BIP – Lesson 2: Possible gene therapies.

With GIP (gene therapy information packet), we take you into the world of gene therapy. In recent years there has been a lot of research into gene therapy. This has provided insight into whether and how gene therapy can help with a treatment for PLN-R14del. We will take you into the world of gene therapy. In this second lesson, we will tell you more about the different types of gene therapy and its possibilities.

Overview gene therapy information packet:

In the previous lesson, we saw that each cell contains a nucleus that contains a kind of cookbook for the cell, the DNA. This DNA contains genes (recipes) that are translated into RNA (messenger bills) so that a protein (dish) can be put together. Furthermore, we have seen that gene therapy has the most potential to cure PLN. In this lesson we will consider different types of gene therapy.

There are different types of gene therapy. Here it is important to distinguish between gene therapy in general and gene editing, which involves modification of DNA. Gene therapy in general works at the RNA level: an extra gene is inserted or a gene is captured. For PLN, three types of treatments are possible (Figure 1): (1) gene editing; (2) the wrong messaging, the wrong RNA capture; (3) general gene therapy that could also work for other heart diseases.

Figure 1 Three types of gene therapy for PLN R14del. In option 1, the DNA is repaired so that the error is gone. In option 2, the wrong RNA is removed so that less broken protein is made. Finally, in option 3, a gene (which can be a healthy PLN gene or another gene that helps the heart) is added.

In this lesson, we first consider gene editing, then gene therapy at the RNA level, and finally how gene therapy is administered. Subsequent lessons will discuss the pros and cons of gene therapy and various gene therapies already used in patients.

Gene editing

A well-known form of gene therapy is CRISPR/Cas9. This technique is actually derived from nature and comes from bacteria. The deletion of a whole amino acid cannot be repaired with CRISPR/Cas9. However, the faulty gene can be completely destroyed so that no more faulty protein is made. Actually, the wrong recipe is discarded so that no more wrong dish is made either. Another form of gene therapy is prime editing in which the change in DNA can be repaired. CRISPR/Cas9 and prime editing are very similar. Basically, three things must happen: first, the site with the change must be traced throughout the genome, second, the DNA is cut, and finally, the error must be repaired (Figure 2). Below we elaborate on these steps with attention to the differences between CRISPR/Cas9 and prime editing:

  • Detecting the error is easier said than done. After all, our DNA consists of about 3,000,000,000 letters! But with a piece of guide RNA of 17-24 letters, it is possible to detect very precisely the location with the mutation;
  • The second step differs between CRISPR/Cas9 and prime editing. CRISPR/Cas9 binds to the DNA and makes a break in both strands of DNA, while prime editing breaks only one strand of DNA. Compare it to a ladder whose side is cut; either on one side or both sides. As you can imagine, the second way is much easier;
  • Finally, the break in the DNA must be repaired. With CRISPR/Cas9, the cell must do this itself. Because there is a break in both strands of DNA, there will be errors in repairing the break (a few pieces of the torn recipe get lost). The result will probably be that the gene is completely broken. This does not sound like a solution, but it can be a good thing. In fact, sometimes no recipe is better than a bad one.
    In prime editing, on the other hand, a protein is included that repairs the error in the DNA using a supplied template. A new temporary recipe is created with the missing ingredient (the amino acid arginine at the 14th position) so that it can be copied into the real recipe (the DNA). So prime editing allows you to very accurately repair all kinds of errors in the DNA. The big advantage is that it allows you to get to the root of the change.

Figure 2 The mechanism behind prime editing and CRISPR/Cas9. Without treatment, the mutated PLN gene is made into the wrong messenger bill and thus the wrong dish. In prime editing, a break is made in the DNA and then a template is used to rewrite the gene. This gets the grocery bill and recipe right again. In CRISPR/Cas9, the mutated gene is actually destroyed. As a result, the prescription also breaks down and is destroyed. Therefore, the wrong recipe is then also no longer made.

Gene therapy at the RNA level

In addition to gene editing, there are several other forms of gene therapy that are potential treatments for PLN. In addition to general forms of gene therapy, this is the removal of wrong PLN RNA. As mentioned, you can compare a wrong gene to a wrong recipe that results in the wrong grocery bill and thus a wrong dish. Suppose two cooks make a big pan of soup together. They both have a copy of the recipe, but one copy contains an error. The cook with the wrong recipe will make the wrong shopping list and therefore cause the whole soup to fail. Back to DNA, you can solve the problem by breaking down the wrong RNA. You can accomplish this in several ways. All these ways have in common that something is attached to the RNA that makes it go to waste.

Finally, there are general forms of gene therapy. We mention two of them here. The first is actually close to RNA capture. In this treatment, additional healthy PLN gene is inserted. Instead of trapping away the broken message tag, you get more of the good message tag. The second option is I-1c, a treatment now being tested in patients. This treatment acts on a protein that binds to PLN and affects the structure of PLN. This inhibits the activity of PLN. Both treatments could also be used for heart diseases not caused by PLN, because PLN is also worse in other heart diseases.

Transportation

One of the big challenges with gene therapy is getting the therapy into the cell. With a “normal” drug, usually a small molecule, the drug itself penetrates the cell. Gene therapy, however, you can’t put it in a pill; it must be administered by injection. If you administer the therapy into the blood without packaging, it will break down quickly and have no effect. Therefore, it must be packaged. There are two types of packaging that are widely studied. These are viruses and fat globules (lipid nano particles).

  • A virus is an envelope containing RNA or DNA. A virus invades a cell, makes new RNA or DNA and thus spreads. We can also modify a virus so that it brings into a cell not its own cargo, but a piece of genetic material we want. In fact, when it comes to viruses for gene therapy, it’s always about AAVs. This is because AAVs have important advantages: (1) there are different types that can invade different tissues; (2) AAVs cannot modify DNA and therefore do not cause side effects there; (3) AAVs are viruses but do not cause disease (as a flu virus does);
  • Fat globules can be made in the lab and modified to do what we want. So in this case, the requirements for gene editing or the RNA for another gene therapy can be put into a fat globule. The fat globule can then be modified so that it primarily invades heart cells. At present, however, it is still difficult to specifically treat the heart.

In addition to these treatments, there are other ways to get gene therapy into a cell such as coupling to an antibody or protein. The PLN Foundation is working with several companies that are investigating this. This keeps all options open, even if one of the transport options turns out not to work.

From this lesson, it has become clear that there are several possible treatments within gene therapy. At the same time, these treatments do have disadvantages. In the next lesson, we will dwell on these disadvantages.