INCLUSION OF A SHRNA TARGETING BCL11A INTO A Β-GLOBIN EXPRESSING
VECTOR ALLOWS CONCURRENT SYNTHESIS OF CURATIVE ADULT AND FETAL
HEMOGLOBIN
Danuta Jarocha1, Silvia Pires Lourenco1, Valentina Ghiaccio1, Amaliris Guerra1, Osheiza Abdulmalik1,
Ping La1, Alexandra Zezulin2, Kim Smith-Whitley1, Janet L. Kwiatkowski1, Virginia Guzikowski1, Yukio
Nakamura3, Tobias Raabe2, Laura Breda1 and Stefano Rivella1,
(1)The Children's Hospital of Philadelphia, Philadelphia
(2)Perelman School of Medicine, University of Pennsylvania, Philadelphia
(3)RIKEN BioResource Center, Tokyo, Japan
Gene therapy is a promising therapeutic approach in many genetic disorders including beta-thalassemia
(b-thal) and sickle cell disease (SCD) with several ongoing clinical trials spanning a wide
range of strategies such as gene replacement and gene editing. However, no lentiviral vector (LV)
based therapy has shown to be effective in the most severe phenotypes of both SCD and b-thal
with VCN levels of 1-2 copies per genome. Here we present a new approach that combines two of
the strategies presently in clinical trial. We aimed to potentiate the beta-globin based vector
ALS10-T87Q (Breda et al, Plos ONE 2012) with expression of a miRNA for the purpose of increasing
HbF levels. The shRNAmiR sequences targeting BCL11A (Guda et al. Molecular Therapy 2015) were
flanked by an optimized backbone termed “miR-E” (Fellmann et al. Cell Reports 2013) and
incorporated in the non-coding regions of ALS10-T87Q. We screened several positions and
secondary structures for the miR-E-BCL11A in ALS10-T87Q and looked for the optimal intronic
location to express the beta-globin mRNA and the shRNAmiR sequence. Screening in HUDEP-2
M#9 cells identified the vector ATM1.1 as the most potent vector, showing the highest concurrent
induction of HbF and transgenic HbA. To evaluate the potential use of this vector in gene therapy
for SCD, we transduced CD34+ hematopoietic progenitor cells isolated from peripheral blood of
three SCD patients. Moreover, we compared the total production of HbF+HbA of ATM1.1 with its
counterpart ALS10-T87Q (a LV expressing the miR-E-BCL11A driven by the core regulatory
elements of ALS10-T87Q). HPLC protein analysis showed a dose-dependent induction of HbF and
HbA for ALS10-T87Q and ATM1.1. Moreover, our results showed that ATM1.1 induces higher level
of curative hemoglobins (HbF+HbA). Western blot analyses confirmed the induction of γ-globin
chains in ATM1.1 treated cells, despite incomplete suppression of BCL11A. The partial knockdown
of BCL11A observed in cells treated with ATM1.1 could be advantageous in light of recent data
suggesting that disruption of BCL11A leads to an erythroid differentiation defect or survival
disadvantage. Additionally, we introduced silent mutations in the coding sequence of the beta-globin
gene, allowing for detection of splicing variants and specific amplification of the transgenic
mRNA. Cells treated with ALS10-T87Q and ATM1.1 showed identical results by RT-PCR and qPCR
indicating that the presence of the miR-EBCL11A in ATM1.1 does not affect the splicing nor lessen
the production of the beta-globin mRNA. Moreover, when exposed to low oxygen tension, cells
transduced with ATM1.1 were less prone to sickle than control SCD patient cells. ATM1.1 treated
cells showed the lowest percentage of sickle-like morphology (58.1%) when compared to cells
transduced with ALS10-T87Q (81.9%). Our results are the proof-of-principal that combined
strategies are feasible and outperform the current single-pronged methods used in clinical trials.
With this approach we expect to generate more powerful and versatile vectors, which can be
successful at treating the most severe genotypes with minimal integrations per genome.
Moreover, many more genetic disorders can benefit from the combination of gene transfer with
RNA interference as one single therapeutic approach.