Rudolph L. Juliano

Conjugates of Antisense Oligonucleotides with the Tat and Antennapedia Cell-Penetrating Peptides: Effects on Cellular Uptake, Binding to Target Sequences, and Biologic Actions

AbstractPurpose. The attainment of effective intracellular delivery remains an important issue for pharmacologic applications of antisense oligonucleotides. Here, we describe the synthesis, binding properties, and biologic properties of peptide-oligonucleotide conjugates comprised of the Tat and Ant cell-penetrating peptides with 2′-O-methyl phosphorothioate oligonucleotides. Methods. The biologic assay used in this study measures the ability of the antisense molecule to correct splicing of an aberrant intron inserted into the Luciferase gene; thus, this assay clearly demonstrates the delivery of functional antisense molecules to the splicing machinery within the nucleus. The binding affinities of the conjugates to their target sequences were measured by surface plasmon resonance (BIAcor) techniques. Results. The peptide-oligonucleotide conjugates progressively entered cells over a period of hours and were detected in cytoplasmic vesicles and in the nucleus. Peptide-oligonucleotide conjugates targeted to the aberrant splice site, but not mismatched controls, caused an increase in Luciferase activity in a dose-responsive manner. The kinetics of Luciferase appearance were consistent with the course of the uptake process for the conjugates. The effects of peptide conjugation on the hybridization characteristics of the oligonucleotides were also examined using surface plasmon resonance. The peptide-oligonucleotide conjugates displayed binding affinities and selectivities similar to those of unconjugated oligonucleotides. Conclusions. Conjugation with cell-penetrating peptides enhances oligonucleotide delivery to the nucleus without interfering with the base-pairing function of antisense oligonucleotides.

Cellular uptake and intracellular fate of antisense oligonucleotides

Antisense oligonucleotides with sequences complementary to a given genetic target can enter cells in sufficient quantities to selectively inhibit gene expression. Thus, they have a potential therapeutic use in preventing undesirable gene expression in diseases such as cancer and AIDS. However, it is remarkable that these molecules, which have high molecular weights and are often charged, gain entry to cells at all. In this article, we review the possible mechanisms by which oligonucleotides enter cells and their subsequent intracellular fates. We also discuss current approaches for improving cellular uptake and delivery of antisense nucleic acids to their intended targets.

Tat-conjugated PAMAM dendrimers as delivery agents for antisense and siRNA oligonucleotides.

PurposePAMAM G5 dendrimer (P) was conjugated to Tat peptide (T), a cell penetrating peptide, in search of an efficient cellular delivery vehicle for antisense and siRNA oligonucleotides.MethodsPAMAM G5 dendrimer was reacted with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt (BODIPY) for visualization to yield the conjugate BP. Bifunctional sulfosuccinimidyl 6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT) was then used to conjugate primary amino groups of BP to cysteine derivatized Tat peptide to give the designed conjugate, BPT. This conjugate was complexed with antisense and siRNA oligonucleotides designed to inhibit MDR1 gene expression. NIH 3T3 MDR cells were used for the evaluation of biological activity of the conjugate.ResultsBoth antisense and siRNA readily formed complexes with the synthesized BPT, introduced into NIH 3T3 MDR cells, and primarily accumulated in intracellular vesicles. MDR1 gene expression was partially inhibited by the antisense–BPT complex and weakly inhibited by the siRNA–BPT complex when both were tested at nontoxic levels of dendrimer. Conjugation with Tat peptide did not improve the delivery efficiency of the dendrimer.ConclusionsDendrimer–oligonucleotide complexes were moderately effective for delivery of antisense and only poorly effective for delivery of siRNA. Conjugation of the dendrimer with the Tat cell penetrating peptide failed to further enhance the effectiveness of the dendrimer.

Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates.

Antisense oligonucleotides are potentially a powerful tool for the therapeutic manipulation of genes associated with cancer. However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells. In this study, we have prepared peptide-oligonucleotide conjugates with the intent of improving intracellular delivery. The phosphorothioate oligonucleotide component of the conjugates was complementary to a site flanking the AUG of the message for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Two types of peptide-antisense oligonucleotide conjugates, but not mismatched control conjugates, provided substantial inhibition of cell surface expression of P-glycoprotein. Surprisingly, the peptide-oligonucleotide conjugates were more potent in the presence of serum than when used under serum-free conditions; this is in striking contrast to most other approaches for intracellular delivery of nucleic acids. Effective inhibition of P-glycoprotein expression was attained with submicromolar concentrations of antisense conjugates under serum-replete conditions. The combination of relatively modest molecular size and good efficacy in the presence of serum proteins suggests that peptide-antisense oligonucleotide conjugates may have significant promise for in vivo therapeutic applications.

Integrin-mediated activation of mitogen-activated protein (MAP) or extracellular signal-related kinase kinase (MEK) and kinase is independent of ras

The integrins are a family of cell surface receptors that mediate adhesive interactions with the extracellular matrix and also generate signals that influence cell growth and differentiation. Ligation and clustering of integrins causes activation and autophosphorylation of focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, and results in the transient activation of p42 and p44 mitogen-activated protein (MAP) kinases. Initial evidence has suggested that the integrin signaling pathway may share common elements with the canonical Ras signal transduction cascade activated by peptide mitogens such as epidermal growth factor (EGF). In this report we demonstrate that Raf-1 and MAP or extracellular signal-related kinase kinase (MEK), key cytoplasmic kinases of the Ras cascade, are activated subsequent to integrin-mediated adhesion of mouse NIH 3T3 fibroblasts. We also show that MAP kinase is downstream of MEK in the integrin signaling pathway. However, in contrast to the receptor tyrosine kinase signaling cascade, integrin-mediated signal transduction seems to be largely independent of Ras. Dominant negative inhibitors of Ras-dependent signaling failed to block integrin-mediated activation of MEK. In addition, while treatment with the peptide mitogen EGF clearly increased GTP-loading of Ras, little effect was observed in response to integrin-dependent cell adhesion. Thus, integrin-mediated activation of MEK and MAP kinase in 3T3 cells occurs primarily by a mechanism that is distinct from the Ras signal transduction cascade.

The delivery of therapeutic oligonucleotides

The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology.

Antisense pharmacodynamics: critical issues in the transport and delivery of antisense oligonucleotides.

This review critically examines current understanding of the kinetics and biodistribution of antisense oligonucleotides, both at the cellular level and at the level of the intact organism. The pharmacodynamic relationships between biodistribution and the ultimate biological effects of antisense agents are considered. The problems and advantages inherent in the use of delivery systems are discussed in the light of further enhancing in vivo pharmacological actions of oligonucleotides.

PAMAM dendrimers as delivery agents for antisense oligonucleotides.

AbstractPurpose. To investigate the potential use of PAMAM dendrimers for the delivery of antisense oligonucleotides into cells under conditions that mimic the in vivo environment. Methods. We used HeLa cells stably transfected with plasmid pLuc/ 705 which has a luciferase gene interrupted by a human β-globin intron mutated at nucleotide 705, thus causing incorrect splicing. An antisense oligonucleotide overlapping the 705 splice site, when delivered effectively, corrects splicing and allows luciferase expression. The ability of dendrimers to deliver oligonucleotides to HeLa Luc/705 cells was evaluated in the absence or presence of serum. Results. PAMAM dendrimers formed stable complexes with oligonucleotides that had modest cytotoxicity and showed substantial delivery activity. The dose of the oligonucleotide, the charge ratio of oligonucleotide to dendrimer, and the size (generation) of the dendrimers were all critical variables for the antisense effect. The physical properties of dendrimer/oligonucleotide complexes were further investigated using sedimentation and gel electrophoresis methods. Effective oligonucleo-tide/generation 5 dendrimer complexes were macromolecular rather than particulate in nature, and were not sedimented at 100,000 RPM. Compared to other types of delivery agents, PAMAM dendrimers were more effective in delivering oligonucleotides into the nucleus of cells in the presence of serum proteins. Conclusions. Our results suggest that PAMAM dendrimers form non-particulate delivery complexes that function in the presence of serum proteins and thus may be suited for in vivo therapeutic applications.

Targeted intracellular delivery of antisense oligonucleotides via conjugation with small-molecule ligands.

Selective delivery of antisense or siRNA oligonucleotides to cells and tissues via receptor-mediated endocytosis is becoming an important approach for oligonucleotide-based pharmacology. In most cases receptor targeting has been attained using antibodies or peptide-type ligands. Thus, there are few examples of delivering oligonucleotides using the plethora of small-molecule receptor-specific ligands that currently exist. In this report we describe a facile approach to the generation of mono- and multivalent conjugates of oligonucleotides with small-molecule ligands. Using the sigma-receptor ligand anisamide as an example, we describe conversion of the ligand to a phosphoramidite and direct incorporation of this moiety into the oligonucleotide by solid-phase DNA synthesis. We generated mono- and trivalent conjugates of anisamide with a splice switching antisense oligonucleotide (SSO) and tested their ability to modify splicing of a reporter gene (luciferase) in tumor cells in culture. The trivalent anisamide-SSO conjugate displayed enhanced cellular uptake and was markedly more effective than an unconjugated SSO or the monovalent conjugate in modifying splicing of the reporter. Significant biological effects were attained in the sub-100 nM concentration range.

Cell-targeting and cell-penetrating peptides for delivery of therapeutic and imaging agents

This review will discuss the basic concepts concerning the use of cell-targeting peptides (CTPs) and cell-penetrating peptides (CPPs) in the context of nanocarrier technology. It deals with the discovery and subsequent evolution of CTPs and CPPs, issues concerning their interactions with cells and their biodistribution in vivo, and their potential advantages and disadvantages as delivery agents. The article also briefly discusses several specific examples of the use of CTPs or CPPs to assist in the delivery of nanoparticles, liposomes, and other nanocarriers.