'''Super Mouse Mutation'''

Super Mouse Mutations and Treatment of Muscular Dystrophy

A summary of the original article "Antisense-induced Myostatin Exon Skipping Leads to Muscle Hypertrophy in Mice Following Octa guanidine Morpholino Oligomer Treatment" by Kang et al. 2010 - Mol Ther. 2011 Jan;19(1):159-64. doi: 10.1038/mt.2010.212. Epub 2010 Oct 5

Muscular Dystrophy

Muscular dystrophy by definition is weakness or loss of muscle mass. It is caused by genetic mutations that prevent the production of a stability protein called dystrophin that helps prevent muscular fiber damage. Often the disorder is inherited but some can occur spontaneously within the mothers egg. There are more than 30 different forms of muscular dystrophy that affect various parts of the body. Each type of dystrophy is expressed at various ages and vary in muscular weakness. Though most appear at infancy or childhood, adult onset is common as well. The overall symptoms of the disease are progressive muscle weakness, the rate and areas affected will vary between types of the disorder. Unfortunately there is no cure for the disorder. Physical therapy and drug therapies can help slow the progression but there is no way to fully stop the progression.

Introduction

Building muscle and fighting muscle disorders such as muscular dystrophy have always been a scientific concern. In this article Myostatin, a growth factor, will be reviewed in its association with skeletal muscle hypertrophy and if knocking out the factor can have positive effects on muscle deficient disorders. Myostatin is a transforming growth factor-β that suppresses muscle cell growth through regulation. Organisms deficient in Myostatin have widespread uncontrolled muscle growth or muscular hypertrophy. Using myostatin inhibitors for disorders such as muscular dystrophy have been found to increase muscle cell mass in treated individuals. Unfortunately, sustaining such treatments is difficult and immune responses to the treatment have been noted. Other methods such as adeno-associated virus vectors delivering recombinant myostatin propeptide gene fragments have also been explored but risks are also associated with this treatment.

In this article RNA-based therapies will be explored using antisense oligonucleotides to hybridize to a sense target and lead to the down regulation of the genes transcription. Using this antisense hybridization, exon skipping can be induced. This exon skipping method has already been found in clinical studies to partially correct the dystrophin mutation in muscular dystrophy, but exploration of exon skipping effects on myostatin is what this paper will explore. The results could constitute an effective treatment for various muscle-wasting conditions.

Results

Summary: Bioinformatics analysis- the collection and analysis of genetic codes, were performed of exon 2 of myostatin. This was done using three tools: Exonic splicing enhancers (ESE) finder, PESX, and Rescue ESE, which helped to identify and locate ESEs, exonic splicing suppressor, or silencer motifs. The location and values for SR protein-binding motifs were found. In addition, certain antisense oligonucleotides were used to purposefully change myostatin pre-mRNA splicing and the locations of ESEs (exonic splicing enhancers) and exonic splicing silencers, which are just short regions of an exon which contribute to alternate splicing were found. High levels of myostatin exon 2 skipping was found in C2C12, and antisense-induced myostatin exon 2 skipping and myostatin knockdown leads to an increase in C2C12 cell proliferation. It was also found that injection of 2’OMePS oligomers that target myostatin exon 2 does ignite exon skipping, and injection of PMOs combined to guanidine dendrimer resulted in exon skipping which influenced the increased muscle mass and myofibre size.

Figure1a: The ESE finder shows the results of three algorithms used to design exon-skipping AOs that target exon 2 of mouse myostatin. The bars indicate the location and values for each of the identified SR protein-binding motifs. Rescue ESE shows the possible positions of exon splicing enhancer sites in relation to exon 2. PESX shows the locations of ESEs (light gray) and ESSs (dark grey)

Figure 1b: RT-PCR on mRNA from C2C12 cells treated with 12 different oligomers. Each of the 12 different oligomers were run twice consecutively (Track 1 and 2, Track 3 and 4, etc.). Tracks 7, 8, 21 and 22 were controls with no AOs.

Figure 2: The anti-sense myostatin exon 2 (skipping with 2'OMePS) leads to an increase in C2C12 cell proliferation. Treating C2C12 cells with 2'OMePS oligomers A3, B3, and D3 resulted in significant cell proliferation while cells treated with C3 and LF2000 (lipofectamine 2000) did not experience substantial change.

Figure 3: Tracks 1 & 2 are 14 day controls. Tracks 15 & 16 are 28 day controls. The controls do not show the lower band due to not having exon 2 skipped. Tracks 3-14 have various OMePs administered to the muscle tissue. The band at 158 base pairs corresponds to the skipped exon 2 product. It is clear that some tissue in tracks 3-14 have the exon 2 skipped. Densitometric evaluation showed particular percentages for each OMeP type: the brighter bands have higher skipped exon percentages.

Figure 4: Since PMO’s have high efficiency in vivo, they were created in this experiment for the most efficient 2’OMePS AOs. C2C12 were treated with different leashed PMOs over a period of 24 hours. Transfections were performed in triplicated andRT-PCR products were loaded on 1.2% agarose gel. Tracks 1-3 contain PMO-A; Tracks 4-6 contain PMO-B; Tracks 7-9 contain PMO-C; Tracks 10-12 contain PMO-D; Tracks 13-15 contain LF2000-control

Figure 5: In part a, weight of soleus muscles using Vivo-PMP treatment increased significantly but weights of EDL did not show any significant change for any treatment. In part b, RT-PCR was carried out on 1 microgram RNA from soleus and EDL muscles, using a 1.2% agarose gel. Track 1 shows the Vivo-OMO treated soleus; track 2 is the control soleus; track 3 is the Vivo-PMO treated EDL; track 4 is control EDL.

Discussion

Knocking down the expression of myostatin should allow skeleton muscle to increase growth. This article discusses using antisense oligonucleotides (AOs) to knockdown expression by skipping exons. These AOs can pair with a portion of the gene and prevent transcription in that area and thereby allowing the exon to be skipped. AOs prevent “uncontrolled insertion in the genome” (Kang et al, 2011), can be regulated and treatment stopped if necessary, and do not have any known unwanted effects as far as toxicity and the immune system.

The researchers were able to show myostatin exon skipping in vitro. The AOs they designed increased cell growth and division in this in vitro experiment. In vivo exon skipping was more difficult. AOs were injected into the muscle and a single muscle did experience exon skipping. However, myostatin in the bloodstream was believed to prevent cell growth.

PMOs (phosphorodiamidate morpholino oligomers) were injected into the tail vein in order to treat the entire body rather than a single muscle. PMOs were used because they are more stable and last longer in vivo. The particular PMO used was chosen because it has been shown to be effective in vitro and it is in a region shared by both mice and humans. Using a region also found in humans will allow it to lead to human clinical uses in the future. Only the soleus muscle had an increase in muscle size. There was no change in the extensor digitorum longus (EDT) muscle tissues. The researchers believe that the dosing they used was not sufficient to increase cell growth in the EDT muscle due to a short half-life of myostatin. They speculate that they will need more frequent dosing to achieve sustained exon skipping.

The experiment proves that in vivo myostatin knockdown can be achieved using AOs, but more research needs to be done in order to determine the exact method that will work best for therapeutic effects. The long term goal is to find treatments for muscle disorders such as age-related muscle loss and muscular dystrophies.

Relevance

This article explores possible treatments for Muscular Disorders by understanding what causes muscular hypertrophy and inserted it into the DNA of muscular dystrophy affected mice. With further research the possibility of therapeutic treatment using a myostatin knockdown could improve the lives of those affected and even extend them. Long term affects will need to be reviewed but these findings could be revolutionary for those affected. Though this may only serve to be another therapeutic treatment, we are one step closer to finding a cure for this genetic disorder.

Materials and Methods

Bioinformatics analysis of the myostatin gene to design AOs reagents.

Three Different bioinformatics algorithms (ESE Finder, PESX, & Rescue ESE) designed antisense reagents. The results defined ESE sites & identified regions of myostatin exon 2. Set of 12 antisense reagents (2’O-methyl RNA, aka 2’OMePS) were designed to target 4 different ESE-rich regions of exon 2 of myostatin. Refer to Figure 1a.

AO reagents. AO reagents stands for antisense oligonucleotides. These reagents are designed so that they bind with their homologues. Twelve 2'OMePS oligomers were obtained from Eurogentec and then PMOs were designed based upon their sequences. Four PMOs were tested and conjugates to octa guanidine dendrimers from Gene Tools.

Cell culture and transfection of C2C12 cells with the designed antisense reagents. The C2C12 cells were maintained in a modified medium that consisted of fetal calf serum, 4 mmol/l l-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. They were incubated at 37°C with 8% CO2. In order to avoid cell differentiation, the cells were split every 24 hours.

RT-PCR analysis of myostatin exon skipping. In the in vitro experiments, RNA was extracted from each well 24 hours are transfection using a QIAshredder/RNeasy extraction kit; whereas, for the in vivo experiements, RNA was extracted from blocks using TRIzol reagent. A microgram of this RNA was reverse transcribed to create complementary DNA. Next, a microliter of PCR products was used as a template for nested PCR. PCR, polymerase chain reaction, is a technique used to analyze short segments of DNA or RNA that focuses on amplifying the sequences of target.

In vitro cell proliferation assay. Proliferation assay (Cell Titer 96 Aqueous One Solution) was preformed on cells transfected with 2'OMePS. 24 hours after seeding, the growth media was replaced with serum-free media. Then the cells were incubated at 37°C for 24 hours. Then 15µl of assay reagent was added to 75µl cells. The plates were then read at 490nm and a statistical analysis on the data from proliferation assay was performed using the individual t-test.

Treatment of mice with PMOs and Vivo-PMOs. The mice used in this experiment were in-house maintained for in vivo experiments and regulated in accordance with Animals Act 1986 for Scientific Procedures. During the experiment, mice were anesthetized for the intramuscular delivery. The experimental group received 3 nmol of 2'OMePS in 25µl normal saline to the 3 TA muscles while the control group only received saline. The whole body weight of each mouse was measured on a weekly basis. The TAs for both the treatment and control groups were removed postmortem after 2 and 4 weeks. These were then weighed and frozen in iso-pentane.

Immunocytochemistry and morphometry. Staining with hematoxylin and eosin was used to determine the muscle sizes while laminin staining was used to analyze muscle fiber size and distribution. The laminin stain used a laminin antibody and biotinylated anti-rabbit immunoglobulin G as a secondary antibody. Sections were then stained and washed with buffers for analysis. Stained cells can be observed in Figure 5d.

References

Kang, J., Malerba, A., Popplewell, L., Foster, K. and Dickson, G. (2010). Antisense-induced Myostatin Exon Skipping Leads to Muscle Hypertrophy in Mice Following Octa guanidine Morpholino Oligomer Treatment. Mol Ther, 19(1), pp.159-164.

Mayoclinic.org. (2016). Muscular dystrophy Lifestyle and home remedies - Mayo Clinic. [online] Available at: http://www.mayoclinic.org/diseases-conditions/muscular-dystrophy/basics/lifestyle-home-remedies/con-20021240 [Accessed 13 Mar. 2016].

National Institute of Neurological Disorders and Stroke. (2016). NINDS Muscular Dystrophy Information Page. [online] Available at: http://www.ninds.nih.gov/disorders/md/md.htm [Accessed 13 Mar. 2016].