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Letters to the Editor
ATM1.1-silent vectors, we were able to discriminate between the SCD and b-globin-T87Q mRNA and that encoded by the silent vectors (Online Supplementary Figure S2A). Amplification of the region encompassing exon 1 and 2 (Figure 1A), performed using oligonucleotides (Online Supplementary Table S1D) that selectively anneal to the silent b-globin mRNA, suggested that the presence of the miR-E-BCL11A in intron 1 did not affect the length and splicing of the portion of the b-globin mRNA ampli- fied in the double vector (Online Supplementary Figure S2B). This indicated no apparent interference between the expression of the transgenic b-globin and the miR-E- BCL11A components.
In order to evaluate the potential use of this vector in gene therapy for SCD, we transduced CD34+-derived hematopoietic progenitor cells isolated from peripheral blood of three patients with SCD. We compared the pro- duction of HbAT87Q and HbF by ATM1.1 with its counter- part ALS10-T87Q. We observed a dose-dependent induc- tion of HbF and HbAT87Q in SCD erythroblasts treated with ATM1.1 (Figure 3A and B). Moreover, our results showed that ATM1.1 outperformed ALS10-T87Q, induc- ing the highest levels of curative hemoglobins (HbAT87Q+HbF) (Figure 3C; Online Supplementary Table S2C). Western blot analysis confirmed the induction of γ-globin chains in cells transduced with ATM1.1 (Figure 3D). While not completely suppressed, the partial knock- down of BCL11A observed in cells treated with ATM1.1 (Figure 3D) could be advantageous. Reported evidence suggests that disruption of BCL11A leads to an erythroid differentiation defect or survival disadvantage.13-14 Gene expression analysis of SCD cells showed comparable expression of transgenic b-globin, irrespectively of whether they carried the silent-b-globin mRNA or not (Online Supplementary Figure S2C). This also indicated that introduction of the silent mutation did not affect the ability of the vectors to produce the b-globin mRNA (Online Supplementary Figure S2C, top panel). Both ATM1.1 and its silent variant presented a significantly increased level of γ-globin mRNA, tightly correlating with the protein yield (Online Supplementary Figure S2A to C). Moreover, the SCD erythroblasts treated with ATM1.1 and exposed to hypoxic conditions showed lower percentage of sickle-like morphology (58.1%) by comparison to parallel specimens transduced with ALS10-T87Q (81.9%) (Figure 3E to F). We analyzed b0/b0 thalassemic patients’ cells and compared the per- centage of total curative hemoglobin produced by ALS10-T87Q and ATM1.1. Untransduced b0/b0 cells showed over 90% of baseline HbF (Figure 3G, left panel). The high level of HbF is in part due to intrinsic features of the culture conditions and lack of b-globin expression in these cells. Upon transduction, we observed that the total content of HbF+HbA was close to 100%, although in different proportions (Figure 3G, left panel). The most informative change was the abundance of a HPLC peak corresponding to α-globin aggregates, which we previ- ously characterized in b0/b0 CD34+ derived erythroid cells.8 ATM1.1-transduced cells showed lower levels of these aggregates when compared to the same cells trans- duced with ALS10-T87Q at similar VCN (Figure 3G, right panel; Online Supplementary Figure S2D). The higher sup- pression of free α-chains suggests that, overall, ATM1.1 was able to produce more curative chains (b+γ).
In conclusion, our results show that HbA and HbF can be elevated simultaneously using a single lentiviral con- struct, as our ATM1.1 vector. The ability of ATM1.1 to induce functional hemoglobin production (HbF+HbA) surpasses that of a vector expressing b-globin alone.
While the results in vitro demonstrate proof-of-principle potency of the vectors used, the level of functional hemo- globin produced in vivo may vary. Future studies will eval- uate this approach analyzing erythroid cells derived from transduced SCD or thalassemic HSC in vivo. These stud- ies will be performed after cloning the best miR-E-BCL11A into an enhanced vector recently devel- oped in our lab.15
Silvia Pires Lourenco,1,2* Danuta Jarocha,1* Valentina Ghiaccio,1 Amaliris Guerra,1 Osheiza Abdulmalik,1
Ping La,1 Alexandra Zezulin,3 Kim Smith-Whitley,1 Janet L. Kwiatkowski,1 Virginia Guzikowski,1 Yukio Nakamura,4 Tobias Raabe,3 Laura Breda1 and Stefano Rivella1
1Department of Pediatrics, Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Graduate Program in Basic and Applied Biology (GABBA), Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal; 3Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA and 4Cell Engineering Division, RIKEN BioResource Research Center, Tsukuba, Japan
*SPL and DJ contributed equally as co-first authors. Correspondence: DANUTA JAROCHA - jarochad@email.chop.edu doi:10.3324/haematol.2020.276634
Received: November 20, 2020.
Accepted: May 3, 2021.
Pre-published: May 27, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: SPL designed the vector and experiments, conducted the experiments, analyzed the data and prepared the manuscript;
DJ analyzed the data and prepared the manuscript; GV provided experimental support and analysis of the data; AG designed the vectors and analyzed the data; OA and LP provided experimental support; S-WK, KJ and GVprepared clinical specimens and analyzed the data; NY provided the Hudep2 cell line; RT helped with M#9 cell line development; BL designed the vectors and analyzed the data;
RS designed the vectors and experiments, analyzed data and revised the final manuscript.
Acknowledgments: we would like to thank Vanessa Carrion for her assistance in identifying the SCD specimens.
References
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