Maximizing Base and Prime Editing Efficiency with Optimized LNP-Based Delivery
Gene editing continues to revolutionize medicine, offering potential cures for genetic
disorders and possibly chronic diseases by precisely modifying genome sequences. However, for this
technology to realize its full potential, efficient delivery of gene-editing tools—such as CRISPR/Cas9
proteins and guide RNAs—is essential. A groundbreaking study published in Nature Biomedical
Engineering introduces an innovative approach to enhancing the safety and efficiency of these tools
by optimizing the delivery of ribonucleoproteins (RNPs) through lipid
nanoparticles (LNPs).1
Evolving gene editing tool delivery approaches
Traditionally, viral vectors like
adenovirus, adeno-associated virus (AAV), and lentivirus have been the go-to options for gene-editing
delivery due to their high transduction efficiency. However, these vectors come with
limitations:1-3
Restricted Packaging Capacity: Large gene-editing tools, such as Streptococcus pyogenes
Cas9 (SpCas9), can exceed the size limits of some viral vectors.
Safety Concerns: The risk of genomic integration and insertional mutagenesis poses
significant therapeutic challenges.
Prolonged Expression Risks: High and sustained expression of editors increases the
likelihood of immunogenic reactions and off-target effects.
Key Properties of Various Viral and Non-Viral Gene Editing Delivery Vehicles
Retrieved with modifications from Bisserier et al.
2022, Figure 2.2
To address these issues, researchers are turning to non-viral delivery methods, including lipid
and virus-like nanoparticles.1,4 These methods allow for greater packaging capacity and the delivery of
fully functional CRISPR/Cas9 RNPs, minimizing risks like insertional mutagenesis and prolonged expression of
editors. However, achieving high efficiency and precision, particularly for in vivo applications, remains a
significant hurdle.
Improving editing precision with new Cas9 proteins
New strategies using advanced Cas9 proteins, such as base editors (e.g., adenine base editors
or ABEs) and prime editors (PEs), are redefining the landscape of gene editing. Unlike conventional
Cas9-based editing, which relies on double-strand breaks (DSBs) and repair pathways, base and prime editors
enable:1
Precise Modifications: Base editors efficiently convert specific base pairs (e.g., C→T
or A→G) without introducing DSBs.
Versatility: Prime editors can perform base substitutions, insertions, and deletions
with minimal off-target effects.
Improved Safety: These approaches reduce unintended consequences like large insertions,
deletions, or chromosomal rearrangements.
These innovations are particularly promising for developing gene-correcting strategies for
monogenic diseases.
Leveraging cell penetrating peptides to improve RNP delivery efficiency
In the recent Nature Biomedical Engineering publication, Hołubowicz and colleagues
initially explored cell-penetrating peptides (CPPs) to enhance
the delivery of gene-editing tools.1 By testing CPPs such as TAT, CPP5, and ANTP, the researchers aimed to
improve RNP delivery to retinal pigment epithelium (RPE) and photoreceptors. While CPPs showed promise for
certain proteins like Cre recombinase, they fell short for ABE and PE RNPs.
However, a non-covalent CPP, 6×His-CM18-PTD4, combined with 10% sucrose to stabilize proteins,
improved delivery efficiency modestly. This approach demonstrated some restoration of retinal function in a
Leber congenital amaurosis (LCA) mouse model but achieved limited editing efficiency (~2% correction in
RPE).
Monitoring the effects of CPPs on ABE8e editing efficiency. (a) The fluorescent rd12 reporter cell line
was developed based on the rd12 reporter construct synthesized by GenScript (mCherry-rd12-eGFP). In
this cell line, correction of the rd12 mutation by ABE results in dual expression of mCherry and eGFP, facilitating the
monitoring of ABE RNP delivery efficiency. (d) Effects of sucrose concentration and the non-covalent
CPP 6×His-CM18-PTD4 (GenScript) on RNP delivery and editing efficiency. Fusion of CPPs to ABE8e failed
at improving RNP delivery efficiency. Retrieved from Holubowicz et al. 2024 Figure 3, with modifications- only panels
a and d are shown.1
Optimizing LNPs for ABE and PE delivery
Recognizing the limitations of CPPs, Hołubowicz and colleagues determined that delivery of ABE RNPs
in vitro and even in vivo could be improved by simply using Lipofectamine 3000, but despite being
an effective approach, in vivo, the reagent was toxic at some concentrations. Therefore, the team shifted
focus to lipid nanoparticles, which could be fine-tuned to improve CRISPR RNP encapsulation, and provided a tried,
tested and safe delivery vehicle.
Having optimized LNP lipid composition to safeguard ABE’s activity and eliminate any potential toxicity
the team found that in vitro this new approach drove efficient delivery, resulting in dual expression of
mCherry and eGFP in mostly all rd12 cells. More important to their ultimate goal, the ABE LNPs were safe
and an efficient delivery vehicle in vivo, rescuing retinal responses significantly above of what they
could achieve with Lipofectamine and approaching normal responses.
Fine tuning the lipid composition and specifically the concentration of DMG-PEG 2000 was critical
to maximize RNP encapsulation, delivery and reassuringly to improve editing precision. Interestingly, the team found
that LNP optimization is not a fit-for-all editing tools, as the optimized formulation for ABE had to be further
fined tunned to achieve maximal encapsulation. Nevertheless, once optimized the LNP formulation enabled efficient
delivery of PE RNPs, supporting high precision of editing in vitro and restoring in vivo retinal
functionality on par with the rescue achieved with ABE RNP LNPs.
A leap forward in therapeutic applications
Hołubowicz and colleagues showcased a groundbreaking achievement: delivering a single dose of ABE or PE
RNPs via their optimized LNP formulations led to remarkable genetic correction, driving measurable physiological and
biochemical improvements. This innovation not only highlights the power of precise gene editing but also signals a
leap forward in therapeutic applications.
Looking ahead, integrating cutting-edge strategies, like tissue-targeting antibodies with optimized
LNPs can help redefining the limits of what’s possible in gene editing. These advancements open new frontiers for
realizing the full therapeutic potential of gene editing, bringing us closer to transforming lives with precision
medicine.
Reference
1. Hołubowicz, R., et al.
(2024). Safer and efficient base editing and prime editing via ribonucleoproteins delivered through
optimized lipid-nanoparticle formulations. Nature Biomedical Engineering. https://www.nature.com/articles/s41551-024-01296-2
2. Bisserier, M., et al.,
(2022). Novel insights into the therapeutic potential of lung-targeted gene transfer in the most common
respiratory diseases. Frontiers in Pharmacology, 13, Article 956423. https://www.mdpi.com/2073-4409/11/6/984
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