Alan Beggs, PhDDirector of the Manton Center for Orphan Disease ResearchSir Edwin and Lady Manton Professor of Pediatrics, Boston Children's HospitalHarvard Medical School, Boston, MA, USAThe challenges that you've heard about are real. Some of them I think we could have foreseen others. There was no way to know until we actually started treating patients in clinic. But we now know that there are immune responses and also responses just to the viral load. As Julie mentioned, we're giving massive doses to these patients on the order of one times ten to the 14 viral genomes per kilogram.Think about the fact that when these capsids are manufactured, there's a certain percentage of empty capsid. The amount of protein that's being delivered to these patients can be massive. One of the approaches to mitigate some of the risk would be to lower the dose. While early studies demonstrated that in order to get adequate delivery to skeletal muscle, you need to give these very large doses. But what if we could engineer a viral capsid that would be potent at lower doses?There has been quite a bit of research in this area that's ongoing, and some new next generation vectors that are just starting to enter the clinic. In particular, there are a class of Myotropic viral vectors or capsids so-called RGD vectors. RGD refers to arginine, glycine, and aspartic acid, which are three residues which, when present at a particular point in the viral capsid proteins interact with integrin receptors that are specific for skeletal muscle. These viral capsids home to skeletal muscle and can deliver their genetic payload at much lower doses. There was one group of these developed in Germany by Theo Grimm's lab.These were the so-called AAV Myos, and simultaneously in Boston at the Broad Institute, a group of capsids was developed that were called Myo AAV. These were both based off of an AAV nine backbone. It's basically an AAV nine legacy vector with these three amino acids changed. Now Solid Biosciences also has their own independently derived vector that I believe is also an RGD vector. These vectors give us the potential then for more efficient and specific delivery to muscle cells.They may or may not target the liver depending on the particular virus. Some of them the risk to the liver is mitigated by delivering a lower dose. You can also develop these vectors in a way that will be liver targeted, that specifically less of it gets delivered to the vectors. These would be really, in my mind potentially third generation vectors.Strategies, there are a number of strategies. You heard about the immunomodulation regimens. I just talked about optimizing vector design. Also, Doctor Parsons mentioned earlier the fact that where you deliver so zolgensma is delivered Intrathecally. We get it to the place we need it, and we're less likely to have off target effects through other tissues.Then improved manufacturing is very important. I mentioned the fact that every viral preparation contains empty capsids. There are ways to minimize the production of empty capsids, and also effective ways to filter out and remove those empty capsids. This is actually a very important aspect that is being developed further by the CMO community. Then in summary, I think it's important to take a holistic approach when we're thinking about the development of AAV based gene therapies for neuromuscular disease.It starts from the fact that for any given disease we're interested in, we need to define the genetic etiology. Since these are gene directed therapies. We need to pay careful attention to the preclinical animal models. How accurately do they really reflect the human condition? Or are there potentially responses in our human patients that we haven't experienced in the animals? It's important to understand the natural history and the patient population.Recognize that there's extensive heterogeneity, not just in age and severity, but also potentially in underlying susceptibilities in our patients. We have a group of toxicities that we know about and can anticipate. But as Julie was saying, you need to be really careful and think about any potential unexpected SAEs. And then finally I mentioned the manufacturing aspect, the development of newer vectors and quality control aspects that go into making a safe and effective therapeutic.In the next part. Doctor Parsons will discuss clinical safety and efficacy observed in AAV mediated gene therapy programs in DMD, SMA, and XLMTM.