Shi, W, Hemminki, A and Bartlett, JS. Capsid modifications overcome low heterogeneous expression of heparan sulfate proteoglycan that limits AAV2-mediated gene transfer and therapeutic efficacy in human ovarian carcinoma. Gynecol Oncol 103: 1054-1062

University of Helsinki, Helsinki, Uusimaa, Finland
Gynecologic Oncology (Impact Factor: 3.77). 01/2007; 103(3):1054-62. DOI: 10.1016/j.ygyno.2006.06.020
Source: PubMed


Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa).
We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay.
All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (alphav integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV.
The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa.

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Available from: Jeffrey S Bartlett, Oct 03, 2015
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    • "Two of these, R585 and R588 are located in a surface loop at the 3-fold axis of symmetry (Kern et al., 2003; Opie et al., 2003). Alterations in this surface loop by insertion of an RGD integrin binding motif is able to ablate heparin binding and increase transduction of human lymphoblastic and chronic myelogenous leukemia cell lines, ovarian carcinoma cells in culture, and in an in vivo xenograft tumor model (Shi et al., 2006). Recently, insertion of atherosclerotic plaque targeting peptides into the AAV2 capsid at amino acid 587 re-targeted the vector (White et al., 2008). "
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    ABSTRACT: Selection of viral vectors by screening viral display peptide libraries is an auspicious approach to improve safety and efficiency of gene vectors. The screening of random AAV peptide libraries occurs via the amplification of viruses from a multitude of potential targeting peptides each presented within an AAV capsid that are internalized into target cells, mediated by the peptide displayed on their surface. The aim of this thesis was the selection of cell type- or tissue-directed gene vectors from random peptide libraries displayed on adeno-associated virus (AAV) and their characterization. Immature malignant blood progenitor cells causing acute myeloid leukemia (AML) are generally considered to be transduction-resistant to most conventional gene vectors. We screened random AAV serotype 2 peptide libraries on AML cells to select vector capsids with optimized leukemia transduction capacity. The screening revealed a distinct peptide sequence motif displayed on the selected viral capsids. The capsid mutant displaying the peptide NQVGSWS transduced the leukemia cell line Kasumi-1 with up to 90% efficiency, in contrast to vectors displaying a random unselected peptide (0.2% efficiency). Transduction assays on a panel of cell lines showed that the NQVGSWS capsid was able to overcome resistance to AAV-transduction especially in hematopoietic cancer cells. We further showed that NQVGSWS transduction of leukemia cells is independent of the primary attachment receptor heparin sulfate proteoglycan that is used for infection by wild-type AAV-2. Finally, leukemia targeted NQVGSWS-AAV vectors harboring a suicide gene conferred selective killing to Kasumi-1 AML cells. Therefore, we concluded that the selected vector capsids are a suitable and valuable tool to target therapeutic genes to AML cells. Screening AAV peptide libraries in vivo provides much more appropriate conditions to select for tissue-targeted gene vectors than mere cell-based in vitro approaches. In the second part of this thesis we developed a PCR-based amplification method allowing for adenovirus independent screening of AAV libraries. We performed in vivo selections applying several kinetic approaches in animals over multiple rounds after intravenous administration. The polyoma middle T-transgenic murine breast cancer and murine lung tissue were used as prototype targets. The peptide sequences of AAV clones yielded distinct sequence motifs unique for the target tissue. Selected capsid mutants conferred gene expression in the target tissue which was not detectable in animals injected with control vectors. However, most of the clones also transduced heart tissue in addition to the target tissue. We therefore conclude that this approach may be particularly useful if the tropism of the intended gene transfer in vivo has to be extended to rather than confined to the tissue of interest, indicating that targeting AAV to certain tissues in vivo seem to require more than one capsid modification. This impact the further development and improvement of AAV peptide libraries. Taken together, the work presented here demonstrates that random AAV displayed peptide libraries can be used to select for improved gene delivery vectors in vitro and, which is entirely novel, in vivo. Our results broaden the knowledge of transduction behavior of vectors isolated from AAV-2 libraries on different targets in vitro and in vivo and showed that such vectors have the potential to be used for therapeutic gene transfer. Die Selektion viraler Vektoren aus randomisierten, auf Viruskapsiden exprimierten Peptidbanken, ist ein vielversprechender Ansatz zur Steigerung der Sicherheit und Effizienz von Genvektoren. Bei AAV Peptidbanken handelt es sich um eine virale Peptidbank mit einer Diversität von 2x108, bei der jeweils ein randomisiertes Peptid in der Rezeptorbindung vermittelten Region des AAV Kapsids präsentiert wird. Ein Screening dieser Peptidbanken auf Zielzellen ermöglicht eine Anreicherung zielzelltransduzierenden Kapsidvarianten. Ziel dieser Arbeit war die Isolierung und Charakterisierung von zelltyp- bzw. gewebespezifisch transduzierender adeno-assoziiert-viraler (AAV) Vektoren mittels Screening randomisierter AAV Peptidbanken. Die zur Entstehung der akuten myeloischen Leukämie (AML) führenden malignen Vorläuferzellen der Hämatopoese, sind durch herkömmliche Vektorsysteme nur in geringem Maße transduzierbar. In der vorliegenden Arbeit wurden mittels Screening randomisierter AAV Peptidbanken auf AML-Zellen virale Kapside mit einem eindeutigen Peptidmotif auf der Kapsidoberfläche angereichert. Rekombinante AAV Vektoren der Kapsidvariante NQVGSWS transduzierten die AML-Zelllinien auf denen sie selektiert worden waren mit einer Effizienz von bis zu 90% (Kontrolle mit unselektiertem Peptid ca. 0,2%). Bei Transduktionsexperimenten auf einer Vielzahl verschiedener Zelllinien zeigte sich, dass die Kasidvariante NQVGSWS die Transduktionsresistenz vor allem in hämatopoetischen Tumorzellen überwindet. In weiteren Versuchen wurde gezeigt, dass der Transduktionsmechanismus von NQVGSWS in Leukämiezellen unabhängig vom natürlichen zellulären Rezeptor Heparansulfat-Proteoglykan ist, welche für die Infektion von Wildtyp-AAV des verwendeten Serotyps 2 benötigt wird. Mittels der Kapsidvariante NQVGSWS wurde ein zielgerichteter zytotoxischer Gentherapieansatz auf Leukämiezellen etabliert und durchgeführt. Hieraus folgerten wir, dass die selektierte NQVGSWS-Mutante ein vielversprechender Vektor zum zielgerichteten Einbringen von therapeutischen Genen in Zellen der akuten myeloischen Leukämie darstellt. Die Selektion von AAV-Peptidbanken in vivo ist im Vergleich zu einem rein zell-basierten in vitro-Ansatz weitaus geeigneterer zur Selektion Gewebe-gerichteter Vektoren. Im zweiten Teil dieser Arbeit wurde daher ein Verfahren entwickelt welches auf PCR-Amplifikation der Peptidinsertinformation basiert. Dieser Ansatz ermöglichte eine in vivo Selektion nach systemischer Gabe der AAV-Banken über mehrere Runden auf Polyoma Mittel-T-induziertem Brustkrebsgewebe und Lungengewebe als Zielorgane im Mausmodell. Abhängig vom jeweiligen Zielorgan wurden verschiedene Vektorkapside angereichert, welche das entsprechende Zielgewebe nach systemischer Gabe transduzierten. Bei Tieren, die mit Kontrollvektoren injiziert wurden, konnte keine Genexpression in den jeweiligen Zielgeweben nachgewiesen werden. Doch war bei den selektierten Vektoren auch neben dem Zielgewebe eine zusätzliche Transduktion des Herzgewebes detektierbar. Diese Befunde werden einen großen Einfluss auf die Weiterentwicklung von AAV-Peptidbanken haben, da die Notwenigkeit deutlich wird, das AAV-Kapsid zusätzlich zu modifizieren, um eine gewebsspezifische Transduktion in vivo zu erreichen. Zusammenfassend zeigen die in dieser Arbeit geschilderten Ergebnisse, dass AAV- Peptidbanken sowohl in vitro als auch in vivo dazu geeignet sind, Genvektoren mit verbesserten Transduktionseigenschaften zu selektieren. Diese hier gewonnenen Erkenntnisse erweitern das grundlegende Verständnis für aus AAV-Peptidbanken isolierte Vektoren und zeigen zugleich, dass diese auch potenziell zum therapeutischen Einsatz geeignet sind.
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