From the Cultural Revolution to the Gene Therapy Revolution

An Interview with Guangping Gao, PhD, a Pioneer of Viral Vector Gene Therapy for Rare Genetic Diseases

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Guangping Gao, PhD
Professor and Director, Horae Gene Therapy Center, University of Massachusetts Medical School

Guangping Gao, PhD, is professor and director of the Horae Gene Therapy Center at the University of Massachusetts Medical School in Worcester, MA. Over the course of three decades, Gao has made profound contributions in the area of adeno-associated virus research, initially working with James M. Wilson, MD, PhD, director of the gene therapy program at the University of Pennsylvania. Gao has received multiple honors in recognition of his service, expertise, and dedication. For example, he was named president (2019–2020) of the American Society of Gene and Cell Therapy.

Gao has published 250+ research papers, six book chapters, and four edited books, and has fulfilled editorial responsibilities for several gene therapy and virology journals, including the Human Gene Therapy, a journal that Gao currently serves as deputy editor-in-chief. Gao recently spoke to Kevin Davies, PhD, executive editor of Human Gene Therapy, about his remarkable life journey and hopes for the future of gene therapy. (The interview originally appeared in Human Gene Therapy, Vol. 31, Nos. 3 and 4, published by Mary Ann Liebert. Kevin Davies, PhD, executive editor of Human Gene Therapy, conducted the interview.)

We will get to your preeminent research and leadership in the gene therapy field, but let’s start at the beginning.

Gao: I grew up in China during the Cultural Revolution. Around 1975, I was compelled to leave my studies and go to the countryside to receive additional “education” from farmers and peasants. My dream about new medicine really starts there. I interacted with farm laborers on a daily basis, and I saw many of them suffer from various diseases and painful conditions.

I was trying my best to use acupuncture and traditional medicine to help them, but I wished I could have some “magic medicine” to make a more substantial impact, particularly for the elderly and people with cancer.

In 1978, I was one of the first generation of students to enter college after the Cultural Revolution. I was admitted to a medical university in Chengdu, Sichuan. I worked on drug development and medicinal chemistry. In 1988, I graduated from the university and got an opportunity to come to the United States, sponsored by the World Health Organization (WHO). I was looking for opportunities to develop the next generation of medicines that I had dreamed about back on the collective farm.

I started my PhD at Miami Children’s Hospital and Florida International University with my mentor, Reuben Matalon, a pediatrician and medical geneticist. He was a prominent researcher on rare diseases such as Tay-Sachs, Hurler, and Gaucher. His major contribution as a geneticist was the discovery of the biochemical defect in an inherited leukodystrophy called Canavan disease.

I remember it well—I published that paper in the early days of Nature Genetics!

Gao: Yes, thank you! I joined his lab in 1989. My assignment was to isolate the genes and the mutations responsible for Canavan disease. Working with my lab mentor, Rajinder Kaul, I discovered the gene and mutations for Canavan disease and published my thesis work in Nature Genetics in 1993.1

After that, I asked myself, what’s my next step? Because we saw many Canavan patients at these centers, we knew exactly what was going wrong with those kids. We had to figure out a way to fix it. In 1993, I decided to look for the next generation of medicine, specifically at the opportunities in gene therapy for genetic disorders. Finally, Jim Wilson accepted me as a postdoctoral fellow at the University of Pennsylvania’s Institute for Human Gene Therapy.

The first task Jim gave me was to create new generations of adenovirus. At that time, adenovirus vector was much hyped because it has a high transduction efficiency. Because we knew adaptive immunity/immunotoxicity is a major issue for adenovirus, we decided to cripple the virus further to make it more replication defective. This might prolong transduction efficiency and stability in tissues.

I spent about two years there, first making a cell line to complement the crippled virus. Then we used that cell line to create the further-crippled virus. (You need to transcomplement its growth with E1 and E4.) They called this third-generation virus at the time. We demonstrated that, yes, virus can reduce liver toxicity in mice and immunotoxicity and prolong expression substantially.

When I published that work in 1996,2 I said to Jim, “I’d like to move on and start my career in industry because I have two kids to raise.” I was 38 at the time. He said, “No! Why leave? I’m going to give you a job.” He told me they were trying to apply the next-generation adenovirus vector for clinical trials. There was a lab called the Human Applications Lab, a GMP facility at Pennsylvania Hospital where scientists were trying to grow the virus for multiple clinical trials, but they could not grow it well.

My career in gene therapy started from there. I spent about two years making the virus work. In the first two weeks, I was able to generate high quantities of virus. Jim was in his office, talking to a reporter from the Philadelphia Inquirer. I told Jim, “I got the virus, and they are 1013 or 1014.” Jim said to the reporter, “Now we can even swim in this gene therapy vector!”

By that time, we were doing several clinical trials in cystic fibrosis, ornithine transcarbamylase (OTC), mesothelioma, and others. By early 1998, we wanted to look for new viruses, the next generation of gene delivery vehicles. I started working with AAV prototypes such as AAV-2, AAV-1, and AAV-5. Those were the first serotypes to attract a lot of interest and development.

Who first identified AAV? Was it discovered serendipitously?

Gao: Yes, it was discovered in 1965 from some adenovirus preps. They called it adeno-associated virus (AAV) because when they purified the adenovirus and looked at it under a microscope, it was a very small virus in the company of the much larger adenovirus.3
I think Arun Srivastava and others sequenced AAV. Nick Muzyczka, Jude Samulski, Barrie Carter, and others started vectorizing—demonstrating you can create a vector in transduced cells very easily. Many groups then demonstrated that AAV can transduce animals in vivo. The difference is that adenovirus only sustains for a maximum of two to four weeks. But AAV—at that time, primarily AAV-2—can sustain for hundreds of days.

My first task with Jim was to figure out how to produce a scalable manufacturing process. I started making cell lines, creating adeno-AAV hybrids. I published a paper in 1998.4 We converted a transfection-infection system into a total infection system that generates tons of AAV. Working with my colleague Guang Qu, we developed a column purification system using heparin-binding columns in early 2000.

Then on September 17, 1999, this tragic event with adenovirus OTC gene therapy happened, and we lost 19-year-old Jesse Gelsinger. For the entire field, it was a drop from a peak to a deep valley. We experienced 10 years of dark ages for gene therapy.
I continued my AAV work. We started the first AAV-2 limb-girdle dystrophy clinical trial with Jerry Mendell (Nationwide Children’s Hospital, Columbus, OH) and colleagues at Penn such as Hansel Stedman and Lee Sweeney. We started the trial using the vector produced with my manufacturing methods under GMP conditions.

After the Gelsinger tragedy, was there added urgency and commitment to establish AAV as an alternative vector?

Gao: Absolutely. We started working with adenovirus, based on the discovery by Yiping Yang (formerly at Duke, now at Ohio State). He discovered immunotoxicity of adenovirus. My job was to reduce that adaptive immunity to adenovirus. But we overlooked this innate immunity, this cytokine storm, which killed Gelsinger.

I had initially started with AAV-2, but we did not really think about AAV-1 and AAV-5, or about discovering new AAVs, until Gelsinger. Then we realized, when you compare the two vectors, adeno is much more efficient. But for immunotoxicity, AAV is much, much better than adeno. Jim and I thought, if we can find a virus as efficient as adeno but without immunotoxicity, that should be the future of gene therapy. Gelsinger was an additional driving force for me to discover new AAVs.

I started work in 2001, and soon we discovered a library of new AAVs in nonhuman primates. We published our first paper in 2002.5 That paper became the hottest paper in the field and gave us new hope to work on the next generation of gene therapy vectors.

How did that discovery come about?

Gao: Back in the winter of 2001, after we found some virus sequences, I presented the PCR data to Jim Wilson at a lab meeting. I could tell his mind was spinning:“Is this real or not?” After the meeting, he said, “Guangping, I think you stepped on a goldmine.”

I started with nonhuman primates. We found that we can detect AAV in any animal. You never run into anyone with absolutely no AAV. It is in any tissue. In any PCR reaction, I always found multiple AAVs. That tells you how diverse [it is], how rapidly AAV is evolving. Then we published our second paper about nonhuman primate viruses, demonstrating AAV evolution.6

At what point did you expand or focus the search for new AAVs in humans?

Gao: You can find AAV everywhere. You can find a different AAV in the same samples. That’s why AAV is amazing to me! As the initial discovery was based on nonhuman primates, I asked Jim in late 2002, “Should we move into human tissues?” He agreed. We discovered AAV-9, which is the first “super virus” for gene therapy from humans, in January 2003.7 Our objective was to develop AAV to be as potent, as efficient, as adenovirus for transduction. But we wanted them to have much less immunogenicity. I think we accomplished that (Figure 1).8

Figure 1. Timeline of major events in adeno-associated virus discovery, characterization, and clinical deployment. Adapted from Wang et al.7

We did not go through the traditional viral isolate characterization. We focused on PCR amplification of the capsid because we realized biology is only determined by the capsid. We didn’t need anything else. We designed PCR primers in the conserved region and amplified through hypervariable regions, generating a new virus capsid with new biology.

When did you move to the University of Massachusetts?

Gao: I moved in 2008. At the time, under the Life Sciences Initiative, then-governor Deval Patrick gave $1 billion to promote biomedicine in the state. Our dean, Terry Flotte, and the chancellor, Michael Collins, wanted to take the momentum to set up three centers in gene therapy, stem cells, and RNA interference. They recruited me from Penn to UMass to set up the gene therapy center.

I continued my AAV discovery, and collaborating with Terry and others—including researchers at the New Iberia (Louisiana) Research Center, a non-human primate facility—we were able to get some primate tissues and start to look for AAV from chimpanzees. We discovered hundreds of AAVs similar to AAV-1, AAV-6, AAV-4, AAV-3, AAV-5, and even AAV-9, which I discovered from humans. I did not know other primates also have AAV-9.

How would you describe the repertoire of AAV vectors? To what degree can researchers adapt these vectors?

Gao: We have now isolated new AAVs from 850 human surgical tissue samples. And we have about 1100 new AAVs. We found large amounts of AAV-2, AAV-3, and AAV-8 in human tissues. My AAV-8 was initially isolated from monkey lymph nodes, but now we see it everywhere in humans. If you talk about the natural reservoir of AAV, I think there is still a lot there.

Of course, now the field has moved to new directions. In addition to a natural reservoir, scientists have started doing directed evolution, rational design, and machine learning. They will complement our original discovery.
In the AAV field now, in the clinic, I’d say 98–99% is still the natural AAV as a gene therapy platform, but there are many other AAVs in development by those other methods.

What are the remaining hurdles? Is manufacturing still a challenge?

Gao: If we want to develop clinical AAV gene therapy and commercialize the drugs, we have to overcome four barriers:

  1. Manufacturing. Currently, if you want to use a gene therapy for eyes, for localized delivery to the brain, you don’t need much. Current technology is good enough. But if you want to do things like Duchenne muscular dystrophy or cross the blood-brain barrier, it may require up to 1016 viruses for each patient. In commercial terms, the current maximum scale is probably 1018. But if you are going to use gene therapy and commercialize the drug, usually you need to be on a scale of 1020. We are at least one or two logs away. Generating large quantities of highly potent virus is the number-one barrier we face in the field. This contributes to a major portion of the high cost of gene therapy.
  2.  Immunotoxicity. As we are giving AAV at much higher doses, preexisting immunity, innate immunity, and adaptive immunity to capsids and transgenes will become an issue. Some immunotoxicity with high-dose injections is starting to show up. We have to manage this.
  3. Choice. People ask me, “Which AAV do you recommend if I want to target the brain?” That’s a hard question because my understanding, based on natural AAV, is you can either have an efficient or inefficient AAV. There is really a lack of a true tissue tropism, a true cell or tissue specificity. It doesn’t matter how you create a new AAV, that is the area we have to fight for. Eventually we will get there. We’ll make a designer AAV for a certain disease and certain targeted tissue.
  4. Expression. When we do gene therapy, we typically think the more expression, the better. Soon, we will realize that sustained expression at a high, superphysiologic levels may not be good. Particularly with some haploinsufficient diseases, you may run into problems.

 

References
1. Kaul R, Gao GP, Balamurugan K, Matalon R. Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease. Nat. Genet. 1993; 5: 118–123.
2. Gao GP, Yang Y, Wilson JM. Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy. J. Virol. 1996; 70: 8934–8943.
3. 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: 257–265.
4. Gao GP, Qu G, Faust LZ, et al. High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus. Hum. Gene Ther. 1998; 9(16): 2353–2362.
5. Gao GP, Alvira MR, Wang L, et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. USA 2002; 99: 11854–11859.
6. Gao G, Alvira MR, Somanathan S, et al. Adeno-associated viruses undergo substantial evolution in primates during natural infections. Proc. Natl. Acad. Sci. USA 2003; 100: 6081–6086.
7. Gao G, Vandenberghe LH, Alvira MR, et al. Clades of adeno-associated viruses are widely disseminated in human tissues. J. Virol. 2004; 78: 6381–6388.
8. Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Disc. 2019; 18: 358–378.

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