Adeno-associated Virus (AAV) Packaging

Efficient transfection of viruses

Ubigene provides lentivirus, adenovirus and adeno-associated virus (AAV) with different sizes and grades of purification. Our products can easily achieve knockout (KO), overexpression and knockdown in vitro and in vivo.

Comparison of different viruses:

Type	Lentivirus	Adenovirus	AAV
Origin	HIV-1	Adenovirus type 5	AAV2
Genome	Two linear and continuous single-stranded RNAs	A linear and continuous double-stranded DNA	A linear and continuous single-stranded DNA
Genome size	~9.2 kb (WT)	~36 kb (WT)	~4.7 kb (WT)
Expression Duration	long-term stable expression	Transient expression	Long-term expression
Integrate to cell genome	Yes	No	Low frequency
Immunogenicity	Low	High	Very low
Capacity	~6.4 kb	~8.3 kb	~4.7 kb
Advantages	· Wide range of applications; · High transduction efficiency; · stable hereditary.	· High transduction efficiency; · Large capacity; · Expandable.	· Low immunogenicity; · High safety; · High specificity.

AAV Packaging Technical Detail

Adeno-associated virus (AAV) is a kind of Parvovirus, which genome is single-stranded DNA. It can infect both divisive and non-divisive cells. Adenoviruses or herpes viruses are usually needed to assist AVV to replicate in vivo. Recombinant AAV (rAAV) is a viral vector that combines the AAV2 genome with capsid protein genomes of different serotypes. CDS or RNAi interference sequences of the target genes can be inserted into the rAAV plasmid. AAV is mainly used in vivo injection because it has low immunogenicity and long-term expression.

Ubigene's AAV will be purified by ultracentrifugation, and its genome copy will be titrated by qPCR. The titer of AAV ranged from 10^12 to 10^13 v. g. / ml.

Different serotypes of AAVs have different transduction efficiency for tissues and cells. There are 10 different AAV serotypes in our packaging service. It is highly recommended that customers should select the corresponding AAV serotypes for different tissues:

Serotype	Tissues
AAV1	SM,CNS,lung,retina,pancreas,heart,liver
AAV2	SM,SNS,liver,kidney,retina
AAV4	CNS,retina,lung,kidney
AAV5	SM,CNS,lung,retina
AAV6	SM,heart,lung,adipose,liver
AAV7	SM,retina,CNS,liver
AAV8	SM,CNS,retina,liver,pancreas,heart,kidney,adipose
AAV9	SM,lung,liver,heart,pancreas,CNS,retina,testes,kidney
AAVrh10	SM,lung,liver,heart,pancreas,CNS,retina,kidney
AAVPHP.B	CNS
AAVDJ/8	SM, liver, CNS, retina
AAVPHP.S	PNS
AAVPHP.eB	CNS（stronger than AAVPHP.B）

Product Specs

Specs	Titer	Turnaround	Application
Standard Size	1*10^12 GC	2-3weeks	Cell transduction
Ultra-purified large size	1*10^13 GC	2-3weeks	In vivo injection

Work Flow and Validation：

Knockout AAV

The gene knockout AAV vector would be constructed and packaged as AAV. After ultracentrifugation purification, it can be used for in vivo injection. Cells infected with AAV can stably express gRNA and Cas9 protein to knockout the target gene.

Gene knockout of AAV vector selection

YKO series	Vector	Reporter gene; Selection marker
AAV YKO plasmid	YKO-AAV001-gRNA	EGFP
AAV YKO plasmid	YKO-AAV002-gRNA	mCherry

Due to the limited capacity of AAV, SaCas9 protein can be used. SaCas9 and gRNA on the same construct can increase transduction efficiency.

Expression AAV

The gene overexpression vector would be constructed and packaged as AAV. After ultracentrifugation purification, it can be used for in vivo injection.

YOE series	Vector	Reporter gene; Selection marker
AAV YOE plasmid	YOE-AAV001	EGFP
AAV YOE plasmid	YOE-AAV002	mCherry

Customers can choose a variety of tissue-specific promoters that can achieve tissue-specific expression in vivo，Click to show shouzhi

Promoter	Description	Tissue	Cell type	Reference
Nes	Rat nestin intron 2 enhancer fused to mouse Hsp68 minimal promoter	Nervous system at embryonic stage	Neural stem/pro-genitor cells	Eur J Neurosci. 9:452 (1997); J Neurosci Res. 59:321 (2000)
Camk2a_short	Mouse α-calcium-calmodulin dep-endent kinase II promoter (1.3 kb)	Brain	Pyramidal neurons	Neuroscience. 161:441 (2009)
SYN1	Human synapsin I promoter	Brain	Mature neurons	Mol Ther. 5:509 (2002)
Nes	Rat neuron-specific enolase promoter	Nervous system	Various neurons	Neuron. 5:187 (1990); J Mol Neurosci. 8:63(1997)
GFAP_short	Human glial fibrillary acidic protein promoter (0.68 kb)	Brain	Astrocytes	Glia. 56:481 (2008)
K14	Human keratin 14 promoter	Epidermis	Keratinocytes	Gene. 153:297 (1995)
cTnT	Chicken cardiac troponin T promoter	Heart	Cardiomyocytes	Am J Physiol Cell Physiol. 286:C556 (2004); Gene Ther. 18: 43 (2011); Circ Res. 115:354 (2014)
alphaM-HC_short	Mouse α-cardiac myosin heavy chain promoter (2.8 kb)	Heart	Cardiomyocytes	Neuroscience. 161:441 (2009)
Myog	Mouse myogenin promoter	Muscle	Myoblasts	Genes Dev. 7:1277 (1993)
MHCK7	Composed of enhancer/promoter regions of murine muscle creatine kinase (MCK) and enhancer region of α-myosin heavy-chain genes.	Genes Dev. 7:1277 (1993)	Differentiated postmitotic stria-ted muscle cells	Mol Ther. 15:320 (2007); Sci Transl Med. 9(418) (2017)
SM22a	Mouse transgelin (a.k.a. Sm22a) promoter	Adult smooth muscle	Vascular smooth muscle cells	J Cell Biol. 132:849 (1996)
EnSM22a	The chimeric vascular smooth muscle-specific enhancer/promoter (The rabbit myosin heavy-chain enh- ancer inserted upstream of a truncated mouse SM22a promoter)	Adult smooth muscle	Vascular smooth muscle cells	J Pharmacol Exp Ther. 329: 775 (2009)
Tie1	Mouse tyrosine kinase with immunoglobulin-like and EGF-like domains 1 promoter	Blood vessel	Endothelial cells	J Cell Sci. 115:2075 (2002)
CD68_short	Human CD68 promoter, short, recommended for in vitro use	Blood	Monocytes and macrophages	Gene Ther. 19:1041 (2012)
Alb	Mouse albumin promoter	Liver	Mature hepatocytes	Genes Dev. 1:268 (1987)
TBG	Human thyroxine-binding globulin promoter	Liver	Hepatocytes	Gene. 506:289 (2012)
MMTV	Mouse mammary tumor virus long terminal repeat (LTR)	Mammary gland	Ductal cells of the salivary gland, mammary epithelial cells	Nucleic Acids Res. 25:4323 (1997)
HIP	Human insulin promoter	Pancreas	β cells	Cell. 52:773 (1998); Proc Natl Acad Sci U S A. 100: 6688 (2003)
Pdx1	Mouse pancreatic and duodenal homeobox 1 promoter	Embryonic develo-ping pancreas and adult pancreatic islets	Pancreatic proge-nitor cells and pancreatic β cells	Development. 129:2447 (2002)
SP-B	Human surfactant protein B promoter	Lung	AT II cells (alveolar type II epithelial cells) and Clara cells (bronchiolar epithelial cells)	J Biol Chem. 270:24852 (1995); Gene Ther. 14:1461 (2007)
CD144	Human cadherin 5 (a.k.a. VE-Cadherin) promoter	Lung, heart, ovary, spleen and kidney glomeruli	Vascular endothelial cells	Oncogene. 24:2992 (2005)

Knockdown AAV

The gene knockdown vector would be constructed and packaged as AAV. After ultracentrifugation purification, it can be used for in vivo injection.

YSH series	Vector	Reporter gene; Selection marker
AAV YSH plasmid	YSH-AAV001-shRNA	EGFP
AAV YSH plasmid	YSH-AAV002-shRNA	mCherry

Customers can choose a variety of tissue-specific promoters that can achieve tissue-specific expression in vivo.

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Adeno-associated Virus (AAV) Packaging

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