Advancements in cell therapy: development of a non-viral gene delivery platform for CAR-T manufacturing

Listen to this webinar hosted by Cell and Gene Therapy Insights about the latest advancements in cell therapy, specifically using homology-independent targeted insertion (HITI) of a therapeutically relevant GD2-CAR transgene into the T cell receptor alpha constant (TRAC) locus using nanoplasmid DNA and CRISPR-Cas9 in primary human T cells.

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The lab of Steven Feldman, PhD, is pioneering non-viral gene delivery methods, which promise to mitigate the limitations of viral vectors.

Speakers

Headshot of Steven Feldman, PhD

Steven Feldman, PhD

Site head and scientific director of the Laboratory for Cell & Gene Medicine at Stanford Center for Cancer Cell Therapy

Dr. Feldman is a pivotal member of the Stanford Center for Cancer Cell Therapy. He serves as the site head and scientific director of Stanford’s GMP facility and leads a team that focuses on the development and manufacture of novel cell therapies for the treatment of cancer, emphasizing more efficient, cost-effective, and safer gene delivery methods, especially testing other methods than traditional viral vectors.

James-Brady

James Brady, PhD

Senior vice president, technical applications and customer support at MaxCyte

Dr. Brady is an experienced biotechnology industry professional with expertise in cell and gene therapy, biologics, and drug discovery. Previously, he was a senior scientist at Genetic Therapy, a Novartis subsidiary, where he worked on lentiviral-based gene therapy treatments and was a group leader at MetaMorphix, managing the company’s transgenic and genetic research programs.

Dr. Brady earned a Master of Business Administration degree in finance from The Johns Hopkins University. He completed his postdoctoral fellowship at the National Eye Institute of the National Institutes of Health in Bethesda, Maryland, after obtaining a PhD in genetics from Indiana University in Bloomington, Indiana.

Headshot of Daniel Nguyen

Daniel Nguyen

Director of global sales development and inside sales at MaxCyte

Mr. Nguyen has extensive experience supporting industry and academic centers with their cell engineering needs from basic research to manufacturing. He has spent the last four years focused on supporting scientists in the areas of cell therapy and CRISPR engineering at MaxCyte.

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Challenges with viral vectors

Viral vectors have been the standard in CAR-T cell manufacturing but present several challenges:

  • High costs: establishing manufacturing suites following current good manufacturing practices (cGMP) or outsourcing virus production is financially burdensome
  • Safety issues: viral vectors can induce immunogenic responses and pose risks associated with random genomic integration
  • Regulatory complexities: meeting regulatory standards requires intricate vector production strategies

Non-viral gene delivery—a scalable, replicable process

The lab at Stanford is pioneering non-viral gene delivery methods, which do not have the same limitations or risks of viral vectors. The team uses electroporation to deliver a CRISPR ribonucleoprotein (RNP) and nanoplasmid DNA to edit the cells through homology-independent targeted integration (HITI). This mechanism of action utilizes CRISPR-Cas9 to create a double-stranded breaks in specific loci, facilitating gene insertion without viral vectors. With minimal off-target effects and high yield HITI achieves functional CAR-T cells with comparable functionality to virally transduced cells in preclinical models.

Role of electroporation in non-viral gene delivery
A significant aspect of Steven's work involves the use of electroporation, particularly leveraging MaxCyte's electroporator instruments. Electroporation is a transfection method that uses electrical pulses to introduce gene-editing machinery and other molecules into cells, and MaxCyte's scalable, consistent technology has been an important, but straightforward, step in this process. Using electroporation has the following benefits:

  • Scalability throughout therapy development: MaxCyte's instrumentation can scale up without changing the protocol conditions. This technology can transfect billions of cells in a single run, ensuring process standardization and consistency.
  • Delivery optimization: MaxCyte provides optimized electroporation protocols for a wide range of cell types and payloads, yielding reproducibly high levels of transfection efficiencies and gene editing.
  • Technical support: MaxCyte's cell engineering experts provide extensive technical and scientific support not only for the electroporation process but for the entire cell therapy development workflow, ensuring success for teams developing their cell therapy.

Challenges with viral vectors

Viral vectors have been the standard in CAR-T cell manufacturing but present several challenges:

  • High costs: establishing manufacturing suites following current good manufacturing practices (cGMP) or outsourcing virus production is financially burdensome
  • Safety issues: viral vectors can induce immunogenic responses and pose risks associated with random genomic integration
  • Regulatory complexities: meeting regulatory standards requires intricate vector production strategies

Non-viral gene delivery—a scalable, replicable process

The lab at Stanford is pioneering non-viral gene delivery methods, which do not have the same limitations or risks of viral vectors. The team uses electroporation to deliver a CRISPR ribonucleoprotein (RNP) and nanoplasmid DNA to edit the cells through homology-independent targeted integration (HITI). This mechanism of action utilizes CRISPR-Cas9 to create a double-stranded breaks in specific loci, facilitating gene insertion without viral vectors. With minimal off-target effects and high yield HITI achieves functional CAR-T cells with comparable functionality to virally transduced cells in preclinical models.

Role of electroporation in non-viral gene delivery
A significant aspect of Steven's work involves the use of electroporation, particularly leveraging MaxCyte's electroporator instruments. Electroporation is a transfection method that uses electrical pulses to introduce gene-editing machinery and other molecules into cells, and MaxCyte's scalable, consistent technology has been an important, but straightforward, step in this process. Using electroporation has the following benefits:

  • Scalability throughout therapy development: MaxCyte's instrumentation can scale up without changing the protocol conditions. This technology can transfect billions of cells in a single run, ensuring process standardization and consistency.
  • Delivery optimization: MaxCyte provides optimized electroporation protocols for a wide range of cell types and payloads, yielding reproducibly high levels of transfection efficiencies and gene editing.
  • Technical support: MaxCyte's cell engineering experts provide extensive technical and scientific support not only for the electroporation process but for the entire cell therapy development workflow, ensuring success for teams developing their cell therapy.