Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 116.89K | Year: 2006
DESCRIPTION (provided by applicant): Gene therapy vectors derived from lentiviruses offer a number of advantages over other gene transfer vectors, and they represent a promising approach for treating a variety of diseases, such as age-related macular degeneration, Parkinson's disease, and blood cell malignancies. Unfortunately, lentiviral vectors are difficult to produce in large numbers due to the lack of stable packaging cell lines and to inefficiencies associated with standard methods of transient transfection. This Phase 1 proposal describes the development of a scalable process for lentiviral vector production that uses MaxCyte's proprietary flow electroporation technology to transfect plasmid DNAs encoding components of bovine immunodeficiency virus (BIV) gene therapy vectors into mammalian cells. The workplan includes optimization of electroporation parameters for loading BIV component plasmids into adherent HEK 293T cells at both small and large scales. These experiments are intended to demonstrate that flow electroporation is superior to other methods (e.g., calcium phosphate precipitation) for transfecting cells that are commonly used in lentivector production. In addition, protocols will be developed for transfecting BIV component plasmids into suspension cells, which offer a number of advantages over adherent cells for manufacturing biological products. The goal is to generate an efficient, suspension cell-based method for lentivirus production that can be scaled up accommodate the needs of clinical-scale testing as well as commercial manufacturing of a lentivirus-based therapeutic product. The results of these studies will provide the foundation for Phase 2 studies to optimize manufacturing protocols that can by used in a GMP facility for producing lentiviral gene therapy vectors on a commercial scale.
Maxcyte Inc. | Date: 2015-08-25
The present invention relates to the transient modification of cells. In particular embodiments, the cells are immune systems, such as PBMC, PBL, T (CD3+ and/or CD8+) and Natural Killer (NK) cells. The modified cells provide a population of cells that express a genetically engineered chimeric receptor which can be administered to a patient therapeutically. The present invention further relates to methods that deliver mRNA coding for the chimeric receptor to unstimulated resting PBMC, PBL, T (CD3+ and/or CD8+) and NK cells and which delivers the mRNA efficiently to the transfected cells and promotes significant target cell killing.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 95.72K | Year: 2003
DESCRIPTION (provided by applicant): A number of highly potent bioactive compounds and therapeutic agents cannot be used at all, or to the full extent of their potential, because the sites of their action are located within biological cells that restrict access of extracellular drugs to their interior. Examples range from relatively simple, low molecular weight entities which cannot cross the cell membrane due to charge or polarity properties, to macromolecules (including gene constructs) which are cannot gain entry to the cell because of their size and net charge. Although several cell-permeation techniques have been designed, none of them is capable of operating at a high-volume/high-throughput industrial level to satisfy many processing needs throughout the broad realm of biotechnology. To address this continuing need we have invented a new approach to electroporation (EP), which we call "Streaming EP". The basic, and fundamentally different, concept of this approach is as follows: cause cells to flow through a pair of very narrow electrodes, which are connected to a DC voltage source. Each cell will be exposed the electric field for the period of time it spends between the electrodes (analogous to pulse width in existing EP devices). This time equals the product of linear velocity of cell flow and the electrode length in the direction of flow. This approach can overcome the volume/throughput limitation of existing methods while retaining all of the known advantages of electroporation over the other cell-loading techniques. We estimate that streaming EP will be able to process 10-50 milliliters of sample per second (up to 200 liters per hour). In addition, every characteristic feature of electroporation - its capability, portability, low cost, and maintenance-free simplicity - can be substantially enhanced in the streaming EP, making it an extremely attractive new-generation technology. The potential of this technology for biomedical, biodefense and clinical applications is unmatched by any existing process or device.
Maxcyte Inc. | Date: 2014-03-14
Methods and compositions are provided involving high producing cell lines. Embodiments concern efficient methods for screening for such cell lines and for creating such cell lines. These cell lines can be used to create large amounts of protein. To quickly generate large quantity of recombinant proteins or vaccines for both pre-clinical study and clinical trials, almost all drug development will face the same challenging obstacle of rapidly generating a high stable producer. Developing and identifying a stable cell line is a critical part of biopharmaceutical development.
Efficient mRNA-based genetic engineering of human NK cells with high-affinity CD16 and CCR7 augments rituximab-induced ADCC against lymphoma and targets NK cell migration toward the lymph node-associated chemokine CCL19
Carlsten M.,U.S. National Institutes of Health |
Levy E.,U.S. National Institutes of Health |
Karambelkar A.,U.S. National Institutes of Health |
Li L.,Maxcyte Inc. |
And 4 more authors.
Frontiers in Immunology | Year: 2016
For more than a decade, investigators have pursued methods to genetically engineer natural killer (NK) cells for use in clinical therapy against cancer. Despite considerable advances in viral transduction of hematopoietic stem cells and T cells, transduction efficiencies for NK cells have remained disappointingly low. Here, we show that NK cells can be genetically reprogramed efficiently using a cGMP-compliant mRNA electroporation method that induces rapid and reproducible transgene expression in nearly all transfected cells, without negatively influencing their viability, phenotype, and cytotoxic function. To study its potential therapeutic application, we used this approach to improve key aspects involved in efficient lymphoma targeting by adoptively infused ex vivo-expanded NK cells. Electroporation of NK cells with mRNA coding for the chemokine receptor CCR7 significantly promoted migration toward the lymph node-associated chemokine CCL19. Further, introduction of mRNA coding for the high-affinity antibody-binding receptor CD16 (CD16-158V) substantially augmented NK cell cytotoxicity against rituximab-coated lymphoma cells. Based on these data, we conclude that this approach can be utilized to genetically modify multiple modalities of NK cells in a highly efficient manner with the potential to improve multiple facets of their in vivo tumor targeting, thus, opening a new arena for the development of more efficacious adoptive NK cell-based cancer immunotherapies. © 2016 Carlsten, Levy, Karambelkar, Li, Reger, Berg, Peshwa and Childs.