altered expression levels of certain genes play a 
 pivotal role in several pathological conditions . for example , 
 in many cancers the upregulation of 
 certain growth factors or growth factor receptors , or the 
 deregulation of intracellular signal transduction pathways , 
 represents key elements in the process of malignant transformation 
 and progression of normal cells towards tumor cells leading to 
 uncontrolled proliferation and decreased apoptosis . since these 
 processes may result in the direct , autocrine stimulation of the 
 tumor cell itself as well as the paracrine stimulation of other 
 cells , including the stimulation of tumor - angiogenesis , many novel 
 therapeutic strategies focus on the reversal of this effect , that 
 is , the inhibition of these proteins or the downregulation of 
 their expression . likewise , several other diseases have been 
 firmly linked to the ( over-)expression of endogenous wildtype or 
 mutated genes . taken together , in addition to strategies based on 
 the inhibition of target proteins , for example , by low molecular 
 weight inhibitors or inhibitory antibodies , this opens an avenue 
 to gene - targeting approaches aiming at decreased expression of the 
 respective gene . the first method to be introduced for the specific inhibition of 
 gene expression was the use of antisense oligonucleotides in the 
 late 1970s [ 1 , 
 2 ] . upon their introduction into a cell , antisense odns are able to hybridize to their target rna leading 
 to the degradation of the rna - dna hybrid double strands through 
 rnaase h , to the inhibition   of the translation of the target 
 mrna due to a steric or conformational obstacle for protein 
 translation and/or to the inhibition of correct splicing . in the 
 early 1980s , the discovery of ribozymes , that is , catalytically 
 active rnas which are able to sequence - specifically cleave a 
 target mrna , further expanded gene - targeting strategies 
 [ 35 ] . subsequently , both methods were extensively 
 studied and further developed with regard to the optimization of targeting 
 efficacies and antisense - odn / ribozyme delivery strategies in vitro and in vivo . most recently , another naturally occurring biological strategy for 
 gene silencing has been discovered and termed rna interference 
 ( rnai ) . since rnai represents a particularly powerful method for 
 specific gene silencing and is able to provide the relatively easy 
 ablation of the expression of any given target gene , it is now 
 commonly used as a tool in biological and biomedical research . this includes the rnai - mediated targeting in vitro and in vivo for 
 functional studies of various genes whose expression is known to 
 be upregulated as well as the development of novel therapeutic 
 approaches based on gene targeting . rnai is an evolutionarily conserved , sequence - specific , 
 posttranscriptional gene silencing phenomenon . it is triggered by 
 double - stranded rna molecules as described first in c elegans by 
 fire et al   who then introduced the name rna 
 interference . these findings also explained earlier observations 
 in petunias which turned white rather than purple upon the 
 introduction of the  purple gene  in form of dsrna 
 , and 
 on gene silencing by antisense oligonucleotides as well as by 
 sense oligonucleotides in c elegans . subsequent studies demonstrated that rnai , while described under different names 
 ( posttranscriptional gene silencing ( ptgs ) , cosuppression , 
 quelling ) , is present in most eukaryotic organisms with the 
 response to dsrna , however , being more complicated in higher organisms . rnai relies on a multistep intracellular pathway which can be 
 roughly divided into two phases , that is , the initiation phase and 
 the effector phase . in the initiation phase , double - stranded rna 
 molecules from endogenous or exogenous origin present in the cell 
 are processed through the cleavage activity of a ribonuclease 
 iii - type protein [ 912 ] into short   2123 nucleotide 
 fragments termed sirnas . these effector sirnas , which contain a 
 symmetric 2 nt overhang at the 3-end as well as a 
 5-phosphate and a 3-hydroxy group , are then in the effector 
 phase incorporated into a nuclease - containing multiprotein 
 complex called risc ( rna - induced silencing complex ) 
 . 
 several structural and biochemical studies have shed light on the 
 processing of double - stranded rna and the formation of the risc 
 complex ( see , eg ,   for a recent review ) . through 
 unwinding of the sirna duplex by an rna helicase activity 
 , this complex becomes activated 
 with the single - stranded sirna guiding the risc complex to its complementary target rna . 
 upon the binding of the sirna through hybridization to its target 
 mrna , the risc complex catalyses the endonucleolytical cleavage of 
 the mrna strand within the target site , which , due to the 
 generation of unprotected rna ends , results in the rapid 
 degradation of the mrna molecule . with the risc complex being recovered for further binding and cleavage cycles , the whole 
 process translates into a net reduction of the specific mrna 
 levels and hence into the decreased expression of the 
 corresponding gene . for an overview of the rnai pathway , see 
 figure 1 . while from this mechanism it becomes obvious that sirna molecules 
 complementary to the target mrna and thus being able to serve as a guide 
 sequence for the risc complex play a pivotal role in this process , they need 
 not be derived from long double - stranded precursor molecules . rather , 
 omitting the initiation phase , they can be delivered directly into the 
 target cell ( figure 1 , upper right arrow ) . several studies have led to the development of guidelines for the 
 generation of sirnas which are optimal in terms of efficacy and 
 specificity [ 12 , 
 16 ] . this includes the initial definition of 
 the preferable length ( 1925 bp ) combined with a low g / c 
 content in the range between 36% and 52% and the requirement 
 of symmetric 2 nt overhangs at the 3-end 
 [ 1618 ] . later studies on synthetic sirna molecules , however , revealed an 
 up to 100-fold higher targeting efficacy in the case of even 
 longer duplexes ( 2530 nucleotides ) which act as a substrate for 
 dicer and which therefore allow the direct incorporation of the 
 newly produced sirnas into the risc complex . as to be expected , intramolecular foldback structures which can result 
 from internal repeats or palindrome sequences decrease the numbers 
 of functional sirna molecules with silencing capability 
 . additional silencing - enhancing criteria include an a in position 3 
 and a g at position 13 of the sense strand , the absence of a c or 
 g at position 19 and , most importantly , a u in position 10 of the 
 sense strand . since nucleotides 1011 represent the site of the 
 risc - mediated cleavage of the target mrna , this indicates that 
 risc is comparable to most other endonucleases in preferentially 
 cleaving 3 of u rather than any other nucleotide 
 [ 20 , 21 ] . furthermore , it was shown more generally that the thermodynamic 
 flexibility of the positions 1519 of the sense strand 
 correlates with the silencing efficacy and that the presence of at 
 least one a / u base pair in this region improves sirna - mediated 
 silencing efficacy due to a decreased internal stability of its 
 3-end . still , different sirna sequences may display differing efficacies , which 
 suggest additional still unknown criteria for optimal sirna selection and 
 emphasize the influence of target mrna accessibility . in fact , several 
 studies also correlate the sirna efficacy with the mrna secondary structure 
 [ 18 , 2227 ] . in conclusion , apart from the selection criteria defined above , the 
 individual screening of different sirnas for highly efficient and specific 
 duplexes , or the pooling of multiple sirnas , is the most effective 
 approach to increase sirna - mediated targeting efficacy . for the design of effective sirnas , several algorithms on publicly 
 accessible web sites are available ( see  
 for review ) . to reduce the risk of nonspecific (  off - target  ) effects of the sirnas , a homology search of the targeting sequence against a gene database is 
 necessary and already incorporated in some of these web sites . nevertheless , 
 it has also been shown that sirnas may cross - react with targets of limited 
 sequence similarity when regions of partial sequence identity between the 
 target mrna and the sirna exist . in fact , in some cases regions comprising 
 of only 1115 contiguous nucleotides of sequence identity were sufficient to 
 induce gene silencing . an additional mechanism that may lead to nonspecific effects in 
 vivo relies on the interferon system [ 3033 ] 
 which is induced when double - stranded rna molecules enter a cell activating 
 a multi - component signalling complex . this effect is particularly 
 true for long dsrna molecules and essentially prevents them from 
 being used as inducers of rna interference in mammalian systems . the development of synthetic sirnas [ 10 , 
 12 , 33 , 
 34 ] largely 
 circumvents this problem since they seem to be too small . however , 
 some synthetic sirnas do induce components of the interferon 
 system which seems to be dependent on their sequence 
 [ 31 , 32 , 
 35 ] 
 as well as , in the case of in vitro transcribed sirnas , on the 5 
 initiating triphosphate . thus , 
 strategies to avoid as far as possible the unwanted interferon 
 response upon application of sirnas in vivo will include a design 
 of sirnas without known interferon - stimulating sequences , the use 
 of the lowest possible sirna dose to still achieve the desired 
 effect and optimized sirna delivery methods . based on the known mechanisms of antisense technology , ribozyme - targeting 
 or rnai , small oligonucleotides or plasmid - based expression vectors can be used 
 to specifically downregulate the expression of a given gene of interest or 
 of pathological relevance in vitro . in principle , this also applies to the 
 in vivo situation leading to novel , potentially relevant therapeutic approaches . for the delivery of therapeutic nucleic acids , viral vectors have 
 been used which have the advantage of high transfection efficacy 
 due to the inherent ability of viruses to transport genetic 
 material into cells . on the other hand , however , viral systems 
 show a limited loading capacity regarding that the genetic material are 
 rather difficult to produce in a larger scale and , most 
 importantly , pose severe safety risks due to their oncogenic 
 potential and their inflammatory and immunogenic effects which 
 prevent them from repeated administration [ 3740 ] . in the light of these problems , concerns , and limitations , nonviral systems 
 have emerged as a promising alternative for gene delivery . main requirements 
 are the protection of their nucleic acid  load  as well as their efficient 
 uptake into the target cells with subsequent release of the dna or rna 
 molecules and , if necessary , their transfer into the nucleus . several 
 strategies can be distinguished , mainly lipofection and polyfection relying 
 on cationic lipids or polymers , respectively ( see , eg , 
 [ 4143 ] ) . the efficient protection against enzymatic or nonenzymatic in fact , while the therapeutic potential of sirnas for the 
 treatment of various diseases is in principle very promising , 
 limitations of transfer vectors may turn out to be rate - limiting 
 in the development of rnai - based therapeutic strategies . one 
 approach to solve this problem is the use of dna expression 
 plasmids which encode palindromic hairpin loops with the desired 
 sequence . upon transcription and folding of the rna , the 
 double - stranded short hairpin rnas ( shrnas ) additionally , an in 
 vitro method has been described recently which is based on the 
 expression of shrnas in e coli and their delivery via bacterial 
 invasion . while 
 all these different dna - based systems 
 offer the advantage of sirna expression with a longer duration and 
 a probably higher level of gene silencing , they still rely on 
 ( viral or nonviral ) delivery of dna molecules and again raise 
 safety issues in vivo . hence , the direct delivery of sirnas 
 molecules , derived from in vitro transcription or chemically 
 synthesized , offers advantages over dna - based strategies and may 
 be preferable for in vivo therapeutic use . in the last years , a large body of studies has been published 
 which describe different strategies for the systemic or local 
 application of sirnas in vivo . the probably largest number of papers focuses 
 the use of unmodified sirnas ( table 1 ) whose 
 administration is often performed iv by hydrodynamic transfection 
 ( high pressure tail vein injection ) . while this method is widely 
 used and in some cases led to efficient target gene inhibition in 
 the liver and , to a lesser extent , in lung , spleen , pancreas , and 
 kidney , it may suffer from certain technical and practical 
 limitations at least in a therapeutical setting since it relies on 
 the rapid iv injection of a comparably large volume ( > = 1 
  ml / mouse / injection , in theory equivalent to a  3 l 
 iv bolus injection in man ) . alternative strategies for the 
 application of naked sirnas include various delivery routes which , 
 however , often provide an only local administration or rely on an 
 administration at least close to the target tissue or target 
 organ , thus restricting the number of target organs which may not 
 be relevant for certain diseases . it should also be noted that 
 several studies described here and below use rather large amounts 
 of sirnas and that upon intravenous injection of sirnas the liver 
 is the primary site of sirna uptake . as an alternative approach 
 for the application of sirnas in vivo , their delivery by 
 liposomes / cationic lipids has been described . for liposome - based 
 sirna formulations , a wide variety of modes of application 
 allowing local or systemic delivery has been used 
 ( table 2 ) . finally , several other strategies for local 
 or systemic sirna administration have been explored , including 
 chemical modifications of sirna molecules , electropulsation , 
 polyamine , or other basic complexes , atelocollagen , virosomes , and 
 certain protein preparations ( table 3 ) . an alternative approach relies on the complexation of unmodified sirna 
 molecules with a cationic polymer , polyethylenimine ( pei ) . polyethylenimines ( peis )    are synthetic polymers available in 
 branched or linear forms ( figure 2 , upper panels ) and 
 in a broad range of molecular weights from < 1000 da to 
 > 1000 kd . commercial pei preparations , although labelled 
 with a defined molecular weight , consist of pei molecules with a 
 broad molecular weight distribution [ 4547 ] . peis possess a high cationic charge density due to a protonable amino group in 
 every third position [ 48 , 
 49 ] . since no quarternary amino 
 groups are present , the cationic charges are generated by 
 protonation of the amino groups and hence are dependent on the ph 
 in the environment ( eg , 20% at ph 7.4 , see 
  for 
 review ) . due to its ability to condense and compact the dna into 
 complexes , which form small colloidal particles allowing efficient 
 cellular uptake through endocytosis , pei has been introduced as a 
 potent dna transfection reagent in a variety of cell lines and in 
 animals for dna delivery ( for review , see [ 51 , 
 52 ] and 
 references therein ) . in fact , in several studies pei has been 
 shown to be able to deliver large dna molecules such as 
 2.3 mb yeast artificial chromosomes ( yacs ) 
  as well 
 as plasmids or small oligonucleotides [ 48 , 
 5456 ] into 
 mammalian cells in vitro and in vivo . the n / p ratio , which 
 indicates the ratio of the nitrogen atoms of pei to dna phosphates 
 in the complex and thus describes the amount of pei used for 
 complex formation independent of its molecular weight , influences 
 the efficiency of dna delivery . a positive net charge of the 
 complexes , resulting from high n / p ratios , inhibits due to 
 electrostatic repulsion their aggregation and improves their 
 solubility in aqueous solutions as well as their interaction with 
 the negatively charged extracellular matrix components and thus 
 their cellular uptake . additionally , 
 the strong buffer capacity , described by the  proton sponge 
 hypothesis  which 
 postulates enhanced transgene delivery by cationic polymer - dna 
 complexes ( polyplexes ) containing h buffering polyamines due 
 to enhanced endosomal cl 
 accumulation and osmotic 
 swelling / lysis , 
 seems to be responsible for the fact 
 that pei - based delivery does not require endosome disruptive 
 agents for lysosomal escape . this tight condensation of the dna 
 molecules as well as the buffering capacity of pei in certain 
 cellular compartments like endosomes and lysosomes also protects 
 dna from degradation [ 48 , 
 49 , 58 , 
 59 ] . peis have been 
 successfully used for nonviral gene delivery in vitro and in vivo . while initial publications showed increased transfection 
 efficacies when using high molecular weight peis 
 , more 
 recent studies demonstrated the advantages of certain low 
 molecular weight peis [ 47 , 
 60 , 
 61 ] . the higher transfection 
 efficacy of low molecular weight peis may be due to a more 
 efficient uptake of the resulting pei / dna complexes , a better 
 intracellular release of the dna and/or lower in vitro 
 cytotoxicity as compared to high molecular weight pei 
 [ 6063 ] 
 . in fact , a decrease in the molecular weight of 
 the pei leads to an increase in complex size which may be 
 favourable at least for in vitro use [ 64 , 
 65 ] . on the other 
 hand , other peis with very low molecular weight ( < 2 kd ) 
 display little or no transfection efficacy even at very high n / p 
 ratios which may be attributed to the fact that a decrease in the 
 molecular weight of pei has been shown to translate into an 
 increasingly lower ability to form small complexes . 
 therefore , low molecular weight peis require higher n / p ratios 
 for optimal transfection efficacies as compared to higher 
 molecular weight peis since higher n / p ratios lead to an increase 
 in compaction with reduced complex sizes and a reduced tendency of 
 the complexes to aggregate due to hydrophobic interactions 
 [ 61 , 63 , 
 64 ] . nevertheless , while several parameters 
 have been extensively studied , some precise determinants for transfection 
 efficacy remain to be elucidated ( see [ 50 , 
 66 ] for review ) . also , the mechanism of the cytotoxic effects of pei complexes is 
 only poorly understood . it may rely on the formation of large 
 aggregates in the range of up to 2 m which , when formed on 
 the cell surface , impairs membrane functions finally leading to 
 cell necrosis . clearly , 
 there is a trend towards low 
 molecular weight peis as rather nontoxic delivery reagents in 
 vitro and in vivo , which combine high biocompatibility and reduced 
 side - effects thus also allowing to employ larger pei / dna complex 
 amounts without significant cytotoxicity . more recently , the use of polyethylenimines has been extended 
 towards the complexation and delivery of rna molecules , especially 
 small rna molecules like 37 nt all - rna ribozymes 
 [ 6769 ] 
 and sirnas   ( figure 2 ) . while 
 chemically unmodified rna molecules are very instable and prone to 
 rapid degradation , the pei complexation has been shown to lead to 
 an almost complete protection against enzymatic or 
 nonenzymatic degradation . in fact , pei - complexed sirnas , which are 
 [ p]-labeled for better detection , remain intact in vitro 
 for several hours even in the presence of rnase a or fetal calf 
 serum at 37c , while non - complexed sirnas are rapidly 
 degraded ( figure 3(a ) ) . this indicates that 
 sirna molecules are efficiently condensed and thus fully covered 
 and protected by pei . indeed , the analysis of pei / sirna complexes 
 by atomic force microscopy showed the absence of free sirnas or 
 sirna molecule ends and thus confirms these findings regarding an 
 efficient complexation ( grzelinski et al , submitted ) . however , 
 while the complex stability seems to be sufficient for sirna 
 protection with all peis tested ( werth et al , in press ; aigner et 
 al , unpublished data ) , several of these complexes do not show any 
 targeting efficacy at all . in fact , only when using certain 
 polyethylenimines , pei / sirna complexes are efficiently delivered 
 into target cells in vitro , where sirnas are released and display 
 bioactivity ( figures 1 and 
 2 ) . in general and 
 as seen before for pei / dna complexes ( see above ) , the transfection 
 efficacy is dependent on the pei used , also indicating that the 
 sirna targeting efficiency mainly depends on the endocytotic 
 uptake of the complex and/or its intracellular 
 decomposition rather than on the in vitro complex stability . good 
 results were obtained with commercially available jetpei 
 while the in vivo jetpei from the same supplier showed only poor 
 sirna delivery efficacies . likewise , a novel low 
 molecular weight pei based on the fractionation of a commercially 
 available polyethylenimine demonstrates high sirna protection and 
 delivery efficacies in vitro ( werth et al , in press ) . under 
 certain conditions , the pei / rna ( sirna or ribozyme ) 
 complexes retain their physical stability and biological 
 activity also after lyophilization ( and werth et al , in 
 press ) . although the pei transfection is only transient , data from 
 our lab show that pei / sirna effects are stable for at least 7 days 
 ( urban - klein and aigner , unpublished results ) . finally , another 
 study has explored the use of sirna nanoplexes comprising of pei 
 that is pegylated with an rgd peptide ligand attached at the 
 distal end of the pei . again , sirna nanoplexes protect sirnas 
 against serum degradation and show in vitro activity . the ultimate goal is the application of sirnas in vivo which has 
 been explored in some studies in different mouse models . ge et al 
 showed that pei - complexed sirnas targeting conserved regions of 
 influenza virus genes are able to prevent and treat influenza 
 virus infection in mice . upon iv injection , pei promoted the 
 delivery of sirnas into the lungs where , either given before or 
 after virus infection , sirna reduced influenza virus production in 
 the lungs . most biological effects of the systemic application of 
 pei - complexed sirnas , however , have been determined in different 
 mouse tumor models and by targeting different proteins which have 
 been shown previously to be tumor - relevant . this includes the 
 epidermal growth factor receptor her-2 ( c - erbb-2/neu ) , the growth 
 factor pleiotrophin ( ptn ) , and vascular endothelial growth factor 
 ( vegf ) and its receptor ( vegf r2 ) , and the fibroblast growth 
 factor - binding protein fgf - bp . the in vivo administration of pei complexed , but not of naked 
 sirnas , through ip or subcutaneous injection resulted in the detection of 
 intact sirnas even hours after injection 
 ( figure 3(b ) ) . radiolabeled sirna molecules were found 
 in several organs including subcutaneous tumors , muscle liver , 
 kidney and , to a smaller extent , lung and brain . it is important 
 to note that the sirnas were actually internalized by the tissues 
 as indicated by the fact that blood was negative for sirnas 
 ( figure 3(b ) ) . overexpression of the her-2 receptor has been observed in a wide variety of 
 human cancers and cancer cell lines . since her-2 displays strong cell 
 growth - stimulating and antiapoptotic effects especially through heterodimer 
 formation with other members of the egfr family , its overexpression has been 
 established as a negative prognostic factor and linked to a more aggressive 
 malignant behaviour of tumors ( eg , ) . consequently , her-2 qualifies as an attractive target molecule for antitumoral treatment 
 strategies including anti - her-2 antibodies , low molecular weight inhibitors , 
 or her-2-specific gene - targeting approaches . in fact , the relevance of her-2 
 ( over-)expression in tumor growth has been established in several in vitro 
 her-2 targeting studies including the use of ribozymes 
 [ 76 , 78 , 
 79 ] 
 or sirnas [ 80 , 
 81 ] . it was 
 demonstrated that her-2 reduction in vitro leads , among others , to the 
 inhibition of cell proliferation and increased apoptosis . the systemic treatment of athymic nude mice bearing subcutaneous skov-3 
 ovarian carcinoma tumor xenografts through ip injection of pei - complexed 
 her-2-specific sirnas led to marked antitumoral effects as seen by a 
 significant reduction tumor growth ( figure 4 ) 
 . this was paralleled by the detection of intact her-2-specific 
 sirnas in the tumors of the specific treatment group already 30 min after 
 administration and for at least 4 h , and by the downregulation of her-2 on 
 mrna and protein levels . another receptor , vegf r2 , was targeted in a study employing self - assembling 
 nanoparticles based on sirnas complexed pei which is pegylated with an rgd 
 peptide ligand attached at the distal end of peg . while the pegylation 
 allows steric stabilization and reduces nonspecific interactions of the 
 complexes , the rgd motif provided tumor selectivity due to their ability to 
 target integrins expressed on activated endothelial cells in the tumor 
 vasculature . upon iv administration into mice bearing subcutaneous n2a 
 neuroblastoma tumor xenografts , a selective tumor uptake and a vegf r2 
 downregulation the receptor ligand , vegf , is a mitogenic and angiogenic growth factor 
 stimulating tumor growth and angiogenesis in several tumors including 
 prostate carcinoma . thus , it may represent attractive target molecule for 
 rnai - based gene - targeting strategies also bearing in mind the double 
 antitumoral effect due to reduction of tumor cell proliferation as well as 
 tumor angiogenesis . the subcutaneous or intraperitoneal injection of 
 vegf - specific sirnas complexed with a novel pei obtained through 
 fractionation of a commercially available pei ( werth et al , in press ) 
 resulted in the reduction of tumor growth due to decreased vegf expression 
 levels ( hbel and aigner , unpublished results ) . the same was true for 
 pei / sirna - mediated targeting of fgf - bp ( dai and aigner , unpublished 
 results ) , which has been established previously as  rate - limiting  for tumor 
 growth and angiogenesis in several tumors ( [ 82 , 
 83 ] ,   see   for review ) . finally , pei / sirna - mediated targeting of pleiotrophin ( ptn ) exerted strong 
 antitumoral effects . ptn is a secreted growth factor which shows mitogenic , 
 chemotactic , angiogenic and transforming activity 
 [ 8593 ] 
 and which is markedly upregulated in several human tumors including cancer 
 of the breast , testis , prostate , pancreas , and lung as well as in melanomas , 
 meningiomas , neuroblastomas , and glioblastomas . the in vivo treatment of nude 
 mice through systemic subcutaneous or ip application of pei - complexed ptn sirnas 
 led to the delivery of intact sirnas into subcutaneous tumor xenografts and a 
 significant inhibition of tumor growth . likewise , in a clinically more 
 relevant orthotopic mouse glioblastoma model with u87 cells growing 
 intracranially , the injection of pei - complexed ptn sirnas into the cns 
 exerted antitumoral effects . this establishes , also in a complex and 
 relevant orthotopic tumor model , the potential of pei / sirna - mediated ptn 
 gene targeting as a novel therapeutic option in gbm , and further extends the 
 modes of delivery of pei / sirna complexes intrathecal strategies as employed 
 in the therapy of glioblastomas with antisense oligonucleotides . only a few years after their discovery , sirnas are catching up with 
 ribozymes and antisense oligonucleotides as efficient tools for 
 gene targeting in vitro and , more recently , also in vivo . this includes the 
 exploration of their potential as therapeutics which will lead to the 
 development of sirna - based therapeutic strategies . their ultimate success , 
 however , will strongly depend on the development of powerful and feasible 
 sirna delivery strategies which need to address several issues including the 
 stability / stabilization of sirna molecules while preserving their efficacy 
 and maintaining their gene - silencing activity , an efficient delivery into 
 the target organ(s ) as well as a sufficiently long sirna half life in the 
 organism and particularly in the target organ . thus , sirna delivery 
 strategies must provide sirna protection and transfection efficacy , the 
 absence of toxic and nonspecific effects , they must be efficacious also 
 when using small amounts of sirnas and must be applicable in various 
 treatment regimens and in various diseases even when this requires to 
 overcome biological barriers after their administration to reach their 
 target tissue or target organ . the research done on dna - based gene delivery , 
 ribozyme - targeting , and antisense technology will facilitate this process 
 since it already provides a basis of established technologies . this is also 
 true for the complexation of sirnas with polyethylenimine , which may 
 represent a promising avenue for sirna applications in vivo . long 
 double - stranded rna molecules ( dsrna ) , which are expressed from 
 dna vectors ( left red arrow ) or directly enter the cell ( center 
 red arrow ) , are processed by the dicer complex resulting in the 
 formation of small inhibitory rnas ( sirnas ) . alternatively , to 
 induce rnai these small 2123 bp duplexes are directly 
 delivered into the cell ( right red arrow ) . the sirnas are 
 incorporated into a nuclease - containing multiprotein complex 
 called risc , which becomes activated upon the atp - dependent 
 unwinding of the sirna duplex by an rna helicase . the now 
 single - stranded sirna guides the risc complex to its complementary 
 target mrna which is then cleaved by the endonucleolytical 
 activity of risc . while the risc complex is recovered for further 
 cycles , the cleaved mrna molecule is rapidly degraded due to its 
 unprotected rna ends . polyethylenimine ( pei)-mediated sirna transfer . upper 
 panel : peis are synthetic linear ( a ) or branched ( b ) 
 polymers with an amino group in every third position . dependent on 
 the ph , some of these amino nitrogens are protonated giving pei a 
 high cationic charge density . lower panel : proposed mechanism of 
 pei - mediated sirna transfer . due to electrostatic interactions , pei is able to complex negatively charged sirnas leading to a 
 compaction and the formation of small colloidal particles which 
 are endocytosed . the  proton sponge effect  exhibited by pei 
 complexes leads to osmotic swelling and ultimately to the 
 disruption of the endosomes . sirnas are protected from degradation 
 due to their tight condensation in the complex and the buffering 
 capacity of pei . upon their release from the pei - based complex , 
 intact sirnas are incorporated into the risc complex and induce 
 rnai ( see figure 1 ) . protection and in vivo delivery of sirnas upon 
 pei complexation . in   ( a ) in vitro 
 protection of sirnas 
 against nucleolytic degradation . [ p ] end - labeled sirnas , 
 complexed ( upper panel ) or not complexed ( lower panel ) with pei , 
 were subjected to treatment with 1 % fetal calf serum at 
 37c . at the time points indicated , the samples were 
 analysed by agarose gel electrophoresis , blotting , and 
 autoradiography . the bands represent full - length sirna 
 molecules indicating that pei complexation leads to the efficient 
 protection of sirnas while noncomplexed sirnas are rapidly 
 degraded . ( b , c ) in vivo delivery of intact sirnas upon pei 
 complexation . [ p]-labeled sirnas , complexed ( + ) or not 
 complexed (  ) with pei , were injected ip into mice bearing 
 subcutaneous skov-3 ovarian carcinoma cell tumor xenografts , and 
 after 30 min ( b ) or 4 h ( b ) total rna 
 from various organ and tissue homogenates was prepared and 
 subjected to agarose gel electrophoresis prior to blotting and 
 autoradiography . the bands represent intact [ p]-labeled 
 sirna molecules which for several hours are mainly found in tumor 
 and muscle as well as in liver and , time - dependently , in kidney . systemic treatment of mice with pei - complexed 
 her-2-specific sirnas leads to reduced growth of subcutaneous 
 skov-3 tumor xenografts due to decreased her-2 expression . 
 in   athymic nude mice bearing subcutaneous 
 tumor xenografts were injected ip with 0.6 nmoles her-2-specific 
 naked ( open circles ) or pei - complexed ( closed circles ) sirnas 23 
 times per week and tumor sizes were evaluated daily from the 
 product of the perpendicular diameters of the tumors . mean + / 
 standard error of the mean ( sem ) is depicted and 
 student 's unpaired t test was used for comparisons between data sets ( * * p < .03 , * * * p < .01 ) . differences in tumor growth reach significance at day 5 indicating 
 the antitumoral effects of the pei - complexed her-2-specific 
 sirnas . studies based on the direct application of sirnas to 
 induce rnai in vivo : administration of unmodified sirnas . studies based on the direct application of sirnas to induce rnai in 
 vivo : administration of sirnas based on liposomes / cationic lipids . studies based on the direct application of sirnas to induce rnai in 
 vivo : other strategies of sirna administration .
rna interference ( rnai ) is a powerful method for specific gene 
 silencing which may also lead to promising novel therapeutic 
 strategies . 
 it is mediated through small interfering rnas ( sirnas ) 
 which sequence - specifically trigger the cleavage and subsequent 
 degradation of their target mrna . 
 one critical factor is the 
 ability to deliver intact sirnas into target cells / organs in vivo . 
 this review highlights the mechanism of rnai and the guidelines 
 for the design of optimal sirnas . 
 it gives an overview of studies 
 based on the systemic or local application of naked sirnas or the 
 use of various nonviral sirna delivery systems . 
 one promising 
 avenue is the the complexation of sirnas with the polyethylenimine 
 ( pei ) , which efficiently stabilizes sirnas and , upon systemic 
 administration , leads to the delivery of the intact sirnas into 
 different organs . 
 the antitumorigenic effects of 
 pei / sirna - mediated in vivo gene - targeting of tumor - relevant 
 proteins like in mouse tumor xenograft models are described .