1. Hyperkalemic Periodic Paralysis
Jake Parker & Israel Amanuel

2. Disease Background
Hyperkalemic Periodic Paralysis (HYPP) is a genetic disease found in quarter horses and humans resulting in faulty sodium channels in skeletal muscle
Disease stems from mutation in the SCN4a gene
SCN4a is an unlinked gene
Occurs when Na channels stay open for too long or not long enough causing too much or not enough depolarization

3. Disease Background
Causes episodes of flaccid weakness, muscle spasms, and temporary inability to move limbs
High levels of serum K+ cause channel let in massive amounts of Na
High potassium diet, rest after exercise, and other environmental conditions can trigger paralysis or contractions
Sudden death can occur following a severe paralytic attack, presumably from heart failure or respiratory muscle paralysis.

4. Origin: “Impressive”
1992: HYPP was linked to popular Quarter Horse Sire named “Impressive”
Born in 1969
355,000 Descendants registered with the American Quarter Horse Association (AQHA)

5. Importance Obvious solution is to stop breeding affected horses
However, all descendents of Impressive are susceptible to disease
Issue: lineage is not always known and horses appear normal so hard to tell without genetic tests(if uneducated on disease) which are expensive
Better quality of life for horse
Lower costs for breeders/owners
Costs money to regulate diet
Vet costs
Devalues race and show horses
Autosomal dominant
High transmission rate to offspring

6. Current Treatment Diet low in potassium
Whole grains
Oats, wheat, barley, corn
High quality grass hay with vitamin A and phosphorus
Potassium wasting diuretics (acetazolamide)
Emergency treatment during attacks
Intravenous solution of 5% dextrose and Calcium gluconate infused slowly
Dextrose aids potassium entry into cell
Calcium antagonizes effects of HYPP
ALL TREATMENTS, NOT CURES

7. Aims
Determine if CRISPR/Cas9 editing tool is effective in excising the mutated SNC4a gene and replacing it with a normal, functional copy.
Determine if the new functional copy of the SNC4a gene prevents leaking of Sodium Channels and prevents effects of HYPP

8. Hypothesis
We hypothesize that the excision and replacement of the mutated SNC4a gene using CRISPR/Cas9 gene editing will eliminate the unregulated, leaky flow of sodium through sodium channels.

9. CRISPR/Cas9 Clustered Regularly Interspaced Short Palindromic Repeats
Complex composed of guide RNA and Cas9 enzyme
Guide RNA (gRNA) is ~20 bp in length and matches beginning and end sequences of target gene
Cas9 cleaves DNA a the specific site matched by gRNA
Repair template contains new sequence or gene and is added into genome using DNA polymerase
Used for alter DNA from 1bp to thousands of bp’s

10. Methods
Embryos from homozygous dominant affected mice will be isolated in vitro to create diseased line
Embryos from homozygous recessive healthy mice will be isolated to create normal line
CRISPR gene editing tool will be used to excise the mutated SCN4a gene and replace with a properly functioning copy of the gene
Functional gene will tagged with GFP allowing corrected cells to be visible in the mouse
CRISPR has been used to treat muscular dystrophy in mice, it is a compatible mechanism

11. Methods Cont....
gRNA will be synthesized for the faulty SCN4a gene, promoter and end sequence
Correct SCN4a gene will be coupled with gene for green fluorescent protein (GFP)
GFP expression will allow muscle tissue samples to be examined and determined if gene replacement was successful
20 Diseased embryos will be injected with CRISPR and the SCN4a gene in vitro
Embryos will be implanted back into female mother for gestation

12. Methods Cont... Monitoring Tissue sampling for presence of GFP
Electromyography
measures muscle tissue activity
Electrodes attached to the skin detect action potentials and nerve impulses
Test at 8 weeks of age and repeat bi-weekly for 24 weeks
2 mmol/L of potassium injected into mice
Standard serum levels are between mmol/L, injection will put mice over normal K level
Induce mice to exercise

13. Anticipated Outcome
95% of treated mice will have GFP on previously mutated SCN4a gene, indicating successful CRISPR editing.
Number of myotonic discharges, doublets, triplets, and spontaneous activity will be reduced 85% in HYPP mice treated with CRISPR gene editing (HYPP-T)
Indicates sodium channels are functioning properly
20 HYPP
20 HYPP-T
20 Normal
20 Sham

14. Outcome cont.. Top to bottom: Normal induced insertional activity
Doublets: two ap with 10-30ms of each other( triplet same but with three)
Myotonic discharge: Firing of action potentials of various frequency and amplitudes due to abnormal muscle fiber membranes
Right:Prolonged insertional activity: discharges lasting longer than 120ms

15. Alternative Methods
For cases where gene editing do not work, or for animals who are living with a disorder an oral dosage of therapeutic resin could be used
A potassium binding polystyrene sulfonate resin binds free potassium ions circulating in serum.
Bound potassium will not be able to pass through channels and prevent a triggering of Na+ influx and depolarization
Sodium polystyrene sulfonate has been used to facilitate potassium secretion in patients with kidney failure.
Would test if binding of excess free potassium decreases a trigger of an HYPP
Could also be used in humans as embryo editing is not occurring

16. Pitfalls
Any advances treatment makes cannot be applied to humans because you can’t edit human embryos
Will not help horses who currently have disease
HYPP is unstudied in mice, possible translation issues to horses
Genetic editing or treatment of SCN4a has not been tested before
Replacing gene could not fix problem
Due to other gene influences on the disease
Large size of SCN4a gene (32.5kb) may affect replacement accuracy

17. Citations
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