David Larocca

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS‐1 cells, resulting in receptor‐mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage‐mediated gene transfer as a novel display phage screening strategy, we transfected COS‐1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS‐1 cells with the targeted FGF2‐phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage‐mediated transduction as a screen for discovering novel ligands.—Larocca, D., Kassner, P. D., Witte, A., Ladner, R. C., Pierce, G., Baird, A. Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage. FASEB J. 13, 727–734 (1999)

Evolving Phage Vectors for Cell Targeted Gene Delivery

We adapted filamentous phage vectors for targeted gene delivery to mammalian cells by inserting a mammalian reporter gene expression cassette (GFP) into the vector backbone and fusing the pIII coat protein to a cell targeting ligand (i.e. FGF2, EGF). Like transfection with animal viral vectors, targeted phage gene delivery is concentration, time, and ligand dependent. Importantly, targeted phage particles are specific for the appropriate target cell surface receptor. Phage have distinct advantages over existing gene therapy vectors because they are simple, economical to produce at high titer, have no intrinsic tropism for mammalian cells, and are relatively simple to genetically modify and evolve. Initially transduction by targeted phage particles was low resulting in foreign gene expression in 1-2% of transfected cells. We increased transduction efficiency by modifying both the transfection protocol and vector design. For example, we stabilized the display of the targeting ligand to create multivalent phagemid-based vectors with transduction efficiencies of up to 45% in certain cell lines when combined with genotoxic treatment. Taken together, these studies establish that the efficiency of phage-mediated gene transfer can be significantly improved through genetic modification. We are currently evolving phage vectors with enhanced cell targeting, increased stability, reduced immunogenicity and other properties suitable for gene therapy.

Receptor-mediated gene transfer by phage-display vectors : applications in functional genomics and gene therapy

Recent studies have demonstrated targeted gene-delivery to mammalian cells using modified phage-display vectors. Specificity is determined by the choice of the genetically displayed targeting ligand. Without targeting, phage particles have virtually no tropism for mammalian cells. Thus, novel ligands can be selected from phage libraries by their ability to deliver a reporter gene to targeted cells. Together with advances in cDNA display technologies, these findings offer new opportunities for the use of phage-display technology in functional genomics. In addition, targeted phage particles have potential as alternative gene therapy vectors that can be further improved using directed evolution.

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection.

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.

A novel serum assay for breast epithelial antigen using a fusion protein

Serum levels of breast epithelial antigens (BrE-Ags) are presently used in the follow-up of breast cancer patients. Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity. A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of the NP5 fusion protein, produced in Escherichia coli, that is composed of beta-galactosidase and polypeptide sequence obtained from a breast carcinoma cell line cDNA library, and anti-human milk fat globule monoclonal antibody Mc5. The fusion protein carries an altered epitope sequence (mimotope) that is similar, but not identical, to that found in the native antigen. This new competitive assay configuration has two essential features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for Mc5 and a competitive step that provides quantitation. Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.

Selection of Internalizing Ligand-Display Phage Using Rolling Circle Amplification for Phage Recovery

Selection of phage libraries against complex living targets such as whole cells or organs can yield valuable targeting ligands without prior knowledge of the targeted receptor. Our previous studies have shown that noninfective multivalent ligand display phagemids internalize into mammalian cells more efficiently than their monovalent counterparts suggesting that cell-based selection of internalizing ligands might be improved using multivalently displayed peptides, antibodies or cDNAs. However, alternative methods of phage recovery are needed to select phage from noninfective libraries. To this end, we reasoned that rolling circle amplification (RCA) of phage DNA could be used to recover noninfective phage. In feasibility studies, we obtained up to 1.5 million-fold enrichment of internalizing EGF-targeted phage using RCA. When RCA was applied to a large random peptide library, eight distinct human prostate carcinoma cell-internalizing peptides were isolated within three selection rounds. These data establish RCA as an alternative to infection for phage recovery that can be used to identify peptides from noninfective phage display libraries or infective libraries under conditions where there is the potential for loss of phage infectivity.

Molecular Analysis of Epitopic Heterogeneity of the Breast Mucin

The breast mucin is a highly glycosylated large molecular weight glycoprotein on the surface of breast epithelial cells, in breast carcinoma cells, and is a major component of the human milk fat globule membrane. By epitope mapping we have determined that 5 different monoclonal antibodies (Mc1, Mc5, BrEl, BrE2, BrE3) raised against human milk fat globule and selected for breast specificity recognize 4 distinct but overlapping linear amino acid sequences encompassing the most hydrophilic region of the 20 amino acid tandem repeat that makes up a large part of polypeptide core of the breast mucin. Although these MoAbs bind to overlapping polypeptide epitopes they have different tissue and tumor specificities in histopathology, differ quantitatively in the binding to breast carcinoma cell lines by flow cytometry, and they have distinct competition patterns for bind-ng to the native antigen on breast carcinoma cells. Even though BrE2 nd BrE3 have the same polypeptide epitope, they differ in their relative binding to breast carcinoma cell as determined by flow cytometry and binding to fusion proteins containing mimotopes produced by cDNA clone isolated from a breast cDNA library. There is considerable variation in the breast mucin mRNA, but the expression of the different epitopes shows little correlation with mRNA levels. Surface expression of an epitope and detection of the epitope in secreted material is associated with its presence on a larger size molecular specie than the exclusively cytoplasmic expression of an epitope. This detailed analysis of the epitopic heterogeneity of an immunodominant region of the tandem repeat segment of the core polypeptide of the breast mucin, possibly involving altered glycosylation, reveals an epitopic heterogenity that may have functional significance and suggests approaches for preparing new monoclonal antibodies with improved qualities for breast cancer diagnosis and/or therapy.

Synthesis of hypomethylated epstein-barr viral DNA is stimulated by dimethylsulfoxide treatment of lymphoblastoid cells

Summary Dimethylsulfoxide, previously described as an inducer of cell differentiation, induced Epstein-Barr viral DNA synthesis but inhibited host cell DNA synthesis in an Epstein-Barr virus producer lymphoblastoid cell line. The viral DNA, whose synthesis was stimulated, was hypomethylated, in contrast to the cellular DNA, which was highly methylated and whose synthesis was inhibited by dimethylsulfoxide treatment of the cells. No alteration of DNA methylation patterns was observed in treated cells. Dimethylsulfoxide therefore appeared to selectively stimulate synthesis of previously hypomethylated DNA while inhibiting synthesis of methylated DNA within this virus-producing cell line.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.

A Novel Serum Assay Using Recombinant Breast Epithelial Mucin Antigen

Serum levels of breast epithelial antigens are presently used in the follow-up of breast cancer patients. Available assays do not have optimal sensitivity and rely on reagents that could vary in their source and purity. A novel competitive solid-phase radioimmunoassay was developed for BrE-Ags that consists of a fusion protein, (comprising a s-galactosidase fusion partner and a polypeptide sequence obtained from a breast carcinoma cell line cDNA library), and anti-human milk fat globule monoclonal antibody Mc5, both produced biosynthetically. The fusion protein carries a modified epitope sequence (mimotope) similar, but not identical, to the one found in the native antigen. This new competitive assay configuration has two features, a solid-phase affinity step that purifies the fusion protein carrying the mimotope for MсS, and a competitive step that provides quantitation. Serum values for this assay show high specificity and sensitivity for breast cancer patients when compared to normal subjects and post-surgical breast cancer patients during their disease-free period.