Data from: Creation of de novo cryptic splicing for ALS/FTD precision medicine

A system enabling the expression of therapeutic proteins specifically in
diseased cells would be transformative, providing greatly increased safety
and the possibility of pre-emptive treatment. Here we describe “TDP-REG”,
a precision medicine approach primarily for amyotrophic lateral sclerosis
(ALS) and frontotemporal dementia (FTD), which exploits the cryptic
splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF)
in order to drive expression specifically in diseased cells. In addition
to modifying existing cryptic exons for this purpose, we develop a
deep-learning-powered algorithm for generating customisable cryptic
splicing events, which can be embedded within virtually any coding
sequence. By placing part of a coding sequence within a novel cryptic
exon, we tightly couple protein expression to TDP-LOF. Protein expression
is activated by TDP-LOF in vitro and in vivo,
including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition
to generating a variety of fluorescent and luminescent reporters, we use
this system to perform TDP-LOF-dependent genomic prime editing to ablate
the UNC13A cryptic donor splice site. Furthermore, we design a
panel of tightly gated, autoregulating vectors encoding a TDP-43/Raver1
fusion protein, which rescue key pathological cryptic splicing events. In
summary, we combine deep-learning and rational design to create
sophisticated splicing sensors, resulting in a platform that provides far
safer therapeutics for neurodegeneration, potentially even enabling
preemptive treatment of at-risk individuals.

一种能够仅在病变细胞中特异性表达治疗性蛋白质的系统将具有变革性意义，可大幅提升治疗安全性，并为抢先治疗提供可能。本文介绍了“TDP-REG”——一种主要针对肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）和额颞叶痴呆（frontotemporal dementia, FTD）的精准医疗策略，其利用TDP-43功能丧失（TDP-43 loss-of-function, TDP-LOF）的细胞中发生的隐秘剪接（cryptic splicing）事件，实现仅在病变细胞中驱动蛋白质表达。
除针对该用途改造现有隐秘外显子外，我们还开发了一种深度学习驱动的算法，用于生成可定制的隐秘剪接事件，这类事件几乎可嵌入任意编码序列（coding sequence）中。通过将部分编码序列置于新型隐秘外显子内，我们将蛋白质表达与TDP-LOF紧密耦联。该蛋白表达可在体外（in vitro）和体内（in vivo）环境中被TDP-LOF激活，包括由胞质TDP-43聚集诱导的TDP-LOF。
除构建多种荧光与发光报告基因外，我们还利用该系统开展了依赖TDP-LOF的基因组先导编辑（genomic prime editing），以敲除UNC13A的隐秘供体剪接位点。此外，我们设计了一组严格门控、自主调控的载体，其编码TDP-43/Raver1融合蛋白，可挽救关键的病理性隐秘剪接事件。
综上，我们结合深度学习与理性设计，构建了精密的剪接传感器，从而打造出可为神经退行性疾病提供更安全治疗方案的平台，甚至有望实现对高危人群的抢先治疗。