Adenovirus vector rescue, production and analysis

Contents

1. Adenovirus: structure and biology
Viral proteins
Replication of adenovirus genome

2. Types of adenoviral vectors

3. Production of adenoviral vectors
3.1 Generation of infectious plasmids
Bacterial transformation
Midiprep
Control gel-Digestion:

3.2 Transfection into producer cell lines
3.3 PEI tranfection of linearized plasmid
3.4 Harvesting of Adenovirus
3.5 Amplification of Ad vectors
3.6 Virus prep

4. Analysis and characterization
4.1 Vector titration
OD260-measurement
Slot-Blot assay

4.2 Infection with different MOI

5. Comparison of the efficiency of different vector systems
5.1 PEI Transfection
Transfection with different N/P ratios
5.2 Transfection vs. transduction efficiency
5.3 Neutralization assay

References

1. Adenovirus: structure and biology

Adenovirus is a non-enveloped, icosahedral virus of 100nm in diameter with a linear, double- stranded DNA genome of 30-40kb. The capsid is composed of 240 hexon capsomeres forming the 20 triangular faces of the icosahedrons, and 12 penton capsomeres with spike-shaped protrusions located at the 12 vertices (Figure 1) (Volpers et al., 2004).

The terminal globular domain of the fibers of the Ad capsid is responsible for the primary virus attachment to the cellular receptor, the coxsackie- and adenovirus-receptor (CAR). Following the initial attachment, the interaction between an RGD-motif exposed in the penton base protein with a cell surface integrin molecule serving as secondary or internalization receptor triggers the virus uptake by endocytosis. The endosomal uptake of the virus and release into the cytoplasm is accompanied by a stepwise dismantling of the capsid, leading to the microtubule-assisted transport and delivery of the core protein-coated viral genome to the nucleus (Volpers et al., 2004).

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Figure 1: Structure of the adenovirus particle (http://viralzone.expasy.org/).

Viral proteins

To detect the different adenoviral proteins with respect to their molecular weight, we performed a SDS-PAGE with several dilutions of an AdEmpty vector, followed by Silver and Coomassie staining of the respective gels (Figure 2).

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Figure 2: Adenovirus proteins. (A) Coomassie staining. (B) Silver staining.

Comparing both staining methods, it becomes obvious that Silver staining is more sensitive, since for example the Hexon protein is visible in the lowest dilution, which is not the case for Coomassie staining. This enables the use of Silver staining to assess if a vector sample is pure and shows that this is the more trustable method.

Replication of adenovirus genome

For understanding the way of producing adenoviral vector, it is important to have a background on the replication of adenovirus genome. The replication depends basically on “inverted terminal repeats” (ITRs) at both ends as cis-acting elements (origin of replication) and one copy of the terminal protein (TP) covalently attached to each 5’ end as initiation primer (Volpers et al., 2004). Therefore, only a linearized genome allows adenoviral replication.

2. Types of adenoviral vectors

Adenoviral vectors (Adv) are among the oldest and most widely used vectors for gene therapy applications. The advantages of this vector type are the extensive packaging capacity, the ability to transduce a variety of different cell types (including non-dividing), and its potential for high-titer preparations.

In first-generation adenovirus vectors the E1 genes are deleted (Figure 3) (Volpers et al., 2004). Since the transactivating and transforming E1 functions are therefore not present in the transduced target cell, the vector does not replicate in these cells under regular conditions. For vector production, the E1 functions have to be provided in trans by a complementing producer cell lines (Schiedner et al., 2000; Volpers et al., 2004). First-generation vectors can have a total transgene capacity of 8 kb.

Second-generation Adv have additional viral gene functions deleted including, for example, E2 or E4, which are complemented by more complex producer cell lines (Volpers et al., 2004). In high-capacity Adv (HC-Adv) or “gutless” Adv, all viral coding sequences are deleted; only the ITRs and the packaging signal remain as viral elements. The cloning capacity is expanded to approximately 37 kb. The lack of viral gene expression from these vectors has been shown to considerably reduce their toxicity and immunogenicity in vivo, and long-term transgene expression (Volpers et al., 2004). Currently, these kinds of vectors are produced in the presence of a replication-defective (first-generation) adenoviral helper virus, which provide in trans all viral gene functions except E1.

Conditionally, replication-competent adenoviruses (no transgene) and Ad vectors (with therapeutic transgene) are intended to replicate and spread exclusively in tumor cells. These oncolytic Ad vectors are characterized by a deletion in the E1A or E1B gene.

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Figure 3: Basics of adevoviral vectors’ production. Modified from (Volpers et al., 2004).

3. Production of adenoviral vectors

Production of Adenovirus vectors involves several steps (Figure 4), and we will describe them here in detail.

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Figure 4: Overview of the production of Adenovirus vectors.

3.1 Generation of infectious plasmids

Production of adenoviral vectors starts with the cloning of an infectious plasmid. It must contain a multiple cloning site for linearization, selection markers needed for growth of plasmid containing bacteria after transformation, the packaging signal and the ITR regions enabling virus replication. Additionally, transgenes are included for gene therapeutical purposes.

Bacterial transformation

After cloning of the infectious plasmid, it is transformed into Escherichia coli (E. coli) for amplification. In our case, we transformed pEGFPN1 in E. coli XL2-blue ultracompetent cells by electroporation, followed by inoculation of kanamycin agar plates. The following day, single clones were picked and incubated in kanamycin containing LB-medium over night to ensure amplification of only transformed bacteria.

Midiprep

The midiprep is a method used to extract and purify plasmid DNA. It involves the following steps: harvesting and lysis of the bacteria, neutralization to precipitate chromosomal DNA and proteins that are pelleted by centrifugation and then discarded; and finally purification of plasmid DNA by ion-exchange chromatography.

We performed the midiprep of pEGFPN1 using a kit from QIAGEN, resulting in the plasmid pGS109#29.

Control gel-Digestion:

After isolating the plasmid DNA, clones of pGS109#29 were digested with the restriction enzyme SwaI and analyzed by gel electrophoresis (Figure 5).

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Figure 5: Control gel of pGS109#29 clones digested with SwaI. Clones 1 and 2 showed the correct fragment size (+), unlike clone 3 (-).

We could verify positive clones containing the transgene by bands on the gel that represent the correct size of both the vector and the insert DNA (Figure 5).

3.2 Transfection into producer cell lines

In our practical course, we generated a first-generation recombinant Ad vector encoding for EGFP (Ad1stGFP). As described above, this kind of vectors is replication-deficient, thus a cell line that provides the missing E1A and E1B genes in trans is required for amplification of this vector (Kreppel et al., 2002). In our case, the E1-complementing producer cell line N52.E6 (N52) was used for this purpose.

3.3 PEI tranfection of linearized plasmid

To start the virus production, N52 cells are Polyethylenimine (PEI) transfected in a small scale with an infectious plasmid. It is essential that this plasmid is linearized, since the Adv needs free ITR to replicate. Therefore, the plasmid pGS109#29 was digested with SwaI prior to transfection. Transfection of a 6cm dish of N52 cells was done with 1, 2 or 4 µg linearized plasmid DNA using a 7.5mM PEI at N/P 30.

3.4 Harvesting of Adenovirus

Morphological changes in cells caused by viral infection are called cytopathic effects (CPE). Examples are rounded and detached cells, or cells that form clusters.

CPE was evaluated with a light microscope and additionally fluorescence was monitored with a fluorescence microscope. Upon significant CPE, cells were harvested and virus was released from the cells by three cycles of freezing in liquid nitrogen and thawing at 37°C. Cell debris was pelleted and supernatant containing virus was transferred to a new tube and stored at -80°C until required.

3.5 Amplification of Ad vectors

For further amplification, a 15cm dish of N52 cells was infected with parts of the raw lysate obtained from the first infection with 1 and 4 µg DNA. After 48 hours, the virus was again harvested as described above. At this point we observed that the supernatant from the second infection with 4 µg DNA was greener than the one from 1 µg, indicating that further amplification is needed for 1 µg DNA but not for 4 µg DNA, whose lysate could infect directly ten 15cm dishes (final infection).

3.6 Virus prep

After several cycles of amplification, enough virus particles were produced to prepare a stock. For this purpose, a severe purification is required to remove cell debris, excess of free vector material and vector transgene products.

After the final infection, the harvested supernatant was added to ultracentrifuge tubes containing a preformed, discontinuous CsCl-gradient (3ml 1.42g/ml CsCl and 5ml 1.27g/ml CsCl). After 2h of ultracentrifugation at 32000rpm, the virus band could be found between the two CsCl solutions at a density of 1.34g/ml. It was collected by puncturing the side of the tube using a 2ml syringe, transferred to a new ultracentrifuge tube containing a 1.34g/ml CsCl and centrifuged for 20h at 32000rpm.

Rearrangement of the transgene is possible during the multiple infection steps, resulting in a virus genome with a different length and thus different density.

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