Molekulare Virologie L U - Helmholtz Zentrum München · Fomivirsen (Vitravene) The first...

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 -

( )

RNA - Inhibition


Aptamere


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

gene3. 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.


siRNA


RNA-Transfer in Zellen

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

Molekulare Virologie L U - Helmholtz Zentrum München · Fomivirsen (Vitravene) The first...

Documents

Transcript of Molekulare Virologie L U - Helmholtz Zentrum München · Fomivirsen (Vitravene) The first...