Non-viral gene delivery for non-small cell lung cancer

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of total lung cancer cases and remains the most frequently diagnosed cancer worldwide, representing 11.4% of global cancer incidences and 18.0% of cancer-related deaths as of 2020 (1,2). Despite advances in targeted therapies and a deeper understanding of tumor biology, the overall cure and survival rates for NSCLC leave room for improvement, particularly for advanced stages of the disease (3). Many oncogenic drivers implicated in NSCLC progression, such as KRAS, EGFR, MYC, VEGF, BCL2, MCL1, and STAT proteins, are considered challenging to target with conventional small-molecule drugs (4-6). Among these, CDC20, Survivin, and STAT5A have drawn increasing attention due to their roles in regulating cell division, inhibiting apoptosis, and promoting tumor proliferation (7-9). One promising strategy to counteract these drivers involves the delivery of therapeutic small interfering RNA (siRNA) to cancer cells, which may suppress oncogenic gene expression by facilitating degradation of the messenger RNA (mRNA) transcripts (10,11). For this approach to be effective, careful selection of a suitable delivery vehicle is essential to ensure that the therapeutic siRNA reaches its target efficiently and exerts its intended biological effect (12,13).

In the article entitled “Cationic lipopolymer based siRNA delivery for experimental lung cancer treatment”, published in Biomaterials Advances, the authors present interesting findings on a novel class of commercially available cationic lipopolymers as nanocarriers for siRNA delivery as an experimental lung cancer treatment (1). In this study, three of these cationic lipopolymer reagents, ALL-Fect, Leu-Fect, and Prime-Fect, were evaluated as siRNA nanocarriers that targeted CDC20, Survivin, and STAT5A, which serve as key regulators of cell cycle progression, apoptosis inhibition, and transcriptional signalling, respectively (1). The authors evaluated these lipopolymers in vitro with three human lung cancer model cell lines: A549 wild type, A549-luc, and Calu-3 (1).

Polyethylenimine (PEI) derivatives have been used as transfection reagents in preclinical research. PEI derivatives may have room for opportunities to enter the clinical stage with molecule re-design and modification (10). Building on this, Kc et al. report data that positions three commercially available lipopolymer formulations, ALL-Fect, Leu-Fect, and Prime-Fect, as potential nanocarriers for siRNA delivery in NSCLC treatment (1). Their work reopens the discussion around delivery platforms that could offer new solutions for targeted nucleic acid therapies. For example, several new PEI-based derivatives, including Tunable Lung-Expressing Nanoparticle Platforms (TULEPs) that append a range of C6–C7 tails onto the amine functional groups of PEI, have been developed for efficient, safe and customizable systemic delivery of mRNA to the lungs (11,14). Other examples include work by Bajaj and colleagues, where they designed cholesterol-modified PEI as an alternative to conventional PEI systems (15). By attaching cholesterol units to low molecular weight PEIs (800, 1,200, and 2,000 Da) through ether linkages, they generated nine distinct lipopolymer variants and evaluated their ability to deliver nucleic acids. With these new advances in PEI-based formulations, we thus foresee a potentially promising research direction that may further pave the way towards utilizing PEI-based derivatives for safe and efficacious nucleic acid therapy.

Toward this end, Kc et al. first screened a library of 45 lipopolymer formulations with a polymer to siRNA ratio of 5 wt/wt % and a final siRNA concentration of 40 nM in wild-type A549 cells in their manuscript. Three top-performing lipopolymers, ALL-Fect, Leu-Fect, and Prime-Fect, were down-selected based on the initial library screen. These three lipopolymers were each simply incubated with siRNA in only aqueous medium such as buffers or growth media at room temperature for 30 min, with a range of polymer to siRNA weight ratios ranging from 2.5 to 7.5. This process yielded nanoparticles with sizes between 150 and 400 nm and surface charges from −15 to 27 mV. The binding and dissociative efficiencies of the three unique cationic lipopolymers were quantitatively measured, which yielded between 0.17 to 1.67 µg/mL and 13.62 to 57.90 U/mL, respectively. Confocal imaging and flow cytometry confirmed that more than 90% of the A549 and Calu-3 cell populations internalised the lipopolymer formulations after 24 h. A single dose of nanoparticles with a polymer/siRNA ratio of 5 resulted in 25–80% gene silencing effect, and up to 80% in cancer cell-killing efficacy by day 3, and with approximately 50% in cancer cell-killing efficacy maintained through day 9. When multiple-dose treatment regimens were conducted every 48 hours over three treatments, the cancer cell-killing effect exceeded 80% on day 9. For therapeutic comparison, lipid nanoparticles (LNPs) composed of D-Lin-MC3-DMA or SM-102, together with DSPC, cholesterol, and DMG-PEG-2000, were tested in parallel with a commercial transfection standard Lipofectamine RNAiMAX. These formulations produced modest cancer cell-killing effect (20–40%) in the same cell lines. The findings position these commercially accessible lipopolymers as promising and potentially adaptable platforms for siRNA-mediated gene silencing in lung cancer therapeutics. ALL-Fect was determined to be the most versatile nanocarrier for siRNA delivery, while siRNA targeting CDC20 mRNA transcripts achieved the highest cancer cell-killing efficacy in all tested cell lines, reaching up to 80% even by day 9. This highlights the complex relationship between tumour biology, cellular RNA transcriptomics and the physicochemical properties of nanocarriers that could influence the outcome of nucleic acid therapeutics.

Positioned within the growing field of nucleic acid therapeutics, this study introduces data that may provide additional context within the greater nucleic acid delivery field (16). For example, in some other work in the nucleic acid delivery field, the lipid and/or polymer components in the nanoparticle formulations may be dissolved in organic solvents, such as ethanol, prior to formulation (17). However, in this work, the three lipopolymers could complex siRNA in aqueous medium without first being dissolved in organic solvents. This finding may represent a promising development in the field of nucleic acid therapeutic manufacturing, given that dialysis may be financially consuming and possibly challenging to implement on large scale. Further, the authors’ approach also does not involve microfluidics, which may further simplify the manufacturing process. Moreover, the authors’ work also demonstrated that these lipopolymers may improve cancer-killing efficacy when compared with select LNP platforms, possibly highlighting their potential for future development of nucleic acid therapeutics. Further, consistent efficacy was observed across three mechanistically distinct target classes, suggesting that this platform could perhaps be agnostic to multiple targets with additional studies.

Building on the in vitro data, additional studies may perhaps continue to pave the way forward for next steps in this research area. For example, additional validation of these materials in animal models that investigate properties such as biodistribution, serum stability, and immunogenicity, amongst others, may perhaps further complement the studies provided in this manuscript. Further, additional types of molecular characterization studies may provide further insight into the long-term potential of these materials. For example, the chemical identity of some cationic polymers may trigger immunological cascades when administered systemically, a process that may result in inflammatory reactions. Further, studies that investigate if or how these polymers potentially interact with red blood cell membranes may also be of interest to study to further understand how these materials interact with their surrounding biological environments (18). Moreover, evaluating the potential clearance mechanics of these lipopolymers in vivo may provide additional context to further understand the clinical potential of these materials. Finally, approaches that take into account the physiological environment of the lungs may also be valuable to consider. For example, the physiology of the lungs introduces several barriers that may be absent or difficult to replicate in cell culture, such as mucus, surfactant proteins, and alveolar macrophages (19).

Building on this, additional manufacturability studies may further complement the work in this manuscript. In this work, the lead lipopolymers were obtained from commercial sources, perhaps paving the way toward their long-term potential use in the clinic. Toward that end, additional studies investigating batch-to-batch consistency may further highlight the potential of this system. Further, adopting these formulations for use in different routes of administration, such as inhalation, may further expand the potential utility of the authors’ delivery systems. For example, aerosol or inhalable formats that target the distal airways could potentially be useful for the potential treatment of various diseases, such as asthma, chronic obstructive pulmonary disease or lung cancer, thus representing a complementary pathway for the development of their nanocarriers (20-22). Further, comparisons with additional controls and benchmarks may also be valuable to consider to contextualize these studies further. For example, in this study, the authors utilize an LNP formulation as a type of benchmark control. Going forward, it may also be of interest to compare their lead formulations to additional controls from the non-viral and viral research sectors that can also target the lungs.

To advance this platform to the clinical stage, several strategic directions could be considered. As one potential approach, in vivo validation using orthotopic models of NSCLC could be considered, with one particular focus on establishing select aspects of clinical pharmacokinetics such as safety profile, drug clearance, biodistribution, immunogenicity, histopathological changes, and efficacy, amongst other possible variables. Additional tests could be further considered for further characterizing delivery mechanism, including properties such as cellular association and endosomal escape, to further explore how these lipopolymers may induce potent gene silencing and cytotoxic effects (23,24). Such studies may also enable further formulation optimization and lipopolymer molecule design for enhanced therapeutic efficacy, potentially paving the way toward next generations of their therapeutic carriers. Further, approaches investigating the impact of whether and how serum proteins may impact the efficacy of their systems may also be of interest, particularly given that the protein corona that forms on nanomedicines may play a key role in their overall mechanism of action and therapeutic efficacy (25).

In conclusion, this work presents a showcase of a cationic lipopolymer-based siRNA delivery system which displays potential for transfecting cells that may provide effective treatment for NSCLC. The preliminary results exhibit binding and dissociation properties between the novel polymers and siRNA, as well as the ability to form nanoparticles that may exhibit efficient cellular uptake. Additionally, complexes formed with each of the lipopolymers and target siRNAs were able to achieve gene silencing and cancer cell growth inhibition in multiple model cell lines. Notably, the three novel polymers studied in this paper display varying performance efficacies depending on the cell type and the siRNA for each corresponding target, highlighting that these platforms may be tunable and customizable for long-term therapeutic use. Building on this, the authors compared the efficacy of their best-performing lipopolymer, ALL-Fect, to a potential LNP benchmark control for delivering target siRNA and inhibiting cancer cell growth in vitro. Going forward, these results pave the way toward additional studies that may further contextualize this work in vitro, including additional mechanistic studies and perhaps cross-validation in additional cell culture models that may represent the pathology of the lungs. This is because translation from in vitro to ex vivo and in vivo systems may require considerable efforts. Furthermore, in vivo studies may further contextualize these findings, particularly given one of the long-term interests of pursuing these nanocarriers as treatments for lung cancer. Moreover, it may also be interesting to explore additional cell lines that may be useful for further reflecting the genetic diversity of NSCLC. Additionally, it may also be of interest to explore siRNA delivery with other vehicles or in conjunction with immunotherapies or kinase inhibitors to further explore this research field. Last, it may also be of interest to explore targeting of their systems. Taken collectively, then, these results not only highlight formulation and evaluation approaches for a given nanocarrier platform for RNA delivery but perhaps more broadly further highlight the potential of RNA nanomedicine for the study and treatment of disease.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Annals of Translational Medicine. The article has undergone external peer review.

Peer Review File: Available at https://atm.amegroups.com/article/view/10.21037/atm-25-127/prf

Funding: This work was supported in part by the National Institute of General Medicine Sciences (NIGMS) award 1R35GM157060 (PI Name = Fenton OS).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-25-127/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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