A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

Kevyn L Hart; Boya Liu; Devin Brown; Beatriz Campo-Fernandez; Kevin Tam; Katherine Orr; Roger P Hollis; Christian Brendel; David A Williams; Donald B Kohn

doi:10.1016/j.omtm.2024.101254

A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

Mol Ther Methods Clin Dev. 2024 Apr 24;32(2):101254. doi: 10.1016/j.omtm.2024.101254. eCollection 2024 Jun 13.

Authors

Kevyn L Hart¹, Boya Liu^{2

3}, Devin Brown⁴, Beatriz Campo-Fernandez⁴, Kevin Tam⁴, Katherine Orr⁵, Roger P Hollis⁴, Christian Brendel^{2

3

6

7}, David A Williams^{2

3

6

7}, Donald B Kohn^{4

8

9

10}

Affiliations

¹ Department of Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
² Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA.
³ Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA.
⁴ Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁵ CSUN-UCLA Stem Cell Scientist Training Program, California State University, Northridge, Northridge, CA 91330, USA.
⁶ Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA.
⁷ Harvard Stem Cell Institute, Harvard University, Boston, MA 02138, USA.
⁸ Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹⁰ The Eli & Edythe Broad Center of Regenerative Medicine & Stem Cell Research, University of California, Los Angeles, Los Angeles, CA 90095, USA.

Abstract

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34⁺ hematopoietic stem and progenitor cells (HSPCs). This LVV contains locus control region cores expressing an anti-sickling β^AS3-globin gene and two microRNA-adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbA^AS3 + HbF), while concurrently decreasing sickle hemoglobin (HbS). The decrease in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34⁺ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at a low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to decrease costs and help improve accessibility to gene therapy for SCD.

Keywords: BCL11A; anti-sickling hemoglobin; gene therapy; hematopoietic stem cells; lentiviral vector; shmiR; sickle cell disease.