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.