NanoScribes: A Superior Prime Editing Delivery System
Summary
This study presents NanoScribes, a virus-like particle (VLP)-based system for delivering Prime Editing (PE) components into human stem cells with high efficiency and safety. Unlike traditional viral methods, NanoScribes avoid genome integration, achieving up to 68% editing efficiency while reducing off-target effects. By utilizing multi-fusogen pseudotyping and Pol II-driven pegRNA expression, this approach enhances cell entry and editing precision, making it a promising tool for gene therapy, regenerative medicine, and personalized treatments.
NanoScribes: A Superior Prime Editing Delivery System
NanoScribes, a virus-like particle (VLP)-based system that efficiently delivers Prime Editing (PE) components into human cells with high precision, outperforming traditional viral-based delivery methods in terms of safety, efficiency, and versatility. The development of NanoScribes represents a significant advancement in genome editing, offering a non-viral, transient delivery method that reduces potential off-target effects and enhances the precision of genetic modifications. Their ability to efficiently deliver prime editing components positions them as a superior alternative to traditional delivery systems, potentially accelerating the development of gene therapies and advancing regenerative medicine.
What is Prime Editing (PE)?
Prime Editing is an advanced gene-editing technique that allows precise changes to DNA without causing double-strand breaks. Unlike traditional CRISPR-Cas9, which relies on cutting DNA and relying on error-prone repair mechanisms, PE can insert, delete, or replace specific DNA sequences with greater accuracy and fewer side effects.
Key Components of Prime Editing:
Prime Editing Guide RNA (pegRNA) – Directs the editing process and contains instructions for the desired DNA change.
Cas9-nickase (H840A) – A modified enzyme that cuts only one strand of DNA, making the process safer.
Reverse Transcriptase (RT) – Synthesizes the new DNA sequence based on the pegRNA template.
The Challenge: Despite its advantages, Prime Editing is difficult to deliver efficiently into human stem cells without causing toxicity or reducing effectiveness.
How do NanoScribes work?
They encapsulate Prime Editing ribonucleoprotein complexes (RNPs) inside non-replicating viral-like particles.
They fuse with target cells, delivering the editing machinery directly into the cytoplasm.
No long-term DNA integration occurs, reducing the risk of unintended mutations or immune responses.
Key Innovations in NanoScribes:
Multi-Fusogen Strategy: Uses different envelope proteins to improve cell entry.
Optimized pegRNA Structure: Improved stability and efficiency of the editing process.
Pol II-Based pegRNA Expression: Allows for higher pegRNA levels, increasing editing success.
Multiplex Editing Capabilities: Can edit multiple genes simultaneously, a step toward advanced gene therapy approaches.
Why NanoScribes are better than other viral delivery methods
1️. No Risk of Genomic Integration (Unlike Lentivirus & AAVs)
Problem with Lentiviruses & AAVs:
Lentiviral vectors integrate their genetic material into the host genome, which can cause unintended mutations or cancerous transformations.
Adeno-associated viruses (AAVs) can create episomes that may persist in cells and disrupt normal gene regulation.
NanoScribes Advantage:
No DNA integration → No risk of insertional mutagenesis.
Delivers pre-formed Prime Editing proteins and guide RNAs directly, avoiding prolonged expression of gene-editing tools.
2️. Larger Cargo Capacity (Better Than AAVs & Adenoviruses)
Problem with AAVs & Adenoviruses:
AAVs have a very limited cargo size (~4.7 kb), which cannot fit the entire Prime Editing machinery in a single vector.
Adenoviruses have a larger capacity (~8.5 kb) but trigger strong immune responses, making them unsuitable for clinical applications.
NanoScribes Advantage:
No cargo size limitations → Can efficiently deliver large Prime Editing components.
No need for complex "split-AAV" systems to fit Prime Editors into small vectors.
3. Higher Editing Efficiency & Multiplexing Capabilities
Problem with mRNA & DNA-Based Delivery:
mRNA-based Prime Editing delivery can be unstable and prone to degradation before reaching the target.
DNA plasmid-based transfection has low efficiency in primary cells and stem cells.
4. Reduced Immune Response & Cytotoxicity
Problem with Adenoviruses & Lentiviruses:
Lentiviruses & adenoviruses can trigger strong immune responses, leading to inflammation and rejection.
AAVs can cause liver toxicity at high doses.
NanoScribes Advantage:
Uses non-replicating virus-like particles (VLPs) → significantly lower immunogenicity.
Delivers transient Prime Editing components, reducing prolonged immune system activation.
5️. Increased Targeting Efficiency with Multi-Fusogen Strategy
Problem with Standard VLPs & Lipid Nanoparticles (LNPs):
Standard VLPs often struggle with cell-type specificity.
Lipid nanoparticles (LNPs) are inefficient for gene delivery in certain primary cells and stem cells.
NanoScribes Advantage:
Uses three different fusogens (VSV-G, BAEV, Syncytin-1) to improve cell targeting and entry efficiency.
More effective in stem cells than other VLP-based systems.
Why Does This Matter?
Gene editing holds tremendous potential for treating genetic diseases, cancer, and regenerative medicine applications. However, safe and efficient delivery remains a major hurdle. NanoScribes could help overcome these challenges by:
Improving safety – No permanent genetic alterations that could trigger immune reactions.
Increasing efficiency – Higher delivery success rates compared to existing methods.
Expanding applications – Usable in stem cells and other hard-to-edit cell types.
Potential Applications:
Treatment of genetic disorders – Potential to correct mutations causing diseases like sickle cell anemia, cystic fibrosis, and muscular dystrophy.
Cancer therapy – Engineering immune cells to recognize and destroy tumors.
Regenerative medicine – Editing stem cells for personalized therapies.
Disease modeling & drug discovery – Creating accurate cell models for testing new treatments.
The Future of Prime Editing & NanoScribes
This research represents a major step toward clinical applications of Prime Editing. Future work will focus on:
Further improving efficiency and expanding the range of targetable genes.
Ensuring long-term safety in animal models and clinical trials.
Scaling up production for therapeutic applications.
Read the full study: https://doi.org/10.1038/s41467-024-55604-0
CSIR-Institute of MIcrobial Technology
