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-β(AS3) -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.

Keywords: Gene therapy; Hematopoietic stem cells; Lentiviral vector; Stem cell enrichment.

Figure 1

Isolation and growth properties of human CD34⁺ and CD34⁺/CD38⁻ cells. (A): Flow cytometry of CD34-enriched cells showing gating strategy used to define CD34⁺/CD38⁺ cells (region P5) and CD34⁺/CD38⁻ cells (region P3). (B): Cell expansion from CD34⁺ and CD34⁺/CD38⁻ cells from cord blood. Cells were cocultured with irradiated MS5 stromal cells in long-term culture medium. The mean fold increase over cell number plated on day 0 is shown at each time point of long-term culture. Data represent cell expansion ±SEM over time (n =3, p <.0001). Abbreviation: APC, allophycocyanin.

Figure 2

Analysis of transduction of CD34⁺ and CD34⁺/CD38⁻ cells with the CCL-β^AS3-FB LV vector. (A): Vector copy number (VCN) ±SEM in transduced CD34⁺ and CD34⁺/CD38⁻ cells (n =9, p =.02). (B): Distribution of hematopoietic colony types (n =80 colonies) formed by nontransduced cord blood (CB) CD34⁺ (NT-CD34⁺), transduced CD34⁺ (CD34⁺), and CD34⁺/CD38⁻ cells. (C): Percentage of plated NT-CD34⁺, CD34⁺, and CD34⁺/CD38⁻ cells that grew into hematopoietic colonies in vitro. Values represent the mean ±SD. (D): Single CFU grown from transduced CD34⁺ (left) and CD34⁺/CD38⁻ (right) CB cells were analyzed for VCN by ddPCR (n =80 colonies). Graph indicates percentages of the CFU that were negative for vector by digital PCR (0 VC/cell) or that had VC/cell of 1–2, 3–4, 5–6, or >6. (E): Vector transduction dose-response for CD34⁺ and CD34⁺/CD38⁻ cells (n =3, p =.05 at 6.6 × 10⁶ TU/ml, p =.002 at 2 × 10⁷ TU/ml). (F): VCN over time in long-term culture (± SEM [n =3]) (time trend difference p =.03, VCN difference p =.004, linear mixed model). Asterisk indicates significance, *, p ≤.05; **, p ≤.01.

Figure 3

Analysis of transduction of CD34⁺ and CD34⁺/CD38⁻ cells by the CCL-MND-GFP LV vector. (A): Comparison of mean vector copy number ±SEM after transduction with a dose range of CCL-MND-GFP LV analyzed by qPCR at day 14 of culture. (B): Representative histogram showing relative GFP expression of transduced CD34⁺ and CD34⁺/CD38⁻ cells. (C): Percentages of GFP+ cells determined by flow cytometry in CCL-MND-GFP-transduced CD34⁺ and CD34⁺/CD38⁻ cells (n =6, p =.02). Abbreviation: GFP, green fluorescent protein.

Figure 4

Erythroid differentiation of CD34⁺ and CD34⁺/CD38⁻ cells transduced by the CCL-β^AS3-FB LV vector. (A): Comparison of vector copy number (VCN) ±SEM during differentiation, at day 14 after transduction (n =3). (B): Percentage of HBBAS3 mRNA expression of all β-globin transcripts per VCN (%AS3/VCN) in erythroid cells differentiated from transduced CD34⁺ and CD34⁺/CD38⁻ cells analyzed by qRT-PCR (n =3).

Figure 5

Role of vector envelope and receptor on transduction by the CCL-β -FB LV vector. LDL receptor expression by CD34 and CD34⁺/CD38⁻ cells on: (A) freshly isolated CD34⁺ cells, (B) CD34⁺ cells at 48 hours of culture in cytokines, (C) freshly isolated CD34⁺/CD38⁻ cells, and (D) CD34⁺/CD38⁻ cells at 48 hours of culture in cytokines. (E): Transduction of CD34⁺ and CD34⁺/CD38⁻ cells with the RD114 pseudotyped CCL-β^AS3-FB LV vector. The graph represents the mean vector copy number of CD34⁺ and CD34⁺/CD38⁻ cells ±SEM analyzed by qPCR at day 14 of culture (n =3). Abbreviation: LDL, low density lipoprotein.

Figure 6

Comparison of engraftment of NOD.Cg-Prkd^scidIl2rg^tm1Wjil/SzJ (NSG) mice. (A): Contribution to human CD45⁺ cell engraftment in NSG mice by transduced, transplanted cell populations. Mock mice were transplanted with nontransduced human CB CD34⁺ cells; control mice were transplanted with transduced CD34⁺ cells; all other mice were transplanted with a combination of CD34⁺/CD38⁻ (1%) and CD34⁺/CD38⁺ cells (99%). Vectors used for transduction (CCLc-UBC-mStrawberry-FB, CCLc-UBC-mCitrine-FB, and CCLc-UBC-mCerulean-FB LV) were alternated among the cell populations for each transplant. BM harvested from NSG mice with human cell engraftment (%huCD45⁺/%huCD45⁺+muCD45⁺ cells) was further analyzed for percent vector expression using flow cytometry. (B): Vector copy number (VCN) of cells analyzed in vivo mouse transplantation. In vivo VCN was analyzed from BM harvested 80–90 days after transplantation into NSG mice using ddPCR with primers and probes specific to each fluorescent reporter. The in vivo VCN/mouse for each population of cells is displayed separately.