Navigating The Evolving Gene Therapy Delivery Landscape

By Cell & Gene Live Editorial Staff

Advancements in gene delivery are transforming medicine, offering new hope for treating various diseases. However, selecting the right delivery system for a gene therapy is a complex decision that requires carefully weighing different safety profiles, targeting capabilities, and manufacturing challenges to find the best balance for the disease being treated and the preferred therapeutic approach. Recent advances and potential breakthroughs on the horizon promise to further advance the safety and efficacy of common gene delivery tools.

Among these tools are lentiviral vectors (LVVs), lipid-based nanoparticles (LNPs), adeno-associated viruses (AAVs), and constructs based on the herpes simplex virus (HSV). Each has benefited from research to fix or circumvent its shortcomings, but more work remains to maximize these tools’ therapeutic potential.

Lentiviral Vectors: In Vivo Use Is Gaining Traction

LVVs are attractive for gene therapy delivery partly because they tend to boast large cargo capacity. While AAV vectors have about a 4.5 kilobase (kb) cargo limit, LVVs can deliver about 8 kb. Another advantage of LVVs is their strength in transducing dividing cells, which other vectors lack. This allows LVVs to be passed on to daughter cells, ensuring continuous gene expression.

The biggest safety hurdles relevant to LVVs — most of which are prepared ex vivo — are not due to the lentivirus itself but arise from how patients are pretreated to allow their stem cells to be reinfused. Essentially, room is made in the patient’s bone marrow through a combination of immunosuppressive agents and, in some cases, high-dose chemotherapy. Research is being dedicated to minimize the intensity of this procedure as well as possibly reduce the pretreatment dose sizes.

Still, a potential immune response to LVVs does not preclude in-vivo application. It is possible different LVV pseudo types could be used to prevent an immune response. Researchers can create mutations in existing lentiviral envelope proteins or craft new virus envelopes that bind to different receptors, allowing them to change tropism. Another option is to insert ligands into lentiviral envelope proteins and use those to target certain cell types. These strategies can minimize immune responses as well as enable different types of targeting. Moreover, because an LVV therapy’s initial dose is intended to be a therapeutic dose, the immune response generated by in vivo delivery should not inhibit the therapy’s ability to enter and to express.

In terms of manufacturing challenges, creating and manufacturing a bespoke LVV product for every patient is a complex process. Time and cost add up quickly when making GMP-grade plasmids, particularly when producing multiple plasmids; LVVs typically are manufactured by processing mammalian cells through a four-plasmid transfection system.

Additional engineering then is required to make the plasmids inducible expression, allowing the manufacturer to introduce the gene of interest by transfection of a single plasmid or to introduce it as part of a producer cell line. In general, moving to suspension cells earlier in process development can help improve efficiency and scalability, since adherence cells are harder to scale up. Because some cytotoxic factors are involved in production, packaging cell lines also can be used to improve efficiency.

Adeno-Associated Viruses: Immune Response Management Options Are Expanding

Adenoviruses generally have a good safety and tolerability profile that facilitates their integration into the standard of care because they do not contribute significantly to toxicity. AAVs typically are useful for indications requiring transient expression — for example, to transitively express vascular endothelial growth factor (VEGF) to restore blood flow to an area of the body. And, as a vector, AAVs are more stable to inject than mRNA. Finally, AAVs are a relatively easy vector to produce with a high titer, in addition to boasting a limited cost of goods and being readily scalable.

However, oncolytic viruses that induce a strong immune system response remain underutilized. While some adenovirus serotypes that potentially reduce preexisting immune responses are available for systemic application, safety concerns surround patients who are administered high doses. Liver toxicity also is a key AAV concern that currently is addressed mainly with immunosuppressive agents.

Intratumoral delivery techniques can minimize overall toxicity to which the body is exposed, compared to systemic delivery. This approach has proven successful in animal models, wherein a strong immune response against the adenovirus has not been induced even after repeated injections. Regardless, adenovirus biodistribution is localized and contained, and the tox profile typically seen in patients is flu-like. Some patients experience fever and chills lasting less than 48 hours, while others exhibit no symptoms at all.

One example of intratumoral delivery is Candel Therapeutics’ CAN-2409 (aglatimagene besadenovec), an adenoviral replication-defective engineered gene construct encoding the thymidine kinase (TK) gene derived from herpes simplex virus (HSV). CAN-2409 is injected directly into the tumor or target tissue and is used in tandem with the prodrug valacyclovir, a generic given orally for 15 days after administration of the virus. The TK activates the prodrug and forms metabolites that integrate into the DNA and induce immunogenic cell death. CAN-2409 is multimodal by virtue of this oncolytic activity combined with proteins in the capsid that are immunogenic and act like a vaccine.

Although only a few viral genes were deleted from the original AAV vectors, researchers can now remove significantly more to create what are called gutless vectors. These modified vectors have most or all viral sequences removed, leaving behind only the essential elements for replication and packaging. As a result, they have a larger capacity for carrying foreign DNA and reduced immunogenicity and toxicity compared to earlier generations of adenovirus vectors. Gutless vectors also are slightly more difficult to manufacture, but modifications to the capsid proteins can improve tropism.

A discussion of AAV manufacture would not be complete without addressing the inadvertent production of some replication competent adenovirus (RCA). This RCA, which does not carry the transgene of interest, can be produced when manufacturing the replication-defective virus using a modified cell line. Developers have seen some success in using an early development cell line as a master cell bank, as it contains very little RCA and new methods of purification have shown promise in reducing RCA levels.

Lipid Nanoparticle Usage Will Grow As Better Assays Emerge

LNPs are useful for applications where re-dosing and/or transient expression are necessary. Since LNPs are essentially non-immunogenic, dose titration is an option if the initial one does not have a significant effect. This is a significant advantage over viral vectors, which often trigger a strong neutralizing antibody response during gene therapy, preventing them from being re-delivered in the same way to increase the dose. However, for LNPs to truly thrive, re-dosing efficacy and efficiency must be more thoroughly proven in clinical settings, and their commercial scalability must improve.

Some LNPs are intended to target non-hepatic tissues, making accumulation in the liver undesirable; for other LNPs designed for liver-specific treatments, the goal is to reduce accumulation in non-hepatic tissues. A potential solution to these targeting challenges is adding targeting, as is altering the lipid composition since the LNP’s surface characteristics can change its targeting. Nevertheless, both options make manufacturing more complex, and payload limitations can potentially hinder the ability of LNPs to meet market demands that require carrying exceptionally large genes or multiple genetic components at once.

Once an LNP is internalized, lysosomal escape is necessary to release the payload into the cytoplasm. Not all targeting approaches are efficient at this, but improvements in lysosomal escape could reduce the required dose, which would then minimize the amount of vector reaching off-target tissues. Additionally, LNPs also would benefit from standardization of purity and reproducibility to improve payload encapsulation.

To achieve these goals, the industry requires more effective in vitro assays for gauging LNP safety and efficacy. These assays should specifically focus on product consistency, particle encapsulation efficiency, and impurity profiling. Unlike viral vectors, which typically are manufactured in mammalian cells, LNPs are manufactured in the absence of such cells and thus have different impurities. So, while viral vector testing targets stability in the form of long-term viability and vector function, LNP assays must seek out instability and degradation products of both the LNP and its cargo.

Herpes Simplex Virus Constructs Chasing Commercial Viability

HSVs’ efficacy replicating and killing tumor cell exposing antigens is well known and, like adenovirus, have a safety and tolerability profile that makes them simple to add to standard of care. However, it has been difficult to demonstrate that HSVs can be a successful commercial enterprise.

For example, talimogene laherparepvec (T-VEC) was the first HSV-1 approved to treat advanced melanoma. Yet, most therapeutic herpes viruses, including T-VEC, are replication-defective because they have a modified or completely removed ICP 34.5 gene, which controls HSV replication. Though capable of replicating in and killing tumor cells, T-VEC needs to be injected individually into each tumor and also expresses the transgene poorly. Therefore,  despite being satisfactory as an oncolytic, T-VEC did not work well as an immune system activator.

Still, lessons learned from T-VEC can be applied to the next generation of HSVs. One class of HSV-based oncolytic viruses is designed to be as replication competent as possible while remaining within established safety parameters. Among these viruses is Candel Therapeutics’ CAN-3110, a replication-competent HSV engineered for selective replication by placing ICP 34.5 under control of a specific nestin promoter.

Other developers have used proteins encoded in the capsid, removing the HSV’s capability to infect all the cells and redirecting it to infect only tumor cells by targeting specific tumor antigens. Another approach to improving the efficacy of HSVs is modifying ICP 47. HSV uses ICP 47 to downregulate MHC-1, which dulls the immune system’s ability to recognize tumor cells. Modification to upregulate MHC-1 enables the immune system to see more tumor cells and therefore mount a better immune response against them.

Because HSV is such a physically large virus, it also can be used as a cargo delivery system. Payload capacity depends on the number of genes deleted from the native form of the HSV. Candel Therapeutics has been able to encode up to five different genes to a single promoter. However, the quantity of genes delivered is less important than which genes are slotted in, since delivery efficiency and expression must be maintained.

In terms of manufacturing, HSV-based platforms are less scalable than adenovirus-based therapies. Candel Therapeutics has experienced better production with modified cell lines versus unmodified and is transitioning from HYPERStack® cell culture vessels to bioreactors to address this challenge. Additionally, because an HSV’s backbone drives biodistribution, a platform approach to production is a viable option. For example, Candel Therapeutics’ enLIGHTEN™ Discovery Platform can be used to create new HSV-based gene constructs. These constructs are designed to modulate the tumor microenvironment in specific solid tumors by design, making it easier to identify druggable properties that correlate with clinical outcomes.

Delivery System Advances Follow Fit-For-Purpose Planning

Ultimately, selecting a gene delivery system — whether a viral vector, an LNP, or another option — depends primarily on the disease a developer aims to treat and their preferred therapeutic approach. For example, is the treatment intended to be permanent, like gene replacement therapy, or a temporary one, such as genome editing? Other critical factors include immune response, clinically proven efficacy, and regulatory understanding of the technology. These considerations are vital for a therapy or delivery system to advance a path toward broader and more frequent use in patient treatment.

To learn more, watch a comprehensive discussion on this topic during the Cell & Gene Live event, Advancing Gene Delivery: LNPs, Adenovirus, Lentivirus, And More.

About The Panelists

Francesca Barone, M.D., Ph.D., serves as Chief Scientific Officer at Candel Therapeutics. She oversees scientific discovery, the development of our novel viral oncolytic immunotherapies and our biomarker strategy across our broad clinical portfolio. Dr. Barone previously served as Vice President and Head of Experimental Medicine at Flagship Pioneering’s Kintai Therapeutics, now Senda Biosciences. She has previously designed experimental medicine clinical trials to support rigorous decision-making across various programs and indications.

Before joining the industry, Dr. Barone held the academic position of Reader in Translational Rheumatology and Academic Director of Business Engagement for the College of Medical and Dental Sciences at the University of Birmingham. While there, she was also the Director of the laboratories for Immuno-phenotyping in the Institute of Translational Medicine. Dr. Barone earned her M.D. and completed a specialization in Rheumatology from the University of Rome, Sapienza, with merit and her Ph.D. from King’s College London.

Karen Kozarsky, Ph.D., has over 30 years of experience in gene therapy, with a primary focus on the preclinical development of gene therapy utilizing adeno-associated virus (AAV) vectors. Dr. Kozarsky has deep experience evaluating potential therapeutic opportunities, identifying new areas, and developing products from the earliest preclinical stages through IND and has also had experience guiding manufacturing efforts. She has been involved in the development of multiple gene therapy products that are currently in clinical trials.

Dr. Kozarsky currently is President of Vector BioPartners, and previously was Co-Founder and Chief Scientific Officer of SwanBio Therapeutics, VP of R&D at REGENXBIO Inc., and Head, Gene Therapy at GlaxoSmithKline. Prior to that, she was a Research Assistant Professor at the University of Pennsylvania in the Institute for Human Gene Therapy and completed postdoctoral fellowships at the University of Michigan in gene therapy and in immunology. Dr. Kozarsky received a Ph.D. in Biology from MIT and a B.A. in Biology from Amherst College.