Exploring the burgeoning role of non-viral approaches in the delivery of nucleic acids
Cell & Gene Therapy Insights 2023; 9(11), 1547–1552
What are you working on right now?
ZG: My professional expertise lies in the realm of nanotechnology and its applications in biomedicine. Having previously worked in academia, my focus was on harnessing the potential of nanotechnology to address various biological and medical challenges. I am passionate about inventing and bringing new technology and medicine to patients.
Currently, I hold a leadership role at AskBio, where I lead the Nonviral Delivery division. Our primary objective is to lead a talented interdisciplinary team to create a sophisticated new delivery system for RNA and DNA payloads—a sort of nanometer-scale ‘Starship Rocket’ platform for shipping nucleic acids cargo. Through this platform, we facilitate the targeted distribution of genetic cargo to specific tissues or cell types, enabling the advancement of genomic medicines, including innovative therapeutic gene editing approaches.
Before my role at AskBio, I spent a quite long time at the University of Michigan, Ann Arbor, and the University of Texas Southwestern Medical Center, Dallas, where I worked with several world-renowned scientists and physicians on the translation of cutting-edge integrated molecular medicine tools for early cancer detection and treatment. This work covered a variety of stages spanning target identification, early discovery, preclinical development, and ‘first-in-human’ clinical trials. Through the years, I have accumulated a highly interdisciplinary scientific background and deep experience in several areas, including drug delivery, molecular imaging, liquid biopsy, and medical devices. As a scientist, I am always humbled to have the opportunity to work with pioneers in the fields. In particular, with all the support I have received from many excellent mentors, colleagues, and teammates, I have received chances to invent several integrated technologies for understanding drug diffusion in the brain extracellular space and improving drug delivery to the central nervous system. These technologies are currently being applied and validated in preclinical models for delivering both viral and non-viral vectors to enable therapeutic gene editing for neurodegenerative diseases and traumatic injuries in the central nervous system Gao Z, David ET, Leong TW, et al. Minimally invasive delivery of peptides to the spinal cord for behavior modulation. bioRxiv 2022; Epub ahead of print. .
AskBio has long been a leader in the AAV vector-driven gene therapy field—can you tell us more about how, where, and why non-viral delivery is gaining a foothold at the company?
ZG: AAV stands out as an effective delivery platform, especially in specific tissues, with several US FDA-approved gene therapy drugs already available. However, despite its success, AAV has its limitations. Notably, patients previously exposed to this virus cannot be re-dosed with AAV. This poses a significant challenge, as immunogenicity from prior exposure can generate neutralizing antibodies against the virus.
The field is actively exploring non-viral delivery (NVD) approaches to overcome AAV’s immunogenicity issues. One noteworthy NVD approach is the use of lipid nanoparticles (LNPs), which has demonstrated success in the development of the COVID-19 vaccines. While redosing through local intramuscular injections has proven feasible, systemic delivery presents more challenges, although progress is underway.
It is important to note that NVD approaches, such as LNPs, are not seen as direct competitors to AAV. Instead, they serve as complementary technologies, addressing the specific challenges of nucleic acids delivery.
Taking a deeper dive into the mechanisms, AAV efficiently delivers DNA cargo due to its natural ability to enter the cell nucleus and release DNA after uncoating. On the other hand, LNPs are known for their effectiveness in delivering RNA cargo, but they face challenges when delivering DNA due to the larger size of the LNP. LNPs can only reach the cytoplasm of cells, not the nucleus. To use LNPs for DNA delivery, the DNA sequence must be engineered to enhance its efficiency in entering the cell nucleus, and significant progress is being made in this direction as well.
How would you frame the opportunity of applying LNP technology in the gene therapy setting?
ZG: The landscape of gene therapy, particularly in the realm of in vivo gene editing, is a dynamic and emerging field, with LNP technology showcasing great potential. In this context, CRISPR/Cas ‘molecular scissors’ can be delivered together with guide RNA in vivo to create small ‘surgical lesions’ at the targeted DNA strand, facilitating the insertion of therapeutic genes into the targeted site in the genome. Notably, for this process, only transient expression of the molecular scissors is required. A prime example is the well-known Streptococcus pyogenes Cas9 (SpCas9).
When delivered with AAV, SpCas9 provides long-term expression of the molecular scissors in cells. However, this extended expression poses risks, as continuous activity may result in unintended DNA cleavage. On the other hand, delivering SpCas9 via an LNP in a messenger RNA (mRNA) format ensures short-term expression. Once the DNA is cleaved, allowing for gene insertion, the Cas protein will no longer be required, mitigating the risk associated with prolonged activity.
LNPs also find application in RNA therapy. While mRNA delivery, exemplified by the success of COVID-19 vaccines, is established, other RNA formats like circular RNA or short interfering RNA hold promise with LNP delivery post-optimization.
Researchers are actively exploring the delivery of gene editing via nanoparticles, despite the myriad of challenges. Recent advancements in this area are noteworthy, and the overarching goal is to enable high specificity, efficacy, and safety through non-viral delivery methods, opening new possibilities in the field of gene therapy.
Can you expand on the specific challenges in implementing LNPs in the therapeutic setting?
ZG: Navigating the challenges in gene therapy delivery is indeed intricate and context-dependent, particularly when considering specific organs or cell types. Notably, the success of local intramuscular injection, exemplified by the COVID-19 vaccine, contrasts with systemic applications where unmodified LNPs tend to preferentially target the liver, homing in on hepatocytes through ApoE-mediated targeting.
In the liver, the efficiency of LNP is evident even without additional targeting elements. However, to enhance specificity, technologies like N-Acetylgalactosamine are employed. N-Acetylgalactosamine, with asialoglycoprotein receptor as its target, which is highly expressed on hepatocytes, facilitates more precise targeted cargo delivery to liver hepatocytes. The advantage lies in the potential to reduce the dose without compromising effectiveness. Nevertheless, there is room for improvement, especially concerning the toxicity of certain ionizable lipids at the high doses required to reach the peak threshold in the liver.
Expanding beyond the liver poses a significant challenge, as the liver tends to act as a drug sink. Noteworthy progress has been made in the lungs, with various labs and companies employing diverse approaches to deliver therapeutics and target specific cell types. However, translation to non-human primates or humans necessitates additional validation.
The spleen, rich in immune cells, is another enticing target. Fine-tuning nanoparticle properties can enable precise targeting of immune cells in the spleen, as demonstrated in the literature Álvarez-Benedicto E, Tian Z, Chatterjee S, et al. Spleen SORT LNP generated in situ CAR T cells extend survival in a mouse model of lymphoreplete B cell lymphoma. Angew Chem. Int. Ed. Engl. 2023; 62(44), e202310395.. Yet, translating these findings to non-human primates and humans remains to be established. While there are potential targets in other organs, the challenges increase. Special routes of administration or local injections may be necessary in these cases to achieve effective delivery. The quest for optimal gene delivery continues, with ongoing research addressing these intricate challenges.
What are the particular challenges and considerations in applying LNPs for DNA as opposed to RNA delivery, and how might they be addressed?
ZG: Foremost among the challenges is the inherent nature of DNA itself. In the realm of in vivo delivery, DNA has a historical association with triggering a robust activation of the innate immune response. This innate immune response is mediated through pathways such as the cyclic guanosine monophosphate–adenosine monophosphate synthase–stimulator of interferon genes signaling pathway, wherein cells possess mechanisms to sense foreign DNA. In addition, foreign DNA can also activate toll-like receptors—a class of pattern recognition receptors that initiate the innate immune response.
Addressing the challenge of delivering DNA to the nucleus is another significant hurdle. While nanoparticle delivery systems lack intrinsic capabilities for nuclear delivery, one approach involves engineering the DNA sequence. For instance, adding a nuclear localization signal can enhance the DNA’s affinity for the nuclear pore complex to facilitate the transport. However, the therapeutic relevance of such modifications requires experimental validation.
Macrophage uptake of nanoparticles poses yet another obstacle. Upon systemic injection, certain macrophages identify nanoparticles as foreign entities and actively remove a substantial portion from the body. Ongoing research is dedicated to minimizing macrophage uptake, representing a critical area of exploration in the pursuit of effective in vivo DNA delivery.
How is the analytical toolkit developing with LNPs specifically in mind, and where is further innovation most needed in this area?
ZG: When encapsulating mRNA in LNPs, several considerations come into play. One critical factor is the stability of mRNA over time, particularly when nanoparticles carrying mRNA are exposed to the bloodstream in vivo. The assessment of mRNA stability is crucial to determine if it can maintain integrity long enough to reach the desired peak threshold. While tools exist for tracking the integrity of mRNA and nanoparticles both ex vivo and in vitro, the ability to monitor these dynamics in vivo within a real system remains a challenge. An analytical toolkit for characterizing the ‘protein corona’ of nanoparticles in a more precise and comprehensive manner is in high demand because this has an important impact on the tissue and cell tropism in vivo. Continued efforts in this direction are essential to enhance our understanding of the in vivo fate of nanoparticles and mRNA.
DNA delivery poses even greater challenges. Current endeavors focus on the delivery of multiple cargoes, with the aim of packaging DNA and mRNA together in a single nanoparticle. The ongoing challenge is to establish an analytic toolkit for identifying the optimal ratio of mRNA to DNA within this packet. Currently, technologies are in the developmental phase, working towards achieving this intricate balance in cargo delivery.
How problematic are freedom to operate issues in the LNP space, and what is the best approach to addressing them for researchers seeking to work in the field?
ZG: Navigating the competitive landscape of the LNP space, particularly in the context of intellectual property, is indeed complex. A focus on developing new ionizable lipids presents a more accessible avenue within the realm of non-viral vectors.
Additionally, alternative vector options, such as polymeric nanoparticles and virus-like particles (VLPs), contribute to the diversity of choices. Each particle type brings its own set of advantages; for instance, VLPs merge the benefits of non-viral vectors with those of viral vectors, demonstrating advantages in brain and spinal cord delivery.
Beyond the traditional LNPs, the inclusion of polymeric nanoparticles, VLPs, and other vectors broadens the spectrum of approaches to address freedom to operate concerns. The diverse array of vectors within this space will be beneficial for diversifying the non-viral platform. Despite the notable successes achieved thus far, it’s important to acknowledge that we are still in the early stages of exploring the vast potential of non-viral delivery systems. The dynamic and competitive nature of the field propels ongoing innovations and advancements.
Finally, can you sum up one or two key goals and priorities that you have for your work in the foreseeable future?
ZG: My primary objective is to propel our liver program forward, leveraging LNPs. With this foundation, advancing this initiative is poised to be an impactful endeavor. Following closely as the second priority is the strategic expansion beyond the liver. This will help us venture into uncharted territories, surpassing the scope of commonly targeted organs. This dual-focus strategy reflects our commitment to pushing the boundaries of non-viral delivery and charting new frontiers in the field. We have a commitment to having non-viral delivery be a pivotal part of the AskBio toolbox serving alongside our AAV platform.
1. Gao Z, David ET, Leong TW, et al. Minimally invasive delivery of peptides to the spinal cord for behavior modulation. bioRxiv 2022; Epub ahead of print. Crossref
2. Álvarez-Benedicto E, Tian Z, Chatterjee S, et al. Spleen SORT LNP generated in situ CAR T cells extend survival in a mouse model of lymphoreplete B cell lymphoma. Angew Chem. Int. Ed. Engl. 2023; 62(44), e202310395. Crossref
Zhenghong Gao is the Director and Head of Nonviral Delivery at Asklepios BioPharmaceutical, Inc. He has established experience in drug delivery, non-viral vector, molecular imaging, and gene therapy, spanning from preclinical discovery, development, and translation, to ‘first-in-human’ clinical study, and is currently focusing on gene editing utilizing non-viral delivery technologies.
Zhenghong Gao PhD
Head of Nonviral Delivery,
Asklepios BioPharmaceutical, Inc. (AskBio)
Authorship & Conflict of Interest
Contributions: The named author takes responsibility for the integrity of the work as a whole, and has given their approval for this version to be published.
Disclosure and potential conflicts of interest: The author has no conflicts of interest.
Funding declaration: The author received no financial support for the research, authorship and/or publication of this article.
Article & Copyright Information
Copyright: Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.
Attribution: Copyright © Gao Z. Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.
Article source: Invited.
Submitted for peer review: Nov 2, 2023; Publication date: Jan 5, 2024.