At AVROBIO, we often talk about how our gene therapy platform, plato^®, includes a variety of cutting-edge technologies that we can bring to bear on our investigational gene therapy programs. This includes the use of proprietary peptide tags designed to help therapeutic protein get to where it needs to go – a kind of fast-track ticket to target specific tissues. We believe this technology can further optimize our approach to lentiviral gene therapy, as it may help the therapeutic protein get to targeted tissues like muscle and the central nervous system (CNS), which is critical for the lysosomal disorders we are focused on at AVROBIO.

Last fall, I wrote about our early work around proprietary GILT tagging for our Pompe disease program. The GILT tag in that program is designed to aid the uptake of therapeutic (or ‘active’) protein not just into target tissues, but even more precisely into the lysosomes of the target cells. We now have an additional program that uses a tag to help us target the devastating lysosomal disorder Hunter syndrome, which primarily affects young boys and causes severe cognitive issues as well as other extensive symptoms throughout the body.

As a quick definition, a tag is simply a peptide (a fragment of a protein) sufficient to initiate binding to a specific receptor on the target tissue, enabling uptake into the cell and then into the lysosomes, which are the cell’s recycling centers. We engineer our gene therapies so that the modified cells will express not just the therapeutic protein on their own, but the protein with a tag attached to one end. The tag serves as an address of sorts, effectively creating a ‘homing’ effect to direct the protein to the places where it is most needed.

The simplicity of the word ‘tag’ belies the trickiness of its design. A tag needs a well-characterized binding site on a receptor that induces the desired action (e.g., being taken up by a cell and transported to the lysosome), as well as a linker to join the peptide to the therapeutic protein. The linker needs to be of an appropriate length and charge, have the right level of flexibility and be in the right position and orientation so as not to disrupt the function of either the tag or the therapeutic protein. All of these elements interact in 3D space, making fusion protein design a real skill.

Helping therapeutic protein get where it needs to go

So, why do we need tags at all? The answer is because for some diseases, inducing endogenous production of active protein is only Step One. Step Two is getting that protein where it needs to go – and at a high enough level. Lentiviral gene therapy is designed to be inherently good at distribution because it’s based on harnessing the natural functionality of hematopoietic stem cells, which differentiate into a range of daughter cells that travel around the whole body and the brain – perfectly automated vehicles for protein delivery.

The AVROBIO gene therapy approach includes a personalized, single cycle of a single conditioning agent named busulfan, which aids engraftment of the gene-modified cells in both the bone marrow. This is designed to provide a continual head-to-toe supply of active protein. But not every tissue or intracellular compartment is equally receptive to receiving that protein. Some need a little help –– which is where tagging comes in as a ‘fast-track ticket’ to the desired destination.

In both our Pompe disease and Hunter syndrome programs, the fusion of a short peptide that binds to a specific receptor may help the therapeutic enzyme get to key tissues like muscle and the CNS. In the case of Pompe disease, we use a glycosylation-independent lysosomal targeting (GILT) tag; in the case of our new Hunter syndrome program, our tag incorporates a novel peptide based on the receptor‐binding domain of human apolipoprotein E (ApoEII).

GILT: a tag that muscles its way into muscle… and the CNS

Pompe disease is a lysosomal disorder caused by a mutation in the GAA gene. The lack of the functional enzyme encoded by GAA results in a toxic buildup of glycogen throughout the body and CNS, causing a wide range of symptoms including progressive weakness and loss of motor function.

Pompe disease is a highly challenging disease to treat. The standard of care is enzyme replacement therapy (ERT), but people living with Pompe disease typically require 20 times more ERT than people living with other lysosomal disorders, such as Fabry disease and Gaucher disease. It is especially difficult to get the functional enzyme into skeletal and cardiac muscle and the CNS in order to reduce the toxic buildup of glycogen.

During preclinical development of our lead vector candidate for Pompe disease, we have seen a significant increase in glycogen reduction through the deployment of a GILT tag.

In a presentation we shared at the American Society of Gene & Cell Therapy (ASGCT) in spring 2020, we demonstrated that vector candidates deploying our proprietary GILT tag drove glycogen levels in a mouse model down to what you would see in wild-type mice unaffected by Pompe disease. We demonstrated a greater than 99% reduction in glycogen in cardiac muscle. Glycogen levels plunged to near wild-type levels in the cerebrum and spinal cord. And our lead vector candidate significantly reduced glycogen in clinically relevant skeletal muscles, including the diaphragm.

Here’s a slide from that presentation:

It’s an exciting finding, indeed. Treatments for Pompe disease have traditionally focused on addressing muscle deterioration, but more recently, the emphasis has shifted to having a highly significant CNS component, which impacts movement as well as other CNS functions. Therefore, a gene therapy capable of addressing both muscle and CNS would be an important advance for the treatment of Pompe disease.

APOEII: CNS efficacy through co-opting a common cell receptor

We’re equally excited about the tagging approach used in our newest program, an investigational gene therapy for Hunter syndrome, or mucopolysaccharidosis type II (MPS II), a rare and often lethal lysosomal disorder. This disease is caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase (I2S) which results in a toxic buildup of large sugar molecules – glycosaminoglycans or GAGs – in multiple organ systems. There are a wide variety of possible symptoms, including serious cognitive deficits, complications in skeletal and connective tissue and in the respiratory and cardiac system.

Based on the presence or absence of progressive neurological involvement as well as behavioral issues, MPS II patients are classified as neuronopathic or attenuated. Neuronopathic patients typically have a plateau of cognition and adaptive development at around four years of age, followed by progressive neurocognitive decline. Neurological findings include deafness, retinal degeneration, seizures and sleep disturbances.  Severe MPSII patients commonly die in their teens due to obstructive airway diseases and cardiac failure. As with Pompe disease and certain other lysosomal disorders, the current standard-of-care is life-long weekly or biweekly infusions of ERT – which can delay some complications but does not halt overall progression of the disease and has not been demonstrated to address cognitive issues. Even with ERT, children with Hunter syndrome face life-limiting symptoms and a significantly reduced life span.

Our planned investigational gene therapy for Hunter syndrome is designed to enable expression of a therapeutic I2S protein linked to two receptor binding domains of ApoE (ApoEII) joined in series. ApoEII natively functions as the major cholesterol transporter in the CNS. We’re hopeful that the use of this tag may improve the efficacy of the planned investigational gene therapy and make a real difference for patients and their families.

Across the industry, other teams are also working on tagged proteins to improve efficacy. For example, the ability of GILT to enhance lysosomal uptake is being explored in conjugation with a more traditional recombinant ERT. Allievex is also pursuing this approach through its tralesinidase alfa clinical program for Sanfilippo syndrome type B. This investigational program uses a type of GILT attached to the active enzyme, which is infused directly into the cerebrospinal fluid at regular intervals.

As we think about the future, there may be opportunities, similar to our approaches to gene therapies for Pompe disease and Hunter syndrome, to deploy a range of different tags. One possibility: isolating cell surface receptors that are highly expressed on cell types of interest and generating novel associated tags that could be linked to the therapeutic protein produced by our gene-modified cells in a ‘plug and play’ manner. As the research moves forward, we’ll be right there at this exciting frontier, looking to harness the latest tools to bring new gene therapies to the world.