Correction of phenotype in a thalassemia mouse model using a nonmyeloablative marrow transplantation regimen

Abstract

Gene therapy, the replacement of normal human beta- or gamma-globin genes into the hematopoietic stem cells of patients with homozygous beta-thalassemia, is a promising therapy for the future. High-level lineage-specific stable globin expression in transduced cells reinfused into patients in an autologous transplantation setting could be curative, if successful. Previous studies have shown high-level donor chimerism in nonmyeloablated non-thalassemic hosts. We have now studied the conditions for stable long-term engraftment of normal cells into a thalassemia mouse model that lead to high-level donor chimerism and correction of the abnormal phenotype. Thalassemic female mice treated with 0 to 300 cGy whole-body irradiation received transplantations of donor cells harvested from wild-type males. Engraftment of male cells was quantitated by Y-chromosome polymerase chain reaction analysis of blood and marrow progenitors, and changes in hemoglobin levels, red cell morphology, and spleen size were measured at various times posttransplantation. High-level stable donor cell engraftment was achieved in mice given 200 cGy and receiving transplants of 2 x 10(7) or more donor cells. The anemia, abnormal peripheral blood smears, and splenomegaly improved in the thalassemic mice that had successful engraftment. These studies demonstrate that stable and successful levels of engraftment of normal cells can correct the thalassemic phenotype without fully myeloablating the host. This animal model should allow us to test the amount of cytoreduction required and the level of engraftment and beta-globin expression needed in autologous transplantation of beta-globin gene-transduced cells to correct the abnormal phenotype in thalassemic mice, and it may be relevant to human clinical trials, as well.

Pretransplant Immunosuppression followed by Reduced-Toxicity Conditioning and Stem Cell Transplantation in High-Risk Thalassemia: A Safe Approach to Disease Control

“…In conclusion, a key success factor in our safe and efficacious program is the sequential use of immunosuppressive pharmacotherapy followed by a reduced-toxicity conditioning program rather than striving for maximum intensity of the conditioning program itself, paired with a relatively high number of hematopoietic progenitor cells (on average >5 × 10 6 CD34 + cells/ kg of recipient weight [5,20]), which resulted in a low incidence of toxicity and durable engraftment in this group of patients with high-risk class 3 thalassemia.…”

Murine marrow cellularity and the concept of stem cell competition: geographic and quantitative determinants in stem cell biology

“…Donor chimerism was calculated as CD45.1/(CD45.1 þ CD45.2) as nucleated red blood cells do not label with CD45.1 or CD45. 2 For lineage analysis, we labeled bone marrow with rat-antimouse Ly6G/GR-1 (8C5 hybridoma), CD45R/B-220 (RA3 hybridoma) or CD3 (T cells) (PharMingen) mAbs. Donor specificity was detected using a mouse-anti-mouse biotin-CD 45.1 mAb (PharMingen).…”

“…In previous studies we, as well as others, have shown high levels of engraftment into nontreated hosts and into minimally treated hosts. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17] The key to high levels of engraftment in these models has generally been infusion of high numbers of donor cells to compete with resident or residual host marrow cells. Data from these studies have suggested that marrow space might not need to be 'opened' by cytotoxic host treatment for stem cell engraftment to occur.…”

Reduced intensity allogeneic umbilical cord blood transplantation in children and adolescent recipients with malignant and non-malignant diseases

“…18,19 Several investigators have shown the potential of non-myeloablative or reduced intensity (RI) conditioning followed by AlloSCT to reduce regimen-related morbidity, regimen-related mortality (RRM) and potentially future late effects in patients with both malignant and nonmalignant disorders. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] A recent review by our group has summarized the results to date following RI conditioning for allogeneic stem cell transplantation (RI-AlloSCT). 35 Recently, successful engraftment has been demonstrated after reduced intensity UCBT (RI-UCBT) in adult recipients.…”