SBIR-STTR Award

Cancer immunotherapy, particularly genetically engineered adoptive T cell transfer, has shown great potential for the treatment of cancer patients. The use of T cells engineered to express a specific T cell receptor (TCRs) or chimeric antigen receptor (CARs) to treat cancer has generated durable cures for many cancer patients and has resulted in the first FDA approved CAR-T therapy to treat childhood acute lymphoblastic leukemia in 2017. Most gene therapies rely on viral methods to genetically modify human primary cells. However, viral delivery method is expensive, poorly reproducible and associated with several safety concerns including insertion in or near genes that may cause malignancy and generation of replication competent virus. Thus, non-viral DNA delivery methods, such as Sleeping Beauty and piggyBac, have been employed to generate CAR T cells. Although these non-viral delivery methods have the advantage of lower cost, immunogenicity, and regulatory considerations, they have been limited by their low transposition efficiency in primary human hematopoietic cells. In this application, we propose to rationally optimize a recently discovered transposon, TcBuster to deliver CARs to T cells. To this end, we further enhance our already very active hyperactive mutants of the TcBuster transposase and optimize the delivery of the transposon into cells. Following optimization of the TcBuster transposon system, we will combine these improvements with our proprietary methods to transfect T-cells efficiently and safely, and test the immunotherapeutic effectiveness of TcBuster delivered CAR into T cells. The successful completion of this project will result in the comprehensive methods to produce CAR T cells delivered by TcBuster which we will license to pharmaceutical companies to produce highly efficient CAR-T immunotherapy. More broadly, these methods could be expanded beyond immunotherapeutic cancer applications to various infectious diseases in which gene delivery by TcBuster in T cells could be advantageous.

Project Terms:
Acute Lymphocytic Leukemia; base; Biotechnology; cancer immunotherapy; Cancer Patient; cancer therapy; Carrying Capacities; CD19 gene; CD3 Antigens; cell killing; Cells; cellular engineering; chimeric antigen receptor; Clinical; Clinical Trials; Communicable Diseases; cost; cost effective; Cyclic GMP; Data; DNA delivery; Effectiveness; Engineering; Ensure; Environment; FDA approved; Gene Delivery; Gene Expression; gene therapy; Gene Transfer; Generations; Genes; Genetic; Genetic Engineering; Hematopoietic; Human; Hyperactive behavior; immunogenicity; Immunotherapeutic agent; Immunotherapy; improved; In Vitro; in vivo; integration site; interest; Leukemia, Lymphocytic, Acute, L1; Licensing; Ligation; Malignant Neoplasms; Mediating; Methods; Mus; mutant; non-viral gene delivery; Pharmacologic Substance; Phase; Protocols documentation; Quality Control; Reproducibility; Safety; safety testing; scale up; Sleeping Beauty; Southern Blotting; Subfamily lentivirinae; Surface Antigens; System; T cell therapy; T-Cell Receptor; T-Lymphocyte; Technology; Technology Transfer; Testing; Time; Transfection; transgene expression; Transgenes; Transgenic Organisms; Transposase; tumor; Viral; Viral Vector; Virus

Cancer immunotherapy, particularly genetically engineered adoptive T cell transfer, has shown great potential for the treatment of cancer patients. The use of T cells engineered to express a specific T cell receptor (TCRs) or chimeric antigen receptor (CARs) to treat cancer has generated durable cures for many cancer patients and has resulted in the first FDA approved CAR-T therapy to treat childhood acute lymphoblastic leukemia in 2017. Most gene therapies rely on viral methods to genetically modify human primary cells. However, viral delivery method is expensive, poorly reproducible and associated with several safety concerns including insertion in or near genes that may cause malignancy and generation of replication competent virus. Thus, non-viral DNA delivery methods, such as Sleeping Beauty and piggyBac, have been employed to generate CAR T cells. Although these non-viral delivery methods have the advantage of lower cost, immunogenicity, and regulatory considerations, they have been limited by their low transposition efficiency in primary human hematopoietic cells. In this application, we propose to rationally optimize a recently discovered transposon, TcBuster to deliver CARs to T cells. To this end, we further enhance our already very active hyperactive mutants of the TcBuster transposase and optimize the delivery of the transposon into cells. Following optimization of the TcBuster transposon system, we will combine these improvements with our proprietary methods to transfect T-cells efficiently and safely, and test the immunotherapeutic effectiveness of TcBuster delivered CAR into T cells. The successful completion of this project will result in the comprehensive methods to produce CAR T cells delivered by TcBuster which we will license to pharmaceutical companies to produce highly efficient CAR-T immunotherapy. More broadly, these methods could be expanded beyond immunotherapeutic cancer applications to various infectious diseases in which gene delivery by TcBuster in T cells could be advantageous.

Public Health Relevance Statement:
This proposal describes a novel method for delivering permanent gene transfer into human primary T cells for achieving safe, efficient and cost-effective cellular based immunotherapy. We propose to rationally optimize the activity of the TcBuster transposonto deliver a chimeric antigen receptor (CAR) safely and efficiently into T cells to improve the immunotherapeutic cancer activity of these cells. The development of such innovative gene delivery methods will not only create an effective CAR-T based cancer therapy, but also allow for the advancement of additional non-cancer T cell based applications such as controlling bacterial, fungal, protozoan or viral infection.

Project Terms:
Acute Lymphocytic Leukemia; Bacterial Infections; base; Biotechnology; cancer immunotherapy; Cancer Patient; cancer therapy; Carrying Capacities; CD19 gene; CD3 Antigens; cell killing; Cell Therapy; Cells; Childhood Acute Lymphocytic Leukemia; chimeric antigen receptor; chimeric antigen receptor T cells; Clinical; Clinical Trials; Communicable Diseases; cost; cost effective; Cyclic GMP; Data; Development; DNA delivery; Effectiveness; engineered T cells; Engineering; Ensure; Environment; FDA approved; Gene Delivery; Gene Expression; gene therapy; Gene Transfer; Generations; Genes; Genetic; Genetic Engineering; Hematopoietic; Human; Hyperactive behavior; immunogenicity; Immunotherapeutic agent; Immunotherapy; improved; In Vitro; in vivo; innovation; integration site; interest; Licensing; Ligation; Malignant Neoplasms; Mediating; Methods; Mus; mutant; Mycoses; non-viral gene delivery; novel; Pharmacologic Substance; Phase; Protocols documentation; Protozoan Infections; Quality Control; Reproducibility; Safety; safety testing; scale up; Sleeping Beauty; Southern Blotting; Subfamily lentivirinae; Surface Antigens; System; T cell therapy; T-Cell Receptor; T-Lymphocyte; Technology; Technology Transfer; Testing; Time; Transfection; transgene expression; Transgenes; Transgenic Organisms; Transposase; tumor; Viral; Viral Vector; Virus; Virus Diseases