Potency Dose and Toxicity

Potency and toxicity is the double-edged sword of dosing that is negotiated for all drugs. For gene therapy, dose can refer to the number of viral particles directly injected into a tumour, the number of stem progenitor cells re-infused into a patient, the number of cells derived from gene-modified stem cells over time or the amount of intracellular expression of a gene therapeutic. The balance of potency and toxicity is well documented in the small-molecule literature, and the relatively...

References

Agrawal S, Zhang R (1997) Pharmacokinetics of oligonucleotides. Ciba Found Symp 209 60-75 Andreopoulos S (2003) Developing drugs for parasitic diseases. Science 300 430-431 Blum, ML, Down JA, Gurnett AM, Carrington M, Turner MJ, Wiley DC (1993) A structural motif in the variant surface glycoproteins of Trypanosoma brucei. Nature 362 603-609 Bonora GM, Ivanova E, Zarytova V, Burcovich B, Veronese FM (1997) Synthesis and characterization of high-molecular mass polyethylene glycol-conjugated...

Creation of Allosteric Ribozymes

As mentioned above, ribozymes can target RNAs where a specific sequence is present for efficient cleavage Fig. 2a. The cleavage of a substrate occurs immediately after the NUX sequence (where N is any base and X is A, C, or U) within the target (Shimayama et al. 1995 Zhou and Taira 1998). In some cases, a cleavable triplet sequence is not available at a suitable position within the target. For example, in the case of certain abnormal chimeric mRNAs, which are generated by chromosomal...

Aptamer Core Structures and Serum Stability

The above-described SELEX experiments led to the identification of high affinity RNA ligands that target the trypanosomal surface and thus suggest several pharmacological approaches. In order to optimize the selected RNAs for their pharmaceutical use, the original molecules have to be converted from in vitro binding reagents to therapeutic compounds, which can be tested for their pharmacological effects in vivo. Within this context an important consideration is the cost of synthesis. Since...

Transcriptome Analysis Facilitated by LNA

Efficient selection of polyadenylated mRNA from eukaryotic cells and tissues is an essential step for a wide selection of functional genomics applications, including full-length complementary (c)DNA library construction and sequencing, Northern and dot blot analyses, gene expression profiling by mi-croarrays, and quantitative RT-PCR. The key to successful selection of intact poly(A)+RNA is a fast extraction of total RNA from cells and tissues using strong denaturing agents to disrupt the cells...

Inhibition of HIV Replication by RNAi

Hiv Replication Cycle

The introduction of combination antiretroviral therapy has resulted in a remarkable improvement of the life expectancy of individuals infected with HIV and has significantly reduced their likelihood of developing AIDS. However, despite this progress, HIV infection remains incurable. Toxicity problems associated with current drug therapies and the emergence of drug resistance clearly indicate the need for alternative therapeutic approaches. Retroviral infection with HIV results in the stable...

RNA Targeting by LNAzymes

DNAzymes are catalytically active DNA molecules that are able to cleave RNA in a sequence-specific manner after binding to complementary sequences. The so-called 10-23 DNAzyme Fig. 3 was found by in vitro selection Santoro and Joyce 1997 . It consists of a 15-nt catalytic core flanked by two binding arms that lead to the sequence selectivity of a given DNAzyme. LNAzymes are LNA-modified DNAzymes, and incorporation of two LNA nucleotides in each of the binding arms yielded an LNAzyme with a...

Detection Methods for Small Molecules and Advantages of Aptamers

Small molecules are involved in innumerable biochemical processes in life and can operate as substrate, catalyst or inhibitor in biochemical reactions. Because of these properties, small molecules are valuable as drugs in medicine or in biotechnological applications. In addition, in all cases the feasibility of quickly and reliably detecting them is essential. Different and versatile techniques have been developed for the detection of small molecules. Classic detection methods are based on...

LNA Hybridization Properties and Structural Features

Lna Dna Rna

We have defined LNA as an oligonucleotide containing at least one LNA monomer, i.e., one 2 -0,4 -C-methylene-P-d-ribofuranosyl nucleotide see Fig. 1 Singh et al. 1998 Koshkin et al. 1998b . The LNA monomers adopt N-type sugar puckers, also termed C3 -endo conformations Singh et al. 1998 Obika et al. 1997 . The vast majority of reports on LNA-mediated RNA targeting have dealt with mixmer LNA oligonucleotides, i.e., LNAs containing a limited number of LNA monomers in combination with other types...

SiRNA Agents Work as Ligands for Toll Like Receptors

Surprisingly, recent studies have indicated that siRNAs can induce global up-regulation ofthe expression of IFN-stimulated genes Bridge et al. 2003 Jackson et al. 2003 Sledz et al. 2003 Kariko et al. 2004 Persengiev et al. 2004 Fig. 5 . This effect was detected with synthetic siRNAs that were transfected into cells, and with siRNAs that were produced within cells by the expression of shRNAs. Both of these papers documented significant non-specific changes in gene expression as a consequence of...

Inhibition of HCV by RNAi

HCV is a major cause of chronic hepatitis and hepatocellular carcinoma. Currently, no vaccines are available for HCV, and several groups have used RNAi to target HCV replication. HCV belongs to the family Flaviviridae, and its genome is encoded by a 9.6-kb RNA of positive polarity. Because there is no cell culture system for HCV replication, all studies have used replicon systems in Huh-7 cells as a model for HCV replication. These replicons support HCV RNA transcription and protein synthesis,...

RNA Aptamers that Recognize the Variant Surface Glycoprotein

The aim of the SELEX experiment published by Lorger and co-workers 2003 was to select RNA aptamers that specifically recognize the VSG protein on the surface of bloodstream-stage African trypanosomes. The bloodstream lifecycle stage represents the infectious phase of the parasite, and VSG is the most abundant surface protein during that period. Since the parasite undergoes antigenic variation, which is characterized by the expression of different VSG variants, a selection scheme was designed to...

Nucleic Acid Aptamers Directed Against Carbohydrates

Nucleic acid interactions with polymeric cellular components, particularly with proteins, are dominated by hydrogen bonds, hydrophobic and ionic interactions between phosphate residues and positively charged amino acids. This is defined by the sequence and tertiary structure of the nucleic acid. Carbohydrate residues are void of basic functional groups that are capable of undergoing ionic interaction with nucleic acids. In addition, they possess a very flexible structure. Such molecules are not...

G V U U Ag

Fig. 1a-c The effects of 2'-amino modification at position 2.1 upon the cleavage activity of a hammerhead ribozyme. a A representative example of multiple turnover reactions of the wild-type ribozyme Wt-URz and the ribozyme with a single 2'-amino group at position 2.1 2'-NH2 2.1 -URz in the presence of 5 mM Mg2 . b The cleavage reaction was performed in the presence of Mn2 . c Summary of the ribozyme catalytic activity following various site-specific modifications at position 2.1. Primary and...

Npcrnas In Vibrio Cholerae

Functional Redundancies and How to Find Them The few examples described here show that the pathogenesis-related RNAs are much more prevalent than previously anticipated. One interesting feature that emerges is the functional redundancy of some of these regulatory RNAs in different pathogenic bacteria Table 2 . It is conceivable that these RNAs are differentially expressed upon different environmental niches conditions or that they have evolved independent regulatory functions i.e., different...