From 2010.igem.org

Therapy using viral vectors is an promising approac. In an first step the plasmids of the AAV-2 Helper-free System were genetically modifyed by converting it into BioBricks and inserting of targeting molecules into the constructs. These plasmids were then used to transfect the producer cell line AAV-293. After an incubation of three days the viral vectors were harvested and used to transduce different target cells. The succesful transduction can then for example be measured by detecting the fluorescence of fluorescent proteins in the target cells

The majority of the modifications that were introduced into the viral vector aimed to allow differential targeting of tumor cell over healthy off-target cells.

Layers of specificity

Employment of viral vectors for means of therapy is idea in the context of personalized medicie that gets more and more interest. In such applications the reduction of side effects and the safety of the patient in general is of the highest priority.

In order to satisfy this requirement we designed our Therapy Vector with several layers of Specificity:

The targeting of the viral vector towards the desired target cell (e.g. tumor cells) is the basic idea behind the emplyment of viral vectors for therapeutical means. There for the natural tropismn has to be knocked down and a desired tropism has to be introduced that allows differential targeting of pathological but not of off-target cells. To fulfill this mission our Virus Construction Kit offers you different solutions.

Off-target cells that were transduced by mistake can be preserved from an undesired therapy effect when the therapeutic gene is controley by a tissue specific promoter. For this mean a promoter has to be used that is as specific for the pathological tissue as possible. We included the human telomerase promoter (phTERT) which is often activated in tumor cells and is there for able to allow differential experssion of a therapeutic geneproduct in pathological cells.

For reasons of safety Therapeutic vector do not directly trigger appoptosis in the successfully targeted cells. To include one further layer of specificity and safety we decided to arm our therapy vector with different prodrug convertases. Neither the single application of the harmless prodrug nor the single expression of the convertase has a noteworthy effect of the transduced cell. Only in cells that express the prodrug convertase and have a sufficient cytoplasmatic concentration of the belonging prodrug apoptosis is triggered. This dependency of the therapy on a prodrug can be employed to protect tissues or other persons that could come in contact with the therapeutical vector. This aspect was specially inportant for the development of a viral vector that is able to infect humans in the context of a undergraduate project for the iGEM competition. Therefor this approach gained our preference over other possibly equivalent arming possibilities described in the tumor therapy with viral vectors.

Introduction to Adeno-Associated Virus Serotype 2

Biology of the AAV-2

Genomic organization

Figure 1: Genomic organization of the wt-AAV-2. The inverted terminal repeats (ITRs) flank the two open reading frames (ORFs). The four-nonstructural proteins encoded from the rep gene are driven by the p5 and p19 promoters, whereas the structural Cap proteins are regulated by the p40 promoter. Additionally, the Assembly Activating Protein (AAP) was found recently within the cap gene.

The Adeno-associated virus serotype-2 (AAV-2) genome is a linear, single-stranded (ss) 4675 bp DNA virus. Due to its small size, gene genomic organization is condensed and gene regulation is complex. The viral nucleotide sequence consist of two open reading frames (ORFs) coding for Rep- and Cap proteins and are flanked on either side by identical inverted terminal repeat (ITR) structures which are palindromic and form hairpin structures (Srivastava et al. 1983).

The ITRs serve as primers for the host cells’ DNA polymerase, which converts the single-stranded virus genome into double-stranded DNA (ds DNA) as a part of the viruses’ replicative cycle. They also play important roles in viral genome integration into and rescue from the hosts genome, the formation of concatamers in the host cell nucleus and encapsidation of the viral genome into preforemd capsids (Berns 1990). Due to these essential functions, the ITR structures cannot be deleted from a viral vector and need to be delivered in cis.

Organization of the Inverted Terminal Repeat Structure

The inverted terminal repeat structures can be subdivided into several palindromic motives: A and A’ form a stem loop which encases B and B’ as well as C and C’. Those motives form both arms of the T-shaped structure. The functional motives on the ITR are two regions that bind Rep 68/78, called Rep-binding elements (RBE on the stem and RBE’ on the B arm) and the terminal resolution site (trs) in which the rep proteins introduce single-stranded nicks. The 3’ OH end of the A motive acts as a primer for DNA replication (Im & Muzyczka 1990) (Lusby et al. 1980).

Figure 2: Organization of the ITRs, which are the only cis-required element in viral genome integration and replication.

Replication

The 3’ OH end of the viral DNA folds onto itself as part of the inverted terminal repeat (ITR) structure and thus serves as a primer for elongation by the host cell’s DNA polymerase. The polymerases strand displacement activity unfolds the opposite ITR structure and elongation continues until the 5’ template end is reached (Lusby et al. 1980).

Figure 3: Figure 3: Schematic overview of AAV-2 replication.

The remaining hairpin structure that served as the origin of replication then acts as a target for the Rep 68/78 protein: It binds to the Rep-binding site (RBS) and unwinds the double-stranded DNA in a way that the terminal resolution site (trs) is being displayed in a single-stranded form on a stem loop. This enables the endonuclease catalytic domain of the Rep protein to introduce a nick of the parental strand at this site, which in turn serves as a new primer for DNA polymerase. The polymerase resolves the hairpin structure through strand displacement and copies the remaining end of the parental strand (Im & Muzyczka 1990) .

Sometimes, nicking does not occur after polymerases have partially copied the virus DNA. In this case, the newly synthesized 3’ end acts as a primer and the host cell’s DNA polymerase copies the whole sequence once again, displacing the ITR strands in the middle of the sequence. This leads to a dsDNA containing the whole virus genome twice, called a duplex dimer (DD). Those dimers can be resolved back to duplex monomers (DM) by the Rep proteins

After replication, the dsDNA separates again forming new ssDNA in (+) and (-) polarity with hairpin structures at its ends. The Rep 40/52 proteins are involved in this process. Newly synthesized copies are either encapsidated into virus capsids or replicated again (Gonçalves, 2005a). Double-stranded genomes are formed as well through annealing of (+) - and (-) single strands. Both mechanisms occur during infection and contribute to transgene expression (Schultz & Chamberlain 2008).

If the double stranded virus DNA exists in an episomal form inside the nucleus, it tends to form linear as well as circular concatamers, which are formed by ligation of duplex monomers(Schultz & Chamberlain 2008).

Viral promoters

Three viral promoters are coordinating gene expression in the wildtype AAV-2. Each promoter regulates different open reading frames (ORFs) of regulatory proteins (p5 and p19 promoter) and structural proteins (p40 promoter). A general overview is provided inError! Reference source not found.. p5 and p19 promoters are repressed in absence of helper proteins provided by Ad or HSV whereas transactivation of p5 and p19 occurs in presence of helper viruses. Furthermore, the larger Rep proteins activate the p40 promoter. Since overexpression of Rep78 leads to cell cycle arrest, high levels of Rep78/68 lead to repression of the p5 promoter.

Figure 4: Regulation of the viral promoters located within the t AAV-2 genome. In the absence of helper viruses, gene expression is suppressed, whereas activation of p5 and p19 occurs in the presence of helper proteins by interacting of Rep proteins with cellular and helper proteins.

p5 promoter

The p5 promoter, located downstream of the rep and cap ORF (Figure 5: The p5 promoter of the wtAAV-2 is located upstream of the rep and cap ORF and contains several elements, which interact with Rep and endogenous proteins.)of the wtAAV-2, regulates gene expression of the two larger non-structural proteins Rep 78 and Rep 68 that are essential in genome replication and viral genome integration into several hotspots of the human chromosome.

Several binding elements for cellular and viral proteins involved in regulation can be found in the p5 promoter (Figure 5) therefore playing an important role in gene transcription, integration and replication, dependent on the presence or absence of helper viruses such as adenovirus (Ad) or herpes simplex virus (HSV) (Murphy et al. 2007). Besides regulation of gene expression, the p5 integration efficient element (p5IEE) containing the rep binding element (RBE) and a terminal resolution site (trs) is responsible for mediating site specific integration into the human genome (Philpott et al. 2002).

Figure 5: The p5 promoter of the wtAAV-2 is located upstream of the rep and cap ORF and contains several elements, which interact with Rep and endogenous proteins.

Containing two consensus sequences for binding immediate early E1A gene product from adenoviruses (Chang et al. 1989), p5 promoter is transactivated in the presence of helper viruses whereas suppression occurs in absence of adenoviral proteins by low levels of Rep proteins (Beaton et al. 1989). Regulating of Rep78/68 by its negative feedback loop is critical since overexpression leads to cell cycle arrest in the S-phase (Berthet et al. 2005) and suppression of cellular promoters (Jing et al. 2001).

p5 TATA-less promoter

In contrast to the natural location of the p5 promoter, the iGEM team Freiburg 2010 provides the RepCap plasmid with a relocated p5 promoter downstream of the RepCap genes (Figure 6). Additionally the p5 promoter lacks the TATA box element (AVIGEN 1997). Those modifications result in an attenuated expression of the larger Rep proteins therefore leading to normal transcription of the Rep proteins driven by p19 promoter and enhanced expression of the Cap proteins, which are under the control of the p40 promoter. Additionally, removing the p5 promoter downstream of the RepCap genes and deletion of the TATA box eliminates contamination with wtAAVs. Hence, alteration of the p5 promoter is useful for enhanced production of recombinant viral particles attenuating repression of Rep78/68 and improving gene transcription of the capsid proteins and Rep proteins involved in genome packaging.

Figure 6: p5 TATA-less promoter is located downstream of the rep and cap ORF.

p19 promoter

p19 promoter drives gene expression of the smaller Rep proteins Rep52 and Rep40. In absence of a helper virus infection the promoter is inactive by repression of all four Rep proteins, but is transactivated by interaction of both the Sp1 site and Rep protein Rep78/68 bound to the Rep binding element (RBE) (Lackner & Muzyczka 2002). By forming a DNA loop (Pereira & Muzyczka 1997) and bringing the two promoters in proximal distance () additional cellular factors bound to p5 promoter interact with the p19 promoter leading to transcriptional activation of Rep52/40 (Lackner & Muzyczka 2002).

Figure 7: Forming of the DNA loop brings the p5 rep binding element in proximal distance to the Sp1 site foud in the p19 promoter.

p40 promoter

The P40 promoter is derived from the adeno-associated virus serotype 2 (AAV2) genome, where it is located at 40 map units. It regulates the transcription of the capsid proteins VP1, VP2 and VP3(Labow, Hermonat, & Berns, 1986; Cassinotti, Weitzand, & Tratschin, 1988).

Several sequence regions have been identified to be important for maximal promoter activity: Two Sp1 sites, which are located 250 (Sp1-50) and 270 (GGT-70) base pairs upstream of the transcriptional start point and to which Sp1 or Sp1-like proteins bind (Pereira & Muzyczka 1997).

Referring to the virus genome, p40 can also be induced through transactivation by the Rep proteins. The Sp1-50, together with the CArG-140 site of the P19 promoter, are the main elements involved in this process. The Rep proteins, which bind to the Rep binding element in the terminal repeat or the P5 promoter, can induce P19 or P40 by interaction with their bound Sp1 proteins thereby forming a DNA-loop (Pereira & Muzyczka 1997). In addition to that, the TATA box, located at 230, is also required for P40 activity. Furthermore the ATF-80 and the AP1-40 elements are also important for maximal promoter induction (Pereira & Muzyczka 1997).

Integration

The AAV-2 is the only known mammalian virus that integrates into a specific location in the human genome in the presence of Rep78/68: Chromosome 19, 19q13.3-qter. The site of integration was termed AAVS1.

The mechanism of Rep-mediated integration into AAVS1 is not yet completely understood and seems to be imprecise and variable. Deletions or insertions often occur in the integration process(Schultz & Chamberlain 2008). Linden et al. (1996) proposed a mechanism that is consistent with the observed patterns in which AAV exists in an integrated form:

AAVS1 bears a Rep-binding site (RBS) which is similar to the RBE in the virus genomes ITR. The Rep proteins are able to simultaneously bind to AAVS1 and the viral RBE, thereby bringing both strands into close proximity towards each other. After binding to the AAVS1 site, Rep acts as an endonuclease, the same way it does when binding to the AAV ITR, introducing a single strand-nick between two thymidine residues close to the binding site. This produces a free 3’-OH end which acts as a primer for the host cells’ DNA polymerase. After replicating the displaced strand, the polymerase switches templates and replicates the AAV DNA, thereby linking AAVS1 and AAV together. Prior to integration, the AAV genome often exists in circular and/or concatameric form, resulting in multiple consecutive AAV copies in the host genome. Another explanation for this phenomenon could be a circularized AAV monomer that is being replicated several times in a rolling-circle manner before being integrated into the host genome.

Another template switch back to the AAVS1 sequence creates a second link between virus and host. This integration mechanism leaves single-stranded gaps that need to be repaired by cellular enzymes before integration is complete. Since successful integration of AAV depends on these cellular repair mechanisms, integration happens more frequently in dividing cells, in which repair functions are more active.

Recently, a sequence in the p5 promoter region that enhances site-specific integration through interaction with Rep78/68 has been identified, this motif was labeled p5 integration enhancer element (p5IEE). Apparently, p5IEE is sufficient to create the AAVS1 - Rep68/78 - Viral DNA -complex necessary for specific integration, even if the ITRs containing RBEs are not present.

Rescue

If the latently infected host cell is superinfected with adenovirus, the integrated virus genome can be rescued from the human chromosome and proceed its lytic lifecycle. Adenovirus gene products act as activators on AAV gene expression, leading to an excision of the viral sequences. Like in the integration process, the Rep 78/68 proteins catalyze the excision by introduction of single strand nicks at the terminal resolution sites within the terminal repeat structures flanking the AAV genome. DNA polymerase, displacing the single-stranded AAV sequence, then elongates the resulting free 3’ OH ends. The incomplete single-stranded AAV sequence missing one terminal repeat primes upon itself at the homologous D motives, allowing DNA polymerase to copy it. This results in full-length, single-stranded AAV molecules, which are being able to re-enter the replicative cycle (Srivastava 2008), (Samulski 1993).

Rep proteins

Overview

The Adeno-associated virus (AAV) consists of two open reading frames (ORF), rep and cap ORF. The four non-structural rep genes are driven by two promoters located at map units 5 (p5 promoter) and 19 (p19 promoter). Rep proteins are involved in genome encapsidation, regulation of gene expression and replication of the viral genome.

Figure 8: Genomic organization of the AAV-2 genome. The rep gene codes for four non-structural proteins – Rep40, Rep52, Rep68 and Rep78 – which are involved in gene regulation, genome encapsidation and viral DNA integration.

The two larger proteins Rep78/68 play an essential role in viral genome integration and regulation of AAV gene expression, whereas the smaller Rep proteins are involved in viral genome encapsidation. Rep proteins act both as repressors and activators of AAV transcription in respect to the absence and presence of helper viruses such as adenoviruses (Ad) or herpes simplex viruses (HSV) by interacting with several cellular proteins (Nash et al. 2009).

Furthermore, in the absence of Rep proteins, as it is the case in recombinant AAVs, integration of the viral genome into the human genome is rare and random. There are several hotspots for integration of wtAAV genomes such as the human chromosome 19q13.42, known as the AAVSI site, but as well some other accessible chromatin regions for preferred integration have been found (5p13.3 and 3p24.3). Integration into the human genome is mediated by the two regulatory proteins Rep68 and Rep78 driven by the AAV p5 promoter. The proteins bind to the Rep binding site (RBS) which is located within the inverted terminal repeats (ITRs). The minimal consensus Rep binding site (RBS) GAGT GAGC is found within the ITRs and in the p5 integration-efficient element (p5IEE) of the p5 promoter (Hüser et al., 2010). Rep78/68 proteins possess DNA-binding (reference), helicase (reference) and site-specific endonuclease activity located within the first 200 amino acids (Davis et al. 2000). Since the N-terminal region is unique to the larger Rep proteins, the two smaller Rep proteins possess other biological functions. Rep52/40 gene expression is driven by the p19 promoter which is located within rep ORF and the proteins are involved in encapsidating the viral genome into the preformed capsids. Gene expression of these proteins is suppressed in absence of adenovirus infection by binding of Rep78/68 to the p5 promoter. Gene expression of p19 and p40 is transacvtivated by the Rep proteins Rep78/68 during coinfection.

Rep 78

Rep78 in a nutshell:

• 78 kDa

• Endonuclease activity

• ATPase and helicase activity

• Regulate viral gene expression

• Involved in genome integration into human chromosome

Regulated by the p5 promoter, Rep78 is the largest non-structural protein found in the wtAAV. Besides regulation of gene expression and viral genome replication, Rep78 has been found to play a functional role in AAV site-specific integration into the human genome (Hüser et al. 2010). In absence of Ad helper viruses, overexpression of Rep78 leads to cell cycle arrest by interacting with cell-cycle regulating phosphatases causing DNA damage by its intrinsic endonuclease activity (Berthet et al. 2005) and induces apoptosis. Due to its ability to bind to the Rep binding site (RBS) in the p5 integration-efficient element (p5IEE) of the p5 promoter, Rep78 mediates gene expression and retain a constant level of Rep proteins by suppressing transcriptional activity of the p5 promoter in absence of Ad viruses (Yue et al. 2010). Interaction of Rep78 with cellular factors such as transcription factors (Lackner & Muzyczka 2002) provides the basis for gene regulation by Rep78 in associated with endogenous molecules.

Rep 68

Rep68 in a nutshell

• 68 kDa

• Endonuclease activity

• ATPase and helicase activity

• Regulate gene expression

• Involved in genome integration into human chromosome

Rep68 is a regulatory protein driven by the p5 promoter with an apparent molecular weight of 68 kDa lacking 92 amino acids from the carboxy terminus due to splicing of mRNA coding for the two larger Rep proteins.

The non-structural protein Rep68 belongs to the superfamily 3 (SF3) helicase found in other small DNA and RNA viruses such as simian virus 40 (SV40) and bovine papillomavirus (Mansilla-Soto et al. 2009). Formation of oligomeric complexes of Rep proteins provides the basis for the functional versatility of the two larger regulatory proteins. The AAA⁺ motor domain is known to function as an initiator for oligomerization of the Rep proteins. The cooperative effect of both domains appears to be further regulated by ATP binding as well as different DNA substrates such as dsDNA and ssDNA. Assembly of different nucleoprotein structures suggest that viral replication and genome integration is regulated and controlled by distinct Rep complexes which means that in presence of dsDNA Rep68 assembles to smaller complexes than in presence of ssDNA resulting in octamers.

Rep52

Rep52 in a nutshell

• 52 kDa

• ATPase and helicase activity

• Involved in genome encapsidation

Rep 52 is under the control of the p19 promoter and shares the same N-terminus with Rep78. It was shown that Rep52 possesses helicase and ATPase activity with 3´-5´polarity (Smith & Kotin 1998). Despite the helicase activity, Rep52 and Rep78 share a putative zinc-finger domain, which suggest interactions with diverse cellular factors (Nash et al. 2009) such as transcription factors (Lackner & Muzyczka 2002) and TATA-binding proteins (Hermonat et al. 1998).

Rep40

Rep40 in a nutshell

• 40 kDa

• ATPase and helicase activity

• Involved in genome encapsidation

The smallest Rep protein (Rep40) possesses helicase and ATPase activity as well, but does not have strict requirements for DNA duplexes containing a 3´single-stranded end. Rep40 helicase activity requires bivalent ions such as Mg²⁺ or Mn²⁺ and is most active using ATP as substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires dimerization for functional helicase activity (Collaco et al. 2003). Rep40/52 proteins are required for translocation of the single-stranded, viral genomes into the preformed capsids proceeding with the 3´end of the DNA (King et al. 2001).

Figure 9: Crystal structure of the SF-3 helicase (PDB: 1SH9).

VP proteins

The AAV capsid consists of 60 capsid protein subunits composed of the three cap proteins VP1, VP2, and VP3, which are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of the VP proteins. VP1, VP2 and VP3 share a common C terminus and stop codon, but begin with a different start codon. The N termini of VP1 and VP2 play important roles in infection and contain motifs that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (NLSs). These elements are conserved in almost all parvoviruses. (Johnson et al. 2010a)

Beschreibung: Cap proteins.png

Figure 10 Genomic organization of the AAV 2 genome. The cap gene codes for the three capsid proteins VP1, Vp2 and VP3, which are responsible for the assembly of the capsid

VP1

Whereas VP1 is translated from the minor spliced mRNA, VP2 and VP3 are translated from the major spliced mRNA. The minor spliced product is approximately 10-fold less abundant than the major spliced mRNA. Thus, there is much less VP1 than VP2 and VP3 resulting in a capsid stoichiometric ratio of 1:1:10. The N terminus of VP1 has an extension of 65 amino acids including an additional extension of 138 N-terminal amino acids forming the unique portion of VP1. It contains a motif of about 70 amino acids that is highly homologous to a phospholipase A2 (PLA2) domain. Furthermore, there are nuclear localization sequences (BR)(+) which are supposed to be necessary for endosomal escape and nuclearentry. (Bleker et al. 2006), (Johnson et al. 2010b), (DiPrimio et al. 2008).

Phospholipases are enzymes that hydrolyze phospholipids into fatty acids and other lipophilic substances and can be found in mammalian tissues but also in insect and snake venom. They are subdivided into four major classes, termed A, B, C and D distinguished by the type of reaction they catalyse whereas the position of hydrolysis on the glycerol backbone defines the class of phospholipase.

Figure 11: The schematic depiction of some AAV2 domains. VP1 contains a phospholipase A2 (PLA2) domain and four basic regions (BR1–4) located at the N-terminus of VP1 – VP3. The HSPG binding domain is generated by the basic residues at positions R484, R487, K532, R585, and R588 which are located near the C-terminus of theVP proteins. The NGR motif 511–513 forms an integrin a5b1 binding domain. Adapted from (Michelfelder & Trepel 2009)

It specifically recognizes and hydrolyzes the sn-2 acyl bond of phospholipids releasing arachidonic acid and lysophospholipids.

Figure 12: adapted from http://www.biochemtech.uni-halle.de/im/1182353660_397_00_800.jpg

The reaction mechanism will be depicted by the following image:

Figure 13 adapted from “Interfacial Enzymology: The Secreted Phospholipase A2-Paradigm”

There are two possible mechanism but the one on the right side has a lower transient state. The mechanism of sPLA2 on the right side is initiated by a His-48/Asp-99/calcium complex within the active site. The sn-2 carbonyl oxygen becomes polarized by the calcium ion while also influencing catalytic water molecule, w5..Via the bridging second water molecule w6 His-48 improves the nucleophilicity of the catalytic water. According to propositions two water molecules are needed to bypass the distance between the catalytic histidine and the ester.. Asp-99 is thought to enhance the basicity of His-48 through hydrogen bonding. Substituting the His-48 with an asparagine maintains wild-typ activity because the functional group on asparagines can function to lower the pKa of the bridging water molecule, too (Berg et al. 2001).

The phospholipase A2 is suggested to mediate membrane disruption of the vesicular compartment, which would allow escape of the virion into the cytosol, although this has not been demonstrated for AAV so far.The propensity of cellular PLA2 to cleave phospholipids is usually regulated by intracellular Ca²⁺ levels as well as phosphorylation of residues near the catalytic domain of the PLA2. Not surprisingly, the N-terminus of VP1 contains a GXG binding site and several phosphorylation sites.

According to the structural modeling of VP1 the N-terminus can translocate through the 5-fold axis of symmetry in the capsid and expose the first 185 residues of VP1 (comment: experimental data also “strongly suggest that N-termini of VP1 harboring the PLA2 domain can be exposed on the capsid surface through the pores at the fivefold symmetry axes” (Girod et al. 2002)(Bleker et al. 2005a). It only takes about 19 amino acids to reach through a phospholipid bilayer so this length would be sufficient for the presentation of the NLS and di-lysine sequences to the cytosol assuming the PLA2 domain had penetrated through an endosomal membrane or Golgi. (Michelfelder & Trepel 2009)

Analysing individual steps in the life cycle of several VP1up mutants and wtAAV-2 lead to the following conclusions: (i) mutations in VP1up did not affect DNA packaging or replication but resulted in a strong reduction of infectivity; (ii) this decrease in virus infectivity correlated with a loss in pvPLA2 activity; (iii) binding to the cell surface and entry into cells was not affected in VP1up mutants; (iv) however, these mutants showed obviously reduced and delayed Rep expression. (Girod et al. 2002) Summarizing these results the pvPLA2 activity is required for a step in the life cycle of the virus following perinuclear accumulation of virions but (Girod et al. 2002) before the onset of early gene expression.

Maybe future work will uncover whether the PLA2 domain in AAV performs optimally in a specific vesicular compartment, prefers a specific phospholipid substrate, operates at multiple cellular membranes such as the endosome and the nuclear envelope, or if its activity is regulated by cellular components.

VP2

The translation of VP2 from the major spliced mRNA is less efficiently compared to the translation of VP3 because it initiates at a Thr codon (ACG). VP2 and VP1 have an extension at the N terminus that remains internal when exposing the capsid to experimental conditions like low pH or heat. The N terminus of VP1 has an extension of 65 amino acids and similar to VP1 it has two functional elements: a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). The exact role of VP2 remains unknown, although the protein is thought to be nonessential for viral assembly and infectivity (Johnson et al. 2010b), (DiPrimio et al. 2008).

VP3

Contained in VP1 and VP2, VP3 is the primary capsid protein that determines the surface topology of the AAV capsid. The capsid in turn dictates antigenicity and tropism. In comparison to the initiation of VP1 and VP2 the initiation of VP3 is because of a Met codon highly efficient (DiPrimio et al. 2008).

Beschreibung: VP1_2_3.png

Figure 14: Genomic organization of the AAV-2 genome. The three capsid proteins VP1, Vp2 and VP3 have a similar C terminus. The N termini of VP1 and VP2 contain a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).

Natrual Tropism and HSPG motif

The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the capsid surface (Trepel, Vectors, et al. 2009).

Beschreibung: Unbenannt

Figure 15: adapted from (Trepel, Vectors, et al. 2009). Schematic depiction of the 5 basic amino acids forming the HSPG motif: R484/R487, K532, R585/587. Other domains or binding motifs are not shown in this picture.

HSPG belongs to the glycosaminoglycanes as well as heparin and consists of heparan sulfate glycosaminoglycan attached to a core protein and can be found on every human cell surface.

Beschreibung: Unbenannt2

Figure 16: adapted from (Sinnis et al. 2007) amidosulfated disaccharid sequence in heparin.

Its acid residues bear negative charges and are therefore prone to electrostatic interactions with e.g. the positively charged HSPG binding motif of AAV-2. Other interactions with polar residues are possible, too.

Regarding AAV-2, two point mutations in AAV-2 (R585A and R588A) are sufficient to eliminate heparin binding (Opie et al. 2003). The biobricks with this knockout are annotated with „HSPG-ko“.

Assembly-activating protein

A gene encoding for the assembly-activating protein (AAP) was recently (in 2010) discovered in the Adeno-associated virus (AAV) serotype 2 genome. Its gene product is conserved among all AAV serotypes, illustrating its essential role in virus life cycle. Its functions comprise transport of the viral structural proteins to the nucleolus and involvement in following capsid assembly.

The AAP gene, located in the Cap coding region, is translated from an alternative open reading frame (ORF) with unconventional start codon. If modifications need to be introduced in the AAV capsid – for example for targeting approaches – the AAP has to be taken in account in order to prevent virus assembly impairment.

Trafficking

For creating efficient AAV2 vectors, precise knowledge of the events following virus transduction is necessary. The subsequent scheme and summary is intended to be an introduction into the complex process of virus transduction.

Figure 17: Adapted from (Hildegard Büning et al. 2008)

After several contacts with cellular structures like heperan sulphate proteoglycan (HSPG) the viral capsid proteins get rearranged. Clathrin-mediated endocytosis and cellular trafficking into thecell’scenter follows. After acidification and following endosomal escape, the viral genome is transferred into the nucleus and replicated (lytic phase) or integrated into the host genome (latent phase).

Before entering the cell, the viral particle has in average 4.4 contacts with the cellular surface.(Seisenberger et al. 2001) The main receptor of AAV2 is heperan sulfate proteoglycan (HSPG). After contact with HSPG the capsid structure gets rearranged (Levy et al. 2009). This is probably essential for interaction with other cofactors, which leads to endocytosis. The factors respectively co-receptors of the cellular surface are known to enhance the initial binding affinity of HSPG: Fibroblast growth factor receptor 1 (FGFR-1), hepatocyte growth factor receptor (HGFR) and laminin receptor. It is known that AAVs affect both: αVβ₅ and αVβ₁integrin. The αVβ₁ -binding site is an asparagine-glycine-arginine motif (Asokan et al. 2006). These integrins interact with intracellular molecules like Rho, Rac and Cdc42 GTPases. Figure 2 depicts the following cascade.

Figure 18: Adapted from (Sanlioglu et al. 2000a)

The initial contact with HSPG, FGFR-1, HGFR and/or laminin is followed by an interaction with αVβ5 and/or αVβ1 which propably leads to an intracellular activation of enzymes involved in the rearrangement of cytoskeletal proteins like actin, via PI3K-pathway (Kapeller & Cantley 1994), (Li et al. 1998). In general, the receptor-mediated endocytosis (RME) is a complex process proteins and co-factors form clathrin coated pits as shown in Figure 16.

Beschreibung: File:Freiburg10 Receptor-mediated endocytosis by clathrin-coated vesicles scheme.PNG

Figure 19:

The adaptor proteins (APs) AP1, AP2, AP3 and AP4 are complexes built of four subunits (Collins et al. 2002), (Asokan et al. 2006). Except for AP2, which requires GTP-bound-Arf1, the APs are linked via phosphatidylinositol (4,5)-bisphosphate (PIP2) to the cell membrane (Robinson 2004). APs recognize short cytoplasmatic motifs like YXX-phi (phi: bulky hydrophobic AA) of transmembrane receptors. In general, the recognition sites (mu-subunits) in the AP-complexes have to be phosphorylated by kinases (Ohno et al. 1995).

The actual scaffold of the endosome is build by the triskelion formed clathrins. The rigide backbone of clathrins is formed by three heavy chains (Ybe et al. 1999) and three light chains are regulating assembly competence (BRODSKY et al. 1991). After building the clathrin scaffold, dynamine is responsible for pinching-off the clathrin-coated pits (CCPs) from the cell’s membrane (Summerford & Samulski 1998) (Sanlioglu et al. 2000b).

Endosomal transport and escape

Still there are possible additional entering pathways, for example knocking down microtubuli and microfilament arrangement does not prevent transduction completely (Kelley 2008). Currently it is thought that endosomal escape happens in the cytoplasma. After pinching off, the endosomes move via motor proteins along microtubuli and microfilaments towards the nuclear area. While trafficking through the cell the early endosoms getting acidulated (Sonntag et al. 2006).

Additional entering pathways were postulated for the virus, for example, it has been shown that proteasomal degradation via ubiquitination hampers transduction efficiency (Douar et al. 2001).

The first viral particles in the nuclear area can be detected after 15 minutes (Seisenberger et al. 2001) and an accumulation of virions takes place after 30 minutes post transfection. After arrival, the viral genomes are transported into the nucleus. It is not entirely clear in which way the transport is accomplished. The viral particles seem to use different pathways to enter the nucleus, either via the nuclear pore complexes with their maximal pore size of 23 nm. In this case, the viral capsid (25 nm diameter) has to be remodeled. Controversial results were published in the past, detecting intact viral particles (lu et al., 2000), but according to Lux et al. no intact capsids were detectable when lower amounts of viral particles were transduced (Lux et al. 2005).

Obviously further investigation of intracellular trafficking is essential for optimizing the AAV2 for medical applications.

Helper Genes

The AAV Helper-Free System by Stratagene (Waldbronn, Germany) is a modularized system for the production of infectious recombinant AAV-2 virion not depending on a coinfection with any helper virus. It is put into practice by the three plasmids pHelper, pAAV-RC, recombinant pAAV vector containing the gene of interest (GOI) and the recombinant cell line AAV-293.

The AAV-2 is a replication-deficient parvovirus, which originally needs a co-infection of adenovirus or herpes virus for replication. To realize a functional replication of AAV-2 without a co-infection, the AAV Helper-Free System allocates the pHelper plasmid and the AAV-293 host cells. The pHelper plasmid encodes for nearly all of the required adenovirus gene products for replication (VA, E2a, E4). The AAV-293 host cells express stably the remaining important replication genes (E1A, E1B).

Due to the fact that AAV-2 needs all relevant replication genes for productive infection and that the important replication-genes are dispersed, the AAV Helper-Free System describes a saver alternative to retroviral or adenoviral gene delivery (Stratagene n.d.).

The AAV-293 host cells contain the E1A and E1B genes. The E1A gene is the first gene to be expressed during an adenovirus infection. The E1A gene produces two different mRNAs resulting in two different proteins. The expressed E1A proteins transactivate and induce transcription of other early genes (like E2 and E4). In this case, E1A proteins do not bind directly onto control regions, but interact with other host proteins, which are binding to those regions (Chang et al. 1989) (Modrow et al. 2003).

Figure 20: Schematic overview of the Helper Free System provided by Stratagene.

In the AAV-2 infection cycle, E1A proteins stimulate the expression of the p19 promoter and the p5 promoter, which are required to begin with the rep-gene transcription of the AAV(Chang et al. 1989) (Tratschin et al. 1984).

The E1B region encodes two polypeptides with overlapping reading frames, the major 21-Mr product and the 55-Mr moiety. It has been shown that only the 55-Mr polypeptide is required for effective helper function. It enables along with the E4orf6, a stable accumulation of AAV-specific cytoplasmic RNA, capsid proteins and DNA replication(Samulski & Shenk 1988). In this context it has to be mentioned, that Stratagene deleted the E4orf6 out of its kit, because of its oncolytic activity. But it has been explained, that deletion of E4orf6 has no effect on virion production efficiency(Clark et al. 1999) .

The pHelper plasmid exists of the E2a gene, the E4 gene and the VA gene as well as a pUC ori and an f1 ori. The E2A gene encodes a 72 kD protein which is produced early in infection (Modrow et al. 2003). One helper function of E2A is to increase the processivity of replication. In the presence of E2A protein, short replication products, which are equivalent to break offs of the elongation strand of the template, are obviously reduced suggestion that E2A supports full-length replication of short substrates. In immune-depletions, co-localizations between the E2A, the AAV Rep protein and the AAV DNA have been shown (Ward et al. 1998) .

It has been reported that E2A has affections on the AAV promoter regulation of spliced p5 and p19 as well as unspliced p40. E2A could also be responsible for the production of the AAV capsid proteins (Carter et al. 1992). To which extend it really takes elementary responsiblitiy for any of the listed functions is not found out yet.

The VAI and VAII genes encode for two RNA-species, with a high GC-percentage and distinct secondary structure. The VAI RNA, which is implicated to have a helper function in AAV, usually plays a fundamental role in adenovirus’ protein expression. There it blocks the phosphorylation of the initiation factor eIF-2, whereby the amino acid chain at the ribosome breaks off. (Modrow et al. 2003)

The expression of the AAV proteins may also be under the VAI adenovirus control. VAI may increase the AAV capsid production, but it also may play a role in RNA metabolism.(West et al. 1987)

The E4 gene exists of seven open reading frames. In this content, the proteins occurring from the gene are named E4-ORF1 up to E4-ORF7. All proteins are under the control of one promoter and arise from alternative splicing. The E4ORF6 is implicated to have a helper function in AAV. It promotes the formation of a dsDNA from the genomic ssDNA of the native virus.

Recombinant Viruses and Mosaic Viruses

Gene Therapy

Treating inherited and acquired diseases such as cancer is still one of the most challenging fields in today’s biomedical research. Ever since Sidney Farber published a study in 1949 about several folic acid antagonists, which prevent tumor progression (FARBER 1949), cancer was treated with chemotherapy, surgery and radiation (Halperin 2006). Nevertheless, due to side effects caused by systemic applications and the lack of specificity, new treatments must be found for improved therapeutic efficacy and enhanced selectivity of the anticancer agents. One promising approach of treating cancer is suicide gene therapy or gene-directed enzyme prodrug therapy (GDEPT) including two steps of treatment: Targeted introduction of a gene encoding for enzymes into tumor cells, followed by the administration of a non-toxic prodrug which is converted into an anti-cancer metabolite.

Figure 21: Schematic overview of gene-directed enzyme prodrug therapy (GDEPT). The suicide gene is introduced into the cancer cells. Administration of the prodrug leads to cell death in the cells expressing the enzyme, which converts the prodrug into the toxic product.

Gene delivery using viral vectors to specifically target cells gained increasing attention in the last years being efficient in combination with suicide gene therapy. Several prodrug/enzyme systems have been reported (Greco & Dachs 2001). Ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV-TK), 5-fluorocytosine/cytosine deaminase (CD) and cyclophosphamide/cytochrome P450 systems have been widely used and their activity has been demonstrated in several preclinical studies (Greco & Dachs 2001).

Using transgenic HSV – thymidine kinase or cytosine deaminase from E. coli for prodrug activation in tumor therapy several advantages can be found. Besides efficient killing of targeted tumor cells, neighboring, non-transduced cells are killed as well, providing an important effect in treating cancer. The bystander phenomenon was first reported by Moolten (1986) showing that HSV-TK negative cells surrounded by HSV-TK positive cells did not survive prodrug treatment.

Figure 22: Efficient tumor killing is desired in cancer treatment. Locally administered prodrugs are converted to toxic metabolites by delivered enzmye in the infected cells. By passive diffusion, gap junction intercellular communication or immune-related response, non-transduced tumor cells are killed as well.

Transfer of toxic molecules between transduced and non-transduced cells can be achieved either through gap junctions (Yang et al. 1998) (Trepel, Stoneham, et al. 2009), via apoptotic bodies (Freeman et al. 1993) or by diffusion of soluble toxic substances (Huber et al. 1993).

Immune Response

Virus infections cause common human disease, including the familiar cold, influenza, mumps and measles. They are also associated with severe diseases, for example with Ebola or Marburg fever, with Hepatitis and AIDS. The immune system protects us from these infections by creating a barrier that prevents viruses from entering the body or by detecting and eliminating them in the corpus.

Every virus lives and reproduces in its own specific host. Reproduction can only take place in cell cytoplasm, whose components are needed in order to compensate for the lack of virus metabolism and biosynthesis-appliance. Viruses, which are located outside the cells, can be detected by antibodies, triggering an immune response by the members of the innate immune system, such as macrophages, neutrophils and natural killer-cells.

Inside a cell the virus can only be detected by cytotoxic T cells: While it uses the cellular machinery for reproduction, some of the viral proteins are degraded by proteasomes and become presented on the cell surface by MHC-I-peptides. These exposed virus components are recognized by cytotoxic CD8+ T cells, which induce death of infected cells. The degraded viral proteins can also be accessible on MHC-II-peptides, which are detected by CD4+ T helper cells, which trigger and enforce the immune response for example by production of specific antibodies.

The Adeno-associated virus (AAV) is not associated with any human disease. Nevertheless, usage of recombinant AAV (rAAV) as therapeutic vector system harbors risks of immune responses.

AAV establishes a latent infection and often integrates at a specific site on q arm of chromosome 19, which is termed AAVSI site (Hernandez et al., 1999). This leads to several obstacles for usage of AAV vectors for therapeutic applications like gene silencing, insertions in gene sequences and immunotoxocity, a dangerous immune response to the vector or the transgene product (Mingozzi & High 2007). Humans are the only natural hosts for AAV-2 besides rhesus macaques. Due to wild-type AAV infections humans keep a population of antigen-specific memory CD8+ T cells (Mingozzi & High 2007). IgG antibodies are predominantly involved in the secondary immune response. 91% of Irish blood donors show a high repertoire of specific IgG1 and IgG2 subclasses and low doses of IgG3 (Madsen et al. 2009).

In vivo studies with AAVlacZ show that AAV vectors induce the secretion of chemokines and cytokines like gamma interferon (IFN-γ) (Zaiss et al. 2002). Studies in vitro show responses of IFN-γ, interleukin 10 (IL-10) and interleukin 13 (IL-13) after stimulation peripheral blood mononuclear cells (PBMC) from donors with AAV-2. This demonstrates a reaction of long-live CD4+ T helper-cells that are reactivated (Madsen et al. 2009). These results reveal that most Europeans are already infected with wildtype-AAV-2. Researchers suggest that more than 30% of mankind is already infected. In vitro studies from the United States support this hypothesis. One group found anti-AAV-antibodies in the blood sera at 80% of randomly chosen volunteers (Moskalenko et al. 2000). Other investigators show that 0,14% of the examined CD8+ T cells purified from PBMC are capsid specific for AAV-2 (Mingozzi, Maus, et al. 2007). These preexisting memory-CD8+ T cells could be responsible for the difference in vector-infusion outcome between humans (the natural host) and other species.

AAV-2 use distinct cellular receptors, e.g. heparin sulfate proteoglycan (HSPG), αVβ5 integrin and human fibroblast growth factor receptor 1 (FGFR1) to become internalized (Favaro et al. 2009). These findings led researchers to the conclusion that the presence of an intact heparinbinding motif and the capsid t-cell responses are correlated. One group ablated the heparin-binding site in AAV-2 and observed no CD8+ T cell response. But it did not seem to influence T helper responses as measured by IgG isotypes and antigen-stimulated secretion of cytokines (Vandenberghe et al. 2006).

Approaches using peptides derived from the sequence of the VP1 viral capsid protein revealed a total of 59 t-cell epitopes. This demonstrates the difficulty to avoid the immune system by modifying the AAV capsid (Madsen et al. 2009). Other approaches in mice reveal that different serotypes of AAV show the ability to cross-react with existing memory-T cells (Sabatino et al. 2005). Also in dogs different AAVs use some common peptides on their surface to activate the immune system (Wang et al. 2010). This shows the high conservation of the epitopes among multiple serotypes of AAV.

While proposing several possible solutions to avoid the immune system, the polymorphic nature of the human MHC and the high conservation of peptides on the surface of different serotypes of AAV may complicate these approaches (Mingozzi, Hasbrouck, et al. 2007). In general it can be said that the immune response to AAV is not severe as caused by other virus-types. This is due to the fact that AAVs fail to trigger inflammatory reactions dendritic cells need to differentiate into professional antigen-presenting cells (Mingozzi, Maus, et al. 2007). These antigen-presenting cells are needed for the activation of CD4+ T helper-cells which are needed for the completely feedback to the immune system. Nevertheless dendritic cells can be activated through the ability of AAV-2 to bind the HSPG binding motif with resultant AAV2 antigen inclusion, processing and MHC-I presentation (Wang et al. 2010). CD4+ T helper-cells can also be activated by other antigen-presenting cells therefore it is conceivable to block CD4+ cells during treatment with AAV. The activation of CD8+-t-cells through CD4+ T cells is depleted and the immune response is even more reduced than within the normal infection process.

Some researchers have found AAV vector DNA in the semen of dogs and fear the risk of germline transmission (Jiang et al. 2006) although these findings are controversially discussed. In a rabbit model it was demonstrated that semen was just positive for vector sequences following intravascular injection but not following intramuscular injection. Infectious vector particles were just detected up to four days after treatment and were undetectable thereafter. So the investigators suggest that AAV-2 presents a low risk of germline transmission for humans and there is no contemplation for male infertility so far (Favaro et al. 2009).

AAV vectors have been used in several phases of clinical trials for Leber’s congenital amaurosis (LCA), hemophilia B, Cystic fibrosis, Arthritis, Muscular dystrophy, Parkinson’s disease, Canavan's disease, Alzheimer's disease, Batten's disease and Hereditary emphysema.

@@ Line 3: / Line 3: @@
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-<div class=WordSection1>
-<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 0cm 1.0pt 0cm;
-margin-left:21.6pt;margin-right:0cm'>
-<p class=MsoTocHeading style='margin-left:0cm;text-indent:0cm'><a
-name="_Toc274911361"></a><a name="_Toc275994040"><span lang=DE>Contents</span></a></p>
+<h1>Introduction</h1>
+<div class="WordSection1">
+<div
+style="border-style: none none solid; border-color: -moz-use-text-color -moz-use-text-color windowtext; border-width: medium medium 1pt; padding: 0cm 0cm 1pt; margin-left: 21.6pt; margin-right: 0cm;">
+<p class="MsoTocHeading" style="margin-left: 0cm; text-indent: 0cm;"><a
+name="_Toc274911361"></a><a name="_Toc275994040"><span lang="DE">Contents</span></a></p>
 </div>
+<p class="MsoToc1"><a href="#_Toc275994657"><span lang="DE">Overview</span><span
-<p class=MsoToc1><a href="#_Toc275994657"><span lang=DE>Overview</span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>.. </span><span
+lang="DE">.. </span><span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>1</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">1</span></a></p>
-<p class=MsoToc2><a href="#_Toc275994658"><span lang=DE>The Experimental System</span><span
+<p class="MsoToc2"><a href="#_Toc275994658"><span lang="DE">The
-lang=DE style='color:windowtext;display:none;text-decoration:none'>.. </span><span
+Experimental System</span><span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>1</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">.. </span><span
-<p class=MsoToc2><a href="#_Toc275994659"><span lang=DE>Layers of specificity</span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>. </span><span
+lang="DE">1</span></a></p>
-lang=DE style='color:windowtext;display:none;text-decoration:none'>2</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994659"><span lang="DE">Layers of
+specificity</span><span
-<p class=MsoToc1><a href="#_Toc275994660">Introduction to Adeno-Associated
+style="color: windowtext; display: none; text-decoration: none;"
-Virus Serotype 2<span lang=DE style='color:windowtext;display:none;text-decoration:
+lang="DE">. </span><span
-none'>. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>4</span></a></p>
+lang="DE">2</span></a></p>
+<p class="MsoToc1"><a href="#_Toc275994660">Introduction to
-<p class=MsoToc2><a href="#_Toc275994661">Biology of the AAV-2<span lang=DE
+Adeno-Associated
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+Virus Serotype 2<span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>4</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
-<p class=MsoToc3><a href="#_Toc275994662">Genomic organization<span lang=DE
+style="color: windowtext; display: none; text-decoration: none;"
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+lang="DE">4</span></a></p>
-lang=DE style='color:windowtext;display:none;text-decoration:none'>4</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994661">Biology of the AAV-2<span
+style="color: windowtext; display: none; text-decoration: none;"
-<p class=MsoToc3><a href="#_Toc275994663">Replication<span lang=DE
+lang="DE">. </span><span
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>5</span></a></p>
+lang="DE">4</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994662">Genomic organization<span
-<p class=MsoToc3><a href="#_Toc275994664">Integration<span lang=DE
+style="color: windowtext; display: none; text-decoration: none;"
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+lang="DE">. </span><span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>11</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">4</span></a></p>
-<p class=MsoToc3><a href="#_Toc275994665">Rescue<span lang=DE style='color:
+<p class="MsoToc3"><a href="#_Toc275994663">Replication<span
-windowtext;display:none;text-decoration:none'>   </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>12</span></a></p>
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-<p class=MsoToc3><a href="#_Toc275994666">Rep proteins<span lang=DE
+lang="DE">5</span></a></p>
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+<p class="MsoToc3"><a href="#_Toc275994664">Integration<span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>12</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
-<p class=MsoToc3><a href="#_Toc275994667">VP proteins<span lang=DE
+style="color: windowtext; display: none; text-decoration: none;"
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+lang="DE">11</span></a></p>
-lang=DE style='color:windowtext;display:none;text-decoration:none'>16</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994665">Rescue<span
+style="color: windowtext; display: none; text-decoration: none;"
-<p class=MsoToc3><a href="#_Toc275994668">Trafficking<span lang=DE
+lang="DE"> </span><span
-style='color:windowtext;display:none;text-decoration:none'>  </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>23</span></a></p>
+lang="DE">12</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994666">Rep proteins<span
-<p class=MsoToc2><a href="#_Toc275994669">Helper Genes<span lang=DE
+style="color: windowtext; display: none; text-decoration: none;"
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+lang="DE">. </span><span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>27</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">12</span></a></p>
-<p class=MsoToc2><a href="#_Toc275994670">Recombinant Viruses and Mosaic
+<p class="MsoToc3"><a href="#_Toc275994667">VP proteins<span
-Viruses<span lang=DE style='color:windowtext;display:none;text-decoration:none'>. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE style='color:windowtext;display:none;text-decoration:none'>29</span></a></p>
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-<p class=MsoToc2><a href="#_Toc275994671"><span lang=DE>Gene Therapy</span><span
+lang="DE">16</span></a></p>
-lang=DE style='color:windowtext;display:none;text-decoration:none'>. </span><span
+<p class="MsoToc3"><a href="#_Toc275994668">Trafficking<span
-lang=DE style='color:windowtext;display:none;text-decoration:none'>30</span></a></p>
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE"> </span><span
-<p class=MsoToc2><a href="#_Toc275994672">Immune Response<span lang=DE
+style="color: windowtext; display: none; text-decoration: none;"
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
+lang="DE">23</span></a></p>
-lang=DE style='color:windowtext;display:none;text-decoration:none'>31</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994669">Helper Genes<span
+style="color: windowtext; display: none; text-decoration: none;"
-<p class=MsoNormal><span lang=DE>&nbsp;</span></p>
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
-<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 0cm 1.0pt 0cm'>
+lang="DE">27</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994670">Recombinant Viruses and
-<h1 style='margin-left:0cm;text-indent:0cm'><span lang=DE>&nbsp;</span></h1>
+Mosaic
+Viruses<span
-<h1 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994657"><span
+style="color: windowtext; display: none; text-decoration: none;"
-lang=DE>Overview</span></a></h1>
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">29</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994671"><span lang="DE">Gene Therapy</span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">30</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994672">Immune Response<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">31</span></a></p>
+<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+<div
+style="border-style: none none solid; border-color: -moz-use-text-color -moz-use-text-color windowtext; border-width: medium medium 1pt; padding: 0cm 0cm 1pt;">
+<h1 style="margin-left: 0cm; text-indent: 0cm;"><span lang="DE">&nbsp;</span></h1>
+<h1 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994657"><span
+lang="DE">Overview</span></a></h1>
 </div>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994658"></a><a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994658"></a><a
+name="_Toc275994041"><span lang="DE">The Experimental System</span></a></h2>
-name="_Toc275994041"><span lang=DE>The Experimental System</span></a></h2>
+<p class="MsoNormal">Therapy using viral vectors is an promising
+approac. In an
-<p class=MsoNormal>Therapy using viral vectors is an promising approac. In an
+first step the plasmids of the AAV-2 Helper-free System were
-first step the plasmids of the AAV-2 Helper-free System were genetically
+genetically
-modifyed by converting it into BioBricks and inserting of targeting molecules
+modifyed by converting it into BioBricks and inserting of targeting
-into the constructs. These plasmids were then used to transfect the producer
+molecules
-cell line AAV-293. After an incubation of three days the viral vectors were
+into the constructs. These plasmids were then used to transfect the
+producer
+cell line AAV-293. After an incubation of three days the viral vectors
+were
 harvested and used to transduce different target cells. The succesful
-transduction can then for example be measured by detecting the fluorescence of
+transduction can then for example be measured by detecting the
+fluorescence of
 fluorescent proteins in the target cells</p>
+<p class="MsoNormal">The majority of the modifications that were
-<p class=MsoNormal>The majority of the modifications that were introduced into
+introduced into
-the viral vector aimed to allow differential targeting of tumor cell over
+the viral vector aimed to allow differential targeting of tumor cell
+over
 healthy off-target cells.</p>
+<p class="MsoNormal"><img style="width: 471px; height: 575px;"
-<p class=MsoNormal><img width=471 height=575 id="Picture 56"
+id="Picture 56"
-src="Introductionwiki-Dateien/image001.png"
+src="https://static.igem.org/mediawiki/2010/3/38/Freiburg10_Experimental_System.png"
-alt="Beschreibung: X:\users\FreiGem\iGEM2010\Volker\g16268.png"></p>
+alt=""></p>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994659"></a><a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994659"></a><a
+name="_Toc275994042"><span lang="DE">Layers of specificity</span></a></h2>
-name="_Toc275994042"><span lang=DE>Layers of specificity</span></a></h2>
+<p class="MsoNormal">Employment of viral vectors for means of therapy
+is idea in
-<p class=MsoNormal>Employment of viral vectors for means of therapy is idea in
+the context of personalized medicie that gets more and more interest.
-the context of personalized medicie that gets more and more interest. In such
+In such
-applications the reduction of side effects and the safety of the patient in
+applications the reduction of side effects and the safety of the
+patient in
 general is of the highest priority.</p>
+<p class="MsoNormal">In order to satisfy this requirement we designed
-<p class=MsoNormal>In order to satisfy this requirement we designed our Therapy
+our Therapy
 Vector with several layers of Specificity:</p>
+<p class="MsoNormal">The targeting of the viral vector towards the
-<p class=MsoNormal>The targeting of the viral vector towards the desired target
+desired target
-cell (e.g. tumor cells) is the basic idea behind the emplyment of viral vectors
+cell (e.g. tumor cells) is the basic idea behind the emplyment of viral
-for therapeutical means. There for the natural tropismn has to be knocked down
+vectors
-and a desired tropism has to be introduced that allows differential targeting
+for therapeutical means. There for the natural tropismn has to be
-of pathological but not of off-target cells. To fulfill this mission our Virus
+knocked down
+and a desired tropism has to be introduced that allows differential
+targeting
+of pathological but not of off-target cells. To fulfill this mission
+our Virus
 Construction Kit offers you different solutions.</p>
+<p class="MsoNormal">Off-target cells that were transduced by mistake
-<p class=MsoNormal>Off-target cells that were transduced by mistake can be
+can be
 preserved from an undesired therapy effect when the therapeutic gene is
-controley by a tissue specific promoter. For this mean a promoter has to be
+controley by a tissue specific promoter. For this mean a promoter has
-used that is as specific for the pathological tissue as possible. We included
+to be
+used that is as specific for the pathological tissue as possible. We
+included
 the human telomerase promoter (<a
 href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K404106">phTERT</a>)
 which is often activated in tumor cells and is there for able to allow
-differential experssion of a therapeutic geneproduct in pathological cells.</p>
+differential experssion of a therapeutic geneproduct in pathological
+cells.</p>
-<p class=MsoNormal>For reasons of safety Therapeutic vector do not directly
+<p class="MsoNormal">For reasons of safety Therapeutic vector do not
-trigger appoptosis in the successfully targeted cells. To include one further
+directly
-layer of specificity and safety we decided to arm our therapy vector with
+trigger appoptosis in the successfully targeted cells. To include one
-different prodrug convertases. Neither the single application of the harmless
+further
-prodrug nor the single expression of the convertase has a noteworthy effect of
+layer of specificity and safety we decided to arm our therapy vector
-the transduced cell. Only in cells that express the prodrug convertase and have
+with
-a sufficient cytoplasmatic concentration of the belonging prodrug apoptosis is
+different prodrug convertases. Neither the single application of the
-triggered. This dependency of the therapy on a prodrug can be employed to
+harmless
+prodrug nor the single expression of the convertase has a noteworthy
+effect of
+the transduced cell. Only in cells that express the prodrug convertase
+and have
+a sufficient cytoplasmatic concentration of the belonging prodrug
+apoptosis is
+triggered. This dependency of the therapy on a prodrug can be employed
+to
 protect tissues or other persons that could come in contact with the
-therapeutical vector. This aspect was specially inportant for the development
+therapeutical vector. This aspect was specially inportant for the
+development
 of a viral vector that is able to infect humans in the context of a
-undergraduate project for the iGEM competition. Therefor this approach gained
+undergraduate project for the iGEM competition. Therefor this approach
-our preference over other possibly equivalent arming possibilities described in
+gained
+our preference over other possibly equivalent arming possibilities
+described in
 the tumor therapy with viral vectors.</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal"><img
+style="border: 0px solid ; width: 463px; height: 512px;"
-<p class=MsoNormal><img border=0 width=463 height=512 id="Picture 57"
+id="Picture 57"
-src="Introductionwiki-Dateien/image002.png"
+src="http://partsregistry.org/wiki/images/b/b5/Freiburg10_Layers_of_Specificity.png"
-alt="Beschreibung: X:\users\FreiGem\iGEM2010\Volker\g16404.png"></p>
+alt=""></p>
 </div>
+<span
-<span lang=DE style='font-size:11.0pt;line-height:150%;font-family:"Calibri","sans-serif"'><br
+style="font-size: 11pt; line-height: 150%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-clear=all style='page-break-before:always'>
+lang="DE"><br style="page-break-before: always;" clear="all">
 </span>
+<div class="WordSection2">
-<div class=WordSection2>
+<div
+style="border-style: none none solid; border-color: -moz-use-text-color -moz-use-text-color windowtext; border-width: medium medium 1pt; padding: 0cm 0cm 1pt;">
-<div style='border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 0cm 1.0pt 0cm'>
+<h1 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994660"></a><a
+name="_Toc275994043">Introduction to </a>Adeno-Associated Virus
-<h1 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994660"></a><a
+Serotype 2</h1>
-name="_Toc275994043">Introduction to </a>Adeno-Associated Virus Serotype 2</h1>
 </div>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994661"></a><a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994661"></a><a
 name="_Toc275994044"></a><a name="_Toc274911362">Biology of the AAV-2</a><a
 name="_Toc274911363"></a></h2>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994662"></a><a
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994662"></a><a
 name="_Toc275994045">Genomic organization</a></h3>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;"
- style='border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:
+align="left" border="1" cellpadding="0" cellspacing="0">
-.8pt'>
+<tbody>
- <tr style='height:191.45pt'>
+<tr style="height: 191.45pt;">
-  <td width=568 valign=top style='width:426.05pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt;height:191.45pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 426.05pt; height: 191.45pt;"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a
+valign="top" width="568">
-  name="_Toc274911364"><img border=0 width=524 height=269 id="Grafik 154"
+<p class="MsoCaption"
-  src="Introductionwiki-Dateien/image003.png"></a></p>
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Toc274911364"><img
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a
+style="border: 0px solid ; width: 524px; height: 269px;" alt=""
-  name="_Ref275946742">Figure </a>1: Genomic organization of the wt-AAV-2. The
+id="Grafik 154"
-  inverted terminal repeats (ITRs) flank the two open reading frames (ORFs).
+src="https://static.igem.org/mediawiki/2010/3/35/Freiburg10_genomic_organisation_wt.png"></a></p>
-  The four-nonstructural proteins encoded from the <i>rep</i> gene are driven
+<p class="MsoCaption"
-  by the p5 and p19 promoters, whereas the structural Cap proteins are
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275946742">Figure
-  regulated by the p40 promoter. Additionally, the Assembly Activating Protein
+</a>1: Genomic organization of the wt-AAV-2. The inverted
-  (AAP) was found recently within the <i>cap</i> gene.</p>
+terminal repeats (ITRs) flank the two open reading frames (ORFs). The
-  </td>
+four-nonstructural proteins encoded from the <i>rep</i> gene are
- </tr>
+driven by the p5 and p19 promoters, whereas the structural Cap proteins
+are regulated by the p40 promoter. Additionally, the Assembly
+Activating Protein (AAP) was found recently within the <i>cap</i> gene.</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">The Adeno-associated virus serotype-2 (AAV-2)
-<p class=MsoNormal>The Adeno-associated virus serotype-2 (AAV-2) genome is a linear,
+genome is a linear,
-single-stranded (ss) 4675 bp DNA virus. Due to its small size, gene genomic
+single-stranded (ss) 4675 bp DNA virus. Due to its small size, gene
-organization is condensed and gene regulation is complex. The viral nucleotide
+genomic
-sequence consist of two open reading frames (ORFs) coding for Rep- and Cap
+organization is condensed and gene regulation is complex. The viral
-proteins and are flanked on either side by identical inverted terminal repeat
+nucleotide
-(ITR) structures which are palindromic and form hairpin structures (Srivastava et al. 1983). </p>
+sequence consist of two open reading frames (ORFs) coding for Rep- and
+Cap
-<p class=MsoNormal>&nbsp;</p>
+proteins and are flanked on either side by identical inverted terminal
+repeat
-<p class=MsoNormal>The ITRs serve as primers for the host cells’ DNA polymerase,
+(ITR) structures which are palindromic and form hairpin structures
-which converts the single-stranded virus genome into double-stranded DNA (ds
+(Srivastava et al. 1983). </p>
-DNA) as a part of the viruses’ replicative cycle. They also play important
+<p class="MsoNormal">&nbsp;</p>
-roles in viral genome integration into and rescue from the hosts genome, the
+<p class="MsoNormal">The ITRs serve as primers for the host cells’ DNA
-formation of concatamers in the host cell nucleus and encapsidation of the
+polymerase,
-viral genome into preforemd capsids (Berns 1990). Due to these essential functions, the ITR structures cannot be deleted from
+which converts the single-stranded virus genome into double-stranded
+DNA (ds
+DNA) as a part of the viruses’ replicative cycle. They also play
+important
+roles in viral genome integration into and rescue from the hosts
+genome, the
+formation of concatamers in the host cell nucleus and encapsidation of
+the
+viral genome into preforemd capsids (Berns 1990). Due to these
+essential functions, the ITR structures cannot be deleted from
 a viral vector and need to be delivered in <i>cis.</i></p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994046">Organization
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994046">Organization
 of the Inverted Terminal </a>Repeat Structure</h4>
+<p class="MsoNormal"><span style="color: black;">The inverted terminal
-<p class=MsoNormal><span style='color:black'>The inverted terminal repeat
+repeat
-structures can be subdivided into several palindromic motives: A and A’ form a
+structures can be subdivided into several palindromic motives: A and A’
-stem loop which encases B and B’ as well as C and C’. Those motives form both
+form a
-arms of the T-shaped structure. The functional motives on the ITR are two
+stem loop which encases B and B’ as well as C and C’. Those motives
-regions that bind Rep 68/78, called Rep-binding elements (RBE on the stem and
+form both
-RBE’ on the B arm) and the terminal resolution site (trs) in which the rep
+arms of the T-shaped structure. The functional motives on the ITR are
-proteins introduce single-stranded nicks. The 3’ OH end of the A motive acts as
+two
-a primer for DNA replication </span><span
+regions that bind Rep 68/78, called Rep-binding elements (RBE on the
-style='color:black'>(Im &amp; Muzyczka 1990)</span><span style='color:black'> </span><span style='color:black'>(Lusby et al. 1980)</span><span style='color:black'>.</span></p>
+stem and
+RBE’ on the B arm) and the terminal resolution site (trs) in which the
-<div align=center>
+rep
+proteins introduce single-stranded nicks. The 3’ OH end of the A motive
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+acts as
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+a primer for DNA replication </span><span style="color: black;">(Im
- -2.25pt'>
+&amp; Muzyczka 1990)</span><span style="color: black;"> </span><span
- <tr style='height:293.05pt'>
+style="color: black;">(Lusby et al. 1980)</span><span
-  <td width=520 valign=top style='width:390.3pt;padding:0cm 5.4pt 0cm 5.4pt;
+style="color: black;">.</span></p>
-  height:293.05pt'>
+<div align="center">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<table class="MsoTableGrid"
-  avoid'><img border=0 width=512 height=384 id="Grafik 11"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-  src="Introductionwiki-Dateien/image004.png"></p>
+align="left" border="0" cellpadding="0" cellspacing="0">
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 2: Organization of the ITRs, which are the only <i>cis</i>-required element in viral
+<tbody>
-  genome integration and replication. </p>
+<tr style="height: 293.05pt;">
-  </td>
+<td style="padding: 0cm 5.4pt; width: 390.3pt; height: 293.05pt;"
- </tr>
+valign="top" width="520">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 512px; height: 384px;" alt=""
+id="Grafik 11"
+src="https://static.igem.org/mediawiki/2010/8/88/Freiburg10_organization_ITRs.png"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 2: Organization
+of the ITRs, which are the only <i>cis</i>-required element in viral
+genome integration and replication. </p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994663"></a><a
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994663"></a><a
 name="_Toc275994047">Replication</a></h3>
+<p class="MsoNormal">The 3’ OH end of the viral DNA folds onto itself
-<p class=MsoNormal>The 3’ OH end of the viral DNA folds onto itself as part of
+as part of
-the inverted terminal repeat (ITR) structure and thus serves as a primer for
+the inverted terminal repeat (ITR) structure and thus serves as a
+primer for
 elongation by the host cell’s DNA polymerase. The polymerases strand
 displacement activity unfolds the opposite ITR structure and elongation
-continues until the 5’ template end is reached <span style='color:black'> </span><span style='color:black'>(Lusby et al. 1980)</span>.</p>
+continues until the 5’ template end is reached <span
+style="color: black;"> </span><span style="color: black;">(Lusby et
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+al. 1980)</span>.</p>
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<table class="MsoTableGrid"
- -2.25pt'>
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
- <tr>
+align="left" border="1" cellpadding="0" cellspacing="0">
-  <td width=614 valign=top style='width:460.5pt;border:solid windowtext 1.0pt;
+<tbody>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<tr>
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<td
-  avoid'><img border=0 width=588 height=413 id="Grafik 369"
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 460.5pt;"
-  src="Introductionwiki-Dateien/image005.png"></p>
+valign="top" width="614">
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 3: Figure 3: Schematic overview of AAV-2 replication.</p>
+<p class="MsoNormal"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
- </tr>
+style="border: 0px solid ; width: 588px; height: 413px;" alt=""
+id="Grafik 369"
+src="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_Overview_AAV-2_replication.png"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 3: Figure 3:
+Schematic overview of AAV-2 replication.</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">The remaining hairpin structure that served as the
+origin of
-<p class=MsoNormal>The remaining hairpin structure that served as the origin of
+replication then acts as a target for the Rep 68/78 protein: It binds
-replication then acts as a target for the Rep 68/78 protein: It binds to the
+to the
-Rep-binding site (RBS) and unwinds the double-stranded DNA in a way that the
+Rep-binding site (RBS) and unwinds the double-stranded DNA in a way
-terminal resolution site (trs) is being displayed in a single-stranded form on
+that the
-a stem loop. This enables the endonuclease catalytic domain of the Rep protein
+terminal resolution site (trs) is being displayed in a single-stranded
-to introduce a nick of the parental strand at this site, which in turn serves
+form on
-as a new primer for DNA polymerase. The polymerase resolves the hairpin structure
+a stem loop. This enables the endonuclease catalytic domain of the Rep
-through strand displacement and copies the remaining end of the parental strand<span
+protein
-style='color:black'> </span><span
+to introduce a nick of the parental strand at this site, which in turn
-style='color:black'>(Im &amp; Muzyczka 1990)</span> .</p>
+serves
+as a new primer for DNA polymerase. The polymerase resolves the hairpin
-<p class=MsoNormal>Sometimes, nicking does not occur after polymerases have
+structure
-partially copied the virus DNA. In this case, the newly synthesized 3’ end acts
+through strand displacement and copies the remaining end of the
-as a primer and the host cell’s DNA polymerase copies the whole sequence once
+parental strand<span style="color: black;"> </span><span
-again, displacing the ITR strands in the middle of the sequence. This leads to
+style="color: black;">(Im &amp; Muzyczka 1990)</span> .</p>
-a dsDNA containing the whole virus genome twice, called a duplex dimer (DD).
+<p class="MsoNormal">Sometimes, nicking does not occur after
-Those dimers can be resolved back to duplex monomers (DM) by the Rep proteins</p>
+polymerases have
+partially copied the virus DNA. In this case, the newly synthesized 3’
-<p class=MsoNormal>After replication, the dsDNA separates again forming new
+end acts
-ssDNA in (+) and (-) polarity with hairpin structures at its ends. The Rep
+as a primer and the host cell’s DNA polymerase copies the whole
-/52 proteins are involved in this process. Newly synthesized copies are
+sequence once
-either encapsidated into virus capsids or replicated again (Gonçalves, 2005a). Double-stranded genomes are formed as well through annealing of (+) - and
+again, displacing the ITR strands in the middle of the sequence. This
-(-) single strands. Both mechanisms occur during infection and contribute to
+leads to
+a dsDNA containing the whole virus genome twice, called a duplex dimer
+(DD).
+Those dimers can be resolved back to duplex monomers (DM) by the Rep
+proteins</p>
+<p class="MsoNormal">After replication, the dsDNA separates again
+forming new
+ssDNA in (+) and (-) polarity with hairpin structures at its ends. The
+Rep
+/52 proteins are involved in this process. Newly synthesized copies
+are
+either encapsidated into virus capsids or replicated again (Gonçalves,
+a). Double-stranded genomes are formed as well through annealing of
+(+) - and
+(-) single strands. Both mechanisms occur during infection and
+contribute to
 transgene expression (Schultz &amp; Chamberlain 2008). </p>
+<p class="MsoNormal">If the double stranded virus DNA exists in an
-<p class=MsoNormal>If the double stranded virus DNA exists in an episomal form
+episomal form
-inside the nucleus, it tends to form linear as well as circular concatamers,
+inside the nucleus, it tends to form linear as well as circular
-which are formed by ligation of duplex monomers(Schultz &amp; Chamberlain 2008). </p>
+concatamers,
+which are formed by ligation of duplex monomers(Schultz &amp;
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994048">Viral
+Chamberlain 2008). </p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994048">Viral
 promoters</a></h4>
+<p class="MsoNormal">Three viral promoters are coordinating gene
-<p class=MsoNormal>Three viral promoters are coordinating gene expression in
+expression in
-the wildtype AAV-2. Each promoter regulates different open reading frames
+the wildtype AAV-2. Each promoter regulates different open reading
-(ORFs) of regulatory proteins (p5 and p19 promoter) and structural proteins
+frames
-(p40 promoter). A general overview is provided in<b>Error! Reference source not
+(ORFs) of regulatory proteins (p5 and p19 promoter) and structural
-found.</b>. p5 and p19 promoters are repressed in absence of helper proteins
+proteins
-provided by Ad or HSV whereas transactivation of p5 and p19 occurs in presence
+(p40 promoter). A general overview is provided in<b>Error! Reference
-of helper viruses. Furthermore, the larger Rep proteins activate the p40
+source not
-promoter. Since overexpression of Rep78 leads to cell cycle arrest, high levels
+found.</b>. p5 and p19 promoters are repressed in absence of helper
+proteins
+provided by Ad or HSV whereas transactivation of p5 and p19 occurs in
+presence
+of helper viruses. Furthermore, the larger Rep proteins activate the
+p40
+promoter. Since overexpression of Rep78 leads to cell cycle arrest,
+high levels
 of Rep78/68 lead to repression of the p5 promoter. </p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+align="left" border="0" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 373.8pt;">
- <tr style='height:373.8pt'>
+<td style="padding: 0cm 5.4pt; width: 284.5pt; height: 373.8pt;"
-  <td width=379 valign=top style='width:284.5pt;padding:0cm 5.4pt 0cm 5.4pt;
+valign="top" width="379">
-  height:373.8pt'>
+<p class="MsoNormal"
-  <p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
-  text-indent:0cm;page-break-after:avoid'><img border=0 width=363 height=432
+align="center"><img
-  id="Grafik 1226" src="Introductionwiki-Dateien/image006.png"></p>
+style="border: 0px solid ; width: 363px; height: 432px;" alt=""
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a
+id="Grafik 1226"
-  name="_Ref275633280"></a><a name="_Ref275633331">Figure </a>4: Regulation of the viral promoters located within the t AAV-2 genome. In the absence of
+src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_regulation_viral_promotors.png"></p>
-  helper viruses, gene expression is suppressed, whereas activation of p5 and
+<p class="MsoCaption"
-  p19 occurs in the presence of helper proteins by interacting of Rep proteins
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275633280"></a><a
-  with cellular and helper proteins.</p>
+name="_Ref275633331">Figure </a>4: Regulation of the viral promoters
-  </td>
+located within the t AAV-2 genome. In the absence of helper viruses,
- </tr>
+gene expression is suppressed, whereas activation of p5 and p19 occurs
+in the presence of helper proteins by interacting of Rep proteins with
+cellular and helper proteins.</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal" style="text-align: center;" align="center">&nbsp;</p>
-<p class=MsoNormal align=center style='text-align:center'>&nbsp;</p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994049"></a><a
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994049"></a><a
 name="_Ref275645430">p5 promoter</a></h5>
+<p class="MsoNormal">The p5 promoter, located downstream of the <i>rep</i>
-<p class=MsoNormal>The p5 promoter, located downstream of the <i>rep</i> and <i>cap</i>
+and <i>cap</i>
-ORF (Figure 5: The p5 promoter of the wtAAV-2 is located upstream of the rep
+ORF (Figure 5: The p5 promoter of the wtAAV-2 is located upstream of
+the rep
 and cap ORF and contains several elements, which interact with Rep and
-endogenous proteins.)of the wtAAV-2, regulates gene expression of the two larger
+endogenous proteins.)of the wtAAV-2, regulates gene expression of the
+two larger
 non-structural proteins Rep 78 and Rep 68 that are essential in genome
-replication and viral genome integration into several hotspots of the human
+replication and viral genome integration into several hotspots of the
+human
 chromosome. </p>
+<p class="MsoNormal">Several binding elements for cellular and viral
-<p class=MsoNormal>Several binding elements for cellular and viral proteins
+proteins
-involved in regulation can be found in the p5 promoter (Figure 5) therefore
+involved in regulation can be found in the p5 promoter (Figure 5)
-playing an important role in gene transcription, integration and replication,
+therefore
-dependent on the presence or absence of helper viruses such as adenovirus (Ad)
+playing an important role in gene transcription, integration and
-or herpes simplex virus (HSV) (Murphy et al. 2007). Besides regulation of gene expression, the p5 integration efficient
+replication,
+dependent on the presence or absence of helper viruses such as
+adenovirus (Ad)
+or herpes simplex virus (HSV) (Murphy et al. 2007). Besides regulation
+of gene expression, the p5 integration efficient
 element (p5IEE) containing the rep binding element (RBE) and a terminal
-resolution site (trs) is responsible for mediating site specific integration
+resolution site (trs) is responsible for mediating site specific
+integration
 into the human genome (Philpott et al. 2002). </p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+align="left" border="0" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 203.8pt;">
- <tr style='height:203.8pt'>
+<td style="padding: 0cm 5.4pt; width: 433.85pt; height: 203.8pt;"
-  <td width=578 valign=top style='width:433.85pt;padding:0cm 5.4pt 0cm 5.4pt;
+valign="top" width="578">
-  height:203.8pt'>
+<p class="MsoNormal"
-  <p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
-  text-indent:0cm;page-break-after:avoid'><img border=0 width=566 height=235
+align="center"><img
-  id="Grafik 1286" src="Introductionwiki-Dateien/image007.png"></p>
+style="border: 0px solid ; width: 566px; height: 235px;" alt=""
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a
+id="Grafik 1286"
-  name="_Ref275633988"></a><a name="_Ref275633992">Figure </a>5: The p5 promoter of the wtAAV-2 is located upstream of the rep and cap ORF and contains
+src="https://static.igem.org/mediawiki/2010/5/56/Freiburg10_p5_promoter_wtAAV2.png"></p>
-  several elements, which interact with Rep and endogenous proteins.</p>
+<p class="MsoCaption"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275633988"></a><a
- </tr>
+name="_Ref275633992">Figure </a>5: The p5 promoter of the wtAAV-2 is
+located upstream of the rep and cap ORF and contains several elements,
+which interact with Rep and endogenous proteins.</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal"><span lang="EN-GB">&nbsp;</span></p>
-<p class=MsoNormal><span lang=EN-GB>&nbsp;</span></p>
+<p class="MsoNormal">Containing two consensus sequences for binding
+immediate
-<p class=MsoNormal>Containing two consensus sequences for binding immediate
+early E1A gene product from adenoviruses (Chang et al. 1989), p5
-early E1A gene product from adenoviruses (Chang et al. 1989), p5 promoter is
+promoter is
-transactivated in the presence of helper viruses whereas suppression occurs in
+transactivated in the presence of helper viruses whereas suppression
-absence of adenoviral proteins by low levels of Rep proteins (Beaton et al. 1989). Regulating of Rep78/68 by its negative feedback loop is critical
+occurs in
-since overexpression leads to cell cycle arrest in the S-phase (Berthet et al. 2005) and suppression of cellular promoters (Jing et al. 2001).</p>
+absence of adenoviral proteins by low levels of Rep proteins (Beaton et
+al. 1989). Regulating of Rep78/68 by its negative feedback loop is
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994050">p5
+critical
+since overexpression leads to cell cycle arrest in the S-phase (Berthet
+et al. 2005) and suppression of cellular promoters (Jing et al. 2001).</p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994050">p5
 TATA-less promoter</a></h5>
+<p class="MsoNormal">In contrast to the natural location of the p5
-<p class=MsoNormal>In contrast to the natural location of the p5 promoter, the
+promoter, the
-iGEM team Freiburg 2010 provides the RepCap plasmid with a relocated p5 promoter
+iGEM team Freiburg 2010 provides the RepCap plasmid with a relocated p5
-downstream of the <i>RepCap</i> genes (Figure 6). Additionally the p5 promoter
+promoter
-lacks the TATA box element (AVIGEN 1997)<b>. </b>Those modifications result in an attenuated expression of the larger
+downstream of the <i>RepCap</i> genes (Figure 6). Additionally the p5
-Rep proteins therefore leading to normal transcription of the Rep proteins
+promoter
-driven by p19 promoter and enhanced expression of the Cap proteins, which are
+lacks the TATA box element (AVIGEN 1997)<b>. </b>Those modifications
-under the control of the p40 promoter. Additionally, removing the p5 promoter
+result in an attenuated expression of the larger
-downstream of the <i>RepCap</i> genes and deletion of the TATA box eliminates
+Rep proteins therefore leading to normal transcription of the Rep
-contamination with wtAAVs. Hence, alteration of the p5 promoter is useful for
+proteins
-enhanced production of recombinant viral particles attenuating repression of
+driven by p19 promoter and enhanced expression of the Cap proteins,
-Rep78/68 and improving gene transcription of the capsid proteins and Rep
+which are
+under the control of the p40 promoter. Additionally, removing the p5
+promoter
+downstream of the <i>RepCap</i> genes and deletion of the TATA box
+eliminates
+contamination with wtAAVs. Hence, alteration of the p5 promoter is
+useful for
+enhanced production of recombinant viral particles attenuating
+repression of
+Rep78/68 and improving gene transcription of the capsid proteins and
+Rep
 proteins involved in genome packaging. </p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+align="left" border="0" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 214.9pt;">
- <tr style='height:214.9pt'>
+<td style="padding: 0cm 5.4pt; width: 236.7pt; height: 214.9pt;"
-  <td width=316 valign=top style='width:236.7pt;padding:0cm 5.4pt 0cm 5.4pt;
+valign="top" width="316">
-  height:214.9pt'>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  avoid'><img border=0 width=300 height=243 id="Grafik 1289"
+style="border: 0px solid ; width: 300px; height: 243px;" alt=""
-  src="Introductionwiki-Dateien/image008.png"></p>
+id="Grafik 1289"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a
+src="https://static.igem.org/mediawiki/2010/0/04/Freiburg10_p5_TATA_less.png"></p>
-  name="_Ref275634090">Figure </a>6: p5 TATA-less promoter is located
+<p class="MsoCaption"
-  downstream of the rep and cap ORF.</p>
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275634090">Figure
-  </td>
+</a>6: p5 TATA-less promoter is located downstream of the rep and
- </tr>
+cap ORF.</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994051"></a><a
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994051"></a><a
 name="_Ref275645443"></a><a name="_Ref275628172">p19</a> promoter</h5>
+<p class="MsoNormal">p19 promoter drives gene expression of the smaller
-<p class=MsoNormal>p19 promoter drives gene expression of the smaller Rep proteins
+Rep proteins
 Rep52 and Rep40. In absence of a helper virus infection the promoter is
-inactive by repression of all four Rep proteins, but is transactivated by
+inactive by repression of all four Rep proteins, but is transactivated
-interaction of both the Sp1 site and Rep protein Rep78/68 bound to the Rep
+by
-binding element (RBE) (Lackner &amp; Muzyczka 2002). By forming a DNA loop (Pereira &amp; Muzyczka 1997) and bringing the two promoters in proximal distance ()
+interaction of both the Sp1 site and Rep protein Rep78/68 bound to the
-additional cellular factors bound to p5 promoter interact with the p19 promoter
+Rep
-leading to transcriptional activation of Rep52/40 (Lackner &amp; Muzyczka 2002). </p>
+binding element (RBE) (Lackner &amp; Muzyczka 2002). By forming a DNA
+loop (Pereira &amp; Muzyczka 1997) and bringing the two promoters in
-<div align=center>
+proximal distance ()
+additional cellular factors bound to p5 promoter interact with the p19
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+promoter
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+leading to transcriptional activation of Rep52/40 (Lackner &amp;
- -2.25pt'>
+Muzyczka 2002). </p>
- <tr style='height:215.65pt'>
+<div align="center">
-  <td width=315 valign=top style='width:235.95pt;padding:0cm 5.4pt 0cm 5.4pt;
+<table class="MsoTableGrid"
-  height:215.65pt'>
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+align="left" border="0" cellpadding="0" cellspacing="0">
-  avoid'><img border=0 width=321 height=250 id="Grafik 1288"
+<tbody>
-  src="Introductionwiki-Dateien/image009.png"
+<tr style="height: 215.65pt;">
-  alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Bea\Rep proteins\Freiburg10_SP1_RBE_Interaction_p5_p19.png"></p>
+<td style="padding: 0cm 5.4pt; width: 235.95pt; height: 215.65pt;"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 7: Forming of the DNA loop brings the p5 rep binding element in proximal distance to
+valign="top" width="315">
-  the Sp1 site foud in the p19 promoter.</p>
+<p class="MsoNormal"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
- </tr>
+style="border: 0px solid ; width: 321px; height: 250px;"
+id="Grafik 1288"
+src="https://static.igem.org/mediawiki/2010/f/f7/Freiburg10_DNA_Loop_P5_to_sp1.png"
+alt=""></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 7: Forming of
+the DNA loop brings the p5 rep binding element in proximal distance to
+the Sp1 site foud in the p19 promoter.</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994052">p40
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994052">p40
 promoter</a></h5>
+<p class="MsoNormal"><span lang="EN-GB">The P40 promoter is derived
-<p class=MsoNormal><span lang=EN-GB>The P40 promoter is derived from the
+from the
-adeno-associated virus serotype 2 (AAV2) genome, where it is located at 40 map
+adeno-associated virus serotype 2 (AAV2) genome, where it is located at
-units. It regulates the transcription of the capsid proteins VP1, VP2 and VP3</span><span lang=EN-GB>(Labow, Hermonat, &amp; Berns, 1986</span><span lang=EN-GB>; </span><span lang=EN-GB>Cassinotti, Weitzand, &amp; Tratschin, 1988)</span><span lang=EN-GB>.
+map
+units. It regulates the transcription of the capsid proteins VP1, VP2
+and VP3</span><span lang="EN-GB">(Labow, Hermonat, &amp; Berns, 1986</span><span
+lang="EN-GB">; </span><span lang="EN-GB">Cassinotti, Weitzand, &amp;
+Tratschin, 1988)</span><span lang="EN-GB">.
 </span></p>
+<p class="MsoNormal"><span lang="EN-GB">Several sequence regions have
-<p class=MsoNormal><span lang=EN-GB>Several sequence regions have been
+been
-identified to be important for maximal promoter activity: Two Sp1 sites, which
+identified to be important for maximal promoter activity: Two Sp1
+sites, which
 are located 250 (Sp1-50) and 270 (GGT-70) base pairs upstream of the
-transcriptional start point and to which Sp1 or Sp1-like proteins bind </span><span lang=EN-GB>(Pereira &amp; Muzyczka 1997)</span><span lang=EN-GB>. </span></p>
+transcriptional start point and to which Sp1 or Sp1-like proteins bind </span><span
+lang="EN-GB">(Pereira &amp; Muzyczka 1997)</span><span lang="EN-GB">. </span></p>
-<p class=MsoNormal><span lang=EN-GB>Referring to the virus genome, p40 can also
+<p class="MsoNormal"><span lang="EN-GB">Referring to the virus genome,
-be induced through transactivation by the Rep proteins. The Sp1-50, together
+p40 can also
-with the CArG-140 site of the P19 promoter, are the main elements involved in
+be induced through transactivation by the Rep proteins. The Sp1-50,
-this process. The Rep proteins, which bind to the Rep binding element in the
+together
-terminal repeat or the P5 promoter, can induce P19 or P40 by interaction with
+with the CArG-140 site of the P19 promoter, are the main elements
+involved in
+this process. The Rep proteins, which bind to the Rep binding element
+in the
+terminal repeat or the P5 promoter, can induce P19 or P40 by
+interaction with
 their bound Sp1 proteins thereby forming a DNA-loop </span><span
-lang=EN-GB>(Pereira &amp; Muzyczka 1997)</span><span lang=EN-GB>. In addition
+lang="EN-GB">(Pereira &amp; Muzyczka 1997)</span><span lang="EN-GB">.
-to that, the TATA box, located at 230, is also required for P40 activity.
+In addition
-Furthermore the ATF-80 and the AP1-40 elements are also important for maximal
+to that, the TATA box, located at 230, is also required for P40
-promoter induction </span><span
+activity.
-lang=EN-GB>(Pereira &amp; Muzyczka 1997)</span><span lang=EN-GB>.</span></p>
+Furthermore the ATF-80 and the AP1-40 elements are also important for
+maximal
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994664"></a><a
+promoter induction </span><span lang="EN-GB">(Pereira &amp; Muzyczka
+)</span><span lang="EN-GB">.</span></p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994664"></a><a
 name="_Toc275994053"></a><a name="_Toc274911366">Integration</a></h3>
+<p class="MsoNormal"><a name="_Toc274911367">The AAV-2 is the only
-<p class=MsoNormal><a name="_Toc274911367">The AAV-2 is the only known
+known
-mammalian virus that integrates into a specific location in the human genome in
+mammalian virus that integrates into a specific location in the human
-the presence of Rep78/68: Chromosome 19, 19q13.3-qter. The site of integration
+genome in
+the presence of Rep78/68: Chromosome 19, 19q13.3-qter. The site of
+integration
 was termed AAVS1. </a></p>
+<p class="MsoNormal">The mechanism of Rep-mediated integration into
-<p class=MsoNormal>The mechanism of Rep-mediated integration into AAVS1 is not
+AAVS1 is not
-yet completely understood and seems to be imprecise and variable. Deletions or
+yet completely understood and seems to be imprecise and variable.
-insertions often occur in the integration process(Schultz &amp; Chamberlain
+Deletions or
-). Linden et al. (1996) proposed a mechanism that is consistent with the
+insertions often occur in the integration process(Schultz &amp;
+Chamberlain
+). Linden et al. (1996) proposed a mechanism that is consistent
+with the
 observed patterns in which AAV exists in an integrated form:</p>
+<p class="MsoNormal">AAVS1 bears a Rep-binding site (RBS) which is
-<p class=MsoNormal>AAVS1 bears a Rep-binding site (RBS) which is similar to the
+similar to the
-RBE in the virus genomes ITR. The Rep proteins are able to simultaneously bind
+RBE in the virus genomes ITR. The Rep proteins are able to
-to AAVS1 and the viral RBE, thereby bringing both strands into close proximity
+simultaneously bind
+to AAVS1 and the viral RBE, thereby bringing both strands into close
+proximity
 towards each other. After binding to the AAVS1 site, Rep acts as an
-endonuclease, the same way it does when binding to the AAV ITR, introducing a
+endonuclease, the same way it does when binding to the AAV ITR,
-single strand-nick between two thymidine residues close to the binding site. This
+introducing a
-produces a free 3’-OH end which acts as a primer for the host cells’ DNA
+single strand-nick between two thymidine residues close to the binding
-polymerase. After replicating the displaced strand, the polymerase switches
+site. This
-templates and replicates the AAV DNA, thereby linking AAVS1 and AAV together.
+produces a free 3’-OH end which acts as a primer for the host cells’
+DNA
+polymerase. After replicating the displaced strand, the polymerase
+switches
+templates and replicates the AAV DNA, thereby linking AAVS1 and AAV
+together.
 Prior to integration, the AAV genome often exists in circular and/or
-concatameric form, resulting in multiple consecutive AAV copies in the host
+concatameric form, resulting in multiple consecutive AAV copies in the
-genome. Another explanation for this phenomenon could be a circularized AAV
+host
-monomer that is being replicated several times in a rolling-circle manner
+genome. Another explanation for this phenomenon could be a circularized
+AAV
+monomer that is being replicated several times in a rolling-circle
+manner
 before being integrated into the host genome. </p>
+<p class="MsoNormal">Another template switch back to the AAVS1 sequence
-<p class=MsoNormal>Another template switch back to the AAVS1 sequence creates a
+creates a
-second link between virus and host. This integration mechanism leaves single-stranded
+second link between virus and host. This integration mechanism leaves
+single-stranded
 gaps that need to be repaired by cellular enzymes before integration is
-complete. Since successful integration of AAV depends on these cellular repair
+complete. Since successful integration of AAV depends on these cellular
-mechanisms, integration happens more frequently in dividing cells, in which
+repair
+mechanisms, integration happens more frequently in dividing cells, in
+which
 repair functions are more active.</p>
+<p class="MsoNormal">Recently, a sequence in the p5 promoter region
-<p class=MsoNormal>Recently, a sequence in the p5 promoter region that enhances
+that enhances
-site-specific integration through interaction with Rep78/68 has been identified,
+site-specific integration through interaction with Rep78/68 has been
-this motif was labeled p5 integration enhancer element (p5IEE). Apparently, p5IEE
+identified,
-is sufficient to create the AAVS1 - Rep68/78 - Viral DNA -complex necessary for
+this motif was labeled p5 integration enhancer element (p5IEE).
+Apparently, p5IEE
+is sufficient to create the AAVS1 - Rep68/78 - Viral DNA -complex
+necessary for
 specific integration, even if the ITRs containing RBEs are not present.</p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994665"></a><a
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994665"></a><a
 name="_Toc275994054">Rescue</a></h3>
+<p class="MsoNormal">If the latently infected host cell is
-<p class=MsoNormal>If the latently infected host cell is superinfected with
+superinfected with
 adenovirus, the integrated virus genome can be rescued from the human
-chromosome and proceed its lytic lifecycle. Adenovirus gene products act as
+chromosome and proceed its lytic lifecycle. Adenovirus gene products
+act as
 activators on AAV gene expression, leading to an excision of the viral
-sequences. Like in the integration process, the Rep 78/68 proteins catalyze the
+sequences. Like in the integration process, the Rep 78/68 proteins
-excision by introduction of single strand nicks at the terminal resolution
+catalyze the
-sites within the terminal repeat structures flanking the AAV genome. DNA
+excision by introduction of single strand nicks at the terminal
-polymerase, displacing the single-stranded AAV sequence, then elongates the
+resolution
-resulting free 3’ OH ends. The incomplete single-stranded AAV sequence missing
+sites within the terminal repeat structures flanking the AAV genome.
-one terminal repeat primes upon itself at the homologous D motives, allowing
+DNA
-DNA polymerase to copy it. This results in full-length, single-stranded AAV molecules,
+polymerase, displacing the single-stranded AAV sequence, then elongates
-which are being able to re-enter the replicative cycle (Srivastava 2008), (Samulski 1993).</p>
+the
+resulting free 3’ OH ends. The incomplete single-stranded AAV sequence
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994666"></a><a
+missing
+one terminal repeat primes upon itself at the homologous D motives,
+allowing
+DNA polymerase to copy it. This results in full-length, single-stranded
+AAV molecules,
+which are being able to re-enter the replicative cycle (Srivastava
+), (Samulski 1993).</p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994666"></a><a
 name="_Toc275994055">Rep proteins</a><a name="_Toc274911368"></a></h3>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994056">Overview</a></h4>
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994056">Overview</a></h4>
+<p class="MsoNormal">The Adeno-associated virus (AAV) consists of two
+open
-<p class=MsoNormal>The Adeno-associated virus (AAV) consists of two open
 reading frames (ORF), <i>rep</i> and <i>cap</i> ORF. The<i> </i>four
-non-structural <i>rep</i> genes are driven by two promoters located at map
+non-structural <i>rep</i> genes are driven by two promoters located at
-units 5 (p5 promoter) and 19 (p19 promoter). Rep proteins are involved in genome encapsidation, regulation of gene expression  and replication of the viral genome. </p>
+map
+units 5 (p5 promoter) and 19 (p19 promoter). Rep proteins are involved
-<div align=center>
+in genome encapsidation, regulation of gene expression and replication
+of the viral genome. </p>
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+<div align="center">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<table class="MsoTableGrid"
- -2.25pt'>
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
- <tr style='height:232.5pt'>
+align="left" border="0" cellpadding="0" cellspacing="0">
-  <td width=533 valign=top style='width:399.65pt;padding:0cm 5.4pt 0cm 5.4pt;
+<tbody>
-  height:232.5pt'>
+<tr style="height: 232.5pt;">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<td style="padding: 0cm 5.4pt; width: 399.65pt; height: 232.5pt;"
-  avoid;text-autospace:none'><img border=0 width=602 height=225 id="Grafik 37"
+valign="top" width="533">
-  src="Introductionwiki-Dateien/image010.png"
+<p class="MsoNormal"
-  alt="Beschreibung: Rep proteins.png"></p>
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>&nbsp;</p>
+style="border: 0px solid ; width: 602px; height: 225px;" id="Grafik 37"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 8: Genomic organization of the AAV-2 genome. The <i>rep</i> gene codes for four
+src="xx" alt=""></p>
-  non-structural proteins – Rep40, Rep52, Rep68 and Rep78 – which are involved
+<p class="MsoCaption"
-  in gene regulation, genome encapsidation and viral DNA integration.</p>
+style="margin-left: 17.85pt; text-indent: 0cm;">&nbsp;</p>
-  </td>
+<p class="MsoCaption"
- </tr>
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 8: Genomic
+organization of the AAV-2 genome. The <i>rep</i> gene codes for four
+non-structural proteins – Rep40, Rep52, Rep68 and Rep78 – which are
+involved in gene regulation, genome encapsidation and viral DNA
+integration.</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">The two larger proteins Rep78/68 play an essential
+role in
-<p class=MsoNormal>The two larger proteins Rep78/68 play an essential role in
+viral genome integration and regulation of AAV gene expression, whereas
-viral genome integration and regulation of AAV gene expression, whereas the
+the
-smaller Rep proteins are involved in viral genome encapsidation. Rep proteins
+smaller Rep proteins are involved in viral genome encapsidation. Rep
-act both as repressors and activators of AAV transcription in respect to the
+proteins
-absence and presence of helper viruses such as adenoviruses (Ad) or herpes
+act both as repressors and activators of AAV transcription in respect
-simplex viruses (HSV) by interacting with several cellular proteins (Nash et al. 2009).</p>
+to the
+absence and presence of helper viruses such as adenoviruses (Ad) or
-<p class=MsoNormal>Furthermore, in the absence of Rep proteins, as it is the
+herpes
-case in recombinant AAVs, integration of the viral genome into the human genome
+simplex viruses (HSV) by interacting with several cellular proteins
-is rare and random. There are several hotspots for integration of wtAAV genomes
+(Nash et al. 2009).</p>
-such as the human chromosome 19q13.42, known as the AAVSI site, but as well some
+<p class="MsoNormal">Furthermore, in the absence of Rep proteins, as it
-other accessible chromatin regions for preferred integration have been found (5p13.3
+is the
+case in recombinant AAVs, integration of the viral genome into the
+human genome
+is rare and random. There are several hotspots for integration of wtAAV
+genomes
+such as the human chromosome 19q13.42, known as the AAVSI site, but as
+well some
+other accessible chromatin regions for preferred integration have been
+found (5p13.3
 and 3p24.3). Integration into the human genome is mediated by the two
-regulatory proteins Rep68 and Rep78 driven by the AAV p5 promoter. The proteins
+regulatory proteins Rep68 and Rep78 driven by the AAV p5 promoter. The
-bind to the Rep binding site (RBS) which is located within the inverted terminal
+proteins
+bind to the Rep binding site (RBS) which is located within the inverted
+terminal
 repeats (ITRs). The minimal consensus Rep binding site (RBS) <span
-style='font-family:"Courier New"'>GAGT GAGC</span> is found within the ITRs and
+style="font-family: &quot;Courier New&quot;;">GAGT GAGC</span> is found within
-in the p5 integration-efficient element (p5IEE) of the p5 promoter (Hüser et al., 2010). Rep78/68 proteins possess DNA-binding (reference), helicase
+the ITRs and
-(reference) and site-specific endonuclease activity located within the first
+in the p5 integration-efficient element (p5IEE) of the p5 promoter
-amino acids (Davis et al. 2000). Since the N-terminal region is unique to
+(Hüser et al., 2010). Rep78/68 proteins possess DNA-binding
-the larger Rep proteins, the two smaller Rep proteins possess other biological
+(reference), helicase
-functions. Rep52/40 gene expression is driven by the p19 promoter which is
+(reference) and site-specific endonuclease activity located within the
-located within <i>rep</i> ORF and the proteins are involved in encapsidating
+first
-the viral genome into the preformed capsids. Gene expression of these proteins
+amino acids (Davis et al. 2000). Since the N-terminal region is
-is suppressed in absence of adenovirus infection by binding of Rep78/68 to the
+unique to
-p5 promoter. Gene expression of p19 and p40 is transacvtivated by the Rep proteins
+the larger Rep proteins, the two smaller Rep proteins possess other
+biological
+functions. Rep52/40 gene expression is driven by the p19 promoter which
+is
+located within <i>rep</i> ORF and the proteins are involved in
+encapsidating
+the viral genome into the preformed capsids. Gene expression of these
+proteins
+is suppressed in absence of adenovirus infection by binding of Rep78/68
+to the
+p5 promoter. Gene expression of p19 and p40 is transacvtivated by the
+Rep proteins
 Rep78/68 during coinfection.</p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994057"></a><a
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994057"></a><a
 name="_Ref275633722"></a><a name="_Toc274911369">Rep 78</a><span
-style='font-family:"Dutch801BT-Bold","serif"'> </span></h5>
+style="font-family: &quot;Dutch801BT-Bold&quot;,&quot;serif&quot;;"> </span></h5>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep78 in a
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  nutshell:</p>
+valign="top" width="619">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     78 kDa</p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Endonuclease
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep78 in a nutshell:</p>
-  activity </p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     ATPase
+style="margin-left: 17.85pt; text-indent: 0cm;">• 78 kDa</p>
-  and helicase activity </p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Regulate
+style="margin-left: 17.85pt; text-indent: 0cm;">• Endonuclease
-  viral gene expression</p>
+activity </p>
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Involved
+<p class="MsoNormal"
-  in genome integration into human chromosome</p>
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
-  </td>
+activity </p>
- </tr>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Regulate viral gene
+expression</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Involved in genome
+integration into human chromosome</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">Regulated by the p5 promoter, Rep78 is the largest
-<p class=MsoNormal>Regulated by the p5 promoter, Rep78 is the largest
 non-structural protein found in the wtAAV. Besides regulation of gene
 expression and viral genome replication, Rep78 has been found to play a
-functional role in AAV site-specific integration into the human genome (Hüser et al. 2010). In absence of Ad helper viruses, overexpression of Rep78 leads to
+functional role in AAV site-specific integration into the human genome
-cell cycle arrest by interacting with cell-cycle regulating phosphatases
+(Hüser et al. 2010). In absence of Ad helper viruses, overexpression of
-causing DNA damage by its intrinsic endonuclease activity (Berthet et al. 2005) and induces apoptosis. Due to its ability to bind to the Rep binding
+Rep78 leads to
-site (RBS) in the p5 integration-efficient element (p5IEE) of the p5 promoter, Rep78
+cell cycle arrest by interacting with cell-cycle regulating
+phosphatases
+causing DNA damage by its intrinsic endonuclease activity (Berthet et
+al. 2005) and induces apoptosis. Due to its ability to bind to the Rep
+binding
+site (RBS) in the p5 integration-efficient element (p5IEE) of the p5
+promoter, Rep78
 mediates gene expression and retain a constant level of Rep proteins by
-suppressing transcriptional activity of the p5 promoter in absence of Ad
+suppressing transcriptional activity of the p5 promoter in absence of
-viruses (Yue et al. 2010). Interaction of Rep78 with cellular factors such as
+Ad
-transcription factors (Lackner &amp; Muzyczka 2002) provides the basis for gene
+viruses (Yue et al. 2010). Interaction of Rep78 with cellular factors
+such as
+transcription factors (Lackner &amp; Muzyczka 2002) provides the basis
+for gene
 regulation by Rep78 in associated with endogenous molecules. </p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994058"></a><a
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994058"></a><a
 name="_Ref275633727"></a><a name="_Toc274911370">Rep 68</a><u> </u></h5>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep68 in a
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  nutshell</p>
+valign="top" width="619">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     68 kDa</p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Endonuclease
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep68 in a nutshell</p>
-  activity </p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     ATPase
+style="margin-left: 17.85pt; text-indent: 0cm;">• 68 kDa</p>
-  and helicase activity </p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Regulate
+style="margin-left: 17.85pt; text-indent: 0cm;">• Endonuclease
-  gene expression</p>
+activity </p>
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Involved
+<p class="MsoNormal"
-  in genome integration into human chromosome</p>
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
-  </td>
+activity </p>
- </tr>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Regulate gene
+expression</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Involved in genome
+integration into human chromosome</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">Rep68 is a regulatory protein driven by the p5
+promoter with
-<p class=MsoNormal>Rep68 is a regulatory protein driven by the p5 promoter with
+an apparent molecular weight of 68 kDa lacking 92 amino acids from the
-an apparent molecular weight of 68 kDa lacking 92 amino acids from the carboxy
+carboxy
 terminus due to splicing of mRNA coding for the two larger Rep proteins.</p>
+<p class="MsoNormal">The non-structural protein Rep68 belongs to the
-<p class=MsoNormal>The non-structural protein Rep68 belongs to the superfamily
+superfamily
-(SF3) helicase found in other small DNA and RNA viruses such as simian virus
+(SF3) helicase found in other small DNA and RNA viruses such as
-(SV40) and bovine papillomavirus (Mansilla-Soto et al. 2009). Formation of oligomeric complexes of Rep proteins provides
+simian virus
-the basis for the functional versatility of the two larger regulatory proteins.
+(SV40) and bovine papillomavirus (Mansilla-Soto et al. 2009).
-The AAA<sup>+</sup> motor domain is known to function as an initiator for
+Formation of oligomeric complexes of Rep proteins provides
-oligomerization of the Rep proteins. The cooperative effect of both domains
+the basis for the functional versatility of the two larger regulatory
+proteins.
+The AAA<sup>+</sup> motor domain is known to function as an initiator
+for
+oligomerization of the Rep proteins. The cooperative effect of both
+domains
 appears to be further regulated by ATP binding as well as different DNA
 substrates such as dsDNA and ssDNA. Assembly of different nucleoprotein
-structures suggest that viral replication and genome integration is regulated
+structures suggest that viral replication and genome integration is
-and controlled by distinct Rep complexes which means that in presence of dsDNA
+regulated
-Rep68 assembles to smaller complexes than in presence of ssDNA resulting in
+and controlled by distinct Rep complexes which means that in presence
+of dsDNA
+Rep68 assembles to smaller complexes than in presence of ssDNA
+resulting in
 octamers. </p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994059"></a><a
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994059"></a><a
 name="_Toc274911372">Rep52</a></h5>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep52 in a
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  nutshell</p>
+valign="top" width="619">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     52 kDa</p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     ATPase
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep52 in a nutshell</p>
-  and helicase activity </p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Involved
+style="margin-left: 17.85pt; text-indent: 0cm;">• 52 kDa</p>
-  in genome encapsidation</p>
+<p class="MsoNormal"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
- </tr>
+activity </p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Involved in genome
+encapsidation</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">Rep 52 is under the control of the p19 promoter
-<p class=MsoNormal>Rep 52 is under the control of the p19 promoter and shares
+and shares
-the same N-terminus with Rep78. It was shown that Rep52 possesses helicase and
+the same N-terminus with Rep78. It was shown that Rep52 possesses
-ATPase activity with 3´-5´polarity (Smith &amp; Kotin 1998). Despite the helicase activity, Rep52 and Rep78 share a putative
+helicase and
-zinc-finger domain, which suggest interactions with diverse cellular factors (Nash et al. 2009) such as transcription factors (Lackner &amp; Muzyczka 2002) and TATA-binding proteins (Hermonat et al. 1998).</p>
+ATPase activity with 3´-5´polarity (Smith &amp; Kotin 1998). Despite
+the helicase activity, Rep52 and Rep78 share a putative
-<h5 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994060"></a><a
+zinc-finger domain, which suggest interactions with diverse cellular
+factors (Nash et al. 2009) such as transcription factors (Lackner &amp;
+Muzyczka 2002) and TATA-binding proteins (Hermonat et al. 1998).</p>
+<h5 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994060"></a><a
 name="_Toc274911371">Rep40</a></h5>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Rep40 in a
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  nutshell</p>
+valign="top" width="619">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     40 kDa</p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     ATPase
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep40 in a nutshell</p>
-  and helicase activity </p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>•     Involved
+style="margin-left: 17.85pt; text-indent: 0cm;">• 40 kDa</p>
-  in genome encapsidation</p>
+<p class="MsoNormal"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
- </tr>
+activity </p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Involved in genome
+encapsidation</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">The smallest Rep protein (Rep40) possesses
-<p class=MsoNormal>The smallest Rep protein (Rep40) possesses helicase and
+helicase and
-ATPase activity as well, but does not have strict requirements for DNA duplexes
+ATPase activity as well, but does not have strict requirements for DNA
-containing a 3´single-stranded end. Rep40 helicase activity requires bivalent
+duplexes
-ions such as Mg<sup>2+</sup> or Mn<sup>2+</sup> and is most active using ATP as
+containing a 3´single-stranded end. Rep40 helicase activity requires
-substrate. Lacking the zinc finger domain, present in Rep52, Rep40 requires
+bivalent
-dimerization for functional helicase activity (Collaco et al. 2003). Rep40/52 proteins are required for translocation of the
+ions such as Mg<sup>2+</sup> or Mn<sup>2+</sup> and is most active
-single-stranded, viral genomes into the preformed capsids proceeding with the
+using ATP as
+substrate. Lacking the zinc finger domain, present in Rep52, Rep40
+requires
+dimerization for functional helicase activity (Collaco et al. 2003).
+Rep40/52 proteins are required for translocation of the
+single-stranded, viral genomes into the preformed capsids proceeding
+with the
 ´end of the DNA (King et al. 2001). </p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+align="left" border="1" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 192.55pt;">
- <tr style='height:192.55pt'>
+<td
-  <td width=285 valign=top style='width:214.0pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 214pt; height: 192.55pt;"
-  padding:0cm 5.4pt 0cm 5.4pt;height:192.55pt'>
+valign="top" width="285">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'><img border=0
+<p class="MsoNormal"
-  width=270 height=232 id="Picture 8"
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
-  src="Introductionwiki-Dateien/image011.jpg"></p>
+style="border: 0px solid ; width: 270px; height: 232px;" alt=""
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 9: Crystal structure of the SF-3 helicase (PDB: 1SH9).</p>
+id="Picture 8"
-  </td>
+src="https://static.igem.org/mediawiki/2010/c/c4/Freiburg10_Crystal_structure_SF3helicase.png"></p>
- </tr>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 9: Crystal
+structure of the SF-3 helicase (PDB: 1SH9).</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal" style="page-break-after: avoid;">&nbsp;</p>
-<p class=MsoNormal style='page-break-after:avoid'>&nbsp;</p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994667"></a><a
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994667"></a><a
 name="_Toc275994061"></a><a name="_Toc274911373">VP proteins</a></h3>
+<p class="MsoNormal">The AAV capsid consists of 60 capsid protein
-<p class=MsoNormal>The AAV capsid consists of 60 capsid protein subunits
+subunits
-composed of the three cap proteins VP1, VP2, and VP3, which are encoded in an
+composed of the three cap proteins VP1, VP2, and VP3, which are encoded
-overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they
+in an
+overlapping reading frame. Arranged in a stoichiometric ratio of
+:1:10, they
 form an icosahedral symmetry. The mRNA encoding for the cap proteins is
-transcribed from p40 and alternative spliced to minor and major products.
+transcribed from p40 and alternative spliced to minor and major
-Alternative splicing and translation initiation of VP2 at a nonconventional ACG
+products.
-initiation codon promote the expression of the VP proteins. VP1, VP2 and VP3 share
+Alternative splicing and translation initiation of VP2 at a
-a common C terminus and stop codon, but begin with a different start codon. The
+nonconventional ACG
-N termini of VP1 and VP2 play important roles in infection and contain motifs
+initiation codon promote the expression of the VP proteins. VP1, VP2
-that are highly homologous to a phospholipase A2 (PLA2) domain and nuclear
+and VP3 share
+a common C terminus and stop codon, but begin with a different start
+codon. The
+N termini of VP1 and VP2 play important roles in infection and contain
+motifs
+that are highly homologous to a phospholipase A2 (PLA2) domain and
+nuclear
 localization signals (NLSs). These elements are conserved in almost all
 parvoviruses. (Johnson et al. 2010a)</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal" style="margin-bottom: 0.0001pt;">&nbsp;</p>
+<table class="MsoTableGrid"
-<p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt'>&nbsp;</p>
+style="border: medium none ; width: 466.1pt; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0" width="621">
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+<tbody>
- width=621 style='width:466.1pt;border-collapse:collapse;border:none;
+<tr>
- margin-left:-2.25pt;margin-right:-2.25pt'>
+<td
- <tr>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 466.1pt;"
-  <td width=621 valign=top style='width:466.1pt;border:solid windowtext 1.0pt;
+valign="top" width="621">
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<table class="MsoTableGrid"
-  <table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-   style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+align="left" border="1" cellpadding="0" cellspacing="0">
-   -2.25pt'>
+<tbody>
-   <tr style='height:134.65pt'>
+<tr style="height: 134.65pt;">
-    <td width=588 valign=top style='width:440.85pt;border:solid windowtext 1.0pt;
+<td
-    padding:0cm 5.4pt 0cm 5.4pt;height:134.65pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 440.85pt; height: 134.65pt;"
-    <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+valign="top" width="588">
-    avoid'><span style='font-size:12.0pt;line-height:150%'><img border=0
+<p class="MsoNormal"
-    width=574 height=179 id="Grafik 39"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><span
-    src="Introductionwiki-Dateien/image012.png"
+style="font-size: 12pt; line-height: 150%;"><img id="Grafik 39"
-    alt="Beschreibung: Cap proteins.png"></span></p>
+src="Introductionwiki-Dateien/image012.png"
-    <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 10 Genomic organization of the AAV 2 genome. The <i>cap</i>  gene codes for the three
+alt="Beschreibung: Cap proteins.png" border="0" height="179"
-    capsid proteins VP1, Vp2 and VP3,  which are responsible for the assembly
+width="574"></span></p>
-    of the capsid </p>
+<p class="MsoCaption"
-    </td>
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 10 Genomic
-   </tr>
+organization of the AAV 2 genome. The <i>cap</i> gene codes for the
-  </table>
+three capsid proteins VP1, Vp2 and VP3, which are responsible for the
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'></p>
+assembly of the capsid </p>
-  </td>
+</td>
- </tr>
+</tr>
+</tbody>
 </table>
+</td>
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994062"></a><a
+</tr>
+</tbody>
+</table>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994062"></a><a
 name="_Toc274911374">VP1</a></h4>
+<p class="MsoNormal">Whereas VP1 is translated from the minor spliced
-<p class=MsoNormal>Whereas VP1 is translated from the minor spliced mRNA, VP2
+mRNA, VP2
-and VP3 are translated from the major spliced mRNA. The minor spliced product
+and VP3 are translated from the major spliced mRNA. The minor spliced
-is approximately 10-fold less abundant than the major spliced mRNA. Thus, there
+product
-is much less VP1 than VP2 and VP3 resulting in a capsid stoichiometric ratio of
+is approximately 10-fold less abundant than the major spliced mRNA.
-:1:10. The N terminus of VP1 has an extension of 65 amino acids including an
+Thus, there
-additional extension of 138 N-terminal amino acids forming the unique portion
+is much less VP1 than VP2 and VP3 resulting in a capsid stoichiometric
-of VP1. It contains a motif of about 70 amino acids that is highly homologous
+ratio of
+:1:10. The N terminus of VP1 has an extension of 65 amino acids
+including an
+additional extension of 138 N-terminal amino acids forming the unique
+portion
+of VP1. It contains a motif of about 70 amino acids that is highly
+homologous
 to a phospholipase A2 (PLA2) domain. Furthermore, there are nuclear
-localization sequences (BR)(+) which are supposed to be necessary for endosomal
+localization sequences (BR)(+) which are supposed to be necessary for
-escape and nuclearentry. <span lang=DE>(Bleker et al. 2006)</span><span
+endosomal
-lang=DE>, </span><span
+escape and nuclearentry. <span lang="DE">(Bleker et al. 2006)</span><span
-lang=DE>(Johnson et al. 2010b)</span><span lang=DE>, </span><span
+lang="DE">, </span><span lang="DE">(Johnson et al. 2010b)</span><span
-lang=DE>(DiPrimio et al. 2008)</span><span lang=DE>.</span></p>
+lang="DE">, </span><span lang="DE">(DiPrimio et al. 2008)</span><span
+lang="DE">.</span></p>
-<p class=MsoNormal><span lang=DE>&nbsp;</span></p>
+<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+<p class="MsoNormal">Phospholipases are enzymes that hydrolyze
-<p class=MsoNormal>Phospholipases are enzymes that hydrolyze phospholipids into
+phospholipids into
-fatty acids and other lipophilic substances and can be found in mammalian
+fatty acids and other lipophilic substances and can be found in
-tissues but also in insect and snake venom. They are subdivided into four major
+mammalian
-classes, termed A, B, C and D distinguished by the type of reaction they
+tissues but also in insect and snake venom. They are subdivided into
-catalyse whereas the position of hydrolysis on the glycerol backbone defines
+four major
+classes, termed A, B, C and D distinguished by the type of reaction
+they
+catalyse whereas the position of hydrolysis on the glycerol backbone
+defines
 the class of phospholipase.</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+align="left" border="1" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr>
- <tr>
+<td
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  padding:0cm 5.4pt 0cm 5.4pt'>
+valign="top" width="619">
-  <p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;
+<p class="MsoNormal"
-  text-indent:0cm;page-break-after:avoid'><img border=0 width=502 height=273
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
-  id="Picture 2064" src="Introductionwiki-Dateien/image013.jpg"></p>
+align="center"><img
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 11: The schematic depiction of some AAV2 domains. VP1 contains a phospholipase A2
+style="border: 0px solid ; width: 502px; height: 273px;" alt=""
-  (PLA2) domain and four basic regions (BR1–4) located at the N-terminus of VP1
+id="Picture 2064"
-  – VP3. The HSPG binding domain is generated by the basic residues at
+src="https://static.igem.org/mediawiki/2010/e/e8/Freiburg10_VP1-3_overview.jpg"></p>
-  positions R484, R487, K532, R585, and R588 which are located near the
+<p class="MsoCaption"
-  C-terminus of theVP proteins. The NGR motif 511–513 forms an integrin <span
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 11: The
-  style='font-family:Symbol'>a</span>5<span style='font-family:Symbol'>b</span>1
+schematic depiction of some AAV2 domains. VP1 contains a phospholipase
-  binding domain. Adapted from (Michelfelder &amp; Trepel 2009)</p>
+A2 (PLA2) domain and four basic regions (BR1–4) located at the
-  </td>
+N-terminus of VP1 – VP3. The HSPG binding domain is generated by the
- </tr>
+basic residues at positions R484, R487, K532, R585, and R588 which are
+located near the C-terminus of theVP proteins. The NGR motif 511–513
+forms an integrin <span style="font-family: Symbol;">a</span>5<span
+style="font-family: Symbol;">b</span>1 binding domain. Adapted from
+(Michelfelder &amp; Trepel 2009)</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">It specifically recognizes and hydrolyzes the sn-2
+acyl bond
-<p class=MsoNormal>It specifically recognizes and hydrolyzes the sn-2 acyl bond
 of phospholipids releasing arachidonic acid and lysophospholipids.</p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+align="left" border="1" cellpadding="0" cellspacing="0">
- -2.25pt'>
+<tbody>
- <tr>
+<tr>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  <p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;
+valign="top" width="619">
-  text-indent:0cm;page-break-after:avoid'><img border=0 width=437 height=227
+<p class="MsoNormal"
-  id="Picture 2065" src="Introductionwiki-Dateien/image014.jpg"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
-  alt="Beschreibung: Unbenannt1"></p>
+align="center"><img
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 12:  adapted from http://www.biochemtech.uni-halle.de/im/1182353660_397_00_800.jpg</p>
+style="border: 0px solid ; width: 437px; height: 227px;"
-  </td>
+id="Picture 2065"
- </tr>
+src="https://static.igem.org/mediawiki/2010/8/87/Freiburg10_PLA2-recognition_site.jpg"
+alt=""></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 12: adapted
+from http://www.biochemtech.uni-halle.de/im/1182353660_397_00_800.jpg</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">The reaction mechanism will be depicted by the
+following
-<p class=MsoNormal>The reaction mechanism will be depicted by the following
 image:</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+align="left" border="1" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr>
- <tr>
+<td
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  padding:0cm 5.4pt 0cm 5.4pt'>
+valign="top" width="619">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'><img border=0
+<p class="MsoNormal"
-  width=604 height=485 id="Picture 2066"
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
-  src="Introductionwiki-Dateien/image015.jpg"></p>
+style="border: 0px solid ; width: 604px; height: 485px;" alt=""
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 13 adapted from “Interfacial Enzymology: The Secreted Phospholipase A2-Paradigm”</p>
+id="Picture 2066"
-  </td>
+src="https://static.igem.org/mediawiki/2010/0/0c/Freiburg10_PLA2-mechanism.PNG"></p>
- </tr>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 13 adapted from
+“Interfacial Enzymology: The Secreted Phospholipase A2-Paradigm”</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">There are two possible mechanism but the one on
+the right
-<p class=MsoNormal>There are two possible mechanism but the one on the right
+side has a lower transient state. The mechanism of sPLA2 on the right
-side has a lower transient state. The mechanism of sPLA2 on the right side is
+side is
-initiated by a His-48/Asp-99/calcium complex within the active site. The sn-2
+initiated by a His-48/Asp-99/calcium complex within the active site.
-carbonyl oxygen becomes polarized by the calcium ion while also influencing
+The sn-2
-catalytic water molecule, w5..Via the bridging second water molecule w6 His-48
+carbonyl oxygen becomes polarized by the calcium ion while also
-improves the nucleophilicity of the catalytic water.  According to propositions
+influencing
-two water molecules are needed to bypass the distance between the catalytic
+catalytic water molecule, w5..Via the bridging second water molecule w6
-histidine and the ester.. Asp-99 is thought to enhance the basicity of His-48
+His-48
-through hydrogen bonding. Substituting the His-48 with an asparagine maintains
+improves the nucleophilicity of the catalytic water. According to
-wild-typ activity because the functional group on asparagines can function to lower
+propositions
+two water molecules are needed to bypass the distance between the
+catalytic
+histidine and the ester.. Asp-99 is thought to enhance the basicity of
+His-48
+through hydrogen bonding. Substituting the His-48 with an asparagine
+maintains
+wild-typ activity because the functional group on asparagines can
+function to lower
 the pKa of the bridging water molecule, too (Berg et al. 2001). </p>
+<p class="MsoNormal"><span style="line-height: 150%;">The phospholipase
-<p class=MsoNormal><span style='line-height:150%'>The phospholipase A2 is suggested
+A2 is suggested
-to mediate membrane disruption of the vesicular compartment, which would allow
+to mediate membrane disruption of the vesicular compartment, which
-escape of the virion into the cytosol, although this has not been demonstrated
+would allow
-for AAV so far.The propensity of cellular PLA2 to cleave phospholipids  is usually
+escape of the virion into the cytosol, although this has not been
-regulated by intracellular Ca<sup>2+</sup> levels as well as phosphorylation of
+demonstrated
+for AAV so far.The propensity of cellular PLA2 to cleave phospholipids
+is usually
+regulated by intracellular Ca<sup>2+</sup> levels as well as
+phosphorylation of
 residues near the catalytic domain of the PLA2. Not surprisingly, the
-N-terminus of VP1 contains  a GXG binding site and several phosphorylation
+N-terminus of VP1 contains a GXG binding site and several
+phosphorylation
 sites.</span></p>
+<p class="MsoNormal"><span style="line-height: 150%;">&nbsp;</span></p>
-<p class=MsoNormal><span style='line-height:150%'>&nbsp;</span></p>
+<p class="MsoNormal"><span style="line-height: 150%;">According to the
+structural
-<p class=MsoNormal><span style='line-height:150%'>According to the structural
+modeling of VP1 the N-terminus can translocate through the 5-fold axis
-modeling of VP1 the N-terminus can translocate through the 5-fold axis of
+of
-symmetry in the capsid and expose the first 185 residues of VP1 (comment:
+symmetry in the capsid and expose the first 185 residues of VP1
-experimental data also “</span><span style='line-height:150%'>strongly suggest
+(comment:
-that N-termini of VP1 </span>harboring the PLA2 domain can be exposed on the
+experimental data also “</span><span style="line-height: 150%;">strongly
-capsid surface through the pores at the fivefold symmetry axes” (Girod et al. 2002)(Bleker et al. 2005a). It only takes about 19 amino acids to reach
+suggest
-through a phospholipid bilayer so this length would be sufficient for the
+that N-termini of VP1 </span>harboring the PLA2 domain can be exposed
-presentation of the NLS and di-lysine sequences to the cytosol assuming the
+on the
-PLA2 domain<span style='line-height:150%'> had penetrated through an endosomal
+capsid surface through the pores at the fivefold symmetry axes” (Girod
+et al. 2002)(Bleker et al. 2005a). It only takes about 19 amino acids
+to reach
+through a phospholipid bilayer so this length would be sufficient for
+the
+presentation of the NLS and di-lysine sequences to the cytosol assuming
+the
+PLA2 domain<span style="line-height: 150%;"> had penetrated through an
+endosomal
 membrane or Golgi. </span><span
-style='font-size:10.0pt;line-height:150%'>(Michelfelder &amp; Trepel 2009)</span></p>
+style="font-size: 10pt; line-height: 150%;">(Michelfelder &amp; Trepel
+)</span></p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal">Analysing individual steps in the life cycle of
-<p class=MsoNormal>Analysing individual steps in the life cycle of several VP1up
+several VP1up
-mutants and wtAAV-2 lead to the following conclusions: (i) mutations in VP1up
+mutants and wtAAV-2 lead to the following conclusions: (i) mutations in
-did not affect DNA packaging or replication but resulted in a strong reduction of
+VP1up
-infectivity; (ii) this decrease in virus infectivity correlated with a loss in
+did not affect DNA packaging or replication but resulted in a strong
-pvPLA2 activity; (iii) binding to the cell surface and entry into cells was not
+reduction of
-affected in VP1up mutants; (iv) however, these mutants showed obviously reduced
+infectivity; (ii) this decrease in virus infectivity correlated with a
-and delayed Rep expression. (Girod et al. 2002) Summarizing these results the pvPLA2 activity is required for a step
+loss in
-in the life cycle of the virus following perinuclear accumulation of virions
+pvPLA2 activity; (iii) binding to the cell surface and entry into cells
+was not
+affected in VP1up mutants; (iv) however, these mutants showed obviously
+reduced
+and delayed Rep expression. (Girod et al. 2002) Summarizing these
+results the pvPLA2 activity is required for a step
+in the life cycle of the virus following perinuclear accumulation of
+virions
 but (Girod et al. 2002) before the onset of early gene expression.</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">Maybe future work will uncover whether the PLA2
+domain in
-<p class=MsoNormal>Maybe future work will uncover whether the PLA2 domain in
+AAV performs optimally in a specific vesicular compartment, prefers a
-AAV performs optimally in a specific vesicular compartment, prefers a specific
+specific
-phospholipid substrate, operates at multiple cellular membranes such as the
+phospholipid substrate, operates at multiple cellular membranes such as
-endosome and the nuclear envelope, or if its activity is regulated by cellular
+the
+endosome and the nuclear envelope, or if its activity is regulated by
+cellular
 components. </p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994063"></a><a
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994063"></a><a
 name="_Toc274911375">VP2</a></h4>
+<p class="MsoNormal">The translation of VP2 from the major spliced mRNA
-<p class=MsoNormal>The translation of VP2 from the major spliced mRNA is less
+is less
-efficiently compared to the translation of VP3 because it initiates at a Thr
+efficiently compared to the translation of VP3 because it initiates at
-codon (ACG). VP2 and VP1 have an extension at the N terminus that remains
+a Thr
-internal when exposing the capsid to experimental conditions like low pH or
+codon (ACG). VP2 and VP1 have an extension at the N terminus that
-heat. The N terminus of VP1 has an extension of 65 amino acids and similar to
+remains
-VP1 it has two functional elements: a phospholipase A2 (PLA2) domain and nuclear
+internal when exposing the capsid to experimental conditions like low
-localization signals (BR)(+). The exact role of VP2 remains unknown, although
+pH or
-the protein is thought to be nonessential for viral assembly and infectivity  (Johnson et al. 2010b), (DiPrimio et al. 2008).</p>
+heat. The N terminus of VP1 has an extension of 65 amino acids and
+similar to
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994064"></a><a
+VP1 it has two functional elements: a phospholipase A2 (PLA2) domain
+and nuclear
+localization signals (BR)(+). The exact role of VP2 remains unknown,
+although
+the protein is thought to be nonessential for viral assembly and
+infectivity (Johnson et al. 2010b), (DiPrimio et al. 2008).</p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994064"></a><a
 name="_Toc274911376">VP</a>3</h4>
+<p class="MsoNormal">Contained in VP1 and VP2, VP3 is the primary
-<p class=MsoNormal>Contained in VP1 and VP2, VP3 is the primary capsid protein
+capsid protein
-that determines the surface topology of the AAV capsid. The capsid in turn
+that determines the surface topology of the AAV capsid. The capsid in
-dictates antigenicity and tropism. In comparison to the initiation of VP1 and
+turn
-VP2 the initiation of VP3 is because of a Met codon highly efficient (DiPrimio et al. 2008). </p>
+dictates antigenicity and tropism. In comparison to the initiation of
+VP1 and
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+VP2 the initiation of VP3 is because of a Met codon highly efficient
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+(DiPrimio et al. 2008). </p>
- -2.25pt'>
+<table class="MsoTableGrid"
- <tr style='height:222.55pt'>
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-  <td width=589 valign=top style='width:441.8pt;border:solid windowtext 1.0pt;
+align="left" border="1" cellpadding="0" cellspacing="0">
-  padding:0cm 5.4pt 0cm 5.4pt;height:222.55pt'>
+<tbody>
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<tr style="height: 222.55pt;">
-  avoid'><img border=0 width=575 height=288 id="Grafik 40"
+<td
-  src="Introductionwiki-Dateien/image016.png" alt="Beschreibung: VP1_2_3.png"></p>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 441.8pt; height: 222.55pt;"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 14: Genomic organization of the AAV-2 genome. The three capsid proteins VP1, Vp2 and
+valign="top" width="589">
-  VP3 have a similar C terminus. The N termini of VP1 and VP2 contain a
+<p class="MsoNormal"
-  phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+).</p>
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  </td>
+id="Grafik 40" src="Introductionwiki-Dateien/image016.png"
- </tr>
+alt="Beschreibung: VP1_2_3.png" border="0" height="288" width="575"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 14: Genomic
+organization of the AAV-2 genome. The three capsid proteins VP1, Vp2
+and VP3 have a similar C terminus. The N termini of VP1 and VP2 contain
+a phospholipase A2 (PLA2) domain and nuclear localization signals
+(BR)(+).</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994065">Natrual
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994065">Natrual
 Tropism and HSPG motif</a></h4>
+<p class="MsoNormal" style="">The primary receptor of AAV-2 is
-<p class=MsoNormal style='text-autospace:none'>The primary receptor of AAV-2 is
+the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006).
-the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its binding motif consists of five amino-acids located on the
+Its binding motif consists of five amino-acids located on the
 capsid surface (Trepel, Vectors, et al. 2009).</p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  avoid;text-autospace:none'><img border=0 width=628 height=211 id="Picture 39"
+valign="top" width="619">
-  src="Introductionwiki-Dateien/image017.png" alt="Beschreibung: Unbenannt"></p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;text-autospace:
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  none'>Figure 15: adapted from (Trepel, Vectors, et al. 2009). Schematic depiction of the 5 basic amino acids forming
+id="Picture 39" src="Introductionwiki-Dateien/image017.png"
-  the HSPG motif: R484/R487, K532, R585/587. Other domains or binding motifs
+alt="Beschreibung: Unbenannt" border="0" height="211" width="628"></p>
-  are not shown in this picture.</p>
+<p class="MsoNormal"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 15: adapted
- </tr>
+from (Trepel, Vectors, et al. 2009). Schematic depiction of the 5 basic
+amino acids forming the HSPG motif: R484/R487, K532, R585/587. Other
+domains or binding motifs are not shown in this picture.</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal" style="">&nbsp;</p>
-<p class=MsoNormal style='text-autospace:none'>&nbsp;</p>
+<p class="MsoNormal" style="">HSPG belongs to the
-<p class=MsoNormal style='text-autospace:none'>HSPG belongs to the
 glycosaminoglycanes as well as heparin and consists of heparan sulfate <span
-lang=EN>glycosaminoglycan attached to a</span> core protein and can be found on
+lang="EN">glycosaminoglycan attached to a</span> core protein and can
+be found on
 every human cell surface. </p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+align="left" border="1" cellpadding="0" cellspacing="0">
- -2.25pt'>
+<tbody>
- <tr>
+<tr>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  <p class=MsoNormal align=center style='margin-left:17.85pt;text-align:center;
+valign="top" width="619">
-  text-indent:0cm;page-break-after:avoid;text-autospace:none'><img border=0
+<p class="MsoNormal"
-  width=510 height=193 id="Picture 37"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
-  src="Introductionwiki-Dateien/image018.png" alt="Beschreibung: Unbenannt2"></p>
+align="center"><img id="Picture 37"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 16: adapted from (Sinnis et al. 2007) amidosulfated disaccharid sequence in heparin.</p>
+src="Introductionwiki-Dateien/image018.png"
-  </td>
+alt="Beschreibung: Unbenannt2" border="0" height="193" width="510"></p>
- </tr>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 16: adapted
+from (Sinnis et al. 2007) amidosulfated disaccharid sequence in heparin.</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal" style="">&nbsp;</p>
-<p class=MsoNormal style='text-autospace:none'>&nbsp;</p>
+<p class="MsoNormal" style="">Its acid residues bear negative
+charges and are therefore prone to electrostatic interactions with e.g.
-<p class=MsoNormal style='text-autospace:none'>Its acid residues bear negative
+the
-charges and are therefore prone to electrostatic interactions with e.g. the
+positively charged HSPG binding motif of AAV-2. Other interactions with
-positively charged HSPG binding motif of AAV-2. Other interactions with polar
+polar
 residues are possible, too.</p>
+<p class="MsoNormal" style="">Regarding AAV-2, two point
-<p class=MsoNormal style='text-autospace:none'>Regarding AAV-2, two point
+mutations in AAV-2 (R585A and R588A) are sufficient to eliminate
-mutations in AAV-2 (R585A and R588A) are sufficient to eliminate heparin binding
+heparin binding
 (Opie et al. 2003). The biobricks with this knockout are annotated with
 „HSPG-ko“.</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994066"><span
+lang="DE">Assembly-activating protein</span></a></h4>
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994066"><span
+<p class="MsoNormal">A gene encoding for the assembly-activating
-lang=DE>Assembly-activating protein</span></a></h4>
+protein (AAP)
+was recently (in 2010) discovered in the Adeno-associated virus (AAV)
-<p class=MsoNormal>A gene encoding for the assembly-activating protein (AAP)
+serotype
-was recently (in 2010) discovered in the Adeno-associated virus (AAV) serotype
+genome. Its gene product is conserved among all AAV serotypes,
-genome. Its gene product is conserved among all AAV serotypes, illustrating
+illustrating
-its essential role in virus life cycle. Its functions comprise transport of the
+its essential role in virus life cycle. Its functions comprise
-viral structural proteins to the nucleolus and involvement in following capsid
+transport of the
+viral structural proteins to the nucleolus and involvement in following
+capsid
 assembly.</p>
+<p class="MsoNormal">The AAP gene, located in the Cap coding region, is
-<p class=MsoNormal>The AAP gene, located in the Cap coding region, is
+translated from an alternative open reading frame (ORF) with
-translated from an alternative open reading frame (ORF) with unconventional
+unconventional
-start codon. If modifications need to be introduced in the AAV capsid – for
+start codon. If modifications need to be introduced in the AAV capsid –
-example for targeting approaches – the AAP has to be taken in account in order
+for
+example for targeting approaches – the AAP has to be taken in account
+in order
 to prevent virus assembly impairment.</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">&nbsp;</p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994668"></a><a
-<p class=MsoNormal>&nbsp;</p>
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994668"></a><a
 name="_Toc275994067">Trafficking</a></h3>
+<p class="MsoNormal">For creating efficient AAV2 vectors, precise
-<p class=MsoNormal>For creating efficient AAV2 vectors, precise knowledge of
+knowledge of
-the events following virus transduction is necessary. The subsequent scheme and
+the events following virus transduction is necessary. The subsequent
-summary is intended to be an introduction into the complex process of virus
+scheme and
+summary is intended to be an introduction into the complex process of
+virus
 transduction. </p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+align="left" border="1" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 354.75pt;">
- <tr style='height:354.75pt'>
+<td
-  <td width=422 valign=top style='width:316.8pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 316.8pt; height: 354.75pt;"
-  padding:0cm 5.4pt 0cm 5.4pt;height:354.75pt'>
+valign="top" width="422">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<p class="MsoNormal"
-  avoid'><img border=0 width=456 height=510 id="Grafik 123"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  src="Introductionwiki-Dateien/image019.png"></p>
+id="Grafik 123" src="Introductionwiki-Dateien/image019.png" border="0"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 17: Adapted from (Hildegard Büning et al. 2008)</p>
+height="510" width="456"></p>
-  </td>
+<p class="MsoCaption"
- </tr>
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 17: Adapted
+from (Hildegard Büning et al. 2008)</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">After several contacts with cellular structures
+like heperan
-<p class=MsoNormal>After several contacts with cellular structures like heperan
 sulphate proteoglycan (HSPG) the viral capsid proteins get rearranged.
-Clathrin-mediated endocytosis and cellular trafficking into thecell’scenter
+Clathrin-mediated endocytosis and cellular trafficking into
-follows. After acidification and following endosomal escape, the viral genome
+thecell’scenter
-is transferred into the nucleus and replicated (lytic phase) or integrated into
+follows. After acidification and following endosomal escape, the viral
+genome
+is transferred into the nucleus and replicated (lytic phase) or
+integrated into
 the host genome (latent phase). </p>
+<p class="MsoNormal"><span style="color: black;">Before entering the
-<p class=MsoNormal><span style='color:black'>Before entering the cell, the
+cell, the
-viral particle has in average 4.4 contacts with the cellular surface.</span><span style='color:black'>(Seisenberger et al. 2001)</span><span style='color:black'>
+viral particle has in average 4.4 contacts with the cellular surface.</span><span
-The main receptor of AAV2 is heperan sulfate proteoglycan (HSPG). After contact
+style="color: black;">(Seisenberger et al. 2001)</span><span
+style="color: black;">
+The main receptor of AAV2 is heperan sulfate proteoglycan (HSPG). After
+contact
 with HSPG the capsid structure gets rearranged </span><span
-style='color:black'>(Levy et al. 2009)</span><span style='color:black'>. This
+style="color: black;">(Levy et al. 2009)</span><span
-is probably essential for interaction with other cofactors, which leads to
+style="color: black;">. This
-endocytosis. The factors respectively co-receptors of the cellular surface are
+is probably essential for interaction with other cofactors, which leads
-known to enhance the initial binding affinity of HSPG: Fibroblast growth factor
+to
-receptor 1 (FGFR-1), hepatocyte growth factor receptor (HGFR) and laminin
+endocytosis. The factors respectively co-receptors of the cellular
-receptor. It is known that AAVs affect both: </span><span lang=DE
+surface are
-style='color:black'>&#945;</span><span style='color:black'>V</span><span
+known to enhance the initial binding affinity of HSPG: Fibroblast
-lang=DE style='color:black'>&#946;</span><sub><span style='color:black'>5</span></sub><span
+growth factor
-style='color:black'>&nbsp;and </span><span lang=DE style='color:black'>&#945;</span><span
+receptor 1 (FGFR-1), hepatocyte growth factor receptor (HGFR) and
-style='color:black'>V</span><span lang=DE style='color:black'>&#946;</span><sub><span
+laminin
-style='color:black'>1</span></sub><span style='color:black'>integrin. The </span><span
+receptor. It is known that AAVs affect both: </span><span
-lang=DE style='color:black'>&#945;</span><span style='color:black'>V</span><span
+style="color: black;" lang="DE">α</span><span style="color: black;">V</span><span
-lang=DE style='color:black'>&#946;</span><sub><span style='color:black'>1</span></sub><span
+style="color: black;" lang="DE">β</span><sub><span
-style='color:black'>&nbsp;-binding site is an asparagine-glycine-arginine motif
+style="color: black;">5</span></sub><span style="color: black;">&nbsp;and
-</span><span
+</span><span style="color: black;" lang="DE">α</span><span
-style='color:black'>(Asokan et al. 2006)</span><span style='color:black'>.
+style="color: black;">V</span><span style="color: black;" lang="DE">β</span><sub><span
-These integrins interact with intracellular molecules like Rho, Rac and Cdc42
+style="color: black;">1</span></sub><span style="color: black;">integrin.
+The </span><span style="color: black;" lang="DE">α</span><span
+style="color: black;">V</span><span style="color: black;" lang="DE">β</span><sub><span
+style="color: black;">1</span></sub><span style="color: black;">&nbsp;-binding
+site is an asparagine-glycine-arginine motif
+</span><span style="color: black;">(Asokan et al. 2006)</span><span
+style="color: black;">.
+These integrins interact with intracellular molecules like Rho, Rac and
+Cdc42
 GTPases. Figure 2 depicts the following cascade.</span></p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+align="left" border="1" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 232.6pt;">
- <tr style='height:232.6pt'>
+<td
-  <td width=427 valign=top style='width:320.35pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 320.35pt; height: 232.6pt;"
-  padding:0cm 5.4pt 0cm 5.4pt;height:232.6pt'>
+valign="top" width="427">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<p class="MsoNormal"
-  avoid'><img border=0 width=420 height=438 id="Grafik 1044"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  src="Introductionwiki-Dateien/image020.png"></p>
+id="Grafik 1044" src="Introductionwiki-Dateien/image020.png" border="0"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 18: Adapted from (Sanlioglu et al. 2000a)</p>
+height="438" width="420"></p>
-  </td>
+<p class="MsoCaption"
- </tr>
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 18: Adapted
+from (Sanlioglu et al. 2000a)</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">The initial contact with HSPG, FGFR-1, HGFR and/or
+laminin
-<p class=MsoNormal>The initial contact with HSPG, FGFR-1, HGFR and/or laminin
+is followed by an interaction with αVβ5 and/or αVβ1 which
-is followed by an interaction with &#945;V&#946;5 and/or &#945;V&#946;1 which
+propably leads to an intracellular activation of enzymes involved in
-propably leads to an intracellular activation of enzymes involved in the
+the
-rearrangement of cytoskeletal proteins like actin, via PI3K-pathway (Kapeller &amp; Cantley 1994), (Li et al. 1998). In general, the receptor-mediated
+rearrangement of cytoskeletal proteins like actin, via PI3K-pathway
-endocytosis (RME) is a complex process proteins and co-factors form clathrin
+(Kapeller &amp; Cantley 1994), (Li et al. 1998). In general, the
+receptor-mediated
+endocytosis (RME) is a complex process proteins and co-factors form
+clathrin
 coated pits as shown in Figure 16.</p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+align="left" border="1" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 249pt;">
- <tr style='height:249.0pt'>
+<td
-  <td width=469 valign=top style='width:351.6pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 351.6pt; height: 249pt;"
-  padding:0cm 5.4pt 0cm 5.4pt;height:249.0pt'>
+valign="top" width="469">
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+<p class="MsoNormal"
-  avoid'><img border=0 width=484 height=330 id="Grafik 59"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  src="Introductionwiki-Dateien/image021.png"
+id="Grafik 59" src="Introductionwiki-Dateien/image021.png"
-  alt="Beschreibung: File:Freiburg10 Receptor-mediated endocytosis by clathrin-coated vesicles scheme.PNG"></p>
+alt="Beschreibung: File:Freiburg10 Receptor-mediated endocytosis by clathrin-coated vesicles scheme.PNG"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><a
+border="0" height="330" width="484"></p>
-  name="_Ref275689978">Figure </a>19:</p>
+<p class="MsoCaption"
-  </td>
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275689978">Figure
- </tr>
+</a>19:</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">The adaptor proteins (APs) AP1, AP2, AP3 and AP4
+are
-<p class=MsoNormal>The adaptor proteins (APs) AP1, AP2, AP3 and AP4 are
+complexes built of four subunits (Collins et al. 2002), (Asokan et al.
-complexes built of four subunits (Collins et al. 2002), (Asokan et al. 2006). Except for AP2, which requires
+). Except for AP2, which requires
-GTP-bound-Arf1, the APs are linked via phosphatidylinositol (4,5)-bisphosphate
+GTP-bound-Arf1, the APs are linked via phosphatidylinositol
-(PIP2) to the cell membrane (Robinson 2004). APs recognize short cytoplasmatic
+(4,5)-bisphosphate
-motifs like YXX-phi (phi: bulky hydrophobic AA) of transmembrane receptors. In general,
+(PIP2) to the cell membrane (Robinson 2004). APs recognize short
-the recognition sites (mu-subunits) in the AP-complexes have to be phosphorylated
+cytoplasmatic
+motifs like YXX-phi (phi: bulky hydrophobic AA) of transmembrane
+receptors. In general,
+the recognition sites (mu-subunits) in the AP-complexes have to be
+phosphorylated
 by kinases (Ohno et al. 1995).</p>
+<p class="MsoNormal">The actual scaffold of the endosome is build by
-<p class=MsoNormal>The actual scaffold of the endosome is build by the
+the
-triskelion formed clathrins. The rigide backbone of clathrins is formed by
+triskelion formed clathrins. The rigide backbone of clathrins is formed
-three heavy chains (Ybe et al. 1999) and three light chains are regulating
+by
+three heavy chains (Ybe et al. 1999) and three light chains are
+regulating
 assembly competence (BRODSKY et al. 1991). After building the clathrin
-scaffold, dynamine is responsible for pinching-off the clathrin-coated pits (CCPs)
+scaffold, dynamine is responsible for pinching-off the clathrin-coated
-from the cell’s membrane (Summerford &amp; Samulski 1998) (Sanlioglu et al. 2000b). </p>
+pits (CCPs)
+from the cell’s membrane (Summerford &amp; Samulski 1998) (Sanlioglu et
-<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994068">Endosomal
+al. 2000b). </p>
+<h4 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994068">Endosomal
 transport and escape</a></h4>
+<p class="MsoNormal">Still there are possible additional entering
-<p class=MsoNormal>Still there are possible additional entering pathways, for
+pathways, for
-example knocking down microtubuli and microfilament arrangement does not
+example knocking down microtubuli and microfilament arrangement does
-prevent transduction completely (Kelley 2008). Currently it is thought that endosomal escape happens in the cytoplasma. After
+not
-pinching off, the endosomes move via motor proteins along microtubuli and
+prevent transduction completely (Kelley 2008). Currently it is thought
-microfilaments towards the nuclear area. While trafficking through the cell the
+that endosomal escape happens in the cytoplasma. After
+pinching off, the endosomes move via motor proteins along microtubuli
+and
+microfilaments towards the nuclear area. While trafficking through the
+cell the
 early endosoms getting acidulated (Sonntag et al. 2006).</p>
+<p class="MsoNormal"><span style="color: black;">Additional entering
-<p class=MsoNormal><span style='color:black'>Additional entering pathways were
+pathways were
-postulated for the virus, for example, it has been shown that proteasomal degradation
+postulated for the virus, for example, it has been shown that
-via ubiquitination hampers transduction efficiency </span><span style='color:black'>(Douar et al. 2001)</span><span style='color:black'>.</span></p>
+proteasomal degradation
+via ubiquitination hampers transduction efficiency </span><span
-<p class=MsoNormal><span style='color:black'>The first viral particles in the
+style="color: black;">(Douar et al. 2001)</span><span
+style="color: black;">.</span></p>
+<p class="MsoNormal"><span style="color: black;">The first viral
+particles in the
 nuclear area can be detected after 15 minutes </span><span
-style='color:black'>(Seisenberger et al. 2001)</span><span style='color:black'>
+style="color: black;">(Seisenberger et al. 2001)</span><span
-and an accumulation of virions takes place after 30 minutes post transfection.</span><span
+style="color: black;">
-style='color:black'> </span><span style='color:black'>After arrival, the viral
+and an accumulation of virions takes place after 30 minutes post
-genomes are transported into the nucleus. It is not entirely clear in which way
+transfection.</span><span style="color: black;"> </span><span
-the transport is accomplished. The viral particles seem to use different
+style="color: black;">After arrival, the viral
-pathways to enter the nucleus, either via the nuclear pore complexes with their
+genomes are transported into the nucleus. It is not entirely clear in
-maximal pore size of 23 nm. In this case, the viral capsid (25 nm diameter) has
+which way
-to be remodeled. Controversial results were published in the past, detecting
+the transport is accomplished. The viral particles seem to use
-intact viral particles (lu et al., 2000), but according to Lux et al. no intact
+different
-capsids were detectable when lower amounts of viral particles were transduced </span><span style='color:black'>(Lux et al. 2005)</span><span style='color:black'>.</span></p>
+pathways to enter the nucleus, either via the nuclear pore complexes
+with their
-<p class=MsoNormal><span style='color:black'>Obviously further investigation of
+maximal pore size of 23 nm. In this case, the viral capsid (25 nm
-intracellular trafficking is essential for optimizing the AAV2 for medical
+diameter) has
+to be remodeled. Controversial results were published in the past,
+detecting
+intact viral particles (lu et al., 2000), but according to Lux et al.
+no intact
+capsids were detectable when lower amounts of viral particles were
+transduced </span><span style="color: black;">(Lux et al. 2005)</span><span
+style="color: black;">.</span></p>
+<p class="MsoNormal"><span style="color: black;">Obviously further
+investigation of
+intracellular trafficking is essential for optimizing the AAV2 for
+medical
 applications.</span></p>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994669"></a><a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994669"></a><a
 name="_Toc275994069">Helper Genes</a></h2>
+<p class="MsoNormal">The AAV Helper-Free System by Stratagene
-<p class=MsoNormal>The AAV Helper-Free System by Stratagene (Waldbronn, Germany)
+(Waldbronn, Germany)
-is a modularized system for the production of infectious recombinant AAV-2
+is a modularized system for the production of infectious recombinant
-virion not depending on a coinfection with any helper virus. It is put into
+AAV-2
-practice by the three plasmids pHelper, pAAV-RC, recombinant pAAV vector
+virion not depending on a coinfection with any helper virus. It is put
-containing the gene of interest (GOI) and the recombinant cell line AAV-293. </p>
+into
+practice by the three plasmids pHelper, pAAV-RC, recombinant pAAV
-<p class=MsoNormal>The AAV-2 is a replication-deficient parvovirus, which
+vector
-originally needs a co-infection of adenovirus or herpes virus for replication. To
+containing the gene of interest (GOI) and the recombinant cell line
-realize a functional replication of AAV-2 without a co-infection, the AAV
+AAV-293. </p>
-Helper-Free System allocates the pHelper plasmid and the AAV-293 host cells.
+<p class="MsoNormal">The AAV-2 is a replication-deficient parvovirus,
-The pHelper plasmid encodes for nearly all of the required adenovirus gene
+which
-products for replication (VA, E2a, E4). The AAV-293 host cells express stably
+originally needs a co-infection of adenovirus or herpes virus for
+replication. To
+realize a functional replication of AAV-2 without a co-infection, the
+AAV
+Helper-Free System allocates the pHelper plasmid and the AAV-293 host
+cells.
+The pHelper plasmid encodes for nearly all of the required adenovirus
+gene
+products for replication (VA, E2a, E4). The AAV-293 host cells express
+stably
 the remaining important replication genes (E1A, E1B). </p>
+<p class="MsoNormal">Due to the fact that AAV-2 needs all relevant
-<p class=MsoNormal>Due to the fact that AAV-2 needs all relevant replication
+replication
-genes for productive infection and that the important replication-genes are
+genes for productive infection and that the important replication-genes
-dispersed, the AAV Helper-Free System describes a saver alternative to retroviral
+are
+dispersed, the AAV Helper-Free System describes a saver alternative to
+retroviral
 or adenoviral gene delivery (Stratagene n.d.).</p>
 <div>
+<table align="left" cellpadding="0" cellspacing="0" hspace="0"
-<table cellspacing=0 cellpadding=0 hspace=0 vspace=0 width=542 align=left>
+vspace="0" width="542">
- <tr>
+<tbody>
-  <td valign=top align=left style='padding-top:0cm;padding-right:7.05pt;
+<tr>
-  padding-bottom:0cm;padding-left:7.05pt'>
+<td style="padding: 0cm 7.05pt;" align="left" valign="top">
-  <p class=MsoCaption>&nbsp;</p>
+<p class="MsoCaption">&nbsp;</p>
-  </td>
+</td>
- </tr>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">The AAV-293 host cells contain the E1A and E1B
-<p class=MsoNormal>The AAV-293 host cells contain the E1A and E1B genes. The
+genes. The
-E1A gene is the first gene to be expressed during an adenovirus infection. The
+E1A gene is the first gene to be expressed during an adenovirus
-E1A gene produces two different mRNAs resulting in two different proteins. The
+infection. The
-expressed E1A proteins transactivate and induce transcription of other early
+E1A gene produces two different mRNAs resulting in two different
-genes (like E2 and E4). In this case, E1A proteins do not bind directly onto
+proteins. The
-control regions, but interact with other host proteins, which are binding to
+expressed E1A proteins transactivate and induce transcription of other
+early
+genes (like E2 and E4). In this case, E1A proteins do not bind directly
+onto
+control regions, but interact with other host proteins, which are
+binding to
 those regions (Chang et al. 1989) (Modrow et al. 2003).</p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none'>
+<tbody>
- <tr style='height:261.85pt'>
+<tr style="height: 261.85pt;">
-  <td width=235 valign=top style='width:176.25pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt;height:261.85pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 176.25pt; height: 261.85pt;"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'><span lang=DE><img
+valign="top" width="235">
-  width=216 height=314 src="Introductionwiki-Dateien/image022.png"></span></p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm'>Figure 20: Schematic overview of the Helper Free System provided by Stratagene.</p>
+style="margin-left: 17.85pt; text-indent: 0cm;"><span lang="DE"><img
-  </td>
+src="Introductionwiki-Dateien/image022.png" height="314" width="216"></span></p>
- </tr>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 20: Schematic
+overview of the Helper Free System provided by Stratagene.</p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">In the AAV-2 infection cycle, E1A proteins
+stimulate the
-<p class=MsoNormal>In the AAV-2 infection cycle, E1A proteins stimulate the
+expression of the p19 promoter and the p5 promoter, which are required
-expression of the p19 promoter and the p5 promoter, which are required to begin
+to begin
-with the <i>rep</i>-gene transcription of the AAV(Chang et al. 1989) (Tratschin et al. 1984).</p>
+with the <i>rep</i>-gene transcription of the AAV(Chang et al. 1989)
+(Tratschin et al. 1984).</p>
-<p class=MsoNormal>The E1B region encodes two polypeptides with overlapping
+<p class="MsoNormal">The E1B region encodes two polypeptides with
-reading frames, the major 21-Mr product and the 55-Mr moiety. It has been shown
+overlapping
-that only the 55-Mr polypeptide is required for effective helper function. It
+reading frames, the major 21-Mr product and the 55-Mr moiety. It has
-enables along with the E4orf6, a stable accumulation of AAV-specific cytoplasmic
+been shown
-RNA, capsid proteins and DNA replication(Samulski &amp; Shenk 1988). In this context it has to be mentioned, that Stratagene
+that only the 55-Mr polypeptide is required for effective helper
-deleted the E4orf6 out of its kit, because of its oncolytic activity. But it
+function. It
-has been explained, that deletion of E4orf6 has no effect on virion production
+enables along with the E4orf6, a stable accumulation of AAV-specific
+cytoplasmic
+RNA, capsid proteins and DNA replication(Samulski &amp; Shenk 1988). In
+this context it has to be mentioned, that Stratagene
+deleted the E4orf6 out of its kit, because of its oncolytic activity.
+But it
+has been explained, that deletion of E4orf6 has no effect on virion
+production
 efficiency(Clark et al. 1999) . </p>
+<p class="MsoNormal">The pHelper plasmid exists of the E2a gene, the E4
-<p class=MsoNormal>The pHelper plasmid exists of the E2a gene, the E4 gene and
+gene and
-the VA gene as well as a pUC ori and an f1 ori. The E2A gene encodes a 72 kD
+the VA gene as well as a pUC ori and an f1 ori. The E2A gene encodes a
-protein which is produced early in infection (Modrow et al. 2003). One helper function of E2A is to increase the processivity of
+kD
-replication. In the presence of E2A protein, short replication products, which
+protein which is produced early in infection (Modrow et al. 2003). One
-are equivalent to break offs of the elongation strand of the template, are
+helper function of E2A is to increase the processivity of
-obviously reduced suggestion that E2A supports full-length replication of short
+replication. In the presence of E2A protein, short replication
-substrates. In immune-depletions, co-localizations between the E2A, the AAV Rep
+products, which
+are equivalent to break offs of the elongation strand of the template,
+are
+obviously reduced suggestion that E2A supports full-length replication
+of short
+substrates. In immune-depletions, co-localizations between the E2A, the
+AAV Rep
 protein and the AAV DNA have been shown (Ward et al. 1998) .</p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
-<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0 align=left
+align="left" border="0" cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+<tbody>
- -2.25pt'>
+<tr style="height: 238.95pt;">
- <tr style='height:238.95pt'>
+<td style="padding: 0cm 5.4pt; width: 346.8pt; height: 238.95pt;"
-  <td width=462 valign=top style='width:346.8pt;padding:0cm 5.4pt 0cm 5.4pt;
+valign="top" width="462">
-  height:238.95pt'>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;">&nbsp;</p>
-  avoid'>&nbsp;</p>
+<p class="MsoCaption"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><img
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
-  width=655 height=258 src="Introductionwiki-Dateien/image023.png"></p>
+src="Introductionwiki-Dateien/image023.png" height="258" width="655"></p>
-  </td>
+</td>
- </tr>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal">It has been reported that E2A has affections on
-<p class=MsoNormal>It has been reported that E2A has affections on the AAV
+the AAV
-promoter regulation of spliced p5 and p19 as well as unspliced p40. E2A could
+promoter regulation of spliced p5 and p19 as well as unspliced p40. E2A
-also be responsible for the production of the AAV capsid proteins (Carter et al. 1992). To which extend it really takes elementary responsiblitiy for any of
+could
+also be responsible for the production of the AAV capsid proteins
+(Carter et al. 1992). To which extend it really takes elementary
+responsiblitiy for any of
 the listed functions is not found out yet.</p>
+<p class="MsoNormal">The VAI and VAII genes encode for two RNA-species,
-<p class=MsoNormal>The VAI and VAII genes encode for two RNA-species, with a
+with a
-high GC-percentage and distinct secondary structure. The VAI RNA, which is
+high GC-percentage and distinct secondary structure. The VAI RNA, which
-implicated to have a helper function in AAV, usually plays a fundamental role
+is
-in adenovirus’ protein expression. There it blocks the phosphorylation of the
+implicated to have a helper function in AAV, usually plays a
-initiation factor eIF-2, whereby the amino acid chain at the ribosome breaks
+fundamental role
+in adenovirus’ protein expression. There it blocks the phosphorylation
+of the
+initiation factor eIF-2, whereby the amino acid chain at the ribosome
+breaks
 off. (Modrow et al. 2003) </p>
+<p class="MsoNormal">The expression of the AAV proteins may also be
-<p class=MsoNormal>The expression of the AAV proteins may also be under the VAI
+under the VAI
-adenovirus control. VAI may increase the AAV capsid production, but it also may
+adenovirus control. VAI may increase the AAV capsid production, but it
+also may
 play a role in RNA metabolism.(West et al. 1987) </p>
+<p class="MsoNormal">The E4 gene exists of seven open reading frames.
-<p class=MsoNormal>The E4 gene exists of seven open reading frames. In this
+In this
-content, the proteins occurring from the gene are named E4-ORF1 up to E4-ORF7.
+content, the proteins occurring from the gene are named E4-ORF1 up to
-All proteins are under the control of one promoter and arise from alternative
+E4-ORF7.
+All proteins are under the control of one promoter and arise from
+alternative
 splicing. The E4ORF6 is implicated to have a helper function in AAV. It
-promotes the formation of a dsDNA from the genomic ssDNA of the native virus.</p>
+promotes the formation of a dsDNA from the genomic ssDNA of the native
+virus.</p>
-<p class=MsoNormal> </p>
+<p class="MsoNormal"> </p>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994670"></a><a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994670"></a><a
+name="_Toc275994070"></a><a name="_Toc274911377">Recombinant Viruses
-name="_Toc275994070"></a><a name="_Toc274911377">Recombinant Viruses and Mosaic
+and Mosaic
 Viruses</a></h2>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994671"></a><a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275994671"></a><a
+name="_Toc275994071"></a><a name="_Toc274911378"><span lang="DE">Gene
-name="_Toc275994071"></a><a name="_Toc274911378"><span lang=DE>Gene Therapy</span></a></h2>
+Therapy</span></a></h2>
+<p class="MsoNormal">Treating inherited and acquired diseases such as
-<p class=MsoNormal>Treating inherited and acquired diseases such as cancer is
+cancer is
-still one of the most challenging fields in today’s biomedical research. Ever
+still one of the most challenging fields in today’s biomedical
-since Sidney Farber published a study in 1949 about several folic acid antagonists,
+research. Ever
+since Sidney Farber published a study in 1949 about several folic acid
+antagonists,
 which prevent tumor progression (FARBER 1949), cancer was treated with
-chemotherapy, surgery and radiation (Halperin 2006). Nevertheless, due to side effects caused by systemic applications and
+chemotherapy, surgery and radiation (Halperin 2006). Nevertheless, due
-the lack of specificity, new treatments must be found for improved therapeutic
+to side effects caused by systemic applications and
-efficacy and enhanced selectivity of the anticancer agents. One promising approach
+the lack of specificity, new treatments must be found for improved
-of treating cancer is suicide gene therapy or gene-directed enzyme prodrug
+therapeutic
-therapy (GDEPT) including two steps of treatment: Targeted introduction of a
+efficacy and enhanced selectivity of the anticancer agents. One
-gene encoding for enzymes into tumor cells, followed by the administration of a
+promising approach
+of treating cancer is suicide gene therapy or gene-directed enzyme
+prodrug
+therapy (GDEPT) including two steps of treatment: Targeted introduction
+of a
+gene encoding for enzymes into tumor cells, followed by the
+administration of a
 non-toxic prodrug which is converted into an anti-cancer metabolite. </p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;"
- style='border-collapse:collapse;border:none;margin-left:4.8pt;margin-right:
+align="left" border="1" cellpadding="0" cellspacing="0">
-.8pt'>
+<tbody>
- <tr>
+<tr>
-  <td width=619 valign=top style='width:464.3pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'><img
+valign="top" width="619">
-  border=0 width=604 height=281 id="Grafik 1155"
+<p class="MsoCaption"
-  src="Introductionwiki-Dateien/image024.png">Figure 21: Schematic overview of
+style="margin-left: 17.85pt; text-indent: 0cm;"><img id="Grafik 1155"
-  gene-directed enzyme prodrug therapy (GDEPT). The suicide gene is introduced
+src="Introductionwiki-Dateien/image024.png" border="0" height="281"
-  into the cancer cells. Administration of the prodrug leads to cell death in
+width="604">Figure 21: Schematic overview of gene-directed enzyme
-  the cells expressing the enzyme, which converts the prodrug into the toxic
+prodrug therapy (GDEPT). The suicide gene is introduced into the cancer
-  product.</p>
+cells. Administration of the prodrug leads to cell death in the cells
-  </td>
+expressing the enzyme, which converts the prodrug into the toxic
- </tr>
+product.</p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">Gene delivery using viral vectors to specifically
-<p class=MsoNormal>Gene delivery using viral vectors to specifically target
+target
 cells gained increasing attention in the last years being efficient in
-combination with suicide gene therapy. Several prodrug/enzyme systems have been
+combination with suicide gene therapy. Several prodrug/enzyme systems
-reported (Greco &amp; Dachs 2001). Ganciclovir (GCV)/herpes simplex virus
+have been
+reported (Greco &amp; Dachs 2001). Ganciclovir (GCV)/herpes simplex
+virus
 thymidine kinase (HSV-TK), 5-fluorocytosine/cytosine deaminase (CD) and
-cyclophosphamide/cytochrome P450 systems have been widely used and their activity
+cyclophosphamide/cytochrome P450 systems have been widely used and
-has been demonstrated in several preclinical studies (Greco &amp; Dachs 2001).</p>
+their activity
+has been demonstrated in several preclinical studies (Greco &amp; Dachs
-<p class=MsoNormal>&nbsp;</p>
+).</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>Using transgenic HSV – thymidine kinase or cytosine
+<p class="MsoNormal">Using transgenic HSV – thymidine kinase or
-deaminase from <i>E. coli</i> for prodrug activation in tumor therapy several
+cytosine
-advantages can be found. Besides efficient killing of targeted tumor cells, neighboring,
+deaminase from <i>E. coli</i> for prodrug activation in tumor therapy
-non-transduced cells are killed as well, providing an important effect in
+several
-treating cancer. The bystander phenomenon was first reported by Moolten (1986) showing that HSV-TK negative cells surrounded by HSV-TK positive cells did not
+advantages can be found. Besides efficient killing of targeted tumor
+cells, neighboring,
+non-transduced cells are killed as well, providing an important effect
+in
+treating cancer. The bystander phenomenon was first reported by Moolten
+(1986) showing that HSV-TK negative cells surrounded by HSV-TK positive
+cells did not
 survive prodrug treatment.</p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 align=left
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
- style='border-collapse:collapse;border:none;margin-left:-2.25pt;margin-right:
+align="left" border="1" cellpadding="0" cellspacing="0">
- -2.25pt'>
+<tbody>
- <tr>
+<tr>
-  <td width=614 valign=top style='width:460.5pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 460.5pt;"
-  <p class=MsoNormal style='margin-left:17.85pt;text-indent:0cm;page-break-after:
+valign="top" width="614">
-  avoid'><img border=0 width=605 height=262 id="Grafik 4"
+<p class="MsoNormal"
-  src="Introductionwiki-Dateien/image025.png"></p>
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
-  <p class=MsoCaption style='margin-left:17.85pt;text-indent:0cm'>Figure 22: Efficient tumor killing is desired in cancer treatment. Locally administered
+id="Grafik 4" src="Introductionwiki-Dateien/image025.png" border="0"
-  prodrugs are converted to toxic metabolites by delivered enzmye in the
+height="262" width="605"></p>
-  infected cells. By passive diffusion, gap junction intercellular
+<p class="MsoCaption"
-  communication or immune-related response, non-transduced tumor cells are
+style="margin-left: 17.85pt; text-indent: 0cm;">Figure 22: Efficient
-  killed as well. </p>
+tumor killing is desired in cancer treatment. Locally administered
-  </td>
+prodrugs are converted to toxic metabolites by delivered enzmye in the
- </tr>
+infected cells. By passive diffusion, gap junction intercellular
+communication or immune-related response, non-transduced tumor cells
+are killed as well. </p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
+<p class="MsoNormal">Transfer of toxic molecules between transduced and
+non-transduced
-<p class=MsoNormal>Transfer of toxic molecules between transduced and non-transduced
+cells can be achieved either through gap junctions (Yang et al. 1998)
-cells can be achieved either through gap junctions (Yang et al. 1998) (Trepel, Stoneham, et al. 2009), via apoptotic bodies (Freeman et al. 1993) or by diffusion of soluble toxic substances (Huber et al. 1993). </p>
+(Trepel, Stoneham, et al. 2009), via apoptotic bodies (Freeman et al.
+) or by diffusion of soluble toxic substances (Huber et al. 1993). </p>
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc274911379"></a><a
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc274911379"></a><a
 name="_Toc275994672"></a><a name="_Toc275994072">Immune Response</a> </h2>
+<p class="MsoNormal">Virus infections cause common human disease,
-<p class=MsoNormal>Virus infections cause common human disease, including the
+including the
-familiar cold, influenza, mumps and measles. They are also associated with
+familiar cold, influenza, mumps and measles. They are also associated
-severe diseases, for example with Ebola or Marburg fever, with Hepatitis and
+with
-AIDS. The immune system protects us from these infections by creating a barrier
+severe diseases, for example with Ebola or Marburg fever, with
-that prevents viruses from entering the body or by detecting and eliminating
+Hepatitis and
+AIDS. The immune system protects us from these infections by creating a
+barrier
+that prevents viruses from entering the body or by detecting and
+eliminating
 them in the corpus. </p>
+<p class="MsoNormal">Every virus lives and reproduces in its own
-<p class=MsoNormal>Every virus lives and reproduces in its own specific host.
+specific host.
-Reproduction can only take place in cell cytoplasm, whose components are needed
+Reproduction can only take place in cell cytoplasm, whose components
+are needed
 in order to compensate for the lack of virus metabolism and
-biosynthesis-appliance. Viruses, which are located outside the cells, can be
+biosynthesis-appliance. Viruses, which are located outside the cells,
-detected by antibodies, triggering an immune response by the members of the
+can be
-innate immune system, such as macrophages, neutrophils and natural killer-cells.</p>
+detected by antibodies, triggering an immune response by the members of
+the
-<p class=MsoNormal>Inside a cell the virus can only be detected by cytotoxic T
+innate immune system, such as macrophages, neutrophils and natural
-cells: While it uses the cellular machinery for reproduction, some of the viral
+killer-cells.</p>
-proteins are degraded by proteasomes and become presented on the cell surface
+<p class="MsoNormal">Inside a cell the virus can only be detected by
-by MHC-I-peptides. These exposed virus components are recognized by cytotoxic
+cytotoxic T
-CD8+ T cells, which induce death of infected cells. The degraded viral proteins
+cells: While it uses the cellular machinery for reproduction, some of
-can also be accessible on MHC-II-peptides, which are detected by CD4+ T helper
+the viral
-cells, which trigger and enforce the immune response for example by production
+proteins are degraded by proteasomes and become presented on the cell
+surface
+by MHC-I-peptides. These exposed virus components are recognized by
+cytotoxic
+CD8+ T cells, which induce death of infected cells. The degraded viral
+proteins
+can also be accessible on MHC-II-peptides, which are detected by CD4+ T
+helper
+cells, which trigger and enforce the immune response for example by
+production
 of specific antibodies. </p>
+<p class="MsoNormal">The Adeno-associated virus (AAV) is not associated
-<p class=MsoNormal>The Adeno-associated virus (AAV) is not associated with any
+with any
-human disease. Nevertheless, usage of recombinant AAV (rAAV) as therapeutic
+human disease. Nevertheless, usage of recombinant AAV (rAAV) as
+therapeutic
 vector system harbors risks of immune responses. </p>
+<p class="MsoNormal">AAV establishes a latent infection and often
-<p class=MsoNormal>AAV establishes a latent infection and often integrates at a
+integrates at a
-specific site on q arm of chromosome 19, which is termed AAVSI site (Hernandez
+specific site on q arm of chromosome 19, which is termed AAVSI site
-et al., 1999). This leads to several obstacles for usage of AAV vectors for
+(Hernandez
-therapeutic applications like gene silencing, insertions in gene sequences and
+et al., 1999). This leads to several obstacles for usage of AAV vectors
-immunotoxocity, a dangerous immune response to the vector or the transgene product
+for
-(Mingozzi &amp; High 2007). Humans are the only natural hosts for AAV-2 besides
+therapeutic applications like gene silencing, insertions in gene
-rhesus macaques. Due to wild-type AAV infections humans keep a population of
+sequences and
-antigen-specific memory CD8+ T cells (Mingozzi &amp; High 2007). IgG antibodies
+immunotoxocity, a dangerous immune response to the vector or the
-are predominantly involved in the secondary immune response. 91% of Irish blood
+transgene product
-donors show a high repertoire of specific IgG1 and IgG2 subclasses and low
+(Mingozzi &amp; High 2007). Humans are the only natural hosts for AAV-2
+besides
+rhesus macaques. Due to wild-type AAV infections humans keep a
+population of
+antigen-specific memory CD8+ T cells (Mingozzi &amp; High 2007). IgG
+antibodies
+are predominantly involved in the secondary immune response. 91% of
+Irish blood
+donors show a high repertoire of specific IgG1 and IgG2 subclasses and
+low
 doses of IgG3 (Madsen et al. 2009). </p>
+<p class="MsoNormal">In vivo studies with AAVlacZ show that AAV vectors
-<p class=MsoNormal>In vivo studies with AAVlacZ show that AAV vectors induce
+induce
-the secretion of chemokines and cytokines like gamma interferon (IFN-&#947;)
+the secretion of chemokines and cytokines like gamma interferon (IFN-γ)
-(Zaiss et al. 2002). Studies in vitro show responses of IFN-&#947;, interleukin
+(Zaiss et al. 2002). Studies in vitro show responses of IFN-γ,
-(IL-10) and interleukin 13 (IL-13) after stimulation peripheral blood
+interleukin
-mononuclear cells (PBMC) from donors with AAV-2. This demonstrates a reaction
+(IL-10) and interleukin 13 (IL-13) after stimulation peripheral
-of long-live CD4+ T helper-cells that are reactivated (Madsen et al. 2009). These
+blood
-results reveal that most Europeans are already infected with wildtype-AAV-2.
+mononuclear cells (PBMC) from donors with AAV-2. This demonstrates a
-Researchers suggest that more than 30% of mankind is already infected. In vitro
+reaction
+of long-live CD4+ T helper-cells that are reactivated (Madsen et al.
+). These
+results reveal that most Europeans are already infected with
+wildtype-AAV-2.
+Researchers suggest that more than 30% of mankind is already infected.
+In vitro
 studies from the United States support this hypothesis. One group found
-anti-AAV-antibodies in the blood sera at 80% of randomly chosen volunteers
+anti-AAV-antibodies in the blood sera at 80% of randomly chosen
-(Moskalenko et al. 2000). Other investigators show that 0,14% of the examined
+volunteers
-CD8+ T cells purified from PBMC are capsid specific for AAV-2 (Mingozzi, Maus,
+(Moskalenko et al. 2000). Other investigators show that 0,14% of the
-et al. 2007). These preexisting memory-CD8+ T cells could be responsible for
+examined
-the difference in vector-infusion outcome between humans (the natural host) and
+CD8+ T cells purified from PBMC are capsid specific for AAV-2
+(Mingozzi, Maus,
+et al. 2007). These preexisting memory-CD8+ T cells could be
+responsible for
+the difference in vector-infusion outcome between humans (the natural
+host) and
 other species. </p>
+<p class="MsoNormal">AAV-2 use distinct cellular receptors, e.g.
-<p class=MsoNormal>AAV-2 use distinct cellular receptors, e.g. heparin sulfate
+heparin sulfate
-proteoglycan (HSPG), &#945;V&#946;5 integrin and human fibroblast growth factor
+proteoglycan (HSPG), αVβ5 integrin and human fibroblast growth factor
-receptor 1 (FGFR1) to become internalized (Favaro et al. 2009). These findings
+receptor 1 (FGFR1) to become internalized (Favaro et al. 2009). These
-led researchers to the conclusion that the presence of an intact heparinbinding
+findings
-motif and the capsid t-cell responses are correlated. One group ablated the
+led researchers to the conclusion that the presence of an intact
-heparin-binding site in AAV-2 and observed no CD8+ T cell response. But it did
+heparinbinding
-not seem to influence T helper responses as measured by IgG isotypes and
+motif and the capsid t-cell responses are correlated. One group ablated
+the
+heparin-binding site in AAV-2 and observed no CD8+ T cell response. But
+it did
+not seem to influence T helper responses as measured by IgG isotypes
+and
 antigen-stimulated secretion of cytokines (Vandenberghe et al. 2006).</p>
+<p class="MsoNormal">Approaches using peptides derived from the
-<p class=MsoNormal>Approaches using peptides derived from the sequence of the
+sequence of the
 VP1 viral capsid protein revealed a total of 59 t-cell epitopes. This
-demonstrates the difficulty to avoid the immune system by modifying the AAV
+demonstrates the difficulty to avoid the immune system by modifying the
-capsid (Madsen et al. 2009). Other approaches in mice reveal that different
+AAV
-serotypes of AAV show the ability to cross-react with existing memory-T cells
+capsid (Madsen et al. 2009). Other approaches in mice reveal that
-(Sabatino et al. 2005). Also in dogs different AAVs use some common peptides on
+different
-their surface to activate the immune system (Wang et al. 2010). This shows the
+serotypes of AAV show the ability to cross-react with existing memory-T
+cells
+(Sabatino et al. 2005). Also in dogs different AAVs use some common
+peptides on
+their surface to activate the immune system (Wang et al. 2010). This
+shows the
 high conservation of the epitopes among multiple serotypes of AAV. </p>
+<p class="MsoNormal">While proposing several possible solutions to
-<p class=MsoNormal>While proposing several possible solutions to avoid the
+avoid the
 immune system, the polymorphic nature of the human MHC and the high
-conservation of peptides on the surface of different serotypes of AAV may
+conservation of peptides on the surface of different serotypes of AAV
-complicate these approaches (Mingozzi, Hasbrouck, et al. 2007). In general it
+may
-can be said that the immune response to AAV is not severe as caused by other
+complicate these approaches (Mingozzi, Hasbrouck, et al. 2007). In
-virus-types. This is due to the fact that AAVs fail to trigger inflammatory
+general it
+can be said that the immune response to AAV is not severe as caused by
+other
+virus-types. This is due to the fact that AAVs fail to trigger
+inflammatory
 reactions dendritic cells need to differentiate into professional
 antigen-presenting cells (Mingozzi, Maus, et al. 2007). These
-antigen-presenting cells are needed for the activation of CD4+ T helper-cells
+antigen-presenting cells are needed for the activation of CD4+ T
-which are needed for the completely feedback to the immune system. Nevertheless
+helper-cells
-dendritic cells can be activated through the ability of AAV-2 to bind the HSPG
+which are needed for the completely feedback to the immune system.
-binding motif with resultant AAV2 antigen inclusion, processing and MHC-I
+Nevertheless
-presentation (Wang et al. 2010). CD4+ T helper-cells can also be activated by other
+dendritic cells can be activated through the ability of AAV-2 to bind
-antigen-presenting cells therefore it is conceivable to block CD4+ cells during
+the HSPG
-treatment with AAV. The activation of CD8+-t-cells through CD4+ T cells is
+binding motif with resultant AAV2 antigen inclusion, processing and
-depleted and the immune response is even more reduced than within the normal
+MHC-I
+presentation (Wang et al. 2010). CD4+ T helper-cells can also be
+activated by other
+antigen-presenting cells therefore it is conceivable to block CD4+
+cells during
+treatment with AAV. The activation of CD8+-t-cells through CD4+ T cells
+is
+depleted and the immune response is even more reduced than within the
+normal
 infection process. </p>
+<p class="MsoNormal">Some researchers have found AAV vector DNA in the
-<p class=MsoNormal>Some researchers have found AAV vector DNA in the semen of
+semen of
-dogs and fear the risk of germline transmission (Jiang et al. 2006) although
+dogs and fear the risk of germline transmission (Jiang et al. 2006)
+although
 these findings are controversially discussed. In a rabbit model it was
-demonstrated that semen was just positive for vector sequences following
+demonstrated that semen was just positive for vector sequences
-intravascular injection but not following intramuscular injection. Infectious
+following
-vector particles were just detected up to four days after treatment and were
+intravascular injection but not following intramuscular injection.
-undetectable thereafter. So the investigators suggest that AAV-2 presents a low
+Infectious
-risk of germline transmission for humans and there is no contemplation for male
+vector particles were just detected up to four days after treatment and
+were
+undetectable thereafter. So the investigators suggest that AAV-2
+presents a low
+risk of germline transmission for humans and there is no contemplation
+for male
 infertility so far (Favaro et al. 2009). </p>
+<p class="MsoNormal">AAV vectors have been used in several phases of
-<p class=MsoNormal>AAV vectors have been used in several phases of clinical
+clinical
-trials for Leber’s congenital amaurosis (LCA), hemophilia B, Cystic fibrosis,
+trials for Leber’s congenital amaurosis (LCA), hemophilia B, Cystic
+fibrosis,
 Arthritis, Muscular dystrophy, Parkinson’s disease, Canavan's disease,
 Alzheimer's disease, Batten's disease and Hereditary emphysema.</p>
+<p class="MsoNormal">&nbsp;</p>
-<p class=MsoNormal>&nbsp;</p>
 </div>
+</body>
 </html>
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Team:Freiburg Bioware/Project/Project Description

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Revision as of 00:36, 28 October 2010

Introduction

Introduction to Adeno-Associated Virus Serotype 2

Organization of the Inverted Terminal Repeat Structure

p19 promoter

VP3