AAV gene therapy development: Immunogenicity and safety aspects

Eveline Molocea and Jörg Schneider

by Eveline Molocea and Jörg Schneider

The evolution of biopharmaceuticals in development follows our ability to understand and manipulate the increasing levels of complexity from proteins and viruses, to cells and organs. Recombinant viruses represent a quantum-leap in complexity of development compared to protein-based biopharmaceuticals see Figure 1. Adeno-associated virus (AAV) is a dominant gene delivery platform that matured in recent years to the level of commercialized products with the potential to cure diseases and conditions for which no treatment options existed previously. However, AAV product developers have to meet many challenges throughout their nonclinical and clinical development, the lately most prominent ones being presented below.

Figure 1 : Development complexity of pharmaceuticals

CpG di-nucleotides and AAV immunogenicity

CpG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5′ → 3′ direction. Bacterial and vertebrate genomic DNA differ in the frequency and methylation of CpG sites, which are relatively abundant in bacteria, but underrepresented and methylated in vertebrates. CpGs are considered pathogen-associated molecular patterns (PAMPs) (1). When unmethylated, CpG sites are recognized by Toll-like receptor 9 (TLR9) which leads to the activation of both the innate and adaptive immune system. Because of this, oligonucleotides containing CpG di-nucleotides are for example used as vaccine adjuvants due to their ability to induce a strong cellular immune response. TLR9 has the potential to recognize unmethylated CpG sites from AAV expression cassettes, which contributes to increased immunogenicity and can have a negative impact on AAV gene therapy efficacy (2; see Figure 2). Results from gene therapy clinical trials in hemophilia B, indicate that codon optimization of the transgene to increase transgene mRNA stabilization and protein expression increases the number of CpG sites (3). This, in turn, correlates with an increased need of immuno-suppressive therapy and a decrease of transgene expression. The contribution of CpGs to immunogenicity is further supported by the observation that CpG-depleted AAV vectors can evade immune detection and establish persistent transgene expression (4).

Figure 2: CpG di-nucleotides contribute to AAV immunogenicity

There are several options to reduce the impact of CpG-mediated immunogenicity. Firstly, to replace CpG with alternative codons encoding for the same amino acid in coding regions. However, for non-coding regions altering CpG may impact DNA conformation and change critical quality attributes of the modified AAV. Secondly, methylation of CpGs to prevent recognition by TLR9 by producing DNA plasmids in methylating bacterial production strains. The impact of methylation on the critical quality attributes also needs to be carefully assessed. Thirdly, co-expression of short non-coding DNA sequences from the AAV vector genome to directly antagonize TLR9 activation and “cloak” the much larger AAV DNA sequence from detection. In preclinical studies, administration of these modified AAV vectors resulted in reduced innate immune and T cell activation with improved gene expression. In macaques, intravitreal injection of the modified vector delayed, but did not fully prevent, development of uveitis. Incorporation of these cloaking sequences may improve the success of AAV-based gene therapies (5).

Dorsal root ganglion inflammation

In preclinical studies the administration of AAV vectors to nonhuman primates (NHP) via blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology (see Figure 3). The pathology is minimal to moderate in most cases, clinically silent in affected animals, and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. DRG mononuclear cell inflammation was sometimes accompanied by neuronal cell body degeneration or loss, leading to concerns over potential safety in humans.

Figure 3 : AAV administration and dorsal root ganglion inflammation in NHPs

A recent publication (6) analyzed 33 nonclinical studies involving a total of 256 NHPs and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, doses, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHPs that received AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. Dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (i.e., <14 days), similar from one to 5 months post-injection, and was less severe after 6 months. Vector purification method had no impact, and all capsids and promoters that were tested resulted in some DRG pathology. Data from five different capsids, five different promoters, and 20 different transgenes suggested that DRG pathology is almost universal after AAV gene therapy in nonclinical studies with NHPs. Most importantly, none of the animals receiving a therapeutic transgene displayed any clinical signs. Incorporation of sensitive techniques such as nerve-conduction velocity testing can show alterations in a minority of animals that correlate with the severity of peripheral nerve axonopathy. The study suggested to monitor sensory neuropathies in the human central nervous system and proposed that high-dose IV clinical studies determine the functional consequences of DRG pathology. It is speculated that the DRG pathology described is due to formation of dsRNA species during AAV transduction and the cytoplasmic dsRNA recognition pathway, which leads to the production of type I IFN-β. This mechanism would apply to protein transgene products and miRNAs (7).

These nonclinical findings raised concerns by the FDA about IV, IT and ICV administration of AAVs and as a result several AAV-related INDs were put on hold. For example, findings in a small NHP study (12 animals) resulted in a partial hold of clinical trials for intrathecal administration of AVXS-101, the gene therapy that won the FDA’s first approval for treating some forms of spinal muscular atrophy (SMA) under the name Zolgensma® (onasemnogene abeparvovec-xioi). The partial hold did not affect the marketing of Zolgensma® or clinical trials assessing intravenous (IV) delivery of AVXS-101. However, the hold affects studies assessing AVXS-101 administered at high doses (6E13 – 2.4E14 vg/patient, intrathecally). As a result of the hold, enrollment in the high dose cohort has been stopped in the Phase I STRONG trial (NCT03381729), an ongoing, open-label, dose-comparison, multi-center trial designed to evaluate the efficacy, safety, and tolerability of one-time intrathecal administration of AVXS-101. The low- and mid-dose cohort enrollment has previously been completed and interim results have been presented here. To date, current clinical experience did not provide evidence that points to DRG occurring in humans – nevertheless DRG-mediated toxicity should be continued to be monitored.

Biopharma Excellence is offering a customized strategic approach to overcome challenges linked to all areas of AAV development to de-risk your gene therapy program. Interested in learning more about how your AAV or any ATMP development program can be optimized in agreement with expectations of regulatory agencies? Get in contact with us.


  1. Krieg AM. CpG motifs: the active ingredient in bacterial extracts? Nature medicine. 2003;9(7):831-5.

  2. Verdera HC, Kuranda K, Mingozzi F. AAV Vector Immunogenicity in Humans: A Long Journey to Successful Gene Transfer. Molecular Therapy. 2020;28(3):723-46.

  3. Wright JF. Codon Modification and PAMPs in Clinical AAV Vectors: The Tortoise or the Hare? Molecular Therapy. 2020;28(3):701-3.

  4. Faust SM, Bell P, Cutler BJ, Ashley SN, Zhu Y, Rabinowitz JE, et al. CpG-depleted adeno-associated virus vectors evade immune detection. The Journal of Clinical Investigation. 2013;123(7):2994-3001.

  5. Chan YK, Wang SK, Chu CJ, Copland DA, Letizia AJ, Verdera HC, et al. Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Science Translational Medicine. 2021;13(580).

  6. Hordeaux J, Buza EL, Dyer C, Goode T, Mitchell TW, Richman L, et al. Adeno-Associated Virus-Induced Dorsal Root Ganglion Pathology. Human gene therapy. 2020;31(15-16):808-18.

  7. Shao W, Earley LF, Chai Z, Chen X, Sun J, He T, et al. Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction. JCI insight. 2018;3(12).

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