Vector-transduced cell tracking feasible in gene therapy-treated ADA-SCID patients
Published Date: 21/12/17Download Full Text Article
medwireNews: Japanese researchers have developed a single cell-based method that enables the tracking of vector-transduced cells in adenosine deaminase deficient-severe combined immunodeficiency (ADA-SCID) patients treated with stem-cell gene therapy.
They explain that sufficient engraftment of transduced cells is needed to achieve clinical improvement, but various parameters, such as the selective advantage of the therapeutic gene, can affect engraftment. “Therefore, the mapping of gene-transduced cell distributions in treated patients is required to evaluate the efficacy of gene therapy”, the team writes.
The method – termed single cell-based droplet digital polymerase chain reaction (PCR) – involves the encapsulation of individual cells along with the PCR mixture into droplets, following which the cells are lysed and the target gene amplified within the droplets. A fluorescent signal indicates the presence of the target gene allowing the identification of vector-positive cells.
Using this system, Toru Uchiyama, from the National Center for Child Health and Development in Tokyo, and fellow investigators evaluated peripheral blood mononuclear cells (PBMCs) and bone marrow samples from two ADA-SCID patients who received retroviral-based gene therapy, in both cases without cytoreductive preconditioning.
Applying single cell-based droplet digital PCR to PMBCs – sorted into CD3+ T cell, CD56+ natural killer (NK) cell and CD19+ B cell subsets by fluorescence-activated cell sorting – showed that the majority of CD3+ T cells were positive for the transduced vector, at rates of 97.1% in patient 1 and 80.8% in patient 2, indicating that “ADA-positive T cells have a strong growth advantage over non-transduced cells.” By contrast, less than 10% of CD56+ NK cells and less than 20% of CD19+ B cells displayed vector integration.
And analysis of bone marrow samples revealed that only around 5–7% of all enucleated cells and approximately 0–2% of CD34+ cells were positive for the vector, highlighting the importance of “securing ‘space’ in the [bone marrow] niche” so that modified haematopoietic stem cells can engraft and differentiate into multiple lineages.
The novel method “enabled the detailed mapping of gene-transduced cells and revealed their complicated distribution in the specific cell fractions of patients’ PB and [bone marrow] samples”, the researchers write in Molecular Therapy: Methods & Clinical Development.
And they conclude that such comprehensive information in gene therapy-treated patients “can be strongly advantageous for determining treatment strategies including conditioning therapy in [stem-cell gene therapy] clinical trials.”
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