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Global Translational Medicine ABE gene therapy for CVDs

Atherosclerotic CVDs are caused by the narrowing engineered to change the editing windows [15,16,58,59] or to
and hardening of the artery walls and the formation enhance the position precision of ABE so that on-target
of plagues . The elevation of low-density lipoprotein editing can be achieved while reducing the bystander
[50]
cholesterol (LDL-C) in blood is a major cause of effects. In addition, machine-learning approaches have
atherosclerosis ; thus, reducing LDL-C is a well-accepted also been exploited to predict the outcome of a given ABE
[51]
strategy to treat atherosclerotic CVDs. PCSK9 is a plasma reaction in silico [60-62] , greatly reducing the costs and efforts
protein that accelerates the removal of LDL receptors in the experimental exploration of a good ABE design for
from the surface of hepatocytes, preventing the uptake a specific application.
[52]
and clearance of blood LDL-C . Most importantly, non-
sense mutations of PCSK9 in the human population are 4.2. Increasing editing efficiency
associated with lower LDL-C levels and a significantly The editing efficiency of ABE is firstly determined by the
reduced risk of coronary heart disease [53,54] , without causing design of the ABE machinery. Codon optimization and
other serious abnormalities. Thus, PCSK9 has served as a the proper installation of nuclear localization sequences
classic therapeutic target to reduce LDL-C for many years. (NLS) have been shown to enhance the performance
[13]
PCSK9 is mainly expressed and secreted by the liver; of ABE . The modulation of the linker amino acids
thus, liver-targeted gene delivery vectors, such as LNPs, between TadA and the Cas9 nickase and the coupling
provide ideal tools to genetically manipulate PCSK9 of ABE with an uracil glycosylase inhibitor (UGI) could
[63]
in vivo. In 2021, Musunuru et al. designed an elegant also improve editing efficiency . Most importantly, the
ABE system to edit the 5’ splice donor sequence in PCSK9 directed evolution of TadA has been shown as a powerful
intron 1, disrupting pre-mRNA splicing, and depositing a approach that continuously increases the editing efficiency
[55]
premature stop codon that ablates PCSK9 expression . of ABEs [9,15,16] .
They used LNPs to deliver ABE8.8 mRNA and the sgRNA In addition to the intrinsic properties of ABE itself, its
targeting PCSK9 into cynomolgus monkeys (Figure 2C) editing efficiency is also influenced by the availability and
and detected about 70% editing rate specifically in the expression levels of ABE components in the cells, which
liver. This one-time treatment results in almost complete is determined by the gene delivery methods. For example,
elimination of blood PCSK9, as well as over 50% reduction LNP has be validated as a robust vector to deliver ABEs
of LDL-C for up to 8 months, suggesting the capacity to to the liver to achieve high editing efficiency . With the
[55]
lower LDL-C with one shot for life . In July 2022, this recent development of new formula, LNPs can also target
[55]
investigational drug has been dosed in the first human the lungs, the spleen, and a couple of other organs , but
[33]
in a phase 1b clinical trial, as a potential treatment whether these new tools will lead to robust editing in these
for heterozygous familial hypercholesterolemia . If organs remains to be examined.
[56]
successful, this game-changing therapy is expected to be
repurposed for other forms of atherosclerotic CVDs in the As compared to LNPs, AAV vectors have been
future. demonstrated to permit base editing in more organs, but
[30]
the editing efficiency is relatively low . The small payload
4. Future challenges of AAV vectors has been the major limiting factor, so
4.1. Identifying and expanding the editable loci in the dual-AAV systems, cells can be edited only when
both AAV vectors transduce the same cell. With the
The first step in designing an ABE study is to determine development of more compact ABE tools and the careful
if the target adenosine is editable. At present, this task is design of AAV vectors, recent studies have started to report
challenged by several factors including the availability of a all-in-one AAV-ABE vectors , which indeed increased
[31]
PAM sequence at the appropriate position, the presence of editing efficiency as compared to the dual-AAV systems.
other adenosines in the editing window that might result However, these vectors have pushed the AAV payload to
in unwanted bystander effects, as well as the sequence the extreme, leaving little space for further modifications
and chromatin neighborhood that might undermine the of these vectors.
editing efficiency.
4.3. Reducing the undesired editing
To overcome these limitations, huge progress has been
made to introduce new Cas proteins with distinct or less The precision of ABE reaction determines the safety of the
restrictive PAM sequences [17,24,25,57] in to ABEs. For example, relevant therapies. Thus, undesired editing by ABEs needs
a recent study equipped TadA8e with multiple distinct Cas9 to be carefully monitored when developing new drugs.
variants and collectively offered editability to about 82% These unwanted editing can be grouped into two types,
adenosines in the human genome . TadA was further namely, the ones on the targeted site and the ones on the
[31]

Volume 2 Issue 1 (2023) 7 https://doi.org/10.36922/gtm.232