Research and development MDMAv2

[surge]

For a few months ago I am developing something I called MDMAv2. Practically, both for personal problems that lead to me to eat antidepressants every day both for pure fun, I started searching for a way to develop a technique to have the effects of the MD we all know without side effects and without incurring problems such as tolerance , not even with chronic intake.
I state that I have no degree, but for a year I dedicate my day of my day to biotechnology and, as I had already said, it has been a few months since I am dedicated to this new project.

The project does not consist of a new molecule, but in developing genetic therapy that goes to add receptors created in the laboratory called DREADDs to neurons of interest. These receptors were designed to activate to molecules that would otherwise be inert as the CNO. For example, adding them only in neurons and excluding heart cells that express the serotonine receptor 2b will not have the cardiotoxicity that has MDMA and avoiding to express them in neuroendocrine neurons are avoided things like teeth grinding. Or again, avoiding expressing them where serotonergic autoreceptors are expressed will avoid negative feedbacks and therefore tolerance and down of the following days. If the normal functioning of the human body are merged, it will remain unchanged since they are used only to drug use.
So the assumption is very simple, we do the injections of genetic therapy to add these dreadds only in the neurons of interest and then when you want to feel the effects just choose one of the many existing molecules on the market to activate them.

However, here is this nice thing I wanted to share with you. If someone wants to help me, I'd love to!

 

[HOOH]

such as tolerance , not even with chronic intake.

I don't know if that's possible with anything, even something as endogenous as DMT, also I don't think inserting DREADDs into humans is going to be plausible in the near future

 

[surge]

I don't know if that's possible with anything, even something as endogenous as DMT, also I don't think inserting DREADDs into humans is going to be plausible in the near future

Well, at the moment DREADDs are possibile in primates and they are trying to use them as an antiepileptic therapy.

 

[Skorpio]

So how are you going to express them homogenously without germline editing. Adenoviruses are a lot harder to work with than most papers let on, and as far as I know there aren't any SERT CRE human lines yet.

I am down to follow you down this rabbit hole though. I've always fancied DREADD humans as wonderfully dystopic in a cyber punk type way.

 

[HOOH]

Well, at the moment DREADDs are possibile in primates and they are trying to use them as an antiepileptic therapy.

How do they swap the normal receptors for DREADDS though

 

[surge]

How do they swap the normal receptors for DREADDS though

They don't swap, they Just add the DREADDs.

 

[HOOH]

They don't swap, they Just add the DREADDs.

Well how do they do that? And selectively, so they don't add the DREADDS onto wrong neurons

 

[surge]

Well how do they do that? And selectively, so they don't add the DREADDS onto wrong neurons

In humans the most plausible approch is with viral vectors, and they use cre-loxp to express DREADDs only in neurons with a given biomarker.

 

[Gaffy]

Screenshot-20210726-133038

Image Screenshot-20210726-133038 hosted in ImgBB

ibb.co

This

 

[Skorpio]

In humans the most plausible approch is with viral vectors, and they use cre-loxp to express DREADDs only in neurons with a given biomarker.

How are you going to get specific neuron populations to express cre?

 

[surge]

Negative feedback and kinase and second-level messenger adjustments do not resolve with this design. I think these types of adjustments are negligible even with daily use, but a biohacker I was talking to who is not in this forum doesn't think the same way. Anyway... I think I'll do some in vitrio experiments to understand of how much tolerance improves by over-expressing the receptors as I said in the first post.

 

[surge]

How are you going to get specific neuron populations to express cre?

Using biomarkers found only in that type of neurons

 

[Skorpio]

Using biomarkers found only in that type of neurons

Yeah but you need them to have cre in that population from germline. How do you propose making say a SERT Cre human? Once you have that, all kinds of genetic manipulation would be feasible, I just don't know how you would get cre in various populations.

Also how would you use dreadds to mimic mdma? MDMA has multiple targets that are not GPCRs (such as SERT, NET, DAT). Dreadds mimic GPCRs, idk of any type of transporter that has external ligand control.

 

[surge]

Yeah but you need them to have cre in that population from germline. How do you propose making say a SERT Cre human? Once you have that, all kinds of genetic manipulation would be feasible, I just don't know how you would get cre in various populations.

Also how would you use dreadds to mimic mdma? MDMA has multiple targets that are not GPCRs (such as SERT, NET, DAT). Dreadds mimic GPCRs, idk of any type of transporter that has external ligand control.

SERT produces increased concentrat of serotonin, and I can mimic that with DREADDs. I Need to experiment how much the receptors are activated with mdma and mimic that with DREADDs. Do you have any opinioni about the remaining regulations?

 

[Skorpio]

SERT produces increased concentrat of serotonin, and I can mimic that with DREADDs. I Need to experiment how much the receptors are activated with mdma and mimic that with DREADDs. Do you have any opinioni about the remaining regulations?

Wait, are you proposing to transfect multiple separate dreadds that mimic the action of multiple serotonin receptors? This would be leaving out dopamine and norepinephrine receptors too. (also you are going to have to get a lot of different cre drivers, as the distribution of different serotonin receptors is heterogenous). Also, these receptors have different activities, with subtypes of each receptor coupling to different g proteins (Gs, Gi, and Gq), which would be an impossible distinction to make using only cre as an expression system.

Infection with viruses is pretty spotty. For every mouse that you see in the paper with a dreadd in a specific circuit there is a score of mice that did not make the cut (as in have a high enough infection rate to generate an effect). Working with humans you really don't have the luxury of do overs.

I still think the thing that renders this work impossible is that cre lines are produced through gene editing at the embryonic state to ensure homogeneity of expression (because that way you need to infect fewer cells, which then duplicate with the same expression level throughout the organism).

Typically crosses (such as mice with multiple genes producing cre) are formed by breeding individual mice with cre in different genes, then finding the offspring with the desired genetics. To do this in humans would be unethical to the degree of the worst human experiments ever performed.

At first when I was typing the response, I felt that it was a task that was completely unfeasible, but a neat thought experiment. After considering the diversity of the receptors you would need to activate, I think it is actually impossible to do (even if you had the technology and allowance to breed cre humans). I do think mimicking some drug effects would be possible (ie mu opioid agonists would only need cre in cell types expressing mu opioid receptors, which could be cool because the dreadds would not couple to beta arrestin), but anything that involves neuotransmitter release (and subsequent activation of whole families of receptors) is pretty much truly impossible.

Like honestly you would be better off engineering from the ground up a form of VMAT2 that can be controlled by a ligand or light or something. And that is also a herculean task.

Edit:

One factor that could be used to increase heterogeneity of receptor signaling would be using both a population of dreadds activated by salvinorin b and one by clozapine n oxide.

This doesn't make the project less impossible, but does provide slightly more tools.

 

[surge]

Wait, are you proposing to transfect multiple separate dreadds that mimic the action of multiple serotonin receptors? This would be leaving out dopamine and norepinephrine receptors too. (also you are going to have to get a lot of different cre drivers, as the distribution of different serotonin receptors is heterogenous). Also, these receptors have different activities, with subtypes of each receptor coupling to different g proteins (Gs, Gi, and Gq), which would be an impossible distinction to make using only cre as an expression system.

Infection with viruses is pretty spotty. For every mouse that you see in the paper with a dreadd in a specific circuit there is a score of mice that did not make the cut (as in have a high enough infection rate to generate an effect). Working with humans you really don't have the luxury of do overs.

I still think the thing that renders this work impossible is that cre lines are produced through gene editing at the embryonic state to ensure homogeneity of expression (because that way you need to infect fewer cells, which then duplicate with the same expression level throughout the organism).

Typically crosses (such as mice with multiple genes producing cre) are formed by breeding individual mice with cre in different genes, then finding the offspring with the desired genetics. To do this in humans would be unethical to the degree of the worst human experiments ever performed.

At first when I was typing the response, I felt that it was a task that was completely unfeasible, but a neat thought experiment. After considering the diversity of the receptors you would need to activate, I think it is actually impossible to do (even if you had the technology and allowance to breed cre humans). I do think mimicking some drug effects would be possible (ie mu opioid agonists would only need cre in cell types expressing mu opioid receptors, which could be cool because the dreadds would not couple to beta arrestin), but anything that involves neuotransmitter release (and subsequent activation of whole families of receptors) is pretty much truly impossible.

Like honestly you would be better off engineering from the ground up a form of VMAT2 that can be controlled by a ligand or light or something. And that is also a herculean task.

Edit:

One factor that could be used to increase heterogeneity of receptor signaling would be using both a population of dreadds activated by salvinorin b and one by clozapine n oxide.

This doesn't make the project less impossible, but does provide slightly more tools.

I need one DREADD for every receptor type. For serotonin I need only two distinct DREADDs, Gi for 5ht1 family and Gq for 5ht2 family. But yes you are right about having multiple CRE, I need a CRE for every cell population expressing a different receptor faimly. Thanks for the suggestion about DREADDs multiplexing, this can be used to obtain different effects with different drugs. I talked only about CNO activated DREADDs for semplicity.

 

[Skorpio]

I need one DREADD for every receptor type. For serotonin I need only two distinct DREADDs, Gi for 5ht1 family and Gs for 5ht2 family. But yes you are right about having multiple CRE, I need a CRE for every cell population expressing a different receptor faimly. Thanks for the suggestion about DREADDs multiplexing, this can be used to obtain different effects with different drugs. I talked only about CNO activated DREADDs for semplicity.

I wonder if the dreadd receptors could be modified to couple beta arrestin or do things like form heterodimers with other gpcrs. I wonder much optimization that would take, and if you could just graft the dreadd binding domains onto receptors with those tendancies and get function? (I feel like ligand binding plays into it somewhat normally, so probably would need to lock the receptor in one state or the other (ie coupling to g proteins or beta arrestin), unless one came up with a range of different flavors of dreadd ligands (like partial agonists)).

 

[surge]

I wonder if the dreadd receptors could be modified to couple beta arrestin or do things like form heterodimers with other gpcrs. I wonder much optimization that would take, and if you could just graft the dreadd binding domains onto receptors with those tendancies and get function? (I feel like ligand binding plays into it somewhat normally, so probably would need to lock the receptor in one state or the other (ie coupling to g proteins or beta arrestin), unless one came up with a range of different flavors of dreadd ligands (like partial agonists)).

In the end DREADDs are simply M3 receptors, so I don't think you can do those things just couse are DREADDs.

Btw I think I found a solution for the second messanger tollerance problems and the difficulty for the gene edit. The secret is to make stem cells and modify them to have DREADDs. This will make it easier than modifying existing neurons and stem cells pass the BBB very easily. In addition, the stem cells will create new neurons with baseline tolerance, so even when the tolerance of the second messenger is reached, it will be enough to repeat the therapy. Someone did this with opioid tolerance and without DREADDs of course https://ascpt.onlinelibrary.wiley.com/doi/am-pdf/10.1002/cpt.959

 

[Skorpio]

In the end DREADDs are simply M3 receptors, so I don't think you can do those things just couse are DREADDs.

Btw I think I found a solution for the second messanger tollerance problems and the difficulty for the gene edit. The secret is to make stem cells and modify them to have DREADDs. This will make it easier than modifying existing neurons and stem cells pass the BBB very easily. In addition, the stem cells will create new neurons with baseline tolerance, so even when the tolerance of the second messenger is reached, it will be enough to repeat the therapy. Someone did this with opioid tolerance and without DREADDs of course https://ascpt.onlinelibrary.wiley.com/doi/am-pdf/10.1002/cpt.959

Growing stem cells into neurons is fairly hard. I have direct experience using cas9 to edit induced pluripotent stem cells, and differentiating them to study disease states in a different organ, and the workflow is like 4 months of daily culture and procedures to get a single indel (which can be pretty non specific, as long as a frame shift is induced to disrupt the gene).

Typically differentiation to specific cell types is fairly finnicky (think a 96 well plate, so that a high failure rate is acceptable).

Stem cells have a lot of quirks when it comes to lentiviral infection, you need to use weak promoters, as they silence viruses with strong promoters like cmv.

I actually think germline editing would be a more robust approach, as you would have the potential to alter pre-exisiting circuits, rather than having a population added into the brain to give the effect.

Serotonergic signaling is very circuit dependant, often you can stimulate very small populations of neurons to get an effect. Opioid withdrawal is tied a lot more to an innapropriate immune response from microglia, which a generalized stem cell infusion has a better chance of treating non specifically.

 

[surge]

I've created a telegram group to research those things @BiohackTheDick Is the username, at the moment there is @ibtisam midlet and another one who is not in the forum (but he will sign up I think). We are all interested in reversing tolerance. Everyone is welcome!!!

https://www.jbc.org/article/S0021-9258(20)40750-1/fulltext DREADDs engineered to not interact with beta arrestin pathways (and so without internalization of the receptor)!!!