As previously reported, we have discovered that a portion of CAMSAP1 must exist for any organism to survive embryogenesis. This proposition is simply due to the results of the initial attempt at creating a mouse model that proved to be embryonic lethal (aka conditional lethal, see Figure 1) as the CRISPR/cas9 cut was made prior to the CH location along the gene (Figure 1, brown stars). However, further analysis showed that there were viable, heterozygous mice that carried the mutation but were unaffected – this proved that only one functioning copy of CAMSAP1 is necessary for normal development.
This initial attempt also left other questions to be answered:
- Does the paternal or maternal mutation create partial protein function throughout Landon’s cells?
- If partial protein function does exist,
- Is it from the paternal or maternal genetic mutation?
- Can a mouse model be created from either the paternal or maternal mutation, depending on which mutation is responsible for the partial protein function?
- If a successful mouse model can be created, can the mutated mouse safely be rescued?
- What types of cells express the highest amount of CAMSAP1 normally, and are Landon’s mutated cells devoid of the appropriate expression levels?
- Can Landon’s cells be rescued via gene therapy?
- Can we analyze Landon’s cells safely to answer these questions?
How Do We Find Out The Answers To These Questions?
Since CAMSAP1 is highly expressed in the nervous system, a brain biopsy would give us some answers but would be far too risky! However, we can create “Induced Pluripotent Stem Cells” (iPSC or iPS). What are iPSCs? Simply put, they are cells that are created from an adult cell (ie. blood cells, skin cells, etc.) that are genetically modified by adding four genes to them (OCT-4, SOX-2, KLF-4, and MYC, also known as “Yamanaka Factors”); however, they are not embryonic stem cells. These genes are specific genetic instructions used to transform or induce adult cells into pluripotent cells (see Video 1). By adding these genes, the adult cells will reverse from their differentiated (specialized) state back to an undifferentiated state. Like embryonic stem cells though, then these cells can be modified to redifferentiate into any cell type in the human body, including cells from the nervous system.
WE ARE EXCITED TO REPORT THAT WE WILL BE WORKING WITH A TEAM OF RESEARCHERS AND PHYSICIANS TO ACHIEVE THIS FEAT!
The plan is to create iPS cells from a simple blood sample from Landon and test the levels of CAMSAP1 in the iPSC samples, as well as an unrelated control sample, to find out whether CAMSAP1 mutations in Landon’s cells lead to partial protein function or gene decay.
Here is an example (Video 2) of the process rescuing of a condition known as Sickle Cell Anemia via gene therapy by way of transforming differentiated skin cells into undifferentiated iPS cells then redifferentiating them into normal red blood cells after the gene was corrected:
If partial protein is recognized, then the hope is to proceed with the creation of the mouse model of the same specific mutation that is responsible for the partial protein function recognized in Landon’s cells. The hope is to create a gene therapy to “rescue/cure” the CAMSAP1 mutation by reintroducing the gene into the various cell types to “rescue/cure” them while in the lab.
How are these cells fixed? A process in a highly dynamic, new, and constantly evolving field of medicine known as gene therapy, can fix, alter, and/or reintroduce a normal functioning copy of a gene into a cell. This process is completed by altering a virus, eliminating its harmful effects, and engineering it to deliver a message to the cell or deliver the corrected copy of the gene to the cell. Afterwards, the cell will respond appropriately and if everything works as designed, the condition is rescued.
Complex Version of the Potential CAMSAP1 Vector
Other Potential For iPSC Creation
While we are simply scratching the proverbial surface of what this could potentially mean for individuals with CAMSAP1 mutations with respect to the creation/development of a curative gene therapy, these iPS cells could also be used to test different drugs to see if there is an existing medication that could mimic the role of CAMSAP1 throughout the body. Furthermore, we can also see if these drugs have any harmful effects on other tissue types (Figure 2).
It has been a while since our last research update, but we have received some very important news regarding the progress of our endeavor. On Tuesday morning, we met with the Director of Development for Dr. Curiel’s laboratory at Washington University in St. Louis School of Medicine.
Our research has determined that a portion of the CAMSAP1 gene is 100% necessary for an organism to survive. The mice from The Jackson Laboratory were shipped to the laboratory at WashU. These mice were then bred with hopes of creating a mouse that had 2 mutated copies of the CAMSAP1 gene. After testing the DNA of all off-spring, none were found to have two mutated copies. However, additional carriers of a CAMSAP1 gene mutation were identified. Because of the location of where the “cuts” were made on the CAMSAP1 gene in our founder mice, homozygous mice (mice carrying 2 copies of the knocked-out/mutated CAMSAP1 gene) did not survive embryogenesis. Hence, this means that a portion of CAMSAP1 is critical for life.
THE SCIENCE AND GENETICS:
To simplify an otherwise complex explanation, genes are sequences of DNA that code/create proteins. Each gene is made up of sections also know as exons. The CAMSAP1 gene is made up of 17 different sections [exons]. CAMSAP1 has been recognized to contain 5 unique locations within these sections [exons] that are unique to the gene. These sections are known as CH, CC1, CC2, CC3, and CKK.
To recap our research to-date, when the “founder mice” were initially created in November 2018, we received 4 different mice with 4 different cuts along the CAMSAP1 gene:
– Mouse 1 had the CAMSAP1 gene cut in the beginning of exon 2
– Mouse 2 had the CAMSAP1 gene cut at the end of exon 2
– Mouse 3 had the CAMSAP1 gene cut in the beginning of exon 3
– Mouse 4 had the CAMSAP1 gene cut at the end of exon 3
After the researchers bred these founder mice with normal mice, it was discovered that 50% of the offspring (born in January 2019) were carriers of the created CAMSAP1 mutation(s).
These mice were then shipped to Washington University in St. Louis School of Medicine. There, the biologists bred the different “families” or lines of mice in hopes of creating a mouse that carried 2 mutated copies of the CAMSAP1 gene. For example, 25% of the offspring of heterozygous mouse 2 (+/-) and heterozygous mouse 4 (+/-) should have offspring that would be effected (-/-) by containing two mutated/altered copies of the CAMSAP1 gene.
It was determined that the 3rd generation offspring did not contain a mouse with 2 mutated copies; however, several more carrier mice were born. This important result means that a section beyond where the cuts/knockouts were made of the CAMSAP1 gene is 100% necessary for survival as mice with 2 mutated copies did not survive embryogenesis. The result is called “conditional lethal”.
As mentioned above, the CAMSAP1 gene has 5 unique locations within it — CH, CC1, CC2, CC3, and CKK. The initial cuts of the gene were made in sections [exons] 2 and 3. This means that CC1, CC2, CC3, and CKK are excluded from these mice as these unique sites are located further down the gene from where the cuts were made. We are still in the process of clarifying where CH is located on this gene to determine if this has also been excluded with these cuts.
Through gene sequencing, it was found that Joe’s mutation is just prior to CC1 in exon 11, and Lauren’s is just behind CC1.
So, where do we go from here? We’ve answered the question that CAMSAP1, at least the portions ahead of where the cuts were made, are necessary for survival. Also, we speculate that perhaps Lauren’s mutation may be the reason for survival as it is behind CC1. We have scheduled a meeting with Dr. Curiel, his team, and other biologists worldwide on Wednesday, 05/22 to discuss next steps.
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🎉 WE DID IT!!! 🎉
THE JACKSON LABORATORY HAS SUCCESSFULLY CREATED THE GENETIC KNOCKOUT CAMSAP1 MOUSE 🐭 MODEL!!!
Despite having zero guarantee that this endeavor would be successful when we began in June 2018, we remained cautiously optimistic, hopeful, and prayerful. Our prayers have been answered!!!
Logistics are being finalized for the mice to arrive at the lab at Washington University in St. Louis School of Medicine in the upcoming days.
Additionally, we’ve initiated communication with the Genetics and Rare Diseases Center (GARD) within the National Institute of Health (NIH). The representative was in awe at how quickly we’ve managed to get to this point and provided additional information/resources to help us progress.
So, why is this a big deal? Drug/gene therapy developmental guidelines indicate that these steps need/should take place prior to beginning any sort of human drug trial:
1. Cell knockout/rescue model: ✔️
2. Small organism knockout/rescue model: ✔️
3. Rodent knockout/rescue model: knockout successful ✔️, but pending rescue…
Going from here, the plan is to create/modify a virus that will serve as a vector to deliver the gene to millions of cells via a single injection 💉.
In our reflection back to 1 year ago (almost to the date), we’d like to share the most defeating words that were said to us:
“…we regret to inform you of this news about your child, but he has a gene mutation of his CAMSAP1 gene. We don’t know what it does, nor has there been much research regarding the gene, but we’ll revisit this gene mutation in two years to see if anything has changed. I’m so sorry. There really isn’t anything that you can do at this time. Have a good rest of your day…”
Words cannot express our gratitude and love to each of you for your unwavering support and trust in us.
“Keep moving, for it may well be that the greatest song has not yet been sung, the greatest book has not been written, the highest mountain has not been climbed. This is your challenge…If you can’t fly, then run. If you can’t run, then walk. If you can’t walk, then crawl, but by all means keep moving.” – Martin Luther King, Jr.
We’ve all heard it at some point in our lives: there are no guarantees in life. Life and medicine are very similar in that manner. Prior to initiating this research project, it was conveyed that there was no guarantee that an affected mouse with a CAMSAP1 gene mutation could be possible or that mice with such mutation would even survive birth.
Researchers and scientists have previously been unable to successfully duplicate disease-specific mouse models for various reasons, including the inability for the affected mice to survive birth, yet we agreed to move forward with this project with cautious optimism and hope.
And, we are certainly moving forward! Today, we were given the best possible news:
Per Katie with The Jackson Laboratory, the four initial founder mice (that were, ironically, born on our wedding anniversary) produced the mice we have all been waiting and hoping for!! Twenty-five (25) second-generation (N1) pups were born with 13 of them having a birth day of 01/13/2019 while the other 12 were born on 01/16/2019! (This, of course, could not have been done without continuing with even more irony as 01/16/2017 was Landon’s original “due date”!)
Katie also gave us a tentative timeline as far as the future of the project is concerned. In about 2 weeks, the 25 N1 mice will undergo a DNA validation process to see which of them are the genetically mutated CAMSAP1 deficient mice. Following that, the mice will then be identified. These mice will then be “phenotyped” or characteristically described by their biologists/technicians at 5 weeks of age, a report will be given to Dr. Curiel’s lab, and then the mice will be shipped to Dr. Curiel’s lab at Washington University School of Medicine in St. Louis, MO.
Thank for your continued support and for following our journey! Stay tuned for more exciting news!
What a great way to start the day! The “pups” that were referenced are the CAMSAP1 mutated pups/baby mice which will display the characteristics/hardships that those affected by a mutated CAMSAP1 gene would otherwise exhibit. We anticipate that they will be maintained at the Jackson Laboratory for about 6 weeks after birth and then be transported to Dr. Curiel’s lab at Washington University Medical School of St. Louis, MO who will test, screen, and perform the gene therapy to “rescue” them with hopefully a single treatment!
All excitement here! Thank you for reading, stay tuned!
…We kind of “cheesed it up” with a countdown for your viewing pleasure 🙂
Today, marked yet another major step towards our goal. Dr. Jana Marcette requested that our team in St. Louis, MO get together and meet to discuss our plan of action as what has seemingly been a dream is quickly becoming a reality! So, Dr. Marcette, Dr. David Curiel, and myself met in Dr. Curiel’s office (12/14/18) to discuss research plans moving forward. We are going to be developing a database that will include the contact information of scientists interested in CAMSAP1 so that we can all readily collaborate. Most importantly, we were given another update by The Jackson Laboratory (Jax)!
To my surprise, upon entering Dr. Curiel’s office, I was handed a packet sent by Jax that contained news that another WONDERFUL research milestone had been reached and completed! Of the 45 pups created from the microinjection process, FOUR of them were identified following genetic screening as “Founder Mice” – the mice that had successfully been genetically created to be carriers of the gene mutation, yet able to transmit the mutation to future generations, but who remain outwardly/phenotypically unaffected.
These CRISPR/Cas9-generated mice were transferred to the Jax mating facility to be bred with “wild-type” mice to create second-generation (N1) offspring. By using wild-type mice, it assures that germline transmission can be established – meaning that future generations may be able to also pass along the CAMSAP1 gene mutation. If the laws of genetics hold true, roughly 50% of the offspring from this mating process will produce effected homozygous CAMSAP1-deficient mice that will be analyzed/phenotyped to understand the effect of any mutations created. I’ve borrowed this image to depict how this process takes place – the progress is currently at the level of the first two mice below (image hyperlinked to source):
Here is an idea of the timeline – we are now at the “Founder report packet” step (per The Jackson Laboratory CAMSAP1 Knockout with CRISPR/Cas9 “Founder Report”):
As mentioned in our November Research Update, the birth of the Founder Mice (N0) occurred on 11/03/2018, our wedding anniversary. If the time line above holds true, even more irony may take place in the near future:
The “Landon-like” N1 pups could potentially be born on 01/04/2018, which is Landon’s birthday…
More updates to come in a few short weeks so please stay tuned. Happy holidays and here is to a promising and successful 2019!
An INCREDIBLE day! A second major research hurdle was crossed yesterday. We received this email from The Jackson Laboratory:
“[We] just got word today that the microinjection for the Camsap1 KO project has successfully produced 45 pups born on 11.3.18. These animals will be weaned and sampled at 3 weeks of age, and then the DNA sequence will be analyzed to identify potential founders mice.
I hope this is good news for you!”
So…what does this mean? First, THIS IS ABSOLUTELY GREAT NEWS!!! The irony? This first generation of mice (called N0) were all born on 11.3.2018, which happens to be Lauren and I’s wedding anniversary!
Simply, the 45 first generation mice will be screened at 3 weeks old to identify the ones that are carriers of CAMSAP1 but unaffected like Lauren and I. Then, the lab will breed those mice. Approximately 25% of their offspring (the second generation, or N1) should have Landon’s exact CAMSAP1 mutation. These N1 mice with Landon’s mutation will be the ones that come to St. Louis to undergo gene therapy/rescue by Dr. Curiel.
Scientifically, to simplify (as much as possible), 45 mice were born following a procedure whereby a microscopic needle was inserted into a developing mouse zygote following initial fertilization. The needle was used to inject the developing cell with the necessary genetic CRISPR/CAS9 enzyme (think of it like a scissors) and guide arm (to guide the scissors to the exact gene) to knockout (KO) or remove the CAMSAP1 gene at the site of Landon’s mutation (see image below linked to and borrowed from The Jackson Laboratory website).
More to updates to come in a few weeks, stay tuned!
The wait is over: We are thrilled to share that we have our first research progress update! 🎉
Before the mouse model research can begin onsite at Washington University in St. Louis School of Medicine, a laboratory in Maine called The Jackson Laboratory is working towards creating mice that carry Landon’s gene mutation. The process of creating Landon’s mice begins at a cellular level and takes months. Please understand that the world of medical research can seem very slow-moving, yet can be very precise and successful. Thus, we must remind ourselves to be patient.
The Jackson Laboratory has acquired the necessary RNA (to simplify, RNA is like one side of a zipper on a coat – two RNAs equals DNA) that will direct a specific enzyme called Cas9 to “cut” the CAMSAP1 gene in the exact location where Landon’s gene has a mutation. This creates a modified gene. The modified gene was then reintroduced to normal mouse cells. These modified mouse cells have been tested and found to have no traces of the CAMSAP1 gene, which mimics Landon’s cells! SUCCESS!!! This will not only help Landon, but it will also help the nearly 500 people worldwide who’ve been identified as having the same mutated CAMSAP1 gene.
The next step is to introduce this exact RNA and Cas9 enzyme into hundreds of mouse zygotes. The zygote is the cell formed when the egg and sperm unite to make a single cell, which will divide and grow up to be a mouse. If this is also successful, mice will be born about 3 weeks later carrying Landon’s mutation!
09/15/2018: A Night at the Mouse Races Event
06/26/2018: Payment delivered to Washington University to purchase a mouse model of CAMSAP1 genetic mutation.
06/17/2018: First fundraiser: T-Shirt campaign kickoff
06/13/2018: Landon’s League Foundation founded as 501(c)3 nonprofit organization.
06/13/2018: Initial quote for a mouse model of CAMSAP1 deficiency received from Jackson Labs.
5/23/2018: Meeting with genetic therapist at Washington University in St. Louis who agreed to research CAMSAP1 in an effort to create a gene therapy for individuals effected by a defect in CAMSAP1.