MegaAAV™ Delivery System

Breaking through the primary limitation of traditional AAV systems, VectorBuilder’s innovative and highly efficient MegaAAV™ split vector system allows for expression of large transgenes that exceed the capacity of a single AAV vector or complex multi-component systems. Our proprietary approach allows developers to design the therapy that works best, not just the one that fits.

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AAV Capacity Limits in Gene Therapy

Traditional AAV vectors are often constrained by limited cargo capacity, restricting delivery to small payloads and significantly hindering therapeutic development. Although dual AAV systems were able to circumvent this by splitting oversized genes across vectors, their clinical potential remains limited due to inefficient delivery and incomplete reconstitution of functional transgenes.

Figure 1. MegaAAV™ efficiently transduces cells in vitro and facilitates functional transgene assembly. HEK293T cells were transduced with either MegaAAV™ or Hybrid AK Dual AAV encoding EGFP at various MOIs. Measurement of EGFP expression and representative fluorescence microscopy images (MOI = 105) were taken at 72 hours post-transduction.

Overcoming AAV Payload Limits

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Utilizing an innovative in vivo recombination strategy, our proprietary multi-AAV delivery system boasts significantly higher transcription levels of delivered genes. With its flexible design, the platform extends beyond dual vectors, enabling delivery of even larger and more complex genes.

Figure 2. MegaAAV™ achieves higher gene transcription levels in vivo. C57BL/6J mice were injected subretinally with equimolar doses of either MegaAAV™ or Hybrid AK Dual AAV encoding EGFP. (A) Representative fluorescence microscopy images of mouse retina samples were taken (magnification: 200x; exposure time: 50 ms) and (B) mRNA extracted at one month post-injection for qPCR analysis.

MegaAAV™ delivery results in functional reconstitution of DMD
Figure 3. Successful reconstitution of DMD in vitro using our MegaAAV™ split vector system. The human DMD gene (~11 kb) was split into three parts (DMD-I, DMD-II, DMD-III) and packaged into separate AAV constructs. HEK293T cells were transduced with either MegaAAV™ containing all three constructs or individual constructs (MOI = 105) and cell lysates harvested at 48 hours post-transduction. Non-transduced cells (NC) and cells transfected with a plasmid encoding full-length DMD (PC) were included as controls. All samples were analyzed by Western blot using antibodies specific to regions within DMD-II and DMD-III. Pink arrows indicate complete in vitro reconstitution of the DMD gene, whereas teal arrows indicate DMD fragments.

Innovating Large Gene Therapy Solutions: A Case Study

Many therapeutic genes implicated in retinal diseases exceed AAV packaging limits, posing a significant challenge for the development of novel AAV-based ocular therapies. Utilizing the MegaAAV™ system, a larger transgene of interest (GOI) was efficiently delivered and functionally reconstituted (full-length GOI; FL-GOI) in target cells, highlighting the potential of this approach for developing novel gene therapies.

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MegaAAV™ enables recovery of visual function
Figure 4. Effective phenotype restoration through MegaAAV™ gene delivery. A human retinal gene (~6 kb) was split into two parts, a 5’GOI containing an N-terminal FLAG tag and a 3’GOI containing a C-terminal HA tag, then packaged into separate AAV8-based constructs. (A) HEK293T cells were transduced with either MegaAAV™ containing both constructs or individual constructs (MOI = 105 ) and cell lysates harvested 72 hours post-transduction. Non-transduced cells (NC) and cells transfected with a plasmid encoding the full-length gene with a C-terminal HA-tag (PC) were included as controls. The pink arrow indicates complete in vitro reconstitution of the retinal gene, whereas the teal arrow indicates gene fragments. (B-C) Two-week old transgenic knockout mice were treated with MegaAAV™ encoding for the same 5’ and 3’ parts of the gene without epitope tags. As indicated by the (B) b-wave (asterisks) of electroretinograms and (C) analyses of cone expression 3 weeks post-injection, both visual function and retinal morphology are largely rescued in the treated mice. Wild-type (WT) and non-treated knockout (KO) mice were also included as controls.
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