Molekulare Virologie LMU · The first FDA-approved antisense drug. Structure of the 21-mer...
Transcript of Molekulare Virologie LMU · The first FDA-approved antisense drug. Structure of the 21-mer...
RNA-Vektoren
LMUMolekulare Virologie
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
?!
RNA-/DNA-Vektoren im Vergleich
http://www.nature.com/nrg/journal/v4/n5/fig_tab/nrg1066_F2.html
(Virale) Vektoren -Systeme und Einsatzfelder
RNA-/DNA-Vektoren im Vergleich
img.medscape.com/.../540/632/nf540632.tab1.gif
Virale Vakzine Vektoren basierend auf RNA-Viren
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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RNA - Inhibition
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
Aptamere
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
Antisense RNA
http://learn.genetics.utah.edu/content/tech/genetherapy/gtapproaches/
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.
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
siRNA
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
RNA-Transfer in Zellen
famulok.chemie.uni-bonn.de/people/mayer/Vorlesung/RNA%20Biochemie%208.pdf -
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
ss-RNA (+)-Vektoren
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
gsbs.utmb.edu/microbook/ch061.htm
The rabies virus genome
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
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
Reoviridae (Rotaviren)
Klinik (Rotavirus A)- Inkubationszeit: 1 - 3 Tage
- Meist inapparent bei Neugeborenen und älteren Kindern
- Apparent v.a. bei Kindern > 3 Monate und < 2 Jahre
- Symptome (3 – 5 Tage):
- Fieber
- Erbrechen
- Bauchschmerzen
- Durchfall
- Bei schweren Verläufen (40%):
- Stationäre Behandlung im Krankenhaus erforderlich
- Dehydration, Lethargie und Kreislaufversagen
- Tödliche Verläufe selten in Industrieländern
- Virusausscheidung bis 3 Tage nach Beschwerdefreiheit
Reoviridae (Rotaviren)Rotavirus Impfstoffe:
Rotarix: - attenuierter Lebendimpstoff - vom häufigsten Serotyp G1P abgeleitet- monovalent- starke Replikation im Darm- zwei Impfdosen (niedrig konzentriert)
Rotateq (Rotashield):- rekombinanter Lebendimpfstoff auf Basis des bovinen Rotavirus (Stamm WC3)
- polyvalent (5 Varianten mit 5 Antigenen der häufigstenhumanpathogenen Rotavirus Serotypen)
- schwächere Replikation im Darm- drei Impfdosen (hoch konzentriert)
Zulassung (Deutschland) in 2006; ab 6. LebenswocheNoch keine STIKO-Empfehlung
98% iger Schutz vor schweren Verläufen
Retroviral Vectors
• Based on:-
– Murine leukaemia virus (MLV)
– Lentiviruses such as HIV, SIV, FIV
– Mouse mammary tumour virus (MMTV)
– Foamy/spuma viruses
Retroviral Genome
Viral RNA
ReverseTranscriptionIntegration
gag pol env
R U5 RU3
Proviral DNA
RU3 U5
gag pol envLTR LTRRU3 U5
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
Packaging CellsRetroviral Vector
RU3 U5RU3 U5geneLTR LTR
ψ
gag pol env
Recombinant Virus
Co-linear integration (always LTR-gene-LTR)
Increased efficiency of expression since transcriptional unit is maintained
Single/low copy number of transferred gene
Clear activity/presence correlation
Titre between 104 and 106 cfu/ml
Lower titre than Adenoviral vectors (~1011)
Pseudotype with VSV-G Protein: results in titre ~1011 cfu/ml
Stability of Virus Particles
Potential use in in vivo gene transfer protocols
Complement mediated inactivation in vivo
Use different species that are less sensitive to complement as basis for packaging lines
Retroviral vectors
Possible recombination with retroviral sequences in packaging cell lines
No recombination in target human cells since retroviral vectors are based on murine retroviruses and show hardly any homology to human retroviruses
May lead to murine RCR associated with cancer or immunode-ficiency
Build safety packaging lines where retroviral genes are on seperate constructs and share little or no sequence homology with vectors
MLV-derived RVs only infect rapidly dividing cells
Useful for targeting some types of tumours and proliferating cells
limits usefulness for some genetic diseases and for differentiated cells
Include HIV MA protein or vprgene sequences to allow infection of quiescent cells
Stable Integration (daughter cells inherit gene)
DNA always present
Possible insertional mutagenesis (activation/inactivation of cellular genes)
Targeting of integration of vectors to specific sites in genome by homologous recombination
Retroviral vectors
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
Risks associated with Retroviruses
• Insertional Integration Leading to– Gene disruption
(tumour suppressor)– Gene activation
(proto-oncogene)
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