Transcript development of adnf14 expressing adeno-associated type 2

DEVELOPMENT OF ADNF14 EXPRESSING ADENO-ASSOCIATED TYPE 2 (AAV) VECTOR WITH MODIFIED
CAPSID RETARGETED TO THE BLOOD BRAIN BARRIER
1Tatiana
Departments of
1Microbiology
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.
M. García, 1Servio H. Ramirez,1Casey Maguire, 1,2Stephen Dewhurst.
and Immunology,
2Cancer
Center, University of Rochester School of Medicine and Dentistry, NY
FIGURE 3. RATIONALE FOR AAV-RC CAPSID GENE MODIFICATION
FIGURE 1. SCHEMATIC REPRESENTATION OF THE ADNF-HA + LUCIFERASE CASSETTE IN PSECTAG
ADNF14
Spacer
HA tag
REP
AGGCCGTGCTGGGAGGAGGATCTGCTCTGCTGAGATCTATTCCTGCTGGAGGAGGATCTTACCCTTACGATGTGCCTGATTACGCTTTA
TCGTCCGGCACGACCCTCCTCCTAGACGAGACGACTCTAGATAAGGACGACCTCCTCCTAGAATGGGAATGCTACACGGACTAATGCGAAATTCGA
XcmI
EconI
AMPr
AAVrc
AAV-RC
7327 bp
PCR product of
UNMODIFIED
CAP GENE
F1/R1 and F2/R2
Overlap PCR
and fill in
SfiI(978)
XhoI(1072)
HindIII(998)
Digest overlap
product
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.
MODIFIED CAP
GENE
B
1kb+
PCR LUCIFERASE
Low DNA marker
Psectag
Psectag+X+H
C1
C1 cut
C2
C2 cut
C3
C3 cut
C4
C4 cut
C5
C5 cut
C6
C6 cut
C7
C7 cut
C8
C8 cut
PRIMER
SEQUENCE
Verification of Luciferase Function from pSECTAG/Luc
Constructs
REVSP8
GCCGCACGGCGTCGGGTCGCCGTCCAACACCGGGTTGCCTCTCT
GGAGGTTGG
FORSP8
CCGGTGTTGGACGGCGACCCGACGCCGTGCGGCAGACAAGCAG
CTACCGCAGATG
TABLE 1 Sequences of primers used to construct AAV-rc/SP5 and AAV-rc/SP8
3
4
4 5
6
C AAV-RC/SP5 CLONES PCR SCREENING
Overlap
PCR
AAV-RC/SP8 CLONES PCR SCREENING
60000
50000
40000
30000
20000
10000
1kb+ladder
ANNEALED
ADNF OLIGOs
0
Mock
PGL3
control
LUC1
LUC2
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 (1+2) and (3+4) from panel A, followed by PCR amplification using outer primers [F1EconI+R2XcmI].
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.
CONCLUSIONS
•
•
GTACATGCCGTGCGTCACCCAGCTGTACCACTCGATCTCGTAGCC
ATTGTTGCCTCTCTGGAGGTTGG
GCCGCACGGCGTCGGGTCGCCGTCCAACACCGGGTTGCCTCTCT
GGAGGTTGG
2
1 2 3
C
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.
R2-XCMI (outer primer) TGTCCTGCCAGACCATGCCTGG
FORSP5
1
1kb
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
F1ECONI (outer primer) GAACCTCTGGGCCTGGTTGAGGAAC
REVSP5
1Kb+
B
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
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 [F1ECONI+REVSP5] and [FORSP5+R2-XCM] were mixed and re-amplified to generate a final PCR
product (the modified cap gene) using primers F1ECON1+R2-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 [F1ECONI+REVSP8] and [FORSP8+R2-XCMI] were mixed and reamplified to generate a final PCR product (the modified cap gene) using primers F1ECON1+R2-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).
A
Relative Light Units (RLU)
A
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).
AAV-RC/SP5 or
AAV-RC/SP8
AAV-RC
MATERIALS AND METHODS
Construction of pSectag/ADNF14-luc Plasmid:
SP5/SP8
•
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
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.