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. 2014 Feb 19:1:12.
doi: 10.1038/mtm.2013.12. eCollection 2014.

Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

Yarong Liu  1 , Young Joo Kim  2 , Man Ji  3 , Jinxu Fang  1 , Natnaree Siriwon  1 , Li I Zhang  2 , Pin Wang  4
Affiliations

Enhancing gene delivery of adeno-associated viruses by cell-permeable peptides

Yarong Liu et al. Mol Ther Methods Clin Dev. .

Abstract

Adeno-associated virus type 2 (AAV2) is considered a promising gene delivery vector and has been extensively applied in several disease models; however, inefficient transduction in various cells and tissues has limited its widespread application in many areas of gene therapy. In this study, we have developed a general, but efficient, strategy to enhance viral transduction, both in vitro and in vivo, by incubating viral particles with cell-permeable peptides (CPPs). We show that CPPs increase internalization of viral particles into cells by facilitating both energy-independent and energy-dependent endocytosis. Moreover, CPPs can significantly enhance the endosomal escape process of viral particles, thus enhancing viral transduction to those cells that have exhibited very low permissiveness to AAV2 infection as a result of impaired intracellular viral processing. We also demonstrated that this approach could be applicable to other AAV serotypes. Thus, the membrane-penetrating ability of CPPs enables us to generate an efficient method for enhanced gene delivery of AAV vectors, potentially facilitating its applicability to human gene therapy.

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Figures

Figure 1
Figure 1
Antp, TAT-HA2, and LAH4 improve adeno-associated virus type 2 (AAV2) transduction in permissive and nonpermissive cells. (a–c) Green flourescent protein (GFP) expression levels of HEK293T cells infected with AAV2 (multiplicity of infection (MOI) = 400) alone or precomplexed with (a) Antp, (b) TAT-HA2, (c) or LAH4. (d–f) GFP expression levels of HepG2 cells infected with AAV2 (MOI = 400) alone or precomplexed with (d) Antp, (e) TAT-HA2, or (f) LAH4. Error bars represent the standard deviation of the mean from triplicate experiments.
Figure 2
Figure 2
Interaction of cell-permeable peptides (CPPs) with adeno-associated virus type 2 (AAV2) facilitates enhanced viral transduction. (a) Cellular cytotoxicity of AAV2-CPP complexes. HEK293T cells were incubated with the AAV2-CPP complexes with increasing concentrations of peptides for 48 hours. Subsequently, cell viability was determined using the XTT assay. (b) Visualization of the AAV2-CPP complexes. AAV2 were incubated with FITC-CPP (green) and subsequently probed with a mouse monoclonal antibody (A20) specific for intact AAV2 (red). Overlaping green and red signals appear as yellow in the merged image. Scale bar represents 5 μm. (c) Quantitative analysis of the number of remaining CPPs per AAV2 particle. The numbers were determined by measuring the spectroscopic property of the purified AAV2-CPP complexes 30 minutes after incubation. (d) green flourescent protein (GFP) expression levels of HEK293T cells infected with increasing multiplicity of infection of AAV2 in the presence of a fixed concentration of CPPs (200 μmol/l). (e) Transduction titers of AAV2 in the presence or in the absence of LAH4 in HEK293T cells and HepG2 cells. (f) GFP expression levels of HEK293T cells infected with AAV2 alone or the AAV2-CPP complexes formed in the presence of increasing concentrations of buffered phosphate (0.1–0.5 mol/l, pH 7.4). All error bars represent the standard deviation of the mean from triplicate experiments.
Figure 3
Figure 3
Cell-permeable peptides (CPPs) enhance adeno-associated virus type 2 (AAV2) uptake by cells. (a,b) Internalization of AAV2 particles in the presence of CPPs to (a) HEK293T cells or (b) HepG2 cells. AAV2 particles were labeled with Alexa488 dye and incubated with CPPs for 30 minutes at 37 °C. Cells were incubated with AAV2-CPP complexes for 30 minutes at 37 °C. The cellular uptake of dye-labeled AAV2 was determined by measuring dye fluorescence using flow cytometry. (c) Internalization kinetics of AAV2 particles in the presence of CPPs. AAV2 particles were labeled with Alexa488 dye and incubated with CPPs for 30 minutes at 37 °C. HEK293T cells were incubated with AAV2-CPP complexes at 37 °C for different indicated time points. After incubation, the cells were washed to remove the unbound complexes, and cellular uptake of dye-labeled AAV2 was determined by measuring dye fluorescence using flow cytometry.
Figure 4
Figure 4
Entry mechanisms of adeno-associated virus type 2 (AAV2)-cell-permeable peptides (CPP) complexes. (a) The internalization of AAV2 or AAV2-CPP complexes in cells after 30 minutes incubation at 37 or 4 °C. (b) The effect of inhibitory drugs on viral transduction of cells infected by AAV2 or AAV2-CPP complexes. HEK293T cells were preincubated with chlorpromazine (CPZ, 30 nmol/l), filipin (15 nmol/l), or Amiloride (1 mmol/l) for 30 minutes at 37 °C. Treated and untreated cells were spin-infected with the AAV2 alone or complexes of AAV2 with peptides (final concentration: 200 μmol/l) for 90 minutes in the presence of drugs. Green flourescent protein (GFP) expression was analyzed 2 days after infection. All error bars represent the standard deviation of the mean from triplicate experiments. Asterisks indicate statistical significance compared to the no drug treatment group (*P < 0.05; **P < 0.01). (c,d) The effect of HSPG receptor blocking by (c) heparin or (d) HSPG antibody on viral transduction mediated by AAV2 or AAV2-CPP complexes. The cells were incubated with heparin (200 µg/ml) or HSPG Ab (1:100) for 30 minutes at 37 °C. The treated and untreated cells were spin-infected with the AAV2 alone or complexes of AAV2 with peptides (final concentration: 200 μmol/l) for 90 minutes. GFP expression was analyzed 2 days after infection. All data are shown as the means of triplicate experiments. Asterisks indicate statistical significance compared to the no drug treatment group (**P < 0.01).
Figure 5
Figure 5
The effect of cell-permeable peptides (CPPs) on the endosomal escape of adeno-associated virus type 2 (AAV2) particles. (a) The effect of bafilomycin-A1 (BAF) on viral transduction mediated by AAV2-CPP complexes. HEK293T cells were preincubated with BAF for 30 minutes at 37 °C. The cells were then spin-infected with AAV2 or AAV2-CPP complexes for 90 minutes in the presence of BAF. GFP expression was analyzed 2 days after infection. All data are shown as the means of triplicate experiments. Asterisks indicate comparisons to the no drug treatment group (*P < 0.05). (b,c) Functional involvement of early and late endosomes in the viral transduction mediated by AAV2-CPP complexes. HEK293T cells were transiently transfected with a wild-type or dominant-negative mutant form of (b) Rab5 or (c) Rab7. Twenty-four hours after transfection, cells were spin-infected with AAV2 or AAV2-CPP complexes for 90 minutes. GFP expression was analyzed 2 days after infection. All data are shown as the means of triplicate experiments. Asterisks indicate statistical significance compared to the wild-type form of Rab treatment groups (*P < 0.05). (d) The effect of CPPs on the endosomal escape of AAV2 particles. AAV2 (107 genome copies per well) or AAV2-CPP complexes (final concentration of CPP: 200 μmol/l) in phosphate-buffered saline (PBS) with different pH values (pH 7.4, 6.0, or 5.0) were placed in the upper compartment of a 24-well transwell plate. After incubation at 37 °C for 12 hours, the AAV2 particles transferred from upper compartment to the lower compartment with pH 7.4 PBS were collected. The intracellular viral genome copies were quantified by quantitative polymerase chain reaction. (e) The GFP expression of NIH3T3 cells infected by AAV2 alone or AAV2-CPP complexes (final concentration of CPP: 200 μmol/l). All error bars represent the standard deviation of the mean from triplicate experiments. Asterisks indicate statistical significance compared to the AAV2 alone treatment group (*P < 0.05; **P < 0.01). (f) Transduction titers of AAV2 in the presence or in the absence of LAH4 (final concentration: 200 μmol/l) in NIH3T3 cells. All data are shown as the means of triplicate experiments.
Figure 6
Figure 6
Cell-permeable peptides (CPPs) enhance viral transduction in primary cells and tissues. (a) The green flourescent protein (GFP) expression levels of bone marrow–derived cells infected by adeno-associated virus type 2 (AAV2) alone or AAV2-CPP complexes. (b) Enhancement of gene delivery of AAV2 in the presence of CPPs in murine mesenchymal stem cells. (c,d) CPPs facilitate gene delivery of AAV2 in mouse cochlear neuron. Mouse cochlear neurons were treated with AAV2-CPP complexes or AAV2 alone for 4 hours, and the medium was replaced by fresh medium. (c) After 7 days, tissues were imaged by fluorescence microscopy. Upper: GFP fluorescence. Lower: Myosin VI image. Scale bar represents 100 μm. (d) Quantification of GFP expression levels in cochlear neuron. To quantify GFP-positive cells, 4 regions of interest (ROI) were randomly chosen per image at ×10 magnification. The data are expressed as % of total area of GFP-positive in the region of AAV-CPP-treated tissues normalized by that of tissues treated by AAV2 alone. All data are shown as the means of triplicate experiments. Asterisks indicate statistical significance compared to the AAV2 alone treatment group (*P < 0.05; **P <0.01).
Figure 7
Figure 7
Cell-permeable peptides (CPPs) facilitate gene delivery of adeno-associated virus type 2 (AAV2) in mouse muscles. (a) Transgene expression in muscle tissues from BALB/c mice injected with AAV2 or AAV2-CPP complexes. Muscle cross sections were taken at day 14 or day 30 after intramuscular injection of AAV2 alone (109 particles/mice) or AAV2-CPP complexes from injected legs. Muscle sections were stained with GFP antibody (red), followed by nuclear staining (blue). Scale bar represents 100 μm. (b) Quantification of GFP expression levels in muscles shown in (a). To quantify GFP-positive cells, four regions of interest (ROI) were randomly chosen per image at x10 magnification. The data are expressed as % of total nuclear area stained by GFP in the region. All error bars represent the standard deviation of the mean from triplicate experiments. Asterisks indicate statistical significance compared to the AAV2 alone treatment group (**P < 0.01). (c) Biodistribution of viral vectors in AAV2-injected and AAV2-LAH4-injected mice. Organs were collected from AAV2-injected and AAV2-LAH4-injected mice 14 days postadministration. AAV2 genome copies were assessed by qPCR assay. Levels of vectors were standardized using primers against housekeeping gene Apob. All error bars represent the standard deviation of the mean from triplicate experiments.
Figure 8
Figure 8
No cytotoxicity of cell-permeable peptides (CPPs) was detected in vivo. (a) Analysis of T lymphocyte infiltration in muscle tissues from BALB/c mice injected with adeno-associated virus type 2 (AAV2) or AAV2-CPP complexes 14 days postadministration. Serial cross-sections were stained with hematoxylin and eosin (H&E), and were immunohistochemically stained with antibodies against CD4 and CD8 (red) followed by nuclear staining (blue). Scale bar represents 100 μm. (b) Analysis of inflammation markers in muscles from mice injected with AAV2 or AAV2-CPP complexes 14 days postadministration. Total RNA was isolated from muscles and mRNA levels of interleukin (IL)-1b, IL-6, and tumor necrosis factor (TNF)-α were assessed by quantitative polymerase chain reaction. 2ΔCt (ΔCt = −Ctcytokine+CtGAPDH) method was used to calculate the relative cytokine expression. All of the data were then normalized by the mean percentage of cytokine expression in AAV2-injected mice. All error bars represent the standard deviation of the mean from triplicate experiments.
Figure 9
Figure 9
Antp, TAT-HA2, and LAH4 improve AAV8 transduction in target cells. (a–c) Green flourescent protein (GFP) expression levels in HEK293T cells infected with AAV8 (multiplicity of infection (MOI) = 1,000) alone or precomplexed with (a) Antp, (b) TAT-HA2, (c) or LAH4. (d–f) GFP expression levels in Huh7 cells infected with AAV8 (multiplicity of infection (MOI) = 1,000) alone or precomplexed with (d) Antp, (e) TAT-HA2, or (f) LAH4. Error bars represent the standard deviation of the mean from triplicate experiments.
Figure 10
Figure 10
Cell-permeable peptides (CPPs) facilitate AAV8-mediated gene delivery in mouse muscles. (a,b) Histochemical and immunofluorescence analyses of muscle tissues from BALB/c mice injected with AAV8 or AAV8-CPP complexes. Muscle cross sections were taken at day 14 after intramuscular injection of AAV8 alone (108 particles/mice) or AAV8-CPP complexes. Muscle sections were stained with hematoxylin and eosin (a), or GFP antibody (red), followed by nuclear staining (blue, b). Scale bar represents 100 μm.
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