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Modification and Labeling of AAV Vector Particles

  • Hildegard BüningEmail author
  • Chelsea M. Bolyard
  • Michael Hallek
  • Jeffrey S. Bartlett
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 807)

Abstract

Adeno-associated virus (AAV) has become a versatile vector platform. In recent years, powerful ­techniques for the generation of tropism-modified vectors (rAAV-targeting vectors) and for investigation of virus–cell interaction were developed. The following chapter describes strategies for insertion of peptide ligands into the viral capsid and the subsequent characterization of capsid mutants, for producing mosaic capsids and for labeling the viral capsid chemically or genetically.

Key words

Labeling Capsid particles Ligands Chemical modification Mosaic 

1 Introduction

Adeno-associated virus (AAV) has a number of attributes that make it an attractive platform for the development of viral gene transfer vectors Traditionally, serotype 2 vectors (rAAV-2) have been used, which can be produced to high titer and purity. These vectors are very stable, mediate long-term transgene expression in vivo and elicit only mild immune responses compared to other viral vectors. However, many clinically relevant cell types are refractory to rAAV-2 transduction. For this reason, serotypes others than rAAV-2 have been engineered as vectors and their potential have more recently been evaluated in both preclinical and clinical studies. However, one shortcoming of natural occurring serotypes, including rAAV-2 is a lack of specificity. Thus, after local application, tissues in the vicinity of the target can be transduced inadvertently. On the other hand, systemically applied AAV vectors accumulate in liver and spleen and are thus not available for transduction of other organs. One strategy to overcome such limitations is to generate receptor/tissue-specific vectors by genetic modification of the viral capsid (targeting). Besides an improved selectivity of cell transduction, lower vector doses are needed, thereby contributing to vector safety. Targeting may also be employed to generate vectors able to transduce cell types that are refractory to infection with natural occurring AAVs.

Most often, targeting is accomplished using site-directed mutagenesis to introduce DNA oligonucleotides encoding small exogenous peptides with receptor-binding specificity into the AAV capsid (cap) open reading frame (ORF). When inserted into appropriate sites, these epitopes are displayed on the capsid surface and can mediate novel cellular interactions. Sites within the capsid protein(s) that accept peptide insertions have been defined by a number of laboratories and include the N-terminal region of VP1 and of VP2, and the loop III and IV regions of VP3 (see Table 1). Importantly, AAV packaging constructs have been generated containing restriction sites in many of these locations (1, 2, 3). This greatly simplifies the generation of modified packaging constructs, since double-stranded DNA oligonucleotides can simply be inserted into these pre-engineered sites. Alternatively, PCR primers need to be designed, and the exogenous DNA sequences inserted by PCR-based site-directed mutagenesis. Both methods are described here. Generally, most sites can tolerate insertions of up to about 15 amino acids while maintaining wild-type levels of vector assembly and DNA packaging. See Note 1 for suggestions on selecting appropriate insertion sites.
Table 1

List of sites that tolerate peptide insertion within the AAV capsid

Serotype

Site (between indicated residues)

Location

Used for targeting

References

AAV1

D590_P591

VP1, VP2, VP3

 

(1)

AAV2

P34_A35

G115_R116

T138_A139

T138_A139

A139_P140

K161_A162

S261_S262

N381_N382

R447_T448

T448_N449

G453_T454

R459_L460

F534_P535

T573_E574

Q584_R585

N587_R588

R588_Q589

A591_T592

A664_K665

VP1

VP1

VP1, VP2

VP2

VP1, VP2

VP1, VP2

VP1, VP2

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

VP1, VP2, VP3

x

xa

x

x

x

x

x

(25)

(3)

(25)

(26)

(3)

(3)

(22)

(22)

(22)

(27)

(28)

(3)

(22)

(22)

(3)

(22)

(5)

(25)

(25)

AAV3

S586_S587

VP1, VP2, VP3

 

(1)

AAV4

S584_N585

VP1, VP2, VP3

 

(1)

AAV5

S575_S576

VP1, VP2, VP3

 

(1)

AAV6

D590_P591

VP1, VP2, VP3

 

(1)

aInsertion within cap ORF, i.e., within VP1 and at the N′-terminus of VP2

In most instances, small flexible linker sequences flanking the inserted epitopes have proven essential in maintaining titer and efficient display of the targeting epitope on the surface of AAV particles. Although there is not one linker sequence that is best for displaying all peptides in all sites, successful epitope display has been demonstrated using: TG-peptide insert-GLS, TG-peptide insert-ALS, TG-peptide insert-LLA, AS-peptide insert-A, and AAA-peptide insert-AA (1, 2, 3, 4, 5, 6) (see Note 2).

Another approach used to generate AAV vectors with novel characteristics is to mix AAV helper plasmids encoding different serotype capsids during vector preparation. This tactic generates mosaic AAV particles. These mosaic particles usually have properties similar to the serotypes from which they were generated, but in some instances these particles can display novel and unexpected behaviors that may be beneficial for some gene transfer applications (7, 8).

The labeling of AAV capsids has been useful for investigating the process of AAV infection. Green fluorescent protein (GFP) is an attractive label because of its high labeling specificity and ease of use. GFP has been fused to the amino-terminus of AAV-2 VP2, and particles incorporating this fusion have been proven extremely useful (9, 10). However, the low valency of VP2 display significantly lessens the signal intensity obtained with this approach. Other protein-based tags that either react covalently or form high affinity complexes with small-molecule probes have yet to be introduced into AAV particles (11, 12). Alternative labeling approaches rely on direct fluorophore conjugation, and although somewhat inefficient have been widely applied and have yielded valuable information regarding intracellular AAV particle trafficking (13, 14, 15, 16, 17) and AAV distribution within tissues (18). Both approaches are described here.

2 Materials

2.1 Genetic Modification of AAV Capsids via Peptide Insertion into Surface-Exposed Loops of AAV Capsids

  1. 1.
    QuikChange® Site-Directed Mutagenesis kit (Agilent Technologies, Stratagene Products), or comparable kit.
    1. (a)

      PfuTurbo DNA Polymerase (2.5 U/μl).

       
    2. (b)

      10× reaction buffer: 100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris–HCl (pH 8.8), 20 mM MgSO4, 1% Triton®-X 100, 1 mg/ml nuclease-free bovine serum albumin (BSA).

       
    3. (c)

      DpnI restriction enzyme (10 U/μl).

       
    4. (d)

      Oligonucleotide control primers, each a 34-mer (100 ng/μl) – see manual for sequence.

       
    5. (e)

      pWhitescript 4.5-kb control plasmid (5 ng/μl).

       
    6. (f)

      dNTP mix.

       
    7. (g)

      XL1-Blue supercompetent cells.

       
    8. (h)

      pUC18 control plasmid (0.1 ng/μl in TE buffer).

       
     
  2. 2.

    DNA oligonucleotides for PCR-based mutagenesis (see Note 3), or complementary DNA oligonucleotides for epitope cloning.

     
  3. 3.

    AAV helper plasmid: pACG2 (19), pAAV-RC (Stratagene, Agilent Technologies) or similar (see Note 4).

     
  4. 4.

    14-ml BD Falcon polypropylene round-bottom tubes (BD Biosciences).

     
  5. 5.

    NZY+ Broth. Prepared by combining 10 g NZ amine (Sigma Aldrich), 5 g yeast extract, and 5 g NaCl in a total volume of 1 L deionized dH2O. Adjust the pH to 7.0 with NaOH, autoclave, and then add the following filter-sterilized supplements prior to use: 12.5 ml 1 M MgCl2, 12.5 ml MgSO4, and 10 ml 2 M glucose.

     
  6. 6.

    LB agar plates containing antibiotic selection (100 μg/ml ampicillin). LB plates are prepared by combining 10 g NaCl, 10 g tryptone, 5 g yeast extract, and 20 g agar (Fisher Bioreagents) in 1 L deionized dH2O. Adjust pH to 7.0 with 5 N NaOH, autoclave to sterilize, cool to 55°C, add 10 ml filter-sterilized ampicillin (10 mg/ml), and pour into Petri dishes.

     
  7. 7.

    LB media containing ampicillin (100 μg/ml). LB media is prepared by combining 10 g NaCl, 10 g tryptone, and 5 g yeast extract in deionized dH2O to a final volume of 1 L. Adjust pH to 7.0 with 5 N NaOH, autoclave, cool to 55°C, and add 10 ml of filter-sterilized ampicillin (10 mg/ml).

     
  8. 8.

    QIAprep Spin MiniPrep kit (QIAGEN, USA), or comparable mini-prep kit.

     
  9. 9.

    Equipment and buffer solutions for DNA agarose gel electrophoresis.

     

2.2 Generation of Mosaic AAV Vector Particles

  1. 1.

    Low passage (<30) human embryonic kidney (HEK) 293 cells (ATCC® number CRL-1573) grown in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS) or cosmic calf serum (Hyclone, Thermo Scientific), and 1% penicillin/streptomycin (10,000 U/ml; Hyclone, Thermo Scientific).

     
  2. 2.

    Two different AAV helper plasmids encoding different AAV serotype capsids (see Note 4).

     
  3. 3.

    AAV vector plasmid DNA containing the transgene of interest (e.g., pAAV-hrGFP, Stratagene, Agilent Technologies).

     
  4. 4.

    Adenovirus helper plasmid DNA, such as pXX6-80 (20), pDG (21), or pHelper vector (Stratagene, Agilent Technologies).

     
  5. 5.
    Calcium phosphate (CaPO4) transfection system (Invitrogen), or
    1. (a)

      tissue culture sterile water.

       
    2. (b)

      2× Hepes-buffered saline (HBS), pH 7.12: 50 mM HEPES, 280 mM NaCl, 1.5 mM Na2HPO4 (see Note 5).

       
    3. (c)

      2 M CaCl2.

       
     
  6. 6.

    100 mM sodium citrate (NaCitrate).

     
  7. 7.

    Dry ice bath: dry ice and 95% ethanol.

     
  8. 8.

    Benzonase (Sigma).

     
  9. 9.
    Iodixanol solution for gradient purification:
    1. (a)

      10× TMSC: 1 M NaCitrate, 100 mM Tris, 25 mM MgCl2, pH 8.0–8.3.

       
    2. (b)

      15% Iodixanol: 50 ml 60% iodixanol (OptiPrep Density Gradient Medium, Sigma-Aldrich), 20 ml 10× TMSC, 130 ml dH2O; filter sterilize; store at 4°C and protect from light.

       
    3. (c)

      25% Iodixanol: 41.7 ml 60% iodixanol, 10 ml 10× TMSC, 49.3 ml dH2O, neutral red (see Note 6); filter sterilize; store at 4°C and protect from light.

       
    4. (d)

      40% Iodixanol: 66.7 ml 60% iodixanol, 10 ml 10× TMSC, 23.3 ml dH2O; filter sterilize; store at 4°C and protect from light.

       
    5. (e)

      60% Iodixanol: 200 ml 60% iodixanol, 5.822 g NaCitrate, 0.888 g Tris–HCl, 0.570 g Tris Base, 0.102 g MgCl2, neutral red; filter sterilize; store at 4°C and protect from light.

       
     
  10. 10.

    Opti-seal™ 30 ml centrifuge tube and protective caps (Beckman-Coulter).

     
  11. 11.

    Ultracentrifuge and rotor (e.g., Beckman Optima ™ LE-80K Ultracentrifuge with Beckman Type 70.1 Ti Rotor).

     

2.3 Characterization of Modified Capsids

2.3.1 Assess Genomic Particle Titer of Vector Preparations by Real-Time qPCR

  1. 1.

    DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), or comparable kit.

     
  2. 2.

    LightCycler FastStart DNA Master Plus SYBR I kit (Roche Diagnostics GmbH, Mannheim, Germany), or comparable kit.

     
  3. 3.

    AAV vector plasmid encoding for the vector genome that was packaged.

     
  4. 4.
    Oligonucleotide Real-Time PCR primers:
    1. (a)
      Example of primer pair for the detection of eGFP:
      • eGFP-forward: 5′-CACAACGTCTATATCATGGC-3′

      • eGFP-backward: 5′-TGTGATCGCGCTTCTC-3′

       
    2. (b)
      Example of primer pair for the detection of β-galactosidase:
      • β-Galactosidase-forward: 5′-ATCCTCTGCATGGTCAGGTC-3′

      • β-Galactosidase-backward: 5′-CTGGGCCTGATTCATTCC-3′

       
     
  5. 5.

    PBS (Invitrogen, Karlsruhe, Germany).

     

2.3.2 Assess Capsid Titer of AAV Preparations by ELISA

  1. 1.

    AAV capsid standard.

     
  2. 2.

    Nunc-Immuno-Plate (Thermo Fisher Scientific, Rochester, USA).

     
  3. 3.

    PBS (Invitrogen, Karlsruhe, Germany).

     
  4. 4.

    Washing buffer: 0.05% Tween20 in PBS.

     
  5. 5.

    Blocking buffer: 3% BSA, 5% sucrose, and 0.05% Tween20 in PBS.

     
  6. 6.

    Capsid-specific antibody (see Note 7).

     
  7. 7.

    Biotin-conjugated secondary antibody, for example biotin-conjugated rabbit anti-mouse antibody (Dianova, Hamburg, Germany).

     
  8. 8.

    Horseradish peroxidase conjugated streptavidin (Dianova, Hamburg, Germany).

     
  9. 9.

    Start Solution: 1 mg tetramethylbenzidine (TMB) in 10 ml 0.1 M NaOAc (pH 6.2) and 100 μl dimethyl sulfoxide (DMSO). Add 1 μl of 30% H2O2 before use.

     
  10. 10.

    1 M H2SO4.

     
  11. 11.

    Alternatively: AAV capsid ELISA (Progen, Heidelberg, Germany), or comparable kit.

     

2.3.3 Determine Transduction Efficiency of AAV Vectors by Flow Cytometry

  1. 1.

    Cell line or primary cells.

     
  2. 2.

    Cell culture multiwell plates.

     
  3. 3.

    Culture medium including supplements.

     
  4. 4.

    PBS (Invitrogen, Karlsruhe, Germany).

     
  5. 5.

    Optional: trypsin (0.05% with EDTA) to harvest adherent cells.

     
  6. 6.

    Optional: heparin (Braun Melsungen AG, Melsungen, Germany), or comparable product to assay for pseudo-­transduction by rAAV-2 preparations.

     

2.4 Tropism and Specificity of Modified AAV Vectors

2.4.1 Monitoring Cell Entry by Real-Time qPCR

  1. 1.

    Cell lines/primary cells expressing targeted receptor.

     
  2. 2.

    Control (nontarget) cell lines.

     
  3. 3.

    Cell culture multiwell plates.

     
  4. 4.

    Culture medium including supplements.

     
  5. 5.

    Trypsin (0.05% with EDTA).

     
  6. 6.

    PBS (Invitrogen, Karlsruhe, Germany).

     
  7. 7.

    DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany), or comparable kit.

     
  8. 8.

    LightCycler FastStart DNA Master Plus SYBR I kit (Roche Diagnostics GmbH, Mannheim, Germany), or comparable kit.

     
  9. 9.

    AAV vector plasmid encoding for the vector genome that was packaged.

     
  10. 10.

    Oligonucleotide Real-Time PCR primer pair (vector genome).

     
  11. 11.

    Oligonucleotide Real-Time PCR primer pair (reference gene).

     
  12. 12.

    LightCycler Relative Quantification Software (Roche Diagnostics GmbH, Mannheim, Germany), or comparable program.

     

2.4.2 Determining Cell Transduction Efficiency by Flow Cytometry

  1. 1.

    Cell lines/primary cells expressing targeted receptor.

     
  2. 2.

    Control (nontarget) cell lines.

     
  3. 3.

    Cell culture multiwell plates.

     
  4. 4.

    Culture medium including supplements.

     
  5. 5.

    Optional: trypsin (0.05% with EDTA) to harvest adherent cells.

     
  6. 6.

    PBS (Invitrogen, Karlsruhe, Germany).

     
  7. 7.

    Competing and noncompeting peptides, proteins, or anti­receptor antibodies to assay for ligand-receptor interaction of modified vectors.

     
  8. 8.

    Heparin (Braun Melsungen AG, Melsungen, Germany), or comparable product to assay for HSPG/heparin binding ability of modified rAAV-2 vectors.

     

2.5 Chemical Labeling of the Capsid

  1. 1.

    Highly purified stock of AAV or rAAV vector (see Note 8).

     
  2. 2.
    The following reagents have been successfully used to fluorescently label AAV particles:
    1. (a)

      Cy-Dye labeling reagents (GE Health Care, CyDye FluoroLink reactive dye).

       
    2. (b)

      Alexa Fluor labeling reagents (Invitrogen, Alexa Fluor protein labeling kit).

       
    3. (c)

      DyLight labeling reagents (Pierce, Thermo Fisher Scientific, DyLight Fluor antibody labeling kit).

       
    4. (d)

      LI-COR labeling reagents (LI-COR, IRDye protein labeling kit).

       
     
  3. 3.
    Appropriate buffer specified by the labeling reagent:
    1. (a)

      CyDye FluorLink; 1.0 M sodium carbonate buffer, pH 9.3.

       
    2. (b)

      Alexa Fluor protein labeling kit; 0.5 M sodium bicarbonate buffer, pH 8.3.

       
    3. (c)

      DyLight Fluor antibody labeling kit; 50 mM sodium borate, pH 8.5.

       
    4. (d)

      IRDye protein labeling kit; 50 mM phosphate buffer, pH 8.5.

       
     
  4. 4.

    G-50 (Sephadex) spin columns.

     
  5. 5.

    TMSC Buffer.

     

2.6 Genetic Labeling

  1. 1.

    Competent E. coli cells or comparable cells for plasmid transformation.

     
  2. 2.

    Plasmid encoding for cap ORF as template for VP2 modification and isolation.

     
  3. 3.

    peGFP-C3 (BD Bioscience, Heidelberg, Germany) or comparable plasmid.

     
  4. 4.
    Oligonucleotide PCR primer pair to amplify VP2 and simultaneously mutate the VP2 start codon. For example:
    1. (a)

      AAV2-VP2-forward: 5′-GAA GCG CGA TCA CAT GGT CC-3′

       
    2. (b)

      AAV2-VP2-backward: 5′-TCA GCG TGG AGA TCG AGT GG-3′

       
     
  5. 5.

    AAV helper plasmid encoding the rep ORF and cap ORF, e.g., pRC (22), pACG2 (19), pAAV-RC (Stratagene, Agilent Technologies) or similar (see Note 4).

     
  6. 6.
    Oligonucleotide PCR primer for site-directed mutagenesis of VP2 start codon in AAV helper plasmid. For example:
    1. (a)

      AAV2-VP2k.o.-forward: 5′-GTTAAGACCGCTCCGGG-3′

       
    2. (b)

      AAV2-VP2k.o.-back: 5′-CCCGGAGCGGTCTTAAC-3′

       
    3. (c)

      AAV2-ligation primer-forward: 5′-AAATCAGGTATGGCTGCCGA-3′

       
    4. (d)

      AAV2-ligation primer-back: 5′-GTTGCCTCTCTGGAGGTT-3′.

       
     
  7. 7.

    T4 polynucleotide kinase.

     
  8. 8.

    Calf intestine phosphatase.

     
  9. 9.

    Restriction endonucleases and buffer (e.g., BglII to linearize peGFP-C3, and BsiWI and EcoNI to isolate cap fragment).

     
  10. 10.

    T4 DNA ligase and buffer.

     
  11. 11.

    Pfu Turbo DNA polymerase for PCR amplification of VP2 and for site directed mutagenesis.

     
  12. 12.

    QiaQuick Gel Extraction Kit (Qiagen, Hilden, Germany).

     
  13. 13.

    LB agar plates containing antibiotic selection (kanamycin at 50 μg/ml or amplicilin at 50–100 μg/ml).

     
  14. 14.

    LB media.

     
  15. 15.

    LB media containing ampicillin at 50–100 μg/ml or kanamycin 50 μg/ml.

     
  16. 16.

    Qiagen MiniPrep kit (Qiagen, Hilden, Germany), or comparable kit.

     
  17. 17.

    HEK293 cells for AAV vector packaging.

     
  18. 18.

    CaCl2 solution (2 M stock solution) and HBS buffer for plasmid transfection into HEK293 cells.

     
  19. 19.

    peGFP-AAV2-VP2 encodes for the eGFP-VP2 fusion protein (Subheading 3.6.1).

     
  20. 20.

    pRC-VP2.k.o. encodes for rep ORF of AAV-2, and VP1 and VP3 of AAV-2 (Subheading 3.6.2).

     
  21. 21.

    AAV vector plasmid containing AAV ITRs and transgene of choice.

     
  22. 22.

    Adenoviral helper plasmid, e.g., pXX6-80 (20) or similar.

     
  23. 23.

    Lysis buffer: 150 mM NaCl and 50 mM Tris/HCl (pH 8.5).

     
  24. 24.

    Benzonase (Merk, Darmstadt, Germany).

     

3 Methods

3.1 Genetic Modification of AAV Capsids via Peptide Insertion into Surface-Exposed Loops of AAV Capsids

Several AAV packaging constructs have been generated containing unique restriction sites engineered into the cap ORF (1, 2, 3). Use of these constructs as starting reagents greatly simplifies the process of epitope insertion, since two 5’ phosphorylated complementary DNA oligonucleotides need only be synthesized with compatible cohesive ends and cloned into these pre-engineered sites. Alternatively, site-directed mutagenesis is used to insert DNA oligo­nucleotides encoding the desired epitopes into the AAV cap ORF. Use of a kit, such as the QuikChange Site-Directed Mutagenesis Kit (Agilent, Stratagene, La Jolla, CA) is recommended. Mutagenesis is performed using a thermostable DNA polymerase (see Note 9) and a temperature cycler. The basic procedure utilizes a supercoiled double-stranded DNA plasmid containing the AAV cap ORF and two synthetic oligonucleotide primers containing the desired insertion (see Fig. 1). The oligonucleotide primers, each complementary to opposite strands of the vector, are extended during temperature cycling by the thermostable DNA polymerase. Incorporation of the oligonucleotide primers generates a mutated packaging construct containing staggered nicks. Following temperature cycling, the product is treated with DpnI. The DpnI endonuclease (target sequence: 5′-Gm6ATC-3′) is specific for methylated and hemimethylated DNA and is used to digest the parental DNA template and to select for the modified DNA. The nicked DNA containing the desired insertion is then transformed into E. coli (e.g., XL1-Blue supercompetent cells). The small amount of starting DNA template required to perform this method, the high fidelity of DNA polymerase, and the low number of thermal cycles all contribute to high efficiency and decreased potential for generating random mutations during the reaction.
Fig. 1.

Design of oligonucleotides for PCR-based site-directed mutagenesis.

3.1.1 Mutagenic Primer Design

For cloning into an AAV helper plasmid containing a pre-engineered restriction site, two complementary DNA oligonucleotides encoding the chosen epitope need to be designed. It may be appropriate to flank the desired epitope with scaffolding sequences, although these may also be encoded by the in-frame restriction site (see Note 10). In addition, nucleotide overhangs to the restriction site need to be added to the 5′ and 3′ end of the DNA oligonucleotides, respectively. Primers should be 5′ phosphorylated and purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Skip to Subheading 3.1.3 for cloning reactions.

3.1.2 Site-Directed Mutagenesis

For site-directed mutagenesis, primers must be designed according to the desired mutation and site of insertion (see Note 3). The 5′ ends of each primer should contain complementary versions of a unique restriction site not present in the parental plasmid. Primers need not be 5′ phosphorylated but must be purified either by FPLC or by PAGE. Failure to purify the primers results in a significant decrease in mutation efficiency.
  1. 1.
    If using the QuikChange® Site Directed Mutagenesis kit (Agilent Technologies, Statagene Products), set up sample and control reactions in thin-walled PCR tubes as follows:

    Control reaction

    1. (a)

      5 μl 10× reaction buffer.

       
    2. (b)

      2 μl (10 ng) dsDNA template (e.g., pWhitescript control plasmid (5 ng/μl)).

       
    3. (c)

      1.25 μl (125 ng) of oligonucleotide primer, forward (e.g., oligonucleotide control primer #1).

       
    4. (d)

      1.25 μl (125 ng) of oligonucleotide primer, reverse (e.g., oligonucleotide control primer #2).

       
    5. (e)

      1 μl dNTP mix (see Note 11).

       
    6. (f)

      40.5 μl ddH2O (to a final volume of 50 μl).

       
    7. (g)

      1 μl PfuTurbo DNA polymerase

      (Total volume 51 μl)

       

    Sample reaction

    1. (a)

      5 μl 10× reaction buffer.

       
    2. (b)

      2 μl (10 ng) dsDNA template, e.g., AAV packaging plasmid (5 ng/μl) (see Note 12).

       
    3. (c)

      1.25 μl (125 ng) of oligonucleotide primer, forward (e.g., mutant forward primer).

       
    4. (d)

      1.25 μl (125 ng) of oligonucleotide primer, reverse (e.g., mutant reverse primer).

       
    5. (e)

      1 μl dNTP mix (see Note 11).

       
    6. (f)

      40.5 μl ddH2O (to a final volume of 50 μl).

       
    7. (g)

      1 μl PfuTurbo DNA polymerase

      (Total volume 51 μl)

       
     
  2. 2.

    Overlay each reaction with mineral oil (∼30 μl) if the thermal cycler to be used does not have a hot-top assembly.

     
  3. 3.
    Cycle each reaction using the following cycling parameters:
    1. (a)

      Denature at 95°C for 30°s.

       
    2. (b)

      Three-step amplification cycle (Run 18 cycles): Denature at 95°C for 30 s, anneal at 55°C for 1 min, and then extend at 68°C for 1 min/kb of plasmid length.

       
    3. (c)

      Maintain the reaction at 4°C after cycling.

       
     
  4. 4.

    Add 1 μl of DpnI endonuclease (10 U/μl) directly to amplification reaction. Be sure to add enzyme below mineral oil overlay, if used. Mix gently and thoroughly by pipetting solution up and down several times.

     
  5. 5.

    Incubate at 37°C for 1 h to eliminate nonmutated parental plasmid template (see Note 13). Skip to Subheading 3.1.4 for transformation of XL1-Blue supercompetent cells.

     

3.1.3 Cloning of DNA Oligonucleotides

  1. 1.

    Resuspend each oligonucleotide at ∼100 μg/ml in TE buffer containing 150 mM NaCl. Mix equimolar amounts of each strand and heat 10 min at 65°C.

     
  2. 2.

    Cool slowly to room temperature by placing the tube at room temperature for 20 min. Check the OD260 to determine final concentration, and check to be sure that the oligonucleotides have completely annealed by running aliquots of each complementary strand, as well as the annealed double-stranded oligonucleotide, on a 4% sieving agarose gel.

     
  3. 3.

    Digest the AAV helper plasmid with the restriction enzyme that recognizes the unique site, engineered for epitope insertion, within the AAV cap ORF, dephosphorylate, gel purify, and determine the final concentration of the linearized DNA.

     
  4. 4.

    Assemble the ligation reaction, as exemplified by the following. Mix the linearized AAV helper plasmid DNA with a 50-fold molar excess of the hybridized oligonucleotides; use ∼400 ng total DNA in 10 μl, for a final concentration of 40 μg/ml. Add 0.4 μl (160 cohesive-end units) T4 DNA ligase and incubate 2 h at 15°C.

     
  5. 5.

    Dilute the ligation mixture fourfold to a concentration of 10 μg/ml.

     

3.1.4 Transformation of Competent Cells

  1. 1.

    Transform 2 μl of the ligation mixture (Subheading 3.1.3), or 1 μl of the Dpn I-treated DNA from each control and sample site-directed mutagenesis reactions (Subheading 3.1.2) into the XL1-Blue supercompetent cells (see Note 14).

     
  2. 2.

    Plate transformation reactions on LB agar plates containing the appropriate antibiotic selection (ampicillin). For mutagenesis and transduction controls using the QuikChange® site directed mutagenesis kit, plate cells on LB-ampicillin plates containing 80 μg/μl X-gal and 20 mM IPTG.

     
  3. 3.

    Incubate the plates overnight (>16 h) at 37°C (see Note 15).

     
  4. 4.

    Select three to five mutant colonies, and incubate them in 3 ml LB broth with ampicillin overnight at 37°C with shaking.

     
  5. 5.

    Extract DNA using QIAprep Spin MiniPrep kit.

     
  6. 6.

    Screen for sequence insertion by restriction endonuclease digestion (see Note 10) and sequence mutant construct to confirm epitope insertion and lack of second site mutations.

     

3.1.5 Production of Capsid-Modified AAV Vectors

  1. 1.

    To produce AAV vectors comprised of modified capsid proteins, use the modified AAV helper plasmid (generated by site-directed mutagenesis, Subheading 3.1.2, or oligonucleotide cloning, Subheading 3.1.3), in place of standard AAV helper plasmid in the triple transfection protocol for AAV vector production (described elsewhere).

     
  2. 2.

    Characterize modified vectors as described below (Subheading 3.3).

     

3.2 Generation of Mosaic AAV Vector Particles

To produce mosaic AAV vectors, transfect low passage HEK293 cells following calcium phosphate transfection protocol. HEK293 cells should be approximately 80% confluent on a 15 cm tissue culture plate.
  1. 1.

    Change media on HEK293 cells 4 h prior to transfection. Add 18 ml of fresh media.

     
  2. 2.
    Add the following DNAs (47 μg total) to CaCl2-buffered dH2O (250 mM final concentration).
    1. (a)

      Adenovirus helper plasmid containing VA, E2A, and E4 genes.

       
    2. (b)

      AAV vector plasmid containing AAV ITRs and transgene of choice.

       
    3. (c)
      The two different AAV helper plasmids (encoding different serotype AAV capsids) at varying ratios (for example, 1:19, 1:3, 1:1, 3:1, 19:1). Assessment of different ratios of one capsid component to another permits appraisal of the importance of each (see Table 2 for example).
      Table 2

      Relative amounts of DNA used to produce mosaic virions

      Plasmid

      Composition

      Amount (μg)/plate

      Adenovirus helper plasmid (e.g., pXX6-80)

       

      27 μg

      Vector plasmid (e.g., AAV-hrGFP)

       

      10 μg

      Serotype A/serotype B helper plasmid

      100% A

      5% B/95% A

      33% B/67%

      50% B/50% A

      67% B/33% A

      95% B/5% A

      10 μg A

      0.5 μg B:9.5 μg A

      2.5 μg B:7.5 μg A

      5.0 μg B:5.0 μg A

      7.5 μg B:2.5 μg A

      9.5 μg B:0.5 μg A

       
     
  3. 3.
    Prepare the DNA-CaCl2 and HBS solutions according to Table 3. Volumes can be scaled based on the number of plates to be used in transfection.
    Table 3

    Components of transfection mixture

    Tube A

    Volume/plate

    Tube B

    Volume/plate

    DNA (47 μg)

    x  μl

    2× HBS

    1,000 μl

    2.5 M CaCl2

    100 μl

      

    Sterile dH2O

    (900  −  x)μl

      

    Total

    1,000 μl

    Total

    1,000 μl

     
  4. 4.

    Using a pipette, slowly add solution from Tube A dropwise to Tube B while bubbling air through 2× HBS solution (Tube B) with another pipette. This is a slow process which should be done over several minutes.

     
  5. 5.

    A fine precipitate should form. Add the precipitate dropwise to the media, 2 ml per 15 cm plate.

     
  6. 6.

    Incubate overnight at 37°C in a humidified CO2 incubator.

     
  7. 7.

    Change the media after 24 h.

     
  8. 8.

    Sixty hours after transfection, harvest cells. Using a cell scraper, dislodge cells, and collect both the media and cells in a 250 ml conical tube. Harvest cells by centrifugation at 500  ×  g for 10 min.

     
  9. 9.

    Resuspend cell pellet in 2 ml of 100 mM NaCitrate per plate. Transfer to a 50 ml Falcon tube (see Note 16).

     
  10. 10.

    Release intracellular viral particles by inducing cell lysis via three consecutive freeze-thaw cycles, consisting of shuttling cell suspension between dry ice/ethanol bath and 37°C water bath (tubes should be vortexed vigorously after each thawing step).

     
  11. 11.

    Clarify the crude lysate by centrifugation at 500  ×  g for 10 min, and reduce viscosity by treating with benzonase (50 U/ml of cell lysate) for 30 min at 37°C, with occasional mixing.

     
  12. 12.

    Pellet cell debris by centrifuging at 5,000  ×  g for 25 min at 18°C.

     
  13. 13.
    Purify viral particles from cell debris via iodixanol density-gradient purification. Prepare an iodixanol step gradient in an Opti-seal 30 ml centrifuge tube.
    1. (a)

      Bring viral vector lysate to 15 ml with NaCitrate if necessary.

       
    2. (b)

      Add the viral vector lysate to an Opti-seal 30 ml centrifuge tube. Underlay the crude viral vector preparation with 6 ml of a 15% iodixanol solution, then 4 ml of a 25% iodixanol solution, 3 ml of 40% iodixanol solution, and 2 ml of 60% iodixanol solution.

       
     
  14. 14.

    Balance centrifuge tubes and ensure that they are full. Bubbles in the tube lead to tube collapse during centrifugation. Cap and seal tubes.

     
  15. 15.

    Centrifuge in a fixed angle rotor (e.g., Beckman 70Ti) with protective caps at 68 K for 1 h at 18°C.

     
  16. 16.

    Remove protective caps and seal. Using an 18 gauge needle, puncture the bottom of the tube and allow the iodixanol fractions to drain; collect the 40% fraction containing AAV.

     
  17. 17.

    Characterize iodixanol-purified vector as described below (Subheading 3.3).

     
  18. 18.

    Further purify AAV vectors as needed and store vectors at −20°C.

     

3.3 Characterization of Modified Capsids

Viral/vector genomes are packaged into preformed capsids. Viral/vector preparations therefore contain both empty and DNA-containing capsids. While the capsid titer (amount of capsids per ml) can be determined by ELISA using anticapsid antibodies, real-time qPCR is commonly used to determine the genomic titer (DNA containing particles per ml) of a preparation. Comparison of the capsid-to-genomic particle ratio of capsid-modified and unmodified vectors reveals whether a capsid modification interferes with packaging.

Furthermore, by assessing the cell entry and transduction efficiencies of capsid-modified vectors on target and nontarget cells conclusions on the tropism can be drawn. Tropism can be further analyzed by conducting cell transductions in the presence and absence of substances interfering with target receptor binding.

3.3.1 Assess Genomic Particle Titer of Vector Preparations by Real-Time qPCR

  1. 1.

    Dissolve 10 μl of AAV vector preparation in 190 μl PBS.

     
  2. 2.

    Isolate vector DNA by DNeasy Blood & Tissue Kit according to the protocol “Purification of total DNA from cells.”

     
  3. 3.

    Elute vector DNA in 200 μl 10 mM Tris–HCL pH 8.0.

     
  4. 4.

    Store DNA at −20°C or proceed to real-time qPCR analysis.

     
  5. 5.

    Prepare at least four PCR standards (106 to 109 AAV vector plasmids per μl).

     
  6. 6.
    If using the LightCycler FastStart DNA MasterPLUS SYBR I kit, set up sample reaction in LightCycler capillary, containing the following reagents:
    1. (a)

      4 μl SYBR Green PCR Master Mix

       
    2. (b)

      1 μl oligonucleotide PCR primer #1 (20 μM)

       
    3. (c)

      1 μl oligonucleotide PCR primer #2 (20 μM)

       
    4. (d)

      12 μl ddH2O

       
     
  7. 7.
    Run real-time PCR; e.g., for quantification of vector genomes coding for eGFP on a LightCycler platform use the following cycling parameters:
    1. (a)

      Denature at 95°C for 5 min.

       
    2. (b)

      Three-step amplification cycle (run 40 cycles): Denature at 95°C for 15 s, anneal at 60°C for 10 s, and then extend at 72°C for 30 s.

       
    3. (c)

      The temperature transition rate should be 20°C/s.

       
     
  8. 8.

    Confirm the specificity of target amplification by melting curve analysis and agarose gel electrophoresis.

     

3.3.2 Assess Capsid Titer of rAAV-2 Vector Preparations by ELISA

  1. 1.

    Prepare a serial dilution of the vector preparation in PBS. The assumed concentration should be between 2  ×  105 and 2  ×  107 capsids per μl (see Note 17).

     
  2. 2.

    Prepare a serial dilution of the capsid standard in PBS.

     
  3. 3.

    Coat the Nunc-Immuno plate with 100 μl of vector dilutions (and standard) per well.

     
  4. 4.

    Incubate overnight at 4°C.

     
  5. 5.

    Perform three washing steps with washing buffer (200 μl per well).

     
  6. 6.

    Add blocking buffer to the wells (200 μl per well).

     
  7. 7.

    Incubate for 2 h at room temperature.

     
  8. 8.

    Remove the blocking buffer.

     
  9. 9.

    Add A20 (anti-AAV2) antibody dilution (1:4 in blocking buffer; 100 μl per well).

     
  10. 10.

    Incubate for 1 h at room temperature.

     
  11. 11.

    Perform three washing steps with washing buffer (200 μl per well).

     
  12. 12.

    Add biotin-conjugated anti-mouse antibody dilution (1:25,000 in blocking buffer; 100 μl per well).

     
  13. 13.

    Incubate for 1 h at room temperature.

     
  14. 14.

    Perform three washing steps with washing buffer (200 μl per well).

     
  15. 15.

    Add horseradish peroxidase-conjugated streptavidin (1:500 in blocking buffer; 100 μl per well).

     
  16. 16.

    Incubate for 1 h at room temperature.

     
  17. 17.

    Perform three washing steps with washing buffer (200 μl per well).

     
  18. 18.

    Perform two washing steps with water (200 μl per well).

     
  19. 19.

    Add Start Solution to the well (100 μl per well).

     
  20. 20.

    Stop color reaction by adding 1 M H2SO4 (50 μl per well).

     
  21. 21.

    Measure the intensity of color reaction (450 nm).

     

3.3.3 Determine Transduction Efficiency of AAV Vectors by Flow Cytometry

  1. 1.

    Suspension cells: Seed the desired number of cells in fresh medium (containing all the required supplements) into a cell culture multiwell plate.

    Adherent cells: Seed cells in fresh medium (containing all the required supplements) into a cell culture multiwell plate. Incubate the cells at 37°C in a humidified CO2 (5%) incubator for 24 h. Prior to transduction, determine the amount of cells per well in one of the wells and change the media in the remaining wells (see Note 18).

     
  2. 2.

    Calculate the amount of vector solution that is needed per well based on the number of cells per well, the genomic particle titer of the rAAV preparation and the desired particle-to-cell ratio.

     
  3. 3.

    Thaw the vector solution on ice.

     
  4. 4.

    Add vector solution to the wells and mix. Do not forget to prepare a negative control (no vector).

     
  5. 5.

    Incubate the cells for 48 h at 37°C in a humidified CO2 (5%) incubator.

     
  6. 6.

    Harvest the cell and wash the cell pellet twice with PBS.

     
  7. 7.

    Resuspend pellet in 500 μl PBS.

     
  8. 8.

    Optional: Incubate cell pellet with antibodies specific for your transgene product according to the manufacturer’s instructions (see Note 19).

     
  9. 9.

    Perform flow cytometry measurements according to manufacturer’s instructions. Negative control should be set as 1%. Count a minimum of 5,000 cells for each sample.

     

3.3.4 Determine Transducing Titer of Vector Preparation

  1. 1.

    Seed permissive cells (see Note 20) in a 12-well cell culture plate (4 cm2) and incubate the cells for 24 h at 37°C in a humidified CO2 (5%) incubator (see Note 18).

     
  2. 2.

    Prepare a serial dilution of vector preparation in medium containing all required supplements (see Note 21).

     
  3. 3.

    Remove the medium from the wells.

     
  4. 4.

    Add 1 ml medium to cells of the negative control.

     
  5. 5.

    Add 500 μl medium to the remaining wells and add 500 μl of each dilution.

     
  6. 6.

    Optional: In order to assay for pseudotransduction by rAAV-2 vector preparations add 425 IU of heparin to the medium of one well (final volume 1 ml) and add 1 μl of the vector preparation to this well.

     
  7. 7.

    Incubate the cell culture plate for 48 h at 37°C in a humidified CO2 (5%) incubator.

     
  8. 8.

    Determine the amount of cells per well by counting the cells of the negative control.

     
  9. 9.

    Measure the percentage of transgene expressing cells by flow cytometry (see above).

     
  10. 10.
    Calculate the transducing titer of the vector solution from the dilution that led to ∼10% transgene expressing cells as shown in the following example:
    1. (a)

      Cells per well: 8  ×  104

       
    2. (b)

      Percentage of transgene expressing cells: 9.5%

       
    3. (c)

      Negative control: 1%

       
    4. (d)

      Vector solution: 0.0003 μl

       
    5. (e)

      [(8  ×  104  ×  (9.5  −  1)]/100)/0.0003  =  transducing particles/μl.

       
     

3.4 Tropism and Specificity of Modified rAAV Vectors

Post-entry barriers may limit successful cell transduction even though vectors are capable of binding and entering target cells. Hence, both cell entry and cell transduction should be determined in order to fully assess the tropism and specificity of a modified vector.

3.4.1 Monitoring of Cell Entry by Real-Time qPCR

  1. 1.

    Seed target and control cells as described above (see Note 18).

     
  2. 2.

    Thaw the vector solutions (different serotypes or modified and unmodified vector) on ice.

     
  3. 3.

    Transduce the cells with equal genomic particle-per-cell ratios of the vectors you would like to compare. Do not forget to prepare a negative control (no vector).

     
  4. 4.

    Incubate the cells for 4 h at 37°C in a humidified CO2 (5%) incubator.

     
  5. 5.

    Harvest the cells and wash the cell pellet twice with PBS (see Note 22).

     
  6. 6.

    Resuspend cell pellet (a maximum 5  ×  106 cells) in 200 μl PBS.

     
  7. 7.

    Isolate vector DNA by DNeasy Blood & Tissue Kit according to the protocol “Purification of total DNA from cells.”

     
  8. 8.

    Elute vector DNA in 200 μl 10 mM Tris–HCl pH 8.0.

     
  9. 9.

    Store at −20°C or proceed to real-time qPCR analysis.

     
  10. 10.

    Prepare at least four PCR standards (106 to 109 AAV vector plasmids per μl).

     
  11. 11.
    If using the LightCylcer FastStart DNA MasterPLUS SYBR I kit, set up sample reaction in LightCycler capillary, containing the following reagents:
    1. (a)

      4 μl SYBR Green PCR Master Mix

       
    2. (b)

      1 μl oligonucleotide primer #1 (20 μM)

       
    3. (c)

      1 μl oligonucleotide primer #2 (20 μM)

       
    4. (d)

      18 μl ddH2O.

       
     
  12. 12.

    Run real-time qPCR according to requirements of PCR reaction.

     
  13. 13.

    Confirm the specificity of target amplification by melting curve analysis and agarose gel electrophoresis.

     
  14. 14.

    Repeat steps 11–13 with oligonucleotide PCR primer pair amplifying a reference (housekeeping) gene.

     
  15. 15.

    Use the LightCycler Relative Quantification Software (Roche Diagnostics GmbH, Mannheim, Germany), or a comparable program to normalize the values obtained for the vector/viral genomes.

     

3.4.2 Determining Cell Transduction Efficiency by Flow Cytometry

  1. 1.

    Seed target and control cells as described above (see Note 18).

     
  2. 2.

    Thaw the vector solutions (different serotypes or modified and unmodified vector) on ice.

     
  3. 3.

    Transduce the cells with equal genomic particle-per-cell ratios of the vectors you would like to compare. Assay at least three different particle-to-cell ratios per cell line. Do not forget to prepare a negative control (no vector).

     
  4. 4.

    Optional: To assess the use of HSPG as cellular receptor, perform cell transductions in the presence of 425 IU of heparin.

     
  5. 5.

    To assess the use of target receptor, perform cell transductions in the presence and absence of competing and noncompeting peptides, proteins, or antireceptor antibodies (see Note 23).

     
  6. 6.

    Add competitor (e.g., heparin or peptides) to the cells prior to vector transduction.

     
  7. 7.

    Incubate the cells for 48 h at 37°C in a humidified CO2 (5%) incubator.

     
  8. 8.

    Harvest the cells and determine the percentage of transgene expressing cells, e.g., by flow cytometry.

     

3.5 Chemical Labeling of the Capsid

  1. 1.

    Buffer exchange. Adjust AAV stock to a concentration of 1.33  ×  1014 particles/ml (1 mg/ml) in the appropriate buffer. Buffer exchange can be accomplished by passing the vector through a desalting column equilibrated with reaction buffer. Alternatively, the vector solution can be dialyzed against the reaction buffer in a small dialysis cassette. If the vector is in a simple PBS buffer free of ammonium ions or primary amines, the pH of the solution can be raised by adding the concentrated buffer (e.g., 1 M potassium phosphate, pH 9 for IRDye labeling). However, the time the virus/vector is maintained at pH 9 should be kept to an absolute minimum.

     
  2. 2.

    Reconstitute the labeling reagent with the diluted AAV stock in 1-ml of reaction buffer. Make sure all of the dye is dissolved. Undissolved dye will retain reactivity and may co-purify with the labeled virus and hamper further characterization.

     
  3. 3.

    Incubate at room temperature for 60 min.

     
  4. 4.

    Purify labeled AAV from unreacted dye by either dialysis against 3 L TMSC buffer using a dialysis chamber (Slide-a-Lyser, 6,000–8,000 MW cutoff, Pierce, Thermo Fisher Scientific), or gel filtration on a G-50 (Sephadex) column equilibrated with TMSC buffer. The benefit of this technique is that purification can be monitored visually.

     
  5. 5.

    Characterize labeled AAV as described above (Subheadings 3.3 and 3.4). The titer of the labeled virus should not have significantly changed. Preparations that show a decrease in titer of more than 50% should be used with caution. Decreases in titer usually come from too high a labeling ratio (dye:particle) or failure to rapidly adjust pH following coupling. To determine the conjugation efficiency, read the absorbance spectrum of the labeled AAV and determine the dye concentration (per recalculated particle number) using the extinction coefficient for the fluorophore (provided by the manufacturer). AAV labeling ratios should be approximately 2 dye molecules per virion/vector for the greatest sensitivity with the least effect on AAV biology.

     

3.6 Genetic Labeling of the Capsid

The N-terminus of VP2 tolerates the insertion of fluorescent proteins. In order to generate AAV capsids that do not contain wild-type VP2, the VP2 start codon has to be deleted in the cap ORF of AAV helper plasmid and in the ORF encoding for the fusion protein.

3.6.1 Cloning of Fluorescent Protein-VP2 N-Terminal Fusion Protein Using peGFP-AAV2-VP2 as Example

  1. 1.
    Perform site directed mutagenesis of the VP2 start codon by PCR. For serotype 2, set up the following sample reaction:
    1. (a)

      Plasmid coding for AAV-2 cap ORF (100 ng)

       
    2. (b)

      Oligonucleotide PCR primer: AAV2-VP2-forward (30 pmol)

       
    3. (c)

      Oligonucleotide PCR primer: AAV2-VP2-backward (30 pmol)

       
    4. (d)

      dNTP mix (see Note 11)

       
    5. (e)

      Pfu Turbo DNA polymerase (1.25 U)

       
    6. (f)

      ddH2O to a final volume of 50 μl

       
    7. (g)
      PCR cycling parameters:
      • Denaturation at 95°C for 5 min.

      • Three-step amplification cycle (run 30 cycles): Denature at 95°C for 1 min, anneal at 53°C for 1 min, and then extend at 72°C for 2 min.

      • Final extension at 72°C for 10 min.

       
     
  2. 2.

    Purify PCR fragment by agarose gel electrophoresis and QiaQuick Gel Extraction Kit, or a comparable kit.

     
  3. 3.

    Phosphorylate the PCR fragment using T4 polynucleotide kinase according to standard protocols.

     
  4. 4.
    Prepare the plasmid backbone using peGFP-C3 as example:
    1. (a)

      Linearize peGFP-C3 (10 μg) with 20 U BglII in 1× reaction buffer in a final volume of 20 μl for 3 h at 37°C.

       
    2. (b)

      Isolate the linearized backbone by agarose gel electrophoreses and QiaQuick Gel Extraction Kit.

       
    3. (c)

      Modify the backbone by DNA polymerase I (Large Klenow Fragment) using 1 U per μg template, 33 μM dNTP (each), and 1× reaction buffer in a final volume of 50 μl.

       
    4. (d)

      Stop the reaction after a 15-min incubation at 25°C by addition of EDTA (final concentration of 10 mM) and a 20-min incubation at 75°C.

       
    5. (e)

      Dephosphorylate the backbone using calf intestine phosphatase (CIAP) according to standard protocols.

       
    6. (f)

      Purify the backbone by QiaQuick Gel Extraction kit.

       
     
  5. 5.

    Ligate PCR fragment and backbone using T4 DNA ligase.

     
  6. 6.

    Transform bacteria according to standard protocols.

     
  7. 7.

    Incubate bacterial suspension in LB medium for 1 h at 37°C.

     
  8. 8.

    Plate transformation reaction on LB agar plates containing the appropriate antibiotic selection.

     
  9. 9.

    Incubate the plates overnight (>16 h) at 37°C.

     
  10. 10.

    Select three to five colonies and incubate them in 3 ml LB with appropriate antibiotic overnight at 37°C with shaking.

     
  11. 11.

    Extract plasmid DNA using Qiagen MiniPrep kit or a comparable kit.

     
  12. 12.

    Screen for in frame VP2 insertion by restriction endonuclease digest and sequencing to confirm insertion and lack of second-site mutation.

     

3.6.2 Site Directed Mutagenesis of VP2 Start Codon of Cap ORF (Cloning of pRC-VP2.k.o.)

  1. 1.
    Perform site directed mutagenesis of the AAV-2 VP2 start codon, followed by PCR-mediated ligation of the fragment. For site-directed mutagenesis, prepare the following two PCR reactions:

    Fragment #1

    1. (a)

      Cap ORF encoding plasmid (100 ng)

       
    2. (b)

      Oligonucleotide PCR primer: AAV2-VP2k.o.-forward (30 pmol).

       
    3. (c)

      Oligonucleotide PCR primer: AAV2-ligation primer-­backward (30 pmol)

       
    4. (d)

      dNTP mix (see Note 11)

       
    5. (e)

      Pfu Turbo DNA polymerase (2.5 U)

       
    6. (f)

      ddH2O to a final volume of 50 μl.

       
    7. (g)
      PCR cycling parameters:
      • Denaturation at 95°C for 5 min.

      • Three-step amplification cycle (run 35 cycles): Denature at 95°C for 30 s, anneal at 50°C for 1 min, and then extend at 72°C for 2 min.

      • Final extension at 72°C for 10 min.

       
    8. (h)

      Purify PCR fragment, e.g., by agarose gel electrophoreses and QiaQuick Gel Extraction according to manufacturer’s instruction.

       

    Fragment #2

    1. (a)

      Cap ORF encoding plasmid (100 ng)

       
    2. (b)

      Oligonucleotide PCR primer: AAV2-ligation primer-forward (30 pmol)

       
    3. (c)

      Oligonucleotide PCR primer: AAV2-VP2k.o.-backward (30 pmol)

       
    4. (d)

      dNTP mix (see Note 11)

       
    5. (e)

      Pfu Turbo DNA polymerase (2.5 U)

       
    6. (f  )

      ddH2O to a final volume of 50 μl

       
    7. (g)
      PCR cycling parameters:
      • Denaturation at 95°C for 5 min.

      • Three-step amplification cycle (run 35 cycles): Denature at 95°C for 30 s, anneal at 50°C for 1 min, and then extend at 72°C for 2 min.

      • Final extension at 72°C for 10 min

       
    8. (h)

      Purify PCR fragment, e.g., by agarose gel electrophoreses and QiaQuick Gel Extraction according to manufacturer’s instruction.

       

    Ligation of fragment #1 and fragment #2

    1. (a)

      Fragment #1 and fragment #2 in ratio of 4:1 (∼100 ng)

       
    2. (b)

      Oligonucleotide PCR primer: AAV2-VP2k.o.-forward (30 pmol)

       
    3. (c)

      Oligonucleotide PCR primer: AAV2-VP2k.o.-backward (30 pmol)

       
    4. (d)

      dNTP mix (see Note 11)

       
    5. (e)

      Pfu Turbo DNA polymerase (2.5 U)

       
    6. (f  )

      ddH2O to a final volume of 50 μl

       
    7. (g)
      PCR cycling parameters:
      • Denaturation at 95°C for 5 min

      • Three-step amplification cycle (run 35 cycles): Denature at 95°C for 30 s, anneal at 50°C for 1 min, and then extend at 72°C for 3 min.

      • Final extension at 72°C for 10 min

       
    8. (h)

      Purify PCR fragment, e.g., by agarose gel electrophoreses and QiaQuick Gel Extraction according to manufacturer’s instruction.

       
     
  2. 2.

    Linearize plasmid backbone (rep and cap ORF encoding AAV helper plasmid, e.g., pRC (22)) by adding 10 U of restriction endonuclease BsiWI to 10 μg of plasmid in 1× reaction buffer adjusted to a final volume of 30 μl.

     
  3. 3.

    Following 3 h of incubation at 55°C, add restriction endonuclease EcoNI (15 U) and incubate for further 3 h at 37°C.

     
  4. 4.

    Dephosphorylate the backbone by CIAP using standard protocols.

     
  5. 5.

    Purify backbone by agarose gel electrophoresis and QiaQuick Gel Extraction Kit according to standard protocols.

     
  6. 6.

    Modify the PCR fragment with BsiWI and EcoNI as described for plasmid backbone with exception of step 4.

     
  7. 7.

    Ligation of backbone and PCR fragment by T4 DNA Ligase

     
  8. 8.

    Transform bacteria, followed by incubation in LB medium for 1 h at 37°C.

     
  9. 9.

    Plate bacterial suspension on LB agar plates containing ampicillin (50–100 μg/ml).

     
  10. 10.

    Incubate the plates overnight (>16 h) at 37°C.

     
  11. 11.

    Select three to five colonies, incubate them in 3 ml LB with ampicillin (final concentration: 50–100 μg/ml) overnight at 37°C with shaking.

     
  12. 12.

    Extract plasmid DNA using Qiagen MiniPrep kit or a comparable kit.

     
  13. 13.

    Screen for fragment insertion by restriction endonuclease digestion.

     
  14. 14.

    Sequence modified plasmid to confirm insertion, mutation of VP2 start codon, and lack of second-site mutations.

     

3.6.3 Generation of eGFP-Tagged rAAV-2 Vector Particles

  1. 1.

    To produce eGFP-tagged rAAV-2 vector particles, transfect low passage HEK293 cells following calcium phosphate transfection protocol (see Note 18).

     
  2. 2.

    Change media 4 h prior to transfection. Adding 18 ml of fresh media.

     
  3. 3.
    Add the following plasmids (1:1:1:1 M ratio; 47 μg total) to CaCl2-buffered dH2O (250 mM final concentration).
    1. (a)

      Adenovirus helper plasmid containing VA, E2A, and E4 genes

       
    2. (b)

      AAV vector plasmid containing AAV ITRs and transgene of choice

       
    3. (c)

      The two different AAV helper plasmids (pRC-VP2-k.o. and peGFP-AAV2-VP2)

       
     
  4. 4.

    Proceed with AAV vector preparation as described above (Subheading 3.2).

     

4 Notes

  1. 1.

    While there are a number of permissive insertion sites within the AAV-2 capsid, we recommend starting with inserting novel targeting ligands following amino acid 587 or 588. Insertions at amino acid 587 block heparan sulfate binding. Insertions at 588 can be engineered to maintain heparan sulfate binding when appropriate linker sequences are incorporated. This is an important consideration when endogenous receptor binding is desired, or for heparin-based purification.

     
  2. 2.

    A major factor in maintaining infectivity following peptide insertion is accurate capsid assembly. Including appropriate linker/scaffolding sequences flanking the inserted epitope can be critical for this process. It has been shown that different linker sequences can greatly influence both assembly and stability of modified AAV capsids.

     
  3. 3.
    It is suggested that a primer design program (e.g., QuikChange® Primer Design Program; available online at http://www.­stratagene.com/qcprimerdesign) be utilized to design the two primers for PCR-based site-directed mutagenesis. The primers should encode the desired targeting epitope flanked by 15 or 20 homologous base pairs on each side of the insertion (see Fig. 1).
    1. (a)

      The targeting epitope should be less than 16 amino acids long, epitopes of 9–12 amino acids are typical. However, an upper limit for epitope length has not been rigorously defined and will depend on insertion site and primary amino acid sequence.

       
    2. (b)

      Only one of the mutagenic primers should contain sequences encoding the desired insertion.

       
    3. (c)

      The primers should anneal to contiguous sequences flanking the insertion site on opposite strands of the plasmid template (AAV helper plasmid containing the AAV cap ORF).

       
    4. (d)

      The 5’ end of each primer should contain a restriction site not present in the plasmid template, and a GC cap of at least 3 nt.

       
    5. (e)

      Primers should contain about 18–25 bases complementary to the plasmid template, with a melting temperature (Tm) of ≥78°C.

       
    6. (f)

      Primers should have a minimum GC content of 40%, and should terminate in one or more G or C bases.

       
    7. (g)

      Primers must maintain appropriate reading frame of AAV cap ORF.

       
     
  4. 4.

    Several different AAV helper plasmids have been described in the literature as for example (19, 23). There is not a single preferred construct. The serotype of encoded AAV cap ORF should be chosen based on the application at hand. The plasmid should only encode the AAV rep and cap ORF, and should not contain adenovirus sequences or AAV terminal repeats.

     
  5. 5.

    CaPO4-DNA transfection is primarily based on pH, thus it is very important for the HBS to be maintained at the proper pH. This may require using several pH meters to ensure accuracy.

     
  6. 6.

    Adding neutral red to the 25% and 60% iodixanol layers allows easier discrimination of the layers. This aids in the isolation of the 40% iodixanol layer in which virus is distributed.

     
  7. 7.

    AAV serotypes differ in the amino acid composition of their capsid and therefore differ in epitopes recognized by the immune system. The antibody A20, e.g., recognizes AAV-2 and AAV-3 capsids (24). A20 supernatant as well as anticapsid antibodies for AAV-1 and AAV-5 are, e.g., available from Progen (Heidelberg, Germany).

     
  8. 8.

    The concentration and purity of the initial AAV stock are of critical importance. If purifying via CsCl gradient, at least 3 successive CsCl gradients are required. Protease digestion during the isolation of AAV should be kept to a minimum. The titer of the AAV stock must be above 1.33  ×  1014 particles/ml for effective labeling. The purity of the final AAV stock must be greater than 99.9% since contaminants will be preferentially labeled with the fluorescent labeling reagents. There should be no cellular proteins or viral proteins other than full-size VP1, VP2, and VP3 evident on overloaded silver-stained SDS-polyacrylamide gels.

     
  9. 9.

    Platinum Pfx DNA polymerase (Stratagene, La Jolla, CA) is preferred due to its high fidelity, and better efficiency and stability when replicating large templates. Several other thermostable DNA polymerases are also available and are acceptable.

     
  10. 10.

    The nonhomologous sequences that flank the insert serve a dual purpose to both encode the critical scaffolding sequences, and contain restriction sites for confirmation of epitope insertion. Designing an AgeI or NgoMIV restriction site can be easily done and these are convenient sites for enzymatic digestion screening, and are absent from commonly used AAV helper plasmids.

     
  11. 11.

    Do not subject the dNTP mixture to repeated freeze/thaw cycles. Thaw the dNTP mixture once, prepare single-use aliquots, and store the aliquots at −20°C. Nucleotide solutions are often optimized for specific buffers and thermostable DNA polymerases; do not substitute dNTP mixtures from different PCR-based site-directed mutagenesis kits.

     
  12. 12.

    For the sample reactions, it is best to set up a series of reactions using increasing amounts of template double-stranded DNA (e.g., 5, 10, 15, 20, 50 ng) while maintaining excess primer concentration.

     
  13. 13.

    Digestion of parental DNA template by DpnI is dependent on methylation. Thus, plasmid expression in dam+ E. coli strains is critical. DNA isolated from dam(−) E. coli strains, such as JM110 and SCS 110 is not suitable for this protocol.

     
  14. 14.

    If using the XL1-Blue supercompetent cells for transformation, make sure to use the specified 14-ml BD Falcon polypropylene tubes. The heat pulse protocol has been optimized for transformation in these tubes according to their thickness and shape.

     
  15. 15.

    The expected number of colonies from the pWhitescript control mutagenesis reaction is between 50 and 800, 80% of which should contain the mutation and appear as blue colonies on the X-gal/IPTG plates. The transformation control (pUC18) should have >250 colonies, with >98% maintaining the blue phenotype.

     
  16. 16.

    Using sodium citrate for cell suspension at this step is preferred because this buffer has been shown to reduce viral particle aggregation.

     
  17. 17.

    The capsid titer is commonly tenfold higher than the genomic titer.

     
  18. 18.

    Cells should be approximately 70% confluent.

     
  19. 19.

    For detection of nonfluorescent intracellular transgene products, cells have to be fixed and permeabilized prior to incubation with antibodies.

     
  20. 20.

    The human carcinoma cell line HeLa (ATCC® number CCL2) is highly permissive for AAV-2 and can therefore be used for determining the transducing titer of rAAV-2 vector preparations. If HeLa cells are used, seed 7  ×  104 cells/well into a 12-well plate.

     
  21. 21.
    Example of serial dilution:
    1. (a)

      Add 2 μl of vector preparation to 1 ml medium (sample A) and mix

       
    2. (b)

      Add 70 μl of sample A to 630 μl medium (sample C) and mix

       
    3. (c)

      Add 70 μl of sample C to 630 μl medium (sample E) and mix

       
    4. (d)

      Add 70 μl of sample E to 630 μl medium (sample G) and mix

       
    5. (e)

      Add 210 μl of sample A to 490 μl medium (sample B) and mix

       
    6. (f)

      Add 70 μl of sample B to 630 μl medium (sample D) and mix

       
    7. (g)

      Add 70 μl of sample D to 630 μl medium (sample F) and mix

       
    8. (h)

      Add 70 μl of sample F to 630 μl medium (sample H) and mix.

       
     
  22. 22.

    In order to remove membrane bound particles treat the vector transduced (adherent as well as suspension) cells with trypsin prior to cell lysis.

     
  23. 23.

    It is suggested that at least three different concentrations of the competitor is used.

     

References

  1. 1.
    Arnold, G. S., Sasser, A. K., Stachler, M. D., and Bartlett, J. S. (2006) Metabolic biotinylation provides a unique platform for the purification and targeting of multiple AAV vector serotypes, Mol Ther 14, 97–106.PubMedCrossRefGoogle Scholar
  2. 2.
    Nicklin, S. A., Buening, H., Dishart, K. L., de Alwis, M., Girod, A., Hacker, U., Thrasher, A. J., Ali, R. R., Hallek, M., and Baker, A. H. (2001) Efficient and selective AAV2-mediated gene transfer directed to human vascular endothelial cells, Mol Ther 4, 174–181.PubMedCrossRefGoogle Scholar
  3. 3.
    Shi, W., Arnold, G. S., and Bartlett, J. S. (2001) Insertional mutagenesis of the adeno-associated virus type 2 (AAV2) capsid gene and generation of AAV2 vectors targeted to alternative cell-surface receptors, Hum Gene Ther 12, 1697–1711.PubMedCrossRefGoogle Scholar
  4. 4.
    Perabo, L., Buning, H., Kofler, D. M., Ried, M. U., Girod, A., Wendtner, C. M., Enssle, J., and Hallek, M. (2003) In vitro selection of viral vectors with modified tropism: the adeno-associated virus display, Mol Ther 8, 151–157.PubMedCrossRefGoogle Scholar
  5. 5.
    Shi, W., and Bartlett, J. S. (2003) RGD inclusion in VP3 provides adeno-associated virus type 2 (AAV2)-based vectors with a heparan sulfate-independent cell entry mechanism, Mol Ther 7, 515–525.PubMedCrossRefGoogle Scholar
  6. 6.
    Stachler, M. D., Chen, I., Ting, A. Y., and Bartlett, J. S. (2008) Site-specific modification of AAV vector particles with biophysical probes and targeting ligands using biotin ligase, Mol Ther 16, 1467–1473.PubMedCrossRefGoogle Scholar
  7. 7.
    Hauck, B., Chen, L., and Xiao, W. (2003) Generation and characterization of chimeric recombinant AAV vectors, Mol Ther 7, 419–425.PubMedCrossRefGoogle Scholar
  8. 8.
    Rabinowitz, J. E., Bowles, D. E., Faust, S. M., Ledford, J. G., Cunningham, S. E., and Samulski, R. J. (2004) Cross-dressing the virion: the transcapsidation of adeno-associated virus serotypes functionally defines subgroups, J Virol 78, 4421–4432.PubMedCrossRefGoogle Scholar
  9. 9.
    Lux, K., Goerlitz, N., Schlemminger, S., Perabo, L., Goldnau, D., Endell, J., Leike, K., Kofler, D. M., Finke, S., Hallek, M., and Buning, H. (2005) Green fluorescent protein-tagged adeno-associated virus particles allow the study of cytosolic and nuclear trafficking, J Virol 79, 11776–11787.PubMedCrossRefGoogle Scholar
  10. 10.
    Warrington, K. H., Jr., Gorbatyuk, O. S., Harrison, J. K., Opie, S. R., Zolotukhin, S., and Muzyczka, N. (2004) Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus, J Virol 78, 6595–6609.PubMedCrossRefGoogle Scholar
  11. 11.
    Keppler, A., Pick, H., Arrivoli, C., Vogel, H., and Johnsson, K. (2004) Labeling of fusion proteins with synthetic fluorophores in live cells, Proc Natl Acad Sci U S A 101, 9955–9959.PubMedCrossRefGoogle Scholar
  12. 12.
    Miller, L. W., Sable, J., Goelet, P., Sheetz, M. P., and Cornish, V. W. (2004) Methotrexate conjugates: a molecular in vivo protein tag, Angew Chem Int Ed Engl 43, 1672–1675.PubMedCrossRefGoogle Scholar
  13. 13.
    Bartlett, J. S., and Samulski, R. J. (1998) Fluorescent viral vectors: a new technique for the pharmacological analysis of gene therapy, Nat Med 4, 635–637.PubMedCrossRefGoogle Scholar
  14. 14.
    Bartlett, J. S., Wilcher, R., and Samulski, R. J. (2000) Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors, J Virol 74, 2777–2785.PubMedCrossRefGoogle Scholar
  15. 15.
    Ren, C., White, A. F., and Ponnazhagan, S. (2007) Notch1 augments intracellular trafficking of adeno-associated virus type 2, J Virol 81, 2069–2073.PubMedCrossRefGoogle Scholar
  16. 16.
    Sanlioglu, S., Benson, P. K., Yang, J., Atkinson, E. M., Reynolds, T., and Engelhardt, J. F. (2000) Endocytosis and nuclear trafficking of adeno-associated virus type 2 are controlled by rac1 and phosphatidylinositol-3 kinase activation, J Virol 74, 9184–9196.PubMedCrossRefGoogle Scholar
  17. 17.
    Seisenberger, G., Ried, M. U., Endress, T., Buning, H., Hallek, M., and Brauchle, C. (2001) Real-time single-molecule imaging of the infection pathway of an adeno-associated virus, Science 294, 1929–1932.PubMedCrossRefGoogle Scholar
  18. 18.
    Bartlett, J. S., Samulski, R. J., and McCown, T. J. (1998) Selective and rapid uptake of adeno-associated virus type 2 in brain, Hum Gene Ther 9, 1181–1186.PubMedCrossRefGoogle Scholar
  19. 19.
    Li, J., Samulski, R. J., and Xiao, X. (1997) Role for highly regulated rep gene expression in adeno-associated virus vector production, J Virol 71, 5236–5243.PubMedGoogle Scholar
  20. 20.
    Xiao, X., Li, J., and Samulski, R. J. (1998) Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus, J Virol 72, 2224–2232.PubMedGoogle Scholar
  21. 21.
    Grimm, D., Kern, A., Rittner, K., and Kleinschmidt, J. A. (1998) Novel tools for production and purification of recombinant adenoassociated virus vectors, Hum Gene Ther 9, 2745–2760.PubMedCrossRefGoogle Scholar
  22. 22.
    Girod, A., Ried, M., Wobus, C., Lahm, H., Leike, K., Kleinschmidt, J., Deleage, G., and Hallek, M. (1999) Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2, Nat Med 5, 1052–1056.PubMedCrossRefGoogle Scholar
  23. 23.
    Rabinowitz, J. E., Rolling, F., Li, C., Conrath, H., Xiao, W., Xiao, X., and Samulski, R. J. (2002) Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity, J Virol 76, 791–801.PubMedCrossRefGoogle Scholar
  24. 24.
    Wobus, C. E., Hugle-Dorr, B., Girod, A., Petersen, G., Hallek, M., and Kleinschmidt, J. A. (2000) Monoclonal antibodies against the adeno-associated virus type 2 (AAV-2) capsid: epitope mapping and identification of capsid domains involved in AAV-2-cell interaction and neutralization of AAV-2 infection, J Virol 74, 9281–9293.PubMedCrossRefGoogle Scholar
  25. 25.
    Wu, P., Xiao, W., Conlon, T., Hughes, J., Agbandje-McKenna, M., Ferkol, T., Flotte, T., and Muzyczka, N. (2000) Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism, J Virol 74, 8635–8647.PubMedCrossRefGoogle Scholar
  26. 26.
    Yang, Q., Mamounas, M., Yu, G., Kennedy, S., Leaker, B., Merson, J., Wong-Staal, F., Yu, M., and Barber, J. R. (1998) Development of novel cell surface CD34-targeted recombinant adenoassociated virus vectors for gene therapy, Hum Gene Ther 9, 1929–1937.PubMedCrossRefGoogle Scholar
  27. 27.
    Grifman, M., Trepel, M., Speece, P., Gilbert, L. B., Arap, W., Pasqualini, R., and Weitzman, M. D. (2001) Incorporation of tumor-­targeting peptides into recombinant adeno-associated virus capsids, Mol Ther 3, 964–975.PubMedCrossRefGoogle Scholar
  28. 28.
    Boucas, J., Lux, K., Huber, A., Schievenbusch, S., von Freyend, M. J., Perabo, L., Quadt-Humme, S., Odenthal, M., Hallek, M., and Buning, H. (2009) Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations, J Gene Med 11, 1103–1113.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Hildegard Büning
    • 1
    • 2
    Email author
  • Chelsea M. Bolyard
    • 3
    • 4
  • Michael Hallek
    • 1
    • 2
  • Jeffrey S. Bartlett
    • 3
    • 5
  1. 1.Department of Internal MedicineUniversity of CologneCologneGermany
  2. 2.Center for Molecular Medicine Cologne (ZMMK)University of CologneCologneGermany
  3. 3.Gene Therapy CenterThe Research Institute at Nationwide Children’s HospitalColumbusUSA
  4. 4.Integrated Biomedical Sciences Graduate Program, School of MedicineThe Ohio State UniversityColumbusUSA
  5. 5.Department of Pediatrics, and Department of Molecular Virology, Immunology, and Medical GeneticsThe Ohio State University ColumbusColumbusUSA

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