By David N. Leff

Gene therapy - the clinical touchstone of biotechnology - scored its first success 10 years ago. In September 1990, a 4-year-old girl born with adenosine deaminase (ADA) deficiency, which crippled her immune system, received ADA replacement genes, delivered by a retroviral vector. That trailblazing experiment partially corrected her condition, as it did for a second small ADA victim, treated in February 1991.

Today, some 400 gene therapy clinical trials are under way worldwide.

Along with increasing technical sophistication, their practitioners confront a spectrum of roadblocks. Notably, the expanding toolkit of gene-delivery vectors, still primarily viral, persist in their stubborn rap sheet of limitations. These include restricted targeting of specific cell types, critical immunogenicity, cramped DNA-packaging space, cell toxicity, recombination problems and high cost.

That's why there's a growing movement among gene therapists to find nonviral substitute vectors for ferrying DNA of interest to its ultimate grail - the nucleus of its target cell. Among such experimental alternatives are naked DNA, sent forth without a chaperoning vector, "biolistic" particle bombardment of constructs by high-velocity "gene guns," penetration by high- or low-voltage electroporation, and polycationic biopolymers, led by a favored compound called polyethylenimine (PEI).

This last transfection polymer, along with other chemical compounds now on numerous drawing boards, are all checkmated by poor efficiency compared to viruses - and by cytotoxicity.

Scientists at the Massachusetts Institute of Technology (MIT) in Cambridge report an end-run around this block in the current Proceedings of the National Academy of Sciences (PNAS), dated Jan. 30, 2001. Its title: "Polymer-based gene delivery with low cytotoxicity by a unique balance of side-chain termini." Its lead author is pharmaceutical chemist David Putnam, a research associate at MIT.

"We were able to get protein levels," Putnam told BioWorld Today, "that were equal to the gold standard, which is polyethylenimin, with much lower toxicity. That's important," he explained, "because delivery is key to the advance of gene therapy, and protein expression is a function of cell viability. If the target cells are killed after the gene is delivered, you have nothing.

"What kills them," Putnam added, "is the cell toxicity of the gene delivery system itself. So the polycationic polymer PEI - which has the highest protein expression levels in the literature - is cytotoxic to cells, even in very small amounts. There are two ways that polycations can induce cytotoxicity," he went on: "One is biophysical, the other, biochemical.

"The exact mechanisms of cytotoxicity are as yet unknown," Putnam allowed, "but biochemically they can activate cell toxicity factors that make the cell grow out of control, or induce apoptosis. On the biophysical side, these nonviral vectors can compromise the fluidity of the cell membrane, which decreases cell viability.

"It's the latter of the two factors," Putnam surmised, "that is functional here - biophysical vs. biochemical. And the reason is that cytotoxicity is prevalent across all cell lines. If it were biochemical, you would have preferential cytotoxicity in certain cells and not in others."

The Great Endosomal Escape Artist

The MIT co-authors' strategy was to do what viral vectors do, but using nonviral, nontoxic polymers instead.

"With viral vectors," Putnam recounted, "you have a nanostructure, which is absorbed by its target cell's surface. That surface then envaginates - sucks itself in like a balloon. The balloon is now inside the cell, and the virus is inside the balloon, which is called an endosome. Viruses are very clever," he went on, "and they've figured out how to escape from that endosome. If one doesn't escape, it's degraded. If it does escape, it's dumped into the cytosol. From there it can translocate to the nucleus, where the gene delivery action gets started.

"PEI was the first polymer to be used for this," Putnam recalled, "that is, mimic the endosomal escape that viruses typically do. PEI does that quite efficiently, but yet it's cytotoxic. What we've done was to use different types of materials, and rationally synthesize them to mediate the endosomal escape - but without the toxicity."

The MIT team carried out in vitro experiments of its synthesized polymer construct with three types of cells. "It was to identify the relationship between the cation [an ion with a positive electrical charge] of our polycationic polylysine structures and the DNA complex," Putnam recounted. "To determine the cytotoxicity of this construct, we grew cells from three different cell lines of potential clinical significance - liver, muscle and macrophage - because we wanted to make sure this polymer wasn't specific to one type of cell.

"Those particular three cells mimic in vivo targets," he related. "Liver might be used to treat hepatitis, hemophilias - those sorts of diseases. Macrophages mimic antigen-presenting cells of the immune system, for areas like DNA-based vaccines. And muscle mimics muscle-cell transfection after intramuscular injection. In these experiments," Putnam observed, "we went head to head with the front-running, nonviral, polymer vector, PEI. The results in each showed, as we reported in PNAS, that we were equal in the amount of protein produced through our vectors, but without the toxicity."

Going For The Brass Ring

"Our goal is ultimately to use these in humans," Putnam observed. "So our next step is to go in vivo. We'll start from the ground up, with mice and rats, based on previous data. We will complex the polycation that we made, with DNA of interest, inject the construct into mice, and look at where it goes. Then we'll try to modify that path. So if we want it to go to the liver, we'll try to target it to that organ. If we want it to go to cancer cells, we'll try to target it to cancer cells."

As for the team's timetable, Putnam vouchsafed, "We'll start on mice as fast as we possibly can. We don't fool around. But it's a long road, and this is only one step along the way. The brass ring would be ours if we can do what viruses do - without using viruses. If we can rationally generate nonviral vectors that achieve what viruses have achieved during the course of evolution," he concluded, "and are able to do it efficiently and commercially viably, then the implications I think are quite outstanding."