Suicide Gene Therapy with a CD33 Targeted AAV6 Vector Expressing an Inducible Caspase-9 Suicide Gene Is Therapeutic in a Xenotransplantation Model of Acute Myeloid Leukemia

Suicide gene therapy for Acute myeloid leukemia (AML) can offer a high therapeutic index if the pro-apoptotic genes can be selectively introduced into cancer cells with high efficiency with no apparent vector-related toxicities in the recipient. Since hematopoietic cells or myeloid cells are particularly intransigent to transduction with gene delivery vectors such as Adeno-associated virus (AAV), we reasoned that a cell-receptor specific AAV that can target the CD33 antigen, over-expressed in leukemic cells may be beneficial.For designing a targeting peptide, we used bioinformatic tools to identify antibody binding region (ABR) from a monoclonal antibody (M195) available against CD33. After further validation, a consensus antigen-binding region comprising a heptamer sequence was selected and inserted into the loop IV region of the best infecting AAV serotype (AAV1-rh10) in leukemic cells, the AAV6 by standard cloning techniques. The AAV6-CD33 vectors thus developed was packaged with a inducible caspase 9 based suicide gene (iCasp9) and further verified for its capsid integrity and functionality. In the next set of studies, we wished to determine if the AAV6-CD33 vectors can enhance suicide gene transfer in AML cells. We evaluated their cytotoxic effect by a luminescence based ATP assay, in a panel of cell lines which were of hematopoietic origin, that either over-express the CD33 antigen (U937) or that do not express CD33 antigen (CEM) or a control hepatic cell line, Huh7. Interestingly, the targeted AAV6 vectors were highly cytotoxic (1.6 fold, 41 vs 67 % viability, p< 0.0004) when compared to AAV6- wild type (WT) vectors in the CD33 positive U937 cells. However, in cells that do not express CD33 antigen, such as CEM or Huh7, AAV6-CD33 vectors showed no advantage over WT-AAV6 vectors in terms of their cytotoxicity. These data demonstrated the specificity of CD33 receptor targeted AAV6 vectors in AML cells in vitro.To further validate the novel AAV6 vector, we developed a Zebrafish xenograft model of AML. Briefly, ~1×10⁵ fluorescently labelled U937 cells per recipient were transplanted into busulfan (20mg/kg bodyweight) conditioned Zebrafish. Four days after engraftment of leukemic cells, fish were randomised to: mock-treated, AAV6-WT iCasp9 vector administered either systemically (retro-orbital,RO) or locally (intra-tumoral,IT) and the receptor targeted AAV vector (AAV6-CD33) administered systemically through the retro-orbital plexus. Ten days later, the rate of tumor growth in vector treated fish was significantly lower in vector treated fishes when compared to mock-treated Zebrafish. The survival was significantly higher in the treatment group that received AAV6-iCasp9 vectors as compared to mock-treated control fishes (~80% vs 15%, p<0.0001). Moreover, Zebrafish in the AAV6-CD33 treatment arm showed better survival as compared to untreated fishes (~100% vs 15%, p<0.0001) or to those treated with wild type AAV6 vectors administered either by RO or IT route (100% vs 77% or 80%). A morphological characterisation and a TUNEL based apoptosis assay further revealed that fishes treated with CD33 targeted AAV6-iCasp9 vectors by systemic administration, had a significantly higher (~39 fold) increase in TUNEL positive cells in comparison to the control group. Interestingly, the receptor targeted AAV6 vectors augmented the in vivocytotoxicity of U937 tumors by at least 3-fold (p<0.0001) when administered retro-orbitally in comparison to AAV6-WT vector administered group. Taken together, our work demonstrates the efficacy and translational potential of CD33-targeted AAV6 vectors for cytotoxic gene therapy in AML.