September 28, 2022
Figure 1. Nanomedicine Possibilities
Nanomedicine offers promising therapeutic solutions for cell and gene therapies, autoimmune diseases, oncology, rare inherited diseases and more. Some new avenues to explore include early disease detection or molecularly tailored treatments for patients who have complex diseases and are not responding to existing therapies. In particular, three areas of genomic medicines: gene editing, gene therapy and mRNA medicines, are currently evolving with a range of new technology platforms providing novel diagnostic and therapeutic solutions. Non-viral gene delivery approaches, such as lipid nanoparticles, have the potential to develop and deliver a range of RNA-based therapeutics and vaccines, through optimization and precise LNP formulations.
Figure 2. Lipid Nanoparticle
The key challenge in implementing cell and gene therapies is the method of delivery. Nucleic acids, both DNA and RNA, face enormous complexities in reaching the target cells. To overcome obstacles like rapid degradation in cells' biological fluids and the inability to accumulate or penetrate even if they reach the target cells, new delivery systems are a necessity for a targeted, safe, and efficient intracellular nucleic acid delivery. Lipid nanoparticles are non-viral delivery systems that can deliver larger payloads with design flexibility, easy manufacture and multi-dosing capabilities. LNPs allow the mixing of lipid excipients which can be modified to deliver a variety of nucleic acid active pharmaceutical ingredients (APIs) to different targets in the body.
Each lipid nanoparticle consists of the following four components.
1. Ionizable lipids that bind nucleic acid and hold a neutral charge under physiological pH to minimize toxicity and shift charge with pH changes to limit cytotoxic effects.
2. Helper lipids aid in LNP stability, intracellular uptake, and endosomal escape.
3. Cholesterol binds apolipoprotein E(ApoE) and mediates endocytosis via low-density lipoprotein (LDL) receptor.
4. PEG-lipids that are hydrophilic lipids create a barrier of water to protect LNPs from aggregating during assembly and increase bloodstream circulation lifetime.
A slight alteration in the chemical structure and the ratio of the four components can alter the LNP properties and delivery efficiency. This advantage of LNPs design flexibility enables overcoming challenges of extrahepatic mRNA delivery and gene editing systems by selective organ targeting (SORT), a strategy to design nanoparticles rationally. As published in the study “On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles,”1 augmenting conventional four-component LNPs for mRNA delivery to the liver with a fifth component (termed a SORT molecule) enabled the delivery of diverse cargoes (nucleic acids and proteins) to achieve gene expression and CRISPR/Cas-based gene editing in therapeutically relevant cell types, including epithelial cells, endothelial cells, B cells, T cells, and hepatocytes. Tuning the nanoparticle’s molecular composition aided in its binding to specific proteins in the serum to enable delivery to the target site and facilitated mRNA biodistribution to the target organ. These properties generally help develop strategies for engineering a wide array of nanomaterials capable of extrahepatic delivery.
Ionizable lipids are the main component of LNPs that enable efficient cellular delivery and overcome payload limitations. In a research study2, Dr. Daniel Rosenblum et al. used ionizable cationic lipids from a novel ionizable amino lipid library to co-encapsulate larger Cas9 mRNA and single-guide RNA (sgRNA). The data for the in vitro gene knockout efficiency proved that CRISPR-LNP (cLNP) formulations targeting the PLK1 gene (a kinase required for mitosis to avoid cell cycle arrest and cell death in dividing cells) could inhibit tumor growth and improve survival in two aggressive cancer models following single or double cLNP administrations. The study demonstrates the potential of using targeted lipid nanoparticles with customized ionizable cationic lipids for targeted gene editing and delivery of novel therapeutics. Therefore, testing and altering lipids in LNPs offer vast opportunities to enable the broader translation of RNA therapeutics and vaccines.
Figure 3. Lipid library
The ionizable lipid is a strategic component of lipid nanoparticles playing a significant role in protecting nucleic acids and facilitating their cytosolic transport. Ionizable lipids have a unique pH sensitivity that allows them to shift charge with pH changes. In acidic pH, they are positively charged to condense nucleic acids into LNPs but are neutral at physiological pH to minimize toxicity. Despite the FDA approval of ionizable lipids for RNA delivery applications, several challenges require consideration to reach the limitless possibilities of genomic therapeutics.
There are five types of ionizable lipids widely used for RNA delivery3.
1. Unsaturated ionizable lipids - Enhances membrane disruption and payload release. However, they do not always correspond with potent in vivo RNA delivery, indicating that both rational design and screening are necessary.
2. Multi-tail ionizable lipids – Offer greater endosome-disrupting ability. They often have stable backbones and low degradability, so their toxicity and immunogenicity can cause hindrance.
3. Ionizable-polymer lipids - Support particle formation through hydrophobic aggregation and can be re-optimized to achieve potent gene silencing. But, even after purification, these lipids comprise a mixture of different substitution compounds, which increase their complexity. Moreover, the toxic polycation core and non-degradable backbone pose extra hurdles for clinical translation.
4. Biodegradable ionizable lipids - Stable at physiological pH but can enzymatically hydrolyze within tissues and cells. However, the position and steric effect of the ester groups can significantly affect ionizable lipid clearance and potency. And the difficulty of synthesis and risk of premature release can limit their applications.
5. Branch-tailed ionizable lipids - Enhances endosomal escape with the potential for integrating different therapeutic modalities (e.g., gene silencing, expression, and editing). Notably, increased tail branching requires a thorough investigation.
However, ionizable lipids that are effective at delivering nanoparticles to cells in culture do not necessarily translate to animal studies. Clinical ionizable lipids are all synthesized in multiple steps, posing scalability challenges. Furthermore, the laborious synthesis process makes it difficult to prepare rationally designed ionizable lipid candidates. Aside from safety and potency, optimizing ionizable lipids for structural properties and additional functionalities such as targeting and immunomodulation is difficult. Decades of research on lipid nanoparticles suggest a need to profile and characterize ionizable lipids to ensure the product quality of vaccines and other mRNA-based therapies. To address this issue, access to a proprietary lipid library that includes systematic categorization of these lipids based on their structures for developing custom formulations can significantly benefit in reaching the full potential of RNA therapeutics.
Dr. Anna Blakney, an Assistant Professor in the Michael Smith Laboratories and School of Biomedical Engineering at UBC, states that LNPs are prominent leaders for RNA delivery, given the success of mRNA COVID-19 vaccines and Onpattro® and that it is essential to optimize LNP for a successful RNA drug delivery experiment. She points out that two primary challenges while working with LNPs are the formulation and the assembly process. The most intricate work is finding the suitable formulation for making LNPs which is both art and science.
As novel ionizable lipids are a new avenue for nanomedicine, it becomes more significant to focus on testing and optimizing to make promising formulations according to their physicochemical attributes (size, size distribution and encapsulation efficiency) and their cellular uptake and in vitro potency. During the formulation design, changing the solvent combination is the central point of intervention while performing analytical tests for physical parameters such as encapsulation or particle size, as well as assays for composition, identity, and purity.
Further, the finished drug product's in vivo performance and quality control require extensive analysis. In the case study, "Optimizing LNP formulations for plasmid expression in iPSCs," formulations had very different in vitro efficacy despite similar physical properties. Microliter formulations containing microgram quantities of mRNA were rapidly produced using the NanoAssemblr® Spark™ microfluidic system to systematically screen compositions against properties and activity. A panel of formulations containing PNI-Ila (a proprietary ionizable cationic lipid) with different helper lipids was created at different N/P ratios, which is the ratio of negatively-charged nucleic acid phosphate groups and positively-chargeable polymer amine groups that can influence many other properties of polymer-based gene delivery vehicles, such as its net surface charge, size, and stability. The N/P ratio is vital to formulation efficacy for plasmid delivery to human iPSC-derived cortical neurons. Analytical testing to identify candidate formulations with optimal performance in this therapeutically relevant cell type revealed that higher N/P ratio formulation had better encapsulation efficiency. However, in vitro, there was a measured decrease in neurite length, showing a negative impact on cell health. Thus, only by performing empirical testing is it possible to know the ideal formulation.
Figure 4.Nanomedicine Optimization
Further optimization, including functionalization of LNPs for developing drugs to target tissues, is likely to improve the efficiency and avoid off-target effects making way for new frontiers in genomic medicine. Although optimizing LNP formulations is complex, Precision NanoSystems provides a wide range of cGMP manufactured lipids to enable researchers and developers to tailor drug delivery systems to perfection. Precision NanoSystems GenVoy Delivery Platform comprises off-the-shelf RUO reagents such as GenVoy-ILM™ and a library of proprietary lipids available as custom formulations for a clear path to the clinic.
Dr. Anna Blakney, “Precision NanoSystems lipid library is an accessible, off-the-shelf system that makes LNP formulations easy to incorporate into my research.”
Precision NanoSystems biopharma service technical expertise team offers assistance in developing the right analytical assays for both viral and non-viral nanomedicines, including drug product identity confirmation, physical characterization, acceptance testing and stability studies, GMP release testing, toxicology testing and raw material testing. As the raw materials for lipid formulations can be costly, optimizing these limited materials with NanoAssemblr® and GenVoy™ technology platforms enable seamless scaling of drug products from preclinical to clinical development with a reduced amount of raw materials, saving cost and time. Precision NanoSystems not only helps scientists and drug developers select appropriate formulations but also offers LNP manufacturing instruments that are scalable to advanced preclinical and clinical scales with NxGen™ NanoAssemblr® technology.
Precision NanoSystems and Replicate Bioscience recently signed a licensing agreement to scale up RNA-based nanomedicines. Accessing its Proprietary Lipid Library will allow Replicate Bioscience to explore diverse chemical spaces to develop novel drug candidates in areas beyond the company’s lead programs. Along with Precision NanoSystems’ technical support, manufacturing systems, and CDMO services, the full suite of end-to-end genomic medicine solutions enables biopharmaceutical companies like Replicate to move investigational products quickly and efficiently through their development.
Learn more about Precision NanoSystems’ Lipid Nanoparticle Portfolio
Have questions about lipid formulations development? Learn more about formulation through our learning platform NanoMedU.
References:
1. Dilliard SA, Cheng Q, Siegwart DJ. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc Natl Acad Sci U S A. 2021 Dec 28;118(52):e2109256118. doi: 10.1073/pnas.2109256118. PMID: 34933999; PMCID: PMC8719871.
2. Rosenblum D, Gutkin A, Kedmi R, Ramishetti S, Veiga N, Jacobi AM, Schubert MS, Friedmann-Morvinski D, Cohen ZR, Behlke MA, Lieberman J, Peer D. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci Adv. 2020 Nov 18;6(47):eabc9450. doi: 10.1126/sciadv.abc9450. PMID: 33208369; PMCID: PMC7673804.
3. Han, X., Zhang, H., Butowska, K. et al. An ionizable lipid toolbox for RNA delivery. Nat Commun 12, 7233 (2021). https://doi.org/10.1038/s41467-021-27493-0
4. Blakney AK, McKay PF, Hu K, Samnuan K, Jain N, Brown A, Thomas A, Rogers P, Polra K, Sallah H, Yeow J, Zhu Y, Stevens MM, Geall A, Shattock RJ. Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines. J Control Release. 2021 Oct 10;338:201-210. doi: 10.1016/j.jconrel.2021.08.029. Epub 2021 Aug 18. PMID: 34418521; PMCID: PMC8412240.
5. Lou G, Anderluzzi G, Schmidt ST, Woods S, Gallorini S, Brazzoli M, Giusti F, Ferlenghi I, Johnson RN, Roberts CW, O'Hagan DT, Baudner BC, Perrie Y. Delivery of self-amplifying mRNA vaccines by cationic lipid nanoparticles: The impact of cationic lipid selection. J Control Release. 2020 Sep 10;325:370-379. doi: 10.1016/j.jconrel.2020.06.027. Epub 2020 Jul 1. PMID: 32619745.
Article
Jul 28, 2022
Article
Jun 09, 2022