Hello, and welcome to the Denali Therapeutics webinar on interim data from our phase I/II study of DNL310 in patients with Hunter syndrome, or MPS II. I am Laura Hansen, Vice President, Investor Relations, and I'd like to thank you for joining our webcast today. Please note that the press release we issued earlier today, and then the slide deck for this webcast are available in the Investor section of our website, denalitherapeutics.com. Before we get started, I'd like to note that the presentations given today and the responses to questions will contain forward-looking statements regarding Denali's future plans, business strategy, product candidates, planned preclinical studies, and clinical trials, among other things. Such statements are subject to numerous important risks, uncertainties, and assumptions. Should any of these risks or uncertainties materialize, or should our assumptions prove to be incorrect, our actual results could differ materially from these forward-looking statements.
These risks, uncertainties, and assumptions are more fully described in our filings with the SEC, including our latest quarterly report on Form 10-Q and our latest annual report on Form 10-K. Any forward-looking statements are based on information available to us as of today. We disclaim any obligation to update any forward-looking statements except as required by law. On the webcast today, I'm joined by members of Denali's management team, Ryan Watts, Chief Executive Officer, Carole Ho, Chief Medical Officer, Alexander Schuth, Chief Operating Officer, and Steve Krognes, Chief Financial Officer. I would like to take a moment to review the agenda and Q&A logistics for today. We have scheduled approximately one hour for the webcast, including presentations and the Q&A session. Ryan will begin with introductory remarks, and Carole will present the interim data from the phase I/II study of DNL310.
Ryan will provide concluding remarks, then begin the Q&A session. If you would like to ask a question at any time during the presentations, you may do so by typing it into the Q&A box. We will do our best to answer as many as possible during the Q&A session. Now I would like to turn the program over to our Chief Executive Officer, Ryan Watts.
Thank you, Laura. Very excited to be here today. Also very excited to share some of our data in our DNL310 program. Actually, extensive data, biomarker data, and clinical data. I'd like to start with our purpose. Denali has set out to defeat degeneration. As we've highlighted in the past, we have a number of ongoing studies or completed studies across all of these therapeutic areas, including rare neurodegenerative diseases, ALS, Parkinson's, and Alzheimer's disease. Today, we'll focus on the rare neurodegenerative diseases, specifically lysosomal storage diseases. We follow a set of principles as we develop each of our medicines. These are our discovery and development principles. First is what we call the degenogenes, genes when mutated that cause neurodegeneration. Today is a highlight of that as we focus on IDS in Hunter syndrome. Second is engineering brain delivery, so inventing medicines that readily cross the blood-brain barrier.
Again, today we'll focus on our Transport Vehicle technology, which is designed to cross the blood-brain barrier for large molecules such as antibodies, enzymes, and antisense oligos. Our third principle is biomarker-driven development, and I think that's a major highlight of today's presentation. We'll show extensive biomarkers as we approach Hunter syndrome. Finally, our goal is to have a patient impact. To increase the likelihood of success, we follow these three principles in each of our programs. Just a reminder that our portfolio is split roughly between small molecules and biotherapeutics, about 50/50. We're focusing on the biotherapeutic portfolio today and our most advanced program in Hunter syndrome, DNL310. We'd like to highlight, however, that we've now advanced DNL126 for Sanfilippo into the IND-enabling stage. Let's talk about biotherapeutics and the blood-brain barrier, and I'll begin with the blood-brain barrier challenge.
The blood-brain barrier is a major obstacle for brain delivery of biotherapeutics, and the way that we've approached it is to use natural transport mechanisms in blood vessels in the brain, and specifically the transferrin receptor, as shown here. We've engineered molecules to latch onto the transferrin receptor, which is constitutively endocytosed, bringing iron into the brain, with the goal of bringing our biotherapeutics across the blood-brain barrier and into the various cell types in the brain. To highlight the technology that we're using, the Transport Vehicle technology, we published two papers last year back-to-back in "Science Translational Medicine." The first paper outlined the invention of the Transport Vehicle technology and its application to antibodies with proof of concept in both small animals as well as non-human primates. The second paper utilizes the Transport Vehicle technology to get an enzyme across the blood-brain barrier for Hunter syndrome.
Here we use the IDS Hunter mouse model, we can show robust reduction in substrate in brain, which correlates with reduction in cerebral spinal fluid, one of the key fluids that we use in our human studies. In summary, the Transport Vehicle achieves high concentrations and broad biodistribution of biotherapeutics in brain. It also can achieve a dose-dependent reduction in brain substrate, we're very excited to show data even at lower doses today for our DNL310 program. Important for us is the broader potential of the Transport Vehicle technology, both in various modalities but also in therapeutic areas. Our initial focus is in neurodegenerative diseases as well as lysosomal storage diseases. However, we see potential in broader neurology, oncology, and infectious disease.
Importantly, we're applying the Transport Vehicle technology to antibodies, enzymes, proteins, and even antisense oligos and are advancing a number of programs in this space. Before I hand it over to Carole, I'd like to summarize the interim data that we'll present today. We're very enthusiastic about the ability to rapidly and durably reduce heparan sulfate in cerebro spinal fluid across all patients tested and across all doses. In addition, we'll be presenting data on exploratory biomarkers of lysosomal function, where we see reduction consistent with improved lysosomal function. We see a high variability in neurofilament observed both pre- and post-treatment. We also show, for the first time, improvement over 24 weeks based on clinician and patient-reported global impression of change at clinical outcome. In addition to this, we see enhanced peripheral activity. All patients are switched from idursulfase to DNL310, and here we're looking at both urine and serum.
We see improvement in activity with DNL310. Importantly, the safety profile is consistent with standard of care replacement therapy. The totality of this data has led us to make the decision to accelerate our program into a phase II/III study. With this, I'm going to hand it over to Carole to go into great detail on this data.
Great. Thank you, Ryan. I'm pleased to be able to present the DNL310 interim phase I/II data. DNL310, as Ryan described, is an IV therapy that uses Denali's Transport Vehicle technology to deliver IDS, the enzyme that's missing or defective in MPS II, to the brain and the body. The technology of a fusion protein of IDS to ETV has a differentiated binding profile to the transferrin receptor, which in preclinical models demonstrates broad distribution through the brain to neurons, astrocytes, and microglia. DNL310 is currently in an ongoing phase I/II study. It's delivered systemically IV once per week, which is the same dosing frequency that standard of care ELAPRASE uses. In this phase I/II study, patients on ELAPRASE switch to DNL310 in order to evaluate the effect of DNL310 on both the body as well as the brain.
The development of DNL310 is intended to replace current standard of care by delivering efficacy in the brain, a current unmet medical need, while also delivering differentiated efficacy to the body. This next slide outlines the study schema for the phase I/II study. This is an open label six-month study followed by an 18-month safety extension in approximately 30 MPS II patients aged 2 - 18- years- old. Eligible patients are either treatment naive or on approved IDS therapy for longer than four months. In the data presented today, all patients were on IDS therapy and were switched to DNL310 without a washout period. There are three cohorts in this study designed to evaluate the safety profile of DNL310 and enable dose selection for our subsequently planned phase II/III study.
The primary endpoints are safety, which includes adverse events, infusion-related reactions, and total urine GAG, which will demonstrate the effect in the periphery. Because patients switch off of IDS. These secondary endpoints and very relevant to the blood-brain barrier crossing capability of this Transport Vehicle include CSF heparan sulfate and then also urine heparan sulfate. Exploratory endpoints include other GAG measures of dermatan sulfate, heparan sulfate, and keratan sulfate, which we'll present today, including lysosomal CSF lipid biomarkers and CSF and serum neurofilaments. Clinical outcome results in cognition, behavior, and global impression are also assessed in this study. Today we will present data on the global impression scale. I'll go through the detail of the cohorts. We'll present data today for Cohort A and B. Cohort A is a within-patient dose escalation cohort and includes neuronopathic patients 5- 10 years old.
Today, we are sharing six-month biomarker data and safety data up to 43 weeks. All patients in Cohort A remain in the safety extension study at 30 mg/ kg. Cohort B now is a dose-finding cohort to evaluate lower dose regimen of DNL310 in both neuronopathic and non-neuronopathic patients aged 2-18. Dosing modifications, as you can see, can be made in Cohort B1 after 12 doses have been administered. Cohort B2 and B3 study stable doses of 7.5 mg/ kg and 15 mg/ kg. All patients in Cohort B will roll over to the safety extension at the end of the study at 15 mg/ kg.
Cohort C is a cohort that will enroll neuronopathic patients younger than four years of age and sibling pairs to further evaluate exploratory clinical endpoints, such as behavior and cognition, in an age before normal development is markedly impacted by this disease. Dose selection in Cohort C will be determined based on ongoing and emerging data from this study. Now I'll share the demographics of the patients that will be reviewed on data in this interim data cut. The demographics are shown here. The median age of both cohorts is six years of age, and all 17 patients, as noted, were on IDS treatment prior to enrollment, except for one patient in Cohort B. These are all neuronopathic patients. There are five patients in Cohort A, all of whom completed the six-month study. Cohort B is ongoing with 12 patients included in this interim analysis.
These 12 patients have completed 12 weekly doses. The largest racial group in this study so far is white, and the majority of patients are non-Hispanic or Latino. This provides a further detail of the data that will be presented today. As noted, safety up to week 43 and 25 in Cohort B. Heparan sulfate, which is our key secondary endpoint in Cohort A up to week 24 and in Cohort B up to week 13. We'll be also presenting lysosomal biomarker data up to week 24 in Cohort A, specifically GM3, BMP, and GlcCer. We'll be presenting Cohort B GM3 data up to week 13.
In addition, we are presenting exploratory neurofilament data on Cohort A up to week 24 and exploratory Clinical Global Impression Change from both an expert clinician as well as a parent/caregiver up to week 24. I'll start with the interim safety summary, which is in the safety population of 17 patients. Very importantly, all patients remain in the study with no discontinuation. All five of the Cohort A patients continue in the safety extension at 30 mg/kg, and of the 12 patients presented today in Cohort B, 10 are continuing in the six-month study period and two have advanced to the safety extension. All of the data presented today was reviewed by an independent data monitoring committee on July 9th, 2021, and importantly recommended continuing the study without any modifications to the study protocol.
All treatment emergent adverse events were mild or moderate except for two severe treatment emergent AEs, which were IRRs in one patient, which I'll describe below. Infusion-related reactions were not unexpectedly the most common treatment emergent adverse event, occurring in approximately 71% of the patients. The majority had mild or moderate IRRs, but one patient had a severe IRR, which was an SAE, which will be detailed below. Of the 12 patients with IRRs, eight required standard interventions to prevent subsequent IRRs, and two required additional dose and infusion rate reductions. Most IRRs to date have occurred between weeks three and six, and of the three of the patients who experienced IRRs and advanced to the safety extension, most of the pre-infusion medications have been discontinued. There have been two patients that have had SAEs related to infusion-related reactions in this study.
One previously reported was a patient who had an SAE at week four that was hospitalized for overnight hospitalization but then was discharged without any further sequelae. The second patient had two SAEs, severe IRRs, that met Sampson criteria of anaphylaxis at weeks three and four. These were managed with pre-infusion medications, dose and infusion rate reductions. Importantly, this patient remains in the study and has now tolerated subsequent weekly doses, including dose increases. Regarding safety laboratories, there were no notable abnormalities or trends in safety laboratory evaluations except for anemia, which was also described previously. There are four patients that have had treatment emergent events of anemia, all were considered not related to drug. These were graded mild in three and moderate in one, and all four are improving or have resolved despite continuing dosing at either 15 mg/kg or 30 mg/kg.
In summary, the weekly IV infusions of DNL310 for Cohort A and B were generally well-tolerated at doses between 3mg/kg-30 mg/kg with a safety profile that is consistent with standard of care therapy. I now will move to the safety biomarker of urine GAG. As a reminder, this was collected in the study to monitor clinical response to DNL310, particularly given patients were washed off of IDS standard of care therapy. Total urine GAGs were measured by a colorimetric CLIA-certified assay and were used in the study to monitor peripheral response to DNL 310. Across Cohorts A and B, total urine GAG levels overall decreased, including in the lowest dose regimen of Cohort B1 at 3 mg/kg. After switching from IDS to DNL310 without a washout period, total urine GAG levels declined almost in all patients, suggesting improved peripheral activity with DNL310.
Consistent with the improvement in the reduction of urine total GAGs, we also assessed whether DNL310 could further reduce elevated levels of heparan sulfate and other peripheral GAGs, including dermatan and keratan sulfate. We have previously published an increase ranging from two to 3.7-fold in all of these three glycosaminoglycans in the serum of MPS patients, which are depicted in orange and red. Notably, these patients are on standard of care enzyme replacement therapy or have received a hematopoietic stem cell transplant. You can see that these are elevated compared to the dots in gray, which are non-MPS pediatric controls. We have observed now with treatment of DNL310 in Cohort A across these three different serum glycosaminoglycans, there is a reduction at week 24. Notably, keratan sulfate is a dominant GAG present in articular and growth plate cartilage where bone grows.
In certain MPS diseases such as MPS IV, where keratan sulfate is the dominant GAG that accumulates, the disease course is characterized by skeletal dysplasia. This reduction in these peripheral GAGs, including keratan sulfate, suggests that DNL310 could have increased potential long-term effects on peripheral clinical endpoints, including bone remodeling. Let's turn to the CNS penetrating capability of this Transport Vehicle technology and DNL310. Importantly, I wanna highlight again a slide that we've shared previously that shows the importance of heparan sulfate in monitoring the effects of blood-brain barrier crossing activity of DNL310. As you can see in the family of MPS disorders, those that have CNS involvement highlighted in light orange, you can see all have heparan sulfate accumulation. For those diseases that do not have central nervous system disorders or cognitive symptoms, you can see that heparan sulfate is not represented here.
Similar to the previous slide, I want to remind everybody about the increase in CSF heparan sulfate that is seen in MPS II patients, even though they are currently on standard of care therapy, as shown in the upper left. There's roughly a 10.7-fold increase in CSF heparan sulfate in MPS II patients compared to controls. As you can see here across Cohorts A, B1, B2, and B3, all 15 patients had normalization of heparan sulfate at the end of the dosing period up until this time, with rapid response in 12 of these patients by week seven. This percent reduction is 90% in Cohort A and ranges between 86%-92% at lower dose regimen, demonstrating the efficiency of DNL310 for crossing the blood-brain barrier and reducing the most important glycosaminoglycan in the CSF that is correlated with clinical symptoms across the MPS disorders.
Dermatan sulfate is also a key substrate of IDS, and so we share the data here again. Again, you see a 31-fold increase in dermatan sulfate in the CSF in MPS patients compared to non-MPS controls. What you can see is that there's a rapid response in 12 patients by week seven in the reduction of dermatan sulfate in the CSF. I think notably, data that we're now presenting in Cohort B also shows that at these lower dose regimen, we continue to have very efficient reduction in CSF dermatan sulfate. To extend the understanding of the effects of DNL310 in the CNS, as you know, we have profiled also lysosomal lipids, which accumulate in cells of both neurons as well as glia in the central nervous system, and we use this as a biomarker to get further evidence of effects in the brain.
As you can see here, GM3, which is a key nervous system ganglioside, this is elevated 3.7-fold in MPS patients compared to non-MPS controls. As you can see here, across Cohorts A and B, 10 of 15 patients achieved normal CSF GM3, including at lower dose regimen, as you can see in Cohort B. This demonstrates that DNL310 is getting into the brain and is reducing these lysosomal lipids that accumulate. We also look at two additional lysosomal lipids that have been associated with lysosomal dysfunction, including BMP and glucosylceramide. As you can see here in natural history cross-sectional data, we do have an increase that is modest in BMP and glucosylceramide, and you can see in both of these biomarkers, there is a mean reduction in these biomarkers at week 24 in Cohort A.
Next, we explored neurofilament, a biomarker of neuronal structure which has not previously been characterized, to our knowledge, in MPS II, aside from publications from Denali. In 2020, Denali was the first to publish cross-sectional data demonstrating an observed increase in CSF and serum neurofilament. We have extended this analysis now to look at natural history data to further characterize the longitudinal trajectory of neurofilament. As you can see in the middle panel, these are three patients from our natural history study that is studying patients with MPS II, and these patients have the opportunity to enroll into Cohort A. Three of these patients in the natural history study are Cohort A patients that then enrolled into Cohort A. As you can see here, there is significant patient variability that is best evidenced by collection of neurofilament in short time frames within a short span.
You can also see that there is a mean increase, a marked mean increase of 94% in the four and a half to six months pre-dose. After treatment, there is a modest increase of 15% and 36% increase in neurofilament in the serum and the CSF. It's within patient variability in neurofilament, as well as the increase in neurofilament that is seen in the natural history study, leads us to conclude that at this time, the utility of neurofilament in MPS II as a treatment biomarker requires further investigation across the field, specifically in generating additional natural history data. Notably, we also looked at another biomarker of neuronal structure, tau, which shows no change in near normal levels over the 24-week dosing period. I'd now like to turn to our data on the cognitive effects based on Clinical Global Impression Change Scale.
Before doing this, I'd like to provide an overview of cognitive milestones in Hunter's patients. As you can see in this graph, this graph shows the correlation between developmental age on the Y-axis and calendar age on the X-axis. As you can see, in normal development, these should track very much along the 45 degree line. You can see that there are two patient populations that are evident in Hunter syndrome. Those that have neural parenchymal involvement that show regression in their developmental age over time, noted in orange, compared to those that may have mild or no cognitive effects or no neurocognitive effects that track along a normal trajectory. As you can see that this change in the trajectory is very evident by age five, and all of the patients that we have studied in Cohort A are above the age of five.
We would expect very little change or regression in their cognitive development over time. Here's the data on the clinical and parent/caregiver Global Impression Change questionnaires, which were used to rank overall MPS II symptoms, cognitive abilities, behavior, and physical abilities on a seven-point scale from very much worse to very much improved. Worsening is depicted in shades of red, and blue is depicting improvement. In Cohort A, all patients were neuronopathic and ranged between five to eight years of age. Interim assessment of clinical outcomes by expert clinicians and caregivers suggests improvement in overall MPS II symptoms, cognitive abilities, behavior, and physical abilities in 5 to 18-year-old patients. In addition to the scales as shown below, we have also extracted excerpts from the comment field in the CGI-C, the expert clinician global impression of change that was entered by the investigators into the database.
These include comments such as language much improved, more complex sentences, now able to hold a pencil, more conscious of conversations surrounding him, physical features that are much improved, building much more complex Lego towers, better stick figure drawings, and use of new words and completing three to four word sentences, less aggressive following directions. In summary, this data suggests that in Cohort A patients who are greater than five years of age, assessment of clinical outcomes suggests improvement in overall MPS II symptoms, cognitive abilities, behavior, and physical abilities as assessed by an expert clinician and parent/caregiver. In summary, the data from the ongoing study of DNL310 in phase I/II supports a well-tolerated safety profile of DNL310 at doses ranging from 3 mg/kg- 30 mg/kg weekly.
DNL310 demonstrates sufficient blood-brain barrier crossing and durable reduction in CSF heparan sulfate and dermatan sulfate over six months, with sustained normal levels in all 15 patients in this data cut. DNL310 effects on GAGs and lysosomal lipids were also observed at all dose levels, including 3 mg/ kg weekly demonstrating activity on lysosomal function. In addition, global impressions suggest improvement in symptoms as assessed by an experienced clinician and caregiver. The utility of neurofilament in MPS II requires continued investigation and additional natural history study data given the variability observed in the marked increases in neurofilament observed in patients rolling over from the natural history study onto the phase I/II study. Our safety profile is consistent with standard of care enzyme replacement therapy in MPS II, with safety data now up to 43 weeks of dosing.
Based on this data, we look forward to enrolling in Cohort C, designed to further explore clinical endpoints, including behavior and cognition in an age range for which treatment effects on development milestones may have the highest likelihood of impact to be observed. In addition, we are accelerating our activities toward the registrational study to start in the first half of 2022 to demonstrate patient benefit in both neuronopathic and non-neuronopathic MPS II. With that, I'll turn it back over to Ryan for conclusions and to start the Q&A.
Excellent. Thank you, Carole. I'd like to summarize, and we'll dive into the Q&A here. In conclusion, I think importantly, we show that one of the hallmark biomarkers of Hunter syndrome, specifically heparan sulfate, we saw a robust and sustained reduction, including normalization. I think importantly, even at low doses, such as 3 mg/ kg, we're seeing normalization. I think this highlights the power of the Transport Vehicle platform and further validates this platform as we use it across other modalities. We also show for the first time global impression scales, improvement in cognition, behavioral, and physical function, including some exploratory biomarkers such as lipids, which are downstream of heparan sulfate. The safety is consistent with standard of care, and then what does this mean in totality?
For us, we're now accelerating the phase II/III study to begin next year, and we're further building out our Transport Vehicle franchise with a focus on the enzyme Transport Vehicle, in addition to other Transport Vehicle-enabled programs, highlighting that we'll be bringing at least two more Transport Vehicle-enabled proteins to the clinic in the next six months. I think with that, we'll take questions. I see that there is a number of questions. Great. Excellent. Maybe we'll start here. Let's see. Let's start with questions related to infusion-related reactions. I think there's about three questions related to this. How do the rates of IRR compare to ELAPRASE? Carole, I'll hand it to you.
As noted, we believe that our current profile is very consistent with standard of care, where per the ELAPRASE label, there is a 57%-69% rate of hypersensitivity reactions that are observed that are in line with what we've observed. In addition, there are severe hypersensitivity reactions involving more than two to three body systems that have been reported in ELAPRASE in approximately 15% of patients. I think just in terms of our IRRs, these were managed by standard infusion-related medications for IRRs, again, we feel that this is very consistent with standard of care.
I think we'll continue on this theme and maybe just ask one or two more questions related to IRRs. How do we gain confidence that these IRRs are not related to the Transport Vehicle, but rather related to ELAPRASE or to IDS?
Yeah. That's a great question, and I think this is something that we've observed and looked at our data very carefully, and I think the best data really that gives us confidence is that these patients in Cohort A remain in the study and at 30 mg/ kg. These are the patients that had the longest dosing, up to 43 weeks. As I've noted, we actually see a decrease in these infusion-related events over time.
Similarly, in Cohort B, those individuals that have had infusion-related reactions continue in the study, and overall, we see that these are very manageable, very much like what we see with standard of care therapy and management of ongoing infusion-related reactions. I think maybe the last thing that I'll say is that just in terms of very importantly looking at the durability of response of reduction in CSF HS, if infusion-related reactions were related to Anti-Drug Antibodies that, for example, decrease the efficacy of the enzyme, we would expect to see changes, and we don't. I think very importantly also with peripheral GAG, urine total GAGs are constantly monitored in the clinic by physicians. Then as well, we have our Mass Spectrometry, very quantitative assay that has shown no increases in these GAGs over time.
Several questions around ADAs, and any new understanding of ADAs to DNL310 or other related effects that we can speak to. Maybe I'll just ask three questions in a row because I think they'll be related. For patients previously on IDS treatment before entering the study, were any positives for Anti-Drug Antibodies found? If so, how did this impact biomarker reductions from the treatment? I think you sort of highlighted this already, Carole, that there was no effect on the biomarkers. Could you characterize the presence of any neutralizing Anti-Drug Antibodies? How does this compare to rates observed with pediatric patients treated with ELAPRASE? Those are three questions around ADAs.
Yeah. ADAs are quite common in this patient population. Yes, there were patients with preexisting Anti-Drug Antibodies, and we've mentioned this previously. For example, in Cohort A, there was one patient that, as you may recall, had a slower reduction in their decline of CSF HS. Very clearly, that patient has normalized at this time. I think the ability to dose higher certainly allows us to dose through these ADAs, and that's something that we're evaluating further in our Cohort B, where we have a more stable dosing across the three doses of 3 mg/kg, 7.5 mg/kg and 15 mg/ kg.
I think I'll just add to that this is heavily influencing our ultimate dose selection as we go forward to the phase II/III. The ability for us to be able to dose higher is critically important because obviously ADAs are par for the course with enzyme replacement therapies. We've definitely seen a correlation between preexisting ADAs and the ability to reduce heparan sulfate. For example, if you look at patients in the B1 Cohort, the one that didn't respond immediately had higher preexisting ADAs, very similar to Cohort A. That ability to have a higher dose is critical. Let's turn our attention to anemia. A question on, do you still believe that anemia events are blood draw related?
Yes, we absolutely do. The patients in Cohort A that initially had reductions in hematocrit, we followed those patients very closely and also actually made protocol modifications to reduce the amount of blood draws that we had over time. As you may recall, also in the early part of the study, there were multiple dose escalations. Cohort A went from 3 mg/kg to 7.5 mg/kg to 15 mg/kg in a short period of time. Every time we did that, we needed to actually draw blood to monitor for PK, ADAs, and other endpoints. What we've seen is that these patients over time, despite dosing up to 30 mg/ kg, have essentially either resolved or near resolved the anemia back to baseline levels. We're quite confident that this is not related to the Transport Vehicle technology.
Okay. Some questions now related to neurofilament and the high variability. The following question, what do you know about variability of neurofilament in different diseases, and the time course of change is dependent on the pathology of the disease in, say, Hunter syndrome versus MS, where you have relapses. What does this teach us about the value in Hunter?
Yeah. That's a great question, and we're certainly learning as we go along the way. I think we're a bit surprised to see the variability and then also the marked increase that we saw in the natural history data. As you noted, we're the first to demonstrate effects or the increase in neurofilament even in this study. We're really characterizing this and sharing this data as we go along. In terms of variability, there is variability, I would say, that can be seen across multiple indications, ALS, AD, that really have profile neurofilament. I think that there's also published data, for example, in COVID, that they looked at neurofilament and MS. I'd say that over short periods of time, there can be quite a bit of variability in neurofilament, which certainly makes it challenging to look at neurofilament as a treatment response biomarker.
I don't know, Ryan, if you want to add to that.
I'd love to add to that. What's interesting is the data that we published on neurofilament showed a very wide spread of neurofilament in both serum and CSF. However, that was just a cross-sectional look at neurofilament. What is I think most interesting to us is that within a single patient, you're seeing high amounts of variability in short periods of time, including this increase, at least in the three patients that subsequently enrolled in the treatment study. Now, I don't know how to compare that. People have generally thought that neurofilament was stable in some of these other diseases, such as ALS. I mean, stable within reason. Obviously, it makes it difficult to interpret, especially with such few patients, these data with that type of variability. Let's stay on the neurofilament question. Is there evidence that heparan sulfate kills neurons slowly?
Removing heparan sulfate might have a long tail of neurofilament fall off, whereas a modest increase would be more consistent with partial correction, likely implicating PK in deeper regions of the brain. Maybe I'll take this one, Carole, and you can add to that. We're just really careful about not over-interpreting the data from this market increase in patients to the modest on treatment. That being said, the other really interesting observation is that tau doesn't really change, and it's near normal levels. A lot of times you're seeing this relationship between tau and neurofilament, which you're not seeing in MPS II. I think just with this variability, we're going to need to get more data to imply something that is asking the question that this is actually evidence of deeper brain penetration.
I think our strongest evidence of brain penetration is obviously the robust effect on heparan sulfate, but also the downstream lysosomal biomarkers, which are fully corrected. It's worth noting that we actually never achieved that level of correction in the animal model that we're seeing in humans. When we looked at brain levels or CSF levels in animal models, probably the maximum reduction we had was about 70%, and it didn't return to normal levels. In this case, I think that my conclusion is that there's a pretty big capacity for transport in the human brain. There's 400 mi worth of blood vessels. Using transferrin receptor is a very efficient way to get across the blood-brain barrier. Just recall that for every neuron, there's an associated capillary. There's only one or two cell body distances you have to travel once you cross that capillary.
I think related to this, and here, we'll ask this question around the preclinical data, which again, I think is very challenging to compare, but we definitely have some insight. How do you believe the preclinical Hunter mouse data is translated for exploratory biomarkers such as neurofilament and clinical outcome? The patients in the study, with exception of one, two-year-old, are a bit older. If you were to start treatment in younger age patients, can you speculate what neurofilament levels would look like over time, perhaps according to what preclinical Hunter models data show? That's, of course, our own data. Was there any notable stabilization or reduction in neurofilament for two-year-old patients? It's a great question, Carole. I'll hand that to you.
Yeah. These are great questions, and I think going back to also the other question about variability in neurofilament over time, I would say that pediatrics, as compared to MS, we have much less data on what the normal trajectory is of neurofilament. I would say that probably the best pediatric data comes from SMA 1 and SMA 2, SMA3. The effect or the correlation between treatment effects are really strong with just SMA 1 and not so much with SMA 2 and SMA3. I think just as we look at these biomarkers as exploratory biomarkers, I think we really have to understand more about the trajectory.
Now going back to the question around starting early, we do certainly think that starting earlier will have a greater impact on the disease, simply because at that earlier stage, you have lost fewer development milestones, and so there may be more of an opportunity to have a greater impact. As far as neurofilament, I think it's still just early days, because even the natural course of neurofilament may be very different in G rays. We know that neurofilament actually starts out much higher, then there's a period of active pruning and remodeling in the brain that the neurofilament starts to go down, and then it increases slowly over age in a normal individual.
I think there's this dynamic period during development that we don't really understand what is happening with neurofilament and when there are changes in the neurocognitive status with treatment intervention, how this affects the remodeling of the brain and therefore effects on neurofilament. It's maybe a long answer, I don't think we know right now how treating a two-year-old with respective neurofilament changes, we could anticipate what that would look like. I would say, the reason that we're going to Cohort A and Cohort C is that from a clinical perspective, we're even more likely to see effects in the younger age range. Given the exploratory data in Cohort A with regard to the behavioral impression scales, we're very enthusiastic to explore further in younger patients.
I think that answers another question based on some of the other presentations at MPS 2021, obviously ongoing today. There appears to be an emerging trend of better responses with earlier treatment. While it may be early with Cohort A subjects, any thoughts on how this may apply to DNL310 treatment? What can we do to facilitate earlier patient identification? I think, Carole, in some ways you've answered that, but maybe just to reemphasize, it would be worthwhile.
Yeah.
Well, I think this is something that we're really enthusiastic around engaging the community in in terms of newborn screening certainly would help identify patients earlier. In some states, that has now been added to the newborn screening panel. For the most part, it is not currently the standard of practice to have newborn screening. I think just as we and others in the field are developing therapies for neurocognitive symptoms, there's certainly awareness, and we're hearing every day from the patient community of individuals that are being diagnosed earlier, and particularly siblings, where one sibling already has the disease and the parents are then seeking diagnosis and treatment for the younger sibling. This is another opportunity to enroll earlier patients.
Definitely think the mantra earlier is better. I will say that we were very excited about the data that we saw, the clinical data treating these more severe patients in Cohort A, albeit a small number of patients. With the understanding of the hazards associated with inter-study comparisons, can you comment on how the six-month DNL310 data may differ from JCR's IDS-TFR molecule JR-141 six-month data. I'll be happy to address that, and Carole, you can add to this. At the mechanistic level, we compare a JR-141-like molecule to DNL310. This paper is actually available on bioRxiv. We presented this data at World.
It's really a question around architecture and brain delivery. I think what's really worth noting in that paper, looking at both brain and CSF reduction at different dose levels, the high differentiation of the Transport Vehicle enabled IDS or ETV:IDS, relative to JR-141-like molecule in terms of architecture. We believe it's related to the affinity and when you're bound tightly to the transferrin receptor not readily crossing the blood-brain barrier. In addition to that, although the assays are different, you can look at percent reductions, right? We, of course, have internal control data. Now, thinking about the clinical data, comparing it, you ask what are the normal levels of a healthy individual versus MPS, and then how does the treatment affect? When we're seeing 90% reduction and normalization, that's in contrast to maybe 30% reduction early on and maybe 60% max reduction.
There's a very clear difference, I think, both how rapid the response is. I think to add to that, even at doses as low as 3 mg/ kg, we're seeing a normalization in at least three out of those four patients. The four patients having higher ADAs, which we can dose at a higher dose. I don't know, Carole, if you want to add to that.
I think that summarizes it very well. I guess I would really just want to emphasize again, it's clinical data where we see a large magnitude of reduction and this normalization to normal levels as opposed to, say, non-neuropathic levels. Then I think just in terms of clinical data, we also are very pleased to see that CSF dermatan sulfate is also substantially reduced. Then just in terms of the overall activity of the enzyme and the penetration into peripheral organ systems, we share the serum data across heparan sulfate, dermatan sulfate, and keratan sulfate, which demonstrates expanded activity. We think that this suggests that the binding profile, as Ryan outlined, is sort of transparent. The receptor binding and the Transport Vehicle is clearly differentiated.
Let's turn to the phase II/III. How do you think about powering a phase II/III study now to show neurocognitive benefits? How long does follow-up need to be, and how large a trial, and what is the basis for the assumptions that we just discussed? Related to that, phase II/III, have you met with the FDA yet? What is the gain in first starting a phase II/III at this point?
Yeah. We've certainly been thinking a lot about the phase II/III study. We have met with global regulatory authorities to discuss this. I think as many of you know, the regulators have spoken at national meetings, including WORLDSymposium, and they've indicated that the FDA would like to see a randomized study, compared to standard of care ELAPRASE. That is factoring into our thinking around the size of the study and the duration of the study that would be required to see a treatment effect. In terms of looking at progression of neurocognitive symptoms, we would anticipate engaging a study of these patients in a randomized setting for a one to two year timeframe.
That being said, we're certainly continuing to push forward with our in Cohort C with open label data to better understand these cognitive and behavioral endpoints and start to understand whether we can see clear evidence of clinical impact even before that we complete the phase II/III study.
Let's turn our attention back to neurofilament in a series of questions that we can get through pretty quickly. Do you have a view on how neurofilament may be impacted by age? Could there be a different outcome in under five?
Yeah. I think we addressed that question earlier in that we don't really understand the natural history of pediatric neurofilament and how not only do we not have a very clear picture of a normal development of how that changes. We do know, again, that it starts high, then goes down, and then as people age into their second decade, it increases again. We don't know how it would be impacted by a treatment paradigm. Again, we do think that in this younger age range, we're more likely to see clinical changes.
There also could be a difference in the nature of the IDS mutation. I think it's really fascinating when you look at our data in the five patients, that there is a real spread between the different levels at baseline of neurofilament, including one patient that's relatively low and stays low, but is deemed neuropathic and saw cognitive benefit. There seems to be a disconnect there. It may be as much the nature of the mutation as it is the age and timing of intervention. Okay. Can you comment on the clearance for neurofilament? Do you have enough data to suggest you are slowing neurofilament growth versus pre-treatment? I think maybe I'll answer this. We're not claiming anything, actually, that we're slowing the reduction. We're showing the data as is. We do not have enough data to be able to claim that.
That is one interpretation of our data. It's a very good point that there's a balance between production and clearance, and neurofilament has a relatively long half-life. What we can tell you is that it's variable between patients and within patients. That's the data that we have. Why does neurofilament increase in the serum in the natural history MPS II patients but is variable in the CSF? What might explain that seeming disconnect? I should make this really clear, and then Carole, if you want to add to this, we only have natural history data for serum. As you might expect, it requires anesthesia to take CSF, and in these observational studies, we're not taking CSF. The data that you see is just in serum, pre and post, and then just in CSF on treatment. I think that answers that.
There's really not a disconnect, it's just that we don't have the comparison.
Ryan, maybe I'll just add to that. They may have been referring to cross-sectional data, which we do have cross-sectional CSF neurofilament data that we show. These are samples that were taken opportunistically before we enrolled our natural history study. Obviously, the limitations of cross-sectional data is exactly why we started enrolling this natural history biomarkers study. This was really, for us, it was a learning to see that in this longitudinal data, there was this marked increase over time, which was not apparent that we would see that from cross-sectional data.
Another question. Has there ever been data showing CSF neurofilament or other biomarkers in CSF with standard enzyme replacement therapy? How does DNL310 compare to those? I think the answer is no. There's no existing data that we're aware of besides the data that we've generated. Obviously the data we generated in our manuscript is on standard of care enzyme replacement therapy as well as stem cell therapy. That's the data. It's cross-sectional but not longitudinal. Another question here. It seems like the plan for Cohort B may be changed a bit. It used to involve dose escalation versus three parts. Why the change?
Yes. We're continuing to learn as we go along in this study and the changes in Cohort B are to better understand the difference between 3 mg/ kg, 7.5 mg/kg and 6 mg/ kg.
And I would say.
I think as you can see, we're seeing clear activity at the lowest dose level.
Yeah. That's the reason for the change, is that because we have activity at all three doses, we've decided to stay on dose longer. Essentially stay on the lower doses longer. Related to lysosomal biomarkers. GM3 data suggests lysosomal correction is incomplete. Do you believe that that result might arise from the intermittent dosing? Will you explore other dose approaches with your improved enzyme? Examples might include gene therapy or cell therapy. Maybe I'll comment on this. You can look at the data, it's either normalized or near normalized. I guess I wouldn't interpret it as incomplete. Interestingly, even at the 3 mg/ kg, we're seeing normalization in GM3. The reason that you may not see it completely all the time is, again, probably some variability in GM3, the way I'd interpret that.
Well, I would agree with that interpretation. Note that I think where there may have been a question of incomplete, we are also looking at Cohort B data with time points as short as seven weeks. We already know from our Cohort A data that the effects on GM3 appear to follow the reduction in CSF GAG, which happens much earlier, which would be logical, that we would see the reduction first in the substrate, then the improvement in lysosomal function.
Okay, here's a question. Any comment on time course for removal of standard of care peripheral treatment, mean, median, and by dose? Maybe this just requires a little bit of clarification. For DNL310, there's a switch from standard of care to DNL310. Basically, they're no longer on standard of care. Basically, DNL310 treats both peripheral and central. I think it's an important point that we're seeing enhanced activity in peripheral biomarkers in addition to normalization in CSF heparan sulfate and robust effects on lysosomal biomarkers. The idea would be that DNL310, we're basically developing it to replace standard of care. Okay. Again, on phase II/III plans in neurofilament, how do you think about setting inclusion criteria with respect to baseline neurofilament levels for the phase II/III trial?
We would not use baseline neurofilament levels for inclusion. I think there's really no data at this time that clearly demonstrates any sort of prognostic or predictive utility of neurofilament as a treatment response or a prognostic biomarker.
Okay. A question here. Is it possible that for CSF neurofilament, there is a variable but dramatic higher progression and increase over time in the natural history for patients on idursulfase? If so, might the much more modest increase in CSF neurofilament and I'd add serum, seeing on DNL310 represent improvement? I think as we commented, that could be one interpretation. I think we don't have enough data to conclude that certainly. I think related to this, could we elaborate more on sort of our thoughts or our hypotheses on the neurofilament data? I don't know, Carole, if you want to summarize your thoughts or hypotheses on the data
Yeah, I think I've mentioned most of these points previously, but I think the variability that we see was surprising in the natural history data. I think, again, just speaks to the importance of having longitudinal data, because these are pediatric patients, again, that are enrolled at different ages, and so baseline values are very different. We knew that actually already from cross-sectional data, but you layer on the variability on top of that. I think it becomes very challenging to make any sort of conclusions at this point. I think this is something that is going to take additional data, and quite a bit more data, including natural history data, which can be very challenging to get. I think there are other programs that have samples that would be great to understand more about the neurofilament levels in well-characterized data sets.
I think until that time, we really can't make any conclusions about the utility of neurofilament.
In the next four minutes, we're going to do some rapid-fire Q&A here. Where do you think DNL310 could fit into the treatment paradigm for MPS II relative to a one-time gene therapy? Maybe I'll answer this, and Carole, you can add to that. At this point, what we're seeing with gene therapy is a highly variable response when you look at, for example, heparan sulfate in the CNS. Also gene therapy targeting the CNS is different than gene therapy targeting the periphery. We view that there's certainly a major role for enzyme replacement therapy until you can have sort of absolute rescue. Our goal is exactly that, to absolutely rescue both in the periphery and in the central nervous system.
Yeah, I think I'll just add to that someday, the vision would be for patients that there's a gene therapy that's a one-time administration, and it addresses both the central and peripheral manifestations of disease. At this point, I think until that happens, this really allows clinicians, if this continues to demonstrate the profile that we've seen, to get weekly therapy that addresses both. In certain gene therapy approaches, there may not be complete efficacy in the periphery, and therefore, patients may have to end up getting two therapies, one for the periphery and then the gene therapy, which would have addressed the central nervous system, where this would address both.
Given the degree in variability in neurofilament you observed in Cohort A patients, how are you thinking about the pooled Cohort AB data? Do you still think there's a potential to observe meaningful reduction with additional time, larger numbers of patients?
Yes, I can answer that. I think six months data, even with more patients, based on what we've seen in the natural history data, variability and increase over time is not going to be informative. I think as we've seen in other therapeutic areas, specifically maybe I'll mention in lysosomal storage disease, for example, CLN2, it may take a long time before you can see these changes in neurofilament. I think based on our data and what we've characterized so far, again, very limited, I think our focus is going to be more on our lysosomal biomarkers, heparan sulfate reduction, and clinical outcome measures.
I think I'll just add that the CLN2 data, the levels are actually substantially higher relative, in terms of fold difference than what we're seeing in MPS. This is related to that. Based on what we know about the pathophysiology of MPS II, do you think GAG levels are more important than neurofilament? Is there a significant neuronal loss in this disease that drives the pathology? I think the last question, it's a great question. We actually don't know if there's significant neuronal loss, and that's obviously why we looked at tau. If there was significant neuronal loss, you would expect that tau would be elevated as it is in other diseases. It may be possible that what you're seeing in elevated neurofilament is some type of neuronal dysfunction or that it's within error in terms of variability.
We most certainly think that GAG levels are the most critical biomarker. It's what's correlated in the periphery with benefit. It's what we believe will correlate in the central nervous system as well. Although sort of perplexing neurofilament data, the totality of the data very strongly supports that DNL310 is crossing the blood-brain barrier, rescuing lysosomal function, and is translating or should be able to translate into clinical benefit. We think that that's certainly the case. To summarize, we're very enthusiastic about where we are with DNL310, and we're expanding the phase I/II study, obviously with C ohort C. We're able to keep patients on the lower dose for longer because we have normalization even at lower doses, again, sort of illustrating the robustness of the TV platform. We look forward to sharing more data as we go forward.
I think with that, we'll thank everyone, and we look forward to connecting more on these data. Take care.
Great. Thank you.