Table 4.

Summary of principal characteristics of methods in CAR T manufacturing.

	Characteristics and peculiarities	Pros/Cons	Technical requirement	Impact of costs
Viral vectors	Gammaretroviral vectors: Infection only in cycling cells Integration near TSS Lentiviral vectors: Infection in cycling/non cycling cells Integration in transcriptional regulatory region	Pros: Stable transduction Long term expression Cons: Limiting insert size Difficulty to scale synthesis up Risk of insertional oncogenesis More immunogenicity	Biosafety Level 2 Trained staff Cryopreserved facility	High costs Limited number of available manufacturing facilities globally, and lot size limitations
Sleeping Beauty	Tc1/mariner DNA Class II TE Cut-and-paste mechanism of insertion Transposon/transposase system Transfection in pre-activated and resting primary cells Close to random integration TA dinucleotide as target site CAG footprint	Pros: Easy to scale synthesis up Large cargo size (up to 100 kb BACs) Versatility Low immunogenicity Cons: Toxicity related to transduction procedure	Electroporation Cryopreserved facility	Relatively low cost Easier manufacturing process
Piggy Bac	PB superfamily DNA Class II TE Cut-and-paste mechanism of insertion Transposon/transposase system Transfection in pre-activated and resting primary cells Preference of integration near TSSs, CpG islands and DNase I hypersensitive sites TTAA dinucleotide as target site No footprint	Pros: Easy to scale synthesis up Large cargo size (200 kb BACs) Versatility Low immunogenicity Higher transposition activity Cons: High risk of gene dysregulation Toxicity related to transduction procedure	Electroporation Cryopreserved facility	Relatively low cost Easier manufacturing process
mRNA	Absence of integration Transient transfection	Pros: Availability of protocols for clinics Versatility and flexibility Safety transient expression (SB100X and PB transposase) Cons (mRNA encoding CAR): Short term potency Need for multiple doses	Electroporation Cation lipids Cationic polymers	High doses of mRNA CAR T are required to achieve efficacy
Nanotechnology	Nanocarriers or lipid nanoparticles coated with ligands ensure encapsulation of non-viral transgenes Ability to reprogram T cells in vivo	Pros: Low toxicity Off -the-shelf process Cons: Limited cargo capacity	Devices for scale up production Specialized staff	Costs of nanoparticles production and costs of encapsulated material