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Meeting Renewed Regulatory Focus on Gene Therapy Safety Standards

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It is not new news that gene therapies represent a rapidly growing new paradigm, both therapeutically and with respect to how they are managed within the larger scope of healthcare. This has wide-ranging impacts on several key areas, including regulation. In terms of the gene therapy regulatory framework, although 2020 brought a mixed bag — six guidance were finalized, and a new draft guidance introduced — some high-profile rejections have rattled the industry. For example, BioMarin’s delay by the Food and Drug Administration (FDA) on the grounds of not having robust enough clinical trial data, and patient deaths in the Audentes’ ASPIRO clinical trial.

These rejections, coupled with Bristol Myers Squibb and bluebird bio being flat-out refused for their latest submissions, suggests that the regulatory burden on the gene therapy industry is going to grow exponentially, and safety is going to come under even greater regulatory scrutiny.

The balancing act between safety, efficacy and manufacturing

Although safety and efficacy are always at the forefront of drug development, the establishment of efficient and scalable manufacturing processes is also important. While such logistical considerations may initially be considered secondary to drug safety and efficacy, these factors are interlinked. Take, for example, the issues that emerged in the ASPIRO trial. This
Phase I/II trial aimed to evaluate the safety and efficacy of the adeno-associated virus (AAV) delivered gene therapy, AT132, in patients with X-linked myotubular myopathy (XLMTM). XLMTM, a rare neuromuscular disease that affects newborn boys, is often fatal – with 50% mortality within the first 18 months of life. Mutation of the myotubularin-1 (MTM1) gene causes progressive muscle weakness, decreased muscle tone and respiratory failure. Disease-modifying treatment is unavailable and current care constitutes symptom-managment.1

AT132 was engineered to deliver a functional copy of the MTM1 gene into skeletal muscle cells to treat XLMTM. Despite positive results in the low-dose cohort, the trial was halted due to participant deaths from liver dysfunction and sepsis in the high-dose arm.2-3 Evidence is mounting regarding the potential risks of high-dose AAVs, with toxicities also observed with high doses of AveXis’s Zolgensma, Solid Biosciences’ SGT-001 and Pfizer’s PF-06939926.4,5

Are directed evolution or novel production cell systems the answer?

How can this Achilles’ heel in an otherwise powerful modality be addressed? One route is re-designing AAVs so that high doses are no longer required to ensure enough drug reaches the target tissue to work. Directed evolution can alter AAV serotypes to allow them to target specific human tissues more closely, eliminating the need for high doses. But manufacturing yields can drop precipitously, with titers reported to be 100 times lower than with standard AAVs.

Gene therapies are now being developed for more common disorders, including
COVID-19. Considering such applications of these therapeutics, a 100-fold decrease in manufacturing capacity would translate into significant delays in getting drugs to patients who may desperately need them.

Another consideration is the trend towards using novel host cell and vector systems in the production of recombinant AAV (rAAV) gene therapies. While traditional transient transfection methods have produced numerous clinical-stage gene therapy candidates, the scale-up of these processes are particularly challenging. For example, ensuring batch-to-batch consistency has proved difficult with yields of more than 500 liters. Therefore, drug makers are exploring other approaches, such as the insect cell and baculovirus system adopted by BioMarin to produce their hemophilia GT candidate. While it provided great scalability – easily achieving batch volumes over 2000 liters, with higher AAV amounts per batch – concerns have been raised in general. Differences in AAV characteristics likely to be functionally and clinically relevant have been observed between AAVs that are generated by different systems, including
post-translational modifications, such as acetylation, glycosylation, phosphorylation and methylation.6 Further, low-level methylation also occurs at differing sites on the rAAV genomes.

Filters: An elegant solution for gene therapy virus-control strategies

Another linchpin requirement with AAVs is the elimination of contaminants, particularly of viral vectors. An example is baculoviruses that are employed in producing AAVs, and adventitious viruses that can infect the production cells, both of which would harm the patient and constitute major safety breaches. To eliminate unwanted viruses from the classical biologic products, such as monoclonal antibodies and recombinant proteins, manufacturers have applied robust downstream methods that remove or destroy viruses. But these methods are unsuitable for AAV-delivered gene therapies, as they would eliminate the vehicle needed to deliver the therapeutic to the target tissue.

As such, gene therapy drug makers are resorting to other approaches, such as virus filters comprising polymeric membrane barriers that separate out desirable AAVs from undesirable viruses by size. While virus filters are applied downstream in classical biologics production, upstream application is considered redundant. But with heightened regulatory focus on gene therapy safety, more developers are including viral filters upstream and downstream, as part of a comprehensive virus control strategy.

The selection of a virus filter with optimal throughput capacity and performance relies on several factors, including viral load, protein concentration, foulants, process interruptions, pressure, operating flux and ionic strength. This complex interplay of factors is being incorporated by manufacturers of filtration and separation products to better meet current and anticipated regulatory requirements faced by gene therapy developers.

As AAV delivered gene therapies are being developed to treat common diseases, regulators and drug developers alike are learning together how best to ensure patient safety throughout the entire gene therapy lifecycle, an approach firmly mirrored by manufacturing vendors.

About the author

Dr Clive Glover, PhD, is the director of strategy at Pall Corporation and leads Pall’s cell and gene therapy business. Previously he was responsible for driving product development efforts around cell therapy at GE Healthcare and has also held positions in marketing and product management at STEMCELL Technologies. Clive holds a PhD in Genetics from the University of British Columbia.


1.       US National Institutes of Health. Genetic and Rare Diseases Information Center (GARD). X-linked myotubular myopathy. https://rarediseases.info.nih.gov/diseases/11925/x-linked-myotubular-myopathy (accessed January 2021).

2.       Editorial. High-dose AAV gene therapy deaths. Nat Biotechnol. 2020; 38: 910. doi:10.1038/s41587-020-0642-9.

3.       Morales L, Gambhir Y, Bennett J, Stedman HH. Broader implications of progressive liver dysfunction and lethal sepsis in two boys following systemic high-dose AAV. Mol Ther. 2020; 28: 1753–1755. doi: 10.1016/j.ymthe.2020.07.009.

4.       Duan D. Systemic AAV micro-dystrophin gene therapy for Duchenne muscular dystrophy. Mol Ther. 2018; 26: 2337–2356. doi: 10.1016/j.ymthe.2018.07.011.

5.       Buscara L, Gross DA, Daniele N. Of rAAV and men: From genetic neuromuscular disorder efficacy and toxicity preclinical studies to clinical trials and back. J Pers Med. 2020 Nov 28;10(4):258–302. doi: 10.3390/jpm10040258.

6.       Rumachik NG, Malaker SA, Poweleit N, et al. Methods matter: Standard production platforms for recombinant AAV produce chemically and functionally distinct vectors. Mol Ther Methods Clin Dev. 2020; 18: 98–118. doi: 10.1016/j.omtm.2020.05.018.