Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy

Christian Brendel; Olivier Negre; Michael Rothe; Swaroopa Guda; Geoff Parsons; Chad Harris; Meaghan McGuinness; Daniela Abriss; Alla Tsytsykova; Denise Klatt; Martin Bentler; Danilo Pellin; Lauryn Christiansen; Axel Schambach; John Manis; Helene Trebeden-Negre; Melissa Bonner; Erica Esrick; Gabor Veres; Myriam Armant; David A Williams

doi:10.1016/j.omtm.2020.03.015

Preclinical Evaluation of a Novel Lentiviral Vector Driving Lineage-Specific BCL11A Knockdown for Sickle Cell Gene Therapy

Mol Ther Methods Clin Dev. 2020 Mar 17:17:589-600. doi: 10.1016/j.omtm.2020.03.015. eCollection 2020 Jun 12.

Authors

Christian Brendel^{1

2

3

4}, Olivier Negre⁵, Michael Rothe⁶, Swaroopa Guda¹, Geoff Parsons⁵, Chad Harris¹, Meaghan McGuinness¹, Daniela Abriss¹, Alla Tsytsykova¹, Denise Klatt^{1

6}, Martin Bentler^{1

6}, Danilo Pellin^{1

2

3}, Lauryn Christiansen⁵, Axel Schambach⁶, John Manis⁷, Helene Trebeden-Negre⁸, Melissa Bonner⁵, Erica Esrick^{1

3}, Gabor Veres⁵, Myriam Armant¹, David A Williams^{1

2

3

4}

Affiliations

¹ Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA, USA.
² Harvard Medical School, Boston, MA, USA.
³ Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.
⁴ Harvard Stem Cell Institute, Cambridge, MA, USA.
⁵ bluebird bio, Inc., Cambridge, MA, USA.
⁶ Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
⁷ Department of Laboratory Medicine, Boston Children's Hospital, Boston, MA, USA.
⁸ Connell & O'Reilly Families Cell Manipulation Core Facility, Dana-Farber Cancer Institute, Boston, MA, USA.

Abstract

In this work we provide preclinical data to support initiation of a first-in-human trial for sickle cell disease (SCD) using an approach that relies on reversal of the developmental fetal-to-adult hemoglobin switch. Erythroid-specific knockdown of BCL11A via a lentiviral-encoded microRNA-adapted short hairpin RNA (shRNA^miR) leads to reactivation of the gamma-globin gene while simultaneously reducing expression of the pathogenic adult sickle β-globin. We generated a refined lentiviral vector (LVV) BCH-BB694 that was developed to overcome poor vector titers observed in the manufacturing scale-up of the original research-grade LVV. Healthy or sickle cell donor CD34⁺ cells transduced with Good Manufacturing Practices (GMP)-grade BCH-BB694 LVV achieved high vector copy numbers (VCNs) >5 and gene marking of >80%, resulting in a 3- to 5-fold induction of fetal hemoglobin (HbF) compared with mock-transduced cells without affecting growth, differentiation, and engraftment of gene-modified cells in vitro or in vivo. In vitro immortalization assays, which are designed to measure vector-mediated genotoxicity, showed no increased immortalization compared with mock-transduced cells. Together these data demonstrate that BCH-BB694 LVV is non-toxic and efficacious in preclinical studies, and can be generated at a clinically relevant scale in a GMP setting at high titer to support clinical testing for the treatment of SCD.

Keywords: BCL11A; HbS; RNA interference; Sickle cell disease; fetal hemoglobin; gene therapy; hemoglobin switch; hemoglobinopathies; lentiviral vector; shRNAmiR.

Grants and funding

U01 HL117720/HL/NHLBI NIH HHS/United States