Nature Biotechnology pp pp 555-558

The problem with traditional genetic engineering techniques in plants is that they are rather messy. Typically, plant transformation methods introduce many copies of foreign genes at random positions on the chromosome leading to variation in the level of foreign gene expression and, occasionally, triggering suppression (silencing) of similar genes that naturally occur in the plant. Now, Chris Baszczynski and his colleagues have adapted a technique, originally developed for gene repair in mammalian cells, that employs small hairpin-shaped molecules made up of RNA and DNA (so called chimeric oligonucleotides) to introduce single base changes into DNA. Using this approach, they have successfully generated herbicide-resistant plants with just a single change in the genetic code.

Chimeric oligonucleotides consist of a short self-complementary stretch of double stranded DNA flanked by a longer stretch of RNA to protect it from degradation in vivo. The DNA sequence corresponds to the region of the gene to be modified, but with a single nucleotide change. When introduced into cells, the chimeric oligonucleotide homes in on the gene of interest with the matching sequence and then triggers the plant's own DNA repair machinery to substitute the oligonucleotide-encoded sequence with the single base alteration for the original plant sequence.

In previous work, Basczynski's team used chimeric oligonucleotides to alter the gene encoding the plant enzyme acetohydroxyacid synthase (AHAS) in masses of plant cells called calli, which as a result acquired resistance to the broad-spectrum herbicide imidazolinone. However, it was not clear whether the results would be reproducible or long lasting in whole plants. In the new study, they regenerate whole transgenic plants from the calli and show that the modified AHAS gene is maintained in subsequent generations.

While the results are promising, researchers still have to overcome the low efficiency of gene conversion (currently only 1 in 10,000 cells are modified) or improve the screening procedures to identify which cells have undergone gene conversion. Nevertheless, if the technique can be made more efficient, it could avoid many of the problems associated with traditional plant transformation used for GM crops.

Nature Biotechnology pp 527 - 532, pp 595 - 598, p 599 and pp 497

Confirming that great things can come in small packages, three teams of scientists have found a way of improving a promising gene therapy vector system. Much of the recent controversy over gene therapy has related to the use of a weakened cold virus, or adenovirus, as a vector to deliver corrective genes. Another system, the adeno-associated virus (AAV), has been proposed as promising alternative because it does not cause deleterious and dangerous immune responses and is capable of inducing prolonged gene expression. Thus far, AAV has not been widely adopted as a vector because it is much smaller than adenoviruses and cannot carry large genes. Now three papers-one by Mark Kay and his colleagues in this issue of Nature Biotechnology and two in Nature Medicine-describe a modification to the AAV system that allows it to deliver larger genes, such as those needed to treat cystic fibrosis, hemophilia A, and certain types of muscular dystrophy. The three teams accomplish this by splitting the gene of interest into two parts and packaging each half into a separate AAV vector.

Kay and his team set about their task by splitting the AAV vector expression cassette, which contains the foreign gene to be delivered and an enhancer/promoter to activate it, into two complementary vectors. One portion contained the enhancer/promoter and a splice donor, and the other contained a splice acceptor and the transgene. They then injected two vectors together into the portal vein that leads to the liver in mice. As a control, they injected other mice with vectors containing the entire enhancer/promoter and transgene together. When they studied the mouse livers, they found that the level of gene expression in mice that received the two separate vectors was 60-70% of that in mice receiving the expression cassette in one piece.

In related papers published this month in Nature Medicine, teams in Iowa and Pittsburgh have gone a step further and shown that this technique can be used to deliver therapeutic genes--this case erythropoietin for anemia and Factor VIII for hemophilia--to their targets.

Nature Biotechnology pp 538 - 543 and pp 494 - 496

By fusing the fluorescent marker green fluorescent protein (GFP) to chemically tagged amino acid sequences that single out proteins for destruction, researchers have found a way of correlating the activity of an important protease, the proteasome, with fluorescence. The novel system not only allows the first studies of proteasome activity in living cells, but also should ultimately enable the rapid identification of drugs that inhibit aberrant proteasome activity in cancer and muscle-wasting diseases.

Proteases are key enzymes that catalyze protein degradation and regulate cellular processes, such as coagulation, inflammation, and cancer progression. One of the most important cellular proteases is the proteasome, a very large protein-degrading complex with a voracious appetite for proteins that are too old, improperly folded, or damaged. In the cell, proteins are tagged for degradation by a cascade of enzymes that add a chain of molecules (ubiquitins) to their ends. Any protein tagged with a ubiquitin chain is immediately recognized by the proteasome and targeted for destruction. Until now, however, there has been no easy method for rapidly identifying inhibitors of proteasome-mediated degradation of ubiquitin-tagged proteins in cells.

Now, Maria Masucci and her colleagues have come up with an assay that reliably assesses proteasome function in cells by correlating proteolytic activity with the fluorescence of fusions between GFP and ubiquitin. In pilot experiments, they show that these GFP fusions are rapidly degraded in cells, the rate of cleavage being dependent on the composition of the amino acid linker between GFP and ubiquitin. They also demonstrate that the fluorescence of GFP fusions is not only enhanced in a dose-dependent fashion by known inhibitors of proteasome activity, but also correlates with the extent of cell death due to proteasome inhibition. Surprisingly, the assay also reveals that protease inhibitors, such as YL3-VS, NP-LLG-VS, and Ritonavir, which had been suggested to act on the proteasome in vitro, do not inhibit the proteasome in intact cells.

A commentary by Arthur Goldberg suggests that the GFP system will prove useful for both validating known and isolating new proteasome inhibitors in drug development. He also suggests it could be refined to allow the identification of inhibitors that act on more specific parts in the proteolysis pathway such as ubiquitination.

Nature Biotechnology pp 509 - 514, pp 515 - 520 and pp 491 - 492

Most cancers grow rapidly and spread because tumor cells are hidden away from the defensive arms of the immune system. Now two groups have taken very different tacts to expose tumor antigens to the body's cancer-destroying cells, cytotoxic T lymphocytes (CTLs), and induce protection against cancer growth. In one approach, Hearn Cho and his colleagues have hooked up immunostimulatory DNA sequences to a tumor antigen, ovalalbumin, to induce potent CTL responses against preestablished tumors. In another, Wilfred Jeffries and his team have induced cancer cells to express tumor-specific antigens on their surfaces by restoring the antigen transport protein TAP1 to TAP-deficient cells. Both approaches show promise for boosting the immune systems ability to fight off cancer.

Cytotoxic T lymphocytes (CTLs) are one of the cancer-destroying weapons used by the body to keep spontaneous tumors in check. These cells are alerted to the presence of tumor cells by a specialized class of cells called antigen-presenting cells (APCs), which display tumor antigens on their surface in the context of major histocompatibility molecules (MHC). With the assistance of T helper cells, APCs turn CTLs into search and destroy killing machines that actively seek out and destroy tumor cells displaying the relevant antigen. The problem with many cancers, however, is that tumor antigens are very often not presented correctly due to poor expression or problems with antigen processing.

Hearn Cho and his colleagues have addressed the problem of low levels of tumor antigen expression by designing a novel vaccine that employs a DNA sequence as an adjuvant to the tumor antigen. Their vaccine comprises a well-studied antigen, ovalbumin, covalently linked to DNA sequences containing cytosine/guanosine-rich motifs (CpG), which are known to be immunostimulatory. After injection into mice, the DNA-ovalbumin vaccine induces a remarkable level of CTL activity, specifically protecting mice against pre-established tumors expressing ovalbumin (but not other tumors). The vaccine appears to stimulate antigen presentation by MHC class I molecules at the cell surface (by an as yet unknown mechanism). As the protective effect does not require helper T cells, the approach may be useful in diseases such as cancer that are characterized by reduced or absent helper T-cell function.

In another approach, Wilfred Jefferies and his colleagues have focused on tumors that lack a protein, termed TAP (transporters associated with antigen processing), that normally shuttles antigens from the cytoplasm into a cellular compartment termed the endoplasmic reticulum (ER), where they are associated with MHC class I molecules. Positing that the reintroduction of the TAP1 molecule would restore the transport pathway and thereby increase tumor antigen presentation, Jefferies and his team engineered a vaccinia virus to contain the TAP1 gene and introduced it into mice bearing tumors deficient in TAP. As they had hoped, mice with TAP-deficient tumor cells infected with virus began to express TAP1, which stimulated CTL killing, and thus had a significantly higher survival rate compared with the control group. Because the TAP vaccine is not dependent on antigen type, the authors suggest it has potential as a general method for increasing immune responses against many different types of tumor.