TY - JOUR
T1 - The history of the HSV amplicon
T2 - From naturally occurring defective genomes to engineered amplicon vectors
AU - Frenkel, Niza
PY - 2006/6
Y1 - 2006/6
N2 - We have derived the HSV amplicon vector in 1981/1982 after elaborate experience with "defective viruses", arising spontaneously in viral stocks propagated at high multiplicities of infection (m.o.i.). The defective viruses were found to contain large concatemeric genomes with repeat units of limited complexity. We employed cloned defective genome repeats to generate the "amplicon" vectors, which in the presence of helper virus replicate to produce packaged large concatemeric genomes, transmissible to uninfected cells. The cloned amplicons were then employed to fine map and analyze the signals essential for amplicon propagation: (i) A DNA replication origin, producing concatemeric genomes by rolling circle replication. Three DNA replication origins were identified in the HSV genome. (ii) Signals termed pac-1 and pac-2, directing a measuring function for coordinate cleavage of the concatemeric genomes and their packaging as fullsize (150 kb) genomes. Using amplicons, foreign genes of large sizes could be linked to less than 1 kb of the cis-acting HSV DNA sequences and become amplified in packaged defective genomes, transmissible to new cells. The transgenes are expressed efficiently, due to sequence reiterations. Large quantities of vectors can be produced in vitro. The amplicons are attractive vectors for use as non-integrating gene delivery vectors. The packaging signals pac-1 and pac-2 are well conserved in different herpesviruses and amplicons with a DNA replication origin and cleavage and packaging signals have been produced in additional herpesviruses. Depending on amplicon-host cell combination, the vectors can be employed with and without mutated helper virus(es) to obtain high gene expression, and desired effect on the target cell. In the absence of helper virus, the defective virus produced is limited for spread in the targeted cells. We expect that new vectors employing state of the art transgenes, will be developed to generate amplicon based concatemeric defective viruses capable of efficient expression of these genes.
AB - We have derived the HSV amplicon vector in 1981/1982 after elaborate experience with "defective viruses", arising spontaneously in viral stocks propagated at high multiplicities of infection (m.o.i.). The defective viruses were found to contain large concatemeric genomes with repeat units of limited complexity. We employed cloned defective genome repeats to generate the "amplicon" vectors, which in the presence of helper virus replicate to produce packaged large concatemeric genomes, transmissible to uninfected cells. The cloned amplicons were then employed to fine map and analyze the signals essential for amplicon propagation: (i) A DNA replication origin, producing concatemeric genomes by rolling circle replication. Three DNA replication origins were identified in the HSV genome. (ii) Signals termed pac-1 and pac-2, directing a measuring function for coordinate cleavage of the concatemeric genomes and their packaging as fullsize (150 kb) genomes. Using amplicons, foreign genes of large sizes could be linked to less than 1 kb of the cis-acting HSV DNA sequences and become amplified in packaged defective genomes, transmissible to new cells. The transgenes are expressed efficiently, due to sequence reiterations. Large quantities of vectors can be produced in vitro. The amplicons are attractive vectors for use as non-integrating gene delivery vectors. The packaging signals pac-1 and pac-2 are well conserved in different herpesviruses and amplicons with a DNA replication origin and cleavage and packaging signals have been produced in additional herpesviruses. Depending on amplicon-host cell combination, the vectors can be employed with and without mutated helper virus(es) to obtain high gene expression, and desired effect on the target cell. In the absence of helper virus, the defective virus produced is limited for spread in the targeted cells. We expect that new vectors employing state of the art transgenes, will be developed to generate amplicon based concatemeric defective viruses capable of efficient expression of these genes.
KW - Amplicon
KW - Cleavage packaging
KW - Concatemer
KW - Foreign gene
KW - Helper virus
KW - Origin
KW - Pac-1
KW - Pac-2
KW - Rolling circle
KW - Transgene
KW - oriL
KW - oriS
UR - http://www.scopus.com/inward/record.url?scp=33745215409&partnerID=8YFLogxK
U2 - 10.2174/156652306777591992
DO - 10.2174/156652306777591992
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AN - SCOPUS:33745215409
VL - 6
SP - 277
EP - 301
JO - Current Gene Therapy
JF - Current Gene Therapy
SN - 1566-5232
IS - 3
ER -