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.

AGGCCGTGCTGGGAGGAGGATCTGCTCTGCTGAGATCTATTCCTGCTGGAGGAGGATCTTACCCTTACGATGTGCCTGATTACGCTTTA TCGTCCGGCACGACCCTCCTCCTAGACGAGACGACTCTAGATAAGGACGACCTCCTCCTAGAATGGGAATGCTACACGGACTAATGCGAAATTCGA

pSECTAGA5166 bp

pCMV

Ig kappa Leader

BGH pA

Ampr

pGL3 LUC5256 bp

LUC

Ampr

MCS HindIIIForwLUC XhoI

BackLUC

SfiI(978)HindIII(998)

XhoI(1072)

ADNF14 Spacer HA tag

PCR Product

Constructs

0

10000

20000

30000

40000

50000

60000

Mock PGL3 control

LUC1 LUC2

Rel

ativ

e L

igh

t U

nit

s (R

LU

)

ANNEALED 1kb+ladder ADNF OLIGOs

AMPr

Digest overlap product

AAVrc

7327 bp

UNMODIFIED CAP GENE

REP

AAV-RC

EconI

XcmI

SP5/SP8

PCR product of

F1/R1 and F2/R2

Overlap PCR and fill in

AAV-RCAAV-RC/SP5 or

AAV-RC/SP8

MODIFIED CAP GENE

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.

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

1Kb+ 1 2 3 4

MATERIALS AND METHODSConstruction 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 [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 re-amplified 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).

 

 

 

 

 

TABLE 1 Sequences of primers used to construct AAV-rc/SP5 and AAV-rc/SP8

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.

PRIMER SEQUENCE

F1ECONI (outer primer) GAACCTCTGGGCCTGGTTGAGGAAC

R2-XCMI (outer primer) TGTCCTGCCAGACCATGCCTGG

REVSP5 GTACATGCCGTGCGTCACCCAGCTGTACCACTCGATCTCGTAGCCATTGTTGCCTCTCTGGAGGTTGG

FORSP5 GCCGCACGGCGTCGGGTCGCCGTCCAACACCGGGTTGCCTCTCTGGAGGTTGG

REVSP8 GCCGCACGGCGTCGGGTCGCCGTCCAACACCGGGTTGCCTCTCTGGAGGTTGG

FORSP8 CCGGTGTTGGACGGCGACCCGACGCCGTGCGGCAGACAAGCAGCTACCGCAGATG

1kb 1 2 3 4 5 6

FIGURE 1. SCHEMATIC REPRESENTATION OF THE ADNF-HA + LUCIFERASE CASSETTE IN PSECTAG FIGURE 3. RATIONALE FOR AAV-RC CAPSID GENE MODIFICATION

Low

DN

A m

arke

r

Pse

ctag

Pse

ctag

+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

1kb+ PCR LUCIFERASEA B C

A B CAAV-RC/SP5 CLONES PCR SCREENING 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

AAV-RC/SP8 CLONES PCR SCREENING 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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.

Verification of Luciferase Function from pSECTAG/Luc

100 bp

OverlapPCR