50 – The use of milk exosomes to increase the expression of SYNGAP1 in SYNGAP1 mice

These are our introductory comments: 

Our talk for today is “The use of milk exosomes to increase the expression of Syngap1 expression in Syngap1 mice”

Dr. Janos Zempleni earned a Ph.D. in nutrition sciences from the Justus-Liebig University in Germany and received postdoctoral training in pediatric nutrition, nutritional biochemistry, and molecular and cell biology in different institutions. Dr. Zempleni joined the University of Nebraska-Lincoln in 2001 where he rose through the ranks and now is a Willa Cather Professor of Molecular Nutrition and founding director of the NIH-funded Nebraska Center for the Prevention of Obesity Diseases through Dietary Molecules.

The Zempleni lab has a long track record of research in nutrition and epigenetics. His laboratory is interested in natural nanoparticles that play an important role in cell-to-cell communication and the regulation of metabolism. Current focus areas in the Zempleni lab focus on the role of exosomes in human milk in the neurological development in infants, interactions between milk exosomes and the gut microbiome, and the use of milk exosomes in the delivery of drugs.

A recorded version of this webinar will be available on the SRF website under Webinars on the Family menu.

By the end of this presentation, you will have the opportunity to get the answer to your questions. We’d love to hear from you – please write your question in the Q&A.

Webinar Overview

Dr. Janos Zempleni is director of the Nebraska Center for the Prevention of Obesity Diseases and has a lab at the University of Nebraska-Lincoln. He starts the webinar by explaining extracellular vesicles, specifically emphasizing exosomes; exosomes are vesicles produced in cells and carry components such as nucleic acids and proteins. He then talks about milk exosomes which can be used for the delivery of therapeutics. The aim of his project is to increase SynGAP expression in the brain by delivering SYNGAP1-coding DNA through milk exosomes. Through his research with mice, he has found that milk exosomes can accumulate in the brain as well as other tissues. However, there are many components required to ensure the milk exosomes make it to the brain and don’t get destroyed. These components include the use of iRFP (near-infrared fluorescent protein) for distribution and expression, RVG (rabies virus glycoprotein) to target the brain, and CD47 protein to ensure macrophages don’t destroy the milk exosomes. The genetically engineered milk exosomes combined with SynGAP1 DNA should result in increased SynGAP expression in the brain. Dr. Zempleni closes by talking about undergoing research where they nasally administer the SYNGAP1 milk exosomes in mice.

Other Relevant Publications by Dr. Zempleni

Small Extracellular Vesicles in Milk Cross the Blood-Brain Barrier in Murine Cerebral Cortex Endothelial Cells and Promote Dendritic Complexity in the Hippocampus and Brain Function in C57BL/6J Mice

Genetically Altered Bovine Milk Exosomes (BMEs) Evade Elimination by Murine Bone Marrow-Derived Macrophages (BMDMs)

THIS IS FOR TRANSCRIPT ONLY:

0:04hello everybody and welcome to today’s webinar  my name is Olga Bothe and i’m a Syngap parent and  

0:09volunteer with SynGAP Research Fund our talk  today discusses the use of milk exosomes to  

0:16increase the expression of SynGAP in SYNGAP1 mice  I have the pleasure to introduce today’s speaker  

0:22Dr Janos Zempleni. Dr Janos Zempleni  earned a PhD in nutrition sciences  

0:28from the justice liebeck university in Germany  and received postdoctoral training in pediatric  

0:33nutrition nutritional biochemistry and molecular  and cell biology in different institutions  

0:40Dr Zempleni joined the University of Nebraska  Lincoln in 2001 where he rose through the ranks  

0:48and now is a willa cather professor of molecular  nutrition and founding director of the NIH funded  

0:54Nebraska center for the prevention of obesity  diseases through dietary molecules the Zempleni  

1:02lab has a long track record of research in  nutrition and epigenetics his lab his laboratory  

1:08is interested in natural nanoparticles that play  an important role in cell to cell communication  

1:14and the regulation of metabolism currently  the Zempleni lab is focusing on the role of  

1:21exosomes in human milk in the neurological  development in infants interactions between  

1:26milk exosomes and gut the gut microbiome and the  use of milk episodes in the delivery of drugs  

1:32a recorded version of this webinar will be  available on the SRF website under webinars under  

1:38the family menu by the end of the presentation  you will have the opportunity to get the answer  

1:44to your questions we’d love to hear from you  please write your question in the Q&A below  

1:50for those of you just joining us welcome and  again our speaker is Dr Janos Zempleni and  

1:55his talk is investigating the functional  single cell biology of SYNGAP1 pathways  

2:01it’s now my pleasure to turn things over to Dr  Zempleni thank you Olga that was a very nice and  

2:07kind introduction before i start my presentation  i would like to make you group a compliment  

2:15i’ve interact with many funding agencies  throughout my career but your group has   been the most pleasant to interact with by far  what comes to mind when when i think of your  

2:27team is you have a very important objective you  have a knowledgeable and invested leadership  

2:33you’re very unbureaucratic which i really like  and you have just pleasant people to interact with  

2:40then I also have one housekeeping item our  university has installed motion sensors in our  

2:45office so if I in my presentation jump up and wave  my arms this is not because i’m going crazy it’s  

2:52just to get the lights back on this may or may  not happen to my presentation so let’s get started  Disclosure

3:00my university requires me to disclose any  potential conflicts of interest that really is not  

3:07turn on my laser front here there really  is not a whole lot to show for it’s just  

3:13my funding agencies and i do serve as  an advisor to a Boston-based company  

3:19named Puretech health they also make exosomes  and drug delivery with the focus on brain cancer  

3:26so it’s really not relevant not directly  relevant to what you and i are working onWhat are exosomes

3:34when i was invited to give this presentation  Syngap parents and leadership informed me that  

3:40the audience will be very diverse so some of you  are scientists some of you parents some of you  

3:46are donors and then maybe just lay public  who wants to chime in and then tune in and  

3:54listen to this presentation so i’m trying to was  trying to set the tone in my presentation for not  

4:02losing anyone on this on this talk also basically  catering to the educated lay public i think so let  

4:09me start off by by telling you a little bit about  extracellular vesicles it’s a group of compounds  

4:16that encompasses a variety of of components  the ones that we are interested in are exosomes  

4:25and there’s spherical particles with the size of  a virus so about 100 nanometers really tiny nut  

4:33tiny tiny particles then there’s another glass  called micro vesicles they’re a little larger  

4:39up to one micrometer which is the equivalent of a  bacterium and these are secreted by healthy cells  

4:46there’s also apoptolic bodies they’re  much much larger five micrometers   and they are shed by cells when they die  so they’re really not relevant in healthy  

4:56biology and for reference the cell mammalian  cell typically is about 10 micrometers large

5:05so exosomes well again there was two two  major classes that biological relevance  

5:13micro vesicles which we are not gonna  focus on today because they just flap   off the plasma membrane it’s tough  to control their cargo and and their  

5:22coal and cell to cell communication is not so well  understood we are going to focusing on exosomes  

5:29that are produced in cells in cell interior  in exosome secreting or donor cells released  

5:37into the extracellular space so this is basically  your bloodstream and then they travel to adjacent  

5:44cells I mean neighboring cells short distances or  can travel very long distances for example from  

5:51the liver to the brain in our case we talk about  even longer distances because we are talking about  

5:58exosomes in milk being absorbed and traveling  to tissues once these exosomes reach their  

6:06recipient cells they either bind to the  cell surface and trigger all kinds of  

6:12cellular responses or they internalize by fusing  the cell membrane release the carbons into the  

6:21cytoplasm of the cell or the internalized and then  later release their carbons when i say carbons you  

6:29can see an exosome right here it’s crazy what  they hold i mean there’s lots of RNA there’s DNA  

6:36there’s proteins there’s lipids it’s really  amazing for these tiny particles what all they  

6:41can hold and what they deliver to these recipient  cells to change the metabolism in recipient cells  

6:49so cartoons are nice this is a real image of  a real cow’s milk exosomes from an electron  

6:58microscope you can see that about you know this  is 200 nanometers about 100 nanometers in size  

7:04and this is how they look like in real life so  research in my lab Olga eluded this a little bit  Research in my lab

7:12i think of having two streams of research  both related to milk exosomes one of them  

7:19is the delivery of drugs and therapeutics  this is what your group is interested in  

7:24and the other stream of research is really make  exosomes human make exosomes in infant nutrition  

7:32and cosmetic exosomes in in  adult nutrition both streams  

7:39confluent merge basically at the interest  or the shared ends of promoting human healthProject aim

7:49so the project aim when i submitted my my proposal  was basically stated as assess whether milk  

7:56exosomes delivers mRNA and blah blah blah blah  and i promise at the beginning of my presentation  

8:03i will keep this very comprehensible and simple  so this big aim can be condensed to just saying  Problem SYNGAP

8:08but we want to increase SynGAP in the brain  and this aim of the proposal really addresses  

8:18a problem to see syngap patients have so there’s  two copies of the Syngap gene in a in a brain  

8:26and in syngap patients you have one functional  copy the solid the line here half half circle and  

8:36then you have one copy that does not function  this is the divide the part of the circuit  

8:44in normal individuals or healthy individuals  you have two functional copies of this in your  

8:51gene and what we want to achieve in this  project is deliver DNA that codes for SynGAP  

9:00to make exosomes to the brain and basically  restore the activity and when the missing activity  

9:08from this copy to just achieve a normal level of  SynGAP in a brain and thereby make brains healthyWhy should this work

9:18so now you want to why should this work is this  crazy idea make exosomes these nanoparticles  

9:24in big for treatment of Syngap so some of you  may remember high school biology or college or  

9:33graduate school biology and you may ask well I  mean these exosomes or anything that enters the  

9:39gut the gastrointestinal lumen is degraded  very rapidly either by gut microbiota or by  

9:48enzymes that are secreted in the gut and what  you can see here there is just reference to  

9:54three papers that showed well exosomes are  really really stable they are not degraded  

9:59in the gut lumen and that is really important  premise for for what we are doing here  

10:04started with a group in Japan in 2012 that  idea was copied by group in Toronto Canada  

10:13and then again copied and say copied not stolen  but copied by group in California they all found  

10:21the same thing these exosomes and their cargos  are very very stable in the GI tract and then  

10:28we added piece of information by showing that  even when you process milk and milk exosomes at  

10:34an industrial level for example homogenizing milk  heat sterilizing milk adjusting the fat content of  

10:43milk and so on these exosomes and their cargos  are very very stable so we really don’t lose  

10:50exosomes in the GI tract and when  processing exosomes at an industrial scale

10:58so first data piece that i’m going to show you now   in our lab we created what we call an exosome  and cargo tracking mouse. This is an extremely  

11:09complicated story so I’m going to spare you  all the details but one thing I have to tell  

11:14you though if this is an exosome produced by  this mouse it is endogenously labeled with  

11:21fluorescent proteins so in other words these  exosomes but this exosome carbon tracking mouse  

11:26they emit light very strong light which  allows us to track these exosomes in tissues

11:35in this particular experiment what we did  in order to demonstrate absorption of milk  

11:40exosomes was to foster normal pups and wild  type pups that do not express these exosomes  

11:48to a dam (mom) that expresses these fluorescent  exosomes and then control experiment we fostered  

11:56these normal pups to a normal dam that does  not express dislabled exosomes that these  

12:04mice nurse for 17 days so normally mice  are weaned at about 21 days so we wanted  

12:10to achieve a fairly long exposure to these milk  exosomes but stop the study before these little  

12:18pups transition slowly to solid foods so 17 days  is right at the end they exclusively rely on milk  

12:25at that point we harvested the tissues from these  pups and the control pups and looked at the light  

12:32in these tissues and now it’s important to  remember these normal pups again they cannot make  

12:40fluorescent exosomes or any fluorescent protein so  any fluorescent fluorescence light that you will  

12:46see in these tissues would have to come from mom’s  miracles this is the only thing that these pups  

12:52have received and then again we just dissected  the tissues and what you can see here  

12:58really a major signal in the brain and then also  quite some accumulation in the liver a little bit  

13:04in the kidney and considerable accumulation in  the intestinal mucosal gut the wall in controls  

13:12you see no signal which is what we expected in  all these tissues just flow auto fluorescence  

13:19I mean background noise in the gut intestinal  reconciliation so this is very conclusive evidence  

13:26that make exosomes transfer from mom to pups  or that medicines in general are absorbed  

13:36and accumulate in tissues including, that’s  relevant for your group, including in the brain.Does this also relate to humans

13:45When I think met the second time with  the folks from the SynGAP Research Fund  

13:52one of your panelists asked me about  yeah so mouse mouse that is nice   but what about make exosomes across species  boundaries first we are dealing with  

14:04milk cow’s milk exosomes delivering cargo syngap  to humans so in other words does this also  

14:11relate to human or other species and the answer  is yes so what we did in this experiment here  

14:18we use cow’s milk and isolated  exosomes from this cow’s milk  

14:24loaded these exosomes with synthetic compounds  and something that we chemically synthesized   in this case it was an RNA with the name  miR-375. Don’t have to remember that one.  

14:35Basically just the nucleic acid with  the fluorescent dye put these labeled  

14:41compounds in our milk exosomes and gave these cows  milk exosomes to mice by all garbage it was an  

14:49overall administration of these exosomes and then  did exactly what we had done with these other pups  

14:55in the previous slide collected tissues and  measured fluorescence in these tissues and again  

15:01for this particular compound you see strong  accumulation in the liver brain kidney and  

15:07also the GI mucosa this is a control mice that i  should have given exosomes and this compound but  

15:16not the common being included or incorporated  in exosomes you can see solutions back on noise  

15:24this one is a controlled experiment that  i don’t want to elaborate on it basically  

15:30just showed us that this compound here was  stable when we measured the tissue signal and  

15:37indicating that we did not just drag the  label that was detached from from the RNA  

15:45so basically this confirms our studies from  the previous mouse experiment saying here these  

15:51exosomes and their powers was accumulating in  tissues i do understand that we in your case are  Milk exosomes in humans

15:59most interested in working with down downstream  at least with human subjects or with patients  

16:05syndicate patients and so we had a previous  publication in which we showed demonstrated  

16:12the availability of milk exosomes in humans  so this was a milk feeding study basically  

16:18that five normal adults come to the lab  had them drink various amounts of milk the  

16:25dose of spawn study ranging between 0.25  liters all the way up to 1 liter and  

16:33i don’t know if you’re used to the metric system  so halfway there’s a large glass of milk one liter  

16:41of milk per day is pushing it i love mix so i  can easily drink a liter per day many of you may  

16:47not but i do but this is a fairly physiological  doses so we’re not trying to do something that is  

16:53really pushing or venturing into the gallons of  milk uh exposure to see a tiny effect and when you  

17:01consume when our adults consumed these amounts  of these volumes of milk you can really see  

17:08the dose response in plasmazones and that’s the  cargo that we’re measuring another micro honey  

17:14you can see the more milk they drink the more  exosome cargo appears in these subjects plasma  

17:22and returns to baseline levels after i think 24  hours but you see down here miami is our negative  

17:29control this is a microwave that’s not present  in milk and since it’s not present in milk you  

17:35would expect there is no increase in plasma  micro on a one level after milk consumption  

17:42the consumption at time zero and obviously  you don’t see any change which was reassuring  

17:49to see because then you could say okay this  is only a specific effect to make consumptionBlood brain barrier

17:56okay next question that that is really relevant  for for this project is the the challenge posed by  

18:06by a barrier that surrounds the brain so the  blood brain barrier that i’m sure all of you  

18:11have heard about it really protects the blade  the brain from the pathogens or any microbial  

18:18end of the entire brain and also excludes certain  metabolites from entering the brain so the  

18:25question for us now for the milk exosomes would  be if it goes back to the slide that i showed   you early on the question now will be what we see  here in the brain is this inside the brain this  

18:38is where we want the exosomes to go in order to  deliver the syngap or is this just exosomes being  

18:45in the blood vessels surrounding the brain and  both would produce the same signal in theory and

18:54what we did in order to answer this question  in the brain versus outside the brain  

18:59was to partner with the investigator duty  southwestern in dallas dr denise ramirez which is  

19:08really fancy technology let me go back to one so  has this fancy technology in which she can really  

19:15create 3D images of brains and these 3D  images and allow her and us to exactly  

19:22to conclude whether something is inside the brain   or just outside the brain so i’m going to show  you now is images from two brains the one that  

19:33had to make examples from mom delivered to the  pubs and then a controlled brain which is not  

19:40of course fluorescent and that’s what the knees  did so this is the part of the brain from these  

19:49fluorescent exosomes you can see  in various regions of the brain   there’s the screen fluorescence this green  light suggesting these exons get into the  

19:58brain tissues that cause the blood plain barrier  and controls i think all of you can see this  

20:05there’s really no green light meaning you know  the negative ones controls were negative so in  

20:11other words this signal is real and these  exosomes actually make it into the brainExosomes in other tissues

20:18so now many of you may wonder but you showed us  these slides and this slide that these exosomes  

20:25do not go exclusively to the brain they accumulate  also in liver and maybe kidney and so on  

20:31so isn’t that a problem doesn’t this distract  from the delivery of exosomes to the brain so  

20:38what is desired it’s actually really exclusive  localization to the brain not so great if exons  

20:46go to other tissues like the liver in this case  because of two reasons first of all accumulation  

20:53in other tissues tissues other than the brain  just take away from from the drug that you  

20:59want to get into the brain and also in some cases  depending on what you put into these exosomes the  

21:07delivery to the wrong tissue could cause adverse  side effects so you really want to get a bs  

21:14specific as possible and not deliver these cargos  to non-target tissues okay so the solution that we  Smart exosomes

21:25have to offer to this problem is the development  of smart exosomes so each tissue has a unique  

21:33most tissues have a unique zip code so you can  deliver for example through zip code zero zero   zero one which is the heart or zero zero zero  five which just a plain i’m making up these  

21:45numbers that are not scientific numbers so but  these tissues have unique zip codes and what  

21:50we will do as i will show you in a minute  we will actually slab the label shipping  

21:58label onto our exosome so this is our mega  exotherm with a single the DNA inside and this  

22:05shipping label or address label will deliver  these exosomes to the tissue that we wanted to go  Shipping labels

22:14and this is not just a wide idea crazy idea  there’s some publications out there that   talk about all these what’s called homing  peptides and shipping labels to multiple tissues i  

22:26know you will not be able to read this uh this is  just part of a table from this paper and there’s  

22:33num i mean hundreds of zip codes or shipping  labels for various tissues so muscle pancreas  

22:40brain kidney and so on so you can just pick  and choose basically from these lists which  

22:47zip code of each shipping label works best for  you and then of course demonstrate that it worksRVG

22:55so in our case what we want to do what we want to  use as a shipping label uh to get our exams to the  

23:03right zip code being the brain is you will put a  protein called rvg it’s rabies virus glycoprotein  

23:13which is called rbg under on the milk exosome and  we picked rbg because of a previous publication  

23:23that showed rpg increased the accumulation of  exosomes in the brain so this is not milk exosomes  

23:31this is mouse exosomes among cell cultures in  which these exomes were changed to have this  

23:39shipping label the rvg and here you can see  the data actually so this is a brain these are  

23:45three brains actually one mouse multiple mice are  given exosomes with the rbg this shipping label  

23:53you can see massive accumulation of the  light in the brain these are exosomes  

24:00without the label you can see that is here they  do go to the brain but not nearly as much as  

24:07the exos carrying the rbg and this is just  a brain that was not treated from a mouse  

24:13that was not treated with any exosomes  so bottom line massive enhancement of  

24:20the excess modulation in the brain if exosomes  were attacked with this rbg shipping label

24:29okay so go back to your high school and college  and graduate school uh training and biology you  

24:39now may ask me well well we all know i mean these  immune cells in the body they they don’t like  

24:45foreign particles especially macrophages they they  eat up these with these particles and any other  

24:52compound that they think should not be here and  of course us delivering cow’s milk nanoparticles  

25:00to mice or to humans they will be recognized  as foreign particles and will be eaten up  

25:06by macrophages last summer we published a paper  which we identified the transporter responsible  

25:13for this uptake and we will leverage our  knowledge and expertise in the field to  

25:23offer a solution to fix this uptake or  elimination issue that these macrophages create  

25:30so the plan for that part is to put a don’t  eat me signal on our mega exosomes so these  

25:37are peptides or proteins that the signal  or telomerico phage you must not eat me  

25:44and one such modification is a protein called  cd47 so we put that on the exosomes and this is a  

25:52experiment that we have already done in  cell cultures we use our make exosomes  

25:58with and without cd47 and measured the uptake  of these exosomes by macrophages and what you  

26:05can see here these are exosomes 100 is to  control exosomes that did not have the cd47  

26:13and if he had exams with the cd47 on the surface  elimination or uptake by the macrophages decreased  

26:21by nearly 60 so this is a massive improvement as  far as avoiding macrophage uptake is concerned

26:32so this is again a quote from this proposal  that i sent to the Syngap Research Fund  

26:40i don’t need to read this out in detail but  basically the plan is to engineer make exomes  

26:47that express both rvg remember this is the brain  homing so that will take the axons to the brain  

26:54and cd47 which is the signal that tells the  macrophages stay away i need to go to the plane

27:02okay so now the logical progression comes from  the audience would be but how do you tell a cow  

27:08to make rbg cd47 positive exosomes because cows  don’t care obviously they they make milk exosomes  

27:17optimized for nutrition of their calves  they’re not worried about drug delivery  

27:23so the solution that we have to offer in this  context is to use cells from the cause other  

27:34cells that actually produce the exosomes and  they’re called mammary alveolar cells mac t cells  

27:42so we actually generated these cells and this  will allow us to actually engineer milk exosomes  

27:49that express rbg and cd47 the one step that  we had to take was to make really sure that  MacT cell exosomes

27:59cow’s milk exosomes and exosomes produced by mac  t cells look alike so they’re very similar and  

28:07there’s not big much of a difference between these  two so we published this paper also last summer  

28:13showing exactly that so looking at the size  cosmetic versus 90 exosomes they’re just  

28:21and they need 100 nanometers so same size  basically the contours shape are the same  

28:28so this is a cosmetic exosome this is a mac  t cell exosome really look look very much  

28:34the same we also looked at carbons i mean what  do they contain and we looked at which tissues  

28:40they accumulate in and both were bhagavata  seminar i’m not going to show you this here

28:47okay now sorry for putting questions in your mouth  but the logical question would be well  

28:55are these mac t cells actually manageable  to genetic engineering can you actually  

29:00change the makeup of exosomes in these mac t  cells that’s really also what we have done in  

29:05this paper that i just mentioned this is actually  how to make t-cells look under a microscope  

29:11and so the question being can we  engineer these mac t cells to produce  

29:18exosomes with the desired modifications i’m going  to show you a light bulb here because that’s  

29:24going to be important in the next slide so we  wanted a test hunt to establish proof of concept  

29:30engineers exosomes to secrete engineered  cells to secrete exosomes that emit light  

29:38and that’s what we have accomplished as you  will see so now i have to work it walk you  Results

29:44through this uh these images so what you see in  the first panel a is normal mac t cells in a dish  

29:53compared to mac t cells that we engineered  to produce these red exosomes and i think  

30:00the difference is obvious and striking so  the normal magnesius no lead production  

30:07and these transform the engineered materials  that’s bright red so they do express the the the  

30:14light bulb basically that that we want to be in  there in order to keep cells happy you know moving  

30:20to panel b in order to keep these cells happy you  have to culture them and soak them in nutrition  

30:27fluids basically so the next thing that we looked  at was the supernatant of these cells the media  

30:34again the normal mac t cell media there was no  fluorescent in the supernatant whereas these  

30:42engineered mac t cell they secreted something in  the media that produced the red light probably  

30:51our light bulb modified exosomes uh because exons  are secreted in the extracellular space as we said  

30:59and that’s what we tested in panel c we spun this  supernatant and now we focus on this one here  

31:07we spun it at increasing g forces and initial  spin is 16000 g and the red light stayed in the  

31:16supernatant which is expected for exosomes as  as you will see in a minute in a second then we  

31:22spun even harder 83 000 g and again i mean most of  the the supernatural most of the red color stayed  

31:30in the supernatant which is what we wanted so that  was exosomes that are tiny and they’re extremely  

31:37tough to to really precipitate by spinning them in  a centrifuge so we increase the the g-force to 130  

31:47000 g looked at the medium so the supernatant  and now you can see all the color is gone  

31:53we collected the pellet which  is not shown here dissolved   dependent in saline and this is what you  see here so the exercise will be spun down  

32:02the producer red light is strong red light saying  okay what we engineered here it’s really exosomes  

32:09so the answer this question would be yeah these  mac t cells we can genetically engineer in a  

32:14very efficient way so just for your reference  130 000g is a crazy force i mean the pilots  

32:23go through training i think they have a g force of  eight so i think this it’s crazy i mean what what  

32:31what you need to apply in order to  precipitate these these exosomes

32:37okay so your question might be okay how  does it does this red bulb help us get  

32:44SynGAP into the brain not not red light well my  answer would be yeah uh that is definitely true  

32:51but we do use uh yeah want to use this irfp this  red fluorescent protein as a tracer so it makes it  

33:00very easy for us to to see where these exosomes  go in the mouse and you have actually seen data  

33:06based on this technology before we have seen  this slide that was actually my exosome sorry  

33:13that had a cd47 remember to don’t eat me signal  plus this label and again i’m competing myself  

33:21there was this almost 60 percent decrease in  exosome uptake by macrophages so very efficient  

33:29as far as don’t eat me is concerned but also  very efficient as fast as label is concernedStrategy

33:36okay now the strategy to be used in  in this single research project uh  

33:42we will create exosomes in mac t cells that  are labeled with both rbg is the brain uh that

33:52shipping label the cd47 the don’t eat me signal  and our label the light bulb so that we can  

34:00track these exosomes in the mouse  so the mouse will actually have  

34:05part of DNA in the circular form actually  but this this DNAside again irfp for  

34:13assessing distribution and expression of our genes  the rbg brain homing cd47 don’t eat me and then  

34:22we will use a technology called pcr to assess  the expression of syngar in the mouse’s brain

34:35the original plan for for this proposal was  to try all the administration in our minds  

34:43then in hindsight i started wondering why we   wouldn’t even supplement this this approach  or change the board to to nasal administration

35:00says something like precision olfactory  delivery these little spray units and you  

35:06actually deliver any medication including  milk exosomes into the nasal cavity  

35:14ideally in the upper nasal region because then  you just won’t step away from entering the brain  

35:20this is all factorable you just have to cause  medications just have to cross a tiny layer of  

35:27cells and right in the brain compared to all you  go to the GI tract has to circle back to the brain  

35:34so we will probably try both from nasal and  all the delivery in our research projectFuture

35:44so that was the point that i thought i  prepared my presentation when a hunch last  

35:51week that reached out to me was i shared the  sportboard or the slideshow with the certain  

35:58last week just for you guys to look at hans came  back saying well can you talk about different  

36:03payloads also so i figured okay i’ll go back to  my presentation change again to to include what’s  

36:09on the horizon so what what is what does the  future hold for for this research uh that allows  

36:16me to address a couple of more questions before  actually talking about what hans was asking so the  

36:24questions that we are facing with cell cultures  like mac tears yeah isn’t the exosome purification  

36:32from cell cultures very unproductive so question  being do you get enough exosomes and the answers  

36:37here that is a challenge because these cells just  don’t secrete a whole lot of exosomes enough to do  

36:44experiments in mice but definitely not enough  to to this large-scale human research study  

36:53and i’ll come back to it in a minute and then  the other question may be why do you want to   lock in on rvg and cd47 in your in your cell  lines and i think that’s a very good question  

37:06because rbg will take these exons to the brain a  person suffering let’s say for pancreatic cancer  

37:14might want to have exosomes and drugs delivered  to pancreas so we don’t really want to lock in  

37:20on certain modifications and cd47 works great  but there’s alternatives so we also playing with  

37:27another protein called cd24 which is has the same  efficacy as cd47 and we will probably try both  

37:36in combination to see if there’s some synergistic  effect that decreases macrophage uptake even more  

37:42so now we do have a really big grand pending  review by the national institutes for health  

37:49that is targeting or designed to develop what i  refer here as a universal goat so this code would  

37:58allow us to change the shipping labels and any any  surface modification like shipping label or cd47  

38:07but put it in a heartbeat so we collect the  existence from these codes and just put put put  

38:15our markers on proteins on there and so this is a  very universal very flexible uh system it’s always  

38:22but it’s scalable because these goats produce  lots of milk as opposed to make exosomes as  

38:27opposed to cell cultures and the reason why we  named codes and that pending current application  

38:36is you can create a transgenic code in two or  three years the transgenic cow takes about eight  

38:43years and the grant application covered a period  of five years and obviously grand trivius would  

38:49not like the idea of us proposing something  that we know a priori it’s not going to work  

38:55within the funding period so this is what  we call our universal code because it  

39:00allows to do any zip code on the exosomes any  payload and then the bad news for you is my

39:10intellectual property people at the university  told me that you cannot disclose the specifics  

39:15because it will compromise the patentability  of this invention so you know this code  

39:22hopefully will be out there in the future  but i cannot exactly tell you but i cannot  

39:28tell you exactly how we actually change the  zip codes on these these code make exosomes  payloads

39:35the question that the hans was asking  was asking is really yeah is there any  

39:41payloads payload is what you put into  these exosomes other than mrna or dna  

39:48that you could actually use in these modified  exosomes and the answer is yes there’s  

39:55at least two other strategies that you could use i  mean so this is the anti-exosome so you could view  

40:01it i’m going to show you two use small interfering  rna which is really tiny snippets of rna that turn  

40:09off genes so in other words tells in in your  case the singular would tell this dysfunctional  

40:16copy of the gene to shut up basically don’t  make what what you’re doing because in this  

40:23non-functional copies that do something so they  produce certain metabolites and there’s diseases  

40:29where these abnormal products are really  contributing to the disease i don’t think  

40:36ever anybody ever looked into this possibility in  the context of Syngap but for many other diseases  

40:43this is a problem it’s actually the main problem  so you want to eliminate the dysfunction of copy  

40:51the the second alternative that  i was going to share with you   that’s getting really really complicated uh in the  lab not for this talk would be the use of CRISPR  

41:03cass and that’s a gene editing tool discovered  not too long ago which allows you to compare the  

41:11dysfunctional genes so in other words you cut out  a certain section of the gene and repair it with  

41:19the normal gene and then all of that you have  two normal copies in the brain again this is very  

41:26involved because all of a sudden you have to load  these exons with proteins with different types of  

41:32RNA and DNA but it’s something that is in progress  and that’s why i want to wanted to share it with  

41:38you okay so now i want to acknowledge my funding  agencies people in the lab as of last November  funding agencies

41:48so your group is named right  here the syngap research fund   really thank you a lot for your for your support  and this is collaborators and lab members  

41:59since my lab got into this exosome field so  that was dating backwards dating back to 2013  

42:062014 then finally an acknowledgement to  Hans slashed he actually was the one who  

42:14introduced me to Syngap reached out to  me and that’s how this whole story took   off so i want to make sure i acknowledge  him for his valuable introduction to the  

42:26singapore research fund people with that i  think i’m at a stage where i’m ready to take   your questions thank you wow thank you very much  yanosh that was that was a tour de force and i  

42:37really appreciate you making it so accessible i  realized it would have been easier for you to um  

42:42just plow through some existing scientific slides  so thank you for spending your time on that  

42:48um since you had that wonderful  picture of hans up i’m gonna um   invite hans up to ask some questions but before  before i do that let me point you to the q and a  

43:00uh do you mind giannis stopping sharing your  screen so people can actually yes more than thatside effects

43:07thank you again all right this is great so while  i’m trying to figure out how to bring up Hans  

43:14if you if you can find the Q&A there’s  a number of questions the first would be   um from one of our volunteers saying would there  be side effects this mRNA treatment given that the  

43:23COVID vaccines also had some i mean you talked  about i mean i realized that’s a bit down the  

43:30path side effects in humans because right now  what just to anchor the audience what we’re  

43:36talking about is proving concept that you can  deliver to the brain and affect Syngap expression   in a mouse and if we got that the  next step would be to say okay  

43:45where do we go from here but do you want to talk  to the yeah side effects here i mean i think  

43:51i touched upon it a little bit in my presence  it’s a good question so i really appreciate that  

43:57but side effects possible yeah i mean because  you you would maybe express SynGAP in a tissue  

44:04that it’s normally not expressed in and you  don’t know what that does we try to minimize  

44:11side effects by the strategies that are shared  with you with the homing peptides so we really  

44:17want to make sure the vast majority of of the  syngap DNA so it’s in our case it’s a plasma  

44:24it’s not mRNA it will produce mRNA but the vast  majority of our RNA or DNA will go to the brain  

44:33but yeah so it remains to be seen i guess is the  answer so we have to just monitor a situation  

44:39and see if there’s any adverse side effects great  that’s a fair question a fair answer i think yeah  

44:45yeah and hans has joined us Hans do you mind if  i just let doc doctors in planning play through  

44:51the rest of Q&A and before i forget i do want to  echo uh what janos said about hans SRF is lucky to  

44:59have someone who is so obsessed with science and  our children and going out there and hunting and  

45:05finding people like professors and plenty because  as i think as the audience has gathered from the   presentation and that impressive list of funders  the 100 000 grant from SRF is not changing the  

45:17fate of the zen pleni lab uh he’s we are we are  fortunate to have um we’re fortunate to have this  

45:24work being done all right Marcos from Germany if  the milk exosomes is used for transportation of  

45:30mRNA how much will cross the blood-brain barrier  are there numbers from former studies again yeah  

45:37it’s a very good question and we do have we have  a very rough estimate about all bioavailability so  

45:43it’s not a brain so all at the minimum as of  today we say 25 of the exosomes are absorbed  

45:51causing the blood brain barrier we do not have  any quantitative data we do know it ends up in  

45:57the brain but we don’t know the percentage  or the mass that that and that’s the brain

46:06and then there was a question about was the  milk raw or processed i think you spoke to   that a little bit with one of your slides yeah  so it really doesn’t matter i mean in our case  

46:16we most of our mixed studies the cow’s milk  study we do with make from the course of a   story so it’s processed we did work somewhere with  romig either from cow or from human milk donors in  

46:28the nutrition arena but yeah typically and i will  not be use processed milk from the grocery store  

46:37that’s great and then ilya hit you with three  questions and ilya is a friend of the fund  

46:43with a child with a different disease so you  can see where he’s going is there any way to   limit where exosomes are expressed much like  AAV when we use promoters how easy is it to  

46:52pivot to other genes they’re already signing up  for more work and lastly could we package more   complex constructs where we knock down a mutant  via sh RNA or similar and over express a gene  

47:05yeah well maybe let me start with the third  question that’s the easy one because i touched  

47:10upon it by the end of my talk yeah yeah if you  have done sir a lot and small hairpin RNA and  

47:17that works like a charm we do this more in the  context of of brain cancer research now in my lab  

47:24where we turn off certain genes  kind of what i showed with this

47:30smiley face turning off telling  this one copy that is dysfunctional  

47:35to stay silent so that is definitely an  option crisper cast i think yeah there is  

47:44lots of talk about that technology in  milk exosomes or in exosomes in general  

47:50but again then packaging becomes an issue because  you will have to get everything i mean all these  

47:55components and building blocks in in one single  exosomes and uh that’s also problem when we do  

48:02our analysis i mean you measure when i talk  about the pellet of exosomes you talk about  

48:08trillions of exosomes and then there’s a  certain percentage that will have one cargo  

48:14another percentage that will have a different  cargo as a data piece it looks fine because it  

48:19says oh yeah we find everything in there but  it’s not like everything is in each and every   exosome which is what you would need and that  becomes a little trickier with the CRISPR cast  

48:29we could engineer some of the components into our  milk exosomes but then i mean the specific guide  

48:39strains and on that would be a little complicated  but it’s something that we’re working on so it’s a  

48:44very good question yeah other genes absolutely  i mean anything that you want to restore  

48:50as far as expression is concerned or knocked down  as far as si on a is concerned we can do that

48:59and then is there a way to limit where exosomes  are expressed much like aav when we use promoters   think the mailing labels we talked about right  yeah yeah that’s also a little challenging we do  

49:10have a transgenic mouse in which we knocked down  the expression of exosomes but today there’s not  

49:19one single gene that you can knock out and inhibit  exosome biogenesis 200 so the gene that we work  

49:27with knocks down exosome biogenesis by i think 80  percent so you still have some residual exosome  

49:34production in these tissues so that’s i think the  challenge i mean technically it’s possible but i  

49:40think that gene has yet to be discovered and that  is the gene that we would actually have to rely on  

49:46if you want to really suppress exosome or restrict  it with certain tissue hands this next one’s yours  

49:54yeah so jano’s fantastic to have you  involved with SRF and this is a project where  

50:01i have to manage my expectations because i’m  that sort of hopeful and one of the questions  

50:10we see host proteins make it into you know  hiv particles that butt off an infected cell  

50:19exosomes that come off the intestine intestinal  mucosa can take a hepatitis e virus with them  

50:27or an enterovirus with them are there other host  proteins that are making it into these exosomes  

50:35and could they be immunogenic that’s a good  question so i don’t know if i know that you  

50:42had to have some other events to do this morning  so at the very beginning of my talk i talked about   some of the cargos and exosomes and there is lots  of proteins in exosomes and you’re absolutely  

50:53right in theory that could elicit an immune  response what we have done in human studies and i  

50:59think that’s probably very helpful in this context  is to to study the immune response in humans after  

51:06exposure to milk exosomes and we did this both in  vivo i’m basically just giving exosomes to human  

51:12subjects but also after isolating immune cells  from human plasma and pvmcs and culturing them in  

51:20the absence or presence of exosomes and measuring  cytokine uh storms and megasms are really very  

51:29non-immunogenic at least in this model and these  model systems can be used but yeah we have to see  

51:35once we really i mean enhance the accumulation  in the brain if there’s any issues with that

51:44and it’s a little bit tangential to the side  here but pasteurization as i’ve seen in your  

51:53publication record disrupts the normal  exosome component to to cow’s milk no  

52:02you remember the paper correct i mean i talked  about this very very early on when we talked about   stability of exosomes in today’s presentation and  it’s typically the homogenization the shearing  

52:14of exosomes and the reason for that is you  apply the shear force and you actually disrupt  

52:19the membrane the pasteurization the heating had  actually no effect you can pasteurize the exosomes  

52:25and you can do fat adjustment and any  other processing and you don’t lose  

52:31the exosomes it was really just a homogenization   that that caused i think a 50 loss of exosomes  okay it’s fascinating work absolutely thank youin vitro vs cow

52:47so the next question comes from Dr Elizabeth  Heller who i’m thrilled is here with us she  

52:52is a member of our scientific advisory board and  and a grantee and an aunt to a syngapian so we are  

53:00very grateful for her and her  many roles in this community   but she asks can you explain the rationale to  make syngap exosomes in vitro versus in cow goat  

53:10is there any benefit to trying both strategies at  the start yeah well the potential benefit for sure  

53:19but as i was trying to say in my presentations if  we wanted to make transgenic goats or cows it’s  

53:27crazy expensive for this this project proposal  named or did i mention this with the universal  

53:33gold i think it’s a five million dollar proposal  NIH and with high risk i mean so it’s that

53:48expanding my given extensive answer there so  it’s NIH does transformative research about award  

53:56it’s big money and it is for high risk high  high return research so saying well we know  

54:0399 of this is not going to work but if it  works it’s a game changer that’s what this  

54:10universal goat is going to be about and yeah  go towards this cover i mean i explained my  

54:15rationale the goat you can do transgenic goat  you can do in two three years a cow eight years  

54:22do this in parallel for this particular project  it’s just not feasible i mean and that’s why the   mac t cells are such a nice model because  we can quickly engineer these mac teasers  

54:32the exosomes are basically milk exosomes  and we can make any modification everyoneSmall interfering RNA

54:48okay and Dr Heller if you want to come up and and  ask more questions more hard questions please just  

54:53just message me Suzanne Jones thank you for this  amazing work back to the small interfering RNA are  

54:59you considering looking into that methodology more  fully in the future seems like a viable solution   for missense patients so i don’t know janos  how close you are to our genetics and doctor  

55:09hello it’ll be better to speak to this but broadly  speaking two-thirds of our population have protein   truncating variants so they’re classic haplo and  i’d say another 20 based on our registry Harvard  

55:19missense mutations which are okay it’s less clear  what’s going on so i think that’s that’s what’s   behind this question yeah this context i think it  would be really good to know if if these mutations  

55:30cause i mean the production of metabolites that  contribute to to the phenotype in single patients  

55:36and as i was saying my talk i i’m not a Syngap  expert so i don’t know if if these mis-sense  

55:43mutations cause anything or cause the production  of some metabolite that contributes to the disease  

55:50if that is the case then we are knocking  down and knocking out that particular allele  

55:57would be perfect it would be much much easier  probably then than what we are trying to do here

56:05um okay and then ilia is back with another  question what is is there a packaging size  

56:10limitation yes this so i mean i don’t know how  how large a molecule can be but i mean again  

56:19these exons have about about 100 nanometers in  size so there’s there’s a physical limitation what  

56:27you can can get into these exosomes our loading  technology is very effective actually we use  

56:34very old-fashioned strategies actually just  ethanol and calcium chloride very basic  

56:40molecular biology from the old ages old days and  it works great i mean it’s i think we get about  

56:47forty percent fifty percent loading efficiency  with this technology but there’s alternatives  

56:52there’s electroporation and then transfection  so but we are happy with what protocol we haveTimeline

57:02and then last question in the chat is from  Marcos what is the timeline for this study  

57:10supposing that what we’re  after here is proof of concept  

57:16so yeah so the timeline this is a one-year  project so yeah that’s that’s my timeline

57:26wonderful i’d be happy but i’m sure hopefully  if this turns out well i mean if we really  

57:33show proof of concept i would love to continue  working with work with your group after this one   year but i think this is a good first step you’re  saying is this leading to something and then we  

57:44can discuss again i guess yeah we would  love to see that hans you’ve got more   yana so are there preclinical examples where  exosomes have modulated the phenotype of  

57:56of an animal or or ipscs let’s say  and their output well if you go to the  

58:04FDA registry or for NIH majesty of clinical  trials there is an increasing number of  

58:11exosomes not necessarily milk exosomes but exosome  trials for delivering that’s typically srna  

58:18and then increasingly now at least at a level  of torque for the crisper cast technology but i  

58:26don’t think this has ever let not let get that to  approved drug or treatment so this is all trials  

58:34in progress yeah it seems like lipid nanoparticles  are very in vogue right now yeah exactly yeah okay

58:45so yeah JJ who is asking an interesting question  what about human breast milk could human milk  

58:52express enough exosome without the immunogenicity  problem yeah that’s a very good question i mean if  

58:57you have the donors willing to give enough milk  and i expect enough exosomes that that actually  

59:04might help if there is an immunogenic potential  i mean like i said we have no evidence to suggest  

59:09that cow’s milk exons cause a problem in this  regard but if they do yeah human make actually not  

59:16a bad idea so one could try that but again then  you run into the issue well i mean if you want  

59:22to genetically engineer this exercise like rbg or  cd47 that is of course not an option with humans  

59:31and that’s where the cow’s milk is or the  code is really the way to go because you can  

59:36engineer them any way you wish okay can may i  ask you one one one sort of broad question that  

59:43isn’t specific to your presentation one of  the things when i started SRF we talked to a   lot of scientists and i heard a lot of things  i didn’t understand and i don’t remember but  

59:52one thing someone said to me  that really stuck with me is   if you want to know where neuroscience research  is going tomorrow look at where cancer research  

1:00:00was yesterday because there’s so much more  money in cancer and so much more science  

1:00:05that you know you can get a sense of the future  in nourishment from looking back a little bit   and so i know that some of your work is is being  used as you mentioned at the top in in brain  

1:00:15cancer without i mean you probably talk about this  for days but can you give us a sense of how to  

1:00:23how to think about where where this might  without making any promises and recognizing   that we’re just talking about a proof of concept  can you just paint for the audience broadly  

1:00:32where exosomes is going broadly based on  what you’ve already seen in the cancer space

1:00:39yes i mean sure sorry it’s

1:00:44first of all i agree with you i mean yeah cancer  if you look at what happened to cancer yesterday  

1:00:50you get a sense for where the field overall is  heading and then I’m really coming myself from  

1:00:56well coaching from nutrition but as far as  drug delivery is concerned our first client  

1:01:02applications are all in the cancer at the brain  cancer field and yeah then i didn’t even think  

1:01:09neurology until hans reached out to me well i  mean this is a cool technology and then maybe and  

1:01:17so yeah the answer is yes where this is going  i don’t i don’t know we have to see if we can  

1:01:23really restore gene expression to normal levels  in the brain or if like that like you said with  

1:01:29the missense mutations maybe it’s good enough  to to eliminate these missense copies i don’t  

1:01:36know i mean that’s something that we definitely  would have to look into but it’s it’s an exciting  

1:01:41field and it’s if you keep up with exosomes  in general it’s almost like the microbiome  

1:01:47there was just a few papers and publications a  few years ago and now there’s hundreds if not  

1:01:52thousands per year and not all of them are about  drug delivery on nutrition and just cell to cell  

1:01:59communication and it’s it’s already it’s a it’s a  crazy feeling it’s very very busy these days yeah  

1:02:06and we’re fortunate that hopefully this proof of  concept will get Syngap in in on that rising tide  

1:02:13so once again thank you from all our families  for for taking this project on doctors and plenty  

1:02:19it was a pleasure to talk with you i was not lying  in my opening statement how much i like your group  

1:02:26that’s very kind of you to say all right  with that give everyone their day back i  

1:02:31appreciate your time professor thank  you thank you you have a nice day bye