R. Michael Linden
- ADJUNCT PROFESSOR Microbiology
B.A., State College of Aargau (Lehrerseminar)
B.S., University of Zurich
Ph.D., University of Zurich
Cornell University Medical College
Finally, our experience with the biology of AAV has placed us into an ideal position to engage in ?"translational?" efforts that are designed to contribute to the development of future therapies. Our approach is two-fold. Recently, we demonstrated that the chromosomal signals required for human site-specific integration are conserved in the mouse genome in a region corresponding to the human target site. Based on this finding we have begun to study AAV-mediated transgene targeting in mouse embryonic stem (ES) cells. The ES system will allow us to ask a range of new questions. Does wild type AAV integration affect the differentiation of ES cells into any or all of the lineages? Furthermore, the need for safe and efficient gene targeting of ES cells has grown since recent developments in stem cell biology have focused considerable attention on the use of cell-based therapies for the treatment of complex diseases. The success of such an approach, however, will require the ability to genetically modify stem cells ex vivo. Therefore, we deem it necessary to investigate whether AAV-based targeted insertion of a transgene into mouse ES cells diminishes concerns about insertional mutagenesis. While in differentiated cells the potential consequences of insertional mutagenesis are apparently negligible, in fast-dividing ES cells, lacking a G1 checkpoint, this concern needs to be addressed. Differentiation assays of ES cells, both in vitro (embryoid body system) and in vivo (transgenic mice), offer the possibility to investigate whether AAVS1 (the human target locus for AAV integration) represents a safe targeting site.
Possibly one of the most intriguing aspects of AAV biology is that it is the only known eukaryotic virus with the unique ability to integrate site-specifically into the human genome (Berns and Linden, 1995; Linden and Berns, 2000; Linden et al., 1996). On this background our laboratory has been active for a number of years in efforts to elucidate the molecular mechanisms underlying AAV2 site-specific integration and, related to this mechanism, DNA replication (Ward et al., 2003; Ward et al., 2001; Ward and Linden, 2000). We have approached these questions from different angles, including the genetic characterization of the human target locus for site-specific integration (Dutheil et al., 2000; Dutheil et al., 2004), the biochemical characterization of the AAV Rep proteins that are responsible for all aspects of the AAV life cycle, including site-specific integration (Smith et al., 1999; Yoon et al., 2001; Yoon-Robarts et al., 2004; Yoon-Robarts and Linden, 2003) and, more recently, the biophysical/structural basis for Rep action (James et al., 2004; James et al., 2003). These efforts have led us to be among the first to define the structure of SF3 helicases, and, as a result, to conclude that these proteins that are frequently found in viruses such as papilloma and polyoma viruses, in fact belong to the AAA+ proteins, a broad family of ATPases that are associated with a variety of functions ranging from membrane fusion, protein degradation and now also functions that are relating to several viral mechanisms. These include, but are not limited to DNA replication and genome packaging. Based on these findings we are now actively engaged in dissecting the biochemical and structural determinants underlying the molecular mechanisms supported by these viral AAA+ (vAAA+) proteins.
In addition, these assays will allow us to study if this chromosomal context permits optimal transgene expression in various lineages and tissues. Once established, this system will allow us to test a variety of lineage specific as well as regulated promoters for their activities within this particular chromosomal environment. In extension of these studies we have also engaged in exploring gene targeting strategies in the to us available human ES cells.\r\n\r\n
During the past years we have spent considerable efforts in establishing a viral vector core that generates and purifies recombinant AAV vectors followed by stringent quality control assessments. At this point these vectors are distributed to a range of collaborators that are actively engaged in gene transfer experiments. These efforts are in support of a multidepartmental translational Mount Sinai program that includes investigators from the Neurobiology, Neuroscience, Psychiatry and Ophthalmology Departments. In addition, gene transfer collaborations include studies on pancreatic islet transplants, liver-mediated gene delivery for a number of monogenic diseases such as lysosomal storage diseases, a program project that is aimed at the developmental aspects of kidney disease as well as several additional exploratory projects. The underlying philosophy to our efforts is to provide our strength to programs and projects that are founded on long-term and in-depth experience in the disease and animal models by our preclinical and clinical collaborators.
In summary, our ongoing studies on the biology of viruses and AAV in particular has provided us with the opportunity to study unique viral and cellular mechanisms and to become part of the efforts in developing strategies that might ultimately become components of future gene and cell-based therapies.
The study of adeno-associated virus (AAV) has brought our laboratory to the intersection of basic virological, genetic and biochemical studies with translational efforts, both in the gene transfer arena and the newly evolving stem cell discipline. In addition, we have committed considerable efforts to the establishment of an AAV vector core, with particular emphasis on scientific and organizational issues that allow for an efficient and appropriate process in providing recombinant AAV to a number of collaborating laboratories.\r\n\r\n
\r\nThe biology of AAV and AAV-based vectors
Adeno-associated viruses (AAVs) have been studied since the early 1960s. In contrast to other human DNA viruses it has become clear that there is no significant correlation between the widespread infection by AAV throughout the population and any known disease entity.
A, Structure-based sequence alignment of representative members of the SF3 family around the B, B?? and C signature motifs. The catalytic residues are shown inred; B?? motif is boxed in orange; the extension to the B?? motif is boxed in yellow. AAV2_Rep: adeno-associated virus type 2 Rep; PPV_NS1: porcine parvovirus NS1; SV40_Tag: simian virus 40 large T antigen; MPOV_Tag: mouse polyomavirus large Tantigen; HPV1a_E1: human papilloma virus type 1a E1; BPV1_E1: bovine papilloma virus type 1a E1.\r\n\r\n
B, Ribbon representation of AAV2 Rep40 highlighting residues lysine404 and lysine 406. The colors used in this representation correspond to Figure 6A. The DNA-sensor loop acts to tether ssDNA binding to motor activity. As depicted in the Rep40 monomer (top), residues K404 and K406 (dark blue) reside on one of two beta- 5 hairpin loops that form the DNA-sensor loop, comprised of the B?? motif. This loop transitions directly into the sensor-1 motif (yellow), which is responsible for detecting the difference between bound ADP and ATP within the ATPase active site. The P-loop, the site of ATP hydrolysis, is also shown (orange). A modeled Rep40 dimer is also shown (bottom), demonstrating the planar arrangement of the pertinent lysine residues, K404 10 and K406.\r\n\r\n
Studies throughout the past several decades have led to an emerging view that AAV might have evolved a possibly optimal relationship with its host through a unique life style that allows the virus to only replicate in cells that are infected by other viruses, which by themselves are deleterious to the host cell. Through this dependency AAV might have overcome an apparent challenge to viral life cycles in general: on one hand viruses depend on their respective hosts for replication, on the other, most viruses hurt the hosts through their replication to various degrees. Through its dependency, AAV will only replicate in cells that are affected by the consequences of helper virus infection. Thus, if our findings from tissue culture studies can be extrapolated to the human host, infection by AAV could indeed be viewed as beneficial to the host in that cells that are infected by adenovirus, herpes viruses and possibly papilloma viruses will die as a result of AAV replication. In light of this aspect it is no surprise that the AAVs appear widespread throughout the vertebrate kingdom.
Ward P, Falkenberg M, Elias P, Weitzman M, Linden RM. Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system. J Virol 2001 Nov; 75(21): 10250-8.
Ward P, Linden RM. A role for Single-Stranded Templates in Cell- Free Adeno-Associated Virus DNA Replication. J Virol 2001; 74(2): 744-54.Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.
Nony P, Tessier J, Chadeuf G, Ward P, Giraud A, Dugast M, Linden RM, Moullier P, Salvetti A. Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences. J Virol 2001 Oct; 75(20): 9991-4.
Linden RM. Gene therapy gets the Beauty treatment. Nat Biotechnol 2002 Oct; 20(10): 987-8.
Ward P, Elias P, Linden RM. Rescue of the adeno-associated virus genome from a plasmid vector: evidence for rescue by replication. J Virol 2003 Nov; 77(21): 11480-90.
James JA, Escalante CR, Yoon-Roberts M, Edwards TA, Linden RM, Aggarwal AK. Crystal structure of the SF3 helicase from adeno-associated virus type 2. Structure (Camb) 2003 Aug; 11(8): 1025-35.
Yoon-Roberts M, Linden RM. Identification of active site residues of the adeno-associated virus type 2 Rep endonuclease. J Biol Chem 2003 Feb; 278(7): 4912-8.
Dutheil N, Yoon-Roberts M, Ward P, Henckaerts E, Skrabanek L, Berns KI, Campagne F, Linden RM. Characterization of the mouse adeno-associated virus AAVS1 ortholog. J Virol 2004 Aug; 78(16): 8917-21.
Yoon-Roberts M, Blouin AG, Bleker S, Kleinschmidt JA, Aggarwal AK, Escalante CR, Linden RM. Residues within the B' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2. J Biol Chem 2004 Nov; 279(48): 50472-81.
James JA, Aggarwal AK, Linden RM, Escalante CR. Structure of adeno-associated virus type 2 Rep40-ADP complex: insight into nucleotide recognition and catalysis by superfamily 3 helicases. Proc Natl Acad Sci U S A 2004 Aug; 101(34): 12455-60.
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Dr.Linden is not currently required to report Industry relationships.
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