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Cell Therapy
Cell Therapies involve reprogramming cells, often immune cells, that are sourced either from the patient (autologous), or from a suitable donor (allogenic). Reprogrammed cells are isolated, expanded and subsequently (re)infused back into the patient.
Lipid Nanoparticles (LNP) offer a flexible way to reprogram cells by enabling delivery of RNA that mediate protein expression, or gene editing. Precision NanoSystems’ Genetic Medicine Toolkit provides essential technologies to facilitate development of RNA-LNP.
Example: How Autologous Chimeric Antigen Receptor T Cells are Engineered with Non-Viral Gene Delivery
1. T cells are isolated from the patient
2. mRNA encoding the CAR designed to recognize biomarkers for the disease (or nearly any protein) are encapsulated into lipid nanoparticles (LNP)
3. LNP mediate delivery of the mRNA into the T cells ex vivo
4. T cells translate the mRNA into CAR proteins, which are presented on the cell surface
5. Engineered CAR T Cells are expanded and given back to the patient where they mount an immune response to the target recognized by the CAR
Genetic Medicine Toolkit for Cell Therapy
Modulating gene expression and engineering cells into Cell Therapies can be enabled by non-viral gene delivery. Precision NanoSystems’ Genetic Medicine Toolkit makes the essential technologies for producing non-viral lipid-based delivery of nucleic acids broadly accessible. Unlike viral vectors, lipid nanoparticles do not require cell culture for production, thus streamlining development and manufacturing.
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Disease Target
Cell Therapy Toolkit
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Genetic Payload Platform
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GenVoy™ Delivery Platform
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NanoAssemblr® Manufacturing Platform
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Drug Development Expertise
Cell Therapy Technology
Precision NanoSystems has developed proprietary lipids and lipid nanoparticle (LNP) compositions for delivering RNA to various cell types. In the study below, LNP formulations were shown to successfully deliver mRNA encoding a reporter gene (GFP) to primary human T cells in vitro. This technology has been adapted to mediate expression of clinically relevant proteins in T cells, such as chimeric antigen receptors (CAR).
In Cell Therapy applications, RNA-LNP are used as reprogramming reagents and are not subject to the same regulatory requirements as a drug product. LNP are easier to produce than viral vectors, which are commonly used to reprogram cells.
Screening identified Proprietary mRNA-LNP formulations that mediated High Transgene Expression and Good Cell Viability in Primary Human T Cells
A) Flow Cytometry Shows Some Formulations Result in High GFP Expression Across Large Fraction of T Cell Population
B) Cell Viability is Unaffected by LNP Treatment and Expression of Transgene, Important for Achieving High Yield of Modified Cells
How to Develop Cell Therapies
Precision NanoSystems’ Framework is designed to guide development of non-viral genetic medicines, such as RNA-LNP, from idea to commercial product. In Cell Therapy, the RNA-LNP is used as a reagent and is not the drug product. Nevertheless, as shown below, aspects of the Framework can be used to guide and accelerate optimization of the RNA-LNP ex vivo reprogramming reagent, which is critical for production of the Cell Therapy product.
Genetic Medicine Framework and Toolkit Supports Development of Cell Therapy Reprogramming Reagents
Example specifications for an RNA-LNP used as a reagent to engineer cells to create a Cell Therapy are shown in the table below.
RNA-LNP Characteristics
General Requirements
Treatment Modality
Ex vivo
RNA Dose
Start with 2µg/1 million T cells
Cell Proliferation Index Post-Treatment
Unaffected
Hydrodynamic Size
70 -120nm
RNA Encapsulation Efficiency
>80%
Transfection Efficiency
(Primary Human T Cells)
>80%
Cell Viability
(Primary Human T Cells)
>80%
1. Target Selection
Antigens are typically selected from biomarkers known to be over-expressed in cancer cells. The scFV antigen binding domain of antibodies raised to bind the cancer biomarker are encoded in an mRNA along with a transmembrane domain and stimulatory and co-stimulatory molecules of the endodomain to engineer the CAR.mRNA provides a convenient platform to mix and match multiple antigen binding domains with multiple stimulatory and co-stimulatory domains to create a library of CARs in silico that can be screened in vitro and in vivo. Furthermore, the ease of encapsulation into non-viral delivery systems such as lipid nanoparticles (LNPs) provides a rapid approach to screening a large library of different CARs.
2. Vector Selection
Expressing exogenous proteins in T Cells: The CAR can be encoded in mRNA and delivered to T Cells ex vivo by lipid nanoparticle. mRNA-LNPs have several advantages over DNA delivered by viral vectors: (1) Cell-free manufacturing is simpler and highly scalable from target validation to commercial scale production. (2) mRNA presents lower risk of genome integration.
Gene Editing: Tools such as CRISPR/Cas9 represent a breakthrough for cell therapy, promising the ability to remove, add or edit genomic DNA with high specificity. There remain numerous challenges to delivering CRISPR components such as the Cas9 endonuclease and the single guide RNA to cells. To date, several groups have demonstrated that the Cas9 endonuclease can be encoded in mRNA, and co-encapsulated with one or more guide RNAs into an LNP and delivered to the liver in vivo [1,2]. This approach gives rise to highly durable gene modulation, while the transient nature of the Cas9 mRNA minimizes the risk of off-target edits caused by persistent Cas9 expression. It is conceivable to employ this approach to enable gene editing in other cell types.
3. Delivery and Formulation TechnologyPrecision NanoSystems has developed a lipid library specifically for gene delivery to a wide range of cells including T Cells. PNI’s lipid nanoparticle (LNP) technology constitutes a synthetic gene delivery tool that does not require the complications of a packaging cell line. This is particularly beneficial for rapidly screening a library of CARs. Additionally, PNI LNPs can be easily adapted to introduce mRNA into a variety of T cell subsets.
GenVoy™ LNP Delivery Technology
Anatomy of an LNP
Versatile Use in Multiple T Cell Subsets
Primary human CD4, CD8, and pan T cells were treated with GFP-mRNA-LNP post activation, resulting in comparable transgene expression.
4. Manufacturing
The NanoAssemblr® manufacturing platform enables the rapid production of RNA-LNP on demand. The NanoAssemblr platform comprises four systems (Spark, Ignite, Blaze and GMP) that use the same proprietary NxGen™ microfluidic architecture to scale production from microliters to hundreds of liters. Non-turbulent microfluidic mixing affords exceptional control over the microenvironment of RNA-LNP formation ensuring high quality product. For Cell Therapy applications, the volume of RNA-LNP reagent needed for cell reprogramming will depend on the workflow of the given application.
NanoAssemblr™ Platform with NxGen Technology
Test batches of up to 20 L/h produced with a single NxGen Mixer while maintaining particle characteristics (size and PDI) and biological activity.
Cell Therapy Development Solutions
Precision NanoSystems is committed to supporting Cell Therapy development with a full stack of accessible technologies and solutions coupled with drug development expertise to accelerate the design, development and manufacturing of promising gene delivery technologies.
GenVoy-ILM™ T Cell Kit for mRNA for Lipid Nanoparticle (LNP)-based Ex Vivo Gene Transfer.
Cell therapy researchers can harness the power of mRNA-LNPs to efficiently generate engineered primary human T cells using a potent non-viral gene delivery method.
GenVoy-ILM™ T Cell Kit for mRNA
Genetic Medicine Development Framework
The Framework provides a roadmap for developing a non-viral gene delivery system
Genetic Medicine Toolkit
Essential technologies for developing non-viral mRNA delivery systems
References
1. Finn, J. D., Smith, A. R., Patel, M. C., et al. (2018). A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. Cell Reports. https://doi.org/10.1016/j.celrep.2018.02.014
2. Miller, J. B., Zhang, S., Kos, P., et al. (2017). Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA. Angewandte Chemie - International Edition. https://doi.org/10.1002/anie.201610209
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