Adenovirus Non-Coding RNA Expression Vector

Overview

The adenovirus non-coding RNA expression vector is a highly efficient vehicle for adenovirus-mediated delivery of non-coding RNAs of interest in several mammalian cell types. Non-coding RNAs include a wide variety of short (<30 nucleotides) and long (>200 nucleotides) functional RNA molecules such as micro RNAs (miRNAs), small interfering RNAs (siRNAs), piwi-interacting RNAs (piRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), large intergenic non-coding RNAs (lincRNAs), intronic long non-coding RNAs (intronic lncRNAs), natural antisense transcripts (NATs), enhancer RNAs (eRNAs) and promoter-associated RNAs (PARs), none of which are translated into proteins, however have been found to play important roles in many cellular processes such as DNA replication, epigenetic regulation, transcriptional and post-transcriptional regulation and translation regulation.

The adenovirus non-coding RNA expression vector uses an RNA polymerase II promoter to drive the expression of the user-selected non-coding RNA gene. This allows the use of tissue-specific, inducible, or variable-strength promoters, enabling a variety of experimental applications. For RNA polymerase II-mediated transcription, the start site is typically in the 3' region of the promoter while the termination site is within the polyA signal sequence. As a result, the transcript generated from this vector does not correspond precisely to the selected non-coding RNA gene, but contains some additional sequences both upstream and downstream. 

The adenovirus non-coding RNA expression vector is first constructed as a plasmid in E. coli. The non-coding RNA of interest along with a user selected promoter is cloned between the two inverted terminal repeats (ITRs) during vector construction. The vector is then transfected into packaging cells, where the region of the vector between the ITRs is packaged into live virus.

When the virus is added to target cells, the DNA cargo is delivered into cells where it enters the nucleus and remains as episomal DNA without integration into the host genome. The non-coding RNA sequence placed in-between the two ITRs during vector construction is introduced into target cells along with the rest of viral genome.

By design, adenoviral vectors lack the E1A, E1B and E3 genes (delta E1 + delta E3). The first two are required for the production of live virus (these two genes are engineered into the genome of packaging cells). As a result, virus produced from the vectors have the important safety feature of being replication incompetent (meaning that they can transduce target cells but cannot replicate in them).

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

References Topic
Cell. 157:77 (2014) Review on non-coding RNAs
Front Genet. 6:2 (2015) Review on functionality of non-coding RNAs
Cell Rep. 14:1867 (2016) Adenovirus-mediated expression of long non-coding RNA
Proc Natl Acad Sci U S A. 91:8802 (1994) The 2nd generation adenovirus vectors
J Gen Virol. 36:59 (1977) A packaging cell line for adenovirus vectors
J Virol. 79:5437 (2005) Replication-competent adenovirus (RCA) formation in 293 Cells
Gene Ther. 3:75 (1996) A cell line for testing RCA

Highlights

The adenovirus non-coding RNA expression vector is derived from the adenovirus serotype 5 (Ad5). It 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.

Advantages

Low risk of host genome disruption: Upon transduction into host cells, adenoviral 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.

Very high viral titer: After our adenoviral vector is transfected into packaging cells to produce live virus, the virus can be further amplified to very high titer by re-infecting packaging cells. This is unlike lentivirus, MMLV retrovirus, or AAV, which cannot be amplified by re-infection. When adenovirus is obtained through our virus packaging service, titer can reach >1011 plaque-forming unit per ml (PFU/ml).

Broad tropism: Cells from commonly used mammalian species such as human, mouse and rat can be transduced with our vector. But some cell types have proven difficult to transduce (see disadvantages below).

Large cargo space: The upper limit size of the adenovirus genome for efficient virus packaging is ~38.7 kb (from 5' ITR to 3' ITR). After excluding the required backbone components for adenovirus gene expression, our vector has about ~7.5 kb of cargo space to accommodate the user's DNA of interest (such as promoter, the non-coding RNA sequence, and fluorescence marker). This is bigger than the ~6.4 kb cargo space in our lentiviral expression vector and is sufficient for most applications.

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.

Safety: The safety of our vector is ensured by the fact that it lacks genes essential for virus production (these genes are engineered into the genome of packaging cells). Virus made from our vector is therefore replication incompetent except when it is used to transduce packaging cells.

Disadvantages

Non-integration of vector DNA: The adenoviral genome does not integrate into the genome of transduced cells. Rather, it exists as episomal DNA, which can be lost over time, especially in dividing cells. 

Difficulty transducing certain cell types: While our adenoviral vectors can transduce many different cell types including non-dividing cells, it is inefficient against certain cell types such as endothelia, smooth muscle, differentiated airway epithelia, peripheral blood cells, neurons, and hematopoietic cells.

Strong immunogenicity: Live virus from adenoviral vectors can elicit strong immune response in animals, thus limiting certain in vivo applications.

Technical complexity: The use of adenoviral 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.

Key components

 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.

Ψ: Adenovirus packaging signal required for the packaging of viral DNA into virus.

Promoter: The promoter that drives your non-coding RNA of interest is placed here.

Non-coding RNA: The non-coding RNA of your interest is placed here.

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

hPGK promoter: Human phosphoglycerate kinase 1 gene promoter. It drives the ubiquitous expression the downstream marker gene.

Marker: A visually detectable gene (such as EGFP). This allows cells transduced with the vector to be selected and/or visualized.

TK pA: Herpes simplex virus thymidine kinase polyadenylation signal. It facilitates transcriptional termination of the upstream ORF.

ΔAd5: Portion of Ad5 genome between the two ITRs minus the E1A, E1B and E3 regions.

3' ITR: 3' inverted terminal repeat.

pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin exist in medium copy numbers in E. coli.

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

PacI: PacI restriction site (PacI is a rare cutter that cuts at TTAATTAA). The two PacI restriction sites on the vector can be used to linearize the vector and remove the vector backbone from the viral sequence, which is necessary for efficient packaging.

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