Engineering a mini-herpesvirus as a general strategy to transduce up to 180 kb of functional self-replicating human mini-chromosomes.
The engineering of therapeutic human artificial episomal chromosomes, HAECs, requires the development of strategies to deliver large functional 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 agents, vectors derived from the human herpes 4 Epstein-Barr virus (EBV) were constructed which transferred up to 180 kb of DNA packaged as infectious virions. Such a transduction strategy was based on a non-oncogenic 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 delivered multimeric linear DNAs as circular episomes up to 160-180 kb in size. Following transduction of Lesch-Nyhan disease cells with a mini-EBV/HPRT, normal human HPRT function was restored in cells carrying large episomal HPRT minigenes. Direct visualization of the therapeutic mini-EBV by fluorescent in situ hybridization (FISH) on metaphase and interphase nuclei indicated that 99% (556/563) of the transduced mini-EBV DNA was episomal with an average copy number of one to two per nucleus. This system should allow the delivery of large genes in common diseases such as hemophilia A and codelivery of multiple genes in cells from polygenic diseases such as cancer. The extrachromosomal mini-EBV-based strategy offers an alternative to integrative or non-replicating gene therapy infectious vectors, which may be generally applicable to other herpesviruses characterized by different tropisms.
Sun
T Q
TQ
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill 27599-7295, USA.
Livanos
E
E
Vos
J M
JM
D001402:B-Lymphocytes; D002875:Chromosomes / Q000235:genetics*; D004261:DNA Replication; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D015316:Genetic Therapy; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D007763:Lac Operon; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics
D012333:RNA, Messenger; D007041:Hypoxanthine Phosphoribosyltransferase