Periodic Reporting for period 1 - inteRNAlizer (Efficient, safe, and cost-efficient RNA delivery vehicles for hard-to-transfect pre-clinical and therapeutic cells.)
Período documentado: 2023-12-01 hasta 2025-05-31
Project Context and Objectives:
Gene delivery represents a fundamental challenge in modern biotechnology and therapeutic development, with applications ranging from basic research to advanced cell therapies. The ability to efficiently introduce genetic material into cells is essential for developing treatments for genetic diseases, cancer immunotherapies, and regenerative medicine approaches. However, current gene delivery methods present significant limitations that hinder both research progress and clinical translation.
Present Challenges and Unmet Needs:
Lentiviruses currently serve as the standard solution for achieving workable transduction efficiencies across diverse cell types. While these viral vectors are effective, they create permanent and random integration into the host cell's genome, which raises significant safety concerns, including the potential for DNA damage and an increased cancer risk. This permanent genetic modification poses significant challenges for therapeutic applications, where transient expression is typically preferred. Alternative non-viral approaches, such as lipid nanoparticles (LNPs), offer improved safety profiles but suffer from limited efficacy, particularly in hard-to-transfect cell types, including differentiated human induced pluripotent stem cells (hiPSCs), post-mitotic cortical neurons, and T cells.
Project Innovation and Objectives:
To address these critical limitations, we have developed VLPs as a genetically controlled, biogenic RNA delivery vehicles that combine the high efficacy of viral systems with the safety advantages of non-viral approaches. We have developed a set of ready-to-use, R&D-grade VLP solutions that can effectively target complex cell types while maintaining Biological Safety Level 1 (BSL-1) safety standards. As a key addition to our RNA delivery capabilities, we have demonstrated that our VLP platform can package CRISPR effectors as ribonucleoprotein complexes (RNPs) for precise gene editing applications. This approach will deliver significant advantages over traditional CRISPR delivery methods by providing a controlled, finite editing time window while eliminating the permanent expression of potentially immunogenic CRISPR components. The transient nature of our RNP delivery system is expected to reduce off-target effects and immune responses, making it particularly suitable for therapeutic applications where precision and safety are paramount.
The anticipated impact and significance:
The VLP technique provides laboratories with enhanced tools for targeting challenging-to-transfect cells, including therapeutic cells. This technology has the potential to accelerate research timelines, reduce experimental costs, and facilitate a safer transition of genetic therapies to clinical use. This advancement has the potential to influence gene delivery approaches, possibly contributing to the development of therapies with improved safety profiles.
Gene delivery represents a fundamental challenge in modern biotechnology and therapeutic development, with applications ranging from basic research to advanced cell therapies. The ability to efficiently introduce genetic material into cells is essential for developing treatments for genetic diseases, cancer immunotherapies, and regenerative medicine approaches. However, current gene delivery methods present significant limitations that hinder both research progress and clinical translation.
Present Challenges and Unmet Needs:
Lentiviruses currently serve as the standard solution for achieving workable transduction efficiencies across diverse cell types. While these viral vectors are effective, they create permanent and random integration into the host cell's genome, which raises significant safety concerns, including the potential for DNA damage and an increased cancer risk. This permanent genetic modification poses significant challenges for therapeutic applications, where transient expression is typically preferred. Alternative non-viral approaches, such as lipid nanoparticles (LNPs), offer improved safety profiles but suffer from limited efficacy, particularly in hard-to-transfect cell types, including differentiated human induced pluripotent stem cells (hiPSCs), post-mitotic cortical neurons, and T cells.
Project Innovation and Objectives:
To address these critical limitations, we have developed VLPs as a genetically controlled, biogenic RNA delivery vehicles that combine the high efficacy of viral systems with the safety advantages of non-viral approaches. We have developed a set of ready-to-use, R&D-grade VLP solutions that can effectively target complex cell types while maintaining Biological Safety Level 1 (BSL-1) safety standards. As a key addition to our RNA delivery capabilities, we have demonstrated that our VLP platform can package CRISPR effectors as ribonucleoprotein complexes (RNPs) for precise gene editing applications. This approach will deliver significant advantages over traditional CRISPR delivery methods by providing a controlled, finite editing time window while eliminating the permanent expression of potentially immunogenic CRISPR components. The transient nature of our RNP delivery system is expected to reduce off-target effects and immune responses, making it particularly suitable for therapeutic applications where precision and safety are paramount.
The anticipated impact and significance:
The VLP technique provides laboratories with enhanced tools for targeting challenging-to-transfect cells, including therapeutic cells. This technology has the potential to accelerate research timelines, reduce experimental costs, and facilitate a safer transition of genetic therapies to clinical use. This advancement has the potential to influence gene delivery approaches, possibly contributing to the development of therapies with improved safety profiles.
Scalability: We scaled up VLP production through optimized ultracentrifugation protocols compatible with GMP-grade manufacturing. We generated comprehensive technical protocols and financial reports detailing estimated costs for GMP-compliant production, positioning the technology for clinical translation.
Quality Controls: We established quality control pipelines using dynamic light scattering, Nanosight analysis, and cryo-electron tomography to verify VLP characteristics. We developed bioluminescence and ELISA-based assays for particle quantification and demonstrated batch-to-batch consistency.
Functional Validation: We optimized VLP concentrations across diverse cell types and encoded constructs, including EMcapsulins (https://doi.org/10.1038/s41587-023-01713-y(se abrirá en una nueva ventana)). Performance benchmarking against commercial reagents demonstrated superior results. We completed functional neuroscience experiments using GCaMP and systematically optimized mRNA and RNP cargo packaging.
Clinical Translation: In collaboration with TUM School of Medicine, we quantified multiplexed knockout efficacies in therapeutic T-cells, addressing clinical market needs for safer gene editing in cell therapy applications.
Publications: We published comprehensive VLP system characterization for mRNA and CRISPR effector delivery: https://doi.org/10.1016/j.cell.2025.03.015(se abrirá en una nueva ventana). We also published a pre-print on VLP-based non-destructive transcriptomics: https://www.biorxiv.org/content/10.1101/2024.11.11.622832v1(se abrirá en una nueva ventana)
IP Portfolio: We filed comprehensive patent applications covering our cellular packaging mechanism for mRNA and CRISPR RNP delivery.
Industry Engagement: We maintain contact with German and international pharmaceutical companies and compete in startup competitions, confirming market interest.
Strategic Development: We secured additional EIC Pathfinder funding for accelerated clinical translation and detailed IP landscaping to differentiate VLP advantages over viral vehicles.
Quality Controls: We established quality control pipelines using dynamic light scattering, Nanosight analysis, and cryo-electron tomography to verify VLP characteristics. We developed bioluminescence and ELISA-based assays for particle quantification and demonstrated batch-to-batch consistency.
Functional Validation: We optimized VLP concentrations across diverse cell types and encoded constructs, including EMcapsulins (https://doi.org/10.1038/s41587-023-01713-y(se abrirá en una nueva ventana)). Performance benchmarking against commercial reagents demonstrated superior results. We completed functional neuroscience experiments using GCaMP and systematically optimized mRNA and RNP cargo packaging.
Clinical Translation: In collaboration with TUM School of Medicine, we quantified multiplexed knockout efficacies in therapeutic T-cells, addressing clinical market needs for safer gene editing in cell therapy applications.
Publications: We published comprehensive VLP system characterization for mRNA and CRISPR effector delivery: https://doi.org/10.1016/j.cell.2025.03.015(se abrirá en una nueva ventana). We also published a pre-print on VLP-based non-destructive transcriptomics: https://www.biorxiv.org/content/10.1101/2024.11.11.622832v1(se abrirá en una nueva ventana)
IP Portfolio: We filed comprehensive patent applications covering our cellular packaging mechanism for mRNA and CRISPR RNP delivery.
Industry Engagement: We maintain contact with German and international pharmaceutical companies and compete in startup competitions, confirming market interest.
Strategic Development: We secured additional EIC Pathfinder funding for accelerated clinical translation and detailed IP landscaping to differentiate VLP advantages over viral vehicles.
Advanced Organoid Applications: We achieved substantial delivery in retinal organoids, with VLPs demonstrating preferential targeting of retinal pigment epithelium and successful reach to photoreceptor cells, providing a foundation for future improvements.
Non-Destructive Transcriptomics Platform: We developed non-destructive transcriptomics by vesicular export (NTVE), enabling real-time RNA expression monitoring in living cells. This technology packages stabilized RNA reporter barcodes via VLPs, allowing multi-time-point transcriptome analysis with high concordance to conventional RNA-seq while enabling longitudinal cellular tracking https://www.biorxiv.org/content/10.1101/2024.11.11.622832v1(se abrirá en una nueva ventana) .
In Vivo Gene Editing: We demonstrated successful mRNA delivery of Cre recombinase and established effective RNP delivery systems for in vivo gene editing. Functional restoration was achieved through gene editing in mouse retinopathy models, with improved safety profiles due to transient RNP effects reducing off-target activity.
Precision Cell Targeting: We developed engineered VLP surface modifications for tunable cell selectivity, successfully targeting specific T-cell populations, distinct neuronal subsets in mouse brain tissue, and retinal photoreceptors.
Non-Destructive Transcriptomics Platform: We developed non-destructive transcriptomics by vesicular export (NTVE), enabling real-time RNA expression monitoring in living cells. This technology packages stabilized RNA reporter barcodes via VLPs, allowing multi-time-point transcriptome analysis with high concordance to conventional RNA-seq while enabling longitudinal cellular tracking https://www.biorxiv.org/content/10.1101/2024.11.11.622832v1(se abrirá en una nueva ventana) .
In Vivo Gene Editing: We demonstrated successful mRNA delivery of Cre recombinase and established effective RNP delivery systems for in vivo gene editing. Functional restoration was achieved through gene editing in mouse retinopathy models, with improved safety profiles due to transient RNP effects reducing off-target activity.
Precision Cell Targeting: We developed engineered VLP surface modifications for tunable cell selectivity, successfully targeting specific T-cell populations, distinct neuronal subsets in mouse brain tissue, and retinal photoreceptors.