Aptamer-Mediated siRNA Targeting - RNA Interference from Biology to Therapeutics - page 209

targeting are the PSMA aptamers [ 44 ]. Several independent groups have demon-
strated anti-PSMA aptamer-mediated siRNA delivery to PSMA-positive cells or
human prostate cancer cells transplanted into nude mice. Chu et al. took advantage
of the noncovalent interaction of streptavidin and biotin to create a modular strepta-
vidin bridge for conjugating the anti-PSMA aptamer (A-9) with lamin A/C or
GAPDH siRNA [ 69 ]. Two biotinylated aptamers and two biotinylated siRNAs were
co-assembled, and effective PSMA receptor-mediated uptake of the aptamer-siRNA
conjugates was observed along with siRNA-mediated specific silencing of the
targeted transcripts in the cultured PSMA-positive cells.
We used a “sticky bridge” strategy to noncovalently assemble an HIV gp120
aptamer with various siRNAs [ 40 ] . The 3 ¢-end of the aptamer and one of the two
siRNA strands were chemically conjugated with complementary GC-rich sticky
sequences, thereby, allowing the aptamer and siRNA portions to be noncovalently
conjugated via Watson-Crick base pairing by simple mixing. Through mixing vari-
ous siRNAs with different aptamers, the “sticky bridge” offers a facile conjugation
approach for multiplex targeted delivery and target downregulation. For example, in
the RNAi-based anti-HIV therapy, rapid emergence of viral escape mutants often
abrogates RNAi efficacy of a single siRNA. Therefore, we combined three different
siRNAs with a single gp120 aptamer: one against the HIV-1 tat/rev transcripts and
two siRNAs targeting the HIV-1 host dependency factors [CD4 and transportin-3
(TNPO3)]. The resulting aptamer—“sticky bridge”—cocktail siRNA conjugates
were specifically internalized into cells expressing the HIV-1 envelope protein and
effectively suppressed viral loads in cell culture and in an HIV-1-infected human-
ized mouse model (Zhou et al. manuscript submitted).

10.3.2 Covalent Aptamer-siRNA Conjugates

Using a different, covalent conjugation approach, Giangrande and coworkers have
developed a simple covalent anti-PSMA aptamer (A-10)-siRNA chimeric RNA
[ 70 ]. In this approach, the aptamer was covalently attached to the sense strand of a
21-mer siRNA portion that was annealed with an unmodified antisense strand. The
resulting chimeric RNA molecule was selectively internalized into cells expressing
PSMA and effectively knocked down expression of the targeted tumor survival
genes PLK1 and BCL2 both in cell culture and in vivo . Recently, this same group
constructed an optimal second-generation chimera with an increased in vivo circula-
tion time and bioavailability [ 71 ]. Based on their previous first-generation chimeric
RNA design, the aptamer portion was truncated from 71 to 39 nt without loss of high
binding affinity. The size of the resulting version (containing the truncated aptamer
and sense strand of siRNA portion) was reduced to 64 nt, making this RNA amena-
ble to milligram-scale chemical synthesis. Moreover, some rational structural
changes of the siRNA portion were added, allowing more efficient incorporation of
the siRNA by the cellular RNAi machinery and increasing the gene silencing efficacy.
Additionally, the sense strand of the siRNA portion was appended with a 20-kDa
polyethylene glycol (PEG) moiety, which substantially increased the circulating