Comparing Viral Vectors for Gene Therapy Delivery

As the market for gene therapies matures, the industry is at a point where focus is on the best method of delivery for the genetic package at the core of a gene therapy product. Today, the two most commonly known methods for delivery are adeno-associated virus (AAV) vectors and lentiviral vectors.

AAV

AAVs, as small viruses (approximately 20–25 nm in diameter, approximately 4.8kb linear single-stranded DNA) in the parvoviridae family, are considered good vectors for gene therapy delivery based on their ability to infect both dividing and non-dividing cells, says Elie Hanania, PhD, vice-president, Process Development, Viral Vector Technologies, Avid Bioservices. Recombinant and engineered AAVs do not integrate into the host genome, but maintain sustained expression for moderate duration, he notes.

Hanania also points out that AAV has a good safety profile (thus far) and can sustain harsh manufacturing conditions while maintaining infectivity because it is small and non-enveloped. There are several serotypes of recombinant AAV (rAAV) available, which provide options for tissue-specific delivery. Furthermore, rAAV can be produced multiple ways based on plasmid transfection or other helper virus infection, Hanania observes.

Emmanuelle Cameau, strategic technology partnership leader, Genomic Medicine, Cytiva, adds that AAV is non-pathogenic to humans, does not cause disease, and has a low immunogenicity, which are crucial factors for the safety of gene therapy patients. In addition, AAV can mediate long-term expression of the therapeutic gene, which is an important factor when treating chronic conditions that require sustained gene expression.

AAV vectors can also be engineered to carry a variety of genetic payloads, including small genes, regulatory elements, and RNA interference molecules, says Cameau, who emphasizes that it is this versatility that allows for customized gene therapy approaches tailored to specific diseases.

LV

Lentiviruses (LVs) also offer the advantage of being able to efficiently infect both dividing and non-dividing cells, allowing them to target different tissue types, such as neurons, muscle cells, and hematopoietic stem cells, says Cameau. “They integrate their genetic material into the host genome, ensuring stable, long-term expression of the therapeutic gene. This is beneficial for conditions requiring sustained gene expression over the lifetime of the patient,” she states.

“LVs can also carry larger genetic payloads compared to many other viral vectors, such as AAV,” Cameau continues. “Like AAV, they can be engineered to include various regulatory elements, promoters, and transgenes, allowing to tailor them to specific therapeutic needs, including tissue-specific expression and controlled gene regulation.”

An added advantage is that lentiviral vectors are generally less immunogenic than other viral vectors, such as adenoviruses; this characteristic reduces the likelihood of an adverse immune response. Moreover, LVs are highly efficient with gene transfer, particularly in stem cells such as hematopoietic stem cells and other progenitor cells, which are essential for therapies targeting blood disorders and other systemic conditions, Cameau explains.

“Lentiviral vectors can also be pseudotyped with different viral envelope proteins to alter their tropism and improve targeting to specific cell types or tissues,” Cameau adds.

Hanania explains that the LV is an enveloped single-stranded RNA virus that belongs to the Retroviridae family. Because of their ability to infect non-dividing cells and integrate into the host cell genome, not only can LVs carry out sustained expression, but they can also pass the ability along to cell progenies. LVs can therefore be used for both ex-vivo gene therapy application (mainly chimeric antigen receptor T cell [CAR-T] therapy and stem cell therapy) and in-vivo gene therapy, with direct injection of the viral vector carrying the desired therapeutic payload into a patient.

“Having the capability of penetrating the nuclear envelope, LVs can infect non-dividing cells (including neurons), hence potential application for neurological-based gene therapy,” Hanania says.

Tackling manufacturing

Manufacturing viral vectors requires consideration of their respective challenges. There are different approaches to manufacturing rAAVs, Hanania points out. “The most common approach is triple plasmid transfection into HEK293 [human embryonic kidney 293] cells mediated by a transfection reagent,” he explains. “Alternatively, rAAV can be produced by infecting Sf9 insect cells with two baculoviruses carrying Rep/Cap and the gene of interest (GOI). Similarly, two herpes simplex viruses harboring Rep/Cap and GOI can be used.”

In addition, wild type adenovirus can be used to render the helper function required to produced rAAV when infecting engineered HeLa (or similar) cell lines, Hanania states. “Being non-enveloped, rAAV is tolerant to harsh purification conditions including low pH, high salt, and high temperature (contaminant virus inactivation if needed),” Hanania adds.

Meanwhile, LVs are classically more difficult to manufacture because they are enveloped viruses, says Cameau. “Enveloped viruses are more delicate and sensitive than non-enveloped [viruses]. Therefore, LVs are sensitive to temperature, pH, foam … which makes them really challenging to process,” she explains.

The LV manufacturing process needs to be designed such that it can mitigate the yield loss all along the way, Cameau says. In comparison, AAV is much easier to produce and purify, even at the largest scales, although empty capsids generation instead of full capsids is a particular challenge of AAV that the industry is currently looking at overcoming, she specifies.

“Third-generation (improved safety profile) LV is typically produced by transfecting HEK293T cells with four plasmids,” explains Hanania. “Being an enveloped virus, temperature, pH, and shear are detrimental and hence purification approaches are typically gentler, with one single-step chromatography and one concentration/buffer exchange step. Furthermore, being enveloped and larger in size compared to AAV, [LV] final sterile filtration is a challenge and may have an impact on titer and yield.”

Hanania further explains that the common challenge in manufacturing for both AAV (rAAV in particular) and LV is the scaling up of the transfection step, which is needed to ensure high titer and yield. However, the industry has made great strides in having commercially available serum-free, chemically defined media to support high density cell cultures. Meanwhile, new and improved transfection reagents have emerged that support more efficient transfection into cells.

“The scale factor and the time element are two focus points to sustain high titer and yield across both platforms. The overall price-tag is still quite high factoring all starting materials (GMP [good manufacturing practice]-grade plasmids, media, transfection reagents, and other associated supplies),” Hanania adds.

Establishing the manufacturing process

Establishing a manufacturing process for either LV vectors or AAV vectors means strategizing from the start to avoid pitfalls and considering the unique needs for each vector platform.

“Unlike production of monoclonal antibodies, where templated approaches are applicable, manufacturing viral vectors has unique (or non-standardized) steps among the various gene therapy programs,” emphasizes Hanania. Common factors for cell thaw and expansion can be generalized, he notes; however, beyond that, optimization is typically required to boost productivity to achieve the desired titers and yield.

This optimization approach applies to plasmid ratios, ratio of DNA to transfection reagents, time of transfection, harvest, filter clarification, lysis (when applicable), host-cell reduction, volume management prior to purification, optimized chromatography approaches, final formulation, and final sterile filtration, Hanania enumerates. These aspects are all areas with impact on final product quantity and quality.

“The above comes at a hefty price of time and cost,” Hanania states, “something to be considered an investment and not ignored as an unnecessary task. Accordingly, process development and optimization need to be factored into the overall program timeline to ensure a robust, reproducible, and CGMP [current GMP]-ready manufacturing process. Once in place, this can be used for campaign runs.”

To achieve an optimized manufacturing process, the manufacturer needs to understand the biology of the viral vector platform, its pros and cons, and the production options (scales and limitation). The manufacturer must also engage a skilled team, whether in-house or at a contract development and manufacturing organization, to help establish a good road map and determine the best approach as well as how to navigate through the process successfully.

Using non-scalable technologies from the start is one major pitfall to avoid, says Cameau. “Whether manufacturing AAV or LV, the manufacturer should ask themselves: What is the final target scale of this process? And [they should] choose from the start the right technologies that will enable that scale, with as little pain as possible,” she explains.

Cameau notes that this approach also applies to reagents and starting materials, such as the right cells (transient or stable or packaging), the right DNA quality in cases other than stable, transfection reagents, and media. “If the application is in vivo, the use of HEK293T cells should be avoided, and preferably use HEK293 cells, as there can be potential risk in using vector potentially carrying the T antigen,” she adds. “Starting with the right biological and physical tools form the beginning will avoid the manufacturer loosing precious time in redeveloping and optimizing a process, thus reducing time to market.”

Choosing the right vector

As both LV and AAV have their own advantages and challenges, what other factors come into play when choosing which platform to use for a gene therapy? According to Cameau, the choice depends on what the final application is. “If the use is in-vivo modification of cells or tissue, then AAV is the safest way to go, provided the size of the GOI does not exceed 4.7 kb and that an AAV serotype can target specifically the selected type of cells or tissue,” she explains.

However, in the case where the GOI is larger than 4.7 kb, drug developers have instead been using LV or other virus types, such as retrovirus. “But the number of LV-based therapies in development for in-vivo applications is significantly minor compared to ex vivo due to the reduced safety aspect of stable integration into the human genome of LV,” says Cameau.

If the application is the transduction of stem cells ex vivo, then LV is usually the viral vector method of choice, as it can package significantly large GOIs and stable integration into the genome makes the cell modification permanent, Cameau adds.

Hanania points out that researchers tend to make a decision based on the ultimate need of the advanced therapy. “Thus far,” he notes, “ex-vivo applications have been utilizing LVs mainly because they can infect non-dividing cells (most CAR-T applications) efficiently. Furthermore, the overall amount of virus needed is not too high at this point, hence large scale (i.e., > 100 L scale) [is] not needed.”

Whereas, for in-vivo applications and tissue-specific delivery, rAAV has been the “go-to” virus, Hanania says. Diverse production options and the robustness of the virus are attractive for many advanced therapies. Meanwhile, production scales nearing the 2000 L range are becoming common for some programs, although 200–500 L is still the desired scale for most early-stage programs, he specifies. However, pay-load size is a major constraint when it comes to rAAV; therefore, Hanania says researchers may opt to choose LV for larger GOIs; although, insertional mutagenesis/oncogenesis is a concern due to LV’s tendency to integrate into the host genome.

“It is important to mention that packaging and producer cell line development have been going on for both rAAV and LV. Great success has been achieved with gamma retrovirus producer cell lines where sustained production and multi-day harvests can be performed. Dealing with cytotoxicity has been one of the major challenges in advancing these initiatives for rAAV and LV. Controlled expression and gene editing/engineering are being used to circumvent some of these issues to come up with practical and functional producer cell lines,” Hanania says.

About the author

Feliza Mirasol is the science editor for BioPharm International.

Article details

BioPharm International®
Vol. 37, No. 7
July/August 2024
Pages: 10–13

Citation

When referring to this article, please cite it as Mirasol, F. Comparing Viral Vectors for Gene Therapy Delivery. BioPharm International 2024, 37 (7) 10–13.