Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells

Abstract

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3 0 and 5 0 arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 Â 10 8 CAR19 T cells per one electroporation of 1 Â 10 7 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, earlyphase CAR T cell products.

Characterizing piggyBat—a transposase for genetic modification of T cells

“…Further modifications, including the use of smaller transposon cassettes and transient transposase expression through mRNA transfection, have also been applied to minimize the risks of potential insertional mutagenesis. 36 The availability of these approaches highlights the need for further optimization of the piggyBat system to satisfy regulatory requirements preceding clinical translation.…”

The Past, Present, and Future of Non-Viral CAR T Cells

“…An alternative approach is the activation of the CAR T receptor by its cognate ligand or specific anti-CAR antibody in the presence of IL-4 and IL-7, which led to selected expansion of functional anti-CD19 CAR T cells, resulting in 90% of CAR positive cells. Moreover, the addition of IL-21 to the IL-4 and IL-7 mixture improves the immunophenotype of CAR T cells with more represented immature stages with less expression of exhaustion molecules such as PD-1, LAG-3, and TIM-3 ( 125 ).…”

“…The linear vector was prepared enzymatically in vitro by PCR whereas mRNA was obtained through in vitro transcription. Electroporated cells were cultivated in presence of IL-4, IL-7, and IL-21 and maintained an early memory immunophenotype at the end of the differentiation ( 125 ). Similarly, the possibility to include the gene of interest flanked by ITRs in doggybone DNA vectors (dbDNA) was investigated.…”

An optimized polymeric delivery system for piggyBac transposition

“…Their ability to mobilize between different locations in the genome has been harnessed for a wide range of applications (Vargas et al, 2016). A comparative study of these three systems revealed the higher transposition efficacy of the piggyBac transposons in addition to being cost‐effective, ability to carry a large cargo and to be able to result in functional fusion proteins (Balasubramanian et al, 2016; Kaštánková et al, 2021; Yusa et al, 2011). Like other transposition systems, piggyBac consists of two main components: a donor DNA transposon pDNA designed to carry the gene of interest with the two inverted terminal repeats (ITR) and a transposase enzyme encoding pDNA (or mRNA).…”

“…Their ability to mobilize between different locations in the genome has been harnessed for a wide range of applications (Vargas et al, 2016). A comparative study of these three systems revealed the higher transposition efficacy of the piggyBac transposons in addition to being cost-effective, ability to carry a large cargo and to be able to result in functional fusion proteins (Balasubramanian et al, 2016;Kaštánková et al, 2021;Yusa et al, 2011).…”