HSV Amplicon Vector Design

A major focus of the Breakefield laboratory is on design and development of HSV amplicon vectors for stable, non-toxic delivery of genes in vivo. These vectors have a transgene capacity of 150 kb allowing them to carry entire genes, including regulatory elements and splice junctions. They express no viral genes and elicit only a mild immune response. They have proven to be highly effective in gene delivery in the nervous system in models of lysosomal storage diseases, ataxia telangiectasia, neurofibromatosis and brain tumors.            

Vectors for gene delivery are derived from herpes simplex virus type 1 (the common cold sore virus).  These virions provide an efficient means to shuttle genes to the cell nucleus. The virions bind to and enter most cells through fusion of the virion envelope with the plasma membrane. Viron capsids and associated tegument proteins are then taken to the cell nucleus via microtubule mediated transport. At the nucleopores the capsid opens and viral DNA is threaded into the nucleus.

The virion has four compartments, all of which can be used as delivery vehicles. The envelope can be modified so that it targets infection to specific cell types (Grandi et al., 2004). Proteins in the tegument, such as VP16, can be fused with  reporter proteins, such as GFP (Bearer et al., 2000) or functional proteins to temporarily alter the physiology of infected cells. Capsid proteins can also be modified as fusion proteins and up to 150 kb of foreign DNA can be incorporated in the capsids for delivery.

In infected cells the viral DNA is replicated from origins of replication, ori, as a rolling circle with 150 kb lengths being placed in the capsids in a capsid-full state with cleavage at pac signals. Plasmid DNA bearing ori and pac signals are packaged as concatenates in virions in the presence of viral functions and can be prepared as helper virus free stocks (Saeki et al., 2003). 

Several modifications have been incorporated into amplicon plasmids to control the fate of the DNA when it enters the cell nucleus. These include incorporation of the latent origin of DNA replication and EBNA1 sequences from Epstein Barr virus that allow replication of amplicon DNA as an extrachromosomal element (Sena-Esteves et al., 2002; Hampl et al., 2003). Further, elements of retrovirus and AAV have been incorporated into amplicon vectors such that infected cells are able to generate retrovirus vectors and integrate genes into the host cell genome at specific sites (Oehmig et al., 2004).