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With the development of biology research, gene therapy has become one of the most revolutionary frontier fields in modern medicine. By precisely regulating gene expression, it directly attacks the genetic roots of diseases, opening up a new path for overcoming complex and refractory diseases. In the whole technical chain of gene therapy, a safe and efficient gene delivery system is the core factor that determines the success or failure of therapy. Adeno-Associated Virus (AAV), with its low immunogenicity, extensive tissue tropism, stable gene integration characteristics and good transduction efficiency in vitro and in vivo, has become an indispensable key vector in the process of gene therapy from laboratory to clinical application.
This paperndiscusses AAV deeply, including its biological characteristics, genome structure, common sequencing methods, application in gene therapy and its future development trend.
AAV belongs to parvovirus family, and its virus particles are icosahedral symmetrical structure with no envelope and the diameter is about 20-26nm. Virus capsid is composed of three structural proteins VP1, VP2 and VP3, which are assembled in a certain proportion, and these proteins protect the single-stranded DNA genome inside the virus. Single-stranded DNA is about 4.7kb in length, and there is an inverted terminal repeat (ITR) at each end of the genome. ITR plays a key role in the process of virus replication, packaging and integration.
The life cycle of AAV is complicated. In the host cell, after AAV infects the cell, its single-stranded DNA genome will enter the nucleus and form a double-stranded DNA template under the action of host cell DNA polymerase. Subsequently, the double-stranded DNA template can transcribe mRNA, and then translate virus protein. Using the replication and transcription system of host cells, AAV carries out genome replication and virus particle assembly. If auxiliary virus exists, AAV can efficiently replicate and produce offspring virus particles; If there is no helper virus, AAV genome will be integrated into a specific region of the host cell genome, in a latent state, waiting for the right time to be reactivated to complete the complete life cycle.
The AAV life cycle and the engineering of a generic rAAV expression cassette (Dhungel et al., 2024)
There are many serotypes of AAV, and different serotypes have different amino acid sequences of capsid proteins, which leads to their different tropism to different tissues and cells. AAV2 has a certain affinity for liver, muscle and other tissues. AAV9 can efficiently transduce myocardial cells and skeletal muscle cells, and has a good ability to cross the blood-brain barrier, which can be used for gene therapy research of central nervous system diseases. The diversity of serotypes provides a variety of options for gene therapy for different tissues and organs.
Compared with other virus vectors, AAV has lower immunogenicity. AAV itself does not encode any toxic or immunogenic protein. When there is no helper virus infection, AAV will not cause obvious immune response after infecting host cells. However, when the host has antibodies against AAV capsid protein in advance, it may be recognized and cleared by the immune system after the virus enters the body, which will affect the effect of gene therapy. In addition, in some cases, AAV vector mediated gene expression products may also trigger the immune response of the body.
The genome of AAV is single-stranded DNA, and the genome length is about 4.7-5.0 kb depending on serotype. Its genome mainly contains two main open reading frames (ORFs), namely rep and cap genes, flanked by ITR.
ITR is about 145 bp in length and consists of a series of short reverse repeats, which can form a hairpin structure. This unique secondary structure plays a key role in the life cycle of AAV. Different serotypes of AAV have certain differences in ITR sequences, but they all have conservative core regions.
ITR is the initial site of viral genome replication. In the process of AAV replication, the Rep protein encoded by the virus combines with ITR to start the DNA replication process. ITR contains the signal that the virus genome is packaged into the virus capsid. Only DNA molecules containing ITR can be effectively packaged into AAV capsids to form infectious virus particles.
Rep gene is large, encoding four kinds of unstructured proteins, namely Rep78, Rep68, Rep52 and Rep40. These proteins are produced by rep gene through different promoters (p5, p19 and p40) and alternative splicing.
Rep protein plays a key role in AAV genome replication. Rep78 and Rep68 can specifically bind to ITR sequence, and start the replication of virus genome through their helicase and endonuclease activities. They also participate in the promotion of DNA replication fork and the regulation of the replication process. Rep protein can regulate the transcription of AAV's own genes, and also affect the gene expression of host cells. Rep protein is involved in the process of packaging virus genome into virus capsid, ensuring that only complete and correct genome is packaged.
Cap gene encodes three structural proteins, namely VP1, VP2 and VP3, which together constitute the viral capsid of AAV. VP1, VP2 and VP3 are transcribed from the same promoter (p40), and different protein products are formed through alternative splicing and different translation initiation sites. In the virus capsid, VP3 is the most abundant, accounting for about 90% of the total capsid protein, VP2 accounts for about 10%, and VP1 is the least, accounting for only about 1%.
VP1, VP2 and VP3 assemble together to form the icosahedral symmetric AAV capsid, which protects the virus genome from nuclease degradation and mediates the combination and infection of the virus with the host cell. The structural protein on the surface of virus capsid determines the tropism of AAV to different host cells. Some serotypes of AAV bind to specific receptors on the surface of host cells through capsid proteins, thus initiating the infection process. In addition, VP1 contains a phospholipase A2 (PLA2) domain, which plays an important role in the process of virus hulling after entering the host cell.
AAV particle with the AAV single-stranded genome (Lopez-Gordo et al., 2024)
AAV is a potential vector in gene therapy, and its genome sequencing is very important for understanding the characteristics of the virus, optimizing the vector design and ensuring the safety and effectiveness of gene therapy. At present, there are many methods for AAV genome sequencing, and each method has its unique advantages and application scope.
For AAV genome sequencing, Sanger sequencing has the remarkable advantage of high accuracy, and the error rate of its sequencing results is low, which can provide high-precision sequence information for AAV genome and can be used as the gold standard for verification of other sequencing methods. However, Sanger sequencing also has some limitations. Its throughput is relatively low, and only one or a few samples can be sequenced at a time, which is inefficient when a large number of AAV samples need to be processed. Moreover, the operation of this method is complicated, which requires multi-step reaction and electrophoretic separation, which has high technical requirements for experimenters and takes a long time in the whole sequencing process.
Illumina sequencing platform has the advantage of Qualcomm, which can generate massive sequencing data in one run, and can simultaneously sequence the genomes of multiple AAV samples, greatly improving the sequencing efficiency, and is suitable for large-scale AAV research projects. In addition, its sequencing cost is relatively low, but it also has some shortcomings, such as relatively short reading length, and it may be difficult to accurately splice and interpret some long repetitive sequences or complex structural regions in AAV genome, which needs to be combined with other methods to assist analysis.
The outstanding advantage of PacBio sequencing is that the reading length is very long, and the average reading length can reach thousands of base pairs or even longer, which has great advantages for the analysis of long repeated sequences, complex gene structures and virus integration sites in AAV genome, and can analyze AAV genome more completely. Moreover, it can directly detect DNA modifications, such as methylation, which is very important for studying the epigenetic regulation mechanism of AAV in host cells. However, the sequencing cost is high, and the accuracy of the data depends on the sequencing depth to a certain extent, so a high sequencing coverage is needed to ensure the reliability of the results.
Cartoon Simplification of Packaging and hybridization of AAV ssDNA (Hanscom et al., 2023)
Nanopore sequencing has the characteristics of portable equipment and real-time sequencing, which can quickly carry out AAV genome sequencing in the field. Its reading length is also long, which can span the complex region of AAV genome and help to obtain complete genomic information. At the same time, this technology has relatively low requirements for samples, and there is no need for complicated library construction steps. However, at present, the sequencing error rate of Nanopore sequencing is relatively high, especially in homopolymer region, which limits its application in the precise analysis of AAV genome, which requires high accuracy.
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As a revolutionary treatment method, gene therapy aims to treat or prevent diseases by changing the genetic material of patients' cells. AAV has become one of the most commonly used vectors in the field of gene therapy because of its unique biological characteristics.
For genetic diseases with functional protein deletion or abnormality caused by gene mutation, AAV vector can be used to introduce normal gene sequence into patient cells to replace the function of defective gene. In the treatment of spinal muscular atrophy (SMA), the normal SMN1 gene was delivered to the motor neurons of patients through AAV9 vector, which successfully improved the muscle function and quality of life of patients.
CRISPR-Cas system has a powerful function in the field of gene editing, but how to deliver it to target cells efficiently is a key problem. AAV vector can be used to carry components of CRISPR-Cas system, Cas9 nuclease and guide RNA (gRNA), so as to realize accurate editing of specific gene sites. This strategy shows great potential in the treatment of monogenic genetic diseases such as sickle cell anemia.
Using AAV vector to express small interfering RNA (siRNA) or short hairpin RNA (shRNA) can specifically inhibit the expression of pathogenic genes. In some tumor gene therapy, AAV-mediated gene silencing technology is used to inhibit the expression of tumor-related genes, thus achieving the purpose of inhibiting tumor growth and metastasis.
Variant tropisms of AAV serotypes (Issa et al., 2023)
At present, AAV gene therapy has been used to treat many diseases, including hereditary blindness, hemophilia and spinal muscular atrophy. With the continuous progress of technology, AAV gene therapy is expected to make a greater breakthrough in the future.
By designing tissue-specific promoters, AAV vectors can deliver genes to target tissues or cell types more accurately. For the treatment of liver diseases, liver-specific promoters can be used to ensure that genes are mainly expressed in liver cells, reduce unnecessary influence on other tissues, improve therapeutic effect and reduce potential side effects.
Develop a more efficient production system to increase the output of AAV carriers. This includes optimizing cell culture conditions, such as selecting a more suitable host cell line, optimizing the medium formula and improving the culture process (such as replacing monolayer culture with suspension culture), so as to realize large-scale and low-cost AAV production.
Develop more advanced purification methods to obtain AAV carriers with higher purity. Traditional purification methods may lead to carrier loss or impurity residue, but the improvement and combined use of new technologies such as affinity chromatography and density gradient centrifugation can remove impurities more effectively, improve the purity and quality of AAV carrier, and ensure the safety and effectiveness of AAV carrier for treatment.
Design AAV vector which can accurately regulate gene expression level. By introducing inducible or adjustable elements, such as regulatory elements that respond to specific small molecules, temperature or light, the time and level of therapeutic gene expression can be accurately controlled. This is especially important for the treatment of diseases that need to adjust the gene expression intensity according to the change of the disease. For example, in neurodegenerative diseases, the expression of neuroprotective genes can be regulated according to the disease progression stage.
Biodistribution on of 10 AAV serotypes across 22 different tissues (Walkey et al., 2024)
To sum up, AAV has become a powerful and widely used gene delivery vector in the field of gene therapy. Its natural characteristics, such as low immunogenicity, stable transduction and targeting to various tissues, make it the first choice in many preclinical and clinical studies. From the structure and function of AAV to its interaction with the host, our understanding of AAV biology is deepening, which also promotes the development of more efficient and safer gene therapy based on AAV.
References
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