Insights The FDA and AAV-Based Gene Therapy Safety: What You...

The FDA and AAV-Based Gene Therapy Safety: What You Should Know


Science fiction author Arthur C. Clarke once commented that “Any sufficiently advanced technology is indistinguishable from magic.” Over the last couple of years, we have witnessed the tremendous potential of adeno-associated virus (AAV)-based gene therapies. Children with spinal muscular atrophy (SMA) who have received Zolgensma, 4 and a half years later are now dancing, swimming, and riding bikes. This was almost unimaginable 10 or 15 years ago, but it was certainly hoped for by all the scientists in our field. We have seen children who were progressively going blind regaining sight, no longer having to “hold on to their friends to walk at night.” Magic. However, even with these successes, there have been a few high-profile safety issues that have occurred, including deaths associated with AAV vector gene therapies that have raised safety concerns among the public and regulators. The objective of this article is to demystify AAV vectors, providing some basic background on the vectors, defining safety concerns, outlining strategies to mitigate safety issues, and discussing recent FDA safety meetings regarding AAV gene therapies.

What Are AAV Vectors?

Wild-type AAVs are some of smallest viruses (approximately 25 nm) with a linear single stranded DNA genome of 4.7 Kb in length with 2,145 nucleotide inverted terminal repeats. The linear DNA has 3 genes encapsulated in 60 outer coat capsid, which include rep (replication) gene encoding 4 proteins required for viral genome replication and packaging, cap (capsid) that encodes the proteins in the capsid, and aap (assembly activation protein) that provides the structure for the capsid.1-3 In order to make AAV viruses usable for gene therapies, they are engineered into recombinant AAVs (rAAVs), where the viral genome is replaced with a promoter, the gene or genes of interest, and a terminator.4 Recombinate AAVs cannot replicate in vivo and need a helper virus to replicate and are not passed on during cell division. These properties make them very safe vehicles to drive long-term gene expression after a single infection.5,6 Presently, there are 11 serotypes (AAV1 through AAV11) cloned, and they can have specific affinity to tissues. For example, AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 are optimal for central nervous system (CNS) disorders.7

What Are Major Considerations When Selecting an AAV Vector?

There are 3 major considerations when selecting the appropriate AAV:

1- Selecting the right capsid and promoter: Selection can improve targeting of cell transduction and increase expression, which has implications for the dosage required for effective therapy.

2- Selecting the best dosing regimen: The choice of dosage can lead to either reduced efficacy when the doses are too low or can lead to toxicities when the dosing is too high. Dosages that are too low could lead to inefficient transduction; dosages that are too high can result in delivery and transduction-related toxicities.8 Inflammatory toxicities have been seen with increasing doses of gene therapies resulting in complement activation, cytopenia, and severe hepatotoxicity. Therefore, it is ideal and safest to have as minimal dose as possible while still being clinically effective. Majority of the deaths associated with AAV vector have been seen at higher doses.                

3- Development of immune responses to the vector (immunogenicity): Many individuals have already been exposed to one or more serotypes of wild type AAV, and thus may have some degree of pre-existing immunity to the vectors used that includes binding antibodies and neutralizing antibodies (NAbs), which may negatively impact clinical efficacy and could increase post-therapy prevent re-administration. The prevalence of pre-existing anti-capsid NAbs varies by AAV serotypes, ranging from approximately 40% for AAV8 to 74% for AAV2.9

Strategies for Mitigating Safety Issues with AAV Vectors

Presently, there are multiple strategies being deployed or developed to mitigate risk associated with AAV vector usage and to increase performance, including excluding individuals with some level of existing NAbs, depleting NAbs, vector engineering removing immunogenic features during vector design lowering therapeutic dosing, and immunosuppression.  

Excluding Patients with Naturalizing Antibodies (Nabs):

Often patients with pre-existing Nabs against the specific AAV capsid are precluded from receiving the gene therapy. This occurs often in clinical trials with 45% excluding patients with a pre-specified Nab levels. With that said, this exclusion in clinical trials is quite variable among therapeutic areas, where approximately 90% of the blood disorder trials exclude patients, and ophthalmologic and CNS clinical trials excluding 10% and 21%, respectively. This represents the current hypothesis that NAbs are of greater concern for systemic delivery of AAV than for targeted therapy to immune privileged sites.5

Tissue-Specific Promoters for AAV Vectors:

For a gene therapy to work, there needs to be a therapeutic gene, promotor, and polyadenylation signal. The promotor’s job is to control the gene expression.10 The traditional promotors have not been tissue-specific, sometimes leading to low expression by silencing the transgene or overexpression, resulting in cell damage and toxicity. One of the trends we anticipate is a movement towards using tissue-specific promotors. Tissue-specific promoters work only on specific cell types and can minimize over- and under expression.

Transgene Optimization:
Modifying the transgene to produce more-effective therapeutic

Proteins is another method beginning to be employed, with the hope that greater therapeutic efficacy will lead to lower doses and better safety with similar efficacy. An example of this is FIX-Padua, a variant of FIX (Factor “9”) with a hyperactivating R338L mutation, which resulted in increasing the efficacy by up to 5–10-fold in hemophilia B patients at lower doses with no liver enzyme elevation in the majority of the patients.10

Better Device Delivery Mechanisms:
Another strategy being employed is the development and use of new devices to deliver more vectors in a more localized manner to increase efficacy and minimize toxicity. We are starting to see this trend, especially in CNS disorders. For example, many gene therapies targeting the CNS, delivering the vector through a bolus injection into the lumbar spine, often with minimal brain penetration and cellular targeting. New approaches being developed use computer models to understand the cerebral spinal flow dynamics in an individual to deliver vector more precisely.11

It is becoming more common to administer immunosuppressive agents as part of a AAV gene therapy. In the first few AAV gene therapies trials, corticosteroids were used reactively after the therapy administration when liver enzymes became elevated. The introduction of the immunosuppressants usually corrected the elevated enzymes. Recently, it has become prevalent to administer immunosuppressants proactively. Common immunosuppressants used are corticosteroids, rapamycin, tacrolimus, mycophenolate, rituximab, eculizumab, and hydroxychloroquine. All of these immunosuppression agents can have significant side effects, so it is important to factor in the safety profile of the immunosuppressant in conjunction with patient clinical presentation before administering.12

FDA on Safety Issues with AAV Vectors

In May of 2020, the US Food and Drug Administration (FDA) issued general guidance for the industry on human gene therapy (GT) for rare diseases. In that guidance, the FDA notes that “pre-existing antibodies to any component of the GT product may pose a potential risk to patient safety and limit its therapeutic potential.” In addition, antibodies to the gene therapy may also limit the re-administration of the therapy to a one-time use. The FDA also stated “Sponsors may choose to exclude patients with pre-existing antibodies to the GT product.” If patients are excluded based on pre-existing antibodies, the sponsor should strongly consider development of a companion diagnostic to detect antibodies to the to the gene therapy. If an in vitro companion diagnostic is needed to appropriately select patients for the clinical study, then there should be coordination of the companion diagnostic marketing application with the biologic license application for the gene therapy.13

In response to a few high-profile adverse events seen in clinical trials and surveillance of AAV gene therapies, on September 2nd and 3rd of 2021, the FDA’s Cellular, Tissue, and Gene Therapies Advisory Committee held a meeting to discuss toxicity seen with AAV vectors. The meeting centered on the following adverse events: hepatotoxicity, thrombotic microangiopathy (TMA), dorsal root ganglion (DRG) toxicities, neurotoxicity – MRI findings, and oncogenicity. The FDA questions to the committee centered around:

Hepatotoxicity: (1) Role of animal studies? (2) Before AAV administrations, how should patients be screened and categorized for risk of liver injury? (3) What strategies could be implemented to prevent or mitigate the risk of liver injury? (4) Beyond weight of the patient, what factors (e.g., level of disease severity) should be considered to determine the vector dose for systemic administration? (5) Considering the risk of toxicities observed in clinical trials with high doses of AAV vectors, should an upper limit be set for the total vector genome dose per subject?

Thrombotic Microangiopathy (TMA): (1) What factors may increase the risk of TMA following AAV vector administration? (2) What strategies could be implemented to prevent or mitigate the risk of AAV vector-mediated TMA? (3) Should an upper limit be set for the total vector dose? (4) Discuss whether an upper limit should be set on the total capsid dose.

Dorsal Root Ganglion (DRG) Toxicities: (1) based on the published data, please discuss the relevance of the non-human primate (NHP) cases of DRG toxicity to human subjects. (2) Please provide recommendations on preclinical study design elements, such as animal species/disease model, age, in-life and post-mortem assessments, and duration of follow-up, post-dose that may contribute to further characterization of DRG toxicity. (3) In addition to periodic neurological examinations, please provide recommendations on other methods to mitigate the risk of DRG toxicity in clinical trials.

Oncogenicity: (1) Discuss the merits and limitations of animal studies to characterize the risk of AAV vector-mediated oncogenicity. (2) Discuss benefit-risk considerations for AAV vector-mediated oncogenesis, such as patient age at the time of treatment, pre-existing liver conditions (e.g., infection with hepatitis B or C virus), and high vector dose.

Although there has not yet been a formal guidance issued from this meeting, the meeting did serve to help define some of the major questions regarding AAV vectors.14

Parting Thoughts The final comments from Peter Marks MD (US FDA) frame the situation well: “I would say the fact that we’re discussing this is evidence that they (gene therapies) very much are becoming a reality. And it’s actually a good sign because with every medical therapy, as it comes along, we have to deal with the side effects that may come up and address them.”14 As Hagrid said to Harry Potter, not all wizards are good. Well, not all AAV gene therapies are bad. AAV gene therapies hold tremendous promise, and we are engaged in the iterative process of refining AAV therapies. We are turning magic into science!

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Deborah Phippard, PhD, Chief Scientific Officer of Precision for Medicine, is a pharma industry veteran and expert at biomarker-driven clinical trial design and execution. Leader of biomarker and drug development programs for pharmaceutical and diagnostics companies, as well as the National Institutes of Health. Spearheaded the discovery of pharmacodynamics biomarkers and novel targets for inflammatory disease therapy. Currently, working on developing, validating, and implementing companion diagnostic assays for neutralizing and total antibodies for multiple gene therapies. 

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Phil Cyr, is Senior Vice President of Customer Solutions for Precision Value & Health with responsibility for cell and gene therapy. Phil has over 26 years of health economics, outcomes research, health policy, and payer experience, including a strong record of conducting published research, conducting health technology appraisal within a U.S. payer, and interacting with global HTAs. Phil and his team have built the health economic evidence and value demonstration strategy for over 15 gene therapies.

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