01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

etroviruses containing the tat22/37 gene and an RRE
decoy in different positions or the tat22/37 and the
RevM10 transdominant negative mutant genes to produce
monoclonal and polyclonal cultures expressing the
integrated genes; none of these recombinant constructs
inhibited virus replication at a high multiplicity of
infection (MOI) and combination of tat and rev mutants
was ineffective in inhibiting HIV-1 replication at both low
and high MOIs; however, at a low MOI, two cell clones
containing tat22/37 and the RRE decoy in 3' position
showed a long lasting protection against virus replication
and in two cell clones, expressing the RevM10 mutant
alone, the HIV-1 replication was efficiently blocked
(Caputo et al, 1997).
IL-16 is secreted by activated CD8 + T lymphocytes
and acts on CD4 + T lymphocytes, monocytes and
eosinophils. Recently, the C-terminal 130-amino acid
portion of IL-16 was shown to suppress HIV-1 replication
in vitro. HIV replication was inhibited by as much as 99%
in HIV-1-susceptible CD4 + Jurkat cells following
transfection and expression of the C-terminal 130-amino
acid portion of IL-16; the mechanism of HIV-1 inhibition
by IL-16 was not at the level of viral entry or reverse
transcription, but at the expression of mRNA (Zhou et al,
1997).
The in vitro antiviral efficacy of two gene therapy
strategies (trans-dominant RevM10 and Gag antisense
RNA) were tested in combination with the clinically
relevant reverse transcriptase inhibitors AZT and ddC or
the protease inhibitor indinavir by Junker et al (1997). The
combination of RevM10 or Gag antisense RNA with
antiviral drugs inhibited HIV-1 replication 10-fold more
effectively than the single antiviral drug regimen alone in
retrovirally transduced human T cell lines after inoculation
with high doses of HIV-1HXB3 in the presence or absence
of inhibitors. The level of anti-HIV-1 activity of the psigag
antisense sequence correlated with the length of the
antisense transcript and maximal anti-HIV efficacy was
observed with complementary sequence more than 1,000
nucleotides long, whereas transcripts less than 400
nucleotides long failed to inhibit HIV-1 replication in a Tcell
line and in primary peripheral blood lymphocytes
(Veres et al, 1996).
The HSV-1 and HSV-2 virion host shutoff gene (vhs),
each of which encodes a protein that accelerates the
degradation of mRNA molecules leading to inhibition of
protein synthesis, was used as a suicide gene for HIV gene
therapy to inhibit replication of HIV; an infectious HIV
proviral clone was cotransfected into HeLa cells together
with the vhs gene under control of the CMV IE promoter;
HSV-1 vhs gene driven by the HIV LTR inhibited HIV
replication more than 44,000-fold in comparison to a
mutant vhs gene (Hamouda et al, 1997).
The specificity of the Vpr protein for the HIV-1 virus
particle was exploited to develop an anti-HIV strategy
Boulikas: An overview on gene therapy
96
targeting the events associated with virus maturation; nine
cleavage sites of the Gag and Gag-Pol precursors were
added to the C terminus of Vpr and the chimeric Vpr
genes were introduced into HIV-1 proviral DNA to assess
their effect on virus infectivity; the chimeric Vpr
containing the cleavage sequences from the junction of
p24 and p2 completely abolished virus infectivity (Serio et
al, 1997).
D. Gene therapeutic strategies for AIDS
A gene therapy strategy to combat acquired
immunodeficiency syndrome (AIDS) in individuals
already infected with HIV-1 has been directed toward
GM-CSF mobilized peripheral blood CD34 + cells isolated
from HIV-1-infected individuals and transduced with
retroviral vectors containing three different anti-HIV-1genes:
(i) the Rev binding domain of the RRE (RRE
decoy) carrying also the Neo R gene, (ii) a double
hammerhead ribozyme vector targeted to cleave the tat
and rev transcripts (L-TR/TAT-neo), and (iii) the RevM10
transdominant negative mutant gene. After selection with
G418, transduced cultures displayed up to 1,000-fold
inhibition of HIV-1 replication following challenge with
HIV-1 (Bauer et al, 1997).
A safe strategy to gene therapy of AIDS aimed at
reducing the virus load in HIV-1-infected individuals was
developed by Nakaya et al (1997). The Rev protein shifts
RNA synthesis to viral transcripts by binding to the RRE
within the env gene. Anti-Rev chimeric RNA-DNA
oligonucleotides, consisting of 29 or 31 nucleotides, were
designed to inhibit the Rev regulatory function and as
decoys on HIV-1 replication; anti-Rev oligonucleotides
containing an RNA "bubble" structure of 13
oligonucleotides (that bound to Rev with high affinity)
were found to reduce more than 90% of the HIV-1
production from infected human T-cell lines and from
healthy donor-derived peripheral blood mononuclear cells;
control oligonucleotides without the bubble structure, that
bound to Rev with considerably less affinity, did not
reduce HIV-1 production (Nakaya et al, 1997).
E. Gene therapy of HIV with ribozymes
Antisense, ribozyme, or RNA aptamers, must be
efficiently transcribed, stabilized against rapid
degradation, folded correctly, and directed to the part of
the cell where they can be most effective. Among (i)
antisense RNA, (ii) hairpin and hammerhead ribozymes,
and (iii) RNA ligands (aptamers) for Tat and Rev RNA
binding proteins, Rev-binding RNAs but not the others,
efficiently blocked HIV-1 gene expression when tested in
expression cassettes based on the human tRNA(met) and
U6 snRNA promoters. In situ localization of both tRNA
and U6 promoter transcripts revealed primarily punctate
nuclear patterns (Good et al, 1997).