| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Address correspondence to Dr. Boon Chin Heng, Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, Singapore 119074; tel 656 874 4630; fax 65 6774 5701; e-mail denhenga{at}nus.edu.sg.
 |
Abstract |
|---|
 Top Abstract References |
|---|
(received 12 April 2005; accepted 18 April 2005)
Keywords: downregulation, interference, intrabody, RNAi, molecuar therapeutics
In recent years there has been growing interest in the use of RNA interference (RNAi) for downregulation of gene expression at the post-transcriptional level. The RNA interference pathway involves processing long double-stranded RNA (dsRNA) into 21- to 25-bp short interfering RNAs (siRNA) by an RNase III-like enzyme called Dicer [1,2]. The siRNA is then incorporated in a multisubunit RNA-induced silencing complex (RISC), which specifically targets homologous cellular mRNA transcripts for degradation [3]. For the purpose of in vitro manipulation it is preferable to utilize short sequences of siRNA rather than long sequences of unprocessed dsRNA. Obviously it is far more technically challenging and expensive to synthesize long dsRNA sequences. Furthermore, direct exposure of mammalian cells to long sequences of dsRNA can induce apoptosis through activation of dsRNA-dependent protein kinase (PKR) and type I interferon response [4]. This problem is less acute or even nonexistent with short siRNA sequences (<30 bp long) [4]. The targeting of mRNA transcripts by siRNA has numerous therapeutic applications, in addition to being a useful tool for research in functional genomics [5]. This approach has been proposed as a new therapeutic strategy for cancer [6] and viral infections [7] and for the rectification of single-gene disorders [8]. To date, the overwhelming majority of studies involve in vitro cell culture models, rather than clinical trials with human patients.
Much interest has also been generated on the potential use of antibodies to target intracellular molecules [9,10]. In effect, this would achieve gene downregulation at the post-translational level. This approach was given impetus by the development of the single chain variable fragment (scFv) antibody format [11], which facilitates the engineering and expression of functional antibodies within the intracellular environment. The recombinant antibody expressed within the confines of the intracellular environment is commonly referred to as an intrabody. The first reported use of an intrabody against an intracellular target was in 1988, when heavy- and light-chain cDNAs of an antibody that neutralized the yeast alcohol dehydrogenase I (ADH I) enzyme were expressed in Saccharomyces cerevisiae by Carlson et al [12]. Since then, numerous studies have demonstrated the tremendous potential of intrabodies for therapeutic applications [9,10], particularly in cancer [13–15] and AIDS [16–18].
The pertinent question that arises is how to choose between these two alternative approaches for therapeutic applications: (a) RNAi-mediated gene-silencing at the post-transcriptional level, or (b) intrabody knockout of protein function at the post-translational level? To answer the question, one must consider the inherent advantages and disadvantages of these two newly-emerging technology platforms.
The non-specific effects elicited by iRNA on target cells are the major technical challenge of the RNA interference approach. This has been demonstrated by several studies [19–22], and poses a formidable barrier to therapeutic applications of iRNA in humans, due to serious safety concerns. Gene expression profiling analysis by Persengiev et al [22] showed that >1,000 genes involved in diverse cellular functions are non-specifically stimulated or repressed in mammalian tissue-culture cells treated with conventional 21-bp iRNAs. The non-specificity of RNAi is thought to arise primarily from non-specific dsRNA-triggered responses that are mediated by interferon-associated pathways, which are absent in invertebrates and plants [19,23]. The use of intrabodies would circumvent this problem, since these can be designed for high specificity to a single target protein.
Another major deficiency of utilizing the RNA interference approach is the relatively short active half-life of iRNAs, which limits their effects on the target cell unless they are expressed via transfected recombinant DNA. In contrast, intrabodies, being proteins, possess a much longer active half-life than RNA, and are much more specific to their target molecules. It would be particularly advantageous to use an intrabody instead of iRNA when the active half-life of the target molecule is long. In that case, gene silencing through iRNA would be slow to produce an effect, whereas the effects of intrabody expression would be almost instantaneous.
In the scenario whereby a target protein has more than one interactive or binding domain, gene repression through RNAi would lead to the loss of multiple functions exhibited by that particular molecule. In contrast, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. This specificity would certainly be advantageous from the therapeutic viewpoint.
Apart from blocking binding interactions with the target molecule, intrabodies can also be designed to modulate the function of the target molecule in other ways. For example, it is possible to design intrabodies to relocate the target molecule to another subcellular location (ie, mitochondria, nucleus, or Golgi apparatus) by incorporation of an appropriate localization signal [24]. Additionally, intrabodies can be designed to promote selective degradation of the target molecule via the ubiquitin-proteosome pathway [25], or to paromote death of the target cell by activating the caspase-3-mediated apoptotic pathway [26]. These modulations could not be accomplished by the RNA interference approach.
Nevertheless, using intrabodies for knockout of protein function at the post-translational level has its inherent disadvantages. For a start, the screening of scFv antibodies through bacteriophage display libraries [27] is far more labor-intensive and time-consuming than the design and use of siRNA. It is almost impossible to rationally design an antibody, and screening the scFv bacteriophage display library is a highly empirical process that is ultimately based on chance. Hence, it is unpredictable whether or not one may end up with a good antibody for a specific target protein.
In contrast, implementation of RNAi is less technically challenging, and there are rational strategies for siRNA design based on well-defined algorithms [28–30], which are constantly being refined and upgraded, particularly in regard to reducing non-specific effects. Moreover, it may be feasible to circumvent the non-specificity of siRNA by using different target sequences.
Hence, it is clear that the siRNA-mediated and the intrabody-mediated knockout of gene function each has inherent advantages and disadvantages for clinical therapeutics. The choice between these two alternative strategies will ultimately depend on the particular pathological model in question, as well as on further technical advances in both of the technology platforms.
|
References |
|---|
|
TopAbstractReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  L. Guglielmi, V. Denis, N. Vezzio-Vie, N. Bec, P. Dariavach, C. Larroque, and P. Martineau Selection for intrabody solubility in mammalian cells using GFP fusions Protein Eng. Des. Sel., December 1, 2011; 24(12): 873 - 881. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Delanote, B. Vanloo, M. Catillon, E. Friederich, J. Vandekerckhove, and J. Gettemans An alpaca single-domain antibody blocks filopodia formation by obstructing L-plastin-mediated F-actin bundling FASEB J, January 1, 2010; 24(1): 105 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Loftus, Z. Yang, N. L. Tran, J. Kloss, C. Viso, M. E. Berens, and C. A. Lipinski The Pyk2 FERM domain as a target to inhibit glioma migration Mol. Cancer Ther., June 1, 2009; 8(6): 1505 - 1514. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Doebis, S. Schu, J. Ladhoff, A. Busch, F. Beyer, J. Reiser, R. F. Nicosia, S. Broesel, H.-D. Volk, and M. Seifert An anti-major histocompatibility complex class I intrabody protects endothelial cells from an attack by immune mediators Cardiovasc Res, November 1, 2006; 72(2): 331 - 338. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
