Improving Gene Therapy Efficiency through the Enrichment of Human Hematopoietic Stem Cells

• Published in: Molecular therapy 2017-09, Vol.25 (9), p.2163-2175
• Main Authors: Masiuk, Katelyn E., Brown, Devin, Laborada, Jennifer, Hollis, Roger P., Urbinati, Fabrizia, Kohn, Donald B.
• Format: Article
• Language: English
• Subjects: ADP-ribosyl Cyclase 1 - metabolism; Animals; Antigens, CD34 - metabolism; Blood; Bone marrow; CD34 antigen; CD38 antigen; Clinical trials; Cloning; Conflicts of interest; Experiments; Flow cytometry; Gene Expression; Gene therapy; Gene Transfer Techniques; Genes, Reporter; Genetic Therapy; Genetic Vectors - genetics; Good Manufacturing Practice; Graft Survival; Hematological diseases; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - metabolism; Humans; Hypothesis testing; Immunomagnetic Separation; Immunophenotyping; Kinases; lentiviral vectors; Lentivirus - genetics; Life span; Mice; Original; Peripheral blood; Purification; Severe combined immunodeficiency; Stem cell transplantation; Stem cells; Transduction, Genetic; Transgenes; Transplants & implants
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2017.05.023

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 × 108 to 1 × 109 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. [Display omitted] Hematopoietic stem cell (HSC) gene therapy is a promising clinical strategy to treat a wide range of genetic blood diseases. Masiuk and colleagues demonstrate a clinically relevant method to improve HSC gene therapy efficiency through isolation and transplantation of highly purified HSCs prior to lentiviral (LV) transduction. Enhanced HSC enrichment allows for a 5- to 10-fold reduction in LV dose and culture scale compared with currently used methods.