Cooper and von Kalle Receive SINF Awards
Laurence Cooper and Christof von Kalle: Genetic modification of T cells for human applications
If you are engaged in gene therapy research and you need to find the location of the introduced genes, Christof von Kalle, M.D., Ph.D. is the “go to guy”. Von Kalle has been looking at inserted genes for most of his career and excels at rooting out the proverbial needle in the haystack. In 2002, von Kalle worked with French researchers at the Necker Hospital for Sick Children in Paris to treat 11 children with severe combined immunodeficiency (SCID). In nine children, researchers were able to establish healthy immune systems from gene therapy. But when one boy developed cancer, von Kalle tracked the disease to the very retrovirus vector used for the therapy. Von Kalle isolated the time period when aberrant T cells began increasing, analyzed those errant T cells and found a clonal outgrowth from one treated cell. Those cells contained sequences of the retrovirus vector, as well as the gene it transported for the therapy. The retrovirus DNA had inserted itself in reverse in a gene, which when mutated, has been linked with leukemia. It was a set-back for the field, but it did not dissuade von Kalle of the importance of continuing to pursue gene therapy as a mechanism to help fight disease.
While von Kalle has been honing his ability to find one gene among many, Laurence Cooper, M.D., Ph.D. has been working on how to pursue gene transfer without the risk of a retrovirus vector. He seems to be on the right track with the use of a bit of ancient fish DNA. Discovered as a result of a Minnesota State Wildlife Fisheries project directing scientists to genetically engineer larger and faster growing game fish, the Sleeping Beauty (SB) transposon system is a 10 million year old, inactive Tc1/mariner salmonoid transposon that researchers synthetically replicated and repaired. Chosen for Cooper’s research because of its ability to transfer information to human cells, the SB transposon, which is simply a sequence of DNA capable of transposing itself into the genome of a cell, acts through a cut and paste mechanism to transfer information. Researchers insert the genetic information they want the transposon to carry and the transposase cuts out the information and pastes the gene in the DNA. Although humans have DNA transposons, they have all been rendered inactive from the accumulation of mutations. In 2010, Cooper gained federal regulatory approval for the first clinical trial using SB transposon to genetically alter T cells enabling them to destroy cancer cells.
For their current SINF awarded project, von Kalle and Cooper are expanding on their respective expertise while working to further the success of gene transfer to treat B-cell malignancies. The SB transposon will be used to introduce a chimeric antigen receptor (CAR) capable of recognizing tumor associated antigens (TAA) on lymphoma and leukemia cells in patients who have undergone hematopoietic stem cell transplantation (HSCT). In addition, the team will determine if they can prevent the SB transposase, the enzyme that enables the transposon to transfer information, from inadvertently integrating into the T cells. This will increase the safety of the process. Finally, the transposon will be programmed to express zinc finger nuclease (ZFN), a restriction enzyme that can target particular sequences in a genome, to take out αβ T cell receptors (TCR). By taking out TCR expression, the team hypothesizes they can prevent graft-versus-host-disease (GVHD), which may occur when donor-derived T cells are infused after allogeneic HSCT. Von Kalle will use his patented, highly-sensitive linear amplification mediated (LAM) polymerase chain reaction (PCR), a method for identifying an unknown or target DNA or RNA sequence to evaluate the ability of SB system to introduce the CAR transgenes without continued presence of the transposase and ZFN to eliminate undesired genes.