Reconstituted influenza virus envelopes as an efficient carrier system for cellular delivery of smal

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Reconstituted influenza virus envelopes as an
efficient carrier system for cellular delivery of
small-interfering RNAsBy Devin DeHaan and Nui
Pholsena
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Why virus envelopes?

• Existing solutions
• siRNA complexed with polyethylenimine (PEI) a
  transfection reagent
• siRNA formed with cationic lipids or collagen
  derivatives
• siRNA entrapped in biodegradable microspheres
• protect siRNA from degradation and prolonging
  the circulation time.
• -not efficient in translocating siRNA into the
  cytosol of the target cells.

• Challenges
• Delivery of siRNAs to cytosol of target cells has
  been inefficient
• 1) siRNAs are degraded by endogenous nucleases
• 2) broad unwanted effects on normal host tissues

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Why virus envelopes?

• Envelopes/membranes protect the genetic material
  (RNA/DNA)
• efficiently delivery to the cytoplasm and the
  nucleus of the host cells

• Influenza virus membrane compositions
• hemagglutinin (HA)
• neuraminidase (NA)

binds to sialic acid residues on glycoplipids
and glycoproteins of target cell membrane?taken
up by endocytosis low pH in endosome?activates
HA fusion-active conformation?mediates fusion of
the viral and the endosomal membrane viral
nucleocapsis get access to the cytosol and virus
replication can proceed
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Objectives

• Virosomes are able to deliver siRNAs to the
  cytosol of target cells
• siRNA duplexes can be encapsulated in virosomes
  that are prepared with cationic lipids and be
  protected from nuclease activity
• Virosomes can fuse with target membranes and
  efficiently deliver the encapsulated siRNAs to a
  variety of cells in vitro thus inducing silencing
  of the target gene
• Virosomes are also suitable for delivering siRNA
  in vivo

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Generation of virosomes

• Constructing the virus envelope
• Solubilization of influenza virus membrane
  envelope with suitable detergent
• (1,2-dihexanoyl-sn-glycero-3-phosphatidylchol
  ine (DCPC))
• Ultracentrifugation of the solubilized virus?a
  clear supernatant
• containing 35-40 of total viral protein
  (mainly HA and NA (by SDS-PAGE test))
• The supernatant was used for reconstitution of
  virosomes.
• Encapsulating siRNA
• siRNA complexed with cationic DODAC
  (N,N-dioleoyl-N,N-dimethylammoniumchloride),
  which is 10-34 of viral phospolipid, and added
  to the solubilized viral membrane prior to
  reconstitution for more efficient encapsulation.
• 10mol DODAC (/- charge ratios of .73)?1.5 of
  added siRNA
• 22mol DODAC (/- charge ratios of 1.6)?2 of
  added siRNA
• 28mol DODAC (/- charge ratios of 2.48)?5 of
  added siRNA
• 34mol DODAC? 35siRNA recovered.
• Virosomes prepared with 34 DODAC were used in
  the experiment.
• Formation of virosomes
• Remove DCPC by dialysis
• Purified the preparation on a discontinuous
  sucrose gradient to remove unwanted materials
  that arent encapsulated or not tightly
  associated with the reconstituted vesicles

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Virosome Composition and Morphology
Composition

• tight association of protein, phospholipid, and
  siRNA.
• all peaked at a sucrose concentration of 30

Morphology

• Spherical and spiky
• Mean size 60 nm (smaller than native virus)

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Analysis of siRNA encapsulation
Was siRNA really encapsulated and By how much?

• Preparations were treated with
• benzonase (an enzyme with nuclease
• activity)
• -Nonencapsulated siRNA were completely
  degraded by the enzyme
• -Encapsulated siRNA was protected from
  degradation
• -Encapsulated siRNA is accessible and
  degraded only when virosomes were solubilized
  with detergent prior to benzonase treatment
  (fourth lane)
• -Comparing siRNA recovered from untreated
  virosomes to benzonase treated virosomes showed
  encapsulated siRNA was protected 80
• Encapsulated siRNA is well shielded from
  extravirosomal proteins and very stable even in
  presence of nucleases.

Above Nuclease protection assay. Free siRNA or
siRNA encapsulated in virosomes was treated with
benzonase in the presence or absence of C12E8 as
indicated. After inhibition of the nuclease
activity by chelation of Mg2, the preparations
were solubilized and analyzed on a 15
polyacrylamide gel. Results of a typical
experiment are shown.
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Evaluation of fusogenic properties

• Virosomes with and without encapsulated siRNA
  were prepared with 10 mol pyrPC
  (1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-
  phosphatidylcholine) incorporated into the
  membrane.
• Why pyrPC?
• Pyrene molecules in high form excimers
  (excited dimers) with an emission maximum at 480
  nm.
• Dilution of the probe (after fusion with an
  unlabeled target membrane) makes the excimers
  fall apart?the drop in excimer fluorescence is a
  measurement of fusion
• Observation
• pH 5.5?HA conformation is fusion-active and fuses
  to a
• final extent of 50-60 (with or without
  siRNA)
• pH 7.4?no fusion of siRNA virosomes were observed
• Exposure of virosomes to low pH without target
• membrane followed by neutralization
  inhibits fusion.

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Delivery of siRNA to cultured cells

• Why?
• To test the toxic effects of virosomes on cells
• Procedure
• Baby Hamster Kidney (BHK21) cells were incubated
  with virosomes for 72 hrs
• The cells were visually inspected on a regular
  basis
• Toleration Rates
• Survival percentages were between 80 and 100
• No cell deterioration

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Determining Virosomes Ability for siRNA Delivery

• Procedure
• BHK21 incubated with virosomes containing siRNA
  labeled with fluorescent labels
• Analyzed with confocal laser scanning microscopy
• Success Rates
• Cells exhibited strong fluorescence
• Cells were incubated with non fluorescent or
  empty virsosomes as a control

Above Microscopical analysis of siRNA delivery.
BHK21 cells (1 X 104/well) were incubated with
10 pmol of FAM-labeled siRNA encapsulated in
virosomes for 90 min. Cells were then fixed and
stained with rhodaminephalloidin to visualize
stress fibers. Specimens were analyzed by
confocal microscopy. Focus was on the cell
surface (a) or on the cell interior (b). Bar10 
 m.
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Graphical Results
Quantification of siRNA delivery by flow
cytometry. (a) BHK21 cells (white 2 X104/well)
and A2780-EGFP cells (hatched 6 X 104/well) were
incubated for 24 h with virosomes containing the
given amounts of Cy5-labeled siRNA.
Cell-associated fluorescence was then measured by
flow cytometry. (b) A2780-EGFP cells (6  
104/well) were transfected with the indicated
amounts of siRNA using L2000 (white), or
fusion-active (hatched) or fusion-inactivated
virosomes (crossed), respectively.
Cell-associated fluorescence was determined as in
(a).
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Inhibition of EGFP neosynthesis by virosome
delivered siRNA

• Why?
• To investigate if siRNA was delivered to cells
  and reached the cytoplasm functionally active
• Procedure
• Madin Darbin canine kidney (MDCK) cells were
  transfected with siRNA directed against mRNA of
  Green Fluorescent Proteins (GFP)
• Results
• Transfection with plasmids containing EGFP
  resulted in high expression
• Cotransfection of cells with siGFP and plasmid
  DNA resulted in very low detection of
  fluorescence
• Exposure of cells to siGFP prior to plasmid
  transfection markedly reduced EGFP expression as
  well

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Graphical Results
Effect of virosome-delivered siGFP on
constitutive EGFP expression. (a) A2780-EGFP
cells (6 x104/well) were transfected with the
indicated amounts of siGFP encapsulated in
virosomes. EGFP fluorescence was measured by flow
cytometry 24, 48, and 72 h later. (b)
Fluorescence distribution curve of cells
transfected for 72 h as in (a). Shaded control
bold line 1 pmol medium line 3 pmol and light
line 10 pmol siRNA. (c) A2780-EGFP cells were
incubated with the indicated amounts of siGFP
using fusion-active virosomes, fusion-inactivated
virosomes, or L2000, respectively, or received
empty virosomes. EGFP expression as the
percentage of untreated control cells 72 h after
transfection is shown. Results are representative
for three experiments performed.
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Mechanism of Virosome mediated siRNA

• Why?
• To determine the action of virosome fusion
• Procedure
• Incubation in low pH to deactiviate HA and then
  neutralization
• Results
• In cells with fusion inactivation no effect was
  seen in expression of GFP
• HA membrane proteins are crucial for
  incorporation of siRNA into the cytoplasm

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Depletion of Constitutively Expressed Proteins by
siRNA Virosomes

• Why?
• To test the effects of inhibition on
  constitutively expressed proteins
• Procedure
• EGFP cells were incubated with increasing amounts
  of siGFP virosomes
• Results
• EGFP cell expression decreased over time
• Cell viability showed no decrease

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Virosome-mediated siRNA delivery in vivo

• Why?
• To test the suitability of virosomes for use in
  vivo
• Procedure
• Injected mice with FAM-labeled siRNA
• Killed mice and examined cell tissue
• Results
• 85.2 of target cells were associated with
  FAM-labeled siRNA

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Discussion

• Results
• Suitable carriers of siRNA
• Delivered siRNA in vitro and in vivo
• Fusion mediatation by HA incorporated in the
  virosome membrane
• Applications
• siRNA-mediated gene therapy for infectious
  diseases, neurological disorders, acute liver
  failure, cancer, sepsis, inflammation, and others
  

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siRNA Delivery

• Other Methods
• Delivery of small siRNA molecules
• Delivery via plasmid vectors encoding small
  hairpin RNA (shRNA)
• Use of viral vectors that encode shRNA or both
  strands of siRNA

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Why Choose Virosomes?

• Advantages
• Virosomes bind to the cellular receptor of
  influenza
• Broad target range
• Active delivery of siRNA into cytoplasm
• Transfection is not effected by serum components
• Safe injections

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Drawbacks/Solutions

• D People who had influenza infections have
  antibodies against virosomes
• S Use virosomes formed from influenza
  strains that havent circulated recently
• D Repeated injections of virosomes might
  lead to HA-specific immune response which might
  impair siRNA delivery
• S Generate virosomes from non-crossreactive
  strains

Target site, mechanism, and application

• Respiratory tract
• -common site of infection by influenza virus
• -poorly immunogenic because of strong tolerance
  mechanism at mucosal sites so repeated
  administration is possible.
• Mechanism bind to and enter the respiratory
  epithelium cells
• Application target respiratory diseases such as
  severe acute respiratory syndrome or (SARS)