Natural Discovery of AAV Serotypes, Directed Evolution of AAV Serotypes, and their Importance to Gene Therapy

As we reach the end of the first month back from break, I think it’s a nice time to continue on our journey into the world of gene therapy. My last blog entry focused on why the adeno-associated virus (AAV) capsid is such an important part of gene therapy, and this time, we’ll focus on how different types of AAV capsid affect therapy efficacy and what researchers are doing to find new types of AAV capsids.

AAV was first discovered in the 1960s by groups of scientists when they found small particles riding along with adenovirus cultures. In 1982, the first AAV serotype, AAV2, was cloned. (For a more in-depth review of AAV history, click here: Birth of a New Therapeutic Platform: 47 Years of Adeno-associated Virus Biology From Virus Discovery to Licensed Gene Therapy). However, it was soon discovered that AAV2 displays natural tropism towards certain tissues. What this means is that once injected into the blood stream, AAV2 automatically moves toward specific areas of the body; in this case, the CNS and kidneys. (For more information on serotype tissue specificity, click here: Adeno-associated Virus (AAV) Guide). This specificity would prove to be a double-edged sword. On the one hand, tissue specificity reduces risks of systemically expressing genes and reduces immunogenicity; on the other hand, this means that unless new capsid types are found to target an organ system, diseases of that system would be impenetrable to gene therapy.

Fortunately, soon after the isolation of AAV2, several other natural AAV serotypes were discovered (such as the, ‘Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy’). These new serotypes were found to have drastically different tropisms from AAV2, which opened up new doors towards creating novel therapies that were more specific, and less immunogenic. However, as hard-working scientists go, you knew that they weren’t just going to let a couple new dozen natural serotypes be discovered. Researchers began to tinker with mixing capsids together to generate new hybrid AAVs, in the hopes of creating new capsids that combined properties that would make them even more efficient. Soon, hybrid capsids like AAV2/5 were created, combining their organ specificities and AAV2’s already well-established production efficiency.

The idea of discovering new AAVs in nature and combining them with other natural AAVs to create hybrids sounds all fun and exciting, but we must take a moment to think. What if we can create an environment that drives AAV evolution towards specific tropisms and/or improved production efficiency? What if we force AAV to adapt to certain set conditions in the lab and create new AAV serotypes that are better than those that evolved in nature? Within the past two decades, the idea of using directed evolution to create new serotypes of AAV has exploded in the field of gene therapy, and multiple methods have been described in literature to achieve this goal (here is a rather well known one: Directed evolution of adeno-associated virus yields enhanced gene delivery vectors). The basic idea of directed evolution is simple: subject AAV to a hostile environment, collect the survivors, let them grow, and repeat until the hardiest one is left. Thus, it became possible to not just find AAV in nature and hybridize them, but it became possible to speed up nature to make our own AAV serotypes.

In summary, with the discovery of new natural AAV serotypes, the hybridisation of these natural serotypes, and by incorporating the directed evolution of AAV; we have created an entire host of AAV agents that are specific to organ systems (and sometimes even to specific cells!). At the same time, it also became possible to develop new techniques that made AAV production costs much cheaper as multiple new serotypes grew much faster in culture. Next time, we will take a look at the beginning of the era of human gene therapy trials.

 

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.

 

Photo Credits: Mehmet Pinari, Creative Commons

Viral Vectors as Containers and Their Importance to Gene Therapy

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.

A) Retrovirus

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?).

B) Adenovirus

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.

Photo Credits: Creative Commons, Capsid

What is Gene Therapy?

By: Danning Li

On December 19th, 2017, Leber’s congenital amaurosis, an inherited disease that causes severe vision loss and blindness in children met its match in the form of voretigene neparvovec (AAV2-hRPE65v2, or Luxturna for ease of pronunciation), the first gene replacement therapy approved by the FDA for sale in the United States. The previous sentence might contain some words that are not part of the current medical curriculum, but they will become an increasingly important part of our future practice as technology develops. Already, the rate of approval for these novel therapies is ramping up. Last year, the FDA approved three new genetic therapies for public sale (for those interested, the FDA news releases are here in chronological order Kymriah, Yescarta, and finally Luxturna). So, for today’s blog entry, with the possible dawn of the genetic therapy age upon us, let us focus on just what a “gene replacement therapy” is, and why it is such a big deal.

Gene Replacement Therapy 

Gene replacement therapy, as its component words suggest, is a treatment that aims to provide a replacement copy of a gene to a patient’s body. It doesn’t matter if the patient doesn’t have this gene, protein or enzyme, or even if the patient has a mutant, non-functional or less-functional version of the enzyme; gene therapy seeks to rectify this issue. The goal of this therapy is for the replacement artificial gene copy to produce a functional enzyme or enzyme subunit that the patient’s own body needs, and can use effectively.

Now, some might be wondering why we don’t just supply the missing enzyme instead, after all, Enzyme Replacement Therapies (ERTs) are well-established treatments for several diseases already. However, the answer to this question is simple: economics. Remember how enzymes are constantly made and broken down in the body naturally? Well, this applies to inherited diseases too since the patient would break down the injected replacement enzymes overtime, and the effects would wear off. Since the replaced enzymes are made in a lab and have to be reinjected into the patient regularly, we would have to constantly produce the purified enzymes and the patient would be on the hook for the rest of their life. This might not sound like such a bad problem, a lot of medications are like this already, right? Unfortunately, it turns out treating someone for decades with regular ERTs cost around USD $9-10 million or more. Insurance companies and OHIP won’t be very happy if a lot of patients started lining up for therapies with these kind of price tags attached.

Looking Towards the Future

Naturally, when faced with such a tantalizingly difficult problem, scientists and physicians thought up a ridiculously simple solution in the 1960s. If making these enzymes outside of the body and then injecting it into the patient is too costly, why don’t we just give the patient’s body the genetic information it needs to make the missing enzymes inherantly? After all, the most efficient enzyme production facility is a living, breathing body that converts regular nutrients into precious enzymes. Even better, if we were lucky enough, and the inserted DNA managed to stick around inside the body somehow, we would have just cured the disease completely, improved the patient’s quality of life, and opened up a completely new world in science. Thus, the idea for gene replacement therapy was born, and brilliant minds around the world would spend the next half a century trying to make this ridiculously simple idea, a reality.

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.

Photo Credits: Creative Commons, Gene Therapy Infographic