Cell Specific Gene Targeting to the CNS Using Engineered Lentiviruses

Viral-mediated gene targeting to specific cells and organs is a major challenge for establishment of an attractive gene therapy regimen. We have developed successfully a gene delivery platform into cells using lentiviral vectors that are pseudotyped with a sindbis mutated envelope and a single chain variable fragment (scFv). Incorporation of scFv onto the surface of viral particles was obtained either via interaction of scFv fused to human Fc antibody domain (scFvFc) with a protein A moiety, inserted to the sindbis E2 envelope protein, or through transient surface display of scFvFc fused with a transmembrane domain of cd28. A third approach facilitated stable surface expression of scFvFc fused with a transmembrane domain of cd28 by expressing it in a lentivector along with an Internal Ribosome Entry Site reporter gene (IRES), thus enhancing the incorporation of scFv to engineered particles. To demonstrate the feasibility of the targeting system, two versions of scFv that specifically target the receptor-binding region of the S1 spike glycoprotein of the Severe Acute Respiratory Syndrome Coronavirus (SARS CoV) strains were used. Despite high similarity of the two S1 antigens, gene transfer was obtained with low background of transduction levels (low levels of unspecific infection), indicating high affinity of the surface displayed scFv to their cognate antigen. Significantly, this delivery platform was exploited for in-vivo targeting of recombinant lentiviruses that incorporate an NG2 antibody to mice hippocampal cells. Colocalization of surface expressed NG2 and lentiviral mediated gene transfer of Zoanthus Green Fluorescent protein (ZsGreen) were detected. Overall, these results demonstrate efficacy of lentiviral vectors as a gene delivery platform. Furthermore, such a system could potentially be used to mark specific cells populations for studying their in-vivo biology under health and disease.

On the formation of the essential structural relationships for In-Cell recording and