Improving Cell and Gene Therapy with Innovative Nonviral Delivery Vectors

The FDA approval of the sickle cell treatment exagamglogene autotemcel (exa-cel; marketed as Casgevy)—the first ever CRISPR therapeutic—last December was a milestone for the large and quickly growing pipeline of gene-editing therapies. There are now an estimated 300-plus CRISPR-based therapies in the pipeline, the majority of which are in preclinical testing.¹ This can usher in a new sense of hope for many patients fighting diseases for which there are few or no good treatments. The explosion of early-stage CRISPR research has fueled a demand for novel drug-delivery vectors to help improve the specificity and efficiency of gene editing, while at the same time reducing cytotoxicity, immunogenicity and the risk of off-target editing.

Innovations in the development of drug-delivery vectors will be pivotal to advancing the CRISPR pipeline, as well as other next-generation treatments, including mRNA vaccines and cell therapies. By boosting the therapeutic efficacy of these medicines, and overcoming the hurdles of first-generation vectors, novel vectors could enhance patient compliance and clinical outcomes, as well. And as disease-altering treatments like gene therapy are made available to patients early in their treatment journeys, health outcomes will improve, leading to potential cures that will save patients the hassle of chronic and often ineffective drug regimens.

Improving the development of genomic medicines will cut costs for healthcare systems around the world. Casgevy was introduced with a list price of $2.2 million—a potential hurdle to patient access.² Tools that improve the efficiency of drug development could lead to reduced costs, expanding access to all eligible patients.

Advances in nonviral vectors

Advances in viral vectors such as adeno-associated viruses (AAVs) have resulted in improved tropism and reductions in immunogenicity, and they continue to be popular for the delivery of gene therapy. But nonviral vectors are showing promise for their potential to deliver a wider range of molecules and improve safety profiles.

Lipid nanoparticles (LNPs) have already proven useful, most recently for the delivery of mRNA vaccines against COVID-19. Emerging alternatives include solid lipid nanoparticles (SLNs), which have crystallized components and may improve stability, as well as nanostructured lipid carriers (NLCs), which were developed to improve drug retention and loading capacity. All have shown early success in delivering hydrophobic and hydrophilic therapeutics, and some are showing promise in facilitating delivery to challenging targets, including the brain.³

Polymer-based nanoparticles made of materials such as polyethyleneimine and chitosan have been shown to efficiently traverse cell membranes and deliver a wide variety of nucleic acid cargos, such as plasmid DNA and mRNA, while at the same time protecting cargos from immune responses and degradation. Researchers have shown they can design polymers to overcome barriers in the tumor microenvironment and achieve targeted delivery.⁴

Another type of nonviral vector that’s showing potential is nucleic acids enhanced with engineered nanotechnologies such as multivalent structures and aptamers. In early studies, these vectors have proven promising for targeting delivery of drugs to many different tissues of the body.⁵

Extracellular vesicles (EVs) such asexosomes and ectosomes are also showing promise for nonviral delivery of gene therapies. EVs are promising due to their low immunogenicity and ability to be targeted to diverse cell types. While some EVs may not need to be modified, they can be engineered to enhance targeting or therapeutic effects.⁶ Additionally, some research groups have shown that they can extend the half-life of EVs in cancer gene therapies, improving the efficacy of delivery.⁷

Several next-generation alternative vectors are being engineered with advantageous traits. For example, some are designed to respond to specific stimuli in their environment, such as pH, temperature and enzyme activity, which may enhance controlled drug release at the target site. These “smart” vesicles may be able to alter their structures in response to environmental changes, further enhancing efficacy and minimizing off-target effects.⁸

The role of flow cytometry

Efficient and robust instruments are enabling research advances in novel drug delivery vectors. For example, next-generation flow cytometers use light scattering to detect nanoparticles such as EVs that are as small as 40 nanometers in size (polystyrene when triggering on violet side scatter). For preparation, high-speed centrifugation technologies provide dependable and quick EV separations with improved process control.

Analytical ultracentrifugation (AUC) is another valuable tool in vector development. AUC systems allow for nanoparticle characterizations that provide insights into such parameters as shape, mass, heterogeneity and purity. This technology, which involves subjecting samples to centrifugal forces that hydrodynamically separate them based on sedimentation and diffusion coefficients, can greatly improve the characterization of LNPs. For example, Novartis scientists used AUC to characterize mRNA payload capacity and the distribution of empty versus full capsids in LNP samples.⁹ They concluded that the technology offered a quick and accurate way to measure both parameters, which are essential for optimizing LNP production.

Flow cytometry instruments offer a wide range of automation options, including automated data acquisition and analysis, as well as automated plate loaders. This reduces the risk of human error and increases consistency and reproducibility of results. Automation also offers researchers the ability to be automatically notified of processes that need to be corrected, so they can respond quickly.

Research advances in nonviral vectors—coupled with flow cytometry innovations and automation that improves workflow efficiencies—promise to enhance cell and gene therapies, CRISPR therapeutics, and mRNA medicines. As these advances move into the mainstream, the promise of genomic innovations solving previously untreatable or rare diseases can become reality for a wider population of patients, renewing hope and hopefully leading to a time when no one is told their disease isn’t treatable.

REFERENCES
1. Casgevy approval unlikely to be followed up by another CRISPR drug in near future, says GlobalData. GlobalData. December 28, 2023. https://www.globaldata.com/media/pharma/casgevy-approval-unlikely-followed-another-crispr-drug-near-future-says-globaldata/
2. Vertex/CRISPR price sickle cell disease gene therapy at $2.2 mln. Reuters. December 8, 2023. https://www.reuters.com/business/healthcare-pharmaceuticals/vertexcrispr-price-sickle-cell-disease-gene-therapy-22-mln-2023-12-08/
3. Xu L, Wang X, et al. Lipid nanoparticles for drug delivery. Advanced NanoBiomed Research. 2021. https://doi.org/10.1002/anbr.202100109
4. Duan, L, Ouyang, K, et al. Nanoparticle delivery of CRISPR/Cas9 for genome editing. 2021. doi: 10.3389/fgene.2021.673286
5. Tan X, Jia F, et al. Nucleic acid-based drug delivery strategies. 2020. 323: 240-252. doi: 10.1016/j.jconrel.2020.03.040
6. Cecchin R, Troyer Z, et al. Extracellular vesicles: The next generation in gene therapy delivery. 2023. 31(5):1225-1230. doi: 10.1016/j.ymthe.2023.01.021
7. Du R, Wang C, et al. Extracellular Vesicles as Delivery Vehicles for Therapeutic Nucleic Acids in Cancer Gene Therapy: Progress and Challenges. Pharmaceutics. 2022. doi: 10.3390/pharmaceutics14102236
8. Municoy S, Álvarez Echazú M, Antezana P. Stimuli-responsive materials for tissue engineering and drug delivery. 2020. 21(13): 4724. doi: 10.3390/ijms21134724
9. Bepperling A, Richter G. Determination of mRNA copy number in degradable lipid nanoparticles via density contrast analytical ultracentrifugation. European Biophysics Journal. 2023. doi: 10.1007/s00249-023-01663-y