In vivo gene editing represents a transformative frontier in therapeutic development, moving beyond simple gene replacement to permanently correct pathogenic mutations directly within host tissues. Adeno-Associated Virus (AAV) remains the vector of choice for long-term tissue-targeted delivery. However, implementing CRISPR/Cas9 systems and advanced base/prime editors in vivo requires overcoming critical hurdles—such as limited vector packaging capacity, potential off-target immunogenicity, and suboptimal editing efficiency in non-dividing terminal cells.
To address these challenges, Protheragen provides an optimized AAV gene editing & in vivo repair platform. By integrating target-optimized AAV-CRISPR/Cas9 architectures and engineered dual-AAV split-prime editor (split-PE) systems, we achieve precise, permanent, and scarless genomic correction across diverse disease targets with minimized off-target risk.
Engineered AAV-CRISPR/Cas9 Systems
Uses optimized promoter-element configurations and compact Cas orthologs (e.g., SaCas9, CjCas9, or engineered SpCas9 variants) to fit functional CRISPR/Cas9 machinery into single or dual AAV packages.
Employs tissue-specific promoters and self-inactivating/transient expression cassettes to limit long-term Cas9 exposure, reducing host immune responses and off-target cleavages.
Fig.1 Mechanism of AAV-CRISPR-Cas Genome Editing. (Kabadi, A. M., et al., 2024)
Dual-AAV Split Prime Editor (Split-PE) Platform
Overcomes the ~6.3 kb size obstacle of Prime Editors (PE) by splitting the editor into N-terminal and C-terminal fragments fused with engineered split-inteins.
Delivered co-dependently by two AAV vectors, the fragments reconstitute into a fully functional Prime Editor inside target cells via fast, traceless protein trans-splicing.
Enables precise single-base transitions, small insertions, or deletions without creating double-strand breaks (DSBs) or requiring donor DNA.
Fig.2 Treatment of eye diseases in mice using dual-AAV split-PE. (Zhi S, et al., 2022)
| Feature | Traditional Gene Replacement | Standard AAV-CRISPR/Cas9 | Dual-AAV Split-PE System |
| Mechanism of Action | Episomal expression of exogenously supplied cDNA. | Targeted DSB generation; Indel/NHEJ knockout or HDR repair. | Precision sequence rewrite without DSBs or donor DNA. |
| Payload Size Constraint | Limited to <4.7 kb payload | Requires compact Cas9 (<4.2 kb) or dual vectors. | Reconstitutes large PE (~6.3 kb) via dual vectors. |
| Double-Strand Breaks (DSBs) | No | Yes (Risk of translocations/indels) | No (Minimized genomic instability) |
| Mutation Targeting Range | Limited to recessively inherited gene defects. | Gene disruption; precise correction via HDR (low efficiency in post-mitotic cells). | All 12 base transitions, precise insertions, deletions, and point mutations. |
| In Vivo Editing Precision | N/A | Variable; potential off-target cleavages | High target fidelity; virtually no detectable off-target activity. |
In silico design, off-target risk modeling, and experimental screening of sgRNA/pegRNA libraries in target cell lines for maximal editing efficiency and sequence fidelity.
Custom split-site selection, split-intein domain optimization, and promoter-regulatory element assembly tailored for dual AAV vector split-PE delivery.
Selection and packaging using tropism-matched wild-type and engineered AAV capsids (AAV8, AAV9, MyoAAV, etc.) to ensure high co-infection rates in target tissues.
cGMP-ready co-production and stringent vector genome packaging ratio determination (1:1 stoichiometric balancing) for dual-vector systems.
Quantification of in vivo editing efficiency using next-generation sequencing (NGS), targeted deep sequencing, and unbiased genome-wide off-target analysis.
Comprehensive translation support including scale-up process development, analytical method validation (AUC, ddPCR, LC-MS), and IND-enabling safety/biodistribution studies tailored for multi-vector gene therapy products.
Our optimized AAV gene editing technology enables permanent gene repair across a wide range of inherited and acquired genetic disorders:
At Protheragen, we combine advanced split-intein engineering, customized AAV expression cassette optimization, and a broad library of organ-targeting capsids to overcome the hurdles of in vivo genomic medicine. Our end-to-end platform supports your pipeline from early gRNA/pegRNA design and dual-AAV vector packaging to rigorous in vivo proof-of-concept testing. Partner with Protheragen to accelerate your AAV gene editing research into safe, precise, and effective clinical candidates.
Contact our AAV gene editing specialists today to discuss your target mutation, design custom split-PE/CRISPR vectors, or request a customized proposal.
References
Our AAVLinkTM platform is at the forefront of adeno-associated viral (AAV) vector development, leveraging our extensive expertise and state-of-the-art technologies to drive innovation in gene therapy.
