Fig 2: AAV9 transduction with codon-optimized PRKN sequences results in increased hParkin protein expression in human PRKN-null iPSC-DA neurons and Prkn KO mice.A PRKN-null iPSC-DA neurons were analyzed 13 days after transduction with AAV9-coPRKN vectors (PK041, PK042, and PK043) and parental WT PRKN AAV9 vector (PK013) at four different doses (3 × 103, 1 × 104, 3 × 104, and 1 × 105 AAV MOI). hParkin protein levels were quantified in AAV-transduced PRKN-null iPSC-DA neurons (n = 3 independent replicates; mean ± SEM). ***p < 0.001 versus PK013 (WT PRKN) at each MOI. B, C AAV9-coPRKN vectors (PK041, PK042, and PK043) and parental WT PRKN vector (PK013) were unilaterally injected into the SN of Prkn KO mice at 1 × 108 VG per brain. B The graph shows VG copy number per μg genomic DNA in the midbrain for each treatment group as indicated on the x-axis (mean ± SD). C The graph shows hParkin protein levels in the midbrain, quantified by ELISA, for each treatment group as indicated on the x-axis (mean ± SEM). *p < 0.05 or **p < 0.01, versus PK013 (WT PRKN).

Fig 3: AAV9 transduction of human PRKN-null iPSC-DA neurons restores Parkin function.PRKN-null iPSC-DA neurons (PRKN KO) were transduced with eight different AAV9 vectors encoding hPRKN under the control of either ubiquitous, neuron-specific, or PRKN promoter at four different doses (3 × 103, 1 × 104, 3 × 104, and 1 × 105 AAV MOI), and after 13 days treated with 20 μM CCCP for 24 h (+) or untreated (−). WT iPSC DA neurons (WT) were used as positive control cells and treated with 20 μM CCCP for 24 h (+) or untreated (−). The quantifications were done using the IN Cell Investigator software by Cytiva. A Quantification of nuclei number of all cells in the counting fields (n = 3 independent replicates; mean ± SEM). *p < 0.05 or **p < 0.01 versus the untreated in PRKN-null control (0, no AAV transduction; -,no CCCP treatment). ##p < 0.01 versus the untreated in WT control (no AAV and no CCCP). The y-axis shows the nuclei number of 16 fields, and the x-axis shows the AAV MOI and treatment groups, (−) indicates untreated controls. B Quantification of poly-pUb-positive area in neurons (n = 3 independent replicates; mean ± SEM). *p < 0.05, **p < 0.01, or ***p < 0.001 versus the untreated PRKN-null control (no AAV and no CCCP). #p < 0.05 or ##p < 0.01 versus the untreated in WT control (no AAV and no CCCP). C Quantification of mitochondrial TOM20-positive area within MAP2-positive neuronal regions (n = 3 independent replicates; mean ± SEM). *p < 0.05, **p < 0.01, or ***p < 0.001 versus the untreated PRKN-null control (no AAV and no CCCP). #p < 0.05 or ##p < 0.01 versus the untreated in WT control (no AAV and no CCCP).

Fig 4: Local delivery of AAV9-PK041 (SYN1-coPRKN CpG5-2) results in DA neuronal protection in the SNpc of Prkn KO mice in the α-Syn PFFs model.AAV9-PK041 was unilaterally injected into the SN of Prkn KO mice at either 0.3 × 108 or 1 × 108 VG per brain, followed by striatal α-Syn PFF injection at the same site (n = 8 or 7, respectively). A Representative images of TH immunohistochemistry staining showing DA neuronal cell bodies and axon fibers (brown signals) in the ipsilateral SN at 1 month after α-Syn PFFs injection. B Quantification of TH-positive DA neurons and total TH-positive area in the ipsilateral SNpc. Data are presented as mean ± SEM. #p < 0.05; compared with vehicle control without α-Syn PFF injection, *p < 0.025; vs. vehicle control following α-Syn PFF injection.

Fig 5: Local delivery of AAV9-PK041 (SYN1-coPRKN CpG5-2) results in DA neuronal protection in the SNpc of 6-OHDA-lesioned Prkn KO mice.AAV9-PK041 (SYN1-coPRKN CpG5-2) was unilaterally injected into the SN of Prkn KO mice at a dose of 1 × 108 VG per brain, followed by 6-OHDA lesion in the MFB of the same side (AAV injection side) (n = 8 / group). A Representative immunohistochemical TH staining in the ipsilateral SN. TH-positive neurons in the SNpc detected by HALO software are shown in red. B Quantification of TH-positive DA neurons in the SNpc on the ipsilateral (Ipsi) or contralateral (Contra) sides of AAV injection. Open bars indicate vehicle-treated controls, gray bars represent 6-OHDA treated groups. The ratio of TH-positive DA neurons between ipsilateral and contralateral sides is presented in the graph labeled Ipsi/Contra. Data are presented as mean ± SEM. *p < 0.05 or **p < 0.01, compared with vehicle control with 6-OHDA. C, D) hParkin protein distribution in the SN was detected by IHC analysis. C Representative image showing hParkin expression (green), TH expression (red), and nuclei (blue) in the midbrain of AAV9-PK041-injected Prkn KO mouse. The image is taken from the anterior side; the right hemisphere corresponds to the AAV and 6-OHDA injection site showing clearly hParkin expression (yellow and green signals). D Images of the SNpc region illustrating hParkin expression in TH-positive neurons of the 6-OHDA-lesioned side (yellow signals), showing images for hParkin-positive cells, TH-positive cells, and nuclei, as well as the merged image.