Digital Droplet PCR Method for the Quantification of AAV Transduction Efficiency in Murine Retina

Summary

This protocol presents how to quantify AAV transduction efficiency in mouse retina using digital droplet PCR (dd-PCR) together with small scale AAV production, intravitreal injection, retinal imaging, and retinal genomic DNA isolation.

Abstract

Many retinal cell biology laboratories now routinely use Adeno-associated viruses (AAVs) for gene editing and regulatory applications. The efficiency of AAV transduction is usually critical, which affects the overall experimental outcomes. One of the main determinants for transduction efficiency is the serotype or variant of the AAV vector. Currently, various artificial AAV serotypes and variants are available with different affinities to host cell surface receptors. For retinal gene therapy, this results in varying degrees of transduction efficiencies for different retinal cell types. In addition, the injection route and the quality of AAV production may also affect the retinal AAV transduction efficiencies. Therefore, it is essential to compare the efficiency of different variants, batches, and methodologies. The digital droplet PCR (dd-PCR) method quantifies the nucleic acids with high precision and allows performing absolute quantification of a given target without any standard or a reference. Using dd-PCR, it is also feasible to assess the transduction efficiencies of AAVs by absolute quantification of AAV genome copy numbers within an injected retina. Here, we provide a straightforward method to quantify the transduction rate of AAVs in retinal cells using dd-PCR. With minor modifications, this methodology can also be the basis for the copy number quantification of mitochondrial DNA as well as assessing the efficiency of base editing, critical for several retinal diseases and gene therapy applications.

Introduction

Adeno associated viruses (AAVs) are now commonly used for a variety of retinal gene therapy studies. AAVs provide a safe and efficient way of gene delivery with less immunogenicity and fewer genome integrations. AAV entry into the target cell occurs through endocytosis, which requires binding of receptors and co-receptors on the cell surface^1,2. Therefore, the transduction efficiency of AAVs for different cell types depends mainly on the capsid and its interactions with the host cell receptors. AAVs have serotypes and each serotype can have distinct cellular/tissue tropisms and transduction efficiencies. There....

Protocol

All experimental protocols were accepted by the Sabanci University ethics committee and experiments were conducted in accordance with the statement of 'The Association for Research in Vision and Ophthalmology' for the use of animals in research

1. Small scale AAV production¹²

1. Culture HEK293T cells using 15 cm plates in complete 10 mL of DMEM/10% FBS until 70-80% confluency.
2. Prepare the transfection mixture with 20 µg of helper plasmid (pHGT1-Adeno1), 7 µg of capsid plasmid and 7 µg of AAV2-CBA-tdTomato-WPRE vector and 136 µL of PEI solution (1 mg/mL) ....

Results

Small scale AAV production is a fast and efficient method that provides vectors for intravitreal injections (Figure 1). Small scale AAV production usually gives titers within the range of 1 x 10¹² GC/ml which is sufficient to detect reporter expression in the retina (Figure 2). Titering of AAV using dd-PCR gives consistent results. ITR2 and WPRE specific primers are routinely used and the starting concentration of each target molecule was calculated w.......

Discussion

In this protocol, we generated two AAV vectors that have different capsid proteins and then titered them accordingly. One of the most crucial steps of this protocol is to produce sufficient amounts of AAVs that will yield detectable reporter expression after the transduction^12,13.

Titering of AAVs is also an important factor to adjust dosages of AAV for intravitreal injections. Once these important criteria are achieved, it is feasi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-Well Semi-Skirted ddPCR plates	BioRad	12001925	ddPCR
Amicon Filter	Millipore	UFC910096	AAV
C1000 TOUCH 96 DEEP WELLS	BioRad	1851197	ddPCR
C57BL/6JRj mice strain	Janvier	C57BL/6JRj	Mice
DG8 gaskets	BioRad	1863009	ddPCR
DG8 Cartridges	BioRad	1864008	ddPCR
DMEM	Lonza	BE12-604Q	AAV
DPBS	PAN BIOTECH	L 1825	AAV
Droplet generation oil eva green	BioRad	1864006	ddPCR
Droplet reader oil	BioRad	1863004	ddPCR
FBS	PAN BIOTECH	p30-3306	AAV
Foil seals for PX1 PCR Plate sealer	BioRad	1814040	ddPCR
Insulin Syringes	BD Medical	320933	Intravitreal injection
Isoflurane	ADEKA ILAC SANAYI VE TICARET	N01AB06	anesthetic
Microinjector MM33	World Precision Instruments	82-42-101-0000	Intravitreal injection
Micron IV	Phoenix Research Labs	Micron IV	Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.
Mydfrin (%2.5 phenylephrine hydrochloride)	Alcon	S01FB01	pupil dilation
Nanofil Syringe 10 μl	World Precision Instruments	NANOFIL	Intravitreal injection
Needle RN G36, 25 mm, PST 2	World Precision Instruments	NF36BL-2	Intravitreal injection
PEI-MAX	Polyscience	24765-1	AAV
Penicillin-Streptomycin	PAN BIOTECH	P06-07100	AAV
Plasmid pHGT1-Adeno1	PlasmidFactory	PF1236	AAV
Pluronic F-68	Gibco	24040032	AAV
PX1 PCR Plate Sealer system	BioRad	1814000	ddPCR
QX200 ddPCR EvaGreen Supermix	BioRad	1864034	ddPCR
QX200 Droplet Reader/QX200 Droplet Generator	BioRad	1864001	ddPCR
SPLITTER FORCEP WATCHER MAKER - LENGTH = 13.5 CM	endostall medical	EJN-160-0155	Retina isolation
Steril Syringe Filter	AISIMO	ASF33PS22S	AAV
Tissue Genomic DNA Kit	EcoSpin	E1070	gDNA isolation
Tobradex (0.3% tobramycin / 0.1% dexamethasone)	Alcon	S01CA01	anti-inflammatory / antibiotic
Tropamid (% 0.5 tropicamide)	Bilim Ilac Sanayi ve Ticaret AS.	S01FA06	pupil dilation
Turbonuclease	Accelagen	N0103L	AAV
Viscotears (carbomer 2 mg/g)	Bausch+Lomb	S01XA20	lubricant eye drop