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The Giovanni Armenise-Harvard Foundation Session 2: Biomedical Research Overview As chairman of the Foundation's Scientific Advisory Committee, one of Dr. Peter Howley's responsibilities is to shape the program for the annual symposium. As he organized individual abstracts to create this year's sessions, Dr. Howley discovered that some of the cutting-edge investigations sponsored by the Foundation aren't easy to categorize. He gathered four of them together for this session, which he dubbed Biomedical Research. The opening presentation dealt with viral vectors for gene therapy, followed by a tour of the enzymatic assembly lines that microbes use to grow antibiotics. The third speaker introduced the first mammalian mutation gene that appears to increase stress resistance and extend lifespan, and the fourth took his listeners inside a "chamber of doom" where proteins are destroyed. As disparate as these topics appear, Dr. Howley said that "they have a similar theme: all are in areas of research that interest me."
Penetrating cells is a do-or-die proposition for viruses, which can't replicate until they've gotten inside host cells. Researchers who are mindful of this special viral skill are seeking to use them as vectors for gene therapy or vaccines. Many viruses, however, aren't well suited to this task because they can only enter dividing cells, they don't penetrate very many cells, or they don't express the transfected gene at high enough levels. To get around these limitations, Dr. Naldini and his colleagues have designed hybrid lentiviral vectors capable of transferring and expressing genes in several rodent tissues in vivo, and in primitive human hematopoietic stem cells ex vivo. They have accomplished this by combining core elements of HIV-1, the pathogen that causes AIDS, with the envelope of a less harmful lentivirus called vesicular stomatitis virus (VSV). The safety profile for these vectors has improved as the researchers cut back on the amount of HIV genetic material they use, they've increased transgene expression through selective use of HIV ltr (long terminal repeat) and packaging signals, and VSV elements permit entry into a variety of cell types. Dr. Naldini's latest and safest vectors inactivate upon transduction and include only a minimal set of HIV genes. In earlier experiments, his group demonstrated efficient delivery and sustained expression of marker genes by these vectors, both in vitro with human donor lymphocytes and in vivo when injected into the brains of adult rats. More recently, Dr. Naldini has been working with a mouse model for metachromatic leukodystrophy (MLD), an inherited liposomal storage disorder that in infants causes rapid, dramatic death as lipids accumulate in the central nervous system and other major organs. His team designed a hybrid lentivirus vector carrying the gene for ASA, the enzyme that is lacking in this condition, which they are testing in two ways. When the vector is injected directly into the brains of MLD mice, there is preliminary evidence that the transgene appears to express well and in a stable fashion. A second set of experiments involves ex vivo reconstitution of hematopoetic stem cells with the transgene, which the researchers speculate will repopulate and replace the missing enzyme in animals. Early results appear promising, and additional research is underway.
Because they live in a bug-eat-bug world, funguses and other microbes have evolved thousands of antibiotics that they use to disable their enemies and competitors. Because many of these substances disrupt only the functions of procaryotic cells, leaving eucaryotic cells alone, doctors use them as invaluable weapons against microbes that cause human disease. All are made by a process that Dr. Walsh calls "assembly line enzymology." His laboratory seeks to understand the molecular logic of antibiotic assembly and "the flip side, by which bacteria produce nonribosomal peptide virulence factors, which allow them to infect you and me." Ultimately, the goal is to use combinatorial elaboration to assemble new antibiotics, block virulence factors, and fight growing problems of antibiotic resistance. At a gestalt level, polyketide antibiotics such as erythromycin and tetracycline and peptides like penicillins and vancomycin look nothing alike. Yet all are templated natural products where assembly instructions come from the domain order of giant megasynthases. These molecules have "way stations," modules that enzymes, such as polyketide or nonribosomal peptide synthetases, use to initiate, elongate, and terminate the natural product chains. At some of these way stations, Dr. Walsh and his colleagues have been able to substitute one module for another, an accomplishment that could lead the way to combinatorial biosynthesis of new antibiotics. If investigators can build a parts list for the antibiotic assembly line, Dr. Walsh predicts that it will be possible to swap modules around and create antibiotics that have not yet been made in nature, but which might be powerful weapons against resistant bacteria.
Although gene mutations that extend lifespan and enhance resistance to environmental stresses such as ultraviolet (UV) light or reactive oxygen species have been identified in C.elegans and other invertebrates, no such genes are known in mammals. In this presentation, Dr. Migliaccio announced that she and her colleagues have found a mutation in the mouse p66shc gene that appears to have such properties. This gene is a splice variation of p52shc/p46shc, a cytoplasmic signal transducer involved in the transmission of mitogenic signals from tyrosine kinases to the Ras oncogene. Unlike p52shc/p46shc, which are known to cause malignant cell changes, p66shc fortunately does not transform fibroblasts. The researchers created a p66shc knockout mouse that retained p52shc/p46shc, then in vitro subjected cells from that mouse to UV stress. After four days, wild-type cells were all dead, whereas the cells with the deletion were alive. Additional in vivo experiments showed the knockout mice to be more resistant to paraquat-induced oxidative stress than wild-type mice. The researchers suspect that p66shc is part of a signal transduction pathway that regulates oxidative stress response, and that hypothesize that disrupting this pathway will protect against this well-known cause of aging. Further support comes from an observational study, in which a group of mice with a double p66shc deletion outlived those who were missing one copy and those with two normal copies of the gene.
For cells, an important part of viability is the prompt and appropriate degradation of intracellular proteins. In mammalian cells, large structures called proteasomes degrade proteins that have been marked for destruction by ubiquitin. Some of the fragments that emerge from this process are converted to amino acids; others are antigenic peptides that trigger cytolytic T-cell activity after presentation on MHC-class 1 molecules. Unlike typical proteases, mammalian 20S and 26S proteasomes degrade proteins in a highly processive fashion that Dr. Goldberg describes as a "bite-chew" model. Once a protein substrate has been labeled by ubiquitin, an ATPase shepherds it into the proteasome's central "chamber of doom," where it will be unfolded and methodically chopped into small pieces. Each molecule is completely chopped up before the proteasome moves on to the next. Dr. Goldberg and his colleagues were surprised to find that all the products of this process are the same size, whether they started out as small polypeptides or big proteins, which they see as an indication that proteolysis continues until the products are small enough to diffuse out of the proteasome, then stops. About 99% of these fragments are smaller than 25 residues, with most in the 3-20 residue range. Only 10-15% of the products are 8-9 residues in length, the size required for MHC-class-1-presentation. Eukaryotic 20S proteasomes contain active sites that cleave proteins in three distinct ways: two cut like chymotrypsin, two like trypsin, and two like caspase. The researchers were surprised to find that instead of acting independently, chymotripsin appears to "bite" the substrate first, which initiates "chewing" by the other processing sites. Caspase substrates signal chymotripsin when it's time to take the next bite. The resulting process is a highly efficient method for destroying abnormal proteins and for alerting the immune system to the presence of viruses and other undesirables. In the future, proteasome inhibitors may hold promise as treatments for cancer and other human diseases. Symposium Topics
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New generations of lentiviral vectors for experimental and human gene
transfer
