AAV miR30-Based shRNA Knockdown Vector

Overview

Our AAV miR30-based shRNA knockdown vector system is a highly efficient viral tool for knocking down expression of target gene(s) in vitro or in vivo. Due to the low immunogenicity and cytotoxicity of AAV, this is the ideal shRNA vector for many animal studies. It utilizes AAV-mediated delivery of a polycistronic expression cassette consisting of one or more miR30-based shRNAs (shRNAmiR) targeting gene(s) of interest and a user-selected ORF, where the vector remains as episomal DNA without integration into the host genome. The shRNAmiR transcript is processed by endogenous, cellular micro-RNA pathways to produce mature shRNAs, which facilitate degradation of target gene mRNAs. 

An AAV miR30-based shRNA knockdown vector is first constructed as a plasmid in E. coli. The polycistronic expression cassette consisting of one or more shRNAmiRs targeting gene(s) of interest and a user-selected ORF is cloned between the two inverted terminal repeats (ITRs). It is then transfected into packaging cells along with helper plasmids, where the region of the vector between the two inverted terminal repeats (ITRs) is packaged into live virus. The shRNAmiR expression cassette placed in-between the two ITRs is introduced into target cells along with the rest of viral genome.

The wild-type AAV genome is a linear single-stranded DNA (ssDNA) with two ITRs forming a hairpin structure on each end. It is therefore also known as ssAAV. In order to express genes on ssAAV vectors in host cells, the ssDNA genome needs to first be converted to double-stranded DNA (dsDNA) through two pathways: 1) synthesis of second-strand DNA by the DNA polymerase machinery of host cells using the existing ssDNA genome as the template and the 3' ITR as the priming site; 2) formation of intermolecular dsDNA between the plus- and minus-strand ssAAV genomes. The former pathway is the dominant one.

AAV genomic DNA forms episomal concatemers in the host cell nucleus. In non-dividing cells, these concatemers can remain for the life of the host cells. In dividing cells, AAV DNA is lost through the dilution effect of cell division, because the episomal DNA does not replicate alongside host cell DNA. Random integration of AAV DNA into the host genome can occur but is extremely rare. This is desirable in many gene therapy settings where the potential oncogenic effect of vector integration can pose a significant concern.

Unlike conventional shRNA vectors, which utilize RNA polymerase III promoters such as U6, miRNA-based shRNA systems are placed under the control of standard RNA polymerase II promoters. This allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications not possible with constitutive U6 promoters.

The ability of RNA polymerase II promoters to efficiently transcribe long transcripts in the miRNA-based shRNA systems also provides additional advantages relative to other knockdown vector systems. Multiple shRNAmiRs can be transcribed as a single polycistron, which is processed to form mature shRNAs within the cell. This allows knockdown of multiple genes or targeting of multiple regions within the same gene using a single transcript. As a result, this vector is available for expressing either single or multiple shRNAmiRs. Secondly, in this vector system, a user-selected protein coding gene is also positioned within the same polycistron as the shRNAmiRs. The expression of this ORF can be used to directly monitor shRNA transcription (if a marker ORF is used) or can be used for other purposes requiring co-expression of an ORF and shRNA(s).

A major practical advantage of AAV is that in most cases AAV can be handled in biosafety level 1 (BSL1) facilities. This is due to AAV being inherently replication-deficient, producing little or no inflammation, and causing no known human disease.

Many strains of AAV have been identified in nature. They are divided into different serotypes based on different antigenicity of the capsid protein on the viral surface. Different serotypes can render the virus with different tissue tropism (i.e. tissue specificity of infection). When our AAV vectors are packaged into virus, different serotypes can be conferred to the virus by using different capsid proteins for the packaging. The table below lists different AAV serotypes and their tissue tropism.

Serotype Tissue tropism
AAV1 Smooth muscle, CNS, lung, retina, inner earpancreas, heart, liver
AAV2 Smooth muscle, CNS, liver, kidney, retina, inner ear
AAV3 Smooth muscle, liver, lung
AAV4 CNS, retina, lung, kidney
AAV5 Smooth muscle, CNS, lung, retina
AAV6 Smooth muscle, heart, lung, adipose, liver
AAV6.2 Lung, liver, inner ear
AAV7 Smooth muscle, retina, CNS, liver
AAV8 Smooth muscle, CNS, retina, inner earliver, pancreas, heart, kidney, adipose
AAV9 Smooth muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney
AAVrh10 Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney
AAV-DJ Liver, heart, kidney, spleen
AAV-DJ/8 Liver, brain, spleen
AAV-PHP.eB CNS
AAV-PHP.S PNS
AAV2-retro Spinal nerves 
AAV2-QuadYF Endothelial cell
AAV2.7m8 Retina, inner ear

Tissue type Recommended AAV serotypes
Smooth muscle AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
CNS AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB
PNS AAV-PHP.S
Brain AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
Retina AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8
Inner ear AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
Lung AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
Liver AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Pancreas AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
Heart AAV1,AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
Kidney AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
Adipose AAV6, AAV8, AAV9
Testes AAV2, AAV9
Spleen AAV-DJ, AAV-DJ/8
Spinal nerves AAV2-retro
Endothelial cells AAV2-QuadYF

For further information about this vector system, please refer to the papers below.

References Topic
Cell Rep. 5:1704 (2013) An Optimized microRNA Backbone for Effective Single-Copy RNAi
Methods Enzymol. 507:229-54 (2012) Review of AAV virology and uses
Curr Opin Pharmacol. 24:59-67 (2015) AAV use in gene therapy, and serotype differences

Highlights

Our AAV miR30-based shRNA knockdown vector incorporates an optimized micro-RNA system for knockdown of target gene(s) and is optimized for high copy number replication in E. coli, high-titer packaging of live virus, efficient transduction of host cells, and high-level transgene expression. This viral vector can be packaged into virus using all known capsid serotypes, is capable of very high transduction efficiency, and presents low safety risk. A user-selected promoter drives expression of a polycistronic expression cassette containing a user-selected ORF and one or more shRNAmiRs with optimized miR30-based sequences to mediate efficient shRNA processing and target gene(s) knockdown.

Advantages

Promoter choice: Unlike standard shRNA systems, which utilize RNA polymerase III promoters such as U6, miR30-based shRNAs can be transcribed by diverse RNA polymerase II promoters. This also enables the use of tissue-specific or inducible promoters.

Multiple shRNA co-expression: Because RNA polymerase II efficiently transcribes long RNAs, multiple shRNAmiRs can be expressed as a polycistron from a single promoter. Therefore, this vector is available for expressing either single or multiple shRNAmiRs.  

Co-expression of a reporter ORF: A user-selected gene of interest or reporter gene ORF is co-expressed with the shRNAmiRs, as a polycistron. This facilitates direct monitoring of shRNA transcription.

Safety: AAV is the safest viral vector system available. AAV is inherently replication-deficient and is not known to cause any human diseases.

Low risk of host genome disruption: Upon transduction into host cells, AAV vectors remain as episomal DNA in the nucleus. The lack of integration into the host genome can be a desirable feature for in vivo human applications, as it reduces the risk of host genome disruption that might lead to cancer.

High viral titer: Our AAV vector can be packaged into high titer virus. When AAV virus is obtained through our virus packaging service, titer can reach >1013 genome copy per ml (GC/ml).

Broad tropism: A wide range of cell and tissue types from commonly used mammalian species such as human, mouse and rat can be readily transduced with our AAV vector when it is packaged into the appropriate serotype. But some cell types may be difficult to transduce, depending on the serotype used (see disadvantages below).

Effectiveness in vitro and in vivo: Our vector is often used to transduce cells in live animals, but it can also be used effectively in vitro.

Disadvantages

Difficulty transducing certain cell types: Our AAV vector system can transduce many different cell types including non-dividing cells when packaged into the appropriate serotype. However, different AAV serotypes have tropism for different cell types, and certain cell types may be hard to transduce by any serotype.

Technical complexity: The use of viral vectors requires the production of live virus in packaging cells followed by the measurement of viral titer. These procedures are technically demanding and time consuming relative to conventional plasmid transfection. These demands can be alleviated by choosing our virus packaging services when ordering your vector.

Key components

Single miR30-shRNA AAV shRNA knockdown vector

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Promoter: Drives transcription of the downstream ORF and shRNAmiR polycistron. This is an RNA polymerase II promoter, rather than an RNA polymerase III promoter such as U6.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest or reporter gene is placed here. This can be used to monitor shRNA expression.

5' miR-30E: An optimized version of the human miR30 5’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.

3' miR-30E: An optimized version of the human miR30 3’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.

miR30-shRNA: This sequence is derived from your target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus and enhances expression of transgenes.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination and polyadenylation of the upstream ORF and shRNAmiR polycistron.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

Multiple miR30-shRNA AAV shRNA knockdown vector

5' ITR: 5' inverted terminal repeat. In wild type virus, 5' ITR and 3' ITR are essentially identical in sequence. They reside on two ends of the viral genome pointing in opposite directions, where they serve as the origin of viral genome replication.

Promoter: Drives transcription of the downstream ORF and shRNAmiR polycistron. This is an RNA polymerase II promoter, rather than an RNA polymerase III promoter such as U6.

Kozak: Kozak consensus sequence. It is placed in front of the start codon of the ORF of interest because it is believed to facilitate translation initiation in eukaryotes.

ORF: The open reading frame of your gene of interest or reporter gene is placed here. This can be used to monitor shRNA expression.

5' miR-30E: An optimized version of the human miR30 5’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.

3' miR-30E: An optimized version of the human miR30 3’ context sequence. Facilitates maturation and processing of the shRNA and separation from the tandemly transcribed ORF and other shRNAs.

miR30-shRNA #1: This sequence is derived from your first target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.

miR30-shRNA #2: This sequence is derived from your second target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.

miR30-shRNA #3: This sequence is derived from your third target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.

miR30-shRNA #4: This sequence is derived from your fourth target sequence and is transcribed to form the stem portion of the “hairpin” structure of the shRNA.

Regulatory element: Allows the user to add the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). WPRE enhances virus stability in packaging cells, leading to higher titer of packaged virus and enhances expression of transgenes.

BGH pA: Bovine growth hormone polyadenylation signal. It facilitates transcriptional termination and polyadenylation the upstream ORF and shRNAmiR polycistron.

3' ITR: 3' inverted terminal repeat. See description for 5’ ITR.

Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.

pUC ori: pUC origin of replication. Plasmids carrying this origin exist in high copy numbers in E. coli.

 

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