AIDSWEEKLY Plus, Monday, 18 August 1997
Daniel J. DeNoon, Senior Editor
German researchers have created a retroviral vector able to deliver therapeutic genes to CD4(+) T cells.
Such intracellular immunization of CD4 cells could be performed in vivo (by direct delivery to patients) or ex vivo (by removing CD4 cells from a patient, treating and expanding them in cell culture, and returning them to the patient).
"This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS," wrote Barbara S. Schnierle of the Institute for Experimental Cancer Research, Freiburg, Germany, and colleagues.
Schnierle et al. reported their discovery in the Proceedings of the National Academy of Sciences ("Pseudotyping of Murine Leukemia Virus with the Envelope Glycoproteins of HIV Generates a Retroviral Vector with Specificity of Infection for CD4 Expressing Cells," PNAS, 1997;94:8640-5).
The MuLV vector is created by removing the MuLV genes needed for viral replication and replacing them with therapeutic genes to create retroviral vector particles. These particles are then used to infect a packaging cell line engineered to encode the RNA for viral proteins needed to assemble viral particles. The RNA for these assembly proteins do not include packaging signals and they therefore do not become part of the new particles.
"This results in the production of helper-free, replication-incompetent, recombinant retroviral vector particles able to transfer therapeutic genes encoded by suitable retroviral vectors," Schnierle et al. noted.
Most clinical trials of intracellular immunization employ as vectors modified versions (pseudotypes) of the Moloney murine leukemia virus (MoMuLV or MuLV). But until now, nobody had been able to create a MuLV pseudotype capable of bearing the HIV envelope glycoprotein on its surface.
At first, Schnierle's group failed as well. They attempted to create particles with the full HIV-1 gp120 envelope glycoprotein on the surface anchored, as in wild-type virus, by the full-length HIV-1 gp41 transmembrane glycoprotein. But HIV-1 gp41 is unusually large for a retroviral transmembrane protein, and it apparently interfered with the assembly of the MuLV particles.
The researchers then turned to a truncated form of gp41 which lacked portions of the protein's carboxyl-terminal cytoplasmic "tail." This short form of gp41 was the key to expressing MuLV pseudoparticles bearing an HIV-1 gp120/gp41 surface molecule. Experiments showed that these particles could infect cells expressing CD4; antibodies to CD4 prevented infection.
"It is conceivable that in vivo or ex vivo transduction of CD4(+) lymphocytes with retroviral vectors ... might help to reduce the virus load in the peripheral blood and/or lymph nodes of infected patients," Schnierle et al. concluded.
"MuLV (HIV-1) pseudotypes also will be useful as a safe and rapid assay system for the analysis of molecules that affect HIV-1 entry and permit the development of drugs that interfere with the HIV-1 infection process."
While this is the first HIV-1/MuLV vector, other research teams have created viral pseudotypes carrying HIV-1 proteins. These include rabies virus pseudotypes (Mebatsion and Conzelmann, PNAS, 1996;93:11366-70) and vesicular stomatitis virus pseudotypes (Owens and Rose, J Virol, 1993;67:360-5; also, in this issue please see the accompanying article on recombinant VSV encoding HIV antigens).
A number of therapeutic genes for intracellular immunization against HIV are under development. These include:
* Dominant negative mutants of HIV transcriptional regulatory genes, particularly rev and tat. These are known as transdominant (or trans-dominant) mutants (e.g., Malim et al., J Exp Med, 1992;176:1197-1201).
* Intracellular anti-HIV antibodies (e.g., Pomerantz et al, 1995 ACTG, see AIDS Weekly Plus, January 8, 1996).
* RNA decoy molecules, the intracellular overexpression of which uses up so much viral protease that little is left over for processing of infectious virions (e.g., Serio et al., PNAS, 1997;94:3346-51).
* Ribozymes, specialized RNA molecules with enzymatic activity. These molecules can be specifically targeted to HIV RNA sequences and can interfere with multiple stages of viral replication (e.g., Sarver et al., Science, 1990;247:1222-5).
* Antisense constructs. Antisense strategies attempt to flood cells with defective RNA or DNA sequences complementary to HIV RNA or DNA. These defective oligonucleotides scramble the genetic messages required for proper expression of the target genes (e.g., Chatterjee et al., Science, 1992;258:1485- 8).
* Thymidine kinase (TK) obliteration. This approach calls for using intracellular immunization to cause HIV infected hematopoietic cells to express the herpes simplex virus (HSV- 1) TK gene. High-level expression of the HSV-1 TK gene renders cells sensitive to the cytopathic effects of acyclovir, a nucleoside analog; the drug is harmless to normal human cells as they do not produce the TK needed to activate the drug (e.g., Caruso and Klatzmann, PNAS, 1992;89:182-6.
* Intracellular expression of anti-HIV cytokines such as interferon alpha-2 (e.g., Bednarik, D.P. et al. PNAS, 1989;86(13):4958-62.
The Schnierle et al. study was supported by Grants 01KV9501/9, 01Kl9406-Teilprojekt 1, and 01KV9550 of the Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie.
The corresponding author for this study is Klaus Cichutek, Abteilung Medizinische Biotechnologie, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, D-63225 Langen, Germany.
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