AAVLINK

AAVLINK is a DNA recombination-based method for large gene delivery using adeno-associated virus (AAV), a leading vector in gene therapy. Genes exceeding packaging limit of AAV are split into two or three fragments and are reconstituted through the following steps:

1. Step 1: Cre recombinase is expressed.
2. Step 2: The splicing acceptor (SA) and 3′ GOI section is translocated downstream to the 5′ end of the GOI and splicing donor (SD), which is catalyzed by Cre.
3. Step 3: The trans-linked AAV genomes allow for the transcription of mRNA containing the entire gene. The full-length GOI is reconstituted following the recombinant intron being spliced out.

Pro: Promoter; LE*-lox: Left element mutant lox sites; RE*-lox: Right element mutant lox sites; Cre: Cre recombinase; SD: Splice donor; SA: Splice acceptor; pA: Polyadenylation signal.

AAVLINK2.0 enables minimization of Cre expression, without compromising gene cargo reconstitution.

Key improvements in AAVLINK2.0 include:

1. Promoter optimization: Use of SCP1 weak promoter to reduce basal Cre expression (AAVLINK1.1).
2. Destabilized Cre: Fusion with UDeg3a, a protein degradation tag, reduces Cre half-life.

SCP1: Weak synthetic promoter; UDeg3a: Protein degradation tag; Cre: Cre recombinase with degradation tag; Other elements: Maintained from AAVLINK1.0 system (LE*-lox, RE*-lox, SD, SA, pA).

A total of 198 genes (193 disease-relevant genes and 5 CRISPR genes) were allocated to bipartite or tripartite AAVLINK vectors depending on their sizes.

The reconstitution of full-length genes was verified at DNA or protein levels, following transfection of HEK293T cells with these constructs.

Key steps in the process include:

1. Allocation of gene fragments into AAVLINK constructs: GOIs with CDS < 6.2 kb were delivered using dual AAVLINK vectors. GOIs with CDS lengths ranging from 6.2 kb to 8 kb were delivered using the triple I vectors (GOIs were split and allocated to two vectors, Cre is provided by the third vector). GOIs with CDS lengths exceeding 8 kb were delivered using the triple II vectors (GOIs were split and allocated to three vectors, Cre is provided by the third vector).
2. Cell transfection: Transfection of HEK293T by AAVLINK constructs
3. Molecular verification: Evaluation of gene reconstitution at DNA and protein levels

Pro: promoter; Cre: Cre recombinase; SD: splice donor; SA: splice acceptor; pA: polyadenylation signal; lox-LE*: left element mutant lox sites (the JT15 or JT1571 sites were used in the resource, JT1571 is a variant of JT15); lox-RE*: right element mutant lox sites (the JTZ17 site was used in the resource); lox-LE:RE: left element and right element mutant lox sites.

1. We strongly recommend sequencing all repository-obtained constructs to verify their identity and ensure the absence of truncations or mutations prior to experimental use.
2. High-quality AAV preparations are essential for in vivo testing.
3. AAVLINK1.0 3' CDS vectors for all disease-associated genes and CRISPRoff-V2 use pCALM1 promoter to offer neuron selectivity in the brain. If non-neuronal expression is required, researchers can either test AAVLINK2.0 with its putative ubiquitous SCP1 promoter or easily incorporate a custom promoter as needed.
4. For applications requiring cell-type-specific expression or precise GOI expression levels, an optimized 5' CDS promoter (the only promoter after reconstitution), either offering cell-type selectivity or desired levels of transcriptional activity, needs to be selected.