Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells - PubMed (original) (raw)
Directed evolution of adeno-associated virus for enhanced gene delivery and gene targeting in human pluripotent stem cells
Prashanth Asuri et al. Mol Ther. 2012 Feb.
Abstract
Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However, natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs), which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities, and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (~50%) to hPSCs, which are importantly accompanied by a considerable increase in gene-targeting frequencies, up to 0.12%. While this level is likely sufficient for numerous applications, we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (>1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus, this study demonstrates that under appropriate selective pressures, AAV vectors can be created to mediate efficient gene targeting in hPSCs, alone or in the presence of ZFN- mediated double-stranded DNA breaks.
Figures
Figure 1
Gene expression in hESCs mediated by various AAV serotypes. HSF-6 cells were infected with numerous natural AAV serotypes at a MOI of 10,000. Transduction efficiency was assessed as the percentage of GFP positive cells measured by flow cytometry 48 hours postinfection. Error bars indicate standard deviation (n = 3). AAV, adeno-associated virus; GFP, green fluorescent protein; hESC, human embryonic stem cell; MOI, multiplicity of infection.
Figure 2
Directed evolution of AAV and hPSC transduction by AAV 1.9. (a) Schematic representation of directed evolution. 1) A viral library is created by mutating the cap gene. 2) Viruses are packaged in HEK293T cells using plasmid transfection, such that each particle is composed of mutant capsid surrounding the cap gene encoding that protein capsid. 3) Viruses are harvested from 293 cells and purified. 4) The viral library is introduced to the HSF-6 hESCs in vitro. 5) Successful viruses are amplified and recovered using adenovirus rescue. 6) Successful clones are enriched through repeated selections. 7) Isolated viral DNA reveals selected cap genes. 8) Selected cap genes are mutated again to serve as a new starting point for selection. (b) Molecular model of the full AAV2 capsid, based on the solved structure, shows the location of the R459G mutation (blue) on the surface of the capsid (VP3 region), near the threefold axis of symmetry and residues known to be important for heparin and FGF receptor binding (pink). (c) AAV-mediated gene expression in hESCs. HSF-6 cells were infected with selected mutants at a MOI of 10,000. Transgene expression was assessed as the percentage of GFP positive cells measured by flow cytometry 48 hours postinfection. Error bars indicate the standard deviation (n = 3), *P < 0.01. (d) Elevating the MOI increases transduction. HSF-6 cells were infected with AAV1.9 at MOIs of 1,000, 10,000, and 100,000. Transgene expression was assessed as the percentage of GFP positive cells measured by flow cytometery 48 hours postinfection. Error bars indicate standard deviation (n = 3), *P < 0.01. (e) AAV1.9-mediated gene expression in hPSCs. HSF-6 hESCs, human dermal fibroblast-derived hiPSCs, human MSC-derived hiPSCs, and H1 hESCs were infected at a MOI of 10,000. Transgene expression was assessed as the percentage of GFP positive cells measured by flow cytometry 48 hours postinfection. Error bars indicate the standard deviation (n = 3), *P < 0.01. (f) In vitro analysis of AAV1.9 tropism. Variant AAV1.9 (white), selected on hESCs, is less infectious on AAV-permissive cell types (HEK293T, CHO) than recombinant AAV2 (black) and AAV6 (gray), but more infectious on HSF-6 hESCs. Error bars indicate standard deviation (n = 3), *P < 0.01. (g) HSF-6 cells infected by AAV1.9 maintain pluripotency marker expression. HSF-6 cells infected with AAV1.9 encoding GFP at an MOI of 100,000 were fixed and immunostained 1 week after infection for the presence of GFP (green) and the pluripotency marker Nanog (red). AAV, adeno-associated virus; FGF, fibroblast growth factor; GFP, green fluorescent protein; hESC, human embryonic stem cell; hiPSC, human induced pluripotent stem cell; MOI, multiplicity of infection; MSC, mesenchymal stem cell.
Figure 3
Mechanistic analysis of transduction by variant AAV. (a) AAV1.9 has a higher affinity for heparin than AAV2 and AAV6. The heparin affinity column chromatogram of recombinant AAV2 (black), AAV6 (gray), and AAV1.9 (white) is shown, where virus was eluted from the column using increasing concentrations of NaCl. Virus was quantified using qPCR. (b) In vitro characterization of AAV1.9 HSPG dependence. CHO (black) and pgsA (white) cells were transduced to demonstrate the decrease in HSPG dependence of AAV1.9 compared to AAV2. Error bars indicate standard deviation (n = 3), *P < 0.01. AAV, adeno-associated virus; hESC, human embryonic stem cell, qPCR, quantitative PCR. (c) In vitro characterization of AAV1.9 binding affinity. AAV1.9 and its parental serotype AAV2 bind hESCs to similar extents, yet AAV1.9 is capable of significantly higher transduction of hESCs. Error bars indicate standard deviation (n = 3), *P < 0.01.
Figure 4
AAV1.9-mediated correction of a nonfunctional GFP expressed in hESCs. (a) Schematic overview depicting the targeting strategy for the GFP gene correction. The defective GFP gene—GFPΔ35, mutated by the insertion of a 35 bp fragment containing a translational stop codon—was integrated into HSF-6 cells using a lentiviral vector. A targeting vector (t37GFP) containing a 5′ truncated GFP coding sequence was packaged into a recombinant AAV vector lacking a promoter. Homologous recombination between donor vector and integrated defective GFPΔ35 gene would correct the 35 bp mutation and result in fluorescent hESCs. (b) Gene targeting frequencies assessed as the percentage of GFP positive cells measured via flow cytometry 72 hours postinfection. Error bars indicate standard deviation (n = 3), *P < 0.01. ND, not detectable by flow cytometry. (c,d) Representative analyses of the targeted correction of GFPΔ35 gene. Genomic DNA from the “corrected” and “uncorrected” HSF-6 cells was amplified using PCR. The 35 bp mutation within the GFPΔ35 gene contains an Xho I site, and therefore only the PCR products from the “uncorrected” cells (sample 2) can be digested using Xho I, whereas the PCR products from the corrected cells (sample 1) show the restoration of the functional GFP gene without the Xho I site. DNA sequencing analysis also shows AAV1.9-mediated correction of the 35 bp mutation in HSF-6 cells originally expressing the mutated GFPΔ35 gene. (e) Time course of AAV-mediated GFPΔ35 correction in HSF-6 cells. Error bars indicate standard deviation (n = 3). (f) Maintenance of an undifferentiated state of gene-corrected, GFP-expressing cells for 30 days postinfection. GFP positive HSF-6 cells, isolated via FACS, were cultured for 30 days, then fixed and probed for the presence of GFP (green), Oct4 (red), and DAPI (blue). (g) Pluripotency of the HSF-6 cells carrying the corrected GFP gene was further confirmed through embryoid body-mediated in vitro differentiation to generate derivatives of all three germ layers, as indicated by the expression of the ectodermal marker β-III tubulin, the mesodermal marker α-smooth muscle actin (α-SMA), and the endodermal marker hepatocyte nuclear factor 3 β (HNF3β) after 24 days of differentiation (Bar = 100 µm). AAV, adeno-associated virus; CMV, cytomegalovirus; DAPI, 4′,6-diamidino-2-phenylindole; FACS, fluorescent-activated cell sorting; GFP, green fluorescent protein; hESC, human embryonic stem cell; IRES, internal ribosome entry site; ITR, inverted terminal repeat; LTR, long terminal repeat.
References
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