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Introduction

Overview

The Experimental System

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).

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.

Team:Freiburg Bioware/Project/Project Description

From 2010.igem.org

Latest revision as of 01:14, 28 October 2010

Introduction

Contents

Overview

The Experimental System

Layers of specificity

Introduction to Adeno-Associated Virus Serotype 2

Biology of the AAV-2

Genomic organization

Organization of the Inverted Terminal Repeat Structure

Replication

Viral promoters

p5 promoter

p5 TATA-less promoter

p19 promoter

p40 promoter

Integration

Rescue

Rep proteins

Overview

Rep 78

Rep 68

Rep52

Rep40

VP proteins

VP1

VP2

VP3

Natrual Tropism and HSPG motif

Assembly-activating protein

Trafficking

Endosomal transport and escape

Helper Genes

Recombinant Viruses and Mosaic Viruses

Gene Therapy

Immune Response

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+<h2>Contents</h2>
+<p class="MsoToc1"><a href="#_Toc275994657"><span lang="DE">Overview</span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">.. </span><span
+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
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">.. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">1</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994659"><span lang="DE">Layers of
+specificity</span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">2</span></a></p>
+<p class="MsoToc1"><a href="#_Toc275994660">Introduction to
+Adeno-Associated
+Virus Serotype 2<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">4</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994661">Biology of the AAV-2<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">4</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994662">Genomic organization<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">4</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994663">Replication<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">5</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994664">Integration<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">11</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994665">Rescue<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE"> </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">12</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994666">Rep proteins<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">12</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994667">VP proteins<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">16</span></a></p>
+<p class="MsoToc3"><a href="#_Toc275994668">Trafficking<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE"> </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">23</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994669">Helper Genes<span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">. </span><span
+style="color: windowtext; display: none; text-decoration: none;"
+lang="DE">27</span></a></p>
+<p class="MsoToc2"><a href="#_Toc275994670">Recombinant Viruses and
+Mosaic
+Viruses<span
+style="color: windowtext; display: none; text-decoration: none;"
+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
+name="_Toc275994041"><span lang="DE">The Experimental System</span></a></h2>
+<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
+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</p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal"><img style="width: 471px; height: 575px;"
+id="Picture 56"
+src="https://static.igem.org/mediawiki/2010/3/38/Freiburg10_Experimental_System.png"
+alt=""></p>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994659"></a><a
+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
+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.</p>
+<p class="MsoNormal">In order to satisfy this requirement we designed
+our Therapy
+Vector with several layers of Specificity:</p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">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 (<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>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal"><img
+style="border: 0px solid ; width: 463px; height: 512px;"
+id="Picture 57"
+src="http://partsregistry.org/wiki/images/b/b5/Freiburg10_Layers_of_Specificity.png"
+alt=""></p>
+<span
+style="font-size: 11pt; line-height: 150%; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+lang="DE"><br style="page-break-before: always;" clear="all">
+</span>
+<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;">
+<h1 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994660"></a><a
+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
+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
+name="_Toc275994045">Genomic organization</a></h3>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 191.45pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 426.05pt; height: 191.45pt;"
+valign="top" width="568">
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Toc274911364"><img
+style="border: 0px solid ; width: 524px; height: 269px;" alt=""
+id="Grafik 154"
+src="https://static.igem.org/mediawiki/2010/3/35/Freiburg10_genomic_organisation_wt.png"></a></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275946742">Figure
+</a>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 <i>rep</i> 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 <i>cap</i> gene.</p>
+</td>
+</tr>
+</tbody>
+</table>
+<p class="MsoNormal">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). </p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal">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 <i>cis.</i></p>
+<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
+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 </span><span 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>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 293.05pt;">
+<td style="padding: 0cm 5.4pt; width: 390.3pt; height: 293.05pt;"
+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>
+<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
+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 <span
+style="color: black;"> </span><span style="color: black;">(Lusby et
+al. 1980)</span>.</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 460.5pt;"
+valign="top" width="614">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+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">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<span style="color: black;"> </span><span
+style="color: black;">(Im &amp; Muzyczka 1990)</span> .</p>
+<p class="MsoNormal">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</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
+episomal form
+inside the nucleus, it tends to form linear as well as circular
+concatamers,
+which are formed by ligation of duplex monomers(Schultz &amp;
+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
+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 in<b>Error! Reference
+source not
+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">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 373.8pt;">
+<td style="padding: 0cm 5.4pt; width: 284.5pt; height: 373.8pt;"
+valign="top" width="379">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
+align="center"><img
+style="border: 0px solid ; width: 363px; height: 432px;" alt=""
+id="Grafik 1226"
+src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_regulation_viral_promotors.png"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><a 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 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.</p>
+</td>
+</tr>
+</tbody>
+</table>
+</div>
+<p class="MsoNormal" style="text-align: center;" align="center">&nbsp;</p>
+<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>
+and <i>cap</i>
+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. </p>
+<p class="MsoNormal">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). </p>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 203.8pt;">
+<td style="padding: 0cm 5.4pt; width: 433.85pt; height: 203.8pt;"
+valign="top" width="578">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
+align="center"><img
+style="border: 0px solid ; width: 566px; height: 235px;" alt=""
+id="Grafik 1286"
+src="https://static.igem.org/mediawiki/2010/5/56/Freiburg10_p5_promoter_wtAAV2.png"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><a 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 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">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).</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
+promoter, the
+iGEM team Freiburg 2010 provides the RepCap plasmid with a relocated p5
+promoter
+downstream of the <i>RepCap</i> genes (Figure 6). Additionally the p5
+promoter
+lacks the TATA box element (AVIGEN 1997)<b>. </b>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 <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">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 214.9pt;">
+<td style="padding: 0cm 5.4pt; width: 236.7pt; height: 214.9pt;"
+valign="top" width="316">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 300px; height: 243px;" alt=""
+id="Grafik 1289"
+src="https://static.igem.org/mediawiki/2010/0/04/Freiburg10_p5_TATA_less.png"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275634090">Figure
+</a>6: p5 TATA-less promoter is located downstream of the rep and
+cap ORF.</p>
+</td>
+</tr>
+</tbody>
+</table>
+</div>
+<p class="MsoNormal">&nbsp;</p>
+<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
+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 &amp; Muzyczka 2002). By forming a DNA
+loop (Pereira &amp; 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 &amp;
+Muzyczka 2002). </p>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 215.65pt;">
+<td style="padding: 0cm 5.4pt; width: 235.95pt; height: 215.65pt;"
+valign="top" width="315">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+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>
+<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
+from the
+adeno-associated virus serotype 2 (AAV2) genome, where it is located at
+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
+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 </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
+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 </span><span
+lang="EN-GB">(Pereira &amp; Muzyczka 1997)</span><span lang="EN-GB">.
+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 </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
+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. </a></p>
+<p class="MsoNormal">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 &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
+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. </p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">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.</p>
+<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
+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
+), (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>
+<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
+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>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 232.5pt;">
+<td style="padding: 0cm 5.4pt; width: 399.65pt; height: 232.5pt;"
+valign="top" width="533">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 602px; height: 225px;" id="Grafik 37"
+src="https://static.igem.org/mediawiki/2010/8/86/Freiburg10_Organisation_cap_proteins.png"
+alt=""></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">&nbsp;</p>
+<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
+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">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).</p>
+<p class="MsoNormal">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) <span
+style="font-family: &quot;Courier New&quot;;">GAGT GAGC</span> 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
+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 <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
+name="_Ref275633722"></a><a name="_Toc274911369">Rep 78</a><span
+style="font-family: &quot;Dutch801BT-Bold&quot;,&quot;serif&quot;;"> </span></h5>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep78 in a nutshell:</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• 78 kDa</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Endonuclease
+activity </p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
+activity </p>
+<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">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 &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
+name="_Ref275633727"></a><a name="_Toc274911370">Rep 68</a><u> </u></h5>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep68 in a nutshell</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• 68 kDa</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• Endonuclease
+activity </p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
+activity </p>
+<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">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.</p>
+<p class="MsoNormal">The non-structural protein Rep68 belongs to the
+superfamily
+(SF3) helicase found in other small DNA and RNA viruses such as
+simian virus
+(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<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
+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
+name="_Toc274911372">Rep52</a></h5>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep52 in a nutshell</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• 52 kDa</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
+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
+and shares
+the same N-terminus with Rep78. It was shown that Rep52 possesses
+helicase and
+ATPase activity with 3´-5´polarity (Smith &amp; 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 &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"
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">Rep40 in a nutshell</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• 40 kDa</p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">• ATPase and helicase
+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
+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 Mg<sup>2+</sup> or Mn<sup>2+</sup> 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
+´end of the DNA (King et al. 2001). </p>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 192.55pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 214pt; height: 192.55pt;"
+valign="top" width="285">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
+style="border: 0px solid ; width: 270px; height: 232px;" alt=""
+id="Picture 8"
+src="https://static.igem.org/mediawiki/2010/c/c4/Freiburg10_Crystal_structure_SF3helicase.png"></p>
+<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>
+<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
+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
+:1:10, 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)</p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal" style="margin-bottom: 0.0001pt;">&nbsp;</p>
+<table class="MsoTableGrid"
+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">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 466.1pt;"
+valign="top" width="621">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 134.65pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 440.85pt; height: 134.65pt;"
+valign="top" width="588">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><span
+style="font-size: 12pt; line-height: 150%;"><img
+style="border: 0px solid ; width: 574px; height: 179px;" id="Grafik 39"
+src="https://static.igem.org/mediawiki/2010/2/20/Freiburg10_Organisation_rep_proteins.png"
+alt="Beschreibung: Cap proteins.png"></span></p>
+<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 capsid proteins VP1, Vp2 and VP3, which are responsible for the
+assembly of the capsid </p>
+</td>
+</tr>
+</tbody>
+</table>
+</td>
+</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
+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
+:1:10. 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. <span lang="DE">(Bleker et al. 2006)</span><span
+lang="DE">, </span><span lang="DE">(Johnson et al. 2010b)</span><span
+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">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.</p>
+<p class="MsoNormal">&nbsp;</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
+align="center"><img
+style="border: 0px solid ; width: 502px; height: 273px;" alt=""
+id="Picture 2064"
+src="https://static.igem.org/mediawiki/2010/e/e8/Freiburg10_VP1-3_overview.jpg"></p>
+<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 (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 <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">It specifically recognizes and hydrolyzes the sn-2
+acyl bond
+of phospholipids releasing arachidonic acid and lysophospholipids.</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
+align="center"><img
+style="border: 0px solid ; width: 437px; height: 227px;"
+id="Picture 2065"
+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">The reaction mechanism will be depicted by the
+following
+image:</p>
+<p class="MsoNormal">&nbsp;</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
+style="border: 0px solid ; width: 604px; height: 485px;" alt=""
+id="Picture 2066"
+src="https://static.igem.org/mediawiki/2010/0/0c/Freiburg10_PLA2-mechanism.PNG"></p>
+<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">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). </p>
+<p class="MsoNormal"><span style="line-height: 150%;">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 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
+sites.</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
+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 “</span><span style="line-height: 150%;">strongly
+suggest
+that N-termini of VP1 </span>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<span style="line-height: 150%;"> had penetrated through an
+endosomal
+membrane or Golgi. </span><span
+style="font-size: 10pt; line-height: 150%;">(Michelfelder &amp; Trepel
+)</span></p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">&nbsp;</p>
+<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
+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).</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
+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). </p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 222.55pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 441.8pt; height: 222.55pt;"
+valign="top" width="589">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 575px; height: 288px;" id="Grafik 40"
+src="https://static.igem.org/mediawiki/2010/5/5a/Freiburg10_vp1vp2vp3.png"
+alt="Beschreibung: VP1_2_3.png"></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>
+<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
+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).</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 628px; height: 211px;"
+id="Picture 39"
+src="https://static.igem.org/mediawiki/2010/8/8a/Freiburg10_Figure15.png"
+alt=""></p>
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;">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.</p>
+</td>
+</tr>
+</tbody>
+</table>
+<p class="MsoNormal" style="">&nbsp;</p>
+<p class="MsoNormal" style="">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
+every human cell surface. </p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-align: center; text-indent: 0cm; page-break-after: avoid;"
+align="center"><img
+style="border: 0px solid ; width: 510px; height: 193px;"
+id="Picture 37"
+src="https://static.igem.org/mediawiki/2010/a/a8/Freiburg10_Freiburg10_amidosulfated_disacharid.png"
+alt="Beschreibung: Unbenannt2"></p>
+<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="">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.</p>
+<p class="MsoNormal" style="">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“.</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>
+<p class="MsoNormal">A gene encoding for the assembly-activating
+protein (AAP)
+was recently (in 2010) discovered in the Adeno-associated virus (AAV)
+serotype
+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.</p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">&nbsp;</p>
+<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
+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. </p>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 354.75pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 316.8pt; height: 354.75pt;"
+valign="top" width="422">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 456px; height: 510px;" alt=""
+id="Grafik 123"
+src="https://static.igem.org/mediawiki/2010/a/a5/Freiburg10_viral_trafficking.png"></p>
+<p class="MsoCaption"
+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">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). </p>
+<p class="MsoNormal"><span style="color: black;">Before entering 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;">
+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
+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: </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;">5</span></sub><span style="color: black;">&nbsp;and
+</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;">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">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 232.6pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 320.35pt; height: 232.6pt;"
+valign="top" width="427">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 420px; height: 438px;" alt=""
+id="Grafik 1044"
+src="https://static.igem.org/mediawiki/2010/1/14/Freiburg10_cellular_surface_HSPG.png"></p>
+<p class="MsoCaption"
+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">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 &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">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 249pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 351.6pt; height: 249pt;"
+valign="top" width="469">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 484px; height: 330px;" id="Grafik 59"
+src="https://static.igem.org/mediawiki/2010/8/87/Freiburg10_Clathrin_triskelions.png"
+alt="Beschreibung: File:Freiburg10 Receptor-mediated endocytosis by clathrin-coated vesicles scheme.PNG"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><a name="_Ref275689978">Figure
+</a>19:</p>
+</td>
+</tr>
+</tbody>
+</table>
+</div>
+<p class="MsoNormal">&nbsp;</p>
+<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.
+). 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).</p>
+<p class="MsoNormal">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 &amp; Samulski 1998) (Sanlioglu et
+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
+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).</p>
+<p class="MsoNormal"><span style="color: black;">Additional entering
+pathways were
+postulated for the virus, for example, it has been shown that
+proteasomal degradation
+via ubiquitination hampers transduction efficiency </span><span
+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;">
+and an accumulation of virions takes place after 30 minutes post
+transfection.</span><span style="color: black;"> </span><span
+style="color: black;">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 </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
+name="_Toc275994069">Helper Genes</a></h2>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">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). </p>
+<p class="MsoNormal">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.)</p>
+<div><br>
+</div>
+<p class="MsoNormal">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).</p>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 261.85pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 176.25pt; height: 261.85pt;"
+valign="top" width="235">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm;"><span lang="DE"><img
+style="width: 216px; height: 314px;" alt=""
+src="https://static.igem.org/mediawiki/2010/9/95/Freiburg10_strategene.png"></span></p>
+<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">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 <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
+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 &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
+gene and
+the VA gene as well as a pUC ori and an f1 ori. The E2A gene encodes a
+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) .</p>
+<div align="center">
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="0" cellpadding="0" cellspacing="0">
+<tbody>
+<tr style="height: 238.95pt;">
+<td style="padding: 0cm 5.4pt; width: 346.8pt; height: 238.95pt;"
+valign="top" width="462">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;">&nbsp;</p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
+style="width: 655px; height: 258px;" alt=""
+src="https://static.igem.org/mediawiki/2010/f/f5/Freiburg10_pHelper.png"></p>
+</td>
+</tr>
+</tbody>
+</table>
+</div>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">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) </p>
+<p class="MsoNormal">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) </p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal"> </p>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994670"></a><a
+name="_Toc275994070"></a><a name="_Toc274911377">Recombinant Viruses
+and Mosaic
+Viruses</a></h2>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275994671"></a><a
+name="_Toc275994071"></a><a name="_Toc274911378"><span lang="DE">Gene
+Therapy</span></a></h2>
+<p class="MsoNormal">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. </p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: 4.8pt; margin-right: 4.8pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 464.3pt;"
+valign="top" width="619">
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;"><img
+style="border: 0px solid ; width: 604px; height: 281px;" alt=""
+id="Grafik 1155"
+src="https://static.igem.org/mediawiki/2010/a/a4/Freiburg10_overview_GDEPT.png">Figure
+: 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.</p>
+</td>
+</tr>
+</tbody>
+</table>
+<p class="MsoNormal">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 &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
+has been demonstrated in several preclinical studies (Greco &amp; Dachs
+).</p>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal">Using transgenic HSV – thymidine kinase or
+cytosine
+deaminase from <i>E. coli</i> 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.</p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse; margin-left: -2.25pt; margin-right: -2.25pt;"
+align="left" border="1" cellpadding="0" cellspacing="0">
+<tbody>
+<tr>
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 460.5pt;"
+valign="top" width="614">
+<p class="MsoNormal"
+style="margin-left: 17.85pt; text-indent: 0cm; page-break-after: avoid;"><img
+style="border: 0px solid ; width: 605px; height: 262px;" alt=""
+id="Grafik 4"
+src="https://static.igem.org/mediawiki/2010/c/c0/Freiburg10_Bystander_effect.png"></p>
+<p class="MsoCaption"
+style="margin-left: 17.85pt; text-indent: 0cm;">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. </p>
+</td>
+</tr>
+</tbody>
+</table>
+<p class="MsoNormal">&nbsp;</p>
+<p class="MsoNormal">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.
+) 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
+name="_Toc275994672"></a><a name="_Toc275994072">Immune Response</a> </h2>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">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 &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
+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
+(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.
+). 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. </p>
+<p class="MsoNormal">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).</p>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">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. </p>
+<p class="MsoNormal">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). </p>
+<p class="MsoNormal">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.</p>
+<p class="MsoNormal">&nbsp;</p>
+</div>
+</body>
+</html>
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