Foamy virus vectors are potent alternatives to lenti- and gamma-retroviral vectors for gene therapy. To construct and optimize gutless feline foamy virus (FFV) replication-deficient (RD) vectors, viral elements essential for optimal efficient marker gene transduction were characterized and fine-mapped and packaging clones constructed. For these purposes, new Gag and Pol expression clones which allow efficient expression of packaging proteins and vectors carrying deletions in coding and non-coding regions of the genome were constructed and functionally evaluated. These studies demonstrate that the 5' major splice donor (5' SD) is indispensable for RD vectors while defined mutations introduced to inactivate the gag start codon improve transgene delivery efficiency. Based on these findings, new gutless FFV vectors were generated yielding un-concentrated vector titers above 10(5) transducing units (TU)/ml. By minimizing the second cis-acting sequence in the pol gene, only 3.8 kb viral sequences are maintained in the novel gutless FFV RD vectors.