Retinitis pigmentosa (RP) is an inherited retinal dystrophy causing progressive and irreversible loss of retinal photoreceptors. Here, we developed a genome-editing tool characterized by the versatility of prime editors (PEs) and unconstrained PAM requirement of a SpCas9 variant (SpRY), referred to as PE SpRY . The diseased retinas of Pde6b-associated RP mouse model were transduced via a dual AAV system packaging PE SpRY for the in vivo genome editing through a non-NGG PAM (GTG). The progressing cell loss was reversed once the mutation was corrected, leading to substantial rescue of photoreceptors and production of functional PDE6β. The treated mice exhibited significant responses in electroretinogram and displayed good performance in both passive and active avoidance tests. Moreover, they presented an apparent improvement in visual stimuli-driven optomotor responses and efficiently completed visually guided water-maze tasks. Together, our study provides convincing evidence for the prevention of vision loss caused by RP-associated gene mutations via unconstrained in vivo prime editing in the degenerating retinas.
In this study, we developed a genome-editing tool that is characterized by the combination of the versatility of PEs and unconstrained PAM requirement of SpRY, referred to as PE SpRY . This construct, together with its paired gRNAs, was delivered into the neural retinas of Pde6b rd10 mouse, a well-known RP mouse model, via a split Npu intein–based dual-AAV (adeno-associated virus) system. Liberated from the restrictions caused by the PAM availability and editing types, the PE SpRY system is herein demonstrated to efficiently correct the mutation with a non-NGG PAM (GTG) and with two types of edits (Pde6b T to C and Pde6b AGA ). Regardless of a complete loss of sight occurring in the age-matched control, the PE SpRY system–elicited genome editing facilitates the apparent preservation of photoreceptors as well as the restoration of compromised PDE6 phosphodiesterase activity, leading to the evident rescue of visual function in Pde6b rd10 mice, which is substantiated by a detailed electroretinogram (ERG) and behavioral assessments.
Retinitis pigmentosa (RP) is characterized by progressing retinal degeneration and represents one of the major causes of blindness throughout the world, with an estimated incidence of one in 4,000 human births ( Hartong et al., 2006 ; Verbakel et al., 2018 ). To date, a variety of mutations in more than 100 genes such as phosphodiesterase 6b (PDE6b) have been found to be associated with this devastating inherited retinal disorder (IRD; Daiger et al., 2013 ; Gagliardi et al., 2019 ; https://sph.uth.edu/retnet/ ). The degeneration in diseased retinas begins with the initial dysfunction of dim light-sensing rod photoreceptors distributed throughout the retina, followed by the onset of secondary death of cone photoreceptors present with the highest density at the center of macula and accounting mainly for the color vision, and leads to severe and irreversible deterioration of vision and eventual blindness.
Instead of using conventional genome-editing tools with restricted targetable loci due to PAM preferences or with limited editing types able to be installed, we took advantage of an engineered Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) fused to a near PAM-less variant of Streptococcus pyogenes Cas9 (SpRY) with H840A mutation introduced, hereafter referred to as PE SpRY (PE with SpRY nickase), for prime editing with unconstrained PAM availability ( Anzalone et al., 2019 ; Walton et al., 2020 ). We first in vitro tested the strategy in an engineered mouse Neuro2a cell line carrying the Pde6b rd10 mutation (referred to as Neuro2a rd10 ), where the native base C is substituted by T to achieve the same sequence compositions as those in Pde6b rd10 mice ( Fig. 1 A ). Based on the previous report ( Anzalone et al., 2019 ), we systematically constructed multiple prime editing guide RNAs (pegRNAs) with single-base resolution around this locus (p1–p14), utilizing recommended lengths of the primer binding site (PBS) and RT template (13–14-nt PBS and 13–15-nt RT templates) in combination with a variety of non-edited strand nicks 51–108-bp away from the target T∙A base pair (n1–n7; Fig. 1, C and D ; and Tables S1 and S2 ). Three regular types of targeted edits at or around the locus from the most common single point edit to the combination edits, including a single nucleotide substitution (T to C, namely an edit back to wild-type, herein referred to as Pde6b T to C ), a silent mutation with double substitutions (TGC to AGA, no change in amino acid composition relative to wild-type, named as Pde6b AGA ), as well as combination edits with a small insertion and substitution (AC insertion and T to C, named as Pde6b ACins, TtoC ; Fig. S1 A ), were assayed and the percentage of correct edits and indels were quantified by high-throughput deep sequencing.
The retinal degeneration in Pde6b rd10 mice is attributed to a change in amino acid composition from arginine residue to cysteine residue at site 560 (R560C) of the protein as a result of an inherited missense mutation from C to T naturally occurring in Pde6b gene on the exon 13 ( Fig. 1 A ; Chang et al., 2007 ). Pde6b encodes a key β subunit of phosphodiesterase (PDE) and exerts a determinant role in initiating rod phototransduction. In Pde6b rd10 mice, degeneration in outer nuclear layer (ONL) herein started from around postnatal day 16 (P16) with rod dying progressively at the initial phase, followed by the secondary cone death, and leaving only a single row of cell bodies in ONL as early as P60 in comparison with the situations in age-matched wild-type and P14 degenerative contexts ( Fig. 1 B ; Chang et al., 2007 ).
In the meantime, we compared the preservative effects in the Pde6b T to C context treated at P14 and P21. Despite the much faster degeneration process in the Pde6b rd10 mice compared with humans, the editing efficiency and rescue effect were significant in Pde6b T to C retinas treated at P21 with an editing frequency averaging 41.91 ± 1.29% ( Fig. S3, L and M ) and a Rhodopsin fluorescence intensity of less than half of those in the retinas treated at P14 ( Fig. S3, N–U ), suggesting the effective prevention from photoreceptor degeneration elicited by PE SpRY -mediated genome editing in the Pde6b rd10 mice.
Since rod death is followed by secondary cone death in the Pde6b rd10 mice, we further evaluated whether cone photoreceptors were preserved by PE SpRY -elicited genome editing. Cone photoreceptors herein were visualized with peanut agglutinin (PNA), a well-known marker for all cone photoreceptor subtypes, through immunohistochemistry. Irrespective of the barely detectable staining in the Pde6b rd10 control mice treated with PE SpRY and gScrambled ( Fig. 4, A–D and I ), the PNA levels exhibited over half of those in wild-type mice in the Pde6b T to C context, and the profile, similar to Rhodopsin, indicated a close correlation with GFP signals ( Fig. 4, E–I ). A closer examination further evidenced that PNA only presented sparsely and was undetectable in most regions in the Pde6b rd10 control mice treated with PE SpRY and gScrambled ( Fig. 4, J–L and P ), whereas PNA appeared bushy and erect in the Pde6b T to C mice ( Fig. 4, M–P ). The concomitant decline of PNA and GFP signal intensities further supported that the cone preservation was a consequence of PE SpRY -elicited genome editing ( Fig. 4, Q–T ).
Having demonstrated the potential that desired combination edits could be installed into the genome through PE SpRY system without stringent PAM restriction, we were motivated to further evaluate whether the strategy could be amenable to the in vivo mutation correction in Pde6b rd10 mice. Since p5n3 with optimized RT template (12 nt) and PBS (14 nt) has shown the best performance in installing desired edits in Neuro2a rd10 cells, we would mainly utilize this setup for two types of targeted correction in Pde6b rd10 mice. One is regular single nucleotide substitution (from T in Pde6b rd10 to C in wild-type, hereafter referred to as Pde6b T to C ) and the other one is a silent edit (relative to wild-type context) from TGC in Pde6b rd10 to AGA (uniformly referred to as Pde6b AGA ), which still encodes Arg residue as wild-type locus ( Fig. S2 A ). As for the delivery, we exploited a split Npu intein–based dual-AAV system widely used for packaging large size of genome editors ( Chen et al., 2020 ; Chew et al., 2016 ; Levy et al., 2020 ; Zhi et al., 2022 ), and the splitting site at 713–714 of SpRY protein would guarantee the accommodation of PE SpRY in AAV vectors ( Fig. S2 A ).
The membrane-associated PDE6 plays an indispensable role in initiating rod phototransduction triggered by light stimulation. The catalytic core of functional PDE6 holoenzyme, PDE6β, along with PDE6α facilitates the hydrolysis of cyclic guanosine monophosphate (cGMP) into GMP, leading to the closure of cGMP-gated cation channels, which causes hyperpolarization and hierarchical signal transmission to the neurons housing in the inner nuclear layer. To assess whether the compromised function of PDE6β was restored in the Pde6b rd10 mice through PE SpRY -elicited genome editing, we measured the PDE activities. In retinal samples from wild-type mice, the PDE activity reached 2.06 ± 0.10 μU/mg at P14 and rose mildly to 2.29 ± 0.03 μU/mg at P120 compared with Pde6b rd10 control mice treated with PE SpRY and gScrambled, in which PDE activities were as low as 0.24 ± 0.01 and 0.11 ± 0.01 μU/mg at P14 and P120, respectively. Instead, PDE activities in samples from Pde6b T to C mice at P14 were the same as those in Pde6b rd10 mice, yet rose to 1.81 ± 0.04 μU/mg, suggesting the restoration of PDE activity to a significant extent after genome editing ( Fig. 5 A ). To examine the genuine involvement of functional PDE6 holoenzyme in hydrolyzing cGMP, we measured the levels of endogenous cGMP in retinas. The samples from wild-type retinas presented a cGMP level as low as 6.51 ± 0.3 nM at P14 and 6.94 ± 0.4 nM at P120, respectively, whereas it reached 10.94 ± 0.3 nM in the Pde6b T to C context at P14. However, the level apparently dropped to 7.20 ± 0.1 nM at P120, providing convincing evidence that the function of PDE6β was substantially restored with the implementation of genome editing by the PE SpRY system in the retinas of Pde6b rd10 mice ( Fig. 5 B ).
In addition, we further examined the active avoidance of these mice via the shuttle box learning test, during which the mice would become much more sensitive to conditioned light due to an electrical shock when they failed to escape ( Fig. 6 I ). In the 5-d training session, the Pde6b T to C mice showed a stepwise improved active avoidance acquisition with a percentage increase from averaging 15.8 ± 4.9% on the first day to 64.5 ± 7.6% at the end of the training (the fifth day; Fig. 6 J ), corresponding to a decline of passive avoidance from averaging 66.0 ± 10.6 to 16.7 ± 2.8% ( Fig. 6 L ), which was nearly to keep up with their age-matched wild-type mouse performance. By contrast, the Pde6b rd10 control mice treated with PE SpRY and gScrambled exhibited rather poor active avoidance performance with an average level of no more than 40% (35.2 ± 8.7%; Fig. 6 J ) and up to 45.1 ± 7.4% passive avoidance ( Fig. 6 L ) and appeared unresponsive in the remaining around 20% of trials, suggesting the difficulty in acquiring correlation between the light and the electrical shock in these mice due to their poor vision. As for the latency, the time to the first active avoidance decreased steadily from around 1,000 to 15.1 ± 7.7 s on the fifth day for the Pde6b T to C mice, which was close to the performance of wild-type ones (3.6 ± 0.6 s; Fig. 6 N ), suggesting they have good knowledge of the setup, despite a relatively mild change in the Pde6b rd10 control treated with PE SpRY and gScrambled. As expected, the time traveled during the training decreased steadily and significantly both in the Pde6b T to C and in the wild-type mice, irrespective of the struggling and somewhat irregular change in the Pde6b rd10 control treated with PE SpRY and gScrambled ( Fig. 6 P ). We then moved forward to test the animal responses 1 and 2 wk after the end of training (d12 and d19), and the performance in the Pde6b T to C mice and in the wild-type ones remained relatively stable or became even better than that in the training session while no substantial changes were detected in the Pde6b rd10 control treated with PE SpRY and gScrambled ( Fig. 6, K, M, O, and Q ).
To provide substantial evidence that the visual function was indeed improved in the Pde6b rd10 mice subjected to PE SpRY system–elicited gene correction, we first carried out behavioral assessments for the passive avoidance via the light–dark transition test. With the luminance increasing from 300 to 900 lux, the Pde6b T to C mice demonstrated an apparent preference for staying at the dark chamber over 80% of time ( Fig. 6 A ), around 70% of distance traveled ( Fig. 6 B ), and up to 84% of rearing time ( Fig. 6 C ) in dark, which were a little inferior to those in wild-type mice (averaging 88.8 ± 2.3%, 86.0 ± 2.1%, and 91.3 ± 2.1%, respectively), whereas much better than those in the Pde6b rd10 mice treated with PE SpRY and gScrambled (averaging 53.6 ± 2.0%, 58.8 ± 1.7%, and 49.5 ± 5.1%, respectively). Meanwhile, the minimal duration in dark significantly kept rising (averaging 3.3 ± 0.5–8.5 ± 2.2 s; Fig. 6 D ) whereas the velocity in the dark declined (averaging 74.4 ± 9.4–33.0 ± 3.5 mm/s; Fig. 6 E ) with the luminance increasing, which was further confirmed by the reduced transition times from dark to light (averaging 10.2 ± 1.3–2.8 ± 0.4; Fig. 6 F ). These performances were very close to those in the wild-type (averaging 11.5 ± 2.5 s, 24.9 ± 4.6 mm/s, and 3.2 ± 0.6 at the luminance of 900 lux) in comparison with the hardly detectable passive avoidance in the Pde6b rd10 mice treated with PE SpRY and gScrambled (averaging 2.6 ± 0.4 s, 86.2 ± 6.8 mm/s, and 9.4 ± 0.7 at the luminance of 900 lux). The preferred movements in dark were more intuitively presented in traveling trajectories ( Fig. 6 G ) and the heat maps recording time spent at distinct regions of the light–dark box ( Fig. 6 H ). On the contrary, the Pde6b rd10 control mice treated with PE SpRY and gScrambled did not exhibit any apparent preference for the dark chamber, not to mention the comparison with the performance of wild-type mice ( Fig. 6, A–H ).
The optomotor responses demonstrated above were essentially contributed by both eyes of each mouse; the conclusion, however, would be generally more convincing if a comparison between paired contralateral eyes yet treated with different vectors is performed. With the aim to address this issue, we only treated one eye with PE SpRY and gPde6b T to C , leaving the contralateral one treated with control vectors, and evaluated the optomotor response levels at the optimal condition with 0.2 cycles/° spatial frequency and 100% contrast. The global OMR profiles appeared comparable in both the left and right eye of the wild-type mouse ( Fig. 8 A , top panel). It is intriguing, however, that the visual stimuli–driven responses in the clockwise direction appeared much more evident than those in the counterclockwise direction when the left eye was treated with PE SpRY and gPde6b T to C , whereas the right one with PE SpRY and gScrambled, and vice versa ( Fig. 8 A , middle and bottom panels). As for the test under the optimized velocity threshold (2–14°/s), the OMR values for the left and right eyes from wild-type context were 2.81 and 2.76, respectively, suggesting the comparable and evident visual function for both eyes. In the degenerative context, the left eye treated with PE SpRY and gPde6b T to C had an OMR value equal to 2.06 versus 1.07, a clear “blindness” indicator, in the right one treated with PE SpRY and gScrambled, which was the case when the visual improvement was occurring exclusively in the right eye ( Fig. 8 B ). The evaluation of visual stimuli–driven responses based on the ΔT was becoming more conclusive with a value of as low as 0 for internal control eyes compared with the obvious visual activities recorded from their contralateral eyes ( Fig. 8 C ). These observations were substantially evidenced by the immunohistochemical assays, in which the rod photoreceptors were only preserved in retinas transduced with PE SpRY and gPde6b T to C vectors, in striking contrast to the situation in those transduced with control vectors ( Fig. 8 D ). In addition, we sought to evaluate whether the visual function could be well sustained in the aged mice after PE SpRY system treatment. Compared with the complete loss of visually driven optomotor responses in age-matched Pde6b rd10 control mice treated with PE SpRY and gScrambled, the visual function in the Pde6b T to C mice remained relatively stable throughout the ages tested (from P150 to P240; Fig. 8 E ), with average ΔT values of no less than 5 s at the 0.2 cycles/° spatial frequency and 100% contrast ( Fig. 8 F ), further suggesting the efficiency and persistence of photoreceptor rescue via PE SpRY system–elicited in vivo genome editing.
Since the difference between T correct (total time animal head moved in the stimulus direction) and T incorrect (total time animal head moved in the opposite direction) in totally blind mice were theoretically close or equal to 0, we, therefore, assessed the visual function with T correct − T incorrect , hereafter named as ΔT, for a much more obviously intuitive method to measure the visual stimuli–driven responses. As expected, in the Pde6b rd10 degenerative context treated with PE SpRY and gScrambled, the optomotor responses were barely detectable at a panel of spatial frequencies and contrast tested ( Fig. 7, I and J ). By contrast, the responses showed parallel changes in the Pde6b T to C mice with those in the wild-type context, with a maximal response at a spatial frequency of 0.2 cycles/° and 100% contrast, respectively. Together, the enhanced optomotor responses suggested an improved visual function in the PE SpRY system–treated mice.
The head movement unrelated to visual stimuli is likely to be minimized when the ideal velocity threshold criteria are set and the range between 2 and 14°/s is regularly recommended based on previous observation ( Kretschmer et al., 2015 ). We thus carried out an investigation on the comparison of visual stimuli–driven responses under these optimal velocity conditions (the boxed ranges colored in magenta and green; Fig. 7, G and H ; and Fig. S5, A–D ). For an intuitive and uniform evaluation of data acquired at different conditions, the response time in either direction (correct or incorrect) at each velocity interval (1°/s in each interval) was normalized to the maximal response time (designated as 1) under each spatial frequency or contrast tested. Compared with those in the opposite direction (negative value, light magenta window), the occurrence of visually driven optomotor responses at 0.2 cycles/° in the Pde6b T to C mice was apparently more frequent and lasted longer (blue bars) in the stimulus direction (positive value, light green window), and the OMR value at 0.2 cycles/° was therefore up to 2.15. Accordingly, OMR values at 0.02, 0.3, and 0.4 cycles/° were 1.66, 1.45, and 1.08, respectively ( Fig. 7 G ). For the contrasts tested, OMR values appeared maximal at 100% contrast and were equal to 2.09, with a steady decline when the contrast levels were decreasing ( Fig. 7 H ). Combined with the observations at different spatial frequencies, the trends of OMR values were essentially consistent with the profiles of the heat maps presented in Fig. 7, E and F , with a particular emphasis on the parallel changes of visual responses between Pde6b T to C and wild-type ( Fig. S5, A and B ) rather than Pde6b rd10 degenerative control mice treated with PE SpRY and gScrambled ( Fig. S5, C and D ).
To globally assess the visually driven optomotor responses, the ratios of response time between the head movements in the correct and incorrect directions, namely OMR (optomotor reflex), were assayed in terms of a wide range of velocity thresholds at defined spatial frequency or contrast levels ( Fig. 7, E and F ). It is anticipated that the overall OMR values in wild-type context appeared relatively higher at a spatial frequency of 0.02 cycles/°, culminating at 0.2 cycles/° as OMR exhibited equal to or higher than 2.0 (presented in red) in most velocity thresholds ( Fig. 7 E ), followed by a decline afterward and drop to a nearly undetectable level at 0.4 cycles/°. By contrast, visual stimuli–driven responses in the Pde6b rd10 degenerative context treated with PE SpRY and gScrambled sustained a barely detectable level at all spatial frequencies tested as OMR values were no higher than 1.2 within most thresholds (in blue). Regardless of a relatively lower OMR at the beginning, the visual stimuli–driven response exhibited an obvious increase at a spatial frequency of 0.2 cycles/° in the Pde6b T to C mice and then declined steadily to an inappreciable level, essentially in line with the trend in wild-type context. As for the contrasts, the visual stimuli–driven response in the Pde6b T to C mice started with the highest level at 100% contrast and tapered down gradually from 50 to 5%, showing a parallel change with the situation in the wild-type mice ( Fig. 7 F ).
It is generally conceivable that mice would appear responsive to different spatial frequencies and contrasts once the vision is functionally restored to a certain degree. To quantitatively determine how much the vision was improved in PE SpRY system–treated mice, we sought to evaluate the visual function by an automated system with optomotor responses accurately and objectively recorded through video tracking algorithms ( Fig. 7, A and B ; Kretschmer et al., 2015 ). Both left and right eyes of wild-type mice exhibited approximately equal contributions to the total optomotor responses elicited by a panel of stimulus at defined spatial frequencies (0.02, 0.1, 0.15, 0.2, 0.3, and 0.4 cycles/°) or contrasts (100, 50, 25, 12.5, 10, and 5%; Fig. 7, C and D ), suggesting a reliable and unbiased detection by the system.
When tested for the visual acuity and contrast sensitivity, both Pde6b T to C and wild-type mice performed best at 0.2 cycles/° spatial frequency or at 100% contrast and demonstrated a clear and visually guided response with the change of conditions tested. In particular, the Pde6b T to C mice were capable of solving the cognitive task when the spatial frequency was as high as around 0.4 cycles/°, which was comparable to the visual acuity shown in wild-type mice ( Fig. 9 G ). This was also the case when the mice were tested at steadily declined contrasts ( Fig. 9 H ). The Pde6b T to C mice were able to pass the test once the contrast was over 25%, which was only a little inferior to the contrast sensitivity indicated in the wild-type mice, not to mention the failure in completing the task at all spatial frequencies and contrast levels tested in the Pde6b rd10 control mice. The performance of Pde6b T to C and wild-type mice was confirmed by their minimal latency time and distance traveled at 0.2 cycles/° spatial frequency ( Fig. 9 J and Fig. S5 I ) and 100% contrast ( Fig. 9 L and Fig. S5 J ), respectively. In contrast, the Pde6b rd10 control mice treated with PE SpRY and gScrambled performed so poorly that their latency time spent in finding the platform was much longer than that of Pde6b T to C and wild-type mice and exhibited irregular profiles as far as both the time and distance traveled were concerned ( Fig. 9, I and K ). These observations were consistent with the contexts of the heat maps reflecting the group-average time traveled at defined positions as well ( Fig. 9 M and Fig. S5 K ). For each of the mice tested in the water maze, both Pde6b T to C and wild-type groups completed the task with a correct rate over 70% compared with the complete failure of all Pde6b rd10 control mice treated with PE SpRY and gScrambled ( Fig. 9 N ). In addition, the test in the Pde6b AGA mice further supported the efficient genome editing elicited by the PE SpRY system ( Fig. S5, L–P ).
The target cells where genome editing is carried out in those reports are mainly restricted to RPE cells which are non-neuronal cells (Choi et al., 2022; Jang et al., 2021; Suh et al., 2021), and the Rpe65 gene encodes an isomerase in the visual cycle that regenerates the active visual chromophore 11-cis-retinal in RPE cells (Redmond et al., 1998). Most of the mutations causing RP and other IRDs are instead associated with neural photoreceptor functions and survival (Daiger et al., 2013; Gagliardi et al., 2019). Thus, given the complexity of neural architecture, the direct utility of genome editing in the neural retinal cells, in particular for unhealthy or dying photoreceptors, would provide much more convincing evidence for the potential applications of these tools in RP treatment. Some studies reported the successful introduction of BEs or PEs into the wild-type retinas for targeted genome editing (Chen et al., 2020; Zhi et al., 2022), which raises further concerns about the effectiveness of genome editing in the retina under the pathological condition. For example, photoreceptor cell death starts as early as P16 in Pde6brd10 mice, and only a single row of cell bodies is left in most regions of ONL at no later than P60. Whether the correction via genome editing enables photoreceptor preservation before all of them die or whether the photoreceptors preserved via genome editing are sustained well enough for the rescue of visual function would be totally different from the issue addressed under normal physiological conditions. Our results clearly indicate that PESpRY-elicited Pde6b gene correction is capable of promoting substantial photoreceptor survival in Pde6brd10 mice and the visual function preserved exhibits comparable or a little inferior to that in wild-type mice. To our knowledge, the present study is the first report systematically exploiting PE-based strategy to implement gene therapy in the RP disease model. In particular, the visual function is found to be sustained well in the treated Pde6brd10 mice as old as P240, which further supports PESpRY as a reliable genome-editing tool in treating RP.
Another point drawing our attention is the limitation of BEs used for the treatment of inherited retinal diseases. As we know, BEs only enable the installation of point mutations in the genome (Gaudelli et al., 2017; Koblan et al., 2021; Komor et al., 2016), and the bystander edits caused by the presence of the multiple C or A within the editing window have to be carefully considered. As expected, the ABE results in limited precision and unintended bystander edits when used in the correction of point mutation at the Rpe65 locus (Suh et al., 2021). To address this issue, PEs are used in the same LCA mouse model and produce an efficient single nucleotide substitution without any detectable indels and bystander effects (Jang et al., 2021). Based on these results, however, two critical issues are worth further consideration. One is whether PEs are able to appear versatile enough for in vivo genome editing when the combination edits are desired or when the inherited disorders are caused by mutations in two or more base pairs or in an even more complicated way. In other words, we expect that PEs exhibit versatility as they are doing in the cell lines (Anzalone et al., 2019) and that they are not restricted by editing types when used for the treatment of retinal diseases resulting from multiple mutations. The other is that all editors used in the above-mentioned studies are SpCas9-based proteins. The stringent PAM requirement would generate significant target sequence restrictions, presumably leading to the low efficiency of genome editing and the limited use of this tool. As demonstrated in the present study, the PESpCas9 is incapable of installing desired edits at Pde6b locus likely due to its PAM preferences, as the selected canonical PAMs and corresponding protospacers either exhibit lower editing efficiency based on the prediction and experimental tests or are far away from the targeted position, resulting in incompatible long-distance RT templates. To provide a solution for these questions, we use PESpRY, combining the advantages of PEs and the PAM-less SpRY variant, to successfully and efficiently introduce combination edits such as Pde6bAGA into the diseased retinas of Pde6brd10 mice and facilitate substantial rescues of both photoreceptors and visual function as well. Of particular importance, all of the 14 PAMs and their protospacers tested appear active, though with differential compatibility, for PESpRY. Among these PAMs, only one (TGG, corresponding to protospacer p7) displays canonical NGG sequence, but the editing efficiencies with other non-NGG PAMs such as GTG, TGC, and CAG (corresponding to protospacer p5, p4, and p9, respectively) appear much higher, suggesting the relaxed or unconstrained PAM requirement for PESpRY. In particular, PESpRY system with GTG as its PAM makes in vivo genome editing for RP mouse model become a reality, which would offer an opportunity for this genome-editing tool for more extensive applications in treating diverse IRDs.
The next point we should pay attention to is the appropriate assessment of the visual function after the correction of mutation causing eye diseases. As generally accepted, whether vision is improved after genome editing or any type of gene therapy relies on detailed behavioral tests. In comparison with previous reports, in which the behaviors were relatively roughly demonstrated after the correction of point mutation via ABEs or PEs in Rpe65-associated LCA mouse model (Jang et al., 2021; Suh et al., 2021), we herein carry out a careful behavioral characterization via a light–dark transition test for passive avoidance, shuttle box learning test for active avoidance, and visually guided optomotor response test, as well as water-maze visual discrimination tasks in Pde6brd10 mice subjected to PESpRY-elicited genome editing. The fully automated recordings and assays for each detail of mouse movements guarantee accurate quantitative assessments for the improved visual function, therefore providing solid evidence for the effects of PESpRY system on the treatment in Pde6b-associated RP mouse model. Despite the more objective evaluation of human vision through well-developed instruments such as Goldmann perimetry, microperimetry, and pupillometry, the introduction of multiluminance mobility testing in the phase 3 trial of Luxturna for the treatment of RPE65-associated LCA (Russell et al., 2017), suggesting the indispensability of intuitively evaluating the visual preservation and restoration via behavioral performance. Thus, the demonstration of functional rescue of vision via genome editing in the Pde6brd10 mice in these real-world simulations provides more conclusive information regarding the preservation of the vision-mediated direction, localization, and motion, and even cognition compared with the histological assay and ERG test.
It is worth noting that non-viral vectors including virus-like particles and lipid nanoparticles are currently emerging as an alternative approach to deliver cargos into the tissues and have demonstrated great potential in treating diseases (Banskota et al., 2022; Raguram et al., 2022). One of the limitations, however, is the size of the non-viral vectors. Typically, the size of AAV is around 20–30 nm whereas those of non-viral vectors generally exceed 100 nm, which precludes the delivery of cargos (DNAs, RNAs, or proteins) into the photoreceptors due to the physical barriers in the eye (such as the outer limiting membranes and the inter-photoreceptor matrix; Trapani et al., 2014). Thus, the non-viral vectors remain to be optimized for their efficiency in transducing neural retinas.