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FILAMENTOUS PHAGE AS A NOVEL TOOL FOR IMAGING AND TREATMENT OF ALZHEIMER’S DISEASE

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FILAMENTOUS PHAGE AS A NOVEL TOOL FOR IMAGING AND TREATMENT OF ALZHEIMER’S DISEASE Powered By Docstoc
					FILAMENTOUS PHAGE AS A NOVEL
     TOOL FOR IMAGING AND
   TREATMENT OF ALZHEIMER’S
           DISEASE

                    Beka Solomon, Ph.D.
                 Professor of Biotechnology
  Chair for Biotechnology of Neurodegenerative Diseases
          George S. Wise Faculty of Life Sciences
                  Tel Aviv University, Israel




                                                          1
Bacteriophages, viruses that infect
 bacteria, are the most numerous
 life forms on earth, and their
 natural contact with human beings
 is not incidental, but rather
 constant and intensive.


                                      2
           Phage therapy
Classic therapy with lytic phages is an
   alternative to antibiotics, and is being
   developed for clinical use by many
   research groups in Europe and the
   USA.
It has been extensively used and
   developed in the former Soviet Union.


                                              3
Unlike lytic phages, which are
 released by cell lysis after
 assembly in the host cell
 cytoplasm, Ff phages are
 continuously extruded through the
 host cell without affecting them.


                                     4
Filamentous bacteriophages are
excellent carriers for the
expression and presentation of
foreign peptides in a variety of
biological systems, so-called
phage libraries


                               5
      Filamentous Phages in
       Alzheimer’s Disease

1. Immunomodulators
2. Delivery vector of antibodies –
   imaging/treatment
3. Anti-aggregating agent of β-amyloid
4. Imaging agent


                                         6
       Phage as
Immunomodulator of Immune
  Response Against Beta-
     Amyloid Peptide


                        7
We developed a series of antigens
  displaying the EFRH epitope on
  filamentous phages, which enabled
  modulating the immune response
  according to the number of EFRH
  copies.

  (Lavie V, et al. J Molec Neurosc (2004) 24:105-113)


                                                        8
     Epitope EFRH, sequenced 3-6 within the -
    amyloid peptide, acts as a regulatory site
    controlling the conformational changes of
    whole -amyloid peptide.

      Locking of the epitope by antibodies
    affects the dynamics of all the molecule,
    preventing self-aggregation as well as
    enabling resolubilization of already formed
    aggregates.

      Frenkel et al. J. Neuroimmunology 1998,1999
                                                    9
  EFRH immunization of hAPP
       transgenic mice
We prepared antigens containing
 between 10-300 copies of different
 numbers of EFRH displayed on the
 phage.
Tg mice model of AD were treated for 6
 months.



                                         10
                              Phage-EFRH Treatment Effect on
                              Amyloid Burden in Tg Mice Brain
                     2.5                     n=4

                      2
Amyloid Burden (%)




                     1.5
                                                      n=5
                      1                                                    n=5

                     0.5
                               n=8
                      0
                           nTg Control   Tg Control    10                  300

                                                            EFRH Coppies


                                                                                 11
No toxic effects were observed
 following EFRH phage
 immunizations in the challenged
 animal sections, including brain,
 liver, kidney, spleen and lung.



                                     12
A Single Chain Variable Fragment
            Molecule




           ScFv molecules origin:

 1. Spleen mRNA of immunized mice
 2. Hybridoma cells expressing a specific
    antibody.
                                            13
     Intranasal administration of
    phage-antibodies or antigens

•   Provides a simple and rapid delivery route
    of therapeutic agents into the CNS due to
    the unique connection between the nose
    and brain.
•   Substances delivered by intranasal
    administration exert their effect on the brain
    rather than on peripheral tissues.



                                                14
 Treatment of Tg Mice Model
    of AD with Phage ScFv

Intranasal administration of 10 μl
  containing 1011 phages per mouse
  took place every 3 weeks for a total
  period of 6 months.


                                     15
Improvement in MWM (Path Length)

    80                                                       *
                                                            n=9
    60           n=9

    40
%




    20                                n=9

     0

    -20
              Non Tg             Tg control            Tg treated

The improvement was measured in path length and was calculated as: (1-
path length at day4/ path length at day1)*100. * P<0.005 from tg control.
                                                                            16
                Amyloid Burden

                                * P<0.0005
          2.5
          2.0
          1.5
      %


                                     *
          1.0
          0.5
          0.0
                   Tg control    Tg treated


Thioflavin-S staining of mice-brain sections
showed that plaque load in the treated mice
was an average of 50% of that in the control
                                           17
tg mice.
Passive immunization with antibodies
  devoid of Fc which may prevent
  over-activation of microglia and
  thus, attenuate auto-antibody
  triggered neuroinflammation.




                                   18
       Phage
as Anti-Aggregating
Beta-Amyloid Agent



                      19
The phage’s long thin filamentous shape
 may be the basis underlying its anti-
 aggregating function.

Modifying the phage’s linear structure
 and rendering it spherical abolished its
 disaggregating activity.



                                        20
In vivo Studies



                  21
   Phage administration schedule to
            PDAPP mice

Intranasal Application                                                                                  Neuropathological
                                                                                                           Evaluation
                I II III IV V           VI         VII       VIII         IX         X         XI      XII        XIII XIV


March   April   May   June       July   August   September   October   November   December   January   February   March   April   May




                                                             Object Recognition Test                              Smell Test
                             I                    II
                       Physiological Monitoring:
                       Blood count & biochemistry
                       Phage clearance
                       Body weight



                                                                                                                                        22
  Memory Assessment (Object Recognition Test)
Day 1 Habituation: 20 min with objects A and B
Day 2 Experiment: 10 min with the old object B and a new object C




Recognition Index (RI), calculated for each mouse, was expressed as the ratio:
tC/(tB + tC) 100 , where tB and tC represent the time spent during trial on old
object B and new object C, respectively.




                                                                           23
Recognition Index in PDAPP Tg Mice

            70

            60                              *
            50

            40
      (%)




            30

            20

            10

             0
                 nTg Control   Tg Control       Phage WT

                  PBS I.N.      PBS I.N.          I.N.




* P< 0.05 from Tg Control
(The one way ANOVA followed by Tukey HSD Post-Hoc comparison test )
                                                                      24
      Assessment of synaptic plasticity by
         synaptophysin measurements




                                                                                                                       25154




                                                                                                                                               25090




                                                                                                                                                                               25098




                                                                                                                                                                                                                            25083



                                                                                                                                                                                                                                            25060
                                                                                                                               25092
                                                         25105

                                                                 25104

                                                                         25103

                                                                                          25102
                                                                                                   25156
                                                                                                               25155




                                                                                                                                       25091




                                                                                                                                                       25101
                                                                                                                                                               25100

                                                                                                                                                                       25099




                                                                                                                                                                                                                                    25062
                                               25106




                                                                                                                                                                                       25097

                                                                                                                                                                                                25096



                                                                                                                                                                                                                    25088
                                                                                                                                                                                                        25089
                               Synaptophysin
                               38 kDa




                              Tubulin
                              55 kDa




                                                       Phage-treated                                                   Control                                 Phage-treated                                         Control




                                                                                                                                                     levels
                                                                                                                                       Synaptophysin levels
Synaptophysin levels in the
                                                                                                   2.5
phage treated group were                                                                                   2
                                                                                                                                                                                                                *




                                                                                 arbitrary units
25% higher compared to the                                                                         1.5

control group.                                                                                             1

                                                                                                   0.5

                                                                                                           0
                                                                                                                                       Tg Control                                                     WT
                                                                                                                                                                                               Phage helper
                                                                                                                                                                                                                        25
Intranasal Immunization Effect on
Astrocytosis in PDAPP Mice Brain
   Staining with anti-GFAP antibody




                                      26
       Evaluation of inflammation markers of
       microgliosis by measuring F4/80 levels



                       50 m

     Control mouse                                     Phage –treated mouse



No statistical difference in                               F4-80 labeling by IHC

                                                 180
microglia levels was                             160


                               arbitrary units
                                                 140
                                                 120

observed between control                         100
                                                  80
                                                  60

and phage-treated group                           40
                                                  20
                                                   0
                                                           Tg Control              Phage


                                                                                           27
  No adverse effects in peripheral
             organs
Kidneys, liver, lungs and spleen histology
were normal, as per:

         Hematoxylin & Eosin

          Congo red

          Hemosiderin staining

          Weight
                                             28
Phage Gadolinium for Plaque
         Imaging




                          29
Intranasal Administration

                  • Bypass the
                    BBB
                  • Ease of
   Gd               administration
                  • No side effects
   Gd

   Gd




                                  30
Phage concentration in the brain and olfactory
bulb, 1, 3, and 24 hours post intranasal
administration to BALB/c mice




                                                 31
    Visualization of the complex

   Theoretically thousands
molecules of Gd-DOTA can
be attached to one
filamentous phage, therefore
high signal can be obtained
with relatively low antibody
concentration.


                                   32
Phages have distinct advantages over
  animal viruses.
The great versatility of the phage system has led to
  the development of novel and improved phage
  delivery vectors, as well as immunomodulation of
  anti-AβP response.

The potential therapeutic phage therapy stems from its
  lack of natural tropism for mammalian cells,
  resulting in no adverse effects.


                                                       33
             CONCLUSIONS
 Linear structure of filamentous phage confers phage
  penetration to the brain and disaggregating
  properties.

 Phages are eliminated from the brain, probably via
  microglia, and secreted from the body in urine and
  feces, without adverse effects.

 The therapeutic potential of phage therapy stems
  from its lack of natural tropism for mammalian cells.

 Intranasal administration of filamentous phage offers
  a convenient and effective way of treating
  Alzheimer’s disease.
                                                      34
TEL-AVIV UNIVERSITY
     Tel-Aviv, Israel
   Dan Frenkel
   Orna Goren
   Vered Lavie
   Maria Becker
   Noa Sharon
   Hemi Dimant
                        35