If gene therapies are to realize their enormous therapeutic and commercial potential, they will need well-designed delivery vehicles—and lots of them. In the near term, most of these vehicles will likely be viral vectors. Viral vectors have a lead on nonviral vectors, which show great promise but still struggle for purchase in clinical trials. So, for some time yet, viral vectors will dominate as the means of delivering gene therapies in vivo. (For ex vivo applications, there may be additional transfection options.)
Viral vectors typically consist of an expression cassette encoding the therapeutic gene as well as a transcription promoter, a transgene, and a polyA signal sequence. The vast majority of the original viral genetic information is removed (96% in the case of adeno-associated viruses, or AAVs), with only short sequences of inverted terminal repeats (ITRs) that are essential for expression, production, and packaging being retained.
Although viral vectors may be tweaked in various ways to enhance their safety and efficacy, they also need to be optimized for commercial-scale production. Consequently, viral vectors could benefit from being engineered in accord with industrial design principles. Industrial design encompasses not just a product’s form and features, but also the product’s manufacturability.
In this article, we will see how various companies are bringing an industrial design sensibility to viral vectors. For the most part, these companies are interested in facilitating gene therapy as we’ve come to know it. That is, they intend to enhance the ability of gene therapies to fix the root cause of a disease by replacing the faulty or missing genes responsible for the condition. In addition, there is interest in future applications. For example, viral vectors could play their part in gene therapies that train the immune system to tolerate foreign tissue. Such gene therapies could help make organ transplants more successful.
The first viral vector to express a foreign gene was created in 1972.1 In the decades since, viral vectors have been used for applications ranging from cell line modification to crop development.
Unfortunately for gene therapy firms, viral vector science has tended to outpace viral vector production technology. Currently available production methods are struggling to meet increased demand, says Mercedes Segura Gally, PhD, vice president of process development at ElevateBio, a cell and gene therapy technology company based in Cambridge, MA.
“The manufacturing processes around viral vectors are complex and still in their infancy when compared with the existing processes for other biologics, such as monoclonal antibodies,” she explains. “Most of the production procedures for widely used vectors such as AAV vectors and lentivirus (LV) vectors are based on transient transfection of adherent cells in 2D culture systems. These procedures simply cannot be easily scaled, and they often lack the high productivity needed to meet growing demand.
“Besides limitations of the upstream production process, the complex biological nature and large size of viral vectors also make it difficult to develop efficient downstream harvest and purification processes. The overall inefficiency of current production processes [and the] lack of reliable in-process analytical toolkits are significant obstacles to the rapid development of processes that are robust and scalable enough to meet increasing clinical vector demand.”
A scalable platform
Capacity limitations have prompted the development of scalable vector production platforms. One such platform, Gally points out, is ElevateBio’s BaseCamp system.
“Our suspension-based, scalable platform for LV and AAV vectors has demonstrated high volumetric productivity of clinically relevant vector yields,” Gally asserts. “[The platform] allows for rapid and efficient transition through the preclinical, clinical, and commercial stages.
“This speed and efficiency can be attributed to our use of standard starting materials, state-of-the-art processing equipment, established unit operation processing steps, and enhanced analytical methods and operation procedures. The increased yields of high-quality vectors combined with shortened development times will help to ultimately drive down vector manufacturing cost of goods and make these therapies more accessible to patients in need.”
Vector design considerations
Vector design is another area seeing a lot of innovation. In recent years, new molecular biology techniques have given scientists the tools to start “designing” vectors like never before.
“Designing gene therapy vectors allows for better target tissue tropism, higher transduction efficiency and transgene expression, lower immunogenicity, reduced genotoxicity, and improved manufacturability,” Gally details. “These metrics are some of the key elements to consider when developing viral-based gene therapies.
“Many novel approaches for capsid engineering have relied on large-scale screening of rationally designed engineering vector libraries or naturally occurring isolates from multiple species. Molecular biology techniques are incorporated to introduce changes to vector sequences that improve the biology activity—such as codon optimization and incorporation of regulatory elements. In parallel, multiplex and high-throughput functional screening methods are employed to facilitate the selection of suitable vectors based on therapeutic application needs.”
This take is shared by Smitha Jagadish, PhD, senior director of neuroscience and pain research at Grünenthal, a pharmaceutical company based in Aachen, Germany. She says that in gene therapy, capsid engineering is a “hot field.”
“Many companies are coming up with next-generation novel capsids that will be the future of gene therapy,” she elaborates. “This goes hand in hand with the current manufacturing processes that need to be improved by orders of magnitude to bring down the costs of these therapies.
“Optimization has to be geared toward best efficacy with the least amount of vector dose and the best safety profile with a given route of administration. The optimized capsid encoding the expression cassette that transduces the cell type and the tissue of interest is then the lead clinical candidate.”
A targeted approach
This revolution in gene therapy vector design is allowing researchers to look at novel applications. For example, scientists at CombiGene, a biotech firm based in Lidingö, Sweden, have used vector design to develop CG01, a treatment candidate for drug-resistant focal epilepsy. It uses an AAV vector to deliver—directly to the part of the brain where epileptic attacks originate—a genetic payload that encodes the combination of neuropeptide Y and its antiepileptic receptor Y2.
Last year, CG01 was licensed from CombiGene by Spark Therapeutics, a gene therapy company based in Philadelphia, PA. At the time, CombiGene CEO Jan Nilsson stated, “We look forward to advancing this potentially transformative therapy together with Spark for the benefit of a patient group in need of better treatments.”
The ability to manipulate the CG01 delivery vector is key to the product’s therapeutic potential, remarks Martin Linhult, PhD, director of chemistry, manufacturing, and control at CombiGene. He explains that for CG01 to be effective against epilepsy, CG01 needs an AAV that can pass the blood-brain barrier and target a small area of the brain. Developing an AAV that can accomplish both these tasks is quite a challenge. Indeed, according to Linhult, it convinced CombiGene of the need to collaborate with experts.
To manufacture the vectors for CG01, CombiGene uses the standard HEK293-based platform, which is widely used in the gene therapy sector for the production of both adenovirus and AAV vectors. As the CG01 program progresses, the vector production platform is likely to be revised, Linhult predicts.
“We do have a dominating platform technology—the triple plasmid in suspension HEK293 cells,” he asserts. “However, the field is quite new, and only a few products have been approved. I think optimization could be done all through the production process.”
Avoiding the immune response
Vector design is also helping industry overcome another major technical challenge: the immune response. “While AAV vectors are delivering a therapeutic gene, the human body’s defenses respond to the engineered viruses just as they would to natural ones—as if they were a potentially deadly threat,” explains Susan Faust, PhD, founder and CEO of NxGEN Vector Solutions. “These defenses have presented a substantial challenge to effective gene therapy: How to deliver the precious genetic cargo to cells without activating the body’s immune response?
“The design of the AAV capsid to escape neutralizing antibody binding and the removal of immunostimulatory unmethylated CpG dinucleotide motifs from the vector genome to avoid Toll-like receptor-9 signaling and immune activation are two strategies to deliver the curative genes without activating the body’s immune system, resulting in efficient transduction and durable transgene expression.”
Improving transplant outcomes
At NxGEN Vector Solutions, vector design is also pivotal to another project—the use of gene therapy to increase the chances of transplant success. “Chronic rejection is a progressive disease, characterized by proliferation of fibroblasts within the graft, which promotes interstitial fibrosis as well as thickening and narrowing of the coronary vessels, a process referred to as transplant-associated vasculopathy,” Faust says. “These pathological changes result in deteriorating graft function for which there is no cure except for retransplantation, and chronic rejection will occur despite the administration of antirejection medication.”
One potential solution is “acquired tolerance,” a process through which a patient can be made selectively unresponsive to the antigens of a given graft while the remainder of the immune defense mechanisms is left intact.2
“Gene therapy was originally developed to treat diseases with a simple Mendelian pattern of inheritance, whereby patients who are missing two copies of a gene are given a new, healthy gene [to bring about] a cure,” Faust observes. “In contrast, acquired tolerance was the goal for our gene therapy platform.”
According to Faust, NxGEN Vector Solutions has already demonstrated the idea’s potential. “We have shown that gene therapy can be employed to modulate the immune system to prevent chronic rejection without the requirement for life-long antirejection medication,” she elaborates. “Chronic rejection occurs despite the administration of antirejection medications, and long-term systemic immunosuppression of patients can lead to kidney damage, diabetes, heart failure, cancer, and increased susceptibility to illness.”
1. Jackson DA, Symons RH, Berg P. Biochemical Method for Inserting New Genetic Information into DNA of Simian Virus 40: Circular SV40 DNA Molecules Containing Lambda Phage Genes and the Galactose Operon of Escherichia coli. Proc. Natl. Acad. Sci. USA 1972; 69(10): 2904–2909. DOI: 10.1073/pnas.69.10.2904.
2. Brent L. The discovery of immunologic tolerance. Hum. Immunol. 1997; 52(2): 75–81. DOI: 10.1016/S0198-8859(96)00289-3.