Melanie Cole, MS (Host): Welcome to UF Health MedEd Cast with UF Health Shands Hospital. I'm Melanie Cole. And today, we're here to highlight gene therapy for hemophilia with AAV vectors. Joining me is Dr. Arun Srivastava. He's the George H. Kitzman Professor of Genetics and Chief of the Division of Cellular and Molecular Therapy in the Department of Pediatrics at the University of Florida College of Medicine.

Welcome, Dr. Srivastava. I'm so glad to have you join us today. I'd like you to start by giving us a little bit of updated information on the current status of hemophilia gene therapy, and tell us a little bit about how that's evolved over the years.

Arun Srivastava, PhD: Well, thank you very much, Melanie, for the kind invitation to talk to you about gene therapy for hemophilia. This has been a long time coming. The good news is that we now have two FDA approved AAV drugs both for hemophilia B and hemophilia A. Hemophilia B drug, which is called Hemgenix, which was approved by the FDA in November of 2022. And for hemophilia A, the drug is called Roctavian, which was approved in June of this year. So ,this is very exciting. But unfortunately, the cost is very high. The Hemgenix costs $3.5 million per patient, and Roctavian costs $2.9 million per patient.

Melanie Cole, MS: Wow. Well, thank you for telling us about that. So expand on that, about the key challenges and limitations associated with the first generation of AAV vectors in hemophilia gene therapy. Help us to understand those limitations.

Arun Srivastava, PhD: So, you know, I've always said this, that AAV evolved as a virus and not as a vector. So, what we have been doing, and I'm just as guilty of doing that as everybody else, we take the naturally-occurring AAV and then try to insert our gene of interest in this particular case, either in the factor IV for hemophilia B or factor VIII for hemophilia. And then, we expect AAV to start performing miracles. I mean, it's quite amazing that even the first generation vectors are actually working. But the problem has been that extremely high doses are needed to achieve efficacy. Trillions and quadrillion particles of AAV vectors are being injected in these patients, which is a phenomenally high dose. And as a result, you know, patients actually have experienced some immune responses because, after all, it is a foreign protein. So in most cases, if not all, patients are also given, prophylactic immunosuppression to suppress that immune response. So, it's clearly not ideal even though there are two FDA approved drugs. So very high doses, problems of immune responses I think make it difficult and, you know, because the production costs are so high because such large doses are needed to achieve efficacy.

Melanie Cole, MS: How do the challenges, doctor, and strategies for gene therapy differ in pediatric patients from those in adults. I'd like you to speak about the implications for early intervention and disease management. And while you're telling us that, explain why and the specific mechanisms through which AAV vectors deliver therapeutic genes for hemophilia treatment.

Arun Srivastava, PhD: There are a couple of questions in there. So, I'll try to answer each one of them separately. First of all, you know, as I said before, AAV evolved as a virus and not as a vector. Actually, AAV is not a very efficient virus to start with. It basically is an inert virus, it just goes in and just sits there quietly, it doesn't want to do anything. But when we use them as a vector, then that's where we run into problems. So both the capsid, which is the outer shell of the virus and inside the gene, both are extremely inefficient in delivering genes. So, what we have been working on is trying to make what we are calling the next generation of AAV vectors, where we have made modifications, both in the capsid structure, which is the code protein. And also, the gene which is inside of the code protein.

So to answer your question about early intervention, actually the one drug that was also approved by the FDA in May of 2019 is called Zolgensma. And this is the drug for a disease called spinal muscular atrophy. These children are basically born almost completely paralyzed, because there's a protein called SMN protein that is missing in the brain. A single injection of AAV vectors actually restores expression of the protein. And now, these children are living happily because average life expectancy of these kids is actually less than two years and now they're up to four or five years old. So, this is quite remarkable. And obviously, there are, you know, four other drugs that I mentioned earlier have been using both adults and children.

So, I'm quite amazed that even the first generation vectors are working. But again, as I said, it's just that the extremely high doses that are being used requires the use of immunosuppression.

Melanie Cole, MS: We'll speak about strategies being developed to mitigate some of the limitations that you've spoken about and why patient selection criteria is also so important in this case.

Arun Srivastava, PhD: So again, this is a two-part question. I'll answer the second part first. So, AAV is a naturally-occurring virus, so roughly 50% of the human population has been exposed to one or more serotypes of AAV virus, which means they have pre-existing antibodies. So when you inject a patient who already has antibodies to the given AAV serotype, it will do no good because as soon as you inject your therapeutic vector, it will be neutralized.

One of the main exclusion criteria for enrollment in any clinical trial, that these patients have to be antibody negative. Seronegative patients are the only ones that are enrolled, which is very unfortunate because the 50% of the population will never be eligible for enrollment in any clinical trial.

And the second problem is, as I said, first generation vectors is essentially a foreign protein. So when it enters the cell, a cell actually sees this as a misfolded protein. So, a cell has a very, very efficient mechanism by which it's actually gets rid of in normal course of cell metabolism, the cell makes certain proteins, which get misfolded. So, the cell has a very efficient mechanism by which it gets rid of it, so it simply degrades it. So incoming AAV vectors, a large fraction of it is being seen by the cell as a misfolded protein, and cell machinery gets activated and degrades it. So, it has two major complications or consequences.

First is that a large fraction of the vector gets degraded in the cytoplasm. It clearly impacts on the transduction efficiency, so a very small fraction of the vector actually gets to the nucleus where it needs to be to encode and then express this gene. And the other problem is that those broken down peptides in the cytoplasm are then presented on the cell surface, which are then picked up by the host immune response. The cytotoxic T cells can then start attacking the cells that have been transduced. So, that's what the problem has been that, you know, we have very inefficient vector, not all of it going to the nucleus to express the gene of interest in this particular case for hemophilia A and B. And then, broken down peptides trigger the immune response.

We are very fortunate that we were able to actually overcome both of these limitations by simply making small changes in the capsid. So, the incoming virus particle is not seen by the cell as a foreign protein and therefore evades degradation in the cytoplasm. And as a result, a large fraction of the incoming virus particles actually makes it all the way to the nucleus so you don't need high doses because a small percent of the fraction would be enough to produce the therapeutic protein. And because it is not being degraded in the cytoplasm, it is not presented on the cell surface, therefore it does not trigger the immune response. So, we have a twofer, we have a vector now, which we are calling the next generation vector. It does not require high doses, it does not induce immune response. You know, what more could you ask for, right?

Melanie Cole, MS: Doctor, this is fascinating. I'd like you to speak about clinical trials and real world experiences and how that's informed our understanding of the long-term durability of AAV-based gene therapy for hemophilia.

Arun Srivastava, PhD: Yeah, I would be happy to. I'll provide a very brief history. AAV vectors come in different flavors. They are called serotypes. So, the first clinical trial was done by actually a company which I actually co-founded is called Avigen, and this company was responsible for doing the first trial using AAV2 serotype vectors. Unfortunately, however, AAV2 vectors are not very efficient, so it was a phase I trial. The main aim for this trial was to establish safety because, you know, AAV vectors had never been injected into the human liver in patients. So yeah, clearly, it was safe initially and it did make low levels of factor IX. But unfortunately, the expression lasted only about eight weeks, because by then, the high dose of the vector triggered, as I said before, an immune response, and the human T cells actually destroyed the hepatocyte that were expressing factor IX.

So, it then turns out that AAV8 was discovered later on. And AAV8 is extremely efficient infecting and transducing the liver. But again, those data were derived from mouse studies. So, AAV8 actually is the gold standard. If you inject AAV8 into the tail vein of a mouse, 100% of the hepatocytes in the liver will get infected and transduce, and they will make high levels of factor IX. Based on those studies, and of course, it was preceded by testing this vector in non-human primates, in monkeys, and it didn't work quite as well as in mice, but it was better than AAV2.

The next clinical trial was actually done with AAV8 vectors, and it was very disappointing that it worked great in mice, but it did not work very well in humans. So, this is another good example that whatever we see in mice doesn't necessarily translate into humans. So, this trial was published in 2011. So yes, these patients did make therapeutic levels of factor IX, but three-year followup showed that this was between 8 to 12%, which is therapeutic, the factor IX level came down to about 5%, which is okay, but it's not really ideal. We'd like to get between 15 to 20 % factor IX.

So, back then, we asked a simple question, you know, which is the best serotype for human liver? And there are 10 different AAV serotype vectors that are currently available. Every lab has those. So, we tested each one of them. And to our great surprise, the one that came out on top was AAV3. AAV3 actually does not infect mouse liver at all. So, that's why there was no interest because if you inject AAV3, nothing happens. And we were very fortunate to have discovered that AAV3 has a remarkable ability to infect only human hepatocytes, but not mouse hepatocytes. And I can go into more detail as to why that is, but suffice it to say that we took it all the way to non-human primates. And so now, we have monkeys that are making more than therapeutic levels of factor IX, anywhere between 50% to 150%, low to moderate doses. So again, this is all preclinical work, but I believe that AAV3 is the vector of choice for treating hemophilia.

Melanie Cole, MS: What about children, Dr. Srivastava, because their livers are still growing and dividing with every cell division? Is it diluted? Tell us a little bit about these trials you were just speaking about and where that fits in the pediatric population.

Arun Srivastava, PhD: That's an excellent question and very insightful question. You're absolutely right. Every single clinical trial for hemophilia, both for hemophilia B and hemophilia A, only adult patients are being enrolled because, as you pointed out, AAV DNA actually remains episomal, which means it's not part of the chromosome. So, when the cell divides, and obviously liver cells divide, in children up to age of 10 to 12, the liver doesn't grow up to its full size until the age of 10 or 12. So for every cell division, the vector genome is diluted out. So, that's the main reason why only adults are being enrolled. So, we are working on this other aspect where we actually are able to deliver factor VIII or factor IX genes, and hopefully, you can do repeat dosing, which is not possible with the AAV vectors, because as soon as you give the first injection to kids, they will make antibodies, which will make the repeat dosing impossible. So, yes, we are working on both A and B treatment for adults as well as in children.

Melanie Cole, MS: So interesting, doctor. Thank you so much for joining us. I'd like you to give us any final thoughts and key takeaways that you would like other providers to take away, where you see this field going, because it's rapidly advancing, very exciting work that you're doing. Let other providers know what you see happening. And if we're looking at a blueprint for future research, what would you like them to take away from this episode?

Arun Srivastava, PhD: Two key issues I think we need to tackle. One is obviously the efficacy. We need to be able to achieve efficacy at a moderate to low doses so that the production costs go down. And this is not prohibitively expensive, the Western world would be able to afford this, but you know, most of the patients are actually in the developing world.

So, I would like to see that the vector production costs go down so much so that it could be made available to the patients who are in need worldwide. And clearly, safety is the main concern. So, again, what is amazing about this virus is actually it's a very mildly immunogenic virus. You give a low dose, you don't have to worry about it. There's no need for immunosuppression, which has its own complications. I am a firm believer that AAV gene therapy is here to stay. It can only get better if we can achieve two things: improve the safety and decrease the cost, which is right now extremely prohibitive.

Melanie Cole, MS: Thank you so much, Dr. Srivastava, for joining us today and sharing your incredible expertise on this topic. And to learn more about this and other healthcare topics at UF Health Shands Hospital, please visit innovation.ufhealth.org. And to listen to more podcasts from our experts, please visit ufhealth.org/medmatters.

That concludes today's episode of UF Health Med EdCast with UF Health Shands Hospital. I'm Melanie Cole. Thanks so much for joining us today.