A new vision for adeno-associated virus delivered gene therapies

AAVs with TLR9-inhibitory sequences incorporated into their genomes allowed significantly higher expression of a linked fluorescent reporter gene in the mouse retina (right) than AAVs that were lacking the sequences (left) following intravitreal administration David Copland/University of Bristol

An international collaboration of leading groups in gene therapy and vision science have developed an adeno-associated virus (AAV) genome-coupled immunomodulation strategy that helps cloak the AAV virus from unwanted immune responses and offers important insights into ocular inflammation. The research led by Harvard University, Harvard Medical School and including the University of Bristol is published in Science Translational Medicine.

In recent years, AAV has been recognised as the leading vehicle for in vivo delivery of therapeutic genes because it is non-pathogenic and efficiently targets many different cell and tissue types. A key challenge of in vivo gene therapies is their potential to cause immune reactions and inflammation, which can affect how well the therapies work or last, and in rare cases can even be life-threatening.

The AAV capsid and genome can both act as immunogenic components. Specifically, the vector genome, which encompasses the therapeutic gene, can activate a protein known as Toll-like receptor 9 (TLR9), a so-called pattern recognition receptor that senses foreign DNA in specialised immune cells. This sensing first triggers an immune response that results in inflammation (innate immunity), and subsequently more specific immune responses (adaptive immunity, in the form of cytotoxic T cells) against the AAV capsid, preventing the therapy from taking effect and posing a potential risk.

The international collaboration developed a "coupled immunomodulation" strategy in which short TLR9-inhibitory sequences are incorporated directly into the much longer AAV genome containing therapeutic DNA sequences. The approach showed broad anti-immunogenic potential and importantly through the University of Bristol ophthalmology team of Professor Andrew Dick, Dr Colin Chu and Dr David Copland it also highlighted that pathways other than TLR9 activation likely contribute to inflammation in the highly immunogenic model of intravitreal AAV injections.

The project was initiated in Professor George Church's group at the Wyss Institute and Harvard Medical School. Professor Church leads the Institute's Synthetic Biology platform and is Professor of Genetics at Harvard Medical School and of Health Sciences and Technology at Harvard and the Massachusetts Institutes of Technology (MIT).

Cloaking AAV using coupled immunomodulation

Dr Ying Kai Chan, first- and co-corresponding author and Chief Scientific Officer at Ally Therapeutics, and currently a Visiting Scholar at the Wyss, said: "We hypothesized that small snippets of DNA that bind and inhibit TLR9 activation, including DNA sequences from the ends of human chromosomes called telomeres, would be a way to cloak the AAV genome from this immune-surveillance mechanism when incorporated directly into it."

The team started by generating a series of synthetic DNA "inflammation-inhibiting oligonucleotide" (IO) sequences that each carry a highly inflammatory portion linked to one of different TLR9-inhibitory sequences and tested their effects on cultured cells. The presence of TLR9-inhibitory sequences dampened the inflammatory response by up to 95 per cent. When directly incorporated as a tandem series into an AAV vector, the IOs dampened innate immune responses in primary human immune cells compared to an unmodified vector.

Andrew Dick, Professor of Ophthalmology from the Bristol Medical School: Translational Health Sciences (THS), added: "Gene therapy is an exciting and advanced treatment for eye disorders. Whilst there are licenced gene therapies for inherited retinal degenerations, there remains a need to develop vectors that subvert inflammatory responses and increase the therapeutic effect of the vector-encoded genes. This work has shown how advanced vector development can potentially improve efficacy and safety, particularly as the field advances to treat multifactorial common diseases."

To test the strategy in AAV in vivo, the researchers administered AAVs as a systemic treatment or locally into muscle tissue of mice. Control viruses lacking IO sequences induced anti-viral interferon responses and the infiltration of innate immune cells in the animals' livers and led to infiltration and activation of cytotoxic T cells in muscle tissues. These effects were absent in mutant mice lacking a functional TLR9 pathway, showing that TLR9 was indeed a key regulator of AAV-induced inflammation. Importantly, the effects were blocked or much reduced in mice that received engineered AAVs containing IO sequences in their genomes, and the coupled immunomodulation strategy enhanced expression of the transgene that the virus delivered, indicative of potentially higher efficacy.

Investigating coupled immunomodulation in the eye

The eye is often described as an immune-privileged site because of the presence of a blood-retina barrier that limits entry of immune cells, and of immune-suppressive factors, an area the team at University of Bristol actively research. Immune privilege is known to be relative and the 'privilege' often relative and in context to disease. Not surprisingly, multiple clinical trials have reported intraocular inflammation following delivery of therapeutically relevant doses of AAV into the eye, demonstrating a limit for immune privilege. Most AAV-based gene therapies in the eye are directly applied to the retina (subretinal injection). AAV delivery to the vitreous cavity (intravitreal injection) of the eye is highly desirable since it would be less invasive and potentially allows for targeting more cells, but to date it appears more inflammatory.

The team in Bristol used in vivo imaging and immune cell characterisation techniques after intravitreal injection of AAV virus in mice, and demonstrated that the incorporation of IO sequences in the virus genome reduced the inflammation and numbers of infiltrating T cell populations in the eye compared to unmodified AAVs. This further coincided with a multifold boost in expression of the vector-encoded reporter gene in the retina. Next, the team studied their coupled immunomodulation strategy in larger animal models.

The findings from the intravitreal toxicity induced by AAV, and the modest response to the TLR9 blocking sequence and to steroids, indicate that there is more than one mechanism leading to toxicity from this injection site. The collaborative research team can now go forward with this understanding and search for additional pathways.

The study represents a critical step in the development of next generation AAV vectors that are safer and more effective.

The study was funded by the Wyss Institute for Biologically Inspired Engineering at Harvard University, National Institutes of Health under grant# RM1 HG008525 and EY026158, European Research Council under grant# 617432, National Eye Research Centre, UK, The Underwood Trust, and Ally Therapeutics.

Dr Chan and Professor Church are both co-founders of Wyss gene therapy start-up company Ally Therapeutics.


'Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses' by Ying Kai Chan, George M. Church et al in Science Translational Medicine

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