Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus-cell fusion.
We have attempted to engineer murine leukemia virus (MuLV)-based retroviral vectors to specifically transduce cells expressing human CD34, an antigen present on the surface of undifferentiated hematopoietic stem cells. A number of chimeric ecotropic MuLV envelope (Env) proteins were constructed that contained anti-CD34 single-chain antibody variable fragments (scFvs). The scFv-Env proteins were generated either by replacing the receptor-binding domain of Env with the scFv or by inserting the scFv into the N terminus of the Env protein. Only chimeric Env proteins with scFv insertions between amino acids 6 and 7 were incorporated into viral particles, and coexpression of native MuLV Env did not rescue incorporation-defective proteins. In addition, the efficiency of incorporation varied with the specific anti-CD34 scFv that was used. Retroviral vectors containing the scFv-Env proteins bound to CD34+ cells and transduced NIH 3T3 cells expressing human CD34 (3T3-CD34 cells) at approximately twice the efficiency of the parental NIH 3T3 cells. However, the introduction of the mutation D84K, which prevents binding to the ecotropic MuLV receptor mcat-1, prevented transduction of both NIH 3T3 and 3T3-CD34 cells. Complementation cell-cell fusion assays [Zhao et al. (1997). J. Virol. 71, 6967-6972] in 3T3-CD34 cells revealed that although the scFv-Env proteins could contribute postbinding entry functions when bound to mcat-1, they were unable to do so when bound to CD34. Taken together, these data suggest that although the interaction with CD34 effectively increased the concentration of virus on 3T3-CD34 cells, entry could occur only through an interaction with mcat-1; CD34 alone was not capable of triggering the appropriate postbinding changes that lead to viral entry.
Benedict
C A
CA
Gene Therapy Laboratories, Norris Cancer Center, University of Southern California School of Medicine, Los Angeles 90033, USA.
Tun
R Y
RY
Rubinstein
D B
DB
Guillaume
T
T
Cannon
P M
PM
Anderson
W F
WF
D016475:3T3 Cells; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D018952:Antigens, CD34 / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D017931:DNA Primers; D015686:Gene Products, env / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D009052:Leukemia Virus, Murine / Q000235:genetics*; D051379:Mice; D014161:Transduction, Genetic
D000911:Antibodies, Monoclonal; D018952:Antigens, CD34; D017931:DNA Primers; D015686:Gene Products, env; D007135:Immunoglobulin Variable Region