Over the years gene therapies have gained significant traction as therapeutic interventions owing to their potential in targeting the underlying cause of a disease at cellular level. Several viral and non-viral vectors are used for introducing genes into cells. Viral agents have a high transferring efficiency; however, these are difficult to handle due to their toxicity. On the other hand, nonviral agents have lower transferring efficiency and less toxicity. Amongst various viral vectors, adeno-associated viral vector is one such gene delivery viral vector which has garnered significant attention in the field. These vectors are the most widely used platform for delivering genes to treat various human disorders. These are non-enveloped virus that may be modified to transfer DNA to target cells. The ability to manufacture recombinant AAV particles devoid of viral genes and yet containing DNA sequences of interest for various therapeutic applications is currently one of the safest gene therapy strategies. Moreover, gene therapies using recombinant AAV are safe and endure in majority of the clinical evaluations. These studies, along with basic biology research and leveraging innovations have aided the field’s expansion. The global adeno-associated viral vectors / AAV vector market is anticipated to grow at a CAGR of around 14%, till 2035, according to Roots Analysis. Driven by the rising demand for vectors in the field of gene therapy, the adeno-associated viral vectors market is expected to grow at a steady rate in the coming decade.
ADENO-ASSOCIATED VIRUS VECTORS OVERVIEW
Adeno-associated virus (AAV) is a small-sized virus of the Parvoviridae family that has a single stranded DNA genome. This virus is capable of infecting a broad range of host cells, including both dividing and non-dividing cells. It is a non-pathogenic virus that does not generate an immune response in most patients. The adeno-associated viral vector genome comprises of inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames (ORFs), namely rep and cap. Each ITR sequence consists of 145 bases that have the ability to form a hairpin structure. These sequences are required for the primase-independent synthesis of a second DNA strand and the integration of the viral DNA into the host cell genome. The rep genes encode proteins that are required for the AAV life cycle and site-specific integration of the viral genome. Cap genes encode the capsid proteins, namely VP1, VP2 and VP3.
STRUCTURE AND DESIGN
The AAV genome is built of single-stranded DNA, which is around 4.8 kilobase long. It consists of ITR (Inverted Terminal Repeat) sequence composed of 145 bases. An important attribute of the sequence is its capability to form a hairpin. This secondary structure offers a free 3′ hydroxyl group for the activation of viral DNA replication via a self-priming strand-displacement process involving leading-strand synthesis and double-stranded replicative intermediates. The ITRs are known to be essential for both adeno-associated viral DNA integration and rescue from the host cell genome (19th chromosome in humans), as well as effective encapsidation of the adeno-associated viral DNA along with formation of fully formed, deoxyribonuclease-resistant AAV particles. In addition, the production of adeno-associated viral vectors requires the use of two plasmids. One contains the rep and cap genes along with certain other helper genes, while the second plasmid contains a cytomegalovirus (CMV) promoter, multiple cloning sites, the SV40 polyA signal and ITR sequences. In terms of arrangement, the rep and cap genes are placed in trans position, while the ITR sequences and the therapeutics genes are arranged cis to the vector.
APPLICATION OF ADENO-ASSOCIATED VIRAL VECTOR
Adeno-associated viral vectors, unlike other viruses, are not pathogenic and have low immunogenicity. These are designed to provide additional safety benefits by omitting all viral genes, including those responsible for chromosomal integration, which reduces the risk of innate immune activation.
- Adeno-associated viral vectors have the ability to infect a wide range of cell types, including muscle, liver, and brain cells. There are several different adeno-associated viral serotypes with minor differences in their viral capsids. It is worth highlighting that new adeno-associated viral capsids are being invented, and recent advancements in adeno-associated virus technology have allowed us to create new and refined capsids with greater specificity.
- Through homology directed repair (HDR), adeno-associated viral vectors can accelerate and improve DNA repair. This property makes them useful for addressing disease-related mutations.
- Adeno-associated viral vectors sustain transgene expression in postmitotic, long-lived cell types for decades.
- Adeno-associated viral vectors offer several advantages as compared to other viruses used. Primarily, the vectors are produced from a non-pathogenic virus that is replication deficient by nature. Vectors are gutless, and hence do not encode for any viral gene. The isolation of various adeno-associated virus serotypes and a variety of capsid variations gives the prospect of developing prime techniques by altering the adeno-associated viral vector capsid. As a result, the anti-capsid neutralizes humoral reactions caused after the initial dose is avoided.
- Adeno-associated viral vectors’ potential to effectively express numerous transgenes, including those coding for soluble proteins, has encouraged their usage for Ab-gene transfer to create neutralizing Ab (NAb) directly in vivo. The use of adeno-associated viral vector enables a prolonged and constant expression of the Ab after a single injection. It is worth highlighting that adeno-associated viral vectors are merely utilized as vehicles to create large amounts of proteins in vivo, similar to gene therapy. Majority of recent investigations in this field have employed natural adeno-associated viral serotypes besides AAV2.
ADVANTAGES OF ADENO-ASSOCIATED VIRAL VECTOR
- Increased Biosafety: All viral genes get deleted to pave way for the therapeutic gene in the adeno-associated viral vector. Removal of these genes enables larger insertion and hence reducing the possibility of an immunological response to viral gene products. The vector has the advantage of being the only non-pathogenic wild-type virus utilized for gene therapy in humans.
- Versatile Features: Further, their genome structure is very complex, making experimental modification easier. A variety of adeno-associated viral serotypes are identified. Their envelope proteins have distinct surface structures, which implies they select different cells as targets (tropism). Because a particular vector genome may readily be packed in the envelope of any adeno-associated viral serotype, there are very few cell types that are not amenable to insertion of adeno-associated viral vector DNA. These versatile characteristics have made AAV vector a vital tool in the progress of gene therapies.
- Low Immunogenicity: Adeno-Associated viral vector elicits a very mild immune response, leading to lack of pathogenicity during gene delivery.
- Wide Array of Infectivity: Adeno-Associated Viral vector can infect both dividing and dormant cells, allowing gene delivery to a wide range of cell types.
- Idea Virus for Researchers: In addition, the adeno-associated virus integrates at a specified location on chromosome 19. This characteristic is beneficial since it allows researchers to determine where the virus integrates. Because this is regulated by the rep gene, targeted integration does not occur when the gene is deleted. Retaining the rep gene in the vector would maintain targeted integration but reduce the insert size, which is very small. As a result, even in the absence of the rep gene, random integration can occur.
CHALLENGES RELATED TO ADENO-ASSOCIATED VIRAL VECTORS
- The anti- adeno-associated viral antibodies produced by patient’s immune systems can neutralize the adeno-associated viral vectors used to treat them.
- Small packaging capacity of the virus vector. This procedure is considered to be comparatively inefficient because of its inability to yield high titers and frequent occurrence of helper cell contamination.
- Hindrance caused by using adeno-associated viral vectors are the ineffective transduction due to the necessary conversion of single-stranded DNA into double-stranded genome.
Adeno-associated virus was discovered more than 50 years ago and has evolved into one of the most extensively used gene delivery vectors in clinical development. On account of its unique biology, simple structure, and lack of disease correlation, adeno-associated viral vector is the most preferred vector for most medical related applications. In spite of these features, several challenges need to be addressed, such as advancement in immunogenicity and overcoming anti-AAV pre-existing immunity so as to improve the persistence of these vectors for longer periods. Moreover, innovating new capsid variants along with targeted evolution could increase transduction efficiency and minimize the immunogenicity. Further, optimized vector manufacturing and new adeno-associated viral variants will evoke future regulatory approvals and advancements in patient’s health.
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