AAV For Gene Therapy


Today, recombinant adeno-associated virus (rAAV) has become the main platform for in vivo gene therapy delivery. The first rAAV gene therapy product, Alipogene Tiparvovec (Glybera) from uniQure, was approved by the European Medicines Agency (EMA) in 2012 for the treatment of lipoprotein lipase deficiency. In 2018, the FDA approved Voretigene neparvovec-rzyl (Luxturna), the first rAAV gene therapy to receive U.S. approval.

overview of rAAV interventional gene therapy clinical trials.

Fig.1 overview of rAAV interventional gene therapy clinical trials. (Wang, D.; et al. 2019)

Fundamentals of AAV vector

AAV For Gene Therapy

AAV belongs to dependoviruses of the family Parvoviridae and are small, non-enveloped DNA viruses with a genome of about 4.7 kb. AAV vector is a gene delivery tool modified from AAV genome. There is an Inverted terminal repeat (ITR) at both ends of the adeno-associated virus genome, in which the D sequence is closely related to the efficient release, selective replication and packaging of the virus genome. There are two open reading frames in the coding region of the genome. They encode four Rep proteins and three Cap proteins, which play roles in genome replication, viral assembly and packaging, respectively1. When designing AAV vector genome, it is necessary to replace the gene sequence of coding region with the target gene and relevant functional fragment, and only the reverse terminal repeat sequence at both ends is retained. Three plasmid co-transfer method was used in the production, which was to co-transfect cells with plasmid containing AAV vector genome, adenovirus co-gene plasmid, and plasmid expressing CAP and REP proteins. There are currently 13 AAV serotypes (AAV1-AAV13) that target different receptors and tissues. When preparing AAV vectors, different serotypes are usually selected according to the disease site and target tissue.

Application of AAV vector in gene therapy

  • Gene replacement

Therapeutic genes are delivered into designated cells and the dysfunctional genes are replaced by homologous recombination of chromosomes. This technique is suitable for the treatment of recessive single gene hereditary diseases2.

  • Gene editing

Gene editing technology can directly repair potential disease genes in the human body. It usually involves two steps: targeted DNA disruption and DNA repair to achieve genomic changes.

The AAV vector can participate in the DNA repair process3, deliver the genome with target sequence homologous recombinant arm and therapeutic gene sequence, and finally achieve gene therapy by simultaneous delivery with the gene encoding Cas9 protein.

  • Gene silencing

RNA interference (RNAi) technology occupies the dominant position of rAAV gene silencing platform, which is an important mechanism of mRNA level regulation.

  • Gene addition

Beyond monogenic diseases, rAAV-mediated gene therapy has the potential to tackle complex genetic diseases and acquired diseases by gene addition. This strategy is currently being tested clinically for HIV infection2.

Immunogenicity of AAV

  • Immune memory for wild-type AAV exists in humans (prior to gene therapy)

When an individual is exposed to endogenous AAV infections, an immune response to the AAV capsids can be mounted. Consequently, a percentage of humans express neutralizing antibodies in the blood that could block gene transfer to cellular targets. In addition, administration of recombinant AAVs (rAAVs) can induce antibodies that can neutralize the transduction of AAV gene therapies, activate the innate immune response, and trigger an adaptive immune response that includes a cellular response that may result in loss of transgene expression4.

Sero-epidemiological studies of human wild-type AAV-neutralizing antibodies have shown that the detection rate of anti-AAV2-neutralizing antibodies is highest, ranging from 30% to 60% of the population. Due to extensive cross-reactivity between AAV serotypes, neutralizing antibodies that identify almost all serotypes can be found in subjects5. The main reason is the homology of amino acid sequence and structure between capsids of different AAV serotypes.

In addition to humoral immunity, cellular immunity against AAV1 and AAV2 was also detected by ELISPOT and flow cytometry6. These AAV-specific T cells are highly cross-reactive to different serotypes and are more prevalent in peripheral immune organs of the spleen than in peripheral blood. Most AAV-specific T cells have a memory phenotype and are detected at similar levels in adults and children.

  • Innate and adaptive immunity induced by AAV vectors

Innate immunity: The AAV vector lacks the sequence encoding the virus, and the main antigenicity comes from the residual contaminants in the purification and production process, viral capsid and transduction gene products, etc. The nucleic acid component of the viral carrier may have an adjuvant-like effect to help activate the host immune system. Through pathogen-associated molecular patterns (PAMP), antigens expressed in viral vectors can be recognized by immune cells' PRR (pattern recognition receptor) to initiate natural immunity. These PRRS can recognize viral nucleic acids, as well as membrane glycoproteins and even chemical messengers. PRR involvement mainly leads to the activation of NF-KB and IRF transcription factors, which play a central role in inducing the expression of pro-inflammatory cytokines or type I IFN, respectively7.

Adaptive immunity: Adaptive immunity occurs after innate immunity by eliminating pathogens through antigen-specific recognition and then establishing immune memory. In establishing an adaptive response, T and B lymphocytes are activated in recognition of the antigen presented by APC. Upon activation, lymphocytes expand and differentiate into effector cells and mediate antigen elimination by inducing humoral immunity or cytotoxic responses. After antigen clearance, memory T and B lymphocytes will be reactivated upon re-exposure to the antigen. It has been demonstrated that gene transfected cells and specialized APCs deliver capsid protein epitopes to cytotoxic CD8+T cells via MHCI-like molecules. Cytotoxic T cells clear AAV transduction cells, leading to inflammation in target organs and reducing the duration and effectiveness of gene transfer. In synchronization with MHCI-class transmission, recognition of capsid derived epitopes on APC surface that bind MHCI-class can activate CD4+T helper cells and promote humoral and cell-mediated immune responses. Clinical trials have shown that the immunogenicity of AAV vectors is to some extent dose-dependent, with low doses of AAV vectors more likely to cause mild inflammation and not a complete loss of transgenic expression8.

  • Immune responses induced by transgene products

During AAV gene transfer, many factors affect the immunogenicity of transgenic products. These can be divided into host-specific factors, associated with the underlying disease or the genetic background of the vector recipient, and vector-specific factors, to group factors related to gene transfer. One key factor determining the level of anti-transgene immune responses is the target organ for gene transfer, which is determined by the combination of the AAV capsid, the vector delivery route, and the tissue specificity of the promoter driving gene expression. In particular, systemic and intramuscular vector administration, with either ubiquitous or muscle-specific promoters, have been shown to be more immunogenic than gene transfer to immune privileged organs, as well as systemic administration with liver-specific promoters9.

Detection of AAV immunity

Although several AVV vector-based gene therapy drugs are currently approved, the immunogenicity of AVV vectors remains an important barrier to their use, both in preclinical and clinical studies.  For example, human and non-human primates exposed to wild-type AAV typically carry anti-capsid neutralizing antibodies (NAbs) that cross-react with several different serotypes used for gene transfer. After vector inoculation, the anti-AAV NAb rapidly develops a high titer that persists for several years, preventing vector re-administration.

The clear definition of the baseline immunological parameters of gene transfer is therefore important to ensure accurate results interpretation in both preclinical and clinical studies. Methods for detecting AAV immunity include cell-based TI tests in vitro, TI tests in vivo (e.g., mice), and total anti-capsid antibody (TAb) tests based on enzyme linked immunosorbent assay10 (ELISA). TAb method may be able to detect low-efficiency NAb below the TI detection threshold, but it may not detect non-antibody neutralizing factors. In vivo and in vitro, TI tests screened samples for anti-AAV, NAb, and other factors that regulate AAV transduction efficiency.


  1. Weitzman, MD.; Linden, RM. Adeno-associated virus biology. Methods Mol Biol. 2011; 807:1-23.
  2. Wang, D.; et al. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019 May;18(5):358-378.
  3. Yang, Y.; et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol. 2016 Mar;34(3):334-8.
  4. Day, JW.; et al. Adeno-associated virus serotype 9 antibodies in patients screened for treatment with onasemnogene abeparvovec. Mol Ther Methods Clin Dev. 2021 Feb 24;21: 76-82.
  5. Calcedo, R.; Wilson, JM. AAV Natural Infection Induces Broad Cross-Neutralizing Antibody Responses to Multiple AAV Serotypes in Chimpanzees. Hum Gene Ther Clin Dev. 2016 Jun;27(2):79-82.
  6. Martino, AT.; et al. Measuring immune responses to recombinant AAV gene transfer. Methods Mol Biol. 2011; 807:259-72.
  7. Trinchieri, G.; Sher, A. Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol. 2007 Mar;7(3):179-90.
  8. Verdera, HC.; et al. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Mol Ther. 2020 Mar 4;28(3):723-746.
  9. Poupiot, J.; et al. Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle. Mol Ther Methods Clin Dev. 2019 Sep 3;15: 83-100.
  10. Falese, L.; et al. Strategy to detect pre-existing immunity to AAV gene therapy. Gene Ther. 2017 Dec;24(12):768-778.

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