That's a complicated question, because there are different types of Rule 63. But honestly, it really depends on how it's written. Most of the time these kinds of things, at least in mainstream media, aren't written with a trans theme in mind. It's either an AU where they were simply born as the opposite sex, or a temporary hurdle to overcome (for example, an evil wizard turned the hero into a woman and now he has to find a way to turn back). Any gender confusion is either played for cheap laughs, or to emphasize the challenge that needs to be overcome (or both). Fanart rarely goes into the details of WHY they're the opposite gender, unless it's explicitly trans art or genderbender fetish art, so you can imagine whatever you want.
In most cases, unless the r63 comes with an obvious trans theme or story, it's safe to just assume it's a non-trans AU version of the character.