Figure3 . Western blot analysis of STAT3 in flexor digitorum
brevis (FDB) muscle after prime editing with constructed pegRNAs
(pegRNA8 and pegRNA10), the ipsilateral extensor digitorum longus (EDL)
muscle was used as untreated control (pegRNA8 vs Untreated control: P =
0.033; pegRNA10 vs Untreated control: P = 0.014; pegRNA8 vs pegRNA10: P
= 0.811).
4. Discussion
In this study, we constructed pegRNA to target mutated leptin receptor
in the db/db mice using FDB muscle electroporation, aiming to evaluate
the in vivo efficiency of pegRNA-mediated genome editing. Our results
demonstrated successful base transversion mediated by the constructed
pegRNA. More importantly, corrected leptin receptor protein functioned
normally as evidenced by downstream STAT3 signaling. These findings
underscore the feasibility of using pegRNA as a valuable tool for
targeted genome editing in vivo. It highlights the potential of PE as a
promising therapeutic approach to manipulate point-mutations in a litany
of diseases, providing new avenues for potential therapeutic
interventions.
While in vivo rescue of the leptin receptor has been performed in db/db
mice [19], we believe our study has several
advantages over the conventional gene delivery approaches. Our study
focused on ”correct” the point mutation in endogenous leptin receptor
gene. Thus, we only mended endogenous leptin receptor gene without
altering its regulatory elements, such as 3’UTR, 5’UTR, etc. Therefore,
the leptin receptor gene expression pattern, expression level, and
tissue distribution in prime editing treated db/db mice should be very
similar as those in wild type animal, which is very challenging in
conventional gene delivery approach. In addition, PE is a
’search-and-replace’ genome editing technology. Unlike CRISPR-Cas9, it
does not require DBSs or donor DNA templates to achieve precise
modifications of the target DNA sequence [6]. In
contrast, base editors, although capable of introducing transition point
mutations without DSBs, currently lack the ability to install
transversion point mutations, precise insertions, or precise deletions[5]. Furthermore, it can inadvertently induce
bystander mutations when multiple target nucleotides exist within the
editing window, and limitations in PAM availability may hinder targeting
certain bases [5]. However, PE does not require a
precisely positioned PAM sequence, thus providing greater flexibility
and precision in targeting specific genomic loci[6]. This targeting flexibility allows for a wide
range of editing possibilities, enabling precise alterations with
enhanced accuracy [6,17]. The success of prime
editing has been evident in numerous studies conducted in diverse cell
types and animal models, underscoring its potential for therapeutic
applications [20-22].
The translation of prime editing into a therapeutic approach in vivo
requires careful consideration and strategic planning. While the
efficiency and precision of prime editing have been demonstrated in
experimental settings, the delivery of prime editing components into
target cells or tissues remains a significant challenge. Various methods
involving viral vectors, nanoparticles, or direct injection have been
explored to ensure delivery [10,23-28]. To further
advance this therapeutic approach, our ongoing efforts are dedicated to
exploring efficient delivery strategies for the constructed pegRNAs into
target cells in vivo and ensuring a desired therapeutic outcome.
5. Conclusions
In conclusion, our present study underscores the efficacy and precision
of prime editing as a potent method for the correction of genetic
mutations within the genome.
Author Contributions: Conceptualization, K.E.L., Y.X., D.L.,
H.Z.; methodology, K.E.L., Y.X. and B.G.; formal analysis, Y.X..;
investigation, K.E.L., Y.X., B.G., J.K., N.K., M.H., Z.Z.; data
curation, Y.X., Z.Z., D.G., and H.Z.; writing—original draft, K.E.L.,
Y.X., Z.Z., D.G., and H.Z.; writing—review and editing, all authors;
supervision, H.Z.; funding acquisition, H.Z.. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was funded by National Institutes of
Health (NIH) (Grant number: EY032583; HL153876; EY030621; AR067766) and
American Heart Association (AHA) (Grant number: 23TPA1142638).
Institutional Review Board Statement: All murine experiments in
this study were performed following The Ohio State University
Institutional Animal Care and Use Committee (IACUC) approved protocols
(#2016A00000017).
Data Availability Statement: The original contributions
presented in the study are included in the article. Further inquiries
can be directed to the corresponding author.
Conflicts of Interest: The authors declare no conflict of
interest.
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