The overall goal of this experiment is to examine the efficacy and toxicity of multiple exon skipping using cocktail antisense oligonucleotides or AONs in a dog model of Duchenne muscular dystrophy or DMD. This method can help answer key question in translational research of antisense exon skipping therapy, such as whether antisense oligonucleotides can induce multiple exon skipping and destroy dystrophy expression in a large animal model. The main advantage of using the dog model in a study is that therapeutic outcomes in larger animals can be more reliably extrapolated to DMD patients.
Though exon skipping is very promising therapy for DMD, it can also be applied to other genetic diseases, such as congenital muscular dystrophy and limb-girdle muscular dystrophy. Demonstrating the in vitro experiments will be done by Dr.Yoshitsugu Aoki, our laboratory chief, and demonstrating the in vivo experiments will be done by Dr.Mutsuki Kuraoka, a postdoctoral fellow with a from my department. After designing AONs according to the text protocol, use 6-well plates to seed one to five times 10 to the third per square centimeter CXMD myoblasts in three milliliters of DMEM with 10%FBS and 1%pen-strep.
Incubate the cells at 37 degrees Celsius until 60 to 80%confluent. When the cells reach the desired confluence, use reduced serum medium to dilute a cationic liposome transfection reagent to a total volume of 100 microliters, and allow the mixture to stand at room temperature for 30 to 45 minutes. Then, with reduced serum medium, dilute the AONs to a final volume of 100 microliters.
Combine the diluted transfection reagent with the diluted AONs, and incubate at room temperature for 10 to 15 minutes. In the meantime, remove the medium from the cells and use fresh medium to wash the cells three times. Next, add 0.8 milliliters of medium to the transfection mixture, and then add the entire solution to the freshly washed cells.
Incubate at 37 degrees Celsius for three hours. Then, add differentiation medium, or DM, to the cells, and incubate for up to 10 days for differentiation to occur. Beginning around day three, check for differentiation.
To perform morpholino transfection of dog myoblasts, culture CXMD myoblasts as demonstrated earlier in this video. Heat cocktail morpholinos at 65 degrees Celsius for 10 minutes. Then add a peptide delivery reagent and adjust the morpholino to a final concentration of three to six micromolar.
Next, use DM to replace the medium in the myoblasts, and add the morpholino to each well at a final concentration of one micromolar. Incubate for 16 to 18 hours. Then, to collect the cells for RNA extraction, add one milliliter of guanidinium thiocyanate-phenol-chloroform to the cells to detach them from the plate.
After incubating the cells at room temperature for 10 minutes, transfer the cells into 1.5-milliliter tubes. Add 200 microliters of chloroform to each tube, and incubate at room temperature for two minutes until three separate layers can be seen, the RNA layer, the protein layer, and the DNA layer. Centrifuge to the tubes at 12, 000 times g and four degrees Celsius for 15 minutes.
Then, remove the top layer of supernatant and transfer to a tube containing 500 microliters of isopropanol. Spin the supernatant tubes before removing the supernatant and saving the RNA pellet. Use ethanol to wash the pellet, and centrifuge at 8, 000 times g and four degrees Celsius for five minutes.
Invert the tube for 15 minutes to evaporate the residual ethanol before adding 15 to 30 microliters of RNase-free water. Then, use UV-Vis spectroscopy at 260 nanometers to quantify the RNA. To carry out RT-PCR, mix together the following reagents and add water to a final volume of 25 microliters.
Place the samples in a thermocycler and run the following program. When the run is complete, store the product at four degrees Celsius or 20 degrees Celsius. To carry out an intramuscular injection, after preparing the animal for surgery and anesthetizing it according to the text protocol, use a scalpel to cut the skin approximately five centimeters longitudinally over the cranial tibial, or CT.To mark the injection sites, use a surgical needle and surgical thread to stitch the deep fascia by making two stitch markers at two-centimeter intervals.
It is very important to mark the injection site with thread as this allows for more accurate biopsy. Next, using a 27-gauge needle, inject the desired concentration of AONs into the muscle, and leave the needle in for one minute. Then, to perform an open muscle biopsy, use a surgical scalpel to remove a piece of muscle tissue approximately two centimeters in length from the CT muscle.
Lay the muscle fascia back over the muscle and use a 000 absorbable thread to close the tissue. Then, use 000 nylon thread to close the skin. Using a needle, administer an intramuscular injection of 0.02 milligrams per kilogram of buprenorphine hydrochloride.
To inject a cocktail containing equal amounts of each AON into a limb vein, while holding the animal according to the text protocol, insert a venous indwelling needle into a limb vein. Using an infusion pump or syringe driver, inject 50 milliliters total at 2.5 milliliters per minute for 20 minutes, following the manufacturer's instructions. Repeat the injections weekly or biweekly, as dystrophin expression will accumulate with repeated injections.
To perform weekly blood tests to examine toxicity, use a needle to collect three milliliters of blood from one of the subcutaneous veins of the fore or hind limb. Following the manufacturer's instructions, include the following assessments. Carry out further analyses according to the text protocol.
Myoblasts were transfected with various 2'OMePS treatment conditions in order to compare the effectiveness of each AON. The gel shown here represents RT-PCR samples from RNA collected four days after treatment. Bands higher on the gel represent out-of-frame DMD products, and were seen in the non-treated Ex8A and Ex8B treated myoblasts.
Ex6A, Ex6B, Ex8A, and the cocktail-treated myoblasts showed in-frame products. cDNA sequencing verified that Exon 6 through 9 had been skipped. As seen in this figure, immunocytochemistry revealed that AON-treated dog cells had increased dystrophin-positive fibers compared to non-treated samples.
To compare the efficacy of various AON treatment conditions, CXMD dogs were injected with Ex6A, or a cocktail of Ex6A, Ex6B, and Ex8A at various dosages. Muscle samples stained for DYS1 show an increase in dystrophin expression in cocktail-treated samples, and dystrophin-positive fibers increased with dystrophin dosage. Following systemic injections, cocktail-treated CXMD dogs showed increased dystrophin expression compared to NT CXMD dogs, both in CT and heart muscle samples.
However, AON-treated skeletal muscles showed much higher expression of dystrophin compared to treated cardiac muscle. In addition, treated CXMD dogs showed improved histopathology with a significant decrease in centrally-nucleated fibers in comparison to NT CXMD dogs. Once mastered, both in vitro and in vivo experiments can be done in a couple of hours if it is performed properly.
While attempting this procedure, it's important to carefully monitor the health condition of the dog. After its development, systemic exon skipping paved the way for researchers in this field to start human clinical trials in DMD patients. After watching this video, you should have a good understanding of how to treat dogs with antisense oligonucleotides.
