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Review of the Application of RNA Interference Technology in the Pharmaceutical Industry
1 Scottish Agricultural College, Allan Watt Building, Bush Estate, Penicuik, EH26 0QE, United Kingdom Correspondence: Address correspondence to: Ian T. Pyrah, Amgen Inc, One Amgen Center Drive, Mailstop 29-2-A, Thousand Oaks, CA, 91320, USA; e-mail:ipyrah{at}amgen.com
Ribonucleic acid (RNA) interference (RNAi) is a recently discovered phenomenon whereby the introduction of double stranded (ds) RNA into the cytoplasm of the cell results in the specific and efficient degradation of complementary messenger (m) RNA and, therefore, reduced protein production. It was discovered by chance during attempts to develop flowers with increased colour intensity. The specific nature of the inhibition of protein production of cells has resulted in an explosion of research to understand and exploit RNAi. The technique is now established in in vitro systems, and much work is focussed in adapting RNAi for in vivo application. The potential of the technology in understanding physiological and pathological processes is significant, while its development as a therapeutic agent holds much promise as targeted agents. This review will describe the basic biological processes that drive RNAi, indicate current areas of areas research, and forecast future areas of development.
Key Words: RNA interference • biotechnology • discovery pathology • drug development • mechanisms of toxicity • molecular pathology Abbreviations: ATP, adenosine triphosphate • CHS, chalcone synthetase • CIA, collagen induced arthritis • CL, cationic liposomes • ds, double stranded • mi, micro • nt, nucleotide • piRNA, piwi interacting RNA • PTGS, post transcriptional gene silencing • RdRp, RNA dependent RNA polymerases • RNA, ribonucleic acid • RNAi, RNA interference • RISC, RNA interfering silencing complex • sh, short hairpin • si, short interfering • siRNA, short interfering RNA • sm, small modulatory
Ribonucleic acid (RNA) interference (RNAi) is a naturally occurring intracellular mechanism, which causes sequence specific posttranscriptional gene silencing. The reaction is triggered by the introduction of double-stranded (ds) RNA into the cytoplasm of the cell, and results in the specific targeted destruction of mRNA and a subsequent reduction in protein production (Elbashir et al., 2001). Its initial discovery (Napoli et al., 1990) was followed by an explosion of research in this field, and by 2002 it had earned the title of "Scientific Breakthrough of the Year" (Couzin, 2002). When successfully manipulated, RNAi can result in the knockdown of single or multiple genes so providing a quick and convenient method of analysing gene function (Dykxhoorn et al., 2003). RNAi libraries have been developed as a useful screening method for assessing functional consequences of inhibiting multiple proteins within given pathways (Devi, 2006). Examples of experimental use include identification of key steps in the p53 pathway (Berns et al., 2004) and verification of the role of Fas in apoptosis regulation in murine liver and kidney (Song et al., 2003; Hamar et al., 2004). In the pharmaceutical industry, investigation into its potential for use in pharmaceutical target validation and as a therapeutic tool is ongoing (Lavery and King, 2003; Lu et al., 2003). As a model therapeutic agent, RNAi has achieved regression of clinical signs in neurodegenerative disease models (Xia et al., 2004) and is currently in phase 1 clinical trials to treat age related macular degeneration (Alnylam Pharmaceutical, Acuity Pharma). It also has potential for use as an antiviral therapeutic with examples of targeted viruses in animal disease models including Herpes simplex virus-2, respiratory syncytial virus, parainfluenza virus, SARS, and influenza A (Cristofaro and Ramratnam, 2006). In addition, RNAi can be used to generate animal disease models, e.g., Parkinson’s disease has been modelled through targeted knockdown of Th within neurons in the mid brain of adult mice. (Hommel et al., 2003). Finally, germline transmission of RNAi in mice has also been achieved (Carmell et al., 2003). This review aims to summarize the current opinion on the mechanisms underlying RNA interference, discuss key in vitro and in vivo existing applications and outline its prospects for future use within the pharmaceutical industry.
The Discovery of RNA Interference The mechanism was recognized as a form of posttranscriptional gene silencing (PTGS) and termed "co-suppression." Further work found that transcripts produced from both loci were immediately degraded in the cytoplasm. In this case, activation of PTGS was thought to be due to the production of aberrant dsRNA by the transgene, which resulted in silencing of the mRNA (Zamore, 2002). A few years later, Fire and his colleagues were investigating the efficiency of injecting single-stranded antisense RNA as a method of gene silencing in the nematode Caenorhabditis elegans by using its ability to hybridise with endogenous mRNA and inhibit translation (Fire et al., 1998). The discovery that introduction of the sense strand also resulted in silencing, and that dsRNA was even more effective at inhibiting the target gene led to the conclusion that both original single-stranded sense and antisense samples may have been initially contaminated with dsRNA (Fire et al., 1998). Similarities between the underlying mechanism in nematodes and co-suppression in plants was soon recognized, and the term RNAi was adopted for both (Ketting and Plasterk, 2000).
The Mechanism of RNA Interference
This complex is able to identify the specific complementary strand of mRNA and degrades it with the help of one of its major components, Argonaute 2 protein (Liu et al., 2004; Song et al., 2004). The result is destruction of the mRNA that is complementary to the antisense strand of the original dsRNA introduced into the cytoplasm, and prevention of translation and protein production. Initial experiments successfully manipulated RNAi in plants and invertebrates through the introduction of long-stranded dsRNA into the cytoplasm. In mammalian cells, however, similar techniques resulted in the initiation of the interferon response and cell death before cleaving by Dicer could occur. It was not until 2001 that Elbashir et al. reported an alternative method of RNAi induction in mammalian cells. Through the direct introduction of siRNAs under 30 base pairs in length, they successfully avoided the interferon response and activated the RISC complex and mRNA destruction in mammalian cells. How the introduction of just a few strands of RNA results in the silencing of a large excess of target mRNA has also been investigated (Zamore, 2002). One explanation suggested the involvement of a family of RNA-dependent RNA polymerases (RdRp), which use the target mRNA as a template, and cleaved primary siRNAs as primers. These produce a population of secondary siRNAs, which lead to an increase in the number of activated RISC complexes formed and mRNA splicing. Members of the RdRp family have been identified in C. elegans, Arabidopsis, Neurospora, and Dictyostelium; but not Drosophila and humans. Other suggested mechanisms include the simple fact that Dicer cuts the dsRNA into shorter lengths, thereby increasing the number of siRNAs 10–20-fold, and that RISC itself acts as a multiple turnover enzyme and uses a catalytic mechanism to recycle already synthesised siRNAs (Plasterk, 2002). The latter two would be feasible explanations inDrosophila and mammals, which lack RdRp and thus replicative mechanisms for RNAi (Harborth et al., 2003). The natural role of RNA interference is thought to be the protection of the plant/nematode from invasion by viral pathogens (Zamore, 2002). On infection, RNA viruses generate double-stranded (ds) RNA molecules either in activation or replication, and it is these molecules that are capable of activating the host RNAi defense mechanism. This results in the specific degradation of the viral RNA so preventing viral multiplication (Voinnet, 2005). This phenomena is highlighted by plants defective in RNAi demonstrating hypersensitivity to viral infection (Ding et al., 2004). Some animal and plant viruses, e.g., Potato virus X, are also found to produce proteins capable of suppressing host-mediated RNA silencing. These proteins have also been shown to be responsible for successful viral spread within the host (Li et al., 2002; Ding et al., 2004). Following this identification of the RNAi phenomenon in plants, invertebrates and mammalian cells, further investigations into its specific properties were carried out (Downward, 2004). Experiments to determine its potential for its systemic spread were performed using nematodes. These were fed, soaked, or injected with a dsRNA rich solution and in all cases this resulted in body-wide RNAi-induced gene silencing. Although the process behind this mechanism was not clear, a transmembrane protein Sid-1 that enables passive cellular uptake of dsRNA was identified that could play an active part in RNAi distribution (Feinberg et al., 2003). RNAi silencing effects have also been observed in progeny in plants and invertebrates but again the underlying mechanism remains undetermined (Napoli et al., 1990; Fire et al., 1998). As a result of these numerous experiments, artificial manipulation of RNAi in cultured mammalian cells has been possible and its full potential for use in vivo hypothesized. It prompted the need for mass production of synthetic siRNAs, which is now the main business of several companies.
SiRNAs and Their Design This work led to the development of a set of empirical guidelines to generate two 21-nt sense and antisense oligoribonucleotides for efficient siRNA (Mittal, 2004; Naito et al., 2004). Success is maximized by achieving looser binding of the 5' end of the antisense strand to its complementary strand in order to promote its binding to RISC, and increasing the specificity of the siRNA sequence to reduce off-target effects (Schwarz et al., 2003). In addition, it is recommended that: each strand must have 2-nt 3' overhangs; A/U base pairing at the 5' end of the sense strand and G/C base pairing at the 5' end of the sense strand; AU richness in the 5' terminal third of the antisense strand; avoidance of introns, 5' and 3' untranslated regions, regions within 75 bases of the start codon and sequences with >50% Guanine and Cysteine content; maximizing sequence divergence from related mRNA. Despite applying these criteria, differences in silencing efficiencies between siRNAs occur, and occurrence of off-target siRNA activity has been recognized as an important factor in siRNA experimental design (Reynolds et al., 2004).
Off-Target Effects of RNA Interference Some studies have shown that as few as 11 contiguous matches between siRNA and the off-target mRNA can result in silencing of protein production (Jackson et al., 2003). In one experiment 16 siRNAs were designed to target the same specific coding region and the expression profiles of each siRNA, then compared. As well as silencing the target gene, it was found that each siRNA showed specific, repeatable, off-target gene silencing with only a small number of gene regulations in common, i.e., the off-target effects were specific to the siRNA, not the target (Jackson et al., 2003). Further work with siRNA targeting luciferase (an exogenous gene) in vivo showed repeatable regulation of off-target endogenous gene expression that also showed a dose-dependent response (Jackson et al., 2003). Some domains of siRNAs appear to be more susceptible to off-target effects than others, and attempts have been made to identify specific patterns that might predict this (Jackson et al., 2003; Du et al., 2005). In the latter study, both the position of the mismatched base pair and the nucleotides involved were shown to influence silencing ability. Substitution of nucleotides at the 3' and 5' termini still resulted in knockdown of the off-target gene, whereas centrally located mismatches were poorly tolerated and gene silencing was abolished. Nucleotides involved in base pairing wobble were Guanine:Uracil (G:U) and Adenine:Cytosine (A:C) (instead of G:C and A:T respectively). These mismatches were well tolerated and resulted in efficient off- target silencing (Du et al., 2005). To reduce the likelihood of off-target effects and identify more unique oligonucleotides, it is suggested to use more sensitive algorithms than the BLAST database, e.g., Smith and Waterman or siDirect (Naito et al., 2004; Reynolds et al., 2004; Snove et al., 2004). The design of such siRNAs has recently been described as one of the "hottest topics in molecular biology" (Yamada et al., 2004).
Other Forms of RNA Interference
Micro (mi)–RNAs
Piwi-Interacting (pi) RNAs
Short-Hairpin (sh) RNAs
Small Modulatory (sm)RNAs
Whether in vitro or in vivo, the RNA interference mechanism can only be initiated once the siRNA has been transported into the cytoplasm of the cell. A successful transfection requires that the siRNA molecule, which carries a net negative charge under normal physiological conditions, must come into contact with and cross a cell membrane that also carries a net negative charge. Several methods of siRNA transfection have been developed for use in vitro and in vivo.
In vitro Transfection Mechanisms and Protocols The most basic approach involves the addition of naked siRNA directly to the cells without the use of a transfection reagent. This was attempted in neurons by Lingor et al. (2004). The siRNA was shown to enter the endosomal compartment of the cells when left in suspension (identified with fluorescent tagging) but its release into the cytoplasm while still fully functional was not demonstrated. Therefore, to ensure functional delivery, the siRNA must either be forced into the cells under high pressure (microinjection) or enter via micropores formed by affecting the charge of the cell membrane with electric pulses (electroporation) (Ambion technotes). High levels of cytotoxicity associated with these methods means that they are generally reserved for the more difficult to transfect cell lines e.g., neurons (McManus et al., 2002; Ambion technotes, undated).
Chemical Transfection Peak mRNA destruction is said to occur within 24–48 hours transfection and is transient with an average duration of 2–3 days (Ambion technotes, undated). The exact length of silencing will depend on the growth rate of the cell line, the level of gene expression and the half-life of the protein being knocked down. The development of other transfection reagents is ongoing. These include: Membrane permeant peptides, which are short amphipathic peptides that translocate across lipid bilayers in an energy-independent manner; Atecollagen, a highly purified, pepsin-treated type 1 collagen from calf dermis which is positively charged; and organic-inorganic hybrid nanoparticles which are poly(ethylene glycol)—block—poly (aspartic acid) with calcium phosphate. All of these have been used to successfully deliver siRNA in vitro (Kakizawa et al., 2004; Minakuchi et al., 2004; Muratovska and Eccles, 2004). There are a number of variables which are known to affect the transfection efficiency and these need to be optimized for each particular cell line. Cell density, concentrations of transfection reagent and siRNA, the time they are left together before adding to the well and the presence of antibiotics or serum in the cell medium will all influence the transfection rate. For example, when using Lipofectamine 2000 (a CL) a cell confluency of 30–50% is recommended and the Lipofectamine and diluted siRNA must be allowed to combine for 30 minutes to achieve optimal transfection efficiency (Dalby et al., 2004).
Vector-Mediated Delivery—Overcoming Transient Silencing In the second, the sense and antisense strands are expressed as a connected ribonucleic acid with several intermediate bases which form a stem loop structure (short hairpin (sh) RNA). This is then presented to Dicer for further cleavage to siRNAs and RISC presentation (Kobayashi et al., 2004). One of the first to describe such a plasmid was Brummelkamp et al. (2002). This particular vector was named pSUPER and it used an RNA polymerase III promotor to express short dsRNA in the form of an inverted repeat sequence containing a hairpin loop. The mammalian cells were transfected using electroporation methods and the targeted protein levels were suppressed for 5–7 days after 10 rounds of cell division. Comparison between these two methods has shown increased efficiency of silencing using shRNA production instead of the tandem type (Hutvagner et al., 2002). Retrovirus vectors have also been developed, and these use either oncoretrovirus or lentivirus vectors (Dyxhoorn et al., 2003). Lentivirus vectors have the added advantage of being able to infect both actively dividing and non-dividing cells. They are also capable of generating transgenic animals as they are resistant to proviral silencing during development (Dyxhoorn et al., 2003).
In vivo Transfection
Naked siRNA
Hydrodynamic Delivery of siRNA The exact mechanism behind the transfection of the gene/siRNA into the cells following hydrodynamic delivery is not fully understood. When delivered via the tail vein, it is suggested that as the injection rate exceeds cardiac output so the introduced fluid accumulates in the superior vena cava. This is then forced out into vessels within organs and subsequently through fenestrae in these vessels into extravascular spaces. The naked siRNA is then brought into contact with the cells of the organ before it is mixed with blood so reducing the chance of nuclease degradation (Hodges and Scheule, 2003). Zhang et al. (2004) further argued that actual transfection of the hepatocyte by this method is the result of "hydroporation." Membrane defects (pores) are generated following the high-pressure delivery and provide a route for introduction of the siRNA into the cell. Injection of a membrane permeability marker (Evans blue) showed that these pores closed up within 10 minutes, by which time the siRNA had entered the cytoplasm and the RNAi mechanism would be underway. Data regarding exact distribution of siRNA molecules following tail vein injection is scant, although initial experiments show a majority of the siRNA distribution is to the liver (Zhang et al., 2004). This can be explained by its proximity to the vena cava and ability to accommodate extra fluid. Following hydrodynamic injection in mice, various side effects have been recorded. The heart rate decreases from 510 beats per minute to 280 and there are transient irregular rhythms lasting for up to 60 seconds (Liu et al., 1999). A concurrent increase in QRS complex size also suggests increased cardiac chamber size. The liver expands and turns whitish soon after injection (Zhang et al., 2004). Clinical side effects included apnoea directly after injection, although gentle massage of the abdomen is said to resolve this (Hodges et al., 2003). This is accompanied by a transient, right–sided, congestive cardiac failure. Histopathological examination showed single cell liver necrosis in half the mice after one day (Liu et al., 1999). The body weight remained elevated for 30 minutes but was back to normal after 2.5 hours due to urination (Liu et al., 1999). Despite all these effects, the survival rate was reported to be 100%, and the method is being widely used for in vivo siRNA delivery in mice. Development of this method of delivery is in early stages. Ethical and animal welfare implications must also be considered as many would regard the intravenous injection of such high volumes of fluids over limited time periods unacceptable. Given the significant side effects, it is highly unlikely that this technique would ever be of use in humans. The use of naked siRNA has been further advanced by the use of stabilization modifications. Unmodified siRNA molecules are susceptible to serum nuclease degradation, and without effective modification they will be immediately degraded on in vivo administration. Czauderna et al. (2003) identified a successful stabilizing 2'-O-methyl modification, which managed not to compromise the efficacy of the siRNA molecule. Layzer et al. (2004) used stability modified 2'–fluoro pyrimidines siRNAs molecules and found over 50% to be intact after 24 hours of suspension in plasma. This compared with complete degradation of normal molecules within 4 hours. The modifications did not affect their silencing capabilities in vitro or in vivo and acted to increase their resistance to nuclease degradation in plasma.
Chemical Modifications Enhancing Transfection in vivo Their potential toxicity is highlighted in a lengthy review by Dass (2004) and includes the induction of acute inflammation following intraocular, intra-articular, and intra-tracheal administration and emboli formation from liposome/siRNA complexes and agglutination of red blood cells in response to their net positive charge. Transient acute toxicities of leukopenia, thrombocytopenia and increases in alanine transaminase, have also been reported (Tousignant et al., 2000). The development of second-generation molecules has helped, increasing stability in serum, improving target organ distribution and penetration, and helping the formation of a homogenous population of complexes with siRNA before injection (Sioud and Sorenson, 2003). siRNA molecules have also been stabilised for in vivo administration with cholesterol conjugation. This has been shown to increase resistance to degradation in vivo and to have good tissue distribution when injected via the tail vein after 24 hours. Using this technique, SiRNA has been detected in the liver, heart, kidney, lung, and adipose tissue (Soutschek et al., 2004). This method was shown to be successful in silencing an endogenous gene (apoB protein) in the liver and jejunum. Plasmid vectors have also been used for in vivo siRNA delivery (Brummelkamp et al., 2002). However, the relative ease of plasmid regeneration did not compensate for general poor transfection efficiency of the plasmid based vectors, and prompted the development of viral-/retroviral-based vectors for shRNA delivery. Vector systems that are based on the adenovirus are limited in that they do not integrate into the host genome (Tomar et al., 2003). In contrast integration of the shRNA expression cassette into the host genome can occur with lentiviral based vector systems, e.g., Moloney murine leukaemia virus, the Murine stem cell virus, and HIV-1 (Mittal, 2004). As transgenes expressed from these viruses are not silenced during development, these vector systems can also be used to generate transgenic animals through the delivery of shRNAs to embryonic stem cells of embryos (Rubinson et al., 2003).
Applications of RNA Interference in Pathology
RNAi in Pharmaceutical Target Validation and Toxicology
RNAi in Mechanistic Pathology
RNAi in Animal Models of Disease
RNAi as a Therapeutic Agent In both cases this was successful in reducing hepatocyte necrosis and inflammation, and protected the mice from future chronic fibrosis. In another experiment siRNA targeting Fas in the kidney was used to reduce ischaemia- reperfusion injury. Hydrodynamic and normal volume intrarenal delivery both successfully reduced Fas protein expression 4-fold. In addition, the pathology of the renal tissue following the ishaemic insult was reported to be much improved compared to the control (Hamar et al., 2004). Following this success one of the first examples of using RNAi to inhibit viruses in vivo was then attempted. Immuno-compromised and immunocompetant mice were hydrodynamically injected with plasmids expressing hepatitis B virus (HBV) and shRNAs targeting HBV. This resulted in a 99% reduction in HBV detection using antibodies to detect HBV core antigen by immunohistochemical methods, and it was suggested that RNAi could be used to treat viral disease in the future (McCaffrey et al., 2003). Since then, several experiments using RNA interference to target respiratory viruses have been attempted. Initially Influenza virus was chosen due to its significant public health issues and lack of a wholly effective vaccine (Tompkins et al., 2004). Proteins were targeted that are highly conserved across several sub types of influenza and which are essential for viral replication. It was found that combined iv hydrodynamic and intranasal (in a lipid carrier) delivery was most effective at specifically inhibiting virus replication at the site of infection. It also reduced lung virus titres in infected animals and protected animals against lethal challenge. Another experiment using slow iv delivery of siRNA complexed with a polycation carrier, and its delivery using DNA vectors iv/intra nasally, also showed a dose dependant reduction in virus production (Ge et al., 2004). Intranasal delivery was also found to be effective when targeting Parainfluenza virus and Respiratory Syncytial virus. Administration of siRNA with and without a vector (Transit TKO) was found to be both protective and therapeutic (Bitko et al., 2005). The ability to induce RNAi across mucosal surfaces is also being explored as a means of treating sexually transmitted disease. Intravaginal delivery of RNAi targeting 2 viral genes have been shown to protect the mice from the otherwise lethal Herpes simplex virus-2 (Palliser et al., 2006). RNAi has also been used to alleviate joint inflammation in experimental animals. siRNA targeting tumor necrosis factor alpha was injected into the knee joints of mice with collagen-induced arthritis (CIA). This was followed by electoporation. The development of arthritis was scored by assessing the inflammation of joints in the mouse paw, and in mice with CIA, joint inflammation was successfully inhibited (Schiffelers et al., 2005). Finally, diminished pain responses have been observed in rats following intrathecal delivery of an siRNA directed against the pain related cation channel P2X3 (Dorn et al., 2004).
The field of RNAi is still rapidly expanding and new discoveries are being made on a daily basis. SiRNAs are currently being used in gene function analysis, target identification and validation and as therapeutic agents. Their potential for use in evaluating target toxicity is significant and warrants further investigation. Although a viable technique for in vitro experimentation, success can still be hampered by problems with intracellular siRNA delivery and effective gene silencing. Low transfection efficiency and excessive cytotoxicity are frequently encountered, and the development of an in vitro transfection method with an improved ratio of transfection to efficacy is a priority. For example, controlled forced intracellular delivery of siRNA by combining nucleotides with magnetic nanoparticles and applying a pulsed magnetic field could reduce the level of cytotoxicity and increase transfection efficiency above that achieved with electroporation of cells exposed to naked nucleic acids. This area is likely to see novel development in the near future. Once delivered to the cell, effective knock-down of protein activity in the cell is influenced by the half-life of the protein under investigation. As RNAi inhibits novel protein production, long half-life proteins may maintain activity for long periods after effective gene silencing. Knowledge of protein half-life is helpful when planning and interpreting RNAi knock-down experiments. Deliveries of RNAi in viral vectors, particularly those that integrate into host genome, allow a prolonged knockdown of protein production. The short-lived nature of inhibition may be of advantage, however, when used as a therapeutic (to allow for rapid reversal following adverse events). Demonstration of effective gene silencing relies on the ability to detect both a reduction in mRNA and protein levels of the specified target in tissues – possibly in limited biopsy material. The availability of reagents to accurately measure reduced protein production may provide a further challenge in the interpretation of experimental results. The transient nature of the inhibition of protein production is of particular concern in dividing cells, where cell division typically results in the loss of RNAi activity. This is of particular concern in vitro, where cell division rates are typically high. Certain in vivo organ systems, for example the gastrointestinal tract, also have high endogenous rates of division and gene silencing in these organ systems would be particularly challenging. Virally mediated integration of RNAi sequences into stem cell would overcome this issue, but would lead to a permanent knock down of protein expression, which may be of advantage for studying in vitro systems, but would carry liability as a therapeutic when adverse events were encountered. The development of efficient in vivo delivery of RNAi is remains problematic. The paucity of publications describing successful in vivo delivery either suggests that either this is technologically demanding or else systems are being developed confidentially to ensure commercial exploitation. Efficient and effective in vivo transient and specific protein inhibition using RNAi clearly has significant potential to generate many therapeutics with associated financial reward. The search for a transfection method that achieves such efficient delivery of siRNA to a majority of body cell types and avoids significant toxicity is a demanding task. The current "gold standard" of hydrodynamic delivery raises issues of adverse events associated with the delivery system. New developments in the area of in vivo delivery are anticipated. Finally, the cost or production of RNAi molecules has been a hurdle for the use, particularly the quantities required for in vivo studies. The cost of RNAi molecules has reduced significantly in recent years, and it appears that this is a trend which will continue. Hopefully, cost should not the barrier to use of this valuable tool. In conclusion, the existing hype and excitement surrounding this mechanism of posttranscriptional gene silencing is justified, but experiments must now concentrate on in vivo validation of the technique to allow the path of discovery to continue.
Toxicologic Pathology, Vol. 35, No. 3,
327-336 (2007)
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Abstract
Introduction
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