beta-thalassemias are the most common single gene disorders and are potentially amenable to gene therapy. However, retroviral vectors carrying the human beta-globin cassette have been notoriously unstable. Recently, considerable progress has been made using lentiviral vectors, which stably transmit the beta-globin expression cassette. Thus far, mouse studies have shown correction of the beta-thalassemia intermedia phenotype and a partial, variable correction of beta-thalassemia major phenotype. We tested a lentiviral vector carrying the human beta-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human thalassemia major, where it would be ultimately relevant. We demonstrated that the vector expressed normal amounts of human beta-globin in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes beta-thalassemia. The gene-corrected human beta-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human beta-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Variability of beta-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal controls. Our results show genetic modification of primitive progenitor cells with correction of the human thalassemia major phenotype.
Only 10% of metastatic melanoma patients survive 5 years, even though many can achieve substantial tumor reduction by surgical resection and/or radiation therapy and/or systemic therapy. An effective, nontoxic, consolidation immunotherapy could benefit such patients. We initiated a randomized trial to compare 2 promising patient-specific immunotherapy cell products. Patients had to have a diagnosis of metastatic melanoma and availability of an autologous melanoma cell line. Patients were stratified by whether their most advanced stage had been regional or distant metastases, and by whether they had measurable disease at the time of treatment, then they were randomized to receive irradiated autologous proliferating tumor cells or autologous dendritic cells (DC) loaded with antigens from such cells. Both products were injected subcutaneously in 500 µg of granulocyte-macrophage colony stimulating factor, weekly for 3 weeks and then monthly for 5 months. Patients in the 2 arms did not differ in baseline characteristics. All patients received prescribed therapy. Treatment was well tolerated. At the time of initial analysis, with no patients lost to follow-up, 50% of patients deceased, and all surviving patients followed for at least 6 months after randomization, survival is superior in the DC arm (hazard ratio, 0.27; 95% confidence interval, 0.098-0.729) with median survival not reached versus 15.9 months, and 2-year survival rates of 72% versus 31% (P=0.007). This trial provides evidence that a DC vaccine is associated with longer survival compared with a tumor cell vaccine, and is consistent with previous data suggesting a survival benefit from this patient-specific immunotherapy.
Personalized vaccine, recognized after the failure of allogenic melanoma whole cell and lysate vaccine phase III trials, involves culturing cells from a patient's own tumor within a short duration and with less passages but with optimized expression of tumor-associated antigens (TAAs). Its feasibility is established by comparing pure cell lines generated from fresh and cryopreserved tissues (n=164) of patients with lymph node (LN) and distant metastases. Stable cell lines (from 67% of specimens) are subcultured after cryopreserving them. Pure cell lines established after eliminating fibroblasts (from 96% of the cell lines) include those from LN (69%), soft tissues including cutaneous (60%), liver (64%), lung (75%), bone (80%), brain (75%), and other sites (73%). Within 3.5 months, stable cell lines (> or =50 million cells) are established from initiating the cell culture. For LN metastases, the duration differs significantly (P2<0.05) between fresh (1.4-3.4 months) and cryopreserved (2.4-4.7 months) tissues. The expression of TAAs varies as follows: Tyrosinase (81%) >Melan-A (80%) >HMB45/gp-100 (75%) >Mel-5/TRP-1 (65%) >MAGE-1 (47%) > S-100 (28%). The number of TAAs per cell line differs between early (<7) and late (>7) passages. Among late passage cell lines, lesser percentage of cell lines express three to six antigens pointing out that early passage (<7) cell lines may be needed for antigen-targeted immunotherapy. This study provides a protocol for establishing cell lines within 2-5 months for personalized vaccine therapy for nodal and organ metastatic melanoma patients.
Significant qualitative differences were noted in serum cytokine, chemokine, and growth factor levels of metastatic melanoma patients versus the normal controls at baseline. The results also demonstrated a significant decrease in the level of angiogenin (P = 0.026) and a significant increase in TARC/CCLl7 (P = 0.008) from week 0 to week 4 which was associated with improved overall survival (P = 0.059). Higher TARC/CCL17 levels were observed by ELISA at week 4 and a log-rank comparison revealed a significant association between high serum TARC/CCL17 levels at week 4 and progression-free survival (P = 0.005). Receiver-operator characteristic analysis revealed that week 4 serum TARC/CCL17 levels were predictive of progression-free and overall survival, indicating that serum TARC/CCL17 might be of predictive value of response to dendritic cell-based anti-melanoma immunotherapy.
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