Targeted Gene Therapy Vectors for Acute Myeloid Leukemia Cells Selected from Random AAV-Displayed Peptide Libraries.

For acute myeloid leukemia, gene therapy may be a valuable tool to treat patients refractory to conventional chemotherapy regimens. However, there are no vectors available that sufficiently and specifically transduce this cell type. Cell type specific receptors can be used to target gene therapy vectors to the cells of interest (Trepel et al., Hum. Gene Ther., 2001). We have developed a screening system based on random peptide libraries displayed on adeno-associated virus type 2 (AAV) (Muller et al., Nat. Biotechnology, 2003). Such libraries consist of up to 108 different viruses that display a targeting peptide with random sequence in a capsid region that mediates binding of the virus particle to cell surface receptors. Targeted AAV gene therapy vectors that specifically transduce the cell types of interest can be selected from such a library. Here we report the selection of a random AAV display peptide library on a panel of acute myeloid leukemia cell lines (Kasumi-1, U937, HL 60). An X7 (X = any amino acid) oligonucleotide insert was cloned in to the AAV-library plasmid followed by the production of AAV library transfer shuttle AAV. To produce the final library, AAV producer cells were infected with shuttle AAV at an MOI of 1 to ensure encoding of displayed peptides by the packaged DNA. The target cells were then infected with the AAV library. Bound particles were internalized and amplified either by superfinfection of the cells with wild type adenovirus type 5 or by a high-sensitivity PCR-system of the random oligonucleotide followed by cloning of the insert and making a preselected library as above. Amplified clones were recovered and subjected to two more rounds of selection. Enriched peptide inserts were analyzed indirectly by DNA sequencing of recovered clones. After three rounds of selection, enrichment of a peptide motif within the selected AAV capsids was observed. The selected viruses were used for production of recombinant AAV vectors harboring a GFP reporter gene. Such recombinant targeted vectors transduced the target leukemia cells they have been selected on (e.g., Kasumi-1 cells) up to 60 times more efficient compared to a random clone of the initial, unselected library. We conclude that our novel targeting strategy can be applied to a great variety of cell types. The clones selected here may be used as valuable tools to target therapeutic genes to acute myeloid leukemia cells.