We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells. We have developed a selection scheme to generate nucleic acid sequences that recognize and directly internalize into mammalian cells without the aid of conventional delivery methods. To demonstrate the generality of the technology, two independent selections with different starting pools were performed against distinct target cells. Each selection yielded a single highly functional sequence, both of which folded into a common core structure. This internalization signal can be adapted for use as a general purpose reagent for transfection into a wide variety of cell types including primary cells.
Abstract Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor’s selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.
Panax ginseng is used in traditional Chinese medicine as a prophylactic, a stimulant, and a treatment for various diseases. In addition, ginseng has been found to enhance the immune system and possess an anti-tumor effect. Ginseng appears to stimulate humoral and cell-mediated immune responses and the subsequent increase in titers of lymphocytes and circulating antibodies. Furthermore, ginseng's cancer-prevention properties seem to be mediated through the increased production of interferons and cytokines, which activate natural killer and cytotoxic T-cells that can served to lyse or inhibit growing tumors. Both clinical and epidemiological studies indicated that Panax ginseng can reduce the incidence of cancer in vivo. These results suggest that ginseng shows antitumor effects as an immunomodulator.
Abstract Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.
The ability to reverse the binding of aptamers to their target proteins has received considerable attention for developing controllable therapeutic agents. Recently, use of aptamers as reversible cell-sorting ligands has also sparked interest. Antibodies are currently utilized for isolating cells expressing a particular cell surface receptor. The inability to remove antibodies from isolated cells following sorting greatly limits their utility for many applications. Previously, we described how a particular aptamer-antidote oligonucleotide pair can isolate cells and clean them. Here, we demonstrate that this approach is generalizable; aptamers can simultaneously recognize more than one cell type during fluorescent activated cell sorting (FACS). Moreover, we describe a novel approach to reverse aptamer binding following cell sorting using a nuclease. This alternative strategy represents a cleaning approach that does not require the generation of antidote oligonucleotides for each aptamer and will greatly reduce the cost and expand the utility of Clean FACS.
Although anti-HIV drugs have been in place for decades, both prophylaxis and a cure remain elusive. Many treatments for HIV infection are associated with severe side effects (which may contribute to poor patient compliance), and resistance mutants frequently arise that render many front-line therapeutics impotent.Nucleic acid–based therapeutics (summarized in Table 1) have been considered as an alternative to the chemical antivirals. Early attempts to develop nucleic acid therapeutics for HIV-1 focused on antisense drugs and RNA decoys or mimetics. More recently these efforts have broadened to more efficacious antisense drugs (siRNAs) and RNA decoys or mimetics (aptamers, reviewed in refs. 1Joshi PJ Fisher TS Prasad VR Anti-HIV inhibitors based on nucleic acids: emergence of aptamers as potent antivirals.Curr Drug Targets Infect Disord. 2003; 3: 255-262Crossref PubMed Scopus (52) Google Scholar,2Scherer L Rossi JJ Weinberg MS Progress and prospects: RNA-based therapies for treatment of HIV infection.Gene Ther. 2007; 14: 1057-1064Crossref PubMed Scopus (70) Google Scholar,3Held DM Kissel JD Patterson JT Nickens DG Burke DH HIV-1 inactivation by nucleic acid aptamers.Front Biosci. 2006; 11: 89-112Crossref PubMed Scopus (83) Google Scholar,4Zhang Z Blank M Schluesener HJ Nucleic acid aptamers in human viral disease.Arch Immunol Ther Exp (Warsz). 2004; 52: 307-315PubMed Google Scholar,5Nielsen MH Pedersen FS Kjems J Molecular strategies to inhibit HIV-1 replication.Retrovirology. 2005; 2: 10Crossref PubMed Scopus (61) Google Scholar). For example, Capodici et al. demonstrated antiviral efficacy with an anti-Gag siRNA in infected U87 cells as well as primary T cells.6Capodici J Kariko K Weissman D Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference.J Immunol. 2002; 169: 5196-5201Crossref PubMed Scopus (281) Google Scholar Several other groups have shown similar effects with siRNA against other HIV targets (reviewed in ref. 7Bennasser Y Yeung ML Jeang KT RNAi therapy for HIV infection: principles and practicalities.BioDrugs. 2007; 21: 17-22Crossref PubMed Scopus (23) Google Scholar). Similarly, aptamers selected against the HIV-1 proteins have shown inhibition of viral replication. For example, anti–reverse transcriptase aptamers have been shown to form a pseudoknot structure that tightly interacts with the polymerase active site.8Tuerk C MacDougal S Gold L RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.Proc Natl Acad Sci USA. 1992; 89: 6988-6992Crossref PubMed Scopus (368) Google Scholar Not only did these aptamers inhibit viral replication, but studies showed that the aptamers could be co-packaged into virions, thereby crippling progeny virions.9Chaloin L Lehmann MJ Sczakiel G Restle T Endogenous expression of a high-affinity pseudoknot RNA aptamer suppresses replication of HIV-1.Nucleic Acids Res. 2002; 30: 4001-4008Crossref PubMed Scopus (106) Google Scholar,10Joshi P Prasad VR Potent inhibition of human immunodeficiency virus type 1 replication by template analog reverse transcriptase inhibitors derived by SELEX (systematic evolution of ligands by exponential enrichment).J Virol. 2002; 76: 6545-6557Crossref PubMed Scopus (79) Google ScholarTable 1Targets of nucleic acid-based therapeuticssiRNADecoysAntisenseRibozymesAptamersViral targetCellular targetTARTARGagRevGagCD4RBERREVifTatEnvCXCR4GagTARRTVifCCR5EnvEnvNCTatCCNT1RREIntRevCDK2LTRgp120NefCDK9CCR5GagLTRSUPT5HTat/RevRNAseHRPAINSam68LIP5PPIARAB9TSG101 Open table in a new tab For all of these nucleic acid drugs, though, one of the chief problems in clinical development has been delivery. Although it is possible for RNA drugs to be transcribed inside cells following gene or template delivery, such methods would face the same safety and regulatory hurdles as many other gene therapy efforts. Alternatively, although nucleic acid drugs can be introduced directly into the body, they are frequently either quickly degraded by rampant serum nucleases or quickly swept out of the system unless appended to larger particles or moieties such as polyethylene glycol or modified with the addition of nonstandard nucleotides and linkages.Anticipating these problems, Hicke and Stephens suggested that nucleic acids might best serve not as therapeutics in their own right but as “escort molecules” for other drugs.11Hicke BJ Stephens AW Escort aptamers: a delivery service for diagnosis and therapy.J Clin Invest. 2000; 106: 923-928Crossref PubMed Google Scholar In recent years this prophetic statement has been validated on a number of occasions. For example, anti-cell aptamers selected in Tan's laboratory have been used to specifically target gold nanoparticles and nanorods to cells.12Huang YF Chang HT Tan W Cancer cell targeting using multiple aptamers conjugated on nanorods.Anal Chem. 2008; 80: 567-572Crossref PubMed Scopus (269) Google Scholar,13Medley CD Smith JE Tang Z Wu Y Bamrungsap S Tan W Gold nanoparticle-based colorimetric assay for the direct detection of cancerous cells.Anal Chem. 2008; 80: 1067-1072Crossref PubMed Scopus (538) Google Scholar An anti–prostate-specific membrane antigen aptamer originally selected by Lupold et al.14Lupold SE Hicke BJ Lin Y Coffey DS Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen.Cancer Res. 2002; 62: 4029-4033PubMed Google Scholar has been used to deliver a variety of compounds to prostate tumor cells expressing this antigen, including nanoparticles, toxins, and even siRNAs.15Chu TC Marks JW Lavery LA Faulkner S Rosenblum MG Ellington AD et al.Aptamer:toxin conjugates that specifically target prostate tumor cells.Cancer Res. 2006; 66: 5989-5992Crossref PubMed Scopus (248) Google Scholar,16Chu TC Twu KY Ellington AD Levy M Aptamer mediated siRNA delivery.Nucleic Acids Res. 2006; 34: e73Crossref PubMed Scopus (390) Google Scholar,17Farokhzad OC Jon S Khademhosseini A Tran TN Lavan DA Langer R Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells.Cancer Res. 2004; 64: 7668-7672Crossref PubMed Scopus (777) Google Scholar,18McNamara JO Andrechek ER Wang Y Viles KD Rempel RE Gilboa A et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (852) Google Scholar In the last instance, introduction of an aptamer-siRNA chimera proved sufficient to reverse tumor growth in a mouse xenograft model.18McNamara JO Andrechek ER Wang Y Viles KD Rempel RE Gilboa A et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (852) Google Scholar The great advantage of these approaches is that not only did they help to obviate the delivery problem, they also provided additional specificity to the therapeutics themselves, with the possibility that both the delivery vehicle and the drug delivered could be tuned to a particular pathogen or malady.John Rossi and his co-workers have now taken these approaches one step further.19Zhou J Li H Li S Zaia J Rossi JJ Novel dual inhibitory function aptamer–siRNA delivery system for HIV-1 therapy.Mol Ther. 2008; 16: 1481-1489Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar Whereas nucleic acid therapeutics can be escorted to cells, it is a priori somewhat difficult to envision how to “escort” nucleic acids to viruses, which typically replicate inside a cell. The elegantly simple solution that the Rossi group has suggested is to let the virus escort the nucleic acid therapeutic into its new cellular prey. In this regard, the nucleic acid drug is no longer an escort per se but a hitchhiker that automatically limits viral spread and load.Modified aptamers against HIVBAL gp120 had previously been selected by William James and co-workers. These aptamers were able to significantly inhibit a broad range of clinical isolates in peripheral blood mononuclear cells, including one viral strain from group O. Analysis of one aptamer's binding suggested that the aptamer did not interfere with the gp120–CD4 interaction. Further deconvolution with gp120 antibodies suggested that the aptamer actually inhibited interactions with the co-receptors CXCR4 and CCR5 (ref. 20Khati M Schüman M Ibrahim J Sattentau Q Gordon S James W Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2′F-RNA aptamers.J Virol. 2003; 77: 12692-12698Crossref PubMed Scopus (147) Google Scholar).Whereas anti-gp120 aptamers should inhibit the interactions between gp120 and co-receptors and therefore reduce viral load, the Rossi group recognized that gp120 presented on the surfaces of infected cells could also be internalized. Because escort aptamers against internalizing receptors had already proven effective,16Chu TC Twu KY Ellington AD Levy M Aptamer mediated siRNA delivery.Nucleic Acids Res. 2006; 34: e73Crossref PubMed Scopus (390) Google Scholar,18McNamara JO Andrechek ER Wang Y Viles KD Rempel RE Gilboa A et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (852) Google Scholar Rossi and colleagues19Zhou J Li H Li S Zaia J Rossi JJ Novel dual inhibitory function aptamer–siRNA delivery system for HIV-1 therapy.Mol Ther. 2008; 16: 1481-1489Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar cleverly saw that HIV could potentially be tricked into mediating its own destruction (Figure 1).The researchers designed hitchhiker constructs that comprised a 2′-fluoro-pyrimidine-modified anti-gp120 aptamer linked to the sense strand of an siRNA directed against the Tat/Rev small messenger RNA. Following transcription, the aptamer-siRNA chimera is hybridized to the unmodified antisense siRNA strand. As had been previously observed, the anti-gp120 aptamer itself inhibited viral adhesion to the cell surface and was able to moderately reduce viral replication in CEM cells, both when virus was pre-incubated with the constructs and when infected cells were challenged with the constructs. With the addition of the siRNA, though, viral replication was nearly obliterated in the pre-incubation experiments and significantly more reduced in viral challenge in infected cells, demonstrating that the constructs directly inhibited viral infectivity as well as inhibiting replication in culture. Both the aptamer by itself and the chimeras were able to inhibit syncytia formation.To account for possible steric interference between the two components, constructs were also tested in which the aptamer and siRNA were separated by four pyrimidine bases. In all versions of the constructs tested, the variant with the added linker fared slightly better in inhibition than its equivalent without the linker. This suggests that accessibility of the different components in such chimeras may influence function. For example, it can be readily imagined that accessibility of the aptamer itself to gp120 could be limited by its siRNA “tail,” whereas accessibility of that siRNA to the RNA-induced silencing complex might be influenced by the presence of the aptamer (although natural “aptamers” with appended siRNAs are already known, such as the VA RNAs from adenovirus21Andersson MG Haasnoot PC Xu N Berenjian S Berkhout B Akusjärvi G Suppression of RNA interference by adenovirus virus-associated RNA.J Virol. 2005; 79: 9556-9565Crossref PubMed Scopus (274) Google Scholar).The wonderful thing about the modularity of the chimera is that it allows individual variables (such as steric interference) to be separately tested and optimized in a way that is difficult to imagine for more amorphous formulations (such as proteoliposomes). For example, the investigators separately tested siRNA constructs that were either 21 or 27 residues long. Dicer cleavage studies in cell extracts showed that processing of the chimeras proceeded from the antisense 5′ end, leaving a blunted 5′ end. Analysis using Remote Analysis Computation for Gene Expression showed that cleavage of the target was approximately 10 nucleotides from the 5′ end of both versions of the siRNA. Therefore, because the two had different 5′ antisense ends, the target cleavage site of one siRNA was offset by about six nucleotides from the other. Consistent with the questions of accessibility we have raised, the authors demonstrated that the 27-mer siRNA chimeras fared better in antiviral efficacy. This is also in concordance with studies that showed that 27-mer siRNAs with 3′ overhangs on the antisense strand were more effective than corresponding 21-mer sequences.22Amarzguioui M Lundberg P Cantin E Hagstrom J Behlke MA Rossi JJ Rational design and in vitro and in vivo delivery of Dicer substrate siRNA.Nat Protoc. 2006; 1: 508-517Crossref PubMed Scopus (107) Google Scholar,23Rose SD Kim DH Amarzguioui M Heidel JD Collingwood MA Davis ME et al.Functional polarity is introduced by Dicer processing of short substrate RNAs.Nucleic Acids Res. 2005; 33: 4140-4156Crossref PubMed Scopus (258) Google ScholarAlthough the work described by the Rossi group focuses on targeting HIV with aptamers and siRNA, the approach sets the tone for modular construction of therapeutics in which either portion of the two-headed antagonist could be substituted by other molecules. For example, other antiviral aptamers have previously been selected, such as aptamers that bind to human cytomegalovirus,24Wang J Jiang H Liu F In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection.RNA. 2000; 6: 571-583Crossref PubMed Scopus (66) Google Scholar and such aptamers could be readily substituted for the anti-HIV-1 hitchhiker. Similarly, the escort moiety doesn't even have to be an RNA, and indeed anti-gp120 antibodies have been used to deliver siRNAs.25Song E Zhu P Lee SK Chowdhury D Kussman S Dykxhoorn DM et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (890) Google Scholar Modularity of cargoes is also a possibility, and toxins or pro-apoptotic peptides could be delivered to kill any newly infected cell before the virus could be further spread.The notion that RNA can deliver RNA has interesting ramifications beyond therapeutics. Indeed, it is curious that nature hasn't previously picked up on this trick. However, just as the engineering of aptamers and aptazymes preceded the discovery of riboswitches, it may be that engineered RNA delivery precedes the discovery of a natural system of RNA-based communication. In this regard, it is interesting to note that RNAs are already being passed around between cells by exosomes.26Valadi H Ekström K Bossios A Sjöstrand M Lee JJ Lötvall JO Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8592) Google Scholar Engineered or natural RNAs could now potentially be used to mediate cell-to-cell communication, directing cargoes to specific cells and thereby triggering responses such as cell growth or even differentiation, or mediating expression profiles. Such cell-to-cell communication might even be carried out with viral particles produced by specially engineered cells, allowing the viral hitchhiker to come full circle and become the driver of a new technology, rather than just the passenger of a pathogen. Although anti-HIV drugs have been in place for decades, both prophylaxis and a cure remain elusive. Many treatments for HIV infection are associated with severe side effects (which may contribute to poor patient compliance), and resistance mutants frequently arise that render many front-line therapeutics impotent. Nucleic acid–based therapeutics (summarized in Table 1) have been considered as an alternative to the chemical antivirals. Early attempts to develop nucleic acid therapeutics for HIV-1 focused on antisense drugs and RNA decoys or mimetics. More recently these efforts have broadened to more efficacious antisense drugs (siRNAs) and RNA decoys or mimetics (aptamers, reviewed in refs. 1Joshi PJ Fisher TS Prasad VR Anti-HIV inhibitors based on nucleic acids: emergence of aptamers as potent antivirals.Curr Drug Targets Infect Disord. 2003; 3: 255-262Crossref PubMed Scopus (52) Google Scholar,2Scherer L Rossi JJ Weinberg MS Progress and prospects: RNA-based therapies for treatment of HIV infection.Gene Ther. 2007; 14: 1057-1064Crossref PubMed Scopus (70) Google Scholar,3Held DM Kissel JD Patterson JT Nickens DG Burke DH HIV-1 inactivation by nucleic acid aptamers.Front Biosci. 2006; 11: 89-112Crossref PubMed Scopus (83) Google Scholar,4Zhang Z Blank M Schluesener HJ Nucleic acid aptamers in human viral disease.Arch Immunol Ther Exp (Warsz). 2004; 52: 307-315PubMed Google Scholar,5Nielsen MH Pedersen FS Kjems J Molecular strategies to inhibit HIV-1 replication.Retrovirology. 2005; 2: 10Crossref PubMed Scopus (61) Google Scholar). For example, Capodici et al. demonstrated antiviral efficacy with an anti-Gag siRNA in infected U87 cells as well as primary T cells.6Capodici J Kariko K Weissman D Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference.J Immunol. 2002; 169: 5196-5201Crossref PubMed Scopus (281) Google Scholar Several other groups have shown similar effects with siRNA against other HIV targets (reviewed in ref. 7Bennasser Y Yeung ML Jeang KT RNAi therapy for HIV infection: principles and practicalities.BioDrugs. 2007; 21: 17-22Crossref PubMed Scopus (23) Google Scholar). Similarly, aptamers selected against the HIV-1 proteins have shown inhibition of viral replication. For example, anti–reverse transcriptase aptamers have been shown to form a pseudoknot structure that tightly interacts with the polymerase active site.8Tuerk C MacDougal S Gold L RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase.Proc Natl Acad Sci USA. 1992; 89: 6988-6992Crossref PubMed Scopus (368) Google Scholar Not only did these aptamers inhibit viral replication, but studies showed that the aptamers could be co-packaged into virions, thereby crippling progeny virions.9Chaloin L Lehmann MJ Sczakiel G Restle T Endogenous expression of a high-affinity pseudoknot RNA aptamer suppresses replication of HIV-1.Nucleic Acids Res. 2002; 30: 4001-4008Crossref PubMed Scopus (106) Google Scholar,10Joshi P Prasad VR Potent inhibition of human immunodeficiency virus type 1 replication by template analog reverse transcriptase inhibitors derived by SELEX (systematic evolution of ligands by exponential enrichment).J Virol. 2002; 76: 6545-6557Crossref PubMed Scopus (79) Google Scholar For all of these nucleic acid drugs, though, one of the chief problems in clinical development has been delivery. Although it is possible for RNA drugs to be transcribed inside cells following gene or template delivery, such methods would face the same safety and regulatory hurdles as many other gene therapy efforts. Alternatively, although nucleic acid drugs can be introduced directly into the body, they are frequently either quickly degraded by rampant serum nucleases or quickly swept out of the system unless appended to larger particles or moieties such as polyethylene glycol or modified with the addition of nonstandard nucleotides and linkages. Anticipating these problems, Hicke and Stephens suggested that nucleic acids might best serve not as therapeutics in their own right but as “escort molecules” for other drugs.11Hicke BJ Stephens AW Escort aptamers: a delivery service for diagnosis and therapy.J Clin Invest. 2000; 106: 923-928Crossref PubMed Google Scholar In recent years this prophetic statement has been validated on a number of occasions. For example, anti-cell aptamers selected in Tan's laboratory have been used to specifically target gold nanoparticles and nanorods to cells.12Huang YF Chang HT Tan W Cancer cell targeting using multiple aptamers conjugated on nanorods.Anal Chem. 2008; 80: 567-572Crossref PubMed Scopus (269) Google Scholar,13Medley CD Smith JE Tang Z Wu Y Bamrungsap S Tan W Gold nanoparticle-based colorimetric assay for the direct detection of cancerous cells.Anal Chem. 2008; 80: 1067-1072Crossref PubMed Scopus (538) Google Scholar An anti–prostate-specific membrane antigen aptamer originally selected by Lupold et al.14Lupold SE Hicke BJ Lin Y Coffey DS Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen.Cancer Res. 2002; 62: 4029-4033PubMed Google Scholar has been used to deliver a variety of compounds to prostate tumor cells expressing this antigen, including nanoparticles, toxins, and even siRNAs.15Chu TC Marks JW Lavery LA Faulkner S Rosenblum MG Ellington AD et al.Aptamer:toxin conjugates that specifically target prostate tumor cells.Cancer Res. 2006; 66: 5989-5992Crossref PubMed Scopus (248) Google Scholar,16Chu TC Twu KY Ellington AD Levy M Aptamer mediated siRNA delivery.Nucleic Acids Res. 2006; 34: e73Crossref PubMed Scopus (390) Google Scholar,17Farokhzad OC Jon S Khademhosseini A Tran TN Lavan DA Langer R Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells.Cancer Res. 2004; 64: 7668-7672Crossref PubMed Scopus (777) Google Scholar,18McNamara JO Andrechek ER Wang Y Viles KD Rempel RE Gilboa A et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (852) Google Scholar In the last instance, introduction of an aptamer-siRNA chimera proved sufficient to reverse tumor growth in a mouse xenograft model.18McNamara JO Andrechek ER Wang Y Viles KD Rempel RE Gilboa A et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (852) Google Scholar The great advantage of these approaches is that not only did they help to obviate the delivery problem, they also provided additional specificity to the therapeutics themselves, with the possibility that both the delivery vehicle and the drug delivered could be tuned to a particular pathogen or malady. John Rossi and his co-workers have now taken these approaches one step further.19Zhou J Li H Li S Zaia J Rossi JJ Novel dual inhibitory function aptamer–siRNA delivery system for HIV-1 therapy.Mol Ther. 2008; 16: 1481-1489Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar Whereas nucleic acid therapeutics can be escorted to cells, it is a priori somewhat difficult to envision how to “escort” nucleic acids to viruses, which typically replicate inside a cell. The elegantly simple solution that the Rossi group has suggested is to let the virus escort the nucleic acid therapeutic into its new cellular prey. In this regard, the nucleic acid drug is no longer an escort per se but a hitchhiker that automatically limits viral spread and load. Modified aptamers against HIVBAL gp120 had previously been selected by William James and co-workers. These aptamers were able to significantly inhibit a broad range of clinical isolates in peripheral blood mononuclear cells, including one viral strain from group O. Analysis of one aptamer's binding suggested that the aptamer did not interfere with the gp120–CD4 interaction. Further deconvolution with gp120 antibodies suggested that the aptamer actually inhibited interactions with the co-receptors CXCR4 and CCR5 (ref. 20Khati M Schüman M Ibrahim J Sattentau Q Gordon S James W Neutralization of infectivity of diverse R5 clinical isolates of human immunodeficiency virus type 1 by gp120-binding 2′F-RNA aptamers.J Virol. 2003; 77: 12692-12698Crossref PubMed Scopus (147) Google Scholar). Whereas anti-gp120 aptamers should inhibit the interactions between gp120 and co-receptors and therefore reduce viral load, the Rossi group recognized that gp120 presented on the surfaces of infected cells could also be internalized. Because escort aptamers against internalizing receptors had already proven effective,16Chu TC Twu KY Ellington AD Levy M Aptamer mediated siRNA delivery.Nucleic Acids Res. 2006; 34: e73Crossref PubMed Scopus (390) Google Scholar,18McNamara JO Andrechek ER Wang Y Viles KD Rempel RE Gilboa A et al.Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (852) Google Scholar Rossi and colleagues19Zhou J Li H Li S Zaia J Rossi JJ Novel dual inhibitory function aptamer–siRNA delivery system for HIV-1 therapy.Mol Ther. 2008; 16: 1481-1489Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar cleverly saw that HIV could potentially be tricked into mediating its own destruction (Figure 1). The researchers designed hitchhiker constructs that comprised a 2′-fluoro-pyrimidine-modified anti-gp120 aptamer linked to the sense strand of an siRNA directed against the Tat/Rev small messenger RNA. Following transcription, the aptamer-siRNA chimera is hybridized to the unmodified antisense siRNA strand. As had been previously observed, the anti-gp120 aptamer itself inhibited viral adhesion to the cell surface and was able to moderately reduce viral replication in CEM cells, both when virus was pre-incubated with the constructs and when infected cells were challenged with the constructs. With the addition of the siRNA, though, viral replication was nearly obliterated in the pre-incubation experiments and significantly more reduced in viral challenge in infected cells, demonstrating that the constructs directly inhibited viral infectivity as well as inhibiting replication in culture. Both the aptamer by itself and the chimeras were able to inhibit syncytia formation. To account for possible steric interference between the two components, constructs were also tested in which the aptamer and siRNA were separated by four pyrimidine bases. In all versions of the constructs tested, the variant with the added linker fared slightly better in inhibition than its equivalent without the linker. This suggests that accessibility of the different components in such chimeras may influence function. For example, it can be readily imagined that accessibility of the aptamer itself to gp120 could be limited by its siRNA “tail,” whereas accessibility of that siRNA to the RNA-induced silencing complex might be influenced by the presence of the aptamer (although natural “aptamers” with appended siRNAs are already known, such as the VA RNAs from adenovirus21Andersson MG Haasnoot PC Xu N Berenjian S Berkhout B Akusjärvi G Suppression of RNA interference by adenovirus virus-associated RNA.J Virol. 2005; 79: 9556-9565Crossref PubMed Scopus (274) Google Scholar). The wonderful thing about the modularity of the chimera is that it allows individual variables (such as steric interference) to be separately tested and optimized in a way that is difficult to imagine for more amorphous formulations (such as proteoliposomes). For example, the investigators separately tested siRNA constructs that were either 21 or 27 residues long. Dicer cleavage studies in cell extracts showed that processing of the chimeras proceeded from the antisense 5′ end, leaving a blunted 5′ end. Analysis using Remote Analysis Computation for Gene Expression showed that cleavage of the target was approximately 10 nucleotides from the 5′ end of both versions of the siRNA. Therefore, because the two had different 5′ antisense ends, the target cleavage site of one siRNA was offset by about six nucleotides from the other. Consistent with the questions of accessibility we have raised, the authors demonstrated that the 27-mer siRNA chimeras fared better in antiviral efficacy. This is also in concordance with studies that showed that 27-mer siRNAs with 3′ overhangs on the antisense strand were more effective than corresponding 21-mer sequences.22Amarzguioui M Lundberg P Cantin E Hagstrom J Behlke MA Rossi JJ Rational design and in vitro and in vivo delivery of Dicer substrate siRNA.Nat Protoc. 2006; 1: 508-517Crossref PubMed Scopus (107) Google Scholar,23Rose SD Kim DH Amarzguioui M Heidel JD Collingwood MA Davis ME et al.Functional polarity is introduced by Dicer processing of short substrate RNAs.Nucleic Acids Res. 2005; 33: 4140-4156Crossref PubMed Scopus (258) Google Scholar Although the work described by the Rossi group focuses on targeting HIV with aptamers and siRNA, the approach sets the tone for modular construction of therapeutics in which either portion of the two-headed antagonist could be substituted by other molecules. For example, other antiviral aptamers have previously been selected, such as aptamers that bind to human cytomegalovirus,24Wang J Jiang H Liu F In vitro selection of novel RNA ligands that bind human cytomegalovirus and block viral infection.RNA. 2000; 6: 571-583Crossref PubMed Scopus (66) Google Scholar and such aptamers could be readily substituted for the anti-HIV-1 hitchhiker. Similarly, the escort moiety doesn't even have to be an RNA, and indeed anti-gp120 antibodies have been used to deliver siRNAs.25Song E Zhu P Lee SK Chowdhury D Kussman S Dykxhoorn DM et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (890) Google Scholar Modularity of cargoes is also a possibility, and toxins or pro-apoptotic peptides could be delivered to kill any newly infected cell before the virus could be further spread. The notion that RNA can deliver RNA has interesting ramifications beyond therapeutics. Indeed, it is curious that nature hasn't previously picked up on this trick. However, just as the engineering of aptamers and aptazymes preceded the discovery of riboswitches, it may be that engineered RNA delivery precedes the discovery of a natural system of RNA-based communication. In this regard, it is interesting to note that RNAs are already being passed around between cells by exosomes.26Valadi H Ekström K Bossios A Sjöstrand M Lee JJ Lötvall JO Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.Nat Cell Biol. 2007; 9: 654-659Crossref PubMed Scopus (8592) Google Scholar Engineered or natural RNAs could now potentially be used to mediate cell-to-cell communication, directing cargoes to specific cells and thereby triggering responses such as cell growth or even differentiation, or mediating expression profiles. Such cell-to-cell communication might even be carried out with viral particles produced by specially engineered cells, allowing the viral hitchhiker to come full circle and become the driver of a new technology, rather than just the passenger of a pathogen.
Actin assembly facilitates vesicle formation in several trafficking pathways, including clathrin-mediated endocytosis (CME). Interestingly, actin does not assemble at all CME sites in mammalian cells. How actin networks are organized with respect to mammalian CME sites and how assembly forces are harnessed, are not fully understood. Here, branched actin network geometry at CME sites was analyzed using three different advanced imaging approaches. When endocytic dynamics of unperturbed CME sites are compared, sites with actin assembly show a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. In addition, N-WASP and the Arp2/3 complex are recruited to one side of CME sites, where they are positioned to stimulate asymmetric actin assembly and force production. We propose that actin assembles preferentially at stalled CME sites where it pulls vesicles into the cell asymmetrically, much as a bottle opener pulls off a bottle cap.
The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.
