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) , procathepsin D (pCD), a zymogen of lysosomal aspartic proteinase cathepsin D , cyclooxygenase-2 (COX-2) , Twist1, the master regulator of epithelial-mesenchymal transition  and Stat3, a member of the signal transducer and activator of transcription family .
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 . Survivin gene was used as target of silencing in hepatocellular carcinoma , gastric cancer , colorectal cancer , prostate cancer , and bladder cancer  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 .STAT3 gene was used as target of silencing for glioma , T-cell leukaemia  and breast cancer , 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 . 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  and the other for bladder cancer ), 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 .
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.  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.  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. and Xia et al. 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.  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.  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. , 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.  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.  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.  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 , 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  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 .
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  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  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  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.  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.  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.  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. , an amino-functionalized multiwalled carbon nanotubes (MWNT-NHt3) reported by Podesta et al. , and other studies will provide more efficient new delivery methods. Vehicles using CD40 siRNA containing immunoliposomes (siILs)  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 . 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
|Technique||siRNA delivery systems||Benefit||Limitations|
|Viral vector||Retivirus Adeno-associated virus Lentivirus||High efficencicy and reproducibility Delivery into primary and nondividing cells Compatible for in vivo dilivery stable expression||Immunogenic Mutagenic Generation and titration of virus paritcles|
|Transfection||Lipid-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|
|Electroperforation||Voltage pulse||Noninvasive Without nonspecific immune stimulations||Pulse conditions need to be optimized Cell damages in the pulse-applied sites|
|Chemical modification||Chemical modification||Modified siRNA showed a better resistance to nuclease degradation than unmodified siRNA||Modified 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.  and Merkel et al.  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.