Zinc finger nuclease proteins cut into helical DNA molecules and make edits at specific locations. Researchers at The Scripps Research Institute have reported a robust, yet surprisingly simple and safe, method for disrupting specific genes in vivo, and highlighted its medical potential by demonstrating a putative alternative to gene therapy for HIV infection. In a study published in Nature Methods, Dr. Carlos Barbas and his team employed the widely used zinc finger nuclease proteins (ZNFs), which bind and cut DNA at very precise locations in the human genome. ZNFs have garnered praise for their potential in disease treatment and biomedical applications, but are, to date, introduced into cells utilizing risky gene therapy techniques. By contrast, the TSRI scientists simply added the ZNF proteins into a lab dish, and found that they crossed cell membranes with a great degree of efficiency and performed gene cutting functions as expected with minimal collateral damage.

ZNFs are a relatively well-established bifurcated artificial protein construct: one part composed of a “zinc finger” structure that can be designed to bind to any DNA sequence and the other composed of a nuclease enzyme that will irreversibly cleave DNA at that sequence. Up until now, scientists had made the assumption that these proteins couldn’t penetrate cell membranes on their own, so the standard methodology has been an adjuvant gene therapy method employing an innocuous virus to “help” a designer ZNF gene into the cell, whereupon the gene codes for a ZNF protein that proceeds with cleavage. The problem with this approach is that viral DNA may not end up being innocuous, and may disrupt a valuable coding region of DNA. Furthermore, because retroviral replication isn’t controlled in gene therapy, production of too many ZNF proteins may result in “off-target” DNA splicing, a known danger of ZNF gene therapy. Among the various HIV treatments is a twice-daily regiment of anti-retroviral drugs, like that pictured in the hands of an Indian HIV patient.

By contrast, the TSRI research team was pleasantly surprised to find that ZNFs don’t need either a helper virus or extra coding for cell membrane penetration; indeed, they carry segments with naturally occurring properties that help them cross the membrane barrier, a much safer and more accurate delivery method. Barbas was, incidentally, the first scientist to publish the technology used today to direct ZNF-mediated gene cleavage back in 1996.

The research team tested their results in a variety of cell types, and found that it works particularly well in human “fibroblast” cells. They hope to harvest these cells and reprogram their gene expression patterns to turn them into stem cells, which can then be further modified with ZNFs and other genetic modifications. When transplanted into patients with a wide variety of disease types, these cells would essentially turn into armies of therapeutic progeny cells over time. Although they could target virtually any disease, Barbas wants to start by disrupting the CCR5 gene in hematopoietic stem cells, a gene that some have postulated could be a target for HIV immunity. By transplanting this population of synthetic anti-HIV cells, the theory is that the body would turn into an incubation factory for HIV-resistant T Cells. When tested for HIV gene therapy, the ZNF methodology also appeared to be safer. While DNA-based methodologies produced ZNF proteins that could be detected for several days, causing off-target damage, ZNFs introduced with the direct exposure method only remained intact for several hours.

“Even a small number of stem cells that carry this HIV-resistance feature could end up completely replacing a patient’s original and vulnerable T cell population,” Barbas said.