Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across
There is an excellent safety record with respect to use of recombinant adeno-associated virus (AAV) vectors in human clinical trials [
Since animal models are not always available for a given disease (or may have an irrelevant phenotype), recent trend for evaluating proof-of-concept of gene therapy in laboratory studies has focused on use of induced pluripotent stem cell- (iPSC-) based cell models from affected individuals [
The evolution of AAV capsid modifications has made available a new repertoire of vectors with different cell-type tropisms which are valuable for controlling transgene level, onset of expression, viral dosage, and organ- or cell-type specificity. In addition, the transduction efficiency
Here, our goal is to establish and optimize a methodological approach to identify which of a variety of AAV serotypes is best suited for applications
Two iPSC cell lines (JBWT2 (PBWT2) and JBWT4 (BMC1)) derived from adult individuals without any known disease were evaluated in these studies and have been previously published showing a complete analysis of pluripotent stem characteristics [
Retinal pigmented epithelium cells were generated from both iPS cell lines and fully characterized as previously published [
For the iPSC-cortical neuron differentiations, on day 3, cells were dissociated using TrypLE (Invitrogen) and plated on GFR Matrigel in HES : MEF-conditioned (90 : 10) medium +20 ng/mL of bFGF and supplemented with ROCK inhibitor (10
Rat cortical neuron experiments were performed using isolated cells from embryonic day 18 Sprague-Dawley rat fetuses. All animal experiments were performed in agreement with the National Institutes of Health guidelines and approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC). Rat fetuses were removed from euthanized timed-pregnant dames via caesarean section. Fetuses were then decapitated, and their brains isolated. Cortical neurons were isolated from 4-6 cortical hemispheres, which were dissociated with 0.25% Trypsin/EDTA (Invitrogen) containing DNase I (Roche) at 37°C. Cells were washed three times using HBSS and suspended in neuronal media: Neurobasal media plus 2% B27, 1 × glutamine, and 1 × pen/strep. Analysis of rat cortical neurons were performed to confirm identity using immunofluorescence of Tju1 (Sigma, 1 : 1000), Satb2 (Abcam, 1 : 500), and CTIP2 (Abcam, 1 : 1000) cortical cell markers and GFAP (SCBT: 1 : 300) as described above. Images were captured using a Nikon Eclipse Ti-S inverted microscope, NIS Elements Advanced Research software and magnification of 10x.
A panel of 11 recombinant AAV serotypes were generated by the Center for Advanced Retinal and Ocular Therapeutic (CAROT) research vector core. Triple transfection of HEK293T cells with pAAV.CMV.C
For transduction experiments, 96-well plates were coated with 1 : 100 GFR Matrigel overnight. Cells were enzymatically detached to create a single-cell suspension prior to plating at a density of approximately 2E4 for iPSC, 8E4 iPSC-RPE, 1E5 iPSC-cortical neuron, and 2E4 rat cortical neuron per well. After 24 to 48 h, cells from a minimum of three wells per dish were disassociated and cell counts were performed. Transduction of rAAV-eGFP serotypes, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV7m8, AAV8, AAV8b, and AAV9, was executed at concentrations of 0, 10,000 (1E4), 100,000 (1E5), and 1,000,000 (1E6) vector genomes per cell (vg/cell) in triplicate. The amount of virus needed to achieve a multiplicity of infection (MOI: 1E4, 1E5, or 1E6 vg/cell) was based on the cell number per well for each AAV serotype. Viral stocks were diluted in 0.001% Pluronic F-68 (formulation buffer) in 1 × PBS when necessary. Representative example of dilution strategy is provided in Supplemental Table
The data of relative fluorescent per cell at three different MOIs were visualized by heat map (i.e., two-dimensional representations in colors). The heat map of each cell type was generated using Seaborn, a visualization library based on Python matplotlib. Fluorescent per cell measurements were parsed from excel datasheets into floating-point vectors with each vector corresponded to all AAV serotypes in each MOI over multiple postinfection measurement times. The colors were based on the relative fluorescent per cell level and ranged from yellow (low) to brown (high) fluorescent intensity. Analysis of variance (ANOVA) (Excel’s Analysis ToolPak) for repeated measures was performed to determine the relationship of cell lines and AAV serotypes on the gene expression effect. To determine the effect of each AAV serotype for each cell line, ANOVA with one factor (i.e., AAV serotype) was used, followed by the post hoc pairwise comparisons (XLSTAT add-ins) with correction for multiple comparisons using Tukey method. For each AAV serotype, the gene expression post-infection across time (48, 72 and 96 h post-infection) was evaluated using repeated measures ANOVA followed by pairwise comparison with correction for multiple comparisons using Tukey method. The differences were considered statistically significant when
Pluripotent stem cells can be used to generate numerous cell types which are candidates for rAAV vector studies and disease modeling. Induced pluripotent stem cells
Schematic of rAAV tropism testing for
Analysis of AAV tropism was performed on iPSCs derived from two individuals, and cell lines were cultured in undifferentiated cell culture conditions to maintain a pluripotent state. Prior to transduction, to ensure cultures were a uniform population of pluripotent stem cells (PSC), cultures were monitored for PSC morphological characteristics (phase) and pluripotent surface markers expression of SSEA3 and SSEA4, by flow cytometry (Figure
iPSC and iPSC-RPE tropism of 11 rAAV serotypes. (a) Representative phase image showing morphology of IPSC cultures and flow cytometry analysis of pluripotency surface marker expression (SSEA3 and SSEA4) of iPS cells. (b) The onset of AAV-eGFP expression in iPSCs at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h posttransduction. There is no significant difference between any two time points (48, 72, and 96 h) after correction for multiple comparisons using Tukey method. (c) Heat map showing relative AAV-GFP expression per cell across all rAAV, dosages, and time in iPSCs. The scale bar shows the intensity of AAV-eGFP expression presented as an arbitrary relative fluorescence unit (A.U.) per cell. (d) Retinal pigmented epithelium (RPE) showing “cobblestone” appearance in phase (10x magnification), and immunofluorescent labeling with expression of ZO-1 (red) and MITF (green), and merged images showing uniform RPE monolayer. Images were captured at 10x magnification. (e) AAV-eGFP expression in RPEs at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h postinfection. Analysis of variance for repeated measures with post hoc pairwise comparisons between time points were performed with statistical significance indicated with
RPE cells have been subject to a number of clinical interventions for the treatment of vision disorders such as retinitis pigmentosa (RP), Leber’s congenital amarosis (LCA), advanced neovascular age-related macular degeneration, and choroidal neovascularization [
RPE cells were able to be maintained on culture dishes for months postinfection and eGFP expression was also maintained; moreover, the level of transgene expression remained consistent with expression measured at 96 h posttransduction for weeks (Supplementary Figure
Cortical neurons were generated from the same iPS cells as described above, and the phenotype of these neurons (day 82+) was determined by immunocytochemical staining with markers of supragranular (layers II/III; Satb2) and infragranular (layer V; CTIP2) neurons prior to AAV transduction experiments. Within these cultures, two different cell types were predominant, either postmitotic pyramidal neurons (Tuj1+,
rAAV tropism of in vitro-derived cortical neurons compared to ex vivo-isolated rat cortical neurons. (a) iPSC-derived cortical neurons show dorsal forebrain identity at 82 days in vitro (DIV). The expression of pyramidal neuron markers, Tuj1 (green)/Satb2 (red) or Tuj1 (green)/CTIP2 (red), is shown. Images were captured at 10x magnification. (b) The onset of GFP expression in iPSC-derived cortical neurons (82-86 DIV) at a dosage of 1E6 vg/cell for all rAAV at 48, 72, and 96 h postinfection. Analysis of variance with post hoc pairwise comparisons between time points were performed with statistical significance indicated with
The fluorescence intensity of eGFP for all rAAV serotypes was significantly lower (~2-4-fold,
Overview of AAV-eGFP transgene expression across four cell types. The illustration shows cell type and serotype comparison between induced pluripotent stem cells (iPSC), iPSC-derived retinal pigmented epithelium (RPE), iPSC-derived cortical neurons, and
Although initial AAV tropism studies focused on AAV2, other serotypes have recently been shown to perform better
Important to note, having the highest level of AAV-eGFP expression will not always provide optimal results as the biology associated with transgene element needs to be considered. The endogenous level and transgene function, transcription factor, structural protein, chaperone, and enzymes, all need to be evaluated during gene augmentation testing. Evaluation of gene expression kinetic profiles also provides insight in the appropriate timing for the evaluation of maximal and sustained AAV-transgene expression which can be used for preclinical and clinical viral preparation validation. Therefore, having a group of rAAVs with variable gene expression kinetic, protein expression profiles, and cell tropisms is essential for selecting the appropriate levels of transgene expression for a given application.
The data used to support the findings of this study are available from the corresponding author upon request.
J.B. holds intellectual property related to the engineered AAV serotype, AAV8b.
The authors declare that they have no conflicts of interest.
TTD, TP, VV, JB, and JAM are responsible for the strategy, conception, and writing of the manuscript. JAM, P.H., and TTD are responsible for the iPSC maintenance and RPE differentiations. TTD, LL, and JAM are responsible for the RPE characterization. JL and HIC are responsible for the iPSC cortical generations. SZ and JP are responsible for the rAAV production. JL and HIC are responsible for the isolation of embryonic rat cortical neurons. TP and HN are responsible for the computational analysis, TTD, TP, VV, JB, and JAM are responsible for the data analysis, interpretation, and contribution to experimental design. All coauthors reviewed and edited the manuscript.
This study was supported in part by the Vietnam Education Foundation (VEF) fellowship (
Supplemental Table 1: representative AAV dosage, titer, and MOI calculation for iPSC, RPE, and human and rat cortical cells. Supplemental Table 2: relative fluorescence intensity (R.F.U.) per cell at 96 h at MOI (1E6 vg/cell) Supplementary Figure 1: stable GFP transgene expression in RPE. (a) The AAVeGFP expression in RPE at d4 (96 h) and d18 for a dosage of 1E6 vg/cell shows similar expression in longer term cultures. There is no statistically significant difference between day 4 and day 18 in any of the AAV. Supplementary Figure 2: RPE and cortical neuron characterization. (a) Flow cytometric analysis of intracellular MITF protein expression. Greater than 95% of RPE expresses MITF. Plot showing MITF positive cells (blue) relative to mouse isotype control antibody (red). (b, c) Immunophenotyping of human and rat cortical neurons and astrocytes showing neuron-specific class III beta-tubulin (TUJ1; green) and glial fibrillary acidic protein (GFAP; red), respectively. Images were captured at 20x magnification (human iPSC cortical) and 10x magnification (rat cortical).