Cell Type-specific Delivery Research Articles

Novel Dual Inhibitory Function Aptamer–siRNA Delivery System for HIV-1 Therapy

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. In this study, we demonstrate cell type-specific delivery of anti-human immunodeficiency virus (anti-HIV) siRNAs through fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1-infected cells, allowing binding and internalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and that the appended siRNA is processed by Dicer; this releases an anti-tat/rev siRNA which, in turn, inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also show that gp120 expressed on the surface of HIV-infected cells can be used for aptamer-mediated delivery of anti-HIV siRNAs.

Presentation of functional foreign peptides on the surface of SV40 virus-like particles

Viral capsids of simian virus 40 (SV40) are highly efficient gene delivery vehicles that infect a broad range of cells and tissues. To develop a controlled, cell type-specific delivery system, we sought to display foreign peptides on the capsid surface by genetically manipulating the major capsid protein Vp1. Here we report the identification of two sites within the surface loops of Vp1 that can accommodate foreign peptides in such a way that the foreign peptides are displayed on the surface of the virus-like particles (VLPs) without interfering with VLP assembly or the packaging of viral DNA. Insertion of Flag-tags but not RGD integrin-binding motifs at these sites strongly inhibited cell attachment of VLPs, which normally associate with host cells through cell surface molecules such as major histocompatibility complex (MHC) class I and ganglioside GM1. Instead, VLPs carrying the RGD motifs bound to integrin in vitro and to the cell surface in an RGD-dependent manner. Thus, insertion of foreign sequences into the surface loops of Vp1 can reduce natural virus–cell interactions and even confer an ability to bind to a new target receptor. This study demonstrates the potential usefulness of this strategy for the development of novel delivery vehicles with different cell tropisms.

Organ- and Cell-Type Specific Delivery of Kinase Inhibitors: A Novel Approach in the Development of Targeted Drugs

During the past years, we have explored the cellular delivery of kinase inhibitors. Kinase inhibitors have selectivity for specific kinases but they lack cellular selectivity. This is exemplified by recent reports on cardiotoxicity of kinase inhibitors used in cancer treatment. We postulate that targeted cellular delivery of kinase inhibitors can improve their safety/toxicity profiles, as will be exemplified by recent published studies. Cell specific delivery of therapeutics is a quickly growing area of investigation. This innovative strategy employs carrier molecules that bind to receptors exposed on the surface of cell types involved in disease processes. Binding and receptor mediated internalization of the carrier facilitates local accumulation of the product in target cells. Upon systemic administration, this may create local drug depots in specific organs, while other tissues are avoided, thus favoring enhanced localized drug efficacy and reduced sideeffects. Synthesis of targeted kinase inhibitor-carrier conjugates was achieved using a new approach, in which kinase inhibitors were bound to a platinum(II) atom, the so-called Universal Linkage System (ULS). We review this novel linkage chemistry and demonstrate the applicability of ULS for drug targeting approaches aiming at angiogenic endothelial cells, hepatic stellate cells, and kidney tubular cells. We will review important issues like drug release mechanism, safety of the linker, and pharmacokinetics of the products in animals. Finally, we review the pharmacological efficacy of the cellular targeted drug conjugates in experimental animal models, especially in renal and liver fibrosis models. Keywords: Signal transduction, kinase inhibitors, drug targeting, intracellular delivery, fibrosis, inflammation, angiogenesis

Organ- and Cell-Type Specific Delivery of Kinase Inhibitors: A Novel Approach in the Development of Targeted Drugs

During the past years, we have explored the cellular delivery of kinase inhibitors. Kinase inhibitors have selectivity for specific kinases but they lack cellular selectivity. This is exemplified by recent reports on cardiotoxicity of kinase inhibitors used in cancer treatment. We postulate that targeted cellular delivery of kinase inhibitors can improve their safety/toxicity profiles, as will be exemplified by recent published studies. Cell specific delivery of therapeutics is a quickly growing area of investigation. This innovative strategy employs carrier molecules that bind to receptors exposed on the surface of cell types involved in disease processes. Binding and receptor mediated internalization of the carrier facilitates local accumulation of the product in target cells. Upon systemic administration, this may create local drug depots in specific organs, while other tissues are avoided, thus favoring enhanced localized drug efficacy and reduced side-effects. Synthesis of targeted kinase inhibitor-carrier conjugates was achieved using a new approach, in which kinase inhibitors were bound to a platinum(II) atom, the so-called Universal Linkage System (ULS). We review this novel linkage chemistry and demonstrate the applicability of ULS for drug targeting approaches aiming at angiogenic endothelial cells, hepatic stellate cells, and kidney tubular cells. We will review important issues like drug release mechanism, safety of the linker, and pharmacokinetics of the products in animals. Finally, we review the pharmacological efficacy of the cellular targeted drug conjugates in experimental animal models, especially in renal and liver fibrosis models.

Novel Cell type-specific aptamer-siRNA delivery system for HIV-1 therapy

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Here we demonstrate cell type-specific delivery of anti-HIV siRNAs via fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and interalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and the appended siRNA is processed by Dicer, releasing an anti-tat/rev siRNA which in turn inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities and that gp120 expressed on the surface of HIV infected cells can be used for aptamer mediated delivery of anti-HIV siRNAs.

Novel Cell type-specific aptamer-siRNA delivery system for HIV-1 therapy

AbstractThe successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Here we demonstrate cell type-specific delivery of anti-HIV siRNAs via fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and interalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and the appended siRNA is processed by Dicer, releasing an anti-tat/rev siRNA which in turn inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities and that gp120 expressed on the surface of HIV infected cells can be used for aptamer mediated delivery of anti-HIV siRNAs.

Exploring cell type-specific internalizing antibodies for targeted delivery of siRNA

A major challenge to the development of therapeutic small interfering RNAs (siRNAs) is specific and efficient in vivo delivery to target cells. Recent studies suggest that cell type-specific gene silencing in vivo can be achieved by combining siRNAs with cell type-specific affinity ligands such as monoclonal antibodies. The antibody-directed siRNA complex enters target cells through receptor endocytosis and is subsequently released to the cytosol to specifically silence target gene expression through biologically conserved RNA interference (RNAi) pathways. Antibody fragments fused with a small basic nucleic-acid-binding protein and antibody fragment-directed nanoimmunoliposomes are two examples of effective delivery vehicles in vivo. The demonstrated specificity of in vivo gene silencing and the lack of nonspecific immune activation and systemic toxicity encourage further development of therapies based on cell type-specific delivery of siRNA.

Cell type–specific delivery of siRNAs with aptamer-siRNA chimeras

Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type-specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.

Transcription Factors in Asthma: Are Transcription Factors a New Target for Asthma Therapy?

The essential features of persistent severe asthma include structural changes in the airway wall (remodelling). It is not known whether these are the sequelae of chronic inflammation or indeed its initiators. Several transcription factors have been implicated in the inflammatory process in asthma, including the glucocorticoid receptor (GR), NFkappaB, Activator Protein-1 (AP-1), Nuclear Factor of Activated T-cells (NF-AT), cyclic AMP Response Element Binding Protein and more recently, the CCAAT/Enhancer Binding Protein (C/EBP), Peroxisome Proliferator-activated Receptor (PPAR) and the bZIP transcription factor, Nrf2. Could a pathological de-regulation of one of these transcription factors explain the broad spectrum of asthma pathology and can their modulation lead to better symptom control? Although some of the transcription factors seem to be valid targets (NFkappaB, Nrf2 or STAT6) or tools (PPARgamma, -alpha and C/EBP-alpha) for new therapeutic approaches, since many transcription factors play a central role in tissue and organ homeostasis, a longterm general suppression or overexpression, would cause severe side effects in other organs. Cell type specific application of decoy or antisense oligonucleotides for NFkappaB, Nrf2 or STAT6, or specific agonists for PPARgamma and -alpha may help to control the inflammatory response in lung epithelial cells and infiltrated immune cells, but additional, unwanted, effects on other resident cells of the lung cannot be excluded and a beneficial effect over known anti-asthma drugs has first to be proven. In order to progress with such novel therapeutic strategies, the only option seems to be to link transcription factor inhibitors/activators to a cell type specific delivery system.

1081. Targeted Nanocapsules for Liver Cell-Type Delivery of Plasmids In Vivo

Top of pageAbstract In contrast to the efficient delivery afforded by viral vectors, the use of nonviral vectors for gene therapy has been hindered by the lack of adequate in vivo delivery systems. Although hydrodynamic push has been used extensively for hepatic plasmid delivery, this method delivers DNA nonselectively to liver cell types. One approach for increasing delivery to a specific cell type is by targeting receptor(s) that are either unique or highly expressed by that cell. Hepatocytes (heps) express aisaloglycoprotein receptors (ASGPr), and liver sinusoidal endothelial cells (LSECs) express hyaluronan receptors (HAr) in high abundance providing ideal targets for ligand- mediated receptor uptake. The aim of this study was to determine if liver cell type specific delivery of DNA could be achieved in vivo using asialoorosomucoid (ASOR) targeting to the hep ASGPr and HA for the LSEC HAr. Using a novel dispersion atomization method (Mol Cell Biochem 274:77, 2005) that forms sub 50 nm capsules with the receptor ligand noncovalently bound to the capsule coating, a red fluorescent protein (DsRed2) reporter plasmid was encapsulated using either ASOR for hep or HA for LSEC uptake. Eight week (wk) old C57/BL6 mice received 100 |[mu]|g of the encapsulated plasmid targeted using ASOR or HA via tail vein injection and were sacrificed 1 wk post-injection. Liver, spleen, kidneys, lung, heart and brain were excised and processed for histology and protein extracts. Immunohistochemical identification of the LSECs in cryosections was done using anti-CD14 antibody (Ab), a marker specific for the discontinuous endothelial cells in the liver and a Cy5 labeled secondary Ab for confocal microscopy. The merged confocal micrographs of the DsRed2 and Cy5 fluorescence demonstrated colocalization of DsRed2 expression and the LSEC specific CD14 marker when HA was used as the targeting ligand. In contrast, there was no detectable colocalization when ASOR was used for targeting DsRed2 to heps. The presence of the DsRed2 protein was confirmed by western blot (WB) analysis of total liver protein extracts using a polyclonal anti-DsRed2 Ab and detection by ECL. There was no detectable DsRed2 expression in the other major organs. Transgenic hemophilia A mice were administered HA nanocapsules containing a Sleeping Beauty (SB) transposon (Tn) expressing B-domain deleted coagulation factor (F) VIII using a plasmid that codelivers an expression cassette for the SB transposase required for genomic Tn insertion. The mice were bled 2 and 5 wks post injection and plasma activated partial thromboplastin time (aPTT) determined. The treated mice had aPTTs of 25.5 |[plusmn]| 3.1 (2 wks) and 26 |[plusmn]| 1.9 sec (5 wks) which were not significantly different from the wild type (wt) aPTT of 23.5 |[plusmn]| 1.3 sec. In contrast, untreated hemophilia A mice had aPTTs of 46.7 |[plusmn]| 3.5 sec (P < 0.001 from treated and wt mice). In conclusion, ASOR and HA targeted nanocapsules can deliver plasmids in vivo to heps or LSECs, respectively. SB-Tns in HA nanocapsules targeted to LSECs provided expression of a clinically relevant gene product, FVIII that improved the phenotype of hemophilia A transgenic mice.

Cell-type-specific gene delivery into neuronal cells in vitro and in vivo

The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain.

Marked Dopaminergic Cell Loss Subsequent to Developmental, Intranigral Expression of Glial Cell Line-Derived Neurotrophic Factor

Glial cell line-derived neurotrophic factor (GDNF) shows potent neuroprotective as well as neurorestorative actions on the adult neurons impacted in animal models of Parkinson's disease (PD). Long-term pharmaco-physiological effects of GDNF on developing dopaminergic (DA) neurons have not yet been explored because of technical difficulties in producing prolonged cell type-specific delivery of this neurotrophic factor in mammalian embryonic brain. The current studies used our previously characterized 9.0-kb tyrosine hydroxylase promoter to produce transgenic mice with neuronal cell type-specific expression of GDNF in substantia nigra pars compacta (SNc) and locus coeruleus (LC). These mice were used to test the parsimonious hypothesis that increased developmental expression of GDNF in SNc and LC would significantly enhance the number of postmitotic adult neurons. To our surprise, adult transgenic mice carrying the TH9.0kb-GDNF hybrid gene showed dramatic reductions in both the numbers and the volumes of SNc-DA and LC-noradrenergic (NA) neurons by quantitative morphometric analysis. The decrease in the number of DA neurons was apparent as early as postnatal day 2, the period before the major naturally occurring apoptotic cell death in midbrain. Aged transgenic mice exhibited no further significant deficits in motor behaviors. These data suggest that continuous, early developmental GDNF expression exerts physiological effects on newly differentiated, immature dopamine neurons that differ from those observed on more mature and adult DA neurons. Further elucidation of the mechanisms underlying differential GDNF actions will greatly improve the pharmacological efficacy of GDNF in fetal neural transplantation as well as adult neuronal gene therapy in PD patients.

Specific CEA-producing colorectal carcinoma cell killing with recombinant adenoviral vector containing cytosine deaminase gene.

To kill CEA positive colorectal carcinoma cells specifically using the E coli cytosine deaminase (CD) suicide gene, a new replication-deficient recombinant adenoviral vector was constructed in which CD gene was controlled under CEA promoter and its in vitro cytotoxic effects were evaluated. Shuttle plasmid containing CD gene and regulatory sequence of the CEA gene was constructed and recombined with the right arm of adenovirus genome DNA in 293 cell strain. Dot blotting and PCR were used to identify positive plaques. The purification of adenovirus was performed with ultra-concentration in CsCl step gradients and the titration was measured with plaque formation assay. Cytotoxic effects were assayed with MTT method, The fifty percent inhibition concentration (IC(50)) of 5-FC was calculated using a curve-fitting parameter. The human colorectal carcinoma cell line, which was CEA-producing, and the CEA-nonproducing Hela cell line were applied in cytological tests. An established recombinant adenovirus vector AdCMVCD, in which the CD gene was controlled under CMV promoter, was used as virus control. Quantitative results were expressed as the mean +/- SD of the mean. Statistical analysis was performed using ANOVA test. The desired recombinant adenovirus vector was named AdCEACD. The results of dot blotting and PCR showed that the recombinant adenovirus contained CEA promoter and CD gene. Virus titer was about 5.0 X 10(14)pfu/L(-1) after purification. The CEA-producing Lovo cells were sensitive to 5-FC and had the same cytotoxic effect after infection with AdCEACD and AdCMVCD (The IC(50) values of 5-FC in parent Lovo cells, Lovo cells infected with 100 M.O.I AdCEACD and Lovo cells infected with 10 M.O.I AdCMVCD were >15000, 216.5+/-38.1 and 128.8+/-25.4 micromol.L(-1), P<0.001, respectively), and the cytotoxicity of 5-FC increased accordingly when the m.o.i of adenoviruses were enhanced (The value of IC(50) of 5-FC was reduced to 27.9+/-4.2 micromol.L(-1) in 1000 M.O.I AdCEACD infected Lovo cells and 24.8+/-7.1 micromol.L(-1) in 100 M.O.I AdCMVCD infected Lovo cells, P<0.05, P<0.01, respectively). The CEA-nonproducing Hela cells had no effect after infection with AdCEACD, but Hela cells had the cytotoxic sensitivity to 5-FC after infection with AdCMVCD (The IC(50) of 5-FC in parent Hele cells and Hela cells infected with AdCMVCD at 10 M.O.I was >15000 and 214.5+/-31.3 micromol.L(-1), P<0.001). AdCEACD/5-FC system also had bystander effect, and the viability was about 30 percent when the proportion of transfected cells was only 10 percent. The recombinant adenovirus vector AdCEACD has the character of cell type-specific gene delivery. The AdCEACD/5-FC system may become a new, potent and specific approach for the gene therapy of CEA-positive neoplasms, especially colon carcinoma.

In vivo cell type-specific gene delivery with retroviral vectors that display single chain antibodies.

Cell type-specific gene delivery will be essential for in vivo gene therapy. Our laboratory has previously developed retroviral vector particles, derived from spleen necrosis virus, SNV, which display the antigen-binding site of an antibody on the viral surface. Such particles infected only human cells in vitro, which expressed a receptor recognized by the antibody. To test cell type-specific gene delivery in vivo, a mouse model system has been developed. Antibiotic resistant human target and non-target cells were injected into the peritoneum of SCID mice. Subsequently, a vector solution containing 106 infectious particles, which display scAs against the human her2neu cell surface protein, was injected. Cells were recovered from the peritoneum, subjected to antibiotic selection, and tested for the expression of a lacZ gene transduced by the retroviral vector. We found that human target cells, which express her2neu, were infected in vivo. However, neither human cells that do not express her2neu, nor normal mouse cells were infected by such viral particles. These data give proof of principle that retroviral vector-mediated, cell type-specific gene delivery can be obtained in vivo.

A genetically engineered spleen necrosis virus-derived retroviral vector that displays the HIV type 1 glycoprotein 120 envelope peptide.

We reported that SNV-derived retroviral vectors, which display single-chain antibodies on the viral surface, enable cell type-specific gene delivery into various human cells. In particular, the SNV cell type-specific gene delivery vector system appears to be well suited to transduce genes into cells of the human hematopoietic system (Jiang et al., J. Virol. 72:10148-10156, 1998). Here, we report the construction of SNV vector particles that display the complete gp120 surface unit of the envelope protein of human immunodeficiency virus type 1 (HIV-1) on the viral surface. The complete gp120-coding region of a T cell-tropic HIV-1 strain (LAI/BRU) was fused to a short peptide spacer coding region [(Gly4Ser)3] linking it to the SNV TM-coding region. The corresponding protein was expressed as a single 145-kDa peptide as expected. This peptide was nontoxic and could be stably expressed in dog D17 SNV-derived packaging cells. Particles harvested from stable packaging lines infected CD4+ human hematopoietic cells with titers exceeding 10(5) CFU/ml supernatant tissue culture medium. Titers in other, CD4- cell lines expressing various coreceptors of HIV-1 were 100-fold lower than titers obtained in CD4+ cells. Specificity of infection was demonstrated by antibody inhibition assays or by preincubating cells with SDF-1alpha, the ligand, which binds to the CXCR4 coreceptor, to which this gp120 binds. Our data indicate that binding of the HIV-1 gp120 to either CD4 or CXCR4 is sufficient to enable infection of human cells with SNV vector particles. We constructed retroviral vector particles that display chimeric HIV-1-SU-SNV-TM proteins plus wild-type SNV envelope on the viral surface. Such particles allowed efficient infection of CD4-positive human T lymphocytes, and, at a lower efficiency, also cells expressing CXCR4 without CD4. These data coincide with our earlier hypothesis that the chimeric envelope is required only to bind the vector particle to a cell surface receptor of the target cell, while membrane fusion is mediated by wild-type Env, which alone is not sufficient to enable infection of human cells.

Diagnosis and clinical significance of parainfluenza virus infections in children.

Cell type specific delivery of drugs by use of nanocapsules (NC) is a promising approach for induction of efficient anti-tumor immunotherapies. Here, we report the generation of IL-2 functionalized NC (HES-IL-2) to target CD25⁺ (IL-2 receptor α chain) T cells. In flow cytometry experiments HES-IL-2 exhibited an enhanced uptake by human CD25⁺ T cells compared to control capsules. The observed uptake was CD25 specific as CD25^high T cells displayed a significantly enhanced NC incorporation compared to CD25⁻ T cells with negligible internalization. In addition blocking of the IL-2 receptor dependent binding by an anti CD25 mAb significantly reduced the HES-IL-2 uptake by human CD25⁺ T cells. Furthermore, comparing and competitive studies of naïve CD25⁻, activated CD25⁺ and regulatory CD25^high human T cells revealed a low incorporation of HES-IL-2 in naïve and a moderate and high uptake, respectively in activated or regulatory T cells. Additionally, in order to preferentially address different T cell populations by means of various IL-2 receptor affinities, we generated HES-IL-2 with distinct reduced amounts of IL-2 on their surface. Intriguingly, in contrast to naïve CD25⁻ and activated CD25⁺ T cells, in which the NC uptake was clearly IL-2 dose-dependent we did not observe any differences when HES-IL-2 with a twofold and tenfold decreased amount of IL-2 were used for studies with regulatory CD25^high T cell, indicating a high IL-2 sensitivity of regulatory T cells for a specific targeting. In addition, <i>in vivo</i> studies in human T cell reconstituted RAG2^−/−γc^−/− mice exhibited a significantly enhanced uptake of HES-D-IL-2 by CD25⁺ T cells. In summary, we generated IL-2 functionalized NC for targeting of human CD4⁺CD25⁺ T cells <i>in vitro</i> and <i>in vivo</i>.

Reply to comments on “The diffusion of chromium in iron and low carbon steels”

One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3′ 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2′ OMe/2′ Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo.