Page 73 - GPD-3-3

Gene & Protein in Disease Gene therapy progress for DEB

showed comparable recovery when treated with a placebo. it from being recognized by the splicing machinery. As
Building on the successful application of B-VEC in treating a result, the exon is removed along with its surrounding
open wounds in DEB patients, further investigations have introns, effectively bypassing the mutation and generating
been conducted to evaluate its potential for ocular use. The a truncated yet functional protein. The specific C7
Wang group 30-32,53 has dedicated years of research to the structure, featuring a collagenous domain encoded by
development of gene replacement therapy for DEB based three repeated amino acids, is ideal for the exon-skipping
on highly branched poly(beta-amino ester)s (HPAEs). They strategy. The feasibility of exon skipping of COL7A1 was
utilize HPAEs as vectors for delivering plasmid/mini-circle confirmed with the truncated mini-C7 function test,
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DNA encoding full COL7A1 cDNA. The effectiveness of and natural exon skipping in DEB patients. As a small
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HPAE-based gene replacement therapy has been validated molecule, AONs are well suited for intracellular delivery
through both in vitro and in vivo experiments. In ongoing and can be applied with various carriers as in vivo
pre-clinical studies, Amryt Pharma Plc. is currently treatment, thereby avoiding ex vivo autologous cell culture.
advancing a non-integrating in vivo treatment (AP103), The topical wound treatment QR-313 entered a phase 1/2
which involves polymer-based delivery of full-length clinical trial using AON-based COL7A1 exon 73 skipping
COL7A1 cDNA through repeated topical applications (ClinicalTrials.gov Identifier: NCT03605069), but the trial
(EMA Orphan designation: EU/3/20/2342). was terminated due to low patient enrolment. The same
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Most RDEB gene replacement therapies developed so sponsor (Phoenicis Therapeutics, Massachusetts, USA) has
far depend on viral carriers, as viral carriers have high gene now started a new phase 1/2 clinical trial (ClinicalTrials.
transduction efficiency. However, these gene replacement gov Identifier: NCT05529134), in which they used an
therapies are still limited by: (1) the transient phenotypic improved formulation (AG21) that increased exon 73
correction, (2) the complex viral production procedure, skipping efficiency threefold compared to the previous
(3) the small gene packaging capacity of viral vectors, version.
and (4) the non-specific expression pattern in transgenic While SMaRT and AONs offer advantages in terms of
epidermis. Progress in gene replacement will be advanced gene size and delivery, effective and safe delivery methods
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using non-viral delivery vectors with capacity for larger still require significant development. Furthermore,
genes, and significantly lower immunogenicity. therapeutic benefits of approaches based on pre-
mRNA regulation face limitations due to their transient
2.2. Pre-mRNA regulation therapeutic effects, necessitating lifelong administration
COL7A1 expression can be reprogrammed at the pre-mRNA for conditions like DEB. In addition, the potential for non-
level. DEB therapies targeting pre-mRNA regulation have specific splicing and mutagenesis remains a concern with
been developed through two main strategies: Spliceosome- SMaRT and AONs. Therefore, continued efforts in refining
mediated RNA trans-splicing (SMaRT) and antisense these strategies are essential to improve their safety and
oligoribonucleotides (AONs)-mediated exon skipping. efficacy for long-term therapeutic use.
SMaRT utilizes the spliceosome within cells to perform 2.3. Gene editing approaches
gene correction specifically within introns at the pre-
RNA stage. This is achieved by introducing a therapeutic 2.3.1. Exon deletion strategy
RNA known as a pre-trans-splicing molecule (PTM) to Exon deletion is similar in principle to exon skipping,
cells, where it binds to an endogenous target pre-mRNA. and this concept was initially proven by Bornert et al.
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The wild-type coding sequence from PTM replaces the by validating the function of a shorter recombinant C7
endogenous target’s pre-mRNA through trans-splicing, protein encoded by a COL7A1 gene lacking exon 13 or 105.
resulting in a reprogrammed mRNA with a new sequence Subsequent studies on exon deletion focused on deleting
that extends from 3’ to 5’. Both 3’ PTM 61,62 and 5’ PTM COL7A1 exon 80, a region highly prevalent to mutation
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have been employed to correct the mutated COL7A1 gene within the Spanish RDEB patient cohort. Gene editing
in DEB treatment through the replacement of exons 1 – 15 tools, including TALENs nuclease and CRISPR/Cas9
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and exons 65 – 118 using SMaRT. nuclease, 39,40,67,68 were used to delete exon 80 which led to
AONs are synthetic polymers of small nucleic acids, the restoration of dermo-epidermal cohesion ex vivo and
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such as 2’-O-methyl phosphorothioates, locked nucleic in vivo. More recently, Du Rand et al. validated the exon
acids, or phosphorodiamidate morpholinos. Their purpose deletion strategy at previously untargeted COL7A1 exons,
is to modulate the splicing of specific exons in pre-mRNA including exons 31, 68, and 109.
that contain disease-causing mutations. By binding to the Gene editing therapy offers the potential for permanent
target exon in a complementary manner, AONs prevent correction of genetic mutations at the DNA level,

Volume 3 Issue 3 (2024) 4 doi: 10.36922/gpd.4047