Hyperkalemic Periodic Paralysis Jake Parker & Israel Amanuel

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

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.

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) http://www.horsetalk.co.nz/2014/10/01/hypp-hyperkalemic-periodic-paralysis/

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 https://www.horsejournals.com/ethical-horse-breeding-equine-genetic-diseases

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

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

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.

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

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

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

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 3.8-5.2 mmol/L, injection will put mice over normal K level Induce mice to exercise

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

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

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

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

Citations http://www.slideshare.net/laxmikantjoshi2/hypokalemic-periodic-paralysishpp https://ghr.nlm.nih.gov/condition/hyperkalemic-periodic-paralysis#definition http://www.ivis.org/proceedings/aaep/2006/pdf/z9100106000347.pdf?origin=pub lication_detail https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255699/?page=3 http://www.omim.org/entry/170500