Construction of a retroviral vector production system with the minimum possibility of a homologous recombination

Abstract

A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia viurs (MLV)-based packaging constructs contained significantly long 5 0 and/or 3 0 untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed. Gene Therapy (2003) 10, 706-711. doi:10.1038/sj.gt.3301892Keywords: retroviral vector; packaging line; homologous recombination; RCR Murine leukemia virus (MLV)-based vectors are the most frequently used gene delivery system, employed in almost 40% of approved protocols and 50% of patients who have been subjected to or are undergoing clinical trials. However, there are many problems that need to be improved in order for MLV-based vectors to become a viable form of gene delivery vehicle in actual clinical settings. In particular, the issue of safety has often been raised because of the possibility of the production of replication-competent retrovirus (RCR). The mechanism of RCR generation still remains poorly understood, but it has been thought to result from homologous recombination between the same nucleotide sequences present in the vector and in the packaging constructs. Indeed, it has been reported that homologous recombination could occur between as very short regions of homology as 7 or 11 bp. 1-3We have recently constructed a series of retroviral vectors that are absent of any viral coding sequences without compromising viral titer or the level of gene expression. 4 These vectors are thought to be safer than other existing vectors harboring viral coding sequences because the probability of homologous recombination between the vector and the packaging genome is much lower with these newly d...

A retroviral vector suitable for ultrasound image-guided gene delivery to mouse brain

“…5,21 Supernatants were collected 48 h after transfection, filtered through a 0.45-mm filter and frozen at À80 1C until used. Concentrated viral stocks were prepared by ultracentrifugation for 90 min in a SW32 rotor (Beckman-Coulter, Fullerton, CA, USA) at 25 000 r.p.m.…”

Kinetics and characteristics of replication-competent revertants derived from self-inactivating foamy virus vectors

“…Since the recombination events leading to RCRs obviously depended on local sequence homologies, it may be possible to suppress recombination events partially as previously described by reducing homologous regions. 11 However, recombination also took place at sites with almost no homology, and FV-derived vectors exclusively carrying a SIN inactivation are expected to be intrinsically not safe enough for the transduction of long-lived cells, for instance stem cells. Therefore, FV SIN vectors also deleted in one or more structural genes, a strategy already used for FV-based vectors, 20,22 should display a degree of biological safety required for application in men and animals; experiments to construct and analyse the corresponding vectors are presently underway.…”

“…[4][5][6] For retroviruses, partial or complete deletion of the U3 promoter of the 3 0 long terminal repeat (LTR) is one way to directly abrogate infectivity resulting in self-inactivating (SIN) retroviral vectors; 7 however, recombination events may lead to replication-competent revertants (RCRs). [8][9][10][11] Such RCRs can be expected to pose, besides the intrinsic insertional mutagenesis potential of retroviral vectors, added risk of altering the expression of cellular genes. Vectors based on apathogenic viruses already possess a substantial degree of biological safety, but under rare conditions genetic manipulation and insertion of therapeutically relevant genes into apparently innocuous viruses can still result in disease potential.…”

Engineering the splice acceptor for improved gene expression and viral titer in an MLV-based retroviral vector

“…Retroviral vectors were transfected to Phoenix Ampho and the FlyA13 packaging line, respectively, using FuGene6 (Roche, Germany) according to the manufacturer's instructions. The levels of CAT activity of transfected cells were determined 2 days after transfection by a standard procedure as previously described by Yu et al 1 To determine the level of gene expression in the PG13 packaging line, 293T cells were transfected with retroviral vectors, together with amphotropic packaging constructs, pVM-GP and pVM-AE, 25 and the cell-free viral supernatants from the transfected cells were used to transduce the PG13 cells at a low MOI of around 0.1. After drug-resistant populations were obtained, the levels of CAT activity of the respective producer lines were determined.…”

“…25 Cell-free viral supernatants were used to transduce PG13 cells at an MOI of o0.1, followed by G418 selection. Drugresistant populations were obtained and the levels of CAT activity were determined.…”