RNAi as Antiviral Therapy: The HIV-1 Case - RNA Interference from Biology to Therapeutics - page 226

cofactors identified in these screens was remarkably small, the overlap of the cellular
pathways involved seems more significant. Interfering with these pathways could,
thus, be of therapeutic use.
Important candidate cofactors may have been missed because several well-
known cofactors were not found in any of the screens. First, cofactors of which the
function is redundant with that of other cellular proteins will not easily be detected.
Second, cofactors that require a nearly complete expression knockdown before an
impact on HIV-1 replication is scored will also be missed. Third, some important
cofactors may simply have been missed because the siRNAs were excluded because
of toxic off-target effects.
The attack on cellular cofactors may have a more general advantage for the attack
on other human pathogenic viruses that may use the same cellular pathways and/or
cellular cofactors. Similar genome-wide screens have been performed for other
human viruses, including influenza virus A, HCV, dengue virus, and West Nile virus
[ 149- 152 ]. When comparing all screens performed for the different viruses, pro-
teins involved in cytoskeleton complexes invariably present themselves as candi-
date cofactors with a broad antiviral impact [ 150 ] . Ten genes that were identi fi ed in
at least one of the HIV-1 screens were also picked up in the HCV screen by Li et al.
[ 151 ] and could, thus, be of double therapeutic value, as an estimated 25-30% of
HIV-infected individuals are coinfected with HCV [ 153 ] .
The use of RNAi against cellular cofactors is not restricted to the gene therapy
setting. A recent publication described the use of an miRNA targeting the human
gene PERK to enhance the immunogenicity of an HIV-1 Env DNA vaccine [ 154 ] .
Normally, PERK has an antiviral function by preventing, in an indirect manner, high
expression levels of viral proteins such as the HIV-1 envelope. Coexpression of
HIV-1 envelope and an engineered miRNA targeting PERK resulted in increased
envelope expression, and a higher Env-specific CD8 + T cell response was measured
in vaccinated mice.

11.4 Appropriate Preclinical Test Systems

Programming of the cellular miRNA pathway with new siRNA specificity is associ-
ated with certain risks. A general problem is that the artificial siRNA molecules can
compete with the endogenous siRNAs, and siRNA overexpression may lead to satu-
ration of the miRNA pathway. As the miRNA pathway is important in the control of
cellular gene expression, one could expect a disturbance in the cellular differentia-
tion program, possibly cell death or even cancer [ 155 ]. Saturation of the miRNA
pathway was reported to cause death when high doses of shRNAs were delivered by
an adeno-associated virus (AAV) vector in mice [ 4, 111- 114, 156 ] . Thus, exoge-
nous RNAi inducers should be expressed at a moderate level. Off-target effects on
unintended mRNAs can occur because an miRNA requires only a seed sequence
complementarity of 7-8 base pairs with a given mRNA [ 157 ] . Furthermore, off-
targeting can not only be elicited by the guide strand but also by the passenger