A presentation of the research that led us to create the first technology of its kind designed for cell macroencapsulation in the human CNS.
Release Therapeutics is thrilled to announce the publication of the research project that led to the establishment of the keystone of our proprietary technology: a novel cell macroencapsulation device for long-term protein delivery validated for clinical applications.
Published in iScience, a Cell Press journal, our paper ‘Engineering a versatile and retrievable cell macroencapsulation device for the delivery of therapeutic proteins’ represents a major breakthrough in therapeutic protein delivery and paves the way for our macroencapsulated cell technology to shape the future of central nervous system (CNS) diseases.
This article follows the enthusiastic response to another article we have published recently, ‘Treating Genetic Diseases of the CNS Without Gene Therapy,’ which sparked a wave of interest in our cutting-edge technology. Below we share an overview of our key findings described in the publication by iScience, which led us to develop our novel macroencapsulation technology specifically designed for sustained protein delivery in humans.
Developing a state-of-the-art device suitable for application in humans
Our primary objective in this project was to develop and validate a cell macroencapsulation device bearing optimised features for clinical translation, specifically for the delivery of therapeutic proteins to treat patients with chronic diseases. In other words, our goal was to develop the first, fully biocompatible device that is scalable, easy to administer, retrieve or replace in humans, protects the encapsulated cells from the host’s immune system and helps maintain their secretion capabilities over the long term.
Building on our previous experience with MVX-ONCO-1, a first-generation, implantable device encapsulating granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cells that we assessed in 50 patients in Phase I and Phase IIa studies,[1] we developed the Myo-P®, a device optimised for long term protein delivery in patients.
Biocompatible and Scalable Design
The Myo-P is a medical-grade cylindrical hollow fibre device, no larger than a pin, essentially consisting of (i) a semi-permeable hollow fibre membrane (HFM) reinforced by a coil and (ii) a retrieval tube on the proximal end of the device.
We selected each material on the basis of its biocompatibility, while the size and geometry of the Myo-P device was designed to allow easy administration (and co-administration of multiple devices), as well as retrieval without invasive surgery.
For the semi-permeable membrane of the HMF, we selected a uniform macroporous surface with selective permeability that protects the encapsulated cells from the host’s immune cells. It enables both the outward diffusion of large and complex proteins released by the encapsulated cells and the inward diffusion of nutrients and oxygen for the survival of the cells, without comprising their viability following implantation. The membrane was reinforced with a helicoid coil for structural support under physical stress, within which we integrated a matrix composed of clinical-grade polyester yarns. The matrix allows greater surface area for suspension cells to grow and survive and a three-dimensional scaffold for adherent cells, such as myoblasts, to attach and fuse together to form myotubes.
In the proximal glue point of the retrieval tube, we embedded a built-in polypropoylene thread to enable physicians to easily and safely remove the Myo-P device from the body. This feature offers a significant advantage, eliminating the need for surgical retrieval in the case of any adverse reactions or loss of function.
Immortalised Myoblast Cell Line
In a complementary, long-term study published in a partner journal of Cell Press,[2] we previously sought to identify a single, well-characterised source of allogeneic human cells with limited immunogenicity to standardise cell macroencapsulation for use in the clinical setting. Though diverse murine and human cell types have been tested in proof-of-concept and exploratory studies, no encapsulated cell line had been specifically established for application in humans until this point.
Drawing on the preclinical success of murine immortalised C2C12 myoblast cell lines and existing evidence of successfully immortalised human myoblasts,[3] we hypothesised that a human myoblast cell line would provide the optimal candidate to achieve our research goal. In particular, we hypothesised that human myoblasts would be able to survive for long periods within a capsule, resisting hypoxia without forming a problematic necrotic core, and release therapeutic recombinant proteins for a prolonged period of time.
We successfully created an immortalised human myoblast cell line by transducing primary myoblasts with lentiviral vectors encoding cyclin-dependent kinase 4 (CDK4) and human telomerase reverse transcriptase (hTERT). Designed to survive and proliferate indefinitely in culture, immortalisation was a crucial step for sustained therapeutic protein production without affecting their myogenic potential.
We then demonstrated that our engineered myoblasts are capable of producing several proteins with well-documented therapeutic effects, including human GM-CSF, anti-CD20, and anti-CTLA4 antibodies, as well as the SARS-CoV-2 spike protein. These proteins were stably secreted over extended periods of time (some for over two years in vitro), providing evidence that our myoblast cell line is capable of sustained production of cytokine adjuvants used in cancer immunotherapy and complex macromolecules currently used as therapies in medical oncology and chronic diseases of the CNS.
In order to test the capacity of the myoblast cell line to withstand metabolic and local constraints in vivo and within the encapsulation device, we implanted capsules loaded with the cell line subcutaneously in immunodeficient mouse models.
Continuous secretion of human GM-CSF was observed for over 2 years in vitro and was detectable for up to 10 months in vivo during this research project. We further observed sustained production of anti-CD20 antibodies (rituximab) for up to 5 months in vivo (the duration of the experiments). These results underscored the potential of our cell macroencapsulation technology for long-term therapeutic protein delivery in clinical settings.
Importantly, in this study, we also demonstrated that the myoblasts retain their viability and ability to produce therapeutic proteins even after freezing inside the capsule, cryopreservation, and thawing, significantly enhancing the manufacturing, storage, and distribution prospects of our technology for clinical applications.
Assessing the Myo-P device in vitro, in vivo and ex vivo
Building on this earlier study, we proceeded to explore the versatility of the Myo-P device with the encapsulation of different secreting cell lines and measure its secretion capabilities in vitro, in vivo and ex vivo.
Identical concentrations of a human GM-CSF-secreting cell lines (K562 cells) were encapsulated in the first-generation and Myo-P devices, respectively, and were implanted subcutaneously in C57/BL6 mice for one week. The Myo-P device consistently demonstrated significantly superior protein delivery over the first-generation device prior to implantation, following explantation and in the adjacent tissue and serum of the mice.
In another experiment, we maintained Myo-P devices containing human myoblast cells secreting human GM-CSF in culture for 33 months, also examining the impact of our standard myoblast freezing and thawing procedures on the potency of the devices loaded with these cell lines. Release of human GM-CSF in culture ranged from 613 to 1173 ng/24 h/capsule over the 33-month period and demonstrated comparable secretion of human GM-CSF from both frozen and non-frozen encapsulated myoblast cells. These results suggest that cytokines can be secreted by Myo-P encapsulated cells for several months and the secretion by the cells encapsulated within the Myo-P device is not hampered post-freezing.
Subsequently, we encapsulated wild type cells (WT), human GM-CSF-secreting cells and anti-CTLA4 antibody secreting cells alone or in combination, at different concentrations in Myo-P devices. The devices were maintained in culture for 21 days and expression of human GM-CSF and anti-CTLA4 antibodies were measured. We then proceeded to implant Myo-P devices loaded with the same combination of cell lines subcutaneously in C57/BL6 mice for one week. The levels of human GM-CSF and anti-CTLA4 antibodies were measured in the serum of the mice at Day 7, demonstrating consistency between cell concentrations, secretion and serum levels. This experiment additionally demonstrated that the Myo-P device effectively protects its cargo cells from external cells and the secretion of large and complex proteins using the Myo-P.
Finally, we implanted Myo-P devices loaded with our proprietary myoblast cell line genetically-engineered to secrete human GM-CSF in an ex vivo human skin model for 7 days. The results suggested that the Myo-P device enables the production of immunomodulatory cytokines, such as GM-CSF, by encapsulated cells for up to seven days in human skin. We also explored the activation and mobilisation of antigen-presenting cells (APCs) by the Myo-P in the same model and assessed the effect of the technology on gene expression over time, compared to a sham device group. Overall, the results of this experiement highlighted the clear increase, mobilisation and activation of two populations of skin APCs with the Myo-P device compared to the sham device.
Looking ahead: Treating genetic diseases of the CNS without gene therapy
Our Cell Press publications mark significant milestones for Release Therapeutics and significantly inform the strategic pivot of our company’s focus towards treating disorders of the central nervous system (CNS) with our proprietary cell macroencapsulation technology.
Facilitating minimally-invasive implantation and retrieval, the state-of-the-art, fully biocompatible design of our miniature Myo-P device offers the opportunity to deliver therapeutic proteins beyond the blood-brain barrier (BBB). Our successful encapsulation of suspension and adherent cells within the Myo-P, capable of releasing diverse therapeutic proteins both in vitro and in vivo over sustained periods of time, creates the advantage of delivering a range of therapeutic proteins in vivo to target chronic, CNS-specific pathologies. Encapsulated cells retained their viability and functionality after freezing and thawing, indicating that the loaded Myo-P can be manufactured, stored, and transported efficiently for clinical applications.
Delivering highly potent therapeutic proteins directly to the brain over the long term opens up new avenues for treating CNS diseases, including genetic disorders, neurodegenerative diseases, and brain cancers.
Release Therapeutics’ lead programme focuses on treating Metachromatic Leukodystrophy (MLD), a rare genetic disorder affecting the CNS, with preclinical development already underway. We are also exploring applications for other lysosomal storage disorders, CNS malignancies, and neurodegenerative diseases.
We look forward to sharing more breakthroughs with you.
The Release Tx Team
[1] MVX-ONCO-1 in advanced refractory cancers: Safety, feasibility, and preliminary efficacy results from all HNSCC patients treated in two ongoing clinical trials. Fernandez et al. Journal of Clinical Oncology, May 28, 2021.
[2] Immortalised Human Myoblast Cell Lines for the Delivery of Therapeutic Proteins Using Encapsulated Cell Technology. Lathuilière et al. Molecular Therapy: Methods & Clinical Development, August 1, 2022.
[3] A subcutaneous cellular implant for passive immunization against amyloid-beta reduces brain amyloid and tau pathologies. Lathuilière et al. Brain, May 2016; Encapsulation matrices for neurotrophic factor-secreting myoblast cells. Li et al. Tissue Eng., April, 2000; Destruction of xenografts but not allografts within cell impermeable membranes. Loudovaris et al. Transplant Proc., October, 1992; Metabolic correction in oligodendrocytes derived from metachromatic leukodystrophy mouse model by using encapsulated recombinant myoblasts. Consiglio et al. J. Neurol. Sci. April 15, 2007.