Adeno-associated viral or AAV vectors for CRISPR-mediated gene editing (Part 39- CRISPR in gene editing and beyond)
Welcome to the 39th part of the multi-part series on applications of CRISPR in gene editing and beyond.
The second type of viral vectors used for CRISPR-mediated gene editing are adeno-associated viral vectors, abbreviated as AAV vectors. The adeno-associated virus was initially discovered as a contaminant of adenovirus preparations; hence the name adeno-associated virus or AAV. Also, AAV is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate.
The genome of the adeno-associated virus is made up of single-stranded DNA, which is about 4.7 kb long. The genome contains only two genes, rep and cap. The rep gene encodes for the proteins that help the AAV to replicate and integrate into the host genome. And the cap gene encodes the expression of adeno-associated viral capsid proteins. These coding sequences are flanked by inverted terminal repeats (ITRs) that are required for genome replication, packaging, and integration of the virus genome into the host cell genome.
When AAV alone infects the host cells, its gene expression is autorepressed, and the genome of the virus is integrated into the 2 kb region of chromosome 19. This allows the virus to stay in a lysogenic or dormant state into the cell until the host cells are infected with the helper virus such as adenovirus. Once a helper virus infects the host cell, the AAV genome can replicate. E1A, E1B, E2A, E4, and viral-associated (VA) genes of helper adenovirus are required for the replication, transcription, and translation to synthesize the AAV genome and capsid proteins. The AAV genome gets assembled and packaged in the capsid proteins to form AAV particles which are then released from the cells along with the helper viruses.
For transforming the AAV into adeno-associated viral vector for gene editing purposes, the two native AAV genes, rep and cap, are removed and are replaced with a transgene that contains instructions and a promoter to produce a normal functional protein. The promoter and transgene are flanked by signals called inverted terminal repeats (ITRs) that allow the transgene to be packaged inside the protein shell of the AAV vector. Then the vector is transfected into the packaging cell lines to generate an adeno-associated virus containing the transgene as its genome. At the same time, the packaging cells are also transfected with a plasmid that contains rep and cap genes. So this plasmid will express the proteins required for the replication of the viral genome and for packaging of it. AAV is dependent on co-infection with other viruses, mainly adenoviruses, in order to replicate and make virus particles. But you would not want to transfect the packaging cells with adenovirus, because adenovirus can cause the lysis of the cells. Therefore the plasmids that contain adenoviral genes required by adeno-associated virus are transfected into the packaging cells. These genes are E1, E2, E4, and viral-associated (VA). So, for getting adeno-associated viral particles with genes of interest, there are 3 plasmids required: the vector with transgene construct, the plasmid that contains rep and cap genes, and the plasmid that contains E1, E2, E4, and viral-associated genes. Even a few other modifications in the vector are done so that the vector does not get integrated into the host cell genome. Instead, it remains in the nucleus in an episomal state.
Now the AAV particles carrying transgene are then delivered to the target cells. This can be accomplished by either injecting or intravenously administering the vector directly to a particular tissue in the body. Upon administration, the vector enters the target cells and delivers the transgene directly to the nucleus. Alternatively, the patient’s target cells can be isolated and then infected with AAV particles. Once the virus attaches to the target cells, it transfers the transgene to the nucleus. Without Rep proteins, transgenes flanked by ITR within recombinant AAV (rAAV) do not integrate into the host genome but instead form circular structures that persist as episomes in the transduced cells’ nuclei.
Upon entering the nucleus, the single-stranded recombinant AAV genome undergoes conversion to a double-stranded form. The second strand synthesis is initiated by the host cell DNA polymerase. The ITRs of AAV contain self-complementary sequences, which are hairpin-like structures; therefore, the ITR at the 3’ end of the genome acts as a primer for host cell DNA polymerase. The double-stranded genome then undergoes circularization, leading to the formation of episomal DNA. The episomal DNA is not integrated into the host cell’s genome, thus, minimizing the risks of insertional mutagenesis and oncogene activation. Finally, the functional transgene gene is expressed, resulting in the production of the corresponding protein.
AAV vectors for CRISPR-mediated gene editing
For AAV-mediated CRISPR-Cas9 gene editing, two transgenes are required that code for gRNA and Cas9 enzyme. Because of the limited size capacity of the AAV vector, i.e., 4.7kb, it has limited use for delivering large transgenes. Thus, it becomes challenging to co-package SpCas9 which is of size 4.1 kb, and sgRNA into an all-in-one AAV vector for genome editing. To circumvent this problem, dual AAV vector system is used, with one vector carrying the Cas9 with the promoter and the other vector carrying the sgRNA driven by the U6 promoter.
The double-stranded breaks produced by the Cas9 enzyme could be repaired either by a non-homologous end-joining pathway or by a homologous directed repair pathway. For the homologous directed repair pathway, donor template DNA with homology arms to the target DNA is inserted into the second vector along with the sgRNA.
Additionally, SpCas9 orthologs have smaller sizes compared to SpCas9. For instance, Staphylococcus aureus-derived Cas9 has a size of 3.16 kb, and Campylobacter jejuni-derived Cas9 has a size of 2.95 kb. Thus SpCas9 orthologues SaCas9 and CjCas9 can be used to pack Cas9 and sgRNA into a single AAV vector. Studies have shown that, CjCas9 can be packaged with multiple gRNAs into a single all-in-one AAV vector.
Adeno-associated viruses are a popular choice for gene delivery systems in research studies due to several features:
(i) Firstly, AAV can infect both dividing and non-dividing cells.
(ii) Further, the AAV elicits only a weak immune response and has never been shown to cause disease in humans, ensuring safety during gene delivery.
(iii) Moreover, over 12 different serotypes of AAV have been identified to date. A serotype is a distinct variation within a virus because of the differences in envelope proteins and interaction with different receptors of host cells. This means that different serotypes can bind to different receptors on various cell types, allowing them to infect cells from multiple tissue types.
As a result, AAVs can be used as excellent in vivo gene delivery systems to target specific tissues in the body. For instance, AAV serotype 2 has the preference to infect skeletal muscles, neurons, vascular smooth muscle cells, and hepatocytes; thus, AAV serotype 2 can be used as an efficient gene-editing system in these tissues.
AAV serotype 1 vector can be used to deliver the CRISPR-Cas system to vascular endothelial cells, as well as the retina, heart, and lungs. AAV-4 exhibits a preference for kidney and heart cells, AAV-5 exhibits a preference for astrocytes, AAV-6 can efficiently deliver transgenes to airway epithelial cells and hepatocytes, and other serotypes of AAV can be used as an efficient gene-editing system for various other cells.
To achieve tissue- or organ-specific gene editing with CRISPR, a promoter specific to the target tissue is used to drive the expression of the CRISPR transgene. For example, the thyroxine-binding globulin TBG promoter is liver-specific and has been used to drive the expression of CRISPR for in vivo genome editing in mouse liver. Similarly, the myosin heavy polypeptide 6 Myh6 promoter is cardiac-specific and has been used for CRISPR-mediated gene editing in the heart. Other tissue-specific promoters can also be used to target other tissues or organs, depending on the research or therapeutic application.
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