The first vector-delivered gene therapies to receive FDA approval for clinical use were Luxturna,1 Spark Therapeutics’ vision loss cure, and Zolgensma,2 Novartis’ spinal muscular atrophy treatment. When the approvals were announced, they represented a triumph of translational science. In addition, they brought about a surge in demand for gene therapy delivery technologies. The surge is ongoing. Currently, it is stimulating innovation in vector design and manufacturing.

Vector technology

Viral vectors, in simple terms, are empty viruses that can be used to introduce genetic material into cells. They play a key role in a range of molecular biology applications, including gene therapy delivery.

Luxturna and Zolgensma both use vectors, specifically, adeno-associated virus (AAV) vectors. AAV vector technologies are the most widely used gene delivery technologies, even for R&D-stage products.

According to a 2021 article by McKinsey & Company analysts, 82% of the gene therapies in development at the time were using AAV vectors.3 The next most popular technology, a system based on lentivirus vectors, is used by just 10% of candidates. Moreover, AAV vectors have been industry’s go-to gene delivery system for therapeutics for decades. “Since their introduction in 1965, AAV vectors have become increasingly popular for biopharmaceutical applications because of their relatively simple structure and lack of disease causation,” explains Weiqiang Liu, head of R&D at Synbio Technologies. The company is a provider of DNA reading, DNA writing, and DNA editing technologies, and it has operations in the United States and China.

Although there are alternatives—the recently approved cancer therapy Adstiladrin, from Ferring Pharmaceuticals, uses an adenovirus vector4—AAV vectors remain the most popular because they are effective. “AAVs can provide high transfection efficiency—up to 100% compared to plasmids, which have significantly lower efficiencies,” Liu observes. “AAVs, then, can be ideal choices for gene therapies.”

He notes that the situation is different in cell line development applications, where lentivirus vectors and plasmid vectors are “more suitable because genome integration is necessary.” Liu adds that AAV vectors are attractive delivery vehicles because AAV genomes are small: “The typical single-stranded DNA AAV genome—composed of Rep and Cap proteins flanked by two 145 base pair inverted terminal repeats for synthesis of complementary DNA strands—is just 4.8 kb in length, yet it contains all the necessary components for successful transfer.”

Vector engineering

Another advantage is that recombinant AAV (rAAV) genomes, unlike the genomes of other vectors, form circular structures in the cytoplasm. According to Liu, these structures, which are called episomes, lend rAAV genomes a favorable safety profile.

“Compared with other gene therapy vectors, such as those derived from lentivirus or even plasmid-based technologies, rAAV vectors are the preferred choice for production,” he explains. “Because of their episomal nature, rAAV genomes are less likely to become integrated into host genomes. This reduces potential risks of transgene expression being affected by chromatin structure at sites of insertion in comparison with other systems where higher levels of integration occur.”

But even AAVs are not completely risk-free. As preclinical studies have shown, AAV-based gene therapies administered at very high dosages may cause immunotoxicity5 and genotoxicity.6 These issues challenge the industry to find ways of “further mitigating AAV toxicity,” Liu insists. He adds that the industry is working on mitigation strategies that would allow it to “take full advantage of the opportunities AAVs offer.”

One strategy is to use recombinant technology and apportion AAV genes onto three separate plasmids. When these plasmids are inserted into a cell, some of the viruses produced are replication incompetent. These can be further processed and turned into vectors. However, there are challenges with this type of engineering.

One potential adverse consequence, Liu points out, is increased immunogenicity. “The rAAV production system can affect rAAV quality and thus immunogenicity,” he elaborates. “Novel strategies must be developed to address the challenges posed by immune responses, and standardized monitoring/measurement methods are needed to ensure reliable results.”

Manufacturing advances

Another problem is that AAV vectors are complex and time consuming to produce. “Manufacturing a research-scale rAAV for cell and gene therapies is no small feat,” Liu remarks. “Lab processes take at least 2 weeks, whereas GMP production can take at least 10 weeks.”

These challenges, combined with limited global manufacturing capacity for vectors, mean that vector supplies might fail to keep up with surging demand. To prevent shortages, manufacturers are proposing or enacting capacity increases. For example, Matica Biotechnology, a contract development and management organization, announced last May that it had opened a 45,000-square-foot facility dedicated to the production of viral vectors and cell-based products used in cell and gene therapies, vaccines, oncolytic therapies, and other genetic medicines.7

“The key is faster delivery without sacrificing quality or safety,” says Byung Se So, PhD, Matica’s CEO. “We have … an advanced, modular system that provides the flexibility to adjust resources required depending on client need and product requirements.”

The potential for supply constraints also concerns industry analysts. For example, McKinsey analysts have warned that limited AAV vector capacity could delay the commercialization of new gene therapies, particularly those intended for larger patient populations.8

Last March, a McKinsey article stated, “The majority of early viral-vector-based therapeutics were developed within the context of rare diseases. [Only small] quantities of viral vectors were required, particularly as most therapies were still in the clinical stage of development. Now, with the shift beyond ultrarare indications, viral vector manufacturing requires rapid expansion to be able to address these diseases in the commercial space.”

This view is shared by Liu. “More than 550 AAV vector-based therapies are being evaluated worldwide,” he says. “Consequently, it is projected that this market will continue growing steadily at an estimated compound annual growth rate of 18% to 2030.”

The big question is whether vector manufacturers can satisfy surging demand. The good news, Liu declares, is that the sector is already working to make production faster and less expensive, particularly with respect to rAAV technology, which remains the “method of choice over the next few years.” He emphasizes that manufacturers are “leveraging cost-effective plasmid production systems and streamlining rAAV bioprocessing solutions to gain greater access to cutting-edge science.”

Engineering tools

Like industry researchers, academic researchers expect that technology will play a key role in helping AAV producers cater to growing demand. “Market demand for AAV vectors is indeed accelerating,” says Logan T. Collins, a doctoral candidate who works in a laboratory led by David T. Curiel, MD, PhD, a professor of cancer biology at the Washington University School of Medicine. “AAVs have previously been approved for treatment of rare diseases with very small patient populations,” Collins adds, “but the field is rapidly moving to the point where AAVs will be employed as part of therapies for ailments affecting far larger numbers of people. This will vastly increase demand for these vectors.

AAV Production diagram
AAV manufacturing poses multiple challenges. Adherent cells are not scalable. Suspension cells are difficult to transfect. Affinity chromatography cannot separate empty capsids from full capsids. Ion-exchange chromatography can lose many full capsids due to poor specificity and the need for extreme pH conditions. These and other challenges can be overcome with synthetic biology technology, say researchers at the Washington University School of Medicine. Possibilities include rationally designed cellular chassis circuits.

“The process of clinical AAV production is highly complex and involves several bottlenecks. Such manufacturing challenges are one of the central reasons that AAV therapies are so exorbitantly expensive.”

Collins recently participated in research that explored how the price of AAV vector production could be reduced through synthetic biology. In general, the research suggests that synthetic biology approaches such as systematic engineering and rational design could lead to more efficient and cost-effective nanofactory platforms.

The research was summarized in a recent paper.9 It noted that synthetic biology principles have already been applied in certain bioprocessing methods, and that these methods could serve as the foundation for more systematic gains in production capacity.

“These [methods] include baculovirus expression vectors which synthesize AAVs inside of insect cells as well as herpes simplex virus platforms for transfecting mammalian cells and providing helper functions,” the article’s authors wrote. “On the more contemporary side, newer synthetic biology technologies have shown promise for upending current paradigms of AAV production. Yeast-based AAV production systems are under development, though these are still limited in their production capacity and may require extensive further engineering to achieve their full potential.”

The authors also indicated that synthetic biology has recently been used to create a self-attenuating helper adenovirus system as a novel approach for AAV production. This system was recently described by researchers from Oxgene and the University of Oxford.10

“Synthetic biology approaches that dramatically enhance AAV yields will be vital to meet the growing demand for these viruses,” Collins maintains, “particularly since they will soon start to be used for diseases with much higher prevalence within the population.”

Machine learning

Another tool that is becoming increasingly important in vector production is capsid engineering, a discipline that focuses on modifying the viral capsid to enhance the vector’s ability to deliver its genetic payload. “Capsid engineering,” Curiel predicts, “will allow AAVs to evade preexisting immunity, to decrease toxicity, and to enhance targeting and transduction. It will allow AAVs to cross the blood-brain barrier more efficiently—and more.”

According to Curiel, innovation in analytics technology and predictive computational modeling has given developers a new suite of vector development tools. “Machine learning–guided capsid engineering,” he says, “seems to be gaining a particularly strong foothold and may soon transform the industry at large.”

1. U.S. Food and Drug Administration. FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss [news release]. Published December 18, 2017. Updated March 16, 2018. Accessed February 6, 2023.
2. U.S. Food and Drug Administration. FDA approves innovative gene therapy to treat pediatric patients with spinal muscular atrophy, a rare disease and leading genetic cause of infant mortality [news release]. Published May 24, 2019. Updated May 24, 2019. Accessed February 6, 2023.
3. Capra E, Godfrey A, Loche A, Smith J. Gene-therapy innovation: Unlocking the promise of viral vectors [online article]. McKinsey & Company. Published May 17, 2021. Accessed February 6, 2023.
4. FDA OKs Bladder Cancer Gene Therapy [online news article]. GEN. Published December16, 2022. Accessed February 6, 2023.
5. Dalwadi DA, Torrens L, Abril-Fornaguera J. et al. Liver Injury Increases the Incidence of HCC following AAV Gene Therapy in Mice. Mol. Ther. 2021; 29(2): 680–690. DOI: 10.1016/j.ymthe.2020.10.018.
6. Nguyen GN, Everett JK, Kafle S, et al. A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells. Nat. Biotechnol. 2021; 39(1): 47–55. DOI: 10.1038/s41587-020-0741-7.
7. Matica Bio Opens New Cell & Gene Therapy GMP Facility in Texas. Published May 3, 2022. Accessed February 6, 2023.
8. Capra E, Gennari A, Loche A, Temps C. Viral-vector therapies at scale: Today’s challenges and future opportunities [online article]. McKinsey & Company. Published March 29, 2022. Accessed February 6, 2023.
9. Collins LT, Ponnazhagan S, Curiel DT. Synthetic Biology Design as a Paradigm Shift toward Manufacturing Affordable Adeno-Associated Virus Gene Therapies. ACS Synth. Biol. 2023; 12(1): 17–26. DOI: 10.1021/acssynbio.2c00589.
10. Su W, Patrício MI, Duffy MR, et al. Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination. Nat. Commun. 2022; 13(1): 1182. DOI: 10.1038/s41467-022-28738-2.

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