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Keith Bupp

Publications and source records attributed to Keith Bupp.

5 recordsLinked to original sources
PubMed2006

Selection of feline leukemia virus envelope proteins from a library by functional association with a murine leukemia virus envelope.

Libraries of feline leukemia virus subgroup A (FeLV-A)-derived envelope (Env) proteins with random peptides incorporated into the cell-targeting region were screened for productive gene delivery to the PC-3 human prostate cell line. In order to increase the efficiency of recovering and testing functional clones, the screen was performed in the presence of a replication-competent 4070A Env-expressing virus under conditions of viral interference. The Env proteins resulting from this library screen were able to mediate gene delivery to 4070A-infected human PC-3, DU145 prostate and TE671 rhabdomyosarcoma cells in the presence, but not absence, of 4070A helper virus. FeLV-A, FeLV-B and Moloney murine leukemia virus (Mo-MuLV) Env proteins were unable to substitute for 4070A Env. Flow cytometry and Western blot analyses indicated increased cell-surface expression and virion incorporation of library-derived Env proteins in the presence of 4070A Env. Interference assays on cells infected with both 4070A and FeLV-B are consistent with the combination of library-derived and 4070A Env proteins utilizing the Pit1 receptor.

Animals↗
PubMed2005

Probing sequence variation in the receptor-targeting domain of feline leukemia virus envelope proteins with peptide display libraries.

Determinants of cellular tropism and receptor targeting lie within a short peptide in the Vr1 region of the envelope (Env) proteins of feline leukemia virus (FeLV) subgroups A and C. Libraries of FeLV Env proteins with random amino acid substitutions in the peptide were screened for their ability to deliver a marker gene to D17 and AH927 cells. Screening on D17 canine cells yielded D17-specific Env proteins that used the FeLV-C receptor. Screening on AH927 cells yielded Env proteins with a broader host range, with maximal titers on AH927 cells and similar or lower titers on other cells. These Env proteins used an unidentified non-FeLV receptor for entry. The A5 isolate obtained from the AH927 screen was readily concentrated to yield titers of 10(5) on human PC-3 prostate tumor cells. The sequence divergence observed among targeting peptides of library-selected Env proteins was greater than that found in parental FeLV isolates. Substitution analyses of a conserved R in the middle of the targeting peptide held constant during screening indicated that maximal titers were obtained only when R was present in both a D17 selected isolate and an AH927 selected isolate. The ability to isolate Env proteins with unique tropisms dependent on the cells on which the library is screened has direct implications for targeting gene delivery vectors.

Amino Acid Sequence↗
PubMed2003

Targeting a retroviral vector in the absence of a known cell-targeting ligand.

An important requirement for many gene therapy applications is to direct therapeutic genes specifically to target cells. Here we describe an improved vector targeting method that does not depend on the use of a known cell-targeting ligand. It entails screening a library of constitutively produced retroviruses with random amino acid substitutions in the cell-targeting region of the envelope proteins for their ability to mediate gene delivery to a target cell. By screening such a library on the ras-transformed 143B human cell line, we have isolated an envelope protein that preferentially targets 143B cells and 293T cells expressing the SV40 T antigen via a novel, unidentified receptor. Furthermore, retroviruses expressing the library-derived envelope protein can be concentrated by centrifugation. This is the first demonstration of a novel concept in vector targeting: the selection of productive retroviral entry via an alternate receptor with modified cellular tropism in the absence of a known cell-targeting moiety. The method is, in principle, applicable even to cells that have not been well characterized, and therefore potentially suitable for targeting many diverse cell types.

Cell Line↗
PubMed2002

Altering retroviral tropism using a random-display envelope library.

Tissue-specific gene delivery is an important aspect of many gene therapy applications. The experiments reported here constitute the first successful demonstration that cell-specific entry can be obtained by screening a random library of retroviral envelope proteins produced from a mammalian cell system. The library consisted of 10(6) different subgroup A feline leukemia virus envelope protein variants with 10 randomly substituted amino acids in the receptor-determining region. Selecting the library for fully functional envelope proteins able to mediate stable gene transfer resulted in the identification of a single envelope protein variant (EF). Subsequent examination of the host range of EF revealed that it was highly specific for D17 canine osteosarcoma cells. This was in contrast to the host ranges of the parental subgroup A and closely related subgroup C envelope proteins. Interference assays on D17 cells further indicated that receptor usage by EF was also altered compared with the A and C envelope proteins. The EF envelope protein thus isolated should be useful for studying gene therapy treatments of osteosarcoma in a large-animal model.

Amino Acid Sequence↗