Comparison of smaller rAAV-HLP-codop-hFVIII constructs. (A) Schematic of smaller codon-optimized hFVIII expression cassettes. Top schematic consists of B domain–deleted variant, rAAV-HLP-codop-BDD-hFVIII, which is ∼5.1 kb in size. It comprises A1-A2-A3-C1-C2 FVIII domains and contains the SQ sequences between the A2 and A3 domains. rAAV-HLP-codop-hFVIII-V1 and rAAV-HLP-codop-hFVIII-V3 are identical to rAAV-HLP-codop-BDD-hFVIII except that peptides V1 or V3 have been placed within the SQ sequence. Both peptides contain the same 6 N-linked glycosylation triplets. (B) Analysis of viral DNA extracted from 5 × 10¹⁰ particles of each of the codon-optimized hFVIII vector preparation following separation on an alkaline agarose gel and run in duplicate showing bands of ∼5 kb, the expected size for the rAAV-HLP-codop-BDD-hFVIII (BDD) and rAAV-HLP-codop-hFVIII-V3 (V). In comparison, a rather diffuse signal was observed for the genomes extracted from AAV8-HLP-codop-hFVIII-N6 (N6), suggesting the packaging of a more heterogeneous proviral species. (Right) Size standards.