Ooh, I've got a few of these.

Nepeta's RP character is a mother, but Nepeta herself doesn't carry any of the traditional/expected traits. Not to mention she gets miffed pretty quickly when the idealized, non-committal motherhood doesn't work out how she's expecting it to go. Add on how well she gets along with the distilled masculinity that is Equius, her merging with Dave, and having a massive crush on somebody who has to date dated zero women, there's plenty of room for a transmasculine reading.

Speaking of Equius, he's what happens when somebody refuses to engage with the Vriskafication. Being stuck with his fists instead of the bow he wants to use (bows being a popular girl's weapon in RPGs) is a metaphor for being stuck in masculinity. His grotesque infatuation with Aradia includes a form of envy. Here is a girl that refuses to be bound by Alternian custom, and he can barely keep his composure because of it. Equius is capable of making the leap. He's even able to make feminine bodies. But time and again he refuses to grant himself what others freely take.

And we can't talk about Jade's gender without going into her home life. Grandpa Harley has views on gender that would make Caliborn proud. Men are supposed to wield massive guns and kill without hesitation. Women (and animals) are trophies to be displayed as proof of your domination over them. And when Jade gets into an argument with his stuffed corpse, the topic happens to be that Jade's weapon isn't big enough.