Systematic optimization of prime editing for the efficient functional correction of CFTR F508del in human airway epithelial cells

Abstract

Prime editing (PE) enables precise and versatile genome editing without requiring double-stranded DNA breaks. Here we describe the systematic optimization of PE systems to efficiently correct human cystic fibrosis (CF) transmembrane conductance regulator (CFTR) F508del, a three-nucleotide deletion that is the predominant cause of CF. By combining six efficiency optimizations for PE—engineered PE guide RNAs, the PEmax architecture, the transient expression of a dominant-negative mismatch repair protein, strategic silent edits, PE6 variants and proximal ‘dead’ single-guide RNAs—we increased correction efficiencies for CFTR F508del from less than 0.5% in HEK293T cells to 58% in immortalized bronchial epithelial cells (a 140-fold improvement) and to 25% in patient-derived airway epithelial cells. The optimizations also resulted in minimal off-target editing, in edit-to-indel ratios 3.5-fold greater than those achieved by nuclease-mediated homology-directed repair, and in the functional restoration of CFTR ion channels to over 50% of wild-type levels (similar to those achieved via combination treatment with elexacaftor, tezacaftor and ivacaftor) in primary airway cells. Our findings support the feasibility of a durable one-time treatment for CF.

Main

Prime editing (PE) enables the replacement of targeted DNA nucleotides with any specified replacement of up to hundreds of nucleotides, thereby enabling a wide variety of substitutions, insertions and deletions in the genomes of living systems^1,2,3,4. The mechanism of PE is inherently resistant both to bystander editing (unwanted editing at the target site)¹ and to off-target editing (unwanted editing away from the target site)^{1,3,5,6,7,8,9,10,11,12,13,14,15,16,17}. In contrast with nuclease-mediated gene editing, prime editors do not require the creation of double-stranded DNA breaks (DSBs), minimizing undesirable outcomes such as uncontrolled insertions and deletions (indels)^2,3,4,18, large deletions^19,20, p53 activation^21,22,23, retrotransposon insertion²⁴ and chromosomal defects^{19,25,26,27,28}. PE does not require co-delivery of donor DNA template, is active in mitotic and non-mitotic cells^{1,5,29,30,31,32,33,34}, and has been successfully performed in vivo in mice^{5,29,32,33,34,35,36,37,38} and in non-human primates³⁹.

Prime editors combine a programmable nickase such as Streptococcus pyogenes Cas9 (SpCas9) H840A nickase with a reverse transcriptase (RT) such as an engineered Moloney murine leukaemia virus RT¹. A PE guide RNA (pegRNA) guides the prime editor protein to its spacer-specified genomic target and also contains a 3′ extension with a primer binding site (PBS) complementary to the nicked target DNA and an RT template (RTT) encoding the desired edited sequence^1,40. When bound to its programmed target sequence, the prime editor–pegRNA complex nicks the target site to create an accessible 3′ end of single-stranded DNA. This target DNA 3′ end hybridizes to the pegRNA’s 3′ PBS, creating a primer–template complex that initiates reverse transcription using the RTT to create a 3′ DNA flap containing the edited DNA sequence¹. The 3′ flap of edited DNA can displace the original sequence and be ligated into the genome, creating a DNA heteroduplex of edited and unedited DNA strands. This editing intermediate is then resolved into a permanent edit on both strands by DNA repair or replication⁴¹. A nickase, RT and pegRNA constitute a ‘PE2-type’ editing system. A ‘PE3-type’ system adds an additional nicking guide RNA (ngRNA) that nicks the unedited strand of the DNA heteroduplex intermediate to enhance editing efficiency by directing mismatch repair (MMR) to remake the unedited strand using the edited strand as a template¹ (Fig. 1a).

PE is well suited to correct pathogenic mutations such as the three-base-pair CTT deletion (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in the loss of phenylalanine 508 in the CFTR protein. This deletion is the most common cause of cystic fibrosis (CF)⁴², an autosomal recessive disorder that affects more than 160,000 people worldwide⁴³. In people with CF, CFTR mutations impair the anion channel activity of CFTR that conducts Cl⁻ and HCO₃⁻ transport across the apical membranes of epithelia-lined secretory organs including the pancreas, gastrointestinal tract and respiratory tract^{44,45,46,47,48}. While over 2,000 CFTR variants have been identified and more than 700 are verified to cause CF, one mutation, CFTR F508del, is present in 85% of patients with CF^42,49. The CFTR F508del protein misfolds, and the majority of the protein undergoes proteasomal degradation^47,50,51,52. If trafficked to the cell membrane, the CFTR F508del channel is functional, albeit with a reduced open probability (P_o)⁵³. These molecular defects have been the target of several breakthrough small-molecule therapies that have greatly enhanced clinical outcomes for patients with CF^{54,55,56,57,58}. While effective and impactful, current small-molecule therapies require daily administration for life at an annual cost of approximately US$300,000 (refs. ^43,59,60). The development of a gene editing strategy to precisely correct the CFTR F508del CTT deletion could offer a path to a durable, one-time treatment for the most common CF-causing mutation.

Here, we describe the development of a PE approach to efficiently correct the CFTR F508del mutation in primary airway epithelial cells from patients with CF and rescue CFTR-dependent anion channel activity. Like others⁸, our initial attempts to correct this mutation with the originally reported PE2 and PE3 systems yielded minimal editing, revealing that this mutation was an unusually challenging one to correct by PE. By systematically applying six recent advances in PE technology, we achieved F508del correction efficiencies of up to 58% in an immortalized human bronchial epithelial cell model and 25% in primary airway epithelial cells from patients with CF. Optimized PE restored CFTR channel function to greater than 50% of wild-type levels in primary airway epithelial cells from patients with CF. These results demonstrate proof-of-concept for the direct therapeutic correction of the most common CF-causing CFTR mutation and serve as a roadmap for engineering PE strategies at difficult-to-edit therapeutic targets.

Results

Initial attempts to correct CFTR F508del with PE2 and PE3

We sought to correct the CFTR F508del mutation soon after we originally reported PE in 2019 (ref. ¹). To efficiently test and optimize PE CFTR F508del correction strategies⁶¹, we first used PE to generate a clonal HEK293T cell line homozygous for this deletion in the endogenous CFTR gene (Supplementary Fig. 1 and Supplementary Tables 1 and 2). Next, we used this cell line with PE2 to screen pegRNAs with combinations of PBS and RTT lengths at two F508del CTT deletion-proximal protospacers with NGG protospacer adjacent motifs (PAMs; NGG1 and NGG2; Supplementary Fig. 2). We observed no detectable correction of CFTR F508del using NGG1 pegRNAs (Extended Data Fig. 1), which we hypothesized was due to the presence of a TTTT sequence on the non-PAM containing strand of the NGG1 protospacer. We speculate that this TTTT may act as an RNA polymerase III transcriptional terminator that prevents complete pegRNA PBS transcription from a U6 promoter, resulting in an incompletely transcribed pegRNA that cannot support PE (Supplementary Fig. 2). We did observe F508del correction when NGG2 pegRNAs were used, but no NGG2 PE2 editing strategies yielded average editing efficiencies greater than 0.2% (Fig. 1b).

To enhance correction of F508del, we performed a PE3 screen of two NGG2 pegRNAs (NGG2 PBS13 RTT29 and NGG2 PBS14 RTT41) against a panel of all available ngRNA protospacers with pegRNA–ngRNA inter-nick distances of approximately 125 bp (Fig. 1c,d). While the best-performing PE3 NGG2 pegRNA offered up to a 2.6-fold improvement in edit installation efficiency over PE2, the maximum mean editing for any NGG2 PE3 edit did not exceed 0.5%, suggesting that PE3 systems alone cannot efficiently correct CFTR F508del.

We hypothesized that the inefficient correction of F508del using NGG1 and NGG2 could be due to inaccessibility of the edit site to Cas effectors due to chromatin state, which has been reported to negatively affect all major forms of mammalian cell genome editing^9,62,63,64. CFTR resides within a topologically associated domain whose chromatin state is tightly controlled by protein interactions with numerous enhancers and insulators embedded within the gene⁶⁵. To distinguish chromatin accessibility from prime editor-specific editing limitations, we targeted both NGG1 and NGG2 with pegRNA-guided ABE8e-SpCas9(D10A) adenine base editor (ABE)⁶⁶ to test if a base editor could edit either protospacer. Reassuringly, we observed efficient A•T-to-G•C editing at adenine bases within the expected editing window of ABE8e at NGG1 and NGG2, averaging up to 45% and 29%, respectively (Fig. 1e), demonstrating that NGG1 and NGG2 were indeed accessible by pegRNA-guided Cas effectors. These results suggest that suboptimal execution of the PE steps that follow target site engagement was primarily responsible for inefficient CFTR F508del correction.

PE advances enhance correction of CFTR F508del

Several advances in PE were developed during the course of this study (Fig. 2a). The first of these improvements was the development of engineered pegRNAs (epegRNAs), which protect pegRNA RTT and PBS sequences from endogenous exonuclease degradation by appending an RNA pseudoknot motif to pegRNA 3′ ends⁴⁰. We also reported that inhibition or evasion of cellular DNA MMR enhances PE purity and efficiency⁴¹ and that PEmax, an architecture-optimized prime editor protein, further increased editing efficiency⁴¹. Recently, the PE6 suite of laboratory-evolved and engineered RTs and prime editor Cas9 domains with enhanced editing capabilities were reported³⁴. Given that these PE enhancements address distinct bottlenecks in the PE mechanism, we sought to combine their capabilities to improve CFTR F508del correction…..