Protein Carrier Adeno-Associated Virus

Figures

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  Figure 1. Insertion of a GFPnb into VP1 enabling packaging of GFP within AAV capsids. (A) Schematic of the modified cap genes and GFP expression cassette used for protein packaging. (B) Model showing the AAV-DJ capsid (gray) containing two GFPnb (dark green) insertions at position H631 and packaging two GFP (light green). The model was generated in PyMol using the AAV-DJ capsid structure obtained from the RCSB Protein Data Bank (PDB) identifier 7KFR. The structure of a VP monomer with a GFPnb insertion at position H631 was predicted using AlphaFold218 and subsequently aligned with two manually selected VP monomers in the AAV-DJ capsid structure. The GFP:GFPnb complex (RCSB PDB identifier 3OGO) was aligned with the two GFPnb insertions within the VP monomers to generate the final image. (C) Representative Western blot of wtAAV-DJ and AAV-DJ capsids incorporating VP1 with a GFPnb inserted at position H631 or H643 produced in cells overexpressing GFP during production.

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  Figure 2. Addition of a c-Myc NLS to GFP and insertion of the GFPnb into VP3 increasing protein packaging efficiency. (A) Representative Western blot comparing the packaging efficiency of wtAAV-DJ capsids and capsids incorporating a GFPnb in VP1 at position H631 in packaging GFP with or without the addition of a c-Myc NLS. (B) Schematic of the two cap genes used for producing AAV-DJ capsids with a GFPnb inserted into VP3 and the expression cassette used for generating GFP tagged with a c-Myc NLS at the C-terminus. (C) Representative Western blot comparing the packaging of GFP tagged with a c-Myc NLS at the C-terminus in the indicated capsid variants. wtAAV-DJ produced in cells overexpressing GFP-NLS protein was included as a negative control.

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  Figure 3. Flow cytometry and live cell imaging of pcAAV-mediated GFP delivery. (A) The delivery of GFP-NLS protein to 293AAV cells by pcAAV packaging GFP-NLS protein was assessed by flow cytometry. 24 h after seeding into a 24-well plate, 293AAV cells were mock transduced or transduced with 250 μL of either wtAAV-DJ or AAV-DJ capsids incorporating the GFPnb in VP3 at position H631 and packaging GFP-NLS protein. The GFP fluorescence intensity for each sample was assessed by flow cytometry 2 h post transduction. (B) The subcellular localization of GFP delivered by AAV-DJ capsids incorporating a GFPnb in VP3 at position H631 and packaging GFP-NLS protein was visualized using fluorescence microscopy. 24 h post seeding into a 96-well glass bottom plate, 293AAV cells were either mock transduced, treated with 1 × 105 molecules recombinant GFP-ALFA protein per cell, or incubated with 15 μL of either wtAAV-DJ or AAV-DJ capsids incorporating a GFPnb at position H631 in VP3 and packaging GFP-NLS protein. 10 h post transduction, the nucleus and cell membrane were labeled and subsequently imaged. In the merged image, the nucleus, GFP, and membrane are colored blue, green, and red, respectively.…

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  Figure 4. Protein packaging in multiple AAV serotypes. Representative Western blot demonstrating packaging of GFP-NLS protein in wtAAV capsids or capsids containing a GFPnb insertion at one of two positions in VP3 for each of the indicated serotypes. The B1 antibody and a GFP antibody were used for visualizing the VP subunits and GFP-NLS, respectively. The dashed line separates the distinct blots.

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  Figure 5. Engineered AAV capsids capable of packaging several proteins, which can retain enzymatic activity. (A) Representative Western blot demonstrating packaging of spCas9 protein with or without a GFP tag in wtAAV-DJ capsids or capsids with a GFPnb inserted into VP1 at position H643. The B1 antibody and a spCas9 antibody were used for visualizing the VP subunits and spCas9, respectively. (B) Representative Western blot demonstrating that GFP tagged with both a c-Myc NLS and an ALFA tag is packaged within capsids containing the ALFAnb inserted into either VP1 or VP3 at either position H631 or H643. The B1 antibody and a GFP antibody were used for visualizing the VP subunits and GFP, respectively. (C) Representative Western blot showing packaging of Cre recombinase protein tagged with an ALFA tag. The B1 antibody and an ALFA tag antibody were used for visualizing the VP subunits and ALFA-tagged Cre, respectively. (D) Representative Western blot demonstrating packaging of the indicated APEX2 constructs in both engineered and wtAAV-DJ capsids. The B1 antibody and V5 antibody were used for visualizing the VP subunits and V5-tagged APEX2, respectively. (E) Fluorescence values per capsid…