Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors

Journal name:
Nature Biotechnology
Year published:
Published online


Prostate cancer cells expressing prostate-specific membrane antigen (PSMA) have been targeted with RNA aptamer–small interfering (si)RNA chimeras, but therapeutic efficacy in vivo was demonstrated only with intratumoral injection. Clinical translation of this approach will require chimeras that are effective when administered systemically and are amenable to chemical synthesis. To these ends, we enhanced the silencing activity and specificity of aptamer-siRNA chimeras by incorporating modifications that enable more efficient processing of the siRNA by the cellular machinery. These included adding 2-nucleotide 3′-overhangs and optimizing the thermodynamic profile and structure of the duplex to favor processing of the siRNA guide strand. We also truncated the aptamer portion of the chimeras to facilitate large-scale chemical synthesis. The optimized chimeras resulted in pronounced regression of PSMA-expressing tumors in athymic mice after systemic administration. Anti-tumor activity was further enhanced by appending a polyethylene glycol moiety, which increased the chimeras' circulating half-life.

At a glance


  1. Optimized PSMA-Plk1 chimeras.
    Figure 1: Optimized PSMA-Plk1 chimeras.

    Blunt, truncated version of first-generation chimera (A10-Plk1) described previously25. The aptamer portion of the chimera has been truncated from 71+ nt to 39 nt. OVH, overhang chimera similar to blunt, but with 2 nt (UU)-overhangs at the 3′ end of the siRNA duplex. G-U, G-U wobble chimera identical to OVH, but with a wobble base pair at the 5′ end of the antisense siRNA strand (silencing/guide strand). Swap, sense and antisense strands of siRNA duplex are reversed. Stem loop, hairpin chimera where the siRNA duplex (stem) is continuous with the aptamer (loop). Structural predictions were generated using RNAstructure V 4.6.

  2. Binding of truncated versions of PSMA A10 aptamer and optimized chimeras to cells expressing PSMA.
    Figure 2: Binding of truncated versions of PSMA A10 aptamer and optimized chimeras to cells expressing PSMA.

    RNAs were end-labeled with 32P. (a) LNCaP cells and PC-3 cells were incubated with either the full-length PSMA aptamer A10 (71 nt) or truncated versions of the PSMA aptamer, A10-3 (57 nt) or A10-3.2 (39 nt). 32P-labeled bound/internalized RNAs were determined by liquid scintillation counter (LSC) or filter binding assay (data not shown). (b) Relative affinity of A10 PSMA aptamer and truncated A10 aptamers to cells expressing PSMA. Varying amounts (0–2 nM) of end-labeled A10, A10-3 and A10-3.2 were incubated with fixed LNCaP cells. Bound counts were determined by filter binding assay. (c) First-generation chimera (A10-Plk1) and optimized chimeras were incubated with either PC-3 cells (black bars) or LNCaP and 22Rv1(1.7). Cells were processed as in a. Bound counts were determined with LSC.

  3. Silencing ability of PSMA chimeras.
    Figure 3: Silencing ability of PSMA chimeras.

    22Rv1(1.7) cells were transfected with 400, 40 or 4 nM of each chimera. Cells were processed for qRT-PCR 24–48 h after transfection. Percent Plk1 expression was normalized to that of mock-transfected (mock) cells. (a) Comparison of silencing efficiencies of the blunt, OVH, G-U Wobble, swap and stem loop chimeras to that of the first-generation chimera (A10-Plk1). (a, inset) Percent Plk1 expression of G-U wobble, swap and stem loop ≤ 1.0 and are depicted on an adjusted y axis. Experiments were performed several times (n = 3). (b) 22Rv1(1.7) cells were treated with either 400 nM or 4 nM of each of the optimized RNA chimeras in the absence of transfection reagent. Cells were processed for qRT-PCR 4 d after treatments.

  4. Analysis of chimera processing by the RNAi machinery.
    Figure 4: Analysis of chimera processing by the RNAi machinery.

    (a) In vitro Dicer processing. The 32P-labeled PSMA-Plk1 chimeras were incubated with recombinant human Dicer enzyme for either 1 or 2 h. The Dicer cleavage or uncleaved (No Dicer) products were visualized after 15% nondenaturing PAGE. (b) Assessment of strand bias: loading of siRNA silencing strand into RISC. Small fragment northern blot of RNA isolated from 22Rv1(1.7) cells transfected with 200 pmols of each of the optimized aptamer-siRNA chimera constructs. Loading of the siRNA silencing strand into RISC protects the siRNA strand from degradation (this can be detected with a specific probe using a modified northern blot assay). The strand that is not loaded is rapidly degraded. U6 RNA was used as a loading control. Duplex, Plk1 siRNA duplex; A10-Plk1, first-generation chimera. Blunt, OVH, G-U, swap and stem loop chimeras are described in Figure 1. Probe controls show hybridization efficiencies of the sense and antisense probes. The varying intensities of unprocessed chimeras (upper bands on blots) are due to differential probe binding to these species and do not reflect their amounts (this same trend was observed when equal amounts of each chimera was directly loaded on gel and processed as described here (data not shown)).

  5. Effect of PSMA-Plk1 chimeras on prostate cancer cell growth.
    Figure 5: Effect of PSMA-Plk1 chimeras on prostate cancer cell growth.

    (a) 22Rv1(1.7) cells were transfected (or treated in the absence of transfection reagent (data not shown)) with either 400 nM or 4 nM of A10-Plk1 or 4 nM of each of the optimized chimeras. 3H-thymidine was added to the media 24 h after transfection and cells were incubated in the presence of 3H-thymidine for another 24 h. The next day cells were lysed with 0.5 N NaOH and incorporated counts determined by liquid scintillation counter. Cisplatin was used as a positive control for this assay. (b) Cell cycle profile of 22Rv1(1.7) cells transfected with 4 nM of each of the optimized chimeras. DNA content of treated cells was determined by flow cytometry 48 h after transfection after staining cells with PI. Nocodazole (Noc) treatment was used as a positive control for this assay to arrest cells in mitosis.

  6. In vivo efficacy of optimized PSMA chimera in a xenograft model of prostate cancer.
    Figure 6: In vivo efficacy of optimized PSMA chimera in a xenograft model of prostate cancer.

    (a) 106 luciferase-expressing (PSMA-positive or PSMA-negative) prostate cancer cells were injected into the flanks of nude (nu/nu) mice 2 weeks before treatment with optimized chimeras. Treatment with the optimized chimeras commenced when tumors reached a volume of ~0.4 cm3. 1 nmol of either blunt, swap or A10-3.2-Con was administered intraperitoneally in mice bearing 22Rv1(1.7) tumors. As a control for specificity, a mouse xenograft model of prostate cancer bearing PSMA-negative prostate cancer cells (PC-3) was also treated with the swap chimera. A total of ten treatments were administered for each treatment group. Treatment occurred every day for 10 consecutive days. Tumors were measured with calipers every other day for the course of the experiment. Saline (PBS) treated animals were used as a control. Animals were euthanized 2–3 d after the last treatment. n ≥ 10 mice per treatment group. Bottom panels: Bioluminescence imaging of 22Rv1(1.7) and PC-3 prostate tumors was carried out after treatment with optimized chimeras (day 10). Examples show tumor growth in four representative animals from each treatment group. Insert indicated by arrow represents bioluminescence imaging images of ~30% of 22Rv1(1.7) tumor–bearing mice treated with the swap chimera that still had palpable tumors (17 out of 48 total tumors) by day 10. All sites represent tumor growth ~25 d after injection of tumor cells. Log-scale heat map (right) of photon flux applies to all panels. (b) Histology of 22Rv1(1.7) and PC-3 tumors treated with the various optimized chimeras. Areas of necrosis (asterisks) were readily detected in swap-treated 22Rv1(1.7) tumors, but not frequently seen in PBS-treated tumors (H&E, 40×). Mitotic figures (arrows) were often detected in tumors from all treatment groups including occasional large bizarre mitoses in swap-treated 22Rv1(1.7) tumors (Hematoxylin, 600×). TUNEL staining was detected in scattered cells throughout the tumor section of each group (TUNEL staining, 600×) and at the interface of viable tissue and necrotic foci (TUNEL staining, 200×). Representative sections from the PBS and swap treatment groups are shown. (c) Assessment of potential chimera-dependent immunostimulatory effects. Serum from mice treated with either saline (PBS), A10-3.2-Con, swap or poly I:C was screened for levels of cytokines INT-a and IL-6 using ELISA. (d) 5′-Rapid amplification of cDNA ends (5′-RACE) PCR analysis to assess siRNA mediated cleavage of Plk1 mRNA in tumors treated with the various PSMA-Plk1 chimeras. (e) Pharmacokinetic profile and efficacy of the swap chimera with polyethylene glycol (PEG). (f) In vivo silencing assessed by quantitative RT-PCR. Plk1 mRNA levels in treated tumors were normalized to GAPDH mRNA levels. Panel on left shows Plk1 levels of tumors (9 tumors/group for this experiment) from animals processed for experiments shown in a and e.

Author information

  1. These authors contributed equally to this work.

    • Justin P Dassie,
    • Xiu-ying Liu &
    • Gregory S Thomas


  1. Department of Internal Medicine, University of Iowa, Iowa City, Iowa, USA.

    • Justin P Dassie,
    • Xiu-ying Liu,
    • Gregory S Thomas,
    • Ryan M Whitaker,
    • Kristina W Thiel,
    • Katie R Stockdale,
    • Anton P McCaffrey,
    • James O McNamara II &
    • Paloma H Giangrande
  2. Molecular and Cellular Biology Program, University of Iowa, Iowa City, Iowa, USA.

    • Justin P Dassie,
    • Gregory S Thomas,
    • Anton P McCaffrey &
    • Paloma H Giangrande
  3. Department of Pathology, University of Iowa, Iowa City, Iowa, USA.

    • David K Meyerholz
  4. Department of Radiation Oncology, University of Iowa, Iowa City, Iowa, USA.

    • Paloma H Giangrande


J.P.D., X.-y.L., G.S.T., R.M.W., K.W.T., K.R.S. performed research; D.K.M. provided expertise and analyzed data; A.P.M. provided expertise and useful discussions; J.O.M. designed research, wrote the manuscript and provided useful discussions; P.H.G. designed, coordinated and performed research, analyzed data and wrote the manuscript.

Competing financial interests

Duke University (P.H.G. and J.O.M.) and the University of Iowa (P.H.G., J.O.M. and A.P.M.) have applied for patents based on this technology.

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