molekulare virologie lmu...the first fda-approved antisense drug. structure of the 21-mer...
TRANSCRIPT
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Virus-Vektoren
LMUMolekulare Virologie
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RNA-/DNA-Vektoren im Vergleich
http://www.nature.com/nrg/journal/v4/n5/fig_tab/nrg1066_F2.html
(Virale) Vektoren -Systeme und Einsatzfelder
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RNA-Vektoren
LMUMolekulare Virologie
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Virale RNA-Vektoren
a) RNA in Funktion einer m-RNA:Proteinexpression (Vakzine, toxisches Protein,therapeutisches Genprodukt, ...)b) RNA als unmittelbar wirkende Substanz:keine Translation (z.B.: Transfektion von Aptameren)
keine Integration (Ausnahme HIV, ...)transiente Expressionhohe Kurzzeitexpressionhohe Virustiterviele ZelltypenWirkort: meist Zytoplasma
LMU
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?!
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Virale Vakzine Vektoren basierend auf RNA-Viren
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
( )
RNA - Inhibition
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
Aptamere
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
Ribozyme gene therapy involves the following steps:
1. Delivery of RNA strands engineered to function as ribozymes.
2. Specific binding of the ribozyme RNA to mRNA encoded by the mutated gene
3. Cleavage of the target mRNA, preventing it from being translated into a protein
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Antisense RNA
http://learn.genetics.utah.edu/content/tech/genetherapy/gtapproaches/
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Antisense-RNA - Fomivirsen (Vitravene)
The first FDA-approved antisense drug. Structure of the 21-mer phosphorothioate, fomivirsen (brand name Vitravene, illustration from [223]). The patient target group for this drug is rather small, and it was taken off the market by the manufacturer in 2002 due to poor sales
Fomivirsen ist ein Antisense-Oligonukleotid und ein Arzneistoff, welcher als Virostatikum zur Behandlung von Infektionen mit dem Cytomegalievirus (CMV) bei Immundefizienz, wie AIDS eingesetzt wird.
Fomivirsen ist ein 21mer Antisense-RNA Phosphorthioat-Oligonukleotid (ISIS 2922)[1] mit einer komplementären Sequenz zur mRNA, der major immediate-early (MIE) transkriptionalen Einheit des humanen Cytomegalievirus (CMV). Durch die Bindung der aRNA an die komplementäre mRNA wird die Translation dieser viralen mRNA blockiert und damit die Genexpression der Proteine der IE2-Region (IE2), IE86 und IE55, verhindert.[2]
Fomivirsen war das erste Antisense-Oligonukleotid das von der FDA zugelassen wurde.
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
siRNA
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
RNA-Transfer in Zellen
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famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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ss-RNA (+)-Vektoren
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http://www.bio.davidson.edu/courses/immunology/Students/spring2006/Jameson/rabies%20structure.bmp
The neuroinvasiveness of the virus results from its ability to migrate to the central nervous system (CNS) through retrograde axonal transport and transynaptic spread. Rabies virus spreads from the postsynaptic site to the presynaptic site via receptor-mediated endocytosis. In retrograde axonal transport, the ribonucleoprotein complexes of the virus are carried by direct attachment to a dynein motor or by encapsulation in vessicles attached to a dynein motor.
Rabiesvirus
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gsbs.utmb.edu/microbook/ch061.htm
The rabies virus genome
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http://cms.frontiersin.org/content/10.3389/neuro.05/001.2009/html/fnana-03-001/images/article/image_n/fnana-03-001-g002.gif
The construction of the RV full-length cDNA vector (pHEP5.0-CVSG) was described previously (Inoue et al., 2004). Three recombinant RV-vectors were newly generated for dual viral tracing by inserting transgenes: the LacZ gene which encodes for β-galactocidase (β-gal) or the gene for green fluorescent protein (GFP) variants, into the multiple insertion site of the virus vector genome (Figure 2). To create a vector which expresses either β-gal or the yellow fluorescent protein named Venus (Nagai et al., 2002), a PCR fragment of LacZ cDNA (Invitrogen, USA) and Venus cDNA containing the Sbf I site, open reading frame, and Sac II were amplified and inserted into the same site of pHEP5.0-CVSG, respectively.
http://cms.frontiersin.org/content/10.3389/neuro.05/001.2009/html/fnana-03-001/fnana-03-001.html
Neuronal tracing of rabies virus
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To evaluate the ability of the rabies virus vectors to infect a cell in which an ongoing infection exists, two vectors expressing different reporter proteins, rHEP5.0-CVSG-β-gal and rHEP5.0-CVSG-Venus, were applied to the same culture dish at different time intervals. First, 300 μl of rHEP5.0-CVSG-Venus (1.0 × 107 focus-forming units (FFU)/ml) was applied. After a certain time delay (0 h, 2 h, 6 h, 12 h), 300 μl of rHEP5.0-CVSG-β-gal (1.0 × 107 FFU/ml) was applied.
Interference studies of double-infected neuron cells
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Retroviral Vectors
• Based on:-
– Murine leukaemia virus (MLV)
– Lentiviruses such as HIV, SIV, FIV
– Mouse mammary tumour virus (MMTV)
– Foamy/spuma viruses
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Retroviral Genome
Viral RNA
ReverseTranscriptionIntegration
gag pol env
R U5 RU3
Proviral DNA
RU3 U5
gag pol envLTR LTRRU3 U5
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Retroviral Vector Principle
Retrovirus
RU3 U5
LTR LTRRU3 U5
Retroviral Vector
RU3 U5
geneLTR LTRRU3 U5
gag pol env
gag pol env
ψ
ψ
Packaging Construct
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Packaging CellsRetroviral Vector
RU3 U5RU3 U5geneLTR LTR
ψ
gag pol env
Recombinant Virus
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Safety features for replication incompetent vectors
•ProCon Vectors
• Deletion of U3 of the 3’ LTR
• Insertion of Inducible/Tissue Specific Promoter
• Replacement of Viral Promoter
U5RU3U5RU3
mRNA
PromoterU5RU3 U5RTG
TG
TG
U5R U5RPromoterPromoter
TG
Mrochen et al., (1997) J.Mol.Med. 75, 820
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Risks associated with Retroviruses
• Insertional Integration Leading to– Gene disruption
(tumour suppressor)– Gene activation
(proto-oncogene)
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DNA (Virus) Vektoren
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Kriterien für die Einteilung von Vektoren:1) Administration
Nackte DNA
Replikationsdefiziente Viren
Replikationskompetente Viren
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Kriterien für die Einteilung von Vektoren:1) Administration – nackte DNA
TransfektionPhysikalische Transfektionsmethoden1) Nadel Injektion in Gewebe / Inhalation 2) Hydrodynamischer Gentransfer3) Mikroinjektion in Nukleus4) „Gene Gun“ Transfer5) ElektroporationChemische Transfektionsmethoden1) Kalziumphosphat Co-Präzipitation 2) Kationische Polymere 3) Kationische Lipide („non-bilayer forming“)4) Liposomen („bilayer forming“)
KondensationPositive Ladung(Membranfusion)
Transfektionsmethoden mit „nuclear localization signals“
Geringe Effizienz des Gentransfers
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Einbringen von Vektoren in Zellen
Transfektion - artifizielles Einbringen von Nukleinsäuren in eukaryotische Zellen- Kunstwort aus TRANSformation + InFEKTION- DNA-Aufnahme durch die Zelle in Form von Salz-Präzipitaten oderals membrangängige Micellen
Calzium-Phosphat-Methode Liposomen-MethodeLiposomen-Methode
www.rz.uni-karlsruhe.de/~dc29/zoologie2/vorlesungsmat/EB1/070207/Protein-Dateien/Protein.ppt
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Kriterien für die Einteilung von Vektoren:1) Administration – Replikationsdefiziente Viren
Transduktion
Beibehaltung der Mechanismen der Ausgangsviren zur Infektion (Transduktion) von Zielzellen
Zusammenbau in z.T. komplexen Produktionssystemen
Häufig immunogen (humorale und zelluläre Immunantwort)
Nicht-integrierende Vektoren sind nur „transient“
Dadurch: sehr hohe Effizienz des Gentransfers
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Kriterien für die Einteilung von Vektoren:1) Administration – Replikationskompetente Viren
Infektion
Anwendung (Studien) v.a. in der Therapie von Tumoren (onkolytische Viren) und in der DNA Vakzinierung
Basieren auf attenuierten bzw. genetisch modifiziertenreplikationskompetenten Viren
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Kriterien für die Einteilung von Vektoren:2) Replikation
(Indirekte) Replikation nach Integration ins Wirtschromosomstabile Expression eines Transgens
Kein Mechanismus zur Replikation vorhandenVerlust des Vektors während der Zellteilungen !transiente Expression eines Transgens
Mechanismen der episomalen Replikation vorhandenstabile Expression eines Transgens
- Kopienzahl des Vektors ?- Zellzyklus (MCM2-7) abhängige Replikation- (mini chromosome maintenance) ?
Achtung: nicht mehr oder selten teilende Zellen !!!
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Plasmidreplicons:
Nicht-virale Plasmidreplicons und künstliche Chromosomen:1) Historisch: Vektoren für die Hefe2) Vektoren für Säugetierzellen
Virale Plasmidreplicons:1) Das Simian Virus 40 (SV40) Plasmidreplicon2) Das Papillomavirus (HPV / BPV) Plasmidreplicon3) Das Epstein-Barr Virus (EBV) Plasmidreplicon4) „Substituted“ EBV Plasmidreplicons
Administration als „nackte“ (Plasmid-) DNA
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Virale Plasmidreplicons:Das Simian Virus 40 (SV40)
Familie: Polyomaviren40 nm großes, ikosaedrisches KapsidNicht umhülltZirkulares, doppelsträngiges DNA Genom (5300 bp)MCM 2-7 unabhängige Replikation (T-Antigen ist replikative Helikase)
5300 bp
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Model für die Untersuchung:- der eukaryontischen DNA Replikation- der Chromatinstruktur (Nukleosomen)- der Genregulation („cis acting“ Enhancer)- des alternativen „splicing“
ab 1976
MCM 2-7 AbhängigkeitNukleäre Retention KopienzahlDNA Replikation
NeinKopienzahl 100-1000
SV40 ORI + T-Antigen
Virale Plasmidreplicons:Das SV40 („high copy number“) Plasmidreplicon
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Anwendung in der Biotechnologie:- Plasmidvektoren mit SV40 ORI und Promoter in cis- Zellinien mit T-Antigen (293T, COS etc.) in trans
Immortalisierung (Transformation) von primären Zellen via T-Antigen („libraries“ !)
Das T-Antigen:- Bindung an SV40 ORI- MCM 2-7 unabhängige, replikative Helikase- Virales Onkoprotein (Inaktivierung von p53 und pRB)- Immunogene Eigenschaften
Virale Plasmidreplicons:Das SV40 („high copy number“) Plasmidreplicon
heute
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Virale Plasmidreplicons:Papillomaviren (HPV / BPV)
Familie: Papillomaviren55 nm großes, ikosaedrisches KapsidNicht umhülltZirkulares, doppelsträngiges DNA Genom (7900 bp)MCM 2-7 unabhängige Replikation (E1 ist replikative Helikase)
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Chromosom
MCM 2-7 AbhängigkeitNukleäre Retention Kopienzahl
DNA Replikation
Nein(E1 +) E2 – Chromsom
(Kopienzahl)~ 50 - 150
BPV ORI + E1 + E2
E2
piggy back
Virale Plasmidreplicons:Das BPV („intermediate copy number“) Plasmidreplicon
E1
1980
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Virale Plasmidreplicons:Das Epstein-Barr Virus (EBV)
ori P ori lyt ori lyt
172 kbp
IR: inverted repeatTR: terminal repeatU: unique regionori P: latenter (Plasmid) Ori ori lyt: lytischer ORI
Familie: Herpesviren160 nm großes, umhülltes VirusIkosaedrisches KapsidDoppelsträngiges DNA Genom (172 kbp):- Linear im Virion- Zirkular nach Rezirkularisierung im ZellkernMCM 2-7 abhängige latente Replikation (ori P) MCM 2-7 unabhängige lytische Replikation (ori lyt)
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MCM 2-7 AbhängigkeitNukleäre Retention KopienzahlDNA Replikation
EBNA
FR 24 x
DS 4 x
ORC / MCM 2-7
Chromosom
JaOriP (FR) + EBNA1 - Chromosom
~10OriP (DS) + EBNA1
EBNA
piggy back
Virale Plasmidreplicons:Das EBV („low copy number“) Plasmidreplicon
EBNA1
EBNA1
ORC / MCM2-7 – RekrutierungReplikation
FR
DS
Nukleäre Retention1985 Latenz-ORI: ori P
FR: family of repeats (24 x)DS:dyad symmetry element (4 x)
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RNA-/DNA-Vektoren im Vergleich
img.medscape.com/.../540/632/nf540632.tab1.gif
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LMU
Fazit: RNA-/DNA-Viren:
RNA-Viren: in der Regel transient(Ausnahme: Retro-/Lentiviren),z.T: onkolytischeher geringe Verpackungskapazität
DNA-Viren: z.T. transient oder persistierend z.T. onkolytischin der Regel eher größere Verpackungskapazitätwenn persistierend, dann Langzeitexpression