Gene Therapy in Action: Adeno-Associated Viral Vectors
Adeno-associated viral vectors, or AAVs, are the tiny shells of viruses. And today they are the most common vessels for delivering gene-based therapies. In this episode, we’ll launch into the past, present, and future of AAVs.
Imagine a rocket ship blasting off from Earth with cargo bound for a distant space station, and you have a pretty good idea what AAVs are all about. But instead of ferrying hardware and supplies, AAVs carry genes.
It’s an achievement nearly six decades in the making. That might seem like a long time to tinker with something smaller than the tiniest single-celled organism. But just like building a rocket ship destined for the deep reaches of space, the development of AAV vectors required patience, persistence, and a few leaps of faith.
In the era before DNA sequencing and gene cloning, scientists in the 1960s realized that AAVs could be a window into understanding genetic variations in viruses – and eventually other organisms, too.
The fact that AAVs were immunologically distinct from other viruses made them curious things.
So in the 1970s, AAV research took off in three directions. One determined that the simple AAV DNA could be rewritten and edited in a lab. The second found that although these small viruses can infect humans, they don’t replicate without a “helper virus” (such as adenovirus). In the absence of another virus, they remain latent, and appear to be of little threat to human health. The third investigated whether AAVs could become vectors for transferring genes from one organism to another.
So now we had proof that adeno-associated viruses could be artificially produced, that they could be hollowed out and filled with other genetic material, and that they could potentially be a vector for delivering genes without harming their new host.
By the 1980s, we had the capability to build lots of viral rocket ships and fill them with genetic cargo, we just needed a destination to send them. Enter the burgeoning field of gene therapy, with its focus on developing treatments for genetic diseases like cystic fibrosis, hemophilia B, Parkinson’s, and more.
Research has continued and today, adeno-associated viral vectors are a mainstay of gene therapy development. While progress may be slow, gene therapy is a science that is aiming for the stars. And with AAV vectors, they are now within our reach.
DDx SEASON 4, EPISODE 4
Gene Therapy in Action: Adeno-Associated Viral Vectors
RAJ: This season of DDx is brought to you by Novartis Gene Therapies.
KIM: Great things come in small packages.
Adeno-associated viral vectors—or AAVs—are the tiny shells of viruses.1 And today they are the most common vessels for delivering gene-based therapies.2
Imagine a rocket ship blasting off from Earth with precious cargo bound for a distant space station, and you have a pretty good idea what AAVs are all about. Only instead of ferrying hardware and supplies for exploring outer space, AAVs carry genes.
For patients with inherited blindness, AAVs can deliver the genetic material they need to see.3
It’s an achievement nearly 6 decades in the making.4
That might seem like a long time to tinker with something smaller than the tiniest single-celled organism.5
But just like building a rocket ship destined for the deep reaches of space, the development of AAV vectors required patience, persistence, and a few leaps of faith.
RAJ: This is DDx, a podcast from Figure 1 about how doctors think.
I’m Dr. Raj Bhardwaj.
This season I’m joined by co-host Kim Handysides as we take a deep dive into gene therapy.
Today we’re talking about an essential delivery mechanism for gene therapy treatments: Adeno-associated viral vectors, or AAVs.
We’ll begin by giving a short history of AAVs and delve into how they work.
KIM: Much like the space program, AAVs trace their origins to early research in the 1960s.
In this case, it all started in 1965, in a laboratory at the University of Pittsburgh. Microbiologist Robert Atchison was peering through his microscope at cell cultures collected from a rhesus monkey.4,6
He was interested in a class of viruses known as adenoviruses.7 But Atchison noticed something else: wherever he found adenoviruses, he also observed smaller, virus-like particles clustered around them.4,7
He began to study these particles, and found that they behaved differently from a normal virus. For one thing, they didn’t seem to replicate very well in human cells or trigger an immune response—they did not cause any illness, unlike the adenoviruses they hung around with.4
Atchison decided to call these particles “adeno-associated viruses,” or AAVs for short.8 And initially, it wasn’t believed that they would prove to be very interesting to science.4
Around the same time, another microbiologist, Wallace Rowe at the National Institutes of Health in Maryland, noticed these AAVs, too.4,8
These AAVs were actually their own class of virus—which we now call “dependoviruses”, because they depend on other viruses to reproduce.4
In the late 1960s, it was thought that these AAVs could be genetic variants of their larger viral cousins – and that’s when they became interesting.4
KIM: In the era before DNA sequencing and gene cloning, scientists in the 1960s realized that AAVs could be a window into understanding genetic variations in viruses—and eventually other organisms, too.4
The fact that AAVs were immunologically distinct from other viruses made them curious things.8
So in the 1970s, AAV research took off in three directions.4
First, molecular biologists led by the British virologist Lionel Crawford investigated the structure of AAV DNA. Crawford reckoned that the simple AAV DNA could be re-written and edited in a lab.4
Second, infectious disease experts at the NIH studied AAVs and found that although these small viruses can infect humans, they don’t replicate without a “helper virus” (such as adenovirus). In the absence of another virus, they remain latent, and appear to be of little threat to human health.4,8,9
And third, geneticists, including Kenneth Berns, at the University of Florida began to investigate whether AAVs could become vectors for transferring genes from one organism to another,4,10,11 like an empty rocket ship that can be loaded with different cargo. Except in the case of AAVs, the cargo would have different genes, to be transported into a living cell.
This all culminated in 1978, when Jude Samulski, then a graduate student at the University of Florida, generated the first cloned AAV.7,11,12
He successfully transferred it to a cell of the E. coli bacterium, where it produced 50 new colonies of AAVs.8
So now we had proof that adeno-associated viruses could be artificially produced, that they could be hollowed out and filled with other genetic material, and that they could potentially be a vector for delivering genes without harming their new host.8,12
After that, AAV research was almost ready for launch.
KIM: Scientists identified three main serotypes in AAVs and sequenced their DNA.8
By the mid-1980s, geneticists had sequenced the second serotype (called AAV2) DNA and were confident that these amazing little particles could be vectors to express genes in a new host organism.4
In other words, we had the capability to build lots of viral rocket ships and fill them with genetic cargo. Now we needed a destination to send them.
Enter the burgeoning field of gene therapy, with its focus on developing treatments for genetic diseases like cystic fibrosis, hemophilia B, Parkinson’s, and more.8
By the 1990s, Jude Samulski had moved to a lab at the University of North Carolina, where he began testing AAVs for their compatibility with human cells. A major breakthrough came when he successfully deployed an AAV vector to bond with a human cell from a lab culture.8
And he further observed that these AAV vectors could be counted on to ensure prolonged gene expression in a therapy—confirming that they can stick around longer in our cells.1 It’s not enough for the rocket ship to reach the space station—we need it to stick around long enough for the cargo to be fully delivered.13,14
Samulski, and one of his graduate students Xiao Xiao, then discovered that AAVs could be made to target specific cell types in the body—they could be customized for efficiency.15
So by the mid-1990s, we now knew how to design and produce AAV vectors16—these genetic rocket ships. They knew how to fill them with cargo, launch them on their journey, navigate their progress, and dock them with a host cell to deliver that cargo of genetic material. Adeno-associated virus vectors were finally ready to move from test tubes in labs, to clinical trials in humans.16
Samulski, working with Dr. Paola Leone at the UNCE Gene Therapy Center,17,18 decided to target a rare genetic condition known as Canavan disease. Canavan disease is a rare form of leukodystrophy with no known treatment. It’s caused by a variation to the aspartocylase (ASPA) gene, which is responsible for producing myelin—the protective fatty tissue around cells in the human central nervous system.8,19
Without this myelination, patients with Canavan disease suffer from degenerative damage to their nerve cells, which in serious cases causes abnormal muscle and brain development, paralysis, seizures—and is often fatal.20
In 2001, the National Institutes of Health (NIH) Recombinant DNA Advisory Committee approved the clinical protocol,18 where Samulski administered cloned ASPA genes via AAV2 vectors into the nerve cells of 10 Canavan patients, via neurosurgery.8,17
His team found that the production of myelin increased dramatically. There was a remarkable slowing of brain tissue atrophy, and participants reported a decline in the frequency of their seizures. Best of all, there were no adverse effects related to the vector.8,17
Seven of the 10 patients had no immunological response at all, and the others were minor.17 This was the first concrete evidence that AAV vectors could deliver gene therapy treatments to human patients, overcoming a disease that was otherwise impossible to solve.
KIM: Today, adeno-associated viral vectors are a mainstay of gene therapy development. They have several important characteristics:
First, they are relatively easy to produce—scientists can now engineer millions at a time.6
Second, they are highly malleable—we can design them to match all different sorts of cells.6
Third, they are non-integrating—the genetic payload inside an AAV vector is unlikely to integrate into the host cell’s DNA. For some diseases, this can mean that genes transferred via AAV vectors have the potential to produce long-term results.9,21-23
But AAVs are not perfect.24 For one thing, they’re tiny. Their cargo capacity is too small to carry larger genes.9
And yes, scientific progress is necessarily slow. For all the amazing potential benefits of AAV vectors to deliver treatments for genetic diseases, science requires a painstaking process of trial after trial to get to the point where a treatment is ready for FDA approval.25
Nevertheless, the rocket ship has blasted off, making course for new worlds. Gene therapy is a science that is aiming for the stars. And with AAV vectors, they are now within our reach.
RAJ: Special thanks to Dr. Jerry Mendell for sharing his expertise in the research of this episode.
This is DDx, a podcast by Figure 1.
Figure 1 is an app that lets doctors share clinical images and knowledge about difficult to diagnose cases.
I’m Dr. Raj Bhardwaj, co-host and story editor of DDx.
You can follow me on Twitter at Raj BhardwajMD.
Head over to figure one dot com slash ddx, where you can find full show notes, photos and speaker bios.
This episode was brought to you by Novartis Gene Therapies.
Thanks for listening.
- Samulski RJ, Muzyczka N. AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annu Rev Virol. 2014;1(1):427-451.
- Hysolli E. Gene Therapy: The Age of AAV. Wyss Institute. Published May 23, 2017. Accessed October 29, 2021. https://wyss.harvard.edu/news/gene-therapy-the-age-of-aav/
- Mendell JR, Al-Zaidy SA, Rodino-Klapac LR, et al. Current clinical applications of in vivo gene therapy with AAVs. Mol Ther. 2021;29(2):464-488.
- Carter BJ. Adeno-associated virus and the development of adeno-associated virus vectors: a historical perspective. Mol Ther. 2004;10(6):981-989.
- Vidyasagar A. Live Science. Published January 5, 2016. Accessed November 16, 2021. https://www.livescience.com/53272-what-is-a-virus.html
- Masset C. Wide Angle: Byrne makes gene therapy click. Pitt Med. Published Winter 2018. Accessed November 1, 2021. https://www.pittmed.health.pitt.edu/story/wide-angle
- Smith RH. Adeno-associated virus integration: virus versus vector. Gene Ther. 2008;15(11):817-822.
- Hastie E, Samulski RJ. Adeno-associated virus at 50: a golden anniversary of discovery, research, and gene therapy success–a personal perspective. Hum Gene Ther. 2015;26(5):257-265.
- Gonçalves MA. Adeno-associated virus: from defective virus to effective vector. Virol J. 2005;2:43.
- Berns KI. My life with adeno-associated virus: a long time spent studying a short genome. DNA Cell Biol. 2013;32(7):342-347.
- Flotte TR. Birth of a new therapeutic platform: 47 years of adeno-associated virus biology from virus discovery to licensed gene therapy. Mol Ther. 2013;21(11):1976-1981.
- Davies K. AAV Jude: An Interview with R. Jude Samulski. Hum Gene Ther. 2021;32(1-2):4-9.
- Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J Virol. 1996;70(11):8098-8108.
- Carroll J. Gene therapy: science in slow motion. Biotechnol Healthc. 2007;4(1):55-60.
- Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol. 1998;72(3):2224-2232.
- Dunbar CE, High KA, Joung JK, Kohn DB, Ozawa K, Sadelain M. Gene therapy comes of age. Science. 2018;359(6372):eaan4672.
- McPhee SW, Janson CG, Li C, et al. Immune responses to AAV in a phase I study for Canavan disease. J Gene Med. 2006;8(5):577-588
- Janson C, McPhee S, Bilaniuk L, et al. Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain. Hum Gene Ther. 2002;13(11):1391-1412.
- Canavan Disease. National Organization for Rare Disorders. Accessed November 16, 2021. https://rarediseases.org/rare-diseases/canavan-disease/
- About Canavan Disease. Canavan Foundation. Accessed November 2, 2021. https://www.canavanfoundation.org/about_canavan_disease
- Naso MF, Tomkowicz B, Perry WL 3rd, Strohl WR. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs. 2017;31(4):317-334.
- Vectors 101. American Society of Gene + Cell Therapy. Accessed November 3, 2021. https://patienteducation.asgct.org/gene-therapy-101/vectors-101
- Manufacturing of Gene Therapies: Ensuring Product Safety and Quality. FDA. Updated September 15, 2020. Accessed November 16, 2021. https://www.fda.gov/media/81682/download
- Qu Y, Liu Y, Noor AF, et al. Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases. Neural Regen Res. 2019;14(6):931-938.
- Clinical Trials Process. American Society of Gene + Cell Therapy. Accessed November 3, 2021. https://patienteducation.asgct.org/gene-therapy-101/clinical-trials-process