Design method for adeno-associated heterotrimeric viral vectors

Summary

A A V coat peptide display library, error-prone P C R and D N A recombination . A A V serotypes with different histophilicity can be generated by natural mutation and recombination, thereby transducing A A V into some difficult-to-sense cell types. Alternatively, heterotrimeric vectors can be constructed from different serotypes using serum homologs, nucleocapsid transplantation, or labeling rescue techniques. Because AAV 2 is the best understood serotype, this serotype is generally used as a template for constructing AAV heterozygous vectors. This chapter focuses on methods for constructing heterotrimeric A A V vectors with expanded histophilicity.

Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".

Operation method

Construction of A A V serotype vectors

Move

Construction of A A V serotype vectors Materials

reagents

benzonase nucleic acid enzyme (S g m a -A ld ric h )

Cellular: human embryonic kidney cell 2; rhinocellular (A T C C)

Cesium chloride (Sigma-Aldrich)

DMEM medium with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma-Aldrich)

Leucovorin Geupeptin) (3p g /m l) dissolved in double distilled water (Sigm a-A ldrich)

Phosphate buffer without C a2V M g 2+ (P B S ) (Sigma-Aldridi)

Plasmid

A A V serotype helper plasmids (e.g., p X R l , p X R 2 , p X R 3 , p X R 4 , and p X R 5 ; U N C carrier core)

Adenovirus helper plasmids (e.g., p X X 6-80; U N C carrier core)

A A V transgene packaging frame (e.g. p T R -C M V -G F P ; U N C vector core)

Superfect transfection kit (Q I A G E N )

T ry p sin -E D T A (Sigm a-A ld rich )

Instrumentation

Centrifugation and Ultracentrifugation System (Sorvall R T 6000B )

Centrifuge tubes (16m m X 76m m ) (Beckman polyallomer Quick-Seal)

Analyzer card (Pierce Slide-A-L yz er) (10 OOOkDa m. w. cutoff)

Ultrasonic Crusher (3 m m probe)

Ultracentrifuge with T L N l O O head (B e c k m a n Optima T L X )

METHODS

1- Digest confluent H E K 293 cells (about 2X ¹⁰⁷ ) on 15 c m plates with trypsin. Dilute with medium at a ratio of I : 3. Reinoculate and overnight.

2- Transfect 7 ug of p T R -C M V - G F P (transgenic packaging frame), 15 pings of P X X 6-80 (adenoviral helper plasmid), and IOug of A A V serotyped helper plasmid (p X R l ~ PX R 5) per plate (5 to 10 plates in total), according to the manufacturer's instructions using the Superfect Transfection Kit.

3 . Harvest cells by cell scraping 48 to 50 h after transfection. Unless otherwise specified, all the following operations are performed on ice.

4.1000r/min (Sorvall R T 6000B ) Centrifuge cells. Resuspend with leupeptin.

5- Cells were broken by ultrasound with 25 bursts at 50% duty and 2 output control setting.

6. lOOOr/min centrifuge to remove cell debris.

7- Add benzonase nuclease to a final concentration of I.2U /ul.

8.37°C |float incubation for 30~60m i n .

9- Add ○ - 6 g of cesium chloride per ml of lysate. Transfer the suspension to a B e c k m a n tube.

1 0 . Centrifuge the mixture 10 0 000r/m in for 4 h using a Beckm an Optima T L X ultracentrifuge with a T L N 1 0 0 turntable.

11 - The highest elution peak in the gradient was determined by spot hybridization using a radiolabeled probe (G F P transgene).

12- Dialyze the elution peaks with Pierce dialysis cards using PBS overnight in an ice chamber and then determine the viral titer (number of vector genes per mL).

Construction of chimeric vectors by nucleocapsid transfer

method

1 . Digest confluent H E K 2 9 3 cells (about 2 X ¹⁰⁷ ) on 15 c m plates with trypsin. Dilute with medium in the ratio i : 3. Reinoculate and leave overnight.

2. Transfect 7 ug of pTR-CMV-GFP (transgenic packaging frame), 15ug of P X X 6-80 (adenoviral helper plasmid), and 10 ug of mixed AAV serotyped helper plasmid (p X RL-5) per plate (5 to 10 plates in total) with the Superfect Transfection Kit according to the manufacturer's instructions.

In order to construct AAV particles with different serotype compositions, the amount of each transfected AAV serotype helper plasmid should be varied according to the estimated ratio. For example, if it is desired to mix AAV1 and AAV2 at a ratio of 19:1, then 9.5 tons of pXRl and 0.5ug of pXR2 should be added to the transfection mixture. 5ug of pXR2.

3- The following steps can be performed as steps 3 to 12 in Scheme 1.

Construction of mosaic plasmids by marker rescue material

reagents

Benzonase (Sigm a-A ldrich)

Cells: Hela cells (A T C C ) or other target cell types for selection/circulation and H E K 293 cells (A T C C )

Cesium Chloride (Sigma-Aldrich)

DMEM medium with 10% fetal bovine serum and 1 % Penicillin/Streptomycin (Sigma-Aldrich)

Ieupeptin (3ug/ml) (Sgma-Aldrich)

Phosphate Buffer Solution (PBS) (Sigma-Aldrich)

Plasmid

AAV-assisted shuttle plasmids (e.g. P X R 2 A N )

The P X R 2A N shuttle plasmid used here has a single A/ " I site at the start of the coat gene and a single N 〇 iI site at the end of the coat gene. This facilitates the cloning of all PCR products directly into the AAV helper plasmid for sequencing and vector construction.

AAV serotype helper plasmids (e.g., p X R l, p X R 2, PX R 3, p X R 4, and p X R 5; U NC vector cores)

Adenovirus helper plasmids (e.g. pX X 6-80; UNC carrier core)

AAV transgenic packaging frames (e.g., p T R -C M V -G F P; UNC carrier core)

AAV serovar plasmids with R e p , C a p , and I T R regions (Unc carrier core)

Required mutant AV serovars (e.g. H/N 3 761 non-infectious AV2 mutant)

Primers for amplification of AAV serotype CAPP protein PCR fragments (e.g. AAV 3b )

Superfect Transfection Kit (Q IA G E N )

Trypsin-EDTA (Sigma-Aldrich)

Virus: Adenovirus along 309 (Unc carrier core)

Instrumentation

The materials and equipment required to construct the inlay carrier are the same as in Option 1.

Methods

1- The H /N 3761 A A V 2 mutant plasmid was digested with P m I I .

2 . Inoculate approximately IO6 H E K 293 cells evenly onto 6 cm plates.

3. Cells were transfected with lM g of digested H /N 3761 A A V 2 mutant plasmid and Ipg A A V 3b P C R fragments using the Superfect Transfection Kit according to the manufacturer's instructions.

4 . Immediately after transfection, 293 cells were transfected along 309 with 4-fold infected replicates of adenovirus. The virus was allowed to adsorb for l h.

5. Replace the virus-containing medium with 5 ml of fresh medium.

6 . Harvest the cells by cell scraping 48~60 h after transfection.

All the following operations are performed on ice unless otherwise specified.

7. Centrifuge cells at 1000 r/min. (SorvallRT 6000B).

8 . Resuspend the cells in PBS and freeze-thaw the cells 3 times to release the virus.

9 - Incubate the lysate at 56°C for 30 m i n to inactivate the adenovirus.

10. Add IOOm I lysate to HeLa cells containing 4-fold infections with plural adenovirus along 309.

Depending on the needs of the study, other cell types may be substituted.

11. Repeat steps 5 to 10.

12-After each cycle, spot hybridization of virus-infected cell lysates was performed to confirm that viable cell type-specific mosaic particles were produced.

13- P C R analysis was used to determine the gene sequences of the mosaic particles obtained in the cycle.

14 - To determine the yield of recombinant AAV, a triple plasmid transfection was performed with plasmids pTR-CMV-GFP, PXX6-80 and the inlay AAV helper plasmid according to Scheme 1.

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