Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

Rive, Craig M.; Yung, Eric; Dreolini, Lisa; Brown, Scott D.; May, Chris G.; Woodsworth, Daniel J.; Holt, Robert A.

doi:10.1016/j.omtm.2022.05.006

Cited by 8 publications

(6 citation statements)

References 52 publications

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“…Our novel approach was developed and validated using three unique plasmids previously generated in-house: BCRxV.TF.1, BCRxV.GagPolRev.1 and BCRxV.VSVG.1 [ 20 ] (Additional file 1 ). The sequence identity of each plasmid had been previously confirmed by Sanger sequencing following the NIH Human Genome finishing standard (> Phred30, double read coverage of every base) [ 21 ].…”

Section: Resultsmentioning

confidence: 99%

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

et al. 2023

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Background Sequence verification is essential for plasmids used as critical reagents or therapeutic products. Typically, high-quality plasmid sequence is achieved through capillary-based Sanger sequencing, requiring customized sets of primers for each plasmid. This process can become expensive, particularly for applications where the validated sequence needs to be produced within a regulated and quality-controlled environment for downstream clinical research applications. Results Here, we describe a cost-effective and accurate plasmid sequencing and consensus generation procedure using the Oxford Nanopore Technologies’ MinION device as an alternative to capillary-based plasmid sequencing options. This procedure can verify the identity of a pure population of plasmid, either confirming it matches the known and expected sequence, or identifying mutations present in the plasmid if any exist. We use a full MinION flow cell per plasmid, maximizing available data and allowing for stringent quality filters. Pseudopairing reads for consensus base calling reduces read error rates from 5.3 to 0.53%, and our pileup consensus approach provides per-base counts and confidence scores, allowing for interpretation of the certainty of the resulting consensus sequences. For pure plasmid samples, we demonstrate 100% accuracy in the resulting consensus sequence, and the sensitivity to detect small mutations such as insertions, deletions, and single nucleotide variants. In test cases where the sequenced pool of plasmids contains subclonal templates, detection sensitivity is similar to that of traditional capillary sequencing. Conclusions Our pipeline can provide significant cost savings compared to outsourcing clinical-grade sequencing of plasmids, making generation of high-quality plasmid sequence for clinical sequence verification more accessible. While other long-read-based methods offer higher-throughput and less cost, our pipeline produces complete and accurate sequence verification for cases where absolute sequence accuracy is required.

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Section: Resultsmentioning

confidence: 99%

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

et al. 2023

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“…CD22sdCAR lentiviral particles were generated using HEK293T (clone 17; ATCC) as previously described. 45 Briefly, envelope, packaging, and transfer plasmids were cotransfected into the packaging cell line HEK293T (clone 17; ATCC) using TransIT-LT1 (catalog no. MIR2305, Mirus).…”

Section: Methodsmentioning

confidence: 99%

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

McComb,

Arbabi-Ghahroudi,

Hay

et al. 2024

Molecular Therapy: Oncology

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“…Transient CAR T cells can be generated in vivo by delivering mRNA encoding the FAP-targeting CAR in lipid nanoparticles (LNPs) ( 104 ). By direct intravenous infusion of replication-incompetent VSV-G-pseudotyped lentiviral particles encoding a CD19-targeting CAR transgene, persistent CAR-transduced CD3 + T cells were produced and complete B cell aplasia was achieved in mice ( 105 ). With this approach, CAR-encoding lentiviruses are directly infused into the patient and viral transduction of T cells occurs spontaneously in vivo .…”

Section: Major Challenges To Overcome For Car T Cell Therapymentioning

confidence: 99%

From bench to bedside: the history and progress of CAR T cell therapy

et al. 2023

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Chimeric antigen receptor (CAR) T cell therapy represents a major breakthrough in cancer care since the approval of tisagenlecleucel by the Food and Drug Administration in 2017 for the treatment of pediatric and young adult patients with relapsed or refractory acute lymphocytic leukemia. As of April 2023, six CAR T cell therapies have been approved, demonstrating unprecedented efficacy in patients with B-cell malignancies and multiple myeloma. However, adverse events such as cytokine release syndrome and immune effector cell-associated neurotoxicity pose significant challenges to CAR T cell therapy. The severity of these adverse events correlates with the pretreatment tumor burden, where a higher tumor burden results in more severe consequences. This observation is supported by the application of CD19-targeted CAR T cell therapy in autoimmune diseases including systemic lupus erythematosus and antisynthetase syndrome. These results indicate that initiating CAR T cell therapy early at low tumor burden or using debulking strategy prior to CAR T cell infusion may reduce the severity of adverse events. In addition, CAR T cell therapy is expensive and has limited effectiveness against solid tumors. In this article, we review the critical steps that led to this groundbreaking therapy and explore ongoing efforts to overcome these challenges. With the promise of more effective and safer CAR T cell therapies in development, we are optimistic that a broader range of cancer patients will benefit from this revolutionary therapy in the foreseeable future.

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Selective B cell depletion upon intravenous infusion of replication-incompetent anti-CD19 CAR lentivirus

Cited by 8 publications

References 52 publications

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

Complete sequence verification of plasmid DNA using the Oxford Nanopore Technologies’ MinION device

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

From bench to bedside: the history and progress of CAR T cell therapy

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