Cellpenetrating Peptide Morpholino Conjugates

Cationic CPPs are promising carriers to enhance the cytoplasmic and nuclear delivery of Morpholinos, referred to hereon as PMOs, for several reasons. First, negatively charged AOs might electrostatically interact with conjugated cationic CPPs with loss of antisense or delivery activity - because of the uncharged nature of PMOs undesired inter- and intra-molecular charge interactions between a CPP and PMO are not a concern. Second, CPP PMO (PPMO) conjugates are simple to use as they can be...

Effects of CPP Sequences on PPMO Efficacy

Length, sequence and amino acid types of a CPP affect efficacy of the resulting PPMO conjugates. A splice-correction reporter assay88 was used to determine the effects of these properties on the effectiveness of CPP to deliver PMO to cell nuclei. This assay uses the ability of steric-blocking AOs to block a splice site created by a mutation to restore normal splicing. The reporter luciferase's coding sequence was interrupted by the human p-globin thalassemic intron 2, which carried a mutated...

Ribozymes

In the early 1980s, the groups of Thomas Cech and Sydney Altman discovered catalytically active ONs and coined the term ribozyme for these ribonucleic acids with enzymatic activity. In the meantime, several classes of ribozymes have been discovered in nature, most of which catalyse intramolecular splicing or cleavage reactions (for reviews, see Schubert and Kurreck28 and Fedor and Williamson29). The present review focuses exclusively on the hammerhead ribozyme, which has the greatest relevance...

Chemical Modifications of CpG Oligonucleotides

Chemical modification of CpG ODNs can be used to enhance stability against nucleases, to improve cellular uptake and compartmentalization, and to modulate binding to TLR9. While there is a plethora of chemical modifications known from antisense ODN research and also on the impact of such modifications on nuclease stability and cell uptake, modulation of binding to and activation of TLR9 has to be elaborated specifically for CpG ODNs. Alterations of the sugar-phosphate backbone have significant...

An Introduction to Aptamers

Over the past two decades, oligonucleotides (ONs) have emerged as a viable alternative to virus-mediated gene complementation, which has proved effective in the treatment of genetic diseases in clinical trials,1,2 but is also associated with pathogenicity and immunogenicity.3,4 Although ON technology is still at an early stage of development, the therapeutic potential of these molecules has been explored extensively in terms of their capacity to correct genetic defects (using single-stranded...

Continuous Synthesis

Several strategies for both manual and automated solid-phase synthesis of PNA have been developed.49 51 They are based on a cycle of chemical reactions, as shown in Figure 4.1, which require PNA monomeric building blocks protected with two orthogonal groups, one for the N-terminus (PG1 in Figure 4.1) and one for the amino function on three out of the four nucleobases (PG2). The two most frequently used compatible combinations of protecting groups PG1 and PG2 are 9-fluorenylmethoxycarbonyl...

Future Prospects

The successful use of RNA mediators of anti-HIV activity in human hematopoietic cells has now been validated by many different investigators. With the development of genetically modified viral vectors that are capable of transducing hematopoietic cells with therapeutic RNA encoding constructs, there will be more proof-of-concept studies in animal models within the next couple of years. Ongoing clinical trials using anti-HIV ribozymes and antisense RNAs have demonstrated the safety of...

Toxicity

The toxicity of PPMO depends on the concentration, treatment time, serum concentration, CPP sequences, cell types and impurities in the PPMO preparation. PPMO toxicity reported in the literature is summarized in Table 3.5. We will discuss some highlights in this section. Concentration-dependent toxicity has been observed for PPMOs both in cell culture and in animals. Animals (mouse and rat) tolerated (RXR)4XB PPMO well with repeated doses at < 10mgkg-1.4,90,96,98,101 At higher doses, animals...

In vitro Inhibition of Growth by AS1411

AS1411 has been tested extensively in tumour cell proliferation and or inhibi- tion experiments in vitro. , ' Original studies showed that GRO29A at a concentration of 15 mmolL-1 induced growth inhibition of prostate (DU-145), breast (MDA-MB-231 and MCF-7) and Henrietta Lack (HeLa) cancer cell lines.15 In most cell lines, growth inhibition was clearly evident after five days of exposure. Further studies showed that a 10 mmol L_1 concentration of either GRO29A or the unmodified GRO29AOH...

The Role of Backbone Modifications in Oligonucleotide Based Strategies

1.2 Antisense Oligonucleotides 3 1.2.2 2'-O-Methyl and 2'-O-Methoxyethyl Ribonucleotides 5 1.2.3 Locked Nucleic Acids, Phosphorodiamidate Morpholino Oligomers and N3' P5' Phosphoramidates 7 1.4 Small Interfering RNAs 11 1.4.1 Introduction of Modified Nucleotides 12 1.4.2 Combination of Different Modifications 13 1.4.3 Terminal Modifications 14 1.5 Aptamers and Decoy Oligonucleotides 15 1.6 Concluding Remarks 18 Acknowledgement 18 References 18

Synthesis of Locked Nucleic Acids Phosphoramidites

Many skilled chemists have optimized the synthesis of LNA phosphoramidites and thereby moved the availability and costs from being a major concern for the commercialization of LNA to being a minor issue that is well in progress. The overall yields of the LNA amidite syntheses were initially very low. The yields of LNA-purine syntheses were 1-5 and the yields for LNA-pyrimidine syntheses were 5-10 . Thanks to the chemistry efforts over the past six years the yields are now in the range 25-40 ....

RNase H Recruitment of LNA

It is well-established that ONs targeting mRNA are most potent if they are designed to recruit RNase H. This ubiquitous enzyme will cleave mRNA when hybridized to a DNA complement. Since LNA nt's adopt an RNA-like structure they will not be able to recruit RNase H however, this can be obtained if a DNA segment is incorporated into the ON as in, e.g., gapmers (Figure 5.4). In a study to test LNA 16-mers the optimal gap size was reported to be in the range 7-10 DNA nt's.22 It was also concluded...

Clinical Phase III Study of SPC2996

Data from the initial Phase I II clinical trial of the Bcl-2 mRNA-directed ON, SPC2996, in patients with advanced CLL was presented at the 2007 Annual Meeting of the American Society of Clinical Oncology. Prior to this first clinical study of SPC2996, comprehensive GLP toxicology studies were conducted. A detailed presentation of the preclinical clinical trial application (CTA) and investigational new drug (IND) supporting studies is beyond the scope of this chapter, but a brief outline of the...

Reagentbased Methods Endo Porter and Ethoxylated Polyethylenimine

Reagent-based delivery methods have been developed to deliver Morpholinos. Endo-Porter is a peptide-based reagent developed to deliver Morpholinos into cells by co-endocytosis followed by release of the endocytosed materials by permeabilization of the acidic endosome. Endo-Porter and a Morpholino are delivered as independent molecules, without conjugation or complexation. This endocytotic entry is more gentle to cells than are lipofection reagents that render the plasma membrane leaky.80...

Hybridization Properties

The inherent property of LNA nucleotide substitutions is the high binding affinity in almost any possible design variant.5,6,13,21,44,50 LNA hybrids Figure 5.3 Structure of a 9-mer LNA-RNA hetero duplex. The LNA oligonucleotide comprises three LNA nucleotides, shown in yellow. Figure 5.3 Structure of a 9-mer LNA-RNA hetero duplex. The LNA oligonucleotide comprises three LNA nucleotides, shown in yellow. obey the WC hydrogen bonding rules and form right-handed helices and classical...

The Spontaneous Vesicle Formation by Ethanol Dilution Method of Encapsulation

The methods described above rely on extrusion steps to ensure the preparation of small, monodisperse liposomes. Expressly developed as an alternative to such techniques, the SNALP method is simple, robust, and fully scalable.13 Originally developed to encapsulate plasmid DNA, subsequent adaptation has allowed the encapsulation of smaller nucleic payloads, such as siRNAs. Liposomes encapsulating siRNA are formed instantaneously by combining the flow streams of an ethanolic lipid solution and an...

Inducible RNAi Systems

Pharmacological control of therapeutic gene expression permits the drug-dependent precise titration of gene-product dosage, intermittent or pulsatile treatment and defined termination of therapy.39 For many biochemical and functional analyses of essential genes, conditional knockout approaches are necessary and conditional RNAi systems may thus be of fundamental impact for use in both experimental and therapeutic applications. For example, knockout of genes essential for embryonic development...

Clinical Studies with AS1411

Based on the original promising preclinical properties of GROs (growth inhibition of cancer cells in vitro at low micromolar concentrations), and subsequent preclinical data that indicated specific potential for AS1411 against a wide range of solid tumours (antitumour activity in vivo in mice bearing human tumour xenografts) and a lack of significant toxicity in rats and dogs, AS1411 was selected for a Phase I, open label, non-randomized, dose escalation clinical study in 30 patients with...

Jai Mata Ka Krappa

Weller, Antisense Nucleic Acid Drug Dev., 1997, 7, 187. 2. R. M. Hudziak, E. Barofsky, D. F. Barofsky, D. L. Weller, S. B. Huang and D. D. Weller, Antisense Nucleic Acid Drug Dev., 1996, 6, 267. 3. D. S. Youngblood, S. A. Hatlevig, J. N. Hassinger, P. L. Iversen and H. M. Moulton, Bioconjugate Chem., 2007, 18, 50. A. Amantana, H. M. Moulton, M. L. Cate, M. T. Reddy, T. Whitehead, J. N. Hassinger, D. S. Youngblood and P. L. Iversen, Bioconjugate Chem., 2007, 18, 1325. J....

References

Wickstrom, J. Biochem. Biophys. Methods, 1990, 20, 259. 2. M. I. Phillips and Y. C. Zhang, Methods Enzymol., 2000, 313, 46. 3. D. A. Brown, S.-H. Kang, S. M. Gryaznov, L. De Dionisio, O. Heidenreich, S. Sullivan, X. Xu and M. I. Neerenberg, J. Biol. Chem., 1994, 43, 26801. 4. A. A. Levin, Biochim. Biophys. Acta, 1999, 1489, 69. 5. C. A. Stein, C. Subasinghe, K. Shinozuka and J. S. Cohen, Nucleic Acids Res., 1988, 16, 3209. 6. P. J. Furdon, Z. Dominski and R....

Early Studies on G3139

Genasense G3139 or oblimersen is an 18-mer phosphorothioate oligodeoxyribonucleotide oligo that is complementary to codons 1-6 of the Bcl-2 messenger RNA mRNA .1 The compound was ultimately derived from a 20-mer phosphorothioate oligo called T1-AS that straddled the Bcl-2 initiation codon region,2 and which thus had significant partial homology to G3139. Pre-B ALL cells line 697 , which lack the t14 18 translocation, were treated under serum-free conditions with this oligo, which, in the time...

Targeting Hypoxiainducible Factor 1 Alpha

Hif-1 is another interesting target for cancer.71,72 Hif-1 is a heterodimer consisting of Hif-1a and Hif-1 p ARNT which are constitutively expressed in the cell. Hif-1 is a major transcriptional factor in the cellular response to hypoxia and regulates a large group of genes, among others vascular endothelial growth factor VEGF , glucose transporter1 Glut1 and multi-drug resistance1 MDR1 . HIF-1a is continually degraded at normoxic conditions t1 2 5 minutes , but is stabilized under hypoxic...

Cationic Lipids

Cationic lipids play two roles in liposomal nucleic acid formulations. First, during the formulation process they promote interaction between the lipid bilayer and the negatively charged nucleic acid, yielding greatly enriched payload concentrations of nucleic acid by comparison with charge-neutral liposomes 40 when using co-extrusion methods, and greater than 95 when using more sophisticated techniques .13 15 Second, cationic lipids lend the particle a net positive charge, facilitating...

The Ethanol Drop Method of Encapsulation

The limited efficiency of passive nucleic acid encapsulation and the pharmacology of the resulting particles are partially addressed by the development of the stabilized antisense lipid particle SALP method of encapsulation Figure 11.1 . SALPs are prepared by dropwise addition or injection of an ethanolic lipid solution to an aqueous solution of nucleic acid generally antisense DNA ONs .19 By utilizing an ionizable aminolipid at an acidic pH, where the aminolipid is fully charged, highly...

Unassisted Cellular Uptake of Oligonucleotides

Cellular internalization of free ONs has frequently been observed. Binding of ASOs to a cell membrane receptor can trigger their endocytotic or pinocytotic uptake,30,31,32 and a number of research groups have investigated uptake of ligand-ASO conjugates. , , In addition, Li and co-workers reported the transport of unassisted ONs through anion channels in cultured bovine adrenal cells.36 Cellular uptake is, however, only the first barrier to be overcome. Following uptake, ONs are initially...

In vivo Biodistribution of AS1411

Biodistribution studies have been done to establish an AS1411 dosing regimen that enables plasma concentrations to be attained in vivo that reflect the low micromolar levels of drug required to kill cells in vitro.8 Firstly, mice bearing subcutaneous lung and renal human tumour xenografts derived from A549 and A498 cells, respectively, were injected with a single intravenous bolus of tritiated AS1411 1, 10 and 25mgkg_1 . Approximately 63 of the radiolabel was recovered in the urine within...

In vivo Antitumour Activity of AS1411

Evidence of the efficacy of AS1411 in vivo was ascertained from a study in nude mice bearing established subcutaneous human tumour xenografts derived from A549 non-small-cell lung cancer cells. AS1411 was administered intravenously Figure 8.3 Effect of AS1411 on the growth in vivo of A A549 and B SKMES human lung carcinoma xenografts grown subcutaneously in nude mice. A Vehicle phosphate-buffered saline or AS1411 5, 10 and 40mgkg intravenously was administered daily for five consecutive days...

Combinatorial Approach 3 Include Cellular Factors as RNAi Targets

We have already discussed that RNAi cannot prevent proviral establishment. In addition, RNAi directed at viral transcripts produced after provirus establishment may not completely silence virus production. Thus, RNAi directed at HIV-1 itself could still allow a very low level of virus spreading. As an alternative to prevent provirus establishment, cellular receptors or co-factors involved in the initial phase of infection can be targeted. In addition, cellular factors involved in infectious...

Lentiviral Vector Development

Of crucial importance for the clinical development of gene therapy strategies is that the vector can be produced to high titres and that the vector is genetically stable. The lentiviral vector is very efficient in transducing CD34 blood stem cells or CD4 T cells. However, this vector system is based on HIV-1, and expression of anti-HIV shRNAs from the lentiviral vector may cause unwanted complications. Indeed, it has been reported that targeting of the Gag-Pol and Rev mRNAs of the packaging...

Calcium Release Assay

To test the inhibitory actions of NOX-E36, THP-1 acute monocytic leukaemia cells which express CCR2 and other chemokine receptors were used in a Ca2 -release assay. Stimulation of THP-1 cells with MCP-1 resulted in a dose-dependent increase of the cytoplasmic calcium concentration Ca2 . Plotting the difference between the maximal and the baseline signals, a dose-response curve for MCP-1 was obtained that indicated a half effective concentration EC50 of about 3nM data not shown . To demonstrate...

Pharmacokinetics and Biodistribution of Liposomal siRNAs Following Systemic Administration

Following intravenous injection, the clearance properties of encapsulated siRNAs can be measured by lipid and or nucleic acid markers. Previous experience shows that when nucleic acids are fully encapsulated in stable liposomes, they remain protected from nuclease degradation, and are cleared from the blood at a rate equal to that of the lipid components.14,15,57 The biodistribution of intact lipid particles can be represented by monitoring radiolabeled lipid markers stably incorporated into...

Anticancer Mechanism of Action of AS1411

As AS1411 is known to target nucleolin and to be internalized efficiently by cancer cells, the possibility that its uptake is mediated by nucleolin was explored.18 The cellular uptake of fluorescein isothiocyanate FITC -labelled AS1411 was greatly enhanced in nucleolin-overexpressing breast carcinoma cells MCF-7 compared to the normal counterpart MCF-10A , which expresses very low levels of nucleolin Figure 8.1 . Hence the cellular internalization of AS1411 depends on nucleolin levels, which,...