Chapter category: Gene Therapy
Safety Considerations in Vector Development
Lentiviral Vector Systems
for Gene Transfer
Edited by: Gary L. BuchschacherISBN: 0-306-47702-5
» Get more information about this book at landesbioscience.com «
Chapter authors:
John C. Kappes and Xiaoyun Wu
The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitations in animal models to evaluate lentiviral vectors for their potential to recombine and induce disease, the vector design itself should ensure against the emergence of RCR in vivo. Issues related to RCR generation and one approach to dealing with this problem are discussed in this chapter. To assess the risk of generating RCR, a highly sensitive biological assay was developed to specifically detect vector recombination in transduced cells. Analysis of lentiviral vector stocks has shown that recombination occurs during reverse transcription in primary target cells. Rejoining of viral protein-coding sequences of the packaging construct and cis-acting sequences of the vector was demonstrated to generate env -minus recombinants (LTR-gag -pol -LTR). Mobilization of recombinant lentiviral genomes was also demonstrated but was dependent on pseudotyping of the vector core with an exogenous envelope protein. 5' sequence analysis has demonstrated that recombinants consist of U3, R, U5, and the y packaging signal joined with an open gag coding region. Analysis of the 3' end has mapped the point of vector recombination to the poly(A) tract of the packaging construct's mRNA. The state-of-the-art third generation packaging construct and Sin vector also have been shown to generate env -minus proviral recombinants capable of mobilizing retroviral DNA when pseudotyped with an exogenous envelope protein. A new class of HIV-based vector (trans-vector) was recently developed that splits the gag -pol component of the packaging construct into two parts: one that expresses Gag/Gag-Pro and another that expresses Pol (RT and IN) fused with Vpr. Unlike other lentiviral vectors, the trans-vector has not been shown to form recombinants capable of DNA mobilization. These results indicate the trans-vector design prevents the generation of env -minus recombinant lentivirus containing a functional gag -pol structure (LTR-gag -pol -LTR), which is absolutely required for retroviral DNA mobilization and the emergence of RCR. Quality assurance based on monitoring for RCR may have limitations as a predictor of safety in vivo, especially in the long term. The demonstration of lentivirus infection via alternative entry mechanisms supports this notion. Therefore, the approach of monitoring trans-vector stocks for env-minus recombinant virus in vitro as a surrogate marker for the possible emergence of RCR in vivo should represent a significant advancement in vector safety quality assurance.
Additional chapters from this book:
Determinants for Lentiviral Infection of Non-Dividing Cells
Marie A. Vodicka
Lentiviruses share the common characteristic of infecting non-dividing target cells, distinguishing them from the oncogenic retroviruses which only productively infect dividing cells. The sear...
Ethical Considerations in the Use of Lentiviral Vectors for Genetic Transfer
Ina Roy
This chapter will outline the various concerns which have been raised in scientific, bioethics, and lay communities about the use of lentiviral vectors for purposes of gene therapy. Many of th...
Prospects for Gene Therapy Using HIV-Based Vectors
Jiing-Kuan Yee and John A. Zaia
Recombinant vectors derived from murine leukemia virus (MLV) have been widely used to introduce genes in human gene therapy clinical trials and have shown the potential for medical application...
Safety Considerations in Vector Development
John C. Kappes and Xiaoyun Wu
The inadvertent production of replication competent retrovirus (RCR) constitutes the principal safety concern for the use of lentiviral vectors in human clinical protocols. Because of limitati...
EIAV, CAEV and Other Lentivirus Vector Systems
John C. Olsen
Lentiviruses that infect non-primates make up a diverse collection of viruses. Although these viruses have some features in common with HIV and other primate viruses, differences in genome org...
FIV Vector Systems
Sybille L. Sauter and Mehdi Gasmi
Why is feline immunodeficiency virus (FIV) such an appealing candidate for gene therapy vector development? Phylogenetic analysis suggests FIV is only distantly related to the primate lentivir...
HIV-2 and SIV Vector Systems
James R. Gilbert and Flossie Wong-Staal
Lentiviral vectors have received much attention in recent years due to their ability to efficiently transduce non-dividing cells. Of the lentiviruses HIV-2 and SIV offer several unique benefit...
HIV-1 Vector Systems
Narasimhachar Srinivasakumar
Human immunodeficiency virus type 1 (HIV-1) based gene transfer systems are gaining in popularity due to their ability to transduce terminally differentiated and non-dividing cells. Oncoretrov...
HIV-1 Replication
Eric O. Freed
In general terms, the replication cycle of lentiviruses, including HIV-1, closely resembles that of other retroviruses.1 There are, however, a number of unique aspects of HIV replic...
Introduction to Retroviruses and Retroviral Vectors
Gary L. Buchschacher, Jr.
As various viral vector systems for gene transfer are developed, interest in using such systems in applied settings continues to grow. This Chapter is designed to provide background informatio...
