Biodistribution analyses of ADA retroviral vector detailed
Published Date: 6/10/17Access Full Text Article
medwireNews: A report published in Human Gene Therapy Clinical Development describes the preclinical safety studies conducted to support the European market authorisation application for GSK2696273, a gammaretroviral-based gene therapy for adenosine deaminase deficient-severe combined immunodeficiency (ADA-SCID).
Lead investigator Alessandro Aiuti (IRCCS San Raffaele Scientific Institute, Milan, Italy) and colleagues explain that “[i]n view of the ultra-rare, often fatal, nature of the disease and limited treatment options, clinical trials were initiated following limited preclinical testing.”
But on the recommendation of the European Medicines Agency (EMA), “a more robust preclinical package to support regulatory review was designed”, one that focussed on the assessment of biodistribution and potential tumourigenicity of GSK2696273, which consists of autologous CD34+ cells transduced with a retroviral vector coding for the human ADA complementary DNA sequence.
In the study, human umbilical cord blood (UCB)-derived CD34+ cells transduced with the ADA gammaretroviral vector were administered by intravenous injection into groups of 10 male and 10 female NOD-SCID gamma (NSG) mice after busulfan conditioning. A control group, comprising the same number of NSG mice of each sex, received mock-transduced UCB CD34+ cells.
Successful engraftment, defined as at least 1% of human CD45+ cells in the bone marrow, occurred in all but six animals given the test or control cells.
After 16 weeks, human CD45+ cells – as detected by fluorescence-activated cell sorting – and human genomes – as assessed by quantitative polymerase chain reaction against the human glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) – were found in peripheral blood samples from the majority of experimental and control animals.
Human cells and human genomes were also detected in the target lymphohaematopoietic organs, the bone marrow and spleen, with no significant differences between the groups.
Such significant differences as were observed – for instance, the significantly higher percentage of human genomes in female experimental than in female mock-transduced mice – “can be considered within normal variation”, say the study authors.
The biodistribution of human genomes in non-target organs was also comparable between vector- and mock-transduced animals, with low to undetectable levels of human GAPDH in the brain, heart, muscle and gonads, while the levels were “slightly higher” in the kidney, lung and liver.
Compared with the post-transduction vector copy number (VCN) of 1.8, the VCN observed in the majority of samples from the experimental mice was lower, at, for instance, an average of 0.49 and 0.30 in 16-week peripheral blood samples from male and female animals, respectively.
But this finding was consistent with previous clinical studies, say Aiuti et al, pointing out that the ADA vector was “never detected in the absence of human GAPDH, supporting the interpretation that vector was present in human cells and not in mouse cells.”
They were also reassured by a detailed analysis of the testes and ovaries of the experimental mice, which showed that the reproductive organs of seven of eight male mice and seven of 10 female mice were below the limit of quantification for VCN analysis. In the remaining animals, less than 0.1% of human DNA was detected and the VCN was similar to that seen in other samples.
These results suggest that “no vector transfer to germline cells has occurred or that such an event can be considered extremely unlikely”, the team comments.
There was one case of lymphoma in a vector-transduced female mouse, but the presence of less than 1% of human CD45+ cells indicated engraftment failure and further analysis showed very low to undetectable levels of human genomes in the lymphoid and non-lymphoid organs, “indicating that the lymphoma was of mouse origin.”
The study authors were unable to further assess the tumourigenicity of GSK2696273 due to the inability to demonstrate long-term engraftment of transduced cells in mice, and in consultation with the regulatory body, “it was agreed that the tumorigenicity risk assessment for GSK2696273 would be based on clinical data and literature on similar vectors.”
The biodistribution data formed part of the marketing authorisation application, which was given a positive opinion by the EMA’s Committee for Medicinal Products for Human Use in April 2016. In May of the same year, the European Commission licensed the product, making GSK2696273 “the first corrective ex vivo gene therapy for children in Europe.”
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