Original Article
Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: Affibody fusion protein

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Abstract

Efficiency of systemically delivered siRNA in gene silencing is compromised due to lack of target-specific delivery and rapid clearance of siRNA by in vivo elimination pathways. We designed a fusion protein consisting of a dsRNA binding domain of transactivation response RNA binding protein (TRBP2) fused to ErbB2 binding affibody (AF) for target specific delivery of siRNA. Designated as TRAF, the fusion protein is stable and binds efficiently and specifically to siRNA, forming homogenous non-aggregated and nuclease-resistant particles that efficiently and selectively transport siRNA into HER-2 overexpressing cancer cells and tissues. Administration of siRNA by TRAF into cells resulted in significant silencing of chosen genes involved in cell proliferation viz. AURKB and ErbB2. Noticeably, intravenous administration of TRAF:siRNA against these genes resulted in remarkable tumor suppression in the SK-OV-3 xenograft mouse model. Our results establish the potential of engineered proteins for specific and systemic delivery of siRNA for cancer therapy.

From the Clinical Editor

The use of siRNA in one of many novel treatments in cancer therapy. However, a major challenge for using siRNA is the lack of specificity and rapid RNA clearance. In this article, the authors designed a tumor targeting fusion protein, which can deliver siRNA specifically. In the experimental xenograft model, it was shown that intravenous administration of this resulted in significant tumor suppression. The results seem to hold promise in future clinical studies.

Graphical Abstract

Recombinant protein with HER-2 targeting and siRNA binding motifs for efficient in vivo siRNA delivery to tumors

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Section snippets

Background

RNA interference holds a great potential in cancer therapy due to its high specificity and ability to efficiently silence a large number of different genes involving a number of distinct cellular pathways.1, 2 This is particularly important for a disease as complex as cancer. However, as in other drug targeting therapies, the major challenge for therapeutic use of siRNA is the need for devising safe, stable and effective carriers capable of delivering low doses of siRNA into the cytoplasm. A

Cell culture

SK-OV-3, MDA-MB-231 and MDA-MB-453 were obtained Medina-Lai Kauwe, University of Southern California Keck School of Medicine, CA. Other cells were obtained from lab stocks. These were maintained in a Dulbecco's Modified Eagle Medium (DMEM): Nutrient Mixture F-12 (DMEM/F-12, Life Technologies) with 10% fetal bovine serum (Life Technologies).

Flow cytometry and confocal microscopy

Cells grown in tissue culture flasks were trypsinized and pelleted. Approximately 50,000 cells were incubated with 40 pmol of FAM-labeled siRNA either alone

Design, purification and structural properties of TRAF

TRAF protein was designed by fusing second domain of TRBP2 (due to its higher affinity) to the DNA sequence of ZHER-2 affibody molecule. To aid proper folding, five glycine codons were inserted between the two domains. During purification, we noticed bacterial RNA associated with TRAF (data not shown) that was removed by adding urea in the lysis buffer. Additionally, 0.1% Triton-X114 was used to remove bacterial endotoxins.29 The purified protein was characterized by SDS-PAGE and mass

Discussion

Efficient delivery of siRNA is a challenge for realizing its gene silencing potential as these approaches involve protection of siRNA from degradation and cellular uptake followed by timely release inside the cell. Besides, it is important for the delivery vehicle to be stable and free from toxicity. Natural or biosimilar entities have shown promising results in delivering siRNA compared to vehicles that are complex or prepared with synthetic materials.1, 3, 5 Moreover, nature has evolved a

Design of TRAF

Cationic polymers and cationic amphiphile structures are considered as gold standard in carrying nucleic acids including siRNA efficiently into the cells in vitro.23 However, electrostatic interaction between siRNA and cationic polymers leads to the formation of heterogeneous aggregates that are susceptible to adhesion of serum proteins, resulting in rapid extravasation from the blood.1, 3 Additionally, cationic polymers such as polyarginine, protamine sulfate and several cell penetrating

Tracking of TRAF:siRNA complexes in cells and in mice

One of the crucial steps in siRNA delivery is its availability to the RISC complex for mediating knockdown of desired genes. Majority of the complexes find their way into the endosomes after taking different entry routes. As endosomes face multiple fates like retrograde or lysosomal route, it is important that siRNA is released from the endosomes into the cytoplasm.46 Recently it has been observed that only 1-3% of the total siRNA internalized is released into the cytoplasm during early

Acknowledgment

The authors acknowledge E. Nathan for helping in developing tumor xenografts, Avinash for tissue cryosectioning, N.R. Chakravarthi for technical assistance in animal live imaging and Nandini for assisting in confocal microscopy.

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      The hybrid TRAF complex effectively binds with the siRNA and selectively transport it into HER2 overexpressing cancer cells. Administration of siRNA by hybrid TRAF into tissues resulted in significant silencing of chosen genes involved in cell proliferation viz. AURKB and ErbB2 and a good candidate for the EbB2 overexpressed carcinoma [111]. Zhang et al., prepared a DNA affibody complex loaded with ∼53 molecules of DOX.

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    We gratefully acknowledge financial support from CSIR (BSC0112). GHD is also grateful to CSIR for a Senior Research Fellowship.

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