Crispr-Cas9 genome editing is one of the most dramatic innovations of the last few decades, causing an uproar among the Australian medical school community. Proposed by Jennifer Doudna and Emmanuelle Charpentier in their seminal 2012 paper, as a way of editing the DNA of living organisms, in 2013, Crispr-Cas9 was shown to be deployable as a tool for editing human and animal DNA. Since then, researchers have proposed that Crispr can be used for the treatment of disease. In 2020, Doudna and Charpentier won the Nobel Prize in Chemistry, “for the development of a method for genome editing”. However, turning those test-tube discoveries into gene editing within the body, has proved challenging. That is now changing.

What is the Crispr-Cas9 Technique?

Crispr-Cas 9 technique has been called a “genetic scissors”. The “Crispr” part of that name refers to “clustered regularly interspaced short palindromic repeats”. Crispr is a family of DNA sequences that exist in the genomes of prokaryotic organisms such as archaea and bacteria. They are derived from the DNA fragments of bacteriophages previously infected by prokaryotes. Crispr is used to identify and kill DNA from other bacteriophages in later infections. They are very important for attaining acquired immunity. About half of sequenced bacterial genomes, and 90% of sequenced archaea contain Crispr. 

Cas9 (Crispr-associated protein 9) refers to an enzyme that uses Crispr to help it identify and cleave certain strands of DNA which are complementary to the Crispr. 

Crispr-Cas9 techniques use Cas9 enzymes and Crispr sequences to edit genomes. In theory, there is the potential to change the course of a disease through gene mutations. Nevertheless, as we have said, it has been difficult to translate theory to real-world results, i.e. getting the gene mutation to occur where it’s needed in the body. Crispr Technologies has tried getting around this by editing cells outside the body. In 2019, a 34-year old patient was cured of sickle cell disease using this approach. There has been progress with HIV treatments, Leber congenital amaurosis, and other diseases. When the gene editing is done outside the body, red blood cells are taken from the body, edited so harmful mutations are corrected, and then those red blood cells are injected back. 

However, in conducting gene editing outside the body, researchers and practitioners incur certain risks and downsides. It is a complex, expensive process that cannot be widely used. The narrowness of use cases is because the majority of diseases are not susceptible to that kind of treatment. So researchers are moving from “ex vivo” (out of body) approaches to “in vivo” (in body) approaches. 

The Success of In Vivo Approaches

In 2021, Intellia Therapeutics was able to conduct an in vivo treatment of transthyretin amyloidosis. Recently, they have gone further, and showed that in vivo treatment can be used to treat hereditary angioedema. The company has also revealed that it was able to reduce the presence of harmful proteins that cause transthyretin amyloidosis by 90%. Although the diseases are caused by different genes, Intellia has shown that Crispr-Cas9 can be successfully used for a variety of diseases. 

The company has not yet published its results in a peer-reviewed journal, and the trials are not sufficiently large for us to draw firm conclusions. However, they are very suggestive that we may now be at the dawn of in vivo gene editing. 

In order to deliver Crispr to where it’s needed, Intellia used lipid nanoparticles to deliver it to the liver, thanks to an IV infusion. The liver then circulates these nanoparticles into the bloodstream. It’s in the liver that lipid nanoparticles are absorbed by hepatocytes, which break them down, allowing Crispr to start editing the genes. Students in Australian Medical Schools will know this intimately: what Intellia is essentially doing is stopping a gene mutation in a harmful protein, from causing and spreading disease in the body. 

When the company was treating hereditary angioedema, the goal was to stop the propagation of the KLKB1 gene, which encodes the Plasma kallikrein protein. An excess of kallikrein causes an overproduction of bradykinin, a peptide that promotes inflammation. Prior to receiving the infusion of Crispr, the patients treated by Intellia had between one and 7 swelling attacks a month. Within the 16-week period in which the patients were observed, the number of attacks was reduced by about 91%.

When the company was treating transthyretin amyloidosis, the goal was to inactivate the propagation of mutations of the TTR gene, a protein coding gene that encodes one of three prealbumins: alpha-1-antitrypsin, transthyretin and orosomucoid. Mutations in the TTR gene are associated with amyloid deposition, which largely affects peripheral nerves or the heart, and in rare cases, they are non-amyloidogenic. These mutations are associated with diseases such as amyloidotic polyneuropathy, euthyroid hyperthyroxinaemia, and amyloidotic vitreous opacities. So, for example, a mutation can lead to heart failure in 200,000 to 500,000 people across the world. Diagnosis often occurs with just two to six years of life left for the patient. 

Intellia’s findings are very exciting, especially because gene editing has permanent effects on the genes, so there is no fear of a disease returning.