Safer ‘cloaked’ gene therapy tools that evade the immune system

safr gene therapy tools to evade immune system

Date: 16th February 2021

Gene therapy is set to revolutionise the treatment of many genetic disorders, and a handful are already approved for use.  Most use adeno-associated viral (AAVs) vectors to deliver the replacement nucleic acid sequence or genes into the cells.  However, such therapies can cause an immune reaction and inflammation, which can affect efficacy, and is caused by the activation of Toll-like receptor 9 (TLR9), a receptor that senses foreign DNA. Now, scientists have engineering ‘cloaked’ AAVs to evade innate immune and inflammatory responses by incorporating short DNA oligonucleotides that antagonise TLR9 activation directly into the vector genome.

AAV capsids and genomes can both act as immunogenic components by activating the pattern recognition receptor, TLR9.  First, the innate immunity is activated resulting in inflammation, followed by the adaptive immune response in the form of cytotoxic T cells.  This can trigger an antiviral state in the cells, which decreases the overall effectiveness of the therapy, can lead to dose-limiting toxicity and in some cases can even be life-threatening.

Now, scientists from Harvard University and Harvard Medical School, US, and the University of Bristol, UK, led by George Church have developed a coupled immunomodulation strategy in which short TLR9-inhibitory sequences which antagonise TLR9 activation were incorporated directly into the AAV genome which also contained the therapeutic DNA sequences.

The team started by generating a series of synthetic DNA ‘inflammation-inhibiting oligonucleotide’ (IO) sequences.  They used specific short single-stranded DNA oligonucleotides that were known to antagonise TLR9 activation, called TLRi, and linked these to highly inflammatory DNA sequences.  They found that in a TLR9 reporter cell assay this combinatorial strategy had a more effective inflammation dampening effects than administering the molecules separately.

In vivo testing

Encouraged by this preliminary data, the team then wanted to evaluate whether the incorporation of the TLR9i sequences within the AAV genome could diminish the innate and adaptive immune responses to gene therapy in vivo.  The team chose a range of clinically relevant target tissues and administration routes in small and large animal models and injected either control viruses lacking the IO sequence or those containing them, AAV-IO, reporter transgenes or therapeutic DNA sequences were also incorporated.

They found that control viruses induced anti-viral interferon responses whereas the AAV-IOs elicited no detectable innate immune response in the liver of mice upon systemic administration, they also saw enhanced transgene reporter expression.  Next, they tested the system under more immunogenic conditions, injecting the therapy intramuscularly and using a uniquely immunogenic capsid.  Whilst the control conditions evoked a strong cytotoxic T cell (CD8+) response, the IO-therapy showed no T cell infiltration in any of the 8 tissue samples.  These promising results led the team to test the system yet further, in an organ that is already a clinical target for gene therapy.

Ocular gene therapy

The eye offers unique advantages as a gene therapy target, as it is highly compartmentalised, accessible, and is a poorly immune responsive organ.  As such, gene therapies for eye conditions have already been approved, and many are in clinical or preclinical trials.  However, AAV dose-dependent intraocular inflammation has been observed, so to investigate the response to AAV-IO therapy for ocular treatments, the team performed injections into the eyes in mice, pigs, and macaques.

Here, they found that there was local inflammation upon intravitreal administration, with optic disc swelling, retinal vascular changes and cellular infiltration in mouse eyes injected with the control AAV.  However, in the eyes treated with AAV-IO, there was reduced ocular T cells numbers, and an enhancement in the reporter transgene-expressing retinal cells both in numbers and expression level/per cell.  A similar reaction was seen upon subretina injection of IO-therapy in pigs, which indicated that the engineered vector carrying TLR9i sequences could avoid eliciting undesirable innate and adaptive immune cell responses in the retina.

Interestingly, in macaques a delay but not prevention of intraocular inflammation after intravitreal AAV-IO administration was observed.  The use of prophylactic systemic steroids did delay inflammation onset modestly in combination with the therapy.  However, whilst a moderate enhancement of transgene expression was detected, the results highlighted that other immune factors aside from TLR9 must contribute to intraocular inflammation in this highly immunogenic route of administration and model.

Conclusions and future applications

The team here have created a therapeutic approach that integrates the immune-inhibitory activity of short TLR9i sequences into gene therapy vectors, ‘cloaking’ them from the immune system and potentially enhancing the expression of therapeutic genes.

This ‘coupled immunomodulation’ strategy may offer a versatile, broadly applicable solution for different AAV therapies, as well as other DNA-based gene transfer methods, and may widen the therapeutic window for gene therapy.  The work represents a crucial advancement in the development of the next generation AAV vectors that are safer and more effective.

Looking to the future the team will be investigating the roles of the other immune factors that may contribute to intraocular inflammation with the aim of developing the technology to circumvent the immune response more efficiently.

There has been a recent surge in gene therapies designed to restore vision and treat eye disorders and injuries. From reprogramming retina cells by restoring youthful DNA methylation patterns, to gene therapy that delivers an adapter molecule, Protrudin, to stimulate axon regeneration of damaged nerve fibres, or the delivery of a highly photosensitive multi-characteristic opsin (MCO1) protein into retina bipolar cells. There is even evidence that unilateral gene therapy can improve the vision in both eyes.  The use of stealth vectors that can evade the immune system may be an attractive delivery for system for many of these types of therapies.

With more efficient ways to evade the immune system the potential for in vivo gene therapy to treat the thousands of genetic disease becomes a more achievable goal, offering hope of improved outcomes for millions of patients.


For more information please see the press release from Bristol University


Chan, Y. K., S. K. Wang, C. J. Chu, D. A. Copland, A. J. Letizia, H. Costa Verdera, J. J. Chiang, M. Sethi, M. K. Wang, W. J. Neidermyer, Y. Chan, E. T. Lim, A. R. Graveline, M. Sanchez, R. F. Boyd, T. S. Vihtelic, R. G. C. O. Inciong, J. M. Slain, P. J. Alphonse, Y. Xue, L. R. Robinson-McCarthy, J. M. Tam, M. H. Jabbar, B. Sahu, J. F. Adeniran, M. Muhuri, P. W. L. Tai, J. Xie, T. B. Krause, A. Vernet, M. Pezone, R. Xiao, T. Liu, W. Wang, H. J. Kaplan, G. Gao, A. D. Dick, F. Mingozzi, M. A. McCall, C. L. Cepko and G. M. Church (2021). “Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses.” Science Translational Medicine 13(580): eabd3438.