Bone marrow harvesting from paediatric patients undergoing haematopoietic stem cell gene therapy

Abstract

Collection of an adequate amount of autologous haematopoietic stem progenitor cells (HSPC) is required for ex vivo manipulation and successful engraftment for certain inherited disorders. Fifty-seven paediatric patients (age 0.5-11.4 years) underwent a bone marrow harvest for the purpose of HSPC gene therapy (GT), including adenosine deaminase-severe combined immunodeficiency (ADA-SCID), Wiskott-Aldrich syndrome (WAS) and metachromatic leukodystrophy (MLD) patients. Total nucleated cells and the percentage and absolute counts of CD34+ cells were calculated at defined steps of the procedure (harvest, CD34+ cell purification, transduction with the gene transfer vector and infusion of the medicinal product). A minimum CD34+ cell dose for infusion was 2 × 10⁶/kg, with an optimal target at 5-10 × 10⁶/kg. Median volume of bone marrow harvested was 34.2 ml/kg (range 14.2-56.6). The number of CD34+ cells collected correlated inversely with weight and age in all patients and particularly in the MLD children group. All patients reached the minimum target dose for infusion: median dose of CD34+ cells/kg infused was 10.3 × 10⁶/kg (3.7-25.9), with no difference among the three groups. Bone marrow harvest of volumes > 30 ml/kg in infants and children with ADA-SCID, WAS and MLD is well tolerated and allows obtaining an adequate dose of a medicinal product for HSPC-GT.

Fig. 1

TNC concentration (a) and TNC ×10⁸/kg (b) in ADA-SCID, WAS and MLD. Each box plot displays the distribution of data based on the five-number summary: minimum, first quartile, median, third quartile and maximum

Fig. 2

CD34+ cell values are expressed as percentages (a), concentration (b) and per kg of body weight (c) in 8 ADA-SCID, 5 WAS and 28 MLD patients. Data measured after collection at the GMP facility are shown

Fig. 3

Relationship between the weight (a) and age (b) of the donor and concentration of TNCs harvested in the three patients subgroups and in all the patients. Relationship between patients’ weight (c) and age (d) with concentration of CD34+ cells in the BM harvest

Fig. 4

Correlation between TNC concentration and volume (a) or volume/kg (b) and between CD34+ cell concentration and volume (c) or volume/kg (d) in the three patients subgroups and in all the patients

Fig. 5

Relationship between selected and infused CD34+ cells in the three patients subgroups and in all the patients

Fig. 6

Cell yield during the consecutive steps of the production process. We calculated cell yield between harvest and selection (a) and cell yield after the transduction step (b)

Fig. 7

Relationship between age of the donor and CD34+ cells (as CD34+ ×10⁶/kg) after purification (a) and after cell manipulation and transduction (b)