Abstract
Current chemotherapeutic regimens for acute myeloid leukemia (AML) have been modestly effective in patients and are associated with poor long-term survival (<30% at 5 years). Viral vector-based suicide gene therapy is an attractive option, if these vectors can target the AML cells with high specificity and efficiency. In this study, we have developed a receptor-specific adeno-associated virus (AAV) based vector to target the CD33 antigen which is overexpressed in leukemic cells. A targeting peptide was rationally designed from the antigen-binding regions of a CD33 monoclonal antibody. This peptide was further expressed on the capsid of the AAV6 vector, since this serotype was most efficient among AAV1-rh10 vectors to infect the pro-monocytic, human myeloid leukemia cells (U937). AAV6-CD33 vectors expressing a suicide gene, the inducible caspase 9 (iCasp9), and its prodrug AP20187 significantly reduced (∼59%) the viability of U937 cells. To further test its efficacy and specificity in vivo, AAV6-CD33 vectors were administered into a xenotransplantation model of AML in zebrafish through systemic delivery. We observed a significant antileukemic effect with AAV6-CD33 vectors, with a markedly higher survival (100% for AAV6-CD33 vectors vs 15% for mock-treated) and a higher number of TUNEL positive apoptotic cells after systemic vector delivery. Taken together, our work demonstrates the efficacy and translational potential of CD33-targeted AAV6 vectors for cytotoxic gene therapy in AML.
Highlights
Acute myeloid leukemia (AML) is a hematological malignancy characterized by the presence of undifferentiated myeloid blasts and is associated with poor prognosis in both adults and children.[1−4] The heterogeneity of AML with its different clinical manifestations, the genetic abnormalities as well as the performance status of the patient strongly influences the choice of therapy
Conventional chemotherapy fails to address the presence of the leukemia initiating compartment in the bulk AML population that is generally resistant to chemotherapy
In the first set of studies, we wished to determine the level of expression of CD33 surface antigen in leukemic cells in anticipation of using them further as suitable models to test the associated virus (AAV) vectors
Summary
Acute myeloid leukemia (AML) is a hematological malignancy characterized by the presence of undifferentiated myeloid blasts and is associated with poor prognosis in both adults and children.[1−4] The heterogeneity of AML with its different clinical manifestations, the genetic abnormalities as well as the performance status of the patient strongly influences the choice of therapy. 647 Imaging Assay, Thermo Fisher, Waltham, MA, USA) (Figure 6D and E) This test has a high sensitivity in detecting the DNA strand break in apoptotic cells and has been widely used as a biomarker to evaluate the efficacy of various therapeutic interventions.[59,60] Our analysis revealed a low baseline number (4.2 ± 2.3) of TUNEL positive cells in tumors harvested from mock-treated fish. An AAV2 vector containing a heptamer [NQVGSWS] identified from a random peptide library and harboring a suicide gene (HSV-TK) conferred selective killing to Kasumi-1 cells in vitro, but its in vivo efficacy and specificity is not known.[20] Taken together, our data suggests that the insertion of a CD33 targeting sequence augments the infectivity and selectivity of AAV6 vectors to AML cells in vitro and in vivo. The fish were subsequently administered with the pro-drug AP20187 and followed up for 10 days after vector administration
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