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Global Translational Medicine ABE gene therapy for CVDs

A

B

Figure 1. The working model of adenine base editing and cytosine base editing. (A) The adenine base editor (ABE) consists of adenosine deaminase
TadA and Cas9 nickase (nCas9). SgRNA guides TadA-nCas9 to target the genomic DNA sequence by complementary base pairing. nCas9 unwinds DNA
and exposes adenine on a single DNA strand for TadA-based editing. nCas9 also cleaves the non-edited DNA strand to facilitate DNA repair. Adenosine
deaminase converts adenosine (A) to inosine (I), which is recognized as guanosine (G) in DNA repairing. Consequently, the ABEs mediate DNA base
editing to convert A·T to G·C. (B) The cytosine base editors (CBEs) consist of cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA
glycosylase inhibitor (UGI). SgRNA guides APOBEC1-nCas9-UGI to target the genomic DNA by complementary base pairing. nCas9 unwinds DNA to
generate an R loop and expose cytidine for APOBEC1-based editing. nCas9 cleaves the non-edited strand to facilitate DNA repair. Cytidine deaminase
converts cytosine (C) to uracil (U), which is recognized as thymine (T) in DNA repairing. UGI inhibits uracil N-glycosylase (UNG) to prevent the reversal
of U·G mismatch back to C·G base pair. Consequently, the CBEs mediate DNA base editing to convert C·G to T·A.

while a broader editing window means the increased gene delivery vectors of ABEs include recombinant adeno-
likelihood of editing the target adenosine, it will also associated virus (rAAV) vectors and lipid nanoparticle
increase the bystander effect by introducing unintended (LNP) vectors.
editing of other nucleotides, particularly other adenosines, rAAVs are viral particles that were engineered from
within the same window.
the adeno-associated virus of the dependovirus genus of
A major strategy to broaden the scope of editable the parvoviruses [26,27] . An rAAV particle is composed of
adenosines is to fuse TadA with a Cas effector protein that a protein capsid and an enclosed single-strand DNA of
uses different or less restrictive PAM sequences. These Cas less than ~5kbp. As a non-pathogenic virus, rAAV can
orthologs can be discovered from the wild microbiome. effectively transduce a number of organs, including the
Good examples include the wide variety of Cas9 and Cas12 heart, with relatively low immunogenicity and toxicity.
family members [21,22] . Cas proteins can also be engineered As of January 1, 2023, six rAAV-based gene therapy drugs
to alter their PAM sequences. Successful examples include have been federally approved for the treatment of different
SpCas9 variants SpCas9-VRQR , which recognizes NGA diseases, building an excellent safety, and effectiveness
[23]
as the PAM. Other commonly used Cas9 variants include record for this new drug format . The trademark names
[28]
SpCas9-NG and SpG , which both use NGN as the of these drugs are Glybera, Luxturna, Zolgensma, Upstaza,
[24]
[25]
PAM. Strikingly, the recently developed SpRY mutant uses Roctavian, and Hemgenix.
the NRN (R means A or G) or NYN (Y means C or T) PAM
and almost completely circumvents the PAM restraints . The coding sequence of ABE7.10 is about 5.4 kbp in
[25]
[9]
length, beyond the packaging capacity of rAAV vectors .
2.3. The gene delivery vector The mainstream solution of this problem harnesses the split
To treat CVDs, the genome editing tools need to be intein system to allow two parts of the proteins to trans-
effectively delivered to the cells that play a primary role in splice into a full-length protein . Therefore, ABE can be
[29]
the disease. At present, the most successful and popular split into two halves, each being delivered by two separate
Volume 2 Issue 1 (2023) 3 https://doi.org/10.36922/gtm.232