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T Cell Mediated Immune Response Against AAV Gene Therapy

Adeno-associated virus (AAV) is a popular vector for gene therapy due to its low immunogenicity and ability to deliver therapeutic genes to target cells. However, recent studies have shown that some individuals may develop a T cell response against AAV capsid proteins, which can limit the effectiveness of AAV-based gene therapies.
  • Wang et al. (2019) found that up to 70% of participants in AAV-based gene therapy clinical trials had circulating CD8+ T cells that recognized specific AAV capsid proteins and were capable of eliminating AAV-transduced cells in vitro. The authors also found that the presence of preexisting AAV capsid-specific CD8+ T cells was associated with reduced transgene expression and therapeutic efficacy in some participants.
  • Mingozzi et al. (2013) investigated CD8+ T cell responses to AAV capsid in healthy human subjects and found that the majority of participants had detectable AAV capsid-specific CD8+ T cell responses. However, these responses did not affect transgene expression or clinical outcomes in a small cohort of patients with hemophilia B who received AAV-based gene therapy.
  • Li et al. (2019) analyzed AAV capsid-specific T cell responses in peripheral blood and cerebrospinal fluid of patients who received AAV-based gene therapies and found that AAV capsid-specific CD8+ T cells were present in both compartments. The authors also found that the presence of preexisting AAV capsid-specific T cells in peripheral blood was associated with a reduction in transgene expression and clinical response.
  • Buchlis et al. (2012) reported successful long-term expression of factor IX in a severe hemophilia B patient who received AAV-mediated gene transfer into skeletal muscle. The authors did not find evidence of immune responses against the AAV capsid or transgene expression over a period of 10 years.
  • Calcedo et al. (2017) investigated the prevalence of preexisting neutralizing antibodies to AAV capsids in large animal models and found that some species, such as dogs and pigs, had high levels of preexisting neutralizing antibodies to certain AAV serotypes. These findings highlight the importance of considering the potential impact of preexisting immunity on the efficacy of AAV-based gene therapies in animal models.
Overall, these studies suggest that the T cell response against AAV capsid proteins can limit the effectiveness of AAV-based gene therapies in some individuals. To overcome this limitation, researchers are exploring strategies to modify AAV vectors to evade T cell recognition or to induce immune tolerance to AAV capsid proteins.

Method for Assessing T-Cell Response Against AAV

The ELIspot assay is a technique used to measure the frequency of antigen-specific T cells in a sample. In the context of AAV capsid-specific T cell responses, the ELIspot assay can be used to detect the presence of AAV capsid-specific T cells in peripheral blood mononuclear cells (PBMCs) or other tissues.

The procedure for the ELIspot assay typically involves the following steps:
  • Coat ELIspot plates with anti-IFN-γ antibody.
  • Isolate PBMCs or other tissue cells from the sample of interest.
  • Incubate the cells with peptides corresponding to AAV capsid epitopes or with AAV capsid protein.
  • Add the stimulated cells to the ELIspot plates and incubate for a period of time.
  • Detect and quantify the number of spots, which represent individual T cells secreting IFN-γ in response to AAV capsid stimulation.
One of the key steps in measuring T-cell response is designing peptides for ELISpot. Designing peptides for EliSpot assays in AAV gene therapy would involve selecting antigenic peptides from the AAV capsid proteins that are known to elicit T-cell responses. These peptides can be predicted using bioinformatics tools that analyze the amino acid sequence of the capsid proteins and identify potential epitopes that can bind to major histocompatibility complex (MHC) molecules and activate T cells.

One commonly used tool for predicting T-cell epitopes is the immune epitope database (IEDB) (https://www.iedb.org/). IEDB provides a comprehensive repository of experimentally validated T-cell epitopes for various antigens, including AAV capsids. Researchers can use IEDB to identify potential epitopes within the AAV capsid proteins, which can then be synthesized and used in EliSpot assays to detect AAV-specific T-cell responses.

Once the peptides have been synthesized, they can be loaded onto ELIspot plates coated with anti-IFN-γ antibodies and incubated with peripheral blood mononuclear cells (PBMCs) isolated from individuals. PBMCs are then stimulated with the peptides, and the resulting T-cell responses are detected by measuring the secretion of IFN-γ using an enzyme-linked immunospot (ELIspot) assay.

The ELIspot assay has been used in various studies to assess AAV capsid-specific T cell responses in humans and animal models. For example, Mingozzi et al. (2013) used the ELIspot assay to evaluate CD8+ T cell responses to AAV capsid in humans, while Li et al. (2019) used the assay to measure AAV capsid-specific T cell responses in the peripheral blood and cerebrospinal fluid of patients with neurological disorders.

Overall, the ELIspot assay is a useful tool for assessing AAV capsid-specific T cell responses and can provide valuable information for the development of safe and effective gene therapies using AAV vectors.

References

Wang, M., et al. (2019). Prevalence and Characterization of Vector-Specific T Cells in Gene Therapy Clinical Trials. Science Translational Medicine, 11(492), eaat9356.
Mingozzi, F., et al. (2013). CD8+ T-cell responses to adeno-associated virus capsid in humans. Nature Medicine, 19(4), 471–473.
Li, C., et al. (2019). Adeno-Associated Virus Capsid-Specific T Cell Responses in Peripheral Blood and Cerebrospinal Fluid. Human Gene Therapy, 30(9), 1107–1122.
Buchlis, G., et al. (2012). Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood, 119(13), 3038–3041.
Calcedo, R., et al. (2017). Pre-existing neutralizing antibodies to adeno-associated virus capsids in large animals other than monkeys may confound in vivo gene therapy studies. Human Gene Therapy Methods, 28(1), 49–59.

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