A New Vision for AAV-delivered Gene Therapies


The AAV virus is cloaked from unwanted immune responses through the broadly applicable AAV genome-coupled immunomodulation strategy and offers important insights into ocular inflammation. 

Adeno-associated virus (AAV) is the leading vector for in vivo delivery of therapeutic genes. This is because it is non-pathogenic and efficiently targets many different cell and tissue types. The FDA approved the AAV-based gene replacement therapies for the treatment of spinal muscular atrophy. They inherited a form of retinal dystrophy, highlighting the promise of its therapeutic modality. One of the major challenges faced with in vivo gene therapies is their potential of causing immune reactions and inflammation, affecting how well the therapies can work and last. In some other cases, it can be life-threatening. 

The AAV capsid and genome can both act as immunogenic components. The vector genome that encompasses the therapeutic gene can activate a protein called Toll-like receptor9 (TLR9). This so-called pattern recognition receptor senses foreign DNA in specialized immune cells in the AAV production addgene. The sensing triggers an immune response that results in inflammation and later more specific immune responses that are adaptive in immunity and in the form of cytotoxic T cells against the AAV capsid, which prevents the therapy from taking effect and posing potential risk.

Some immunomodulation strategies were developed that allow short TLR9-inhibitory sequences to be incorporated directly into the AAV genome, are much longer, and contain therapeutic DNA sequences. The approach revealed broad anti-immunogenic potential in investigating different tissues such as mice, mice, pigs’ ocular tissues, and non-human primates. Pathways other than TLR9 activation were also highlighted and likely contributed to the inflammation in the highly immunogenic model intravitreal of AAV injections in macaques. 

Cloaking AAV through coupled immunomodulation

Small snippets of DNA were hypothesized, binding and inhibiting TLR9 activation, including DNA sequences from the ends of human chromosomes known as telomeres. And this would be a way of cloaking the AAV genome from the immune-surveillance mechanism after it has been incorporated directly into it. A series of synthetic DNA was generated into the inflammation-inhibiting oligonucleotide (IO) sequences that carry a highly inflammatory portion linked to one of the different TLR9-inhibitory sequences. Their effects were tested on cultured cells having addgene AAV purification

The inflammatory responses were dampened up to 95% as a result of TLR9-inhibitory sequences that is present. When it was directly incorporated as a tandem series into an AAV vector, the IOs dampened the innate immune responses in the primary human immune cells compared to an unmodified vector.

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In testing the strategy of the AAV in vivo, AAVs were administered as a systemic treatment or locally into the muscle tissue of mice. Control viruses that lack the IO sequences induced anti-viral interferon responses and the infiltration of innate immune cells in the animals’ livers. This led to the infiltration and activation of cytotoxic T cells in the muscle tissues. 

These effects were absent in mutant mice that lack a functional TLR9 pathway that shows TLR9 was indeed a key regulator of AAV-induced inflammation. The effects were blocked in mice that received engineered AAVs containing IO sequences in their genomes. The coupled immunomodulation strategy enhanced the expression of the transgene that the virus delivered, and it’s indicative of potentially higher efficacy. 

The result of Coupled immunomodulation Investigated in the eye

The eye is seen as an immune-privileged site due to a blood-retina brier that limits the entry of immune cells and immune-suppressive factors. Intraocular inflammation that followed the delivery of therapeutically relevant doses of AAV into the eye demonstrated a limit for the immune privilege. The eye’s retina receives the direct application of AAV-based gene therapies, a subretinal injection. AAV that is delivered into the eye’s vitreous cavity is desirable because it will be less invasive and potentially allow for targeting more cells. Unfortunately, the AAV delivery to the vitreous cavity is highly inflammatory.

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In the AAV expression system, using the in vivo imaging and immune cell characterization techniques after intravitreal injection of AAV virus in mice, there was a demonstration of IO sequences incorporation in the virus genome that reduced the numbers of the infiltrating T cell populations in the eye when compared to unmodified AAVs. A coupled immunomodulation strategy was also carried out using pigs through subretinal injections. 

In macaque monkeys, through intravitreal injections, a strategy was also carried out. The result of the strategy was an ameliorated distinct pathology triggered by the control of AAV viruses in pigs and the inclusion of shortening photoreceptor cells essential for high-acuity vision. There was also a substantial reversal in the infiltration of the photoreceptor layer of the retina by the immune cells with microglia and T cells. 

With production and purification, immunosuppressive effects were not so obvious in macaques that received engineered and control AAVs through intravitreal injections. Although, the coupled immunomodulation approach delayed the clinical uveitis symptoms that were triggered by the control virus.

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