A new kind of recombinant herpes simplex virus type 1 (HSV 1) was constructed. This recombinant, named HSV1 LaL, contained an unique packaging signal (“a" sequence) flanked by two loxP sites in parallel orientat...A new kind of recombinant herpes simplex virus type 1 (HSV 1) was constructed. This recombinant, named HSV1 LaL, contained an unique packaging signal (“a" sequence) flanked by two loxP sites in parallel orientation, named LaL, while the original packaging signals of HSV 1 were deleted. Based on a set of cosmids containing the entire HSV 1 genome except the “a" sequence, the LaL was inserted into HSV 1 UL44 gene on one of the cosmids, cos56, generating cos56/LaL. By co transfecting cos56/LaL with the other cosmids, HSV1 LaL was generated in the cells by recombination. By introducing cos56/LaL or HSV1 LaL respectively into E.coli or BHK cells that expressed Cre recombinase, LaLs on both of them were excised by Cre, which was proved by PCR detection. To study the potential use as helper virus in packaging amplicon vector, HSV1 LaL was compared with a control virus HSV1 lacZ that contained a lacZ gene in the UL44 gene. The titer of amplicon virus generated from HSV1 LaL infected BHK/Cre cells was basically the same as that from HSV1 lacZ infected cells, however,the former contained about 10 fold less helper virus than the later, while HSV1 LaL showed the same replication rate as HSV1 lacZ on standard cells, like BHK 21.展开更多
A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion ...A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 amplicon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-liter rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.展开更多
文摘A new kind of recombinant herpes simplex virus type 1 (HSV 1) was constructed. This recombinant, named HSV1 LaL, contained an unique packaging signal (“a" sequence) flanked by two loxP sites in parallel orientation, named LaL, while the original packaging signals of HSV 1 were deleted. Based on a set of cosmids containing the entire HSV 1 genome except the “a" sequence, the LaL was inserted into HSV 1 UL44 gene on one of the cosmids, cos56, generating cos56/LaL. By co transfecting cos56/LaL with the other cosmids, HSV1 LaL was generated in the cells by recombination. By introducing cos56/LaL or HSV1 LaL respectively into E.coli or BHK cells that expressed Cre recombinase, LaLs on both of them were excised by Cre, which was proved by PCR detection. To study the potential use as helper virus in packaging amplicon vector, HSV1 LaL was compared with a control virus HSV1 lacZ that contained a lacZ gene in the UL44 gene. The titer of amplicon virus generated from HSV1 LaL infected BHK/Cre cells was basically the same as that from HSV1 lacZ infected cells, however,the former contained about 10 fold less helper virus than the later, while HSV1 LaL showed the same replication rate as HSV1 lacZ on standard cells, like BHK 21.
基金Project supported by the National High Technology Development Program National Climbing Project
文摘A novel packaging system for producing recombinant adeno-associated virus (rAAV) vector was described. Instead of the conventional method for rAAV production by two-plasmid co-transfection followed by superinfec-tion with adenovirus 5, an HSV-1 amplicon system expressing AAV-2 rep and cap genes from their native promoters was used to provide complete helper functions for rAAV replicating and packaging. This HSV-1 amplicon stock consisted of two kinds of infectious HSV-1 virions, a replicating-defective HSV-1 amplicon pseudovirus harboring multi-copies of AAV-2 rep and cap gene and a temperature-sensitive HSV-1 mutant strain ts-KOS. High-liter rAAV was generated with this new packaging system. This packaging system gives a simple and scaleable process for rAAV production.
