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Retroviral-Fibronectin Interactions inTransduction of Mammalian Cells

DAVID A. WILLIAMSa

Section of Pediatric Hematology/Oncology, Herman B Wells Center for PediatricResearch, Riley Hospital for Children, Howard Hughes Medical Institute,Indiana University School of Medicine, 1044 W. Walnut Street -R4 402,Indianapolis, Indiana 46202, USA

ABSTRACT: Hematopoiesis occurs in a complex environment in the medul-lary cavity in close proximity to stromal cells, fibroblasts, endothelial cells andmatrix molecules. Hematopoietic cell interactions in this environment appearto involve both integrin and proteoglycan-mediated cell-cell and cell-matrix in-teractions. Genetic transduction of hematopoietic stem cells via retroviral vec-tors has been hampered by low efficiency of gene transfer. Recently,hematopoietic stem cell adhesion to the extracellular matrix molecule fibronec-tin has been shown to increase transduction of these target cells using retrovi-rus vectors. The mechanism of increased transduction appears to involvecolocalization of virus particles and target cells. These data are reviewed in thispaper.

Hematopoiesis occurs in a complex hematopoietic microenvironment made up ofendothelial cells, fibroblasts, macrophages and matrix proteins.1 Hematopoietic celladhesion in this environment is mediated via several cell-cell and cell-matrix inter-actions, which can involve both integrin- and proteoglycan-mediated processes.Primitive hematopoietic stem cells capable on long-term reconstitution in vivo ad-here to the extracellular matrix molecule fibronectin (FN) via the integrins very lateantigen (VLA)-42–4 and VLA-5,4a and also via cell surface proteoglycans.5 Se-quences mediating adhesion via VLA-5 are located in the central cell binding do-main of fibronectin, while VLA-4 interacts with a sequence termed connectingsegment-1 (CS-1) expressed via an alternative spliced mRNA of fibronectin. Cellsurface proteoglycans are known to interact with heparin binding domains located intype III repeats 12–14 (FIG. 1).

Genetic transduction of more primitive hematopoietic stem cells of large animalsvia retroviral vectors has previously been inefficient, limiting the application of genetransfer methods to therapeutic trials.6 Inefficient transduction of these cells is likelydue to multiple properties of hematopoietic stem cells, including the presence of alarge number of these cells in quiescent state, a low density of receptors for the bind-ing of retroviruses and the inability to manipulate stem cells in vitro for prolongedperiods of time without loss of repopulating capacity. In this regard, new approachesto gene transfer into hematopoietic stem cells of large animals have included: 1) theuse of different viral pseudotypes in an effort to utilize viruses with increased recep-

aPhone, 317/274-8960; fax, 317/274-8679; e-mail, dwilliam@iupui.edu

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tor density on the target cells; 2) the use of viruses that do not require cell divisionfor integration; 3) the use of in vivo selection methods to select and amplify a smallpopulation of transduced cells; 4) methods to increase the level of viral receptor ex-pression on target cells; and 5) methods to improve ex vivo manipulation of cellsand/or modify virus/cell interactions.

Previous work by Moore et al.7 and by Nolta et al.8 has shown increased genetransfer into hematopoietic cells adherent to bone marrow-derived stromal cells, al-though the mechanism of this increased transduction is unclear. In other cell sys-tems, integrin-mediated inside-out signaling has been shown to affect theproliferation of cells. Since our laboratory2 and other investigators3,4 have demon-strated expression of integrins on primitive hematopoietic cells, we have examinedthe effect of the adhesion of hematopoietic and other cells to fibronectin on genetransfer efficiency. Human CD34+ bone marrow or peripheral blood cells adhere tofibronectin fragments that contain the central cell binding domain, CS-1, and typeIII repeats 12–14 called CH-296 (FIG. 1). Infection of cells adherent to this fragmentincreases transduction 4–5-fold compared to nonadherent cells.9 This increase intransduction requires prestimulation with cytokines, apparently to increase the num-

FIGURE 1. Composition of recombinant FN fragments derived from sequences locatedwithin the A chain of FN. The binding sites for the integrins VLA-5 and VLA-4 are markedas CELL and CS-1, respectively. The CS-1 site is composed of the first 25 amino acids ofthe alternatively spliced IIICS region. The binding site for proteoglycans is marked as HE-PARIN for the heparin-binding domain spanning the type III repeats 12–14 (III 12–14).

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ber of cells in cycle, but also to increase the binding of the target cells to CH-296.By using this method, transduction of CD34+ bone marrow cell-derived colony-forming units (CFU) reaches 80%, while transduction of granulocyte colony-stimu-lating factor mobilized peripheral blood CD34-derived progenitors is as high as70%.10 More primitive CD34+, CD38− cells, a fraction believed to contain long-termrepopulating cells, is transduced at a frequency of 10–15%, based on CFU analysis.

Although these assays demonstrate a significant improvement in transduction ofprogenitor and primitive cell populations analyzed in vitro, a more difficult targetpopulation for retroviral infection and integration is the in vivo repopulating cell. Toanalyze transduction of cells in this hematopoietic compartment, bone marrow cellsderived from 5-fluorouracil-treated mice were either cocultivated directly on retro-viral producer cell lines, infected with supernatant virus in the absence of producercells or infected with supernatant after the target cells were adhered to the car-boxy-terminal fragment of fibronectin containing the CS-1 sequence and the type IIIrepeats 12–14. The vector used, phosphoglycerate kinase–human adenosine deami-nase (PGK-hADA), expresses the hADA cDNA via the phosphoglycerate kinasepromoter.11 Expression of the hADA transgene can be detected by in situ gel analy-sis and the level of expression compared to endogenous murine ADA expression. In-fected cells were harvested and infused into lethally irradiated mice. One year aftertransplantation, no expression of hADA was detectable in mice transplanted withcells infected with supernatant alone. In contrast, expression of hADA was easily de-tectable in mice transplanted with cells infected either by cocultivation or by the useof supernatant on FN-adherent cells.12 Thus these studies demonstrate that efficientinfection of long lived stem cells is possible in the presence of FN without the needfor cocultivation. Recently, Keim et al.13 extended these studies and demonstratedthat FN is superior to cocultivation in the transduction of primate repopulating cells.

In addition to a FN fragment containing the CS-1 sequence and type III repeats,we also utilized recombinant FN fragments that contain combinations of all threecell binding domains. Surprisingly, we found that transduction of murine stem cellswas facilitated on a fragment, CH-271, containing only the cell binding domain andtype III repeats, but not on a fragment, H-271, containing the type III repeats.14

These data, along with our previous data showing that colocalization of target cellsand virus is the mechanism by which FN facilitates gene transfer, suggested that re-populating murine stem cells adhere to the cell binding domain of FN. Such bindinghas not previously been demonstrated for reconstituting stem cell populations. Ad-hesion assays were performed using recombinant FN fragments with either murinebone marrow or granulocyte colony-stimulating factor (G-CSF) mobilized peripher-al blood cells. Analysis of adherent cells populations included colony-forming unit(CFU) and nonobese diabetic/severe combined immunodeficiency (NOD/SCID) en-graftment assays (human) and high proliferative potential–colony-forming cells(HPP-CFC) and competitive long-term repopulation assays (mouse). Data fromthese experiments demonstrate that both CFU and HPP-CFC largely adhere to thetype III repeats in combination with CS-1, as previously demonstrated for murineand human cells. Surprisingly, however, a population of NOD/SCID engrafting hu-man cells and long-term repopulating murine cells adhere to the cell binding do-main. This adhesion was specific and via VLA-5, since inhibition of adhesion couldbe demonstrated with anti-VLA-5 monoclonal antibodies.4a

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Additional studies in both CD34+ primary cells and hematopoietic cell lines showthat transduction of cell targets on FN is not affected by the apparent multiplicity ofinfection (titer of virus: cell number) over several logs. This result, and the ability topre-incubate virus on FN, increasing the apparent titer subsequently presented to thetarget cells (‘pre-loading’) suggests that saturation of the retroviral receptor whileusing FN is possible. In addition, specific targeting of cells within a mixed popula-tion may also be possible by exploiting unique cell surface receptors present on thetarget cell and utilizing chimeric FN fragments containing both retoviral binding se-quences and cell-specific ligands. These applications are currently being investigat-ed in our laboratory. For instance, cells that express VLA-4 but not VLA-5 aretransduced when CS-1, but not the cell binding domain is present in on the FN mol-ecule; while cells expressing VLA-5 but not VLA-4 are transduced on FN containingthe cell binding domain but not CS-1.14 Finally, incubation of primary T lympho-cytes with antibodies to CD3 and CD28 increases the adhesion of these cells to bothVLA-5 and VLA-4. By utilizing a FN fragment that contains both ligands (CH-296),up to 90% of primary T cells can be transduced, eliminating the need for selection.15

Since adhesion via integrins may affect cell cycle progression, or the position incell cycle may affect adhesion, the interaction of stem cells with FN during retroviralinfection requires further analysis to investigate the affects of adhesion on stem cellsurvival and repopulating capacity. However, the data presented here demonstratehighly efficient transduction of human and murine hematopoietic cells, routinelyyielding transduction efficiencies of 50–70%, and eliminating the use of cocultiva-tion, polycations and extended exposure to growth factors and stroma. The use of re-combinant FN is currently being studied in human Phase I trials.

REFERENCES

1. YODER, M.C. & D.A. WILLIAMS. 1995. Matrix molecule interactions with hematopoie-tic stem cells. Exp. Hematol. 23: 961–967.

2. WILLIAMS, D.A., M. RIOS, C. STEPHENS & V. PATEL. 1991. Fibronectin and VLA-4 inhaematopoietic stem cell–microenvironment interactions. Nature 352: 438–441.

3. VERFAILLIE, C.M., J.B. MCCARTHY & P.B. MCGLAVE. 1991. Differentiation of primi-tive human nultipotent hematopoietic progenitors is accompanied by alterations intheir interaction with fibronectin. J. Exp. Med. 174: 693–703.

4. PAPAYANNOPOULOU, T. & B. NAKAMOTO. 1993. Peripheralization of hematopoieticprogenitors in primates treated with anti-VLAÃ Ã4Ä Ä integrin. Proc. Natl. Acad.Sci. USA 90: 9374–9378.

4a. VAN DER LOO, J.C., X. XIAO, D. MCMILLIN, K. HASHINO, I. KATO & D.A. WILLIAMS.1998. VLA-5 is expressed by mouse and human long-term repopulating hematopoieticcells and mediates adhesion to extracellular matrix protein fibronectin. J. Clin. Invest.102: 1051–1061.

5. MINGUELL, J.J., C. HARDY & M. TAVASSOLI. 1992. Membrane associated chondroitinsulfate proteoglycan and fibronectin mediate the binding of hemopoietic progenitorcells to stromal cells. Exp. Cell Res. 201: 200–207.

6. MORITZ, T. & D.A. WILLIAMS. 1994. Somatic gene therapy. In Scientific Basis ofTransfusion Medicine. K. Anderson, Ed.: 872–888. Churchill Livingstone. Philadel-phia.

7. MOORE, K.A., A.B. DEISSEROTH, C.L. READING, D.E. WILLIAMS & J.W. BELMONT.1992. Stromal support enhances cell-free retroviral vector transduction of humanbone marrow long-term culture-initiating cells. Blood 79: 1393–1399.

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8. NOLTA, J.A., E.M. SMOGORZEWSKA & D.B. KOHN. 1995. Analysis of optimal condi-tions for retroviral-mediated transduction of primitive human hematopoietic cells.Blood 86: 101–110.

9. MORITZ, T., V.P. PATEL & D.A. WILLIAMS. 1994. Bone marrow extracelolular matrixmolecules improve gene transfer into human hematopoietic cells via retroviral vec-tors. J. Clin. Invest. 93: 1451–1457.

10. HANENBERG, H., K. HASHINO, H. KONISHI, R.A. HOCK, I. KATO & D.A. WILLIAMS.1997. Optimization of fibronectin-assisted retroviral gene transfer into humanCD34+ hematopoietic cells. Hum. Gene Ther. 8: 2193–2206.

11. LIM, B., D.A. WILLIAMS & S. H. ORKIN. 1987. Retrovirus-mediated gene transfer ofhuman adenosine deaminase: Expression of functional enzyme in murine hemato-poietic stem cells in vivo. Mol. Cell. Biol. 7: 3459–3465.

12. MORITZ, T., P. DUTT, X.L. XIAO, D. CARSTANJEN, T. VIK, H. HANENBERG & D.A.WILLIAMS. 1996. Fibronectin improves transduction of reconstituting hematopoie-tic stem cells by retroviral vectors: evidence of direct viral binding to chymotrypticcarboxy-terminal fragments. Blood 88: 855–862.

13. KIEM, H.P., J. MORRIS, S. HEYWARD, L. PETERSON, J. POTTER, A.D. MILLER & R. G.ANDREWS. 1997. Gene transfer into baboon hematopoietic repopulating cells usingrecombinant human fibronectin fragment CH-296. Blood 90(Suppl.): 236a.

14. HANENBERG, H., X.L. XIAO, D. DILLOO, K. HASHINO, I. KATO & D.A. WILLIAMS.1996. Colocalization of retrovirus and target cells on specific fibronectin frag-ments increases genetic transduction of mammalian cells. Nat. Med. 2(8): 876–882.

15. POLLOK, K.E., H. HANENBERG, T.W. NOBLITT, W.L. SCHROEDER, I. KATO, D. EMAN-UEL & D.A. WILLIAMS. 1998. High-efficiency gene transfer into normal and ade-nosine deaminase-deficient T-lymphocytes is mediated by transduction onrecombinant fibronectin fragments. J. Virol. 72: 4882–4892.

DISCUSSION

G. KELLER (Howard Hughes Medical Institute): Does binding to fibronectin in-duce a signal in the cell?

WILLIAMS: We cannot answer that directly yet. That is what Gillian Bradford,who joined the laboratory from Ivan Bertincello’s group, is examining. But based onother cell systems, that is what you would hypothesize. Binding of the integrin itselfprobably sends a signal that relates to differentiation and proliferation, but probablythe binding actually colocalizes signaling molecules related to growth factor recep-tors. Therefore, one would hypothesize that this probably is an important and com-plex system by which the cell amplifies signals from growth factors. That is clearlywhere we are fucusing our studies right now. We have some evidence that this is animportant interaction. If one takes cells in the murine system, Sca+Lin− cells, and in-cubates them in vitro either on or off fibronectin and very low concentrations ofgrowth factor, i.e., not the concentrations you normally would use to do progenitorassays, and then tests to see how long the reconstituting cells survive, it is clear thatwith cells adherent to fibronectin, survival is maintained for prolonged periods in vit-ro versus nonadherent cells. That is the key observation in our laboratory that tellsus that this is an important aspect of biology to look at.

T. PAPAYANNOPOULOU (University of Washington): Does the CH-296 inducemobilization of stem/progenitor cells into the blood?

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WILLIAMS: We looked at the spleen, but we have not looked at the blood. We de-cided to look at the spleen of these animals. CH-296 injected in vivo does perturbhematopoiesis in the spleen, increasing the number of CFUs contained within thespleen.