Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Summary

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields >1 x 10¹³ vector genomes/mL, providing vector quantities appropriate for large animal studies.

Abstract

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Introduction

Gene therapy utilizing adeno-associated virus (AAV) vectors has made huge strides over the past three decades^1,2. Demonstrated improvements in a diverse range of genetic diseases, including congenital blindness, hemophilia, and diseases of the musculoskeletal and central nervous system, have brought AAV gene therapy to the forefront of clinical research^3,4. In 2012, the European Medicines Agency (EMA) approved Glybera, an AAV1 vector expressing lipoprotein lipase (LPL) for the treatment of LPL deficiency, making it the first marketing authorization for....

Protocol

1. Double plasmid transfection of HEK293 cells in cell stacks

1. Thaw a cryo-vial of HEK293 cells in a bead bath set at 37 °C.
   NOTE: Pre-warm complete DMEM to 37 °C while cells are thawing to ensure the cold temperature does not shock cells when plating. Ensure the cells have a low passage number, ideally less than 20, to ensure optimal growth and transfection efficiency. Ensure that the cells are certified to be mycoplasma-free.
2. Transfer the contents of the cryo-vial dropwise into a 15 mL conical tube containing 10 mL of pre-warmed complete DMEM and centrifuge the cells at 500 x g for 5 min.
3. Aspirate the media, and then resu....

Results

Translation from small rodent models to larger animal models and eventual clinical application presents a significant challenge due to the large amount of AAV required to transduce larger animals and achieve therapeutic effects. To compare transduction efficiency of the rationally designed AAV6.2FF capsid, previously demonstrated a 101-fold increase in transduction efficiency in murine muscle cells compared to AAV6³, mice, hamsters, and lambs were all administered AAV6.2FF expressing a human monoc.......

Discussion

The production of recombinant AAV (rAAV) vectors described in this paper uses common materials, reagents, and equipment found in majority of the molecular biology research labs and facilities. This paper allows for high-quality in vitro and in vivo grade rAAV to be produced by the reader. Above all, this protocol for rAAV production, compared to more tedious protocols involving cesium chloride purification, is efficient and avoids the use of ultracentrifugation. Once HEK293 cells have been transfected, .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm filter	Millipore Sigma	S2GPU05RE
0.25% Trypsin	Fisher Scientific	SM2001C
1-Butanol	Thermo Fisher Scientific	A399-4	CAUTION. Use under a laminar flow hood. Wear gloves
10 chamber cellstack	Corning	3271
1L PETG bottle	Thermo Fisher Scientific	2019-1000
30% Acrylamide/Bis Solution	Bio-Rad	1610158
96-well skirted plate	FroggaBio	FS-96
Adhesive plate seals	Thermo Fisher Scientific	08-408-240
Ammonium persulfate (APS)	Bio-Rad	161-0700	CAUTION. Use under a laminar flow hood. Wear gloves
Blood and Tissue Clean up Kit	Qiagen	69506	Use for DNA clean up in section 4.6 of protocol
Bromophenol blue	Fisher Scientific	B392-5	CAUTION. Use under a laminar flow hood. Wear gloves
Cell Culture Dishes	Greiner bio-one	7000232	15 cm plates
Culture Conical Tube	Thermo Fisher Scientific	339650	15 mL conical tube
Culture Conical Tube	Fisher Scientific	14955240	50 mL conical tube
Dulbecco's Modified Eagle Medium (DMEM) with 1000 mg/L D-glucose, L-glutamine	Cytiva Life Sciences	SH30022.01
ECL Western Blotting Substrate	Thermo Fisher Scientific	32209
Ethanol	Greenfield	P016EA95	Dilute ethyl alcohol(95% vol) to 20% for section 3.7.4 and 70% for section 6.1.1.1
Fetal Bovine Serum (FBS)	Thermo Fisher Scientific	SH30396.03
Glacial acetic acid	Fisher Scientific	A38-500	CAUTION. Use under a laminar flow hood. Wear gloves
Glycerol	Fisher Scientific	BP229-1
Glycine	Fisher Scientific	BP381-500
HBSS with Mg2+ and Ca2+	Thermo Fisher Scientific	SH302268.02
HBSS without Mg2+ and Ca2+	Thermo Fisher Scientific	SH30588.02
HEK293 cells	American Tissue Culture Collection	CRL-1573	Upon receipt, thaw the cells and culture as described in manufacturer’s protocol. Once cells have been minimally passaged and are growing well, freeze a subfraction for future in aliquots and store in liquid nitrogen. Always use cells below passage number 30. Once cultured cells have been passaged more than 30 times, it is recommended to restart a culture from the stored aliquots
HEK293 host cell protein ELISA kit	Cygnus Technologies	F650S	Follow manufacturer’s instructions
Heparin sulfate column	Cytiva Life Sciences	17040703
Kimwipe	Thermo Fisher Scientific	KC34120
L-glutamine (200 mM)	Thermo Fisher Scientific	SH30034.02
Large Volume Centrifuge Tube Support Cushion	Corning	CLS431124	Support cushion must be used with large volume centrifuge tubes uless the centrifuge rotor has the approriate V-bottom cushions
Large Volume Centrifuge Tubes	Corning	CLS431123-6EA	500 mL centrifuge tubes
MgCl2	Thermo Fisher Scientific	7791-18-6
Microcentrifuge tube	Fisher Scientific	05-408-129	1.5 mL microcentrifuge tube, sterilize prior to use
Molecular Grade Water	Cytiva Life Sciences	SH30538.03
N-Lauroylsarcosine sodium salt	Sigma Aldrich	L5125	CAUTION. Wear gloves
NaCl	Thermo Fisher Scientific	BP35810
Optimem, reduced serum medium	Thermo Fisher Scientific	31985070
Pasteur pipets	Fisher Scientific	13-678-20D	Sterilize prior to use
PBS (10x)	Thermo Fisher Scientific	70011044	Dilute to 1x for use on cells
Penicillin-Streptomycin Solution	Cytiva Life Sciences	SV30010
pHelper plasmid	De novo design or obtained from plasmid repository	NA
Pipet basin	Thermo Fisher Scientific	13-681-502	Purchase sterile pipet basins
Polyethylene glycol tert-octylphenyl ether (Triton X-100)	Thermo Fisher Scientific	9002-93-1	CAUTION. Wear gloves
Polyethylenimine (PEI)	Polyscience	24765-1	Follow manufacturer’s instructions to produce a 1L solution. 0.22μm filter and store at 4°C
Polypropylene semi-skirted PCR Plate	FroggaBio	WS-96
Polysorbate 20 (Tween 20)	Thermo Fisher Scientific	BP337-100	CAUTION. Wear gloves
polyvinylidene difluoride (PVDF) membrane	Cytiva Life Sciences	10600023	Use forceps to manipulate. Wear gloves.
Primary antibody	Progen	65158
Protein Ladder	FroggaBio	PM008-0500
Proteinase K	Thermo Fisher Scientific	AM2546
pTrangene plasmid	De novo design or obtained from plasmid repository	NA	Must contain SV40 polyA in genome to be compatible with AAV titration in section 5.0
Pump tubing	Cole-Parmer	RK-96440-14	Optimize length of tubing and containment of virus in fractions E1-E5
RQ1 Dnase 10 Reaction Buffer	Promega	M6101	Use at 10x concentration in protocol from section 4.0
RQ1 Rnase-free Dnase	Promega	M6101
Sample dilutent	Cygnus Technologies	I700	Must be purchased separately for use with HEK293 host cell protein ELISA kit
Secondary antibody, HRP	Thermo Fisher Scientific	G-21040
Skim milk powder	Oxoid	LP0033B
Sodium dodecyl sulfate (SDS)	Thermo Fisher Scientific	28312	CAUTION. Use under a laminar flow hood. Wear gloves
Sodium hydroxide (NaOH)	Thermo Fisher Scientific	SS266-4
SV40 polyA primer probe	IDT		Use sequence in Table X for quote from IDT for synthesis
Tetramethylethylenediamine (TEMED)	Thermo Fisher Scientific	15524010	CAUTION. Use under a laminar flow hood. Wear gloves
Tris Base	Fisher Scientific	BP152-5
Trypan blue	Bio-Rad	1450021
Ultra-Filter	Millipore Sigma	UFC810024	Ultra-4 Centrifugal 10K device must be used, as it has a 10000 molecular weight cutoff
Universal Nuclease for cell lysis	Thermo Fisher Scientific	88702
Universal qPCR master mix	NEB	M3003L
Whatman Paper	Millipore Sigma	WHA1001325
β-mercaptoethanol	Fisher Scientific	21985023	CAUTION. Use under a laminar flow hood. Wear gloves
CAUTION: Refer to the Materials Table for guidelines on the use of dangerous chemicals.