De uno de los CT con edición génica que comenté en la entrada del día 27 de enero de 2020 "Los PD-1 y CCR-5 los objetivos más deseados en los CT de edición génica", el ClinicalTrials.gov NCT03399448, se ha presentado resultados de fase 1.
En el artículo: CRISPR-engineered T cells in patients with refractory cancer de Stadtmauer et al, y entre ellos Carl H June, se describe el procedimiento de elaboración de NY-ESO-1 transduced CRISPR 3X edited cells (NYCE) y los resultados desde el punto de vista, sobre todo, de la seguridad.
El producto consiste en linfocitos T del paciente, modificados mediante CRISPR y tres gRNA para eliminar tres genes codificadores de los receptores TRAC, TRBC Y PD1. Además, mediante vector viral modifican el gen NY-ESO-1
Copio del artículo:
Experimental design
Guide RNAs (gRNA)
The genomic gRNA target sequences with PAM in bold were: TRAC1 and TRAC2: 5’-TGTGCTAGACATGAGGTCTATGG-3’, TRBC: 5’-GGAGAATGACGAGTGGACCCAGG-3’, and PDCD1: 5’-GGCGCCCTGGCCAGTCGTCTGGG-3
Lentiviral vector manufacturing
The 8F TCR recognizes the HLA-A*0201 SLLMWITQC epitope on NY-ESO-1 and LAGE-1. The 8F TCR was isolated from a T cell clone obtained from patient after vaccination with NY-ESO-1 peptide. The TCR sequences were cloned into a transfer plasmid that contains the EF-1α promoter, a cPPT sequence, a rev response element and a woodchuck hepatitis virus posttranscriptional regulatory element
El proceso de manufactura se refleja en este gráfico
Y el protocolo del CT
En resumen:
In conclusion, our phase I human pilot study has confirmed that multiplex CRISPR-Cas9 editing of the human genome is possible at clinical scale. We note that while the initial clinical results are safe, experience with more patients given infusions with higher editing efficiencies and longer observation after infusion will be required to fully assess the safety of this approach. The potential rejection of infused cells due to pre-existing immune responses to Cas9 (28, 29) does not appear to be a barrier to the application of this promising technology. Finally, it is important to note that our manufacturing was based on the reagents available in 2016, when our protocol had been reviewed by the NIH Recombinant DNA Advisory Committee and received approval. Our Investigational New Drug Application was subsequently reviewed and accepted by the U.S. FDA. There has been rapid progress in the field since that time, with the development of reagents that should increase efficiencies and decrease off-target editing using CRISPR-based technology (50).
Of the three patients that were infused with CRISPR-Cas9 engineered T cells, two patients had refractory advanced myeloma and one patient had a refractory metastatic sarcoma not responding to multiple prior therapies
En una entrada anterior mencioné a Carl H June y Emily Whitehead, actores con diversos papeles en el desarrollo de las CAR-T, el primero como médico y la segunda como paciente
En el artículo: CRISPR-engineered T cells in patients with refractory cancer de Stadtmauer et al, y entre ellos Carl H June, se describe el procedimiento de elaboración de NY-ESO-1 transduced CRISPR 3X edited cells (NYCE) y los resultados desde el punto de vista, sobre todo, de la seguridad.
El producto consiste en linfocitos T del paciente, modificados mediante CRISPR y tres gRNA para eliminar tres genes codificadores de los receptores TRAC, TRBC Y PD1. Además, mediante vector viral modifican el gen NY-ESO-1
Copio del artículo:
Experimental design
Guide RNAs (gRNA)
The genomic gRNA target sequences with PAM in bold were: TRAC1 and TRAC2: 5’-TGTGCTAGACATGAGGTCTATGG-3’, TRBC: 5’-GGAGAATGACGAGTGGACCCAGG-3’, and PDCD1: 5’-GGCGCCCTGGCCAGTCGTCTGGG-3
Lentiviral vector manufacturing
The 8F TCR recognizes the HLA-A*0201 SLLMWITQC epitope on NY-ESO-1 and LAGE-1. The 8F TCR was isolated from a T cell clone obtained from patient after vaccination with NY-ESO-1 peptide. The TCR sequences were cloned into a transfer plasmid that contains the EF-1α promoter, a cPPT sequence, a rev response element and a woodchuck hepatitis virus posttranscriptional regulatory element
El proceso de manufactura se refleja en este gráfico
Y el protocolo del CT
En resumen:
In conclusion, our phase I human pilot study has confirmed that multiplex CRISPR-Cas9 editing of the human genome is possible at clinical scale. We note that while the initial clinical results are safe, experience with more patients given infusions with higher editing efficiencies and longer observation after infusion will be required to fully assess the safety of this approach. The potential rejection of infused cells due to pre-existing immune responses to Cas9 (28, 29) does not appear to be a barrier to the application of this promising technology. Finally, it is important to note that our manufacturing was based on the reagents available in 2016, when our protocol had been reviewed by the NIH Recombinant DNA Advisory Committee and received approval. Our Investigational New Drug Application was subsequently reviewed and accepted by the U.S. FDA. There has been rapid progress in the field since that time, with the development of reagents that should increase efficiencies and decrease off-target editing using CRISPR-based technology (50).
Of the three patients that were infused with CRISPR-Cas9 engineered T cells, two patients had refractory advanced myeloma and one patient had a refractory metastatic sarcoma not responding to multiple prior therapies
En una entrada anterior mencioné a Carl H June y Emily Whitehead, actores con diversos papeles en el desarrollo de las CAR-T, el primero como médico y la segunda como paciente
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