Research and development MDMAv2

[surge]

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.

I saw it clearly with the tolerance of Adenylyl cyclase and apparently it is necessary to prevent it. It is as strong as the tolerance of the receptor and therefore with DREADD tolerance is not resolved but only the side effects of MDMA that derive from the lack of selectivity are resolved. This means that MDMAv2 relies totally on stem cells to resolve the tolerance of the whole cell and it is not possible to do without it...

UPDATE: lol it is possible to engineer dreadds to not sensitize Adenylyl cyclase https://sci-hub.do/https://www.sciencedirect.com/science/article/abs/pii/S0014299915300133

 

[Skorpio]

H

UPDATE: lol it is possible to engineer dreadds to not sensitize Adenylyl cyclase

How so? Are you referring to the disinhibitory pka phosphorylation of AC V/VI? I think that is a feedback mechanism; I don't quite get how you could prevent that with only dreadd modifications.

 

[surge]

H

How so? Are you referring to the disinhibitory pka phosphorylation of AC V/VI? I think that is a feedback mechanism; I don't quite get how you could prevent that with only dreadd modifications.

I think it is possible to engineer the DREADDs with random mutagenesis to not release the Gβγ subunit and so inhibiting part of the sensitization.

 

[surge]

Anyway I'm thinking about expressing a PKA antibody with a gene switch-on to mimic decrease in cAMP and thus bypassing all tolerances factors.

 

[Skorpio]

Anyway I'm thinking about expressing a PKA antibody with a gene switch-on to mimic decrease in cAMP and thus bypassing all tolerances factors.

How would it get in cells.

Also a neutralizing cell-permeable pka antibody would be profoundly toxic.

PKA has so many roles in cells it is really really hard to dissect the desirable effects of PKA activation from the less desirable effects.

A majority of drugs target g protein coupled receptors. This is partially because they are located on the cell surface so more readily accessible by a larger range of compounds, but also because the vast diversity of GPCRs, and their subsequent expression in different contexts allows for modulation of PKA signaling (among other 2nd messenger targets) in a much more nuanced manner than global inhibition or activation.

PKA inhibitor drugs exist, but don't see use in the clinic even in the context of chemotherapy where pan-ERK inhibition is commonplace.

Conversely forskolin, which activates adenylyl cyclase really lacks utility as a medicinal/recreational drug because it's effects of increasing PKA signaling are untargeted. This results in side effects occurring before beneficial effects can be reached.