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Sep 1, 2012 (Vol. 32, No. 15)

Honing In On Gene Delivery Targets

  • Vector Agnosticism

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    Two ZFNs bound to DNA [Jeff Miller, Sangamo BioSciences]

    Sangamo BioSciences, too, used an adenovirus for Phase I trials of its SB-728-T for HIV/AIDS therapy, but in a sense that doesn’t really matter. Because the genes that they deliver need only to be transiently transcribed to achieve their aim, “we have the luxury of using gene delivery approaches that are also transient in their nature,” said Philip Gregory, D.Phil., CSO and vp for research.

    They use whatever makes sense for the application: for T cells a chimeric adenoviral vector designed to recognize T cells has worked best, they electroporate plasmid DNA or mRNA into bone marrow stem cells, and they use AAV vector to transfect the liver. “We can be quite agnostic in terms of vector, and quite inclusive because we don’t require a persistence in that tissue.”

    The nonpersistence and nonintegration of vector means, among other things, that they need not worry about the random integration of vector (and all that entails in terms of transgene or endogenous gene silencing or endogenous gene activation). Nor is the immune response to vector or transgene an issue, as these wash out as the cells divide.

    In the trials, T cells were treated ex vivo by delivering a transgene encoding both monomers of a zinc finger nuclease (ZFN) in a single cistron. The obligate heterodimeric protein—each monomer composed of a zinc finger DNA sequence-specific recognition domain and a nuclease domain taken from FOK1—recognizes and creates a double-stranded break in the CCR5 gene. The cell’s error-prone nonhomologous end joining (NHEJ) pathway then repairs the break, frequently creating a disruption during the process.

    Researchers relied on the observation that the CCR5 protein seems to be virtually necessary for HIV entry into the host cell in that people who are homozygous mutant for CCR5 are naturally resistant to HIV infection. And, in what is seen as a proof-of-concept for gene therapy, an HIV patient with leukemia was “cured” of HIV after receiving a bone marrow transplant from a CCR5-mutant donor.

    Dr. Gregory has no expectation of 100% protection from HIV—the majority of T cells will remain infectable. Only about 10% of the 10–30 billion T cells being returned to the patient will be CCR5-/-, yet those billion-plus T cells are possibly sufficient to protect the entire T-cell repertoire with a CCR5 bi-allelic modification, he said. “So we should potentially be able to protect every cell that recognizes any known epitope in the patient.”

  • Transfecting New Genes Into the Liver

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    AAV8, a second-generation AAV technology, was discovered in James Wilson’s lab at UPenn.

    In the mid-80s, a six-year-old girl with familial hypercholesterolemia (FH) was given a liver transplant, planting a seed in the mind of James Wilson, M.D., Ph.D., “that if you replaced the LDL receptor in the liver, that you could correct the systemic metabolic defect in hypercholesterolemia.” He worked for years on ex vivo gene therapy using retrovirus and another 10 years on adenovirus before realizing that while that vector could transfer genes to the liver, it was extremely toxic “and we could not engineer the virus to avoid significant toxicity.”

    By this time AAV—which didn’t elicit immune responses or inflammation toxicity—was being developed for in vivo gene therapy, but “only 1–2% of the liver cells took the gene up”—too little to correct diseases like homozygous FH, recalled Dr. Wilson, by now at the University of Pennsylvania, where he is currently a professor in the department of pathology and laboratory medicine, and the director of the gene therapy program. He is also the editor of Human Gene Therapy, a peer-reviewed journal published by Mary Ann Liebert, Inc.

    Dr. Wilson used PCR to find new AAV isolates from humans and nonhuman primate genomes, many of which were 100 times more efficient than the original isolates, with AAV8 being the “most attractive for targeting liver.”

    Using sophisticated mouse models of homozygous FH, Dr. Wilson’s work has progressed to late preclinical stages: “We’ve had a pre-IND meeting with FDA. We’re just completing the toxicology experiments, and hope to move into the clinic sometime in the next year,” Dr. Wilson noted. A recent Phase I study by another group using AAV8 to express Factor IX in hemophiliacs “has further encouraged us that AAV8 will be useful in targeting human liver by transfecting new genes into the hepatocytes.”


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