Page 74 - GPD-3-3

Gene & Protein in Disease Gene therapy progress for DEB

addressing the root cause of diseases. However, the need iPSCs remain unknown. A selection marker-free HDR
for customized methods for specific mutations poses a approach reported by Bonafont et al. obtained 35% to
75
significant economic burden, particularly for DEB, which 50% DNA correction frequency in primary RDEBK cells.
lacks specific mutation hotspots. The exon deletion strategy Although bulk populations of RDEBK cells edited by this
can target all mutations within the same exon, so the same approach were able to achieve dermo-epidermal adhesion
therapeutic agent can benefit a larger number of patients. upon transplantation onto nude mice, co-expression of
However, this approach requires simultaneous targeting truncated C7 was also detected, which has the potential to
and editing of two genetic sites, increasing complexity and impede triple-helix formation and hence proper secretion
efficiency challenges. of C7. HDR-guided gene correction represents an ideal
gene editing strategy; however, its efficiency is often too
2.3.2. Gene reframing low to achieve the desired therapeutic effect. To overcome
In exon deletion therapy, the excised targeted exon is this limitation, advancements in both gene editing tools
repaired through non-homologous end joining (NHEJ) and delivery methods are necessary.
to restore DNA integrity. NHEJ is more frequently used
for gene reframing and gene knockout by inducing indels 2.3.4. Base editing
(inserts or deletions) in the target genomic loci. Through With the rapid CRISPR/Cas-based gene editing tool
NHEJ, Takashima et al. achieved 34% corrected gene development, genome editing can be achieved by single-base
70
reframing from a recurrent frameshift mutation that editing. Base editing tools enable site-specific modifications
resulted in premature termination codons in RDEB without causing DSB or requiring an exogenous donor
fibroblasts (RDEBF). However, not all NHEJ-mediated template. These modifications can mediate either C-G to
COL7A1 variants produce a functional protein; screening T-A conversions (cytosine base editors, CBEs) or A-T to
is subsequently required for the gene-corrected single G-C conversions (adenine base editors, ABEs). Osborn
77
clones. More recently, Kocher et al. reported a predictable et al. electroporated mRNA of ABEmax (the latest
41
44
CRISPR/Cas9-mediated COL7A1 reframing by precisely advanced version of ABEs) and sgRNAs into primary
inserting a single adenine in the sense strand at the target RDEBF and iPSCs derived from the RDEBF for correcting
site and restored C7 expression in more than 70% of mutations of c.553C>T and c.1573C>T. Although the
RDEBK cells. Such high-efficiency approaches could be mutation correction efficiency was only 23.8% and 8.2%
considered for epidermal engraftment with bulk-treated at c.533 and c.1573 targets in genomic DNA, respectively,
samples, obviating the need for single-cell expansion. the functionality of edited cells was observed both in vitro
and in vivo. Another group using ABEmax for base editing
2.3.3. Gene correction by homology-directed repair in fibroblasts also achieved similar correction efficiency
(HDR) for the mutations of c.2005C>T and c.3631C>T. More
46
In contrast to NHEJ-mediated gene editing, homology- recently, Sheriff et al. achieved 94.6% gene correction
78
directed repair (HDR) with a homologous sequence as a efficiency by electroporating mRNA of ABE8e. Besides
template provides precise and locus-specific correction the ABEs, CBEs have also been tested for restoration of
of mutations. TALEN nuclease, meganuclease, and C7 expression in primary fibroblasts and iPSCs harboring
72
71
CRISPR/Cas9 nuclease 73-76 have all been used to create a mutation of c.425A>G. Base editors offer higher gene
45
either double-strand breaks (DSB) or single-strand breaks, editing efficiency compared to HDR-based gene editing, as
for gene correction of mutations in COL7A1. HDR-based they do not require DSB or donor DNA for precise editing.
gene editing not only requires a DNA break at the targeted However, the use of base editors is also accompanied by
loci but also needs to replace the mutated gene with the limitations, including unwanted on-target bystander edits
exogenous template. Therefore, achieving high efficiency and off-target effects. 79
of gene editing is a considerable challenge, and requires
antibiotic cassettes and recombination systems such as Cre/ 2.3.5. Prime editing (PE)
loxP to eliminate the cassettes for isolating correctly edited PE provides another alternative solution for precise repair
clones. Moreover, the cells for HDR editing also need to by fusing an engineered reverse transcriptase to the Cas9
have unlimited self-renewal capacity, such as induced nickase domain and working with a special prime editing
pluripotent stem cells (iPSCs). Once gene correction has guide RNA (pegRNA) to encode a reverse transcription
been confirmed, the iPSCs are differentiated into epidermal template. The advantages of Cas9 nickase and pegRNA
keratinocytes and/or fibroblasts for transplantation. make this technique more efficient in gene correction
However, the long-term skin regeneration capacity and over the traditional HDR-base correction of pathogenic
safety profile of keratinocytes and fibroblasts derived from mutations, as it does not require DSB and provides the

Volume 3 Issue 3 (2024) 5 doi: 10.36922/gpd.4047