8/8/2019 Production of SiRNA for in Vitro and in Vivo Applications

1/2

Production of siRNA for in vitro and in vivo applications

The last few years have seen a great deal of applications of RNA molecules. Sequence-specific genesilencing using small interfering RNA (siRNA) is a Nobel prizewinning technology that is now being

evaluated in clinical trials as a potentially novel therapeutic strategy. As biological researchers move toin vivo experiments ; there are a few challenges facing siRNA therapeutics, that focus on the deliverystrategies for synthetic siRNA duplexes in vivo, as this remains one of the most important issues to be

resolved. We at Bio-Synthesis like to highlight the importance of understanding the

genocompatibility/toxicogenomics of siRNA delivery reagents in terms of their impact on gene-

silencing activity and specificity. Collectively, this information is essential for the selection of optimallyacting siRNA delivery system combinations for the many proposed applications of RNA interference.

It should be noted that siRNAs used for preliminary in vivo studies do not have to be synthesized under full

cGMP conditions, however they do need to be assembled and processed under clean, asceptic conditionsfree of contaminants that could affect the viability of the cell culture/experimental animals being used.

Potential Contaminants

The protecting groups utilized during the chemical assembly of synthetic RNA are removed during the

ammonium based deprotection and purification steps and analogous to GMP, a residual solvent analysisis useful. Whereas toxic heavy metals are not used during the synthetic steps, their presence is

minimized by using EDTA in the HPLC buffers (EDTA is a good chelator and will remove any heavy

metal that may be present). The RNAs are obtained as the ammonium salts after synthesis and isdesirable to convert them to the sodium form, by using a sodium chloride gradient in the presence of

sodium phosphate buffer. Nonetheless the main concern one has to deal with is the presence ofendotoxins, which when present in small amounts can cause severe inflammation and even death inlaboratory animals.

Endotoxin and Septic Shock

Endotoxin detection took significance early on protein expression work, as the main expression vehicles

were of bacterial origin. Endotoxins are lipopolysaccharides (LPS) present in the outer membrane of thecell wall of Gram negative bacteria. Toxicity is associated with the lipid component (Lipid A) and

immunogenicity is associated with the polysaccharide components. The cell wall antigens (Oantigens) of Gram-negative bacteria are components of LPS. LPS elicits a variety of inflammatory

responses in mammals and it activates complement by the alternative (properdin) pathway, so it may bea part of the pathology of Gram-negative bacterial infections.

In 1986 a plasma protein namedLPS binding protein (LBP) helped elucidate the mechanism by which

the LPS-LBP complex binds to CD14 receptors on monocytes and macrophages. Subsequente

612 E. Main Street Lewisville, TX 75057www.biosyn.com

Phone: (972) 420-8505 Toll Free: (800) 227-0627 Fax (972) 420-0442

8/8/2019 Production of SiRNA for in Vitro and in Vivo Applications

2/2

interaction with Toll-like receptor 4(TLR4) results in the release of cytokines(IL-1,6 and 8), TNF andplatelet activating factor; all of which cause the secretion of prostaglandins, activation of the

complement and coagulation cascades; ultimately all this leads to acute inflammation, hemorrhage andseptic shock. It is clear then, that any in vivo experiment can be affected by the presence of undesiredendotoxins.

Purification Methods and Endotoxin Controls

In order to keep an endotoxin free environment, the following standard operating procedures are

implemented:

1. An ultrapure clean water system is in place; double distilled, reverse osmosis, ion exhangepurified water is filtered tru activated carbon and 0.002 micron pore size hollow filters.

2. Bio-Synthesis, Bio-Active siRNAs are purified tru HPLC using EDTA containing buffers3. 3. All glassware is autoclaved and washed with 0.2 M sodium hydroxide and rinsed with sterile

water

4. Ultra pure water is continuosly checked by external labs for endotoxin contamination5. Our threshold values are set to be