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    Home > Active Ingredient News > Antitumor Therapy > Recombinant adeno-associated virus vector (rAAV) and the new observation of tumor risk

    Recombinant adeno-associated virus vector (rAAV) and the new observation of tumor risk

    • Last Update: 2021-09-30
    • Source: Internet
    • Author: User
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    Click on the picture and sign up for the conference September 24, 2021/eMedClub News/--The relationship between rAAV and tumor risk is currently one of the hottest topics in gene therapy
    .

    Xiao Weidong’s research group, Richard Jude Samulski and Professor Roland Herzog presented new insights in Molecular Therapy (29(9): 2637-2639, 2021).
    They believe that a type of viral impurity in rAAV packaging-SBG (snapback genome) may be a tumor The resulting risk factors
    .

    In the carrier, these molecules may be a mouse poop, which breaks a pot of soup
    .

    In 2012, McCarty published a study on adeno-associated virus (AAV) vector injection in mice that can cause tumorigenesis in "Molecular Therapy" magazine [1]
    .

    In this study, they used the characteristics of the self-complimentary AAV (scAAV) vector to design a blank vector (scAAV-CBA-null) that contains only the CBA promoter without the overexpression gene and the polyA tail, and also contains the CBA promoter.
    The seed contains an expression vector (scAAV-CBA) of over-expressed genes and polyA tail
    .

    The structural characteristics of the two vectors are shown in Figure 1A.
    The former is used as the experimental group and the latter is used as the control group.
    The tumor-susceptible mouse strain C3H/HeJ or the severe immunodeficiency mouse model SCID are injected respectively
    .

    The results of the study showed that the incidence of tumors in the two mouse models injected with scAAV-CBA-null was significantly increased; at the same time, the site of integration of the vector is closely related to common protocarcinoma and tumor suppressor genes
    .

    Therefore, they speculated that the occurrence of liver cancer may be caused by the integration of the scAAV-CBA-null vector into the host chromosome to cause transcription to read through and activate the proto-oncogene, or the integration of the vector may have an enhancer effect on the transcription of the affected gene
    .

    Surprisingly, Xiao Weidong's group discovered that the vector they called SBG, as a type of viral impurity, can be naturally produced during the preparation of rAAV
    .

    As we all know, the rAAV vector packs a single-stranded DNA genome, which can be positive or negative strands, accounting for a similar proportion
    .

    They speculate that SBG is produced when the complementary positive and negative strands are connected during the replication of the viral genome (Figure 1B)
    .

    The presence of SBG was detected in all vectors prepared from different methods, which seems unavoidable (Figure 1C)
    .

    The difference between SBGs is only the length of the complementary sequence.
    Among them, the promoter-only one with complementary promoter sequence is very similar to the rAAV blank vector used in the McCarty study in terms of genome structure
    .

    This finding suggests that when rAAV packaging is carried out in the conventional design of a vector that uses a promoter to drive the expression of the target gene, a SBG configuration similar to the scAAV-CBA-null empty vector can be generated; SBG can finally pass its own ITR packaging signal Into the capsid, thereby producing a genome that only carries a promoter and other viral vector impurities with incomplete genomes
    .

    At the same time, Xiao Weidong's group also found that when different production systems were used to prepare different vectors, promoter-only SBG types accounted for different proportions in all different rAAV genomes (Figure 1C)
    .

    The promoter of rAAV with CB promoter seems to be the hot spot for forming SBG structure.
    Among vector molecules containing CB promoter, viral vectors with a genome smaller than 1kb, that is, vectors with only promoters, occupy about 40% (Figure 1C)
    .

    However, the total abundance of these smaller SBGs depends on the specific design and production method of each vector (Figure 1C)
    .

    Since SBG has similar physical and biochemical properties to vectors containing a complete expression framework, they may be difficult to simply remove through downstream processes
    .

    In addition to the promoter-only structure mentioned above, SBG has other types, so not all SBG structures are genetically toxic after integration
    .

    But the author suggests that rAAV should better characterize these by-products before use
    .

    When there are many genotypes in SBG that contain the entire promoter but lack the polyA sequence, the risk of gene read-through after the vector is integrated after chromosome integration will increase, and the genotoxicity will also increase
    .

    This situation is particularly noteworthy when using tissue-specific promoters, because it limits the potential harm to the target tissue or target cell type
    .

    Judging from the current results, the level of this unwanted SBG produced during vector preparation depends on the nucleotide composition of the promoter, the strength of the promoter and its size
    .

    The author believes that the main reason why rAAV is associated with the risk of tumor formation is SBG produced during the production of rAAV
    .

    However, the formation of cancer has not been observed in clinical trials.
    The reason may be that the number of SBGs in the final carrier is relatively small, and the target cells that are easy to form tumors are also required [2]
    .

    Nevertheless, the molecular forms of these "promoter-only" vectors ubiquitous in rAAV may lead to potential risks of genotoxicity still require special attention
    .

    In addition, the use of strong promoters may also create additional risks in the host cell
    .

    Previous studies have also shown that vector dosage, enhancer/promoter selection, and gene delivery timing are all key factors related to the pathogenesis of hepatocellular carcinoma (HCC) [3-4]
    .

    Increasing the dosage will increase the risk of tumor formation
    .

    The authors suggest that in order to eliminate these potentially risky rAAVs carrying defective genomes, vector design and technology development, including the analysis of purified vectors, require special attention
    .

    The author further demonstrated a method of reducing risks and enhancing carrier safety through optimized design (Figure 1D)
    .

    They simply changed the direction of the expression framework in the scAAV genome, that is, placing the expression framework promoter on the side of the mutant ITR, and promoter-only SBG would not be produced
    .

    The reason is that the replication of the rAAV genome starts from the 3'end of the complete ITR
    .

    Some principles that should be followed in clinical vector design in previous studies, such as removing CpG elements, replacing open reading frames, and unintentional splicing sites, can be used with the new discovery of SBG to optimize vector design and reduce potential risks
    .

    Whether rAAV vectors have carcinogenic risks has always been a highly controversial topic in the field of human gene therapy
    .

    Most of the wild-type AAV (wtAAV) is considered to be non-pathogenic [5], but there are some reports of insertion mutations caused by wtAAV [6-9]
    .

    DNA detection of wtAAV in tumor and non-tumor liver tissues of 1461 patients found that wtAAV was inserted in about 30 non-cirrhotic livers [6]
    .

    So far, rAAV has been tested in 223 clinical trials, two of which have been approved by the FDA for the treatment of LCA and SMA, and other clinical trials are also following up
    .

    However, it is particularly vigilant that in many clinical treatments, rAAV is delivered to cells in extremely high doses (such as striatum, slow-contracting muscle, Muller cells, etc.
    ).
    The number of these rAAV vectors far exceeds the level of wtAAV
    .

    In addition, rAAV is usually designed to express high-level transgenes with no incubation period, while wtAAV in the cell maintains a stable latent infection through the inhibition of the p5 promoter by the YY1 protein [10]
    .

    It is not yet clear how the difference between rAAV and wtAAV will affect the interaction between rAAV and the host, so the true safety of rAAV for gene therapy needs to be strengthened
    .

    In a study, the rAAV vector was found to cause HCC in neonatal type VII mucopolysaccharidosis mice and even normal mice[11-12]
    .

    The carcinogenic events of rAAV found in some studies using mouse models are similar to those found in Dr.
    McCarty's research [13-14]
    .

    Another study also revealed that the integration of rAAV can cause the over-expression of microRNAs and Rtl1 in its proximal end to cause HCC[3-4]
    .

    However, there are many other studies using rAAV that have not observed tumors in mouse models
    .

    A recent study on a dog model of hemophilia A found that in dogs treated with rAAV, although no malignant tumors were observed during the 10-year follow-up period, the hepatocytes carrying the therapeutic clotting factor VIII gene have The phenomenon of amplification [15]
    .

    Finally, the author proposes that if the promoter-only SBG in the vectors used in these studies is analyzed, it will be very useful for further evaluation of the safety of rAAV
    .

    ▲ Figure 1.
    Subgenomic AAV vector molecules with increased risk for cancer formation by “readthrough” gene activation.
    (A) Illustration of known vector designs that induce genotoxicity as described previously in Molecular Therapy (CBA, CMV enhancer/chicken beta actin promoter).
    (B) Illustration of SBG formed during rAAV production and of the dimer that is formed upon 2nd stranded DNA conversion.
    (C) Summary of the amount of SBG molecules in rAAV preparations.
    The SBG ratio indicates the number of molecules with SBG configuration divided by the number of all sequenced rAAV genomes in the same size range (based on Pacbio Single Molecules analysis of pooled vector DNA).
    For example, SBG in size range 4,000–5,000 means the complementary region is 2,000–2,500 nucleotides.
    Q20:long reads with = Q20 (99%) single-molecule accuracy generated using the circular consensus sequencing analysis method.
    SEQUEL sequencing was used, except for the one RSII sequencing library indicated with asterisks (*), which had shorter reads.
    (D) Comparison of two scAAV vector designs.
    Placing the promoter in close proximity to the mutant ITR (which lacks the terminal resolution site) produces dimeric forms beneficial for transgene expression (with 2 copies of the enhancer in close proximity) and generates only “harmless” SBGs lacking a promoter.
    Vectors with either orientation were produced and sequenced.
    Problematic SBG contents was determined and graphed.
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