Cystic Fibrosis Doctor Discusses Phage Therapy

0:04 Okay, great. Today, Um, I have a guest host, um, and I'll let Bekah Dedrick from the CF Foundation briefly introduce herself.

0:15 Yes, thanks, thanks, Joe. Um, Bekah Dedrick from the Cystic Fibrosis Foundation. I'm a research director there, in charge of the, uh, the academic anti-infective portfolio. And I have a background in phage research for, um, NTM uh infections. So great to be here, Joe. Um, and I should say that any, uh, opinions that are expressed are mine and and not of the Cystic Fibrosis Foundation.

0:43 Great thanks, Bekah. Um, all right. Today our guest is Jerry Nick from the National Jewish Hospital in Colorado. And just to start out, Jerry, maybe you can tell us when and how you first got involved with phage therapy?

1:01 Um, yeah, thank you, uh, thanks so much for inviting me to be on the podcast and, uh, to talk about phage. Um, so I'm, um, I'm an adult pulmonologist, I'm the director of the adult CF program at National Jewish. Um, and so my, uh, my introduction to phage was definitely on the clinical side. Um, we have, uh, the largest adult CF population in the United States, and also, uh, we're a a referral site for nontuberculous mycobacteria.

1:36 So lots of cases of, uh, Mycobacterium abscessus and Mycobacterium avium, um, and uh, a very active research uh program as well. And so it was, um, I think it was right around early 2020, um, that, uh, it was right after Graham and Bekah had successfully provided phage uh for the the case at Great Ormond Street Hospital, um, of the the young woman with CF and disseminated Mycobacterium abscessus, and she had had a a successful response.

2:13 And uh, Graham had reached out to us and to other programs as well, and and asked if we had isolates from subjects who might benefit from phage. So we sent, uh, a couple isolates to Graham, um, in early or late mid 2020, um, and a few months went by, and then Bekah, uh, reached out to us with, uh, the exciting news that two, um, two lytic phages had been found, uh, for one of our patients.

2:45 And by then it was, we were right in the middle of COVID, and it was, uh, it was not a great time to to jump into a new venture like this. But, um, we do have a lot of support here. And and uh, this individual was was really, um, you know, not doing well, um, so we decided to to take the leap and to and to jump into phage therapy.

3:16 Yeah, so, um, I remember that time distinctly, and, um, I'm wondering, you know, based on that first patient? And you know, we'll, we'll get into, uh, the POSTSTAMP uh trial that you're, that you're doing with Graham, um, later on in the in the podcast. But I'm wondering how you have seen phage therapy change when you've been treating patients? So from that very first patient that we sort of collaborated together, ha, have there been any significant changes over time in the patients that you've treated more recently?

3:55 Just thinking about dosage, delivery, like anything that you can think of.

4:00 Yeah, no, I mean, it's a good question. I think, you know, right now, uh, the for us, it looks a lot the same, the process looks a lot the same. From the clinical standpoint that, you know, the the various steps that we go through to, you know, look for a match to, um, get approval, regulatory approval, uh, to get the pharmacy piece in place, um, that part hasn't changed, you know what

4:32 What has changed is that we've adopted, uh, a standardized protocol for the the treatment, um, so we, we're, we're starting, you know, with a protocol in place, um, and with a a research plan in place. Um, so we're not, we're not starting from scratch in that sense. But, but a lot of it has not changed. And you could, you know, very much argue that it needs to change.

5:00 But at the moment, it's it's very similar to how it was in 2020.

5:06 Yeah, go ahead.

5:07 Oh, sorry, sorry, Joe, just thinking about, you know, our. So that case was published in Cell in 2022. I believe, um, you know, that patient ended up was on the transplant list, right, and was treated with two phage and ended up being able to get the lung transplant. And then the explanted lungs did not have any NTM. Is that correct? Tell me if I’m wrong.

5:40 Yeah, no, that’s correct.

5:42 And did that—were you—were you surprised by that? Did you expect that? I mean, what was your initial reaction to the the phage therapy, uh, working? Yeah, right,

5:54 Yeah, no, I mean. It was, um, it was a long course, you know, of treatment, over a year, a year before the transplant.

6:02 And so the we, really, we had—this was a individual that has been under the care of the Colorado CF program his entire life. So we have followed him, you know, for, oh, probably a decade at the adult program. And and had—he had been in all of our trials, so we had a lot of of research background, he had participated in several trials already.

6:30 Um, and we had a lot of samples and biomarkers available, uh, on him. So we, you know, we didn't see a big culture response, probably for the first six months, but we did see markers… There was an effect; there was lysis of the mycobacteria. And then, um, when he started a CFTR modulator, um, that's when we started to see the cultures turn negative.

7:00 But we still saw episodic positive cultures, um, within several months of the transplant. So when, when we got the explanted lungs, you know, we did a very rigorous uh screen of all the lobes, uh, lavaging and homogenizing, um, and taking just raw sputum samples. And I think close to 20 cultures, and they were all negative. And subsequently, we've—we followed him throughout his post transplant course and and he never did have evidence of mycobacterium again.

7:36 Um, and we have a quite a bit more data now on that post transplant course that, um, that we—we are confident that he had no viable mycobacteria post transplant. Um, but, uh, you know, that was a surprise because it was he had very advanced, uh, CF lung disease and and cavitary uh, Mycobacterium abscessus.

8:01 Yup.

8:03 So, following up on something you said, um, to the first question about when you first got involved, you said there were a couple patients that you sent isolates to Graham's lab from. And I'm, I'm wondering, were those patients who were having, uh, especially a hard time clearing the infection with standard antibiotics, um, and maybe showing some signs of antibiotic resistance,

8:32 yeah, both of those were treatment refractory, that they they had both been on on uh treatment for several years. Uh, and really, you know, progression of of the infection without any kind of, you know, evidence of response. Um, so, I mean, we, you know, we thought that that was what was called for, you know? With regards to we're gonna, we're gonna do phage therapy. Then, you know, we wanted to pick the the most difficult cases.

9:03 And and that's something that I think, you know, could be discussed, and maybe that's not the right approach. But um, that's how those two were selected.

9:12 right? And has that, for lack of a better term, evolved at all in the time between when those first two patients were picked out, and are you still pretty much just picking people who have, uh, treatment-refractory infections for phage therapy?

9:33 Yeah, so the the trial that we're doing now is, um, is limited to treatment refractory. They have to have received at least

9:43 a year of standard guideline-based therapy before we offer phage. But I think, you know, there is thought that that's waiting too long. But the reality is, you know, it's such a big lift on the clinical side that we prioritize the most difficult cases. And people that may be responding to standard therapy, you know,

10:17 we're less likely to embark on phage therapy.

10:22 Right.

10:23 Go ahead.

10:25 Sorry, I'm just wondering if our listeners know about how, when patients start standardized treatments. Can you can you elaborate on how many positive samples are needed, when treatment takes place, what those treatments are and why patients may be—why those treatments aren't the best?

10:50 Yeah, no, it's it's a really good question. So with people with CF, they're a vulnerable population to mycobacteria. But most of the time we don't need to treat. For whatever reason, the infection seems indolent. And we just kind of watchful waiting. But in the patients that do progress to NTM disease, they have to have at least two positive cultures, and then they have to demonstrate clinical decline with—

11:20 both with CAT scans and clinical features. And so the treatment guidelines for abscessus, for the most part, would try to find four active drugs and start with an induction phase for twelve weeks and then a continuation phase for a year. And so there's just a lot of toxicity with that. And there's just a lot of burden both for the patient and for the healthcare system as well.

11:54 And the initial response is not terrible. You know, if you, if you follow the guidelines, you know, 50 to 60% of patients will appear to eradicate it. But there is this group that just does not respond, and often we don't know why they don't respond because sometimes it is drug resistance, but sometimes that's not apparent, you know, that they should be responding better and they don't. And

12:23 that's the group that we're targeting with the phage therapy.

12:27 So this keeps—we keep not getting to the next question, but there's some interesting follow-ups. When you said you use

12:34 the term active for active drugs, what is the—in this case, what is the definition of active? Is that it kills it in a test tube? Or, um,

12:45 Yeah, so that we have a—our lab's a national reference lab, so we have ex—expanded drug susceptibility testing. And and so, you know, we, we go through the list and, you know, try to start with drugs that seem like they're susceptible, you know, on the benchtop, um, and there, you know, there's not a super great correlation with some of these drugs. But—but especially with macrolides and aminoglycosides, if it doesn't work on the benchtop, then then the chance of response clinically is low.

13:18 So, um, we try to, you know, if if it looks like it's resistant, we don't usually try to use that drug in the patient.

13:28 Makes sense, um, so what, um, research do you think is most needed to improve your ability to successfully treat patients? Um, and I guess, in that context, can you discuss the POSTSTAMP trial and its role in filling those gaps?

13:46 Yeah, so the the idea behind POSTSTAMP was, you know, we were really struck by the by, uh, Bekah's publication of the first 20 cases that were treated with phage. And these were consecutive cases, so there was no bias or cherry picking. Um, and so there—there was just a huge range of response, which is fine, I mean, we're used to variability in response. But each of the patients had an absolutely unique treatment plan as well.

14:19 With regard to dose and frequency, and route of administration, and number of phages, underlying disease, every, every person was a unique situation. Um, so it it became, you know, as a clinician, you know, you—you think that things like, um, dose and route and frequency and and number of phages might be important. It, it may not be important, but if it is important, then that was the first thing we could standardize.

14:51 So the POSTSTAMP trial was aimed to standardize the treatment course so that we could compare outcomes with people that had a similar treatment course. Um, and so we published our protocol, the one that was successfully applied for our first case. The CF Foundation has sponsored a trial to enroll people using that protocol. It's actually technically an observational trial, so we're just observing the response to people who are being enrolled with the same protocol.

15:30 The same background of disease, the same underlying CF condition, uh, the same experience of failing a year of treatment, so we've tried to re reduce the variability in the patient selection and in the drug administration, so we can have a better idea, really, what is the success rate? Uh, for phage,

15:52 And is there a control group that doesn't get phage?

15:56 um, yeah, we have a, uh, basically, the group of patients that, uh, qualify for the trial, uh, are enrolled. And then the ones that don't have a lytic match are observed as a as a contemporary comparison group. Um, it's not a control, there's no placebo, you know, so no one, no one thinks they're getting phage and they're not. But they were, they were selected for having the same criteria.

16:26 Initially, they just didn't have a lytic phage available. And so we follow them along clinically, um, and uh, with our biomarker as well.

16:37 And to be clear, the the patients that are on phage and POSTSTAMP, they continue with their antibiotics that they're, that they're on.

16:46 Yeah, so the the protocol, uh, requires them to continue on guideline based therapy, uh, in addition to phage. And they've had to have already had it for, um, a year prior.

17:01 Okay, and and do we know when the readout of that will be?

17:06 Uh, so the, you know, it's been very staggered, um, and so we haven't, you know, we haven't really formally analyzed, you know, the results. Uh, you know, we started a patient last week on it, you know, and the first, the first patient finished over a year—the the protocol is a year of treatment and then a year of follow up. So, um, you know, the results are kind of trickling in, but I think I think we should, we, you know, we do wanna start revealing some of these results.

17:38 I think, you know, the the protocol, you know, I I think, has a lot of room for improvement, you know, but it's it's the kind of the first step. We, you know, we're not—you know, we don't think this will be the ultimate treatment protocol, but I think, you know, we're at a point that we could start to analyze some of the results.

17:58 And I think some of our…

18:01 Just to be clear, are they all getting the same route of administration?

18:06 So the yeah, I should, I should back up a little bit. The protocol is IV phage twice daily for a year. Um, and again, this was based on, this was based on recommendations that we initially received, you know, from Graham, and then the success that we had had with that one case. Um, and so it's a it's an onerous protocol, for sure. And I, you know, and I think, you know, we hear a lot, you know, people are more interested in nebulized.

18:39 Um, we ourselves wonder if twice a day is really needed. Um, you know, and and I, you know, I would. Personally, I think the modifications I would make would be less frequent, larger dose. Um, and, uh, you know, looking at the inhaled uh route as well, um, but there's, there's a lot of challenges to inhaled therapy too.

19:07 and that is IV route of administration for for these patients, is that unusual? Uh, is there a way that they can do that at home?

19:16 Yeah, so this, um, population, you know, especially the treatment-refractory abscessus population, typically gets IV drugs. So all of our, all of the subjects that we've enrolled already had, uh, long-term IV access, and they were all familiar with home IV administration. So the phage is distributed in a syringe. Um, so they just do a quick push twice a day, uh, usually through a med port, but we've had people that use a PICC line as well.

19:48 Um, and so that that's not a huge obstacle, um, but, uh, you know, I think, you know, in other circumstances, having long-term IV access is an obstacle.

20:07 I was gonna ask if you, what if anything, you can tell us about the immune response to the phages that you observed?

20:15 I was gonna go there too.

20:20 Yeah, no, it seems, in our experience, we haven't systematically—we—we collect serum every month, along with a lot of other markers. Uh, the ones that we've tested so far have all developed neutralizing antibodies, and, uh, a variable, or almost all of them have have developed neutralizing antibodies. Uh, and sometimes very quickly, sometimes within the first three to four weeks, uh, often it's within the first three or four months.

20:53 Uh, with our initial case, uh, it was delayed, and uh, neutralizing antibodies appeared against one of the phages, but not the other phage. Um, but it seems like, for the most part, uh, neutralizing antibodies can be expected when you have such a long duration of therapy.

21:14 yeah, and that's what I was gonna bring up. So, um, I'm, I'm, I'm guessing that a lot of our audience is is used to thinking about phage therapy in short periods of time. A week or two weeks, or maybe a month at the most, and we're talking about a year or more of phage therapy for NTM infections. Do you think that's necessary, and why do you think it is or isn't necessary?

21:44 Yeah, so, yeah, it's a good question. I think that the experience with mycobacteria is that, um, the response has always taken several months, uh, before it's evident, at least from culture data. Um, and I think it's because the mycobacteria is in low numbers and it's hard to reach, and it divides very slowly, and it has both a biofilm lifestyle and an intracellular lifestyle. And so the the actual encounter of a viable bacteria with a viable phage is a very rare event.

22:26 Um, and that it—it just takes a lot of cumulative exposure before the phage finds the bacteria that's replicating or in the right phase of life to be susceptible and gets through all the mucus and gets through the cell and and all of that. Um, and so I th—I think that's why it takes so long. Um, a lot of these treatment refractory mycobacteria, they divide very, very slowly, to the point, to the point you can hardly grow them in the lab, that they divide so slowly.

23:00 Um, and so I think that's a virulence mechanism that they're, that may be part of the reason that the phage has to be given over such a long duration. But there's also, you know, cell wall phenotypes, the rough and smooth morphotype, that that seems protective against phage. And um, there's just a lot of obstacles to overcome, and I think that's why it's taken so long to see response.

23:28 Yeah, I, I was gonna touch on that too. So, um, you know, the rough, uh, morphology of M. abscessus? Um, it seems that, you know, phage can infect those that morphotype, but the smooth, right? Um, there hasn't been, correct me if I'm wrong—a patient that had a smooth morphotype that was treated with phage that had a successful or or clinical response, or a successful outcome.

23:55 Uh, is that correct?

23:58 Uh, well, at least in the published literature…

24:04 We've heard of an example, um, but, um, I think, you know, especially with Graham's library, there haven't been any matches that I'm aware of against a smooth morphotype.

24:19 So you alluded to earlier that there isn't a placebo group; there's a group of people whose bacteria you can't find a match for. Do you exclude people with smooth isolates because you know you won't find a match, or do you only look for matches with people who have rough isolates?

24:46 Yeah, that, you know, that's, that's a good question. I I think there is a bias toward rough, although, you know, from the from the clinical standpoint, most of these treatment refractory cases have been infected for years and they tend to be rough or a mix of rough and smooth. Um, it's much less common for, you know, a long term chronic infection to be a smooth. But I I think that, you know, within the community, it's kind of known that the smooth are unlikely to have a match with the Hatfull library.

25:19 Um, so there, there may be a bias in that regard.

25:23 Yeah, no, I mean, I guess I was just kind of worried. And I assume that you had obviously thought of this. But that you could have a bias towards smooth strains in the no phage arm of this study, and that might be biasing things. But

25:39 Yeah, I mean, we have ten sites on the study, so, you know, I, I'm—I'm not that familiar with all of the cases. Um, um, but that is a that is a good question. If if our control, it's not, it's not really a control arm, but it's a comparison arm, is largely smooth, um, we'll have to see how that pans out.

26:02 Jerry, I'm curious if if we were in a world where we had lots of lytic phages that infected and killed smooth isolates, do you think that patients clinicians would then want to treat patients, say, earlier, uh, in infection? And um, would patients feel more comfortable being treated earlier? Or if, or would that be something that they would desire? Because, you know, there really aren't any, uh, horrible uh reactions to phage as compared to the antibiotics that they typically take.

26:41 Yeah, I mean, I think, you know, I I think the availability of phage is important. But I mean, right now, the barriers are just this sum of all the parts of the regulatory and the the physical, uh, steps that have to be accomplished to get phage therapy in place, you know, to actually administer it to a patient. Um, that it's—it's really, very, very difficult for people outside of academic centers that have research teams and research infrastructure, and, and, you know, coordinators and and things of that nature.

27:23 You know, for for most people that are in practice, that are seeing patients all day long, you know, there's—it's just too much of a burden to lift, you know, they, they, they'd love to do it. The patients are very interested, you know, we get many questions every week about phage therapy, the patients are up for it. It's just the the—there's just too much to push through for most, for most busy practitioners, it's just too much of a lift. Um, and I think that's where we're at, you know, with regards to to really expanding it into the mycobacteria field.

28:03 so can you, um, sort of dig a little deeper into the regulatory hurdles? You sort of alluded that to them? Um, so for the person who's not part of a big academic center, where do the regulatory hurdles rear their head?

28:24 Yeah, so, I mean, the really the first thing, and it's the dumbest thing. But just getting an isolate to a academic lab is beyond the capacity of most clinicians, you know, cause they're using a centralized lab that is could very well be in another state. And there's no pathway for them to to reach out to that lab and say, Hey, you know that that culture that you did, you know, three weeks ago or three months ago?

28:54 Could you just go ahead and send that over to Graham Hatfull at the University of Pittsburgh? I mean, that is, that is like, you might as well ask them to send it to Mars, you know, I mean, there.

29:05 Th—that is just not gonna happen because there's no reimbursement.

29:09 They would have to have a material transfer agreement in place. Um, they may not even have that culture anymore, it might have been discarded. Um, so right there, you've eliminated probably two thirds of practitioners from even, you know, screening. But then then, if you can get it to an academic lab like Graham's lab, it, then they have to screen. They may or may not have a phage.

29:34 If they do have a phage, then you have to apply to the to the FDA for an IND. But you have to simultaneously apply to your local IRB for, uh, IRB approval to give it from a compassionate use standpoint. But then you also, like, if you're involved in research, you also have to have a separate IRB, uh, to apply to do research. Because the compassionate care is not a research activity, it's a it's a clinical activity.

30:02 And then you need a protocol in place to administer the phage, you need a protocol in place for the research piece, and then you need a material transfer agreement with your university to get the phage back to you. And then you need to identify a pharmacy that's willing to administer it without cost, because there's no reimbursement for the the phage to be given. And then you need, of course, the the individual to be still healthy enough to receive it.

30:30 For our protocol, you need long term IV access, and then you need, like an infusion center to give the first dose. And you need someone to pick it up every two weeks, cause it has a shelf life of about two weeks, and then you need a way to monitor them, you know, and so it's. There's just so many pieces, you know, and people, people have enthusiasm to do it, but they just don't have the time or the resources, you know, to do all that.

30:57 Um, so that's why it's limited.

31:02 That's helpful, because, I mean, often it's just sort of bandied about that, you know, there are people out there who tend to label the FDA as the bad guy. Um, but it sounds like, from what you said, there's a whole bunch of things that are causing the issue, and maybe the FDA is a fairly small part of that.

31:30 Yeah, no, I would agree with that. I think, actually, the FDA has streamlined it. They, uh, my understanding is they approve over 95 percent of applications. Uh, they have, like a website now, that that is very user friendly. Um, they've actually been very, very responsive to our emails. Um, so, yeah, I would not blame the FDA. Uh, I mean, they, they're probably the easiest part of this whole thing.

31:58 Yeah, no, that's good. I mean, uh, well, I'll confess. I have friends in the FDA and I think they do a good job. But I also, like, you know, the number of times, you know, this podcast has been going on for a while. and a number of times we put a podcast on. and someone will comment, you know, oh my god, the FDA is causing all these problems.

32:17 And if it wasn't for them, everyone in the US who needed phage therapy would be getting it. And, uh, I, my impression from talking to people like you who actually do this, that's not usually the case.

32:30 Yeah, that's not the bottleneck. Um, for, at least for us, but but it's just the sum of the parts, you know?

32:37 Yeah, yeah, no, understood.

32:40 yeah. So so where do you—where do you hope to be in, say, five years from now, when you're treating patients with phage therapy? Yeah,

32:51 Yeah, and I think, you know, what I would like to see is, you know, a protocol that's been refined. Um, I think, you know, I think this is—the right approach for most people is to to have a standardized protocol. You know that, I mean, in the absence of of any data, to be using a standardized protocol, that's that has had a track record of success. I think, you know, I'd like to just have more experience available, you know, for the community to to review cases and see what worked and what didn't work.

33:25 Um, but I think a lot of it is I, I—I think it is important to have a a protocol. And that success can be linked to a certain extent, you know, to the route and the duration and the dose, and and um, the time of of, you know, the frequency of delivery, you know that that probably does have some impact. I mean, I I think, you know, if you're a true phage believer, you could say it doesn't matter.

33:56 The phage will will find the bacteria and it doesn't matter how you give it, but but I think there probably is a a, there probably is a better protocol than other protocols.

34:12 Yeah, um, and I guess looking sort of farther in the future, uh, we—we usually ask people five years, 15 years, and and I know it's sort of hard to parse those out. But like, in the really long term, do you think, where do you think we might be able to get to?

34:32 Yeah, I mean, I think from our standpoint, what we're really interested in is is markers of response. Um, that we would, you know, like, we know already that the phage is active, you know, because they they screened it on the benchtop. But then to know very early in the treatment phase if it's working, uh, you know, to see if we, if there's markers of lysis, if there's markers, you know, that we're making progress.

35:01 Not wait for the cultures to turn negative, um, and if if it's not working, then be able to adapt more quickly. Or even even to the point where we could—we can have enough knowledge about what causes treatment failure, that that we could say, okay, we've got a lytic phage. But this in this individual, this is not gonna be successful. And cause we can know what mechanisms cause treatment failure.

35:28 And and so not embark on a a futile treatment, you know, if we understood, you know why it doesn't work, and that, you know, the the problem is now that, you know, when it works, you know, it's, it's great, but if it doesn't work, you know, we really often don't know why it doesn't work. And and it's off, and it's often much, much later that we have any of those clues.

35:51 And and to try to to to predict that earlier, even before this treatment started,

35:59 And do

36:00 would you?

36:00 you think that we could predict that better if we had, uh, more accurate ways of screening phage susceptibility in vitro? And what I mean by that is media that more closely resembles the environment of the lung, a CF lung or a non CF lung. Um, or or other specific in vitro, um, assays. Do you think that that would lead to more in vivo success?

36:33 Yeah, and I think that there's, there's potential, you know, that you could, like, you could envision a mouse model that you took the exact phage isolate combination and tested it in a mouse before you put it in the in the subject. Um, I mean, that might be one approach, like you said, if it, if you had a media that was more like a airway liquid, you know, surface media, rather than, you know, just, you know, what's being, you know, kind of recapitulated.

37:04 Now is more like a systemic infection. Uh, I think if you added in combinations of antibiotics that are being administered to the patient, you could see if there's synergy with the phage or or there could be antagonism with the phage. Uh, you know, so you could, you could add in different key elements to treatment failure early on and see, you know, I think the antibiotic piece is a huge piece.

37:30 You know that we we assume the antibiotics are beneficial or additive, or even synergistic with a phage, but there have been cases where they're antagonistic to the phage. Um,

37:45 Yeah, and I think a barrier there is time, right? I mean, when you're sending a clinical isolate, typically that that patient isn't in a good spot, right? Um, and so you're trying as quickly as you can to find a match. Um, and so maybe those, uh, those, you know, assays that you should do aren't, aren't normally done. Um, but it would be good to to think about that and and think about how we can sort of, um, predict what would happen in vivo by doing the appropriate in in vitro assays.

38:21 Yeah, and I, I guess I—I was wondering, uh, do you have any plans? Or have you done any looking? Maybe like quantitative PCR, just to see if one of the phages looks like it's being replicated in the sputum? Um, or anything like that?

38:44 yeah, I mean, when we've looked in the sputum, we've seen very, very little evidence of of the phage, either presence or, um, you know, plaque assays, um, but we have, we haven't done a lot with the plaque assays. Um, the PCR, we just haven't recovered much of it. And that's, you know, we're really—we are very worried about, you know, are we dosing high enough? You know, there was, there's been, you know, some really strong work from Miriam Braunstein at CSU.

39:16 That they've looked at, um, the effect of dosing on intracellular killing. And and there is—there's a threshold where if you're below it, you don't see much killing, and if you're above it, then you do see killing. And and I just worry we're not getting enough phage to the site of infection, uh, to get through all the barriers, um, and get it to the infection.

39:43 and to increase that dosage, that puts, uh, that really is relying on good manufacturing, getting enough phage in the manufacturing process to be able to go in at a higher dose. And that could be a limiting factor.

40:00 Yeah, no, I've talked to Graham about it. It's, you know, and you know, better than I do that—that to scale it up even two or three logs is like, you know, you'd have to have like an oil refinery or something to run it.

40:15 It's not easy.

40:17 But then you think about, like, okay, so we can't get more of it. But what if we gave it all as one dose a week, you know, just combined all the doses and gave…

40:29 That's a way we could push the dose by giving it less often, potentially.

40:36 Because maybe then they'd get where they needed to be, and then they self amplify, right? If they found the bacteria, yeah, that's that would be interesting,

40:45 yeah, but then we, if we give it less frequently, then we might miss the the dividing point or whatever. I mean, do you think the do you think that mycobacterium is more susceptible to phage when it's dividing, or—or is there a point in the in the mycobacterial life cycle that it's more susceptible to phage?

41:06 Um, I think there there was a paper, um, semi recently, uh, um, and from from Graham's lab, where they show that, uh, specific, uh phage infect or adhere to the dividing ar—the areas with new cell wall. And so, uh, you know, there's not—there's not a whole lot of data about this, but that piece does suggest to me, at least, that, that, that, that's where the phage are attaching, at least some of them.

41:44 Um, so I think phages are different, so we have to, we have to think about that. Not a whole lot is known about receptors for the mycobacteria phages. So I I think there's a lot of basic science that still needs to be done. Um, but definitely, I think that could be—that could be an issue, right? If you need dividing cells and they're dividing so slowly, I think that could be a reason why we need to treat, uh, NTM infections for a long period of time.

42:13 With all our patients that receive phage, the later isolates' growth rate has gotten so slow, you know, like it's, it's like it's selecting for the slowest growing, slowest dividing possible isolate. Um,

42:32 yeah. And and and do you think those were always there, or do you think they sort of evolved into that?

42:43 yeah, no, that's a really good question. Cause we've done, we've done whole genome sequencing on all of them and and developed these timescale phylogenies, and they're, you know, they don't. The genetic change isn't a lot, but there is, there is a selection kind of pattern. Um, but it, you know, it's a very heterogeneous. You know, there's different parts of the lung, and it's hard to know, like if there was always a really slow grower that was there or not. Um, but that's—those are the last ones that we've recovered from—all of these individuals have grown so slowly.

43:21 do you have any sense of whether the technique is the the when you sequence them, if the technique is sensitive enough that if the bacteria had had a phage injected into them, that maybe didn't do anything, but you would detect, you'd get a sequencing read from them. Um, even if it hadn't managed to inj, you know, lead to a lytic cycle.

43:50 Yeah, no, that's interesting. I, you know, I don't know if if the software we've used would pick up a new prophage or not, but yeah,

43:59 I'm not talking about prophage, I'm talking about phage D, like naked phage DNA that wasn't replicating, cause it hit the wrong cell cycle or something.

44:08 Yeah, no, that's really interesting.

44:14 But, you know, it's hard to do, obviously.

44:19 yeah. And and that's a good point about the prophages, that kind of that kind of left my mind for a moment. So, um, one of, I think, the more interesting things about, um, Mycobacterium abscessus specifically is that there's lots of prophages in those genomes. And, um, I'm just wondering, have you seen, as you've, as you've looked at patient isolates over time during phage therapy? Do you see any changes in in phage content?

44:49 Like, is it, is it pretty much the same, or do they evolve? Do they lose some? Do they gain some? And

44:57 Yeah, I mean, we haven't looked too much at that, um, in the cases that have been given phage therapy. But, um, we have those sequences, you know, so we can… Uh, it'd be really interesting to go back and look. A lot of our phylogenetic analysis is based on the core genome, and that would typically not include the prophage.

45:24 Okay, well, um, I know we talked about this a little bit, but what do you feel are the biggest challenges that prevent you from getting phage to your patients? I know you talked about all the paperwork. Um, is there anything else you wanted to mention there?

45:45 Yeah, I mean, I I think it is, um, you know, within our medical system, we're just so busy that, you know, there's no—there's no reimbursement, you know, for any of this, or compensation, or, you know, funding, really, unless you have a grant, uh, to do any of this. And so it just doesn't get done, you know, basically, and I think, you know, and and I think probably we're not gonna expect to see this from insurance companies to support this activity, you know?

46:22 So it would be—it would be like phage programs within institutions that would say, okay, we're gonna support this, we're gonna support the effort that's needed. We need a pharmacist to do this work, we need a nurse to do this work, we need a physician to do this work, and we're gonna support it. Um, and, and, you know, for for whatever reasons, you know, uh, both, you know, o, obviously, you know, for care of patients, but also for research reasons, that they're gonna support this work, uh.

46:54 But right now there's just not a mechanism to support it. So, you know, you're asking, you know, people to do it, you know, on their weekends and stuff like that, and and they just may not have the time to do it. And and that's just kind of the reality, you know, unfortunately, of it. Um,

47:11 Yeah, I mean, that's why it's so great that people like you are so dedicated in doing what you're doing. Do you have any sense of whether? I mean, there are a fair number of clinical trials ongoing, including things like UTIs and stuff like that. Do you have a sense that if there was a clear, successful clinical trial, even if it were for, say, UTIs or something like that?

47:38 Would that change the picture at all? Or do you think insurance companies are still gonna say, until there's been a clinical trial with the exact phages in the, you know, for mycobacteria phages, that we're not gonna support that?

47:54 yeah, no,

47:55 No,

47:56 I mean, I think, yeah, I mean, I—I personally don't think there's a commercial path for mycobacteriophage therapy. That it, you know, these patients, these cases are too scattered, and they're, um, they're just not, uh, enough similarity between them to to conduct a a phase 3 clinical trial, let alone a phase 1 and a phase 2 clinical trial, you know? So to get to that point, you know, based on my understanding of the process, I don't—I don't think we can get there with mycobacteriophages. I think I think we're gonna be in this same, um, kind of pathway of compassionate use, IND, uh, individual case kind of thing. But, uh, you know, if there was a successful phage trial that led to commercialization, even for a different indication?

48:52 I think I think that would lead to downstream interest. You know that that there may be mechanisms that that this could be financially viable, even though it's not a a. We can't write a prescription for it, but but someone could still, you know, support it financially. Um, I don't know exactly how to do that, but um, yeah, but success breeds success, you know, like it'd be really great to have a successful trial.

49:26 yeah, uh, definitely. Um, all right. Well, I, if, give you a chance to give us any final thoughts that we, I know—we asked you a ton of questions, so maybe we got most of the information out of you. But if you have anything else you wanna tell us,

49:45 Yeah, no, I mean, I I think, you know, it's—it's just a very exciting time. And and there's just, uh, you know, it's just a matter of kind of organizing the resources that are out there. I think that there's just m the the actually, the phage biology is way ahead of the of the clinical side, you know, and that and the people that are that are seeing patients, you know, they want to understand this and they want to access it.

50:17 But but it's just, uh, you know, the they don't really know what, how to go about it, you know? And that's that's why, if we organize and and and really try to take advantage of every case of phage therapy to learn from it, uh, that's how we can move things forward. I mean, I have this, I have this idea about, like, if you think about, um, Alexander Fleming discovering penicillin and having this huge amount of penicillin.

50:49 And then sending it out all around the world in 1942, with absolutely no instructions on how to use it, you know?

50:58 hm,

50:58 So some people would be, you know, injecting it, some people would be swallowing it, some people would be inhaling it, some people would be rubbing it on their skin. And then they'd be reporting back. It worked great, it didn't work at all, you know, and and we wouldn't have any idea, you know, th. It would be exactly the same as where we're at with phage, that people, they get this drug, they know it works, but they don't know what to do with it.

51:26 Um, and that's what, you know, they, that's where we have to get, you know, to the point where we can say, okay, don't rub it on your skin, don't, you know, put it in your ear, you know, you can put it IV, you can inhale it, you know, that kind of thing. And and to give them the direction. And so, I mean, I think that's that's the next step for for a lot of, you know, for a better adaption of of adoption of phage, you know, for this, a disease like mycobacteria.