The choice of negative control antisense oligonucleotides dramatically impacts downstream analysis depending on the cellular background.

BACKGROUND: The lymphatic and the blood vasculature are closely related systems that collaborate to ensure the organism's physiological function. Despite their common developmental origin, they present distinct functional fates in adulthood that rely on robust lineage-specific regulatory programs. The recent technological boost in sequencing approaches unveiled long noncoding RNAs (lncRNAs) as prominent regulatory players of various gene expression levels in a cell-type-specific manner. RESULTS: To investigate the potential roles of lncRNAs in vascular biology, we performed antisense oligonucleotide (ASO) knockdowns of lncRNA candidates specifically expressed either in human lymphatic or blood vascular endothelial cells (LECs or BECs) followed by Cap Analysis of Gene Expression (CAGE-Seq). Here, we describe the quality control steps adopted in our analysis pipeline before determining the knockdown effects of three ASOs per lncRNA target on the LEC or BEC transcriptomes. In this regard, we especially observed that the choice of negative control ASOs can dramatically impact the conclusions drawn from the analysis depending on the cellular background. CONCLUSION: In conclusion, the comparison of negative control ASO effects on the targeted cell type transcriptomes highlights the essential need to select a proper control set of multiple negative control ASO based on the investigated cell types.

Making a Morpholino Experiment Work: Controls, Favoring Specificity, Improving Efficacy, Storage, and Dose.

A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.

The role of the toxicologic pathologist in the preclinical safety evaluation of biotechnology-derived pharmaceuticals.

Biotechnology-derived pharmaceuticals, or biopharmaceuticals, represent a special class of complex, high molecular weight products, such as monoclonal antibodies, recombinant proteins, and nucleic acids. With these compounds, it is not appropriate to follow conventional safety testing programs, and the preclinical "package" for each biopharmaceutical needs to be individually designed. In addition to standard histopathology, the use of molecular pathology techniques is often required either in conventional animal studies or in in vitro tests. In this review, the safety evaluation of biopharmaceuticals is discussed from the perspective of the toxicologic pathologist, and appropriate examples are given of the use of molecular pathology procedures. Examples include the use of in situ hybridization to localize gene therapy vectors, the assessment of vector integration into genomic DNA by the polymerase chain reaction (PCR), and the use of immunohistochemistry to evaluate the potential cross-reactivity of monoclonal antibodies. In situ PCR techniques may allow for confirmation of the germ cell localization of nucleic acids and may therefore facilitate the risk assessment of germline transmission. Increased involvement with biopharmaceuticals will present challenging opportunities for the toxicologic pathologist and will allow for much greater use of molecular techniques, which have a critical role in the preclinical development of these compounds.

Recent developments and perspectives of biotechnology-derived products.

Recent advances in molecular biology especially in the area of rDNA, gene transfer, polymerase chain reaction and hybridoma techniques have provided the necessary molecular tools for the development of a new class of biopharmaceuticals. These biopharmaceuticals include antisense drugs, carbohydrate-based macromolecules and agents that interfere with apoptosis pathways. Cytokines and other immunomodulators also represent an exciting class of new therapeutic entities. The safety evaluation, efficacy, manufacturing and quality control of these complex biopharmaceuticals represent a challenge to the pharmacologist and toxicologist. Finally, the regulatory issues associated with the new biopharmaceuticals need to be addressed to insure the safety of these evolving therapeutic substances.

Regulatory concerns for the chemistry, manufacturing, and controls of oligonucleotide therapeutics for use in clinical studies.

It is important to remember that while a new class of therapeutic agents like oligonucleotides may introduce novel concerns, the basic regulatory issues regarding the chemistry, manufacturing, and controls of drug substances and drug products must be addressed. This article focuses on information that should be included in an Investigational New Drug Application (IND), a request to use an investigational drug in clinical studies. The regulatory challenge presented with oligonucleotide therapeutics is to prove the identity of the oligonucleotide, and demonstrate its quality, purity, and strength/potency using both those characteristics that are the same as all other drugs, as well as those that are unique. Most of the discussion will concern issues that are unique to oligonucleotides, or those topics that deserve more detailed attention than would be needed for more typical small molecule drugs. Regulatory issues will need to be evaluated so that safety concerns are addressed while not imposing undue burden on the sponsors of investigational drugs.