AIDSWEEKLY Plus, Monday, 15 September 1997
Daniel J. DeNoon, Senior Editor
A new vector greatly improves long-term intracellular immunization against HIV.
Intracellular immunization against HIV - treatment of infection by genetically engineering antimicrobial resistance into a person's cells - is already being tested in preliminary clinical trials. But a major problem with the technique has been suboptimal expression of the antiviral genes.
Now University of Michigan researchers Udaykumar Ranga, Gary J. Nabel, and colleagues report development of an improved gene-delivery vector that includes CD4 specific regulatory elements and viral regulatory regions.
"This vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone," Ranga et al. reported. "Expression of an inhibitory form of the [HIV-1] Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4(+) peripheral blood mononuclear cells [PBMC]."
Ranga et al. reported their findings in the Journal of Virology ("Cell and Viral Regulatory Elements Enhance the Expression and Function of a Human Immunodeficiency Virus Inhibitory Gene," J Virol, 1997;71(9):7020-9).
The authors previously created a vector (pBL(CD4)-CAT) combining a 339-base-pair CD4 enhancer element with the CD4 promoter sequence (Killeen, N. et al., EMBO J, 1993;12:1547- 53). Although this vector maintained long-term, lineage- specific gene expression in transfected cells, it could only weakly activate gene expression.
Randa et al. were able to improve this vector in two ways:
* Addition of the Tat activation response (TAR) of the HIV-1 long terminal repeat sequence (LTR) to the CD4 promoter made the vector responsive to activation by HIV-1 Tat protein. The researchers suggested that at low levels of Tat expression, this element might add to the anti-HIV effect by acting as a Tat decoy, binding to the protein and preventing it from acting on replicating virus.
* Addition of a cytomegalovirus (CMV) IE enhancer element upstream of the CD4 enhancer improved gene expression 100- fold. The CMV IE enhancer contains binding sites for several transcription-enhancing factors including NF-k(beta) and Ap1. These factors can be induced by mitogenic or antigenic activation; such activation is continuously present in HIV infection.
Inclusion of these elements resulted in the optimized vector pK7cI.
"Data indicate that pK7cI can also function in acute viral infection," Randa et al. wrote. "Specific features of HIV replication and cellular gene expression can be exploited to develop potentially more effective genetic interventions in AIDS. ... Combinations of antiviral genes and appropriate regulatory elements may optimize the effects of gene products which may inhibit HIV replication and contribute to gene therapy of AIDS."
Rev M10 is the product of a nonfunctional mutant HIV-1 rev gene with a dominant phenotype. When cells expressing Rev M10 are infected with HIV, the virus preferentially interacts with the mutant Rev and virus replication comes to a halt.
Clinical trials of this strategy are proceeding slowly. Early trials showed that the intervention was safe and did not increase HIV replication. Continuing trials focus on improving Rev 10 expression.
Future trials of intracellular immunization against HIV likely will combine several anti-HIV genes such as TAR and Rev response element (RRE) decoys, ribozymes, toxic gene products, intracellular antibodies, antisense genes, and transdominant mutants of other HIV structural and regulatory proteins.
This work was supported in part by NIH grant AI 36606.
The corresponding author for this study is Gary J. Nabel, Departments of Internal Medicine and Biological Chemistry. Howard Hughes Medical Institute, University of Michigan Medical Center, 1150 W. Medical Center Dr., 4520 MSRBI, Ann Arbor, Michigan 48109-0650. Phone: (313) 647-4798; fax: (313) 647-4730; e-mail: <gnabel@umich.edu>.
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