Using Precise Gene Editing and Cell Reprogramming as New Approaches to Model and Treat Genetic Heart Diseases

PE1 is the original prime editing system, consisting of a prime editor protein and a prime editing gRNA (pegRNA), which besides the spacer and scaffold sequences of regular gRNAs it has (i) a primer binding site (PBS) that hybridizes with the DNA flap generated after target DNA nicking, (ii) the edit of interest, and (iii) a reverse transcriptase template (RTT). This prime editor is a fusion between the Streptococcus pyogenes Cas9 H840A nickase and the wild-type reverse transcriptase (RT) from the Moloney murine leukaemia virus (MMLV). An optimized PE2 variant was generated by substituting the wild-type for an engineered MMLV RT variant whose five mutations improve thermostability, RNA–DNA template affinity and DNA synthesis processivity [24]. Similar to the gRNA-Cas9 complex, the pegRNA-prime editor complex binds first to the PAM associated with the intended genomic target site and, after spacer-protospacer hybridization, nicking of the PAM-containing DNA strand by the Cas9.H840A moiety yields a 3’-ended flap that anneals with the PBS of the pegRNA. Subsequently, the annealed product creates a primer for RT-mediated DNA synthesis over the RTT sequence and encoded edit of interest, resulting in a 3' DNA flap that anneals to the complementary genomic DNA. Finally, through endogenous DNA repair mechanisms, the edit of interest is permanently installed at the genomic target site completing the prime editing process [24]. With the further demand for efficient editing, various PE systems have been developed in recent years as shown in Table 1 [25].

Beyond the aforementioned PE2 construct bearing an optimized MMLV RT region for improving the efficiency of reverse transcription [24], further and fast developments are yielding novel PE variants that include PE*, PEmax, and PEΔRnaseH. As previously shown for S. pyogenes Cas9 nucleases [26], prime editing can profit from the addition of nuclear localization signals (NLSs). For instance, PE* has enhanced nuclear localization, hence performance, owing to the addition of two extra NLSs to the both termini of PE2 [27]. PEmax has in turn a codon-optimized RT sequence and two additional mutations in its Cas9.H840A moiety for increased nicking activity [28]. Finally, via the removal of the prime editing-dispensable RNaseH domain, PEΔRNaseH displays a reduced size while maintaining editing efficiency [29, 30], which permits its delivery via carriers with limited cargo capacity, e.g., commonly used adeno-associated viral vectors [31].

In addition to optimizing the construction of PE proteins, adding extra components can further enhance their capabilities. For instance, HyPE2 incorporates the Rad51 DNA-binding domain that is hypothesized to promote DNA/RNA hybrid formation by binding to ssDNA and RNA and, in doing so, enhancing reverse transcription during prime editing. The HyPE2 construct improves PE efficiency by a median of 1.5-fold across various genomic sites and is particularly effective in genomic loci where PE2 demonstrates lower than 1% editing efficiency, achieving significant improvements at up to 34% of target sequences [32].

IN-PE2 enhances prime editing by including dual peptides, NFATC2IP and IGF1, thereby increasing prime editing outcomes across various cell lines and target sites. Velimirovic and co-researchers constructed two constructs, IN-GFP-PE2 and CTRL-GFP-PE2, and found that mESCs possess 1.58 fold higher amounts of IN-GFP-PE2 than of CTRL-GFP-PE2 with degradation occurring at a similar rate. These observations suggested that the two additional peptides increase either transcription or translation of the PE2 enzyme, offering an explanation for the increased activity of IN-PE2 [33]. The PE modality dubbed PE3 builds upon the PE2 system via the addition of a gRNA to direct the induction of another nick on the non-edited strand. This secondary gRNA-directed nick locates at an offset position from the primary nick directed by the pegRNA, enhancing in the process of editing efficiency by promoting the newly edited strand to serve as a template for DNA repair. PE3 and PE3b differ on the location of the additional secondary nick. In particular, in the latter approach, secondary nicking can only take place after edit incorporation as to minizine DSB formation via concomitant nicking of top and bottom DNA strands. Indeed, the PE3 modality can significantly enhance editing efficiency but, typically, it increases the rate of indels due to non-homologous end joining repair of DSBs created by concomitant nicking of top and bottom DNA strands, researchers thus need to consider the balance between editing efficiency and side-effects when selecting specific PE modalities [24]. The PE4 and PE5 systems build on PE2 and PE3 components, respectively, by incorporating a mismatch repair (MMR)-inhibiting protein consisting of a dominant-negative form of the human MLH1 protein (MLH1dn). By temporarily suppressing the cellular MMR pathway, MLH1dn enhances editing efficiency as this pathway tends to resolve mismatches in heteroduplex prime-editing intermediates consisting of edited and unedited strands [28].

Through Phage-Assisted Continuous Evolution (PACE) and protein engineering, smaller prime editor variants (516–810 bp coding sequences) were obtained, capable of yielding an editing efficiency improvement of up to 22-fold [34].The PE6 series employs PACE to significantly enhance the compactness of prime editing compared to PEmax and PEΔRnaseH.The PE6a-g variants have improved delivery vehicle compatibilities and editing efficacy in vivo, with one variant achieving a 24-fold improvement in loxP insertion efficiency in the murine brain cortex [34]. The PE7 system incorporates the RNA-binding protein La to enhance the interaction with pegRNAs, improving overall editing outcomes [35]. These diverse prime editors provide various options for achieving heightened genome editing efficiencies in different experimental contexts.

The development of paired or dual prime-editing systems, involving the use of two pegRNAs, represents a pivotal set of technologies offering precise and more versatile prime editing options. Indeed, these systems leverage the strengths of prime editing by incorporating dual pegRNAs that, by working in concert, expand the range of feasible genetic modifications from single base-pair substitutions and small insertions and deletions to larger-scale chromosomal edits. For instance, the Homologous 3′ Extensions Mediated Prime Editor (HOPE) uses paired pegRNAs encoding the same edits on both reverse transcribed DNA strands achieving efficient editing and improved product purity over that obtained with the PE3 system [36]. TwinPE employs two pegRNAs to template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, enabling the programmable replacement or excision of DNA sequences at endogenous sites without double-strand breaks. TwinPE can also be combined with site-specific serine recombinases for targeted integration of large donor DNA into recombinase recognition sequences programmed by dual pegRNAs and, thereby, expand the range of precision exogenous gene insertion strategies. For instance, the combination of TwinPE and BxbI, a serine recombinase, successfully inserted a 5.6 kb DNA sequence into three genomic loci, exhibiting an editing efficiency of 2.5–6.8% [37]. Similarly, by fusing Cas9, reverse transcriptases and large serine integrases, Programmable Addition via Site-specific Targeting Elements (PASTE) achieves targeted gene insertions at efficiencies of ~ 4–5% for large cargos in primary human hepatocytes and T cells [38]. Moreover, GRAND, PRIME-Del, and Bi-PE employ a pair of pegRNAs with reverse transcription templates complementary to each other that are nonhomologous to the target DNA [39–41]. PRIME-Del allows for deletions of up to 10 kb with significantly higher precision and fewer unintended off-target effects than that resulting from using the CRISPR-Cas9 system [40]. PE-Cas9-based deletion and repair (PEDAR) uses dual pegRNAs and a regular Cas9 nuclease fused to a reverse transcriptase for creating large genomic deletions and for replacing DNA fragments (1-10 kb) with an intended exogenous sequence (up to 60 bp). PEDAR was used in a tyrosinemia I mouse model derived by replacing a 19-bp sequence with a ~ 1.3-kb neo-expression cassette at exon 5 of the Fah gene. In particular, Jiang and co-researchers designed two pegRNAs, aimed at deleting the large insertion and inserting the missing 19-bp Fah gene fragment. One week later, they detected a 0.76 ± 0.25% correction rate in PEDAR-treated mice, but no correction in Cas9-treated mice [42]. Additionally, equally building on fusion constructs between Cas9 nucleases and reverse transcriptases, prime editor nuclease-mediated translocation and inversion (PETI) and WT-PE were shown to be capable of generating large genomic deletions and defined chromosomal translocations with efficiencies comparable to that achieve with regular Cas9 nuclease [43, 44].

The continuous innovation in prime editing systems highlights the rapid advancements in the field of gene editing. Each system offers distinct advantages tailored to specific editing requirements, showcasing a remarkable diversity in established and potential applications that can in principle extend to therapeutic gene correction and multiplexing genetic engineering of multicellular organisms.

PE represents a significant advancement in the genome editing field, offering distinct advantages over other CRISPR-based tools (see Table 2). Firstly, unlike CRISPR-Cas9-based gene editing, PE achieves DNA editing without creating DSBs, thereby minimizing the risk of undesired outcomes at on-target and off-target sites. Such outcomes include deletions, duplications and translocations at the DNA level and aneuploidy and chromothripsis at the cellular level [25]. Secondly, as it is more flexible, PE is applicable to a broader range of genetic targets and diseases, particularly those requiring multiple edits beyond simple base-pair substitutions [45]. The precision of PE is enhanced by its unique mechanism, which involves the innovative combination of RTT and PBS sequences in a single pegRNA that license specific hybridization steps between RNA and DNA templates. Prime editing offers unprecedented specificity owing to the required multitier complementarity between pegRNA sequences (i.e., spacer, PBS and RTT) and target DNA. These multiple hybridization requirements ensure high-fidelity base incorporation and accurate gene correction [24]. In summary, the versatility, precision, and reduced off-target effects offered by PE enhance the safety and efficacy prospects of these technologies for therapeutic applications.

PE has effectively corrected small insertions, deletions, and substitutions in various cell types. For correcting point mutation in the Duchenne muscular dystrophy (DMD) gene, PE efficiencies ranged from 21% to 38% in HEK293T cells and 22% in myoblasts [46–49]. Introduction of prime editing complexes via high-capacity adenovector particles can further enhance the performance of the editing process [50], including at defective DMD alleles in human myoblasts and iPSC-derived cardiomyocytes [51]. Additionally, a large-scale deletion in the DMD gene, spanning from exon 17 through 55, was successfully achieved using WT-PE [44].

Lipid nanoparticles (LNPs) delivering chemically-modified pegRNA and prime editor mRNA were used in HAP1 reporter cells to achieve an editing efficiency of 54% [52]. PE-mediated gene editing was successful in iPSC-CMs, achieving notable editing endpoints in cardiomyocytes. For instance, in DMD exon 51–deleted human iPSCs (ΔEx51 iPSCs), PE3-mediated modification of splice donor sites in the dystrophin gene was used to complete a -GT insertion [53]. This approach achieved gene editing efficiencies of up to 54%. After differentiation, the edited ΔEx51 iPSC-CMs were confirmed to have restored dystrophin protein expression when compared to control iPSC-CMs. In the case of the RBM20 R636S mutation, up to 40% correction was observed, releasing hypertrophic cardiomyopathy (HCM) symptoms [54].

PE has been applied in vivo through viral vector delivery methods. Adenoviral vectors (Advs) delivered PE3 components into a phenylketonuria mouse model yielding up to 11.1% editing efficiencies resulting in 2.0%-6.0% of the wild-type Pah enzyme activity in treated mice [55]. Moreover, Liu and colleagues developed a dual-AAV (adeno-associated viral vector) encoding a split-PE system that retains 75% editing activity of that achieved with the full-length PE construct [56]. These findings underscore the robust capabilities of prime editing for targeted and precise genetic modifications both in vitro and in vivo, opening up new possibilities for treating a wide range of genetic cardiac diseases.