A New and Potent Method to Overcome AAV Cargo Limits for Large Gene Delivery

A research team led by Dr. Zhonghua Lu at the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, recently published a groundbreaking study in Cell titled “AAVLINK: A potent DNA-recombination method for large cargo delivery in gene therapy.” The work introduces AAVLINK, a new adeno-associated virus (AAV)–based delivery strategy that utilizes Cre/lox-mediated intermolecular DNA recombination to overcome the conventional 4.7 kb packaging limit. This innovation enables the efficient reconstitution and expression of large genes and CRISPR tools. PackGene is proud to have provided the high-quality AAV products and services that contributed to the success of this study.

 The Challenge of Large Gene Delivery

Gene therapy has emerged as a transformative approach for treating both inherited and acquired diseases. Among available delivery systems, recombinant AAV (rAAV) is one of the most widely used vectors due to its non-pathogenic nature, its ability to transduce both dividing and non-dividing cells, and its capacity for long-term gene expression with a low integration frequency and minimal risk of insertional mutagenesis.

Several rAAV-based therapies are already approved for retinal degeneration, hemophilia, neuromuscular disorders, and aromatic L-amino acid decarboxylase deficiency. However, a major limitation of rAAV is its packaging capacity (~4.7 kb). After accounting for ITRs and regulatory elements, the effective coding capacity is typically under 5 kb. Many disease-causing genes—including SHANK3, SCN1A, and CEP290—exceed this limit.

However, the therapeutic potential of rAAV is often restricted by its limited packaging capacity. To address this, researchers have developed various split-vector strategies, including DNA trans-splicing, RNA trans-splicing, and intein-based protein trans-splicing. Despite their utility, DNA and RNA-based approaches often exhibit modest reconstruction efficiency, while intein-based protein reconstitution depends heavily on correct protein folding and the identification of optimal split sites. Furthermore, these methods frequently generate truncated protein fragments, which may lead to potential dominant-negative effects or reduced therapeutic efficacy.

What is AAVLINK?

AAVLINK is a DNA-recombination-driven assembly strategy that leverages Cre recombinase and engineered lox sites to catalyze precise intermolecular recombination between separate rAAV genomes—then uses mRNA splicing to produce a full-length transcript.

In the Cell paper, the authors report that AAVLINK provides:

• High gene reconstitution efficiency
• Flexible split-site choices (relative to many protein-splicing designs)
• Marked reduction in truncated protein byproducts

How AAVLINK works (dual-vector concept)

A practical way to understand AAVLINK is as a 3-step sequence inside the co-transduced target cell (Figure 1A-C):

1. Co-delivery of two AAV genomes

   • Vector A carries: a promoter, 5′ portion of the gene of interest (GOI), a splice donor (SD), and an engineered lox site.
   • Vector B carries: a promoter driving Cre, an engineered lox site embedded within an artificial intron, a splice acceptor (SA), the 3′ portion of the GOI, and a poly(A) signal.

2. Cre/lox intermolecular DNA recombination (“translocation linkage”)
   Cre recombines the engineered lox sites so that the 5′-GOI(SD) segment becomes linked to the SA-3′GOI segment in the correct order.
3. Intron splicing → full-length mRNA → full-length protein
   After recombination, the transcript is spliced to generate a continuous GOI coding sequence for expression.

AAVLINK also incorporates design logic intended to minimize truncated byproducts, by preventing productive translation from incomplete fragments (e.g., avoiding a functional poly(A) on the 5′ fragment and placing the 3′ fragment in a context that is not efficiently translated until correct recombination/splicing occurs).

Standard loxP/loxP recombination can be reversible because substrate and product lox sites remain equivalent. AAVLINK uses asymmetric mutant lox pairs—reported as loxJT15 (LE mutant) and loxJTZ17 (RE mutant)—that bias the reaction toward the desired product and make the reverse reaction far less favorable after recombination (because the product includes a double-mutant lox that is a poor Cre substrate).

Figure 1A-C. Design and optimization of AAVLINK

AAVLINK successfully reconstructed split EYFP across multiple models—ranging from in vitro cells to mouse and cynomolgus monkey brains—showing fluorescence only when the dual-vector system and Cre were combined (Fig 1E-G). These results highlight AAVLINK’s primary advantages: exceptional DNA recombination efficiency, high accuracy, and a clean expression profile free from significant by-products.

Figure 1E-G. rAAV delivery of EYFP split in two using AAVLINK in vitro (E), in mouse brains (F), and in macaque brains (G)

Scaling up: three-vector AAVLINK for larger genes

To accommodate genes exceeding dual-vector capacity, the team developed a triple-vector AAVLINK system. This approach utilizes two pairs of orthogonal lox sites (including lox2272 mutations) to mediate precise three-part assembly while preventing cross-reactivity.

Validation using a split TagRFP gene confirmed precise DNA and mRNA reconstruction in HEK293T cells, with Western blots showing full-length protein expression and no detectable truncated by-products. In vivo experiments demonstrated efficient TagRFP expression in the cerebral cortex of both mice and cynomolgus monkeys, triggered only when all three vectors and Cre were present (Figure 2). By bypassing the 4.7 kb AAV limit, this triple-vector system expands delivery capacity to approximately 11 kb, sufficient to cover the majority of genes associated with human genetic diseases.

Figure 2. Triple-vector delivery of gene cargoes by AAVLINK

AAVLINK vs. Intein: Superior Performance and Flexibility

The research team systematically compared AAVLINK with the common intein-mediated protein trans-splicing strategy. While inteins are highly sensitive to sequences near split sites, AAVLINK offers greater flexibility, requiring only a consensus exonic AG/G site that can be further optimized via synonymous mutations.

Key Performance Advantages:

• Dual-Vector Efficiency: In luciferase reporter assays across 10 split sites, AAVLINK’s peak signal was 23.3 times higher than the intein method, with an overall average improvement of 25.8-fold (Figures 3A–3C).
• Triple-Vector Breakthrough: In triple-vector systems, AAVLINK outperformed inteins by an average of 245.5 times, providing significantly higher proportions of full-length protein with minimal truncated by-products (Figures 3D–3F).
• In Vivo Validation: In mouse models, AAVLINK achieved 4.5 times the overall efficiency of intein strategies after four weeks of expression (Figures 3G–3I).

Therapeutic Gene Benchmarking:

• Shank3 (5.4 kb): AAVLINK demonstrated 36.6 times higher reconstruction efficiency than inteins, yielding a clean expression profile (Figures 3J–3M).
• CEP290 (7.4 kb): Triple-vector AAVLINK delivery resulted in 83.4% full-length protein, whereas the intein method yielded a negligible 0.2% (Figures 3N–3R).

Collectively, these results (Figure 3) establish AAVLINK as a vastly more effective platform for large gene delivery, offering both higher therapeutic yields and superior molecular precision compared to existing protein-splicing technologies.

Figure 3. AAVLINK vs. intein-based protein reconstitution benchmarks

Therapeutic Potential: Restoring Function in Autism and Epilepsy Models

The study extended these findings to therapeutic applications, targeting two major conditions caused by oversized genes: Autism Spectrum Disorder (via Shank3) and Dravet Syndrome (via SCN1A).

1. Rescuing Behavioral Deficits in Shank3 Mutant Mice

To address the synaptic dysfunction associated with Shank3 deletion, the team used a dual-vector AAVLINK system to deliver the 5.4 kb gene into the striatum of 4-week-old mutant mice.

• Molecular Success: Stable full-length protein expression was confirmed with intact N- and C-terminals (Figures 4A–4D).
• Behavioral Recovery: Treated mice exhibited significant improvements in motor coordination and a reduction in stereotypic behaviors, highlighting AAVLINK’s ability to restore functional protein levels in the brain (Figures 4E, 4F).

2. Alleviating Seizures in Dravet Syndrome Models

Dravet syndrome, caused by SCN1A haploinsufficiency, requires the delivery of a 6 kb coding sequence—well beyond standard AAV limits. Using systemic co-delivery of AAVLINK vectors:

• Physiological Impact: The treatment successfully reconstructed Nav1.1 protein throughout the brain, significantly increasing mouse survival rates and raising the threshold for hyperthermia-induced seizures (Figures 4G–4K).
• Electrophysiological Correction: Analysis showed that SCN1A replacement normalized hippocampal neuron excitability and restored the function of fast-spiking interneurons, leading to a marked reduction in epilepsy-related EEG activity.

Despite the inherent challenges of systemic co-transduction, AAVLINK achieved sufficient gene reconstruction to provide clear therapeutic benefits, demonstrating its robustness for in vivo clinical applications.

Figure 4. AAVLINK-mediated gene replacement therapy in mouse models of PMS and Dravet syndrome

Efficient Delivery of Large CRISPR-Cas Tools

AAVLINK also overcomes the packaging constraints that limit the use of oversized CRISPR-Cas technologies in gene therapy. The study validated AAVLINK’s ability to deliver a wide array of genome-editing and transcriptional tools:

• Precision Gene Editing: Split SpCas9 was successfully reconstructed, achieving precise deletions in PSEN1 in cell culture and inducing double-strand breaks at the Rosa26 locus in mouse livers (Figures 5A–5C, 5J).
• High-Efficiency Base Editing: Large base editors ABE8e and YE1-BE4max were delivered with up to 80% editing efficiency in cells. In vivo delivery of ABE8e successfully targeted Pcsk9 in mice, reducing its expression (Figures 5D–5F, 5K).
• Transcriptional Modulation: AAVLINK effectively delivered dCas9-based regulators, including CRISPRoff-V2 for gene repression and dSpCas9-VPH for targeted gene activation—the latter of which successfully upregulated Scn1a in the mouse hippocampus (Figures 5G–5I, 5L).

These results demonstrate AAVLINK’s broad potential as a universal delivery platform for complex, large-scale genetic engineering tools in vivo.

Figure 5. Delivery of CRISPR tools by AAVLINK

AAVLINK 2.0: Optimizing Biosafety through Minimal Cre Expression

To address potential biosafety concerns regarding exogenous Cre expression, the study introduced AAVLINK 2.0, a highly optimized version designed to minimize Cre levels without compromising therapeutic efficacy.

• Promoter Optimization: By replacing strong promoters with the weak SCP1 promoter, the team maintained efficient gene reconstruction while producing extremely low Cre levels (AAVLINK 1.1).
• Enhanced Protein Turnover: The team further refined the system by fusing a UDeg3a destabilization tag to the Cre C-terminus. This modification significantly shortened the Cre protein half-life, making it nearly undetectable across all time points (AAVLINK 2.0).
• In Vivo Safety Profile: Experimental results in both mice and cynomolgus monkeys showed that while AAVLINK 2.0 achieved robust target gene expression, Cre remained undetectable via immunohistochemistry and Western blot.

These optimizations highlight the significant biosafety advantages of AAVLINK 2.0, mitigating the risks of long-term Cre exposure and enhancing its prospects for clinical and non-human primate translation.

 The AAVLINK large-gene resource bank

To accelerate adoption, the authors constructed a vector bank covering:

• 198 total payloads (193 disease-relevant large genes + 5 CRISPR tool genes)
• A strong emphasis on ASD-associated genes (many with coding sequences >4 kb), plus additional disease genes

Most genes required only a single split design to achieve successful reconstitution, underscoring the robustness of AAVLINK, although reconstitution efficiency still varied among different split sites, suggesting that split-site optimization can further improve performance for some genes.​

Practical tips and limitations

The AAVLINK repository highlights several actionable considerations:

• Sequence-verify constructs received from any repository before use (to confirm identity and rule out truncations/mutations).
• High-quality AAV preparations are critical for in vivo testing.
• Many split strategies—including AAVLINK—depend on co-transduction, which can be less efficient in large animals after systemic dosing.
• Promoter choice matters: in AAVLINK designs, the 5′-fragment promoter becomes the dominant/only promoter after reconstitution, so it should be selected to match the intended cell type and expression level.
• Some AAVLINK1.0 neuron-targeted configurations use a neuron-selective promoter (e.g., pCALM1) in specific vector components; if non-neuronal expression is needed, users may test AAVLINK2.0 (SCP1) or substitute a custom promoter.

Key Takeaways

AAVLINK expands what is feasible with AAV by shifting large-cargo reconstitution toward high-efficiency, Cre/lox-driven DNA recombination coupled to splicing, reducing truncated byproducts and improving robustness across split sites. With AAVLINK2.0 (minimized Cre) and a validated large-gene/CRISPR vector bank, the platform is positioned as a broadly usable toolkit for large-gene diseases and next-generation in vivo genome engineering. AAVLINK, intein-based split-AAV, and StitchR are three complementary strategies developed to push beyond the native cargo limit of AAV vectors for large-gene delivery. Intein-based methods instead split the protein itself and rely on protein trans-splicing to ligate fragments in transduced cells, offering a protein-centric solution but with stricter constraints on split-site choice and higher risk of by-products. StitchR operates at the RNA level, using ribozyme-guided mRNA trans-ligation to join split transcripts into a seamless open reading frame, providing “scarless” coding sequences but requiring careful junction engineering and context-specific optimization (Table 1).

Table 1. Comparison of AAVLINK, intein-based split-AAV, and StitchR

Taken together, this comparison highlights that AAVLINK currently offers the most alternative solution for large-gene delivery, combining high reconstitution efficiency, low levels of truncated products, and flexible split-site selection across dual- and triple-AAV formats.

Reference:

Jianbang Lin et al, AAVLINK: A potent DNA-recombination method for large cargo delivery in gene therapy, Cell (2026).