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Lentiviral vector (LV)-based hematopoietic stem cell (HSC) gene therapy is becoming a promising clinical strategy for the treatment of genetic blood diseases. However, the current approach of modifying 1 × 10 8 to 1 × 10 9 CD34 + cells per patient requires large amounts of LV, which is expensive and technically challenging to produce at clinical scale. Modification of bulk CD34 + cells uses LV inefficiently, because the majority of CD34 + cells are short-term progenitors with a limited post-transplant lifespan. Here, we utilized a clinically relevant, immunomagnetic bead (IB)-based method to purify CD34 + CD38 − cells from human bone marrow (BM) and mobilized peripheral blood (mPB). IB purification of CD34 + CD38 − cells enriched severe combined immune deficiency (SCID) repopulating cell (SRC) frequency an additional 12-fold beyond standard CD34 + purification and did not affect gene marking of long-term HSCs. Transplant of purified CD34 + CD38 − cells led to delayed myeloid reconstitution, which could be rescued by the addition of non-transduced CD38 + cells. Importantly, LV modification and transplantation of IB-purified CD34 + CD38 − cells/non-modified CD38 + cells into immune-deficient mice achieved long-term gene-marked engraftment comparable with modification of bulk CD34 + cells, while utilizing ∼7-fold less LV. Thus, we demonstrate a translatable method to improve the clinical and commercial viability of gene therapy for genetic blood cell diseases.

Sickle cell disease (SCD, OMIM 603903) is one the most common inherited blood disorders worldwide, caused by a single amino acid substitution (E6V) in the β-globin chain. SCD is associated with abnormal red cell morphology, hemolytic anemia, vascular occlusion, severe pain, progressive organ damage and reduced life expectancy. Gene therapy by transplantation of autologous, genetically corrected hematopoietic stem cells could be a therapeutic alternative, particularly in patients lacking an allogeneic bone marrow donor. Recent attempts to treat β-thalassemia and SCD with gene therapy showed encouraging results as well as potential limits of a technology based on complex lentiviral vectors for stem cell transduction due to the vector size and complexity, and limited globin synthesis. Designing an efficient vector that combines high-level globin expression with high titer and infectivity remains a formidable challenge. The GLOBE vector carries a β-globin gene under the control of its promoter and a reduced version of the β-globin locus control region (LCR); it was designed to reduce the size and complexity of the LCR while maintaining its enhancer and chromatin opening activity (Miccio et al., PNAS 2008). Based on the GLOBE design, we developed novel LVs for SCD gene therapy, carrying a modified transgene encoding an anti-sickling β-globin gene with 3 amino acid substitutions (AS3), alternative β-globin promoters and a short sequence (FB) with insulating and enhancer-blocking activity in the LTR. The vectors are produced at high titer and reproducibly transduce 60-70% of human BM CD34+ cell-derived clonogenic progenitors at an average VCN of 3. The GLOBE vectors were compared to the Lenti/βAS3-FB vector (Romero, Urbinati et al. JCI 2013), previously developed for a gene therapy clinical trial of SCD. We will present data comparing the different vectors for transduction efficiency in BM CD34+ cells from healthy donors and SCD patients, βAS3-globin transgene expression and anti-sickling activity, in vivo repopulation activity in NSG mice and vector integration profiles in repopulating cells before and after transplantation. This study provides a comprehensive analysis of different LV vectors, useful to determine the optimal candidate for gene therapy of SCD that can provide high transduction of Hematopoietic Stem and Progenitor Cells (HSPCs) as well as maintaining an adequate expression of the anti-sickling globin gene.