ENGINEERING A MINI-HERPESVIRUS AS A GENERAL STRATEGY TO TRANSDUCE UP TO 180 KB OF FUNCTIONAL SELF-REPLICATING HUMAN MINICHROMOSOMES

The engineering of therapeutic human artificial episomal chromosomes, HAECs, requires the development of strategies to deliver large functio nal self-replicating extrachromosomal DNA in target cells. Members of the herpesviral family are among the largest episomal double-stranded DNA viruses. As model systems of this family of endemic infectious age nts, vectors derived from the human herpes 4 Epstein-Bar virus (EBV) w ere constructed which transferred up to 180 kb of DNA packaged as infe ctious virions. Such a transduction strategy was based on a non-oncoge nic helper-dependent mini-EBV carrying minimal cis elements for latent replication and virus production. After exposure of human B lymphoma and lymphoblastoid cells to mini-EBVs transducing lacZ and human HPRT minigenes, stable cell transformants were selected which carried the d elivered multimeric linear DNAs as circular episomes up to 160-180 kb in size. Following transduction of Lesch-Nyhan disease cells with a mi ni-EBV/HPRT, normal human HPRT function was restored in cells carrying large episomal HPRT minigenes. Direct visualization of the therapeuti c mini-EBV by fluorescent in situ hybridization (FISH) on metaphase an d interphase nuclei indicated that 99% (556/563) of the transduced min i-EBV DNA was episomal with an average copy number of one to two per n ucleus. This system should allow the delivery of large genes in common diseases such as hemophilia A and codelivery of multiple genes in cel ls from polygenic diseases such as cancer. The extrachromosomal mini-E BV-based strategy offers an alternative to integrative or non-replicat ing gene therapy infectious vectors, which may be generally applicable to other herpesviruses characterized by different tropisms.