Plasmid constructions
The donor plasmid pFastBac1 (pFB) was supplied in a commercial kit with the Tn7-bacmid DNA (Bac-to-Bac® system) (ThermoFisher Scientific, MA). It contains a selective gene coding for ampicillin (AmpR) and the two Tn7 sites left (L) and right (R) that are surrounding a polylinker to clone the gene of interest and another selective gene coding for gentamicin (GmR). We have generated a first construct containing the cytomegalovirus (CMV) promoter, the enhanced green fluorescent protein (eGFP) reporter gene and a human β-globin (HBB) polyadenylation signal, all flanked by two flop-oriented ITRs of AAV serotype 2 derived from the pSub-201 plasmid (Samulski, 1987) (Supplementary Fig. 1 ). The ITR upstream of the CMV promoter lacks 15 bp in the external A region, and the ITR downstream of the polyA is truncated by 17 bp with respect of the wild-type ITR2. The second construct was kindly provided by R.M. Kotin (NIH, Bethesda, USA) and carries the coding genes “Rep2” and “Cap8” that follows the design described by Smith and colleagues allowing production of rAAV vectors using a dual baculovirus system (Smith et al., 2009). We additionally generated Anc80 construct changing the ATG start codon of Anc80 capsid orf in ACG and the CAG start codon of the putative assembly-activating protein (AAP) in CTG as described in the patent number WO2019067982A3. The optimized Anc80 orf was cloned between the p10 baculovirus promoter and the herpes simplex virus (HSV) thymidine kinase (tk) polyadenylation signal sequence in the donor plasmid allowing the expression of AAV2 Rep78 and Rep52 proteins under the control of the polyhedrin promoter and followed by the simian virus SV40 polyadenylation signal sequence. The related baculovirus constructs were also generated with the donor plasmid pBac-1 supplied in a commercial kit with the HR-bacmid DNA (BacMagic-2, Merck or flashBAC GOLD, Oxford Expression Technologies), containing a selective gene coding for ampicillin (AmpR) and a polylinker to clone the gene of interest which is surrounded by the two wild-type homologous baculoviral sequences lef2/orf603 and orf1629 . The BacMagic-2 and flashBAC GOLD kits contain chi-a and v-cath deleted parental bacmid. The pBac-1 is linearized by enzymatic digestion with the “PmeI/AclI” or “PmeI/DrdI” restriction endonucleases before the transfection in Sf9 cells. All these plasmids were validated by Sanger sequencing and subsequently used for the generation of the recombinant baculoviruses.