MegaAAV™ Delivery System

Figure 3. Successful reconstitution of DMD in vitro upon using 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 per AAV) and cell lysates were harvested 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.

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-terminus FLAG tag and a 3’GOI containing a C-terminus 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 per AAV) and cell lysates harvested 72 hours post-transduction. Non-transduced cells (NC) and cells transfected with a plasmid encoding the C-terminus HA-tagged full-length gene (PC) were included as controls. The pink arrow indicate complete in vitro reconstitution of the retinal gene, whereas teal arrows indicate gene fragments. 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 (dashed boxes) 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. Control wild-type (WT) and non-treated knockout (KO) mice were also observed.