Navigating the prime editing strategy to treat cardiovascular genetic disorders in transforming heart health

ABSTRACT

Introduction

After understanding the genetic basis of cardiovascular disorders, the discovery of prime editing (PE), has opened new horizons for finding their cures. PE strategy is the most versatile editing tool to change cardiac genetic background for therapeutic interventions. The optimization of elements, prediction of efficiency, and discovery of the involved genes regulating the process have not been completed. The large size of the cargo and multi-elementary structure makes the in vivo heart delivery challenging.

Areas covered

Updated from recent published studies, the fundamentals of the PEs, their application in cardiology, potentials, shortcomings, and the future perspectives for the treatment of cardiac-related genetic disorders will be discussed.

Expert Opinion

The ideal PE for the heart should be tissue-specific, regulatable, less immunogenic, high transducing, and safe. However, low efficiency, sup-optimal PE architecture, the large size of required elements, the unclear role of transcriptomics on the process, unpredictable off-target effects, and its context-dependency are subjects that need to be considered. It is also of great importance to see how beneficial or detrimental cell cycle or epigenomic modifier is to bring changes into cardiac cells. The PE delivery is challenging due to the size, multi-component properties of the editors and liver sink.

KEYWORDS:

• Gene editing
• prime editing
• cardiac diseases
• viral vectors
• non-viral vectors

1. Introduction

Gene therapy has a great potential for treating hereditary illnesses, including the possibilities to treat genetic disorders by removing mutations. ‘The intentional, expected permanent, and specific alteration of the DNA sequence of the cellular genome, for a clinical purpose,’ is proposed to define gene therapy according to Sherkow et al. [1]. In 1990, a foreign gene was injected into a child’s immune cells to create the first gene therapy trial to be approved by the FDA [2]. DNA insertion into the host genome was the initial method of the early gene therapy experiments [3]. Since then, the biomedical sciences have undergone a revolution, biotechnology has experienced many innovations, and clinical promise for the therapeutic correction of genetic abnormalities has been demonstrated by technologies that can produce desired sequence modifications at specific locations in the DNA of living cells. Any targeted DNA can be changed to any other sequence with a high yield, a few unwanted byproducts at the target locus, and a few unexpected alterations to off-target genomic loci using the optimal gene-editing methods.

In the 2000s, technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and integrase-homing endonuclease fusion proteins that allowed the introduction of changes at particular target sites in the genome were created [4–6]. Therefore, a long-term objective of the life sciences has been to create gene-editing techniques with high efficiency, adaptability, safety, and sequence specificity.

CRISPR-associated (Cas) nucleases, base editors, and prime editors are the three classes of Cas9 programmable technologies to edit the genomes of mammalian cells at a wide range of target sites found after the seminal discovery of the guide RNA-programmable CRISPR systems [7–10]. Although the fundamentals are similar in these three classes, their potential, efficiency, and limitations vary.

2. Cas9 nucleases-based gene editing technologies

The 2012 discovery of CRISPR/Cas9 denoted a significant turning point in the development of gene manipulation. Usually, different Cas nucleases are used to cause a double-strand DNA break (DSB) at a specific location in the genome. A guide RNA (gRNA) directs the Cas nuclease to the appropriate genomic point to cause target alteration. This guide is a single-strand RNA complementary to one strand adjacent to the target site and consists of 18–24 nucleotides (nt) [11–14]. The Cas-gRNA complex is triggered when the Cas nuclease detects a protospacer adjacent motif (PAM) and gRNA hybrid to the corresponding DNA target sequence [7]. When Cas9 recognizes this trinucleotide, it will cut 3nt upstream of the recognized PAM [15]. Finally, the cell will then carry out mechanisms, such as non-homologous end joining (NHEJ), homology-directed repair (HDR), and microhomology-mediated end joining (MMEJ), to fix the changed sequence [16,17]. Nucleases can be co-delivered with an exogenous donor DNA template that has the desired edit flanked by a sequence homologous to the genomic target location to accomplish specific DNA alterations [18,19]. Cellular HDR can then rejoin the DNA template into the DSB location after DSB formation. Although in theory, practically any form of editing (such as point mutations, insertions, or deletions) can be made using this method, HDR is primarily active in mitotic cells and is often outcompeted by end-joining for processing of DSBs [20–22].

Base editing has been developed based on the improvements in the field of CRISPR/Cas9 technology and because of the need for short but precise gene modifications. Cytosine base editors (CBEs), adenine base editors (ABEs), and C to G base editors (CGBEs) are the three variations of this technology that have been developed to transverse single nucleotide mutations [23–25]. These editors convert all targeted base pairs in a specific window to their opposites, for as CBE, which converts all C•G base pairs to T•A base pairs. The advantage of this approach over CRISPR/Cas9 is that base editing neither needs an exogenous donor DNA template nor a DSB as the system employs a modified D10A nickase Cas9, which reduces the number of undesired indels and unfavorable effects of DSBs [26–33]. Because base editing may specifically target a single codon, it also allows for a far more accurate correction of the mutation. Base editors are composed of a programmable DNA-binding protein, such as a transcription activator-like effector (TALE) repeat array or a catalytically impaired Cas nuclease, joined to a deaminase enzyme that changes one base into another base [23,24,27,34–36]. As the CRISPR-Cas method, gRNA directs the CRISPR-base editor fusion to find a specific sequence in the genomic DNA. Single-stranded DNA (ssDNA) is displaced at the target site by the base editor’s Cas protein domain, which also activates the tethered enzyme that specifically deaminates the accessible target nucleotide [23,24,35–37]. In summary, BEs have some advantages over classic Cas9-based editions such as precision and a point mutation creation. However, they suffer from the small window size and possible bystander edition near the target size. The comparison of all Cas9-mediated edition strategies is shown in Table 1.

Table 1. Genetics of selected inherited cardiovascular diseases.

Disease	Phenotype	Mutated genes
Inherited cardiomyopathies	Arrhythmogenic cardiomyopathy	DSC2, DSG2, DSP, JUP, PKP2, PLN, TMEM43 [38–41]
	Dilated cardiomyopathy(DCM)	BAG3, DES, DMD, DSP, EYA4, FLNC, LMNA, MYH7, PLN, RBM20, SCN5A, TNNC1, TNNI3, TNNT2, TTN, TPM1, VCL [42–47]
	Hypertrophic cardiomyopathy(HCM)	ACTC1, MYBPC3, MYH7, MYL2, MYL3, TNNI3, TNNT2, TPM1 [48–50]
	Non-compacted left ventricular hypertrophy	PRKAG2, TTN, LMNA, RBM20, DSC2 [51–53]
Inherited arrhythmia syndromes	Brugada syndrome	SCN5A [54–56]
	Catecholaminergic polymorphic ventricular tachycardia	RYR2, CASQ2 [57]
	Long QT syndrome	KCNQ1, KCNH2, SCN5A [58]
Familial hypercholesterolemia	Familial hypercholesterolemia	LDLR, APOB, PCSK9 [59,60]
Familial arthropathies	Thoracic aortic aneurysms, Loey-Deitz, Marfan syndrome	ACTA2, COL3A1, FBN1, LOX, MYH11, MYLK, PRKG1 SMAD3, TGFB2, TGFBR1, TGFBR2 [61]

3. Prime editing technology

Given the need for precise editing, the initial idea of prime editing came from Liu ‘s group, when they combined the potential of the retroviral reverse transcriptase (RT) with the Cas9 nickase activity. They employed the RT-mediated reverse transcription on the provided RNA template to create any kind of mutation at target loci without DSBs [62]. As the first step, they constructed the initial prime editor (PE1) by fusing a SpCas9 nickase to the wild-type RT from Moloney murine leukemia virus (MMLV) [62]. The SpCas9 does not contain an active HNH domain due to H840A substitution, therefore instead of DSB, only a nick is established by enzyme 3 nucleotides upstream of the PAM site. The programming of the PE machine is performed using a long tripartite RNA, prime editing RNA (pegRNA), which consists of a gRNA, a scaffold, and a unique 3ʹ extension region. The gRNA contains a spacer sequence to direct the editing machine on the target loci and has a similar characteristic to CRISPR RNA. The 3ʹ extension contains a primer binding site (or prime binding site, PBS) and RT template (RTT) containing an edition complement sequence surrounded by left and right homology arms. It provides a desirable template for RT polymerization to bring edition into the newly synthesized DNA strand. The PBS at 3ʹ extreme is complementary to the spacer sequence on the PAM-strand, named as proto-spacer, and has an important role in on-target edition efficiency by the editor proteins [62].

The process of ‘search and replace’ editing starts when pegRNA and cas9/RT fusion are assembled in the nucleus (Figure 1). In this form, Cas9 covers all spacer sequence to protect it from exonuclease. The 5ʹ pegRNA, spacer part, binds to the complementary target site on the non-PAM strand followed by 3ʹ extensions annealing to the target point through PBS. The structure of Cas9 which is associated with the hybridization of pegRNA to the complementary sequences on the target site induces a single R loop formation in which the editor machine can cleave the displaced target strand at three nucleotides upstream of the PAM sequence, designated as number 0 position to permit true numbering of the surrounding nucleotides as + for upstream or – for downstream (Figure 1(a)).

Figure 1. Graphical description of the prime editing process.

(a) The prime editor machine is composed of a Cas9/RT complex protein and pegRNA. The Cas9 nickase-RT fusion works together on the target loci to cut the PAM strand. The location of the PAM is indicated by the red DNA bases and scissors. (b) The resulting 3′-overhang can be primed by RT on the RTT template to install the edition on a newly synthesized DNA strand. (c) There is an equilibration between 3′-flap and 5′-flap, however, (d) 3′-flap insertion into new heteroduplex DNA might be fixed into the genome by further repair. The details of each strategy have been discussed in the text.

The resulting nick releases a 3ʹ-OH end which can prime RT enzyme polymerization to copy the RTT template and extend a new synthesizing strand, while PBS is still annealed (Figure 1(b)). The DNA polymerization by RT at the prime site generates either 3ʹ or 5ʹ overhangs due to prime extension and strand displacement, respectively. The 3ʹ flap with the desired edit equilibrates with the original unedited 5´flap. If the 5ʹ flap is excised by host endonuclease, 3ʹ flap containing the intended edits and PAM strand homology arms anneal with a unedited strand (Figure 1(c and d)) to produce a heteroduplex DNA with edit [62]. On the other hand, excision of the 3ʹ flap removes the edition and permits the displaced 5ʹ flap to be ligated into dsDNA. It has been shown that after fixing the 3ʹ flap into the heteroduplex DNA form, an induced repair by an extra nick outside the target structure can induce coping the inserted edit to the non-target strand to enhance pure edition on both strands. Including an additional sgRNA to the editor machine, as named PE3 generation, down or upstream of the edit site bases the repair system toward the installation of the edit on both strands [63].

3.1. The advantages of prime editing

Prime editing technology has several advantages over other methods of gene editing. In the case of the CRISPR-Cas9 method, DSBs generated by nucleases can bring some large mutations including deletions, translocations, and chromothripsis [63]. Because cardiac cells are quiescent, stress by genome edition might push them into an irreversible path of damage and apoptosis. In contrast to CRISPR-Cas9, BEs are a very precise method of changing nucleotides with substantially greater efficiency. Theoretically, it might leave few indel bystander changes and a more controlled edition. Most cardiac genetic diseases arise from a single nucleotide mutation that can be targeted through BEs. Therefore, BEs can be employed as a suitable method in heart-related genetic disorders without large undesired consequences of DSBs. However, similar bases in the small window of base edition (4–5 nucleotide) are subjected to unintended conversion and may lead to bystander mutations. In most cases, this undesired change is a silent effect, but if this bystander edit creates non-synonymous amino acid or stop codon at the sequence which would have a significant impact on the cell. Additionally, BEs can induce off-target mutations in DNA strands even though much less compared to the CRISPR-Cas9 method. The nonspecific activity of deaminase has been shown to induce Cas9-independent bystander mutations on DNA and interestingly on RNA [63]. PAM dependency on BEs is also another shortage that restricts the targeting scope of the machine to a maximum of 15 nucleotides from a PAM site. Finally, this method permits only six out of 12 possible point mutations to be corrected without the capability for insertion and deletion of any nucleotide(s).

The PEs are the optimal strategies to bring editions into cardiac tissues, owing to a set of advantages over the other methods. In the PE complex, precision genome manipulation of all 12 possible point mutations, insertions, and deletions is practical due to the relevant template that is designed in the pegRNAs. Moreover, multiple base substitutions, at different positions inside the PE window (near 33 bp from the PAM site), medium-size insertions (<40 nucleotides), large deletions (up to 100 bp by classic variants and up to some kbps by dual-pegRNA variants), and the combination of some genetic alterations are applicable using generations of PEs. The higher the window size, the more targeting scope PEs have as compared to BEs. In other words, the manipulation that is achievable by PEs might not be reproduced by BE or Cas9-mediated HDR, and it is suggested that more than 90% of all human genetic defects could be targeted by it. Despite gRNA that contains only 20 nucleotide spacer sequence for the specificity of CRISPR-Cas9 and BEs, pegRNA contains 3 elements: Spacer, RTT, and PBS that hybridize to double strand DNA, therefore making the cleavage reaction much more specific in PEs. However, bystander mutation could arise due to the genetic context, rate of efficiency, and sub-optimal activity of the editor proteins in mammalian cells. Moreover, as PEs do not need DSBs or homology-directed repair, they can introduce any kind of mutation to the cardiac cells that are in the G1 state of the cell cycle without leaving cell cycle arrest after the change [63].

3.2. PE generations/variants for cardiac gene editing

After the discovery of the so far most precise strategy of ‘searching and replacing’ PE, many efforts were made to improve the initial version. Although the primary wild-type PE1 machine has theoretically the potential of creating all 24 possible mutations alongside the genome, low efficiency, typically <5% at targeted allele, and the possibility of off-target effects was a great drawback [64]. To enhance the efficiency, new generations and variants of PE have been developed by modifications on a) PE editor machine, b) pegRNA/sgRNA, and c) repair system.

As the main PE generations, PE2-PE5 have been developed through fine-tuning of modifications at the amino acid sequence of MuLV-RT, nCas9, and employment of an additional sgRNA. They first introduced five amino acid mutations (D200N/L, T306K, W313F, T330P, L603W,) into the MuLV-RT structure of PE2 to improve its DNA-RNA hybridization strength, thermo-stability, and enzyme processivity. Even though PE2 has increased efficiency, it is still dependent on an endogenous repair system to establish an edition at a non-PAM strand of loci. Therefore, to enforce the repair machine to copy the edited sequence on the unedited strand, PE3 with an extra single guide RNA (sgRNA) was included to provide a distal nick to prompt the repair system to copy the edit on the non-PAM strand [65]. The second nick by sgRNA might increase the rate of indels due to double-strand break and over-functioning of the repair system [63]. Therefore, a version of PE3, PE3b with sequence-matched sgRNA to the edited point of the target sequence was optimized with better specificity. However, the superior efficiency of PE3 over PE2 to create edition don’t inspire scientists to apply it for all cases, especially for pluripotent stem cells manipulation and creation of animal models due to higher unwanted indels and substitutions [66]. To further optimize the strategy, a new improved PE2 version, PEmax with different RT/Cas9 codon usage, increasing the number of NLS signals, and modification in the composition of the peptide linker was developed. The PEmax version showed superior activity compared to the original architecture of the PE2 and PE3 generations when tested in several loci in HeLa cells [63]. Currently, the PEmax version combined with enhanced pegRNAs (epegRNA) is used as the gold standard for any newly developed versions of editing. In David Liu group, most recently the sixth generation with seven variants (PE6a-g) has been developed based on viral and retroelement’s RT species with smaller size and through phage-assisted evolution of RT/spCas9 proteins [67]. They also introduced an algorithm to simplify the use of these variants based on edition situation and structure of pegRNA.

Nelson et al. designed a new form of pegRNA with an incorporated structural RNA motif to the 3′-terminus. The addition of this element to pegRNA 3′-end improves structural stability and reduces degradation by nuclear exonucleases [68]. They demonstrated that the PEmax-epegRNA version increased efficiency 3–4-fold in different cell lines and primary human fibroblast without affecting the rate of bystander mutations.

The range of prime editing activity from the PAM site is <40-nt and in the best condition <10-nt, which is a limit, especially for PE3b. To expand the range, new types of Cas9 variants with non-NGG PAMs or alternative nuclease sites have also been investigated [69]. Some studies also developed compact and less-PAM-dependent versions of Cas9 which are suitable for efficient packaging into small capacity vectors. Mbakam et al., in a study on c.8713C>T mutation, located in exon 59 of the DMD, tried to increase the edition rate by PE3 and use of a PAM-independent Cas-9, but their effort failed on 293T, with nearly 10% efficiency. They also brought the edition site closer to PAM, however, it also has not shown a significant increase in efficiency (5.5%) [70]. The results showed that editing efficiency is affected by many unknown factors that remain to be cleared.

Due to the cell status of myocardial cells, MMR system is continually active, and MLH1 protein is also expressed at a higher level compared to other tissues. Regarding the assumed importance of the cellular repair system in PE, Chen et al. showed that MMR elements strongly suppress PE efficiency through the reversion of the intended edit and disruption of the 3′ extension flap. Therefore, they developed a dominant negative MMR protein (MLH1dn) in the PE expression cassette, PE4 (PE2max+MLH1dn) and PE5 (PE3max+MLH1dn), twice as effective in vitro [63]. The results indicated that blocking of MLH1 has the most dominant impact on enhancing of PE editing outcome. Therefore, it is expected that applying PE4 and PE5 or similar generations with preventive capability for MMR could increase the editing rate on cardiac cells. However, Xu et al. found that overexpression of other repair factors, such as flap endonuclease 1 (FEN1) and the DNA ligase 1 (LIG1) also enhanced the efficiency of prime editing. FEN1 cleaves the 5′-flap generated by DNA displacement during the Okazaki fragment extension [71]. This idea remains to be tested on cardiac cells.

The accessibility of genomic loci for the PE complex is another parameter that can be regulated for enhancing the edition. Park et al., with a vision of open regional chromatin, used proximal dead sgRNA (dsgRNA) and chromatin-modulating peptides at various target loci to perform gene knockout in mice. It has shown that histone deacetylase inhibitor (HDACi) molecules significantly boost the efficiency of PE3 for indel-type modification at endogenous genes [72].

Several types of modifications at pegRNA have been shown to be effective in increasing the edition efficiency by PEs. Nelson et al. described pegRNA 3´-end modification through their engineering by the addition of viral or synthetic structured RNA motifs. They showed that two structured RNA motifs including evopreQ₁ (prequeosine₁-1 riboswitch aptamer) and knot (frameshifting pseudoknot of MuLV) are suitable candidates to be added at the 3´-end and prevent degradation of the pegRNA [68]. The addition of other stem-loop structures, such as MS2, PP7, Csy4, and BoxB to the 3′-end of the pegRNA can increase the edition even more compared to the epegRNA method [73,74]. Also, silent mutations complementary to the PAM site, or on-purpose changes in the RTT template avoid retargeting the PAM site by nCas9 or prevent the MMR repair system from reverting the edited sequence to the original one, respectively [75,76].

In a new class of PEs, a pair of pegRNAs are employed to bring long insertion/deletion edition into genome. The long-range variants of PEs are recently shown to have the ability of changing wide range of mutations in several cell types. In the pioneering work, a long-range edition strategy was developed by using a pair of pegRNA targeting strands at different sites. Upon transcription of RTT, the homology sequences hybridize together and ligate. The new method, PRIME-Del can delete up to 10kb, with up to 30% efficiency [77]. Anzalone et al. designed TwinPE prime editing by two pegRNAs for precise long-range modification in human genome sites. This strategy employed two opposite strand-targeting pegRNAs with complementary 3′-flaps that target the same site. The hybridization of the resultant 3′-flaps together replaces the original sequence with newly edited DNA strands [78]. Other methods such as GRAND, homologous 3′extension mediated prime editor system (HOPE) [79], Bi-PE, PASTE, PAINT3.0, and precise and specific deletion, and repair (PEDAR) have been tested for their efficiency in the insertion or deletion of long sequences into endogenous loci of different cell lines [80]. These PEs might help to edit a part of CHD-associated copy number variations such as Del22q11 and del8p23, which cause deletion of cardiac transcription factor GATA4 [81]. However, this type of large-scale modification is too young to become a candidate for further investigation in the field of cardiology.

3.3. Inherited cardiac diseases targetable by gene editing tools

Genome editing technologies have the potential to correct the suffering gene of bad mutations. In the early era of CRISPR-Cas9 discovery, the disease-related genes/exons were targeted to introduce indels by NHEJ repair and inducing a frameshift around the malfunctioning variants. Ablation of exon expression thus eliminates the disorder-related protein. However, any strategies for removal or correction of the dominant-negative variants need some parameters to merit effectiveness criteria. The precision factor is important as it must correct only one nucleotide, in most cases. The targeting strategy should ensure the correction of the affected nucleotide but not the wild allele. Moreover, in cardiac tissue with low turnover, the cellular response stress and DNA damage should be avoided due to off-target effects and large genetic alterations, to keep healthy cell mass intact [82].

On the other hand, correction of some other malfunctioning genes needs restoration of protein expression at least to some extent. The resumption of expression can be achieved by gene therapy or in some cases by genome editing. Duchenne muscular dystrophy (DMD), X-linked Emery-Dreifuss muscular dystrophy (EDMD), and Barth syndrome are attractive candidates for gene editing [83]. As an example, DMD arises by loss-of-function mutation encoding dystrophin which is necessary for heart muscle integrity. Features of DMD that make it suited to CRISPR-Cas9 editing include 1) an X-linked pattern that permits correction of the gene in boys carrying a single mutant allele, 2) the repetitive pattern of rod domains for deletion of one motif without affecting the whole protein structure, 3) low-level physiological needs of dystrophin for the maintenance of muscle activity. Thus, the CRISPR-Cas9 strategy can easily reach the therapeutic benefit of corrected protein without affecting wild alleles. However, a protein structure cannot always tolerate an exon deletion or a big alteration which leaves the function intact. Therefore, a more precise method of editing should be considered for other cases of genetic defects. We might consider the CRISPR-Cas9 HDR method for the correction of loss-of-function and dominant-negative variants. However, most rehappident cells in the heart and vascular system are in the G1 phase, while HDR occurrence is possible only during the S or G2 phases of the cell cycle in dividing cells [82]. Despite this, a few recent reports are acclaiming HDR-mediated correction of the gene in adult and neonatal mouse myocadiac cells following injection of an extraordinarily high dose of Adeno-associated virus (AAV)-prime editor. They have reported that intraventricular or subcutaneous injection of AAV could induce HDR with a precise and safe pattern at post-mitotic cardiomyocytes, which was not seen through other routes of injection [82]. Despite later development to CRISPR, base editing progression is very promising in animal models and has even entered to a clinical trial in 2022. BEs can bring a mutation into the genome to correct a damaging autosomal mutation or to induce exon skipping. The mouse models of Hypertrophic cardiomyopathy (HCM), carrying a pathogenic MYH6 R404Q variant have been successfully targeted by BEs [84]. Also, a single nucleotide mutant of the LMNA gene, encoding malfunctioning lamin, was efficiently ablated by receiving an indel mutation causing a frameshift. To restore loss-of-function mutations for dystrophin expression in ∆Ex51 iPSC-derived cardiomyocytes, a BEs design for exon skipping has been applied [85].

The most successful story of BEs is the ablation of the expression of PCSK9 (proprotein convertase subtilisin/kexin type 9) with for a consequent LDL (low-density lipoprotein) level control in patients with familial hypercholesterolemia. After preclinical studies in mice and primates, this BE experiment was approved for a clinical testing in 2022 [86]. In this study, Verve Therapeutics initiated the study by lipid-based intravenously delivered particles carrying an ABE editor mRNA as well as gRNA to disrupt PCSK9 expression in the liver. In the primate model, they observed a significant reduction of PCSK9 and LDL levels, durable for at least 8 months, just by a single infusion of lipid nanoparticles [87]. However, the Verve initial trial was halted later on by the FDA for clarification of the edition potency in humans, mitigating the risk of germ cell edition and deep sequencing analysis of the off-target effects in non-target cells. There are other successes of targeting lipid-modulating genes such as angiopoietin-related protein 3 (ANGPTL3) and LDL-R that have already tested in the mice model [88]. The prevention of HCM by genome editing in hypertrophic mice in two consequent studies showed the possibility for the correction of monogenic myosin heavy chain (MYH7) p.R403Q variant in iPSC and humanized mice model [89,90]. Despite the high efficiency for some purposes, BEs can correct some variants like RBM20 mutations due to a limited activity window, and lack of a proper PAM sequence near the target site. In other words, BEs always require precise positioning of the editor machinery on the edit window. However, more flexible Cas9 variants have been developed to use alternate PAMs [91]. Moreover, the same bases in an editing window can be wrongly identified as true targets and deaminated by the base editor. In a recent attempt, a modified base editor with a narrower editing window has been developed [92]. In addition, BEs have been shown to exhibit a new expectable deaminase activity on RNA transcripts, the natural targets in eukaryotic cells cause unpredicted RNA off-target effects with uncontrolled consequences. More importantly, as BEs can introduce a nick on the non-target strand, excision of the edited strand by the host repair system might create a DSB and indel generation. Therefore, the ablation of the base repair system could be applicable to reduce the indels by this strategy.

Regarding the PEs potential and advantages over other editing technologies, it is currently considered as a new tool to correct inherited heart diseases. Most inherited cardiac diseases could be targeted by PEs. Although autosomal recessive, X-linked, and maternal mitochondrial disorders have been reported, autosomal dominant features are most frequently associated with inherited cardiovascular diseases (Table 1). Many cardiac genetic disorders, such as HCM, dilated cardiomyopathy (DCM), and arrhythmogenic cardiomyopathy are inherited in an autosomal dominant manner, although autosomal recessive and X-linked, particularly loss-of-function disorders, have also been described. Based on recent efforts and the versatility of the tools, most inherited cardiac diseases can potentially be curable by PEs. Also, this method can be used to correct dominant-negative mutations and to restore loss-of-function variants [82].

3.4. Current state of prime editing application for treating heart diseases

PE is the most versatile and precise tool to rely on for curing many cardiac genetic diseases in the future [93]. Due to the early state of the technology, PEs have not been investigated either in human cardiomyocyte cell models or in mouse heart disease models in vivo. The pioneering gene editing study in the heart investigated the correction efficacy of different CRISPR-based methods to reverse some pre-defined changes in an isogenic genetic background. Surun et al. showed that after experimental setup, the reversion of GFP to BFP at the AAVS1-eGFP site went up to 7.5% edition in a human iPSCs model using RNA delivery [94].

A few studies have evaluated the potential of prime editing in introducing small genetic changes in hiPSC cells from normal individuals and patients. Nishiyama et al. [93] employed a modified version of PE3b, epegRNA, to correct 1906C>A mutation in RBM20^R636S. The transfection of editor plasmid by nucleofection in this study showed 40% A-to-C editing efficiency in homozygous R636S-affected hiPSC cells. Interestingly, deep sequencing confirmed a good safety profile for this strategy and the absence of potential off-target effects. Some mutations in RNA binding motif protein 20 (RBM20) induce aberration in protein localization and cause familial dilated cardiomyopathy. The editing result of BEs in this study was more encouraging than PE, even though only the BEs results have been published from the mouse model.

DMD is an X-linked genetic disease that causes cardiomyopathy due to a mutation in dystrophin. Even with a high variety of mutations, key variants have been recognized to be spanning the termini of exons 43 and 53 which yield a truncated malfunctioning dystrophin. Since removing the bad exons retains the protein functionality, CRISPR-based strategies have been evaluated in this model from early after discovery [95]. Similarly, after the invention of PEs, efforts to apply the methodology to DMD also started. In a study by Chemello et al. [85], they used a PE3 editor machine to create a ΔExon51 genotype for restoring DMD protein expression in hiPSC-derived cardiomyocytes. This genetic correction reframed the coding sequence of the protein to produce a functional version and restored a rhythmic order in treated cells. This precise correction strategy had not been reported for DMD before, and they succeeded to edit target site by insertion of GT nucleotides with >50% efficiency. They also showed a relative expression of DMD in edited cells population up to 40% of the healthy iPSC-derived cardiomyocytes. The group tested the ABS strategy by AAV delivery in mice to edit bases in the DMD mouse model with promising results [84]. The results of another study on iPSC (not myocardial ones) by Li et al. [96] emphasized the capability of prime editing in introducing heterozygous edition compared to CRISPR-Cas9 technology. They used a combination of mRNA expressing PE machine and synthetic pegRNA fragment based on PE3 for their assessment. The method showed a superior result compared to the traditional CRISPR method.

Very recently Liu^, s group optimized the first in vivo heart gene editing on some pre-evaluated loci by v3em PE3-AAV9. They showed that retro-orbital administration of split AAV9 containing editor machine with specific promoters established near 11% edition in the heart tissue. This study highlighted the importance of the stability of pegRNA, the expression level of the editor machinery, and the type of editor (PEmax). However, even with this modification, the muscle editing efficiency was very low [97]. This study also highlighted more efficient editions in the liver and brain than in the heart tissue of the mouse model (48%, 42%, and 11% efficiency). However, many other cardiovascular disorders have not been tested for PE in vitro. For instance, cardiac arrhythmia related to a malfunction of phospholamban, hPLN-R14del allele, is a potential target for prime editing, but not BEs. Recently, CRISPR-based disruption of the pathogenic PLN-R14del allele by AAV9 reduced sustained ventricular tachycardia in the humanized PLN-R14del mouse model [98].

Inherited cardiomyopathies, HCM, and DCM are also candidates suitable for PE genome editing. Their genes are diverse with different heritage patterns that would influence the selected PE methods. Recently, one dose of an adenine base editor delivered by a dual-AAV9 system to treat mice with a heterozygous HCM pathogenic variant myosin R403Q was shown to correct the pathogenic mutation in ≥70% of cardiomyocytes [90]. However, PE strategy remains open for research on most of aforementioned cardiac models. Finally, atherosclerosis is another key disorder where several CRISPR-based and BE studies to disrupt the lipid metabolism gene, Pcsk9, are undergoing at pre-clinical studies [82]. Therefore, evaluation of different strategies for targeting this gene by PEs should be possible in the mean future.

3.5. Prime editing for modeling genetic cardiac diseases

The new advances in whole-exome sequencing allow identification of disease-causing variations and to cure them by genome editing. Also, screening and evaluation of the effect of disease-causing mutations in cell models and relevant animals is also beneficial in the field. The CRISPR-Cas9 system has already been employed for the efficient preparing of cell and animal cardiac models with noncoding, structural, copy number, and gene variants [82]. However, unwanted changes during the DSB at the genomic backbone of the models might be challenging and even misleading results.

The ability of PE to introduce any kind of change from a few nucleotides to large fragments also opens the window for the creation of cell and animal models of cardiovascular diseases such as DMD as shown by Zhou et al. [99], and Happi Mbakam et al. [100]. To study the physiology of cardiac genetic diseases, a couple of genetic diseases including DCM, HCM, long QT syndrome (LQTS), and laminopathy have been established by CRISPR-based gene editing of iPSC [101]. However, no example is yet available based on the PE strategy.

4. Challenges in the prime editing of the heart

Despite the diverse capacity of prime editing, its application in the cardiovascular system has not been very common compared to other methods of editing. The delivery of a CRISPR-based edits into the cardiovascular system remains challenging but AAV vectors are the most popular vectors so far [102]. However, the application of AAVs that are well-optimized for heart delivery is restricted due to the large size of the PE machinery (>6–8 kbp). Even the pioneering in vivo study in mice revealed a low efficiency of AAVs for prime editing compared to other tissues [97]. The other issue for cardiac gene editing is the liver that is accessible during heart infusion and vector delivery. Liver is a site that always plays a sink role for viral and non-viral vectors, therefore de-targeting of delivery cargos would be important. On the other hand, liver could be a target of genome editing for correction of other tissue’s genetic disorders. As the first trial to study edition on a heart disease, familial hypercholesterolemia, was set by an AAV-based delivery system into the liver [103].

Viral vectors are the most efficient delivery vectors to the cardiovascular system. However, in terms of editing strategies, the high risk of off-target editing due to the long-expression and risk of vector integration in human genome are of importance [64]. Persistent expression of RT and the possibility of integration might induce tumorigenesis or unintended sequence [64]. Modes of RNA, DNA, and protein delivery likely offer great potential and further optimization of the vehicles for safe and efficient clinical application [65]

The cell cycle status of cardiomyocytes is another factor that limits the efficiency of prime editing [104]. In the early stage after birth, the heart is permissive to all kinds of genome editions, due to the mitotic and proliferative properties. However, after fetal development, heart cell proliferation is ceased by differentiating factors and turnover is ~0.04% in the first year of life and even less in adulthood [105]. The differentiated cardiac cells are very resistant to genetic modification by CRISPR-based editing, as they are no longer dividing. CRISPR methods need the S and G2 phases of the cell cycle for their activity. In theory, it has been suggested that inducing cells to enter the G1-S state or inhibiting the NHEJ process could increase the CRISPR-based edition [104]. The G1 state suggested as the best point for applying PE in any cells, but most of cardiomyocytes are spent in G0 phase. It should be noted that the artificial pushing into G1/mitotic phases could perturb the normal function of the differentiated myocardial cells [104]. Many studies have indicated that a small portion of heart cells reenter the cell cycle following mechanical injury [106]. However, lack of cytokinesis leads to genome polyploidization but not to the generation of new offspring. Therefore, manipulation of cell status not only has no special benefit for providing a better milieu for the edition, but it might also affect other consequences of the prime editing process [106].

Regarding the cardiomyocytes cell status, the epigenomics of these cells could be a barrier to access genome for PE activity in many loci. Recently, several efforts have done to open the regional chromatin for better PE targeting using proximal dead sgRNA (dsgRNA), chromatin-modulating peptides (CMPs) and histone deacetylase inhibitor (HDACi) molecules [72]. However, the use of these components for cell cycle or chromatin structure might not be safe enough in cardiac tissue.

Innate immune induction is another issue that might arise following the use of viral and plasmid vectors. The backbone of bacterial plasmids, all the viral vector components, and more importantly PE components contain innate and adaptive immune stimulators that can be detected by sensors in the cytoplasm and nucleus. The sensing of pathogen-associated molecular patterns (PAMPs) could trigger different pathways of innate immunity, and ultimately interferon (IFN) and inflammation induction. Therefore, measures to control the innate immune response against PE components and delivery vectors should be considered in vivo protocols. The transient expression of the PE machine with the RNP complex by lipid nanoparticles (LNPs) and virus-like particles (VLPs) could improve the non-inflammatory profile of the method given the temporary effect to the immune cells [106].

As foreign proteins, the fusion of Cas9-RT triggers specific humoral and cytotoxic T-cell immune responses. It has been demonstrated that local and systemic administration of AAV-CRISPR in dystrophic dog induced an immune response causing the elimination of CRISPR-restored dystrophin and significant muscle inflammation [107]. The use of any kind of gene editing raises concerns about foreign proteins, such as Cas9 and RT. The continuous activity of Cas9 and RT in the nucleus could cause unintended genomic mutations and risk of tumorigenesis or off-target effects. To reduce the Cas9-related concerns, Cui et al. suggested selective inhibitors of nuclear export (SINEs), which can prevent the nuclear export of Cas9 mRNA [83]. In addition, some viral factors with the natural potency to inactivate Cas9 have recently been discovered such as bacteriophage anti-CRISPRs (ACRs). ACRs are naturally produced by bacteriophages to protect them from bacterial CRISPR-Cas system. Therefore, these molecules can be applied to control the timing of the CRISPR activity in any editing protocol [108]. Also, several protein modifiers, such as Bcl-xL-interacting peptide (BH3) and pomalidomide have recently been discovered to bring the Cas9 activity under the control, and they could be adopted for navigation of CRISPR-based technologies [109].

5. Cardiac delivery of editor machinery

Although prime editing technology grows very fast in different areas, a suitable delivery to the target site is of great concern for clinical applications (Figure 2). Viral vectors are suitable delivery tools for most tissues like the heart and endothelial system. Adenoviruses, AAVs, and retroviruses have been widely used for CRISPR-based editions in vivo [65]. However, the application of these vectors in PE could be challenging due to the limited capacity of some vectors. The total size of the editor protein (>6kb), MLH1 dominant negative (in PE3, PE5 versions), pegRNA cassette, and sgRNA assembly together are a great challenge to be packaged in the single adenovirus, AAVs, or lentivirus vectors and even in plasmids [65]. The ideal delivery vector for PE should include all the components from protein-coding sequences to non-coding RNAs. The all-in-one vector expressing all the editing elements would be optional for clinical applications, but putting different elements under different promoters could be problematic.

Figure 2. Prime editing for genetic cardiac disorders. (a) Viral and non-viral delivery methods can be employed in “all-in-one” or in dual vector systems to transfer PEs into the nucleus of cardiac cells. (b) The scope of prime editing for correction of cardiac genetic disorders. Prime editing can cover the treatment of nearly all possible genetic defects by nucleotide mutations, sequence disruptions, exon deletions, and even insertions.

Wang et al. developed a high-capacity adenoviral vector containing all the PE elements in one package. They showed that Advp.PE2.pegRNA vector permits high efficacy delivery (90% edition frequency) and screening of PE reagents in cells independently of replication or transformation status [110]. In the cardiac system, the first-generation HAd5 serotype is the most widely used for gene therapy applications. The encouraging results have been reported regarding the successful delivery of angiogenesis genes by this vector [111]. However, cardiac-targeted vectors for systemic delivery are demanding [112]. Bock et al. systemically administered HAd-5 encoding PE components as well as U6-pegRNA cassette into newborn mice. To fit a big editor machinery into the adenoviral vector, they developed a smaller PE lacking the RNase H domain [65]. In a model of phenylketonuria mouse, they edited the deficiency for Dnmt1 and Pah (phenylalanine hydroxylase) locus with 58% and 11.1%, respectively.

Lentiviral vectors are among the candidates for the delivery of the PE cargo in one package to human cells. However, given the long-lasting expression of genes by lentiviral vectors, adverse events due to their integration should be considered. Integration-deficient lentiviral vectors should be safer and less oncogenic than original vectors but still preventing genotoxicity and oncogenicity need to be solved before further steps [113].

Non-viral delivery of plasmid DNA, RNA-based constructs, and RNP complex can also be employed to overcome the capacity limit of viral vectors for the transduction of prime editors (Figure 2). While different nanoparticle structures have been described for gene delivery to the heart, formation of the protein corona, sequestration by resident phagocytes, kidney filtration, weak extravasation, and penetration into the heart, inefficient endosomal escape, and nonspecific uptake by different cardiac cells are considered as drawbacks need to be overcome [114]. Plasmids containing all PE cassettes are also practical to manipulate, construct, and use for tissue-specific expression. Due to the lack of packaging limit, the capacity of naked plasmids is larger than the usual viral transfer construct. As a result, they are suited for designing all-in-one PE constructs for the early steps of pegRNA screening or even in the clinic. Dirkx et al. constructed PE2Max, PE3Max, and PE4Max-based all-in-one plasmid (pAIO) under the control of EF1a promoter which exhibited suitable transfection efficiency as well as editing efficiency in ihPSC compared to the multi-plasmid method [115,116]. Plasmid-based delivery of the editor machinery has been used in cardiovascular system modeling and therapy [84]. The majority of research that has been focused on PE strategy has been developed through the DNA plasmid constructs. Another strategy that can circumvent the plasmid delivery challenges is a minicircle. This structure has less immune toxicity compared to plasmids, due to the removal of bacterial elements from the backbone. It has been tested in several heart disease models in naked or gel mixture forms [117].

The direct delivery of the prepared RNP complex of pegRNA and Cas9-RT protein is another method to send all the PE components simultaneously into the cell. The fast-working manner, safe, and transient expression of the PE-RNP compared to plasmids or viral vectors makes it very promising for clinical use. Delivering BE RNPs has been shown to lead to less off-target variations in different tissues [64], though the specific targeting of the complex to cells is impossible. The strategy also showed benefits over the CRISPR-Cas9 delivery with acceptable safety [118]. The virus-like particles (VLPs) are capsid structures of viruses without any infectious genome. The capability of engineering the capsid and the possibility of packaging any plasmid, mRNA or RNP complex into the structure makes VLPs very interesting for safe delivery of any kind of gene editors. The combination of mRNA/VLPs or RNP/VLPs could easily be designed as an all-in-one construct for the delivery of PE or BE. Recently, a new generation of BE editor VLPs was evaluated in a mouse model with pathogenic Pcsk9, which is involved in cholesterol homeostasis. A single injection of this construct established a therapeutic level of BE to reduce serum Pcsk9 levels up to 78% through liver editing. The targeting of this complex has also been managed by pseudo-envelope from glycoprotein VSV-G [64]. A thesis at Michigan University about the all-in-one VLP prime editor described this approach for the first time in different cell lines [119].

In nearly all published studies, a few constructed architectures for multiplex prime editing and different plasmids have often been used. However, Yuan and Gao et al. developed an array containing tandem transfer RNA (tRNA)-sgRNAs for targeting several targets. This type of array can be employed for combining pegRNAs and sgRNAs or other elements in one construct to enhance efficiency, using dual pegRNA for larger editions and to compact the size of the required elements [120].

The second strategy for the delivery of PE to tissue is the dual/split vectors strategy, which is based on the use of two separate AAVs or plasmids. The delivery of two AAVs expressing two separated parts of the PE machinery (either dual AAV or inter-split strategy) could provide enough space to carry long sequences such as BEs and PEs (Figure 2(a)). AAV-expressing split or dual-part of BEs showed the successful gene modification in mouse model of progeria and [121] dystrophic mice [122]. AAV-BE has also been developed to induce exon skipping and restore loss-of-function mutations such as DMD exon 51 deletions (∆Ex51) in iPSC-derived cardiomyocytes [82]. This AAV delivery strategy also has been used in large animals for the ablation of the PCSK9 gene by exon skipping to reduce LDL cholesterol levels [87]. In the dual strategy, the chance of co-transduction rate of target cell is lower than a single AAV system [123]. The pioneer study for PE in vivo in adult mice employed dual AAV intein-split PE3 variants [97]. They constructed a split plasmids system to test the efficiency on 293 cells then for in vivo study then they moved to dual split AAVs which showed near 11% edition rate.

6. Future perspective

PE holds great promise for curing genetic cardiovascular disorders. However, due to infancy, many challenges, such as low efficiency, lack of animal studies, possible safety medicine issues, and lack of universal delivery methods are still big challenges to PE development [83].

The safety issue is a big concern of gene editing in cardiovascular systems. The lack of relevant cardiac models to measure off-target events is an obstacle to predicting the safety profile of selected PEs. Several platforms are used to analyze genome-wide effects and for prediction of protocols [124]. However, the stability of editing and long-term consequences in the large animal genome have not been completed, that led a halt on LNP-encapsulated BE during a clinical trial for familial hypercholesterolemia [102].

The transduction rate of the heart and peripheral muscles hardly surpasses 10–20% efficiency and factors like transgene properties, volume per injection, and matrix binding properties of vectors will affect the final efficacy. In this situation, secretory proteins could achieve the therapeutic dose in tissue, but this is not the case for RNA and protein components of the PE machinery [125]. The ideal delivery vector for prime editing of the cardiovascular system should contain no genotoxicity, immunogenicity, or long-term expression and must be conducted under specific promoters. The regulated delivery method for controlling the expression of PE elements in cardiovascular tissues is demanding. The development of elements to regulate the duration of PE machinery activity and expression is also critical for enhancing safety [83]. Direct intramyocardial injection, vector targeting, and expression control of cassette by insertion of cardiac muscle-specific promoters would be the most rational and simplest way to regulate the PE expression in the heart. Although the polII-based expression has been evaluated in a few PE studies, the employment of many natural promoters including cardiac sodium-calcium exchanger (NCX1), ventricle-specific myosin light chain-2 (MLC 2-v), cardiac troponin T promoter (cTnT) can restrict the PE activity in cardiac cells [126,127]. In addition, another level of gene targeting could be achieved through the addition of siRNA-mediated silencing sequences or promoter-targeted ncRNAs into cassettes to limit and concentrate the expression in the cardiovascular system [128]. Moreover, different strategies for spatiotemporal control of Cas9/RT expression, such as using bio-responsive delivery carriers, employment of regulatory small-molecules, and optical/thermal/ultrasonic/magnetic activation methods can be applied to the machinery to increase safety [129].

The adaptive immunity to foreign element of PE, such as Cas9, RT, and viral vectors is a safety challenge for clinical use, as a part of the human population has already been infected by the relevant microbial sources [130]. However, it has shown that editing strategy for cardiac arrhythmias would promote if PE gene expression were avoided from resident immune cells and also timing [131]. Innate immunity is also a perpetual barrier to efficient in vivo delivery of PEs complex. Therefore, measures to control innate immune response and transient expression through RNPs, LNPs, or VLPs might impede inflammatory reactions given the temporal touch of immunity [64].

The efficient delivery and accurate targeting of the cardiac cells is a challenging issue for sending any editing machinery into cells. The targeted delivery to cardiomyocytes, fibroblasts, and endothelial cells could be improved by the incorporation of ligands specific to cardiac cell receptors and specific promoters. Precise targeting could improve the rate of delivery, reduce off-target effects, and limit the PE expression in immune cells. Viral capsid manipulation by altering attachment ligands, incorporation of cardiac specific molecules, and generation of hybrid vectors with tropism to target cells could increase the target efficiency. However, due to the capacity limit size of some vectors, such as AAVs and adenoviruses, other options like helper-dependent adenovirus vectors, LNPs, and VLPs should be reconsidered. The application of AAVs and lentiviral vectors should be restricted in prime editing technology because of the integration possibility of these viral vectors, and prolonged-expression that exposes the genome to hazardous off-target effects and unpredicted results [64]. The LNP carrier was recently employed in a clinical trial of genome editing, however, designing a non-liver targeted vector is a goal in the future [102]. The short half-life of LNP and VLP-based vectors is an advantage due to reduced off-target effects. Also, restriction of Cas9 nickase to cardiac tissue could be possible by using siRNA blocking effect [102].

The use of prime editing is not restricted to genetic defects such as non-genetic arrhythmia treatment but can also be approached for targeting amino acid residues on key domains of functional proteins. Lebek et al. showed that the BEs strategy can remove oxidation-sensitive methionine residues on CaMKII protein kinase [132]. As identical CaMKII residues are functioning in ventricular arrhythmogenesis in DMD and atrial fibrillation, the prime editing has the potential to ablate a wider array of molecules related to arrhythmia disorders [132]. This kind of PE-based modification of proteins permits a wide range of change over most cellular pathways with less side effects.

In contrast to the difficulty of CRISPR-based HDR replacement of genes in iPS cells to create cardiac disease model, prime editing can precisely introduce a specific residue via fragment replacement of mutation without the need for DSBs and bystander effect on other locations. Therefore, the utility of this approach in modeling cells and animals in the future could be very helpful in the field of cardiology [133]. However, the lack of a handy cardiac cell line for easy manipulation of genome is another problem that could slow down the progress of PE method in heart.

Besides many unknown factors related to transcriptomics and genomics, local chromatin structure, epigenetic status, and architecture of adjacent sequences of the target site could affect the accessibility of the loci and edition efficiency. Moreover, phases of the cell cycle, type of cell, and even tissue mixture might impact the rate of on-target efficacy [101]. Understanding the correlation between these factors and edition rate in heart cells could circumvent challenges during the selection of several options to hit the run at in vivo model. Regarding the cardiomyocytes cell status, epigenomics could be a barrier to accessibility of genome for PE activity in many loci. Recently, several efforts have performed to open the chromatin for better PE targeting using proximal dead sgRNA (dsgRNA), chromatin-modulating peptides, and histone deacetylase inhibitor (HDACi) molecules [72]. However, the use of these components for cell cycle progression or chromatin opening might not be safe in cardiac tissue.

In conclusion, gene editing technology, particularly prime editing, needs more investigation to achieve sustainable efficiency for in vivo application. In addition, it remains to be investigated whether PEs have the capability to restore the functionality or more importantly, to prevent chronic cardiac diseases for a life in animal models.

7. Expert opinion

For chronic hereditary heart failure, the PE approach may be a good alternative to the long-term medications now in use. The benefits and limitations of PE in the field of cardiology were discussed in this review.

Despite PE’s potency for accurate and versatile gene editing of the heart and other tissues, due to its poor in vivo efficiency, much research into the strategy’s enhancement is demanded. The sup-optimal structure, and large architecture size in terms of editor machinery should be taken into consideration for better applications.

The enhancement of PE is an endless process that needs more investigation to reach a universal machinery for all edition purposes. The methods for improvement to achieve a universal PE version for cardiac cells edition were outlined in our review.

One of the obstacles to comprehending the impact of PE on the heart and moving forward with preclinical research is the scarcity of myocardial cell and genetic animal models, especially for long-term preventive studies. Only a few studies on animals have been done to predict the effectiveness of PE on heart diseases. Nevertheless, PE is an excellent method for development of animal and cell models of genetic cardiac disorders. PE would be a precise approach to modify progenitor cells prior to engrafting into the cardiac niche in the future of cell therapy for heart regeneration.

The correct heart delivery of PE machinery would be a major concern for the future. Given the low rate of in vivo edition and the poor outcomes from both viral and non-viral vectors, along with the recent improvements in the mRNA/LNP platform’s manufacturing, size capacity, and safety, this platform could be the first choice soon. In addition, the complexity, durability, and immunogenicity of viral vectors hamper their safe clinical use as a PE delivery system particularly for heart tissue. Moreover, any physical method such as cardiac vein retro-injections that facilitates the accessibility of myocardial cells is also beneficial and should be considered more.

The cardiomyocyte physiology should be softly affected by delivery vector during the edition process, therefore transient exposure to LNPs and short-term gene expression seems to be the most favorable ones. Also, the expression time should be conducted based on specific promotes, miRNA, and other molecular switches. Handling the tunable expression for a multi-component machinery like PE also needs special design to achieve a balance between elements. The U6 promoter directed expression has been the method of choice for pegRNA and sgRNA expression, until now. An ideal PE delivery should be targeted specifically to myocardial cells to increase efficiency and to avoid immune cells transduction.

On the other side, long-lived cardiomyocytes cells should be prepared for better PE performance by modification of cell cycle status and epigenetic. In the future, fine-tuning chromatin and epigenetic changes will be crucial to enhancing the accessibility of editing machines to the target regions. Determining the relative benefits and risks of using cell cycle or epigenomic modifiers to modify cardiac cells is also crucial. Utilizing siRNA, shRNA, various medication classes, and epigenetic modifiers may offer a novel avenue for priming cells to be more receptive to gene editing.

Deciphering the cellular essential components involved in the PE process using transcriptomics would enable customized medicine to support the identification and prediction of the most effective PE approach for everyone. To reach a sustained edition rate per person, personalized PE would offer a more suited PE technique with less vector injection and other elements. In addition, prediction approaches, particularly those based on the combined transcriptome and epigenetic data, could be useful. In the future, based on the cost and significance of the PE method, it’s affordable to screen everyone’s transcriptomics for prediction of efficiency as well as to avoid some risk related to type of edition.

The PE will reach clinical use in few years for remedy of many human diseases, such as cardiac malfunction, however, other point that must be investigated is whether PEs are stable enough to permanently restore gene functioning in animal models in term of heart diseases.

Article highlights

• As the most versatile and precise editing strategy, prime editing can bring a durable cure for most genetic cardiac diseases.

• Several generations of prime editors have been developed that can correct most cardiac genetic disorders.

• Despite fast progression, the optimization of PE elements and heart-specific delivery methods need further studies to be functional in vivo.

• The most important challenge of PE is the delivery method due to its large size and current limitations of viral and non-viral vectors.

• Non-viral delivery such as RNP and VLP have advantages of safety, transient expression, and low immune induction that can be employed for PE transduction.

• Myocardial cells are different from other cells due to their quiescent status, and long half-life, therefore genome editing by PE is a promising approach but needs improvements.

Abbreviations

AAV	=	Adeno-associated virus
BFP	=	Blue fluorescent protein
BEs	=	base editors
CRISPR	=	clustered regularly interspaced short palindromic repeats
Cas9	=	CRISPR-associated nuclease 9
CHD	=	Chronic heart disease
DCM	=	dilated cardiomyopathy
DMD	=	Duchene muscular dystrophy
DSB	=	double-strand DNA break
DsDNA	=	Double-stranded DNA
GFP	=	Green fluorescent protein
hiPSC	=	human induced pluripotent stem cell
HCM	=	hypertrophic cardiomyopathy
HDR	=	homology-directed repair,
IPSCs	=	induced pluripotent stem cells
Kbp	=	kilo base pair
LNP	=	Lipid nanoparticle
LDL	=	Low-density lipoprotein
MMEJ	=	microhomology-mediated end joining
NHEJ	=	non-homologous end joining
PAM	=	protospacer adjacent motif
PBS	=	primer binding site
pegRNA	=	prime editing guide RNA
PEs	=	Prime editors
RNP	=	ribonucleoprotein complexes
RT	=	reverse transcriptase
RTT	=	reverse transcriptase template
SNP	=	single nucleotide polymorphism
sgRNA	=	single guide RNA
VLP	=	virus-like particle

Declaration of interests

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Additional information

Funding

The study was supported by the ERC advanced grant Flagship program of the Finnish Medical Research Council and Horizon European Country grant.