Improving cell and gene therapy safety and performance using next-generation Nanoplasmid vectors

Williams, James A.; Paez, Patrick A

doi:10.1016/j.omtn.2023.04.003

Cited by 12 publications

(3 citation statements)

References 71 publications

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“…Minimal-backbone Nanoplasmid transposon vectors and Nanoplasmid HDR donor template vectors for CRISPR/Cas9 mediated gene editing demonstrated superior cell viability and gene integration frequencies during non-viral CAR-T cell manufacturing over traditional pUC plasmids and linearized templates (Clinical Trial NCT03288493 ). 55 , 64 , 65 , 66 We electroporated this template along with a CRISPR-Cas9 RNP specific for the TRAC locus and cultured the cells for 7 days. Robust gene editing at clinical or benchtop scale was observed by flow cytometry, as greater than 90% of T cells lacked expression of the endogenous TCR given the CRISPR-mediated knockout of the TRAC gene ( Figure 1 B) when compared with untransfected T cells (clinical: 98.2% [0.7] or benchtop: 90.7% [1.0] versus untransfected: 8.6% [5.1], p < 0.001, respectively).…”

Section: Resultsmentioning

confidence: 99%

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Cappabianca,

Pham,

Forsberg

et al. 2024

Molecular Therapy - Methods & Clinical Development

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Section: Resultsmentioning

confidence: 99%

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Cappabianca,

Pham,

Forsberg

et al. 2024

Molecular Therapy - Methods & Clinical Development

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“…Two decades of intense worldwide efforts to develop the novel approaches towards clinical utility have led to remarkable progress for a number of important diseases and in a few cases their entry into clinical practice. Whereas the liver and hepatocytes emerged as rather easily accessible target for RNAi or ASO strategies, once breakthrough molecular design and delivery discoveries (Figure 5) were made, efficient and selective targeting of many other organs and, in particular, proper targeting of specific cell population therein [248][249][250][251][252] has not yet been successfully applied in clinical trials.…”

Section: State-of-the-art In Nucleic Acid-based Therapeutics and Thei...mentioning

confidence: 99%

Exploration of the Noncoding Genome for Human-Specific Therapeutic Targets—Recent Insights at Molecular and Cellular Level

Poller,

Sahoo,

Hajjar

et al. 2023

Cells

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While it is well known that 98–99% of the human genome does not encode proteins, but are nevertheless transcriptionally active and give rise to a broad spectrum of noncoding RNAs [ncRNAs] with complex regulatory and structural functions, specific functions have so far been assigned to only a tiny fraction of all known transcripts. On the other hand, the striking observation of an overwhelmingly growing fraction of ncRNAs, in contrast to an only modest increase in the number of protein-coding genes, during evolution from simple organisms to humans, strongly suggests critical but so far essentially unexplored roles of the noncoding genome for human health and disease pathogenesis. Research into the vast realm of the noncoding genome during the past decades thus lead to a profoundly enhanced appreciation of the multi-level complexity of the human genome. Here, we address a few of the many huge remaining knowledge gaps and consider some newly emerging questions and concepts of research. We attempt to provide an up-to-date assessment of recent insights obtained by molecular and cell biological methods, and by the application of systems biology approaches. Specifically, we discuss current data regarding two topics of high current interest: (1) By which mechanisms could evolutionary recent ncRNAs with critical regulatory functions in a broad spectrum of cell types (neural, immune, cardiovascular) constitute novel therapeutic targets in human diseases? (2) Since noncoding genome evolution is causally linked to brain evolution, and given the profound interactions between brain and immune system, could human-specific brain-expressed ncRNAs play a direct or indirect (immune-mediated) role in human diseases? Synergistic with remarkable recent progress regarding delivery, efficacy, and safety of nucleic acid-based therapies, the ongoing large-scale exploration of the noncoding genome for human-specific therapeutic targets is encouraging to proceed with the development and clinical evaluation of novel therapeutic pathways suggested by these research fields.

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“…Mesenchymal stem cells were transfected with large- or small-sized plasmid, either at equimolar or equimass concentrations, and the transfection efficiency is directly linked to the physical size of plasmid, a larger plasmid being more toxic and harder to transfect than a small plasmid ( 6 ). Recent research found that using the minicircle or nanoplasmid technique could further reduce or remove bacterial-derived elements on the constructs and increase the efficiency of transfection and expression ( 7 , 8 ). However, modifying the currently available plasmids to reduce the size is a feasible and practical way.…”

Section: Introductionmentioning

confidence: 99%

Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10

Li,

Hu,

Hua

et al. 2023

Microbiol Spectr

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Antibiotic resistance screening is crucial in the construction of recombinant plasmids. The dfrB10 gene, a short-size trimethoprim (TMP)-resistance type B dihydrofolate reductase gene, is a promising antibiotic drug screening marker for plasmid construction in molecular genetic research. Currently, available plasmid backbones (without the resistance gene) were amplified using PCR, and then we ligated these products with dfrB10 gene fragments via homologous recombination to construct new plasmid vectors with TMP resistance. The cloning, expression, and gene editing functions of the new plasmids based on their different purposes were verified. By successfully replacing the original resistance genes of pUC19, pET28a, pcDNA3.1, and pX330 plasmids with dfrB10 , we constructed a series of plasmids with TMP resistance. The change of the resistance selection marker to TMP did not affect the function of the plasmids compared to the original ones. Furthermore, a short (988 bp) TMP-resistant miniature tool plasmid, pTMi, was constructed based on the pUC19 plasmid. With the use of the dfrB10 gene as a resistance marker under TMP drug screening, various size-reduced tool plasmids can be routinely constructed. These plasmids have similar functions to the original ones and can be used for research applications in molecular genetics. IMPORTANCE Plasmid size is one of the factors affecting transfection efficacy in most of the molecular genetic research studies. One effective approach for reducing plasmid size is to replace relatively large, conventional antibiotic resistance genes with the short-size dfrB10 gene. The successful construct of a series of dfrB10- based tool plasmids and their functional validation, via comparison with original plasmids, suggest that dfrB10 is a potent drug resistance selection marker. The antibiotic trimethoprim offers convenient usage comparable to that of ampicillin or kanamycin. Additionally, fluorescence analysis has demonstrated the compatibility of TMP with protein expression in various host cells. Based on these findings, TMP- dfrB10 could be an alternative choice for future use in molecular genetic research studies that require miniature plasmids to achieve optimal results.

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Improving cell and gene therapy safety and performance using next-generation Nanoplasmid vectors

Cited by 12 publications

References 71 publications

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Metabolic priming of GD2 TRAC-CAR T cells during manufacturing promotes memory phenotypes while enhancing persistence

Exploration of the Noncoding Genome for Human-Specific Therapeutic Targets—Recent Insights at Molecular and Cellular Level

Construction and functional verification of size-reduced plasmids based on TMP resistance gene dfrB10

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