Research Models
Rajeev Dhawan, PhD

Overcoming Barriers to Testing Gene Therapies in Animal Models

Screening assays can help developers pre-select the right animals for their AAV vectors

It may be hard to wrap your head around the notion that you can use viruses to help treat and cure disease, but that is essentially what viral vectors do.

Adeno-associated viruses (AAVs)—harmless cold viruses ubiquitous in nature—are critical components in the development of gene therapies for patients. In the past five years, US regulators approved two landmark gene therapies, one for a rare, inherited form of blindness and the other for a genetic condition that progressively destroys motor neurons. Both were delivered using AAVs. No doubt there will be more AAV-based treatments coming. In the US clinical trials database , there are more than 200 gene therapy trials employing a viral vector—the vast majority of which are AAV vectors.

Still, while viral vectors have led the way in many clinical and preclinical successes, a number of challenges limit their true potential . Among these is the fact that the immune systems of people could have been previously exposed to one or many of the 13 known AAV serotypes and done what our immune systems are trained to do – induce antibodies that neutralize them. In short, the viruses being used to shuttle the gene therapies into cells can unwittingly interfere with their clinical success.

This is not just a problem for patients. Animals including mice and some large animal species we work with to test gene therapies can also have pre-existing immunity to the viruses used as vectors, undermining the potential efficacy of the drug being tested, and biasing the outcome of the study. Fortunately, scientists have developed a workaround to this problem.

Breakthrough using AAV

AAVs are the most common vector used in gene therapy, and we have learned a lot about them since they were first discovered in the 1960s. We know the most about AAV2 which serves as a prototype for the AAV family and is currently the one mostly commonly used in gene therapies. There are 13 known serotypes of AAV. These serotypes differ in their ability to infect certain tissues, or cell types. This makes AAV a useful system for preferentially transferring a gene therapy to a specific cell type.

As the chart below shows, not every AAV serotype infects every organ. When conducting animal studies—and for that matter human trials – it is important to know whether neutralizing antibodies for a specific AAV are present in their blood because it can impact whether they are a good candidate for being dosed.

AAV Serotype by Tropism

Antibody Screening Assays

Let’s say you want to do a study on a kidney or diabetes drug. An ideal viral vector for this candidate might be the AAV2 serotype. You need to know if the animals you are working with have neutralizing or non-neutralizing antibodies induced following pre-exposure to the serotype, because when the neutralizing antibodies are present they can bind to AAV, reducing delivery of the gene therapy and thereby lowering the efficacy of the drug being tested.

If scientists could prevent antibodies from neutralizing AAV, it could greatly enhance the dose of the gene therapy delivered and expand the animals who can receive it. What we can do is develop assays that tell you whether the animal is producing antibodies against the drug, and if those antibodies are neutralizing it.

For instance, one can design in vitro tests that both screen for the presence of pre-existing neutralizing antibodies in animals via a qualitative NAb screening assay at different dilutions, and if present, measure the concentration or titer of antibodies in their blood or tissue via a quantitative NAb titer assay. If you detect that an animal has a high presence of neutralizing antibodies against a particular AAV serotype, it means that you likely you cannot work with the animal. For those with lower titers, you may still be able to work with the animal, but it could mean the levels of the drug need to be adjusted.

Charles River Lab will soon be offering serology and PCR-based antibody screening assays for the AAV2 and AAV9 serotypes followed by expansion into remaining serotypes for AAV gene therapy research.

At the end of the day, being able to identify the presence of these neutralizing antibodies early will save significant time and money in the development of gene therapies, and in the long run help patients.

Rajeev Dhawan, PhD, is Director of Bioassay Services for Charles River Laboratories' Research Models Services division. His group is currently responsible for developing reagents for routine serological screening of SPF animal for infectious agents.