A SMALL AND EFFICIENT DIMERIZATION PACKAGING SIGNAL OF RAT VL30 RNA AND ITS USE IN MURINE LEUKEMIA VIRUS-VL30-DERIVED VECTORS FOR GENE-TRANSFER/

Retroviral genomes consist of two identical RNA molecules associated a t their 5' ends by the dimer linkage structure located in the packagin g element (Psi or E) necessary for RNA dimerization in vitro and packa ging in vivo. In murine leukemia virus (MLV)-derived vectors designed for gene transfer, the Psi+ sequence of 600 nucleotides directs the pa ckaging of recombinant RNAs into MLV virions produced by helper cells. By using in vitro RNA dimerization as a screening system, a sequence of rat VL30 RNA located next to the 5' end of the Harvey mouse sarcoma virus genome and as small as 67 nucleotides was found to form stable dimeric RNA. In addition, a purine-rich sequence located at the 5' end of this VL30 RNA seems to be critical for RNA dimerization. When this VL30 element was extended by 107 nucleotides at its 3' end and insert ed into an MLV-derived vector lacking MLV Psi+, it directed the effici ent encapsidation of recombinant RNAs into MLV virions. Because this V L30 packaging signal is smaller and more efficient in packaging recomb inant RNAs than the MLV Psi+ and does not contain gag or glyco-gag cod ing sequences, its use in MLV-derived vectors should render even more unlikely recombinations which could generate replication-competent vir uses. Therefore, utilization of the rat VL30 packaging sequence should improve the biological safety of MLV vectors for human gene transfer.