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Nature Protocols 2, 523 - 531 (2007)
Published online: 15 March 2007 | doi:10.1038/nprot.2007.51

Subject Categories: Isolation, purification and separation | Nanotechnology | Genetic modification | Microbiology and virology

Design and construction of targeted AAVP vectors for mammalian cell transduction

Amin Hajitou1, Roberto Rangel1, Martin Trepel2, Suren Soghomonyan3, Juri G Gelovani3, Mian M Alauddin3, Renata Pasqualini1 & Wadih Arap1

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic–eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approx1010–1011 transducing units per mul) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

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Introduction

The use of new genetic systems for the study of currently intractable biological questions will require the development of ligand-directed (targeted) vectors that can be systemically delivered. Over the past decade, common approaches at targeted gene delivery have typically relied on ablation of the native tropism of mammalian viruses, redirection to alternative receptors or both1, 2, 3, 4, 5, 6, 7, 8. Incorporation of homing peptides selected from bacteriophage (phage) display library screenings into mammalian viral vectors has been attempted, but such strategy has the potential to alter the structure of the capsid, affect the targeting attributes of the ligand peptides or even prevent the display within a viable viral capsid altogether9, 10, 11, 12, 13. In contrast, phage have no intrinsic tropism for mammalian cells14, 15 and can mediate modest gene expression in mammalian cells after genetic manipulation16, 17, 18, 19. In theory, phage-based vectors have some potential advantages over animal viruses for mammalian cell-targeted delivery of transgenes. First, there are no known natural receptors for phage (which have evolved as prokaryotic viruses) on mammalian cells14, 15. However, receptor-mediated internalization by mammalian cells occurs if phage vectors are genetically modified to display specific ligands, such as fibroblast growth factor (FGF2), anti-ErbB2 scFv F5 antibody and integrin-binding peptides16, 17, 18, 19, 20. Moreover, bacteriophage have long been administered to humans, from its antibacterial use in the environment during the preantibiotic era14 to the very recent Food and Drug Administration approval of certain phage preparations as antibacterial food additives21. Indeed, feasibility clinical trials have shown that the selection of phage libraries in cancer patients can yield ligand–receptor systems22, 23 and that serial library administration can be accomplished without major untoward clinical effects24. Finally, unlike mammalian viruses, phage do not require further context modification of their capsid because the targeting peptides are actually selected and isolated directly as homing to specific cell-surface receptors22, 25, 26, 27, 28, 29, 30.

Despite these potential advantages, phage-based vectors have inherently been considered poor gene delivery vehicles. As a working hypothesis, we proposed that the rate-limiting step might be mechanistically related to the post-targeting fate of the single-stranded DNA of the phage genome1. In an attempt to improve phage as targeted vectors for mammalian cells, we reasoned that the genetic incorporation of compatible cis-elements (such as inverted terminal repeats (ITRs)) from a mammalian—yet single-stranded—DNA virus such as recombinant adeno-associated virus (AAV) would improve post-targeting transgene expression. Thus, we have developed ligand-directed vectors as a hybrid between AAV and phage (termed AAVP). In our targeted AAVP prototype vector1, the targeted phage displays an RGD-4C peptide that binds to alphav integrins26, 27, 29, 31, with the mammalian transgene cassette flanked by full-length ITRs of AAV serotype 2. We reported1 that the improved mammalian transduction efficiency by targeted AAVP over conventional phage-based vectors is associated with an improved fate of the delivered transgene, through maintenance of the entire mammalian transgene cassette, better persistence of episomal DNA and formation of concatamers of the transgene cassette1. Here, we detail how to insert an AAV transgene cassette into the backbone phage vector genome to generate targeted AAVP hybrid constructs (Steps 1–18) and how to produce, purify and titrate the vector preparations (Steps 19–26). We also describe a standard protocol for AAVP-mediated mammalian cell transduction, both in tissue culture and in targeted tissues in vivo after systemic administration. Briefly, DNA olignonucleotide sequences encoding peptide ligands are inserted into the SfiI site of the gene of the pIII minor coat protein of fUSE5-MCS (multicloning site)-based filamentous phage1, 32, 33. Phage produced in this manner display 3–5 copies of the specific peptide32. The fUSE5-MCS-based filamentous phage display vector is then genetically modified to generate the corresponding targeted AAVP vector, by inserting a recombinant AAV (carrying the desirable promoter/transgene cassette) into an intergenomic region of the phage genome. This strategy also serves to construct non-targeted control vectors (either displaying no peptides or displaying mutant/scrambled versions of the peptide). Targeted and control AAVP particles are amplified, isolated and purified by adapting the protocols used for phage30, 32. AAVP particles are then resuspended in phosphate-buffered saline (PBS; pH 7.4) and recentrifuged to remove residual bacterial debris. Next, AAVP particles in suspension are sterile-filtered through 0.45-mum pores, then titrated by infection of host bacteria for colony counting on Luria–Bertani (LB) agar plates under a double antibiotic selection and expressed as bacterial transducing units (TU). Transduction of mammalian cells in culture is performed by incubation with the targeted AAVP for 4 h in serum-free medium with a ratio of at least 106 TU per cell. Transgene expression begins 48–72 h later and reaches a maximum level by 1 week. Typically, non-targeted AAVP vectors or AAVP displaying mutated and/or scrambled versions of the targeting peptide serve as negative controls for the ligand-directed (i.e., targeting) experiments; either a corresponding version of a targeted phage vector "AAV-less" or a targeted AAVP containing an "ITR-less" or mutant ITR serves as a suitable control for post-internalization (i.e., integration, concatemerization) experiments.

Under these conditions, approx10–20% of cells are transduced in culture. Specificity can be demonstrated by blocking the interaction of the targeted AAVP by preincubating the target cells with the corresponding synthetic peptide. In our prototype RGD-4C AAVP, transduction inhibition was dose-dependent, greater than 99% inhibition was observed and incubation with nonspecific negative control peptides had no detectable effects1. This data set showcases an example of targeted AAVP transduction of mammalian cells expressing a specific receptor and mediated by an established ligand–receptor system. Transduction of a target tissue in vivo will vary among ligand–receptors but one can attempt to use increasing doses of targeted AAVP, starting at 1010 TU per mouse, administered intravenously (tail vein). Transgene expression should be monitored and its detection can be expected starting from day 3 after delivery. Depending on the specific reporter used, one can determine gene expression initiation in the target and maximal expression timing. Evidently, different organs will require different ligand peptides and particular conditions should be anticipated for optimal temporal and spatial transduction. In an unpublished work, we have shown systemic targeted tissue-specific transduction of the lung tissue in wild-type mice via a homing peptide34 that targets membrane dipeptidase in the lung endothelium35; we have also tested a tumor-homing peptide25 that targets MMP-2 and MMP-9 to transduce human tumor xenografts with phage-based vectors. In addition to the suicide gene therapy strategy previously reported1, other targeted AAVP including applications for delivery of therapeutic genes (unpublished observations) will follow.

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Materials

Reagents

  • Plasmids:fUSE51, 32, 33, 36 phage plasmid, accession number AF218364fd-tet1, 32, 33, 36 phage plasmid, accession number AF217317fMCS1, 36 phage plasmid, accession number AF218733fUSE5-MCS1 phage plasmidpAAV-GFP plasmid (Stratagene)pMOD-Luc-Sh (Luciferase reporter; InvivoGen)pCMVbeta (beta-galactosidase reporter; Clontech)
  • Bacterial strains:XL1-Blue MR supercompetent cells (Stratagene) Escherichia coli (MC1061 and k91Kan) (see ref. 36)
  • LB–tetracycline plates, LB–tetracycline–kanamycin plates and media
  • Terrific broth (TB) medium
  • SOC medium (Invitrogen)
  • Polyethylene glycol (PEG)/NaCl
  • QIAquick Nucleotide removal kit (Qiagen)
  • Essential restriction enzymes: BglI, BglII, HindIII, PstI, PvuII, SacI, SfiI and XhoI. Other restriction enzymes will be required depending on the specific sequences of the construct
  • Phosphatase alkaline (Roche)
  • T4 DNA ligase with ligase buffer (Invitrogen)
  • Rapid DNA ligation kit (Roche)
  • Agarose, electrophoresis (IscBioExpress)
  • E-Gel 0.8% agarose (Invitrogen)
  • QIAquick Gel extraction kit (Qiagen)
  • Phosphorylated DNA linkers (NEB)
  • Taq-DNA polymerase with supplied buffer (Promega)
  • 100 mM dNTPs (Fisher)
  • Oligonucleotides (Sigma-Genosys) (see Table 1)
  • DMSO
    Caution DMSO is readily absorbed through the skin. When handling DMSO, wear appropriate gloves, safety glasses and use a pipeting aid under a safety chemical hood.
  • Transfection reagent: Fugene (Roche)
  • Human embryonic kidney cells (HEK293; ATCC)
  • HEK293 cells are maintained in Dulbecco's modified Eagle's medium (Gibco), supplemented with 10% fetal bovine serum (Gibco), L-glutamine and penicillin/streptomycin
  • D-Luciferin potassium salt (InvivoGen)
  • Immunocompetent and immunodeficient mice (Harlan–Sprague–Dawley)
    Critical Mice must be used according to the national and institutional guidelines concerning use of animals.

Equipment

  • Fluorescent microscope (Olympus or equivalent).
  • Fluorescence-activated cell sorting (FACS) to analyze and sort the cells by using a BD FACS Vantage (Becton-Dickinson)
  • 0.22 mum filter units (Corning)
  • 0.45 mum filter units (Corning)
  • 24-well tissue culture plates
  • Tissue culture incubator at 5% CO2 for HEK293 cells and other cell lines
  • Shaker at 37 °C
  • Centrifuge Sorvall SA-600 and SLA-3000 rotors
  • Cell electroporator (Life Technologies)
  • In vivo Bioluminescence Imaging (BLI) System 200 (IVIS200; Xenogen)
  • PCR machine (Eppendorf)
  • DNA electrophoresis equipment (Bio-Rad)

Reagent setup

  • PEG/NaCl solution stock 500 g of PEG and 584.5 g of NaCl in 2,380 ml H2O (double-distilled) and store at 4 °C for up to 1 year.
  • TB medium 9.6 g of tryptone, 19.2 g of yeast extract and 3.2 ml of glycerol in 1 liter of H2O. Autoclave, add 100 ml of TB supplements and 50 mg of kanamycin
  • TB supplements To prepare 1 liter of TB supplements, add 23.1 g of KH2PO4 and 125.4 g of K2HPO4 in 1 liter of H2O (double distilled). Filter through 0.22 mum filter unit and store at 4 °C.

Equipment setup

  • Whole-body BLI of luciferase Mice are first anesthetized inside a clear plexiglass box by using an anesthetic gas admixture (2% isofluorane, 98% oxygen) and are then transferred to nose cones attached to the manifold in the imaging chamber. The imaging time is 5 min per side (dorsal/ventral), depending on the experiment. Imaging parameters are as follows: image acquisition time, 1 min; binning, 2; no filter; f/stop (aperture size), 1. Regions of interest can be defined manually over the tumors or target tissues for measuring signal intensities, expressed as photons s- 1 cm- 2sr- 1.

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Procedure

Overview

  1. Insertion of the targeted peptide into the pIII coat protein of fUSE5-MCS phageGenerate phage clones displaying targeting peptides by cloning the corresponding oligonucleotide sequences flanked by BglI restrictions sites into the SfiI site of the gene for the pIII coat protein of fUSE5-MCS. The fUSE5-MCS plasmid can also be generated by replacing the 5.4 kb BamHI–SacII fragment of the fUSE5 with the 4.1 kb BamHI–SacII fragment from the fMCS plasmid that contains an MCS.
  2. Design and convert the synthetic oligonucleotide templates flanked by BglI restriction sites (500 ng) to double-stranded DNA by PCR amplification. We use the following 57 bp oligonucleotide as a template: 5'-CACTCG GCCGACGGGGCTXXXXXXXXXXXXXXXXXXXXXGGGGCCGCTGGG GC CGAA-3'. This template is flanked by the BglI restriction sites (GCCNNNNNGGC: underlined). The bold nucleotides in the above template sequence indicate the annealing of the sense and antisense primers, respectively. The nucleotide sequence encoding the displayed peptide is marked "X".
  3. Finally, we use primer set A (see Table 1) and 2.5 U of Taq DNA polymerase (Promega) in 20 mul. Use the following setup: 94 °C for 2 min, followed by 35 cycles at 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, followed by 72 °C for 5 min, held at 4 °C and stored at - 20 °C until ready for Step 3.
    Critical step For effective PCR, it is recommended to add DMSO (2% final) to weaken hydrogen bonding and prevent formation of hairpin structures.
  4. Purify and elute the double-stranded DNA sequences containing BglI restriction sites by using a QIAquick nucleotide removal kit.
  5. Digest oligonucleotides with BglI restriction enzyme for 4 h at 37 °C, then inactivate BglI activity by incubation at 65 °C for 20 min. Digestion with BglI restriction enzyme generates the following sticky ends: 5'- GGGCTXXXXXXXXXXXXXXXXXXXXXGGGGCCGCTG-3' 3'-TGCCCCGAXXXXXXXXXXXXXXXXXXXXXCCCCGGC-5'.
  6. Digest the fUSE5-MCS plasmid with SfiI restriction enzyme for 1–2 h at 50 °C. Run the approx9.5-kb linearized fUSE5-MCS plasmid on an agarose gel or E-Gel 0.8% agarose to confirm digestion. The fUSE 5 backbone vector contains two SfiI restriction sites36 (GGCCNNNN/NGGCC) in the pIII gene. After digestion, the SfiI restriction enzyme will generate non-identical, non-complementary three bases 3'-overhanging ends36. This will allow directional cloning after removal of the stuffer that lies between these sites in the phage vector. A schematic representation of the final vector sequences is shown below.

    fUSE5      Stuffer      fUSE5

    5'-TCGGCCGACG  TGGCCTGGCCTCTG   GGGCCGAA-3'

    3'-AGCCGGC   TGCACCGGACCGGA  GACCCCGGCTT-5'

    Troubleshooting
  7. Ligate the SfiI-digested fUSE5-MCS vector plasmid with the BglI-digested oligonucleotides by using the Rapid Ligation Kit for 5–10 min at room temperature (25 °C, Fig. 1). It is recommended not to inactivate SfiI and not to dephosphorylate the linearized fUSE5-MCS plasmid, as this markedly reduces the efficiency of ligation. Ligate immediately after digestion. Also, set up a reaction with the fUSE5-MCS plasmid alone and no insert to estimate the bacterial transformation due to autoligation of the fUSE5-MCS plasmid and/or the presence of non-digested fUSE5-MCS plasmid.
    Figure 1: Targeted AAVP vectors.
    Figure 1 : Targeted AAVP vectors.

    (a) Cloning scheme for generation of targeted AAVP and control vectors. The most convenient procedure is to first clone the BglI-digested oligonucleotide sequence corresponding to the targeting peptide within the SfiI site of the gene for the pIII coat protein. Next, the PvuII-digested ITR-flanking transgene cassette (rAAV) is cloned into the PuvII site in the MCS of the targeted fUSE5-MCS plasmid or in the cohesive restriction sites after addition of the corresponding linkers. (b) Binding of the targeted AAVP particle to a specific cell-surface receptor in the target tissue and internalization after systemic administration. Alternative cloning approaches are discussed in the text.

    Full size image (63 KB)

  8. Use 2 mul of the ligation product to transform MC1061 or XL1-Blue MR bacteria according to the manufacturer's instructions. Incubate transformed bacteria on LB–tetracycline plates for 24 h at 37 °C.
  9. Verify the correct insertion and nucleotide sequence by PCR of the bacterial colonies generated. Pick single colonies (at least ten single colonies plus colonies from vector alone as controls) in 20 mul of medium. Use the primer set B (see Table 1) and 2 U of Taq DNA polymerase (Promega) in 20 mul. Use the following setup: 94 °C for 3 min, followed by 35 cycles at 94 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s.
    Critical step It is recommended to add 2% DMSO to weaken hydrogen bonding and prevent formation of hairpin structures.
  10. Run 2 mul of PCR product on a 2% agarose gel or E-Gel 0.8% agarose to verify the insertion. Include the product of an fUSE5-MCS without insert, which will be smaller in size on the gel.
  11. Insertion of recombinant (r) AAV into the MCS of the fUSE5-MCS plasmid displaying the targeting peptidePrepare the rAAV carrying the trangene of interest. Remove and replace GFP from the pAAV-GFP plasmid with the transgene of interest if applicable. GFP can also be replaced with a GFP variant for maximal fluorescence such as eGFP (enhanced GFP, catalog number 6084-1 from ClonTech). Thus, the transgene of interest must be flanked by restriction sites compatible with the MCS of the pAAV plasmid.
  12. Remove the transgene cassette flanked by the ITRs from the pAAV plasmid created in Step 10. Use the PvuII restriction enzyme that digests adjacent to the ITRs and gel-purify the released transgene-ITR-segment using the gel extraction kit (Fig. 1). Note that expression cassettes of interest should not have any PvuII site. Otherwise, alternative strategies should be used. Then, use an rAAV plasmid with a restriction map compatible with that of the expression cassette of interest. For instance, some AAV plasmids from Stratagene (or other commercial outfits) have convenient rare cutter restriction enzyme sites such as SbfI or Sse8387I adjacent to each PvuII site. Sometimes, these can be used for AAV vector genome insertion into the phage vector backbone if other genetic elements permit this utilization.
  13. The targeted fUSE5-MCS plasmid has a unique PvuII site in the MCS. Digest with PvuII to linearize and run on an agarose gel or on an E-Gel 0.8% agarose to confirm digestion.
  14. Dephosphorylate the targeted fUSE5-MCS plasmid vector by using phosphatase alkaline according to the manufacturer's instructions.
    Critical step It is recommended to dephosphorylate before ligation to reduce background as the fUSE5-MCS plasmid autoligates.
  15. Ligate the ITR-flanked transgene cassette into the PvuII-linearized fUSE5-MCS plasmid for 4 h at 23 °C or 24 h overnight at 16 °C using T4 DNA ligase (Fig. 1).Troubleshooting
  16. Digestion with PvuII generates blunt ends. Linkers containing recognition sequences of enzymes can be added to the PvuII-recovered ITR-flanked cassette to produce compatible cohesive ends.

    Linkers to BglII, HindIII, PstI, SacI and XhoI can be used. After determining which restriction site is most appropriate for the construction, add the corresponding restriction enzyme to digest the linkers flanking the ITR transgene cassette. Ligate into the compatible cohesive restriction sites (BglII, HindIII, PstI, SacI and XhoI) of the MCS of the fUSE5-MCS plasmid.

    Troubleshooting
  17. Use 2 mul of the ligation product to transform MC1061 or XL1-Blue MR bacteria according to the manufacturer's instructions. Plate bacteria on LB-tetracycline and grow overnight at 37 °C for 24 h.
  18. Pick ten colonies, grow overnight in 5 ml LB and purify the plasmid DNA by using a QIAprep miniprep kit protocol for low-copy plasmids. Identify the positive clones by enzymatic restriction digestions.
  19. Transgene expression can be analyzed at this stage from the AAVP plasmids generated to confirm that the ITR-flanked transgene cassette is functional when inserted into the phage genome (in the context of AAVP). Therefore, transfect human embryonic kidney (HEK293) cells with the AAVP DNA plasmids by using the Fugene transfection reagent (Roche). Different experimental approaches, such as western blots or immunostainings, can be used to detect transgene expression depending on the gene of interest. For reporter GFP-, beta-galactosidase- or luciferase-containing cassettes, expression can be analyzed as described in detail in Step 31.
  20. Production, purification and titration of AAVP particlesAAVP particles can be amplified either by growing MC1061 colonies (option A) or by infecting and growing K91Kan cells (option B). Following amplification, AAVP particles are isolated and purified from the culture supernatant following a modified phage purification protocol30, 32 by using the steps given below. It is important to note that option A is faster because E. coli MC1061 can be used to generate AAVP constructs. This strategy offers the advantage to directly grow positive clones to produce phage particles and to skip the infection step needed for option B. However, E. coli K91Kan (option B) are pilus-positive F+ bacteria and can therefore be infected by the newly produced phage particles during the overnight growth; this phenomenon will result in higher titers compared to MC1061 (option A), which are F- bacteria.
    1. Growth of MC1061 colonies
      1. Amplify AAVP particles directly by growing the MC1061 colonies in LB plus tetracycline overnight at 37 °C.
    2. Infection and growth of K91Kan cells
      1. Incubate 1 ml of growing K91Kan E. coli bacteria with 1010 TU of AAVP for 1 h at room temperature in TB medium. Then, grow in 500 ml at 37 °C in the presence of tetracycline and kanamycin.
  21. After overnight growth, centrifuge cultures at 6,000g for 20 min at 4 °C and collect the supernatant. Repeat centrifugation to remove residual bacterial debris.
  22. Add PEG/NaCl (15% of the supernatant volume) solution to the supernatant to precipitate the AAVP phage particles. Incubate for 2 h on ice.
  23. Centrifuge suspension at 10,000g for 30 min at 4 °C. A white pellet should be obtained. Discard the supernatant and centrifuge again for 10 min. Carefully decant the supernatant.
  24. Resuspend the AAVP pellet in 10 ml of sterile PBS with agitation at 37°C for 30 min.
  25. Repeat the precipitation with PEG/NaCl (15% of the supernatant volume) solution for 30 min on ice. Then, centrifuge at 14,000g for 30 min at 4 °C and resuspend in an adequate volume of PBS depending on the size of the pellet.
  26. Transfer the solution to an Eppendorf tube, centrifuge at approx13,000g for 10 min at room temperature, transfer to a new tube and recentrifuge to remove residual bacteria and debris.
  27. Filter the resulting supernatant containing the AAVP particles in suspension through a 0.45-mum filter. Then, titrate by infection of K91Kan bacteria for 20 min at room temperature and plaque assay according to the standard protocols30, 32. The AAVP titers are expressed as bacterial TUs per mul. Also, one must keep in mind that bacterial TU and multiplicity of infection are entirely different entities and should not be confused with one another.
    Critical step E. coli K91Kan bacteria infection for more than 20 min might generate higher titers due to the newly produced AAVP particles. When comparing two different construct versions of AAVP for transduction efficiency, it is recommended to titrate the preparations side by side to ensure that same doses are compared. Indeed, the titers may vary from one AAVP version to another, depending on the ligand used, the size of the transgene of interest and size of the AAV mammalian cassette. These parameters can affect the coating of the virus in host bacteria during production. Viability of bacteria also plays a role, and it is recommended to infect a log-phase growing bacteria with an optical density ranging between 1.6 and 2.0 at a wavelength of 600 nm (OD600).Pause Point AAVP titers are relatively stable and the preparations can be stored at 4 °C for long periods of time (several months) without any significant decrease in the titers. For longer storage times, one should check the titer of the preparation before use.Troubleshooting
  28. Transduction of mammalian cells in culture by targeted AAVP vector and specific inhibition by using synthetic peptides—day 1: cell seedingTo transduce mammalian cells by targeted AAVP, it is recommended to incubate at least 106 AAVP TUs per cell. Transduction can be performed in any tissue culture dish or flask; however, as an initial experiment to confirm transduction of mammalian cells, it is recommended to work with a 24-well plate to avoid the use of large amounts of AAVP. Therefore, seed 4 times 104 cells in each well of the 24-well plate in a final volume of 0.5 ml of complete medium. Incubate overnight at 37 °C.
    Critical step Factors such as concentration of cells in the edge of the well can affect the efficiency of transduction. Therefore, after seeding, observe the cells under the microscope to determine whether the cells are concentrated in the edge. Then to solve this problem, gently tilt or rock the plates to move the cells from the edge, until they are homogeneously distributed.
  29. Analyze the efficiency of transduction of the cell line in vitro for each targeted AAVP. This can be carried out using a GFP reporter gene, as well as other reporter genes (e.g., luciferase, beta-galactosidase). The thymidine kinase of herpes simplex virus type I (HSV1-tk) can also be used as a reporter gene37, 38. Note that transduction with AAVP particles is an approach different from transfection using a naked plasmid containing a reporter cassette. Transfection with naked plasmid DNA is a general, nonspecific strategy aimed at introducing naked DNA into cells, with transgene expression starting after a few hours. However, transduction with a targeted AAVP virus is designed to transduce a specific cell line expressing a specific receptor. It is a process that occurs via a ligand-directed mechanism by binding of the targeting ligand displayed on AAVP virus to its specific receptor on the cell surface. Escape of AAVP from endosomes, the uncoating of the virus and conversion of single- to double-stranded DNA (transcriptionally active) do take time. This causes a delayed initiation of transgene expression from AAVP, starting at day 3 and peaking 7–10 days later. Thus, plasmid transfection and transduction with AAVP are two distinct strategies with different experimental aims. The choice of the cell line will depend on the ligand used and expression of the corresponding receptor on the cell surface. Thus, efficiency of transduction will vary from one cell line to another. For example, HEK293 cells or KS1767 Kaposi sarcoma cells highly express alphav-integrin receptors1, 27 and are suitable cell lines to use for transduction by AAVP displaying the alphav integrin-binding peptides.
  30. Day 2: targeted AAVP infectionWhen cells are 50–60% confluent, they are ready for infection. Incubate the cells with the AAVP vectors at 106 TU per cell in a 0.2 ml total volume of tissue culture medium without serum at 37 °C for 4 h. Manually tilt after 30 min, then continue to gently tilt every 15 min, during incubation.
    Critical step Incubation in the presence of serum markedly decreases the transduction efficiency of RGD-4C-targeted AAVP vector. Serum-mediated decrease of transduction efficiency has already been reported for other gene delivery vectors such as adenoviral vectors or liposomes and for plasmid transfection procedures such as lipofection39, 40. Moreover, groups that used RGD-targeted adenoviral vectors reported infection of cells in vitro in the absence of serum41. We have not tested AAVP displaying other ligands.
  31. After 4 h of transduction, add 0.3 ml of 10% serum-supplemented medium to each well to have a final volume of 0.5 ml. Incubate the cells at 37 °C and renew the medium every 2 days.
  32. Analyze the cells for transgene expression at 48–72 h after transduction. A number of options are available for this, depending on which reporter gene system is used: for example, GFP (option A), beta-galactosidase (option B) or luciferase (option C). Maximum levels of gene expression are usually reached by 7–10 days (Fig. 2). At this stage, as we previously reported1, the transduced cells can be used to perform rescue experiments to verify the ITR structure integrity and functional ability to rescue rAAV particles from transduced cells (see Box 1).
    Figure 2: In vitro mammalian cell transduction with targeted AAVP particles.
    Figure 2 : In vitro mammalian cell transduction with targeted AAVP particles.

    (a) MDA-MB-435 breast tumor cells27, 29 were incubated with either targeted RGD-4C AAVP-betagal displaying the alphav-integrin binding cyclic CDCRGDCFC peptide (termed RGD-4C)31 or non-targeted AAVP-betagal as a control. betagal expression was evaluated by using an anti-betagal antibody (Sigma). The left panel shows only Texas red-positive cells; the right panel shows the total number of cells in identical fields (green fluorescence). (b) Quantitative analysis of cell transduction by targeted or control AAVP. AAVP vectors were incubated with tumor cells as described above. An anti-betagal antibody was used for staining; gene expression was detected by immunofluorescence and results are expressed as percentage of betagal-positive cells. In each case, s.e.m. was calculated after counting ten fields under the microscope in three independent experiments.

    Full size image (67 KB)

    1. GFP detection
      1. For GFP detection, analyze the cells by FACS or count cells by using a standard fluorescence microscope.
    2. Detection of beta-galactosidase expression
      1. Detection of beta-galactosidase expression can be performed by X-gal staining42. beta-Gal activity in cell lysates can also be detected by the Galacto-Star chemiluminescent reporter gene system (Tropix).
    3. Detection of Luciferase expression
      1. Detect luciferase expression in vitro by incubating the cells with the D-luciferin substrate. Aspirate the medium from the cultured cells and add D-luciferin (150 mug ml- 1 final). Monitor expression with a luminometer, a scintillation counter or a BLI system.
  33. After maximum gene expression is obtained, split the transduced cells.
    Critical step Splitting the cells before maximum gene expression is achieved and transferring them into a larger tissue culture dish will stimulate cells to divide and cause dilution of transduced cells within a large background population of non-transduced cells. To circumvent this problem, use a specific selection genetic marker to allow selection of only transduced cells.
  34. To determine whether the AAVP particle is transfecting via the correct receptor, carry out a peptide inhibition experiment. For peptide inhibition experiments, seed the cells at 4 times 104 cells per well as in Step 27 and incubate with either 1 mg ml- 1 of the specific targeting or control peptides for 30 min at 37 °C. After 30 min, wash the cells and add 106 TU of AAVP per cell for 4 h in serum-free medium as in Step 29. After incubation, add medium supplemented with 10% FCS. Negative control peptide (scrambled and/or mutant sequence) versions of the targeting peptide can be included. Cells are analyzed for transgene expression similar to conventionally transduced mammalian cells (Step 31).
  35. In vivo transduction of target tissues by AAVP vector after systemic administrationAnalysis of transgene expression in vivo was optimized for AAVP targeting systemic gene delivery to murine and human tumors established in mice, but can be applied to target normal tissues or perhaps other pathologically affected tissues. It is important to determine the suitable targeted AAVP dose to be administered to the animal to achieve an efficient and specific transgene expression within the target tissue. Indeed, each target tissue may require a different dose to achieve transduction and transgene expression in vivo, depending on the level of expression of the targeted receptor, the ligand used and promoter activity in the tissue. Thus, it is recommended to test different doses and analyze transgene expression over a time course to determine initiation and maximal expression (see Boxes 2 and 3).Troubleshooting
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Troubleshooting

Troubleshooting advice can be found in Table 2.


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Anticipated results

The protocols described here should result in successful cloning and application of AAVP-based vectors. AAVP preparations of 1–5 times 1010 TU mul- 1 are routinely obtained and are consistent with the titers generated with the parental targeted phage. This shows that insertion of a mammalian transgene cassette in the bacteriophage genome does not affect vector production in bacteria. Transgene expression in cells in culture starts at 48–72 h after incubation with the targeted AAVP vector and reaches maximum levels by days 7–10 (Fig. 2). In vivo, transgene expression is detectable in target tissues at day 3 after intravenous delivery of targeted AAVP vectors, then it increases gradually until days 7–10 (Fig. 3). We previously followed transgene expression until day 10 post-AAVP administration1; if required, further reporter detection may be accomplished. For long-term detection of transgene expression, real-time RT-PCR may be used. In recent work (unpublished observation), we have followed transgene expression using BLI of luciferase in a tumor model for 5–6 weeks. Transgene expression started at day 3 after vector delivery and was still detectable at adequate levels upon experiment termination due to tumor burden.

Figure 3: In vivo transduction and molecular-genetic imaging of tumors in mice after systemic delivery of targeted AAVP.
Figure 3 : In vivo transduction and molecular-genetic imaging of tumors in mice after systemic delivery of targeted AAVP.

Targeted RGD-4C AAVP vectors or controls are intravenously administered to tumor-bearing mice. (a) Immunofluoresence analysis of GFP expression in tumors at 1 week after intravenous administration of RGD-4C AAVP-GFP or control non-targeted AAVP-GFP. (b) BLI of luciferase expression in living mice at 1 week after intravenous delivery of RGD-4C AAVP-Luc carrying the gene for firefly luciferase or AAVP control (non-targeted AAVP) or AAVP control (displaying CDCGFDCRC, a scrambled RGD-4C version).

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Acknowledgments

We thank Marco Arap, David Bier, Carlotta Cavazos, Carol M. Johnston, Erkki Koivunen, Darwin Lee, Frank C. Marini, Bradley H. Restel, Karen Schmidt, Yan Sun and Claudia Zompetta for advice and assistance. This work was funded by grants from the NIH (including the SPORE) and DOD (including the IMPACT) and by awards from the Gillson-Longenbaugh, the Keck Foundation and the Prostate Cancer Foundation (to R.P. and W.A.). A.H. received a Léon Fredericq award.

Competing interests statement: 

The authors declare no competing financial interests.

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  1. Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.
  2. Department of Hematology and Oncology and Institute for Molecular Medicine and Cell Research, University of Freiburg Medical Center, Hugstetter Strasse 55, D-79106 Freiburg, Germany.
  3. Department of Experimental Diagnostic Imaging, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Correspondence to: Renata Pasqualini1 e-mail: rpasqual@mdanderson.org

Correspondence to: Wadih Arap1 e-mail: warap@mdanderson.org

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