The AAV non-coding RNA expression vector is a highly efficient vehicle for in vitro and in vivo delivery of non-coding RNAs of interest. 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 AAV 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 AAV non-coding RNA expression vector is first constructed as a plasmid in E. coli. 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 non-coding RNA of interest 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.
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. Due to their low immunogenicity in host organisms, AAV is the ideal viral vector for many animal studies.
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.
List by Serotype
List by Tissue Type
||Smooth muscle, skeletal muscle, CNS, brain, lung, retina, inner ear, pancreas, heart, liver
||Smooth muscle, CNS, brain, liver, pancreas, kidney, retina, inner ear, testes
||Smooth muscle, liver, lung
||CNS, retina, lung, kidney, heart
||Smooth muscle, CNS, brain, lung, retina, heart
||Smooth muscle, heart, lung, pancreas, adipose, liver
||Lung, liver, inner ear
||Smooth muscle, retina, CNS, brain, liver
||Smooth muscle, CNS, brain, retina, inner ear, liver, pancreas, heart, kidney, adipose
||Smooth muscle, skeletal muscle, lung, liver, heart, pancreas, CNS, retina, inner ear, testes, kidney, adipose
||Smooth muscle, lung, liver, heart, pancreas, CNS, retina, kidney
||Liver, heart, kidney, spleen
||Liver, brain, spleen, kidney
||Retina, inner ear
||Recommended AAV serotypes
||AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10
||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV-PHP.eB
||AAV1, AAV2, AAV5, AAV7, AAV8, AAV-DJ/8
||AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV2.7m8
||AAV1, AAV2, AAV6.2, AAV8, AAV9, AAV2.7m8
||AAV1, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV9, AAVrh10
||AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
||AAV1, AAV2, AAV6, AAV8, AAV9, AAVrh10
||AAV1,AAV4, AAV5, AAV6, AAV8, AAV9, AAVrh10, AAV-DJ
||AAV2, AAV4, AAV8, AAV9, AAVrh10, AAV-DJ, AAV-DJ/8
||AAV6, AAV8, AAV9
For further information about this vector system, please refer to the papers below.