Silencing the Immunogenicity of AAV Vectors 

Recombinant adeno-associated viral (AAV) vectors are an appealing delivery strategy for in vivo gene therapy but face a formidable challenge: avoiding detection by an ever-watchful immune system (1,2). Efforts to compensate for the immune response to these virus particles have included immunosuppressive drugs and engineering the AAV vector to be especially potent to minimize its effective dosage. These methods, however, come with their own challenges and do not directly solve for the propensity of AAV vectors to induce immune responses.  

A recent study introduced a new approach to reduce the inherent immunogenicity of AAV vectors (2). Researchers strategically swapped out amino acids in the AAV capsid to remove the specific sequences recognized by T-cells that elicit the most pronounced immune response. As a result, they significantly reduced T-cell mediated immunogenicity and toxicity of the AAV vector without compromising its performance.  

Read on to get more of the study details, which include the use of NanoLuc® luciferase and Nano-Glo® Fluorofurimazine In Vivo Substrate for in vivo bioluminescent imaging of the AAV variants’ distribution and transduction efficiency in mice. 

A teal colored ribbon model of a AAV virus capsid floats against a black background.

Rational Immunosilencing” 

The researchers focused their efforts on the AAV9 serotype, which, among the various known AAV serotypes, is known for its ability to effectively infect a range of tissue types and has a relatively favorable immune profile, among other benefits (1,3). Modifying the AAV9 capsid to rationally suppress its immunogenicity required three key steps: 

  1. Identify which amino acid sequences in the AAV9 capsid were immunodominant epitopes. Immunodominant epitopes are specific regions in an antigen that are recognized by the immune system and stimulate the strongest immune response relative to other antigens. The researchers first prepared 242 unique peptides that covered the entire amino acid sequence of the AAV9 capsid protein, VP1. Then, they measured the immune response these peptides elicited in human peripheral blood mononuclear (PBMC) cells expanded with the AAV9 capsid. The strongest immune response was stimulated by peptides 103–105, which correspond to VP1 amino acids 307–327. This epitope is recognized by CD4+ T cells and promiscuously binds HLA-DP (a cell-surface receptor that presents antigens to CD4+ T cells).  
  1. Examine which of the amino acids in the immunodominant epitope are conserved across AAV serotypes. To potentially identify amino acids in the immunodominant epitope that may be amenable to mutation because they aren’t conserved across AAV serotypes, the researched turned to sequence alignment. Sequence alignment of 12 AAV serotypes showed that amino acids 307–325 in the AAV9 immunodominant epitope are conserved. However, serotype AAV5 contained five non-conserved amino acids. The researchers found that corresponding AAV5 peptides did not stimulate an immune response from PBMCs expanded with AAV5 capsid. Using this information, they planned to strategically modify the AAV9 immunodominant epitope’s sequence based on the non-conserved amino acids in AAV5, hypothesizing that this could decrease the immunogenicity of the AAV9 vector while maintaining its function.
  1. Predict which modifications to the AAV9 amino acid sequence would result in binding fewer HLA molecules. The researchers used the Immune Epitope Database binding algorithms to predict the binding affinity of modified AAV9 peptide sequences to HLA alleles. The modifications were based on the previously identified non-conserved amino acids in AAV5. Based on these findings, they prepared two of the peptide variants that showed reduced binding affinity. One replaced two AAV9 residues with amino acids corresponding to the AAV5 (F315V and L317I); the other replaced five (K311R, R312S, N314R, F315V and L317I) 

The mutations introduced in the AAV9 epitope variants are not on the surface of the AAV9 capsid and do not include amino acids required for the virus particle’s function. The researchers therefore predicted that, in addition to reducing the immune response, AAV9 capsids with these modified sequences would have similar potency and functionality as the original vector.  

Putting the “Immunosilenced” AAV Vector to the Test

To quantify the behavior of the modified AAV9 capsids, the researchers loaded the AAVs with a NanoLuc® reporter gene. While preparing the viral particles with the modified AAV9 capsid, they observed no differences in the quality or properties of the particles compared with particles prepared using the parent AAV9 capsid. Furthermore, the modifications didn’t impact the vector’s ability to deliver the NanoLuc® reporter to cells, nor did they have any impact on the ability of anti-AAV antibodies to neutralize the vectors.  

After these in vitro experiments, the researchers injected the mutated AAV9 vectors carrying NanoLuc® reporter gene into mice. They measured luciferase expression over the course of several weeks with subsequent injections of the NanoLuc® luciferase in vivo substrate, NanoGlo® Fluorofurimazine, and detection via live bioluminescent imaging. Luciferase expression levels in mice injected with mutated AAV9 vectors were similar to mice injected with the parental AAV9 vector. What’s more, luciferase expression was stable for 29 days post injection.

Nano-Glo® Fluorofurimazine In Vivo Substrate (FFz) is designed specifically for in vivo detection of NanoLuc® luciferase. Find other studies using FFz for live imaging on the product page.

AAV vectors can show selectivity for certain tissues and organs, a property known as tropism. The researchers found the parental AAV9 vector strongly expressed the NanoLuc® reporter gene in mouse liver, heart and thymus. With the AAV5 vector, expression in these organs was limited and restricted to mouse liver and lung. In contrast, the mutated AAV9 vectors showed similar distribution and expression levels to the parental AAV9 vector.

Finally, the researchers found restimulation by mutant AAV9 peptides elicited lower levels of immune activation from PBMCs previously expanded with the mutated AAV9 vectors, unlike PBMCs expanded with wild type AAV9.

Taken together, these results demonstrate that the researchers were able to rationally modify an AAV vector to avoid undesirable immune responses while maintaining vector function. They believe their approach could be used to “immunosilence” other AAV vectors and improve in vivo gene therapies.

For more tools that enable AAV research, explore our portfolio of AAV solutions and their applications.


  1. Mingozzi, F. and High, K.A. (2011) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat. Rev. Genet. 12, 341. doi: 10.1038/nrg2988
  1. Bing, S.J. et al. (2023) Rational immunosilencing of a promiscuous T-cell epitope in the capsid of an adeno-associated virus. Nat. Biomed. Eng. 8, 193. doi: 10.1038/s41551-023-01129-8
  1. DiMattia, M.A., et al. (2012) Structural Insight into th Unique Properties of Adeno-Associated Virus Serotype 9. Journal of Virology. 86, 6947. doi: 10.1128/JVI.07232-11
The following two tabs change content below.
Jordan Nutting
Jordan is formerly a science writer at Promega Corporation. She earned her PhD in Chemistry at the University of Wisconsin-Madison and worked as a science reporter at the Milwaukee Journal Sentinel as a AAAS Mass Media Fellow. Jordan loves reading and is always looking for book recommendations. In her spare time, Jordan also enjoys knitting, going on hikes and gardening.

Leave a Reply

This site uses Akismet to reduce spam. Learn how your comment data is processed.