Chapter 1: Lysosomal Disorders and the Potential for Gene Therapy

Nicola Longo MD, PhD, and Mark Roberts, MD

 Drs. Longo and Roberts discussed the current status of gene therapies in rare neuromuscular disorders in this eight-part podcast series. This is derived from the symposium that was presented at World Symposium 2025 in San Diego, California on February 4th through 7th, 2025, and is intended for healthcare professionals only.

This podcast includes information about investigational compounds that do not yet have a regulatory approval or authorization for a specific indication. The safety and efficacy of the agents under investigation have not been established, and contents of this podcast shall not be used in any manner to directly or indirectly promote or sell the product for unapproved uses.

The views, thoughts, and opinions expressed in this presentation belong solely to the author and are subject to change without notice. The contents of this presentation do not constitute an endorsement of any product or indication by Astellas.

Mark Roberts, MD

I’m going to give an overview of what is gene therapy, emphasizing the current challenges and the development issues and needs that there will be as we try and enable gene therapy for our patients, particularly those with lysosomal storage disorders.

I’m going to try and make a case for why lysosomal storage disorders are an extremely good group of conditions for the potential benefits of gene modifying therapies. Firstly, whilst we all recognize that these conditions are inherently individually rare, they’re certainly severe. Collectively, with over 70 LSD disorders, 1 in 5,000 may be afflicted by these conditions ultimately in their life and can be detected, for example, by newborn screening programs.

Secondly, there’s certainly a significant clinical burden with these patients with the current standard of care, so a large unmet need exists. Existing enzyme replacement therapies have undoubtedly changed the natural history of many of these conditions, but there are limitations and often initial benefits and later deteriorations.

Unfortunately, for most lysosomal storage disorders, it’s only symptomatic treatments and indeed, care that is available for these patients with no specific treatment. Thirdly, these conditions are extremely well-characterized, monogenic singleton and problems of inborn errors of metabolism. We know the functional protein that is deficient in these conditions. Because of that, and knowing that these are critical for lysosomal function, and using preclinical models, we can model the potential benefits of gene therapies very well in a number of systems, including, of course, soon, muscle chip experiments as well.

Finally, with these conditions, they may potentially be really useful targets whilst not perhaps curing the condition, at least ameliorating the phenotype, and enabling the addition of other treatments as well, potentially. I’ve noted, some of these therapies can be directly delivered to certain tissues, so muscle tissue, which is my main interest, but also, crucially, the central nervous system, which is very important when we consider ameliorated phenotypes, for example, treated by enzyme replacement therapy, but where the children who become the adults have significant learning disability as a major component to their problems.

Chapter 2: Vectors, Different Strategies, Modes of Administration, and Targets

Mark Roberts, MD

Now in the broader sense, gene replacement therapy seeks to actually deliver genetic material directly into the host cell to influence gene expression. In the most simple idea, one of course has a vector, this is most commonly but not exclusively a virus, which can then be given intravenously for example, and can hope to potentially correct the condition within the individual cells using novel transgenes. Suitable candidate conditions for this as examples of genetic conditions are now well understood. And crucially, this applies not only towards some more recessive, but dominant and even accident conditions.

Across the piece, one can see for example, mitochondrial problems, spinal muscular atrophy as is well known, X-linked myotubular myopathy, Duchenne muscular dystrophy, a very common condition affecting one in 3000 male individuals, Pompe disease of course, an important focus of the meeting here, but other very common conditions, for example, cystic fibrosis, immunological conditions and perhaps obviously very crucial in early work on gene therapy, hemophilia.

Let’s now think about the approaches to gene therapy. One can seek to work at the DNA level and gene replacement. In essence, one is trying to put a new transgene through into the nucleus that will ultimately be transcribed and translated and produce the important functional protein that is lost. Gene editing which is a very exciting new technology or CRISPR technology actually seeks to actually modify in vivo the actual mutations that are responsible for the pathogenic production of abnormal proteins and correcting these and actually producing a more normalized protein.

But of course there are also RNA approaches where one seeks to actually repair the mRNA transcripts copied from the mutated gene. For example, this may be a novel approach that could be extremely useful in myotonic dystrophy, a multisystem condition. When we talk about the viral vectors, predominantly we’re talking about viruses. Those such as adenoviruses and AAV viruses which have the virtue of not integrating into the host genome or at least not in a large amount, and those which deliberately seek to integrate into host genome such as retroviral or lentiviral systems that may be particularly useful for ex vivo systems.

There are of course other ways to get genetic payloads into the nucleus, various polymers, nanoparticles and even cell penetrating peptides. Nanoparticles in particular is certainly on the ascendant. That being said, in a recent review of the clinical trials in gene therapy, it was certainly the viral vectors that stood out both in direct gene replacement with lentivirus and AAV, but also actually as delivery systems, for example, for gene editing. An example of what one is seeking to do with AAV, so of course one seeking to remove the native DNA, insert the new transgene directly into the vector and of course keen to make sure that there’s a high transmission into the capsid producing a recombinant AAV, which then can be given as a treatment and hopefully produce a therapeutic increase in the functional protein that is deficit in the disorder.

How then are we going to get these gene therapies in? We’ve also heard allusions to tropism being particularly important.

These early pictures will remind you somewhat of the mode of action of enzyme replacement therapy. We need good cell surface receptors that bind to the viral vector in situ and these are typically again glycosylated receptors. The vector is then taken up into the endosome. It’s broken down in the endosome and does not go through the autophagic system and into the lysosome per se, but permeates through the nuclear pore and uncoats and crucially produces an episome, the hope therefore being of non integration into the host DNA. Of course, there’s then mRNA transcription and ultimately from transcription and moving out into the cytoplasm translation and producing a functional protein.

Crucially, this functional protein is produced within the cells. So this really becomes an endogenous treatment. If we compare and contrast the two main methods of gene replacement therapy, the in vivo approach gives the vector directly into the patient through the intravenous circulation. The hope is and indeed many models are showing this, this leads to a transaction of a long acting cell type in which integration within the nuclear DNA per se is not actually required. Typically, the vector of course is AAV.

But another approach is the ex vivo approach, where in essence cells are removed from the patient, they are then transfected with the lentivirus typically and this produces a transgene within these cells produced externally and then reinfused into the patient, but importantly with immunological treatment to prevent any rejection of these cells and potentially integration into a stem cell system that ensures again a durability of response.

Turning to the in vivo approaches, in essence the replacement treatment can be administered by multiple routes. The systemic administration has the major advantage of targeting multiple different systems, but it does of course require a large vector load and these are expensive to generate. There’s obviously a risk of immunogenicity and potentially, and hence the need for long term monitoring of these patients given gene therapies, off target effects that could not perhaps have been predicted. This can be given of course and most commonly is through the intravenous route, but of course bearing in mind that muscle itself is a highly vascular tissue, one can in principle deliver these intramuscularly.

But one appreciates that many of the conditions that we’re interested in at this meeting are very organ specific and for example CNS specific in their issues. So albeit more invasively, one can target the treatments. For example, giving it directly into a ventricular system to tackle CNS disease, giving it into the eye to tackle retinopathy, giving it even into the acoustic nerve to tackle deafness, for example, in mitochondrial diseases. Although more challenging, it can of course be given directly through into the heart, albeit with huge trepidation and the excellent interventional cardiologists.

Pulmonary delivery systems can be really useful. Again, a little bit like the inhalers we use in asthma, directly targeting the main tissue involvement. When we think about how we might want to design a gene therapy, we obviously need to be thinking whether we want to be targeting the liver either as a site of production or as a site of disease, the muscle itself or of course the central nervous system.

To do this of course, we need to deliberately develop viral vectors that show great tropism for those individual tissue cells, but also crucially to be thinking about unique promoters that are available in those tissues, for example muscle promoters to ensure that one gets full delivery and indeed more importantly transcription and translation than into the product in question. Using AAV one really can try and target very specific systems and tissue specific promoters are being developed. This of course takes a lot of work and underlies why it can take so long in the preclinical work to develop these viral vectors.

In the next part, Dr. Roberts will discuss immune responses and other safety concerns related to gene therapies.

Chapter 3: Immune Responses and Other Safety Concerns Related to Gene Therapies

Mark Roberts, MD

Undoubtedly, the immune system is a major issue in these patients. It would be fantastic if we could immunotolerize our patients and indeed prevent the rejection of the therapy. We’ve talked about the fact that these are viral vectors and of course there may be high seroprevalence of antibodies to these viral vectors, and it’s very important in the pre-screening of patients who might be eligible to understand that at the beginning. These of course can have developed over the years and of course can be part of immunological memory and therefore extremely difficult and probably impractical to actually shift.

On giving the treatment though as I think we’re all aware there is this problem of the innate immunity and potential therefore for acute toxicities and then a learned or adaptive response with cytotoxic T cells and antibodies which may of course become high tighter neutralizing antibodies and potentially antibodies not only against the viral vector, even the functional protein, even the transgene are all theoretical possibilities with time. The capsid, the transgene, and even the protein product can all potentially induce an immunological event. Of course, all of these would lead to both potential patient changes and then a lack of efficacy of the treatment.

Indeed, there have been some serious and indeed fatal problems in the gene therapy development program as I think we’re all aware. Though many of these are thankfully been overcome. Spinal muscular atrophy has a gene therapy which is licensed, but there were early patients who actually had significant problems. A patient of just 6 months of age who developed kidney failure, two other patients who actually developed liver failure.

In Duchenne muscular dystrophy, a very common condition, again there were significant issues and crucially in these patients who all have cardiomyopathy, it was heart failure and cardiac arrest that were big concerns and pulmonary edema and this was seen even with a CRISPR-based technology and is perhaps is best known but has been addressed the excellent myotubular myopathy patients, four patients died and crucially quite a long time after the gene therapy emphasizing the need to monitor these patients extremely carefully and these patients died of cholestatic liver failure albeit that they had a degree of liver dysfunction.

That’s changed our screening of course of patients, we’re now all looking in myotubular patients for liver involvement and Rett syndrome as well. Now these immunoprophylaxis treatment regimes to hopefully try and reduce the immunological reaction against the gene are certainly evolving.

Pretreatment with rituximab can certainly be a very useful treatment. Certainly if you compare as this good study has the use of steroids alone to those patients receiving agents such as steroids, but also rituximab and sirolimus, one sees a much lower incidence of these immunological reactions accepting that these treatments in their own right have some risk to clinical patients.

This is just a summary of some of the other immunosuppressive regimes used in other disorders, for example, spinal muscular atrophy, but Pompe and MPS as examples of LSDs. Certainly these regimes will continue to evolve and are going to be very important in seeking to make sure that these treatments are effective. It reminds me somewhat of what’s happened with enzyme replacement therapy that the use of these immunological strategies in infants has revolutionized the utility of those treatments in early patients.

In the next part, Dr. Roberts will discuss lessons learned from gene therapy trials.

Chapter 4: Lessons Learnt from Gene Therapy Trials

Mark Roberts, MD

When we think about the challenges of actually doing clinical trials with these gene therapies, there’s a huge development stage in terms of picking the right viral vector with the right surface receptor. That’s a major piece of work. That can often take years. The preclinical work is obviously very important as indeed is understanding the natural history because it’s really not practical to do placebo-controlled trials of gene therapies.

In contrast to other studies, when we turn to phase 1 and phase 2, you’ll notice that the patient numbers are often quite small. One is having to think carefully about surrogate measurements of response. Especially when in phase 3 studies, we may be thinking about withdrawing the existing, for example, enzyme replacement therapy because we believe the gene therapy will then be effective.

That’s just a few snapshots of where we’ve come and there’s a lot more work to be done.

In the next part, Dr. Longo will discuss the current treatment landscape and limitations in lysosomal disorders.

Chapter 5: Current Treatment Landscape and Limitations

Nicola Longo MD, PhD

What I want to do today, is just place gene replacement therapy within the current landscape of lysosomal storage disorder treatment therapy. Gene therapy obviously has the potential of treating lysosomal disorder to correct the root cause of lysosomal storage disorder. The gene is defective, and what happen is that you can potentially either fix the gene or bypass the lack of the genetic product. But there are already therapies that are existing and are functioning. Obviously, in many cases, the lysosomal disorder is caused by defective production of an enzyme, which is defective.

We can either replace the enzyme with enzyme replacement therapy, or provide chaperone for specific mutations that retain the synthesis of the enzyme, that however is not very functional. Another avenue that it is being reported is the utilization of substrate reduction therapy. A substrate accumulates, you prevent the synthesis of the substrate to reduce the accumulation of toxic material. What we know now is that this is not enough to produce many lysosomal disorders. In many cases, the lysosomal disorder result sometime in impairment of intracellular trafficking, and sometime in the function of other organelles.

At the end, it results in the activation of the macrophagic system and inflammation. Already we have some therapy acting at this level. The end result of lysosomal storage disorder, there will be cell suffering and cell death, leading to a progression of the disease, and morbidity and mortality. Now, what therapy do we have available already? Obviously, hematopoietic stem cell transplantation has been around for quite some time.

It has been the same thing that we do with gene therapy, except that instead of reintroducing the gene of the subject, we place gene of a subject who is not affected of the disease. This therapy has been proven effective in cases of MPS-1 and alpha-mannosidosis. But in many cases this has to be given way before symptoms start to be affected.

Enzyme replacement therapy has been around for quite some time, starting with Gaucher disease, and now that it is available for a list of diseases that are there, so it’s like Fabry, Gaucher, Pompe, different types of mucopolysaccharidosis, alpha-mannosidosis, acid lipase deficiency, 1 neuronal ceroid lipofuscinosis, and Niemann-Pick type A and B.

Obviously, the advantage of this therapy, they give back the enzyme that it is defective. But the disadvantage that many time they cannot enter specialized areas such as the brain. There is already the second generation of enzyme replacement therapy that it is available. With this second generation, some of the newer drugs are more effective in terms of cellular uptake, or in terms of having a prolonged half-life and prolonged activity.

Then there are pharmacological chaperone therapy, and the one which is FDA approved is migalastat for Fabry disease, under study is ambroxol for Gaucher disease. The disadvantage of this therapy that only a selected number of mutations respond to this therapy.

Substrate reduction therapy has been introduced for Gaucher disease many years ago with miglustat, and it was followed by eliglustat. Both of them are effective, and some of them more effective than other, simply because of the fewer side effects of eliglustat as compared to miglustat. But at the same time, eliglustat does not pass the blood brain barrier.

Finally, the newer agents that are already administered, N-acetyl-L-leucine and arimoclomol, both approved for Niemann-Pick type C, they act more on the downstream effect of the lysosomal storage disorder, either by stabilizing neuronal cell activity or by reducing the inflammation that is present in the brain.

Now what are the limitations of the current therapy for lysosomal storage disorder? Obviously one of the great limitations of bone marrow transplant is that you need to have a suitable donor. Somebody whose HLA match with you and can donate the blood. That still remains a big problem, especially in certain ethnic group. The surrounding way would be to do exactly what Dr. Roberts has shown, to do ex vivo gene therapy. Where you take the CD34 cell from the subject, you introduce the defective gene, or you fix the defective gene, and then you put it back doing an autologous bone marrow transplant, that obviously would be accepted much better by the body.

In terms of enzyme replacement therapy, the problem is that many times it is inefficient in cross correcting different cells. In other words, some cells will pick it up, but sometimes it doesn’t reach certain tissue, and those tissue will be refractory to the action of enzyme replacement therapy. The other thing is that they require a lifelong infusion, in some cases once a week, in some cases every 2 weeks. That obviously it is a big burden on patients and their family.

All the enzyme replacement therapy pose a risk of immune reaction every time that you do that. Obviously this needs to be mitigated either by the use of pre-medication, or sometimes with the need of having available drugs to rescue anaphylaxis.

Last, many of them… All of them that we have currently available, are unable to cross the blood brain barrier. So the way around it is the localized administration, with administration, for example, within the brain or within the central nervous system with different ways. Pharmacological chaperone are a wonderful drug. However, again, the number of mutations that they can correct is very limited, and that greatly limits efficacy of this type of approach.

Finally, substrate reduction therapy is effective, but the problem is that many of the compound whose synthesis is inhibited are really essential for the functioning of cellular membrane and other cellular functions. In other words, the therapeutic interval is very limited. We have to give a certain amount, but not too much, because otherwise we might impair some basic cellular functions that are essential for life. Obviously it is possible to bypass part of this problem by delivering them only to the tissue that needs them by using lipid nanoparticle.

In the next part, Dr. Longo will discuss gene replacement therapy in lysosomal disorders.

Chapter 6: Gene Replacement Therapy in Lysosomal Disorders

Nicola Longo MD, PhD

Let’s go back a second to gene therapy. Gene therapy obviously has the potential of answering many of the questions that we still have open in lysosomal disorder because they could restore the activity of the lysosome pretty much in the whole body, or at least in multiple tissues. As you have seen, gene therapy can be done ex vivo where we take cells from the affected patient, we correct the gene, or we put an extra gene that it is functional. Then we put them back by doing a bone marrow transplant, basically creating space for the cells that have been genetically modified to correct the lysosomal defect. The biggest approach this is done usually by lentiviruses that they integrate inside the genome.

The second type of approach is with in vivo gene therapy where it is given systemically, and in most cases this is done utilizing adeno-associated virus. There are different types that have affinity for different tissues that allow them to direct them where the gene is actually necessary. Obviously that allows you to target the delivery of the gene. In addition, by using specific promoter, you try to restrict expression of the gene in the tissue that need them. This is very important because, for example, if you express this gene in the dendritic cell that are involved in the immune reaction, you have an increased chance of having a bad reaction to the gene therapy. You want the gene therapy to be restricted to the tissue and the organ that needs them.

Finally, there are more and more examples of in vivo administration restricted to certain tissue. Obviously, delivery to the CNS is important because many gene therapies do not cross the blood-brain barrier, even though some of the newest ones seem to have access to it as well, even when given systemically. There are also examples of administration of gene therapy directly inside the eye or directly inside the cochlea to correct, in the last case, some type of hearing loss, and in some of these approaches seem to be already relatively successful.

In the next part, Doctor Longo will discuss ongoing gene therapies in lysosomal disorders.

Nicola Longo MD, PhD

I’m going to present to discuss some example of ongoing gene therapy for lysosomal disorder. There are gene therapy in development for both Fabry disease and some of this involve ex vivo gene therapy, many others involve systemic administration with an AAV, Gaucher disease type 1 that affect the periphery, and Gaucher disease type 2, where the replacement should occur within the central nervous system because this condition affects the brain. There is already one approved gene therapy for lysosomal disorder, which is for the early onset metachromatic leukodystrophy. This has been approved both in Europe and now even in the United States, which consists of ex vivo gene therapy with the administration of an extra gene that restore the function of the defective enzyme. Now there are many others that are ongoing for the same indication. There are gene therapy programs for GM1 and GM2 gangliosidosis, and at least one for Krabbe disease. It is important to know that some of these condition are actually included in the recommended uniform screening panel. Basically, we would have access to patients in a timely manner for some of these conditions. Then there are several gene therapy under development for the mucopolysaccharidoses, including MPS-IH, MPS-II, MPS-IIIA and MPS-IV.

There are different type of lysosomal disorders, the one caused by mutation, integral membrane protein, not enzyme within the lysosome, but protein that are present on the membrane of the lysosome. This gene therapy that have been tested, it is for cystinosis, that it is caused by a defective lysosomal and for Danon disease, which is caused by a deficiency of an integral membrane part. Finally, one lysosomal disorder, which obviously seems a metabolic condition, but it is really not, is glycogen storage disease type 2 or Pompe disease, in which there is the intralysosomal accumulation of glycogen. There are several ongoing clinical trials to try to correct the problem in this condition.

Now, I’m going to discuss some of the most advanced program in the lysosomal storage disorder. This include one for Fabry, which is on an accelerated approval pathway with phase 1 and 2 data, one for Gaucher disease type 1. Obviously, I’m going to discuss the one that has been already approved for metachromatic leukodystrophy. There is one for Hunter syndrome, and the difference of the one for Hunter syndrome, it is an example of the direct administration of gene therapy within the central nervous system.

Finally, there is one ongoing for glycogen storage disease type 2 or Pompe disease in adult patients. In gene therapy for metachromatic leukodystrophy, it was the first gene therapy approved for lysosomal disorder in human, and this requires harvesting the CD34 cell from affected patient and then introducing the [inaudible 00:04:32] gene back in this cell, and then placing them back inside the patient again. This has been very effective in patients who were treated early, and obviously, the treatment needs to occur before there is irreversible brain damage in this patient.

One of the things to keep in mind that all of the gene therapy that we are being tested will require prolonged surveillance evidence of patient receiving it. All of the programs usually include about 15 years of follow-up of this patient to make sure that there are no unwanted side effects that are not evident immediately. Now there are currently a phase 3 clinical trial for the late juvenile form of metachromatic leukodystrophy that it is enrolled, tried to expand the indication of gene therapy to people who have a milder form of the disease.

There are gene therapy for Fabry and Gaucher. The previous one was ex vivo antiviral gene therapy. The one for Fabry and Gaucher, they are systemically administered, the one that I’m going to discuss, and they involve adeno-associated viruses. Usually, this therapy are given to stable patients that don’t have a risk of deterioration. Basically, the one in Fabry disease has caused a significant improvement, actually, in the glomerular filtration in a relatively short time. Obviously one need to make sure that that continues to be the case with longer clinical trial and expanding that to a larger number of patients.

The one for Gaucher disease, there have already been two different trials, one to identify the effective dose, the second one to look for efficacy. Many of these patients have been able to discontinue the concurrent either enzyme replacement therapy or substance reduction therapy. Obviously, they are expected to progress into the trial and possibly to provide positive result.

In the case of Hunter syndrome, the difference is the adeno-associated viral vector is delivered by direct injection into the central nervous system. What we know so far is that this therapy is effective in reducing some of the biomarkers for Hunter syndrome. In this specific case, the non-reducing end of the glycosaminoglycan that accumulate in this condition, indicating that there is at least biological activity with actual biological action in the central nervous system. This is obviously both well for thinking that there might be an effect on cognition and development. So far, the patients are relatively stable. More importantly, many of these patients remain very stable, and they seem to be doing very well. Obviously, there is not much of an alternative in this disease because the current therapy does not cross the blood-brain barrier, and this offer the opportunity to this patient for normal development.

Finally, there is an ongoing clinical trial for late-onset Pompe disease. In this condition, the gene therapy is given to adults on a stable dose of enzyme replacement therapy, and many of the patients who have received this gene therapy have been able to discontinue the enzyme replacement therapy and remain stable in terms of pulmonary function and motor activity. Obviously, the fact that they have discontinued enzyme replacement therapy has impacted in a positive way their quality of life and their overall outcome.

In the next part, Dr. Roberts and Longo will discuss treatment with gene therapies.

Chapter 8: Gene Therapy Discussion and Q&A

Nicola Longo MD, PhD and Mark Roberts, MD

Question: Can one administer AAV-mediated gene therapy repeatedly?

Mark Roberts, MD: I think the traditional view would have been no. One can think of gene therapy as a silver bullet. Hopefully, it will reach its target. But if it’s not effective, that bullet has been shot, the immunological response has occurred, and it means redosing, at least with that particular vector, may become difficult. But this situation is changing and evolving as we have better understanding of immunological modulation for repeat testing. We were discussing this yesterday evening, weren’t we, Professor Longo?

Nicola Longo MD, PhD: Correct. Basically, the current AAV-based gene therapy cannot be readministered. It is either effective, or it doesn’t work. The other thing is that even though in theory, one could utilize a different AAV vector with different immunogenicity, there is many times cross-reactivity among the different adenovirus, adeno-associated viruses. Now, there are approaches in animal models in which you give a strong immune suppression to prevent the creation of the immune response against the adeno-associated virus, and at least in the animal model, it has been possible to give some of the gene therapy repeatedly.

The second approach that is being tested is with gene correction therapy, in which by using an RNA guide and the CRISPR/Cas9 system delivered by lipid nanoparticles, you basically correct some of the effective genetic information. Obviously, since this is done by lipid nanoparticles and not by an AAV, the immunity that you create is really not there. You can give this one repeatedly, and in theory, it can be given more than one time. But again, you are absolutely correct. The current gene therapy cannot be given twice, and either it works or it doesn’t work.

Question: Will gene-therapy-treated patients be able to go back to the standard of care or enzyme replacement therapy?

Mark Roberts, MD: I think when we’re talking to patients about the potential benefits of gene therapy and the amelioration of the requirement to have these infusions on a regular basis of ERT, the hope is that will work, but they need to be reassured that we can potentially go back to the ERT. Gene therapy is an important treatment, but we don’t know the destination of the patient at the beginning, and we have to make it available to them to go back to ERT.

One of the crucial questions, of course, though, is the basis of the immunological reaction that perhaps prevented the gene therapy being effective. If it’s against the viral vector, well, okay. If it’s against the transgene, not great. If it’s against the functional protein, that becomes more difficult. It is somewhat, I think at this time, to be fair to say to patients, think of gene therapy as a trial treatment. It is somewhat a leap of faith and an important observation, of course, for the patient community, but just be aware there may be downsides.

Nicola Longo MD, PhD: They totally agree with Dr. Roberts. In general, they should be able to go back to enzyme replacement therapy if the gene therapy is not effective. However, what we are starting to appreciate is that we need to understand the immune response, not just to the enzyme replacement therapy, but also to gene therapy. What this field is doing is forcing geneticists to deal with the immune response. I feel that historically has not been dealt together. The two things need to be integrated. The advantage of the gene therapy is that the protein is produced endogenously. There should be the development of some degree of tolerance with time in the body towards the endogenous continuous production of a protein.

Now, will that happen all the time? I still do not know. Again, we need to understand much better what is the integration of the immune system with the response to gene therapy in the ongoing clinical trials.

Question: What are the potential factors that impact durability of gene therapy?

Mark Roberts, MD: If one takes the in-vivo approach, where, of course, we’re seeking to have the episome producing the DNA there within the cell, there is the possibility that the episome may not be retained, and that could impact the durability of the response. By contrast, the in-vitro response, where, of course, one’s hoping very much for inoculation, if you like, of the host genome, may be a more durable response, but it’s far more intrusive as a treatment approach. Effectively, you’re giving an autologous haematopoietic stem cell transplant.

Nicola Longo MD, PhD: We already have evidence of the ex-vivo gene therapy that it is of long duration, and it remains effective for quite some time, in a way that it is comparable for many aspects to a standard bone marrow transplant. We know much less about the AAV. One of the concerns that is around is the fact that if you deliver AAV therapy to young children where organs are still developing and multiplying, there is the possible loss of the gene correction in daughter cell that are generated, obviously, by cell division. We still do not know enough about it, but it is a theoretical possibility that still it is in our mind. Should we be doing gene therapy in organ that divide very rapidly in children, or should we limit our trial to adults?

For now, many of the trials you have seen have been done in adults, but some of them have been done in children. However, the one done in children, many times they affect the central nervous system that it is not an organ that divides very rapidly.

Question: Could you clarify for patients in gene therapy clinical trials who have discontinued ERT over a long time, what was that transition like? Did patients stay on enzyme replacement therapy while in the trial and then taper off or another approach?

Nicola Longo MD, PhD: Basically, when do we choose to discontinue enzyme replacement therapy in patients who have received gene therapy? In my own experience, basically, there were data emerging from the biological testing. What you do many times, you measure the activity of the enzyme, either in serum or in the cell, or you use additional biomarkers that usually are utilized to track progression of the disease. When you have a consistent reduction with maintenance of body function, in the clinical trial that I did, there was maintenance of the respiratory function and the 6-minute walk test. You stop the enzyme replacement therapy, and then you keep monitoring all of the same biomarker with the idea that if you have a worsening, then you would go back on enzyme replacement therapy.

Obviously, there was no worsening. The patient was feeling happy and didn’t have to go back to clinic every 2 weeks to do the infusion. There was actually an increase in physiological function of the patient. The patient decided not to go back to ERT, and they stay there. Now, will that happen that way all the time? I do not know, but at the same time, I think that if you have good biomarkers, good endpoint that you can assess to determine the functional effect of the gene therapy, you reassure whether something is effective or not effective.

Question: Can you comment on the use of non-viral vectors for lysosomal storage diseases, especially after the technology is developed for the COVID vaccines?

Mark Roberts, MD: I think this is a very exciting area, and we’ve already alluded to the fact that many intravenously delivered viral vectors will not get through into the central nervous system. Nanotechnology and new nanoparticles offer the possibility of delivery into the CNS without the need for invasive technologies, for example, directly into the ventricular system.

I personally think that’s going to be an extremely important step because if one thinks, for example, about the model of Pompe disease, and as we’ve all seen, the huge amelioration of the natural history with enzyme replacement therapy, we expect with gene therapy, we’ll see a similar issue, but we have to think about the CNS involvement, and those children who are coming through into our practice with learning needs. Nanotechnology might be helpful, or two different gene therapies, one to target CNS involvement and one more peripheral neuromuscular involvement, might be something that really leads us closer to the amelioration. Not a cure of the disease, but closer to the amelioration of the condition in the longer term and not just in the short term.

Chapter 1: Lysosomal Disorders and the Potential for Gene Therapy

Chapter 2: Vectors, Different Strategies, Modes of Administration, and Targets

Chapter 3: Immune Responses and Other Safety Concerns Related to Gene Therapies

Chapter 4: Lessons Learnt from Gene Therapy Trials

Chapter 5: Current Treatment Landscape and Limitations

Chapter 6: Gene Replacement Therapy in Lysosomal Disorders

Chapter 7: Ongoing Gene Therapies in Lysosomal Disorders

Chapter 8: Gene Therapy Discussion and Q&A