Gene Therapy, Not Just for Superheroes?

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It is interesting writing a blog… People give you the best advice! “Can you make it more relatable?” “Why not write it like Deadpool’s inner monologue!”.  Wait, what? How does that make sense? How can I rationalize that into some kind of story? Well the topic of the blog is supposed to be gene therapy. Ok. This makes sense. Some of our favorite super heroes have undergone some genetic alteration, either intentionally, like Captain America or Deadpool, or unintentionally, such as Hulk (with a mutation created by a combination of events including gamma irradiation) or Spiderman (where the genetic changes are introduced via a vector … the radioactive spider, which itself had been altered by a process that is less clear).  Do these characters and their stories have any bearing on reality and what is the future of gene therapy? It is always fun to gaze into the crystal ball and make some predictions. Perhaps some perspective on the past and the progress that has already been made can help us in that quest.


The biological revolution began with Pasteur’s germ theory in the 1860s, step forward almost a century to elucidation of the DNA structure and its confirmation as the genetic material. What can we expect a hundred years from this seminal discovery, another 30 years from now or what we might consider the near future? Well the progress has been amazing. The molecular biology revolution and the sequencing of the human genome around the turn of the millennia ushered in a new age that enabled a genetic understanding of human disease. This is the key that facilitates gene therapy or to put it another way gene therapy fulfills and delivers on the promise that the sequence of the human genome started.


There are successful gene therapies already approved and in use. The approved therapies can be divided into two categories in vivo (where the genetic changes are created inside the body) and ex vivo (outside the body). There have already been four FDA approvals for the ex vivo CAR T-cell cancer therapies. However, here we will focus on gene function replacement therapies of which two have been approved by the FDA, Luxturna* and Zolgensma*. These important life-changing medicines and their FDA approvals pave the way for future gene therapies. Cynically these two approvals may be considered “low hanging fruit”. Zolgensma, for the treatment of spinal muscular atrophy for patients under two years of age has a high reward to risk as the disease is fatal and as infants are treated smaller doses are required. Luxturna for the treatment of retinal dystrophy is injected directly into the eye. This lowers risk as the eye is relatively small, requiring low doses and moreover the eye is sequestered from the immune system and is considered immune privileged. So, going back to the crystal ball how are gene therapies going to tackle more widespread applications, or give us super powers? Well there are multiple challenges, the two approved therapies are for monogenic diseases (controlled by a single gene), but the majority of human diseases are polygenic (controlled by multiple genes). Supplying multiple genes adds a challenge as the gene therapy vector of choice (that is used for the approved drugs Zolgensma and Luxturna), adeno-associated virus (AAV) can only accommodate relatively small genetic payloads, 4700 bases, probably not enough to encode for more than one gene. Maybe dosing of several AAV therapies concurrently could treat polygenic disease. But, the challenge is AAV can still invoke an unwanted immune response, and this can only currently be controlled by limiting the total viral dose. The immune response is exacerbated because currently the production of AAV results in a large proportion of viruses, as much as 95%, that do not contain the DNA payload. These so called “empty” vectors can still cause an immune response: But, as they have no payload, they do not have the desired therapeutic effect. Reducing the proportion of empty vectors becomes a key step in the production of effective therapies. We can predict in the future that the biology around vector production will become better understood and through virus and host cell optimization a much higher proportion of “full” vectors will be produced. In the meantime, the challenge is to lower the proportion of empty vectors in the final product.  This needs to happen during the manufacturing process and this is no mean feat. The empty and full viruses appear very similar. A slight difference in density can be exploited to perform separation by ultracentrifugation, but this is not considered a scalable technique and it is difficult to imagine purifying the amounts of virus required for a mainstream application this way. Alternatively, the presence of DNA inside the vector results in a small change of charge that can be exploited via chromatography. This though is apparently easier said than done. My team, the real super heroes, (I kind of hope they are not reading this) have been working on this for the last two years and have come up with some novel solutions using our Mustang® Q membrane chromatography that work for a range of AAV serotypes. We see in the literature that gradient elutions are often employed for this separation, but often the empty and the full capsids are not completely resolved.  It is then very difficult to decide where to cut the peaks, choosing what material to keep and what to discard.  We have had success employing small conductivity steps, of around 1 mS/cm. This elution strategy results in a series of discrete and reproducible elution peaks that can be more readily assessed for enrichment of full capsids.


Adding to the challenge of full capsid enrichment is the ability of AAV to package non-therapeutic DNA, this could also pose a threat to patient safety and will likely have to be addressed before AAV can breakthrough to mainstream applications. We can predict that through modification of the capsid structure, the DNA to be packaged and perhaps even the host cell line that packaging of the correct DNA will be more efficient. Or perhaps, by breaking the virus manufacturing system the cell has into its critical constitutive components it will be possible to manufacture virus in the absence of non-therapeutic DNA.


With these improvements we can also see the rise of gene editing and epigenetic control, modifying the expression of genes through their promoter and enhancer elements through either DNA modification or adjustment to histone acetylation and methylation that change the DNA structure and subsequent gene expression. When we realize these capabilities Homo sapiens will have control over their genome making disease and even aging history. So, no super powers just yet, infact it is difficult to imagine going much beyond where human evolution has taken us already. But still there are, and have been, some pretty interesting humans already. Shaquille O’ Neal vs. Stephen Hawking? Imagine being able to take the best genetic elements of those super men.  I wonder at what point the focus of the scientific dialogue will shift, and to think of Jurassic Park… “whether or not they could, they didn't stop to think if they should”.


To learn more about how to approach the separation of empty and full AAV capsids you can listen to a podcast I participated in which you can find here.

Learn more about Zolgensma.


* Deadpool, Captain America, The Hulk and Spiderman are trademarks of Marvel Characters Inc. Luxturna is a trademark of Spark Therapeutics Inc. Zolgensma is a trademark of Novartis AG. Jurassic Park is a trademark of Universal City Studios LLC.

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Mark Schofield, Senior Manager, Research and Development

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