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autoimmune hepatitis, by injecting siRNA against the Fas gene [36]. siRNA was

injected into mice via the hydrodynamic tail vein injection. The mRNA and

protein levels of Fas were signiﬁcantly reduced in the hepatocytes of mouse and

the effects persisted for 10 days (Fig. 13.2). Fulminant hepatitis was induced in

animals by injecting agonistic Fas-speciﬁc antibody; 82% of the animals treated

with siRNA silencing Fas survived for 10 days, as compared to control animals,

that died within 3 days (Fig.13.3). This shows that effective gene silencing can be

FIGURE 13.2 RPA for Fas mRNA expression in hepatocytes from mice that were

untreated (lane 1) or were injected 24 h earlier with saline (lane 2), GFP (sequence 1)

siRNA (lane 3) or Fas (sequence 1) siRNA (lane 4). Silencing of Fas expression in mice

treated with Fas siRNA is maintained 5 (lane 5) or 10 days (lane 6) later. Expression of

other genes involved in the Fas pathway and housekeeping genes (L32 and Gapd) were

unaffected (names of the corresponding proteins are listed at right). Similar results were

obtained in three independent experiments. The graph shows results of densitometric

quantiﬁcation of the Fas/GAPDH ratios in three mice per condition. Fas mRNA

levels in hepatocytes are signiﬁcantly lower (



P < 0.001) at all times in mice treated

with Fas siRNA mice than in control mice. (Reproduced with permission from

Reference 36.)

346

BIOMEDICAL NANOSTRUCTURES

achieved in hepatocytes via the hydrodynamic tail vein injection and silencing

effects are not diluted as the hepatocytes are mostly nondividing cells.

siRNA use can also potentiate/sensitize diseases to ﬁrst line agents. Such

studies have been shown with siRNAs potentiating the effects of imatini b

(Ab1-kinase speciﬁc competitive inhibitor). siRNAs were used to decrease

the protein levels of Bcr/Ab1, thus potentiating the effects of imatinib [41]. The

major hurdle in cancer treatment with chemotherapeutic agents is the

development of drug resistance, owing to the expression of MDR1

(a multidrug transporter encoding P-glycoprotein). In vitro RNAi targeting

of MDR1 has been shown to overcome resistance to daunorubicin by over 90%

in pancreatic and gastric carcinomas [42]. Currently, there are numerous studies

with siRNA as a potential therapeutic in different stages of clinical studies.

13.5.3 RNA Transfection–Delivering siRNA Inside Cells

13.5.3.1 In vitro Efﬁcient gene silencing can be achieved by trans fecting

the cells in vitro either using chemically synthesized siRNA or by transfecting

the cells with transcript encoding plasmid DNA. Either way, the molecules to

be introduced into cellssiRNA/plasmid DNAare hydrophilic and poly-

anionic, and need some kind of transfecting agent/vector to achieve sufﬁcient

levels inside the cell. However, introducing chemically synthesized siRNA

molecules may be less complicated than transfecting cells with transcript

encoding plasmid DNA. The difference lies in the fact that siRNA molecules

require cytoplasmic delivery for gene silencing; however, intracellular produc-

tion of siRNA from plasmid DNA requires nuclear delivery of the plasmids.

FIGURE 13.3 Survival advantage of mice injected with Fas siRNA as compared to

saline or GFP siRNA after challenge by intraperitoneal injection with Fas-speciﬁc

antibody and observation for 10 day before sacriﬁce. Sequences that silenced Fas by

80% protected against fulminant hepatitis, whereas sequences that did not silence or

silenced inefﬁciently provided no protection.



P < 0.0001. &, GFP (dt) (n = 10); ~,

GFP (CU) (n = 15); !, saline (n = 15); ^, Fas (sequence 1) (n = 20); , Fas (sequence

2) (n = 10); &, Fas (sequence 4) (n = 10); ~ Fas (sequence 5) (n = 10);

, Fas

(sequence 6) (n = 10). (Reproduced with permission from Reference 36.)

CONTROLLING CELL BEHAVIOR VIA DNA AND RNA TRANSFECTIONS 347

Further, gene silencing achieved by transfecting mammalian cells with siRNA

duplexes is transient and depends on the number of siRNA molecules

transfected into the cells. The mammalian cell lacks the enzyme: RNA-

dependent RNA polymerases (which can amplify the siRNA molecules inside a

cell). Thus, the number of effective siRNA molecules present inside cells gets

diluted with each cell division. Omi et al. have shown that siRNA activity lasts

for 37 days in proliferating cells, but can persist for more than 3 weeks in

terminally differentiated cells (such as in neurons) [43]. Gene expression can be

knocked down in mammalian cell lines, primary cells, and even in embryonic

stem cells [44].

Elbashir et al. were the ﬁrst to report the transfection of siRNA duplexes in

cultured mammalian cells [26]. siRNA duplexes were synthesized against the

luciferase gene and cotransfected with the plasmid encoding luciferase. The

transfections were performed with cationic liposomes in a variety of

mammalian cell linesNIH/3T3, COS-7, and HeLa S3 cells. Expression of

luciferase gene was monitored 20 h after the transfection, and it was found that

the siRNA duplexes can speciﬁcally inhibit the gene expression, but to a

different extent in different cell lines. Another important ﬁnding of these

experiments was that siRNAs are extraordinarily potent reagents for mediating

gene silencing as they were found to be effective at concentrations that are

several orders lower than that required for conventional antisense reagents.

siRNA can be transfected into mammalian cells using lipid-based

formulations, conjugating with peptides, or by electroporation. Membrane

permeant peptides (MPPs) are amphipathic peptides that can translocate

across the lipid bilayers of cells in an energy-independent manner. Thiol-

containing siRNAs were conjugated to MPPs (penetratin, transportan) v ia

disulﬁde bonds [45]. The peptides allowed efﬁcient cytoplasmic delivery of

siRNAs, since upon entering the cells the disulﬁde bonds are reduced, releasing

the siRNA molecules. MPP-siRNAs successfully silenced the expression of

GFP and luciferase genes in stably transfected cell lines. Chinese hamster ovary

cells (CHO-AA8-Luc Tet-Off) that stably expressed luciferase gene were used

for the experiments to compare the transfection efﬁciency of MPP-siRNA and

lipofectamine-delivered siRNAs. Lipofectamine-siRNA transfected cells

showed a decrease in luciferase activity by 36%, 2 days after the transfection;

however, the luciferase levels returned to the basal levels on the third day. On

the contrary, penetratin-delivered siRNAs decreased the luciferase levels by

53% on second day after treatment and maintained these low levels of gene

expression on the third day as well. These results indicate that MPPs have

higher transfection efﬁciencies for siRNA as compared to lipofectamine in this

cell line.

13.5.3.2 In vivo The ﬁrst method to successfully deliver siRNA in vivo was

the high pressure tail vein injection of siRNAs (in physiological solution) in

mice. This method of rapid hydrodynamic infusion delivers siRNA to the

highly vascularized organs, like liver, lung, kidney, spleen and pancreas.

348 BIOMEDICAL NANOSTRUCTURES

Almost 90% reduction in target gene expression was observed in the liver of

mice, though the effect was transient, lasting for about a week. Numerous

studies have used this method to introduce siRNA/shRNA into rodents,

achieving efﬁcient delivery and silencing of target gene expression. Though

quite successful for introducing siRNA in rodents, this method is not a

clinically feasible option for humans.

Sorensen et al. were the ﬁrst to show that cationic liposome-mediated

delivery of synthetic siRNA molecules can speciﬁcally inhibit the expression of

exogenous and endogenous gene in adult mice [38]. Cationic liposomes

(DOTAP based) complexed with a plasmid encoding green ﬂuorescent protein

(GFP) and its cognate siRNA was injected intravenously into adult mice. GFP

gene express ion was found to be signiﬁcantly inhibited with the siRNA in the

liver and spleen of mice, 3 days after the injection, as compared to the

mismatched/inactive siRNA as control. Furthermore, the therape utic efﬁcacy

of synthetic siRNA molecules was investiga ted in vivo in a mouse model of

sepsis. BALB/c mice were intraperitoneally pretreated with anti-TNF-a

siRNA, prior to the injection of a lethal dose of lipopolysaccharide (LPS)

capable of inducing septic shock. A signiﬁcant protective effect of siRNA was

found in terms of the number of mice surviving the septic shock, as compared

to the inactive siRNA. Further this effect was attributed to the decreased levels

of TNF-a as a result of the speciﬁc inhibition of TNF-a gene expression with

siRNA.

Since high concentrations of siRNA can induce the nonspeciﬁc immune

stimulation response, it is important that the vectors for in vivo delivery of

siRNA can deliver siRNA even at low concentrations to the target site. Such

an experiment was performed using a lipid-based vector for the delivery of

siRNA to mouse brain. JetSI

(a mixture of cationic lipids) was used to

complex plasmid DNA encoding luciferase gene and picomolar concentrations

of siRNA against this gene [46]. These polyplexes were injected into the mouse

brain using stereotaxic intracerebr oventricular injections. A formulation of

JetSI

with another fusogenic lipid dioleoylphosphatidylethanolamine

(DOPE) was found to efﬁciently inhibit the expression of target gene in vivo,

even at picomolar concentrations of siRNA. The gene silencing effect was

found to be dose dependent and speciﬁc.

Most of the in vivo studies have been performed with rodents, but one recent

study has demonstrated that RNAi-mediated gene silencing can be successfully

achieved in nonhuman primates as well, thus highlighting the potential of

siRNA as a therapeutic via systemic delivery in species higher than roden ts.

siRNA targeting the gene expressing apolipoprotein B (ApoB) was adminis-

tered, as a liposomal formu lation via a bolus intravenous injection into

cynomolgus monkeys [47]. mRNA levels of the target gene in the liver were

found to be reduced within 48 h of the injection. Signiﬁcant reduction in the

levels of ApoB protein was observed as early as 24 h after siRNA injection with

the effects lasting for 11 days. Furthermore, there was no evidence of

complement activation, proinﬂammatory cytokine production, or toxicities in

CONTROLLING CELL BEHAVIOR VIA DNA AND RNA TRANSFECTIONS 349

the animals. The only detected changes in the primates as a result of siRNA

liposomal injections were the transient increase in the liver enzymes, which

normalized to the basal levels within 6 days of treatment.

siRNAs can be complexed with polycationic polymers to form polyplexes.

Self-assembling nanoparticles with siRNA were prepared with PEI that is

PEGylated with an ArgGlyAsp (RGD) peptide ligand attached at the distal

end of the PEG [48]. The use of RGD ligand allows targeting of integrins

expressed on tumor neovasculature, thus selectively delivering siRNA to the

tumor tissue. The polymer was prepared with three functio nalities in order to

obtain targeted self-assembling nanoplexes: (1) branched PEIas the cationic

complexing polymer, (2) PEGsteric protective surface layer, and (3) RGD

peptidetargeting ligand. The nanoplexes were used to successfully deliver

siRNA inhibiting the vascular endothelial grow th factor receptor-2 (VEGF

R2) expression, to the tumor neovasculature, and thereby inhibiting tumor

angiogenesis. siRNA carrying nanoplexes were injected every 3 days into mice

carrying subcutaneous tumor (developed by injec ting N2A cells) by tail vein

injection. There was a selective tumor uptake of the nanoplexes showing a

sequence-speciﬁc inhibition of VEGF R2 expression and inhibition of both

tumor angiogenesis and tumor growth (Fig.13.4).

Chemical modiﬁcations of siR NA have also been attempted to improve the

delivery of siRNA without using any other transfecting agents. Lipophilic

siRNAs were synthesized by covalently conjugating the 5

- ends of RNA with

derivatives of cholest erol, lithocholic acid, or lauric acid [49]. These constructs

were shown to improve the delivery of siRNA to liver cells and downregulate

the expression of a LacZ expression construct. In another study, the 3

-endof

siRNA was conjugated to cholesterol by means of a pyrrolidine linker.

This improved the resistance of siRNA to nuclease degradation, increased

its stability in blood circulation, and also, increased the uptake of siRNA

by liver.

Recombinant viral vectors, such as AAV, can mediate the delivery and long-

term silencing of a gene in both dividing and nondividing mammalian cells.

siRNA-producing lentivirus can transduce nondividing cells and has been used

to deliver siRNA into embryon ic stem cells to create knockdown mice. Kunath

et al. used viral constructs expressing shRNA for Ras GTPase-activating

protein to transfect mouse embryonic stem cells [50]. The resulting mice

showed defects similar to that of a knockout developed by the conventional

homologous recombination. Despite the advantage of viral vectors to deliver

siRNA to nontransfectable cell s, their potential for therapeutic applications is

reduced by the fact that this delivery method is more prone to nonspeciﬁc

interferon response, owing to high expression of shRNA. Viral vectors

expressing siRNA have been shown to markedly diminish the expression of

exogenous and endogenous genes in vitro and in vivo in brain and in liver [51].

Recombinant adenoviral vectors were generated for intracellular expression of

siRNA against GFP (siGFP) and b-glucouronidase. HeLa cells were infected

with the viral vectors expressing siRNA against b-glucouronidase, and lysed

350 BIOMEDICAL NANOSTRUCTURES

72 h after treatment. This resulted in a speciﬁc reduction of b-glucouronidas e

gene expression, leading to a 60% reduction in b-glucouronidase activity. For

in vivo studies, adenoviral vectors expressing siGFP were injected into the brain

striatal regions of transgenic mice expressing eGFP. Effective gene silencing for

up to 5 days was observed after a local injection of siRNA-producing AAV in

mouse brain.

FIGURE 13.4 Tumor growth inhibition by VEGF R2 siRNA nanoplexes. (a) Tumor

growth inhibition by siRNA RPP nanoplexes. Mice were inoculated with N2A tumor

cells and left untreated (open squares) or treated every 3 days by tail vein injection with

RPP-nanoplexes with siLacZ (ﬁlled squares) or siVEGF R2 (ﬁlled circles) at a dose of

40 mg per mouse. Treatment was started at the time point that the tumors became

palpable (20 mm

). Only VEGF R2-sequence-speciﬁc siRNA inhibited tumor growth,

whereas treatment with LacZ siRNA did not affect tumor growth rate as compared with

untreated controls (n = 5). (bd) Neovascularization in tumors treated with siRNA

RPP nanoplexes. Representative tumors excised at the end of the tumor growth

inhibition experiment (a) were examined using low magniﬁcation light microscopy.

Transillumination of tumor and surrounding skin tissue shows strong neovasculariza-

tion in mice left untreated (b) and mice treated with RPP nanoplexes with siRNA-LacZ

(c). In contrast, mice treated with RPP nanoplexes with VEGF R2 siRNA showed low

neovascularization and erratic branching of blood vessels (d). Asterisks indicate tumor

tissue. Bar = 2 mm. (Reproduced with permission from Reference 48.)

CONTROLLING CELL BEHAVIOR VIA DNA AND RNA TRANSFECTIONS 351

13.5.4 Issues

13.5.4.1 Speciﬁcity Nonspeciﬁc inhibition of gene expression is a

problem with the use of dsRNAs for RNAi. dsRNAs are known to activate

the dsRNA-dependent protein kinase (PKR), which results in a general

inhibition of total protein synthesis and the nons peciﬁc degradation of mRNAs

present in the cells. However, the use of synthetic siRNA molecules is

capable of bypassing the activation of this nonspeciﬁc pathway. Still there

are numerous reports indicating the potential of siRNA molecules to produce

some nonspeciﬁc and off-target effects. This may be due to pa rtial homology

of the siRNA sequences to mRNA sequences, other than the target

mRNA [52,53]. Most nonspeciﬁc responses can be minimized with careful

design of siRNA sequences. The nonspeciﬁc effects of siRNA on gene

expression depend on the siRNA concentration, speciﬁc sequence, delivery

method, and the cell type [54]. High siRNA concentrations enhance nonspeciﬁc

effects.

13.5.4.2 Resistance Cells can develop resistance to the effect of siRNA

by loss of genes required for RISC complex formation or by selection of

suppressors that inhibit degradation [35]. Further, not all mRNA sequences

can be targeted by siRNAs, due to lack of accessibility of the target sequences

hidden by RNA-binding proteins or complex secondary structures. Recent

reports have indicated that siRNA-resistant viral strains (HIV) can emerge in

as few as 25 days [55]. To reduce the chances of such resistance, multiple

sequences per target and multiple targets per viral genome would be required.

On the contrary, so far there are no reports of resistance developing in

mammalian cells upon introduction of siRNA.

13.5.4.3 Stability For successful in vivo use, siRNA molecules must have

sufﬁcient stability in systemic circulation and should have desirable pharma-

cokinetic properties. The molecules should bind blood proteins to an extent so

that immediate loss by excretion is prevented, but at the same time, this degree

of binding should not be toxic [56]. However, studies have shown that siRNA

degradation peaks 3648 h after introduction and diminishes around 96 h.

Biodistribution of radiolabeled siRNA was studied by intravenous and

intraperitoneal injections in mice [57]. siRNA primarily accumulated in the

liver and kidney and was also detected in the heart, spleen, and lung. These

biodistribution patterns demand an improvement in the pharmacokinetic

properties of siRNA for success ful in vivo gene silencing.

Chemical modiﬁcations are in pursuit for increasing the stability, of siRNA

while maintaining an adequate gene-silencing activity for therapeutic applica-

tions. Such chemical modiﬁcations can be made throughout the siRNA duplex

or at selected one or two bases, to alter the chemical properties of siRNA. This

can potentially help to increase the resistance of siRNA to nuclease enzymes,

impart serum stability, and also alter the biodistribution patterns. Further, by

352 BIOMEDICAL NANOSTRUCTURES

directing the biodistribution of siRNA to target tissues, the nonspeciﬁc effects

can also be effectively reduced. The introduction of phosphorothioate linkages

enhance nuclease resistance and improve serum stability [57]. However, the use

of such modiﬁcations often results in cellular and in vivo toxicity, also their

effect on in vivo potency of siRNA is yet to be established. Chemical

modiﬁcations to the sugar moiety of the siRNA sequence can also signiﬁcantly

alter thermal stability, and, potentially affect the gene silencing activity of

siRNA. Jayasena and coworkers have demonstrated that functional and

nonfunctional siRNAs show distinct thermodynamic proﬁles. Several 2

modiﬁcations have been tested for their effects on gene expression in cultured

cells. Gene silencing activity is retained after partial substitution of bases with

-ﬂuoro bases [58].

13.6 CONCLUSIONS

Advances in molecular biology and polymer chemistry have enabled

controlling cell behavior via DNA/RNA transfections. This has steered

research toward identifying new genes responsible for diseas e induction/

progression. DNA/RNA transfections have now become a routine research

tool, though its applications as a therapeutic intervention is limited by the

availability of safe and efﬁcient transfection methods.

ACKNOWLEDGMENTS

Grant support from the National Institutes of Health (R01 EB003975) and a

predoctoral fellowship to JKV from the American Heart Association,

Heartland Afﬁliate (Award # 0515489Z).

REFERENCES

1. Li Z, et al. Murine leukemia induced by retroviral gene marking. Science 2002;

296:497.

2. Vasir JK and Labhasetwar V. Polymeric nanoparticles for gene delivery. Expert

Opin Drug Deliv 2006;3:325344.

3. Wiethoff CM and Middaugh CR. Barriers to nonviral gene delivery. J Pharm Sci

2003;92:203217.

4. Lechardeur D and Lukacs GL. Intracellular barriers to non-viral gene transfer.

Curr Gene Ther 2002;2:183194.

5. Pouton CW and Seymour LW. Key issues in non-viral gene delivery. Adv Drug

Deliv Rev 2001;46:187203.

6. Lee EJ and Jameson JL. Gene therapy of pituitary diseases. J Endocrinol 2005;

185:353362.

CONTROLLING CELL BEHAVIOR VIA DNA AND RNA TRANSFECTIONS 353

7. Ohno T, et al. Gene therapy for vascular smooth muscle cell proliferation after

arterial injury. Science 1994;265:781784.

8. Lee RJ, et al. VEGF gene delivery to myocardium: deleterious effects of

unregulated expression. Circulation 2000;102:898901.

9. Bettinger T, Carlisle RC, Read ML, Ogris M, Seymour LW. Peptide-mediated

RNA delivery: a novel approach for enhanced transfection of primary and post-

mitotic cells. Nucleic Acids Res 2001;29:38823891.

10. Sweeney P, et al. Efﬁcient therapeutic gene delivery after systemic administration of

a novel polyethyleneimine/DNA vector in an orthotopic bladder cancer model.

Cancer Res 2003;63:40174020.

11. Vernejoul F, et al. Antitumor effect of in vivo somatostatin receptor subtype 2 gene

transfer in primary and metastatic pancreatic cancer models. Cancer Res 2002;

62:61246131.

12. Prabha S and Labhasetwar V. Nanoparticle-mediated wild-type p53 gene delivery

results in sustained antiproliferative activity in breast cancer cells. Mol Pharm

2004;1:211219.

13. Cohen H, et al. Sustained delivery and expression of DNA encapsulated in

polymeric nanoparticles. Gene Ther 2000;7:18961905.

14. Bernkop-Schnurch A and Krajicek ME. Mucoadhesive polymers as platforms for

peroral peptide delivery and absorption: synthesis and evaluation of different

chitosanEDTA conjugates. J Control Release 1998;50:215223.

15. Artursson P, Lindmark T, Davis SS, Illum L. Effect of chitosan on the permeability

of monolayers of intestinal epithelial cells (Caco-2). Pharm Res 1994;11:13581361.

16. Roy K, Mao HQ, Huang SK, Leong KW. Oral gene delivery with chitosanDNA

nanoparticles generates immunologic protection in a murine model of peanut

allergy. Nat Med 1999;5:387391.

17. Roman S, Lindeman R, O’Toole G, Poole MD. Gene therapy in plastic and

reconstructive surgery. Curr Gene Ther 2005;5:8199.

18. Baltzer AW and Lieberman JR. Regional gene therapy to enhance bone repair.

Gene Ther 2004;11:344350.

19. Madeddu P. Therapeutic angiogenesis and vasculogenesis for tissue regeneration.

Exp Physiol 2005;90:315326.

20. Kasahara H, et al. Biodegradable gelatin hydrogel potentiates the angiogenic effect

of ﬁbroblast growth factor 4 plasmid in rabbit hindlimb ischemia. J Am Coll

Cardiol 2003;41:10561062.

21. Nagaya N, et al. Hybrid cellgene therapy for pulmonary hypertension based on

phagocytosing action of endothelial progenitor cells. Circulation 2003;108:889895.

22. Michiels F, et al. Arrayed adenoviral expression libraries for functional screening.

Nat Biotechnol 2002;20:1154  1157.

23. Ziauddin J and Sabatini DM. Microarrays of cells expressing deﬁned cDNAs.

Nature 2001;411:107110.

24. Honma K, et al. Atelocollagen-based gene transfer in cells allows high-throughput

screening of gene functions. Biochem Biophys Res Commun 2001;289:10751081.

25. Fire A, et al. Potent and speciﬁc genetic interference by double-stranded RNA in

Caenorhabditis elegans. Nature 1998;391:806811.

354

BIOMEDICAL NANOSTRUCTURES

26. Elbashir SM, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in

cultured mammalian cells. Nature 2001;411:494498.

27. Reynolds A, et al. Rational siRNA design for RNA interference. Nat Biotechnol

2004;22:326330.

28. Couzin J. Breakthrough of the year. Small RNAs make big splash. Science

2002;298:22962297.

29. Paddison PJ, et al. A resource for large-scale RNA-interference-based screens in

mammals. Nature 2004;428:427431.

30. Berns K, et al. A large-scale RNAi screen in human cells identiﬁes new components

of the p53 pathway. Nature 2004;428:431437.

31. Hemann MT, et al. An epi-allelic series of p53 hypomorphs created by stable RNAi

produces distinct tumor phenotypes in vivo. Nat Genet 2003;33:396400.

32. Dorsett Y and Tuschl T. siRNAs: applications in functional genomics and potential

as therapeutics. Nat Rev Drug Discov 2004;3:318329.

33. Wall NR and Shi Y. Small RNA: can RNA interference be exploited for therapy?

Lancet 2003;362:14011403.

34. Gupta S, Schoer RA, Egan JE, Hannon GJ, Mittal V. Inducible, reversible, and

stable RNA interference in mammalian cells. Proc Natl Acad Sci USA

2004;101:19271932.

35. Ryther RC, Flynt AS, Phillips JA, 3rd, Patton JG. siRNA therapeutics: big

potential from small RNAs. Gene Ther 2005;12:511.

36. Song E, et al. RNA interference targeting Fas protects mice from fulminant

hepatitis. Nat Med 2003;9:347351.

37. Song E, et al. Sustained small interfering RNA-mediated human immunodeﬁciency

virus type 1 inhibition in primary macrophages. J Virol 2003;77:71747181.

38. Sorensen DR, Leirdal M, Sioud M. Gene silencing by systemic delivery of synthetic

siRNAs in adult mice. J Mol Biol 2003;327:761766.

39. Yoshinouchi M, et al. In vitro and in vivo growth suppression of human

papillomavirus 16-positive cervical cancer cells by E6 siRNA. Mol Ther

2003;8:762768.

40. Reich SJ, et al. Small interfering RNA (siRNA) targeting VEGF effectively inhibits

ocular neovascularization in a mouse model. Mol Vis 2003;9:210216.

41. Wohlbold L, et al. Inhibition of bcrabl gene expression by small interfering RNA

sensitizes for imatinib mesylate (STI571). Blood 2003;102:22362239.

42. Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing of leukemic cells with

a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002;21:

57165724.

43. Omi K, Tokunaga K, Hohjoh H. Long-lasting RNAi activity in mammalian

neurons. FEBS Lett 2004;558:8995.

44. Leung RK and Whittaker PA. RNA interference: from gene silencing to gene-

speciﬁc therapeutics. Pharmacol Ther 2005;107:222239.

45. Muratovska A and Eccles MR. Conjugate for efﬁcient delivery of short interfering

RNA (siRNA) into mammalian cells. FEBS Lett 2004;558:6368.

46. Hassani Z, et al. Lipid-mediated siRNA delivery down-regulates exogenous gene

expression in the mouse brain at picomolar levels. J Gene Med 2005;7:198207.

CONTROLLING CELL BEHAVIOR VIA DNA AND RNA TRANSFECTIONS 355