Initial recombinant AAV production system

Transcription

1 AAV vectors

2 Initial recombinant AAV production system Construction of plasmids encoding raav genomes in which wild-type sequences necessary for genome replication and packaging (i.e., the AAV ITRs) frame a transgene instead of the AAV rep and cap genes. These constructs are transfected into packaging cells together with a rep and cap expression plasmid leading to the production of raav particles. Helper activities required for the activation and support of the productive phase of the AAV life cycle are introduced by infection of the cells with wild-type Ad. Cellular DNA polymerase activities together with the Rep78 and Rep68 proteins lead to DNA replication. This DNA is incorporated in the single-stranded format into preformed empty capsids through the activities of the Rep52 and Rep40 proteins.

3 Initial recombinant AAV production system The resulting raav virions are released from the producer cells together with helper Ad particles. Sequential heat treatment and buoyant density centrifugation in the cesium chloride gradient allows the selective elimination of the helper virus from the final raav preparation.

4 Viral-Based System for High-Efficiency Gene Delivery General features of AAV: High-efficiency gene delivery - high transduction efficiency Broad host range including both dividing and nondividing cells Many cell types are difficult or impossible to transfect and extensive time is required in tissue culture to generate a stable cell line. AAV helper-free system: Helper-free system - no adenovirus required gene Safe delivery system for long-term gene expression.

5 AAV Helper-Free System Vectors described here have been used successfully in human phase I clinical trials1 and use of these vectors to generate cell-based assays for high-throughput screening has increased due to efficient stable cell line production over a broad range of cell types.

6 AAV Helper-Free System

7 Production of AAV particles using the AAV Helper-Free System I. Cloning the gene of interest into an appropriate plasmid vector. For most applications, the DNA of interest is cloned into one of the ITR/MCS containing vectors (paav-mcs or paav-ires-hrgfp). The inverted terminal repeat (ITR) sequences present in these vectors provide all of the cis-acting elements necessary for AAV-2 replication and packaging. II. The recombinant expression plasmid is co-transfected into the AAV-293 cells with phelper (carrying adenovirus-derived genes) and paav-rc (carrying AAV-2 replication and capsid genes), which supply all of the trans-acting factors required for AAV replication and packaging in the AAV-293 cells. Recombinant AAV-2 viral particles are prepared from infected AAV-293 cells and may then be used to infect a variety of mammalian cells. III. Upon infection of the host cell, the single-stranded virus must become doublestranded in order for gene expression to occur. This is a limiting step in recombinant gene expression and can be accelerated using adenovirus superinfection or etoposides like camptothecin or sodium butyrate. However, agents to accelerate gene expression can be toxic to the target cells. The use of etoposides is therefore only recommended for short-term use or in obtaining viral titers.

8 AAV Helper-Free System allows the production of AAV-2 virions without the use of a helper virus. Most of the adenovirus gene products required for the production of infective AAV particles are supplied on the plasmid phelper (i.e. E2A, E4, and VA RNA genes) that is co-transfected into cells with human AAV-2 vector DNA. The remaining adenoviral gene product is supplied by the AAV-293 host cells (HEK 293-derived cells), which stably express the adenovirus E1 gene. The wild-type AAV-2 genome consists of the viral rep and cap genes (encoding replication and capsid genes, respectively), flanked by ITRs that contain all the cisacting elements necessary for replication and packaging. In the AAV Helper-Free System, the rep and cap genes have been removed from the viral vector that contains AAV-2 ITRs and are supplied in trans on the plasmid paav-rc. The removal of the AAV rep and cap genes allows for insertion of a transgene (up to 3 kb) in the viral genome. AAV-2 ITR-containing plasmids (paav-mcs, paav-lacz and paav-hrgfp and paav-ires-hrgfp) do not share any regions of homology with the rep/cap-gene containing plasmid (paav-rc), preventing the production of wild-type AAV-2 through recombination.

9 The AAV Helper-Free System produces recombinant viral titers of 10 7 particles/ml of primary virus stock. viral AAV-2 is especially valuable for long-term gene expression due to the ability of AAV- 2 to replicate epichromosomally under replication permissive conditions. Slowly dividing or non-dividing cells can maintain the epichromosomal AAV-2 genome over time, and gene expression will be stable while the AAV-2 genome is maintained. Rapidly dividing cells will lose epichromosomal genome after cell replication and will, therefore, lose gene expression. Viral integration into the genome can occur, however, integration events are rare. The frequency of integration events may increase if an extremely high multiplicity of infection is used or if the cell is infected in the presence of adenoviral replicase.

10

11

12 AAV-293 Cells The AAV-293 cells provided are derived from the commonly used HEK293 cell line, but produce higher viral titers. HEK293 cells are human embryonic kidney cells that have been transformed by sheared adenovirus type 5 DNA. AAV-293 cells, like HEK293 cells, produce E1 in trans, allowing the production of infectious virus particles when cells are transfected with E1- deleted adenovirus vectors or when co-transfected with the three AAV Helper-Free System plasmids. AAV-293 cells do not adhere well to tissue culture dishes and have a tendency to clump. When exchanging solutions, gently pipette down the side of the dish and not directly onto the cells to prevent disruption of the cell monolayer. It is important to maintain the AAV-293 cells at 50% confluence to ensure the integrity of the stock, maintaining the higher titer production phenotype. It is important to prepare a liquid nitrogen stock of early passage cell aliquots for long-range experiments.

13 The paav-mcs and the paav-ires-hrgfp vectors contain the CMV promoter and other elements for high-level gene expression in mammalian cells when a transgene is cloned into the multiple cloning site (MCS). Both vectors also contain AAV-2 inverted terminal repeats (ITRs), which direct viral replication and packaging.

14 The paav-ires-hrgfp vector contains a bicistronic expression cassette in which the GFP is expressed as a second open reading frame that is translated from the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES). GFP fluorescence may be used: * to measure the titer of the recombinant virus stock directly * to ascertain the infection efficiency for the desired target cell type * as an expression marker of the inserted transgene.

15 paav-ires-hrgfp features the 3 FLAG sequence at the C-terminus of the MCS. Foreign genes cloned in-frame with the tag may be detected or purified using an anti- FLAG antibody. This feature is very useful when antibodies against the protein encoded by the transgene are unavailable. The markerless paav-mcs vector is recommended for inserts approaching 3 kb in size, while paav-ires-hrgfp can accommodate inserts 1.7 kb.

16 The paav-rc plasmid contains the AAV-2 rep and cap genes, encoding replication proteins and viral capsid structural proteins, respectively. Establishing the proper expression levels of rep and cap gene products is an important step in achieving high-titer virus production. paav-rc directs Rep and Cap expression from two different promoters, achieving optimal expression levels for each set of gene products.

17 The phelper plasmid contains the subset of adenovirus genes, VA, E2A and E4, which are necessary for high-titer AAV production in the AAV-293 cells. Adenovirus proteins E1A and E1B, which are also required for AAV-2 production, are stably expressed in the AAV-293 cells.

18 Preparing Viral Stocks Viral particles are present in both intact cells and the growth medium. Preparation of the viral stock from the combined suspension of cells plus growth medium results in the greatest yield of virus. 1. Prepare a dry ice-ethanol bath and a 37 C water bath. 2. Transfer the transfected cells plus DMEM growth medium to a 15-ml conical tubes. 3. Subject the cell suspension to four rounds of freeze/thaw by alternating the tubes between the dry ice-ethanol bath and the 37 C water bath, vortexing briefly after each thaw. 4. Collect cellular debris by centrifugation at 10,000 g for 10 minutes at room temperature. 5. Transfer the supernatant (primary virus stock) to a fresh tube. Viral stocks can be stored for more than one year at 80 C.

19 Principle of the purification procedure First release much of the virus from the transfected cells by multiple cycles of freezing and thawing. The cellular debris is removed by centrifugation leaving the viable virus particles in the supernatant. Supernatant is treated with DNase or Benzonase. The supernatant is further purified by passing it through a 0.45 micron filter. After adding a buffer, the virus solution is slowly passed over a treated filter which adsorbs the virus particles, allowing much of the cellular debris to pass through the filter. Following a wash to remove any bound debris, the virus is eluted off the filter with an elution buffer.

20 Initial Growth of cels in tisue culture vesels HEK293 cells or their variants can be grown in tissue culture treated flasks. For the production of AAV, cells should be at a relatively early passage level. Cells should not remain confluent for more than a few days. Cell that have remained confluent and unpassed for a more than several days can be passed at least one time at a low seeding density to reset the cells into an active growing state. Cells should be seeded into the tissue culture flask at approximately 4 x 10 4 cells per cm 2. Recommended media: DMEM, high glucose with 4 mm glutamine and 10% Fetal Calf Serum. The cell monolayer will become nearly confluent within approximately 2 to 4 days. You will transfect the cultures according to your own protocol with multiple plasmids. After transfection, harvest the cultures within 2 to 5 days by gently shaking or pipetting the cells off of the culture dish.

21 Monitoring Virus Production It is possible to monitor the progress of AAV particle production in the culture by observing phenotypic changes to the AAV-293 cell culture. The most obvious sign of viral production is a color change in the medium from red to orange or yellow. As viral production proceeds, some of the cells will round up and detach from the plate, and can be seen floating in the medium. Generally, three days posttransfection is the optimal time to prepare AAV stocks. negative control 3 days after transfection without the removal of media and floating cells. 3 days after transfection following the removal of media and floating cells.

22 Step 1: Harvest and Centrifugation of Infected Cel Lysate At harvest, pool all the cell lysate and media into one capped vessel and freeze and thaw at least three times. After the third thaw, pour the entire cell lysate into a centrifuge tube or bottle. Spin at 2500 to 2800 rpm for 30 minutes. Collect the supernatant into another clean bottle; discard the pellet. The solution should be free of observable debris. If any flecks of debris are still observed, spin the supernatant again. Before using the bottle top filter unit to clarify the crude virus solution and before running the virus over the purification filter, you can remove much of the contaminating DNA by adding DNase II to the unpurified virus solution.

23 Step 2: Prepare the Bottle Top Filter Unit Place the single glass fiber pre-filter on top of the membrane in the top of the filter unit. Attach the bottle top filter to a vacuum source and pre-wet the filters with approximately 25 ml of PBS or media. This pre-wetting adheres the pre-filter to the 0.45 micron filter. Step 3: Clarify Infected Cel Lysate Pour the spun virus supernatant into the top of the filter unit. Filter all of the spun virus supernatant into the receptor vessel. If it becomes full, disconnect the vacuum source from the filter unit, unscrew the top filter from the bottle and pour the filtrate into a clean new bottle that you have prepared. Reattach the screw-top filter to the bottle and finish the filtration of the virus supernatant.

24 Step 4: Dilute Infected Cel Lysate Measure the volume of the filtered supernatant. Determine one ninth the volume of the filtered supernatant. You will use one part Dilution Buffer 1 to nine parts supernatant (for example: If you have 180 ml of filtered supernatant, add 20 ml of Dilution Buffer). Mix gently but thoroughly. You are now ready to use the virus purification filter assembly. Step 5: Purification Procedure Attach the purification filter assembly to a syringe filled with sterile PBS. Wet the interior of the purification filter assembly by passing about 5 ml of sterile PBS through the filter. Detach the syringe and prepare to attach the tubing assembly to the purification filter assembly.

25 Attach the Large/Small purification filter assembly to the tubing and syringe. Pass the diluted virus supernatant through the purification filter assembly as follows: Pull the virus supernatant through the filters After the first syringe is full of media flow-through, remove the syringe. Empty the syringe into a waste bottle Reattach the syringe to the tubing and continue to pull the remaining virus supernatant through the purification filter, discarding each syringe-full until the entire volume has been filtered. DISCONNECT the two filters and discard the LARGE filter.

26 Step 6: Elute Virus from the Filter Remove the tubing from both sides of the SMALL purification filter. Obtain two new 3-5 ml syringes. Draw at least 1.5 ml of Elution Buffer into one syringe. Only 1.5 ml is sufficient to elute all bound virus, however, you may use more if you desire your virus to be more diluted. Attach the two syringes to the SMALL filter. Pass the elution buffer slowly from the first syringe through the filter into the second syringe. Slowly pass the elution buffer back to the first syringe. Your virus is now in the syringe. The virus is now in a salt solution and can be aliquoted and stored at 4 o C. For long-term storage, 15% glycerol can be added and aliquots stored at -20 o C.

27 Clincial-grade reagents The switch from centrifugation to affinity column purification has greatly simplified vector purification and provides a rapid, efficient, inexpensive procedure to isolate virus of high purity. Furthermore, these procedures can be easily adapted to large-scale, batch purifications.

28 Schematic representation of three different AAV vectors a) Standard raav cassette contains a constitutive promoter or tissue-specific promoter and a polya sequence. Incorporation of WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) and an intron are optional. b) Regulatable raav vector with a bidirectional promoter, based on the Tet-ON system. The transactivator (rtta) is expressed under the control of the CMV promoter. It binds the tetracycline responsive element (TRE) only upon tetracycline addition and stimulates the expression of transgene. The Tet promoter is composed of tetracycline response element and CMV minimal promoter. c) Bicistronic raav vector expresses two genes encoded by the same mrna under the control of a promoter. Capped mrna initiates the translation of the first gene, while the second gene is translated from the IRES.

29 Schematic representation of the trans-splicing system One vector contains a promoter, the 5' portion of the gene and a splice donor sequence (SD). Second vector contains a splice acceptor sequence (SA), the 3' portion of the gene and a polya sequence. Only head-to-tail heterodimer leads to the reconstitution of an intact mrna following transcription and splicing and results in the expression of a functional protein.

30 Good luck!