Potential clinical applications of siRNA technique: benefits and limitations


Getu Zhaori, The Editorial Office, Chinese Medical Journal, English Edition, 42 Dongsi Xidajie, Beijing 100710, China. Tel.:/fax: +8610 64 116960; e-mail: zhaorig@126.com


Eur J Clin Invest 2011; 41 (2): 221–232


Background  RNA interference (RNAi) has become the method of choice for researchers wishing to target specific genes for silencing and has provided immense potential as therapeutic tools. This narrative review article aimed to understand potential benefits and limitations of RNAi technique for clinical application and in vivo studies through reading the articles published during the recent 3 years.

Materials and methods  Medline database was searched by using ‘siRNA’ or ‘RNAi’ and ‘in vivo’ with limits of dates ‘published in the last 3 years’, language ‘English’ and article type ‘clinical trial’ for obtaining articles on in vivo studies on the use of RNAi technique. Characteristics of clinical trials on siRNA registered at the http://www.ClinicalTrials.gov were analysed.

Results  The only three clinical studies published so far and many in vivo studies in animals showed that the RNAi technique is safe and effective in treatment of cancers of many organ/systems and various other diseases including viral infection, arterial restenosis and some hereditary diseases with considerable benefits such as high specificity, many possible routes of administration and possibility of silencing multiple genes at the same time. Limitations and uncertainty include efficiency of cellular uptake, specific guidance to the target tissue or cell, long-term safety, sustained efficacy and rapid clearance from the body.

Conclusions  RNAi technique will become an important and potent weapon for fighting against various diseases. RNAi technique has benefits and limitations in its potential clinical applications. Overcoming the obstacles is still a formidable task.


RNA interference (RNAi), a natural phenomenon of sequence-specific gene silencing, had already been developed as a technology of silencing almost any selected gene that are widely studied for using in two major categories: investigating physiological and pathophysiological mechanisms and treatment of various diseases, including especially diseases for which there are no specific and very effective treatments, such as cancers, hereditary diseases and infectious diseases caused by pathogens for which there are neither vaccines nor very effective therapies, e.g., certain viruses, including respiratory syncytial virus (RSV), hepatitis C virus, human immunodeficiency virus, coxsackievirus and parasites, such as schistosoma. RNAi technology is believed to have a very good prospect for clinical application in the treatment of many diseases [1,2]. For such a clinically highly attractive technique, clinicians desperately wish to have this entirely new therapeutic approach in hand to combat many diseases that are currently hard to manage. Although thousands of papers on RNAi are published per year during the recent decade, there are only three published clinical trial reports, one of which was conducted on a single subject. Therefore, it may be still too early to talk about clinical application. However, we may understand some advances from reading reports on in vivo studies, because, when compared with in vitro studies, the in vivo studies are conducted in an environment that is much closer to that in the human body although the host animals used are mainly immunologically disabled mice.

The purpose of this article is to understand the potential benefits and limitations of RNAi technique for clinical application through reading literature and answering such questions as what diseases of what organ/systems have been frequently studied for potential application of RNAi technique, what are the major target genes for silencing, how were the effects of the RNA interfering agents (short interfering RNAs, siRNAs, or vectors expressing siRNA), how are the results of pharmacokinetic studies on siRNAs, are RNAi agents safe, etc. from the perspective of clinicians.

Sources of information

We used ‘small or short interfering RNA (siRNA)’or ‘RNA interference (RNAi)’or ‘small hairpin RNA (shRNA)’ and ‘in vivo’ as search terms with limits of type of article ‘clinical trial’, dates ‘published in the last 3 years’ and languages ‘English’ for obtaining articles on in vivo studies on the use of RNAi technique in the Medline database. Characteristics of clinical trials on siRNA registered at the http://www.ClinicalTrials.gov were also analysed.

As a result of the search for in vivo studies, 381 articles were displayed. After reading the abstracts of all these articles, it was finally found that only 89 of them were original studies that included in vivo investigations and the major purpose of the study was to test the efficacy and safety of siRNA, instead of using siRNA as one of the methods to study pathogenesis or mechanisms of diseases or other therapeutic approaches. Another search at Medline was performed for reports of clinical trials on siRNA.

We read the articles with the questions mentioned earlier in mind and provide possible answers based on the findings of the studies to the questions as follows.


Clinical trials

Published clinical studies on the use of siRNA technique.  The literature search at Medline by using ‘siRNA AND clinical AND trial’ as search terms with limits of article type ‘clinical trials’, species ‘human’ and language ‘English’ without any other limits, retrieved 21 articles, and it was found that only three of them were reports of clinical trials. One was a very recently published randomized, double-blind, placebo controlled trial on nasal spray of an siRNA (ALN-RSV01) against RSV applied in 85 of 88 adult volunteers before and after they were experimentally infected with wild type RSV [3]. The intranasal ALN-RSV01 showed a very good safety profile, and a significant decrease (38%, = 0·009) in the number of infected and a significant increase (95%) in the number of uninfected subjects. No rebound in acquisition of infection was observed. The authors also demonstrated that the antiviral effect of ALN-RSV01 was independent of pre-existing antibody to RSV and intranasal proinflammatory cytokines. No serious adverse events occurred. However, if a control group treated with a nonsequence-specific small RNA molecule which in molecular size similar to that of the ALN-RSV01 had been assigned, it might have been able to exclude the possibility of so-called ‘off-target’ or nonspecific effect [4,5]; on the other hand, if there had been a follow-up for a longer period of time, the concern about the possibility of later occurring side effects would have been dispelled.

The second published clinical trial was a study on treatment of a hereditary disease of skin, pachyonychia congenita (PC), caused by mutation of the gene K6a. This phase Ib clinical trial was conducted on a single patient. The therapeutic siRNA (TD101) was designed to silence the mutated K6a gene. The intradermal injection of the siRNA was performed on one side of the patient and the other side was used as control [6]. Interestingly, this was a double-blind, vehicle-controlled, dose-escalation trial. The regression of callus was seen on the siRNA-treated foot but not on the control foot. However, because of the nature of the disease treated, no systemic application of the siRNA was necessary; therefore, no evaluation of systemic effects or side effects of the siRNA was possible. On the other hand, although this study in a single patient was very well designed and showed a good therapeutic effect, further clinical studies of a certain sample size with a control group are needed.

The third and the most recently published clinical trial was a prospective, open-label, single-dose, dose-escalation phase 1 study, which aimed to assess the effect of Sirna-027, a small interfering RNA targeting vascular endothelial growth factor receptor-1, on treatment of choroidal neovascularization (CNV) resulting from neovascular age-related macular degeneration (AMD) [7]. Twenty-six eyes of 26 patients with a median age of 82 years and CNV resulting from AMD who had previous treatments with other therapies were treated at two academic retinal practices. Patients received intravitreal injection (IVT) of a single dose of Sirna-027 (100, 200, 400, 800, 1200, or 1600 μg per eye). The results showed that IVT of a single dose of Sirna-027 from 100 to 1600 μg was well tolerated in patients with AMD, with no dose-limiting toxicity found. Adverse events were mild to moderate in severity. Adjusted mean foveal thickness decreased within 2 weeks after the study treatment.

Clinical trials registered at http://www.ClinicalTrials.gov but unpublished.  The trend of clinical application of the RNAi technique may also be reflected by the status of ongoing or completed but unpublished clinical trials. The website http://www.ClinicalTrials.gov (Accessed on 1 August 2010) displayed 21 clinical studies on siRNA on a recent search at the Internet by using the search terms ‘siRNA OR RNAi OR shRNA’ (Table 1). The diseases these trials studied include AMD in four trials, neuroblastoma in 2, solid tumour in 4, renal disease in 2 (acute renal failure in 1 and kidney transplantation delayed graft function in 1), pachyonychia congenita in 1, metastatic melanoma in 1, chronic myeloid leukaemia in 1, diabetic macular oedema in 1, chronic optic nerve atrophy in 1, hypercholesterolemia in 1, pre-eclampsia in 1, and cell differentiation 1. If we classify these trials into subspecialties, six are on ophthalmology, four on oncology, two on nephrology. Of the 21 trials, eight were completed, three were terminated, six were recruiting, three were active but not recruiting and one is not yet recruiting. Of the eight completed trials, two were studies on AMD, one on pachyonychia, one on chronic myeloid leukaemia, one on diabetic macular oedema and two on neuroblastoma. Of the 21 clinical trials, 17 aimed to determine the clinical safety/tolerability and the dose-limiting toxicities and to characterize the pharmacokinetics (PK) of the siRNA. The other four shed light on the pathogenesis of the targeted genes and further determine whether the siRNA could be a target of gene therapy. Among the 17 clinical trials, there are two vaccines including a novel immune-based cancer therapy using gene-silenced dendritic cells and lentivirus-transduced T-cell immunotherapy by inducing three forms of anti-HIV RNA. Although some of the studies were completed even couple of years ago, the corresponding publications are still not available except for the study on pachyonychia congenita. As many as six of the clinical trials were on ophthalmologic disorders and one of the siRNA preparations used was bevasiranib, and its safety in treatment of AMD was demonstrated in both preclinical and clinical studies but its efficacy may need further studies [8,9].

Table 1.   Twenty-one clinical trials on siRNA registered at http://www.Clinicaltrials.gov
 ProductTargeted geneSponsorsLocationDisease/conditionRoute of administrationPhaseStage
  1. IV, intravenous injecton; IVT, intravitreal injection; AMD, age-related macular degeneration; CNV, choroidal neovascularization; VEGF, vascular endothelial growth factor.

 1TD101Keratins, K6aPachyonychia Congenita ProjectUSAPachyonychia congenitaIntradermalPhase ICompleted
 2Sirna-027VEGF receptor-1Allergan|Sirna Therapeutics Inc.USAAMD/CNVIVTPhase I/Phase IICompleted
 3AGN211745VEGF receptor-1AllerganUSAAMDIVTPhase IITerminated
 4Proteasome siRNA and tumour antigen RNA-transfected dendritic cellsImmunoproteasomeDuke UniversityUSAMelanomaVaccinationPhase IRecruiting
 5SV40 vectors carrying siRNATyrosine kinaseHadassah Medical OrganizationIsraelChronic myeloid leukaemia (CML) Not knownCompleted
 6CALAA-01M2 subunit of ribonucleotide reductaseCalando PharmaceuticalsUSASolid tumour cancersIVPhase IRecruiting
 7Atu027An siRNA formulationSilence Therapeutics AGGermanyAdvanced solid cancerIVPhase IRecruiting
 8BevasiranibVEGFOpko Health, Inc.USADiabetic macular oedemaIVTPhase IICompleted
 9QPI-1007Not foundQuark PharmaceuticalsUSANeuroblastomaIVTPhase IRecruiting
10PRO-040201Not foundTekmira Pharmaceuticals CorporationUSAHypercholesterolemia Phase ITerminated
11IL-10National Taiwan University HospitalTaiwanPreeclampsia Not knownTerminated
12AHR siRNAAromatic hydrocarbon receptorNational Taiwan University HospitalTaiwanNeuroblastoma Not knownCompleted
13Bevasiranib/ranibizumabVEGFOpko Health, IncNot knownAMDIVTPhase IIINot yet recruiting
14I5NPNot foundQuark PharmaceuticalsUSAKidney injury/acute renal failure undergoing major cardiovascular surgeryIVPhase IActive, not recruiting
15I5NPNot foundQuark PharmaceuticalsUSADelayed Graft Function/Kidney TransplantIVPhase I/IIActive, not recruiting
16TBX3TBX3University of California, IrvineUSAHuman ES cell differentiation Not knownActive, not recruiting
17GlycosyltransferasesNational Taiwan University HospitalTaiwanNeuroblastoma Not knownCompleted
18BevasiranibVEGFOpko Health, Inc.USAWet AMDIVTPhase IICompleted
19pHIV7-shI-TAR-CCR5RZ treated CD4 cellsHIV-1 tat/rev (shI), a decoy for HIV TAT-activated RNA (TAR), and a ribozyme that targets the host T cell CCR5 cytokine receptor (CCR5RZ)City of Hope Medical CenterUSAHIV-1 infections Phase 0Recruiting
20ALN-VSP02Not foundAlnylam PharmaceuticalsUSASolid tumoursIVPhase IRecruiting
21Autologous FANG vaccineTGFβ1 and TGFβ2Gradalis, Inc.USASolid tumoursVaccinationPhase IRecruiting

Diseases investigated in the in vivo studies

Many clinicians must be interested in knowing in what particular fields of clinical medicine the RNAi technique is most frequently studied and will probably be clinically applied soon. Although this technique of sequence-specific gene silencing has been studied in various fields of medicine, cancer is the most frequently studied field. Of the 89 reports on in vivo investigations, 54 (60·1%) were studies on cancer. Among these studies on cancer, seven were on breast cancer, four were on pancreatic cancer, four on prostate cancer, four on gynecological cancers, three on lung cancer, three on gastric cancer, three on colorectal cancer, two on leukaemia, two on meningioma, two on head and neck cancer, one on hepatocellular carcinoma, one on glioma and very few studies on melanoma, multiple myeloma, neuroblastoma, etc.

Of the studies on noncancer diseases, three were on diabetes, two on restenosis of artery after balloon injury, two on pain, one on hepatitis B, one on hepatitis C, one on RSV infection and one on Schistosoma japanicum. No more than one report which included in vivo study on siRNA technique was found for each of the following diseases/conditions: Huntington disease, myocardial ischaemia–reperfusion injury, Schistosoma mansoni, pachyonychia congenita, and collagen-induced arthritis. Ten of the 89 reports were on studies on delivery of siRNA.

Target genes used for treatment of cancer

For the studies on treatment of cancers with RNAi technique, what are the most frequently targeted genes? What issues were found to be the most important?

Development of cancer, growth, invasion, angiogenesis and metastasis of cancer cells involve molecules with various functions (oncogenes, proteins of different functions, including enzymes, cell cycle regulators, cytokines, etc.) and molecular mechanisms in the body. It is interesting to see that for the same cancers, silencing of many different target genes of proteins has been studied and the effects of the treatment approaches are encouraging. Examples of such proteins for breast cancer include serine protease urokinase-type plasminogen activator (uPA), matrix metalloproteinases (MMPs) [10], procathepsin D (pCD), a zymogen of lysosomal aspartic proteinase cathepsin D [11], cyclooxygenase-2 (COX-2) [12], Twist1, the master regulator of epithelial-mesenchymal transition [13] and Stat3, a member of the signal transducer and activator of transcription family [14].

Many of the clinicians may not be very much interested in knowing the detailed mechanisms of genes involved in development, growth, metastasis of cancers; however, they may be interested in knowing which genes are most frequently studied for the treatment of cancers with siRNA. Selection of appropriate target genes is the most important event in the potential success of siRNA cancer therapies. Candidate targets include genes associated with cell proliferation, invasion, metastasis, angiogenesis, and drug resistance, and preferably the protein products of such genes are expressed only in cancer cells. Furthermore, silencing of such genes must not affect the normal functions of normal cells. Survivin is identified as one of the most cancer-specific proteins. Biologically, survivin has been shown to inhibit apoptosis, enhance proliferation and promote angiogenesis and it is upregulated in almost all human tumours. Because of its upregulation in malignancy and its key role played in apoptosis, proliferation and angiogenesis, survivin is currently attracting considerable attention as a new target for anticancer therapies [15]. Survivin gene was used as target of silencing in hepatocellular carcinoma [16], gastric cancer [17], colorectal cancer [18], prostate cancer [19], and bladder cancer [20] and showed significant inhibitory effects on growth, invasiveness and metastasis of cancers. Significant inhibitory effects on cancer cells and cancer growth may be one of the reasons why the authors selected survivin and the other genes reported by these articles. As siRNA technique is a sequence-specific gene silencing technique, it may not be suitable for targeting cancer-inhibiting genes, such as P51 or P21. The transition of the above-mentioned findings to the clinic is therefore rational and currently ongoing.

STAT proteins play a central role in determining whether immune responses in the tumour microenvironment inhibit or promote cancer. Persistently activated STAT3 increases tumour cell proliferation, survival and invasion and mediates tumour-promoting inflammation while suppressing antitumour immunity. Consequently, STAT3 has dual role in tumour inflammation and immunity and therefore STAT3 is a promising target to redirect inflammation for cancer therapy [21].STAT3 gene was used as target of silencing for glioma [22], T-cell leukaemia [23] and breast cancer [14], and the results showed efficient therapy.

Angiogenesis is fundamental to the process of tumour growth, invasion and metastasis, and it has been targeted effectively with pharmacologic strategies. The vascular endothelial growth factor (VEGF) family of ligands and receptors are well established as key regulators of these processes. Activated VEGF-receptor pathway results in signalling cascades that promote endothelial cell growth, migration, differentiation and survival from pre-existing vasculature [24]. Targeting angiogenesis has been validated in cancer therapy. Although only 2 of the 54 in vivo studies set VEGF gene as target for silencing (one for nonspecified cancer [25] and the other for bladder cancer [20]), silencing of this gene may be needed in the treatment of most of cancers. A study on siRNA targeted against VEGF-C alone provides convincing evidence, which showed significantly reduced number of lymphatics and growth of colorectal cancer in mice model [26].

Because the growth, migration, invasion and metastasis of cancers involve many mechanisms, some of the researchers designed a few siRNAs for silencing two or more target genes and obtained good tumour inhibitory effects in the suppression of growth and metastasis of cancers. Tummalapalli et al. [27] used cathepsin B and MMP-9 genes as targets for malignant meningioma and observed a significant regression of pre-established orthotopic tumours. Shi et al. [28] constructed recombinant vector plasmids that could express siRNAs targeted to silence either K-ras or Akt2 alone or both genes simultaneously and tested the plasmids in vitro in Panc-1 cells as well as in vivo on Panc-1 tumour in nude mice to see whether synergistic effects can be achieved by combined silencing of the two oncogenes. The results of both in vitro and in vivo experiments showed that simultaneous silencing of the two genes did have a synergistic effect in the inhibition of the tumour cell growth and the combined silencing was more effective and efficient than silencing either oncogene alone. This is probably one of the major benefits of RNAi technique in treatment of cancer, which allows inhibition of multiple oncogenes or genes of relevant proteins that are involved in the development of cancers; therefore, it is possible to develop double-specific or multiple-specific RNAi therapeutic approaches not only for cancers but may also for other diseases.

It is difficult to find out which RNAi agents against particular target gene(s) have better therapeutic effects because there are almost no studies that compared the effects of RNAi targeting different genes.

Routes of administration of the siRNA Agents

Researchers selected one or more of the genes of the proteins that play important roles in development, growth and metastasis as targets for silencing and designed accordingly the siRNAs or shRNAs, tested their silencing effect in vitro, and on the basis of the in vitro evidences of effectiveness, the researchers conducted in vivo animal studies and the majority of which were conducted in nude mice or mice with severe combined immunodeficiency (SCID mice). The cells of human cancer cell lines or the tissue of human cancer were transplanted to the mice. The siRNA or shRNA, usually carried by a certain vector (replication disabled viruses, such as adenovirus or lentivirus or liposomes or other types of carriers), is delivered through different paths, including systemic delivery through the tail vein (TV), high-pressure or low-pressure tail vein injection (HPTV or LPTV), or local delivery through intraperitoneal (IP), intranasal (IN), intramuscular (IM), intratracheal (ITr), intrathecal (ITh), intratumoral (ITu), intraliver (IL), intraretinal (IR), intratesticular (ITe) or subcutaneous (SC) administration even inhalation and other routes. Currently, siRNAs in most of clinical trials are directly administered to local target sites such as the eye, the skin and the lung (for which the siRNA was administered by means of inhalation), thereby avoiding the complexity of systemic delivery, which is an advantage. However, most tissues are not easily accessible and for many cancers, such as multiple diffuse type liver and pancreatic cancers, intracranial cancers, direct injection is very difficult or impossible. Therefore, the development of effective and safe systemic delivery approaches is necessary.

The optimal in vivo systemic delivery systems should be biodegradable, biocompatible and nonimmunogenic. Furthermore, the systems should provide efficient delivery of active double-stranded siRNA into target cells or tissues. Finally, the delivery systems must provide target tissue-specific distribution after systemic administration, avoiding rapid hepatic or renal clearance. Although there are a few reports of new siRNA delivery system (including amino-functionalized multiwalled carbon nanotubes (MWNT-NHt3)[29,30], only the reports of Zheng et al.[31] and Xia et al.[32] represent the development of antibody ‘guided’ delivery of siRNA to specific cell or tissue. The results of Zheng et al.’s study showed that the siRNA conjugated with monoclonal antibody accumulated in the spleen and lymph nodes where there are plenty of dendritic cells after early concentration in the liver and was not cleared by the kidney as soon as most of the unconjugated siRNA did. The expression of the CD40, the product of the target gene, on dendritic cells was significantly reduced by the antibody-guided siRNA. Xia et al. used a monoclonal antibody to human insulin receptor bridged with siRNA via biotin and avidin. The siRNA was effectively delivered to the cytosol of cells with the receptors, and the expression of the target gene was reduced by more than 90%. Both studies included various control experiments that support cell-specific delivery of the siRNAs and strong silencing of the target genes. Therefore, cancer cell-specific or tissue-specific monoclonal antibody conjugated siRNA delivery system should be further and widely studied. The possibility of administering siRNA via multiple routes is a great benefit of this technique when compared to many chemotherapeutic agents that can only be given through IV injection or infusion.

In vivo effects of siRNAs in treatment of cancer

Although numerous studies have demonstrated the sequence-specific gene silencing effects of RNAi technique, these effects must be tested in the bodies of animals as well as in human body for proof of the efficacy and safety of the technique. The in vivo animal studies for such purposes described in the literature published during the recent few years mostly used nude mice or SCID mice to allow the xenograft tumours to form, grow and progress without being rejected, and on the other hand to provide an environment for testing the efficacy and safety of the RNAi constructs or synthesized siRNAs or shRNAs. Such an in vivo environment is very important, because there are many anatomical and physiological features in common between human being and the nude or SCID mice, except for immune functions which are almost completely absent in those mice. The researchers make model of various human tumours in these mice by inoculating or implanting human cancer cell lines or tumour cells into a corresponding organ or tissue where the tumours originally develop in human body (orthotopic) or a site where the tumours do not usually develop primarily in human body (ectopic). The researchers apply different methods to measure the growth of the tumour, measure the volume and weight postmortem or estimate the size of tumour by using imaging techniques. In many studies, the researchers did histopathological examinations of the tumours or any part of the body. Immunohistochemical staining is often used for understanding changes in the expression of proteins of particular interests. The following are examples describing the effects of a few studies on the treatment of cancers and other types of diseases. Only a few studies included extensive parameters for safety issues, for example, tests for liver and kidney functions, histopathological changes of the major organs.

Kunigal et al. [14] demonstrated that siRNA targeting Stat3 (pRNAi-Stat3) significantly suppressed tumour growth compared with controls in nude mice orthotopic breast tumour model. Treatment with the pRNAi-Stat3 for 3 weeks reduced the tumour size by more than 60% compared to the control and vector treatment, and 6 weeks after treatment with the siRNA, complete regression of the tumour occurred. The authors demonstrated that the tumour regression was mainly because of the apoptosis of the tumour cells.

Some of the studies explored the effect of combined treatment of cancer with chemotherapy and RNAi approach. Li et al. [13] demonstrated in an athymic mice breast cancer model that combined use of adriamycin with twist1-targeted RNAi agent (pSilencer-twist1) could significantly reduce the volume and weight of the tumour by 76·8% and 78·2%, respectively. The survival (110 days) of the mice with the tumour treated with combined therapy was significantly longer than those treated with either drug alone (77 and 48 days).

In the in vivo study of Kunigal et al. [10], the vectors expressing siRNAs targeting uPA receptor (puPAR) and MMP9 (pMMP-9) reduced the growth of the tumour significantly by 70% and 60%, respectively, while the siRNA targeting both uPAR and MMP-9 (pUM) could cause total regression of the tumour.

In a study on meningioma, Tummalapalli et al. [33] used the same RNAi preparations as those used by Kunigal et al. to study their effects in the treatment of meningioma that was grown intracranially in nude mice by inoculating IOMM-Lee cells. Compared with the controls, the tumour size in the mice of pUM-treated group showed almost complete regression of the tumour, but brain sections of mice in pUR or pM group showed only about 50% regression of the tumour. Importantly, the effects of the treatments in this study were tested in pre-established tumour, which as a model may be closer to the clinical situations when compared to some studies where the researchers use cells of cancer cell lines which were transfected or pretreated with certain constructs that express siRNA or control RNA and then injected or infused into the nude mice (See below).

In contrast to the above-mentioned study on pre-established tumour, Ohri et al. [11] studied the effects of siRNA targeted procathepsin D (pCD) in metastasis of breast cancer by intravenous infusion of MDA-MB-231 cells either transfected or not transfected with siRNA targeting pCD. All the animals injected with controls cells or negative control shRNA developed colonies in various organ/tissues and all died; however, of those injected with specific RNAi preparations (shRNA-6 and -16), only 10–20% developed colonies and the survival rate was 100%. This study suggested that the shRNAs could effectively suppress these cancer cells and prevent metastasis from occurring at least through blood circulation.

For lung cancer, Ren et al. [34] reported a study that included in vivo experiments in BALB/c athymic mice on the effects of plasmid expressing siRNA for silencing HER2/neu oncogene in treatment of nonsmall cell lung cancer (NSCLC). The HER2/neu-targeted siRNA accounted for an inhibition of 50% in tumour volume and 43% in the ratio of tumour weight to mouse weight. Immunohistochemical analysis confirmed an almost complete blockade of HER2/neu expression in tumour tissues developed from tumour cells transfected with HER2/neu-specific siRNA.

More studies on lung cancer [35,36], gastric cancer [37,38], colorectal cancer [18,39,40], pancreatic cancer [28,41,42], prostate cancer [43], ovarian cancer [44,45] and many more showed similar results of the RNAi technique in partial or complete regression of tumour growth. Many of the studies had control siRNAs such as scrambled siRNA [11] or siRNA targeting an irrelevant gene and empty vector as controls, which are very important in confirming specificity of the technique.

It is important to point out that a few studies [16,46] focused on reduction/reversal of chemoresistance of certain cancers by using siRNA technique. In one of these studies, the half inhibitory concentration (IC50) of gemcitabine against a pancreatic cancer cell line was decreased 40-folds, and the tumour size in both was reduced by approximately 50% or more. The results of these studies suggest that siRNA technique has potential applicability in chemosensitization of certain cancers.

The in vivo studies on the treatment of cancers obtained very promising and encouraging effects that were evaluated comprehensively by using macroscopic, microscopic, immunohistochemical and imaging techniques. These results reflect success in many different aspects, including the selected target gene was relevant, the vehicle or vector for the delivery was appropriate and efficient, the route of administration was suitable, the siRNA/shRNA was finally delivered into the cells where the target genes locate and the dose of the siRNA/shRNA was sufficient to silence the target genes. Failure at any of these steps will not be able to suppress the growth of the tumour, downregulate the expression of the target genes, etc. The possibility of nonspecific or off-target effects that presumably can also have some effects similar to what was primarily expected to achieve or demonstrate by different controls.

It is very interesting to know how the RNAi techniques (e.g., HER2/neu-targeted siRNA for breast cancer) should be evaluated when they are compared with the existing chemotherapy or targeted therapy (e.g., trastuzumab for breast cancer). However, no reports on studies that compared these two techniques either clinically or in animal experiments. There are some reports describing side effects, including cardiac toxicity of trastuzumab in clinical studies [47].

Studies on siRNA targeting genes of infectious agents

Viruses, especially those for which there are no specifically effective methods of prevention and treatment, have been studied as targets of the RNAi technique. Among the in vivo studies we retrieved, there are four reports on infectious diseases, including hepatitis C virus (HCV, 1), hepatitis B virus (HBV, 1), RSV (1), coxsackievirus (1) and Schistosoma japonicum (1). The in vivo study in transgenic mice that can be induced to express HCV structural proteins [48] showed that intravenous injection of the shRNA targeting the viral gene could specifically suppress viral protein expression in the liver of the mice. The study on the treatment of HBV infection in ICR mice [49] showed that intravenous injection of specific endoribonuclease prepared siRNA (esiRNAs) could dose-dependently and significantly reduced HBsAg secretion by 88% and 92%, respectively. The in vivo study on the treatment of RSV infection in BALB/C mice [50] showed that ALN-RSV01, the same siRNA against the N protein gene of the virus used in the clinical trials mentioned earlier, showed a dose-dependent inhibition of viral replication in the lung, reduction in viral titre by 2·5–3·0 Log10 PFU g−1 lung tissue. Interestingly, the authors of that study observed that ALN-RSV01 given at lower dose for multiple times per day was significantly better than higher dose given once a day. There has been no effective antiviral therapy for coxsackievirus-caused myocarditis. Fechner et al. [51] developed an shRNA targeting RNA polymerase of coxsackievirus B3 and generated an AAV vector-based complex AAV2.9-shRdRp. Intravenous treatment with this shRNA of coxsackievirus B3-induced myocarditis in mice significantly improved cardiac function.

Studies on cardiological and other diseases

Coronary artery restenosis is a common problem that occurs after percutaneous coronary intervention (PCI). Studies by Hlawaty et al. [52,53] showed that siRNA targeting MMP-2 injected into carotid artery of rabbits could significantly reduce the expression of MMP-2. A study by Natarajan et al. [54] demonstrated that silencing of prolyl-4 hydroxylase-2 (P4HA2) gene by siRNA significantly reduced chemokines and ICAM-1 expression when compared with the controls in postischaemic hearts, which reduced the infiltration of polymorphonuclear leucocytes and myocardial infarct size (> 60%).

Other diseases/conditions studied for the treatment with RNAi technique included diabetes, alpha-1 antitrypsin deficiency, Huntington disease, pain, etc. The fields where RNAi technique can potentially be applied are very broad.

Delivery of siRNA preparations in vivo

The ‘packaging’ and delivery of siRNA or shRNA have been a focus of studies on this technique, because these small molecules are vulnerable to being destroyed by the nuclease existing in blood or other body fluids, and they will be able to function as a therapeutic only when their molecules arrive at sites where the cells bearing the target genes are. Therefore, the first important issue is how to pack and protect the molecules effectively without affecting their activities. The most commonly used routes of delivery among the in vivo studies are intravenous, subcutaneous, intratumoral and intraperitoneal injections, although a few studies used inhalation of aerosolized siRNA, intracranial injection, etc. For injection through different routes, the commonly used vehicles include viruses, liposome, cationic polymers and nonionic polymers, etc. The recent advances in studies on vehicles for siRNA delivery will provide more efficient delivery of siRNA and shRNA. The in vivo experiments of delivering siRNA by using liposome vehicle by Mikhaylova et al. [12] were performed in SCID mice subcutaneously inoculated with cell suspension of human breast cancer cell line MDA-MB-231 in an extremely delicate study. The siRNA targeting against COX-2 incorporated into liposome DDP intravenously injected to the mice showed intratumour delivery.

A new generation of nucleic acid carrier, bioresponsive endosomolytic PEG-PLLDMMAn-Mel siRNA conjugate developed by Meyer et al. [29], an amino-functionalized multiwalled carbon nanotubes (MWNT-NHt3) reported by Podesta et al. [30], and other studies will provide more efficient new delivery methods. Vehicles using CD40 siRNA containing immunoliposomes (siILs) [31] even can specifically deliver the siRNA into dendritic cells, and combination of a receptor-specific targeting ligand, such as the monoclonal antibody against human insulin receptor (HIRMAb) and avidin–biotin technology, allows for high-affinity capture of the monobiotinylated siRNA by the targeting mAb [32]. Such techniques will make cell- or tissue-specific delivery of the siRNAs possible clinically. The benefits and limitations of different siRNA delivery systems are shown in Table 2.

Table 2.   Benefits and limitations of different siRNA delivery systems
TechniquesiRNA delivery systemsBenefitLimitations
Viral vectorRetivirus
Adeno-associated virus
High efficencicy and reproducibility
Delivery into primary and nondividing cells
Compatible for in vivo dilivery stable expression
Generation and titration of virus paritcles
TransfectionLipid-based siRNA delivery
Polymer-mediated siRNA delivery
Conjugates and targeted delivery of siRNA
High efficencicy and reproducibility
Easy to use in most cell type
Lack of cell-type specificity
With toxicity associated with lipid-based siRNA delivery
Immune response
Nonspecificity of targeting
Particle size control
ElectroperforationVoltage pulseNoninvasive
Without nonspecific immune stimulations
Pulse conditions need to be optimized
Cell damages in the pulse-applied sites
Chemical modificationChemical modificationModified siRNA showed a better resistance to nuclease degradation than unmodified siRNAModified RNAi activity was reduced
Unsafe molecules was maybe produced by the degradation of a modified siRNA.

Pharmacokinetic and biodistribution studies

Thorough pharmacokinetic studies are necessary for any new drug before its clinical use because such studies provides basis for dosage profile, length of treatment course and many other important clinically relevant issues such as stability and storage. It seems that there are no many pharmacokinetic studies for naked or various vector-based siRNA or shRNA or RNAi constructs. Recent pharmacokinetic studies reported by Malek et al. [55] and Merkel et al. [56] provide valuable data of pharmacokinetics and biodistribution of PEI (PEG)- siRNA complex formulations. Radioactively (32P) labelled polyethylenimine (PEI) complexed siRNAs was intravenously injected in Malek’s study. The results showed that major site of biodistribution were liver, lung, spleen and kidney, and such study was also helpful in choosing more promising PEI. In the study by Merkel et al., the gamma-emitter (111In or 99mTc) labelled siRNA complexes were intravenously injected to BALB/C mice, and single photon emission computed tomography (SPECT) imaging was performed. Dual isotope SPECT and gamma camera imaging are very useful to follow biodistribution of polyplexes especially for kinetics of distribution.

Since there are and will be many more different formulations which all need in-depth pharmacokinetic and biodistribution studies. Lack of such studies may be one of the limitations in clinical application of RNAi technique. Therefore, how to deal with issues of absorption, distribution, metabolism and excretion of siRNA are significant obstacles.


Many researchers currently focus on the discovery and development of therapeutics using the principle of RNAi for the treatment of a variety of diseases. The promise of RNAi, as a powerful new approach for treatment of diseases, has propelled early-stage clinical testing of siRNAs for many diseases. No siRNA drug has been approved by FDA although the three clinical trials of siRNA showed safe and no patient experienced a serious adverse event. Over the recent years, weaknesses in the field of RNAi have been surfaced. The RNAi technology can silence both the target gene and the other genes with a similar enough sequence. Not only is there that off-target issue, but siRNAs are prone to activating the immune system to respond. Minimizing the off-target effects of RNAi therapeutics is critical for controlling unwanted side effect and potential safety concerns. Overcoming the obstacles for achieving this is still a formidable task.


In conclusion, in vivo studies on application of RNAi technique published during the recent few years have shown great achievements in many diseases, especially in studies on cancers of many different organ/systems. New generation of delivery system for siRNA has been developed; ‘guided’ delivery of siRNA became possible. Pharmacokinetic studies, although few in number at present, have set important paradigm for further investigations. Other problems that have to be solved before the techniques of RNAi are clinically used include the following: toxicity and safety, e.g., does an siRNA has off-target effect? Does it silence any gene(s) that should not be silenced? How are the vehicles and siRNA/shRNA metabolized? Are their intermediate metabolites toxic or harmful to the body? Many of these questions have not been solved; therefore, these are also some transient limitations of the technique.

Since there are already a couple of reports on clinical trials on application of siRNA for the treatment of diseases and more are underway, RNAi technique will very soon become an important and potent weapon in the clinicians’ hands for fighting against various diseases/disorders that are currently hard to manage.

Sources of funding



Department of Gastroenterology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China (S.-H. Chen); The Editorial Office, Chinese Medical Journal, English Edition, 42 Dongsi Xidajie, Beijing 100710, China (G. Zhaori).