What is an AAV plasmid?
Adeno-associated viruses (AAV) are small viruses originally discovered as contaminants of adenovirus stocks. One major advantage of using AAV for research is that it is replication-limited and typically not known to cause disease in humans.
Recombinant adeno-associated virus (AAV) is a versatile and popular viral vector used for in vitro and in vivo gene delivery.
How do you make a plasmid AAV?
Why are AAV vectors used?
Adeno-associated virus (AAV) vectors have been frequently used for gene therapy applications due to the lack of pathogenicity and toxicity, ability to infect both dividing and non-dividing cells, and long-term transgene expression profiles. Furthermore, the safety profile in various clinical trials has been good.
When AAV infects a cell alone, its gene expression is repressed (AAV does not replicate), and its genome is incorporated into the host genome (into human chromosome 19). In rare cases, lysis can occur without a helper virus, but usually AAV can not replicate and kill a cell on its own.
What is a Repcap plasmid?
Each rep/cap plasmid contains the gene sequence for AAV serotype 2 replication (rep) protein paired with either serotype 2, 5 or 6 capsid (cap) protein respectively.
Though AAV is not known to cause disease in humans, precautions must be taken due to the possibility of insertional mutagenesis. In addition, helper viruses used to trigger AAV replication may cause disease. Numerous serotypes of adeno-associated virus with different cell tropisms have been isolated.
What is AAV expression cassette?
AAV viral vectors are produced from packaging cell lines following transfection of the AAV construct and the co-infection with a helper virus, such as adenovirus (Ad) or Herpes Simplex Virus (HSV) or via a single infection with a recombinant helper viral vector containing the rAAV genome.
What cells are used for AAV production?
Current AAV expression systems avoid using helper viruses and include the plasmid pHelper instead8, containing essential genes such as E2A and E4. Human embryonic kidney cells (HEK) 293T cells, which express SV40 large T antigen, supply additional necessary proteins9.19-Sept-2019
AAV workflow There are multiple strategies to develop AAV vectors, but typical steps include plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, and fill and finish.
How does AAV gene transfer work?
How does AAV work? Simply put, AAV is transformed from a naturally occurring virus into a delivery mechanism for gene therapy. The viral DNA is replaced with new DNA, and it becomes a precisely coded vector and is no longer considered a virus, as most of the viral components have been replaced.
In many cases, AAV vectors integrate into the host cell genome, which can be important for certain applications, but can also have unwanted consequences.
What does helper plasmid do?
In the context of genetic transformation of plants, a helper plasmid is a plasmid present in Agrobacterium that provides functions required by the bacteria for transferring foreign DNA to a plant cell. They have been extremely important in plant genetic engineering.
AAV, or adeno-associated virus, is currently the main viral vector that researchers use and further develop for gene therapy because it is considered to be non-pathogenic to humans and because it has been successfully altered to prevent its integration into the genome, thus eliminating DNA damage and unpredictable 23-May-2017
Is AAV a viral vector?
AAV as a virus AAV belongs to the genus Dependoparvovirus within the family Parvoviridae. Its life cycle is dependent on the presence of a helper virus, such as AdV, hence its name and taxonomy classification. AAV is found in multiple vertebrate species, including human and non-human primates (NHPs).01-Feb-2019
Timing will highly depend on the capsid type and on the tissue you're infecting. Waiting ~2 weeks is a good starting point for many tissues. AAV-mediated gene expression has been reported to be quite stable, lasting for several years in human clinical trials and in dogs (Wonjo et al., 2013).26-Jul-2016
How does AAV enter nucleus?
Studies of AAV biology reveal that AAV accumulates in the perinuclear region of cells, presumably unable traffic into the nucleus. The mechanism by which AAV enters the nucleus is largely unknown.
The onset of expression can occur as early as 16-24 hours after infection. The high immune response from the target cells are the main limitation of adenoviral systems.Adenovirus vs. AAV.
Why does AAV need adenovirus?
AAV contains two genes, rep and cap, that are required for its replication. With adenoviruses as helpers, the adenoviral E1 and E4 gene products stimulate AAV replication. Furthermore, E4 is required for synthesis of the second DNA strand to produce a double-stranded and functioning transgene molecule.
What is an AAV plasmid?