Retroviral display of antibody fragments; interdomain spacing strongly influences vector infectivity

• Five different single-chain antibody fragments (scFv) against human cell-surface antigens were displayed on murine ecotropic retroviral vectors by fusing them to the Moloney SU envelope glycoprotein. The spacing between the scFv and the SU glycoprotein was varied by fusing the scFv to residue +7 or to residue +1 of Moloney SU and by inserting linker sequences of different lengths between the domains. All of the chimeric envelopes were efficiently incorporated into vector particles and could bind to human cells through their displayed antibody fragments, but did not infect them. The spacing between the scFvs and the SU glycoproteins had no significant effect on the efficiency of envelope expression or viral incorporation and did not affect the binding properties of the chimeric envelopes, nor did it influence the efficiency of targeted gene delivery to human cells by scFv-displaying vectors. However, on murine fibroblasts the infectivity of vectors incorporating the chimeric envelopes was strongly influenced by the length of the interdomain spacer. The titers were very low when the single-chain antibodies were fused through a tripeptide linker to SU residue +7 and were greatly enhanced (up to 10(5)-fold) when they were fused to SU residue +1 through a heptapeptide linker. These results point to the importance of steric interactions between the domains of chimeric envelope glycoproteins and may have implications for retroviral vector design for human gene therapy.

Ämnesord

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Medicinsk genetik (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Medical Genetics (hsv//eng)

Nyckelord

Amino Acid Sequence

Animals

Antigen-Antibody Reactions

Antigens, Surface

Base Sequence

CD3 Complex

Cells, Cultured

Cloning, Molecular

Fibroblasts

Flow Cytometry

Gene Expression Regulation

Genetic Therapy

Genetic Vectors

Glycoproteins

Humans

Immunoblotting

Introns

Mice

Molecular Sequence Data

Moloney murine leukemia virus

Mutagenesis, Insertional

Plasmids

Journal Article

Research Support, Non-U.S. Gov't

Publikations- och innehållstyp

art (ämneskategori)

ref (ämneskategori)