|[Frontiers in Bioscience 1, d241-247, September 1, 1996]|
HERPES SIMPLEX VIRUS: A TOOL FOR NEUROSCIENTISTS
Frank J. Jenkins1 and Sharon L. Turner2
1 Department of Pathology, School of Medicine; 1Department of
Infectious Diseases and Microbiology, School of Public Health;
Received 08/02/96; Accepted 08/08/96; On-line 09/01/96
The advances of modern molecular biology and in vivo gene therapy have challenged neuroscientists with the potential prospect of gene manipulation in postmitotic neurons. The ability to alter gene expression in these cells would open the door towards potential therapies for several disorders such as Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis. Gene therapy using viral-based vectors has received considerable attention and represents a major focus of ongoing research in many laboratories. Viral vectors using several different human viruses such as adenoviruses, retroviruses and herpes viruses are currently being developed. Gene therapy directed towards neuronal cells however, presents unique problems. These problems include the genetic manipulation of post-mitotic (i.e., non-dividing) cells, the ability to specifically infect neurons, long-term maintenance of the vector DNA and expression of the target gene within the neuronal cells. Herpesviruses, particularly herpes simplex virus type 1, have unique characteristics of infection, replication and pathogenesis which make them potentially ideal candidates for the development of viral vectors capable of altering endogenous gene expression or delivery of foreign genes both in vivo and in vitro. The reader is directed to several reviews on these subjects (38-41).
Herpes viruses have several advantages which lend to their ability to act as neuronal vectors. The HSV genome has been sequenced in its entirety and is rather extensively studied (9). As a result of many years of intense research, a general knowledge exists of which genes and DNA sequences may be deleted and at which sites foreign DNA may be inserted into the DNA genome (10). These studies also have defined the minimal requirements for viral replication and packaging (41). HSV-based vector strategies rely on the ability of HSV to infect neuronal cells and to establish a latent infection. Latency is defined as a state in which viral DNA is maintained within the cell nucleus in the absence of any viral replication. During latency, viral gene expression is largely absent with the exception of the latency-associated transcripts (LAT's) which may remain transcriptionally active (8).
The two main strategies for HSV-based vectors in use today are genetically-engineered viruses and plasmid derived "amplicon" vectors. The first strategy involves the construction of recombinant viruses containing deletions in one or more viral genes whose expression is essential for viral replication (for reviews, see 38-39). These viruses are incapable of producing a productive viral infection (i.e., they are replication incompetent) in normal cells and require a complementing cell line (a cell line that can supply the deleted protein(s) to the virus in trans) for replication. Foreign genes can be inserted into theses mutated viral genomes with the goal of producing a virus vector that will infect the target cell (i.e., neurons), and express the foreign gene without killing the cell (due to viral replication). The second strategy involves the use of plasmid derived vectors containing HSV-1 origins of DNA replication and DNA packaging signals which enable multiple copies of the vector genomes to be packaged into helper virus virions (for reviews see 40, 41). Helper viruses can be either recombinant viruses containing a deletion within an essential viral gene or viruses containing temperature-sensitive mutations that prevent replication at 37°C (normal body temperature). In the case of the former, the replication of the helper virus and packaging of the amplicon vector DNA must occur in a cell line capable of complementing the mutations in the helper virus. Plasmid-derived vectors (amplicons) are advantageous because the DNA constructs can be easily manipulated to test endogenous, foreign, antisense or promoter gene expression in the target cell. Although the efficiency of delivery of these multiple copy vectors is high, the primary disadvantage of this system is the fluctuating helper virus to amplicon ratios with passage, which may result in some infected cells not receiving the amplicon genome. Viral titers must be monitored to ensure high amplicon delivery and experimental reproducibility in the absence of wild-type recombinants (41).
Regardless of the vector system used, two primary goals must be achieved to enable long-term gene expression in neuronal cells. The first goal involves the construct of mutant vectors which themselves are noncytotoxic to cells. Several studies have noted active expression of a foreign gene by HSV vector constructs which subsequently became inactivated (41-43). Reasons for this are not completely apparent, but evidence suggests that the inactivation is a result of cytotoxic effects induced by vector systems.
The second goal involves designing stable, active promoters capable of expressing appropriate levels of the foreign protein. The specific promoter involved in individual therapies may change according to the type, status and activity of the neuronal cell of interest. Originally, strong promoter systems such as the human cytomegalovirus IE promoter, the SV40 enhancer, and the RSV LTR were used to drive gene expression. Although such promoter systems were capable of expression they were only active transiently (1 week) and did not result in long-term gene expression (38, 39). Neuronal specific promoters (such as the neurofilament and neuronal-specific enolase promoters) which are believed to be constitutively active in neurons, also produced only transient expression in several HSV vector constructs (38, 39).
During HSV latency the only viral transcripts consistently detected are the latency associated transcripts (LATs). The possibility that the LATs are constitutively expressed in latently infected neurons has made them strong candidates for long-term gene expression in neuronal systems. This hypothesis along with the goal of understanding possible functions and implications of the latent transcripts, has led to a vast literature focused on understanding LAT transcription. The identification of transcriptional activators and suppression mechanisms which may determine functionality in any promoter system is a difficult task considering the modulation which occurs in specific cell types and culture systems. Recently, it has been shown that plasmid derived vectors utilizing HSV-1 promoters are resistant to short-term inactivation and capable of long-term gene expression (44). One possible explanation could be the high copy number of amplicon molecules delivered to individual cells (45). Another explanation stems from data suggesting low level IE gene activity during latency (46). Regardless of the process of sustained activity IE promoters may serve as useful promoter systems in experimental gene transfer vectors.