Universal Prime Editing Therapeutic Strategy for RyR1-Related Myopathies: A Protective Mutation Rescues Leaky RyR1 Channel

Abstract

RyR1-related myopathies (RyR1-RMs) include a wide range of genetic disorders that result from mutations in the RYR1 gene. Pathogenic variants lead to defective intracellular calcium homeostasis and muscle dysfunction. Fixing intracellular calcium leaks by stabilizing the RyR1 calcium channel has been identified as a promising therapeutic target. Gene therapy via prime editing also holds great promise as it can cure diseases by correcting genetic mutations. However, as more than 700 variants have been identified in the RYR1 gene, a universal treatment would be a more suitable solution for patients. Our investigation into the RyR1-S2843A mutation has yielded promising results. Using a calcium leak assay, we determined that the S2843A mutation was protective when combined with pathogenic mutations and significantly reduced the Ca²⁺ leak of the RyR1 channel. Our study demonstrated that prime editing can efficiently introduce the protective S2843A mutation. In vitro experiments using the RNA electroporation of the prime editing components in human myoblasts achieved a 31% introduction of this mutation. This article lays the foundation for a new therapeutic approach for RyR1-RM, where a unique once-in-a-lifetime prime editing treatment could potentially be universally applied to all patients with a leaky RyR1 channel.

Keywords: CRISPR/Cas9; RYR1 gene; RyR1-RM; calcium channel; calcium leak; prime editing; protective mutation.

Figure 1

Impact of Ser2843 phosphorylation on RyR1 mechanism. (a) Calstabin in complex with a subunit of the RyR1 channel stabilizes its closed conformation. The action of cyclic AMP (cAMP)-activated protein kinase A (PKA) phosphorylates Ser2843 displaces calstabin and destabilizes the channel closed state, resulting in a leaky channel. (b) Ala2843 cannot be phosphorylated. Calstabin remains in complex with the channel, reducing the probability of channel opening.

Figure 2

Calcium leak from RyR1 expressing HEK microsomes. (a) Ca²⁺ leak measured in microsomes from HEK cells expressing WT-RyR1, RyR1-R1667C + 1714del, RyR1-R1667C + L1714del + S2843A, and RYR1-R1667C + 1714del + S107. The graph of one trial is represented. The graphs of the four other trials are in Figure S1. (b) Bar graphs represent the quantification of the increase in Fluo-4 signals over the first 5 s after the addition of thapsigargin (change in signal/5 s = change/s). The 5 s time interval was selected as it provides the most precise measurement of the calcium ‘leak’, as reflected by the slope of the initial calcium release following thapsigargin treatment. Subsequently, cytosolic calcium levels stabilize, establishing a new equilibrium. Five trials of this experiment were performed. Thus, N = 5 for each group. ** represents a p-value < 0.01 vs. WT, and **** represents a p-value < 0.0001 vs. WT. †† represents a p-value < 0.01 vs. designated condition, and ††† represents a p-value < 0.001 vs. designated condition. ns represents a non-significative difference between (R1167C + L1714del + S2843A) and (R1167C + L1714del + S107).

Figure 3

Introduction of the S2843A mutation in the RYR1 gene by prime editing. (a–c) Sequence of the section of the RYR1 gene containing the S2843 codon. The coding nucleotides are in capital letters, and those in the intron are in lowercase. Note that the sense strand is on the top of the figure. Three spacers were tested. The normal 2843 codon is TCA-coding for serine. To introduce the protective mutation S2843A, this codon is mutated to GCA-coding for alanine. (a) Spacer 1 uses an NGG PAM. The intended T>G substitution is at +12 from the cut site. The PAM (5′-CGG-3′) is in the sense strand, and it is the antisense strand that will be nicked by the SpCas9 nickase part of the PE. To introduce the protective mutation, a C must be inserted into the RTT sequence so that the reverse transcriptase synthesizes a new sense strand containing a 5′-GCA-3′ alanine sense codon. In this case, the PAM was not disrupted. (b) Spacer 2 uses an NGAN PAM. The intended T>G substitution is at +6 from the cut site. The PAM (5′-TGAT-3′) is in the antisense strand, and it is the sense strand that will be nicked by the SpCas9 nickase part of the PE. To introduce the protective mutation, a G must be inserted into the RTT sequence so that the reverse transcriptase synthesizes a new antisense strand containing a 5′-CGT-3′ alanine antisense codon. Note that the introduction of this mutation disrupts the PAM at the same time. It was tested with the PE-VQR and the PE-NG. (c) Spacer 3 uses an NGG PAM. The intended T>G substitution is at +16 from the cut site. The PAM (5′-GGG-3′) is in the antisense strand, and it is the sense strand that will be nicked by the SpCas9 nickase part of the PE. To introduce the protective mutation, a G must be inserted into the RTT sequence so that the reverse transcriptase synthesizes a new antisense strand containing a 5′-CGT-3′ alanine antisense codon. A mutation disrupting the PAM was also introduced (5′-GGG-3′ to 5′-GAG-3′). (d) Introduction of the S2843A mutation in the RYR1 gene by prime editing in HEK293T cells. Transfection was carried out through the lipofection of plasmid DNA coding for the prime editing components (PE3). The mean of Spacer 1 includes the results of all the combinations of RTT-PBS lengths tested with Spacer 1 (see Table S1). The mean of Spacer 2 includes the results of all the combinations of the RTT-PBS lengths tested with Spacer 2 (see Table S1). Only one combination of the RTT-PBS length was performed with Spacer 3, and N = 3 replicates were performed. One-way ANOVA was used as a statistical test. **** represents a p-value < 0.0001. Efficiencies with Spacer 1 and Spacer 2 (with PE-VQR) were not significantly different from that of the negative control (Ctrl-). The efficiency of the epegRNA with Spacer 3 is significantly different from all other conditions with a p-value > 0.0001. (e) Introduction of the S2843A mutation in the RYR1 gene by prime editing in human myoblasts. The S2843A mutation is at +16 from the cut site. The PAM at +5 was also disrupted. Transfection was carried out through the electroporation of plasmid DNA coding for the prime editing components (PE3). N = 7 replicates were performed. One-way ANOVA was used as a statistical test. * represents a p-value < 0.05, *** represents a p-value < 0.001, and **** represents a p-value < 0.0001.

Figure 4

RTT sequence exhibiting the silent (same-sense) mutation combinations from the epegRNA targeting the introduction of the S2843A mutation. The S2843A mutation (+16) is in blue. The PAM silent mutation (+5) is in green. By counting from right to left, the position of the mutation from the cut site can be calculated. The +8 silent mutation is in red. The +11 silent mutation is in purple. The +14 silent mutation is in yellow. RTT+ represents the original RTT used in Figure 3d (Spacer 3) and 3e. The WT sequence is the one found in the human genome.

Figure 5

Effects of different combinations of silent mutations on the efficiency of prime editing to disrupt the PAM and to insert the S2843A mutation in the RYR1 gene. (a) Prime editing efficiency to introduce the S2843A mutation in the RYR1 gene (+16 from the cut site) and to disrupt the PAM (+5 from the cut site) in wildtype myoblasts with the PE3 strategy. The + condition represents the epegRNA with only the +5 (PAM disruption) and +16 (S2843A) edits. Combinations of silent mutations for conditions A to J are specified in Figure 4. The—condition represents the negative control. The experiment was performed in biological duplicates. Tukey’s multiple comparison test was performed. p-values are represented in (b,c). (b) p-value of multiple comparisons of different epegRNA configurations for PAM disruption efficiency. (c) p-value of multiple comparisons of different epegRNA configurations for S2843A introduction efficiency. * represents a significant difference with a p-value of 0.05; **, of 0.01; ***, of 0.001; and ****, of 0.0001.

Figure 6

Impact of the +11 (A>G) silent mutation added in epegRNAs on prime editing efficiency. (a) Comparison of epegRNAs without the A>G mutation at +11 (+, A, B, F, and G) with those containing it (C, D, E, H, I, and J) to induce the C>T mutation in PAM at +5. (b) Comparison of epegRNAs containing the A>G mutation in +11 with those without it to induce the S2843A mutation (n = 10 for the group without the mutation in +11 and n = 12 for the group with the mutation in +11). An unpaired t-test was performed, and **** represents a significant difference with a p-value < 0.0001.

Figure 7

Comparison of different prime editing strategies (PE3, PE5, PE5max, and PE6) to disrupt the PAM and to introduce the S2843A mutation into the RYR1 gene. (a) Efficiency of different prime editing strategies to disrupt the PAM in WT myoblasts by plasmid electroporation. (b) Efficiency of different prime editing strategies to introduce the S2843A mutation into the RYR1 gene in WT myoblasts by plasmid electroporation. EpegRNAs B, F, and G were used with the PE3, PE5, PE5max, and PE6 editing strategies. The—condition represents the negative control. The experiment was performed in biological duplicates. Tukey’s multiple comparison test was performed within each group (groups are the different epegRNAs: B, F, G, and -. * represents a significant difference with a p-value of 0.05; **, of 0.01; ***, of 0.001 and ****, of 0.0001. In (a), conditions within groups were not significantly different.

Figure 8

Introduction of the S2843A mutation in the RYR1 gene by prime editing. The PE3 components were delivered by plasmid DNA or RNA by electroporation in WT myoblasts. RNA ½ Qty represents the following quantities: 0.5 µg PE mRNA, 2.3 µg of epegRNA, and 0.9 µg of nsgRNA. RNA 1X Qty represents the following quantities: 1 µg PE mRNA, 4.6 µg of epegRNA, and 1.8 µg of nsgRNA. RNA 2X Qty represents the following quantities: 2 µg PE mRNA, 9.2 µg of epegRNA, and 3.6 µg of nsgRNA. One-way ANOVA and Tukey’s multiple comparison test were used as a statistical test. All conditions are significantly different from the negative control (Ctrl-). ** represents a significant difference with a p-value of 0.01; ***, of 0.001 and ****, of 0.0001.