aav vector production  (Addgene inc)

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: Modeling of PPRPIPM and MPIPRPP peptides as part of VR-VIII (GH12/GH13) of AAV2 VPs and characterization of vector production (A) The PPRPIPM or MPIPRPP peptide was inserted into VP1, VP2, and VP3 of AAV2 at I-587, flanked by alanine-serine-alanine residues at the 5′ end and two alanine residues at the 3′ end. (B and C) Overlay of the tertiary structure of the variable region (VR) VIII loop of (B) AAV.PPR (orange, AlphaFold prediction) or (C) AAV.MPI (blue, AlphaFold prediction) with AAV2 (green, PDB ID 6IH9 ). (D) Western blot using the B1 antibody, which detects the C-terminus of capsid proteins (VP1: 87 kDa; VP2: 72 kDa; VP3: 62 kDa) in their linear form, to visualize the capsid composition following SDS-PAGE and western blotting of vector preparations (5E9 capsids per lane). (E) Packaging efficiency, expressed as the ratio of capsid titer to genomic particle titer, was determined by ELISA and qPCR, respectively. Scatter dot blot: mean (SD) n = 3; ns: not significant; one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Plasmid Preparation, Western Blot, SDS Page, Enzyme-linked Immunosorbent Assay, Dot Blot, Comparison

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: AAV.MPI transduces auditory cell lines more efficiently than AAV.PPR and AAV2 (A and B) Percentage of transgene-expressing HEI-OC1 cells (A) and SV-k1 cells (B) after transduction with the indicated vector preparations at a GOI of 500 and 2,500 at 24 hours post transduction (hpt). Bars, mean (SD), n = 3; ns: not significant, ∗ p < 0.05, ∗∗∗ p < 0.001; two-way ANOVA with Bonferroni post hoc test. GOI, genomic particles of infection.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Expressing, Transduction, Plasmid Preparation, Infection

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: AAV.MPI and AAV.PPR exhibit reduced affinity to HSPG compared with AAV2 (A–C) Surface model of heparin docking in the heparan sulfate proteoglycan-binding pocket of AAV2 (A) and the corresponding region of AAV.PPR (B) and AAV.MPI (C) VP trimers, using VP3 as the basis. Additionally, close-ups of heparin interactions with the VR-VIII loop, including crucial residues, are shown. (A) Heparin contacts R585, R588, R484, and K532, with the HSPG-binding motif constituted by two AAV capsid subunits. Panel A was adapted from Meumann et al. and is shown for comparison. (B and C) For AAV.MPI and AAV.PPR, the model predicts heparin binding to R585 and R588 but interference with heparin binding to K532 and R484 due to the peptide insertion between N587 and R588, i.e., at I-587. (D) Heparin affinity chromatography. AAV.PPR, AAV.MPI, and AAV2 vectors were loaded onto a heparin affinity column, and flow-through (FT) and wash (WS) fractions were collected. AAV vectors were gradually eluted with increasing concentrations of sodium chloride (NaCl) (0.2–1.1 M) in PBS/MgCl 2 /KCl. Vector genome-containing particles in the collected fractions were quantified by qPCR. Bars, mean (SD), n = 3. (E) Heparin competition assay. AAV-PPR, AAV.MPI, and AAV2 vectors were pre-incubated with increasing heparin concentrations (0–24 U/ml) for 30 min at RT and then administered to HEI-OC1 cultures at a GOI of 500. At 48 hpt, eGFP expression levels were measured by flow cytometry and normalized to no-heparin controls. Bars, mean (SD), n = 3. GOI, genomic particles of infection.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Binding Assay, Comparison, Affinity Chromatography, Affinity Column, Plasmid Preparation, Competitive Binding Assay, Incubation, Expressing, Flow Cytometry, Infection

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: AAV.MPI capsid efficiently transduces multiple cell types in the adult mouse cochlea One-month-old C57BL/6J mice were injected with AAV2-CMV-dTomato or AAV.MPI-CMV-dTomato vectors via canalostomy (PSCC) or left untreated , and cochleae were analyzed 7 days post injection (dpi). (A) Schematic of the anatomy of a cochlear cross-section. IHC, inner hair cell; OHC, outer hair cells; DC, Deiters’ cells; PC, pillar cells; HC, Hensen’s cells; BC, Boettcher cells; CC, Claudius’ cells; BM, basilar membrane; TM, tectorial membrane; RM, Reissner membrane; NF, nerve fibers; SGN, spiral ganglion neurons; SV, stria vascularis; RC, root cells. Created with Biorender. (B) Representative cross-section from a cochlea injected with AAV2 vectors (8.6E8 vg) (C–F) Representative cross-section from a cochlea injected with AAV.MPI vectors (1.0E9 vg) showing the apical (C), middle (D), and basal (E) turns of the cochlea, and a close-up of the organ of Corti (F). Red indicates dTomato-expressing cells, and blue indicates DAPI-stained nuclei. Scale bars, 100 μm. vg, vector genome-containing AAV particles.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Injection, Membrane, Expressing, Staining, Plasmid Preparation

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: AAV.MPI shows SGN transduction in in vivo -injected cochleae at different doses One-month-old C57BL/6J mice were injected with AAV.MPI-CMV-dTomato vector at doses of 1E9 vg (A–B), 1E8 vg (C–D), or 1E7 vg (E–F) using canalostomy (PSCC), and cochleae were analyzed 7 days post injection. (A–F) Representative cross-sections of the apical (A, C, E) and middle (B, D, F) turns of the cochleae. (G ad H) Quantification of the normalized mean fluorescence of the vector-encoded reporter protein per mm 2 from three cross-sections for each condition in IHCs, OHCs, and SGNs of the apical (G) and middle (H) cochlear turns. Bars, mean (SD). Red indicates dTomato expressing cells, and blue indicates DAPI-stained nuclei. Scale bars, 100 μm. vg, vector genome-containing AAV particles.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Transduction, In Vivo, Injection, Plasmid Preparation, Fluorescence, Expressing, Staining

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: Compared to AAV2, AAV.MPI shows distinct features, including enhanced uncoating, that lead to improved transduction efficiency (A) Intracellular vector copy numbers of AAV2 and AAV.MPI in HEI-OC1 cells after 24 h (GOI 5,000). DNA was extracted from whole lysates and analyzed by qPCR using transgene-specific primers. Bars, mean (SD), n = 3, ∗∗ p < 0.01, unpaired t test. (B) Transgene expression of AAV2 and AAV.MPI on HEI-OC1 cells after 24 h (GOI 5,000), analyzed by flow cytometry. Mean fluorescence intensity (MFI, median) was multiplied by the number of transgene-positive cells. Bars, mean (SD), n = 3, technical triplicates, ∗∗∗∗ p < 0.0001, unpaired t test. (C) Amount of intracellular vector copies of AAV2 and AAV.MPI in HEI-OC1 cells at the indicated time points (GOI 5,000). Genomic DNA was extracted from whole lysates and analyzed by qPCR using transgene-specific primers. Bars, mean (SD), n = 3, ns: not significant, ∗ p < 0.05, ∗∗ p < 0.01, two-way ANOVA with Bonferroni post hoc test. (D) Transgene expression of AAV2 and AAV.MPI in HEI-OC1 cells at the indicated time points (GOI 5,000), analyzed by flow cytometry. MFI median was multiplied by the number of transgene-positive cells for each time point and vector. Bars, mean (SD), n = 3, ns: not significant, ∗∗∗∗ p < 0.0001, two-way ANOVA with Bonferroni post hoc test. (E) In vitro uncoating in the nuclear fraction of HEI-OC1 cells treated with AAV2 and AAV.MPI after 12 and 24 h. Isolated DNA was treated with T5 exonuclease, and the ratio of episomal to total DNA was analyzed by qPCR using transgene-specific primers. Bars, mean (SD), n = 3, technical triplicates, ns: not significant, ∗∗ p < 0.01, two-way ANOVA with Bonferroni post hoc test. (F) Capsid destabilization assay for AAV2 and AAV.MPI. Vector preparations were incubated for 30 min at the specified temperatures, followed by native dot blot using the B1 antibody to detect disintegrated capsids. Representative image of n = 3. (G) Co-detection of vector DNA with vector capsids and vector capsid proteins. NHF cells were transduced with either AAV2 or AAV.MPI (GOI 20,000). At 24 hpt, cells were fixed and processed for multicolor IF analysis combined with FISH. Intact capsids (green) or capsid proteins (yellow) were detected using either an antibody against intact AAV2 capsids or an antibody against VP1, VP2, and VP3. AAV2 DNA (red) was detected with an Alexa Fluor (AF) 647-labeled, amine-modified DNA probe that binds to the AAV2 genome. Nuclei were counterstained with DAPI (blue). (H) Image-based quantification of the complete uncoating rate of AAV2 and AAV.MPI, determined as the ratio of capsid-DNA+/capsid+DNA+ of 50 individual cells for each vector. Bars, mean (SD), ∗∗∗ p < 0.001, unpaired t test. (J) Indirect uncoating assay of vector-treated (8E8 vg) murine cochlea at 1, 3, and 7 days post injection. Cochleae were isolated, DNA was extracted and treated with T5 exonuclease or mock-treated. Samples were analyzed by qPCR using transgene-specific primers and normalized to horseradish peroxidase ( hprt ) and control samples from not injected mice. Bars, mean (SD), fold-change of uncoating in AAV.MPI injected cochleae to AAV2, n = 3, ns: not significant, ∗∗ p < 0.01, two-way ANOVA with Bonferroni post hoc test. GOI, genomic particles of infection.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Transduction, Plasmid Preparation, Expressing, Flow Cytometry, Fluorescence, In Vitro, Isolation, Incubation, Dot Blot, Labeling, Modification, Injection, Control, Infection

Journal: Molecular Therapy Advances

Article Title: Capsid-engineered AAV vector overcomes a key intracellular barrier and efficiently transduces spiral ganglion neurons in adult mice

doi: 10.1016/j.omta.2026.201669

Figure Lengend Snippet: Delivery of BDNF by AAV.MPI results in enhanced spiral ganglion survival Adult C57BL/6J mice were deafened using an aminoglycoside and loop diuretic and subsequently received AAV.MPI-BDNF (9E8 vg per inner ear) via canalostomy (PSCC) three months post-deafening. Cochleae were collected three months after vector administration. (A) TUJ-1 positive neurons (green) with associated neurites are seen in representative cochlear cross-sections from both the apical and basal turn of deafened AAV.MPI-BDNF-treated mice (left). Few surviving neurons are observed in the deafened, vector-untreated contralateral spiral ganglion (middle). Degeneration is more marked in the untreated basal turn, with degeneration of all neurites but only a few surviving neuronal cell bodies. In contrast, the untreated wild-type control cochlea (right) shows abundant neurons and intact neurites throughout. (B) Representative immunofluorescent staining with an anti-BDNF antibody shows robust labeling on the AAV.MPI-BDNF-treated side, with minimal fluorescent signal on the vector-untreated side (arrow). (C) There is a statistically significant survival of neurons in both the apical ( p < 0.01) and basal ( p < 0.05) spiral ganglion of deafened AAV.MPI-BDNF-treated mice compared with deafened, vector-untreated mice. Bars, mean (SEM), n = 5 mice per cohort, ∗ p < 0.05, ∗∗ p < 0.01 two-tailed unpaired t test with Welch’s correction.

Article Snippet: For the generation of AAV.MPI and AAV.PPR encoding helper plasmids for AAV vector production, sense and antisense oligonucleotides representing the respective peptide sequencing, flanked by linkers and restriction enzymes cutting sites for AscI and MluI, were synthesized (Eurofins Genomics GmbH) and inserted into pRC’99 , to produce pRC.MPI and pRC.PPR, respectively.

Techniques: Plasmid Preparation, Control, Staining, Labeling, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: pLannotate: engineered plasmid annotation

doi: 10.1093/nar/gkab374

Figure Lengend Snippet: Annotation results for three engineered plasmids. Each panel compares output from the pLannotate web server, the SnapGene viewer desktop program, and the PlasMapper web server. SnapGene and PlasMapper both have options to display generic open reading frames above a length cutoff that do not match specific features in their databases. These ORFs are displayed as thin arrows for SnapGene results and pink arrows for PlasMapper results. pLannotate displays features in different colors based on their type if they are matches to the GenoLIB feature database and in tan if they are matches to proteins in Swiss-Prot. Fragmentary matches are shown as unfilled arrows. Labels were manually edited to improve legibility for all tools. ( A ) Plasmid 7M8 is used for adeno-associated virus (AAV) vector production. ( B ) Plasmid pAM5505 is a helper plasmid for engineering cyanobacteria via conjugal transfer of DNA. ( C ) Plasmid pTECH-chPyIRS(IPYE) contains an engineered aminoacyl-tRNA synthetase enzyme. Note the partial TcR open-reading frame downstream of the tet promoter that is discussed in the text

Article Snippet: The 7M8 plasmid for adeno-associated virus (AAV) vector production (Addgene #64839) was constructed by Dalkara et al. ( ).

Techniques: Plasmid Preparation