Abstract
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Science Wire
Clathrin-Mediated Cell Entry Is Needed for Efficient rAAV Transduction
Recombinant adeno-associated viral (rAAV) vectors bear unquestionable potential for gene therapeutic applications, as rAAV is capable of mediating persistent expression of transgenes while generally lacking toxicity. Knowledge of AAV biology has burgeoned over the past two decades, making rational vector design emerge as an attractive strategy for targeting specific tissues.
Heparan sulfate proteoglycan (HSPG) is the primary receptor for the most widely studied AAV serotype, AAV2. On cell surface binding, integrin coreceptors are recruited and AAV2 entry occurs by clathrin-mediated endocytosis. The residues responsible for HSPG binding to AAV2 are known, and their disruption by engineered expression of peptide ligands represents one instance in which capsid modifications have been successfully exploited to detarget vector from its typical accumulation sites. 1
One interesting finding is that certain peptide insertions into the HSPG-binding site can also restore binding to HSPG through charge interactions. In the journal Gene Therapy, Uhrig and colleagues describe potential differences in intracellular processing of AAV in identified peptide insertion mutants that differ in HSPG-binding capacity. 2 Intriguingly, the article reports that HSPG ligand binding at the cell surface influenced the endocytic route by which AAV2 is internalized. Mutants incapable of binding to HSPG entered the cell in clathrin-coated pits, whereas HSPG-binding mutants entered a variety of cell types independently of clathrin or caveolin. Unexpectedly, only vector entering via clathrin pathways achieved efficient transgene expression levels. Mechanistically, these data suggest that AAV2 might not properly escape from late endosomes when entering in this “unconventional” manner.
The finding that employment of a specific cellular entry route might be necessary for proper rAAV transduction highlights how crucial it is to consider that the successful development of efficient targeting vectors will require a thorough understanding of not only binding and entry, but of all the subsequent steps of AAV processing, nuclear translocation, and eventual gene expression. (rmt)
References
Mending a Broken Heart
By Frank Jacobs, Ph.D. (Science Wire Contributing Author)
In a report published by the journal Nature (Smart et al., 2011), an international group of scientists reported their discovery of an endogenous protein that can activate resident stem cells within the heart to regenerate damaged muscle tissue.
Coronary artery disease and concomitantly, heart failure, remain a leading cause of death and morbidity in the United States and in many countries throughout the world. Heart muscle cells, or cardiomyocytes, are irreparably damaged by a heart attack. Unfortunately, these cells are not capable of generating de novo heart muscle tissue. For the heart to maintain proper function, these damaged cardiomyocytes must be replaced. Several studies have attempted to regenerate the heart muscle via injections of various types of embryonic or adult stem cells, although results have been limited or inconsistent (Janssens, 2011). To overcome these limitations, Dr. Paul Riley, a British Heart Foundation researcher at University College London, has focused on the identification of resident stem cells within the injured myocardium. Indeed, during embryonic development, the epicardium, a thin layer of cells surrounding the outer perimeter of the heart, contains a potent population of cardiac stem cells (so-called embryonal epicardial stem cells or EPDCs) that gives rise to different lineages in the adult heart. Unfortunately, in the adult heart these cells reside in a dormant state and remain largely inactive. In a previous study, Dr. Riley had shown that thymosin β4 (Tβ4), a protein found in many tissues and that regulates cell structure and mobility, can reactivate these adult heart stem cells and stimulate the formation of new blood vessels after myocardial infarction in mice (Smart et al., 2007).
In the current study, the authors started by injecting healthy mice with Tβ4 every day for 1 week, after which an artery on the heart was surgically closed in order to mimic a myocardial infarction. Mice survive this procedure, making it possible to study the way their hearts respond to Tβ4 treatment. Although mice that did not receive Tβ4 did show increased activation of resident stem cells within the myocardium, the effect was modest and the activated cells did not initiate a cardiogenic program. In contrast, in the mice infused with Tβ4, the heart progenitor stem cells became activated, after which they migrated from the epicardium to the area of damaged muscle tissue and regained functional characteristics of mature cardiomyocytes. As a result, Tβ4-treated mice showed a significant reduction in myocardial damage and major improvements in heart function when compared with controls. These observations underscore the potency of endogenous cardiac stem cells in the regeneration of the myocardium. Further studies aimed at delineating the underlying mechanisms by which Tβ4 primes these stem cells are ongoing. The authors thus conclude that the identification of a bona fide source of myocardial progenitors is a significant step toward resident cell-based therapies for acute myocardial infarctions in human patients.
References
Finally, a T Cell Vaccine That Can Control SIV Infection
By Roberto Calcedo, Ph.D. (Science Wire Contributing Author)
Failure to prevent and control HIV infection in the STEP and other high-profile clinical trials, using adenovirus 5/HIV or DNA/HIV vaccines that induce strong HIV-specific T cell responses, has redirected efforts from developing T cell-mediated vaccines to developing therapies to block HIV infection. Several groups have already reported a new generation of effective and broadly reactive HIV-neutralizing antibodies.
A study from the group of Dr. Louis J. Picker at the Oregon National Primate Research Center, published in Nature (Hansen et al., 2011), has given a refreshing push to the development of HIV T cell-mediated vaccines. The group, using rhesus cytomegalovirus (RhCMV) as a vector in a prime–boost regimen in RhCMV+ rhesus macaques, demonstrated stringent control of highly pathogenic SIVMAC239 infection after mucosal challenge. Residual SIV infection was barely detectable in gastrointestinal and associated lymph nodes, peripheral lymph nodes, and other hematolymphoid tissues for more than 1 year after SIVMAC239 challenge. The data indicated that the tight control observed in ∼50% of the vaccinated monkeys was due to the high frequency of SIV-specific CD8+ T cells with an effector memory phenotype that are indefinitely maintained in mucosal portals of entry and potential sites of distant viral spread, protecting without anamnestic expansion. A control group of rhesus macaques vaccinated with DNA/human AdV5 developed an SIV-specific central memory CD8+ T cell response that required expansion to show effector function and was not able to control SIV infection. This study reopens the door for T cell vaccine development against HIV/AIDS and presents a new and powerful approach using CMV vector as a vaccine carrier.