RNAi as Antiviral Therapy: The HIV-1 Case - RNA Interference from Biology to Therapeutics - page 223

11.2 Viral Escape and Combinatorial RNAi Approaches

Potent and sequence-specific HIV-1 inhibition has been reported with RNAi-
inducing reagents in infected cell cultures, but it soon became apparent that HIV-1
is prone to viral escape when a single shRNA inhibitor is applied [ 64, 69, 85- 89 ] .
Prolonged culturing in the presence of HIV-1 should be performed to test the likeli-
hood of viral escape, which is difficult to predict. Care should be taken not to mis-
interpret the results of such escape studies. For instance, the appearance of point
mutations in the viral target sequence forms definitive proof of viral escape, and in
fact, it demonstrates the exquisite sequence-specificity of the RNAi mechanism.
However, sometimes viral breakthrough is observed without the acquisition of
apparent escape mutations. First, mutations may be selected outside the actual target
sequence. These changes can nevertheless cause resistance by triggering a confor-
mational switch in the RNA such that the target becomes inaccessible to the RNAi
machinery [ 64, 65 ]. Second, no changes may be apparent despite phenotypic viral
escape, which may in fact represent false breakthrough replication due to subopti-
mal virus inhibition [ 70, 84 ]. We previously discussed the complexities of properly
interpreting HIV-1 evolution studies [ 90- 94 ]. Thus, both detailed phenotypic and
genotypic analyses are required to satisfactorily address the issue of viral escape.
The ease of HIV-1 escape mimics what occurs in patients treated with a single
antiretroviral drug, but we know that combinatorial drug regimes can prevent viral
escape and therapy failure. Thus, also an RNAi therapeutic attempt should tackle
the virus with multiple shRNA inhibitors at the same time. Such a combinatorial
RNAi attack can target the virus at multiple genome positions [ 83 ], but one can add
an attack against host-encoded cofactors, as will be discussed later. One could also
combine RNAi molecules with other RNA effector molecules such as decoys and
ribozymes [ 95, 96 ]. Different RNA-based inhibitors can also be combined in a sin-
gle transcript such as the conjugate of an antiviral aptamer that binds the HIV-1
envelope protein and an antiviral siRNA [ 97 ]. The aptamer not only blocks the
envelope protein on virion particles, but it also selectively ferries the siRNA to HIV-
infected cells that express the envelope protein on their surface. This conjugate
demonstrated good antiviral activity in the preclinical model of the humanized
mouse, although uncertainty remains about the efficiency of the intracellular deliv-
ery of the siRNA [ 98 ]. Another elegant solution to avoid viral escape is the use of
the second-generation shRNAs that specifically target viral escape variants [ 99 ] .
However, the shear endless number of possible viral escape routes may limit the
feasibility of this approach [ 84 ]. In fact, we recently demonstrated the power of the
second-generation concept by effectively blocking common viral escape routes, but
little therapeutic benefit was achieved because HIV-1 selected alternative escape
routes [ 100 ]. RNAi can also be used to specifically block the evolution of drug-
resistant virus variants that evolve under pressure by the regular antiretroviral drugs,
e.g., inhibitors of the viral protease enzyme [ 101 ] .