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. 2021 Jan 1;106(1):74-86.
doi: 10.3324/haematol.2019.238261.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Valentina Capo  1 , Sara Penna  1 , Ivan Merelli  2 , Matteo Barcella  1 , Serena Scala  1 , Luca Basso-Ricci  1 , Elena Draghici  1 , Eleonora Palagano  3 , Erika Zonari  1 , Giacomo Desantis  1 , Paolo Uva  4 , Roberto Cusano  4 , Lucia Sergi Sergi  1 , Laura Crisafulli  3 , Despina Moshous  5 , Polina Stepensky  6 , Katarzyna Drabko  7 , Zühre Kaya  8 , Ekrem Unal  9 , Alper Gezdirici  10 , Giuseppe Menna  11 , Marta Serafini  12 , Alessandro Aiuti  1 , Silvia Laura Locatelli  13 , Carmelo Carlo-Stella  14 , Ansgar S Schulz  15 , Francesca Ficara  16 , Cristina Sobacchi  16 , Bernhard Gentner  1 , Anna Villa  1
Affiliations

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis

Valentina Capo et al. Haematologica. .

Abstract

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

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Figures

Figure 1.
Figure 1.
Circulating CD34+ cells. (A) The graph shows the percentage of CD34+ in the peripheral blood (PB) of ARO patients, divided in the indicated age groups. (B) Pie charts show distribution of 25 subsets within the CD45+ gate in ARO patient PB (n=5), healthy donor PB (n=4) and bone marrow (BM) (n=7). Black outer ticks indicate 100 cells/L. Percentages indicate the frequency of hematopoietic stem and progenitor cell (HSPC) population (CD34+ LIN–) on total CD45+ cells. Stacked bar graphs indicate the absolute cell count/L of the HSPC compartment, composed of ten primitive subsets: CMP: common myeloid progenitors; DC: dendritic cells; Ep: erythroid progenitors; ETP: early T progenitors; GMP: granulocyte-monocyte progenitors; HSC: hematopoietic stem cells; iPMN: immature polymorphonucleated cells; MEP: megakaryo-erythroid progenitors; MKp: megakaryocyte progenitors; MLP: multi-lymphoid progenitors; MPP: multipotent progenitors; NKT: natural killer-T cells; NK: natural killer cells; PMN: mature polymorphonucleated cells. (C-E) Graphs show cell count/L of HSC (C), MPP (D), and MLP (E) in ARO patient PB, healthy donor PB and BM. Data show mean±standard deviation (A and B) or mean±standard error of mean (C-E). Statistical significance was determined by non-parametric Mann Whitney test (A) or Kruskal-Wallis test with Dunn’s multiple comparison post test (C-E).
Figure 2.
Figure 2.
RNA sequencing. (A) Principal component analysis of ARO, cord blood (CB) and mobilized peripheral blood (mPB) samples. (B) Heatmaps show unsupervised hierarchical clustering on differentially expressed genes (DEG) of ARO patients versus CB (left) or mPB (right). (C) Heatmap shows normalized enrichment score (NES) from gene set enrichment analysis (GSEA) in the indicated categories.
Figure 3.
Figure 3.
Transduction and expansion. (A) Schematic representation of the two vector constructs driving TCIRG1 expression: the PGK.TCIRG1 vector and PGK.TCIRG1/dNGFR bidirectional vector. LTR: long terminal repeat; PGK: phosphoglycerate kinase promoter; TCIRG1: T-cell immune regulator 1; dNGFR: deleted nerve growth factor receptor; CMV: cytomegalovirus promoter. (B) Time-line of transduction, expansion and in vivo and in vitro studies. (C) Absolute counts of hematopoietic progenitor colonies obtained in MethoCult cultures. BFU-E: burst-forming unit of erythroid cells; CFU-GM: colony-forming unit of granulocyte/ macrophage. Data show mean±standard deviation.
Figure 4.
Figure 4.
Hematopoietic stem and progenitor cells (HSPC) expansion. (A) Fold expansion, calculated as a ratio between total CD34+ cells at the start of the culture and at the end of expansion phase in untransduced (UT) or transduced (PGK.TCIRG1 and PGK.TCIRG1/dNGFR) patient cells. (B) Absolute counts of the CD34high CD90+ endothelial protein C receptor-positive (EPCR+) population, which is enriched for the long-term hematopoietic stem cells (HSC), at day 0 (freshly isolated CD34+ cells) and day 7 (end of expansion phase). Each color represents a different untransduced patient, cultured with 35 or 100 nM UM171, as indicated in figure. (C) Frequency of the CD90+ EPCR+ population on the CD34high cells at day 0 and at day 7. As in (A), each color represents a different untransduced patient, cultured with 35 or 100 nM UM171, as indicated in figure. Statistical significance was determined by non-parametric one-way ANOVA with Dunns post test (A) or non-parametric Mann Whitney test (B and C). *P<0.05, **P<0.01. Data show mean±standard deviation.
Figure 5.
Figure 5.
Osteoclast bone resorption. (A) Immunofluorescence staining of osteoclasts differentiated from CD34+ cells and cultured on bone slices. Images show the merge result of alendronate-AF-488 (resorption pits in green), phalloidin-TRITC (actin rings in red) and TO-PRO-3 (nuclei in blue). (Top) Representative images of osteoclasts obtained from mobilized peripheral blood (mPB) CD34+ cells from two healthy donors. (Bottom) Representative images of untransduced (UT) or transduced (PGK.TCIRG1) osteoclasts from seven ARO patients. Vector copy numbers (VCN) of the transduced cells are indicated for each patient. Images were acquired with Leica TCS SP5 laser scanning confocal, equipped with HC PL FLUOTAR 10X (NA 0.3) dry and Leica Application Suite Advanced Fluorescence (LASAF) software. (B) Toluidine blue staining of bone slices that were previously cultured with osteoclasts from healthy donor mPB CD34+ cells and ARO patient UT or transduced (PGK.TCIRG1) cells. VCN of the transduced cells are shown for each patient. Images were acquired with Nikon ECLIPSE E600 microscope equipped with Nikon DSRi2 camera, using Plan Fluor 4x/0.13 objective and NIS-Elements F 4.30.01 software. (C) C-terminal telopeptide fragment of type I collagen (CTX-I) quantification in the osteoclast culture supernatant at day 0, day 8, and day 21. Healthy donor mPB (mPB HD) CD34+ cells in white, ARO patient untransduced (ARO UT) cells in gray and ARO patient transduced (ARO PGK.TCIRG1) in black. Statistical significance was determined by two-way ANOVA with Bonferroni post-test (C). ****P<0.0001. All data show mean±standard.
Figure 6.
Figure 6.
Transplants in primary NOD scid gamma common chain (NSG) recipients. (A) Percentage of human hematopoietic (huCD45+) cells in the peripheral blood (PB) of primary NSG recipients over time. Each color represents a different patient, as indicated in figure. UM171 dosage used for CD34+ cell expansion is also indicated. Continuous line indicates untransduced (UT) cells, dotted line indicates cells transduced with PGK.TCIRG1 vector and dashed line indicates cells transduced with PGK.TCIRG1/dNGFR vector. (B) Percentage of human hematopoietic (huCD45+) cells on total CD45+ cells in spleen, bone marrow (BM) and thymus at sacrifice. (C) Percentage of CD19+ (B lymphocytes, in blue), CD3+ (T lymphocytes, in green), CD13+ (myeloid cells, in pink), and CD34+ (hematopoietic stem and progenitor cells, in yellow) subsets on the total human CD45+ cells. (D) Vector copy number (VCN) of in vitro transplanted cells and in tissues (spleen, BM and thymus). Data show mean (A) or mean±standard deviation (B and C).
Figure 7.
Figure 7.
Transplants in secondary NOD scid gamma common chain (NSG) recipients. (A) Percentage of human hematopoietic (huCD45+) cells in the peripheral blood (PB) of secondary NSG recipients over time. Each color represents a different patient, as indicated in figure. Continuous line indicates untransduced (UT) cells, dotted line indicates cells transduced with PGK.TCIRG1 vector and dashed line indicates cells transduced with PGK.TCIRG1/dNGFR vector. (B) Percentage of human hematopoietic (huCD45+) cells on total CD45+ cells in spleen, bone marrow (BM) and thymus at sacrifice. (C) Percentage of CD19+ (B lymphocytes, in blue), CD3+ (T lymphocytes, in green), CD13+ (myeloid cells, in pink), and CD34+ (hematopoietic stem and progenitor cells, in yellow) subsets on the total human CD45+ cells. (D) Vector copy number (VCN) of in vitro transplanted cells and in tissues (spleen, BM and thymus). Data show mean (D) or mean±standard deviation (B and C).
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Funding: This research was supported by a grant from the Telethon Foundation (TGT16C05) to AV and partially by a fellowship from the European Calcified Tissue Society (ECTS) to VC.