% of that made by ALS10-T87Q at VCN=1 (Online Supplementary Figure S1C). However, the total production of HbAT87Q +HbF by ATM1 vector was suboptimal, as the combination of the miR-E-BCL11A with the b-globin gene did not lead to an overall increase of therapeutic hemoglobins (HbAT87Q+HbF) compared to ALS10-T87Q (Figure 1F; Online Supplementary Figure S1D to E).
In order to overcome this limitation, we modified the sequence of miR-E-BCL11A within intron 1, generating additional vectors named ATM1.1, ATM1.2, ATM1.3 and ATM1.4 (Figure 2A; Online Supplementary Table S1A). We tested two different modifications of the guide and one modification of the loop of the miR scaffold itself. ATM1.1 differs from ATM1 in the last four bases of the guide to adapt the shift from shRNA to miR-based design, as indicated in the legend of Figure 2A. These changes ensure higher 5’ passenger stability for efficient guide recognition and incorporation into RISC complex as previously shown.9
Among these constructs, ATM1.1 produced the highest HbAT87Q and HbF expression levels (Figure 2B and C, top and bottom panels). ATM1.1 produced between ~25% and ~47% more therapeutic hemoglobins (HbAT87Q+HbF) compared to the HbAT87Q produced by ALS10-T87Q, at low and high VCN, respectively (Figure 2D, top and bottom panels). Using a mixed-effects linear regression model, the increase was quantified in ~33% more HbAT87Q+HbF in ATM1.1 compared to the HbAT87Q made by ALS10-T87Q (looking at similar levels of VCN; Figure 2E; Online Supplementary Table S1B). Western blot analysis confirmed the simultaneous reduction of BCL11A and the increase of γ-globin protein levels in M#9 samples treated with ATM1.1 (Figure 2F).
In order to verify that the mRNA or DNA of the ATM1.1 vector did not recombine due to the inclusion of the miR-E-BCL11A, both during integration and expres- sion of the transcript, we introduced silent mutations in exon1 and exon2 of the b-globin gene (Silb-globin gene) to distinguish, in human cells, DNA and mRNA of the endogenous and the transgenic Silb-globin sequences
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