Chimeric antigen receptor-modified T (CAR-T) cells are living therapies that are genetically engineered to express CAR molecules targeting antigens found on tumor cells or target cells of interest. Unfortunately, an obstacle for the field of CAR-T cell therapy is limited CAR T cell persistence after infusion into cancer patients. Thus, genetic engineering strategies to improve CAR T cell persistence will be central to the success of these gene-modified cellular therapies.
CAR-T cell therapies mostly depend on stable CAR expression upon delivery by viral and nonviral gene transfer systems. There are three key types of vectors used for clinical applications: σ-retroviral vectors, lentiviral vectors and the transposon/transposase system. Messenger RNA transfer-mediated gene expression is another method to induct CARs into cells while avoiding long-term expression.
Initially, σ-retroviral vectors were used to provide stable CD19 CAR expression. Currently, they are used in about a fifth of all clinical trials requiring gene transfer delivery. Besides high gene expression, another favorable feature of retroviral vectors is the availability of multiple stable packaging cell lines with wide tropism. Long-term patient follow-up studies have demonstrated the safety profile of retroviral vectors in adoptive T-cell therapies.
Lentiviral vectors are being extensively used as they can transduce nondividing cells and show a safer genomic integration profile at least in genetically modified hematopoietic stem cells. Just like σ-retroviral vectors, lentiviral vectors show high gene transfer efficiency and drive stable level of CAR expression.
Yet, there are many obstacles for scaling up the lentiviral vector production platform. The obstacles include the lack of widely available stable vector packaging systems due to the intrinsic fusogenic property of the commonly used VSV-G envelop, lot size limitation, and lot-to-lot variability brought about by the current multi-plasmids transient transaction procedure.
Both the viral vectors necessitate intensive and expensive biosafety testing.
In comparison, new plasmid-based transposon/transposase system is being used to introduce anti-CD19 CAR into T cells by electroporation. The benefits of this system include: simple manufacturing procedure, relatively low cost, and straightforward release testing.
Integration is random, causing a potential oncogenic risk secondary to mutagenesis. Current anti-CD19 CAR-T cell trials using the sleeping beauty transposon/transposase system reveals low T-cell toxicity. However, the efficacy of CAR-T cells produced by this method is yet to be demonstrated.
Messenger RNA (mRNA) Transfer
Messenger RNA (mRNA) transfer offers a cytoplasmic expression system that permits transient expression of the transgene. At variance to stable and permanent expression of the transgene introduced by viral transduction or plasmid DNA transfection, in vitro transcribed mRNA can be inserted into cells by electroporation or by endocytosis.
No genomic integration events occur in this method and hence the problems of genotoxicity and potential generation of replication-competent retrovirus are eliminated.
RNA transfection enables the expression of the transgene for nearly one week, and can be used for delivering mRNA for TCR/CAR, chemokine receptors, and cytokines. This technique is advantageous to screen potentially toxic CAR molecules that could cross-react with normal tissues.
Not long ago, a proof-of-concept study demonstrated that the electroporation of transcription activator-like effector nucleases specific for disrupting TCRα and CD52 molecules enable the production of “off-the-shelf” CAR-T cells from third-party healthy donors.
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