VacciGenex

Revolutionizing cardiovascular disease care through gene-editing medicines

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The heart disease epidemic is one to not discriminate, affecting anyone and everyone. It is responsible for nearly one-third of all deaths globally. In the United States alone, someone dies from a heart attack every 34 seconds (source), resulting in approximately 2,200 deaths per day. By 2035, it is estimated that over 45% of all Americans will have developed some form of cardiovascular disease (source).

Familial hypercholesterolemia (FH) is an inherited genetic disorder that increases the risk of developing cardiovascular diseases. Approximately 40 million people worldwide are affected by FH (source). This condition operates on an autosomal dominant genetic pattern, resulting in elevated levels of LDL (low-density lipoprotein), also known as ‘bad”, cholesterol in the bloodstream. Individuals with FH are unable to remove the excessive levels of bad cholesterol, leading to its accumulation in the arteries and impeding blood flow to the heart. This accumulation leads to an increased susceptibility to cardiovascular diseases, compounding the risk of fatality. Research indicates that around 85% of men with FH will experience a heart attack by the time they reach sixty years old (source).

High cholesterol causing a blockage in the artery (source)

As of now, there is no cure available for Familial Hypercholesterolemia. However, there is a range of prescribed medications and treatment strategies dedicated to reducing elevated cholesterol levels.

Here are some current medications available:

1. Statins: work by blocking the liver’s ability to produce cholesterol, which in turn reduces the amount of LDL-cholesterol in the blood. The dosage and potency of statins vary depending on the individual’s cholesterol levels and other medical factors (source).
2. Ezetimibe: works by preventing the absorption of dietary cholesterol from the intestines, which can help to reduce LDL-cholesterol levels by around 18 to 25 percent (source).

In some cases, liver transplants may be recommended for individuals with severe FH. The liver is responsible for producing cholesterol and a new liver can produce cholesterol at a lower rate than the original liver (source).

Along with prescribed medications and treatments, a healthy lifestyle can also help to manage FH. This includes a balanced diet low in saturated and trans fats, regular exercise, and avoiding smoking and excessive alcohol consumption (source).

Overall, a combination of prescribed medications, treatments, and a healthy lifestyle can help individuals manage their cholesterol levels and reduce the risk of developing heart disease and stroke. Despite the available treatments, their effectiveness is limited and often accompanied by numerous side effects. This is primarily due to two factors. One, the medications and treatments are not tailored to the specific needs of the individual. Two, they only treat the symptoms of FH and not its root cause as FH is a genetic disorder.

Introducing VacciGenex

VacciGenex has made it our mission to revolutionize cardiovascular disease care through gene-editing medicines. A current vaccine focus is to create a gene-editing medicine to cure Familial Hypercholesterolemia.

Our vaccine targets the three genes that mostly affect patients with this disease; PCSK9 found in chromosome 1, APOB found in chromosome 2 and LDLR found in chromosome 19.

The three most common mutations in each gene type other than the vaccine targets are the LDLR W23X, APOB R3500Q, and PCSK9 D374Y mutations.

1. The LDLR gene, located on the short arm of chromosome 19 (19p13.1–13.3), plays a crucial role in managing the production of the LDL receptor in the body. This receptor binds to LDL particles as a means of regulating cholesterol levels. In cases of the W23X mutation, where a premature stop codon occurs within the LDLR gene, it results in the production of a truncated and non-functional protein. This mutation is associated with impaired LDL receptor function (source).
2. The APOB gene, located on the short arm of chromosome 2 (2p24.1), encodes the information necessary for the synthesis of two variants of the apolipoprotein B protein. This protein is integral in the formation of chylomicrons, which play a role in lipid transport. The specific mutation we are addressing, known as the R3500Q mutation, leads to an abnormally shortened version of the protein (source).
3. The PCSK9 gene, located on chromosome 1 (1p32), regulates the quantity of LDL receptors within the body. The D374Y mutation in this gene affects the breakdown of low-density lipoprotein (LDL) receptors, which can influence cholesterol regulation (source).

Our solution is designed to target these mutations and restore the normal function of the affected genes, resulting in decreased levels of LDL in the blood. This approach can potentially prevent or delay the development of heart disease in individuals with FH. By addressing the underlying genetic causes of the condition, we can offer a more effective and long-lasting treatment for patients with FH.

Preliminary Testing Process

Before patients can receive the gene-editing vaccine, they must meet certain criteria for the test. Patients must go through three single-gene tests — a test that only sequences the exons of these three genes; LDLR, APOB, and PCSK9.

Here is the procedure for the testing done:

1. Sample collection: A small sample of blood, saliva, or tissue is collected from the individual undergoing the test.
2. DNA extraction: The DNA is isolated from the sample and purified to remove any contaminants.
3. Amplification: The DNA is amplified or replicated using a technique called polymerase chain reaction (PCR), which produces many copies of the specific gene of interest.
4. Sequencing: The DNA sequence of the amplified gene is determined using a technique called Sanger sequencing, which reads the sequence of the nucleotide bases (A, C, T, G) in the gene.
5. Analysis: The sequence data is compared to a reference sequence of the gene to identify any changes or mutations that may be present.
6. Interpretation: The results are interpreted by a genetic counsellor or medical professional who can explain the implications of the findings and provide guidance on further testing or treatment.

This testing will understand which gene is mutated because, in most FH patients, only one out of the 3 genes is mutated causing the condition. Only the crisper for the specific genes mutated will be carried out and put into the vaccine.

The Vaccine

The innovative approach of using CRISPR-Cas9 technology embedded in lipid nanoparticles to treat Familial Hypercholesterolemia represents a major breakthrough in the field of genetic medicine. CRISPR-Cas9 is a highly precise gene-editing technology that allows scientists to make targeted changes to DNA sequences in cells. This technology has shown great promise in treating a range of genetic diseases, including FH.

Our vaccine uses CRISPR-Cas9 technology to edit and change the mutations in all three of the genes that are responsible for FH. The goal is to restore the normal function of these genes, thereby reducing the levels of LDL cholesterol in the blood. By using CRISPR-Cas9 to correct genetic mutations, we believe that we can cure patients with the disease.

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CRISPR-Cas9

The evolution of the gene-editing process originated from a bacterial defence mechanism designed to counter invading bacteria. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. These repeats are short DNA sequences that are repeated multiple times in a cluster, with each repeat separated by short segments of non-repeating DNA called spacers. The CRISPR system also includes a set of proteins, including the Cas9 protein, which can cut DNA at specific loci (source).

When a bacteria is infected by a virus, it uses a small piece of the viral DNA in its own CRISPR system as a spacer. This allows the bacteria to recognize and defend against the virus in the future by using the Cas9 protein to cut the viral DNA at specific loci.

Now this technology has been developed to be used in living cells to edit genome sequences. By designing a Guide RNA (gRNA) that matches a specific DNA sequence, they can use the Cas9 protein to cut the DNA at that locus. This can be used to remove, replace, or add specific genes to an organism’s DNA.

There are 2 components of this process: a Cas9 enzyme (can cut DNA) and a gRNA (guides the cas9 to the area) (source).

Essentially the Cas9 attaches, unzips DNA, and matches it to its RNA at the PAM (the specific site that wanted to be cut). DNA pairs up with the cut ends to make sure there are no errors in the DNA repair process and replaces the original sequence with a new version (source).

RNA-guided Cas9 recognizes the PAM sequence (source)

Gene-Editing Process

Here are the steps to this procedure:

1. Identify a target site and design a gRNA.
2. FH is usually triggered by a mutation in one of these genes (sometimes, a more severe form of FH can be caused by two of these genes):

• LDLR w23x — substitution of adenine for guanine at position 131 of cDNA source.
• APOB R3500Q — substitution of the amino acid arginine (R) with the amino acid glutamine (Q) at position 3500 of the polypeptide chain.
• PCSK9 D374Y — substitution of G→T nucleotide variant present on the K1173 haplotype variant resulting in the non-synonymous.

3. Based on which genes are affected, using their chromosomal site, we can design the complementary RNA strand.

4. Creation of gRNA.

5. The gRNA will be delivered to the target cells along with the base editor.

A specific concern for injectable vaccines using crisper technology is the off-target effects it can have. We have found three main gRNAs that will minimize the risk of getting off-target effects.

Made using Synthego

• LDLR guides RNA with the least off-target effects
• PCSK9 guides RNA with the least off-target effects
• APOB guides RNA with the least off-target effects

We have ensured that we are only using the gRNA strand that has the least off-target effects on the body.

Delivering the Vaccine

To deliver the CRISPR-Cas9 to specific cells, we will use lipid nanoparticles. Lipid nanoparticles are natural substances composed of lipids that are commonly used to deliver drugs and other substances to cells (source). In our vaccine, the lipid nanoparticles are designed to target the cells that make up the liver, which is where much of the LDL cholesterol in the body is produced.

The use of lipid nanoparticles as a delivery mechanism for the CRISPR-Cas9 is a key component of our vaccine’s effectiveness. These particles help to protect the CRISPR-Cas9 from degradation in the body and allow it to reach its target cells. Once inside the liver cells, CRISPR-Cas9 can then edit the DNA sequence of the cells to correct the genetic mutations responsible for FH (source).

Lipid nanoparticle encompassing the CRISPR-Cas9 (source)

Since cells reproduce quickly, patients can be cured of FH after taking the injection. The edited cells will continue to reproduce, passing on the corrected DNA sequence to new cells as they divide. Over time, the number of cells with the corrected DNA sequence will increase, leading to a reduction in the levels of LDL cholesterol in the body.

By correcting the underlying genetic mutations responsible for the disease, we hope to cure the condition and prevent the development of heart disease in affected individuals. With further research and development, we plan to leverage this technology to create further vaccines to treat a range of other genetic disorders and conditions, offering new hope for patients and their families.

Challenges

The technology for this vaccine is ready. We now need to conduct trials to test how the vaccine will interact with the body once it is injected.

Main concerns:

1. Off-target effects: Although we have identified the gRNA strands with the least off-target effects for our vaccines, we still need to test the effects of the vaccine editing genes in unintended cells.
2. Delivery of cas9 to the liver: Further tests will need to be conducted to maximize the efficiency and precision of delivering the cas9 directly to the liver.

Economic Benefits & Partnerships:

Verve Therapeutics is a biotechnology company that specializes in the treatment of cardiovascular diseases, specifically the subtype of atherosclerotic cardiovascular disease (ASCVD). (Source) Recently, this company has been focusing on targeting genes such as PCSK9 and ANGPTL3 in order to lower the cholesterol density in the bloodstream (LDL-C levels). In late July of 2022, an individual from New Zealand was injected with base-editing medicine in order to change a base in the genes that are in charge of producing bad cholesterol. (Source). It was reported that the individual did not face any adverse side effects after receiving the single dose. While there is no available data on the success rate of this test, it is said that when testing with non-human primates, there was a success rate of 70%. (Source)

We hope to partner with Verve Therapeutics as they are currently testing to use gene editing to cure FH. Our product also will align with their mission to cure FH. We are also proposing a vaccine version and curing the other two genes other companies are currently not working on.

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VacciGenex’s gene-editing vaccine offers a future for revolutionizing cardiovascular disease care. By targeting the genetic basis of Familial Hypercholesterolemia, this innovation could significantly impact disease treatment, providing hope for patients and families affected by this widespread condition.

Thank you to my teammates Anya and Iliana for making this project possible!

Go check out our additional content related to this project down below 👇

VacciGenex Official Website