1. Can We Use Stem Cells and Gene Therapy to Cure Retinitis Pigmentosa?
By Brendan Creemer
COSMOS 2015 Cluster 1: Biotechnology

2. Background Things to go over: What is retinitis pigmentosa?
What are stem cells?
The type of gene therapy we will use is CRISPR
What is CRISPR?
Liposomes are the method we will use to insert the genes into the cell
What are liposomes?

3. What is Retinitis Pigmentosa?
Retinitis pigmentosa is a genetic disorder that causes progressive vision loss throughout life.
It is caused by a genetic mutation that causes the retinal cells to die over time, causing night blindness at first and eventually leading to total blindness.
Usher Syndrome: A type of retinitis pigmentosa that also includes hearing loss
Rarer than retinitis pigmentosa
Hearing loss is taken care of, yet the vision part is still a problem.

4. What are stem cells?
Definition: Stem cells are cells that are undifferentiated- that is, not a specific type of cell.
Types of Stem Cells:
Pluripotent- can become any type of cell except for embryonic stem cells
Totipotent- can become any type of cell
Multipotent- can become only a few types of closely related cells
Unipotent- can only be one type of cell
Induced Pluripotent- pluripotent stem cells derived from adult skin cells
Picture on next slide

5. Why are Embryonic Stem Cells Pluripotent?

6. Stem Cell Advantages and Disadvantages
Type
Definition
Advantages
Disadvantages
Embryonic Stem Cells
Cells from an embryo- can become any type of cell (pluripotent)
Can become any type of cell
-Ethical issues
-Immune rejection
Multipotent Stem Cell
Can be a bunch of cell types
No immune rejection- can come from your own tissue
No ethical issues
Can only become a restricted amount of cells, have mutation in DNA unless treated
Induced Pluripotent Stem Cell
Adult skin cells transformed into stem cells- can be any type of cell
-Can become ANY type of cell
-No immune rejection
-No ethical issues
Have same mutation that patient has unless treated
Can form tumors if improperly differentiated/isolated

7. What is CRISPR?
CRISPR (clustered regularly interspaced short palindromic repeats) is a method of taking out a certain part of a genome and rewriting it in a different way.
First discovered in bacteria as one sequence of DNA being repeated over and over with unique sequences in between them
Turns out it’s a defense mechanism against viruses- a bit of the viral DNA is stored in the bacteria so it recognizes it and releases an enzyme called CRISPR Cas9 (CRISPR associated protein) to cut up the viral DNA
Picture on next slide

8. CRISPR Process

9. Why is CRISPR Useful?
CRISPR is useful in gene therapy. It can be used to cut up a faulty part of a gene and insert the normal copy of it into the cell.
Normal restriction enzymes would cut up random parts of the DNA, destroying it. The CRISPR Cas enzyme only cuts up one piece of DNA. It can cut up pieces up to 20 base-pairs long. All you have to do is give it an RNA template and it will cut up that part of the genome- and ONLY that part of the genome.
To get the RNA template for the part of the genome you want to cut, use an online tool to design the target sequence and it will be mailed to you in a few days.
To repair the genome: Use Homology Directed Repair/HDR (Non-Homologous End Joining/NHEJ repair isn’t perfect and disrupts the genome with insertions or deletions). HDR repairs the genome the way you want it to be without any errors
Picture on next slide

10. Mechanism of CRISPR in Gene Therapy

11. What are Liposomes?
Liposomes: Small vesicles made of cell membrane material.
They attach themselves to cells and dump their contents into cells.
Mainly used for drug delivery into cells.
Can also be used for gene therapy- dumping genes (DNA) into cells.
They can dump the CRISPR Cas9 enzyme into cells to edit the genome.

12. To Cure Retinitis Pigmentosa
Gene therapy will change the genes of the cell, allowing them to see.
Problem: The cells are dead, changing the genes in the dead cells won’t help anything.
Use stem cells to grow a new retina.
Use induced pluripotent stem cells because embryonic stem cells raise ethical issues and are at risk of immune rejection.
Problem: Induced pluripotent stem cells have the same mutation- will die from mutation.
Use gene therapy (CRISPR) to fix mutation in these stem cells.
To insert CRISPR Cas9 into cells, use liposomes
Selectable marker: Green fluorescent protein (GFP) gene- allows cells to glow green under UV light

13. Which Type of RP Will We Work With?
Usher Syndrome is rarer than retinitis pigmentosa, so that is what we will work with.
There are several types of Usher Syndrome, but one of the rarer subtypes is Usher Syndrome Type 1F.
Gene: Protocadherin-15 (PCDH15) is very long (1955 amino acids base-pairs), so viral vectors will not work- it is too large.
Use CRISPR instead- only affects mutated part of the gene- does not require the whole gene.
The mutation: One nucleotide is replaced with another- most common type is replacement of amino acid arginine with a stop codon

14. The Experiment
Question: Are induced pluripotent stem cells and CRISPR the cure for Usher Syndrome Type 1F?
Hypothesis: Taking skin cells, transforming them into stem cells, using liposomes to deliver the CRISPR materials and the selectable marker, allowing the CRISPR to change the mutation, turning the retinal cells back into stem cells, and injecting them into both eyes of an affected mouse will help fix its vision.
Test Subjects: We will use four different mice:
Mouse 1: Not affected by mutation, will not be tested on (control)
Mouse 2: Affected by mutation, will be tested on (trial 1)
Mouse 3: Affected by mutation, will be tested on (trial 2)
Mouse 4: Affected by mutation, will not be tested on (control)

15. Procedure Part 1- Stem Cells
Take skin cells from Mice 2 and 3 (leave Mice 1 and 4 alone)
Grow skin cells in culture in two petri dishes (one for use, one as a backup)
Turn these skin cells into stem cells by:
Adding the genes necessary for transformation
To add the genes, use an adenovirus to inject them into the cells
Inject viruses into one of the petri dishes
Confirm they are all stem cells by using a microscope
If not, then take some cells from the backup dish, grow them on a new dish, and try again
inject skin cells with adenovirus confirm existence of stem cells

16. Procedure Part 2- CRISPR Insertion
Once you have confirmed that you have only stem cells:
Take some stem cells and grow them on another plate as a backup
Insert the following ingredients into a liposome:
CRISPR Cas9 enzyme (with RNA template of mutated part of DNA- 20 base-pairs long)
repair template (with selectable marker)
Mix liposomes with cells
Give cells time to transform and grow
CRISPR enzyme RNA template insert into liposome add to cells

17. Procedure Part 3- CRISPR Isolation
Once the cells have been growing for at least 24 hours:
Take the dish into a dark room and shine an ultraviolet light on it
Take the time to locate the green-glowing colonies of cells on the plate
Isolate some of the glowing cells and put them on a new plate
If no cells glow, take some cells from the backup plate from Part 2 and try again
Remove some glowing cells put them on a new plate

18. Procedure Part 4- Retinal Cells
Once the transformed stem cells have been isolated:
Prepare a plate with FBS-Differentiation medium (turns stem cells into retinal cells)
Move some stem cells onto the plate
Give the stem cells a chance to grow and differentiate into retinal cells
Confirm they are retinal cells by viewing them under a microscope
Once you have confirmed they are retinal cells, inject some into both eyes of the mouse
Do the whole procedure over for the other mouse (Recommended: Do both at the same time)
prepare plate w/medium add stem cells

19. Procedure Part 5- Testing Mouse Vision
Give the mice a few weeks for the retinal cells to grow new retinas
Prepare a mini obstacle course in a plastic box with a light at the end for the mice to find
Get a video camera ready
Place a mouse at the start of the course- start recording immediately
When the mouse reaches the end of the course, stop recording
Repeat for the other three mice
Keep track of which recording is of which mouse

20. Expected Results
Observe the videos of the mice- if the mice make it through the course easily enough, they have good vision. If they have trouble, they do not have good vision.
Mouse 1 (no mutation, not tested)- should make it through easily
Mouse 2(mutation, tested)- should make it through pretty easily
Mouse 3(mutation, tested)- should make it through pretty easily
Mouse 4(mutation, not tested)- should have a hard time making it through
Mouse Mouse Mouse Mouse 4

21. Potential Errors- How to Avoid Them
Errors may include:
Tumors: Skin cells may not change properly into stem cells, resulting in cancer cells
Extra DNA mutations: CRISPR Cas9 may accidentally cut up other parts of DNA, resulting in some extra mutations
How to avoid these errors:
Make sure that the stem cells you made are actually stem cells. Give them time to develop and observe them under a microscope for a few days until you are absolutely sure. If they are anything but stem cells, throw these away and start again (remember, make a backup plate)
Make the target DNA sequence as unique as possible. To do so, order an RNA template with as many base pairs as possible

22. Significance of Results
If induced pluripotent stem cells and CRISPR successfully restore vision in affected mice without any complications, then we can use this same technique on humans. This means we could use this to restore vision in affected people. If we can successfully avoid any errors, we can get this cure approved by the FDA

23. Sources <http://hmg.oxfordjournals.org/content/23/R1/R9.full>
<
<
<
<
<
<
<
<
<
<
<
<
<
<
<
Osakada, Fumitada. "Toward the Generation of Rod and Cone Photoreceptors from Mouse, Monkey, and Human Embryonic Stem Cells." Nature Biotechnology (2008): Web. 27 July 2015