Although aptamers have extremely high specificity[1], it cannot be said for certain that the aptamer may introduce other adverse effects in the body, mice or humans.
Unmodified antisense oligonucleotides, regardless if they are aptamers or not, distribute rapidly and broadly from the plasma compartment to the tissues, with the highest concentrations in the liver, kidney, lymph nodes, spleen, and bone marrow[2]. Thus, we must make sure that the ostamer will not cause adverse effects to other cells and systems.
Histopathological examinations on the vital organs, including the brain/cerebellum/cerebral vessels, heart/aortic root, kidneys, livers, lungs/bronchi, spleen, adrenal glands, thymus, thyroid/parathyroid, prostate glands, testicles, ovaries and uterus/cervix, were conducted in healthy rats after administering the drug twice per week for six weeks.
Microscopic examination revealed normal cell structures, and that no lesion or pathological changes were present in the organs listed above. Further evaluations will be performed in the clinical trials, which we are currently in the process of applying for.
We have used various cell lines, like MC3T3-E1 subclone 4 (i.e mouse pre-osteoblasts), RAW 264.7 (macrophages) and vascular smooth muscle cells (part of the aorta/smooth muscles), as part of our analysis to determine the efficacy of the aptamer drug. We will culture the cells, transfect cells with corresponding expression plasmid and treat cells with our drug. We will extract mRNA from the cells after treatment and analyze the expression levels of genes.
These cell lines and experiments conducted has been evaluated by our team to carry little to no risk of contaminating the environment and/or ourselves. To ensure this, we will bleach and autoclave cells and wastes after all experiments and treat them as biological wastes and dispose of them correctly.
As our project focuses on increasing bone anabolic potential to alleviate the effects brought about by osteogenesis imperfecta in mice, we have incorporated two types of mice into our experiments:
Reasons for using these two types of organisms are listed as follows:
The main source of hazard that will result from this are bites from the mice during the injection of various reagents and the extraction of bones. We will use a restraining device/cage and use protective equipment including laboratory and gloves during operation.
The Research Ethics Committee of Hong Kong Baptist University is responsible for the safety and security of biology labs. We have applied official research ethics from them.
We will use TRIzol in mRNA extraction. The following steps will be taken for first aid measures.
Collect liquids in leak-proof containers such as flasks or bottles. Liquid waste containers designed to withstand autoclaving temperatures must be used when steam sterilization is utilized. To allow pressure equalization, they must not be sealed.
Non-sharp, solid laboratory waste (empty plastic cell culture flasks and petri dishes, agar plates,empty plastic tubes, gloves, wrappers, absorbent tissues, etc.) which may be, or is known to be, contaminated with viable biological agents should be collected in a yellow Bio Waste plastic 20 litre pail. These plastic pails display the biohazard warning symbol and are available from Environmental Protection Services (EPS) 416.946.3473.
For laboratories generating large volumes of agar gel in disposable petri dishes and tubes requiring sterilization, such waste should be collected in a yellow Bio Waste plastic 20 litre pail in the laboratory.
Autoclavable bags filled with plasticware containing agar gel tend to leak fluids during and after the sterilization process. The pail will contain the liquids released by the agar gel. After sealing the pail and filling in the tag, the pail must be placed beside other waste awaiting removal by EPS Environmental Protection Technicians.
Any waste that has been autoclaved in Autoclave bags at the University must be double bagged, twist tied or taped shut and both Biosafety Certificate number and room number marked visibly on them before they will be collected by the Environmental Protection Technicians.
When necessary or for safety reasons, inactivate the biological agents by employing either chemical disinfection or steam sterilization procedures. NOTE: Although chemical disinfectants play a useful role in many situations where decontamination is required, when they are used to sterilize waste, the investigator must assure the Biosafety Committee that the routines and methods achieve the desired objective.
Autoclaving (steam sterilization) is the preferred (and generally regarded as the most reliable) method of sterilizing biological waste. Depending on the volume of waste to be sterilized, it may be necessary to extend the duration of exposure to high temperature steam under pressure.
Steam sterilization is not recommended for laboratory waste contaminated with or containing a combination of viable biological agents and significant amounts of hazardous chemical or radioactive materials.
Containers of liquid waste must be placed into an autoclavable tray or pan of sufficient capacity to contain all liquid in the event of vessel failure or breakage inside the autoclave chamber. Use extreme caution when handling autoclaved liquids since they are hot and may boil over.
Autoclavable bags of solid waste should be closed but not sealed airtight to allow steam penetration before they are placed into the autoclave chamber. After autoclaving andcooling, these bags of autoclaved waste must be double bagged, twist tied or taped shut and both Biosafety Certificate number and room number marked visibly on them.
Further details may be found on our Safety Form.
[1] Osborne S et al. "Aptamers as therapeutic and diagnostic reagents: problems and prospects". Current Opinion in Chemical Biology, vol. 1,1 (1997): 5-9. [2] Levin A et al. "Basic Principles of the Pharmacokinetics of Antisense Oligonucleotide Drugs". Antisense Drug Technology (2007): 183-215. [3] Moghadasian, M H et al. “Pathophysiology of apolipoprotein E deficiency in mice: relevance to apo E-related disorders in humans.” FASEB journal: official publication of the Federation of American Societies for Experimental Biology vol. 15,14 (2001): 2623-30. [4] As according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).
