By: Danning Li
Hello everyone and welcome back to the big wide world of gene replacement therapy and medicine! On our last blog post, we tackled the general idea of gene replacement therapy, which was namely to put a working copy of a gene into a patient’s body, so that the gene product could be produced permanently. This idea of course sounds incredibly simple, but considering the lack-luster implementation of gene replacement therapy in medicine today, why hasn’t this idea become widespread? Well, this blog post is going to tackle that exact question, and look at some of the challenges facing gene therapy today.
To start off, the main problem in gene replacement therapy is that the human body really hates taking in undigested DNA (if anyone wants to read about extracellular DNA in the body, here’s a good paper: The Origin and Properties of Extracellular DNA: From PAMP to DAMP). So, automatically, the idea of oral gene therapy pills is difficult to implement, since stomach acid and digestive enzymes would rapidly degrade incoming DNA into individual base pairs or base pair components. At the same time, injecting DNA directly into the blood stream or local tissue would meet a different problem; the immune system, which would rapidly detect the foreign DNA and then degrade it into all of its components (click here to see how the innate immune system detects DNA). Therefore, this leaves scientists and physicians with a problem, how can we create a therapy that can sneak past the immune system to deliver our uncompromised DNA to our target tissue?
Infective Solutions: The Virus
First thought: viruses, a common solution to a scientist’s dilemma. By the 1970s, it was already known that viruses are natural agents at injecting their genetic information into host cells for viral reproduction purposes. The goal then became to find a good viral candidate that could somehow be used as a gene delivery system to the human body. To be considered a good candidate, the virus must meet several criteria: it must be able to deliver the genetic information efficiently, it must be minimally immunogenic, it must be non-replicative, and it should have a low risk of insertion into the host genome (this article is a good summary of viral vectors for gene therapy).
The process to meet these goals requires the original viral DNA to be removed, and only the viral capsid to be used. Without going into a lot of details, since the virus has been rendered non-replicative, the production of the viruses would have to be split into multiple parts; this means that the DNA plasmid of our target gene would be given alongside our packaged plasmid expressing the viral capsid protein using a co-transfection protocol (triple transfection is the newer technique and has much better yield than double transfection). However, the question remains, just what kind of virus should be used? After all, there are so many types of viruses to choose from! For now, we will focus on 3 types of viruses that are of interest: retroviruses, adenoviruses, and adeno-associated viruses.
As medical students, we have all heard of retroviruses, with HIV being the most widely known member of the retroviral family. For gene therapy purposes, lentivirus, a subtype of retrovirus, is used instead of a standard retrovirus, since a lentivirus can infect non-dividing cells. The good and bad thing about a lentivirus is that it will insert its own genome into the host genome-this is great because once the insertion occurs, the cell will have the DNA forever (no backsies)-but on the negative side, inserting DNA into random places within the human body will disrupt normal gene function and could lead to cancer. This was unfortunately shown when children given an experimental gene therapy to cure X-linked SCID-XI syndrome developed leukemia due to random insertion inducing mutagenesis (they were cured of their SCID-XI syndrome though, so mission success with unfortunate side-effect?).
Our second virus of interest is the adenovirus, a virus responsible for many infections in the respiratory tract, among others. This early candidate for gene therapy however, had the tendency to spread throughout the body and become immunogenic. This was shown sadly in 1999, when Jesse Gelsinger died due to an immune reaction to adenovirus in a trial to cure ornithine transcaramylase, a metabolic disease that affects ammonia elimination,.
C) Adeno-associated Virus (AAV)
Finally, we arrive at our last viral candidate, the adeno-associated virus or AAV. The name is pretty straight forward, AAV depends on adenoviruses to reproduce (it’s like a virus of a virus). In fact, when AAV was first visualized under electron microscopy, scientists weren’t quite sure what it was due to its small physical size. Since AAVs are naturally non-replicative, they are minimally immunogenic, and there are no diseases that are known to be caused by AAV. The major limit of AAV usage however is their small size, since only about 2.5kb worth of DNA can be delivered with one virus, which significant limits the amount of treatable diseases with this strategy, since human genes are quite large in base pairs. However, since safety is the major concern of viral gene delivery, much of the focus on gene replacement therapy has focused on developing good recombinant AAV (rAAV) platforms because of their high safety features.
To conclude, I hope this post provided you a brief understanding of some of the current challenges in delivering gene replacement therapy in medicine and next time, our topic will be on the different types of rAAV vectors (delivery gene mechanisms), and how the choice of this rAAV subtype (there are a lot) is important for therapy development.
Author: Danning Li
Danning Li completed his BSc. majoring in Physiology at McGill University. Afterwards, he worked for two years on developing a gene replacement therapy for Canavan Disease, a rare inherited leukodystrophy, at the Horae Gene Therapy Center at the University of Massachusetts Medical School. Now a medical student at Schulich, he wants to bring attention to the interesting genetic therapies that will become available in the not so distant future.