No RNA Extraction: Real-Time RT-qPCR Works Anyway!

Hi all,
welcome back to our bioanalytical ligand binding assay album for the Biotechnology scene. Today we would like to point you to the outstanding Merck & Co paper collection on siRNA drug development research. The major finding of Wang Z et al (2011), J Pharm Toxicol Methods 63: 174-9 was the observation the stem-loop RT primer technology originally developed by Applied Biosystems for the microRNA (miRNA) and siRNA quantification, can also be utilized to analyze microRNA- (miRNA-) and siRNA-mediated mRNA knockdown. No expensive total RNA extraction from tissues is needed anymore. Additionally, a unique primer set could be designed to quantify target mRNA in liver homogenates from human, monkey, mouse, and rat. Excellent!
Finally, we would like to announce the completion of the Real-Time RT-qPCR Assay site, which summarizes published assay development activities on microRNA and siRNA quantification.
Enjoy the reading, your team.

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RNActive® Vaccines Complexed to Protamine

Dear all,
numerous research activities with a strong focus on nucleic acid chemistry-based therapeutics have to face the same problem: how to transport these poly-anionic compounds into the cell? The most applied technology is the complexation of siRNA (1), microRNA (2), antagomirs (3), antisense (4), or mRNA vaccines (5) with cationic lipids. In fact, a once promising approach has turned into a disillusioning obstacle.
The first mRNA transcript vaccine ever studied in a human clinical trial was CV9103 (6), a transcript encoding for four proteins in a row, and complexed with the cationic peptide protamine (7). The idea behind was to produce an intracellular shuttle for antigen expression encoded by the transcript, and also to generate an immunostimulatory agent. The most preferred and also effective ratio in either case was uncovered to be mRNA:protamine = 2:1 (8), resulting in a component containing both complexed and free (=naked) transcripts.
In another very recent clinical trial the mRNA vaccines were electroporated into autologous dendritic cells, and reinjected into 34 cancer patients (9), an innovative approach which could reduce GMP production and packaging costs. Interestingly, the same four therapeutic proteins were included as for CV9103.
And finally, there are an increasing number of publications dealing with the injection of just naked, but chemically stabilized transcript vaccines, so simple (10). Strong immune response can also be stimulated by simply changing the route of administration for naked mRNA vaccines (11).

Enjoy your weekend,
Your team

(1) Adami RC et al (2011), Mol Ther 19(6): 1141-51 (a Marina Biotech DiLA2 publication).
(2) Wu Y et al (2011), Mol Pharm doi:10.1021/mp2002076
(3) Liu XQ et al (2011), Mol Pharm 8(1): 250-9.
(4) Chiu SJ et al (2006), J Control Release 112(2): 199-207 (a Genasence® paper)
(5) Perche F et al (2011), Nanomedicine doi:10.1016/j.nano.2010.12.010
(6) Weide B et al (2009), J Immunother 32(5): 498-507.
(7) Fotin-Mleczek and Voss S (2010), Patent WO 2010/037408 A1.
(8) Fotin-Mleczek M et al (2011), J Immunother 34(1), 1-15.
(9) Wilgenhof S et al (2011), J Immunother 34(5): 448-56.
(10) Kormann MSD et al (2011), Nature Biotech 29: 154-157 (an Ethris publication)
(11) Lorenzi JCC et al (2010), BMC Biotechnol 10: 77

RNActive® is a trademark of CureVac GmbH.
Genasence® is a trademark owned by Genta Inc.

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Solid Phase vs. Liquid Phase Ligand Capturing

As in real life, often there are different ways to get the same result. Almost the same, I´d better say, because for the bioanalytical ligand binding assay developer it is indeed a difference whether the ligand is captured in the liquid phase, or immobilized on a solid phase (like a microwell plate or magnetic bead).

Well, this guy is crackbrained, you might say, talking about somehow confidential assay development topics on the bioanalytical blog, so everybody can switch off his mind, and on top of that saves time for his own assay development project. Good point, I would answer, but all the informations I share with you here are already published in the PubMed database. If I hypothesize you can read and write, you will come across this post topic on your own, will you? So what´s the point? It is a difference you develop your own bioanalytical ligand binding assays for research purposes, or you have to develop custom ligand binding assays to support preclinical/clinical drug development of investigative novel drugs (IND). In the latter case strictly compliance to Good Laboratory Practice (GLP) regulations is obligatory, is a must.

Certainly we developed bioanalytical assays for the biotech industry, and stumbled across stones we couldn´t foresee. The devil is in the details. But we won´t tell you.

Now let´s focus on the two ligand capturing strategies I mentioned in the post headline. Either the ligand can be captured in the liquid phase, followed by immobilization of the catcher on microwell plates or magnetic beads. Or the catcher is first immobilized on a solid phase, and the ligand is captured afterwards. Please notice the catcher is pinched in a molecular sandwich.

We believe the immuno qPCR (iqPCR) technology is one of the bioanalytical ligand binding assays which will revolutionize the assay development world. You hardly can find a more sensitive bioanalytical assay with a broader detection window for peptide, hormone, protein detection: the ancient enzyme-linked immuno sorbent assay (ELISA) for many applications is a discontinued model, at least e.g. for antibody detection assays (ADA).

Another application of the immuno qPCR technology is the detection of human pathogens in food samples (1). Staphylococcus aureus enterotoxins are not only one of the main poisons in food, but the methicillin resistant variant (MRSA) is also the main cause of serious bacterial infections in hospitals worldwide. The more sensitive the assay detection limit is, the earlier the pathogen is detected, and can be combatted.

Aspergillus funghi also produce toxigenic substances which even can cause cancer. A recent publication on immuno qPCR (iqPCR) mediated aflatoxin detection demonstrates the capturing of aflatoxin B1 by monoclonal or polyclonal antibodies in solution, followed by complex immobilization on protein G coupled magnetic beads (2). The detection limit is greatly enhanced if aflatoxin is captured in the liquid phase by a monoclonal antibody before immobilization.

The sandwich hybridization assay is a powerful and sensitive bioanalytical assay for the detection of virtually any nucleic acid ligand. Briefly, the ligand can either be captured by a short complementary probe in solution, followed by solid phase immobilization of the capture probe to streptavidin microtiter plates (3). The ligand is then detected by another short complementary probe which also can hybridize to the ligand. Sirna Therapeutics´s Heptazyme drug at that time was hybridized to both probes in a one-step liquid phase capturing, and immobilized afterwards (3), producing reliable results of outstanding bioanalytical assay sensitivity. The ribozyme is a single-stranded nucleic acid drug, and certainly it can improve assay sensitivity if both the capture and detect probe are added to the sample at once.

For the enzyme-linked immuno sorbent assay (ELISA), these solid phase assays are a consequent advancement of the classic radioimmunoassays (RIA), which are a combination of liquid phase ligand capturing with the immunoprecipitation procedure. Antibody performance in sandwich solid phase ELISAs can provide a better assay accuracy, when compared to RIA (4). Additionally, the antibody is more stable for long-term storage when immobilized to microwell-plates.

1. Panneerseelan L & Muriana PM (2009), J Fod Prot 72(12): 2538-46.
2. Babu D & Muriana PM (2011), J Microbiol Methods,
3. Brown-Augsburger P et al (2004), J Pharm Biomed Anal 34(1): 129-39.
4. Schramm W et al (1987), Clin Chem 33(8): 1331-7.

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Upcoming Drugs: In Vivo Gene Transfer With Messenger RNA

Dear all,

the world of nucleic acid therapeutics is fascinating. A novel advancement in this field develops messenger RNA as a tool for in vivo gene transfer, either for antigen expression in vaccination procedures, or to improve, and even supplement homologous gene expression for disease treatment. It is obvious the messenger RNA has to be chemically stabilized somehow, in order to suppress or at least to delay in vivo degradation.
There are several ways to achieve this goal: modifications of the ribose backbone are already in the clinic (with the great disadvantage this modification can hardly be incorporated into the GMP manufacturing process by in vitro transcription). The use of naturally occuring nucleosides like pseudouridine (1), 2-thiouridine (2), or 5-methylcytidine is a promising approach to produce large amounts of biologically stabilized in vitro transcripts enzymatically. The 5-methylcytidine modification is that “famous” methylation of genomic DNA for pre-transcriptional gene silencing (monoclonal antibodies to this modification are used in immunoprecipitation of genomic DNA).
Another approach uses no chemical modification at all, but the in vitro transcripts are complexed with the cationic peptide protamine (3). Both components form a complex which improves biological stability of the messenger RNA in vivo, but on the other hand represses translation of the messenger RNA. Read our latest summary on novel and innovative messenger RNA therapeutics.

Enjoy your weekend, your team.

(1) Karikó K et al (2008), Mol Ther 16(11): 1833-40.
(2) Kormann MSD et al (2011), Nature Biotech 29(2): 154-7.
(3) Scheel B et al (2005), Eur J Immunol 35: 1557-66.

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Haemolytic Uremic Syndrome (HUS) Epidemy in Germany

Dear all,
for all people living outside Germany you probably might not came across the HUS outbreak in the north of Germany, caused by multidrug resistant enterohaemorrhagic Escherichia coli (EHEC) strain O104:H4. The dramatic epidemy caused 29 deaths (as of June 10, 2011), which is an incredible number for an industrialized country like Germany. More than 2.800 new infected people were registered by the German Robert-Koch-Institute, 722 of these show serious symptoms for the haemolytic uremic syndrome (HUS), as of June 10, 2011.
This demonstrates once again the urgent need for novel and innovative therapeutic approaches to fight microbial pathogen attacks. Novel therapeutics like Patent US 7,939,492 and Patent US 7,850,975 are welcome.
Today we released some informations about the second-most popular ligand binding assay usually performed in the liquid phase (comes right behind the RT-qPCR, qPCR assays on top): the limulus amebocyte lysate (LAL) endotoxin detection assay, applied by the pharmaceutical industry and manufacturers to ensure endotoxin-free products.

On Monday we have a sort of Bank Holiday in Germany, so we´ll be back on Thursday next week. Have a nice weekend, your team

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