Nick Tsimplis

In my previous post on Zolgensma, I wrote about how this incredible drug is able to treat such a complex genetic disease by modifying the DNA of patients. With the future of medicine being likely to involve the widespread use of gene editing I think it is interesting to explore where we are now and where we might be in the future. But first lets start with why there is a need for these therapies.

Why use gene editing therapies?

A great number of diseases have a genetic cause. Cancer is caused by mutations in the DNA causing uncontrolled cell growth. Cystic fibrosis is caused by a genetic defect resulting in the inability to produce a protein for making mucus less viscous. In both cases therapy has been focused on relieving symptoms or, in the case of cancer, killing the cancerous cells in an non-targeted manner. The ability to make changes to the DNA of patients has the potential to be a true therapy for these and many other diseases.

However, an issue arises immediately. How do you get the DNA through the cell membrane? In bacteria, it is often as simple as mixing the cells with the DNA to be inserted and heating. Similar methods have been used for transfer to mammalian cells. These use electrodes or ultrasound to increase the permeability of the cell membrane or a lipid capsule to carry the DNA across the cell membrane. Direct injection into cells has also had some success. But all these techniques are not efficient compared to what has been up to now the main DNA transfer method.

Viral vectors

How do they work?

Viruses are built to transfer their genetic information to a host’s DNA. They can thought of as a string of DNA (or RNA) surrounded by protein case called the capsid. On the surface of the capsid there is often a lipid coat that contains several more proteins scattered around which have a critical role in attaching and infecting the target cell.

However, virsuses are usually pathogenic and have the propensity to self-replicate at levels which kill the infected cell. As result great efforts have been made to develop viruses that do not cause disease and do not replicate. It is also critical that the virus does not cause an immune response. What has arrisen from this work is the recombinant Adeno-associated virus (rAAV) class of viral vectors of which AAV9 the vector for Zolgensma, is a member.

The AAV9 DNA contains only essential genes that are needed for the virus to survive and replicate. This means that is incredibly small at only 4,700 bases in size. For comparison the human genome contains over 3 million.

For use in gene therapy all that is needed is the therapuetic gene and 2 other genes responsible for producing 7 proteins. The first is called rep and it produces 4 proteins that are involved in replicating the virus DNA and packaging it into new viruses. The other gene is cap and this contains the information for producing the proteins that form the capsid. The image below shows the structure of a AAV6 virus capsid with each structural proteins coloured differently.

Issues with viral DNA transfer

By using a virus with a small genome it reduces the chance of unwanted modifications. However there are cases unwanted viral genetic material being incorporated into the host genome. In 2003 it was reported that 2 recipients of a viral gene therapy developed leukemia. This was caused by the insertion of a DNA near a gene that produces a protein called LM02. LM02 is known to be produced in large quantities in a number of blood cancers.

Whilst the use of AAV class of viral vectors reduces the chance of an immune response it still causes issues in therapy. In an early mouse experiment for a potential treatment for Parkinson’s disease it was found that after initial treatment on one half of a mouse brain there was reduced effectiveness of the same treatment on the opposite half. However, immune responses do vary depending on the virus used, the target organ and the dosing schedule and form.

It is important to remember that a viral vector is the delivery method for the therapy. Once the DNA has entered the cell it is up to other factors to ensure that the gene editing is accurate and efficient. To do so viral mechanisms of DNA transfer can be used. This can have varying levels of efficiency and is limited in the size of the gene that can be transferred. Luckily in the last few years experiments have started being performed which has the potential of being a real game changer.

CRISPR

Clustered regularly interspaced short palindromic repeats, or CRISPR, is a relatively recent development in the gene editing field. It utilises a defense mechanism that bacterial have developed to protect against viruses. It was observed that following viral infection the DNA of Streptococcus thermophilus bacteria would now contain a short stretches of the viral genome. When the same virus would infect the cell a molecule acelled CRISPR-RNA, crRNA, would be produced. This matches the DNA of the virus and directs a DNA cutting protein, Cas9, to come into contact with it. By cutting the viral DNA the bacteria is able to quickly degrade it and prevent the virus infecting and replicating.

It was later shown that by modifying the crRNA this process can be directed to cut any DNA sequence in a very targeted and specific manner. Once cut the sequence is repaired by cell but due to the nature of the cut this often introduces or removes parts of the sequence. As a result the gene in which the cut has been directed becomes deactivated. Alternatively, a DNA segment can also be injected into the cell that acts template for repair. This is less efficient than the previous technique but does allow for modification rather than just deactivation. This presents great opportunities for making large and targeted changes to genes responsible for the development several diseases.

CRISPR in cancer

Cancer is the uncontrolled growth and multiplication of a defective cell. This is usually as a result of the over production of a protein that enhances cell growth or the reduced production of a protein that reduces it. The cause of this is usually damage to the DNA of the cell. As a result CRISPR has been investigated for repairing damaged DNA in cancerous cells. It has also been used cause damage to the DNA of cells to identify which are responsible for causing cancer.

In 2018, it was reported that a modified CRISPR method, ChaCha, could be used to direct immune cells against cancerous cells. This worked by coupling CRISPR to a group of receptors called G-protein coupled receptors (GPCRs) that respond to a wide variety of external signals. The researchers showed that this can be used to direct changes to specific genes. They suggest that this could be used create modified immune cells that only become active when near a tumour. This could potentially reduce the side effects usually associated with anti-cancer therapy.

CRISPR in Parkinson’s

Gene editing therapies have been investigated as being a possible cure for some forms of Parkinson’s disease. Parkinson’s is charicterised by the death of dopamine sensitive neurons in the brain. This is sometime the result of damage to the gene that produces a protein called synuclein. Using CRISPR to deactivate this gene has been shown to be sufficient to reduce the onset of Parkinson’s in animal models.

CRISPR in anti-bacterial therapy

It’s not just human cells that are the target. In 2015, it was reported in Nature Biotechnology that a CRISPR system using a bacteriophage vector could be used to kill Staphylococcus aureus bacteria that had developed resistance to antibiotics. This worked by directly targeting the gene responsible for conferring resistance to the antibiotic methicillin. Much like the phage therapy, this did not kill every single bacterium it reduces the resistant population sufficiently for the infection to be cleared.

The potential advantages of the CRISPR method is the even more specific targeting of bacteria, as was shown in the 2015 paper where only resistant bacteria were eradicated. This reduces potential damage to the patient’s bacterial flora. However, due to the lack of new phage production during treatment the treatment dose has to be much higher than the phage therapy.

At this point the number number of gene editing therapies is relatively small but with the development of CRISPR this is likely to change. Whilst it may be a few years until these now experimental techniques make it to the clinical it does give us a glimpse into a future where widespread treatment of genetic disease is a reality.