Enrichment of Human Hematopoietic Stem/Progenitor Cells Facilitates Transduction for Stem Cell Gene Therapy

Abstract

Autologous hematopoietic stem cell (HSC) gene therapy for sickle cell disease has the potential to treat this illness without the major immunological complications associated with allogeneic transplantation. However, transduction efficiency by β-globin lentiviral vectors using CD34-enriched cell populations is suboptimal and large vector production batches may be needed for clinical trials. Transducing a cell population more enriched for HSC could greatly reduce vector needs and, potentially, increase transduction efficiency. CD34+/CD38− cells, comprising ~1%–3% of all CD34+ cells, were isolated from healthy cord blood CD34+ cells by fluorescence-activated cell sorting and transduced with a lentiviral vector expressing an antisickling form of beta-globin (CCL-βAS3-FB). Isolated CD34+/CD38− cells were able to generate progeny over an extended period of long-term culture (LTC) compared to the CD34+ cells and required up to 40-fold less vector for transduction compared to bulk CD34+ preparations containing an equivalent number of CD34+/CD38− cells. Transduction of isolated CD34+/CD38− cells was comparable to CD34+ cells measured by quantitative PCR at day 14 with reduced vector needs, and average vector copy/cell remained higher over time for LTC initiated from CD34+/38− cells. Following in vitro erythroid differentiation, HBBAS3 mRNA expression was similar in cultures derived from CD34+/CD38− cells or unfractionated CD34+ cells. In vivo studies showed equivalent engraftment of transduced CD34+/CD38− cells when transplanted in competition with 100-fold more CD34+/CD38+ cells. This work provides initial evidence for the beneficial effects from isolating human CD34+/CD38− cells to use significantly less vector and potentially improve transduction for HSC gene therapy.

Disruption of the BCL11A Erythroid Enhancer Reactivates Fetal Hemoglobin in Erythroid Cells of Patients with β-Thalassemia Major

“…Under these conditions, we have now shown, by using both normal and thalassemic samples, that we can not only significantly expand ex vivo cells with an engraftable edited HSC phenotype within 5 days but also efficiently edit HSCs after expansion. Although, by this method, larger-scale transfections and higher amounts of ZFN mRNA are required, the expanded, more primitive CD34+/CD38− cells, if originally insufficient in numbers, could be purified and edited efficiently in isolation without the co-presence of large numbers of the CD34+/CD38+ population 45 . As recently proposed, 4 the edited HSCs could then be co-transplanted with unedited progenitor or precursor cells.…”

Genetic Tagging During Human Mesoderm Differentiation Reveals Tripotent Lateral Plate Mesodermal Progenitors

“…FUGW (carrying the EGFP reporter) was used in all transductions except for the negative control experiment (Fig. D) in which mCitrine, mCerulean and mStrawberry were expressed in pCCLc‐UBC‐reporter‐PRE‐FB‐2xUSE . Vector production and titer were as described .…”

Optimizing autologous cell grafts to improve stem cell gene therapy

“…The group of D. Kohn showed increased susceptibility to transduction with markedly less vector amounts by further purification of HSCs beyond the standard CD34+cell enriched fraction, through the isolation and transduction with lenti-viral vectors of CD34 + /CD38 - cells as compared with the unfractionated CD34 + cells[41]. These sorted and gene-modified CD34 + /CD38 - cells were approximately 100 fold more competitive for engraftment than their CD34 + /CD38 + counterparts when xenotransplanted into NSG mice.…”