Immunotherapy for Infectious Diseases

Gene Therapy for HIV-1 Infection 241
Transcription from the HIV-1 LTR promoter is dependent on the Tat protein. Mutant
Tat proteins, which still bind to the nascent viral RNA but are unable to further trigger
RNA elongation of transcription, greatly reduce the production of HIV-1 RNAs and
consequently the production of progeny virus (7). In addition, mutant Gag proteins
lead to abnormal virus core assembly and have also been shown to inhibit HIV-1 replication
(8).
In a similar way, mutant Rev proteins interfere with regulated posttranscriptional
events and also greatly reduce the efficiency of virus replication in an infected cell. In
particular, one Rev mutant, termed RevM10, has been shown to efficiently block HIV-
1 replication and has become a standard to measure the inhibitory effect of other anti-
HIV-1 genetic antivirals. Clinical trials are now under way to test long-term expression
and therapeutic effects of RevM10 in AIDS patients (see also Clinical Trials below).
Toxic Genes
The production of progeny virus can also be reduced by endowing HIV-1 target cells
with toxic genes. Such genes were inserted downstream of the HIV-1 LTR promoter
and only become activated immediately upon HIV-1 infection, when the viral Tat protein
is expressed. The activation of the toxic gene leads to immediate cell death, and
therefore no new progeny virus particles can be produced. In vitro experiments have
shown that the production of HIV-1 virus particles was indeed reduced, if target cells
were endowed with genes coding for the herpes simplex virus (HSV) thymidine kinase
or a mutant form of the bacterial diphtheria toxin protein (9).
CD4 and HIV-1 Coreceptors as Decoys
Efforts have been made to block HIV-1 replication by modifying the main envelope
docking molecules CD4, CXCR-4, or CCR-5. For example, a chimeric CD4 coding
gene has been constructed, which contained an endoplasmatic reticulum (ER) retention
sequence, derived from the T-cell receptor (TCR) CD3-� chain. Thus, this mutant CD4
molecule remained inside the endoplasmatic reticulum (ER) and was able to prevent
HIV-1 envelope maturation by binding to the HIV-1 envelope in the ER and blocking
its transport to the cell surface. Consequently, formation of infectious particles could
not take place. In other experiments, soluble CD4 has been used to block the envelope
of free extracellular virus particles and to prevent binding to fresh target cells (5).
It has been reported that individuals who have a mutant form of CCR-5, a coreceptor
used by macrophage-tropic strains of HIV-1, are naturally protected against HIV-1
infection. Such mutant CCR-5 appears to be retained in the ER and therefore is not
available at the cell surface for HIV-1 entry. Thus, efforts are being made to phenotypically
knock out wild-type CCR-5 in HIV-1-infected individuals to make their
macrophages resistant to CCR-5-tropic HIV-1 infection. Macrophages have been transduced
with a genetically modified chemokine gene, which expresses a modified protein
targeted to the ER. In the ER, the modified chemokine binds to CCR-5, preventing
its transport to the cell surface.
Single-Chain Antibodies
Single-chain antibodies (scA, also termed single-chain variable fragments [scFv])
were originally developed for E. coli expression to bypass the costly production of
monoclonal antibodies in tissue culture or mice. They comprise only the variable