DEVELOPMENT OF ADNF14 EXPRESSING ADENOASSOCIATED TYPE 2 AAV VECTOR WITH MODIFIED CAPSID RETARGETED T

1
DEVELOPMENT OF ADNF14 EXPRESSING ADENO-ASSOCIATED
TYPE 2 (AAV) VECTOR WITH MODIFIED CAPSID
RETARGETED TO THE BLOOD BRAIN BARRIER 1Tatiana
M. García, 1Servio H. Ramirez,1Casey Maguire,
1,2Stephen Dewhurst. Departments of
1Microbiology and Immunology, 2Cancer Center,
University of Rochester School of Medicine and
Dentistry, NY



ABSTRACT HIV-associated dementia (HAD) is a
frequent manifestation of late-stage HIV
infection and is characterized by an array of
cognitive and motor deficits that arise in
10-15 of HIV positive individuals. HIV
infection in the brain occurs in macrophages and
microglia, but not in neurons therefore, it is
believed that the neuronal damage and neuronal
loss occur via an indirect pathway that is
mediated by the release of cellular and viral
toxins from activated and/or HIV-infected
macrophages and microglia. One such toxin is HIV
Tat, which can be released from intact
HIV-infected cells and can directly contribute to
the demise of primary neurons both in vivo and in
vitro. Recently, we showed that several
neurotrophins can protect against the neurotoxic
effects of Tat, including a short peptide derived
from the Activity-Dependent Neurotrophic Factor
(ADNF). Biologically active forms of this
peptide can be either nine or fourteen amino
acids in length (ADNF9, ADNF14), and exhibit
protective efficacy at concentrations at or below
1 pM. In light of this, we wished to construct
and test a recombinant Adeno-Associated Virus
(AAV) vector which could express this peptide.
In order to do this, some key features needed to
be incorporated into the design of the vector
construct. First, the peptide must have a
secretory signal to allow for autocrine and
paracrine action. Second, in order for the
peptide to be detectable, it was necessary to add
a short antigenic epitope from the influenza
virus Hemagglutinin (HA) protein. The final ADNF
construct was then either expressed as a simple
HA-fusion, or else it was fused to firefly
Luciferase so that the secreted product could be
readily detected and quantitated, over a wide
dynamic range with very high sensitivity. In this
case, a furin protease cleavage site was
incorporated between the ADNF-HA fusion peptide,
and the Luciferase protein, in order to allow for
the removal of the Luciferase moiety, in the
event that it should be found to interfere with
the biological activity of the ADNF-HA peptide.
This final expression cassette has been
generated, but sequence confirmation is presently
pending. Upon verification, the cassette will be
subcloned into the AAV expression vector.
In a related project, we have also
been engaged in the construction of modified AAV
vectors which may have an enhanced ability to
cross the blood-brain barrier (BBB) and enter the
central nervous system. For this purpose, we
have exploited the fact that novel peptides can
be inserted into certain regions of the AAV VP3
capsid protein, resulting in the formation of
virus particles that possess a modified host cell
tropism. Here we demonstrate the cloning approach
for insertion of novel BBB-targeting peptides
into the AAV cap gene. These peptides,
designated Sp5 and Sp8, were provided to us by
Supratek Inc. (Montréal, Canada) and derived
using a phage display selection system that
harvested phage clones which were able to cross a
tight cell monolayer in vitro. Once these AAV
clones have been generated, our next goal will be
to test whether the Sp5 and Sp8 peptides can
allow more efficient uptake of AAV vectors into
the brain, for subsequent delivery of
neuroprotective mediators such as the ADNF-HA
peptide and its derivatives.
FIGURE 3. RATIONALE FOR AAV-RC CAPSID GENE
MODIFICATION
FIGURE 1. SCHEMATIC REPRESENTATION OF THE
ADNF-HA LUCIFERASE CASSETTE IN PSECTAG
AGGCCGTGCTGGGAGGAGGATCTGCTCTGCTGAGATCTATTCC
TGCTGGAGGAGGATCTTACCCTTACGATGTGCCTGATTACGCTTTA
TCGTCCGGCACGACCCTCCTCCTAGACGAGACGACTCTAGATAAGGA
CGACCTCCTCCTAGAATGGGAATGCTACACGGACTAATGCGAAATTCGA
AMPr
B
A
C
1kb 1 2 3 4 5 6
1Kb 1 2 3 4
AAV-RC/SP5 CLONES PCR SCREENING 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15
MATERIALS AND METHODS Construction of
pSectag/ADNF14-luc Plasmid The goal of this
cloning procedure is to engineer a construct that
would provide ADNF14 with a secretory signal, an
HA detection tag and a luciferase reporter.
First, two oligos bearing complementary sequences
were designed to contain the codon optimized
ADNF14, a spacer (GGGS) and a hemagglutinin tag
(YPYDVPDYA). ADNF14 (VLGGGSALLRSIPA) is derived
from activity-dependent neurotrophic factor, and
it is biologically functional as a
neuroprotective peptide at very low
concentrations (1 pM). Upon hybridization these
oligos were then subcloned into pSecTag2
(Invitrogen, Ca) using restriction sites SfiI and
HindIII, located in both the oligos and the
subcloning vector. The pSecTag2 expression
plasmidtm allows for incorporation of a secretory
signal (V-J2-C region of the Ig kappa chain) into
a gene of interest or in this case the ADNF14
peptide. In parallel the luciferase gene from
plasmid pGL3 (Promega, WI) was amplified via PCR
using the following primers ForwLuc AAAAAAAAGCT
TCAGAGTGAGAAGAATGGAAGAC and BackLuc
AAAAAACCCGGGTTACACGGCGATCT. The PCR product were
next digested with restriction enzymes HindIII
and XhoI for subsequent cloning into the pSectag
plasmid. Note that the primers also provided the
amplified luciferase gene with a FURIN site
(RVRR). Construction of Sp5 and Sp8 Modified
AAV-RC pAAV-RC plasmid contains the AAV-2 rep
and cap genes, encoding replication proteins and
viral capsid structural proteins respectively
(Stratagene). The plasmids AAV-rc /SP5 and
AAV-rc /SP8 contain the full-length wild type
AAV2 cap gene in addition to the Sp5 and
Sp8-encoding sequences which were inserted at
position 587 of VP3. The following cloning
procedure ensures in frame insertion of the novel
peptides at the desired location in the cap gene.
First, two PCR products were generated for each
of the modifying sequences (Sp5 and Sp8). In the
case of AAV-rc/SP5, the two overlapping PCR
products of the primer sets F1ECONIREVSP5 and
FORSP5R2-XCM were mixed and re-amplified to
generate a final PCR product (the modified cap
gene) using primers F1ECON1R2-XCM (Table 1).
The same strategy as described above was applied
for the generation of AAV-rc/SP8, where the two
overlapping PCR products of the primer sets
F1ECONIREVSP8 and FORSP8R2-XCMI were mixed
and re-amplified to generate a final PCR product
(the modified cap gene) using primers
F1ECON1R2-XCM (Table 1). Finally, the resultant
PCR product and the AAV-rc plasmid were digested
with restriction enzymes EcoN1 and XcmI, mixed
and then ligated with T4 DNA ligase
(NEB).           TABLE 1 Sequences of
primers used to construct AAV-rc/SP5 and
AAV-rc/SP8
B
C
A
1kb PCR LUCIFERASE
Verification of Luciferase Function from
pSECTAG/Luc
Overlap PCR
Constructs
AAV-RC/SP8 CLONES PCR SCREENING 1 2 3
4 5 6 7 8 9 10 11 12 13 14 15
60000
50000
40000
Relative Light Units (RLU)
30000
FIGURE 5. A. Lanes 1 and 2 are the overlapping
PCR products of F1EconI-REVSP5 and of
FORSP5-R2XcmI respectively. Lanes 3 and 4 are
the overlapping PCR products of F1EconI-REVSP8
and of FORSP8-R2XcmI respectively. B. Lanes 1, 2
and 3 correspond to the final PCR products
generated upon mixing of the products from lanes
(12) and (34) from panel A, followed by PCR
amplification using outer primers
F1EconIR2XcmI. The expected size of the final
overlap PCR products are 1544 bp (Sp5 lanes 1-3)
and 1477bp (Sp8 lanes 4-6). These PCR products
were extracted, purified and digested with EconI
and XcmI for insertion into AAV-rc the resulting
ligation products were transformed into E. coli
cells and analysed in panel C. The transformants
were examined by colony PCR amplification using
primers that only hybridize to the inserted Sp5
or Sp8 sequences.
20000
10000
0
LUC2
LUC1
PGL3 control
Mock
FIGURE 2. A. PCR of Luciferase gene from pGL3
using primers ForwLUCHindIII and BackLUCXhoI. The
PCR fragment size is 1656bp. The fragments were
extracted and purified. B. Ligation of PCR
luciferase product with pSectag yielded
transformants which were picke, mini-prepped and
examined by restriction digestion with HindIII
and XhoI. The figure show positive clones 1, 2
and 3 (either uncut, or following restriction
digestion) the other clones shown are negative.
Positive clones produced two fragments upon
restriction digestion a fragment of 1656bp
(Luciferase) and another fragment of 5092bp
corresponding to the rest of the Psectag vector.
Clones 1 and 2 were chosen for subsequent cloning
steps. C. HEK293Cells were transfected with 1.6
?g/?l of DNA for all the samples PGL-3 control
plasmid as positive control, a mock transfection
as negative control and the two positive clones
of pSecTag-LUC 1 and 2 using LIPOfectamine 2000.
Firefly Luciferase activity in cell lysates was
determined with the use of a Luciferase assay kit
from Promega and a Lumicount spectrometer from
Packard.

• CONCLUSIONS
• Construction of an hybrid protein expression
  cassette, corresponding to an in-frame fusion of
  firely Luciferase with a cell secretion moiety.
• Demonstration that the Luciferase portion of this
  hybrid protein is secreted into the medium of
  plasmid-transfected HEK293 cells, and that it
  retains enzymatic activity at close-to-normal
  levels.
• Insertion of two novel brain-targeting peptides
  (Sp5, Sp8) into the AAV cap gene.
• FUTURE DIRECTIONS
• Insertion of the ADNF-HA peptide into the
  pSecTag-Luciferase Vector, followed by subcloning
  into the AAV vector.
• Analysis of whether the Sp5 and Sp8-modified AAV
  vector constructs can more efficiently enter the
  brain, following systemic delivery into rats.
• Analysis of whether the secreted ADNF-HA or
  ADNF-HA-luciferase fusion proteins, when
  expressed by AAV vectors, can successfully
  protect neurons from the effects of exposure to
  HIV Tat.

ANNEALED
1kbladder ADNF OLIGOs
FIGURE 3. ADNF14 oligos were annealed using STE
buffer (High salt concentration buffer), brought
to 100?C for 10 minutes, and then slowly cooled
to room Temp, to allow annealing.
100 bp