Reference standards for accurate validation and optimization of assays that determine integrated lentiviral vector copy number in transduced cells.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti.

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as ß-actin, ef-1ß, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes.

Highly efficient, biocompatible, and fast nucleic acid delivery methods are essential for biomedical applications and research. At present, two main strategies are used to this end. In non-viral transfection liposome- or polymer-based formulations are used to transfer cargo into cells via endocytosis, whereas viral carriers enable direct nucleic acid delivery into the cell cytoplasm. Here, we introduce a new generation of liposomes for nucleic acid delivery, which immediately fuse with the cellular plasma membrane upon contact to transfer the functional nucleic acid directly into the cell cytoplasm. For maximum fusion efficiency combined with high cargo transfer, nucleic acids had to be complexed and partially neutralized before incorporation into fusogenic liposomes. Among the various neutralization agents tested, small, linear, and positively charged polymers yielded the best complex properties. Systematic variation of liposomal composition and nucleic acid complexation identified surface charge as well as particle size as essential parameters for cargo-liposome interaction and subsequent fusion induction. Optimized protocols were tested for the efficient transfer of different kinds of nucleic acids like plasmid DNA, messenger RNA, and short-interfering RNA into various mammalian cells in culture and into primary tissues.

Lot-to-Lot Variation in Adeno-Associated Virus Serotype 9 (AAV9) Preparations.

Viral vectors are complex drugs that pose a particular challenge for manufacturing. Previous studies have shown that, unlike small-molecule drugs, vector preparations do not yield a collection of identical particles. Instead, a mixture of particles that vary in capsid stoichiometry and impurities is created, which may differ from lot to lot. The consequences of this are unclear, but conflicting reports regarding the biological properties of vectors, including transduction patterns, suggest that this variability may have an effect. However, other variables, including differences in animal strains and techniques, make it difficult to identify a cause. Here, we report lot-to-lot variation in spinal cord gray matter transduction following intrathecal delivery of self-complementary adeno-associated virus serotype 9 vectors. Eleven lots of vector were evaluated from six vector cores, including one preclinical/Good Laboratory Practice lot. Eight of the lots, including the preclinical lot, failed to transduce the gray matter, whereas the other three provided robust transduction. The cause for this variation is unknown, but it did not correlate with vector titer, buffer, or purification method. These results highlight the need to identify the cause of this variation and to develop improved production and quality control methods to ensure lot-to-lot consistency of vector potency.

The Impact of Alkyl-Chain Purity on Lipid-Based Nucleic Acid Delivery Systems - Is the Utilization of Lipid Components with Technical Grade Justified?

The physicochemical properties and transfection efficacies of two samples of a cationic lipid have been investigated and compared in 2D (monolayers at the air/liquid interface) and 3D (aqueous bulk dispersions) model systems using different techniques. The samples differ only in their chain composition due to the purity of the oleylamine (chain precursor). Lipid 8 (using the oleylamine of technical grade for cost-efficient synthesis) shows lateral phase separation in the Langmuir layers. However, the amount of attached DNA, determined by IRRAS, is for both samples the same. In 3D systems, lipid 8 p forms cubic phases, which disappear after addition of DNA. At physiological temperatures, both lipids (alone and in mixture with cholesterol) assemble to lamellar aggregates and exhibit comparable DNA delivery efficiency. This study demonstrates that non-lamellar structures are not compulsory for high transfection rates. The results legitimate the utilization of oleyl chains of technical grade in the synthesis of cationic transfection lipids.

Synthesis, characterization and evaluation of transfection efficiency of dexamethasone conjugated poly(propyleneimine) nanocarriers for gene delivery#.

CONTEXT: Polypropylenimine (PPI), a cationic dendrimer with defined structure and positive surface charge, is a potent non-viral vector. Dexamethasone (Dexa) conveys to the nucleus through interaction with its intracellular receptor. OBJECTIVE: This study develops efficient and non-toxic gene carriers through conjugation of Dexa at various percentages (5, 10 and 20%) to the fourth and the fifth generation PPIs (PPIG4s and PPIG5s). MATERIALS AND METHODS: The 21-OH group of Dexa (0.536 mmol) was modified with methanesulfonyl chloride (0.644 mmol) to activate it (Dexa-mesylate), and then it was conjugated to PPIs using Traut's reagent. After dialysis (48 h) and lyophilization, the physicochemical characteristics of products (PPI-Dexa) including zeta potential, size, buffering capacity and DNA condensing capability were investigated and compared with unmodified PPIs. Moreover, the cytotoxicity and transfection activity of the Dexa-modified PPIs were assessed using Neuro2A cells. RESULTS: Transfection of PPIG4 was close to PEI 25 kDa. Although the addition of Dexa to PPIG4s did not improve their transfection, their cytotoxicity was improved; especially in the carrier to DNA weight ratios (C/P) of one and two. The Dexa conjugation to PPIG5s enhanced their transfection at C/P ratio of one in both 5% (1.3-fold) and 10% (1.6-fold) Dexa grafting, of which the best result was observed in PPIG5-Dexa 10% at C/P ratio of one. DISCUSSION AND CONCLUSIONS: The modification of PPIs with Dexa is a promising approach to improve their cytotoxicity and transfection. The higher optimization of physicochemical characteristics, the better cell transfection and toxicity will be achieved.

Functional DNA Delivery Enabled by Lipid-Modified Charge-Altering Releasable Transporters (CARTs).

Safe and effective DNA delivery systems are required to enable or enhance clinical strategies and research involving gene therapy and DNA vaccinations. To address this delivery problem, a series of charge-altering releasable transporters (CARTs) with varied lipid content were prepared and evaluated for plasmid DNA (pDNA) delivery into cultured cells. These lipid-modified CART co-oligomers were synthesized in only two steps via sequential organocatalytic ring-opening polymerization of lipid-containing cyclic carbonate monomers and morpholinone monomers. Lipid variations of the CARTs substantially impacted the delivery efficiency of pDNA, with oleyl- and linoleyl-based CARTs showing enhanced performance relative to the commercial transfection agent Lipofectamine 2000 (L2000). The best-performing oleyl CART was carried forward to study stable luciferase transfection with a Sleeping Beauty ( SB) transposon system. The oleyl CART outperformed the L2000 positive control with respect to stable transfection efficiency. CART-pDNA complexes represent a new DNA delivery system for research and clinical applications.

Cationic starch/pDNA nanocomplexes assembly and their nanostructure changes on gene transfection efficiency.

This study aims at developing biocompatible starch based gene carriers with good gene delivery and transfection efficacy. By controlling the molecular weight and aggregation behavior of spermine modified cationic starch (CS) molecules, nanocomplexes spontaneously formed through electrostatic interaction using CS and plasmid pAcGFP1-C1 (pDNA) displaying different structural changes (particle size, zeta potential, shape, compactness) response to the simulated intracellular pH variation. Results indicated that CS2 with weight average molecular weight (Mw) of 6.337 × 104 g/mol displayed relatively higher transfection efficacy (~30%) in HepG2 cells than others and revealed significantly low cytotoxicity. By simulating the intracellular pH variation, Dynamic Light Scattering (DLS) and Small Angle X-ray Scattering (SAXS) results demonstrated that CS2 could bind to pDNA tightly and form nanocomplexes with smaller and compact internal aggregate structure at acidic conditions, which facilitated the effective pDNA protection under endosome pH change, while larger and loose internal aggregate structure at physiological pH which promoted the disintegration of CS2/pDNA nanocomplexes. Therefore, CS with suitable Mw of around 6.0 × 104 g/mol represents a potential gene carrier for gene delivery. This study also demonstrated that controlling the internal nanostructure change of polymer/gene nanocomplexes could provide guidance in designing effective starch based gene carriers.

A Scalable and Accurate Method for Quantifying Vector Genomes of Recombinant Adeno-Associated Viruses in Crude Lysate.

Increasing interest and application of recombinant adeno-associated viruses (rAAVs) in basic and clinical research have urged efforts to improve rAAV production quality and yield. Standard vector production workflows call for genome titration of purified vectors at the endpoint of production to assess yield. Unfortunately, quality control measures for preparations during mid-production steps and economical means to assess the fidelity of multiple batches of rAAV preparations are lacking. Here we describe a scalable and accurate method for the direct quantitative polymerase chain reaction (qPCR) titration of rAAV genomes in crude lysate. Lysate samples are pretreated with DNase I to remove vector and packaging plasmid DNAs, followed by proteinase K to release endonuclease-resistant packaged viral genomes and to proteolyze factors inherent to crude lysates that can impinge upon quantitative PCR efficiencies. We show that this method is precise, scalable, and applicable for vector genome titrations of both single-stranded and self-complementary AAV genomes irrespective of serotype differences-a major limitation for standard lysate transduction methods that indirectly screen for vector packaging efficiency. Our described method therefore represents a significant improvement to rAAV vector production in terms of alleviating time and cost burdens, in-process quality control assessment, batch/lot monitoring in large-scale preparations, and good manufacturing practices.

Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells.

Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by >60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.

Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains.

Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non-human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca2+/calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates (NHPs). These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.

Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells.

Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with vesicular stomatitis virus glycoprotein were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The elongation factor 1α short (EFS) promoter mediated the most robust expression, whereas expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full-length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE.EFS and CBX3.EFS vectors maintained highest transgene expression in a larger fraction of cells as compared with all other constructs tested here. The function of UCOE diminished, but did not fully counteract, vector silencing and possibilities for improvements remain. Nevertheless, the CBX3.EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.

Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent.

The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2-3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.

pH and reduction dual-responsive dipeptide cationic lipids with α-tocopherol hydrophobic tail for efficient gene delivery.

A series of tocopherol-based cationic lipid 3a-3f bearing a pH-sensitive imidazole moiety in the dipeptide headgroup and a reduction-responsive disulfide linkage were designed and synthesized. Acid-base titration of these lipids showed good buffering capacities. The liposomes formed from 3 and co-lipid 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) could efficiently bind and condense DNA into nanoparticles. Gel binding and HPLC assays confirmed the encapsulated DNA could release from lipoplexes 3 upon addition of 10 mM glutathione (GSH). MTT assays in HEK 293 cells demonstrated that lipoplexes 3 had low cytotoxicity. The in vitro gene transfection studies showed cationic dipeptide headgroups clearly affected the transfection efficiency (TE), and arginine-histidine based dipeptide lipid 3f give the best TE, which was 30.4 times higher than Lipofectamine 3000 in the presence of 10% serum. Cell-uptake assays indicated that basic amino acid containing dipeptide cationic lipids exhibited more efficient cell uptake than serine and aromatic amino acids based dipeptide lipids. Confocal laser scanning microscopy (CLSM) studies corroborated that 3 could efficiently deliver and release DNA into the nuclei of HeLa cells. These results suggest that tocopherol-based dipeptide cationic lipids with pH and reduction dual-sensitive characteristics might be promising non-viral gene delivery vectors.

Cationic lipids bearing succinic-based, acyclic and macrocyclic hydrophobic domains: Synthetic studies and in vitro gene transfer.

In this communication we describe the construction of four succinic-based cationic lipids, their formulation with plasmid DNA (pDNA), and an evaluation of their in vitro gene delivery into Chinese hamster ovarian (CHO-K1) cells. The cationic lipids employed in this work possess either a dimethylamine or trimethylamine headgroup, and a macrocyclic or an acyclic hydrophobic domain composed of, or derived from two 16-atom, succinic-based acyl chains. The synthesized lipids and a co-lipid of neutral charge, either cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were formulated in an overall 3:2 cationic-to-neutral lipid molar ratio, then complexed with plasmid DNA (pDNA). The relative transfection performance was evaluated via a comparison between matched versus mismatched formulations defined by the rigidity relationship between the lipids employed. Gel electrophoresis was used to characterize the binding of the lipid formulations with plasmid DNA and the relative degree of plasmid degradation using a DNase I degradation assay. Small angle X-ray diffraction (SAXD) was employed to characterize the packing morphology of the lipid-DNA complexes. In general, the succinic unit embedded within the hydrophobic domain of the cationic lipids was found to improve lipid hydration. The transfection assays revealed a general trend in which mismatched formulations that employed a rigid lipid combined with a non-rigid (or flexible) lipid, outperformed the matched formulations. The results from this work suggest that the design of the cationic lipid structure and the composition of the lipoplex formulation play key roles in governing the transfection performance of nonviral gene delivery agents.

Four Novel Splice-Switch Reporter Cell Lines: Distinct Impact of Oligonucleotide Chemistry and Delivery Vector on Biological Activity.

New advances in oligonucleotide (ON) chemistry emerge continuously, and over the last few years, several aspects of ON delivery have been improved. However, clear knowledge regarding how certain chemistries behave alone, or in combination with various delivery vectors, is limited. Moreover, characterization is frequently limited to a single reporter cell line and, when different cell types are studied, experiments are commonly not carried out under similar conditions, hampering comparative analysis. To address this, we have developed a small "tissue" library of new, stable, pLuc/705 splice-switching reporter cell lines (named HuH7_705, U-2 OS_705, C2C12_705, and Neuro-2a_705). Our data show that, indeed, the cell type used in activity screenings influences the efficiency of ONs of different chemistry (phosphorothioate with locked nucleic acid or 2'-O-methyl with or without N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine). Likewise, the delivery method, Lipofectamine® 2000, PepFect14 nanoparticles, or "naked" uptake, also demonstrates cell-type-dependent outcomes. Taken together, these cell lines can potentially become useful tools for future in vitro evaluation of new nucleic acid-based oligomers as well as delivery compounds for splice-switching approaches and cell-specific therapies.

Bioreducible Gene Delivery Vector Capable of Self-Scavenging the Intracellular-Generated ROS Exhibiting High Gene Transfection.

Cationic polymer vectors have received increasing attention for gene delivery in biotechnology over the past 2 decades, but few polymer vectors were used in clinical applications due to their low gene transfection efficacy. One of the major reasons is that the conventional cationic polymers can induce the increasing of intracellular reactive oxygen species (ROS) concentration and oxidative stress, which reduces the gene transfection efficacy. Here, we create a novel class of thioether dendron-branched polymer conjugate and self-assemble this conjugate into bioreducible cationic nanomicelles with disulfide bond connecting the thioether core to the cationic shell. The obtained nanomicelles have a unique ROS self-scavenging ability, thereby dramatically improving gene transfection efficacy.

Synthesis of Amphiphilic Poly(ß-amino ester) for Efficiently Minicircle DNA Delivery in Vivo.

Minicircle DNA (mcDNA) is a kind of enhanced nonviral DNA vector with excellent profiles in biosafety and transgene expression. Herein, we reported a novel amphiphilic polymer comprising polyethylenimine(PEI) modified Poly(ß-amino ester) PEI-PBAE(C16) for efficient mcDNA delivery in vivo. The synthesized polymer could condense mcDNA into nanoscaled structure and exhibited efficient gene transfection ability without detectable cytotoxicity. Importantly, when injected into mouse intraperitoneally, these PEI-PBAE(C16) nanocomplexes were able to result in high level of trangene expression which lasted at least 72 h. Overall, these results demonstrated the PEI-PBAE(C16) can mediate effective and safe gene delivery in vivo with clinical application potential.

Cell-Targeting Cationic Gene Delivery System Based on a Modular Design Rationale.

En route to target cells, a gene carrier faces multiple extra- and intracellular hurdles that would affect delivery efficacy. Although diverse strategies have been proposed to functionalize gene carriers for individually overcoming these barriers, it is challenging to generate a single multifunctional gene carrier capable of surmounting all these barriers. Aiming at this challenge, we have developed a supramolecular modular approach to fabricate a multifunctional cationic gene delivery system. It consists of two prefunctionalized modules: (1) a host module: a polymer (PCD-SS-PDMAEMA) composed of poly(ß-cyclodextrin) backbone and disulfide-linked PDMAEMA arms, expectedly acting to compact DNA and release DNA upon cleavage of disulfide linkers in reductive microenvironment; and (2) a guest module: adamantyl and folate terminated PEG (Ad-PEG-FA), expectedly functioning to reduce nonspecific interactions, improve biocompatibility, and provide folate-mediated cellular targeting specificity. Through the host-guest interaction between ß-cyclodextrin units of the "host" module and adamantyl groups of the "guest" module, the PCD-SS-PDMAEMA-1 (host) and Ad-PEG-FA (guest) self-assemble forming a supramolecular pseudocopolymer (PCD-SS-PDMAEMA-1/PEG-FA). Our comprehensive analyses demonstrate that the functions preassigned to the two building modules are well realized. The gene carrier effectively compacts DNA into stable nanosized polyplexes resistant to enzymatic digestion, triggers DNA release in reducing environment, possesses significantly improved hemocompatibility, and specifically targets folate-receptor positive cells. Most importantly, endowed with these predesigned functions, the PCD-SS-PDMAEMA-1/PEG-FA supramolecular gene carrier exhibits excellent transfection efficacy for both pDNA and siRNA. Thus, this work represents a proof-of-concept example showing the efficiency and convenience of an adaptable, modular approach for conferring multiple functions to a single supramolecular gene carrier toward effective in vivo delivery of therapeutic nucleic acids.

Establishing guidelines for CAR-T cells: challenges and considerations.

T cells, genetically modified by chimeric antigen receptors (CAR-T), are endowed with specificity to a desired antigen and are cytotoxic to cells expressing the targeted antigen. CAR-T-based cancer immunotherapy is a promising therapy for curing hematological malignancy, such as acute lymphoid leukemia, and is promising for extending their efficacy to defeat solid tumors. To date, dozens of different CAR-T cells have been evaluated in clinical trials to treat tumors; this necessitates the establishment of guidelines for the production and application of CAR-T cells. However, it is challenging to standardize CAR-T cancer therapy because it involves a combination of gene therapy and cell therapy. In this review, we compare the existing guidelines for CAR-T cells and discuss the challenges and considerations for establishing guidance for CAR-T-based cancer immunotherapy.