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Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers.

Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, Rothman AM, Hernandez FJ, McNamara JO, Giangrande PH - Nucleic Acids Res. (2012)

Bottom Line: RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2.We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression.The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

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Affinity and specificity analysis of aptamer binding to recombinant HER2 using Surface Plasmon Resonance (SPR). (A) High-affinity interaction between immobilized aptamers (E1, B1 and C1) and rHer2 recombinant protein. Four different rHer2 protein concentrations were analyzed (5–40 nM) and the KD was reported for each aptamer. The aptamer binding profiles were fitted using Langmuir fitting model (with mass transfer), as determined by BIA evaluation 4.1 software, black lines. (B) Evaluation of the rHer2 protein specificity using three non-specific targets at 40 nM concentration each (b-hHer2; c-hEphA2 and d-BSA), over the immobilized aptamers.
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gks294-F3: Affinity and specificity analysis of aptamer binding to recombinant HER2 using Surface Plasmon Resonance (SPR). (A) High-affinity interaction between immobilized aptamers (E1, B1 and C1) and rHer2 recombinant protein. Four different rHer2 protein concentrations were analyzed (5–40 nM) and the KD was reported for each aptamer. The aptamer binding profiles were fitted using Langmuir fitting model (with mass transfer), as determined by BIA evaluation 4.1 software, black lines. (B) Evaluation of the rHer2 protein specificity using three non-specific targets at 40 nM concentration each (b-hHer2; c-hEphA2 and d-BSA), over the immobilized aptamers.

Mentions: To address whether the RNA aptamers identified by the cell-based cell-internalization SELEX approach bound directly to HER2, we performed surface plasmon resonance (SPR, BIAcore) on selective RNA aptamers (B1, C1 and E1) using recombinant purified rat HER2 protein (Figure 3). Importantly, all three RNA sequences analyzed, bound with low nanomolar affinity (Figure 3A) and specificity (Figure 3B) to the purified recombinant rat HER2 protein. The respective affinities of the RNA aptamers for purified, recombinant rat HER2 protein are as follows: C1 (KD = 45.8 nM) > E1 (KD = 60.8 nM) > B1 (KD = 85.2 nM) (Figure 3A). Interestingly, the binding affinities of B1, C1 and D1 correlate with the degree of aptamer cell-internalization as observed by flow cytometry (Figure 2E, bottom panel). Binding specificity was demonstrated by examining the binding of B1, C1 and D1 aptamers to analogous receptor tyrosine kinases (RTKs) (e.g. human HER2 and human EphA2) (Figure 3B). All three RNAs tested bound solely to the rat isoform of HER2 and not to the human isoform of the receptor (indicating specificity for the rat isoform) or to a different RTK (human EphA2) (Figure 3B). In addition, for aptamers C1 and E1, specificity for the rat HER2 isoform was confirmed using N202.1A cells expressing human HER2 (data not shown).Figure 3.


Delivery of chemo-sensitizing siRNAs to HER2+-breast cancer cells using RNA aptamers.

Thiel KW, Hernandez LI, Dassie JP, Thiel WH, Liu X, Stockdale KR, Rothman AM, Hernandez FJ, McNamara JO, Giangrande PH - Nucleic Acids Res. (2012)

Affinity and specificity analysis of aptamer binding to recombinant HER2 using Surface Plasmon Resonance (SPR). (A) High-affinity interaction between immobilized aptamers (E1, B1 and C1) and rHer2 recombinant protein. Four different rHer2 protein concentrations were analyzed (5–40 nM) and the KD was reported for each aptamer. The aptamer binding profiles were fitted using Langmuir fitting model (with mass transfer), as determined by BIA evaluation 4.1 software, black lines. (B) Evaluation of the rHer2 protein specificity using three non-specific targets at 40 nM concentration each (b-hHer2; c-hEphA2 and d-BSA), over the immobilized aptamers.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401474&req=5

gks294-F3: Affinity and specificity analysis of aptamer binding to recombinant HER2 using Surface Plasmon Resonance (SPR). (A) High-affinity interaction between immobilized aptamers (E1, B1 and C1) and rHer2 recombinant protein. Four different rHer2 protein concentrations were analyzed (5–40 nM) and the KD was reported for each aptamer. The aptamer binding profiles were fitted using Langmuir fitting model (with mass transfer), as determined by BIA evaluation 4.1 software, black lines. (B) Evaluation of the rHer2 protein specificity using three non-specific targets at 40 nM concentration each (b-hHer2; c-hEphA2 and d-BSA), over the immobilized aptamers.
Mentions: To address whether the RNA aptamers identified by the cell-based cell-internalization SELEX approach bound directly to HER2, we performed surface plasmon resonance (SPR, BIAcore) on selective RNA aptamers (B1, C1 and E1) using recombinant purified rat HER2 protein (Figure 3). Importantly, all three RNA sequences analyzed, bound with low nanomolar affinity (Figure 3A) and specificity (Figure 3B) to the purified recombinant rat HER2 protein. The respective affinities of the RNA aptamers for purified, recombinant rat HER2 protein are as follows: C1 (KD = 45.8 nM) > E1 (KD = 60.8 nM) > B1 (KD = 85.2 nM) (Figure 3A). Interestingly, the binding affinities of B1, C1 and D1 correlate with the degree of aptamer cell-internalization as observed by flow cytometry (Figure 2E, bottom panel). Binding specificity was demonstrated by examining the binding of B1, C1 and D1 aptamers to analogous receptor tyrosine kinases (RTKs) (e.g. human HER2 and human EphA2) (Figure 3B). All three RNAs tested bound solely to the rat isoform of HER2 and not to the human isoform of the receptor (indicating specificity for the rat isoform) or to a different RTK (human EphA2) (Figure 3B). In addition, for aptamers C1 and E1, specificity for the rat HER2 isoform was confirmed using N202.1A cells expressing human HER2 (data not shown).Figure 3.

Bottom Line: RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2.We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression.The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA.

ABSTRACT
Human epidermal growth factor receptor 2 (HER2) expression in breast cancer is associated with an aggressive phenotype and poor prognosis, making it an appealing therapeutic target. Trastuzumab, an HER2 antibody-based inhibitor, is currently the leading targeted treatment for HER2(+)-breast cancers. Unfortunately, many patients inevitably develop resistance to the therapy, highlighting the need for alternative targeted therapeutic options. In this study, we used a novel, cell-based selection approach for isolating 'cell-type specific', 'cell-internalizing RNA ligands (aptamers)' capable of delivering therapeutic small interfering RNAs (siRNAs) to HER2-expressing breast cancer cells. RNA aptamers with the greatest specificity and internalization potential were covalently linked to siRNAs targeting the anti-apoptotic gene, Bcl-2. We demonstrate that, when applied to cells, the HER2 aptamer-Bcl-2 siRNA conjugates selectively internalize into HER2(+)-cells and silence Bcl-2 gene expression. Importantly, Bcl-2 silencing sensitizes these cells to chemotherapy (cisplatin) suggesting a potential new therapeutic approach for treating breast cancers with HER2(+)-status. In summary, we describe a novel cell-based selection methodology that enables the identification of cell-internalizing RNA aptamers for targeting therapeutic siRNAs to HER2-expressing breast cancer cells. The future refinement of this technology may promote the widespread use of RNA-based reagents for targeted therapeutic applications.

Show MeSH
Related in: MedlinePlus